U.S. patent application number 15/598262 was filed with the patent office on 2017-10-26 for methods of treating cancer.
The applicant listed for this patent is Epizyme, Inc.. Invention is credited to Heike KEILHACK, Sarah K. KNUTSON, Natalie WARHOLIC.
Application Number | 20170305890 15/598262 |
Document ID | / |
Family ID | 49519104 |
Filed Date | 2017-10-26 |
United States Patent
Application |
20170305890 |
Kind Code |
A1 |
KNUTSON; Sarah K. ; et
al. |
October 26, 2017 |
METHODS OF TREATING CANCER
Abstract
The present invention relates to methods of treating cancer by
administering the EZH2 inhibitor compounds and pharmaceutical
compositions to subjects in need thereof. The present invention
also relates to the use of such compounds for research or other
non-therapeutic purposes.
Inventors: |
KNUTSON; Sarah K.; (Lincoln,
MA) ; WARHOLIC; Natalie; (Brighton, MA) ;
KEILHACK; Heike; (Belmont, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Epizyme, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
49519104 |
Appl. No.: |
15/598262 |
Filed: |
May 17, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14054646 |
Oct 15, 2013 |
9688665 |
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15598262 |
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61786277 |
Mar 14, 2013 |
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61780703 |
Mar 13, 2013 |
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61758972 |
Jan 31, 2013 |
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61714140 |
Oct 15, 2012 |
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61714045 |
Oct 15, 2012 |
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61714145 |
Oct 15, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 11/00 20180101;
A61P 15/00 20180101; A61P 7/00 20180101; A61K 31/5377 20130101;
C07D 211/86 20130101; A61P 13/08 20180101; A61K 31/4545 20130101;
A61P 25/00 20180101; C07D 405/14 20130101; A61P 1/18 20180101; A61P
35/02 20180101; C07D 405/12 20130101; C12Q 1/6886 20130101; A61K
31/4412 20130101; A61P 35/00 20180101; A61P 43/00 20180101; A61P
13/12 20180101 |
International
Class: |
C07D 405/14 20060101
C07D405/14; C07D 405/12 20060101 C07D405/12; C07D 211/86 20060101
C07D211/86; A61K 31/5377 20060101 A61K031/5377; A61K 31/4545
20060101 A61K031/4545; C12Q 1/68 20060101 C12Q001/68; A61K 31/4412
20060101 A61K031/4412 |
Claims
1. A method comprising administering to a subject having
epithelioid sarcoma a therapeutically effective amount of a
compound being of Formula (V) or a pharmaceutically acceptable salt
thereof: ##STR00051## wherein R.sub.2, R.sub.4 and R.sub.12 are
each, independently C.sub.1-6 alkyl; R.sub.6 is C.sub.6-C.sub.10
aryl or 5- or 6-membered heteroaryl, each of which is optionally
substituted with one or more -Q.sub.2-T.sub.2, wherein Q.sub.2 is a
bond or C.sub.1-C.sub.3 alkyl linker optionally substituted with
halo, cyano, hydroxyl or C.sub.1-C.sub.6 alkoxy, and T.sub.2 is H,
halo, cyano, --OR.sub.a, --NR.sub.aR.sub.b,
--(NR.sub.aR.sub.bR.sub.c).sup.+A.sup.-, --C(O)R.sub.a,
--C(O)OR.sub.a, --C(O)NR.sub.aR.sub.b, --NR.sub.bC(O)R.sub.a,
--NR.sub.bC(O)OR.sub.a, --S(O).sub.2R.sub.a,
--S(O).sub.2NR.sub.aR.sub.b, or R.sub.S2, in which each of R.sub.a,
R.sub.b, and R.sub.c, independently is H or R.sub.S3, A.sup.- is a
pharmaceutically acceptable anion, each of R.sub.S2 and R.sub.S3,
independently, is C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered
heterocycloalkyl, or 5- or 6-membered heteroaryl, or R.sub.a and
R.sub.b, together with the N atom to which they are attached, form
a 4 to 12-membered heterocycloalkyl ring having 0 or 1 additional
heteroatom, and each of R.sub.S2, R.sub.S3, and the 4 to
12-membered heterocycloalkyl ring formed by R.sub.a and R.sub.b, is
optionally substituted with one or more -Q.sub.3-T.sub.3, wherein
Q.sub.3 is a bond or C.sub.1-C.sub.3 alkyl linker each optionally
substituted with halo, cyano, hydroxyl or C.sub.1-C.sub.6 alkoxy,
and T.sub.3 is selected from the group consisting of halo, cyano,
C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10
aryl, 4 to 12-membered heterocycloalkyl, 5- or 6-membered
heteroaryl, OR.sub.d, COOR.sub.d, --S(O).sub.2R.sub.d,
--NR.sub.dR.sub.e, and --C(O)NR.sub.dR.sub.c, each of R.sub.d and
R.sub.e independently being H or C.sub.1-C.sub.6 alkyl, or
-Q.sub.3-T.sub.3 is oxo; or any two neighboring -Q.sub.2-T.sub.2,
together with the atoms to which they are attached form a 5- or
6-membered ring optionally containing 1-4 heteroatoms selected from
N, O and S and optionally substituted with one or more substituents
selected from the group consisting of halo, hydroxyl, COOH,
C(O)O--C.sub.1-C.sub.6 alkyl, cyano, C.sub.1-C.sub.6 alkoxyl,
amino, mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6
alkylamino, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to
12-membered heterocycloalkyl, and 5- or 6-membered heteroaryl;
R.sub.7 is -Q.sub.4-T.sub.4, in which Q.sub.4 is a bond,
C.sub.1-C.sub.4 alkyl linker, or C.sub.2-C.sub.4 alkenyl linker,
each linker optionally substituted with halo, cyano, hydroxyl or
C.sub.1-C.sub.6 alkoxy, and T.sub.4 is H, halo, cyano,
NR.sub.fR.sub.g, --OR.sub.f, --C(O)R.sub.f, --C(O)OR.sub.f,
--C(O)NR.sub.fR.sub.g, --C(O)NR.sub.fOR.sub.g,
--NR.sub.fC(O)R.sub.g, --S(O).sub.2R.sub.f, or R.sub.S4, in which
each of R.sub.f and R.sub.g, independently is H or R.sub.S5, each
of R.sub.S4 and R.sub.S5, independently is C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered
heterocycloalkyl, or 5- or 6-membered heteroaryl, and each of
R.sub.S4 and R.sub.S5 is optionally substituted with one or more
-Q.sub.5-T.sub.5, wherein Q.sub.5 is a bond, C(O), C(O)NR.sub.k,
NR.sub.kC(O), S(O).sub.2, or C.sub.1-C.sub.3 alkyl linker, R.sub.k
being H or C.sub.1-C.sub.6 alkyl, and T.sub.5 is H, halo,
C.sub.1-C.sub.6 alkyl, hydroxyl, cyano, C.sub.1-C.sub.6 alkoxyl,
amino, mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6
alkylamino, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to
12-membered heterocycloalkyl, 5- or 6-membered heteroaryl, or
S(O).sub.qR.sub.q in which q is 0, 1, or 2 and R.sub.q is
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to
12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl, and
T.sub.5 is optionally substituted with one or more substituents
selected from the group consisting of halo, C.sub.1-C.sub.6 alkyl,
hydroxyl, cyano, C.sub.1-C.sub.6 alkoxyl, amino,
mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6 alkylamino,
C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered
heterocycloalkyl, and 5- or 6-membered heteroaryl except when
T.sub.5 is H, halo, hydroxyl, or cyano; or -Q.sub.5-T.sub.5 is oxo;
and R.sub.8 is H, halo, hydroxyl, COOH, cyano, R.sub.S6, OR.sub.S6,
or COOR.sub.S6, in which R.sub.S6 is C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.8
cycloalkyl, 4 to 12-membered heterocycloalkyl, amino,
mono-C.sub.1-C.sub.6 alkylamino, or di-C.sub.1-C.sub.6 alkylamino,
and R.sub.S6 is optionally substituted with one or more
substituents selected from the group consisting of halo, hydroxyl,
COOH, C(O)O--C.sub.1-C.sub.6 alkyl, cyano, C.sub.1-C.sub.6 alkoxyl,
amino, mono-C.sub.1-C.sub.6 alkylamino, and di-C.sub.1-C.sub.6
alkylamino; or R.sub.7 and R.sub.8, together with the N atom to
which they are attached, form a 4 to 11-membered heterocycloalkyl
ring having 0 to 2 additional heteroatoms, and the 4 to 11-membered
heterocycloalkyl ring formed by R.sub.7 and R.sub.8 is optionally
substituted with one or more -Q.sub.6-T.sub.6, wherein Q.sub.6 is a
bond, C(O), C(O)NR.sub.m, NR.sub.mC(O), S(O).sub.2, or
C.sub.1-C.sub.3 alkyl linker, R.sub.m being H or C.sub.1-C.sub.6
alkyl, and T.sub.6 is H, halo, C.sub.1-C.sub.6 alkyl, hydroxyl,
cyano, C.sub.1-C.sub.6 alkoxyl, amino, mono-C.sub.1-C.sub.6
alkylamino, di-C.sub.1-C.sub.6 alkylamino, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered
heterocycloalkyl, 5- or 6-membered heteroaryl, or S(O).sub.pR.sub.p
in which p is 0, 1, or 2 and R.sub.p is C1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered
heterocycloalkyl, or 5- or 6-membered heteroaryl, and T.sub.6 is
optionally substituted with one or more substituents selected from
the group consisting of halo, C.sub.1-C.sub.6 alkyl, hydroxyl,
cyano, C.sub.1-C.sub.6 alkoxyl, amino, mono-C.sub.1-C.sub.6
alkylamino, di-C.sub.1-C.sub.6 alkylamino, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered
heterocycloalkyl, and 5- or 6-membered heteroaryl except when
T.sub.6 is H, halo, hydroxyl, or cyano; or -Q.sub.6-T.sub.6 is
oxo.
2. The method of claim 1, wherein R.sub.6 is C.sub.6-C.sub.10 aryl
or 5- or 6-membered heteroaryl, each of which is optionally,
independently substituted with one or more -Q.sub.2-T.sub.2,
wherein Q.sub.2 is a bond or C.sub.1-C.sub.3 alkyl linker, and
T.sub.2 is H, halo, cyano, --OR.sub.a, --NR.sub.aR.sub.b,
--(NR.sub.aR.sub.bR.sub.c).sup.+A.sup.-, --C(O)NR.sub.aR.sub.b,
--NR.sub.bC(O)R.sub.a, --S(O).sub.2R.sub.a, or R.sub.S2, in which
each of R.sub.a and R.sub.b, independently is H or R.sub.S3, each
of R.sub.S2 and R.sub.S3, independently, is C.sub.1-C.sub.6 alkyl,
or R.sub.a and R.sub.b, together with the N atom to which they are
attached, form a 4 to 7-membered heterocycloalkyl ring having 0 or
1 additional heteroatom, and each of R.sub.S2, R.sub.S3, and the 4
to 7-membered heterocycloalkyl ring formed by R.sub.a and R.sub.b,
is optionally, independently substituted with one or more
-Q.sub.3-T.sub.3, wherein Q.sub.3 is a bond or C.sub.1-C.sub.3
alkyl linker and T.sub.3 is selected from the group consisting of
halo, C.sub.1-C.sub.6 alkyl, 4 to 7-membered heterocycloalkyl,
OR.sub.d, --S(O).sub.2R.sub.d, and --NR.sub.dR.sub.e, each of
R.sub.d and R.sub.e independently being H or C.sub.1-C.sub.6 alkyl,
or -Q.sub.3-T.sub.3 is oxo; or any two neighboring
-Q.sub.2-T.sub.2, together with the atoms to which they are
attached form a 5- or 6-membered ring optionally containing 1-4
heteroatoms selected from N, O and S.
3. The method of claim 1, wherein the compound is of Formula (VI)
or a pharmaceutically acceptable salt thereof: ##STR00052## wherein
Q.sub.2 is a bond or methyl linker, T.sub.2 is H, halo, --OR.sub.a,
--NR.sub.aR.sub.b, --(NR.sub.aR.sub.bR.sub.c).sup.+A.sup.-, or
--S(O).sub.2NR.sub.aR.sub.b, R.sub.7 is piperidinyl,
tetrahydropyran, cyclopentyl, or cyclohexyl, each optionally
substituted with one -Q.sub.5-T.sub.5 and R.sub.8 is ethyl.
4. The method of claim 1, wherein the compound is of Formula (VIa)
or a pharmaceutically acceptable salt thereof: ##STR00053## wherein
each of R.sub.a and R.sub.b, independently is H or R.sub.S3,
R.sub.S3 being C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.10 aryl, 4 to 12-membered heterocycloalkyl, or 5- or
6-membered heteroaryl, or R.sub.a and R.sub.b, together with the N
atom to which they are attached, form a 4 to 12-membered
heterocycloalkyl ring having 0 or 1 additional heteroatom, and each
of R.sub.S3 and the 4 to 12-membered heterocycloalkyl ring formed
by R.sub.a and R.sub.b, is optionally substituted with one or more
-Q.sub.3-T.sub.3, wherein Q.sub.3 is a bond or C.sub.1-C.sub.3
alkyl linker each optionally substituted with halo, cyano, hydroxyl
or C.sub.1-C.sub.6 alkoxy, and T.sub.3 is selected from the group
consisting of halo, cyano, C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered
heterocycloalkyl, 5- or 6-membered heteroaryl, OR.sub.d,
COOR.sub.d, --S(O).sub.2R.sub.d, --NR.sub.dR.sub.e, and
--C(O)NR.sub.dR.sub.e, each of R.sub.d and R.sub.e independently
being H or C.sub.1-C.sub.6 alkyl, or -Q.sub.3-T.sub.3 is oxo;
R.sub.7 is -Q.sub.4-T.sub.4, in which Q.sub.4 is a bond,
C.sub.1-C.sub.4 alkyl linker, or C.sub.2-C.sub.4 alkenyl linker,
each linker optionally substituted with halo, cyano, hydroxyl or
C.sub.1-C.sub.6 alkoxy, and T.sub.4 is H, halo, cyano,
NR.sub.fR.sub.g, --OR.sub.f, --C(O)R.sub.f, --C(O)OR.sub.f,
--C(O)NR.sub.fR.sub.g, --C(O)NR.sub.fOR.sub.g,
--NR.sub.fC(O)R.sub.g, --S(O).sub.2R.sub.f, or R.sub.S4, in which
each of R.sub.f and R.sub.g, independently is H or R.sub.S5, each
of R.sub.S4 and R.sub.S5, independently is C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 7-membered
heterocycloalkyl, or 5- or 6-membered heteroaryl, and each of
R.sub.S4 and R.sub.S5 is optionally substituted with one or more
-Q.sub.5-T.sub.5, wherein Q.sub.5 is a bond, C(O), C(O)NR.sub.k,
NR.sub.kC(O), S(O).sub.2, or C.sub.1-C.sub.3 alkyl linker, R.sub.k
being H or C.sub.1-C.sub.6 alkyl, and T.sub.5 is H, halo,
C.sub.1-C.sub.6 alkyl, hydroxyl, cyano, C.sub.1-C.sub.6 alkoxyl,
amino, mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6
alkylamino, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to
7-membered heterocycloalkyl, 5- or 6-membered heteroaryl, or
S(O).sub.qR.sub.q in which q is 0, 1, or 2 and R.sub.q is
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, C.sub.3-C.sub.8cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to
7-membered heterocycloalkyl, or 5- or 6-membered heteroaryl, and
T.sub.5 is optionally substituted with one or more substituents
selected from the group consisting of halo, C.sub.1-C.sub.6 alkyl,
hydroxyl, cyano, C.sub.1-C.sub.6 alkoxyl, amino,
mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6 alkylamino,
C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 7-membered
heterocycloalkyl, and 5- or 6-membered heteroaryl except when
T.sub.5 is H, halo, hydroxyl, or cyano; or -Q.sub.5-T.sub.5 is oxo;
provided that R.sub.7 is not H; and R.sub.8 is H, halo, hydroxyl,
COOH, cyano, R.sub.S6, OR.sub.S6, or COOR.sub.S6, in which R.sub.S6
is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, amino, mono-C.sub.1-C.sub.6 alkylamino, or
di-C.sub.1-C.sub.6 alkylamino, and R.sub.S6 is optionally
substituted with one or more substituents selected from the group
consisting of halo, hydroxyl, COOH, C(O)O--C.sub.1-C.sub.6 alkyl,
cyano, C.sub.1-C.sub.6 alkoxyl, amino, mono-C.sub.1-C.sub.6
alkylamino, and di-C.sub.1-C.sub.6 alkylamino; or R.sub.7 and
R.sub.8, together with the N atom to which they are attached, form
a 4 to 11-membered heterocycloalkyl ring which has 0 to 2
additional heteroatoms and is optionally substituted with one or
more -Q.sub.6-T.sub.6, wherein Q.sub.6 is a bond, C(O),
C(O)NR.sub.m, NR.sub.mC(O), S(O).sub.2, or C.sub.1-C.sub.3 alkyl
linker, R.sub.m being H or C.sub.1-C.sub.6 alkyl, and T.sub.6 is H,
halo, C.sub.1-C.sub.6 alkyl, hydroxyl, cyano, C.sub.1-C.sub.6
alkoxyl, amino, mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6
alkylamino, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to
7-membered heterocycloalkyl, 5- or 6-membered heteroaryl, or
S(O).sub.pR.sub.p in which p is 0, 1, or 2 and R.sub.p is
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to
7-membered heterocycloalkyl, or 5- or 6-membered heteroaryl, and
T.sub.6 is optionally substituted with one or more substituents
selected from the group consisting of halo, C.sub.1-C.sub.6 alkyl,
hydroxyl, cyano, C.sub.1-C.sub.6 alkoxyl, amino,
mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6 alkylamino,
C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 7-membered
heterocycloalkyl, and 5- or 6-membered heteroaryl except when
T.sub.6 is H, halo, hydroxyl, or cyano; or -Q.sub.6-T.sub.6 is
oxo.
5. The method of claim 4, wherein R.sub.a and R.sub.b, together
with the N atom to which they are attached, form a 4 to 7-membered
heterocycloalkyl ring having 0 or 1 additional heteroatoms to the N
atom and the ring is optionally substituted with one or more
-Q.sub.3-T.sub.3, wherein the heterocycloalkyl is azetidinyl,
pyrrolidinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl,
isoxazolidinyl, triazolidinyl, piperidinyl,
1,2,3,6-tetrahydropyridinyl, piperazinyl, or morpholinyl.
6. The method of claim 5, wherein R7 is C3-C8 cycloalkyl or 4 to
7-membered heterocycloalkyl, each optionally substituted with one
or more -Q5-T5.
7. The method of claim 6, wherein R7 is piperidinyl,
tetrahydropyran, tetrahydro-2H-thiopyranyl, cyclopentyl,
cyclohexyl, pyrrolidinyl, or cycloheptyl, each optionally
substituted with one or more -Q5-T5.
8. The method of claim 7, wherein R.sub.8 is H or C1-C6 alkyl which
is optionally substituted with one or more substituents selected
from the group consisting of halo, hydroxyl, COOH,
C(O)O--C.sub.1-C.sub.6 alkyl, cyano, C.sub.1-C.sub.6 alkoxyl,
amino, mono-C.sub.1-C.sub.6 alkylamino, and di-C.sub.1-C.sub.6
alkylamino.
9. The method of claim 1, wherein the subject has a deletion or
loss of function in at least one component of the SWI/SNF
complex.
10. The method of claim 9, wherein the subject has a deletion of
INI1.
11. The method of claim 1, wherein the epithelioid sarcoma is
resistant or refractory to at least one prior therapy.
12. The method of claim 1, wherein the compound is administered
orally.
13. The method of claim 12, wherein the compound is administered in
an amount of from about 0.1 mg/day to about 10 g/day.
14. The method of claim 12, wherein the compound is administered in
an amount of from about 0.1 mg/day to about 3 g/day.
15. The method of claim 12, wherein the compound is administered in
an amount of from about 0.1 mg/day to about 1 g/day.
16. The method of claim 12, wherein the compound is administered
twice daily.
17. A method comprising administering to a subject having
epithelioid sarcoma a therapeutically effective amount of a
compound being ##STR00054## or a pharmaceutically acceptable salt
thereof.
18. The method of claim 17, wherein the subject has a deletion or
loss of function in at least one component of the SWI/SNF
complex.
19. The method of claim 18, wherein the subject has a deletion of
INI1.
20. The method of claim 17, wherein the epithelioid sarcoma is
resistant or refractory to at least one prior therapy.
21. The method of claim 17, wherein the pharmaceutically acceptable
salt is a hydrobromic acid salt.
22. The method of claim 17, wherein the pharmaceutically acceptable
salt is a hydrochloric acid salt.
23. The method of claim 17, wherein the compound is administered
orally.
24. The method of claim 23, wherein the compound is administered in
an amount of from about 0.1 mg/day to about 10 g/day.
25. The method of claim 23, wherein the compound is administered in
an amount of from about 0.1 mg/day to about 3 g/day.
26. The method of claim 23, wherein the compound is administered in
an amount of from about 0.1 mg/day to about 1 g/day.
27. The method of claim 23, wherein the compound is administered
twice daily.
Description
RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 14/054,646, filed Oct. 15, 2013 (now U.S. Pat.
No. 9,688,665), which claims priority to, and the benefit of U.S.
Provisional Application Nos. 61/714,045, filed Oct. 15, 2012,
61/758,972, filed Jan. 31, 2013, 61/714,140, filed Oct. 15, 2012,
61/714,145, filed Oct. 15, 2012, 61/780,703, filed Mar. 13, 2013,
and 61/786,277, filed Mar. 14, 2013. The entire contents of each of
these provisional applications are incorporated herein by reference
in their entireties.
INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING
[0002] The contents of the text file named "41478-513001US
ST25.txt", which was created on Jan. 10, 2014 and is 141 KB in
size, are hereby incorporated by reference in their entireties.
FIELD OF INVENTION
[0003] The present invention relates generally to the field of
cancer treatment, and in particular, the treatment of cancer
associated with the SWI/SNF complex (i.e., SWI/SNF mediated
cancer). More particularly, the present invention provides methods
and compositions which treat, alleviate, prevent, diminish or
otherwise ameliorate the symptoms of cancer associated with the
SWI/SNF complex.
BACKGROUND OF THE INVENTION
[0004] Disease-associated chromatin-modifying enzymes (e.g., EZH2)
play a role in diseases such as proliferative disorders, metabolic
disorders, and blood disorders. Thus, there is a need for the
development of small molecules that are capable of modulating the
activity of EZH2.
SUMMARY OF THE INVENTION
[0005] The present invention provides a method for treating or
alleviating a symptom of a SWI/SNF-associated cancer in a subject
by administering to a subject in need thereof a therapeutically
effective amount of an EZH2 inhibitor, where the subject has a
cancer selected from the group consisting of brain and central
nervous system cancer, head and neck cancer, kidney cancer, ovarian
cancer, pancreatic cancer, leukemia, lung cancer, lymphoma,
myeloma, sarcoma, breast cancer, and prostate cancer. For example,
the SWI/SNF-associated cancer is characterized by reduced
expression and/or loss of function of the SWI/SNF complex or one or
more components of the SWI/SNF complex.
[0006] For example, the subject has a cancer selected from the
group consisting of medulloblastoma, malignant rhabdoid tumor, and
atypical teratoid/rhabdoid tumor.
[0007] For example, the one or more components are selected from
the group consisting of SNF5, ATRX, and ARID1A.
[0008] For example, the loss of function is caused by a loss of
function mutation resulting from a point mutation, a deletion,
and/or an insertion.
[0009] For example, the subject has a deletion of SNF5.
[0010] For example, the subject has a mutation of ATRX selected
from the group consisting of a substitution of asparagine (N) for
the wild type residue lysine (K) at amino acid position 688 of SEQ
ID NO: 5 (K688N), and a substitution of isoleucine (I) for the wild
type residue methionine (M) at amino acid position 366 of SEQ ID
NO: 5 (M366I).
[0011] For example, subject has a mutation of ARID1A selected from
the group consisting of a nonsense mutation for the wild type
residue cysteine (C) at amino acid position 884 of SEQ ID NO: 11
(C884*), a substitution of lysine (K) for the wild type residue
glutamic acid (E) at amino acid position 966 (E966K), a nonsense
mutation for the wild type residue glutamine (Q) at amino acid
position 1411 of SEQ ID NO: 11 (Q1411*), a frame shift mutation at
the wild type residue phenylalanine (F) at amino acid position 1720
of SEQ ID NO: 11 (F1720fs), a frame shift mutation after the wild
type residue glycine (G) at amino acid position 1847 of SEQ ID NO:
11 (G1847fs), a frame shift mutation at the wild type residue
cysteine (C) at amino acid position 1874 of SEQ ID NO: 11
(C1874fs), a substitution of glutamic acid (E) for the wild type
residue aspartic acid (D) at amino acid position 1957 (D1957E), a
nonsense mutation for the wild type residue glutamine (Q) at amino
acid position 1430 of SEQ ID NO: 11 (Q1430*), a frame shift
mutation at the wild type residue arginine (R) at amino acid
position 1721 of SEQ ID NO: 11 (R1721fs), a substitution of
glutamic acid (E) for the wild type residue glycine (G) at amino
acid position 1255 (G1255E), a frame shift mutation at the wild
type residue glycine (G) at amino acid position 284 of SEQ ID NO:
11 (G284fs), a nonsense mutation for the wild type residue arginine
(R) at amino acid position 1722 of SEQ ID NO: 11 (R1722*), a frame
shift mutation at the wild type residue methionine (M) at amino
acid position 274 of SEQ ID NO: 11 (M274fs), a frame shift mutation
at the wild type residue glycine (G) at amino acid position 1847 of
SEQ ID NO: 11 (G1847fs), a frame shift mutation at the wild type
residue P at amino acid position 559 of SEQ ID NO: 11 (P559fs), a
nonsense mutation for the wild type residue arginine (R) at amino
acid position 1276 of SEQ ID NO: 11 (R1276*), a frame shift
mutation at the wild type residue glutamine (Q) at amino acid
position 2176 of SEQ ID NO: 11 (Q2176fs), a frame shift mutation at
the wild type residue histidine (H) at amino acid position 203 of
SEQ ID NO: 11 (H203fs), a frame shift mutation at the wild type
residue alanine (A) at amino acid position 591 of SEQ ID NO: 11
(A591fs), a nonsense mutation for the wild type residue glutamine
(Q) at amino acid position 1322 of SEQ ID NO: 11 (Q1322*), a
nonsense mutation for the wild type residue serine (S) at amino
acid position 2264 of SEQ ID NO: 11 (S2264*), a nonsense mutation
for the wild type residue glutamine (Q) at amino acid position 586
of SEQ ID NO: 11 (Q586*), a frame shift mutation at the wild type
residue glutamine (Q) at amino acid position 548 of SEQ ID NO: 11
(Q548fs), and a frame shift mutation at the wild type residue
asparagine (N) at amino acid position 756 of SEQ ID NO: 11
(N756fs).
[0012] The present invention also provides a method of treating or
alleviating a symptom of a SWI/SNF-associated cancer in a subject
in need thereof by (a) determining the expression level of at least
one gene selected from the group consisting of neuronal
differentiation genes, cell cycle inhibition genes and tumor
suppressor genes in a sample obtained from the subject; (b)
selecting the subject having a decreased expression level of at
least one gene in step a; and (c) administering to the subject
selected in step b an effective amount of an EZH2 inhibitor,
thereby treating or alleviating a symptom of cancer in the
subject.
[0013] The present invention further provides a method of treating
or alleviating a symptom of a SWI/SNF-associated cancer in a
subject in need thereof by (a) determining the expression level of
at least one gene selected from the group consisting of hedgehog
pathway genes, myc pathway genes and histone methyltransferase
genesin a sample obtained from the subject; (b) selecting the
subject having an increased expression level of at least one gene
in step a; and (c) administering to the subject selected in step b
an effective amount of an EZH2 inhibitor, thereby treating or
alleviating a symptom of cancer in the subject.
[0014] For example, the cancer can be medulloblastoma, malignant
rhabdoid tumor or atypical teratoid rhabdoid tumor.
[0015] For example, the neuronal differentiation gene is CD133,
DOCK4, or PTPRK.
[0016] For example, the cell cycle inhibition gene is CKDN1A or
CDKN2A.
[0017] For example, the tumor suppressor gene is BIN1.
[0018] For example, the hedgehog pathway gene is GLI1 or PTCH1.
[0019] For example, the myc pathway gene is MYC.
[0020] For example, the histone methyltransferase gene is EZH2.
[0021] The present invention also provides a method of inducing
neuronal differentiation, cell cycle inhibition or tumor
suppression by contacting a cell with an EZH2 inhibitor. The EZH2
inhibitor may be in an amount sufficient to increase expression of
at least one gene selected from the group consisting of CD133,
DOCK4, PTPRK, CKDN1A, CDKN2A and BIN1.
[0022] The present invention also provides a method of inhibiting
hedgehog signaling by contacting a cell with an EZH2 inhibitor. The
EZH2 inhibitor can be in an amount sufficient to reduce expression
of GLI1 and/or PTCH1.
[0023] The present invention also provides a method of inducing
gene expression by contacting a cell with an EZH2 inhibitor. The
EZH2 inhibitor can be in an amount sufficient to induce neuronal
differentiation, cell cycle inhibition and/or tumor suppression.
For example, the gene can be CD133, DOCK4, PTPRK, CKDN1A, CKDN2A or
BIN1.
[0024] The present invention also provides a method of inhibiting
gene expression by contacting a cell with an EZH2 inhibitor. The
EZH2 inhibitor is in an amount sufficient to inhibit hedgehog
signaling. For example, the gene can be GLI1 or PTCH1.
[0025] For example, the cell may have loss of function of SNF5,
ARID1A, ATRX, and/or a component of the SWI/SNF complex.
[0026] For example, the loss of function is caused by a deletion of
SNF5.
[0027] For example, the cell is a cancer cell. The cancer can be
medulloblastoma, malignant rhabdoid tumor or atypical teratoid
rhabdoid tumor.
[0028] For example, the EZH2 inhibitor is Compound A having the
following formula:
##STR00001##
stereoisomers thereof, or pharmaceutically acceptable salts or
solvates thereof.
[0029] For example, the EZH2 inhibitor is Compound B having the
following formula:
##STR00002##
stereoisomers thereof, or pharmaceutically acceptable salts or
solvates thereof.
[0030] For example, the EZH2 inhibitor is Compound C having the
following formula:
##STR00003##
stereoisomers thereof, or pharmaceutically acceptable salts or
solvates thereof.
[0031] For example, the EZH2 inhibitor is Compound D having the
following formula:
##STR00004##
stereoisomers thereof, or pharmaceutically acceptable salts or
solvates thereof.
[0032] For example, the EZH2 inhibitor is Compound E having the
following formula:
##STR00005##
stereoisomers thereof, or pharmaceutically acceptable salts or
solvates thereof.
[0033] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. In the
specification, the singular forms also include the plural unless
the context clearly dictates otherwise. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of the present invention, suitable
methods and materials are described below. All publications, patent
applications, patents and other references mentioned herein are
incorporated by reference. The references cited herein are not
admitted to be prior art to the claimed invention. In the case of
conflict, the present specification, including definitions, will
control. In addition, the materials, methods and examples are
illustrative only and are not intended to be limiting.
[0034] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTIONS OF FIGURES
[0035] FIGS. 1A and 1B are a series of Western blot analyses of
cell lines with wild type (RD and SJCRH30) and mutant SNF5.
[0036] FIGS. 2A-2E are a series of graphs establishing that SNF5
mutant cell lines A204 (FIG. 2C), G401 (FIG. 2D) and G402 (FIG. 2E)
selectively respond to EZH2 compound (Compound E) compared to wild
type cell lines RD (FIG. 2A) and SJCRH30 (FIG. 2B).
[0037] FIGS. 3A-3D are a series of bar graphs showing that G401 SNF
mutant cell line is responding to Compound E after 7 days in soft
agar compared to wild type cells RD. FIG. 3A shows cell line RD
(5,000 cells/well). FIG. 3B shows G401 cells (5,000
cells/well).
[0038] FIG. 3C shows G401 cells in 2D growth. FIG. 3D shows G401
cells (10,000 cells/well).
[0039] FIGS. 4A-4D are four graphs showing that G401 SNF5 mutant
cell line is sensitive to Compound A in vitro. Wild type cell lines
SJCRH30 (FIG. 4A) and RD (FIG. 4C) and SNF5 mutant cell lines G401
(FIG. 4B) and A204 (FIG. 4D) were pretreated for 7 days with
indicated concentrations of Compound A and replated on day 0. Cell
viability was determined by CellTiter-Glo.RTM. Luminescent Cell
Viability Assay.
[0040] FIGS. 5A-5E are a series of graphs showing durable
regressions in G401 xenografts (malignant rhabdoid tumor model)
with Compound A treatment. FIG. 5A shows tumor regressions induced
by Compound A at the indicated doses. FIG. 5B shows tumor
regressions induced by twice daily administration of Compound A at
the indicated doses. Data represent the mean values.+-.SEM (n=8).
Compound administration was stopped on day 28. FIG. 5C shows the
EZH2 target inhibition in G401 xenograft tumor tissue collected
from a parallel cohort of mice on day 21. Each point shows the
ratio of H3K27Me3 to total H3. Horizontal lines represent group
mean values. BLLQ=below lower limit of quantification. FIGS. 5D and
5E show immunohistochemical staining of tumor histone methylation
of tumor samples from the vehicle treated (FIG. 5D) and Compound A
treated (FIG. 5E) (at 125 mg/kg) mice.
[0041] FIG. 6 is a graph showing the locations of ATRX mutations
identified in SCLC cell lines.
[0042] FIG. 7A is a graph showing that LNCAP prostate cancer cells
display dose-dependent cell growth inhibition with Compound E
treatment in vitro.
[0043] FIG. 7B is a graph showing IC50 value of Compound E at day
11 and day 14 for WSU-DLCL2 and LNCAP cells.
[0044] FIGS. 8A-8C are three graphs establishing that ATRX mutant
SCLC lines NCI-H446 (FIG. 8A), SW1271 (FIG. 8B) and NCI-H841 (FIG.
8C) are responding to Compound E.
[0045] FIGS. 9A-9C are three microscopy images showing that SCLC
line NCI-H841 changes morphology after treatment with vehicle (FIG.
9A) or Compound E at concentration of 4.1E-02 uM (FIG. 9B) or 3.3
uM (FIG. 9C).
[0046] FIGS. 10A-10F are a series of graphs showing effects of
Compound A on cellular global histone methylation and cell
viability. FIG. 10A shows the chemical structure of Compound A.
FIG. 10B illustrates the concentration-dependent inhibition of
cellular H3K27Me3 levels in G401 and RD cells. FIGS. 10C through
10F illustrate the selective inhibition of proliferation of
SMARCB1-deleted G401 cells by Compound A in vitro (measured by ATP
content). G401 (FIGS. 10C and 10D) and RD cells (FIGS. 10E and 10F)
were re-plated at the original seeding densities on day 7. Each
point represents the mean for each concentration (n=3).
[0047] FIGS. 11A and 11B are a series of graphs showing biochemical
mechanism of action studies. The IC.sub.50 value of Compound A
increases with increasing SAM concentration (FIG. 11A) and is
minimally affected by increasing oligonucleosome concentration
(FIG. 11B), indicating SAM-competitive and
nucleosome-noncompetitive mechanism of action.
[0048] FIGS. 12A and 12B are a series of immunoblots demonstrating
verification of SMARCB1 and EZH2 expression in cell lines and
specificity of Compound A for inhibition of cellular histone
methylation. FIG. 12A shows cell lysates analyzed by immunoblot
with antibodies specific to SMARCB1, EZH2 and Actin (loading
control). FIG. 12B illustrates selective inhibition of cellular
H3K27 methylation in G401 and RD cells. Cells were incubated with
Compound A for 4 days, and acid-extracted histones were analyzed by
immunoblot.
[0049] FIGS. 13A and 13B are a series of bar graphs demonstrating
that Compound A induces G.sub.1 arrest and apoptosis in
SMARCB1-deleted MRT cells. Cell cycle analysis (by flow cytometry)
and determination of apoptosis (by TUNEL assay) in RD (FIG. 13A) or
G401 cells (FIG. 13B) during incubation with either vehicle or 1
.mu.M Compound A for up to 14 days. G.sub.1 arrest was observed as
of day 7 and apoptosis was induced as of day 11. Data are
represented as mean values.+-.SEM (n=2). The DMSO control values
shown are the average.+-.SEM from each time point. Cells were split
and re-plated on days 4, 7 and 11 at the original seeding
density.
[0050] FIGS. 14A-14L are a series of graphs showing that Compound A
induces changes in expression of SMARCB1 regulated genes and cell
morphology. FIG. 14A shows the basal expression of SMARCB1
regulated genes in G401 SMARCB1-deleted cells, relative to RD
control cells (measured by qPCR, n=2). FIGS. 14B-14K show the
expression of GLI1 (FIG. 14B), PTCh1 (FIG. 14C), DOCK4 (FIG. 14D),
CD133 (FIG. 14E), PTPRK (FIG. 14F), BIN1 (FIG. 14G), CDKN1A (FIG.
14H), CDKN2A (FIG. 14I), EZH2 (FIG. 14J), and MYC (FIG. 14K) genes
in G401 and RD cells. The cells were incubated with either DMSO or
1 .mu.M Compound A for 2, 4 and 7 days. Gene expression was
determined by qPCR (n=2) and is expressed relative to the DMSO
control of each time point. FIG. 14L illustrates the morphology of
G402 cells that were incubated with either DMSO (left panel) or 1
.mu.M Compound A (right panel) for 14 days. Cells were split and
re-plated to the original seeding density on day 7.
[0051] FIGS. 15A-15D are a series of graphs demonstrating body
weights, tumor regressions and plasma levels in G401 xenograft
bearing mice treated with Compound A. FIG. 15A shows body weights
that were determined twice a week for animals treated with Compound
A on a BID schedule for 28 days. Data are presented as mean
values.+-.SEM (n=16 until day 21, n=8 from day 22 to 60). FIG. 15B
shows tumor regressions induced by twice daily (BID) administration
of Compound A for 21 days at the indicated doses (mean
values.+-.SEM, n=16). *p<0.05, **p<0.01, repeated measures
ANOVA, Dunnett's post-test vs. vehicle. FIG. 15C shows the tumor
weights of 8 mice euthanized on day 21. ****p<0.0001, Fisher's
exact test. FIG. 15D shows plasma levels of Compound A. Plasma was
collected 5 min before and 3 h after dosing of Compound A on day
21, and compound levels were measured by LC-MS/MS. Animals were
euthanized, and tumors were collected 3 h after dosing on day 21.
Tumor homogenates were generated and subjected to LC-MS/MS analysis
to determine Compound A concentrations. Note that tumor compound
levels could not be determined from all animals especially in the
higher dose groups because the xenografts were too small on day 21.
Dots represent values for the individual animals; horizontal lines
represent group mean values.
[0052] FIGS. 16A-16F are a series of graphs showing that Compound A
eradicates SMARCB1-deleted MRT xenografts in SCID mice. FIG. 16A
illustrates tumor regressions induced by twice daily (BID)
administration of Compound A for 28 days at the indicated doses.
Compound administration was stopped on day 28 and tumors were
allowed to re-grow until they reached 2000 mm.sup.3 (data shown as
mean values.+-.SEM, n=8). FIG. 16B shows the EZH2 target inhibition
in G401 xenograft tumor tissue collected from mice euthanized on
day 21. Each point shows the ratio of H3K27Me3 to total H3,
measured by ELISA. Horizontal lines represent group mean values;
grey symbols are values outside of the ELISA standard curve. FIGS.
16C-16F summarize the change in gene expression in G401 xenograft
tumor tissue collected from mice treated with Compound A for 21
days. The graphs correspond to genes CD133 (FIG. 16C), PTPRK (FIG.
16D), DOCK4 (FIG. 16E), and GLI1 (FIG. 16F), respectively. Data are
presented as fold change compared to vehicle.+-.SEM (n=6, n=4 for
500 mg/kg group). *p<0.05, **p<0.01, ****p<0.0001, vs.
vehicle, Fisher's exact test.
DETAILED DESCRIPTION OF THE INVENTION
[0053] The present invention is based in part upon the discovery
that EZH2 inhibitors can effectively treat SWI/SNF-associated
cancers that are characterized by altered expressions and/or loss
of function of certain biomarkers or genes. Specifically, tumors or
tumor cells having altered expressions and/or loss of function of
selected biomarkers or genesare sensitive to the EZH2 inhibitors of
the present invention. Accordingly, the present invention provides
methods of treating or alleviating a symptom of cancers in a
subject by administering a therapeutically effective amount of an
EZH2 inhibitor to the subject, particular treating cancers
associated with altered expression and/or loss of function of
certain biomarkers or genes. For example, the biomarker is one
component of the SWI/SNF complex. For example, the gene is selected
from the group consisting of neuronal differentiation genes, cell
cycle gene inhibition genes, tumor suppressor genes, hedgehog
pathway genes, myc pathway genes and histone methyltransferase
genes.
[0054] The SWI/SNF complex in human includes at least
evolutionarily conserved core subunits and variant subunits.
Evolutionarily conserved core subunits include SNF5 (also called
SMARCB1, INI1 or BAF47), SMARCA4 (also known as BRM/SWI2-related
gene 1, BRG1), BAF155, and BAF170. Variant subunits include BAF53
(A or B), BAF60 (A, B or C), BAF 57, BAF45 (A, B, C, or D). Other
subunits include ARIDI1A (also known as SMARCF1), ARID1B, SMARCA2
(also known as brahma homologue, BRM), ATRX, BAF200, BAF180 (also
known as PBRM1), and bromodomain-containing 7 (BRD7). The at least
one component of the SWI/SNF complex can by any component of the
complex, for example, the component/subunit described herein or
known in the art.
[0055] In any methods presented herein, neuronal differentiation
gene may be, but is not limited to, CD133 (also called PROM1),
DOCK4, PTPRK, PROM2, LHX1, LHX6, LHX9, PAX6, PAX7, VEFGA, FZD3B,
FYN, HIF1A, HTRA2, EVX1, CCDC64, or GFAP.
[0056] In any methods presented herein, cell cycle inhibition gene
may be, but is not limited to, CKDN1A, CDKN2A, MEN1, CHEK1, IRF6,
ALOX15B, CYP27B1, DBC1, NME6, GMNN, HEXIM1, LATS1, MYC, HRAS,
TGFB1, IFNG, WNT1, TP53, THBS1, INHBA, IL8, IRF1, TPR, BMP2, BMP4,
ETS1, HPGD, BMP7, GATA3, NR2F2, APC, PTPN3, CALR, IL12A, IL12B,
PML, CDKN2B, CDKN2C, CDKN1B, SOX2, TAF6, DNA2, PLK1, TERF1, GAS1,
CDKN2D, MLF1, PTEN, TGFB2, SMAD3, FOXO4, CDK6, TFAP4, MAP2K1,
NOTCH2, FOXC1, DLG1, MAD2L1, ATM, NAE1, DGKZ, FHL1, SCRIB, BTG3,
PTPRK, RPS6KA2, STK11, CDKN3, TBRG1, CDC73, THAP5, CRLF3, DCUN1D3,
MYOCD, PAF1, LILRB1, UHMK1, PNPT1, USP47, HEXIM2, CDK5RAP1, NKX3-1,
TIPIN, PCBP4, USP44, RBM38, CDT1, RGCC, RNF167, CLSPN, CHMP1A,
WDR6, TCF7L2, LATS2, RASSF1, MLTK, MAD2L2, FBXO5, ING4, or
TRIM35.
[0057] In any methods presented herein, tumor suppressor gene may
be, but is not limited to, BIN1. As used herein, the term "tumor
suppressor gene" has its commonly understood meaning in the art,
i.e. a gene whose expression and normal function act to suppress
the neoplastic phenotype or induce apoptosis, or both. In some
embodiments, tumor suppressor genes include cell cycle inhibition
genes. Exemplary categories of tumor suppressors based on their
functions include, but not limited to:
(1) genes that inhibit cell cycles; (2) genes that are coupling the
cell cycle to DNA damage. When there is damaged DNA in the cell,
the cell should not divide. If the damage can be repaired, the cell
cycle can continue. If the damage cannot be repaired, the cell
should initiate apoptosis (programmed cell death); (3) genes that
prevent tumor cells from dispersing, block loss of contact
inhibition, and inhibit metastasis. These genes and their encoded
proteins are also known as metastasis suppressors; and (4) DNA
repair proteins. Mutations in these genes increase the risk of
cancer.
[0058] In any methods presented herein, hedgehog signaling pathway
gene may be, but is not limited to, GLI1, PTCH1, SUFU, KIF7, GLI2,
BMP4, MAP3K10, SHH, TCTN3, DYRK2, PTCHD1, or SMO.
[0059] In any methods presented herein, myc pathway gene may be,
but is not limited to, MYC NMI, NFYC, NFYB, Cyclin T1, RuvB-like 1,
GTF2I, BRCA1, T-cell lymphoma invasion and metastasis-inducing
protein 1, ACTL6A, PCAF, MYCBP2, MAPK8, Bc1-2, Transcription
initiation protein SPT3 homolog, SAP130, DNMT3A, mothers against
decapentaplegic homolog 3, MAX, mothers against decapentaplegic
homolog 2, MYCBP, HTATIP, ZBTB17, Transformation/transcription
domain-associated protein, TADA2L, PFDNS, MAPK1, TFAP2A, P73, TAF9,
YY1, SMARCB1, SMARCA4, MLH1, EP400 or let-7.
[0060] In any methods presented herein, histone methyltransferase
gene may be, but is not limited to, EZH2.
[0061] Compounds of the present invention inhibit the histone
methyltransferase activity of EZH2 or a mutant thereof and,
accordingly, in one aspect of the invention, compounds disclosed
herein are candidates for treating or preventing certain conditions
and diseases. The present invention provides methods for treating,
preventing or alleviating a symptom of cancer or a precancerous
condition. The method includes administering to a subject in need
thereof, a therapeutically effective amount of a compound of the
present invention, or a pharmaceutically acceptable salt,
polymorph, solvate, or stereoisomeror thereof. Exemplary cancers
that may be treated include medulloblastoma, oligodendroglioma,
ovarian clear cell adenocarcinoma, ovarian endomethrioid
adenocarcinoma, ovarian serous adenocarcinoma, pancreatic ductal
adenocarcinoma, pancreatic endocrine tumor, malignant rhabdoid
tumor, astrocytoma, atypical teratoid rhabdoid tumor, choroid
plexus carcinoma, choroid plexus papilloma, ependymoma,
glioblastoma, meningioma, neuroglial tumor, oligoastrocytoma,
oligodendroglioma, pineoblastoma, carcinosarcoma, chordoma,
extragonadal germ cell tumor, extrarenal rhabdoid tumor,
schwannoma, skin squamous cell carcinoma, chondrosarcoma, clear
cell sarcoma of soft tissue, ewing sarcoma, gastrointestinal
stromal tumor, osteosarcoma, rhabdomyosarcoma, epithelioid sarcoma,
renal medullo carcinoma, diffuse large B-cell lymphoma, follicular
lymphoma and not otherwise specified (NOS) sarcoma. Alternatively,
cancers to be treated by the compounds of the present invention are
non NHL cancers.
[0062] The present invention further provides the use of a compound
of the present invention, or a pharmaceutically acceptable salt,
polymorph or solvate thereof in the treatment of cancer or
precancer, or, for the preparation of a medicament useful for the
treatment of such cancer or pre-cancer. Exemplary cancers that may
be treated include medulloblastoma, oligodendroglioma, ovarian
clear cell adenocarcinoma, ovarian endomethrioid adenocarcinoma,
ovarian serous adenocarcinoma, pancreatic ductal adenocarcinoma,
pancreatic endocrine tumor, malignant rhabdoid tumor, astrocytoma,
atypical teratoid rhabdoid tumor, choroid plexus carcinoma, choroid
plexus papilloma, ependymoma, glioblastoma, meningioma, neuroglial
tumor, oligoastrocytoma, oligodendroglioma, pineoblastoma,
carcinosarcoma, chordoma, extragonadal germ cell tumor, extrarenal
rhabdoid tumor, schwannoma, skin squamous cell carcinoma,
chondrosarcoma, clear cell sarcoma of soft tissue, ewing sarcoma,
gastrointestinal stromal tumor, osteosarcoma, rhabdomyosarcoma,
epithelioid sarcoma, renal medullo carcinoma, diffuse large B-cell
lymphoma, follicular lymphoma and not otherwise specified (NOS)
sarcoma. Alternatively, the compound of the present invention can
be used for the treatment of non NHL cancers, or, for the
preparation of a medicament useful for the treatment of non NHL
cancers.
[0063] The compounds of this invention can be used to modulate
protein (e.g., histone) methylation, e.g., to modulate histone
methyltransferase or histone demethylase enzyme activity. The
compounds of the invention can be used in vivo or in vitro for
modulating protein methylation. Based upon the surprising discovery
that methylation regulation by EZH2 involves in tumor formation,
particular tumors bearing altered expression and/or loss of
function of selected biomarkers/genes, the compounds described
herein are suitable candidates for treating these diseases, i.e.,
to decrease methylation or restore methylation to roughly its level
in counterpart normal cells.
[0064] In some embodiments, compounds of the present invention can
selectively inhibit proliferation of the SWI/SNF complex associated
tumor or tumor cells (as shown in FIGS. 1-9). Accordingly, the
present invention provides methods for treating, preventing or
alleviating a symptom of the SWI/SNF complex associated cancer or a
precancerous condition by a compound of the present invention, or a
pharmaceutically acceptable salt, polymorph or solvate thereof. The
present invention further provides the use of a compound of the
present invention, or a pharmaceutically acceptable salt, polymorph
or solvate thereof in the treatment of the SWI/SNF complex
associated cancer or a precancer condition, or, for the preparation
of a medicament useful for the treatment of such cancer or
pre-cancer.
[0065] Also provided in the present invention are methods for
determining responsiveness of a subject having a cancer to an EZH2
inhibitor. The method includes the steps of obtaining a sample (a
nucleic acid sample or a protein sample) from the subject and
detecting reduced expression, haploinsufficiency, and/or loss of
function of at least one component of the SWI/SNF complex,
detecting the expression and/or function of this component, and the
presence of such reduced expression, haploinsufficiency, and/or
loss of function indicates that the subject is responsive to the
EZH2 inhibitor. The term "sample" means any biological sample
derived from the subject, includes but is not limited to, cells,
tissues samples, body fluids (including, but not limited to, mucus,
blood, plasma, serum, urine, saliva, and semen), tumor cells, and
tumor tissues. Samples can be provided by the subject under
treatment or testing. Alternatively samples can be obtained by the
physician according to routine practice in the art.
[0066] The present invention also provides methods for determining
predisposition of a subject to a cancer or a precancerous condition
by obtaining a sample from the subject and detecting reduced
expression, haploinsufficiency, and/or loss of function of at least
one component of the SWI/SNF complex, and the presence of such
reduced expression, haploinsufficiency, and/or loss of function
indicates that the subject is predisposed to (i.e., having higher
risk of) developing the cancer or the precancerous condition
compared to a subject without such loss of function of the at least
one component of the SWI/SNF complex.
[0067] The term "predisposed" as used herein in relation to cancer
or a precancerous condition is to be understood to mean the
increased probability (e.g., at least 1%, 5%, 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or more increase in
probability) that a subject with reduced expression,
haploinsufficiency, and/or loss of function of at least one
component of the SWI/SNF complex, will suffer cancer or a
precancerous condition, as compared to the probability that another
subject not having reduced expression, haploinsufficiency, and/or
loss of function of at least one component of the SWI/SNF complex,
will suffer cancer or a precancerous condition, under circumstances
where other risk factors (e.g., chemical/environment, food, and
smoking history, etc.) for having cancer or a precancerous
condition between the subjects are the same.
[0068] "Risk" in the context of the present invention, relates to
the probability that an event will occur over a specific time
period and can mean a subject's "absolute" risk or "relative" risk.
Absolute risk can be measured with reference to either actual
observation post-measurement for the relevant time cohort, or with
reference to index values developed from statistically valid
historical cohorts that have been followed for the relevant time
period. Relative risk refers to the ratio of absolute risks of a
subject compared either to the absolute risks of low risk cohorts
or an average population risk, which can vary by how clinical risk
factors are assessed. Odds ratios, the proportion of positive
events to negative events for a given test result, are also
commonly used (odds are according to the formula p/(1-p) where p is
the probability of event and (1-p) is the probability of no event)
to no-conversion.
[0069] Accordingly, the present invention provides personalized
medicine, treatment and/or cancer management for a subject by
genetic screening of reduced expression, haploinsufficiency, and/or
loss of function of at least one component of the SWI/SNF complex
in the subject. For example, the present invention provides methods
for treating, preventing or alleviating a symptom of cancer or a
precancerous condition by determining responsiveness of the subject
to an EZH2 inhibitor and when the subject is responsive to the EZH2
inhibitor, administering to the subject a therapeutically effective
amount of the EZH2 inhibitor, or a pharmaceutically acceptable
salt, solvate, or stereoisomeror thereof. The responsiveness is
determined by obtaining a sample from the subject and detecting
reduced expression, haploinsufficiency, and/or loss of function of
at least one component of the SWI/SNF complex (such as SNF5, ARID1A
or ATRX), and the presence of such loss of function indicates that
the subject is responsive to the EZH2 inhibitor.
[0070] In other example, the present invention provides methods of
cancer management in a subject by determining predisposition of the
subject to a cancer or a precancerous condition periodically. The
methods include steps of obtaining a sample from the subject and
detecting reduced expression, haploinsufficiency, and/or loss of
function of at least one component of the SWI/SNF complex, and the
presence of such reduced expression, haploinsufficiency, and/or
loss of function indicates that the subject is predisposed to
developing the cancer or the precancerous condition compared to a
subject without such reduced expression, haploinsufficiency, and/or
loss of function of the at least one component of the SWI/SNF
complex.
[0071] In merely illustrative embodiments, the methods of treatment
presented herein include steps of (a) collecting a nucleic acid
sample or a protein sample from a biological sample obtained from a
subject, (b) measuring the expression level or function level of a
component of the SWI/SNF complex in the sample, (c) measuring the
expression level or function level of the component of the SWI/SNF
in a control sample; (d) comparing the expression level or the
function level of the component measured in step (b) in the tested
sample to the expression level or the function level of the
component measured in step (c) in the control sample (or a
reference value); (e) identifying the subject as a candidate for
treatment when the expression level or the function level of the
component measured in step (b) is reduced or lost (e.g.,
haploinsufficiency or loss of function) compared to the expression
level or the function level of the component measured in step (c);
and (f) administering a therapeutically effective amount of an EZH2
inhibitor to the subject identified in step (e) or selecting a
treatment regimen for the subject identified in step (e). The
expression level or the function level of component in the subject
sample is reduced, for example, 10%, 25%, 50% or 1-, 2-, 5- or more
fold compared to the expression level or the function level of the
component in the control sample. Any suitable methods known in the
art can be utilized to measure the expression level or the function
level of the component of the SWI/SNF complex. In some embodiments,
the subject has malignant rhabdoid tumor, medulloblastoma or
atypical teratoid rhabdoid tumor. In some embodiments, the
component is SNF5, ARID1A or ATRX.
[0072] For example, the identified subject can be treated with the
standard of care treatment as described in the most current
National Comprehensive Cancer Network (NCCN) guidelines.
[0073] For example, a control sample is obtained from a healthy,
normal subject. Alternatively, a control sample is obtained from a
subject who is not suffering, has not been diagnosed, or is not at
risk of developing cancer associated with the SWI/SNF complex.
[0074] In one preferred aspect, the present invention provides a
method for treating or alleviating a symptom of cancer in a subject
by determining responsiveness of the subject to an EZH2 inhibitor
and administering to the subject a therapeutically effective amount
of the EZH2 inhibitor if the subject is responsive to the EZH2
inhibitor and the subject has a cancer selected from the group
consisting of brain and CNS cancer, kidney cancer, ovarian cancer,
pancreatic cancer, leukemia, lymphoma, myeloma, and/or sarcoma.
Such responsiveness is determined by obtaining a sample from the
subject and detecting reduced expression, haploinsufficiency,
and/or loss of function of SNF5, ARID1A, and/or ATRX, and the
presence of the reduced expression, haploinsufficiency, and/or loss
of function indicates the subject is responsive to the EZH2
inhibitor.
[0075] In another preferred aspect, the present invention provides
a method for treating or alleviating a symptom of malignant
rhabdoid tumor in a subject by determining responsiveness of the
subject to an EZH2 inhibitor and administering to the subject a
therapeutically effective amount of the EZH2 inhibitor if the
subject is responsive to the EZH2 inhibitor. Such responsiveness is
determined by obtaining a sample from the subject and detecting
reduced expression, haploinsufficiency, and/or loss of function of
SNF5, ARID1A, and/or ATRX, and the presence of the reduced
expression, haploinsufficiency, and/or loss of function indicates
the subject is responsive to the EZH2 inhibitor.
[0076] In another preferred aspect, the present invention provides
a method for treating or alleviating a symptom of medulloblastoma
in a subject by determining responsiveness of the subject to an
EZH2 inhibitor and administering to the subject a therapeutically
effective amount of the EZH2 inhibitor if the subject is responsive
to the EZH2 inhibitor. Such responsiveness is determined by
obtaining a sample from the subject and detecting reduced
expression, haploinsufficiency, and/or loss of function of SNF5,
ARID1A, and/or ATRX, and the presence of the reduced expression,
haploinsufficiency, and/or loss of function indicates the subject
is responsive to the EZH2 inhibitor.
[0077] In another preferred aspect, the present invention provides
a method for treating or alleviating a symptom of atypical teratoid
rhabdoid tumor in a subject by determining responsiveness of the
subject to an EZH2 inhibitor and administering to the subject a
therapeutically effective amount of the EZH2 inhibitor if the
subject is responsive to the EZH2 inhibitor. Such responsiveness is
determined by obtaining a sample from the subject and detecting
reduced expression, haploinsufficiency, and/or loss of function of
SNF5, ARID1A, and/or ATRX, and the presence of the reduced
expression, haploinsufficiency, and/or loss of function indicates
the subject is responsive to the EZH2 inhibitor.
[0078] Malignant rhabdoid tumors (MRTs) and atypical teratoid
rhabdoid tumors (ATRTs) are extremely aggressive pediatric cancers
of the brain, kidney, and soft tissues that are highly malignant,
locally invasive, frequently metastatic, and particularly lethal.
They are typically diploid and lack genomic aberrations; however,
they are characterized by an almost complete penetrance of loss of
SMARCB1 (also called SNF5, INI1 or BAF47), a core component of the
SWI/SNF chromatin remodeling complex. The biallelic inactivation of
SMARCB1 is in essence the sole genetic event in MRTs and ATRTs
which suggests a driver role for this genetic aberration.
[0079] Without being bound by any theory, a compound of the present
invention specifically inhibits cellular H3K27 methylation leading
to selective apoptotic killing of SMARCB1 mutant MRT cells. For
example, in vitro treatment of SMARCB1-deleted MRT cell lines with
Compound A induced strong anti-proliferative effects with IC.sub.50
values in the nM range; while the control (wild-type) cell lines
were minimally affected (FIG. 10C and table 6). Furthermore, the
compound of the present invention induces genes of neuronal
differentiation, cell cycle inhibition and tumor suppression while
suppressing expression of hedgehog pathway genes, MYC and EZH2. For
example, Compound A treatment of G401 SMARCB1-deleted cells for up
to 7 days strongly induced expression of CD133, DOCK4 and PTPRK and
up-regulated cell cycle inhibitors CDKN1A and CDKN2A and tumor
suppressor BIN1, all in a time-dependent manner (FIG. 14B).
Simultaneously, the expression of hedgehog pathway genes, MYC and
EZH2 were reduced. Notably, G402 SMARCB1-deleted cells exposed to
Compound A for 14 days assumed a neuron-like morphology (FIG.
14C).
[0080] Accordingly, the present invention further provides methods
of treating or alleviating a symptom of cancer in a subject in need
thereof by (a) determining the expression level of at least one
gene selected from the group consisting of neuronal differentiation
genes, cell cycle inhibition genes and tumor suppressor genes in a
sample obtained from the subject; (b) selecting a subject having a
decreased expression level of at least one gene in step (a); and
(c) administering to the subject selected in step (b) an effective
amount of a compound of the invention, thus treating or alleviating
a symptom of cancer in the subject.
[0081] The present invention also provides methods of treating or
alleviating a symptom of cancer in a subject in need thereof by (a)
determining the expression level of at least one gene selected from
the group consisting of hedgehog pathway genes, myc pathway genes
and histone methyltransferase genes in a sample obtained from the
subject; (b) selecting a subject having an increased expression
level of at least one gene in step (a); and (c) administering to
the subject selected in step (b) an effective amount of a compound
of the invention, thus treating or alleviating a symptom of cancer
in the subject.
[0082] Also provided herein are methods of selecting a cancer
therapy for a subject in need thereof by (a) determining the
expression level of at least one gene selected from the group
consisting of neuronal differentiation genes, cell cycle inhibition
genes, and tumor suppressor genes in a sample obtained from the
subject, and (b) selecting a cancer therapy when the subject has a
decreased expression level of at least one gene in step (a), where
the cancer therapy includes the administration of an effective
amount of a compound of the invention to the subject.
[0083] The present invention further provides methods of selecting
a cancer therapy for a subject in need thereof by (a) determining
the expression level of at least one gene selected from the group
consisting of hedgehog pathway genes, myc pathway genes and histone
methyltransferase genes in a sample obtained from the subject, and
(b) selecting a cancer therapy when the subject has an increased
expression level of at least one gene in step (a), where the cancer
therapy includes the administration of an effective amount of a
compound of the invention to the subject.
[0084] In merely illustrative embodiments, the methods presented
herein may include the steps of (a) collecting a nucleic acid or a
protein sample from a biological sample obtained from a subject,
(b) measuring the expression level of at least one gene selected
from the group consisting of neuronal differentiation genes, cell
cycle inhibition genes, and tumor suppressor genes in the sample,
(c) measuring the expression level of the same gene(s) in a control
sample; (d) comparing the expression level of the gene measured in
step (b) in the tested sample to the expression level of the gene
measured in step (c) in the control sample (or to a reference
value); (e) identifying the subject as a candidate for treatment
when the expression level of the component measured in step (b) is
reduced compared to the expression level of the gene measured in
step (c); and (f) administering a therapeutically effective amount
of an EZH2 inhibitor to the subject identified in step (e) or
selecting a treatment regimen for the subject identified in step
(e). The expression level of the gene in the tested subject is
reduced, for example, 10%, 25%, 50% or 1-, 2-, 5- or more fold
compared to the expression level of the gene in the control
sample.
[0085] In merely illustrative embodiments, the methods presented
herein may include the steps of (a) collecting a nucleic acid or a
protein sample from a biological sample obtained from a subject,
(b) measuring the expression level of at least one gene selected
from the group consisting of hedgehog pathway genes, myc pathway
genes and histone methyltransferase genes in the sample, (c)
measuring the expression level of the same gene(s) in a control
sample; (d) comparing the expression level of the gene measured in
step (b) in the tested sample to the expression level of the gene
measured in step (c) in the control sample (or to a reference
value); (e) identifying the subject as a candidate for treatment
when the expression level of the component measured in step (b) is
increased compared to the expression level of the gene measured in
step (c); and (f) administering a therapeutically effective amount
of an EZH2 inhibitor to the subject identified in step (e) or
selecting a treatment regimen for the subject identified in step
(e). The expression level of the gene in the tested subject is
increased, for example, 10%, 25%, 50% or 1-, 2-, 5- or more fold
compared to the expression level of the gene in the control
sample.
[0086] The term "expression level" refers to protein, RNA, or mRNA
level of a particular gene of interest. Any methods known in the
art can be utilized to determine the expression level of a
particular gene of interest. Examples include, but are not limited
to, reverse transcription and amplification assays (such as PCR,
ligation RT-PCR or quantitative RT-PCT), hybridization assays,
Northern blotting, dot blotting, in situ hybridization, gel
electrophoresis, capillary electrophoresis, column chromatography,
Western blotting, immunohistochemistry, immunostaining, or mass
spectrometry. Assays can be performed directly on biological
samples or on protein/nucleic acids isolated from the samples. It
is routine practice in the relevant art to carry out these assays.
For example, the measuring step in any method described herein
includes contacting the nucleic acid sample from the biological
sample obtained from the subject with one or more primers that
specifically hybridize to the gene of interest presented herein.
Alternatively, the measuring step of any method described herein
includes contacting the protein sample from the biological sample
obtained from the subject with one or more antibodies that bind to
the biomarker of the interest presented herein.
[0087] A decreased expression level of a particular gene means a
decrease in its expression level by at least 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 100%, 200%, 300%, 400%, 500%, 1000%, 1500%, or more
compared to a reference value or the expression level of this gene
measured in a different (or previous) sample obtained from the same
subject.
[0088] An increased expression level of a particular gene means an
increase in its expression level by at least 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 100%, 200%, 300%, 400%, 500%, 1000%, 1500%, or more
compared to a reference value or the expression level of this gene
measured in a different (or previous) sample obtained from the same
subject.
[0089] A "reference or baseline level/value" as used herein can be
used interchangeably and is meant to be relative to a number or
value derived from population studies, including without
limitation, such subjects having similar age range, disease status
(e.g., stage), subjects in the same or similar ethnic group, or
relative to the starting sample of a subject undergoing treatment
for cancer. Such reference values can be derived from statistical
analyses and/or risk prediction data of populations obtained from
mathematical algorithms and computed indices of cancer. Reference
indices can also be constructed and used using algorithms and other
methods of statistical and structural classification.
[0090] In some embodiments of the present invention, the reference
or baseline value is the expression level of a particular gene of
interest in a control sample derived from one or more healthy
subjects or subjects who have not been diagnosed with any
cancer.
[0091] In some embodiments of the present invention, the reference
or baseline value is the expression level of a particular gene of
interest in a sample obtained from the same subject prior to any
cancer treatment. In other embodiments of the present invention,
the reference or baseline value is the expression level of a
particular gene of interest in a sample obtained from the same
subject during a cancer treatment. Alternatively, the reference or
baseline value is a prior measurement of the expression level of a
particular gene of interest in a previously obtained sample from
the same subject or from a subject having similar age range,
disease status (e.g., stage) to the tested subject.
[0092] In some embodiments, an effective amount means an amount
sufficient to increase the expression level of at least one gene
which is decreased in the subject prior to the treatment or an
amount sufficient to alleviate one or more symptoms of cancer. For
example, an effective amount is an amount sufficient to increase
the expression level of at least one gene selected from the group
consisting of neuronal differentiation genes, cell cycle inhibition
genes, and tumor suppressor genes by at least 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 100%, 200%, 300%, 400%, 500%, 1000%, 1500%, or more
compared to a reference value or the expression level without the
treatment of any compound.
[0093] In some embodiments, an effective amount means an amount
sufficient to decrease the expression level of at least one gene
which is increased in the subject prior to the treatment or an
amount sufficient to alleviate one or more symptoms of cancer. For
example, an effective amount is an amount sufficient to decrease
the expression level of at least one gene selected from the group
consisting of hedgehog pathway genes, MYC and EZH2 by at least 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 1000%,
1500%, or more compared to a reference value or the expression
level without the treatment of any compound.
[0094] The precise effective amount for a subject will depend upon
the subject's body weight, size, and health; the nature and extent
of the condition; and the therapeutic selected for administration.
An effective amount for a given situation can be determined by
routine experimentation that is within the skill and judgment of
the clinician.
[0095] The present invention further provides a method of
determining efficacy of a cancer treatment in a subject in need
thereof by (a) measuring the expression level of at least one gene
selected from the group consisting of neuronal differentiation
genes, cell cycle inhibition genes, and tumor suppressor genes in a
sample obtained from the subject, (b) comparing the expression
level of at least one gene in step (a) to a reference value or a
prior measurement, and (c) determining the efficacy of the cancer
treatment based on the comparison step. An exemplary cancer
treatment is administering a compound of the invention to the
tested subject.
[0096] The treatment is effective when the tested subject has an
increased expression of at least one gene selected from the group
consisting of neuronal differentiation genes, cell cycle inhibition
genes and tumor suppressor genes 1) compared to a reference value
or a prior measurement; or 2) over the period of time being
monitored, such as 1, 2, 3, 4, 5, 6, or 7 days, or 1, 2, 3, 4, 5,
6, 7, 8, 9, 10 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12
months or longer. When the existing treatment is not effective, a
new treatment or an increased dosage of the existing treatment (for
example, increasing the dosage of the compound administered to the
subject) should be sought for the tested subject.
[0097] The present invention also provides a method of determining
efficacy of a cancer treatment in a subject in need thereof by (a)
measuring the expression level of at least one gene selected from
the group consisting of hedgehog pathway genes, myc pathway genes
and histone methyltransferase genes in a sample obtained from the
subject, (b) comparing the expression level of at least one gene in
step (a) to a reference value or a prior measurement, and (c)
determining the efficacy of the cancer treatment based on the
comparison step. An exemplary cancer treatment is administering an
EZH2 inhibitor of the invention to the tested subject.
[0098] For example, the treatment is effective when the tested
subject has a decreased expression of at least one gene selected
from the group consisting of hedgehog pathway genes, myc pathway
genes and histone methyltransferase genes 1) compared to a
reference value or a prior measurement; or 2) over the period of
time being monitored, such as 1, 2, 3, 4, 5, 6, or 7 days, or 1, 2,
3, 4, 5, 6, 7, 8, 9, 10 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12 months or longer. When the existing treatment is not
effective, a new treatment or an increased dosage of the existing
treatment (for example, increasing the dosage of the compound
administered to the subject) should be sought for the tested
subject.
[0099] In any methods presented herein, cancer is selected from the
group consisting of brain and central nervous system (CNS) cancer,
head and neck cancer, kidney cancer, ovarian cancer, pancreatic
cancer, leukemia, lung cancer, lymphoma, myeloma, sarcoma, breast
cancer, and prostate cancer. Preferably, cancer is selected from
the group consisting of medulloblastoma, oligodendroglioma, ovarian
clear cell adenocarcinoma, ovarian endomethrioid adenocarcinoma,
ovarian serous adenocarcinoma, pancreatic ductal adenocarcinoma,
pancreatic endocrine tumor, malignant rhabdoid tumor, astrocytoma,
atypical teratoid rhabdoid tumor, choroid plexus carcinoma, choroid
plexus papilloma, ependymoma, glioblastoma, meningioma, neuroglial
tumor, oligoastrocytoma, oligodendroglioma, pineoblastoma,
carcinosarcoma, chordoma, extragonadal germ cell tumor, extrarenal
rhabdoid tumor, schwannoma, skin squamous cell carcinoma,
chondrosarcoma, clear cell sarcoma of soft tissue, ewing sarcoma,
gastrointestinal stromal tumor, osteosarcoma, rhabdomyosarcoma,
epithelioid sarcoma, renal medullo carcinoma, diffuse large B-cell
lymphoma, follicular lymphoma and not otherwise specified (NOS)
sarcoma. More preferably, cancer is medulloblastoma, malignant
rhabdoid tumor, or atypical teratoid rhabdoid tumor.
[0100] As used herein, the term "responsiveness" is interchangeable
with terms "responsive", "sensitive", and "sensitivity", and it is
meant that a subject is showing therapeutic responses when
administered an EZH inhibitor, e.g., tumor cells or tumor tissues
of the subject undergo apoptosis and/or necrosis, and/or display
reduced growing, dividing, or proliferation. This term is also
meant that a subject will or has a higher probability, relative to
the population at large, of showing therapeutic responses when
administered an EZH inhibitor, e.g., tumor cells or tumor tissues
of the subject undergo apoptosis and/or necrosis, and/or display
reduced growing, dividing, or proliferation.
[0101] As used herein, a "subject" is interchangeable with a
"subject in need thereof", both of which refer to a subject having
a disorder in which EZH2-mediated protein methylation plays a part,
or a subject having an increased risk of developing such disorder
relative to the population at large. A subject in need thereof may
be a subject having a disorder associated with SWI/SNF complex. A
subject in need thereof can have a precancerous condition.
Preferably, a subject in need thereof has cancer. A subject in need
thereof can have cancer associated with SWI/SNF complex. A subject
in need thereof can have cancer associated with loss of function in
at least one component of SWI/SNF complex. In a preferred aspect, a
subject in need thereof has one or more cancers selected from the
group consisting of brain and central nervous system (CNS) cancer,
head and neck cancer, kidney cancer, ovarian cancer, pancreatic
cancer, leukemia, lung cancer, lymphoma, myeloma, sarcoma, breast
cancer, and prostate cancer. Preferably, a subject in need thereof
has medulloblastoma, oligodendroglioma, ovarian clear cell
adenocarcinoma, ovarian endomethrioid adenocarcinoma, ovarian
serous adenocarcinoma, pancreatic ductal adenocarcinoma, pancreatic
endocrine tumor, malignant rhabdoid tumor, astrocytoma, atypical
teratoid rhabdoid tumor, choroid plexus carcinoma, choroid plexus
papilloma, ependymoma, glioblastoma, meningioma, neuroglial tumor,
oligoastrocytoma, oligodendroglioma, pineoblastoma, carcinosarcoma,
chordoma, extragonadal germ cell tumor, extrarenal rhabdoid tumor,
schwannoma, skin squamous cell carcinoma, chondrosarcoma, clear
cell sarcoma of soft tissue, ewing sarcoma, gastrointestinal
stromal tumor, osteosarcoma, rhabdomyosarcoma, epithelioid sarcoma,
renal medullo carcinoma, diffuse large B-cell lymphoma, follicular
lymphoma and not otherwise specified (NOS) sarcoma. Alternatively,
a subject in need thereof has a non NHL cancer.
[0102] As used herein, a "subject" includes a mammal. The mammal
can be e.g., a human or appropriate non-human mammal, such as
primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep or a
pig. The subject can also be a bird or fowl. In one embodiment, the
mammal is a human. A subject can be male or female.
[0103] A subject in need thereof can be one who has not been
previously diagnosed or identified as having cancer or a
precancerous condition. A subject in need thereof can be one who
has been previously diagnosed or identified as having cancer or a
precancerous condition. A subject in need thereof can also be one
who is having (suffering from) cancer or a precancerous condition.
Alternatively, a subject in need thereof can be one who has a risk
of developing such disorder relative to the population at large
(i.e., a subject who is predisposed to developing such disorder
relative to the population at large).
[0104] Optionally a subject in need thereof has already undergone,
is undergoing or will undergo, at least one therapeutic
intervention for the cancer or precancerous condition.
[0105] A subject in need thereof may have refractory cancer on most
recent therapy. "Refractory cancer" means cancer that does not
respond to treatment. The cancer may be resistant at the beginning
of treatment or it may become resistant during treatment.
Refractory cancer is also called resistant cancer. In some
embodiments, the subject in need thereof has cancer recurrence
following remission on most recent therapy. In some embodiments,
the subject in need thereof received and failed all known effective
therapies for cancer treatment. In some embodiments, the subject in
need thereof received at least one prior therapy.
[0106] A subject in need thereof may be one who had, is having or
is predisposed to developing a cancer or a precancerous condition
associated with the SWI/SNF complex. A subject in need thereof may
be one who had, is having or is predisposed to developing cancer or
a precancerous condition associated with loss of function of at
least one component of the SWI/SNF complex. In a preferred aspect,
a subject in need thereof is one who had, is having or is
predisposed to developing one or more cancers selected from the
group consisting of brain and central nervous system (CNS) cancer,
head and neck cancer, kidney cancer, ovarian cancer, pancreatic
cancer, leukemia, lung cancer, lymphoma, myeloma, sarcoma, breast
cancer, and prostate cancer. Preferably, a subject in need thereof
is one who had, is having or is predisposed to developing brain and
CNS cancer, kidney cancer, ovarian cancer, pancreatic cancer,
leukemia, lymphoma, myeloma, and/or sarcoma. Exemplary brain and
central CNS cancer includes medulloblastoma, oligodendroglioma,
atypical teratoid rhabdoid tumor, choroid plexus carcinoma, choroid
plexus papilloma, ependymoma, glioblastoma, meningioma, neuroglial
tumor, oligoastrocytoma, oligodendroglioma, and pineoblastoma.
Exemplary ovarian cancer includes ovarian clear cell
adenocarcinoma, ovarian endomethrioid adenocarcinoma, and ovarian
serous adenocarcinoma. Exemplary pancreatic cancer includes
pancreatic ductal adenocarcinoma and pancreatic endocrine tumor.
Exemplary sarcoma includes chondrosarcoma, clear cell sarcoma of
soft tissue, ewing sarcoma, gastrointestinal stromal tumor,
osteosarcoma, rhabdomyosarcoma, and not otherwise specified (NOS)
sarcoma. Alternatively, cancers to be treated by the compounds of
the present invention are non NHL cancers.
[0107] Alternatively, a subject in need thereof is one who had, is
having or is predisposed to developing one or more cancers selected
from the group consisting of medulloblastoma, oligodendroglioma,
ovarian clear cell adenocarcinoma, ovarian endomethrioid
adenocarcinoma, ovarian serous adenocarcinoma, pancreatic ductal
adenocarcinoma, pancreatic endocrine tumor, malignant rhabdoid
tumor, astrocytoma, atypical teratoid rhabdoid tumor, choroid
plexus carcinoma, choroid plexus papilloma, ependymoma,
glioblastoma, meningioma, neuroglial tumor, oligoastrocytoma,
oligodendroglioma, pineoblastoma, carcinosarcoma, chordoma,
extragonadal germ cell tumor, extrarenal rhabdoid tumor,
schwannoma, skin squamous cell carcinoma, chondrosarcoma, clear
cell sarcoma of soft tissue, ewing sarcoma, gastrointestinal
stromal tumor, osteosarcoma, rhabdomyosarcoma, and not otherwise
specified (NOS) sarcoma. Preferably, a subject is one who had, is
having or is predisposed to developing medulloblastoma, ovarian
clear cell adenocarcinoma, ovarian endomethrioid adenocarcinoma,
pancreatic ductal adenocarcinoma, malignant rhabdoid tumor,
atypical teratoid rhabdoid tumor, choroid plexus carcinoma, choroid
plexus papilloma, glioblastoma, meningioma, pineoblastoma,
carcinosarcoma, extrarenal rhabdoid tumor, schwannoma, skin
squamous cell carcinoma, chondrosarcoma, ewing sarcoma, epithelioid
sarcoma, renal medullo carcinoma, diffuse large B-cell lymphoma,
follicular lymphoma and/or NOS sarcoma. More preferably, a subject
in need thereof is one who had, is having or is predisposed to
developing malignant rhabdoid tumor, medulloblastoma and/or
atypical teratoid rhabdoid tumor.
[0108] In some embodiments of the present invention, a subject in
need thereof has a decreased expression level of at least one gene
selected from the group consisting of neuronal differentiation
genes, cell cycle inhibition genes, and tumor suppressor genes.
[0109] In some embodiments, a subject in need thereof has an
increased expression level of at least one gene selected from the
group consisting of hedgehog pathway genes, myc pathway genes and
histone methyltransferase genes.
[0110] In some embodiments of the present invention, a subject in
need thereof has loss of function of at least one component/subunit
of the SWI/SNF complex. Alternatively, a subject in need thereof
has reduced expression or haploinsufficiency of at least one
component/subunit of the SWI/SNF complex. In certain embodiments, a
subject in need thereof has loss of function of SNF5 subunit.
[0111] In any method of the present invention, a subject in need
thereof may have reduced expression, haploinsufficiency or loss of
function of at least one signaling component downstream of SWI/SNF
complex. Such downstream component includes, but is not limited to,
polycomb complex (PcG) and its targets.
[0112] As used herein, the term "loss of function" refers to less
or no function of a gene product/protein compared to the wild type.
Loss of function of a SWI/SNF complex component means the
component/subunit or the entire SWI/SNF complex has less or no
biological function compared to the wild type component/subunit or
the entire SWI/SNF complex, respectively. Loss of function can be
caused by transcriptional, post-transcription, or post
translational mechanisms. In one aspect of the present invention,
loss of function is caused by loss of function mutation resulted
from a point mutation (e.g., a substitution, a missense mutation,
or a nonsense mutation), an insertion, and/or a deletion in a
polypeptide of a SWI/SNF complex component or a nucleic acid
sequence encoding a polypeptide of a SWI/SNF complex component. The
mutations referred herein are somatic mutations. The term "somatic
mutation" refers to a deleterious alteration in at least one gene
allele that is not found in every cell of the body, but is found
only in isolated cells. A characteristic of the somatic mutations
as used herein is, that they are restricted to particular tissues
or even parts of tissues or cells within a tissue and are not
present in the whole organism harboring the tissues or cells. The
term "wild-type" refers to a gene or gene product that has the
characteristics of that gene or gene product when isolated from a
naturally occurring source. A wild-type gene is that which is most
frequently observed in a population and is thus arbitrarily
designed the "normal" or "wild-type" form of the gene.
[0113] Accordingly, a loss of function mutation or a reduced
expression can be detected using any suitable method available in
the art. For example, a loss of function mutation can be detected
by measuring the biological function of a gene product, such as the
ATP-dependent chromatin remodeling activity of the SWI/SNF complex.
Alternatively, a loss of function mutation can be determined by
detecting any alternation in a nucleic acid sequence encoding a
component of the SWI/SNF complex. For example, a nucleic acid
sequence encoding a component of the SWI/SNF complex having a loss
of function mutation can be detected by whole-genome resequencing
or target region resequencing (the latter also known as targeted
resequencing) using suitably selected sources of DNA and polymerase
chain reaction (PCR) primers in accordance with methods well known
in the art. The method typically and generally entails the steps of
genomic DNA purification, PCR amplification to amplify the region
of interest, cycle sequencing, sequencing reaction cleanup,
capillary electrophoresis, and/or data analysis. Alternatively or
in addition, the method may include the use of microarray-based
targeted region genomic DNA capture and/or sequencing. Kits,
reagents, and methods for selecting appropriate PCR primers and
performing resequencing are commercially available, for example,
from Applied Biosystems, Agilent, and NimbleGen (Roche Diagnostics
GmbH). Alternatively or in addition, a nucleic acid sequence
encoding a SWI/SNF polypeptide having a loss of function mutation
may be detected using a Southern blot in accordance with methods
well known in the art. Optionally, a loss of function mutation can
be detected by measuring the absence of the expression of a
component polypeptide or by measuring the expression of the mutant
component polypeptide. Detection of (mutant) polypeptide expression
can be carried out with any suitable immunoassay in the art, such
as Western blot analysis.
[0114] Human nucleic acid and amino acid sequence of components of
the SWI/SNF complex have previously been described. See, e.g.,
GenBank Accession Nos NP_003064.2, NM_003073.3, NP_001007469.1, and
NM_001007468.1 for SNF5, GenBank Accession Nos NM_000489.3,
NP_000480.2, NM_138270.2, and NP_612114.1 for ATRX, GenBank
Accession Nos NP_006006.3, NM_006015.4, NP_624361.1, and
NM_139135.2 for ARID1A, each of which is incorporated herein by
reference in its entirety.
[0115] Spectrum of hSNF5 somatic mutations in human has also been
described in Sevenet et al., Human Molecular Genetics, 8:
2359-2368, 1999, which is incorporated herein by reference in its
entirety.
[0116] A subject in need thereof may have reduced expression,
haploinsufficiency, and/or loss of function of SNF5. For example, a
subject can comprise a deletion of SNF5 in SNF5 polypeptide or a
nucleic acid sequence encoding a SNF5 polypeptide.
TABLE-US-00001 SWI/SNF-related matrix-associated actin-dependent
regulator of chromatin subfamily B member 1 isoform a (SMARCB1,
also called SNF5) [Homo sapiens] (SEQ ID NO: 1) 1 mmmmalsktf
gqkpvkfqle ddgefymigs evgnylrmfr gslykrypsl wrrlatveer 61
kkivasshgk ktkpntkdhg yttlatsvtl lkaseveeil dgndekykav sistepptyl
121 reqkakrnsq wvptlpnssh hldavpcstt inrnrmgrdk krtfplcfdd
hdpavihena 181 sqpevlvpir ldmeidgqkl rdaftwnmne klmtpemfse
ilcddldlnp ltfvpaiasa 241 irqqiesypt dsiledqsdq rviiklnihv
gnislvdqfe wdmsekensp ekfalklcse 301 lglggefvtt iaysirgqls
whqktyafse nplptveiai rntgdadqwc plletltdae 361 mekkirdqdr
ntrrmrrlan tapaw
TABLE-US-00002 Homo sapiens SWI/SNF related, matrix associated,
actin dependent regulator of chromatin, subfamily b, member 1
(SMARCB1, also called SNF5), transcript variant 1, mRNA (SEQ ID NO:
2) 1 aacgccagcg cctgcgcact gagggcggcc tggtcgtcgt ctgcggcggc
ggcggcggct 61 gaggagcccg gctgaggcgc cagtacccgg cccggtccgc
atttcgcctt ccggcttcgg 121 tttccctcgg cccagcacgc cccggccccg
ccccagccct cctgatccct cgcagcccgg 181 ctccggccgc ccgcctctgc
cgccgcaatg atgatgatgg cgctgagcaa gaccttcggg 241 cagaagcccg
tgaagttcca gctggaggac gacggcgagt tctacatgat cggctccgag 301
gtgggaaact acctccgtat gttccgaggt tctctgtaca agagataccc ctcactctgg
361 aggcgactag ccactgtgga agagaggaag aaaatagttg catcgtcaca
tggtaaaaaa 421 acaaaaccta acactaagga tcacggatac acgactctag
ccaccagtgt gaccctgtta 481 aaagcctcgg aagtggaaga gattctggat
ggcaacgatg agaagtacaa ggctgtgtcc 541 atcagcacag agccccccac
ctacctcagg gaacagaagg ccaagaggaa cagccagtgg 601 gtacccaccc
tgcccaacag ctcccaccac ttagatgccg tgccatgctc cacaaccatc 661
aacaggaacc gcatgggccg agacaagaag agaaccttcc ccctttgctt tgatgaccat
721 gacccagctg tgatccatga gaacgcatct cagcccgagg tgctggtccc
catccggctg 781 gacatggaga tcgatgggca gaagctgcga gacgccttca
cctggaacat gaatgagaag 841 ttgatgacgc ctgagatgtt ttcagaaatc
ctctgtgacg atctggattt gaacccgctg 901 acgtttgtgc cagccatcgc
ctctgccatc agacagcaga tcgagtccta ccccacggac 961 agcatcctgg
aggaccagtc agaccagcgc gtcatcatca agctgaacat ccatgtggga 1021
aacatttccc tggtggacca gtttgagtgg gacatgtcag agaaggagaa ctcaccagag
1081 aagtttgccc tgaagctgtg ctcggagctg gggttgggcg gggagtttgt
caccaccatc 1141 gcatacagca tccggggaca gctgagctgg catcagaaga
cctacgcctt cagcgagaac 1201 cctctgccca cagtggagat tgccatccgg
aacacgggcg atgcggacca gtggtgccca 1261 ctgctggaga ctctgacaga
cgctgagatg gagaagaaga tccgcgacca ggacaggaac 1321 acgaggcgga
tgaggcgtct tgccaacacg gccccggcct ggtaaccagc ccatcagcac 1381
acggctccca cggagcatct cagaagattg ggccgcctct cctccatctt ctggcaagga
1441 cagaggcgag gggacagccc agcgccatcc tgaggatcgg gtgggggtgg
agtgggggct 1501 tccaggtggc ccttcccggc acacattcca tttgttgagc
cccagtcctg ccccccaccc 1561 caccctccct acccctcccc agtctctggg
gtcaggaaga aaccttattt taggttgtgt 1621 tttgtttttg tataggagcc
ccaggcaggg ctagtaacag tttttaaata aaaggcaaca 1681 ggtcatgttc
aatttcttca acaaaaaaaa aaaaaaa
TABLE-US-00003 SWI/SNF-related matrix-associated actin-dependent
regulator of chromatin subfamily B member 1 isoform b [Homo
sapiens] (SMARCB1, also called SNF5) (SEQ ID NO: 3) 1 mmmmalsktf
gqkpvkfqle ddgefymigs evgnylrmfr gslykrypsl wrrlatveer 61
kkivasshdh gyttlatsvt llkaseveei ldgndekyka vsisteppty lreqkakrns
121 qwvptlpnss hhldavpcst tinrnrmgrd kkrtfplcfd dhdpavihen
asqpevlvpi 181 rldmeidgqk lrdaftwnmn eklmtpemfs eilcddldln
pltfvpaias airqqiesyp 241 tdsiledqsd qrviiklnih vgnislvdqf
ewdmsekens pekfalklcs elglggefvt 301 tiaysirgql swhqktyafs
enplptveia irntgdadqw cplletltda emekkirdqd 361 rntrrmrrla
ntapaw
TABLE-US-00004 Homo sapiens SWI/SNF related, matrix associated,
actin dependent regulator of chromatin, subfamily b, member 1
(SMARCB1, also called SNF5), transcript variant 2, mRNA (SEQ ID NO:
4) 1 aacgccagcg cctgcgcact gagggcggcc tggtcgtcgt ctgcggcggc
ggcggcggct 61 gaggagcccg gctgaggcgc cagtacccgg cccggtccgc
atttcgcctt ccggcttcgg 121 tttccctcgg cccagcacgc cccggccccg
ccccagccct cctgatccct cgcagcccgg 181 ctccggccgc ccgcctctgc
cgccgcaatg atgatgatgg cgctgagcaa gaccttcggg 241 cagaagcccg
tgaagttcca gctggaggac gacggcgagt tctacatgat cggctccgag 301
gtgggaaact acctccgtat gttccgaggt tctctgtaca agagataccc ctcactctgg
361 aggcgactag ccactgtgga agagaggaag aaaatagttg catcgtcaca
tgatcacgga 421 tacacgactc tagccaccag tgtgaccctg ttaaaagcct
cggaagtgga agagattctg 481 gatggcaacg atgagaagta caaggctgtg
tccatcagca cagagccccc cacctacctc 541 agggaacaga aggccaagag
gaacagccag tgggtaccca ccctgcccaa cagctcccac 601 cacttagatg
ccgtgccatg ctccacaacc atcaacagga accgcatggg ccgagacaag 661
aagagaacct tccccctttg ctttgatgac catgacccag ctgtgatcca tgagaacgca
721 tctcagcccg aggtgctggt ccccatccgg ctggacatgg agatcgatgg
gcagaagctg 781 cgagacgcct tcacctggaa catgaatgag aagttgatga
cgcctgagat gttttcagaa 841 atcctctgtg acgatctgga tttgaacccg
ctgacgtttg tgccagccat cgcctctgcc 901 atcagacagc agatcgagtc
ctaccccacg gacagcatcc tggaggacca gtcagaccag 961 cgcgtcatca
tcaagctgaa catccatgtg ggaaacattt ccctggtgga ccagtttgag 1021
tgggacatgt cagagaagga gaactcacca gagaagtttg ccctgaagct gtgctcggag
1081 ctggggttgg gcggggagtt tgtcaccacc atcgcataca gcatccgggg
acagctgagc 1141 tggcatcaga agacctacgc cttcagcgag aaccctctgc
ccacagtgga gattgccatc 1201 cggaacacgg gcgatgcgga ccagtggtgc
ccactgctgg agactctgac agacgctgag 1261 atggagaaga agatccgcga
ccaggacagg aacacgaggc ggatgaggcg tcttgccaac 1321 acggccccgg
cctggtaacc agcccatcag cacacggctc ccacggagca tctcagaaga 1381
ttgggccgcc tctcctccat cttctggcaa ggacagaggc gaggggacag cccagcgcca
1441 tcctgaggat cgggtggggg tggagtgggg gcttccaggt ggcccttccc
ggcacacatt 1501 ccatttgttg agccccagtc ctgcccccca ccccaccctc
cctacccctc cccagtctct 1561 ggggtcagga agaaacctta ttttaggttg
tgttttgttt ttgtatagga gccccaggca 1621 gggctagtaa cagtttttaa
ataaaaggca acaggtcatg ttcaatttct tcaacaaaaa 1681 aaaaaaaaaa
[0117] A subject in need thereof may have reduced expression,
haploinsufficiency, and/or loss of function of ATRX. For example, a
subject can comprise a mutation selected from the group consisting
of a substitution of asparagine (N) for the wild type residue
lysine (K) at amino acid position 688 of SEQ ID NO: 5 (K688N), and
a substitution of isoleucine (I) for the wild type residue
methionine (M) at amino acid position 366 of SEQ ID NO: 5
(M366I).
TABLE-US-00005 Homo sapiens alpha thalassemia/mental retardation
syndrome X-linked (ATRX) isoform 1 (SEQ ID NO: 5) 1 mtaepmsesk
lntlvqklhd flahsseese etsspprlam nqntdkisgs gsnsdmmens 61
keegtsssek skssgssrsk rkpsivtkyv esddekpldd etvnedasne nsenditmqs
121 lpkgtvivqp epvlnedkdd fkgpefrsrs kmktenlkkr gedglhgivs
ctacgqqvnh 181 fqkdsiyrhp slqvlicknc fkyymsddis rdsdgmdeqc
rwcaeggnli ccdfchnafc 241 kkcilrnlgr kelstimden nqwycyichp
eplldlvtac nsvfenleql lqqnkkkikv 301 dseksnkvye htsrfspkkt
ssncngeekk lddscsgsvt ysysalivpk emikkakkli 361 ettanmnssy
vkflkqatdn seissatklr qlkafksvla dikkahlale edlnsefram 421
davnkekntk ehkvidakfe tkarkgekpc alekkdisks eaklsrkqvd sehmhqnvpt
481 eeqrtnkstg gehkksdrke epqyepants edldmdivsv pssvpedife
nletamevqs 541 svdhqgdgss gteqevesss vklnisskdn rggiksktta
kvtkelyvkl tpvslsnspi 601 kgadcqevpq dkdgykscgl npklekcglg
qensdnehlv enevslllee sdlrrsprvk 661 ttplrrptet npvtsnsdee
cnetvkekqk lsvpvrkkdk rnssdsaidn pkpnklpksk 721 qsetvdqnsd
sdemlailke vsrmshssss dtdineihtn hktlydlktq agkddkgkrk 781
rksstsgsdf dtkkgksaks siiskkkrqt qsessnydse lekeiksmsk igaarttkkr
841 ipntkdfdss edekhskkgm dnqghknlkt sqegssddae rkqeretfss
aegtvdkdtt 901 imelrdrlpk kqqasastdg vdklsgkeqs ftslevrkva
etkekskhlk tktckkvqdg 961 lsdiaekflk kdqsdetsed dkkqskkgte
ekkkpsdfkk kvikmeqqye sssdgteklp 1021 ereeichfpk gikqikngtt
dgekkskkir dktskkkdel sdyaekstgk gdscdssedk 1081 kskngaygre
kkrckllgks srkrqdcsss dtekysmked gcnssdkrlk rielrerrnl 1141
sskrntkeiq sgssssdaee ssednkkkkq rtsskkkavi vkekkrnslr tstkrkqadi
1201 tsssssdied ddqnsigegs sdeqkikpvt enlvlsshtg fcqssgdeal
sksvpvtvdd 1261 ddddndpenr iakkmlleei kanlssdedg ssddepeegk
krtgkqneen pgdeeaknqv 1321 nsesdsdsee skkpryrhrl lrhkltvsdg
esgeekktkp kehkevkgrn rrkvssedse 1381 dsdfqesgvs eevsesedeq
rprtrsakka eleenqrsyk qkkkrrrikv qedsssenks 1441 nseeeeeeke
eeeeeeeeee eeeedendds kspgkgrkki rkilkddklr tetqnalkee 1501
eerrkriaer erereklrev ieiedasptk cpittklvld edeetkeplv qvhrnmvikl
1561 kphqvdgvqf mwdcccesvk ktkkspgsgc ilahcmglgk tlqvvsflht
vllcdkldfs 1621 talvvcplnt alnwmnefek wqeglkddek levselatvk
rpqersymlq rwqedggvmi 1681 igyemyrnla qgrnvksrkl keifnkalvd
pgpdfvvcde ghilkneasa vskamnsirs 1741 rrriiltgtp lqnnlieyhc
mvnfikenll gsikefrnrf inpiqngqca dstmvdvrvm 1801 kkrahilyem
lagcvqrkdy taltkflppk heyvlavrmt siqcklyqyy ldhltgvgnn 1861
seggrgkaga klfqdfqmls riwthpwclq ldyiskenkg yfdedsmdef iasdsdetsm
1921 slssddytkk kkkgkkgkkd ssssgsgsdn dvevikvwns rsrgggegnv
detgnnpsvs 1981 lkleeskats ssnpsspapd wykdfvtdad aevlehsgkm
vllfeilrma eeigdkvlvf 2041 sqslisldli edflelasre ktedkdkpli
ykgegkwlrn idyyrldgst taqsrkkwae 2101 efndetnvrg rlfiistkag
slginlvaan rviifdaswn psydiqsifr vyrfgqtkpv 2161 yvyrflaqgt
medkiydrqv tkqslsfrvv dqqqverhft mneltelytf epdllddpns 2221
ekkkkrdtpm lpkdtilael lqihkehivg yhehdslldh keeeelteee rkaawaeyea
2281 ekkgltmrfn iptgtnlppv sfnsqtpyip fnlgalsams nqqledlinq
grekvveatn 2341 svtavriqpl ediisavwke nmnlseaqvq alalsrqasq
eldvkrreai yndvltkqqm 2401 liscvqrilm nrrlqqqynq qqqqqmtyqq
atlghlmmpk ppnlimnpsn yqqidmrgmy 2461 qpvaggmqpp plqrapppmr
sknpgpsqgk sm
TABLE-US-00006 Homo sapiens alpha thalassemia/mental retardation
syndrome X-linked (ATRX), transcript variant 1, mRNA (SEQ ID NO: 6)
1 aattctcctg cctgagcctc ggcccaacaa aatggcggcg gcagcggtgt cgctttgttt
61 ccgcggctcc tgcggcggtg gcagtggtag cggcctttga gctgtgggga
ggttccagca 121 gcagctacag tgacgactaa gactccagtg catttctatc
gtaaccgggc gcgggggagc 181 gcagatcggc gcccagcaat cacagaagcc
gacaaggcgt tcaagcgaaa acatgaccgc 241 tgagcccatg agtgaaagca
agttgaatac attggtgcag aagcttcatg acttccttgc 301 acactcatca
gaagaatctg aagaaacaag ttctcctcca cgacttgcaa tgaatcaaaa 361
cacagataaa atcagtggtt ctggaagtaa ctctgatatg atggaaaaca gcaaggaaga
421 gggaactagc tcttcagaaa aatccaagtc ttcaggatcg tcacgatcaa
agaggaaacc 481 ttcaattgta acaaagtatg tagaatcaga tgatgaaaaa
cctttggatg atgaaactgt 541 aaatgaagat gcgtctaatg aaaattcaga
aaatgatatt actatgcaga gcttgccaaa 601 aggtacagtg attgtacagc
cagagccagt gctgaatgaa gacaaagatg attttaaagg 661 gcctgaattt
agaagcagaa gtaaaatgaa aactgaaaat ctcaaaaaac gcggagaaga 721
tgggcttcat gggattgtga gctgcactgc ttgtggacaa caggtcaatc attttcaaaa
781 agattccatt tatagacacc cttcattgca agttcttatt tgtaagaatt
gctttaagta 841 ttacatgagt gatgatatta gccgtgactc agatggaatg
gatgaacaat gtaggtggtg 901 tgcggaaggt ggaaacttga tttgttgtga
cttttgccat aatgctttct gcaagaaatg 961 cattctacgc aaccttggtc
gaaaggagtt gtccacaata atggatgaaa acaaccaatg 1021 gtattgctac
atttgtcacc cagagccttt gttggacttg gtcactgcat gtaacagcgt 1081
atttgagaat ttagaacagt tgttgcagca aaataagaag aagataaaag ttgacagtga
1141 aaagagtaat aaagtatatg aacatacatc cagattttct ccaaagaaga
ctagttcaaa 1201 ttgtaatgga gaagaaaaga aattagatga ttcctgttct
ggctctgtaa cctactctta 1261 ttccgcacta attgtgccca aagagatgat
taagaaggca aaaaaactga ttgagaccac 1321 agccaacatg aactccagtt
atgttaaatt tttaaagcag gcaacagata attcagaaat 1381 cagttctgct
acaaaattac gtcagcttaa ggcttttaag tctgtgttgg ctgatattaa 1441
gaaggctcat cttgcattgg aagaagactt aaattccgag tttcgagcga tggatgctgt
1501 aaacaaagag aaaaatacca aagagcataa agtcatagat gctaagtttg
aaacaaaagc 1561 acgaaaagga gaaaaacctt gtgctttgga aaagaaggat
atttcaaagt cagaagctaa 1621 actttcaaga aaacaggtag atagtgagca
catgcatcag aatgttccaa cagaggaaca 1681 aagaacaaat aaaagtaccg
gtggtgaaca taagaaatct gatagaaaag aagaacctca 1741 atatgaacct
gccaacactt ctgaagattt agacatggat attgtgtctg ttccttcctc 1801
agttccagaa gacatttttg agaatcttga gactgctatg gaagttcaga gttcagttga
1861 tcatcaaggg gatggcagca gtggaactga acaagaagtg gagagttcat
ctgtaaaatt 1921 aaatatttct tcaaaagaca acagaggagg tattaaatca
aaaactacag ctaaagtaac 1981 aaaagaatta tatgttaaac tcactcctgt
ttccctttct aattccccaa ttaaaggtgc 2041 tgattgtcag gaagttccac
aagataaaga tggctataaa agttgtggtc tgaaccccaa 2101 gttagagaaa
tgtggacttg gacaggaaaa cagtgataat gagcatttgg ttgaaaatga 2161
agtttcatta cttttagagg aatctgatct tcgaagatcc ccacgtgtaa agactacacc
2221 cttgaggcga ccgacagaaa ctaaccctgt aacatctaat tcagatgaag
aatgtaatga 2281 aacagttaag gagaaacaaa aactatcagt tccagtgaga
aaaaaggata agcgtaattc 2341 ttctgacagt gctatagata atcctaagcc
taataaattg ccaaaatcta agcaatcaga 2401 gactgtggat caaaattcag
attctgatga aatgctagca atcctcaaag aggtgagcag 2461 gatgagtcac
agttcttctt cagatactga tattaatgaa attcatacaa accataagac 2521
tttgtatgat ttaaagactc aggcggggaa agatgataaa ggaaaaagga aacgaaaaag
2581 ttctacatct ggctcagatt ttgatactaa aaagggcaaa tcagctaaga
gctctataat 2641 ttctaaaaag aaacgacaaa cccagtctga gtcttctaat
tatgactcag aattagaaaa 2701 agagataaag agcatgagta aaattggtgc
tgccagaacc accaaaaaaa gaattccaaa 2761 tacaaaagat tttgactctt
ctgaagatga gaaacacagc aaaaaaggaa tggataatca 2821 agggcacaaa
aatttgaaga cctcacaaga aggatcatct gatgatgctg aaagaaaaca 2881
agagagagag actttctctt cagcagaagg cacagttgat aaagacacga ccatcatgga
2941 attaagagat cgacttccta agaagcagca agcaagtgct tccactgatg
gtgtcgataa 3001 gctttctggg aaagagcaga gttttacttc tttggaagtt
agaaaagttg ctgaaactaa 3061 agaaaagagc aagcatctca aaaccaaaac
atgtaaaaaa gtacaggatg gcttatctga 3121 tattgcagag aaattcctaa
agaaagacca gagcgatgaa acttctgaag atgataaaaa 3181 gcagagcaaa
aagggaactg aagaaaaaaa gaaaccttca gactttaaga aaaaagtaat 3241
taaaatggaa caacagtatg aatcttcatc tgatggcact gaaaagttac ctgagcgaga
3301 agaaatttgt cattttccta agggcataaa acaaattaag aatggaacaa
ctgatggaga 3361 aaagaaaagt aaaaaaataa gagataaaac ttctaaaaag
aaggatgaat tatctgatta 3421 tgctgagaag tcaacaggga aaggagatag
ttgtgactct tcagaggata aaaagagtaa 3481 gaatggagca tatggtagag
agaagaaaag gtgcaagttg cttggaaaga gttcaaggaa 3541 gagacaagat
tgttcatcat ctgatactga gaaatattcc atgaaagaag atggttgtaa 3601
ctcttctgat aagagactga aaagaataga attgagggaa agaagaaatt taagttcaaa
3661 gagaaatact aaggaaatac aaagtggctc atcatcatct gatgctgagg
aaagttctga 3721 agataataaa aagaagaagc aaagaacttc atctaaaaag
aaggcagtca ttgtcaagga 3781 gaaaaagaga aactccctaa gaacaagcac
taaaaggaag caagctgaca ttacatcctc 3841 atcttcttct gatatagaag
atgatgatca gaattctata ggtgagggaa gcagcgatga 3901 acagaaaatt
aagcctgtga ctgaaaattt agtgctgtct tcacatactg gattttgcca 3961
atcttcagga gatgaagcct tatctaaatc agtgcctgtc acagtggatg atgatgatga
4021 cgacaatgat cctgagaata gaattgccaa gaagatgctt ttagaagaaa
ttaaagccaa 4081 tctttcctct gatgaggatg gatcttcaga tgatgagcca
gaagaaggga aaaaaagaac 4141 tggaaaacaa aatgaagaaa acccaggaga
tgaggaagca aaaaatcaag tcaattctga 4201 atcagattca gattctgaag
aatctaagaa gccaagatac agacataggc ttttgcggca 4261 caaattgact
gtgagtgacg gagaatctgg agaagaaaaa aagacaaagc ctaaagagca 4321
taaagaagtc aaaggcagaa acagaagaaa ggtgagcagt gaagattcag aagattctga
4381 ttttcaggaa tcaggagtta gtgaagaagt tagtgaatcc gaagatgaac
agcggcccag 4441 aacaaggtct gcaaagaaag cagagttgga agaaaatcag
cggagctata aacagaaaaa 4501 gaaaaggcga cgtattaagg ttcaagaaga
ttcatccagt gaaaacaaga gtaattctga 4561 ggaagaagag gaggaaaaag
aagaggagga ggaagaggag gaggaggagg aagaggagga 4621 ggaagatgaa
aatgatgatt ccaagtctcc tggaaaaggc agaaagaaaa ttcggaagat 4681
tcttaaagat gataaactga gaacagaaac acaaaatgct cttaaggaag aggaagagag
4741 acgaaaacgt attgctgaga gggagcgtga gcgagaaaaa ttgagagagg
tgatagaaat 4801 tgaagatgct tcacccacca agtgtccaat aacaaccaag
ttggttttag atgaagatga 4861 agaaaccaaa gaacctttag tgcaggttca
tagaaatatg gttatcaaat tgaaacccca 4921 tcaagtagat ggtgttcagt
ttatgtggga ttgctgctgt gagtctgtga aaaaaacaaa 4981 gaaatctcca
ggttcaggat gcattcttgc ccactgtatg ggccttggta agactttaca 5041
ggtggtaagt tttcttcata cagttctttt gtgtgacaaa ctggatttca gcacggcgtt
5101 agtggtttgt cctcttaata ctgctttgaa ttggatgaat gaatttgaga
agtggcaaga 5161 gggattaaaa gatgatgaga agcttgaggt ttctgaatta
gcaactgtga aacgtcctca 5221 ggagagaagc tacatgctgc agaggtggca
agaagatggt ggtgttatga tcataggcta 5281 tgagatgtat agaaatcttg
ctcaaggaag gaatgtgaag agtcggaaac ttaaagaaat 5341 atttaacaaa
gctttggttg atccaggccc tgattttgtt gtttgtgatg aaggccatat 5401
tctaaaaaat gaagcatctg ctgtttctaa agctatgaat tctatacgat caaggaggag
5461 gattatttta acaggaacac cacttcaaaa taacctaatt gagtatcatt
gtatggttaa 5521 ttttatcaag gaaaatttac ttggatccat taaggagttc
aggaatagat ttataaatcc 5581 aattcaaaat ggtcagtgtg cagattctac
catggtagat gtcagagtga tgaaaaaacg 5641 tgctcacatt ctctatgaga
tgttagctgg atgtgttcag aggaaagatt atacagcatt 5701 aacaaaattc
ttgcctccaa aacacgaata tgtgttagct gtgagaatga cttctattca 5761
gtgcaagctc tatcagtact acttagatca cttaacaggt gtgggcaata atagtgaagg
5821 tggaagagga aaggcaggtg caaagctttt ccaagatttt cagatgttaa
gtagaatatg 5881 gactcatcct tggtgtttgc agctagacta cattagcaaa
gaaaataagg gttattttga 5941 tgaagacagt atggatgaat ttatagcctc
agattctgat gaaacctcca tgagtttaag 6001 ctccgatgat tatacaaaaa
agaagaaaaa agggaaaaag gggaaaaaag atagtagctc 6061 aagtggaagt
ggcagtgaca atgatgttga agtgattaag gtctggaatt caagatctcg 6121
gggaggtggt gaaggaaatg tggatgaaac aggaaacaat ccttctgttt ctttaaaact
6181 ggaagaaagt aaagctactt cttcttctaa tccaagcagc ccagctccag
actggtacaa 6241 agattttgtt acagatgctg atgctgaggt tttagagcat
tctgggaaaa tggtacttct 6301 ctttgaaatt cttcgaatgg cagaggaaat
tggggataaa gtccttgttt tcagccagtc 6361 cctcatatct ctggacttga
ttgaagattt tcttgaatta gctagtaggg agaagacaga 6421 agataaagat
aaacccctta tttataaagg tgaggggaag tggcttcgaa acattgacta 6481
ttaccgttta gatggttcca ctactgcaca gtcaaggaag aagtgggctg aagaatttaa
6541 tgatgaaact aatgtgagag gacgattatt tatcatttct actaaagcag
gatctctagg 6601 aattaatctg gtagctgcta atcgagtaat tatattcgac
gcttcttgga atccatctta 6661 tgacatccag agtatattca gagtttatcg
ctttggacaa actaagcctg tttatgtata 6721 taggttctta gctcagggaa
ccatggaaga taagatttat gatcggcaag taactaagca 6781 gtcactgtct
tttcgagttg ttgatcagca gcaggtggag cgtcatttta ctatgaatga 6841
gcttactgaa ctttatactt ttgagccaga cttattagat gaccctaatt cagaaaagaa
6901 gaagaagagg gatactccca tgctgccaaa ggataccata cttgcagagc
tccttcagat 6961 acataaagaa cacattgtag gataccatga acatgattct
cttttggacc acaaagaaga 7021 agaagagttg actgaagaag aaagaaaagc
agcttgggct gagtatgaag cagagaagaa 7081 gggactgacc atgcgtttca
acataccaac tgggaccaat ttaccccctg tcagtttcaa 7141 ctctcaaact
ccttatattc ctttcaattt gggagccctg tcagcaatga gtaatcaaca 7201
gctggaggac ctcattaatc aaggaagaga aaaagttgta gaagcaacaa acagtgtgac
7261 agcagtgagg attcaacctc ttgaggatat aatttcagct gtatggaagg
agaacatgaa 7321 tctctcagag gcccaagtac aggcgttagc attaagtaga
caagccagcc aggagcttga
7381 tgttaaacga agagaagcaa tctacaatga tgtattgaca aaacaacaga
tgttaatcag 7441 ctgtgttcag cgaatactta tgaacagaag gctccagcag
cagtacaatc agcagcaaca 7501 gcaacaaatg acttatcaac aagcaacact
gggtcacctc atgatgccaa agcccccaaa 7561 tttgatcatg aatccttcta
actaccagca gattgatatg agaggaatgt atcagccagt 7621 ggctggtggt
atgcagccac caccattaca gcgtgcacca cccccaatga gaagcaaaaa 7681
tccaggacct tcccaaggga aatcaatgtg attttgcact aaaagcttaa tggattgtta
7741 aaatcataga aagatctttt atttttttag gaatcaatga cttaacagaa
ctcaactgta 7801 taaatagttt ggtcccctta aatgccaatc ttccatatta
gttttacttt tttttttttt 7861 aaatagggca taccatttct tcctgacatt
tgtcagtgat gttgcctaga atcttcttac 7921 acacgctgag tacagaagat
atttcaaatt gttttcagtg aaaacaagtc cttccataat 7981 agtaacaact
ccacagattt cctctctaaa tttttatgcc tgcttttagc aaccataaaa 8041
ttgtcataaa attaataaat ttaggaaaga ataaagattt atatattcat tctttacata
8101 taaaaacaca cagctgagtt cttagagttg attcctcaag ttatgaaata
cttttgtact 8161 taatccattt cttgattaaa gtgattgaaa tggttttaat
gttcttttga ctgaagtctg 8221 aaactgggct cctgctttat tgtctctgtg
actgaaagtt agaaactgag ggttatcttt 8281 gacacagaat tgtgtgcaat
attcttaaat actactgctc taaaagttgg agaagtcttg 8341 cagttatctt
agcattgtat aaacagcctt aagtatagcc taagaagaga attccttttt 8401
cttctttagt ccttctgcca ttttttattt tcagttatat gtgctgaaat aattactggt
8461 aaaatttcag ggttgtggat tatcttccac acatgaattt tctctctcct
ggcacgaata 8521 taaagcacat ctcttaactg catggtgcca gtgctaatgc
ttcatcctgt tgctggcagt 8581 gggatgtgga cttagaaaat caagttctag
cattttagta ggttaacact gaagttgtgg 8641 ttgttaggtt cacaccctgt
tttataaaca acatcaaaat ggcagaacca ttgctgactt 8701 taggttcaca
tgaggaatgt acttttaaca attcccagta ctatcagtat tgtgaaataa 8761
ttcctctgaa agataagaat cactggcttc tatgcgcttc ttttctctca tcatcatgtt
8821 cttttacccc agtttcctta cattttttta aattgtttca gagtttgttt
tttttttagt 8881 ttagattgtg aggcaattat taaatcaaaa ttaattcatc
caatacccct ttactagaag 8941 ttttactaga aaatgtatta cattttattt
tttcttaatc cagttctgca aaaatgacct 9001 ataaatttat tcatgtacaa
ttttggttac ttgaattgtt aaagaaaaca ttgtttttga 9061 ctatgggagt
caactcaaca tggcagaacc atttttgaga tgatgataca acaggtagtg 9121
aaacagctta agaattccaa aaaaaaaaaa aaaaaaaaaa aaaagaaaac tgggtttggg
9181 ctttgcttta ggtatcactg gattagaatg agtttaacat tagctaaaac
tgctttgagt 9241 tgtttggatg attaagagat tgccattttt atcttggaag
aactagtggt aaaacatcca 9301 agagcactag gattgtgata cagaatttgt
gaggtttggt ggatccacgc ccctctcccc 9361 cactttccca tgatgaaata
tcactaataa atcctgtata tttagatatt atgctagcca 9421 tgtaatcaga
tttatttaat tgggtggggc aggtgtgtat ttactttaga aaaaatgaaa 9481
aagacaagat ttatgagaaa tatttgaagg cagtacactc tggccaactg ttaccagttg
9541 gtatttctac aagttcagaa tattttaaac ctgatttact agacctggga
attttcaaca 9601 tggtctaatt atttactcaa agacatagat gtgaaaattt
taggcaacct tctaaatctt 9661 tttcaccatg gatgaaacta taacttaaag
aataatactt agaagggtta attggaaatc 9721 agagtttgaa ataaaacttg
gaccactttg tatacactct tctcacttga cattttagct 9781 atataatatg
tactttgagt ataacatcaa gctttaacaa atatttaaag acaaaaaaat 9841
cacgtcagta aaatactaaa aggctcattt ttatatttgt tttagatgtt ttaaatagtt
9901 gcaatggatt aaaaatgatg atttaaaatg ttgcttgtaa tacagttttg
cctgctaaat 9961 tctccacatt ttgtaacctg ttttatttct ttgggtgtaa
agcgtttttg cttagtattg 10021 tgatattgta tatgttttgt cccagttgta
tagtaatgtt tcagtccatc atccagcttt 10081 ggctgctgaa atcatacagc
tgtgaagact tgcctttgtt tctgttagac tgcttttcag 10141 ttctgtattg
agtatcttaa gtactgtaga aaagatgtca cttcttcctt taaggctgtt 10201
ttgtaatata tataaggact ggaattgtgt ttttaaagaa aagcattcaa gtatgacaat
10261 atactatctg tgttttcacc attcaaagtg ctgtttagta gttgaaactt
aaactattta 10321 atgtcattta ataaagtgac caaaatgtgt tgtgctcttt
attgtatttt cacagctttg 10381 aaaatctgtg cacatactgt ttcatagaaa
atgtatagct tttgttgtcc tatataatgg 10441 tggttctttt gcacatttag
ttatttaata ttgagaggtc acgaagtttg gttattgaat 10501 ctgttatata
ctaaattctg taaagggaga tctctcatct caaaaagaat ttacatacca 10561
ggaagtccat gtgtgtttgt gttagttttg gatgtctttg tgtaatccag ccccatttcc
10621 tgtttcccaa cagctgtaac actcatttta agtcaagcag ggctaccaac
ccacacttga 10681 tagaaaagct gcttaccatt cagaagcttc cttattacct
ggcctccaaa tgagctgaat 10741 attttgtagc cttcccttag ctatgttcat
tttccctcca ttatcataaa atcagatcga 10801 tatttatgtg ccccaaacaa
aactttaaga gcagttacat tctgtcccag tagcccttgt 10861 ttcctttgag
agtagcatgt tgtgaggcta tagagactta ttctaccagt aaaacaggtc 10921
aatcctttta catgtttatt atactaaaaa ttatgttcag ggtatttact actttatttc
10981 accagactca gtctcaagtg acttggctat ctccaaatca gatctaccct
tagagaataa 11041 acatttttct accgttattt tttttcaagt ctataatctg
agccagtccc aaaggagtga 11101 tcaagtttca gaaatgcttt catcttcaca
acattttata tatactatta tatggggtga 11161 ataaagtttt aaatccgaaa
tataaaaaaa aaaaaaaaaa aa
TABLE-US-00007 Homo sapiens alpha thalassemia/mental retardation
syndrome X-linked (ATRX) isoform 2 (SEQ ID NO: 7) 1 mtaepmsesk
lntlvqklhd flahsseese etsspprlam nqntdkisgs gsnsdmmens 61
keegtsssek skssgssrsk rkpsivtkyv esddekpldd etvnedasne nsenditmqs
121 lpkedglhgi vsctacgqqv nhfqkdsiyr hpslqvlick ncfkyymsdd
isrdsdgmde 181 qcrwcaeggn liccdfchna fckkcilrnl grkelstimd
ennqwycyic hpeplldlvt 241 acnsvfenle qllqqnkkki kvdseksnkv
yehtsrfspk ktssncngee kklddscsgs 301 vtysysaliv pkemikkakk
liettanmns syvkflkqat dnseissatk lrqlkafksv 361 ladikkahla
leedlnsefr amdavnkekn tkehkvidak fetkarkgek pcalekkdis 421
kseaklsrkq vdsehmhqnv pteeqrtnks tggehkksdr keepqyepan tsedldmdiv
481 svpssvpedi fenletamev qssvdhqgdg ssgteqeves ssvklnissk
dnrggikskt 541 takvtkelyv kltpvslsns pikgadcqev pqdkdgyksc
glnpklekcg lgqensdneh 601 lvenevslll eesdlrrspr vkttplrrpt
etnpvtsnsd eecnetvkek qklsvpvrkk 661 dkrnssdsai dnpkpnklpk
skqsetvdqn sdsdemlail kevsrmshss ssdtdineih 721 tnhktlydlk
tqagkddkgk rkrksstsgs dfdtkkgksa kssiiskkkr qtqsessnyd 781
selekeiksm skigaarttk kripntkdfd ssedekhskk gmdnqghknl ktsqegssdd
841 aerkqeretf ssaegtvdkd ttimelrdrl pkkqqasast dgvdklsgke
qsftslevrk 901 vaetkekskh lktktckkvq dglsdiaekf lkkdqsdets
eddkkqskkg teekkkpsdf 961 kkkvikmeqq yesssdgtek lpereeichf
pkgikqikng ttdgekkskk irdktskkkd 1021 elsdyaekst gkgdscdsse
dkkskngayg rekkrckllg kssrkrqdcs ssdtekysmk 1081 edgcnssdkr
lkrielrerr nlsskrntke iqsgssssda eessednkkk kqrtsskkka 1141
vivkekkrns lrtstkrkqa ditsssssdi edddqnsige gssdeqkikp vtenlvlssh
1201 tgfcqssgde alsksvpvtv ddddddndpe nriakkmlle eikanlssde
dgssddepee 1261 gkkrtgkqne enpgdeeakn qvnsesdsds eeskkpryrh
rllrhkltvs dgesgeekkt 1321 kpkehkevkg rnrrkvssed sedsdfqesg
vseevsesed eqrprtrsak kaeleenqrs 1381 ykqkkkrrri kvqedsssen
ksnseeeeee keeeeeeeee eeeeeedend dskspgkgrk 1441 kirkilkddk
lrtetqnalk eeeerrkria ererereklr evieiedasp tkcpittklv 1501
ldedeetkep lvqvhrnmvi klkphqvdgv qfmwdccces vkktkkspgs gcilahcmgl
1561 gktlqvvsfl htvllcdkld fstalvvcpl ntalnwmnef ekwqeglkdd
eklevselat 1621 vkrpqersym lqrwqedggv miigyemyrn laqgrnvksr
klkeifnkal vdpgpdfvvc 1681 deghilknea savskamnsi rsrrriiltg
tplqnnliey hcmvnfiken llgsikefrn 1741 rfinpiqngq cadstmvdvr
vmkkrahily emlagcvqrk dytaltkflp pkheyvlavr 1801 mtsiqcklyq
yyldhltgvg nnseggrgka gaklfqdfqm lsriwthpwc lqldyisken 1861
kgyfdedsmd efiasdsdet smslssddyt kkkkkgkkgk kdssssgsgs dndvevikvw
1921 nsrsrgggeg nvdetgnnps vslkleeska tsssnpsspa pdwykdfvtd
adaevlehsg 1981 kmvllfeilr maeeigdkvl vfsqslisld liedflelas
rektedkdkp liykgegkwl 2041 rnidyyrldg sttaqsrkkw aeefndetnv
rgrlfiistk agslginlva anrviifdas 2101 wnpsydiqsi frvyrfgqtk
pvyvyrflaq gtmedkiydr qvtkqslsfr vvdqqqverh 2161 ftmneltely
tfepdllddp nsekkkkrdt pmlpkdtila ellqihkehi vgyhehdsll 2221
dhkeeeelte eerkaawaey eaekkgltmr fniptgtnlp pvsfnsqtpy ipfnlgalsa
2281 msnqqledli nqgrekvvea tnsvtavriq plediisavw kenmnlseaq
vqalalsrqa 2341 sqeldvkrre aiyndvltkq qmliscvqri lmnrrlqqqy
nqqqqqqmty qqatlghlmm 2401 pkppnlimnp snyqqidmrg myqpvaggmq
ppplqrappp mrsknpgpsq gksm
TABLE-US-00008 Homo sapiens alpha thalassemia/mental retardation
syndrome X-linked (ATRX), transcript variant 2, mRNA (SEQ ID NO: 8)
1 aattctcctg cctgagcctc ggcccaacaa aatggcggcg gcagcggtgt cgctttgttt
61 ccgcggctcc tgcggcggtg gcagtggtag cggcctttga gctgtgggga
ggttccagca 121 gcagctacag tgacgactaa gactccagtg catttctatc
gtaaccgggc gcgggggagc 181 gcagatcggc gcccagcaat cacagaagcc
gacaaggcgt tcaagcgaaa acatgaccgc 241 tgagcccatg agtgaaagca
agttgaatac attggtgcag aagcttcatg acttccttgc 301 acactcatca
gaagaatctg aagaaacaag ttctcctcca cgacttgcaa tgaatcaaaa 361
cacagataaa atcagtggtt ctggaagtaa ctctgatatg atggaaaaca gcaaggaaga
421 gggaactagc tcttcagaaa aatccaagtc ttcaggatcg tcacgatcaa
agaggaaacc 481 ttcaattgta acaaagtatg tagaatcaga tgatgaaaaa
cctttggatg atgaaactgt 541 aaatgaagat gcgtctaatg aaaattcaga
aaatgatatt actatgcaga gcttgccaaa 601 agaagatggg cttcatggga
ttgtgagctg cactgcttgt ggacaacagg tcaatcattt 661 tcaaaaagat
tccatttata gacacccttc attgcaagtt cttatttgta agaattgctt 721
taagtattac atgagtgatg atattagccg tgactcagat ggaatggatg aacaatgtag
781 gtggtgtgcg gaaggtggaa acttgatttg ttgtgacttt tgccataatg
ctttctgcaa 841 gaaatgcatt ctacgcaacc ttggtcgaaa ggagttgtcc
acaataatgg atgaaaacaa 901 ccaatggtat tgctacattt gtcacccaga
gcctttgttg gacttggtca ctgcatgtaa 961 cagcgtattt gagaatttag
aacagttgtt gcagcaaaat aagaagaaga taaaagttga 1021 cagtgaaaag
agtaataaag tatatgaaca tacatccaga ttttctccaa agaagactag 1081
ttcaaattgt aatggagaag aaaagaaatt agatgattcc tgttctggct ctgtaaccta
1141 ctcttattcc gcactaattg tgcccaaaga gatgattaag aaggcaaaaa
aactgattga 1201 gaccacagcc aacatgaact ccagttatgt taaattttta
aagcaggcaa cagataattc 1261 agaaatcagt tctgctacaa aattacgtca
gcttaaggct tttaagtctg tgttggctga 1321 tattaagaag gctcatcttg
cattggaaga agacttaaat tccgagtttc gagcgatgga 1381 tgctgtaaac
aaagagaaaa ataccaaaga gcataaagtc atagatgcta agtttgaaac 1441
aaaagcacga aaaggagaaa aaccttgtgc tttggaaaag aaggatattt caaagtcaga
1501 agctaaactt tcaagaaaac aggtagatag tgagcacatg catcagaatg
ttccaacaga 1561 ggaacaaaga acaaataaaa gtaccggtgg tgaacataag
aaatctgata gaaaagaaga 1621 acctcaatat gaacctgcca acacttctga
agatttagac atggatattg tgtctgttcc 1681 ttcctcagtt ccagaagaca
tttttgagaa tcttgagact gctatggaag ttcagagttc 1741 agttgatcat
caaggggatg gcagcagtgg aactgaacaa gaagtggaga gttcatctgt 1801
aaaattaaat atttcttcaa aagacaacag aggaggtatt aaatcaaaaa ctacagctaa
1861 agtaacaaaa gaattatatg ttaaactcac tcctgtttcc ctttctaatt
ccccaattaa 1921 aggtgctgat tgtcaggaag ttccacaaga taaagatggc
tataaaagtt gtggtctgaa 1981 ccccaagtta gagaaatgtg gacttggaca
ggaaaacagt gataatgagc atttggttga 2041 aaatgaagtt tcattacttt
tagaggaatc tgatcttcga agatccccac gtgtaaagac 2101 tacacccttg
aggcgaccga cagaaactaa ccctgtaaca tctaattcag atgaagaatg 2161
taatgaaaca gttaaggaga aacaaaaact atcagttcca gtgagaaaaa aggataagcg
2221 taattcttct gacagtgcta tagataatcc taagcctaat aaattgccaa
aatctaagca 2281 atcagagact gtggatcaaa attcagattc tgatgaaatg
ctagcaatcc tcaaagaggt 2341 gagcaggatg agtcacagtt cttcttcaga
tactgatatt aatgaaattc atacaaacca 2401 taagactttg tatgatttaa
agactcaggc ggggaaagat gataaaggaa aaaggaaacg 2461 aaaaagttct
acatctggct cagattttga tactaaaaag ggcaaatcag ctaagagctc 2521
tataatttct aaaaagaaac gacaaaccca gtctgagtct tctaattatg actcagaatt
2581 agaaaaagag ataaagagca tgagtaaaat tggtgctgcc agaaccacca
aaaaaagaat 2641 tccaaataca aaagattttg actcttctga agatgagaaa
cacagcaaaa aaggaatgga 2701 taatcaaggg cacaaaaatt tgaagacctc
acaagaagga tcatctgatg atgctgaaag 2761 aaaacaagag agagagactt
tctcttcagc agaaggcaca gttgataaag acacgaccat 2821 catggaatta
agagatcgac ttcctaagaa gcagcaagca agtgcttcca ctgatggtgt 2881
cgataagctt tctgggaaag agcagagttt tacttctttg gaagttagaa aagttgctga
2941 aactaaagaa aagagcaagc atctcaaaac caaaacatgt aaaaaagtac
aggatggctt 3001 atctgatatt gcagagaaat tcctaaagaa agaccagagc
gatgaaactt ctgaagatga 3061 taaaaagcag agcaaaaagg gaactgaaga
aaaaaagaaa ccttcagact ttaagaaaaa 3121 agtaattaaa atggaacaac
agtatgaatc ttcatctgat ggcactgaaa agttacctga 3181 gcgagaagaa
atttgtcatt ttcctaaggg cataaaacaa attaagaatg gaacaactga 3241
tggagaaaag aaaagtaaaa aaataagaga taaaacttct aaaaagaagg atgaattatc
3301 tgattatgct gagaagtcaa cagggaaagg agatagttgt gactcttcag
aggataaaaa 3361 gagtaagaat ggagcatatg gtagagagaa gaaaaggtgc
aagttgcttg gaaagagttc 3421 aaggaagaga caagattgtt catcatctga
tactgagaaa tattccatga aagaagatgg 3481 ttgtaactct tctgataaga
gactgaaaag aatagaattg agggaaagaa gaaatttaag 3541 ttcaaagaga
aatactaagg aaatacaaag tggctcatca tcatctgatg ctgaggaaag 3601
ttctgaagat aataaaaaga agaagcaaag aacttcatct aaaaagaagg cagtcattgt
3661 caaggagaaa aagagaaact ccctaagaac aagcactaaa aggaagcaag
ctgacattac 3721 atcctcatct tcttctgata tagaagatga tgatcagaat
tctataggtg agggaagcag 3781 cgatgaacag aaaattaagc ctgtgactga
aaatttagtg ctgtcttcac atactggatt 3841 ttgccaatct tcaggagatg
aagccttatc taaatcagtg cctgtcacag tggatgatga 3901 tgatgacgac
aatgatcctg agaatagaat tgccaagaag atgcttttag aagaaattaa 3961
agccaatctt tcctctgatg aggatggatc ttcagatgat gagccagaag aagggaaaaa
4021 aagaactgga aaacaaaatg aagaaaaccc aggagatgag gaagcaaaaa
atcaagtcaa 4081 ttctgaatca gattcagatt ctgaagaatc taagaagcca
agatacagac ataggctttt 4141 gcggcacaaa ttgactgtga gtgacggaga
atctggagaa gaaaaaaaga caaagcctaa 4201 agagcataaa gaagtcaaag
gcagaaacag aagaaaggtg agcagtgaag attcagaaga 4261 ttctgatttt
caggaatcag gagttagtga agaagttagt gaatccgaag atgaacagcg 4321
gcccagaaca aggtctgcaa agaaagcaga gttggaagaa aatcagcgga gctataaaca
4381 gaaaaagaaa aggcgacgta ttaaggttca agaagattca tccagtgaaa
acaagagtaa 4441 ttctgaggaa gaagaggagg aaaaagaaga ggaggaggaa
gaggaggagg aggaggaaga 4501 ggaggaggaa gatgaaaatg atgattccaa
gtctcctgga aaaggcagaa agaaaattcg 4561 gaagattctt aaagatgata
aactgagaac agaaacacaa aatgctctta aggaagagga 4621 agagagacga
aaacgtattg ctgagaggga gcgtgagcga gaaaaattga gagaggtgat 4681
agaaattgaa gatgcttcac ccaccaagtg tccaataaca accaagttgg ttttagatga
4741 agatgaagaa accaaagaac ctttagtgca ggttcataga aatatggtta
tcaaattgaa 4801 accccatcaa gtagatggtg ttcagtttat gtgggattgc
tgctgtgagt ctgtgaaaaa 4861 aacaaagaaa tctccaggtt caggatgcat
tcttgcccac tgtatgggcc ttggtaagac 4921 tttacaggtg gtaagttttc
ttcatacagt tcttttgtgt gacaaactgg atttcagcac 4981 ggcgttagtg
gtttgtcctc ttaatactgc tttgaattgg atgaatgaat ttgagaagtg 5041
gcaagaggga ttaaaagatg atgagaagct tgaggtttct gaattagcaa ctgtgaaacg
5101 tcctcaggag agaagctaca tgctgcagag gtggcaagaa gatggtggtg
ttatgatcat 5161 aggctatgag atgtatagaa atcttgctca aggaaggaat
gtgaagagtc ggaaacttaa 5221 agaaatattt aacaaagctt tggttgatcc
aggccctgat tttgttgttt gtgatgaagg 5281 ccatattcta aaaaatgaag
catctgctgt ttctaaagct atgaattcta tacgatcaag 5341 gaggaggatt
attttaacag gaacaccact tcaaaataac ctaattgagt atcattgtat 5401
ggttaatttt atcaaggaaa atttacttgg atccattaag gagttcagga atagatttat
5461 aaatccaatt caaaatggtc agtgtgcaga ttctaccatg gtagatgtca
gagtgatgaa 5521 aaaacgtgct cacattctct atgagatgtt agctggatgt
gttcagagga aagattatac 5581 agcattaaca aaattcttgc ctccaaaaca
cgaatatgtg ttagctgtga gaatgacttc 5641 tattcagtgc aagctctatc
agtactactt agatcactta acaggtgtgg gcaataatag 5701 tgaaggtgga
agaggaaagg caggtgcaaa gcttttccaa gattttcaga tgttaagtag 5761
aatatggact catccttggt gtttgcagct agactacatt agcaaagaaa ataagggtta
5821 ttttgatgaa gacagtatgg atgaatttat agcctcagat tctgatgaaa
cctccatgag 5881 tttaagctcc gatgattata caaaaaagaa gaaaaaaggg
aaaaagggga aaaaagatag 5941 tagctcaagt ggaagtggca gtgacaatga
tgttgaagtg attaaggtct ggaattcaag 6001 atctcgggga ggtggtgaag
gaaatgtgga tgaaacagga aacaatcctt ctgtttcttt 6061 aaaactggaa
gaaagtaaag ctacttcttc ttctaatcca agcagcccag ctccagactg 6121
gtacaaagat tttgttacag atgctgatgc tgaggtttta gagcattctg ggaaaatggt
6181 acttctcttt gaaattcttc gaatggcaga ggaaattggg gataaagtcc
ttgttttcag 6241 ccagtccctc atatctctgg acttgattga agattttctt
gaattagcta gtagggagaa 6301 gacagaagat aaagataaac cccttattta
taaaggtgag gggaagtggc ttcgaaacat 6361 tgactattac cgtttagatg
gttccactac tgcacagtca aggaagaagt gggctgaaga 6421 atttaatgat
gaaactaatg tgagaggacg attatttatc atttctacta aagcaggatc 6481
tctaggaatt aatctggtag ctgctaatcg agtaattata ttcgacgctt cttggaatcc
6541 atcttatgac atccagagta tattcagagt ttatcgcttt ggacaaacta
agcctgttta 6601 tgtatatagg ttcttagctc agggaaccat ggaagataag
atttatgatc ggcaagtaac 6661 taagcagtca ctgtcttttc gagttgttga
tcagcagcag gtggagcgtc attttactat 6721 gaatgagctt actgaacttt
atacttttga gccagactta ttagatgacc ctaattcaga 6781 aaagaagaag
aagagggata ctcccatgct gccaaaggat accatacttg cagagctcct 6841
tcagatacat aaagaacaca ttgtaggata ccatgaacat gattctcttt tggaccacaa
6901 agaagaagaa gagttgactg aagaagaaag aaaagcagct tgggctgagt
atgaagcaga 6961 gaagaaggga ctgaccatgc gtttcaacat accaactggg
accaatttac cccctgtcag 7021 tttcaactct caaactcctt atattccttt
caatttggga gccctgtcag caatgagtaa 7081 tcaacagctg gaggacctca
ttaatcaagg aagagaaaaa gttgtagaag caacaaacag 7141 tgtgacagca
gtgaggattc aacctcttga ggatataatt tcagctgtat ggaaggagaa 7201
catgaatctc tcagaggccc aagtacaggc gttagcatta agtagacaag ccagccagga
7261 gcttgatgtt aaacgaagag aagcaatcta caatgatgta ttgacaaaac
aacagatgtt 7321 aatcagctgt gttcagcgaa tacttatgaa cagaaggctc
cagcagcagt acaatcagca
7381 gcaacagcaa caaatgactt atcaacaagc aacactgggt cacctcatga
tgccaaagcc 7441 cccaaatttg atcatgaatc cttctaacta ccagcagatt
gatatgagag gaatgtatca 7501 gccagtggct ggtggtatgc agccaccacc
attacagcgt gcaccacccc caatgagaag 7561 caaaaatcca ggaccttccc
aagggaaatc aatgtgattt tgcactaaaa gcttaatgga 7621 ttgttaaaat
catagaaaga tcttttattt ttttaggaat caatgactta acagaactca 7681
actgtataaa tagtttggtc cccttaaatg ccaatcttcc atattagttt tacttttttt
7741 ttttttaaat agggcatacc atttcttcct gacatttgtc agtgatgttg
cctagaatct 7801 tcttacacac gctgagtaca gaagatattt caaattgttt
tcagtgaaaa caagtccttc 7861 cataatagta acaactccac agatttcctc
tctaaatttt tatgcctgct tttagcaacc 7921 ataaaattgt cataaaatta
ataaatttag gaaagaataa agatttatat attcattctt 7981 tacatataaa
aacacacagc tgagttctta gagttgattc ctcaagttat gaaatacttt 8041
tgtacttaat ccatttcttg attaaagtga ttgaaatggt tttaatgttc ttttgactga
8101 agtctgaaac tgggctcctg ctttattgtc tctgtgactg aaagttagaa
actgagggtt 8161 atctttgaca cagaattgtg tgcaatattc ttaaatacta
ctgctctaaa agttggagaa 8221 gtcttgcagt tatcttagca ttgtataaac
agccttaagt atagcctaag aagagaattc 8281 ctttttcttc tttagtcctt
ctgccatttt ttattttcag ttatatgtgc tgaaataatt 8341 actggtaaaa
tttcagggtt gtggattatc ttccacacat gaattttctc tctcctggca 8401
cgaatataaa gcacatctct taactgcatg gtgccagtgc taatgcttca tcctgttgct
8461 ggcagtggga tgtggactta gaaaatcaag ttctagcatt ttagtaggtt
aacactgaag 8521 ttgtggttgt taggttcaca ccctgtttta taaacaacat
caaaatggca gaaccattgc 8581 tgactttagg ttcacatgag gaatgtactt
ttaacaattc ccagtactat cagtattgtg 8641 aaataattcc tctgaaagat
aagaatcact ggcttctatg cgcttctttt ctctcatcat 8701 catgttcttt
taccccagtt tccttacatt tttttaaatt gtttcagagt ttgttttttt 8761
tttagtttag attgtgaggc aattattaaa tcaaaattaa ttcatccaat acccctttac
8821 tagaagtttt actagaaaat gtattacatt ttattttttc ttaatccagt
tctgcaaaaa 8881 tgacctataa atttattcat gtacaatttt ggttacttga
attgttaaag aaaacattgt 8941 ttttgactat gggagtcaac tcaacatggc
agaaccattt ttgagatgat gatacaacag 9001 gtagtgaaac agcttaagaa
ttccaaaaaa aaaaaaaaaa aaaaaaaaaa gaaaactggg 9061 tttgggcttt
gctttaggta tcactggatt agaatgagtt taacattagc taaaactgct 9121
ttgagttgtt tggatgatta agagattgcc atttttatct tggaagaact agtggtaaaa
9181 catccaagag cactaggatt gtgatacaga atttgtgagg tttggtggat
ccacgcccct 9241 ctcccccact ttcccatgat gaaatatcac taataaatcc
tgtatattta gatattatgc 9301 tagccatgta atcagattta tttaattggg
tggggcaggt gtgtatttac tttagaaaaa 9361 atgaaaaaga caagatttat
gagaaatatt tgaaggcagt acactctggc caactgttac 9421 cagttggtat
ttctacaagt tcagaatatt ttaaacctga tttactagac ctgggaattt 9481
tcaacatggt ctaattattt actcaaagac atagatgtga aaattttagg caaccttcta
9541 aatctttttc accatggatg aaactataac ttaaagaata atacttagaa
gggttaattg 9601 gaaatcagag tttgaaataa aacttggacc actttgtata
cactcttctc acttgacatt 9661 ttagctatat aatatgtact ttgagtataa
catcaagctt taacaaatat ttaaagacaa 9721 aaaaatcacg tcagtaaaat
actaaaaggc tcatttttat atttgtttta gatgttttaa 9781 atagttgcaa
tggattaaaa atgatgattt aaaatgttgc ttgtaataca gttttgcctg 9841
ctaaattctc cacattttgt aacctgtttt atttctttgg gtgtaaagcg tttttgctta
9901 gtattgtgat attgtatatg ttttgtccca gttgtatagt aatgtttcag
tccatcatcc 9961 agctttggct gctgaaatca tacagctgtg aagacttgcc
tttgtttctg ttagactgct 10021 tttcagttct gtattgagta tcttaagtac
tgtagaaaag atgtcacttc ttcctttaag 10081 gctgttttgt aatatatata
aggactggaa ttgtgttttt aaagaaaagc attcaagtat 10141 gacaatatac
tatctgtgtt ttcaccattc aaagtgctgt ttagtagttg aaacttaaac 10201
tatttaatgt catttaataa agtgaccaaa atgtgttgtg ctctttattg tattttcaca
10261 gctttgaaaa tctgtgcaca tactgtttca tagaaaatgt atagcttttg
ttgtcctata 10321 taatggtggt tcttttgcac atttagttat ttaatattga
gaggtcacga agtttggtta 10381 ttgaatctgt tatatactaa attctgtaaa
gggagatctc tcatctcaaa aagaatttac 10441 ataccaggaa gtccatgtgt
gtttgtgtta gttttggatg tctttgtgta atccagcccc 10501 atttcctgtt
tcccaacagc tgtaacactc attttaagtc aagcagggct accaacccac 10561
acttgataga aaagctgctt accattcaga agcttcctta ttacctggcc tccaaatgag
10621 ctgaatattt tgtagccttc ccttagctat gttcattttc cctccattat
cataaaatca 10681 gatcgatatt tatgtgcccc aaacaaaact ttaagagcag
ttacattctg tcccagtagc 10741 ccttgtttcc tttgagagta gcatgttgtg
aggctataga gacttattct accagtaaaa 10801 caggtcaatc cttttacatg
tttattatac taaaaattat gttcagggta tttactactt 10861 tatttcacca
gactcagtct caagtgactt ggctatctcc aaatcagatc tacccttaga 10921
gaataaacat ttttctaccg ttattttttt tcaagtctat aatctgagcc agtcccaaag
10981 gagtgatcaa gtttcagaaa tgctttcatc ttcacaacat tttatatata
ctattatatg 11041 gggtgaataa agttttaaat ccgaaatata aaaaaaaaaa
aaaaaaaa
[0118] A subject in need thereof may have reduced expression,
haploinsufficiency, and/or loss of function of ARID1A. For example,
a subject may comprise a mutation selected from the group
consisting of a nonsense mutation for the wild type residue
cysteine (C) at amino acid position 884 of SEQ ID NO: 11 (C884*), a
substitution of lysine (K) for the wild type residue glutamic acid
(E) at amino acid position 966 (E966K), a nonsense mutation for the
wild type residue glutamine (Q) at amino acid position 1411 of SEQ
ID NO: 11 (Q1411*), a frame shift mutation at the wild type residue
phenylalanine (F) at amino acid position 1720 of SEQ ID NO: 11
(F1720fs), a frame shift mutation after the wild type residue
glycine (G) at amino acid position 1847 of SEQ ID NO: 11 (G1847fs),
a frame shift mutation at the wild type residue cysteine (C) at
amino acid position 1874 of SEQ ID NO: 11 (C1874fs), a substitution
of glutamic acid (E) for the wild type residue aspartic acid (D) at
amino acid position 1957 (D1957E), a nonsense mutation for the wild
type residue glutamine (Q) at amino acid position 1430 of SEQ ID
NO: 11 (Q1430*), a frame shift mutation at the wild type residue
arginine (R) at amino acid position 1721 of SEQ ID NO: 11
(R1721fs), a substitution of glutamic acid (E) for the wild type
residue glycine (G) at amino acid position 1255 (G1255E), a frame
shift mutation at the wild type residue glycine (G) at amino acid
position 284 of SEQ ID NO: 11 (G284fs), a nonsense mutation for the
wild type residue arginine (R) at amino acid position 1722 of SEQ
ID NO: 11 (R1722*), a frame shift mutation at the wild type residue
methionine (M) at amino acid position 274 of SEQ ID NO: 11
(M274fs), a frame shift mutation at the wild type residue glycine
(G) at amino acid position 1847 of SEQ ID NO: 11 (G1847fs), a frame
shift mutation at the wild type residue P at amino acid position
559 of SEQ ID NO: 11 (P559fs), a nonsense mutation for the wild
type residue arginine (R) at amino acid position 1276 of SEQ ID NO:
11 (R1276*), a frame shift mutation at the wild type residue
glutamine (Q) at amino acid position 2176 of SEQ ID NO: 11
(Q2176fs), a frame shift mutation at the wild type residue
histidine (H) at amino acid position 203 of SEQ ID NO: 11 (H203fs),
a frame shift mutation at the wild type residue alanine (A) at
amino acid position 591 of SEQ ID NO: 11 (A591fs), a nonsense
mutation for the wild type residue glutamine (Q) at amino acid
position 1322 of SEQ ID NO: 11 (Q1322*), a nonsense mutation for
the wild type residue serine (S) at amino acid position 2264 of SEQ
ID NO: 11 (S2264*), a nonsense mutation for the wild type residue
glutamine (Q) at amino acid position 586 of SEQ ID NO: 11 (Q586*),
a frame shift mutation at the wild type residue glutamine (Q) at
amino acid position 548 of SEQ ID NO: 11 (Q548fs), and a frame
shift mutation at the wild type residue asparagine (N) at amino
acid position 756 of SEQ ID NO: 11 (N756fs). "*" used herein refers
to a stop codon. "fs" used herein refers to a frame shift.
TABLE-US-00009 AT-rich interactive domain-containing protein 1A
(ARID1A) isoform a [Homo sapiens] (SEQ ID NO: 9) 1 maaqvapaaa
sslgnppppp pselkkaeqq qreeaggeaa aaaaaergem kaaagqeseg 61
pavgppqplg kelqdgaesn gggggggags gggpgaepdl knsngnagpr palnnnltep
121 pggggggssd gvgapphsaa aalpppaygf gqpygrspsa vaaaaaavfh
qqhggqqspg 181 laalqsgggg glepyagpqq nshdhgfpnh qynsyypnrs
aypppapaya lssprggtpg 241 sgaaaaagsk pppsssasas sssssfaqqr
fgamggggps aagggtpqpt atptlnqllt 301 spssargyqg ypggdysggp
qdggagkgpa dmasqcwgaa aaaaaaaaas ggaqqrshha 361 pmspgssggg
gqplartpqp sspmdqmgkm rpqpyggtnp ysqqqgppsg pqqghgypgq 421
pygsqtpqry pmtmqgraqs amgglsytqq ippygqqgps gygqqgqtpy ynqqsphpqq
481 qqppysqqpp sqtphaqpsy qqqpqsqppq lqssqppysq qpsqpphqqs
papypsqqst 541 tqqhpqsqpp ysqpqaqspy qqqqpqqpap stlsqqaayp
qpqsqqsqqt aysqqrfppp 601 qelsqdsfgs qassapsmts skggqedmnl
slqsrpsslp dlsgsiddlp mgtegalspg 661 vstsgisssq geqsnpaqsp
fsphtsphlp girgpspspv gspasvaqsr sgplspaavp 721 gnqmpprpps
gqsdsimhps mnqssiaqdr gymqrnpqmp qysspqpgsa lsprqpsggq 781
ihtgmgsyqq nsmgsygpqg gqygpqggyp rqpnynalpn anypsagmag ginpmgaggq
841 mhgqpgippy gtlppgrmsh asmgnrpygp nmanmppqvg sgmcpppggm
nrktqetava 901 mhvaansiqn rppgypnmnq ggmmgtgppy gqginsmagm
inpqgppysm ggtmannsag 961 maaspemmgl gdvkltpatk mnnkadgtpk
teskskksss stttnekitk lyelggeper 1021 kmwvdrylaf teekamgmtn
lpavgrkpld lyrlyvsvke iggltqvnkn kkwrelatnl 1081 nvgtsssaas
slkkqyiqcl yafeckierg edpppdifaa adskksqpki qppspagsgs 1141
mqgpqtpqst sssmaeggdl kpptpastph sqipplpgms rsnsvgiqda fndgsdstfq
1201 krnsmtpnpg yqpsmntsdm mgrmsyepnk dpygsmrkap gsdpfmssgq
gpnggmgdpy 1261 sraagpglgn vamgprqhyp yggpydrvrt epgigpegnm
stgapqpnlm psnpdsgmys 1321 psryppqqqq qqqqrhdsyg nqfstqgtps
gspfpsqqtt myqqqqqnyk rpmdgtygpp 1381 akrhegemys vpystgqgqp
qqqqlppaqp qpasqqqaaq pspqqdvynq ygnaypatat 1441 aaterrpagg
pqnqfpfqfg rdrvsappgt naqqnmppqm mggpiqasae vaqqgtmwqg 1501
rndmtynyan rqstgsapqg payhgvnrtd emlhtdqran hegswpshgt rqppygpsap
1561 vppmtrppps nyqpppsmqn hipqvsspap lprpmenrts pskspflhsg
mkmqkagppv 1621 pashiapapv qppmirrdit fppgsveatq pvlkqrrrlt
mkdigtpeaw rvmmslksgl 1681 laestwaldt inillyddns imtfnlsqlp
gllellveyf rrclieifgi lkeyevgdpg 1741 qrtlldpgrf skvsspapme
ggeeeeellg pkleeeeeee vvendeeiaf sgkdkpasen 1801 seekliskfd
klpvkivqkn dpfvvdcsdk lgrvqefdsg llhwrigggd ttehiqthfe 1861
sktellpsrp hapcppaprk hvttaegtpg ttdqegpppd gppekritat mddmlstrss
1921 tltedgakss eaikesskfp fgispaqshr nikiledeph skdetplctl
ldwqdslakr 1981 cvcvsntirs lsfvpgndfe mskhpgllli lgklillhhk
hperkqaplt yekeeeqdqg 2041 vscnkvewww dclemlrent lvtlanisgq
ldlspypesi clpvldgllh wavcpsaeaq 2101 dpfstlgpna vlspqrlvle
tlsklsiqdn nvdlilatpp fsrleklyst mvrflsdrkn 2161 pvcremavvl
lanlaqgdsl aaraiavqkg signllgfle dslaatqfqq sqasllhmqn 2221
ppfeptsvdm mrraaralla lakvdenhse ftlyesrlld isvsplmnsl vsqvicdvlf
2281 ligqs
TABLE-US-00010 Homo sapiens AT rich interactive domain 1A
(SWI-like) (ARID1A), transcript variant 1, mRNA (SEQ ID NO: 10) 1
cagaaagcgg agagtcacag cggggccagg ccctggggag cggagcctcc accgcccccc
61 tcattcccag gcaagggctt ggggggaatg agccgggaga gccgggtccc
gagcctacag 121 agccgggagc agctgagccg ccggcgcctc ggccgccgcc
gccgcctcct cctcctccgc 181 cgccgccagc ccggagcctg agccggcggg
gcggggggga gaggagcgag cgcagcgcag 241 cagcggagcc ccgcgaggcc
cgcccgggcg ggtggggagg gcagcccggg ggactgggcc 301 ccggggcggg
gtgggagggg gggagaagac gaagacaggg ccgggtctct ccgcggacga 361
gacagcgggg atcatggccg cgcaggtcgc ccccgccgcc gccagcagcc tgggcaaccc
421 gccgccgccg ccgccctcgg agctgaagaa agccgagcag cagcagcggg
aggaggcggg 481 gggcgaggcg gcggcggcgg cagcggccga gcgcggggaa
atgaaggcag ccgccgggca 541 ggaaagcgag ggccccgccg tggggccgcc
gcagccgctg ggaaaggagc tgcaggacgg 601 ggccgagagc aatgggggtg
gcggcggcgg cggagccggc agcggcggcg ggcccggcgc 661 ggagccggac
ctgaagaact cgaacgggaa cgcgggccct aggcccgccc tgaacaataa 721
cctcacggag ccgcccggcg gcggcggtgg cggcagcagc gatggggtgg gggcgcctcc
781 tcactcagcc gcggccgcct tgccgccccc agcctacggc ttcgggcaac
cctacggccg 841 gagcccgtct gccgtcgccg ccgccgcggc cgccgtcttc
caccaacaac atggcggaca 901 acaaagccct ggcctggcag cgctgcagag
cggcggcggc gggggcctgg agccctacgc 961 ggggccccag cagaactctc
acgaccacgg cttccccaac caccagtaca actcctacta 1021 ccccaaccgc
agcgcctacc ccccgcccgc cccggcctac gcgctgagct ccccgagagg 1081
tggcactccg ggctccggcg cggcggcggc tgccggctcc aagccgcctc cctcctccag
1141 cgcctccgcc tcctcgtcgt cttcgtcctt cgctcagcag cgcttcgggg
ccatgggggg 1201 aggcggcccc tccgcggccg gcgggggaac tccccagccc
accgccaccc ccaccctcaa 1261 ccaactgctc acgtcgccca gctcggcccg
gggctaccag ggctaccccg ggggcgacta 1321 cagtggcggg ccccaggacg
ggggcgccgg caagggcccg gcggacatgg cctcgcagtg 1381 ttggggggct
gcggcggcgg cagctgcggc ggcggccgcc tcgggagggg cccaacaaag 1441
gagccaccac gcgcccatga gccccgggag cagcggcggc ggggggcagc cgctcgcccg
1501 gacccctcag ccatccagtc caatggatca gatgggcaag atgagacctc
agccatatgg 1561 cgggactaac ccatactcgc agcaacaggg acctccgtca
ggaccgcagc aaggacatgg 1621 gtacccaggg cagccatacg ggtcccagac
cccgcagcgg tacccgatga ccatgcaggg 1681 ccgggcgcag agtgccatgg
gcggcctctc ttatacacag cagattcctc cttatggaca 1741 acaaggcccc
agcgggtatg gtcaacaggg ccagactcca tattacaacc agcaaagtcc 1801
tcaccctcag cagcagcagc caccctactc ccagcaacca ccgtcccaga cccctcatgc
1861 ccaaccttcg tatcagcagc agccacagtc tcaaccacca cagctccagt
cctctcagcc 1921 tccatactcc cagcagccat cccagcctcc acatcagcag
tccccggctc catacccctc 1981 ccagcagtcg acgacacagc agcaccccca
gagccagccc ccctactcac agccacaggc 2041 tcagtctcct taccagcagc
agcaacctca gcagccagca ccctcgacgc tctcccagca 2101 ggctgcgtat
cctcagcccc agtctcagca gtcccagcaa actgcctatt cccagcagcg 2161
cttccctcca ccgcaggagc tatctcaaga ttcatttggg tctcaggcat cctcagcccc
2221 ctcaatgacc tccagtaagg gagggcaaga agatatgaac ctgagccttc
agtcaagacc 2281 ctccagcttg cctgatctat ctggttcaat agatgacctc
cccatgggga cagaaggagc 2341 tctgagtcct ggagtgagca catcagggat
ttccagcagc caaggagagc agagtaatcc 2401 agctcagtct cctttctctc
ctcatacctc ccctcacctg cctggcatcc gaggcccttc 2461 cccgtcccct
gttggctctc ccgccagtgt tgctcagtct cgctcaggac cactctcgcc 2521
tgctgcagtg ccaggcaacc agatgccacc tcggccaccc agtggccagt cggacagcat
2581 catgcatcct tccatgaacc aatcaagcat tgcccaagat cgaggttata
tgcagaggaa 2641 cccccagatg ccccagtaca gttcccccca gcccggctca
gccttatctc cgcgtcagcc 2701 ttccggagga cagatacaca caggcatggg
ctcctaccag cagaactcca tggggagcta 2761 tggtccccag gggggtcagt
atggcccaca aggtggctac cccaggcagc caaactataa 2821 tgccttgccc
aatgccaact accccagtgc aggcatggct ggaggcataa accccatggg 2881
tgccggaggt caaatgcatg gacagcctgg catcccacct tatggcacac tccctccagg
2941 gaggatgagt cacgcctcca tgggcaaccg gccttatggc cctaacatgg
ccaatatgcc 3001 acctcaggtt gggtcaggga tgtgtccccc accagggggc
atgaaccgga aaacccaaga 3061 aactgctgtc gccatgcatg ttgctgccaa
ctctatccaa aacaggccgc caggctaccc 3121 caatatgaat caagggggca
tgatgggaac tggacctcct tatggacaag ggattaatag 3181 tatggctggc
atgatcaacc ctcagggacc cccatattcc atgggtggaa ccatggccaa 3241
caattctgca gggatggcag ccagcccaga gatgatgggc cttggggatg taaagttaac
3301 tccagccacc aaaatgaaca acaaggcaga tgggacaccc aagacagaat
ccaaatccaa 3361 gaaatccagt tcttctacta caaccaatga gaagatcacc
aagttgtatg agctgggtgg 3421 tgagcctgag aggaagatgt gggtggaccg
ttatctggcc ttcactgagg agaaggccat 3481 gggcatgaca aatctgcctg
ctgtgggtag gaaacctctg gacctctatc gcctctatgt 3541 gtctgtgaag
gagattggtg gattgactca ggtcaacaag aacaaaaaat ggcgggaact 3601
tgcaaccaac ctcaatgtgg gcacatcaag cagtgctgcc agctccttga aaaagcagta
3661 tatccagtgt ctctatgcct ttgaatgcaa gattgaacgg ggagaagacc
ctcccccaga 3721 catctttgca gctgctgatt ccaagaagtc ccagcccaag
atccagcctc cctctcctgc 3781 gggatcagga tctatgcagg ggccccagac
tccccagtca accagcagtt ccatggcaga 3841 aggaggagac ttaaagccac
caactccagc atccacacca cacagtcaga tccccccatt 3901 gccaggcatg
agcaggagca attcagttgg gatccaggat gcctttaatg atggaagtga 3961
ctccacattc cagaagcgga attccatgac tccaaaccct gggtatcagc ccagtatgaa
4021 tacctctgac atgatggggc gcatgtccta tgagccaaat aaggatcctt
atggcagcat 4081 gaggaaagct ccagggagtg atcccttcat gtcctcaggg
cagggcccca acggcgggat 4141 gggtgacccc tacagtcgtg ctgccggccc
tgggctagga aatgtggcga tgggaccacg 4201 acagcactat ccctatggag
gtccttatga cagagtgagg acggagcctg gaatagggcc 4261 tgagggaaac
atgagcactg gggccccaca gccgaatctc atgccttcca acccagactc 4321
ggggatgtat tctcctagcc gctacccccc gcagcagcag cagcagcagc agcaacgaca
4381 tgattcctat ggcaatcagt tctccaccca aggcacccct tctggcagcc
ccttccccag 4441 ccagcagact acaatgtatc aacagcaaca gcagaattac
aagcggccaa tggatggcac 4501 atatggccct cctgccaagc ggcacgaagg
ggagatgtac agcgtgccat acagcactgg 4561 gcaggggcag cctcagcagc
agcagttgcc cccagcccag ccccagcctg ccagccagca 4621 acaagctgcc
cagccttccc ctcagcaaga tgtatacaac cagtatggca atgcctatcc 4681
tgccactgcc acagctgcta ctgagcgccg accagcaggc ggcccccaga accaatttcc
4741 attccagttt ggccgagacc gtgtctctgc accccctggc accaatgccc
agcaaaacat 4801 gccaccacaa atgatgggcg gccccataca ggcatcagct
gaggttgctc agcaaggcac 4861 catgtggcag gggcgtaatg acatgaccta
taattatgcc aacaggcaga gcacgggctc 4921 tgccccccag ggccccgcct
atcatggcgt gaaccgaaca gatgaaatgc tgcacacaga 4981 tcagagggcc
aaccacgaag gctcgtggcc ttcccatggc acacgccagc ccccatatgg 5041
tccctctgcc cctgtgcccc ccatgacaag gccccctcca tctaactacc agcccccacc
5101 aagcatgcag aatcacattc ctcaggtatc cagccctgct cccctgcccc
ggccaatgga 5161 gaaccgcacc tctcctagca agtctccatt cctgcactct
gggatgaaaa tgcagaaggc 5221 aggtccccca gtacctgcct cgcacatagc
acctgcccct gtgcagcccc ccatgattcg 5281 gcgggatatc accttcccac
ctggctctgt tgaagccaca cagcctgtgt tgaagcagag 5341 gaggcggctc
acaatgaaag acattggaac cccggaggca tggcgggtaa tgatgtccct 5401
caagtctggt ctcctggcag agagcacatg ggcattagat accatcaaca tcctgctgta
5461 tgatgacaac agcatcatga ccttcaacct cagtcagctc ccagggttgc
tagagctcct 5521 tgtagaatat ttccgacgat gcctgattga gatctttggc
attttaaagg agtatgaggt 5581 gggtgaccca ggacagagaa cgctactgga
tcctgggagg ttcagcaagg tgtctagtcc 5641 agctcccatg gagggtgggg
aagaagaaga agaacttcta ggtcctaaac tagaagagga 5701 agaagaagag
gaagtagttg aaaatgatga ggagatagcc ttttcaggca aggacaagcc 5761
agcttcagag aatagtgagg agaagctgat cagtaagttt gacaagcttc cagtaaagat
5821 cgtacagaag aatgatccat ttgtggtgga ctgctcagat aagcttgggc
gtgtgcagga 5881 gtttgacagt ggcctgctgc actggcggat tggtgggggg
gacaccactg agcatatcca 5941 gacccacttc gagagcaaga cagagctgct
gccttcccgg cctcacgcac cctgcccacc 6001 agcccctcgg aagcatgtga
caacagcaga gggtacacca gggacaacag accaggaggg 6061 gcccccacct
gatggacctc cagaaaaacg gatcacagcc actatggatg acatgttgtc 6121
tactcggtct agcaccttga ccgaggatgg agctaagagt tcagaggcca tcaaggagag
6181 cagcaagttt ccatttggca ttagcccagc acagagccac cggaacatca
agatcctaga 6241 ggacgaaccc cacagtaagg atgagacccc actgtgtacc
cttctggact ggcaggattc 6301 tcttgccaag cgctgcgtct gtgtgtccaa
taccattcga agcctgtcat ttgtgccagg 6361 caatgacttt gagatgtcca
aacacccagg gctgctgctc atcctgggca agctgatcct 6421 gctgcaccac
aagcacccag aacggaagca ggcaccacta acttatgaaa aggaggagga 6481
acaggaccaa ggggtgagct gcaacaaagt ggagtggtgg tgggactgct tggagatgct
6541 ccgggaaaac accttggtta cactcgccaa catctcgggg cagttggacc
tatctccata 6601 ccccgagagc atttgcctgc ctgtcctgga cggactccta
cactgggcag tttgcccttc 6661 agctgaagcc caggacccct tttccaccct
gggccccaat gccgtccttt ccccgcagag 6721 actggtcttg gaaaccctca
gcaaactcag catccaggac aacaatgtgg acctgattct 6781 ggccacaccc
cccttcagcc gcctggagaa gttgtatagc actatggtgc gcttcctcag 6841
tgaccgaaag aacccggtgt gccgggagat ggctgtggta ctgctggcca acctggctca
6901 gggggacagc ctggcagctc gtgccattgc agtgcagaag ggcagtatcg
gcaacctcct 6961 gggcttccta gaggacagcc ttgccgccac acagttccag
cagagccagg ccagcctcct 7021 ccacatgcag aacccaccct ttgagccaac
tagtgtggac atgatgcggc gggctgcccg 7081 cgcgctgctt gccttggcca
aggtggacga gaaccactca gagtttactc tgtacgaatc 7141 acggctgttg
gacatctcgg tatcaccgtt gatgaactca ttggtttcac aagtcatttg 7201
tgatgtactg tttttgattg gccagtcatg acagccgtgg gacacctccc ccccccgtgt
7261 gtgtgtgcgt gtgtggagaa cttagaaact gactgttgcc ctttatttat
gcaaaaccac 7321 ctcagaatcc agtttaccct gtgctgtcca gcttctccct
tgggaaaaag tctctcctgt
7381 ttctctctcc tccttccacc tcccctccct ccatcacctc acgcctttct
gttccttgtc 7441 ctcaccttac tcccctcagg accctacccc accctctttg
aaaagacaaa gctctgccta 7501 catagaagac tttttttatt ttaaccaaag
ttactgttgt ttacagtgag tttggggaaa 7561 aaaaataaaa taaaaatggc
tttcccagtc cttgcatcaa cgggatgcca catttcataa 7621 ctgtttttaa
tggtaaaaaa aaaaaaaaaa aatacaaaaa aaaattctga aggacaaaaa 7681
aggtgactgc tgaactgtgt gtggtttatt gttgtacatt cacaatcttg caggagccaa
7741 gaagttcgca gttgtgaaca gaccctgttc actggagagg cctgtgcagt
agagtgtaga 7801 ccctttcatg tactgtactg tacacctgat actgtaaaca
tactgtaata ataatgtctc 7861 acatggaaac agaaaacgct gggtcagcag
caagctgtag tttttaaaaa tgtttttagt 7921 taaacgttga ggagaaaaaa
aaaaaaggct tttcccccaa agtatcatgt gtgaacctac 7981 aacaccctga
cctctttctc tcctccttga ttgtatgaat aaccctgaga tcacctctta 8041
gaactggttt taacctttag ctgcagcggc tacgctgcca cgtgtgtata tatatgacgt
8101 tgtacattgc acataccctt ggatccccac agtttggtcc tcctcccagc
taccccttta 8161 tagtatgacg agttaacaag ttggtgacct gcacaaagcg
agacacagct atttaatctc 8221 ttgccagata tcgcccctct tggtgcgatg
ctgtacaggt ctctgtaaaa agtccttgct 8281 gtctcagcag ccaatcaact
tatagtttat ttttttctgg gtttttgttt tgttttgttt 8341 tctttctaat
cgaggtgtga aaaagttcta ggttcagttg aagttctgat gaagaaacac 8401
aattgagatt ttttcagtga taaaatctgc atatttgtat ttcaacaatg tagctaaaac
8461 ttgatgtaaa ttcctccttt ttttcctttt ttggcttaat gaatatcatt
tattcagtat 8521 gaaatcttta tactatatgt tccacgtgtt aagaataaat
gtacattaaa tcttggtaag 8581 acttt
TABLE-US-00011 AT-rich interactive domain-containing protein 1A
(ARID1A) isoform b (SEQ ID NO: 11) 1 maaqvapaaa sslgnppppp
pselkkaeqq qreeaggeaa aaaaaergem kaaagqeseg 61 pavgppqplg
kelqdgaesn gggggggags gggpgaepdl knsngnagpr palnnnltep 121
pggggggssd gvgapphsaa aalpppaygf gqpygrspsa vaaaaaavfh qqhggqqspg
181 laalqsgggg glepyagpqq nshdhgfpnh qynsyypnrs aypppapaya
lssprggtpg 241 sgaaaaagsk pppsssasas sssssfaqqr fgamggggps
aagggtpqpt atptlnqllt 301 spssargyqg ypggdysggp qdggagkgpa
dmasqcwgaa aaaaaaaaas ggaqqrshha 361 pmspgssggg gqplartpqp
sspmdqmgkm rpqpyggtnp ysqqqgppsg pqqghgypgq 421 pygsqtpqry
pmtmqgraqs amgglsytqq ippygqqgps gygqqgqtpy ynqqsphpqq 481
qqppysqqpp sqtphaqpsy qqqpqsqppq lqssqppysq qpsqpphqqs papypsqqst
541 tqqhpqsqpp ysqpqaqspy qqqqpqqpap stlsqqaayp qpqsqqsqqt
aysqqrfppp 601 qelsqdsfgs qassapsmts skggqedmnl slqsrpsslp
dlsgsiddlp mgtegalspg 661 vstsgisssq geqsnpaqsp fsphtsphlp
girgpspspv gspasvaqsr sgplspaavp 721 gnqmpprpps gqsdsimhps
mnqssiaqdr gymqrnpqmp qysspqpgsa lsprqpsggq 781 ihtgmgsyqq
nsmgsygpqg gqygpqggyp rqpnynalpn anypsagmag ginpmgaggq 841
mhgqpgippy gtlppgrmsh asmgnrpygp nmanmppqvg sgmcpppggm nrktqetava
901 mhvaansiqn rppgypnmnq ggmmgtgppy gqginsmagm inpqgppysm
ggtmannsag 961 maaspemmgl gdvkltpatk mnnkadgtpk teskskksss
stttnekitk lyelggeper 1021 kmwvdrylaf teekamgmtn lpavgrkpld
lyrlyvsvke iggltqvnkn kkwrelatnl 1081 nvgtsssaas slkkqyiqcl
yafeckierg edpppdifaa adskksqpki qppspagsgs 1141 mqgpqtpqst
sssmaeggdl kpptpastph sqipplpgms rsnsvgiqda fndgsdstfq 1201
krnsmtpnpg yqpsmntsdm mgrmsyepnk dpygsmrkap gsdpfmssgq gpnggmgdpy
1261 sraagpglgn vamgprqhyp yggpydrvrt epgigpegnm stgapqpnlm
psnpdsgmys 1321 psryppqqqq qqqqrhdsyg nqfstqgtps gspfpsqqtt
myqqqqqvss paplprpmen 1381 rtspskspfl hsgmkmqkag ppvpashiap
apvqppmirr ditfppgsve atqpvlkqrr 1441 rltmkdigtp eawrvmmslk
sgllaestwa ldtinillyd dnsimtfnls qlpgllellv 1501 eyfrrcliei
fgilkeyevg dpgqrtlldp grfskvsspa pmeggeeeee llgpkleeee 1561
eeevvendee iafsgkdkpa senseeklis kfdklpvkiv qkndpfvvdc sdklgrvqef
1621 dsgllhwrig ggdttehiqt hfesktellp srphapcppa prkhvttaeg
tpgttdqegp 1681 ppdgppekri tatmddmlst rsstltedga ksseaikess
kfpfgispaq shrnikiled 1741 ephskdetpl ctlldwqdsl akrcvcvsnt
irslsfvpgn dfemskhpgl llilgklill 1801 hhkhperkqa pltyekeeeq
dqgvscnkve wwwdclemlr entlvtlani sgqldlspyp 1861 esiclpvldg
llhwavcpsa eaqdpfstlg pnavlspqrl vletlsklsi qdnnvdlila 1921
tppfsrlekl ystmvrflsd rknpvcrema vvllanlaqg dslaaraiav qkgsignllg
1981 fledslaatq fqqsqasllh mqnppfepts vdmmrraara llalakvden
hseftlyesr 2041 lldisvsplm nslvsqvicd vlfligqs
TABLE-US-00012 Homo sapiens AT rich interactive domain 1A
(SWI-like) (ARID1A), transcript variant 2, mRNA (SEQ ID NO: 12) 1
cagaaagcgg agagtcacag cggggccagg ccctggggag cggagcctcc accgcccccc
61 tcattcccag gcaagggctt ggggggaatg agccgggaga gccgggtccc
gagcctacag 121 agccgggagc agctgagccg ccggcgcctc ggccgccgcc
gccgcctcct cctcctccgc 181 cgccgccagc ccggagcctg agccggcggg
gcggggggga gaggagcgag cgcagcgcag 241 cagcggagcc ccgcgaggcc
cgcccgggcg ggtggggagg gcagcccggg ggactgggcc 301 ccggggcggg
gtgggagggg gggagaagac gaagacaggg ccgggtctct ccgcggacga 361
gacagcgggg atcatggccg cgcaggtcgc ccccgccgcc gccagcagcc tgggcaaccc
421 gccgccgccg ccgccctcgg agctgaagaa agccgagcag cagcagcggg
aggaggcggg 481 gggcgaggcg gcggcggcgg cagcggccga gcgcggggaa
atgaaggcag ccgccgggca 541 ggaaagcgag ggccccgccg tggggccgcc
gcagccgctg ggaaaggagc tgcaggacgg 601 ggccgagagc aatgggggtg
gcggcggcgg cggagccggc agcggcggcg ggcccggcgc 661 ggagccggac
ctgaagaact cgaacgggaa cgcgggccct aggcccgccc tgaacaataa 721
cctcacggag ccgcccggcg gcggcggtgg cggcagcagc gatggggtgg gggcgcctcc
781 tcactcagcc gcggccgcct tgccgccccc agcctacggc ttcgggcaac
cctacggccg 841 gagcccgtct gccgtcgccg ccgccgcggc cgccgtcttc
caccaacaac atggcggaca 901 acaaagccct ggcctggcag cgctgcagag
cggcggcggc gggggcctgg agccctacgc 961 ggggccccag cagaactctc
acgaccacgg cttccccaac caccagtaca actcctacta 1021 ccccaaccgc
agcgcctacc ccccgcccgc cccggcctac gcgctgagct ccccgagagg 1081
tggcactccg ggctccggcg cggcggcggc tgccggctcc aagccgcctc cctcctccag
1141 cgcctccgcc tcctcgtcgt cttcgtcctt cgctcagcag cgcttcgggg
ccatgggggg 1201 aggcggcccc tccgcggccg gcgggggaac tccccagccc
accgccaccc ccaccctcaa 1261 ccaactgctc acgtcgccca gctcggcccg
gggctaccag ggctaccccg ggggcgacta 1321 cagtggcggg ccccaggacg
ggggcgccgg caagggcccg gcggacatgg cctcgcagtg 1381 ttggggggct
gcggcggcgg cagctgcggc ggcggccgcc tcgggagggg cccaacaaag 1441
gagccaccac gcgcccatga gccccgggag cagcggcggc ggggggcagc cgctcgcccg
1501 gacccctcag ccatccagtc caatggatca gatgggcaag atgagacctc
agccatatgg 1561 cgggactaac ccatactcgc agcaacaggg acctccgtca
ggaccgcagc aaggacatgg 1621 gtacccaggg cagccatacg ggtcccagac
cccgcagcgg tacccgatga ccatgcaggg 1681 ccgggcgcag agtgccatgg
gcggcctctc ttatacacag cagattcctc cttatggaca 1741 acaaggcccc
agcgggtatg gtcaacaggg ccagactcca tattacaacc agcaaagtcc 1801
tcaccctcag cagcagcagc caccctactc ccagcaacca ccgtcccaga cccctcatgc
1861 ccaaccttcg tatcagcagc agccacagtc tcaaccacca cagctccagt
cctctcagcc 1921 tccatactcc cagcagccat cccagcctcc acatcagcag
tccccggctc catacccctc 1981 ccagcagtcg acgacacagc agcaccccca
gagccagccc ccctactcac agccacaggc 2041 tcagtctcct taccagcagc
agcaacctca gcagccagca ccctcgacgc tctcccagca 2101 ggctgcgtat
cctcagcccc agtctcagca gtcccagcaa actgcctatt cccagcagcg 2161
cttccctcca ccgcaggagc tatctcaaga ttcatttggg tctcaggcat cctcagcccc
2221 ctcaatgacc tccagtaagg gagggcaaga agatatgaac ctgagccttc
agtcaagacc 2281 ctccagcttg cctgatctat ctggttcaat agatgacctc
cccatgggga cagaaggagc 2341 tctgagtcct ggagtgagca catcagggat
ttccagcagc caaggagagc agagtaatcc 2401 agctcagtct cctttctctc
ctcatacctc ccctcacctg cctggcatcc gaggcccttc 2461 cccgtcccct
gttggctctc ccgccagtgt tgctcagtct cgctcaggac cactctcgcc 2521
tgctgcagtg ccaggcaacc agatgccacc tcggccaccc agtggccagt cggacagcat
2581 catgcatcct tccatgaacc aatcaagcat tgcccaagat cgaggttata
tgcagaggaa 2641 cccccagatg ccccagtaca gttcccccca gcccggctca
gccttatctc cgcgtcagcc 2701 ttccggagga cagatacaca caggcatggg
ctcctaccag cagaactcca tggggagcta 2761 tggtccccag gggggtcagt
atggcccaca aggtggctac cccaggcagc caaactataa 2821 tgccttgccc
aatgccaact accccagtgc aggcatggct ggaggcataa accccatggg 2881
tgccggaggt caaatgcatg gacagcctgg catcccacct tatggcacac tccctccagg
2941 gaggatgagt cacgcctcca tgggcaaccg gccttatggc cctaacatgg
ccaatatgcc 3001 acctcaggtt gggtcaggga tgtgtccccc accagggggc
atgaaccgga aaacccaaga 3061 aactgctgtc gccatgcatg ttgctgccaa
ctctatccaa aacaggccgc caggctaccc 3121 caatatgaat caagggggca
tgatgggaac tggacctcct tatggacaag ggattaatag 3181 tatggctggc
atgatcaacc ctcagggacc cccatattcc atgggtggaa ccatggccaa 3241
caattctgca gggatggcag ccagcccaga gatgatgggc cttggggatg taaagttaac
3301 tccagccacc aaaatgaaca acaaggcaga tgggacaccc aagacagaat
ccaaatccaa 3361 gaaatccagt tcttctacta caaccaatga gaagatcacc
aagttgtatg agctgggtgg 3421 tgagcctgag aggaagatgt gggtggaccg
ttatctggcc ttcactgagg agaaggccat 3481 gggcatgaca aatctgcctg
ctgtgggtag gaaacctctg gacctctatc gcctctatgt 3541 gtctgtgaag
gagattggtg gattgactca ggtcaacaag aacaaaaaat ggcgggaact 3601
tgcaaccaac ctcaatgtgg gcacatcaag cagtgctgcc agctccttga aaaagcagta
3661 tatccagtgt ctctatgcct ttgaatgcaa gattgaacgg ggagaagacc
ctcccccaga 3721 catctttgca gctgctgatt ccaagaagtc ccagcccaag
atccagcctc cctctcctgc 3781 gggatcagga tctatgcagg ggccccagac
tccccagtca accagcagtt ccatggcaga 3841 aggaggagac ttaaagccac
caactccagc atccacacca cacagtcaga tccccccatt 3901 gccaggcatg
agcaggagca attcagttgg gatccaggat gcctttaatg atggaagtga 3961
ctccacattc cagaagcgga attccatgac tccaaaccct gggtatcagc ccagtatgaa
4021 tacctctgac atgatggggc gcatgtccta tgagccaaat aaggatcctt
atggcagcat 4081 gaggaaagct ccagggagtg atcccttcat gtcctcaggg
cagggcccca acggcgggat 4141 gggtgacccc tacagtcgtg ctgccggccc
tgggctagga aatgtggcga tgggaccacg 4201 acagcactat ccctatggag
gtccttatga cagagtgagg acggagcctg gaatagggcc 4261 tgagggaaac
atgagcactg gggccccaca gccgaatctc atgccttcca acccagactc 4321
ggggatgtat tctcctagcc gctacccccc gcagcagcag cagcagcagc agcaacgaca
4381 tgattcctat ggcaatcagt tctccaccca aggcacccct tctggcagcc
ccttccccag 4441 ccagcagact acaatgtatc aacagcaaca gcaggtatcc
agccctgctc ccctgccccg 4501 gccaatggag aaccgcacct ctcctagcaa
gtctccattc ctgcactctg ggatgaaaat 4561 gcagaaggca ggtcccccag
tacctgcctc gcacatagca cctgcccctg tgcagccccc 4621 catgattcgg
cgggatatca ccttcccacc tggctctgtt gaagccacac agcctgtgtt 4681
gaagcagagg aggcggctca caatgaaaga cattggaacc ccggaggcat ggcgggtaat
4741 gatgtccctc aagtctggtc tcctggcaga gagcacatgg gcattagata
ccatcaacat 4801 cctgctgtat gatgacaaca gcatcatgac cttcaacctc
agtcagctcc cagggttgct 4861 agagctcctt gtagaatatt tccgacgatg
cctgattgag atctttggca ttttaaagga 4921 gtatgaggtg ggtgacccag
gacagagaac gctactggat cctgggaggt tcagcaaggt 4981 gtctagtcca
gctcccatgg agggtgggga agaagaagaa gaacttctag gtcctaaact 5041
agaagaggaa gaagaagagg aagtagttga aaatgatgag gagatagcct tttcaggcaa
5101 ggacaagcca gcttcagaga atagtgagga gaagctgatc agtaagtttg
acaagcttcc 5161 agtaaagatc gtacagaaga atgatccatt tgtggtggac
tgctcagata agcttgggcg 5221 tgtgcaggag tttgacagtg gcctgctgca
ctggcggatt ggtggggggg acaccactga 5281 gcatatccag acccacttcg
agagcaagac agagctgctg ccttcccggc ctcacgcacc 5341 ctgcccacca
gcccctcgga agcatgtgac aacagcagag ggtacaccag ggacaacaga 5401
ccaggagggg cccccacctg atggacctcc agaaaaacgg atcacagcca ctatggatga
5461 catgttgtct actcggtcta gcaccttgac cgaggatgga gctaagagtt
cagaggccat 5521 caaggagagc agcaagtttc catttggcat tagcccagca
cagagccacc ggaacatcaa 5581 gatcctagag gacgaacccc acagtaagga
tgagacccca ctgtgtaccc ttctggactg 5641 gcaggattct cttgccaagc
gctgcgtctg tgtgtccaat accattcgaa gcctgtcatt 5701 tgtgccaggc
aatgactttg agatgtccaa acacccaggg ctgctgctca tcctgggcaa 5761
gctgatcctg ctgcaccaca agcacccaga acggaagcag gcaccactaa cttatgaaaa
5821 ggaggaggaa caggaccaag gggtgagctg caacaaagtg gagtggtggt
gggactgctt 5881 ggagatgctc cgggaaaaca ccttggttac actcgccaac
atctcggggc agttggacct 5941 atctccatac cccgagagca tttgcctgcc
tgtcctggac ggactcctac actgggcagt 6001 ttgcccttca gctgaagccc
aggacccctt ttccaccctg ggccccaatg ccgtcctttc 6061 cccgcagaga
ctggtcttgg aaaccctcag caaactcagc atccaggaca acaatgtgga 6121
cctgattctg gccacacccc ccttcagccg cctggagaag ttgtatagca ctatggtgcg
6181 cttcctcagt gaccgaaaga acccggtgtg ccgggagatg gctgtggtac
tgctggccaa 6241 cctggctcag ggggacagcc tggcagctcg tgccattgca
gtgcagaagg gcagtatcgg 6301 caacctcctg ggcttcctag aggacagcct
tgccgccaca cagttccagc agagccaggc 6361 cagcctcctc cacatgcaga
acccaccctt tgagccaact agtgtggaca tgatgcggcg 6421 ggctgcccgc
gcgctgcttg ccttggccaa ggtggacgag aaccactcag agtttactct 6481
gtacgaatca cggctgttgg acatctcggt atcaccgttg atgaactcat tggtttcaca
6541 agtcatttgt gatgtactgt ttttgattgg ccagtcatga cagccgtggg
acacctcccc 6601 cccccgtgtg tgtgtgcgtg tgtggagaac ttagaaactg
actgttgccc tttatttatg 6661 caaaaccacc tcagaatcca gtttaccctg
tgctgtccag cttctccctt gggaaaaagt 6721 ctctcctgtt tctctctcct
ccttccacct cccctccctc catcacctca cgcctttctg 6781 ttccttgtcc
tcaccttact cccctcagga ccctacccca ccctctttga aaagacaaag 6841
ctctgcctac atagaagact ttttttattt taaccaaagt tactgttgtt tacagtgagt
6901 ttggggaaaa aaaataaaat aaaaatggct ttcccagtcc ttgcatcaac
gggatgccac 6961 atttcataac tgtttttaat ggtaaaaaaa aaaaaaaaaa
atacaaaaaa aaattctgaa 7021 ggacaaaaaa ggtgactgct gaactgtgtg
tggtttattg ttgtacattc acaatcttgc 7081 aggagccaag aagttcgcag
ttgtgaacag accctgttca ctggagaggc ctgtgcagta 7141 gagtgtagac
cctttcatgt actgtactgt acacctgata ctgtaaacat actgtaataa 7201
taatgtctca catggaaaca gaaaacgctg ggtcagcagc aagctgtagt ttttaaaaat
7261 gtttttagtt aaacgttgag gagaaaaaaa aaaaaggctt ttcccccaaa
gtatcatgtg 7321 tgaacctaca acaccctgac ctctttctct cctccttgat
tgtatgaata accctgagat
7381 cacctcttag aactggtttt aacctttagc tgcagcggct acgctgccac
gtgtgtatat 7441 atatgacgtt gtacattgca catacccttg gatccccaca
gtttggtcct cctcccagct 7501 acccctttat agtatgacga gttaacaagt
tggtgacctg cacaaagcga gacacagcta 7561 tttaatctct tgccagatat
cgcccctctt ggtgcgatgc tgtacaggtc tctgtaaaaa 7621 gtccttgctg
tctcagcagc caatcaactt atagtttatt tttttctggg tttttgtttt 7681
gttttgtttt ctttctaatc gaggtgtgaa aaagttctag gttcagttga agttctgatg
7741 aagaaacaca attgagattt tttcagtgat aaaatctgca tatttgtatt
tcaacaatgt 7801 agctaaaact tgatgtaaat tcctcctttt tttccttttt
tggcttaatg aatatcattt 7861 attcagtatg aaatctttat actatatgtt
ccacgtgtta agaataaatg tacattaaat 7921 cttggtaaga cttt
[0119] The present invention also provides methods of inducing
neuronal differentiation by contacting a cell with a compound
(i.e., an EZH2 inhibitor) of the invention. Preferably, the
compound is in an amount sufficient to increase expression of at
least one gene selected from the group consisting of CD133 (also
called PROM1), DOCK4, PTPRK, PROM2, LHX1, LHX6, LHX9, PAX6, PAX7,
VEFGA, FZD3B, FYN, HIF1A, HTRA2, EVX1, CCDC64, and GFAP.
[0120] The term "inducing neuronal differentiation" used herein
refers to causing a cell to develop into a cell of the neuronal
lineage as a result of a direct or intentional effect on the
cell.
[0121] The present invention also provides methods of inducing cell
cycle inhibition by contacting a cell with a compound of the
invention. Preferably, the compound is in an amount sufficient to
increase expression of at least one gene selected from the group
consisting of CKDN1A, CDKN2A, MEN1, CHEK1, IRF6, ALOX15B, CYP27B1,
DBC1, NME6, GMNN, HEXIM1, LATS1, MYC, HRAS, TGFB1, IFNG, WNT1,
TP53, THBS1, INHBA, IL8, IRF1, TPR, BMP2, BMP4, ETS1, HPGD, BMP7,
GATA3, NR2F2, APC, PTPN3, CALR, IL12A, IL12B, PML, CDKN2B, CDKN2C,
CDKN1B, SOX2, TAF6, DNA2, PLK1, TERF1, GAS1, CDKN2D, MLF1, PTEN,
TGFB2, SMAD3, FOXO4, CDK6, TFAP4, MAP2K1, NOTCH2, FOXC1, DLG1,
MAD2L1, ATM, NAE1, DGKZ, FHL1, SCRIB, BTG3, PTPRK, RPS6KA2, STK11,
CDKN3, TBRG1, CDC73, THAP5, CRLF3, DCUN1D3, MYOCD, PAF1, LILRB1,
UHMK1, PNPT1, USP47, HEXIM2, CDK5RAP1, NKX3-1, TIPIN, PCBP4, USP44,
RBM38, CDT1, RGCC, RNF167, CLSPN, CHMP1A, WDR6, TCF7L2, LATS2,
RASSF1, MLTK, MAD2L2, FBXO5, ING4, and TRIM35.
[0122] The term "inducing cell cycle inhibition" used herein refers
to causing an accumulation or an arrest at any phase during cell
division and/or duplication.
[0123] The present invention also provides methods of inducing
tumor suppression by contacting a cell with a compound of the
invention. Preferably, the compound is in an amount sufficient to
increase expression of BIN1 or any tumor suppressors.
[0124] The term "inducing tumor suppression" may include, but is
not limited to, a reduction in size of a tumor, a reduction in
tumor volume, a decrease in number of tumors, a decrease in number
of metastatic lesions in other tissues or organs distant from the
primary tumor site, an increase in average survival time of a
population of treated subjects in comparison to a population
receiving carrier alone, an increase in average survival time of a
population of treated subjects in comparison to a population of
untreated subjects, an increase in average survival time of a
population of treated subjects in comparison to a population
receiving monotherapy with a drug that is not a compound of the
present invention, a decrease in the mortality rate of a population
of treated subjects in comparison to a population receiving carrier
alone, a decrease in tumor growth rate, or a decrease in tumor
regrowth rate.
[0125] The present invention also provides methods of inhibiting
hedgehog signaling by contacting a cell with a compound of the
invention. Preferably, the compound is in an amount sufficient to
reduce expression of at least one gene selected from the group
consisting of GLI1, PTCH1, SUFU, KIF7, GLI2, BMP4, MAP3K10, SHH,
TCTN3, DYRK2, PTCHD1, and SMO.
[0126] The phrase "inhibiting hedgehog signaling" means the
hedgehog signaling strength (intensity) with a compound treatment
is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%,
400%, 500%, 1000%, 1500%, or more compared to the hedgehog
signaling strength (intensity) without any compound treatment.
[0127] The present invention also provides methods of inducing a
gene expression by contacting a cell with a compound of the
invention. Preferably, the compound is in an amount sufficient to
induce neuronal differentiation, cell cycle inhibition and/or tumor
suppression. Such gene is selected from the group consisting of
CD133 (also called PROM1), DOCK4, PTPRK, PROM2, LHX1, LHX6, LHX9,
PAX6, PAX7, VEFGA, FZD3B, FYN, HIF1A, HTRA2, EVX1, CCDC64, GFAP,
CKDN1A, CDKN2A, MEN1, CHEK1, IRF6, ALOX15B, CYP27B1, DBC1, NME6,
GMNN, HEXIM1, LATS1, MYC, HRAS, TGFB1, IFNG, WNT1, TP53, THBS1,
INHBA, IL8, IRF1, TPR, BMP2, BMP4, ETS1, HPGD, BMP7, GATA3, NR2F2,
APC, PTPN3, CALR, IL12A, IL12B, PML, CDKN2B, CDKN2C, CDKN1B, SOX2,
TAF6, DNA2, PLK1, TERF1, GAS1, CDKN2D, MLF1, PTEN, TGFB2, SMAD3,
FOXO4, CDK6, TFAP4, MAP2K1, NOTCH2, FOXC1, DLG1, MAD2L1, ATM, NAE1,
DGKZ, FHL1, SCRIB, BTG3, PTPRK, RPS6KA2, STK11, CDKN3, TBRG1,
CDC73, THAP5, CRLF3, DCUN1D3, MYOCD, PAF1, LILRB1, UHMK1, PNPT1,
USP47, HEXIM2, CDK5RAP1, NKX3-1, TIPIN, PCBP4, USP44, RBM38, CDT1,
RGCC, RNF167, CLSPN, CHMP1A, WDR6, TCF7L2, LATS2, RASSF1, MLTK,
MAD2L2, FBXO5, ING4, TRIM35, BIN1 and any tumor suppressors.
[0128] The phrase "inducing a gene expression" means the expression
level of a particular gene of interest is increased by at least 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 1000%,
1500%, or more compared to the expression level of this gene
without any compound treatment.
[0129] The present invention also provides methods of inhibiting a
gene expression comprising contacting a cell with a compound of the
invention. Preferably, the compound is in an amount sufficient to
inhibit hedgehog signaling. Such gene is GLI1, PTCH1, SUFU, KIF7,
GLI2, BMP4, MAP3K10, SHH, TCTN3, DYRK2, PTCHD1, or SMO.
[0130] The phrase "inhibiting a gene expression" means the
expression level of a particular gene of interest is reduced by at
least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%,
1000%, 1500%, or more compared to the expression level of this gene
without any compound treatment.
[0131] Neuronal differentiation, cell cycle inhibition, tumor
suppression and hedgehog signaling inhibition can be determined by
any methods known in the art.
[0132] As used herein, a cell refers to any cell that can be
obtained and used by a method described herein. For example, a cell
may be obtained from a cell culture. Alternatively, a cell may be
isolated from a subject. A cell may also refer to a cell of a
subject.
[0133] A cell may comprise loss of function of SNF5, ARID1A, ATRX,
and/or a component of the SWI/SNF complex. Preferably, a cell may
comprise a deletion of SNF5.
[0134] A cell may be a cancer cell, where the cancer is selected
from the group consisting of medulloblastoma, oligodendroglioma,
ovarian clear cell adenocarcinoma, ovarian endomethrioid
adenocarcinoma, ovarian serous adenocarcinoma, pancreatic ductal
adenocarcinoma, pancreatic endocrine tumor, malignant rhabdoid
tumor, astrocytoma, atypical teratoid rhabdoid tumor, choroid
plexus carcinoma, choroid plexus papilloma, ependymoma,
glioblastoma, meningioma, neuroglial tumor, oligoastrocytoma,
oligodendroglioma, pineoblastoma, carcinosarcoma, chordoma,
extragonadal germ cell tumor, extrarenal rhabdoid tumor,
schwannoma, skin squamous cell carcinoma, chondrosarcoma, clear
cell sarcoma of soft tissue, ewing sarcoma, gastrointestinal
stromal tumor, osteosarcoma, rhabdomyosarcoma, epithelioid sarcoma,
renal medullo carcinoma, diffuse large B-cell lymphoma, follicular
lymphoma and not otherwise specified (NOS) sarcoma. More preferably
a cell is a cancer cell of medulloblastoma, malignant rhabdoid
tumor, or atypical teratoid rhabdoid tumor.
[0135] A cancer that is to be treated can be staged according to
the American Joint Committee on Cancer (AJCC) TNM classification
system, where the tumor (T) has been assigned a stage of TX, T1,
T1mic, T1a, T1b, T1c, T2, T3, T4, T4a, T4b, T4c, or T4d; and where
the regional lymph nodes (N) have been assigned a stage of NX, N0,
N1, N2, N2a, N2b, N3, N3a, N3b, or N3c; and where distant
metastasis (M) can be assigned a stage of MX, M0, or M1. A cancer
that is to be treated can be staged according to an American Joint
Committee on Cancer (AJCC) classification as Stage I, Stage IIA,
Stage IIB, Stage IIIA, Stage IIIB, Stage IIIC, or Stage IV. A
cancer that is to be treated can be assigned a grade according to
an AJCC classification as Grade GX (e.g., grade cannot be
assessed), Grade 1, Grade 2, Grade 3 or Grade 4. A cancer that is
to be treated can be staged according to an AJCC pathologic
classification (pN) of pNX, pN0, PN0 (I-), PN0 (I+), PN0 (mol-),
PN0 (mol+), PN1, PN1(mi), PN1a, PN1b, PN1c, pN2, pN2a, pN2b, pN3,
pN3a, pN3b, or pN3c.
[0136] A cancer that is to be treated can be evaluated by DNA
cytometry, flow cytometry, or image cytometry. A cancer that is to
be treated can be typed as having 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, or 90% of cells in the synthesis stage of cell division
(e.g., in S phase of cell division). A cancer that is to be treated
can be typed as having a low S-phase fraction or a high S-phase
fraction.
[0137] As used herein, a "normal cell" is a cell that cannot be
classified as part of a "cell proliferative disorder". A normal
cell lacks unregulated or abnormal growth, or both, that can lead
to the development of an unwanted condition or disease. Preferably,
a normal cell possesses normally functioning cell cycle checkpoint
control mechanisms.
[0138] As used herein, "contacting a cell" refers to a condition in
which a compound or other composition of matter is in direct
contact with a cell, or is close enough to induce a desired
biological effect in a cell.
[0139] As used herein, "monotherapy" refers to the administration
of a single active or therapeutic compound to a subject in need
thereof. Preferably, monotherapy will involve administration of a
therapeutically effective amount of an active compound. For
example, cancer monotherapy with one of the compound of the present
invention, or a pharmaceutically acceptable salt, polymorph,
solvate, analog or derivative thereof, to a subject in need of
treatment of cancer. Monotherapy may be contrasted with combination
therapy, in which a combination of multiple active compounds is
administered, preferably with each component of the combination
present in a therapeutically effective amount. In one aspect,
monotherapy with a compound of the present invention, or a
pharmaceutically acceptable salt, polymorph or solvate thereof, is
more effective than combination therapy in inducing a desired
biological effect.
[0140] As used herein, "treating" or "treat" describes the
management and care of a patient for the purpose of combating a
disease, condition, or disorder and includes the administration of
a compound of the present invention, or a pharmaceutically
acceptable salt, polymorph or solvate thereof, to alleviate one or
more symptoms or complications of a disease, condition or disorder,
or to eliminate the disease, condition or disorder. The term
"treat" can also include treatment of a cell in vitro or an animal
model.
[0141] A compound of the present invention, or a pharmaceutically
acceptable salt, polymorph or solvate thereof, can also be used to
prevent a disease, condition or disorder, or used to identify
suitable candidates for such purposes. As used herein, "preventing"
or "prevent" describes reducing or eliminating the onset of the
symptoms or complications of the disease, condition or
disorder.
[0142] As used herein, the term "alleviate" is meant to describe a
process by which the severity of a sign or symptom of a disorder is
decreased. Importantly, a sign or symptom can be alleviated without
being eliminated. In a preferred embodiment, the administration of
pharmaceutical compositions of the invention leads to the
elimination of a sign or symptom, however, elimination is not
required. Effective dosages are expected to decrease the severity
of a sign or symptom. For instance, a sign or symptom of a disorder
such as cancer, which can occur in multiple locations, is
alleviated if the severity of the cancer is decreased within at
least one of multiple locations.
[0143] As used herein, the term "severity" is meant to describe the
potential of cancer to transform from a precancerous, or benign,
state into a malignant state. Alternatively, or in addition,
severity is meant to describe a cancer stage, for example,
according to the TNM system (accepted by the International Union
Against Cancer (UICC) and the American Joint Committee on Cancer
(AJCC)) or by other art-recognized methods. Cancer stage refers to
the extent or severity of the cancer, based on factors such as the
location of the primary tumor, tumor size, number of tumors, and
lymph node involvement (spread of cancer into lymph nodes).
Alternatively, or in addition, severity is meant to describe the
tumor grade by art-recognized methods (see, National Cancer
Institute, www.cancer.gov). Tumor grade is a system used to
classify cancer cells in terms of how abnormal they look under a
microscope and how quickly the tumor is likely to grow and spread.
Many factors are considered when determining tumor grade, including
the structure and growth pattern of the cells. The specific factors
used to determine tumor grade vary with each type of cancer.
Severity also describes a histologic grade, also called
differentiation, which refers to how much the tumor cells resemble
normal cells of the same tissue type (see, National Cancer
Institute, www.cancer.gov). Furthermore, severity describes a
nuclear grade, which refers to the size and shape of the nucleus in
tumor cells and the percentage of tumor cells that are dividing
(see, National Cancer Institute, www.cancer.gov).
[0144] In another aspect of the invention, severity describes the
degree to which a tumor has secreted growth factors, degraded the
extracellular matrix, become vascularized, lost adhesion to
juxtaposed tissues, or metastasized. Moreover, severity describes
the number of locations to which a primary tumor has metastasized.
Finally, severity includes the difficulty of treating tumors of
varying types and locations. For example, inoperable tumors, those
cancers which have greater access to multiple body systems
(hematological and immunological tumors), and those which are the
most resistant to traditional treatments are considered most
severe. In these situations, prolonging the life expectancy of the
subject and/or reducing pain, decreasing the proportion of
cancerous cells or restricting cells to one system, and improving
cancer stage/tumor grade/histological grade/nuclear grade are
considered alleviating a sign or symptom of the cancer.
[0145] As used herein the term "symptom" is defined as an
indication of disease, illness, injury, or that something is not
right in the body. Symptoms are felt or noticed by the individual
experiencing the symptom, but may not easily be noticed by others.
Others are defined as non-health-care professionals.
[0146] As used herein the term "sign" is also defined as an
indication that something is not right in the body. But signs are
defined as things that can be seen by a doctor, nurse, or other
health care professional.
[0147] Cancer is a group of diseases that may cause almost any sign
or symptom. The signs and symptoms will depend on where the cancer
is, the size of the cancer, and how much it affects the nearby
organs or structures. If a cancer spreads (metastasizes), then
symptoms may appear in different parts of the body.
[0148] Treating cancer can result in a reduction in size of a
tumor. A reduction in size of a tumor may also be referred to as
"tumor regression". Preferably, after treatment, tumor size is
reduced by 5% or greater relative to its size prior to treatment;
more preferably, tumor size is reduced by 10% or greater; more
preferably, reduced by 20% or greater; more preferably, reduced by
30% or greater; more preferably, reduced by 40% or greater; even
more preferably, reduced by 50% or greater; and most preferably,
reduced by greater than 75% or greater. Size of a tumor may be
measured by any reproducible means of measurement. The size of a
tumor may be measured as a diameter of the tumor.
[0149] Treating cancer can result in a reduction in tumor volume.
Preferably, after treatment, tumor volume is reduced by 5% or
greater relative to its size prior to treatment; more preferably,
tumor volume is reduced by 10% or greater; more preferably, reduced
by 20% or greater; more preferably, reduced by 30% or greater; more
preferably, reduced by 40% or greater; even more preferably,
reduced by 50% or greater; and most preferably, reduced by greater
than 75% or greater. Tumor volume may be measured by any
reproducible means of measurement.
[0150] Treating cancer results in a decrease in number of tumors.
Preferably, after treatment, tumor number is reduced by 5% or
greater relative to number prior to treatment; more preferably,
tumor number is reduced by 10% or greater; more preferably, reduced
by 20% or greater; more preferably, reduced by 30% or greater; more
preferably, reduced by 40% or greater; even more preferably,
reduced by 50% or greater; and most preferably, reduced by greater
than 75%. Number of tumors may be measured by any reproducible
means of measurement. The number of tumors may be measured by
counting tumors visible to the naked eye or at a specified
magnification. Preferably, the specified magnification is 2.times.,
3.times., 4.times., 5.times., 10.times., or 50.times..
[0151] Treating cancer can result in a decrease in number of
metastatic lesions in other tissues or organs distant from the
primary tumor site. Preferably, after treatment, the number of
metastatic lesions is reduced by 5% or greater relative to number
prior to treatment; more preferably, the number of metastatic
lesions is reduced by 10% or greater; more preferably, reduced by
20% or greater; more preferably, reduced by 30% or greater; more
preferably, reduced by 40% or greater; even more preferably,
reduced by 50% or greater; and most preferably, reduced by greater
than 75%. The number of metastatic lesions may be measured by any
reproducible means of measurement. The number of metastatic lesions
may be measured by counting metastatic lesions visible to the naked
eye or at a specified magnification. Preferably, the specified
magnification is 2.times., 3.times., 4.times., 5.times., 10.times.,
or 50.times..
[0152] Treating cancer can result in an increase in average
survival time of a population of treated subjects in comparison to
a population receiving carrier alone. Preferably, the average
survival time is increased by more than 30 days; more preferably,
by more than 60 days; more preferably, by more than 90 days; and
most preferably, by more than 120 days. An increase in average
survival time of a population may be measured by any reproducible
means. An increase in average survival time of a population may be
measured, for example, by calculating for a population the average
length of survival following initiation of treatment with an active
compound. An increase in average survival time of a population may
also be measured, for example, by calculating for a population the
average length of survival following completion of a first round of
treatment with an active compound.
[0153] Treating cancer can result in an increase in average
survival time of a population of treated subjects in comparison to
a population of untreated subjects. Preferably, the average
survival time is increased by more than 30 days; more preferably,
by more than 60 days; more preferably, by more than 90 days; and
most preferably, by more than 120 days. An increase in average
survival time of a population may be measured by any reproducible
means. An increase in average survival time of a population may be
measured, for example, by calculating for a population the average
length of survival following initiation of treatment with an active
compound. An increase in average survival time of a population may
also be measured, for example, by calculating for a population the
average length of survival following completion of a first round of
treatment with an active compound.
[0154] Treating cancer can result in increase in average survival
time of a population of treated subjects in comparison to a
population receiving monotherapy with a drug that is not a compound
of the present invention, or a pharmaceutically acceptable salt,
polymorph, solvate, analog or derivative thereof. Preferably, the
average survival time is increased by more than 30 days; more
preferably, by more than 60 days; more preferably, by more than 90
days; and most preferably, by more than 120 days. An increase in
average survival time of a population may be measured by any
reproducible means. An increase in average survival time of a
population may be measured, for example, by calculating for a
population the average length of survival following initiation of
treatment with an active compound. An increase in average survival
time of a population may also be measured, for example, by
calculating for a population the average length of survival
following completion of a first round of treatment with an active
compound.
[0155] Treating cancer can result in a decrease in the mortality
rate of a population of treated subjects in comparison to a
population receiving carrier alone. Treating cancer can result in a
decrease in the mortality rate of a population of treated subjects
in comparison to an untreated population. Treating cancer can
result in a decrease in the mortality rate of a population of
treated subjects in comparison to a population receiving
monotherapy with a drug that is not a compound of the present
invention, or a pharmaceutically acceptable salt, polymorph,
solvate, analog or derivative thereof. Preferably, the mortality
rate is decreased by more than 2%; more preferably, by more than
5%; more preferably, by more than 10%; and most preferably, by more
than 25%. A decrease in the mortality rate of a population of
treated subjects may be measured by any reproducible means. A
decrease in the mortality rate of a population may be measured, for
example, by calculating for a population the average number of
disease-related deaths per unit time following initiation of
treatment with an active compound. A decrease in the mortality rate
of a population may also be measured, for example, by calculating
for a population the average number of disease-related deaths per
unit time following completion of a first round of treatment with
an active compound.
[0156] Treating cancer can result in a decrease in tumor growth
rate. Preferably, after treatment, tumor growth rate is reduced by
at least 5% relative to number prior to treatment; more preferably,
tumor growth rate is reduced by at least 10%; more preferably,
reduced by at least 20%; more preferably, reduced by at least 30%;
more preferably, reduced by at least 40%; more preferably, reduced
by at least 50%; even more preferably, reduced by at least 50%; and
most preferably, reduced by at least 75%. Tumor growth rate may be
measured by any reproducible means of measurement. Tumor growth
rate can be measured according to a change in tumor diameter per
unit time.
[0157] Treating cancer can result in a decrease in tumor regrowth.
Preferably, after treatment, tumor regrowth is less than 5%; more
preferably, tumor regrowth is less than 10%; more preferably, less
than 20%; more preferably, less than 30%; more preferably, less
than 40%; more preferably, less than 50%; even more preferably,
less than 50%; and most preferably, less than 75%. Tumor regrowth
may be measured by any reproducible means of measurement. Tumor
regrowth is measured, for example, by measuring an increase in the
diameter of a tumor after a prior tumor shrinkage that followed
treatment. A decrease in tumor regrowth is indicated by failure of
tumors to reoccur after treatment has stopped.
[0158] Treating cancer can result in a reduction in the rate of
cellular proliferation. Preferably, after treatment, the rate of
cellular proliferation is reduced by at least 5%; more preferably,
by at least 10%; more preferably, by at least 20%; more preferably,
by at least 30%; more preferably, by at least 40%; more preferably,
by at least 50%; even more preferably, by at least 50%; and most
preferably, by at least 75%. The rate of cellular proliferation may
be measured by any reproducible means of measurement. The rate of
cellular proliferation is measured, for example, by measuring the
number of dividing cells in a tissue sample per unit time.
[0159] Treating cancer can result in a reduction in the proportion
of proliferating cells. Preferably, after treatment, the proportion
of proliferating cells is reduced by at least 5%; more preferably,
by at least 10%; more preferably, by at least 20%; more preferably,
by at least 30%; more preferably, by at least 40%; more preferably,
by at least 50%; even more preferably, by at least 50%; and most
preferably, by at least 75%. The proportion of proliferating cells
may be measured by any reproducible means of measurement.
Preferably, the proportion of proliferating cells is measured, for
example, by quantifying the number of dividing cells relative to
the number of nondividing cells in a tissue sample. The proportion
of proliferating cells can be equivalent to the mitotic index.
[0160] Treating cancer can result in a decrease in size of an area
or zone of cellular proliferation. Preferably, after treatment,
size of an area or zone of cellular proliferation is reduced by at
least 5% relative to its size prior to treatment; more preferably,
reduced by at least 10%; more preferably, reduced by at least 20%;
more preferably, reduced by at least 30%; more preferably, reduced
by at least 40%; more preferably, reduced by at least 50%; even
more preferably, reduced by at least 50%; and most preferably,
reduced by at least 75%. Size of an area or zone of cellular
proliferation may be measured by any reproducible means of
measurement. The size of an area or zone of cellular proliferation
may be measured as a diameter or width of an area or zone of
cellular proliferation.
[0161] Treating cancer can result in a decrease in the number or
proportion of cells having an abnormal appearance or morphology.
Preferably, after treatment, the number of cells having an abnormal
morphology is reduced by at least 5% relative to its size prior to
treatment; more preferably, reduced by at least 10%; more
preferably, reduced by at least 20%; more preferably, reduced by at
least 30%; more preferably, reduced by at least 40%; more
preferably, reduced by at least 50%; even more preferably, reduced
by at least 50%; and most preferably, reduced by at least 75%. An
abnormal cellular appearance or morphology may be measured by any
reproducible means of measurement. An abnormal cellular morphology
can be measured by microscopy, e.g., using an inverted tissue
culture microscope. An abnormal cellular morphology can take the
form of nuclear pleiomorphism.
[0162] Treating cancer can result in cell death, and preferably,
cell death results in a decrease of at least 10% in number of cells
in a population. More preferably, cell death means a decrease of at
least 20%; more preferably, a decrease of at least 30%; more
preferably, a decrease of at least 40%; more preferably, a decrease
of at least 50%; most preferably, a decrease of at least 75%.
Number of cells in a population may be measured by any reproducible
means. A number of cells in a population can be measured by
fluorescence activated cell sorting (FACS), immunofluorescence
microscopy and light microscopy. Methods of measuring cell death
are as shown in Li et al., Proc Natl Acad Sci USA. 100(5): 2674-8,
2003. In an aspect, cell death occurs by apoptosis.
[0163] As used herein, the term "selectively" means tending to
occur at a higher frequency in one population than in another
population. The compared populations can be cell populations.
Preferably, a compound of the present invention, or a
pharmaceutically acceptable salt, polymorph or solvate thereof,
acts selectively on a cancer or precancerous cell but not on a
normal cell. Preferably, a compound of the present invention, or a
pharmaceutically acceptable salt, polymorph or solvate thereof,
acts selectively to modulate one molecular target (e.g., a target
protein methyltransferase) but does not significantly modulate
another molecular target (e.g., a non-target protein
methyltransferase). The invention also provides a method for
selectively inhibiting the activity of an enzyme, such as a protein
methyltransferase. Preferably, an event occurs selectively in
population A relative to population B if it occurs greater than two
times more frequently in population A as compared to population B.
An event occurs selectively if it occurs greater than five times
more frequently in population A. An event occurs selectively if it
occurs greater than ten times more frequently in population A; more
preferably, greater than fifty times; even more preferably, greater
than 100 times; and most preferably, greater than 1000 times more
frequently in population A as compared to population B. For
example, cell death would be said to occur selectively in cancer
cells if it occurred greater than twice as frequently in cancer
cells as compared to normal cells.
[0164] A compound of the present invention, or a pharmaceutically
acceptable salt, polymorph or solvate thereof, can modulate the
activity of a molecular target (e.g., a target protein
methyltransferase). Modulating refers to stimulating or inhibiting
an activity of a molecular target. Preferably, a compound of the
present invention, or a pharmaceutically acceptable salt, polymorph
or solvate thereof, modulates the activity of a molecular target if
it stimulates or inhibits the activity of the molecular target by
at least 2-fold relative to the activity of the molecular target
under the same conditions but lacking only the presence of said
compound. More preferably, a compound of the present invention, or
a pharmaceutically acceptable salt, polymorph or solvate thereof,
modulates the activity of a molecular target if it stimulates or
inhibits the activity of the molecular target by at least 5-fold,
at least 10-fold, at least 20-fold, at least 50-fold, at least
100-fold relative to the activity of the molecular target under the
same conditions but lacking only the presence of said compound. The
activity of a molecular target may be measured by any reproducible
means. The activity of a molecular target may be measured in vitro
or in vivo. For example, the activity of a molecular target may be
measured in vitro by an enzymatic activity assay or a DNA binding
assay, or the activity of a molecular target may be measured in
vivo by assaying for expression of a reporter gene.
[0165] A compound of the present invention, or a pharmaceutically
acceptable salt, polymorph or solvate thereof, does not
significantly modulate the activity of a molecular target if the
addition of the compound does not stimulate or inhibit the activity
of the molecular target by greater than 10% relative to the
activity of the molecular target under the same conditions but
lacking only the presence of said compound.
[0166] As used herein, the term "isozyme selective" means
preferential inhibition or stimulation of a first isoform of an
enzyme in comparison to a second isoform of an enzyme (e.g.,
preferential inhibition or stimulation of a protein
methyltransferase isozyme alpha in comparison to a protein
methyltransferase isozyme beta). Preferably, a compound of the
present invention, or a pharmaceutically acceptable salt, polymorph
or solvate thereof, demonstrates a minimum of a fourfold
differential, preferably a tenfold differential, more preferably a
fifty fold differential, in the dosage required to achieve a
biological effect. Preferably, a compound of the present invention,
or a pharmaceutically acceptable salt, polymorph or solvate
thereof, demonstrates this differential across the range of
inhibition, and the differential is exemplified at the IC.sub.50,
i.e., a 50% inhibition, for a molecular target of interest.
[0167] Administering a compound of the present invention, or a
pharmaceutically acceptable salt, polymorph or solvate thereof, to
a cell or a subject in need thereof can result in modulation (i.e.,
stimulation or inhibition) of an activity of a protein
methyltransferase of interest.
[0168] Detection of methylation of H3-K27, formation of
trimethylated H3-K27, conversion of monomethylated H3-K27 to
dimethylated H3-K27, or conversion of dimethylated H3-K27 to
trimethylated H3-K27 can be accomplished using any suitable method.
Exemplary methods can be found in US20120071418, the contents of
which are incorporated herein by reference.
[0169] Administering a compound of the present invention, or a
pharmaceutically acceptable salt, polymorph or solvate thereof, to
a cell or a subject in need thereof results in modulation (i.e.,
stimulation or inhibition) of an activity of an intracellular
target (e.g., substrate). Several intracellular targets can be
modulated with the compounds of the present invention, including,
but not limited to, protein methyltrasferase.
[0170] Preferably, an effective amount of a compound of the present
invention, or a pharmaceutically acceptable salt, polymorph or
solvate thereof, is not significantly cytotoxic to normal cells. A
therapeutically effective amount of a compound is not significantly
cytotoxic to normal cells if administration of the compound in a
therapeutically effective amount does not induce cell death in
greater than 10% of normal cells. A therapeutically effective
amount of a compound does not significantly affect the viability of
normal cells if administration of the compound in a therapeutically
effective amount does not induce cell death in greater than 10% of
normal cells. In an aspect, cell death occurs by apoptosis.
[0171] Contacting a cell with a compound of the present invention,
or a pharmaceutically acceptable salt, polymorph or solvate
thereof, can induce or activate cell death selectively in cancer
cells. Administering to a subject in need thereof a compound of the
present invention, or a pharmaceutically acceptable salt, polymorph
or solvate thereof, can induce or activate cell death selectively
in cancer cells. Contacting a cell with a compound of the present
invention, or a pharmaceutically acceptable salt, polymorph or
solvate thereof, can induce cell death selectively in one or more
cells affected by a cell proliferative disorder. Preferably,
administering to a subject in need thereof a compound of the
present invention, or a pharmaceutically acceptable salt, polymorph
or solvate thereof, induces cell death selectively in one or more
cells affected by a cell proliferative disorder.
[0172] One skilled in the art may refer to general reference texts
for detailed descriptions of known techniques discussed herein or
equivalent techniques. These texts include Ausubel et al., Current
Protocols in Molecular Biology, John Wiley and Sons, Inc. (2005);
Sambrook et al., Molecular Cloning, A Laboratory Manual (3.sup.rd
edition), Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
(2000); Coligan et al., Current Protocols in Immunology, John Wiley
& Sons, N.Y.; Enna et al., Current Protocols in Pharmacology,
John Wiley & Sons, N.Y.; Fingl et al., The Pharmacological
Basis of Therapeutics (1975), Remington's Pharmaceutical Sciences,
Mack Publishing Co., Easton, Pa., 18.sup.th edition (1990). These
texts can, of course, also be referred to in making or using an
aspect of the invention.
[0173] A compound (i.e., an EZH2 inhibitor) that can be used in any
methods described herein may have the following Formula I:
##STR00006##
or a pharmaceutically acceptable salt thereof; wherein
[0174] R.sup.701 is H, F, OR.sup.707, NHR.sup.707,
--(C.ident.C)--(CH.sub.2).sub.n7--R.sup.708, phenyl, 5- or
6-membered heteroaryl, C.sub.3-8 cycloalkyl, or 4-7 membered
heterocycloalkyl containing 1-3 heteroatoms, wherein the phenyl, 5-
or 6-membered heteroaryl, C.sub.3-8 cycloalkyl or 4-7 membered
heterocycloalkyl each independently is optionally substituted with
one or more groups selected from halo, C.sub.1-3 alkyl, OH,
O--C.sub.1-6 alkyl, NH--C.sub.1-6 alkyl, and, C.sub.1-3 alkyl
substituted with C.sub.3-8 cycloalkyl or 4-7 membered
heterocycloalkyl containing 1-3 heteroatoms, wherein each of the
O--C.sub.1-6 alkyl and NH--C.sub.1-6 alkyl is optionally
substituted with hydroxyl, O--C.sub.1-3 alkyl or NH--C.sub.1-3
alkyl, each of the O--C.sub.1-3 alkyl and NH--C.sub.1-3 alkyl being
optionally further substituted with O--C.sub.1-3 alkyl or
NH--C.sub.1-3 alkyl;
[0175] each of R.sup.702 and R.sup.703, independently is H, halo,
C.sub.1-4 alkyl, C.sub.1-6 alkoxyl or C.sub.6-C.sub.10 aryloxy,
each optionally substituted with one or more halo;
[0176] each of R.sup.704 and R.sup.705, independently is C.sub.1-4
alkyl;
[0177] R.sup.706 is cyclohexyl substituted by N(C.sub.1-4
alkyl).sub.2 wherein one or both of the C.sub.1-4 alkyl is
substituted with C.sub.1-6 alkoxy; or R.sup.706 is
tetrahydropyranyl;
[0178] R.sup.707 is C.sub.1-4 alkyl optionally substituted with one
or more groups selected from hydroxyl, C.sub.1-4 alkoxy, amino,
mono- or di-C.sub.1-4 alkylamino, C.sub.3-8 cycloalkyl, and 4-7
membered heterocycloalkyl containing 1-3 heteroatoms, wherein the
C.sub.3-8 cycloalkyl or 4-7 membered heterocycloalkyl each
independently is further optionally substituted with C.sub.1-3
alkyl;
[0179] R.sup.708 is C.sub.1-4 alkyl optionally substituted with one
or more groups selected from OH, halo, and C.sub.1-4 alkoxy, 4-7
membered heterocycloalkyl containing 1-3 heteroatoms, or
O--C.sub.1-6 alkyl, wherein the 4-7 membered heterocycloalkyl can
be optionally further substituted with OH or C.sub.1-6 alkyl;
and
[0180] n.sub.7 is 0, 1 or 2.
[0181] For example, R.sup.706 is cyclohexyl substituted by
N(C.sub.1-4 alkyl).sub.2 wherein one of the C.sub.1-4 alkyl is
unsubstituted and the other is substituted with methoxy.
[0182] For example, R.sup.706 is
##STR00007##
[0183] For example, the compound is of Formula II:
##STR00008##
[0184] For example, R.sup.702 is methyl or isopropyl and R.sup.703
is methyl or methoxyl.
[0185] For example, R.sup.704 is methyl.
[0186] For example, R.sup.701 is OR.sup.707 and R.sup.707 is
C.sub.1-3 alkyl optionally substituted with OCH.sub.3 or
morpholine.
[0187] For example, R.sup.701 is H or F.
[0188] For example, R.sup.701 is tetrahydropyranyl, phenyl,
pyridyl, pyrimidyl, pyrazinyl, imidazolyl, or pyrazolyl, each of
which is optionally substituted with methyl, methoxy, ethyl
substituted with morpholine, or --OCH.sub.2CH.sub.2OCH.sub.3.
[0189] For example, R.sup.708 is morpholine, piperidine,
piperazine, pyrrolidine, diazepane, or azetidine, each of which is
optionally substituted with OH or C.sub.1-6 alkyl.
[0190] For example, R.sup.708 is morpholine
[0191] For example, R.sup.708 is piperazine substituted with
C.sub.1-6 alkyl.
[0192] For example, R.sup.708 is methyl, t-butyl or
C(CH.sub.3).sub.2OH.
[0193] A compound (i.e., an EZH2 inhibitor) that can be used in any
methods described herein may have the following Formula III:
##STR00009##
or a pharmaceutically acceptable salt thereof.
[0194] In this formula:
[0195] R.sup.801 is C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.3-8 cycloalkyl, 4-7 membered heterocycloalkyl
containing 1-3 heteroatoms, phenyl or 5- or 6-membered heteroaryl,
each of which is substituted with O--C.sub.1-6 alkyl-R.sub.x or
NH--C.sub.1-6 alkyl-R.sub.x, wherein R.sub.x is hydroxyl,
O--C.sub.1-3 alkyl or NH--C.sub.1-3 alkyl, and R.sub.x is
optionally further substituted with O--C.sub.1-3 alkyl or
NH--C.sub.1-3 alkyl except when R.sub.x is hydroxyl; or R.sup.801
is phenyl substituted with -Q.sub.2-T.sub.2, wherein Q.sub.2 is a
bond or C.sub.1-C.sub.3 alkyl linker optionally substituted with
halo, cyano, hydroxyl or C.sub.1-C.sub.6 alkoxy, and T.sub.2 is
optionally substituted 4- to 12-membered heterocycloalkyl; and
R.sup.801 is optionally further substituted;
[0196] each of R.sup.802 and R.sup.803, independently is H, halo,
C.sub.1-4 alkyl, C.sub.1-6 alkoxyl or C.sub.6-C.sub.10 aryloxy,
each optionally substituted with one or more halo;
[0197] each of R.sup.804 and R.sup.805, independently is C.sub.1-4
alkyl; and R.sup.806 is -Q.sub.x-T.sub.x, wherein Q.sub.x is a bond
or C.sub.1-4 alkyl linker, T.sub.x is H, optionally substituted
C.sub.1-4 alkyl, optionally substituted C.sub.3-C.sub.8 cycloalkyl
or optionally substituted 4- to 14-membered heterocycloalkyl.
[0198] For example, each of Q.sub.x and Q.sub.2 independently is a
bond or methyl linker, and each of T.sub.x and T.sub.2
independently is tetrahydropyranyl, piperidinyl substituted by 1,
2, or 3 C.sub.1-4 alkyl groups, or cyclohexyl substituted by
N(C.sub.1-4 alkyl).sub.2 wherein one or both of the C.sub.1-4 alkyl
is optionally substituted with C.sub.1-6 alkoxy;
[0199] For example, R.sup.806 is cyclohexyl substituted by
N(C.sub.1-4 alkyl).sub.2 or R.sup.806 is tetrahydropyranyl.
##STR00010##
[0200] For example, R.sup.806 is
[0201] For example, R.sup.801 is phenyl or 5- or 6-membered
heteroaryl substituted with O--C.sub.1-6 alkyl-R.sub.x, or
R.sup.801 is phenylsubstituted with CH.sub.2-tetrahydropyranyl.
[0202] For example, a compound of the present invention is of
Formula IVa or IVb:
##STR00011##
wherein Z' is CH or N, and R.sup.807 is C.sub.2-3
alkyl-R.sub.x.
[0203] For example, R.sup.807 is --CH.sub.2CH.sub.2OH,
--CH.sub.2CH.sub.2OCH.sub.3, or
--CH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3.
[0204] For example, R.sup.802 is methyl or isopropyl and R.sup.803
is methyl or methoxyl.
[0205] For example, R.sup.804 is methyl.
[0206] A compound of the present invention may have the following
Formula (V):
##STR00012##
or a pharmaceutically acceptable salt or ester thereof.
[0207] In this formula:
[0208] R.sub.2, R.sub.4 and R.sub.12 are each independently
C.sub.1-6 alkyl;
[0209] R.sub.6 is C.sub.6-C.sub.10 aryl or 5- or 6-membered
heteroaryl, each of which is optionally substituted with one or
more -Q.sub.2-T.sub.2, wherein Q.sub.2 is a bond or C.sub.1-C.sub.3
alkyl linker optionally substituted with halo, cyano, hydroxyl or
C.sub.1-C.sub.6 alkoxy, and T.sub.2 is H, halo, cyano, --OR.sub.a,
--NR.sub.aR.sub.b, --(NR.sub.aR.sub.bR.sub.c).sup.+A.sup.-,
--C(O)R.sub.a, --C(O)OR.sub.a, --C(O)NR.sub.aR.sub.b,
--NR.sub.bC(O)R.sub.a, --NR.sub.bC(O)OR.sub.a, --S(O).sub.2R.sub.a,
--S(O).sub.2NR.sub.aR.sub.b, or R.sub.S2, in which each of R.sub.a,
R.sub.b, and R.sub.c, independently is H or R.sub.S3, A.sup.- is a
pharmaceutically acceptable anion, each of R.sub.S2 and R.sub.S3,
independently, is C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered
heterocycloalkyl, or 5- or 6-membered heteroaryl, or R.sub.a and
R.sub.b, together with the N atom to which they are attached, form
a 4 to 12-membered heterocycloalkyl ring having 0 or 1 additional
heteroatom, and each of R.sub.S2, R.sub.S3, and the 4 to
12-membered heterocycloalkyl ring formed by R.sub.a and R.sub.b, is
optionally substituted with one or more -Q.sub.3-T.sub.3, wherein
Q.sub.3 is a bond or C.sub.1-C.sub.3 alkyl linker each optionally
substituted with halo, cyano, hydroxyl or C.sub.1-C.sub.6 alkoxy,
and T.sub.3 is selected from the group consisting of halo, cyano,
C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10
aryl, 4 to 12-membered heterocycloalkyl, 5- or 6-membered
heteroaryl, OR.sub.d, COOR.sub.d, --S(O).sub.2R.sub.a,
--NR.sub.dR.sub.e, and --C(O)NR.sub.dR.sub.e, each of R.sub.d and
R.sub.e independently being H or C.sub.1-C.sub.6 alkyl, or
-Q.sub.3-T.sub.3 is oxo; or any two neighboring -Q.sub.2-T.sub.2,
together with the atoms to which they are attached form a 5- or
6-membered ring optionally containing 1-4 heteroatoms selected from
N, O and S and optionally substituted with one or more substituents
selected from the group consisting of halo, hydroxyl, COOH,
C(O)O--C.sub.1-C.sub.6 alkyl, cyano, C.sub.1-C.sub.6 alkoxyl,
amino, mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6
alkylamino, C.sub.3-C.sub.6 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to
12-membered heterocycloalkyl, and 5- or 6-membered heteroaryl;
[0210] R.sub.7 is -Q.sub.4-T.sub.4, in which Q.sub.4 is a bond,
C.sub.1-C.sub.4 alkyl linker, or C.sub.2-C.sub.4 alkenyl linker,
each linker optionally substituted with halo, cyano, hydroxyl or
C.sub.1-C.sub.6 alkoxy, and T.sub.4 is H, halo, cyano,
NR.sub.fR.sub.g, --OR.sub.f, --C(O)R.sub.f, --C(O)OR.sub.f,
--C(O)NR.sub.fR.sub.g, --C(O)NR.sub.fOR.sub.g,
--NR.sub.fC(O)R.sub.g, --S(O).sub.2R.sub.f, or R.sub.S4, in which
each of R.sub.f and R.sub.g, independently is H or R.sub.S5, each
of R.sub.S4 and R.sub.S5, independently is C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.5
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered
heterocycloalkyl, or 5- or 6-membered heteroaryl, and each of
R.sub.S4 and R.sub.S5 is optionally substituted with one or more
-Q.sub.5-T.sub.5, wherein Q.sub.5 is a bond, C(O), C(O)NR.sub.k,
NR.sub.kC(O), S(O).sub.2, or C.sub.1-C.sub.3 alkyl linker, R.sub.k
being H or C.sub.1-C.sub.6 alkyl, and T.sub.5 is H, halo,
C.sub.1-C.sub.6 alkyl, hydroxyl, cyano, C.sub.1-C.sub.6 alkoxyl,
amino, mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6
alkylamino, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to
12-membered heterocycloalkyl, 5- or 6-membered heteroaryl, or
S(O).sub.qR.sub.q in which q is 0, 1, or 2 and R.sub.q is
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, C.sub.3-C.sub.5 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to
12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl, and
T.sub.5 is optionally substituted with one or more substituents
selected from the group consisting of halo, C.sub.1-C.sub.6 alkyl,
hydroxyl, cyano, C.sub.1-C.sub.6 alkoxyl, amino,
mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6 alkylamino,
C.sub.3-C.sub.5 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered
heterocycloalkyl, and 5- or 6-membered heteroaryl except when
T.sub.5 is H, halo, hydroxyl, or cyano; or -Q.sub.5-T.sub.5 is oxo;
and
[0211] R.sub.8 is H, halo, hydroxyl, COOH, cyano, R.sub.S6,
OR.sub.S6, or COOR.sub.S6, in which R.sub.S6 is C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl,
C.sub.3-C.sub.8 cycloalkyl, 4 to 12-membered heterocycloalkyl,
amino, mono-C.sub.1-C.sub.6 alkylamino, or di-C.sub.1-C.sub.6
alkylamino, and R.sub.S6 is optionally substituted with one or more
substituents selected from the group consisting of halo, hydroxyl,
COOH, C(O)O--C.sub.1-C.sub.6 alkyl, cyano, C.sub.1-C.sub.6 alkoxyl,
amino, mono-C.sub.1-C.sub.6 alkylamino, and di-C.sub.1-C.sub.6
alkylamino; or R.sub.7 and R.sub.8, together with the N atom to
which they are attached, form a 4 to 11-membered heterocycloalkyl
ring having 0 to 2 additional heteroatoms, and the 4 to 11-membered
heterocycloalkyl ring formed by R7 and R8 is optionally substituted
with one or more -Q.sub.6-T.sub.6, wherein Q.sub.6 is a bond, C(O),
C(O)NR.sub.m, NR.sub.mC(O), S(O).sub.2, or C.sub.1-C.sub.3 alkyl
linker, R.sub.m being H or C.sub.1-C.sub.6 alkyl, and T.sub.6 is H,
halo, C.sub.1-C.sub.6 alkyl, hydroxyl, cyano, C.sub.1-C.sub.6
alkoxyl, amino, mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6
alkylamino, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to
12-membered heterocycloalkyl, 5- or 6-membered heteroaryl, or
S(O).sub.pR.sub.p in which p is 0, 1, or 2 and R.sub.p is
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to
12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl, and
T6 is optionally substituted with one or more substituents selected
from the group consisting of halo, C.sub.1-C.sub.6 alkyl, hydroxyl,
cyano, C.sub.1-C.sub.6 alkoxyl, amino, mono-C.sub.1-C.sub.6
alkylamino, di-C.sub.1-C.sub.6 alkylamino, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered
heterocycloalkyl, and 5- or 6-membered heteroaryl except when
T.sub.6 is H, halo, hydroxyl, or cyano; or -Q.sub.6-T.sub.6 is
oxo.
[0212] For example, R.sub.6 is C.sub.6-C.sub.10 aryl or 5- or
6-membered heteroaryl, each of which is optionally, independently
substituted with one or more -Q.sub.2-T.sub.2, wherein Q.sub.2 is a
bond or C.sub.1-C.sub.3 alkyl linker, and T.sub.2 is H, halo,
cyano, --OR.sub.a, --NR.sub.aR.sub.b,
--(NR.sub.aR.sub.bR.sub.c).sup.+A.sup.-, --C(O)NR.sub.aR.sub.b,
--NR.sub.bC(O)R.sub.a, --S(O).sub.2R.sub.a, or R.sub.S2, in which
each of R.sub.a and R.sub.b, independently is H or R.sub.S3, each
of R.sub.S2 and R.sub.S3, independently, is C.sub.1-C.sub.6 alkyl,
or R.sub.a and R.sub.b, together with the N atom to which they are
attached, form a 4 to 7-membered heterocycloalkyl ring having 0 or
1 additional heteroatom, and each of R.sub.S2, R.sub.S3, and the 4
to 7-membered heterocycloalkyl ring formed by R.sub.a and R.sub.b,
is optionally, independently substituted with one or more
-Q.sub.3-T.sub.3, wherein Q.sub.3 is a bond or C.sub.1-C.sub.3
alkyl linker and T.sub.3 is selected from the group consisting of
halo, C.sub.1-C.sub.6 alkyl, 4 to 7-membered heterocycloalkyl,
OR.sub.d, --S(O).sub.2R.sub.d, and --NR.sub.dR.sub.e, each of
R.sub.d and R.sub.e independently being H or C.sub.1-C.sub.6 alkyl,
or -Q.sub.3-T.sub.3 is oxo; or any two neighboring
-Q.sub.2-T.sub.2, together with the atoms to which they are
attached form a 5- or 6-membered ring optionally containing 1-4
heteroatoms selected from N, O and S.
[0213] For example, the compound of the present invention is of
Formula (VI):
##STR00013##
or a pharmaceutically acceptable salt thereof, wherein Q.sub.2 is a
bond or methyl linker, T.sub.2 is H, halo, --OR.sub.a,
--NR.sub.aR.sub.b, --(NR.sub.aR.sub.bR.sub.c).sup.+A.sup.-, or
--S(O).sub.2NR.sub.aR.sub.b, R.sub.7 is piperidinyl,
tetrahydropyran, cyclopentyl, or cyclohexyl, each optionally
substituted with one -Q.sub.5-T.sub.5 and R.sub.8 is ethyl.
[0214] A compound of the present invention may have the following
Formula (VIa):
##STR00014##
wherein
[0215] each of R.sub.a and R.sub.b, independently is H or R.sub.S3,
R.sub.S3 being C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.5 cycloalkyl,
C.sub.6-C.sub.10 aryl, 4 to 12-membered heterocycloalkyl, or 5- or
6-membered heteroaryl, or R.sub.a and R.sub.b, together with the N
atom to which they are attached, form a 4 to 12-membered
heterocycloalkyl ring having 0 or 1 additional heteroatom, and each
of R.sub.S3 and the 4 to 12-membered heterocycloalkyl ring formed
by R.sub.a and R.sub.b, is optionally substituted with one or more
-Q.sub.3-T.sub.3, wherein Q.sub.3 is a bond or C.sub.1-C.sub.3
alkyl linker each optionally substituted with halo, cyano, hydroxyl
or C.sub.1-C.sub.6 alkoxy, and T.sub.3 is selected from the group
consisting of halo, cyano, C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered
heterocycloalkyl, 5- or 6-membered heteroaryl, OR.sub.d,
COOR.sub.d, --S(O).sub.2R.sub.d, --NR.sub.dR.sub.e, and
--C(O)NR.sub.dR.sub.e, each of R.sub.d and R.sub.e independently
being H or C.sub.1-C.sub.6 alkyl, or -Q.sub.3-T.sub.3 is oxo;
[0216] R.sub.7 is -Q.sub.4-T.sub.4, in which Q.sub.4 is a bond,
C.sub.1-C.sub.4 alkyl linker, or C.sub.2-C.sub.4 alkenyl linker,
each linker optionally substituted with halo, cyano, hydroxyl or
C.sub.1-C.sub.6 alkoxy, and T.sub.4 is H, halo, cyano,
NR.sub.fR.sub.g, --OR.sub.f, --C(O)R.sub.f, --C(O)OR.sub.f,
--C(O)NR.sub.fR.sub.g, --C(O)NR.sub.fOR.sub.g,
--NR.sub.fC(O)R.sub.g, --S(O).sub.2R.sub.f, or R.sub.S4, in which
each of R.sub.f and R.sub.g, independently is H or R.sub.S5, each
of R.sub.S4 and R.sub.S5, independently is C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 7-membered
heterocycloalkyl, or 5- or 6-membered heteroaryl, and each of
R.sub.S4 and R.sub.S5 is optionally substituted with one or more
-Q.sub.5-T.sub.5, wherein Q.sub.5 is a bond, C(O), C(O)NR.sub.k,
NR.sub.kC(O), S(O).sub.2, or C.sub.1-C.sub.3 alkyl linker, R.sub.k
being H or C.sub.1-C.sub.6 alkyl, and T.sub.5 is H, halo,
C.sub.1-C.sub.6 alkyl, hydroxyl, cyano, C.sub.1-C.sub.6 alkoxyl,
amino, mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6
alkylamino, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to
7-membered heterocycloalkyl, 5- or 6-membered heteroaryl, or
S(O).sub.qR.sub.q in which q is 0, 1, or 2 and R.sub.q is
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to
7-membered heterocycloalkyl, or 5- or 6-membered heteroaryl, and
T.sub.5 is optionally substituted with one or more substituents
selected from the group consisting of halo, C.sub.1-C.sub.6 alkyl,
hydroxyl, cyano, C.sub.1-C.sub.6 alkoxyl, amino,
mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6 alkylamino,
C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 7-membered
heterocycloalkyl, and 5- or 6-membered heteroaryl except when
T.sub.5 is H, halo, hydroxyl, or cyano; or -Q.sub.5-T.sub.5 is oxo;
provided that R.sub.7 is not H; and
[0217] R.sub.8 is H, halo, hydroxyl, COOH, cyano, R.sub.S6,
OR.sub.S6, or COOR.sub.S6, in which R.sub.S6 is C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, amino,
mono-C.sub.1-C.sub.6 alkylamino, or di-C.sub.1-C.sub.6 alkylamino,
and R.sub.S6 is optionally substituted with one or more
substituents selected from the group consisting of halo, hydroxyl,
COOH, C(O)O--C.sub.1-C.sub.6 alkyl, cyano, C.sub.1-C.sub.6 alkoxyl,
amino, mono-C.sub.1-C.sub.6 alkylamino, and di-C.sub.1-C.sub.6
alkylamino; or R.sub.7 and R.sub.8, together with the N atom to
which they are attached, form a 4 to 11-membered heterocycloalkyl
ring which has 0 to 2 additional heteroatoms and is optionally
substituted with one or more -Q.sub.6-T.sub.6, wherein Q.sub.6 is a
bond, C(O), C(O)NR.sub.m, NR.sub.mC(O), S(O).sub.2, or
C.sub.1-C.sub.3 alkyl linker, R.sub.m being H or C.sub.1-C.sub.6
alkyl, and T.sub.6 is H, halo, C.sub.1-C.sub.6 alkyl, hydroxyl,
cyano, C.sub.1-C.sub.6 alkoxyl, amino, mono-C.sub.1-C.sub.6
alkylamino, di-C.sub.1-C.sub.6 alkylamino, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 7-membered
heterocycloalkyl, 5- or 6-membered heteroaryl, or S(O).sub.pR.sub.p
in which p is 0, 1, or 2 and R.sub.p is C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.5
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 7-membered
heterocycloalkyl, or 5- or 6-membered heteroaryl, and T6 is
optionally substituted with one or more substituents selected from
the group consisting of halo, C.sub.1-C.sub.6 alkyl, hydroxyl,
cyano, C.sub.1-C.sub.6 alkoxyl, amino, mono-C.sub.1-C.sub.6
alkylamino, di-C.sub.1-C.sub.6 alkylamino, C.sub.3-C.sub.5
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 7-membered
heterocycloalkyl, and 5- or 6-membered heteroaryl except when
T.sub.6 is H, halo, hydroxyl, or cyano; or -Q.sub.6-T.sub.6 is
oxo.
[0218] For example, R.sub.a and R.sub.b, together with the N atom
to which they are attached, form a 4 to 7-membered heterocycloalkyl
ring having 0 or 1 additional heteroatoms to the N atom and the
ring is optionally substituted with one or more -Q.sub.3-T.sub.3,
wherein the heterocycloalkyl is azetidinyl, pyrrolidinyl,
imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl,
triazolidinyl, piperidinyl, 1,2,3,6-tetrahydropyridinyl,
piperazinyl, or morpholinyl.
[0219] For example, R.sub.7 is C.sub.3-C.sub.5 cycloalkyl or 4 to
7-membered heterocycloalkyl, each optionally substituted with one
or more -Q.sub.5-T.sub.5.
[0220] For example, R.sub.7 is piperidinyl, tetrahydropyran,
tetrahydro-2H-thiopyranyl, cyclopentyl, cyclohexyl, pyrrolidinyl,
or cycloheptyl, each optionally substituted with one or more
-Q.sub.5-T.sub.5.
[0221] For example, R.sub.8 is H or C.sub.1-C.sub.6 alkyl which is
optionally substituted with one or more substituents selected from
the group consisting of halo, hydroxyl, COOH,
C(O)O--C.sub.1-C.sub.6 alkyl, cyano, C.sub.1-C.sub.6 alkoxyl,
amino, mono-C.sub.1-C.sub.6 alkylamino, and di-C.sub.1-C.sub.6
alkylamino.
[0222] In some embodiments, a compound that can be used in any
methods presented here is:
##STR00015##
stereoisomers thereof or pharmaceutically acceptable salt or
solvate thereof
[0223] In some embodiments, a compound that can be used in any
methods presented here is:
##STR00016## ##STR00017##
stereoisomers thereof or pharmaceutically acceptable salts and
solvates thereof.
[0224] In some embodiments, a compound that can be used in any
methods presented here is:
##STR00018##
stereoisomers thereof or pharmaceutically acceptable salts and
solvates thereof.
[0225] In some embodiments, the compounds suitable for use in the
method of this invention include compounds of Formula (VII):
##STR00019##
wherein,
[0226] V.sup.1 is N or CR.sup.7,
[0227] V.sup.2 is N or CR.sup.2, provided when V.sup.1 is N,
V.sup.2 is N,
[0228] X and Z are selected independently from the group consisting
of hydrogen, (C.sub.1-C.sub.8)alkyl, (C.sub.2-C.sub.8)alkenyl,
(C.sub.2-C.sub.8)alkynyl, unsubstituted or substituted
(C.sub.3-C.sub.8)cycloalkyl, unsubstituted or substituted
(C.sub.3-C.sub.8)cycloalkyl-(C.sub.1-C.sub.8)alkyl or
--(C.sub.2-C.sub.8)alkenyl, unsubstituted or substituted
(C.sub.5-C.sub.8)cycloalkenyl, unsubstituted or substituted
(C.sub.5-C.sub.8)cycloalkenyl-(C.sub.1-C.sub.8)alkyl or
--(C.sub.2-C.sub.8)alkenyl, (C.sub.6-C.sub.10)bicycloalkyl,
unsubstituted or substituted heterocycloalkyl, unsubstituted or
substituted heterocycloalkyl-(C.sub.1-C.sub.8)alkyl or
--(C.sub.2-C.sub.8)alkenyl, unsubstituted or substituted aryl,
unsubstituted or substituted aryl-(C.sub.1-C.sub.8)alkyl or
--(C.sub.2-C.sub.8)alkenyl, unsubstituted or substituted
heteroaryl, unsubstituted or substituted
heteroaryl-(C.sub.1-C.sub.8)alkyl or --(C.sub.2-C.sub.8)alkenyl,
halo, cyano,
--COR.sup.a, --CO.sub.2R.sup.a, --CONR.sup.aR.sup.b,
--CONR.sup.aNR.sup.aR.sup.b, --SR.sup.a, --SOR.sup.a,
--SO.sub.2R.sup.a, --SO.sub.2NR.sup.aR.sup.b, nitro,
--NR.sup.aR.sup.b, --NR.sup.aC(O)R.sup.b,
--NR.sup.aC(O)NR.sup.aR.sup.b, --NR.sup.aC(O)OR.sup.a,
--NR.sup.aSO.sub.2R.sup.b, --NR.sup.aSO.sub.2NR.sup.aR.sup.b,
--NR.sup.aNR.sup.aR.sup.b, --NR.sup.aNR.sup.aC(O)R.sup.b,
--NR.sup.aNR.sup.aC(O)NR.sup.aR.sup.b,
--NR.sup.aNR.sup.aC(O)OR.sup.a, --OR.sup.a, --OC(O)R.sup.a, and
--OC(O)NR.sup.aR.sup.b;
[0229] Y is H or halo;
[0230] R.sup.1 is (C.sub.1-C.sub.8)alkyl, (C.sub.2-C.sub.8)alkenyl,
(C.sub.2-C.sub.8)alkynyl, unsubstituted or substituted
(C.sub.3-C.sub.8)cycloalkyl, unsubstituted or substituted
(C.sub.3-C.sub.8)cycloalkyl-(C.sub.1-C.sub.8)alkyl or
--(C.sub.2-C.sub.8)alkenyl, unsubstituted or substituted
(C.sub.5-C.sub.8)cycloalkenyl, unsubstituted or substituted
(C.sub.5-C.sub.8)cycloalkenyl-(C.sub.1-C.sub.8)alkyl or
--(C.sub.2-C.sub.8)alkenyl, unsubstituted or substituted
(C.sub.6-C.sub.10)bicycloalkyl, unsubstituted or substituted
heterocycloalkyl or --(C.sub.2-C.sub.8)alkenyl, unsubstituted or
substituted heterocycloalkyl-(C.sub.1-C.sub.8)alkyl, unsubstituted
or substituted aryl, unsubstituted or substituted
aryl-(C.sub.1-C.sub.8)alkyl or --(C.sub.2-C.sub.8)alkenyl,
unsubstituted or substituted heteroaryl, unsubstituted or
substituted heteroaryl-(C.sub.1-C.sub.8)alkyl or
--(C.sub.2-C.sub.8)alkenyl, --COR.sup.a, --CO.sub.2R.sup.a,
--CONR.sup.aR.sup.b, --CONR.sup.aNR.sup.aR.sup.b;
[0231] R.sup.2 is hydrogen, (C.sub.1-C.sub.8)alkyl,
trifluoromethyl, alkoxy, or halo, in which said
(C.sub.1-C.sub.8)alkyl is optionally substituted with one to two
groups selected from amino and (C.sub.1-C.sub.3)alkylamino;
[0232] R.sup.7 is hydrogen, (C.sub.1-C.sub.3)alkyl, or alkoxy;
[0233] R.sup.3 is hydrogen, (C.sub.1-C.sub.8)alkyl, cyano,
trifluoromethyl, --NR.sup.aR.sup.b, or halo;
[0234] R.sup.6 is selected from the group consisting of hydrogen,
halo, (C.sub.1-C.sub.8)alkyl, (C.sub.2-C.sub.8)alkenyl,
(C.sub.2-C.sub.8)alkynyl, unsubstituted or substituted
(C.sub.3-C.sub.8)cycloalkyl, unsubstituted or substituted
(C.sub.3-C.sub.8)cycloalkyl-(C.sub.1-C.sub.8)alkyl, unsubstituted
or substituted (C.sub.5-C.sub.8)cycloalkenyl, unsubstituted or
substituted (C.sub.5-C.sub.8)cycloalkenyl(C.sub.1-C.sub.8)alkyl,
(C.sub.6-C.sub.10)bicycloalkyl, unsubstituted or substituted
heterocycloalkyl, unsubstituted or substituted
heterocycloalkyl-(C.sub.1-C.sub.8)alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted
aryl-(C.sub.1-C.sub.8)alkyl, unsubstituted or substituted
heteroaryl, unsubstituted or substituted
heteroaryl-(C.sub.1-C.sub.8)alkyl, cyano, --COR.sup.a,
--CO.sub.2R.sup.a,
--CONR.sup.aR.sup.b, --CONR.sup.aNR.sup.aR.sup.b, --SR.sup.a,
--SOR.sup.a, --SO.sub.2R.sup.a, --SO.sub.2NR.sup.aR.sup.b, nitro,
--NR.sup.aR.sup.b, --NR.sup.aC(O)R.sup.b,
--NR.sup.aC(O)NR.sup.aR.sup.b, --NR.sup.aC(O)OR.sup.a,
--NR.sup.aSO.sub.2R.sup.b, --NR.sup.aSO.sub.2NR.sup.aR.sup.b,
--NR.sup.aNR.sup.aR.sup.b, --NR.sup.aNR.sup.aC(O)R.sup.b,
--NR.sup.aNR.sup.aC(O)NR.sup.aR.sup.b, --NR.sup.aC(O)OR.sup.a,
--NR.sup.aSO.sub.2R.sup.b, --NR.sup.aSO.sub.2NR.sup.aR.sup.b,
--NR.sup.aNR.sup.aR.sup.b, --NR.sup.aNR.sup.aC(O)R.sup.b,
--NR.sup.aNR.sup.aC(O)NR.sup.aR.sup.b,
--NR.sup.aNR.sup.aC(O)OR.sup.a, --OR.sup.a, --OC(O)R.sup.a,
--OC(O)NR.sup.aR.sup.b;
[0235] wherein any (C.sub.1-C.sub.8)alkyl,
(C.sub.2-C.sub.8)alkenyl, (C.sub.2-C.sub.8)alkynyl, cycloalkyl,
cycloalkenyl, bicycloalkyl, heterocycloalkyl, aryl, or heteroaryl
group is optionally substituted by 1, 2 or 3 groups independently
selected from the group consisting of
--O(C.sub.1-C.sub.6)alkyl(R.sup.c).sub.1-2,
--S(C.sub.1-C.sub.6)alkyl(R.sup.c).sub.1-2,
--(C.sub.1-C.sub.6)alkyl(R.sup.c).sub.1-2,
--(C.sub.1-C.sub.8)alkyl-heterocycloalkyl,
(C.sub.3-C.sub.8)cycloalkyl-heterocycloalkyl, halo,
(C.sub.1-C.sub.6)alkyl, (C.sub.3-C.sub.8)cycloalkyl,
(C.sub.5-C.sub.8)cycloalkenyl, (C.sub.1-C.sub.6)haloalkyl, cyano,
--COR.sup.a, --CO.sub.2R.sup.a, --CONR.sup.aR.sup.b, --SR.sup.a,
--SOR.sup.a, --SO.sub.2R.sup.a, --SO.sub.2NR.sup.aR.sup.b, nitro,
--NR.sup.aR.sup.b, --NR.sup.aC(O)R.sup.b,
--NR.sup.aC(O)NR.sup.aR.sup.b, --NR.sup.aC(O)OR.sup.a,
--NR.sup.aSO.sub.2R.sup.b, --NR.sup.aSO.sub.2NR.sup.aR.sup.b,
--OR.sup.a, --OC(O)R.sup.a, OC(O)NR.sup.aR.sup.b, heterocycloalkyl,
aryl, heteroaryl, aryl(C.sub.1-C.sub.4)alkyl, and
heteroaryl(C.sub.1-C.sub.4)alkyl; [0236] wherein any aryl or
heteroaryl moiety of said aryl, heteroaryl,
aryl(C.sub.1-C.sub.4)alkyl, or heteroaryl(C.sub.1-C.sub.4)alkyl is
optionally substituted by 1, 2 or 3 groups independently selected
from the group consisting of halo, (C.sub.1-C.sub.6)alkyl,
(C.sub.3-C.sub.8)cycloalkyl, (C.sub.5-C.sub.8)cycloalkenyl,
(C.sub.1-C.sub.6)haloalkyl, cyano, --COR.sup.a, --CO.sub.2R.sup.a,
--CONR.sup.aR.sup.b, --SR.sup.a, [0237] --SOR.sup.a,
--SO.sub.2R.sup.a, --SO.sub.2NR.sup.aR.sup.b, nitro,
--NR.sup.aR.sup.b, --NR.sup.aC(O)R.sup.b,
--NR.sup.aC(O)NR.sup.aR.sup.b, --NR.sup.aC(O)OR.sup.a,
--NR.sup.aSO.sub.2R.sup.b, --NR.sup.aSO.sub.2NR.sup.aR.sup.b,
--OR.sup.a, --OC(O)R.sup.a, and --OC(O)NR.sup.aR.sup.b;
[0238] R.sup.a and R.sup.b are each independently hydrogen,
(C.sub.1-C.sub.8)alkyl, (C.sub.2-C.sub.8)alkenyl,
(C.sub.2-C.sub.8)alkynyl, (C.sub.3-C.sub.8)cycloalkyl,
(C.sub.5-C.sub.8)cycloalkenyl, (C.sub.6-C.sub.10)bicycloalkyl,
heterocycloalkyl, aryl, or heteroaryl, wherein said
(C.sub.1-C.sub.8)alkyl, (C.sub.2-C.sub.8)alkenyl,
(C.sub.2-C.sub.8)alkynyl, cycloalkyl, cycloalkenyl, bicycloalkyl,
heterocycloalkyl, aryl or heteroaryl group is optionally
substituted by 1, 2 or 3 groups independently selected from halo,
hydroxyl, (C.sub.1-C.sub.4)alkoxy, amino,
(C.sub.1-C.sub.4)alkylamino,
((C.sub.1-C.sub.4)alkyl)((C.sub.1-C.sub.4)alkyl)amino, --CO.sub.2H,
--CO.sub.2(C.sub.1-C.sub.4)alkyl, --CONH.sub.2,
--CONH(C.sub.1-C.sub.4)alkyl,
--CON((C.sub.1-C.sub.4)alkyl)((C.sub.1-C.sub.4)alkyl),
--SO.sub.2(C.sub.1-C.sub.4)alkyl, --SO.sub.2NH.sub.2,
--SO.sub.2NH(C.sub.1-C.sub.4)alkyl, and
SO.sub.2N((C.sub.1-C.sub.4)alkyl)((C.sub.1-C.sub.4)alkyl);
[0239] or R.sup.a and R.sup.b taken together with the nitrogen to
which they are attached represent a 5-8 membered saturated or
unsaturated ring, optionally containing an additional heteroatom
selected from oxygen, nitrogen, and sulfur, wherein said ring is
optionally substituted by 1, 2 or 3 groups independently selected
from (C.sub.1-C.sub.4)alkyl, (C.sub.1-C.sub.4)haloalkyl, amino,
(C.sub.1-C.sub.4)alkylamino,
((C.sub.1-C.sub.4)alkyl)((C.sub.1-C.sub.4)alkyl)amino, hydroxyl,
oxo, (C.sub.1-C.sub.4)alkoxy, and
(C.sub.1-C.sub.4)alkoxy(C.sub.1-C.sub.4)alkyl, wherein said ring is
optionally fused to a (C.sub.3-C.sub.8)cycloalkyl,
heterocycloalkyl, aryl, or heteroaryl ring;
[0240] or R.sup.a and R.sup.b taken together with the nitrogen to
which they are attached represent a 6- to 10-membered bridged
bicyclic ring system optionally fused to a
(C.sub.3-C.sub.8)cycloalkyl, heterocycloalkyl, aryl, or heteroaryl
ring;
[0241] each R.sup.c is independently (C.sub.1-C.sub.4)alkylamino,
--NR.sup.aSO.sub.2R.sup.b, --SOR.sup.a, --SO.sub.2R.sup.a,
--NR.sup.aC(O)OR.sup.a, --NR.sup.aR.sup.b, or
--CO.sub.2R.sup.a;
[0242] or a salt thereof.
[0243] Subgroups of the compounds encompassed by the general
structure of Formula (I) are represented as follows:
[0244] Subgroup A of Formula (VII)
[0245] X and Z are selected from the group consisting of
(C.sub.1-C.sub.8)alkyl, (C.sub.3-C.sub.8)cycloalkyl,
heterocycloalkyl, aryl, heteroaryl, --NR.sup.aR.sup.b, and
--OR.sup.a;
[0246] Y is H or F;
[0247] R.sup.1 is selected from the group consisting of
(C.sub.1-C.sub.8)alkyl, (C.sub.3-C.sub.8)cycloalkyl,
heterocycloalkyl, aryl, and heteroaryl;
[0248] R.sup.2 is hydrogen, (C.sub.1-C.sub.8)alkyl,
trifluoromethyl, alkoxy, or halo, in which said
(C.sub.1-C.sub.8)alkyl is optionally substituted with one to two
groups selected from amino and (C.sub.1-C.sub.3)alkylamino;
[0249] R.sup.7 is hydrogen, (C.sub.1-C.sub.3)alkyl, or alkoxy;
[0250] R.sup.3 is selected from the group consisting of hydrogen,
(C.sub.1-C.sub.8)alkyl, cyano, trifluoromethyl, --NR.sup.aR.sup.b,
and halo;
[0251] R.sup.6 is selected from the group consisting of hydrogen,
halo, cyano, trifluoromethyl, amino, (C.sub.1-C.sub.8)alkyl,
(C.sub.3-C.sub.8)cycloalkyl, aryl, heteroaryl, acylamino;
(C.sub.2-C.sub.8)alkynyl, arylalkynyl, heteroarylalkynyl;
--SO.sub.2R.sup.a; --SO.sub.2NR.sup.aR.sup.b and
--NR.sup.aSO.sub.2R.sup.b; [0252] wherein any
(C.sub.1-C.sub.8)alkyl, (C.sub.3-C.sub.8)cycloalkyl,
(C.sub.2-C.sub.8)alkynyl, arylalkynyl, heteroarylalkynyl group is
optionally substituted by 1, 2 or 3 groups independently selected
from --O(C.sub.1-C.sub.6)alkyl(R.sup.c).sub.1-2,
--S(C.sub.1-C.sub.6)alkyl(R.sup.c).sub.1-2,
--(C.sub.1-C.sub.6)alkyl(R.sup.c).sub.1-2,
--(C.sub.1-C.sub.8)alkyl-heterocycloalkyl,
(C.sub.3-C.sub.8)cycloalkyl-heterocycloalkyl, halo,
(C.sub.1-C.sub.6)alkyl, (C.sub.3-C.sub.8)cycloalkyl,
(C.sub.5-C.sub.8)cycloalkenyl, (C.sub.1-C.sub.6)haloalkyl, cyano,
--COR.sup.a, --CO.sub.2R.sup.a, --CONR.sup.aR.sup.b, --SR.sup.a,
--SOR.sup.a, --SO.sub.2R.sup.a, --SO.sub.2NR.sup.aR.sup.b, nitro,
--NR.sup.aR.sup.b, --NR.sup.aC(O)R.sup.b,
--NR.sup.aC(O)NR.sup.aR.sup.b, --NR.sup.aC(O)OR.sup.a,
--NR.sup.aSO.sub.2R.sup.b, --NR.sup.aSO.sub.2NR.sup.aR.sup.b,
--OR.sup.a, --OC(O)R.sup.a, --OC(O)NR.sup.aR.sup.b,
heterocycloalkyl, aryl, heteroaryl, aryl(C.sub.1-C.sub.4)alkyl, and
heteroaryl(C.sub.1-C.sub.4)alkyl;
[0253] R.sup.a and R.sup.b are each independently hydrogen,
(C.sub.1-C.sub.8)alkyl, (C.sub.2-C.sub.8)alkenyl,
(C.sub.2-C.sub.8)alkynyl, (C.sub.3-C.sub.8)cycloalkyl,
(C.sub.5-C.sub.8)cycloalkenyl, (C.sub.6-C.sub.10)bicycloalkyl,
heterocycloalkyl, aryl, or heteroaryl, wherein said
(C.sub.1-C.sub.8)alkyl, (C.sub.2-C.sub.8)alkenyl,
(C.sub.2-C.sub.8)alkynyl, cycloalkyl, cycloalkenyl, bicycloalkyl,
heterocycloalkyl, aryl or heteroaryl group is optionally
substituted by 1, 2 or 3 groups independently selected from halo,
hydroxyl, (C.sub.1-C.sub.4)alkoxy, amino,
(C.sub.1-C.sub.4)alkylamino,
((C.sub.1-C.sub.4)alkyl)((C.sub.1-C.sub.4)alkyl)amino, --CO.sub.2H,
--CO.sub.2(C.sub.1-C.sub.4)alkyl, --CONH.sub.2,
--CONH(C.sub.1-C.sub.4)alkyl,
--CON((C.sub.1-C.sub.4)alkyl)((C.sub.1-C.sub.4)alkyl),
--SO.sub.2(C.sub.1-C.sub.4)alkyl, --SO.sub.2NH.sub.2,
--SO.sub.2NH(C.sub.1-C.sub.4)alkyl, and
--SO.sub.2N((C.sub.1-C.sub.4)alkyl)((C.sub.1-C.sub.4)alkyl);
[0254] or R.sup.a and R.sup.b taken together with the nitrogen to
which they are attached represent a 5-8 membered saturated or
unsaturated ring, optionally containing an additional heteroatom
selected from oxygen, nitrogen, and sulfur, wherein said ring is
optionally substituted by 1, 2 or 3 groups independently selected
from (C.sub.1-C.sub.4)alkyl, (C.sub.1-C.sub.4)haloalkyl, amino,
(C.sub.1-C.sub.4)alkylamino,
((C.sub.1-C.sub.4)alkyl)((C.sub.1-C.sub.4)alkyl)amino, hydroxyl,
oxo, (C.sub.1-C.sub.4)alkoxy, and
(C.sub.1-C.sub.4)alkoxy(C.sub.1-C.sub.4)alkyl, wherein said ring is
optionally fused to a (C.sub.3-C.sub.8)cycloalkyl,
heterocycloalkyl, aryl, or heteroaryl ring;
[0255] or R.sup.a and R.sup.b taken together with the nitrogen to
which they are attached represent a 6- to 10-membered bridged
bicyclic ring system optionally fused to a
(C.sub.3-C.sub.8)cycloalkyl, heterocycloalkyl, aryl, or heteroaryl
ring. An aryl or heteroaryl group in this particular subgroup A is
selected independently from the group consisting of furan,
thiophene, pyrrole, oxazole, thiazole, imidazole, pyrazole,
oxadiazole, thiadiazole, triazole, tetrazole, benzofuran,
benzothiophene, benzoxazole, benzothiazole, phenyl, pyridine,
pyridazine, pyrimidine, pyrazine, triazine, tetrazine, quinoline,
cinnoline, quinazoline, quinoxaline, and naphthyridine or another
aryl or heteroaryl group as follows:
##STR00020##
wherein in (1),
[0256] A is O, NH, or S; B is CH or N, and C is hydrogen or
C.sub.1-C.sub.8 alkyl; or
##STR00021##
wherein in (2),
[0257] D is N or C optionally substituted by hydrogen or
C.sub.1-C.sub.8 alkyl; or
##STR00022##
wherein in (3),
[0258] E is NH or CH.sub.2; F is O or CO; and G is NH or CH.sub.2;
or
##STR00023##
wherein in (4),
[0259] J is O, S or CO; or
##STR00024##
wherein in (5),
[0260] Q is CH or N;
[0261] M is CH or N; and
[0262] L/(5) is hydrogen, halo, amino, cyano,
(C.sub.1-C.sub.8)alkyl, (C.sub.3-C.sub.8)cycloalkyl, --COR.sup.a,
--CO.sub.2R.sup.a, --CONR.sup.aR.sup.b,
--CONR.sup.aNR.sup.aR.sup.b, --SO.sub.2R.sup.a,
--SO.sub.2NR.sup.aR.sup.b, --NR.sup.aR.sup.b,
--NR.sup.aC(O)R.sup.b, --NR.sup.aSO.sub.2R.sup.b,
--NR.sup.aSO.sub.2NR.sup.aR.sup.b, --NR.sup.aNR.sup.aR.sup.b,
--NR.sup.aNR.sup.aC(O)R.sup.b,
--NR.sup.aNR.sup.aC(O)NR.sup.aR.sup.b, or --OR.sup.a, [0263]
wherein any (C.sub.1-C.sub.8)alkyl or (C.sub.3-C.sub.8)cycloalkyl
group is optionally substituted by 1, 2 or 3 groups independently
selected from (C.sub.1-C.sub.6)alkyl, (C.sub.3-C.sub.8)cycloalkyl,
(C.sub.5-C.sub.8)cycloalkenyl, (C.sub.1-C.sub.6)haloalkyl, cyano,
--COR.sup.a, --CO.sub.2R.sup.a, --CONR.sup.aR.sup.b, --SR.sup.a,
--SOR.sup.a, --SO.sub.2R.sup.a, --SO.sub.2NR.sup.aR.sup.b, nitro,
--NR.sup.aR.sup.b, --NR.sup.aC(O)R.sup.b,
--NR.sup.aC(O)NR.sup.aR.sup.b, --NR.sup.aC(O)OR.sup.a,
--NR.sup.aSO.sub.2R.sup.b, --NR.sup.aSO.sub.2NR.sup.aR.sup.b,
--OR.sup.a, --OC(O)R.sup.a, and --OC(O)NR.sup.aR.sup.b; wherein
R.sup.a and R.sup.b are defined as above; or
##STR00025##
[0263] wherein in (6),
[0264] L/(6) is NH or CH.sub.2; or
##STR00026##
wherein in 7, [0265] M/(7) is hydrogen, halo, amino, cyano,
(C.sub.1-C.sub.8)alkyl, (C.sub.3-C.sub.8)cycloalkyl,
heterocycloalkyl, --COR.sup.a, --CO.sub.2R.sup.a,
--CONR.sup.aR.sup.b, --CONR.sup.aNR.sup.aR.sup.b,
--SO.sub.2R.sup.a, --SO.sub.2NR.sup.aR.sup.b, --NR.sup.aR.sup.b,
--NR.sup.aC(O)R.sup.b, --NR.sup.aSO.sub.2R.sup.b,
--NR.sup.aSO.sub.2NR.sup.aR.sup.b, --NR.sup.aNR.sup.aR.sup.b,
--NR.sup.aNR.sup.aC(O)R.sup.b,
--NR.sup.aNR.sup.aC(O)NR.sup.aR.sup.b, or --OR.sup.a, [0266]
wherein any (C.sub.1-C.sub.8)alkyl, (C.sub.3-C.sub.8)cycloalkyl, or
heterocycloalkyl group is optionally substituted by 1, 2 or 3
groups independently selected from (C.sub.1-C.sub.6)alkyl,
(C.sub.3-C.sub.8)cycloalkyl, (C.sub.5-C.sub.8)cycloalkenyl,
(C.sub.1-C.sub.6)haloalkyl, cyano, --COR.sup.a, --CO.sub.2R.sup.a,
--CONR.sup.aR.sup.b, --SR.sup.a, --SOR.sup.a, --SO.sub.2R.sup.a,
--SO.sub.2NR.sup.aR.sup.b, nitro, --NR.sup.aR.sup.b,
--NR.sup.aC(O)R.sup.b, --NR.sup.aC(O)NR.sup.aR.sup.b,
--NR.sup.aC(O)OR.sup.a, --NR.sup.aSO.sub.2R.sup.b,
--NR.sup.aSO.sub.2NR.sup.aR.sup.b, -0R.sup.a, --OC(O)R.sup.a, and
--OC(O)NR.sup.aR.sup.b; wherein R.sup.a and R.sup.b are defined as
above; or
##STR00027##
[0266] wherein in (8),
[0267] P is CH.sub.2, NH, O, or S; Q/(8) is CH or N; and n is 0-2;
or
##STR00028##
wherein in (9),
[0268] S/(9) and T/(9) is C, or S/(9) is C and T/(9) is N, or S/(9)
is N and T/(9) is C;
[0269] R is hydrogen, amino, methyl, trifluoromethyl, or halo;
[0270] U is hydrogen, halo, amino, cyano, nitro, trifluoromethyl,
(C.sub.1-C.sub.8)alkyl, (C.sub.3-C.sub.8)cycloalkyl, --COR.sup.a,
--CO.sub.2R.sup.a, --CONR.sup.aR.sup.b, --SO.sub.2R.sup.a,
--SO.sub.2NR.sup.aR.sup.b, --NR.sup.aR.sup.b,
--NR.sup.aC(O)R.sub.b, --NR.sup.aSO.sub.2R.sup.b,
--NR.sup.aSO.sub.2NR.sup.aR.sup.b, --NR.sup.aNR.sup.aR.sup.b,
--NR.sup.aNR.sup.aC(O)R.sup.b, --OR.sup.a, or 4-(1H-pyrazol-4-yl),
[0271] wherein any (C.sub.1-C.sub.8)alkyl or
(C.sub.3-C.sub.8)cycloalkyl group is optionally substituted by 1, 2
or 3 groups independently selected from (C.sub.1-C.sub.6)alkyl,
(C.sub.3-C.sub.8)cycloalkyl, (C.sub.5-C.sub.8)cycloalkenyl,
(C.sub.1-C.sub.6)haloalkyl, cyano, --COR.sup.a, --CO.sub.2R.sup.a,
--CONR.sup.aR.sup.b, --SR.sup.a, SOR.sup.a, --SO.sub.2R.sup.a,
--SO.sub.2NR.sup.aR.sup.b, nitro, --NR.sup.aR.sup.b,
--NR.sup.aC(O)R.sup.b, --NR.sup.aC(O)NR.sup.aR.sup.b,
--NR.sup.aC(O)OR.sup.a, --NR.sup.aSO.sub.2R.sup.b,
--NR.sup.aSO.sub.2NR.sup.aR.sup.b, --OR.sup.a, --OC(O)R.sup.a, and
--OC(O)NR.sup.aR.sup.b; wherein R.sup.a and R.sup.b are defined as
above.
[0272] Subgroup B of Formula (VII)
[0273] X and Z are selected independently from the group consisting
of (C.sub.1-C.sub.8)alkyl, (C.sub.3-C.sub.8)cycloalkyl,
heterocycloalkyl, aryl, heteroaryl, --NR.sup.aR.sup.b, and
--OR.sup.a;
[0274] Y is H;
[0275] R.sup.1 is (C.sub.1-C.sub.8)alkyl,
(C.sub.3-C.sub.8)cycloalkyl, or heterocycloalkyl;
[0276] R.sup.2 is hydrogen, (C.sub.1-C.sub.3)alkyl, or halo, in
which said (C.sub.1-C.sub.3)alkyl is optionally substituted with
one to two groups selected from amino and
(C.sub.1-C.sub.3)alkylamino;
[0277] R.sup.7 is hydrogen, (C.sub.1-C.sub.3)alkyl, or alkoxy;
[0278] R.sup.3 is hydrogen, (C.sub.1-C.sub.8)alkyl or halo;
[0279] R.sup.6 is hydrogen, halo, cyano, trifluoromethyl, amino,
(C.sub.1-C.sub.8)alkyl, (C.sub.3-C.sub.8)cycloalkyl, aryl,
heteroaryl, acylamino; (C.sub.2-C.sub.8)alkynyl, arylalkynyl,
heteroarylalkynyl, --SO.sub.2R.sup.a, --SO.sub.2NR.sup.aR.sup.b,
or
--NR.sup.aSO.sub.2R.sup.b; [0280] wherein any
(C.sub.1-C.sub.8)alkyl, (C.sub.3-C.sub.8)cycloalkyl,
(C.sub.2-C.sub.8)alkynyl, arylalkynyl, or heteroarylalkynyl group
is optionally substituted by 1, 2 or 3 groups independently
selected from halo, (C.sub.1-C.sub.6)alkyl,
(C.sub.3-C.sub.8)cycloalkyl, (C.sub.5-C.sub.8)cycloalkenyl,
(C.sub.1-C.sub.6)haloalkyl, cyano, --COR.sup.a, --CO.sub.2R.sup.a,
--CONR.sup.aR.sup.b, --SR.sup.a, --SOR.sup.a, --SO.sub.2R.sup.a,
--SO.sub.2NR.sup.aR.sup.b, nitro, --NR.sup.aR.sup.b,
--NR.sup.aC(O)R.sup.b, --NR.sup.aC(O)NR.sup.aR.sup.b,
--NR.sup.aC(O)OR.sup.a, --NR.sup.aSO.sub.2R.sup.b,
--NR.sup.aSO.sub.2NR.sup.aR.sup.b, --OR.sup.a, --OC(O)R.sup.a,
--OC(O)NR.sup.aR.sup.b, heterocycloalkyl, aryl, heteroaryl,
aryl(C.sub.1-C.sub.4)alkyl, and
heteroaryl(C.sub.1-C.sub.4)alkyl;
[0281] R.sup.a and R.sup.b are each independently hydrogen,
(C.sub.1-C.sub.8)alkyl, (C.sub.2-C.sub.8)alkenyl,
(C.sub.2-C.sub.8)alkynyl, (C.sub.3-C.sub.8)cycloalkyl,
(C.sub.5-C.sub.8)cycloalkenyl, (C.sub.6-C.sub.10)bicycloalkyl,
heterocycloalkyl, aryl, or heteroaryl, wherein said
(C.sub.1-C.sub.8)alkyl, (C.sub.2-C.sub.8)alkenyl,
(C.sub.2-C.sub.8)alkynyl, cycloalkyl, cycloalkenyl, bicycloalkyl,
heterocycloalkyl, aryl or heteroaryl group is optionally
substituted by 1, 2 or 3 groups independently selected from halo,
hydroxyl, (C.sub.1-C.sub.4)alkoxy, amino,
(C.sub.1-C.sub.4)alkylamino,
((C.sub.1-C.sub.4)alkyl)((C.sub.1-C.sub.4)alkyl)amino, --CO.sub.2H,
--CO.sub.2(C.sub.1-C.sub.4)alkyl, --CONH.sub.2,
--CONH(C.sub.1-C.sub.4)alkyl,
--CON((C.sub.1-C.sub.4)alkyl)((C.sub.1-C.sub.4)alkyl),
--SO.sub.2(C.sub.1-C.sub.4)alkyl, --SO.sub.2NH.sub.2,
--SO.sub.2NH(C.sub.1-C.sub.4)alkyl, and
--SO.sub.2N((C.sub.1-C.sub.4)alkyl)((C.sub.1-C.sub.4)alkyl);
[0282] or R.sup.a and R.sup.b taken together with the nitrogen to
which they are attached represent a 5-8 membered saturated or
unsaturated ring, optionally containing an additional heteroatom
selected from oxygen, nitrogen, and sulfur, wherein said ring is
optionally substituted by 1, 2 or 3 groups independently selected
from (C.sub.1-C.sub.4)alkyl, (C.sub.1-C.sub.4)haloalkyl, amino,
(C.sub.1-C.sub.4)alkylamino,
((C.sub.1-C.sub.4)alkyl)((C.sub.1-C.sub.4)alkyl)amino, hydroxyl,
oxo, (C.sub.1-C.sub.4)alkoxy, and
(C.sub.1-C.sub.4)alkoxy(C.sub.1-C.sub.4)alkyl, wherein said ring is
optionally fused to a (C.sub.3-C.sub.8)cycloalkyl,
heterocycloalkyl, aryl, or heteroaryl ring;
[0283] or R.sup.a and R.sup.b taken together with the nitrogen to
which they are attached represent a 6- to 10-membered bridged
bicyclic ring system optionally fused to a
(C.sub.3-C.sub.8)cycloalkyl, heterocycloalkyl, aryl, or heteroaryl
ring. Aryl and heteroaryl in this definition are selected from the
group consisting of furan, thiophene, pyrrole, oxazole, thiazole,
imidazole, pyrazole, oxadiazole, thiadiazole, triazole, tetrazole,
benzofuran, benzothiophene, benzoxazole, benzothiazole, phenyl,
pyridine, pyridazine, pyrimidine, pyrazine, triazine, tetrazine,
quinoline, cinnoline, quinazoline, quinoxaline, and naphthyridine
or a compound of another aryl or heteroaryl group as follows:
##STR00029##
wherein in (1),
[0284] A is O, NH, or S; B is CH or N, and C is hydrogen or C1-C8
alkyl; or
##STR00030##
wherein in (2),
[0285] D is N or C optionally substituted by hydrogen or C1-C8
alkyl; or
##STR00031##
wherein in (3),
[0286] E is NH or CH.sub.2; F is O or CO; and G is NH or CH.sub.2;
or
##STR00032##
wherein in (4),
[0287] J is O, S or CO; or
##STR00033##
wherein in (5),
[0288] Q is CH or N;
[0289] M is CH or N; and
[0290] L/(5) is hydrogen, halo, amino, cyano,
(C.sub.1-C.sub.8)alkyl, (C.sub.3-C.sub.8)cycloalkyl, --COR.sup.a,
--CO.sub.2R.sup.a, --CONR.sup.aR.sup.b,
--CONR.sup.aNR.sup.aR.sup.b, --SO.sub.2R.sup.a,
--SO.sub.2NR.sup.aR.sup.b, --NR.sup.aR.sub.b,
--NR.sup.aC(O)R.sup.b, --NR.sup.aSO.sub.2R.sup.b,
--NR.sup.aSO.sub.2NR.sup.aR.sup.b, --NR.sup.aNR.sup.aR.sup.b,
--NR.sup.aNR.sup.aC(O)R.sup.b,
--NR.sup.aNR.sup.aC(O)NR.sup.aR.sup.b, or --OR.sup.a, [0291]
wherein any (C.sub.1-C.sub.8)alkyl, (C.sub.3-C.sub.8)cycloalkyl,
group is optionally substituted by 1, 2 or 3 groups independently
selected from (C.sub.1-C.sub.6)alkyl, (C.sub.3-C.sub.8)cycloalkyl,
(C.sub.5-C.sub.8)cycloalkenyl, (C.sub.1-C.sub.6)haloalkyl, cyano,
--COR.sup.a, --CO.sub.2R.sup.a, --CONR.sup.aR.sup.b, --SR.sup.a,
--SOR.sup.a, --SO.sub.2R.sup.a, --SO.sub.2NR.sup.aR.sup.b, nitro,
--NR.sup.aR.sup.b, --NR.sup.aC(O)R.sup.b,
--NR.sup.aC(O)NR.sup.aR.sup.b, --NR.sup.aC(O)OR.sup.a,
NR.sup.aSO.sub.2R.sup.b, --NR.sup.aSO.sub.2NR.sup.aR.sup.b,
-0R.sup.a, --OC(O)R.sup.a, and --OC(O)NR.sup.aR.sup.b, wherein
R.sup.a and R.sup.b are defined as above; or
##STR00034##
[0291] wherein in (6),
[0292] L/(6) is NH or CH.sub.2; or
##STR00035##
wherein in (7), [0293] M/(7) is hydrogen, halo, amino, cyano,
(C.sub.1-C.sub.8)alkyl, (C.sub.3-C.sub.8)cycloalkyl,
heterocycloalkyl, --COR.sup.a, --CO.sub.2R.sup.a,
--CONR.sup.aR.sup.b, --CONR.sup.aNR.sup.aR.sup.b,
--SO.sub.2R.sup.a, --SO.sub.2NR.sup.aR.sup.b, --NR.sup.aR.sup.b,
--NR.sup.aC(O)R.sup.b, --NR.sup.aSO.sub.2R.sup.b,
--NR.sup.aSO.sub.2NR.sup.aR.sup.b, --NR.sup.aNR.sup.aR.sup.b,
--NR.sup.aNR.sup.aC(O)R.sup.b,
--NR.sup.aNR.sup.aC(O)NR.sup.aR.sup.b, or --OR.sup.a, [0294]
wherein any (C.sub.1-C.sub.8)alkyl, (C.sub.3-C.sub.8)cycloalkyl,
heterocycloalkyl group is optionally substituted by 1, 2 or 3
groups independently selected from (C.sub.1-C.sub.6)alkyl,
(C.sub.3-C.sub.8)cycloalkyl, (C.sub.5-C.sub.8)cycloalkenyl,
(C.sub.1-C.sub.6)haloalkyl, cyano, --COR.sup.a, --CO.sub.2R.sup.a,
--CONR.sup.aR.sup.b, --SR.sup.a, --SOR.sup.a, --SO.sub.2R.sup.a,
--SO.sub.2NR.sup.aR.sup.b, nitro, --NR.sup.aR.sup.b,
--NR.sup.aC(O)R.sup.b, NR.sup.aC(O)NR.sup.aR.sup.b,
--NR.sup.aC(O)OR.sup.a, --NR.sup.aSO.sub.2R.sup.b,
--NR.sup.aSO.sub.2NR.sup.aR.sup.b, -0R.sup.a, --OC(O)R.sup.a,
--OC(O)NR.sup.aR.sup.b; wherein R.sup.a and R.sup.b are defined as
above; or
##STR00036##
[0294] wherein in (8),
[0295] P is CH.sub.2, NH, O, or S; Q/(8) is CH or N; and n is 0-2;
or
##STR00037##
wherein in (9),
[0296] S/(9) and T/(9) is C, or S/(9) is C and T/(9) is N, or S/(9)
is N and T/(9) is C;
[0297] R is hydrogen, amino, methyl, trifluoromethyl, halo;
[0298] U is hydrogen, halo, amino, cyano, nitro, trifluoromethyl,
(C.sub.1-C.sub.8)alkyl, (C.sub.3-C.sub.8)cycloalkyl, --COR.sup.a,
--CO.sub.2R.sup.a, --CONR.sup.aR.sup.b, --SO.sub.2R.sup.a,
--SO.sub.2NR.sup.aR.sup.b, --NR.sup.aR.sup.b,
--NR.sup.aC(O)R.sup.b, --NR.sup.aSO.sub.2R.sup.b,
--NR.sup.aSO.sub.2NR.sup.aR.sup.b, --NR.sup.aNR.sup.aR.sup.b,
--NR.sup.aNR.sup.aC(O)R.sub.b, --OR.sup.a, or 4-(1H-pyrazol-4-yl),
[0299] wherein any (C.sub.1-C.sub.8)alkyl, or
(C.sub.3-C.sub.8)cycloalkyl group is optionally substituted by 1, 2
or 3 groups independently selected from (C.sub.1-C.sub.6)alkyl,
(C.sub.3-C.sub.8)cycloalkyl, (C.sub.5-C.sub.8)cycloalkenyl,
(C.sub.1-C.sub.6)haloalkyl, cyano, --COR.sup.a, --CO.sub.2R.sup.a,
--CONR.sup.aR.sup.b, --SOR.sup.a, --SO.sub.2R.sup.a,
--SO.sub.2NR.sup.aR.sup.b, nitro, --NR.sup.aR.sup.b,
--NR.sup.aC(O)R.sup.b, --NR.sup.aC(O)NR.sup.aR.sup.b,
--NR.sup.aC(O)OR.sup.a, --NR.sup.aSO.sub.2R.sup.b,
--NR.sup.aSO.sub.2NR.sup.aR.sup.b, --OR.sup.a, --OC(O)R.sup.a, and
--OC(O)NR.sup.aR.sup.b, wherein R.sup.a and R.sup.b are defined as
above.
[0300] In some embodiments, the EZH2 inhibitor is:
##STR00038##
stereoisomers thereof or pharmaceutically acceptable salt or
solvate thereof
[0301] In some embodiments, the EZH2 inhibitor is
##STR00039##
[0302] stereoisomers thereof or pharmaceutically acceptable salt or
solvate thereof.
[0303] The compounds described herein can be synthesized according
to any method known in the art. For example, the compounds having
the Formula (VII) can be synthesized according to the method
described in WO 2011/140325; WO 2011/140324; and WO 2012/005805,
each of which is incorporated by reference in its entirety.
[0304] As used herein, "alkyl", "C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5 or C.sub.6 alkyl" or "C.sub.1-6 alkyl" is intended
to include C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5 or C.sub.6
straight chain (linear) saturated aliphatic hydrocarbon groups and
C.sub.3, C.sub.4, C.sub.5 or C.sub.6 branched saturated aliphatic
hydrocarbon groups. For example, C.sub.1-C.sub.6 alkyl is intended
to include C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5 and C.sub.6
alkyl groups. Examples of alkyl include, moieties having from one
to six carbon atoms, such as, but not limited to, methyl, ethyl,
n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, s-pentyl
or n-hexyl.
[0305] In certain embodiments, a straight chain or branched alkyl
has six or fewer carbon atoms (e.g., C.sub.1-C.sub.6 for straight
chain, C.sub.3-C.sub.6 for branched chain), and in another
embodiment, a straight chain or branched alkyl has four or fewer
carbon atoms.
[0306] As used herein, the term "cycloalkyl" refers to a saturated
or unsaturated nonaromatic hydrocarbon mono- or multi-ring (e.g.,
fused, bridged, or spiro rings) system having 3 to 30 carbon atoms
(e.g., C.sub.3-C.sub.10). Examples of cycloalkyl include, but are
not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl, cyclooctyl, cyclopentenyl, cyclohexenyl,
cycloheptenyl, and adamantyl. The term "heterocycloalkyl" refers to
a saturated or unsaturated nonaromatic 3-8 membered monocyclic,
7-12 membered bicyclic (fused, bridged, or spiro rings), or 11-14
membered tricyclic ring system (fused, bridged, or spiro rings)
having one or more heteroatoms (such as O, N, S, or Se), unless
specified otherwise. Examples of heterocycloalkyl groups include,
but are not limited to, piperidinyl, piperazinyl, pyrrolidinyl,
dioxanyl, tetrahydrofuranyl, isoindolinyl, indolinyl,
imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl,
triazolidinyl, tetrahyrofuranyl, oxiranyl, azetidinyl, oxetanyl,
thietanyl, 1,2,3,6-tetrahydropyridinyl, tetrahydropyranyl,
dihydropyranyl, pyranyl, morpholinyl, 1,4-diazepanyl,
1,4-oxazepanyl, 2-oxa-5-azabicyclo[2.2.1]heptanyl,
diazabicyclo[2.2.1]heptanyl, 2-oxa-6-azaspiro[3.3]heptanyl,
2,6-diazaspiro[3.3]heptanyl, 1,4-dioxa-8-azaspiro[4.5]decanyl and
the like.
[0307] The term "optionally substituted alkyl" refers to
unsubstituted alkyl or alkyl having designated substituents
replacing one or more hydrogen atoms on one or more carbons of the
hydrocarbon backbone. Such substituents can include, for example,
alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
amino (including alkylamino, dialkylamino, arylamino, diarylamino
and alkylarylamino), acylamino (including alkylcarbonylamino,
arylcarbonylamino, carbamoyl and ureido), amidino, imino,
sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,
alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moiety.
[0308] An "arylalkyl" or an "aralkyl" moiety is an alkyl
substituted with an aryl (e.g., phenylmethyl (benzyl)). An
"alkylaryl" moiety is an aryl substituted with an alkyl (e.g.,
methylphenyl).
[0309] As used herein, "alkyl linker" is intended to include
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5 or C.sub.6 straight
chain (linear) saturated divalent aliphatic hydrocarbon groups and
C.sub.3, C.sub.4, C.sub.5 or C.sub.6 branched saturated aliphatic
hydrocarbon groups. For example, C.sub.1-C.sub.6 alkyl linker is
intended to include C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5 and
C.sub.6 alkyl linker groups. Examples of alkyl linker include,
moieties having from one to six carbon atoms, such as, but not
limited to, methyl (--CH.sub.2--), ethyl (--CH.sub.2CH.sub.2--),
n-propyl (--CH.sub.2CH.sub.2CH.sub.2--), i-propyl
(--CHCH.sub.3CH.sub.2--), n-butyl
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), s-butyl
(--CHCH.sub.3CH.sub.2CH.sub.2--), i-butyl
(--C(CH.sub.3).sub.2CH.sub.2--), n-pentyl
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), s-pentyl
(--CHCH.sub.3CH.sub.2CH.sub.2CH.sub.2--) or n-hexyl
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--).
[0310] "Alkenyl" includes unsaturated aliphatic groups analogous in
length and possible substitution to the alkyls described above, but
that contain at least one double bond. For example, the term
"alkenyl" includes straight chain alkenyl groups (e.g., ethenyl,
propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl,
decenyl), and branched alkenyl groups. In certain embodiments, a
straight chain or branched alkenyl group has six or fewer carbon
atoms in its backbone (e.g., C.sub.2-C.sub.6 for straight chain,
C.sub.3-C.sub.6 for branched chain). The term "C.sub.2-C.sub.6"
includes alkenyl groups containing two to six carbon atoms. The
term "C.sub.3-C.sub.6" includes alkenyl groups containing three to
six carbon atoms.
[0311] The term "optionally substituted alkenyl" refers to
unsubstituted alkenyl or alkenyl having designated substituents
replacing one or more hydrogen atoms on one or more hydrocarbon
backbone carbon atoms. Such substituents can include, for example,
alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
amino (including alkylamino, dialkylamino, arylamino, diarylamino
and alkylarylamino), acylamino (including alkylcarbonylamino,
arylcarbonylamino, carbamoyl and ureido), amidino, imino,
sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,
alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, heterocyclyl, alkylaryl, or an aromatic or
heteroaromatic moiety.
[0312] "Alkynyl" includes unsaturated aliphatic groups analogous in
length and possible substitution to the alkyls described above, but
which contain at least one triple bond. For example, "alkynyl"
includes straight chain alkynyl groups (e.g., ethynyl, propynyl,
butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl),
and branched alkynyl groups. In certain embodiments, a straight
chain or branched alkynyl group has six or fewer carbon atoms in
its backbone (e.g., C.sub.2-C.sub.6 for straight chain,
C.sub.3-C.sub.6 for branched chain). The term "C.sub.2-C.sub.6"
includes alkynyl groups containing two to six carbon atoms. The
term "C.sub.3-C.sub.6" includes alkynyl groups containing three to
six carbon atoms.
[0313] The term "optionally substituted alkynyl" refers to
unsubstituted alkynyl or alkynyl having designated substituents
replacing one or more hydrogen atoms on one or more hydrocarbon
backbone carbon atoms. Such substituents can include, for example,
alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
amino (including alkylamino, dialkylamino, arylamino, diarylamino
and alkylarylamino), acylamino (including alkylcarbonylamino,
arylcarbonylamino, carbamoyl and ureido), amidino, imino,
sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,
alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moiety.
[0314] Other optionally substituted moieties (such as optionally
substituted cycloalkyl, heterocycloalkyl, aryl, or heteroaryl)
include both the unsubstituted moieties and the moieties having one
or more of the designated substituents. For example, substituted
heterocycloalkyl includes those substituted with one or more alkyl
groups, such as 2,2,6,6-tetramethyl-piperidinyl and
2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridinyl.
[0315] "Aryl" includes groups with aromaticity, including
"conjugated," or multicyclic systems with at least one aromatic
ring and do not contain any heteroatom in the ring structure.
Examples include phenyl, benzyl, 1,2,3,4-tetrahydronaphthalenyl,
etc.
[0316] "Heteroaryl" groups are aryl groups, as defined above,
except having from one to four heteroatoms in the ring structure,
and may also be referred to as "aryl heterocycles" or
"heteroaromatics." As used herein, the term "heteroaryl" is
intended to include a stable 5-, 6-, or 7-membered monocyclic or
7-, 8-, 9-, 10-, 11- or 12-membered bicyclic aromatic heterocyclic
ring which consists of carbon atoms and one or more heteroatoms,
e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, or e.g. 2,
3, 4, 5, or 6 heteroatoms, independently selected from the group
consisting of nitrogen, oxygen and sulfur. The nitrogen atom may be
substituted or unsubstituted (i.e., N or NR wherein R is H or other
substituents, as defined). The nitrogen and sulfur heteroatoms may
optionally be oxidized (i.e., N.fwdarw.O and S(O).sub.p, where p=1
or 2). It is to be noted that total number of S and O atoms in the
aromatic heterocycle is not more than 1.
[0317] Examples of heteroaryl groups include pyrrole, furan,
thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole,
pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine,
pyrimidine, and the like.
[0318] Furthermore, the terms "aryl" and "heteroaryl" include
multicyclic aryl and heteroaryl groups, e.g., tricyclic, bicyclic,
e.g., naphthalene, benzoxazole, benzodioxazole, benzothiazole,
benzoimidazole, benzothiophene, methylenedioxyphenyl, quinoline,
isoquinoline, naphthrydine, indole, benzofuran, purine, benzofuran,
deazapurine, indolizine.
[0319] In the case of multicyclic aromatic rings, only one of the
rings needs to be aromatic (e.g., 2,3-dihydroindole), although all
of the rings may be aromatic (e.g., quinoline). The second ring can
also be fused or bridged.
[0320] The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring
can be substituted at one or more ring positions (e.g., the
ring-forming carbon or heteroatom such as N) with such substituents
as described above, for example, alkyl, alkenyl, alkynyl, halogen,
hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl,
alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate,
phosphonato, phosphinato, amino (including alkylamino,
dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino
(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and
ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,
thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,
sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,
alkylaryl, or an aromatic or heteroaromatic moiety. Aryl and
heteroaryl groups can also be fused or bridged with alicyclic or
heterocyclic rings, which are not aromatic so as to form a
multicyclic system (e.g., tetralin, methylenedioxyphenyl).
[0321] As used herein, "carbocycle" or "carbocyclic ring" is
intended to include any stable monocyclic, bicyclic or tricyclic
ring having the specified number of carbons, any of which may be
saturated, unsaturated, or aromatic. Carbocycle includes cycloalkyl
and aryl. For example, a C.sub.3-C.sub.14 carbocycle is intended to
include a monocyclic, bicyclic or tricyclic ring having 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms. Examples of carbocycles
include, but are not limited to, cyclopropyl, cyclobutyl,
cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl,
cycloheptenyl, cycloheptyl, cycloheptenyl, adamantyl, cyclooctyl,
cyclooctenyl, cyclooctadienyl, fluorenyl, phenyl, naphthyl,
indanyl, adamantyl and tetrahydronaphthyl. Bridged rings are also
included in the definition of carbocycle, including, for example,
[3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane
and [2.2.2]bicyclooctane. A bridged ring occurs when one or more
carbon atoms link two non-adjacent carbon atoms. In one embodiment,
bridge rings are one or two carbon atoms. It is noted that a bridge
always converts a monocyclic ring into a tricyclic ring. When a
ring is bridged, the substituents recited for the ring may also be
present on the bridge. Fused (e.g., naphthyl, tetrahydronaphthyl)
and spiro rings are also included.
[0322] As used herein, "heterocycle" or "heterocyclic group"
includes any ring structure (saturated, unsaturated, or aromatic)
which contains at least one ring heteroatom (e.g., N, O or S).
Heterocycle includes heterocycloalkyl and heteroaryl. Examples of
heterocycles include, but are not limited to, morpholine,
pyrrolidine, tetrahydrothiophene, piperidine, piperazine, oxetane,
pyran, tetrahydropyran, azetidine, and tetrahydrofuran.
[0323] Examples of heterocyclic groups include, but are not limited
to, acridinyl, azocinyl, benzimidazolyl, benzofuranyl,
benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl,
benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl,
benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl,
carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl,
2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran,
furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl,
1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl,
3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl,
isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,
methylenedioxyphenyl, morpholinyl, naphthyridinyl,
octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,
1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,
1,2,4-oxadiazol5(4H)-one, oxazolidinyl, oxazolyl, oxindolyl,
pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl,
phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,
piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,
pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl,
pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole,
pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl,
pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl,
quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,
tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,
tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,
1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,
thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,
thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl,
1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl and
xanthenyl.
[0324] The term "substituted," as used herein, means that any one
or more hydrogen atoms on the designated atom is replaced with a
selection from the indicated groups, provided that the designated
atom's normal valency is not exceeded, and that the substitution
results in a stable compound. When a substituent is oxo or keto
(i.e., .dbd.O), then 2 hydrogen atoms on the atom are replaced.
Keto substituents are not present on aromatic moieties. Ring double
bonds, as used herein, are double bonds that are formed between two
adjacent ring atoms (e.g., C.dbd.C, C.dbd.N or N.dbd.N). "Stable
compound" and "stable structure" are meant to indicate a compound
that is sufficiently robust to survive isolation to a useful degree
of purity from a reaction mixture, and formulation into an
efficacious therapeutic agent.
[0325] When a bond to a substituent is shown to cross a bond
connecting two atoms in a ring, then such substituent may be bonded
to any atom in the ring. When a substituent is listed without
indicating the atom via which such substituent is bonded to the
rest of the compound of a given formula, then such substituent may
be bonded via any atom in such formula. Combinations of
substituents and/or variables are permissible, but only if such
combinations result in stable compounds.
[0326] When any variable (e.g., R.sub.1) occurs more than one time
in any constituent or formula for a compound, its definition at
each occurrence is independent of its definition at every other
occurrence. Thus, for example, if a group is shown to be
substituted with 0-2 R.sub.1 moieties, then the group may
optionally be substituted with up to two R.sub.1 moieties and
R.sub.1 at each occurrence is selected independently from the
definition of R.sub.1. Also, combinations of substituents and/or
variables are permissible, but only if such combinations result in
stable compounds.
[0327] The term "hydroxy" or "hydroxyl" includes groups with an
--OH or --O.sup.-.
[0328] As used herein, "halo" or "halogen" refers to fluoro,
chloro, bromo and iodo. The term "perhalogenated" generally refers
to a moiety wherein all hydrogen atoms are replaced by halogen
atoms. The term "haloalkyl" or "haloalkoxyl" refers to an alkyl or
alkoxyl substituted with one or more halogen atoms.
[0329] The term "carbonyl" includes compounds and moieties which
contain a carbon connected with a double bond to an oxygen atom.
Examples of moieties containing a carbonyl include, but are not
limited to, aldehydes, ketones, carboxylic acids, amides, esters,
anhydrides, etc.
[0330] The term "carboxyl" refers to --COOH or its C.sub.1-C.sub.6
alkyl ester.
[0331] "Acyl" includes moieties that contain the acyl radical
(R--C(O)--) or a carbonyl group. "Substituted acyl" includes acyl
groups where one or more of the hydrogen atoms are replaced by, for
example, alkyl groups, alkynyl groups, halogen, hydroxyl,
alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, amino (including alkylamino,
dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino
(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and
ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,
thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,
sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,
alkylaryl, or an aromatic or heteroaromatic moiety.
[0332] "Aroyl" includes moieties with an aryl or heteroaromatic
moiety bound to a carbonyl group. Examples of aroyl groups include
phenylcarboxy, naphthyl carboxy, etc.
[0333] "Alkoxyalkyl," "alkylaminoalkyl," and "thioalkoxyalkyl"
include alkyl groups, as described above, wherein oxygen, nitrogen,
or sulfur atoms replace one or more hydrocarbon backbone carbon
atoms.
[0334] The term "alkoxy" or "alkoxyl" includes substituted and
unsubstituted alkyl, alkenyl and alkynyl groups covalently linked
to an oxygen atom. Examples of alkoxy groups or alkoxyl radicals
include, but are not limited to, methoxy, ethoxy, isopropyloxy,
propoxy, butoxy and pentoxy groups. Examples of substituted alkoxy
groups include halogenated alkoxy groups. The alkoxy groups can be
substituted with groups such as alkenyl, alkynyl, halogen,
hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, amino (including alkylamino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties.
Examples of halogen substituted alkoxy groups include, but are not
limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy,
chloromethoxy, dichloromethoxy and trichloromethoxy.
[0335] The term "ether" or "alkoxy" includes compounds or moieties
which contain an oxygen bonded to two carbon atoms or heteroatoms.
For example, the term includes "alkoxyalkyl," which refers to an
alkyl, alkenyl, or alkynyl group covalently bonded to an oxygen
atom which is covalently bonded to an alkyl group.
[0336] The term "ester" includes compounds or moieties which
contain a carbon or a heteroatom bound to an oxygen atom which is
bonded to the carbon of a carbonyl group. The term "ester" includes
alkoxycarboxy groups such as methoxycarbonyl, ethoxycarbonyl,
propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, etc.
[0337] The term "thioalkyl" includes compounds or moieties which
contain an alkyl group connected with a sulfur atom. The thioalkyl
groups can be substituted with groups such as alkyl, alkenyl,
alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, carboxyacid,
alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,
alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl,
alkoxyl, amino (including alkylamino, dialkylamino, arylamino,
diarylamino and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moieties.
[0338] The term "thiocarbonyl" or "thiocarboxy" includes compounds
and moieties which contain a carbon connected with a double bond to
a sulfur atom.
[0339] The term "thioether" includes moieties which contain a
sulfur atom bonded to two carbon atoms or heteroatoms. Examples of
thioethers include, but are not limited to alkthioalkyls,
alkthioalkenyls, and alkthioalkynyls. The term "alkthioalkyls"
include moieties with an alkyl, alkenyl, or alkynyl group bonded to
a sulfur atom which is bonded to an alkyl group. Similarly, the
term "alkthioalkenyls" refers to moieties wherein an alkyl, alkenyl
or alkynyl group is bonded to a sulfur atom which is covalently
bonded to an alkenyl group; and alkthioalkynyls" refers to moieties
wherein an alkyl, alkenyl or alkynyl group is bonded to a sulfur
atom which is covalently bonded to an alkynyl group.
[0340] As used herein, "amine" or "amino" refers to unsubstituted
or substituted --NH.sub.2. "Alkylamino" includes groups of
compounds wherein nitrogen of --NH.sub.2 is bound to at least one
alkyl group. Examples of alkylamino groups include benzylamino,
methylamino, ethylamino, phenethylamino, etc. "Dialkylamino"
includes groups wherein the nitrogen of --NH.sub.2 is bound to at
least two additional alkyl groups. Examples of dialkylamino groups
include, but are not limited to, dimethylamino and diethylamino.
"Arylamino" and "diarylamino" include groups wherein the nitrogen
is bound to at least one or two aryl groups, respectively.
"Aminoaryl" and "aminoaryloxy" refer to aryl and aryloxy
substituted with amino. "Alkylarylamino," "alkylaminoaryl" or
"arylaminoalkyl" refers to an amino group which is bound to at
least one alkyl group and at least one aryl group. "Alkaminoalkyl"
refers to an alkyl, alkenyl, or alkynyl group bound to a nitrogen
atom which is also bound to an alkyl group. "Acylamino" includes
groups wherein nitrogen is bound to an acyl group. Examples of
acylamino include, but are not limited to, alkylcarbonylamino,
arylcarbonylamino, carbamoyl and ureido groups.
[0341] The term "amide" or "aminocarboxy" includes compounds or
moieties that contain a nitrogen atom that is bound to the carbon
of a carbonyl or a thiocarbonyl group. The term includes
"alkaminocarboxy" groups that include alkyl, alkenyl or alkynyl
groups bound to an amino group which is bound to the carbon of a
carbonyl or thiocarbonyl group. It also includes "arylaminocarboxy"
groups that include aryl or heteroaryl moieties bound to an amino
group that is bound to the carbon of a carbonyl or thiocarbonyl
group. The terms "alkylaminocarboxy", "alkenylaminocarboxy",
"alkynylaminocarboxy" and "arylaminocarboxy" include moieties
wherein alkyl, alkenyl, alkynyl and aryl moieties, respectively,
are bound to a nitrogen atom which is in turn bound to the carbon
of a carbonyl group. Amides can be substituted with substituents
such as straight chain alkyl, branched alkyl, cycloalkyl, aryl,
heteroaryl or heterocycle. Substituents on amide groups may be
further substituted.
[0342] In the present specification, the structural formula of the
compound represents a certain isomer for convenience in some cases,
but the present invention includes all isomers, such as geometrical
isomers, optical isomers based on an asymmetrical carbon,
stereoisomers, tautomers, and the like, it being understood that
not all isomers may have the same level of activity. In addition, a
crystal polymorphism may be present for the compounds represented
by the formula. It is noted that any crystal form, crystal form
mixture, or anhydride or hydrate thereof is included in the scope
of the present invention. Furthermore, so-called metabolite which
is produced by degradation of the present compound in vivo is
included in the scope of the present invention.
[0343] "Isomerism" means compounds that have identical molecular
formulae but differ in the sequence of bonding of their atoms or in
the arrangement of their atoms in space. Isomers that differ in the
arrangement of their atoms in space are termed "stereoisomers."
Stereoisomers that are not mirror images of one another are termed
"diastereoisomers," and stereoisomers that are non-superimposable
mirror images of each other are termed "enantiomers" or sometimes
optical isomers. A mixture containing equal amounts of individual
enantiomeric forms of opposite chirality is termed a "racemic
mixture."
[0344] A carbon atom bonded to four nonidentical substituents is
termed a "chiral center."
[0345] "Chiral isomer" means a compound with at least one chiral
center. Compounds with more than one chiral center may exist either
as an individual diastereomer or as a mixture of diastereomers,
termed "diastereomeric mixture." When one chiral center is present,
a stereoisomer may be characterized by the absolute configuration
(R or S) of that chiral center. Absolute configuration refers to
the arrangement in space of the substituents attached to the chiral
center. The substituents attached to the chiral center under
consideration are ranked in accordance with the Sequence Rule of
Cahn, Ingold and Prelog. (Cahn et al., Angew. Chem. Inter. Edit.
1966, 5, 385; errata 511; Cahn et al., Angew. Chem. 1966, 78, 413;
Cahn and Ingold, J. Chem. Soc. 1951 (London), 612; Cahn et al.,
Experientia 1956, 12, 81; Cahn, J. Chem. Educ. 1964, 41, 116).
[0346] "Geometric isomer" means the diastereomers that owe their
existence to hindered rotation about double bonds or a cycloalkyl
linker (e.g., 1,3-cylcobutyl). These configurations are
differentiated in their names by the prefixes cis and trans, or Z
and E, which indicate that the groups are on the same or opposite
side of the double bond in the molecule according to the
Cahn-Ingold-Prelog rules.
[0347] It is to be understood that the compounds of the present
invention may be depicted as different chiral isomers or geometric
isomers. It should also be understood that when compounds have
chiral isomeric or geometric isomeric forms, all isomeric forms are
intended to be included in the scope of the present invention, and
the naming of the compounds does not exclude any isomeric forms, it
being understood that not all isomers may have the same level of
activity.
[0348] Furthermore, the structures and other compounds discussed in
this invention include all atropic isomers thereof, it being
understood that not all atropic isomers may have the same level of
activity. "Atropic isomers" are a type of stereoisomer in which the
atoms of two isomers are arranged differently in space. Atropic
isomers owe their existence to a restricted rotation caused by
hindrance of rotation of large groups about a central bond. Such
atropic isomers typically exist as a mixture, however as a result
of recent advances in chromatography techniques, it has been
possible to separate mixtures of two atropic isomers in select
cases.
[0349] "Tautomer" is one of two or more structural isomers that
exist in equilibrium and is readily converted from one isomeric
form to another. This conversion results in the formal migration of
a hydrogen atom accompanied by a switch of adjacent conjugated
double bonds. Tautomers exist as a mixture of a tautomeric set in
solution. In solutions where tautomerization is possible, a
chemical equilibrium of the tautomers will be reached. The exact
ratio of the tautomers depends on several factors, including
temperature, solvent and pH. The concept of tautomers that are
interconvertable by tautomerizations is called tautomerism.
[0350] Of the various types of tautomerism that are possible, two
are commonly observed. In keto-enol tautomerism a simultaneous
shift of electrons and a hydrogen atom occurs. Ring-chain
tautomerism arises as a result of the aldehyde group (--CHO) in a
sugar chain molecule reacting with one of the hydroxy groups (--OH)
in the same molecule to give it a cyclic (ring-shaped) form as
exhibited by glucose.
[0351] Common tautomeric pairs are: ketone-enol, amide-nitrile,
lactam-lactim, amide-imidic acid tautomerism in heterocyclic rings
(e.g., in nucleobases such as guanine, thymine and cytosine),
imine-enamine and enamine-enamine. An example of keto-enol
equilibria is between pyridin-2(1H)-ones and the corresponding
pyridin-2-ols, as shown below.
##STR00040##
[0352] It is to be understood that the compounds of the present
invention may be depicted as different tautomers. It should also be
understood that when compounds have tautomeric forms, all
tautomeric forms are intended to be included in the scope of the
present invention, and the naming of the compounds does not exclude
any tautomer form. It will be understood that certain tautomers may
have a higher level of activity than others.
[0353] The term "crystal polymorphs", "polymorphs" or "crystal
forms" means crystal structures in which a compound (or a salt or
solvate thereof) can crystallize in different crystal packing
arrangements, all of which have the same elemental composition.
Different crystal forms usually have different X-ray diffraction
patterns, infrared spectral, melting points, density hardness,
crystal shape, optical and electrical properties, stability and
solubility. Recrystallization solvent, rate of crystallization,
storage temperature, and other factors may cause one crystal form
to dominate. Crystal polymorphs of the compounds can be prepared by
crystallization under different conditions.
[0354] The compounds of any of Formulae disclosed herein include
the compounds themselves, as well as their salts or their solvates,
if applicable. A salt, for example, can be formed between an anion
and a positively charged group (e.g., amino) on an aryl- or
heteroaryl-substituted benzene compound. Suitable anions include
chloride, bromide, iodide, sulfate, bisulfate, sulfamate, nitrate,
phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate,
glucuronate, glutarate, malate, maleate, succinate, fumarate,
tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, and
acetate (e.g., trifluoroacetate). The term "pharmaceutically
acceptable anion" refers to an anion suitable for forming a
pharmaceutically acceptable salt. Likewise, a salt can also be
formed between a cation and a negatively charged group (e.g.,
carboxylate) on an aryl- or heteroaryl-substituted benzene
compound. Suitable cations include sodium ion, potassium ion,
magnesium ion, calcium ion, and an ammonium cation such as
tetramethylammonium ion. The aryl- or heteroaryl-substituted
benzene compounds also include those salts containing quaternary
nitrogen atoms.
[0355] Additionally, the compounds of the present invention, for
example, the salts of the compounds, can exist in either hydrated
or unhydrated (the anhydrous) form or as solvates with other
solvent molecules. Nonlimiting examples of hydrates include
monohydrates, dihydrates, etc. Nonlimiting examples of solvates
include ethanol solvates, acetone solvates, etc.
[0356] "Solvate" means solvent addition forms that contain either
stoichiometric or non stoichiometric amounts of solvent. Some
compounds have a tendency to trap a fixed molar ratio of solvent
molecules in the crystalline solid state, thus forming a solvate.
If the solvent is water the solvate formed is a hydrate; and if the
solvent is alcohol, the solvate formed is an alcoholate. Hydrates
are formed by the combination of one or more molecules of water
with one molecule of the substance in which the water retains its
molecular state as H2O.
[0357] As used herein, the term "analog" refers to a chemical
compound that is structurally similar to another but differs
slightly in composition (as in the replacement of one atom by an
atom of a different element or in the presence of a particular
functional group, or the replacement of one functional group by
another functional group). Thus, an analog is a compound that is
similar or comparable in function and appearance, but not in
structure or origin to the reference compound.
[0358] As defined herein, the term "derivative" refers to compounds
that have a common core structure, and are substituted with various
groups as described herein. For example, all of the compounds
represented by Formula (I) are aryl- or heteroaryl-substituted
benzene compounds, and have Formula (I) as a common core.
[0359] The term "bioisostere" refers to a compound resulting from
the exchange of an atom or of a group of atoms with another,
broadly similar, atom or group of atoms. The objective of a
bioisosteric replacement is to create a new compound with similar
biological properties to the parent compound. The bioisosteric
replacement may be physicochemically or topologically based.
Examples of carboxylic acid bioisosteres include, but are not
limited to, acyl sulfonimides, tetrazoles, sulfonates and
phosphonates. See, e.g., Patani and LaVoie, Chem. Rev. 96,
3147-3176, 1996.
[0360] The present invention is intended to include all isotopes of
atoms occurring in the present compounds. Isotopes include those
atoms having the same atomic number but different mass numbers. By
way of general example and without limitation, isotopes of hydrogen
include tritium and deuterium, and isotopes of carbon include C-13
and C-14.
[0361] The present invention provides methods for the synthesis of
the compounds of any Formula disclosed herein. The present
invention also provides detailed methods for the synthesis of
various disclosed compounds of the present invention according to
the following schemes as shown in the Examples.
[0362] Throughout the description, where compositions are described
as having, including, or comprising specific components, it is
contemplated that compositions also consist essentially of, or
consist of, the recited components. Similarly, where methods or
processes are described as having, including, or comprising
specific process steps, the processes also consist essentially of,
or consist of, the recited processing steps. Further, it should be
understood that the order of steps or order for performing certain
actions is immaterial so long as the invention remains operable.
Moreover, two or more steps or actions can be conducted
simultaneously.
[0363] The synthetic processes of the invention can tolerate a wide
variety of functional groups, therefore various substituted
starting materials can be used. The processes generally provide the
desired final compound at or near the end of the overall process,
although it may be desirable in certain instances to further
convert the compound to a pharmaceutically acceptable salt,
polymorph or solvate thereof
[0364] Compounds of the present invention can be prepared in a
variety of ways using commercially available starting materials,
compounds known in the literature, or from readily prepared
intermediates, by employing standard synthetic methods and
procedures either known to those skilled in the art, or which will
be apparent to the skilled artisan in light of the teachings
herein. Standard synthetic methods and procedures for the
preparation of organic molecules and functional group
transformations and manipulations can be obtained from the relevant
scientific literature or from standard textbooks in the field.
Although not limited to any one or several sources, classic texts
such as Smith, M. B., March, J., March's Advanced Organic
Chemistry: Reactions, Mechanisms, and Structure, 5.sup.th edition,
John Wiley & Sons: New York, 2001; Greene, T. W., Wuts, P. G.
M., Protective Groups in Organic Synthesis, 3.sup.rd edition, John
Wiley & Sons: New York, 1999; R. Larock, Comprehensive Organic
Transformations, VCH Publishers (1989); L. Fieser and M. Fieser,
Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and
Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for
Organic Synthesis, John Wiley and Sons (1995), incorporated by
reference herein, are useful and recognized reference textbooks of
organic synthesis known to those in the art. The following
descriptions of synthetic methods are designed to illustrate, but
not to limit, general procedures for the preparation of compounds
of the present invention.
[0365] Compounds of the present invention can be conveniently
prepared by a variety of methods familiar to those skilled in the
art. The compounds of this invention with any Formula disclosed
herein may be prepared according to the procedures illustrated in
Schemes 1-10 below, from commercially available starting materials
or starting materials which can be prepared using literature
procedures. The Z and R groups (such as R.sub.2, R.sub.3, R.sub.4,
R.sub.6, R.sub.7, R.sub.8, and R.sub.12) in Schemes 1-10 are as
defined in any of Formulae disclosed herein, unless otherwise
specified.
[0366] One of ordinary skill in the art will note that, during the
reaction sequences and synthetic schemes described herein, the
order of certain steps may be changed, such as the introduction and
removal of protecting groups.
[0367] One of ordinary skill in the art will recognize that certain
groups may require protection from the reaction conditions via the
use of protecting groups. Protecting groups may also be used to
differentiate similar functional groups in molecules. A list of
protecting groups and how to introduce and remove these groups can
be found in Greene, T. W., Wuts, P. G. M., Protective Groups in
Organic Synthesis, 3.sup.rd edition, John Wiley & Sons: New
York, 1999.
[0368] Preferred protecting groups include, but are not limited
to:
[0369] For a hydroxyl moiety: TBS, benzyl, THP, Ac
[0370] For carboxylic acids: benzyl ester, methyl ester, ethyl
ester, allyl ester
[0371] For amines: Cbz, BOC, DMB
[0372] For diols: Ac (.times.2) TBS (.times.2), or when taken
together acetonides
[0373] For thiols: Ac
[0374] For benzimidazoles: SEM, benzyl, PMB, DMB
[0375] For aldehydes: di-alkyl acetals such as dimethoxy acetal or
diethyl acetyl.
[0376] In the reaction schemes described herein, multiple
stereoisomers may be produced. When no particular stereoisomer is
indicated, it is understood to mean all possible stereoisomers that
could be produced from the reaction. A person of ordinary skill in
the art will recognize that the reactions can be optimized to give
one isomer preferentially, or new schemes may be devised to produce
a single isomer. If mixtures are produced, techniques such as
preparative thin layer chromatography, preparative HPLC,
preparative chiral HPLC, or preparative SFC may be used to separate
the isomers.
[0377] The following abbreviations are used throughout the
specification and are defined below:
[0378] Ac acetyl
[0379] AcOH acetic acid
[0380] aq. aqueous
[0381] BID or b.i.d. bis in die (twice a day)
[0382] BOC tert-butoxy carbonyl
[0383] Cbz benzyloxy carbonyl
[0384] CDCl.sub.3 deuterated chloroform
[0385] CH.sub.2Cl.sub.2 dichloromethane
[0386] DCM dichloromethane
[0387] DMB 2,4 dimethoxy benzyl
[0388] DMF N,N-Dimethylformamide
[0389] DMSO Dimethyl sulfoxide
[0390] EA or EtOAc Ethyl acetate
[0391] EDC or EDCI
N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide
[0392] ESI- Electrospray negative mode
[0393] ESI+ Electrospray positive mode
[0394] EtOH ethanol
[0395] h hours
[0396] H.sub.2O water
[0397] HOBt 1-Hydroxybenzotriazole
[0398] HCl hydrogen chloride or hydrochloric acid
[0399] HPLC High performance liquid chromatography
[0400] K.sub.2CO.sub.3 potassium carbonate
[0401] LC/MS or LC-MS Liquid chromatography mass spectrum
[0402] M Molar
[0403] MeCN Acetonitrile
[0404] min minutes
[0405] Na.sub.2CO.sub.3 sodium carbonate
[0406] Na.sub.2SO.sub.4 sodium sulfate
[0407] NaHCO.sub.3 sodium bicarbonate
[0408] NaHMDs Sodium hexamethyldisilazide
[0409] NaOH sodium hydroxide
[0410] NaHCO.sub.3 sodium bicarbonate
[0411] Na.sub.2SO.sub.4 sodium sulfate
[0412] NMR Nuclear Magnetic Resonance
[0413] Pd(OH).sub.2 Palladium dihydroxide
[0414] PMB para methoxybenzyl
[0415] p.o. per os (oral administration)
[0416] ppm parts per million
[0417] prep HPLC preparative High Performance Liquid
Chromatography
[0418] PYBOP (Benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate
[0419] Rt or RT Room temperature
[0420] TBME tert-Butyl methyl ether
[0421] TFA trifluoroacetic acid
[0422] THF tetrahydrofuran
[0423] THP tetrahydropyran
[0424] The present invention also provides pharmaceutical
compositions comprising a compound of any Formula disclosed herein
in combination with at least one pharmaceutically acceptable
excipient or carrier.
[0425] A "pharmaceutical composition" is a formulation containing
the compounds of the present invention in a form suitable for
administration to a subject. In one embodiment, the pharmaceutical
composition is in bulk or in unit dosage form. The unit dosage form
is any of a variety of forms, including, for example, a capsule, an
IV bag, a tablet, a single pump on an aerosol inhaler or a vial.
The quantity of active ingredient (e.g., a formulation of the
disclosed compound or salt, hydrate, solvate or isomer thereof) in
a unit dose of composition is an effective amount and is varied
according to the particular treatment involved. One skilled in the
art will appreciate that it is sometimes necessary to make routine
variations to the dosage depending on the age and condition of the
patient. The dosage will also depend on the route of
administration. A variety of routes are contemplated, including
oral, pulmonary, rectal, parenteral, transdermal, subcutaneous,
intravenous, intramuscular, intraperitoneal, inhalational, buccal,
sublingual, intrapleural, intrathecal, intranasal, and the like.
Dosage forms for the topical or transdermal administration of a
compound of this invention include powders, sprays, ointments,
pastes, creams, lotions, gels, solutions, patches and inhalants. In
one embodiment, the active compound is mixed under sterile
conditions with a pharmaceutically acceptable carrier, and with any
preservatives, buffers, or propellants that are required.
[0426] As used herein, the phrase "pharmaceutically acceptable"
refers to those compounds, anions, cations, materials,
compositions, carriers, and/or dosage forms which are, within the
scope of sound medical judgment, suitable for use in contact with
the tissues of human beings and animals without excessive toxicity,
irritation, allergic response, or other problem or complication,
commensurate with a reasonable benefit/risk ratio.
[0427] "Pharmaceutically acceptable excipient" means an excipient
that is useful in preparing a pharmaceutical composition that is
generally safe, non-toxic and neither biologically nor otherwise
undesirable, and includes excipient that is acceptable for
veterinary use as well as human pharmaceutical use. A
"pharmaceutically acceptable excipient" as used in the
specification and claims includes both one and more than one such
excipient.
[0428] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical), and transmucosal administration. Solutions
or suspensions used for parenteral, intradermal, or subcutaneous
application can include the following components: a sterile diluent
such as water for injection, saline solution, fixed oils,
polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates, and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
The pH can be adjusted with acids or bases, such as hydrochloric
acid or sodium hydroxide. The parenteral preparation can be
enclosed in ampoules, disposable syringes or multiple dose vials
made of glass or plastic.
[0429] A compound or pharmaceutical composition of the invention
can be administered to a subject in many of the well-known methods
currently used for chemotherapeutic treatment. For example, for
treatment of cancers, a compound of the invention may be injected
directly into tumors, injected into the blood stream or body
cavities or taken orally or applied through the skin with patches.
The dose chosen should be sufficient to constitute effective
treatment but not so high as to cause unacceptable side effects.
The state of the disease condition (e.g., cancer, precancer, and
the like) and the health of the patient should preferably be
closely monitored during and for a reasonable period after
treatment.
[0430] The term "therapeutically effective amount", as used herein,
refers to an amount of a pharmaceutical agent to treat, ameliorate,
or prevent an identified disease or condition, or to exhibit a
detectable therapeutic or inhibitory effect. The effect can be
detected by any assay method known in the art. The precise
effective amount for a subject will depend upon the subject's body
weight, size, and health; the nature and extent of the condition;
and the therapeutic or combination of therapeutics selected for
administration. Therapeutically effective amounts for a given
situation can be determined by routine experimentation that is
within the skill and judgment of the clinician. In a preferred
aspect, the disease or condition to be treated is cancer. In
another aspect, the disease or condition to be treated is a cell
proliferative disorder.
[0431] For any compound, the therapeutically effective amount can
be estimated initially either in cell culture assays, e.g., of
neoplastic cells, or in animal models, usually rats, mice, rabbits,
dogs, or pigs. The animal model may also be used to determine the
appropriate concentration range and route of administration. Such
information can then be used to determine useful doses and routes
for administration in humans. Therapeutic/prophylactic efficacy and
toxicity may be determined by standard pharmaceutical procedures in
cell cultures or experimental animals, e.g., ED.sub.50 (the dose
therapeutically effective in 50% of the population) and LD.sub.50
(the dose lethal to 50% of the population). The dose ratio between
toxic and therapeutic effects is the therapeutic index, and it can
be expressed as the ratio, LD.sub.50/ED.sub.50. Pharmaceutical
compositions that exhibit large therapeutic indices are preferred.
The dosage may vary within this range depending upon the dosage
form employed, sensitivity of the patient, and the route of
administration.
[0432] Dosage and administration are adjusted to provide sufficient
levels of the active agent(s) or to maintain the desired effect.
Factors which may be taken into account include the severity of the
disease state, general health of the subject, age, weight, and
gender of the subject, diet, time and frequency of administration,
drug combination(s), reaction sensitivities, and tolerance/response
to therapy. Long-acting pharmaceutical compositions may be
administered every 3 to 4 days, every week, or once every two weeks
depending on half-life and clearance rate of the particular
formulation.
[0433] The pharmaceutical compositions containing active compounds
of the present invention may be manufactured in a manner that is
generally known, e.g., by means of conventional mixing, dissolving,
granulating, dragee-making, levigating, emulsifying, encapsulating,
entrapping, or lyophilizing processes. Pharmaceutical compositions
may be formulated in a conventional manner using one or more
pharmaceutically acceptable carriers comprising excipients and/or
auxiliaries that facilitate processing of the active compounds into
preparations that can be used pharmaceutically. Of course, the
appropriate formulation is dependent upon the route of
administration chosen.
[0434] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringeability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol and sorbitol, and sodium chloride in
the composition. Prolonged absorption of the injectable
compositions can be brought about by including in the composition
an agent which delays absorption, for example, aluminum
monostearate and gelatin.
[0435] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, methods of preparation are vacuum
drying and freeze-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof
[0436] Oral compositions generally include an inert diluent or an
edible pharmaceutically acceptable carrier. They can be enclosed in
gelatin capsules or compressed into tablets. For the purpose of
oral therapeutic administration, the active compound can be
incorporated with excipients and used in the form of tablets,
troches, or capsules. Oral compositions can also be prepared using
a fluid carrier for use as a mouthwash, wherein the compound in the
fluid carrier is applied orally and swished and expectorated or
swallowed. Pharmaceutically compatible binding agents, and/or
adjuvant materials can be included as part of the composition. The
tablets, pills, capsules, troches and the like can contain any of
the following ingredients, or compounds of a similar nature: a
binder such as microcrystalline cellulose, gum tragacanth or
gelatin; an excipient such as starch or lactose, a disintegrating
agent such as alginic acid, Primogel, or corn starch; a lubricant
such as magnesium stearate or Sterotes; a glidant such as colloidal
silicon dioxide; a sweetening agent such as sucrose or saccharin;
or a flavoring agent such as peppermint, methyl salicylate, or
orange flavoring.
[0437] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser, which contains a suitable propellant, e.g., a gas
such as carbon dioxide, or a nebulizer.
[0438] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0439] The active compounds can be prepared with pharmaceutically
acceptable carriers that will protect the compound against rapid
elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions (including liposomes targeted to infected cells with
monoclonal antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art, for
example, as described in U.S. Pat. No. 4,522,811.
[0440] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved.
[0441] In therapeutic applications, the dosages of the
pharmaceutical compositions used in accordance with the invention
vary depending on the agent, the age, weight, and clinical
condition of the recipient patient, and the experience and judgment
of the clinician or practitioner administering the therapy, among
other factors affecting the selected dosage. Generally, the dose
should be sufficient to result in slowing, and preferably
regressing, the growth of the tumors and also preferably causing
complete regression of the cancer. Dosages can range from about
0.01 mg/kg per day to about 5000 mg/kg per day. In preferred
aspects, dosages can range from about 1 mg/kg per day to about 1000
mg/kg per day. In an aspect, the dose will be in the range of about
0.1 mg/day to about 50 g/day; about 0.1 mg/day to about 25 g/day;
about 0.1 mg/day to about 10 g/day; about 0.1 mg to about 3 g/day;
or about 0.1 mg to about 1 g/day, in single, divided, or continuous
doses (which dose may be adjusted for the patient's weight in kg,
body surface area in m.sup.2, and age in years). An effective
amount of a pharmaceutical agent is that which provides an
objectively identifiable improvement as noted by the clinician or
other qualified observer. For example, regression of a tumor in a
patient may be measured with reference to the diameter of a tumor.
Decrease in the diameter of a tumor indicates regression.
Regression is also indicated by failure of tumors to reoccur after
treatment has stopped. As used herein, the term "dosage effective
manner" refers to amount of an active compound to produce the
desired biological effect in a subject or cell.
[0442] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0443] The compounds of the present invention are capable of
further forming salts. All of these forms are also contemplated
within the scope of the claimed invention.
[0444] As used herein, "pharmaceutically acceptable salts" refer to
derivatives of the compounds of the present invention wherein the
parent compound is modified by making acid or base salts thereof.
Examples of pharmaceutically acceptable salts include, but are not
limited to, mineral or organic acid salts of basic residues such as
amines, alkali or organic salts of acidic residues such as
carboxylic acids, and the like. The pharmaceutically acceptable
salts include the conventional non-toxic salts or the quaternary
ammonium salts of the parent compound formed, for example, from
non-toxic inorganic or organic acids. For example, such
conventional non-toxic salts include, but are not limited to, those
derived from inorganic and organic acids selected from
2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic,
benzene sulfonic, benzoic, bicarbonic, carbonic, citric, edetic,
ethane disulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic,
gluconic, glutamic, glycolic, glycollyarsanilic, hexylresorcinic,
hydrabamic, hydrobromic, hydrochloric, hydroiodic, hydroxymaleic,
hydroxynaphthoic, isethionic, lactic, lactobionic, lauryl sulfonic,
maleic, malic, mandelic, methane sulfonic, napsylic, nitric,
oxalic, pamoic, pantothenic, phenylacetic, phosphoric,
polygalacturonic, propionic, salicyclic, stearic, subacetic,
succinic, sulfamic, sulfanilic, sulfuric, tannic, tartaric, toluene
sulfonic, and the commonly occurring amine acids, e.g., glycine,
alanine, phenylalanine, arginine, etc.
[0445] Other examples of pharmaceutically acceptable salts include
hexanoic acid, cyclopentane propionic acid, pyruvic acid, malonic
acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid,
4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,
4-toluenesulfonic acid, camphorsulfonic acid,
4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylic acid,
3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic
acid, muconic acid, and the like. The present invention also
encompasses salts formed when an acidic proton present in the
parent compound either is replaced by a metal ion, e.g., an alkali
metal ion, an alkaline earth ion, or an aluminum ion; or
coordinates with an organic base such as ethanolamine,
diethanolamine, triethanolamine, tromethamine, N-methylglucamine,
and the like. In the salt form, it is understood that the ratio of
the compound to the cation or anion of the salt can be 1:1, or any
ration other than 1:1, e.g., 3:1, 2:1, 1:2, or 1:3.
[0446] It should be understood that all references to
pharmaceutically acceptable salts include solvent addition forms
(solvates) or crystal forms (polymorphs) as defined herein, of the
same salt.
[0447] The compounds of the present invention can also be prepared
as esters, for example, pharmaceutically acceptable esters. For
example, a carboxylic acid function group in a compound can be
converted to its corresponding ester, e.g., a methyl, ethyl or
other ester. Also, an alcohol group in a compound can be converted
to its corresponding ester, e.g., acetate, propionate or other
ester.
[0448] The compounds, or pharmaceutically acceptable salts or
solvates thereof, are administered orally, nasally, transdermally,
pulmonary, inhalationally, buccally, sublingually,
intraperintoneally, subcutaneously, intramuscularly, intravenously,
rectally, intrapleurally, intrathecally and parenterally. In one
embodiment, the compound is administered orally. One skilled in the
art will recognize the advantages of certain routes of
administration.
[0449] The dosage regimen utilizing the compounds is selected in
accordance with a variety of factors including type, species, age,
weight, sex and medical condition of the patient; the severity of
the condition to be treated; the route of administration; the renal
and hepatic function of the patient; and the particular compound or
salt thereof employed. An ordinarily skilled physician or
veterinarian can readily determine and prescribe the effective
amount of the drug required to prevent, counter, or arrest the
progress of the condition.
[0450] Techniques for formulation and administration of the
disclosed compounds of the invention can be found in Remington: the
Science and Practice of Pharmacy, 19.sup.th edition, Mack
Publishing Co., Easton, Pa. (1995). In an embodiment, the compounds
described herein, and the pharmaceutically acceptable salts
thereof, are used in pharmaceutical preparations in combination
with a pharmaceutically acceptable carrier or diluent. Suitable
pharmaceutically acceptable carriers include inert solid fillers or
diluents and sterile aqueous or organic solutions. The compounds
will be present in such pharmaceutical compositions in amounts
sufficient to provide the desired dosage amount in the range
described herein.
[0451] All percentages and ratios used herein, unless otherwise
indicated, are by weight. Other features and advantages of the
present invention are apparent from the different examples. The
provided examples illustrate different components and methodology
useful in practicing the present invention. The examples do not
limit the claimed invention. Based on the present disclosure the
skilled artisan can identify and employ other components and
methodology useful for practicing the present invention.
[0452] In the synthetic schemes described herein, compounds may be
drawn with one particular configuration for simplicity. Such
particular configurations are not to be construed as limiting the
invention to one or another isomer, tautomer, regioisomer or
stereoisomer, nor does it exclude mixtures of isomers, tautomers,
regioisomers or stereoisomers; however, it will be understood that
a given isomer, tautomer, regioisomer or stereoisomer may have a
higher level of activity than another isomer, tautomer, regioisomer
or stereoisomer.
[0453] Compounds designed, selected and/or optimized by methods
described above, once produced, can be characterized using a
variety of assays known to those skilled in the art to determine
whether the compounds have biological activity. For example, the
molecules can be characterized by conventional assays, including
but not limited to those assays described below, to determine
whether they have a predicted activity, binding activity and/or
binding specificity.
[0454] Furthermore, high-throughput screening can be used to speed
up analysis using such assays. As a result, it can be possible to
rapidly screen the molecules described herein for activity, using
techniques known in the art. General methodologies for performing
high-throughput screening are described, for example, in Devlin
(1998) High Throughput Screening, Marcel Dekker; and U.S. Pat. No.
5,763,263. High-throughput assays can use one or more different
assay techniques including, but not limited to, those described
below.
[0455] An EZH2 inhibitor of the present invention may, if desired,
be presented in a kit (e.g., a pack or dispenser device) which may
contain one or more unit dosage forms containing the EZH2
inhibitor. The pack may for example comprise metal or plastic foil,
such as a blister pack. The pack or dispenser device may be
accompanied by instructions for administration. Compositions
comprising an EZH2 inhibitor of the invention formulated in a
compatible pharmaceutical carrier may also be prepared, placed in
an appropriate container, and labeled for treatment of an indicated
condition. Instructions for use may also be provided.
[0456] Also provided herein are kits comprising a plurality of
methylation detection reagents that detect the methylated H3-K27.
For example, the kit includes mono-methylated H3-K27, di-methylated
H3-K27 and tri-methylated H3-K27 detection reagents. The detection
reagent is for example antibodies or fragments thereof, polypeptide
or aptamers.
[0457] A kit may also include reagents for detecting loss of
function of at least one component of the SWI/SNF complex, e.g.,
nucleic acids that specifically identify a mutant component nucleic
acid sequence by having homologous nucleic acid sequences, such as
oligonucleotide sequences, complementary to a portion of the mutant
component nucleic acid sequence or antibodies to proteins encoded
by the wild type and/or mutant component nucleic acids packaged
together in the form of a kit. The oligonucleotides can be
fragments of the component gene. For example the oligonucleotides
can be 200, 150, 100, 50, 25, 10 or less nucleotides in length. The
kit may contain in separate containers an aptamer or an antibody,
control formulations (positive and/or negative), and/or a
detectable label such as fluorescein, green fluorescent protein,
rhodamine, cyanine dyes, Alexa dyes, luciferase, radiolabels, among
others. In addition, reagents for detecting the biological activity
of the SWI/SNF complex (such as its chromatin remodeling activity)
may be included in the kit.
[0458] Instructions (e.g., written, tape, VCR, CD-ROM, etc.) for
carrying out the assay may be included in the kit. The assay may
for example be in the form of a Western Blot analysis,
Immunohistochemistry (IHC), immunofluorescence (IF), sequencing and
Mass spectrometry (MS) as known in the art.
Example 1: Durable Tumor Regression in Genetically Altered
Lymphomas and Malignant Rhabdoid Tumors by Inhibition of EZH2
[0459] Compound a is a Potent and Selective Inhibitor of EZH2:
[0460] Cell free biochemical assays that included radiolabeled SAM
and either chicken erythrocyte oligonucleosomes or peptides
corresponding to H3K27 as substrates showed that Compound A
selectively inhibited the activity of human PRC2 containing
wild-type EZH2 with an inhibition constant (Ki) value of 2.5.+-.0.5
nmol/L and IC.sub.50 values of 11.+-.5 nM (nucleosome assay) or
16.+-.12 nM (peptide assay). The IC.sub.50 values were similar for
human and rat EZH2 enzymes as well as for EZH2 proteins bearing all
known lymphoma change-of-function mutations. The IC.sub.50 value of
Compound A increased with increasing concentration of SAM, but was
minimally affected by increasing the amount of oligonucleosome
which is consistent with a SAM-competitive and
nucleosome-noncompetitive modality of inhibition. In order to
demonstrate HMT selectivity, inhibition by Compound A against a
panel of HMTs other than EZH2 encompassing both lysine and arginine
HMTs was assessed. Compound A displayed a 35-fold selectivity
versus EZH1 and greater than 4500-fold selectivity relative to the
14 other HMTs tested.
[0461] Compound a Specifically Inhibits Cellular H3K27 Methylation
in Cells:
[0462] When WSU-DLCL2 EZH2 Y641F mutant lymphoma cells were
incubated with Compound A for 4 days, a concentration-dependent
reduction in global H3K27Me3 levels was observed with an average
IC.sub.50 value of 0.26 .mu.M (H3K27Me3 levels determined by
ELISA). When studying the kinetics of methylation inhibition, the
half-life of H3K27Me3 was approximately 1 day as 90% inhibition was
only achieved after 3 to 4 days of incubation. When OCI-LY19 EZH2
wild-type lymphoma cells were incubated with 2.7 .mu.M Compound A
for 4 days, the only methyl marks affected were the H3K27Me1,
H3K27Me2 and H3K27Me3, the three known products of PRC2 catalysis.
Incubation with Compound A also resulted in an increase in H3K27
acetylation. The ability of Compound A to reduce global H3K27
trimethylation levels was further tested in several other human
lymphoma cell lines including lines expressing either wild-type or
mutant EZH2. Compound A reduced H3K27Me3 with similar potency in
all cell lines independent of the EZH2 status (Table 1).
[0463] Compound a Leads to Selective Killing of Lymphoma Cell Lines
Bearing EZH2 Point Mutations:
[0464] Incubation of WSU-DLCL2 EZH2 Y641F mutant cells with
Compound A lead to anti-proliferative effects with an average
IC.sub.50 value of 0.28.+-.0.14 .mu.M in a 6 day proliferation
assay. The kinetics of the effect of Compound A on viable cell
number was further tested over an extended period of 11 days. The
antiproliferative effect of Compound A was apparent after WSU-DLCL2
cells had been exposed to compound for longer than 4 days,
consistent with the kinetics of Compound A-mediated cellular H3K27
methylation inhibition. The IC.sub.50 value for Compound A
inhibition of proliferation of WSU-DLCL2 cells in the 11-day assay
(0.0086 Table 1) was lower when compared with results obtained with
a 6-day proliferation assay, suggesting increased sensitivity with
longer incubation periods. In contrast to the WSU-DLCL2 cells, the
growth of OCI-LY19 human lymphoma cells (EZH2 wild type for residue
Y641) over 11 days was not significantly affected, despite
comparable IC.sub.50 values for H3K27Me3 inhibition for both cell
lines (Table 1). In order to identify a concentration at which
cells stop proliferating considering the entire incubation period
of 11 days, the lowest cytotoxic concentration (LCC) for a
particular cell line was calculated. The LCC value for WSU-DLCL2
EZH2 Y641F mutant human lymphoma cells was significantly lower when
compared with OCI-LY19 cells that are wild type for EZH2 (Table 1).
This context specific cell killing was further supported by results
from 11-day proliferation assays with an extended lymphoma cell
line panel. All cell lines harboring an EZH2 mutation, with the
exception of the RL cell line (EZH2 Y641N), were more sensitive to
the antiproliferative effects of Compound A when compared with cell
lines with wild-type EZH2 (Table 1). The Pfeiffer cell line (EZH2
A677G) showed a 20 to 300 fold increase in sensitivity to Compound
A, as measured by IC.sub.50 value and LCC, respectively, over the
Y641 mutant cell lines. Next the minimum time of compound exposure
necessary for sustained cell killing was investigated by washout
experiments. The LCC values on day 11 or 14 for WSU-DLCL2 cells
that were either incubated with Compound A for 7 days (followed by
7 days of compound washout) or continuously for 14 days were
similar (Table 2). Drug exposure for only 4 days, however, was not
sufficient to induce LCC values similar to continuous
incubation.
[0465] Compound a Induces G.sub.1 Arrest and Apoptosis in EZH2
Mutant Lymphoma Cells:
[0466] Next, the effects of incubation with Compound A (1 .mu.M)
for 7 days on cell cycle progression and apoptosis in WSU-DLCL2
cells were assessed. An increase in the percentage of cells in
G.sub.1 phase, and a decrease in the percentage of cells in S phase
and G2/M phase was apparent after 2 days of Compound A incubation.
The maximum effect was achieved after 4 days. There was no apparent
increase in the sub-G.sub.1 fraction suggesting that apoptosis was
not induced by Compound A incubation for 7 days. This is in
agreement with the growth curves of WSU-DLCL2 cells in the presence
of Compound A indicating that cytotoxic effects were observed only
after 7 days of incubation. Following incubation of WSU-DLCL2 cells
with Compound A for up to 14 days, the fraction of apoptotic cells
determined by TUNEL assay was significantly increased on day 14
compared to vehicle, indicating that Compound A-mediated cell death
occurred through the induction of apoptosis.
[0467] Oral Administration of Compound a Leads to EZH2 Target
Inhibition in EZH2 Mutant Xenograft Models in Mice:
[0468] The effect of oral dosing of Compound A on systemic compound
exposure and in vivo target inhibition in mice bearing EZH2 mutant
lymphoma xenografts was investigated. First, SCID mice implanted
subcutaneously with WSU-DLCL2 xenografts were orally dosed with
Compound A for 4 or 7 days. Measuring Compound A plasma levels
either 5 minutes before or 3 hours after the last dose revealed a
clear dose dependent increase in exposure. Only animals dosed at
160 mg/kg TID or 213 mg/kg BID maintained mean compound levels in
plasma above the LCC for WSU-DLCL2 cells throughout a dosing cycle
(1652 ng/mL, with mouse plasma protein binding considered).
Compound determination in homogenates from tumors collected 3 hours
after the last dose revealed that only for the highest dose groups
compound levels in the 2 compartments were similar. When H3K27Me3
levels in tumors were analyzed, dose dependent EZH2 target
inhibition was observed. H3K27Me3 inhibition was less in tumors
from mice dosed at 213 mg/kg QD, suggesting that maintaining a
plasma concentration above LCC throughout a dosing cycle is
required for optimal target inhibition. Dosing for 4 days at 160
mg/kg TID resulted in slightly lower target inhibition than dosing
for 7 days at the same dose and schedule, indicating that prolonged
dosing increased the degree of target inhibition in WSU-DLCL2
tumors. A similar 7-day study in nude mice implanted subcutaneously
with KARAPS-422 xenografts assessing both BID and QD schedules was
performed. Compound A induced a dose-dependent reduction of tumor
H3K27Me3 levels at both regimens.
[0469] Compound a Induces Significant Antitumor Effects in Several
EZH2 Mutant Lymphoma Xenografts:
[0470] When WSU-DLCL2 EZH2 Y641F mutant xenograft tumor bearing
SCID mice were treated with Compound A for 28 days, dose-dependent
tumor growth inhibition, 58% at the highest dose of 150 mg/kg TID,
was observed. Only animals administered the highest dose maintained
mean Compound A plasma levels above LCC for WSU-DLCL2 cells
throughout the dosing cycle. Dosing of Compound A for 28 days led
to a relative compound accumulation in tumor tissue compared with
plasma, in contrast to what was observed with 7-day dosing. ELISA
analysis of histones from tumors collected on day 28 indicated
dose-dependent target inhibition. H3K27Me3 levels in WSU-DLCL2
xenografts were lower in mice dosed for 28 days compared with 7
days indicating that prolonged administration of Compound A
increased the degree of target inhibition. In KARPAS-422 EZH2 Y461N
mutant xenografts, 28-day dosing of Compound A on a BID schedule
had much more dramatic effects. Tumor growth inhibition was
observed at doses as low as 80.5 mg/kg BID, but higher doses
eradicated the xenografts, and no re-growth was observed for up to
90 days after cessation of dosing. When intermittent dosing
schedules were investigated in KARPAS-422 xenograft bearing mice,
Compound A again showed significant dose-dependent antitumor
effects with two cycles of 7-day on/7-day off and 21 day on/7 day
off schedules. For all dosing schedules, tumor growth inhibition
and complete regressions were observed at 90 and 361 mg/kg BID,
respectively. The Pfeiffer EZH2 A677G mutant xenograft model was
the most sensitive tumor model, as suggested by the potent
anti-proliferative effects of Compound A on this cell line in
vitro. All Compound A dose groups (QD schedule) except the lowest
one (30 mg/kg QD) showed complete tumor regressions in all animals.
Again, tumor re-growth was not observed until the end of the study
(36 days after stopping Compound A administration). Although tumor
re-growth was observed at 30 mg/kg QD, this very low dose induced
tumor stasis during the administration period. Due to tolerability
issues dosing was stopped on day 12 for mice administered 1140
mg/kg QD; still, durable complete regressions were observed in this
group that were only exposed to Compound A for 12 days.
[0471] Compound a Selectively Kills SMARCB1 Mutant MRT Cells In
Vitro and In Vivo:
[0472] Whether EZH2 inhibition had any effects on the growth and
survival of SMARCB1-deleted MRT cells was tested. Incubating
SMARCB1-deleted MRT cell lines G401 and A204 with Compound A in a
14-day proliferation assay in vitro induced strong
anti-proliferative effects with IC.sub.50 values in the nM range
while the control cell lines RD and SJCRH30 which expressed SMARCB1
were minimally affected (Table 3). Dosing of SCID mice bearing
subcutaneous G401 xenografts with Compound A at 266 or 532 mg/kg
BID for 28 days eliminated those extremely fast growing tumors.
Similar to the KARPAS-422 and Pfeiffer EZH2 mutant NHL xenograft
models re-growth was not observed at study end, 32 days after
dosing stop. Compound A dosed at 133 mg/kg induced stasis during
the administration period, and produced a significant tumor growth
delay compared to vehicle after dosing stop. Tumors that were
harvested from subsets of mice from each group on day 21 showed
strong EZH2 target inhibition at all doses.
[0473] Compound a Inhibits 113K27 Methylation in Nontumor Tissues
in a Dose Dependent Manner:
[0474] The data described above demonstrate that Compound A
represents a new treatment modality for SWI/SNF driven cancers and
MRTs. Measuring pharmacodynamic biomarker modulation post-dose is
often performed in early clinical trials to assess the degree of
target inhibition that is predicted to produce a response based on
data from preclinical models. Since the collection of post-dose
tumor biopsies is often not possible, easier accessible surrogate
tissues such as peripheral blood mononuclear cells (PBMCs), skin or
bone marrow are often collected instead. To test EZH2 target
inhibition in surrogate tissues male and female Sprague Dawley rats
were orally administered 100, 300, or 1000 mg/kg Compound A for 28
days, and PBMCs, bone marrow and skin samples were collected at
study end. Plasma levels of Compound A increased dose-dependently
in both male and female rats, and the plasma levels were generally
higher in females compared with those in males. Due to tolerability
issues, females in the 1000 mg/kg group had to be euthanized on day
23. Dose-dependent target inhibition was observed in PBMCs and bone
marrow from rats dosed with Compound A, as measured by ELISA. The
degree of target inhibition was less pronounced for PBMCs from
females that were dosed for 22 days compared with males that were
dosed for 28 days (same dose of 1000 mg/kg). A dose dependent
reduction in H3K27Me3 positive cells was observed in the epidermis
of skin of Compound A-dosed rats, as assessed by an IHC assay. The
maximum effect was observed at the highest dose, and was already
evident after 22 days of Compound A administration.
[0475] Compound A displayed similar properties as other EZH2
inhibitors in vitro, such as very high specificity for EZH2 in
biochemical assays when compared with other HMTs and specific
inhibition of cellular H3K27 methylation leading to context
specific killing of EZH2 mutated NHL cell lines. However, this
compound achieved an approximately 10-fold increase in potency,
reflected by decreased K.sub.i and IC.sub.50 values determined in
biochemical and cell-functional assays. In addition, Compound A
showed excellent oral bioavailability when administered to rodents
which lead to dose dependent EZH2 target inhibition in xenograft
tumor and nontumor tissues. Importantly, dosing of Compound A
induced significant antitumor effects in mice bearing EZH2 mutant
lymphoma xenografts. The responses ranged from tumor eradication
(no regrowth after dosing cessation) to dose-dependent tumor growth
inhibition. The delayed onset of antitumor activity (after 4 to 7
days) was consistent with the kinetics of methylation inhibition
and antiproliferative activity induced by incubation of cells with
Compound A in vitro. Keeping Compound A plasma levels above LCC
throughout a dosing cycle was necessary for the WSU-DLCL2 xenograft
model to induce maximal target inhibition and antitumor response.
The other two lymphoma xenograft models (KARPAS-422 and Pfeiffer),
however, were extremely sensitive to Compound A administration, and
keeping plasma levels above LCC was not necessary. Pfeiffer EZH2
A677G mutant xenograft tumors disappeared permanently with very low
doses or short dosing periods, suggesting that patients with this
type of genetically defined NHL would have a significant treatment
effect with Compound A.
[0476] MRTs are extremely aggressive pediatric cancers of the
brain, kidney, and soft tissues that are highly malignant, locally
invasive, frequently metastatic, and particularly lethal, but they
are typically diploid and lack genomic aberrations. They are,
however, characterized by an almost complete penetrance of loss of
expression of the SMARCB1, a core component of the SWI/SNF
chromatin remodeling complex. The biallelic inactivation of
SMARCB1, for instance induced by mutations, is in essence the sole
genetic event in MRTs which suggests a driver role for this genetic
aberration. Through genetic studies it has been suggested that PRC2
and SWI/SNF antagonistically regulate gene expression around the
RB, Cyclin D1 and MYC pathways. Here, it has been demonstrated
pharmacological EZH2 inhibition induced antiproliferative effects
in SMARCB1 deleted MRT cell lines and permanently eradicated MRT
xenografts in mice. This confirms the dependency of such cancers,
in which EZH2 itself is not genetically altered, on PRC2
activity.
[0477] Compound A represents a new treatment modality for
genetically defined subsets of NHL and for MRTs. The ability to
measure dose-dependent changes in H3K27Me3 levels in skin, PBMCs
and bone marrow portends the use of signal from these surrogate
tissues as a non-invasive pharmacodynamics biomarker in human
clinical trials.
TABLE-US-00013 TABLE 1 IC.sub.50 Values for Methylation and
Proliferation as well as LCC Values for Compound A in Human
Lymphoma Cell Lines Methylation Proliferation EZH2 IC.sub.50
IC.sub.50 Cell Line Status (nmol/L).sup.a (.mu.mol/L).sup.b LCC
(.mu.mol/L).sup.b DOHH-2 Wild Type ND 1.7 >10 Farage Wild Type
ND 0.099 >10 OCI-LY19 Wild Type 8 6.2 10-25 Toledo Wild Type ND
7.6 >10 Karpas-422 Y641N 90 0.0018 0.12 Pfeiffer A677G 2 0.00049
0.0005 RL Y641N 22 5.8 >25 SU-DHL-10 Y641F ND 0.0058 0.14
SU-DHL-6 Y641N 20 0.0047 0.21 WSU-DLCL2 Y641F 9 0.0086 0.17
.sup.aDerived after incubation for 4 days by immunoblot. Values
represent the result from one experiment. .sup.bDerived after
incubation for 11 days. Compound incubations for each experiment
were performed in triplicate, and values represent one experiment
for all cell lines except OCI-LY19, Pfeiffer, and WSU-DLCL2. For
the remaining three cell lines, values represent the mean from the
following number of experiments: OCI-LY19 n = 9; Pfeiffer n = 2 and
WSU-DLCL2 n = 15.
TABLE-US-00014 TABLE 2 LCC Values for Compound A for WSU-DLCL2
Human Lymphoma Cells Dosed Either Continuously or After Compound
Washout Day 11 Day 14 WSU-DLCL2 Washout LCC (.mu.M) LCC (.mu.M) No
Washout 0.17 0.11 4-day Compound A; 11-day Washout 0.36 0.42 7-day
Compound A; 7-day Washout 0.19 0.075 Values represent the mean of
duplicate experiments with three replicates per incubation
concentration within the experiments.
TABLE-US-00015 TABLE 3 IC.sub.50 Values for Compound A for SMARCB1
Negative MRT Cell Lines and SMARCB1 Positive Control Cell Lines
Proliferation Proliferation Cell Line SMARCB1 Status IC.sub.50
(.mu.M), day 7 IC.sub.50 (.mu.M), day 14 RD Wild Type 9.2 5.2
SJCRH30 Wild Type 6.1 8.8 G401 Mutant 0.087 0.042 A204 Mutant 3.2
0.14 Values represent the mean of duplicate experiments with three
replicates per incubation concentration within the experiments.
Example 2: Durable Tumor Regression in Genetically Altered
Malignant Rhabdoid Tumors by Inhibition of EZH2
[0478] Compound a is a Potent and Selective Inhibitor of EZH2:
[0479] Compound A was developed through iterative medicinal
chemistry (FIG. 10A). Compound A inhibited the activity of human
PRC2 containing wild-type EZH2 with an inhibition constant (Ki)
value of 2.5.+-.0.5 nM, and similar potency was observed for EZH2
proteins bearing all known lymphoma change-of-function mutations
(Table 5). The compound was found to be SAM-competitive and
nucleosome-noncompetitive by steady state kinetic studies (FIG.
11). Inhibition by Compound A against a panel of HMTs other than
EZH2 encompassing both lysine and arginine HMTs was also assessed.
Compound A displayed a 35-fold selectivity versus EZH1 and
>4500-fold selectivity relative to 14 other HMTs tested (Table
5).
TABLE-US-00016 TABLE 4 Histone Methyltransferase Inhibition by
Compound A % Inhibition at 1 .mu.M Enzyme Assay IC.sub.50 (nM)
Compound A.sup.a CARM1 >50,000.sup.b 5 .+-. 3 DOT1L
>50,000.sup.c 2 .+-. 8 EHMT1 >50,000.sup.c 6 .+-. 6 EHMT2
>50,000.sup.c 7 .+-. 3 EZH1.sup.d,e 392 .+-. 72.sup.f 98 .+-. 1
EZH2 Peptide Assay.sup.d,e 11 .+-. 5.sup.f ND EZH2 Nucleosome
Assay.sup.d 16 .+-. 12.sup.f 100 .+-. 1 A677G EZH2.sup.d,e 2.sup.b
ND A687V EZH2.sup.d,e 2.sup.b ND Y641F EZH2.sup.d,e 14 .+-. 5.sup.f
ND Y641C EZH2.sup.d,e 16.sup.c ND Y641H EZH2.sup.d,e 6.sup.c ND
Y641N EZH2.sup.d,e 38.sup.b ND Y641S EZH2.sup.d,e 6.sup.c ND rat
EZH2.sup.d,e 4.sup.c ND PRMT1 >50,000.sup.c 5 .+-. 4 PRMT3 ND 2
.+-. 2 PRMT5/MEP50 >50,000.sup.c 2 .+-. 6 PRMT6 ND 3 .+-. 3
PRMT8 >50,000.sup.c 7 .+-. 3 SETD7 ND 4 .+-. 3 SMYD2
>50,000.sup.c 1 .+-. 2 SMYD3 ND 0 .+-. 5 WHSC1 >100,000.sup.c
8 .+-. 3 WHSC1L1 >100,000.sup.c 9 .+-. 8 .sup.aValues represent
the mean and standard deviation of duplicate experiments determined
at 10 .mu.mol/L Compound A. .sup.bValues represent the mean of
duplicate experiments with two replicates per experiment.
.sup.cValues represent one experiment with two replicates per
experiment. .sup.dAll EZH1 and EZH2 proteins were assayed in the
context of 4 PRC2 components (EZH1/2, SUZ12, RBAP48, EED).
.sup.eAssaved with H3K27 peptides as substrates.
[0480] Compound a Specifically Inhibits Cellular 113K27 Methylation
Leading to Selective Apoptotic Killing of SMARCB1 Mutant MRT
Cells:
[0481] A panel of SMARCB1 deficient MRT cells and SMARM wild-type
control cells (confirmed by immunoblot, FIG. 12A) were treated with
Compound A for 4 days, resulting in concentration-dependent
reductions in global H3K27Me3 levels (FIG. 10B and table 6).
Treatment of either wild-type or mutant cells resulted in
diminution only of methyl marks on H3K27, with no other histone
methyl marks being affected (FIG. 12B). In vitro treatment of
SMARCB1-deleted MRT cell lines with Compound A induced strong
anti-proliferative effects with IC.sub.50 values in the nM range;
while the control (wild-type) cell lines were minimally affected
(FIG. 10C and table 6). Antiproliferative effects were apparent in
SMARCB1-deleted MRT cells after 7 days of compound exposure, but
required 14 days of exposure for maximal activity. The effects of
incubation with Compound A (1 .mu.M) for 14 days on cell cycle
progression and apoptosis in G401 and RD cells were also assessed.
Compound A incubation of RD SMARCB1 wild-type cells showed no
changes in cell cycle or apoptosis compared to the DMSO control
(FIG. 13A). In contrast, G401 SMARCB1-deleted cells showed an
increase in the percentage of cells in G.sub.1 phase, and a
concomitant decrease in S phase and G2/M phase after 7 days (FIG.
13B). There was no apparent increase in the sub-G.sub.1 fraction
through day 7, suggesting that apoptosis was not yet induced by
that time. This coincides with the growth curves of G401 cells in
the presence of Compound A that display cytotoxicity only after 7
days of incubation (FIG. 10C). Following Compound A treatment of
G401 cells for up to 14 days, the fraction of cells in sub-G.sub.1
as well as apoptotic cells determined by TUNEL assay increased in a
time dependent manner through days 11 and 14, indicating that
Compound A-mediated cell death occurred through the induction of
apoptosis (FIG. 13B).
TABLE-US-00017 TABLE 6 SMARCB1 Proliferation IC.sub.50 on Cell Line
Status Methylation IC.sub.50 (nM).sup.a Day 14 (nM).sup.b G401
mutant 2.7 135 A204 mutant 1.4 590 G402 mutant 1.7 144 KYM-1 mutant
4.3 32 RD wild-type 5.6 6100, >10000.sup.c 293 wild-type 2.4
>10000 SJCRH30 wild-type 4.9 5100, >10000.sup.c .sup.aDerived
after incubation for 4 days, extraction of histones, immunoblot and
densitometry. Values represent the mean from two experiments.
.sup.bCompound incubations for each experiment were performed in
triplicate, and values represent the mean of 2 experiments for all
cell lines. .sup.cMean calculation of duplicate experiment not
possible.
[0482] Compound a Induces Genes of Neuronal Differentiation and
Cell Cycle Inhibition while Suppressing Expression of Hedgehog
Pathway Genes, MYC and EZH2:
[0483] It has been suggested that SMARCB1 loss drives cancer
formation through simultaneous epigenetic perturbation of key
cancer pathways. The present data confirmed the previously
described reduced expression of genes important for neuronal
differentiation (CD133, DOCK4, PTPRK), cell cycle inhibition
(CDKN2A) and tumor suppression (BIN1), as well as increased
expression of the hedgehog pathway gene GLI1 in SMARCB1-deleted
G401 cells compared to control cells (FIG. 14A). Compound A
treatment of G401 cells for up to 7 days strongly induced
expression of CD133, DOCK4 and PTPRK and up-regulated cell cycle
inhibitors CDKN1A and CDKN2A and tumor suppressor BIN1, all in a
time-dependent manner (FIG. 14B). Simultaneously, the expression of
hedgehog pathway genes, MYC and EZH2 were reduced. Notably, G402
SMARCB1-deleted cells exposed to Compound A for 14 days assumed a
neuron-like morphology (FIG. 14C). In contrast, Compound A
incubation of RD control cells had minimal effect on expression of
the above-mentioned genes.
[0484] Compound a Eradicates SMARCB1 Mutant MRT Xenografts:
[0485] Oral dosing of Compound A led to systemic compound exposure,
in vivo target inhibition and antitumor activity in mice bearing
SMARCB1-deleted MRT xenografts. A study in SCID mice bearing
subcutaneous G401 xenografts was performed where animals were dosed
for 21 days with Compound A. Half of the mice per group were
euthanized on day 21 to collect blood and tissues, while the
remaining animals were treated for an additional 7 days and then
left without dosing for another 32 days. Compound A was well
tolerated at all doses with minimal effect on body weight (FIG.
15A). Dosing at 250 or 500 mg/kg twice daily (BID) for 21 to 28
days practically eliminated the fast-growing G401 tumors (FIGS.
15B, 14C and 16A). Re-growth was not observed for 32 days after
dose cessation. Compound A dosed at 125 mg/kg induced tumor stasis
during the administration period, and produced a significant tumor
growth delay compared to vehicle after the dosing period. Measuring
Compound A plasma levels either 5 min before or 3 h after dosing on
day 21 revealed a clear dose-dependent increase in systemic
exposure (FIG. 15D). Tumors that were harvested from subsets of
mice from each group on day 21 showed strong inhibition of
H3K27me3, correlating with the antitumor activity (maximum effect
achieved at 250 mg/kg, FIG. 16B). In addition, dose-dependent
changes in the expression of CD133, PTPRK, DOCK4 and GLI1 were
detected in the G401 xenograft tumors (FIG. 16C).
[0486] The present data demonstrate that pharmacological inhibition
of EZH2 induced antiproliferative effects specifically in
SMARCB1-deleted MRT cell lines and permanently eradicated MRT
xenografts in mice. This confirms the dependency of such cancers on
PRC2 activity, despite the fact that EZH2 itself is not genetically
altered in this context. Data presented herein show that in the
context of SMARCB1-deleted MRT, inhibition of EZH2 functions as a
SMARCB1 surrogate and de-represses neuronal differentiation genes,
cell cycle inhibitors and tumor suppressors while reducing GLI1,
PTCH1, MYC and EZH2. The sum of the effects of Compound A mediated
EZH2 inhibition on several cancer pathways is the cause for the
dramatic and permanent anti-tumor activity seen in MRT models.
Thus, Compound A represents a new treatment modality for these
lethal childhood tumors.
[0487] Furthermore, since several members of the SWI/SNF complex
are genetically altered in other cancer types besides MRT, it is
conceivable that EZH2 also plays a role in tumor maintenance and
survival in a spectrum of cancer types. Combined with recent
reports demonstrating the effectiveness of EZH2 inhibitors in
selective killing of EZH2 mutant bearing non-Hodgkin lymphomas, the
present data demonstrate that small molecule-based inhibition of
EZH2 is an effective mechanism of therapeutic intervention in a
variety of hematologic and solid tumors for which genetic
alterations--either target-directed or indirect--confer a
proliferative dependency on EZH2 enzymatic activity.
Example 3: Material and Methods
[0488] Cell Culture:
[0489] Cell lines 293T, RD, SJCRH30, A204, G401, G402, and KYM-1.
293T (CRL-11268), RD (CRL-136), SJCRH30 (CRL-2061), A204 (HTB-82),
G401 (CRL-1441), and G402 (CRL-1440) were obtained from ATCC. KYM-1
(JCRB0627) was obtained from JCRB. 293T and RD cells were cultured
in DMEM+10% FBS. SJCRH30 cells were cultured in RPMI+10% FBS. A204,
G401, and G402 cells were cultured in McCoys 5a+10% FBS. KYM-1
cells were cultured in DMEM/Ham's F12+10% FBS.
[0490] Western Blots Analysis:
[0491] Histones were acid extracted as previously described (Daigle
et al., Blood. 2013 Aug. 8; 122(6):1017-25). Western blots for acid
extracted histones were performed as previously described (Knutson
et al., Proc Natl Acad Sci USA. 2013 May 7; 110(19):7922-7). Whole
cell lysates (WCL) were prepared using a modified RIPA buffer
(10.times.RIPA Lysis Buffer (Millipore #20-188), 0.1% SDS
(Invitrogen AM9823), protease mini-tablet (Roche #1836153)). Cells
were pelleted, washed with ice cold PBS, resuspended in ice cold
RIPA buffer, and incubated on ice for 5 minutes. Lysates were
sonicated 3.times. for 10 sec at 50% power, then incubated on ice
for 10 minutes. Lysates were then centrifuged at max speed for 15
minutes at 4 degrees in a table top centrifuge. Clarified lysates
were aliquoted to a fresh tube, and protein concentrations for WCL
were determined by BCA assay (Pierce). Ten micrograms of each
lysate was fractionated on 10-20% Tris-Glycine gel (Biorad),
transferred using iBlot (7 minutes on program 3, using
Nitrocellulose transfer stacks), and probed with the following
antibodies in Odyssey blocking buffer: SNF5 (CST #8745), EZH2 (CST
#5246), and Beta-actin (CST #3700).
[0492] In Vitro Cell Assays:
[0493] For the adherent cell line proliferation assays (all cell
lines except KYM-1, which was analyzed as previously described for
suspension cell lines (Daigle et al., Blood. 2013 Aug. 8;
122(6):1017-25), plating densities for each cell line were
determined based on growth curves (measured by ATP viability) and
density over a 7 day timecourse. On the day before compound
treatment, cells were plated in either 96-well plates in triplicate
(for the day 0-7 timecourse) or 6-well plates (for replating on day
7 for the remainder of the timecourse). On Day 0, cells were either
untreated, DMSO-treated, or treated with Compound A starting at 10
uM and decreasing in either 3- or 4-fold dilutions. Plates were
read on Day 0, Day 4, and Day 7 using CellTiter-Glo.RTM. (Promega),
with compound/media being replenished on Day 4. On Day 7, the
6-well plates were trypsinized, centrifuged, and resuspended in
fresh media for counting by Vi-Cell. Cells from each treatment were
replated at the original density in 96-well plates in triplicate.
Cells were allowed to adhere to the plate overnight, and cells were
treated as on Day 0. On Day 7, 11 and 14, plates were read using
CellTiter-Glo.RTM., with compound/media being replenished on Day
11. Averages of triplicates were used to plot proliferation over
the timecourse, and calculate IC50 values. For cell cycle and
apoptosis, G401 and RD cells were plated in 15 cm dishes in
duplicate at a density of 1.times.10.sup.6 cells per plate. Cells
were incubated with Compound A at 1 uM, in a total of 25 mL, over a
course of 14 days, with cells being split back to original plating
density on day 4, 7, and 11. Cell cycle analysis and TUNEL assay
were performed using a Guava.RTM. flow cytometer, following the
manufacturer's protocol.
[0494] Gene Expression Analysis:
[0495] G401 and RD cells were plated in T-75 flasks at 175,000
cells/flask and 117,000 cells/flask respectively and allowed to
adhere overnight. On Day 0, cells were treated in duplicates with
DMSO or 1 uM Compound A. Cells were harvested and pelleted on Day
2, 4, and 7 with media and compound being replenished on Day 4.
Tumor tissue from the G401 xenograft animals dosed for 21 days
(vehicle, 125 mg/kg, and 250 mg/kg (6 animals each) and 500 mg/kg
(4 animals) Compound A dose groups) were used for gene expression
analysis. Total mRNA was extracted from cell pellets and tumor
tissue using the RNeasy Mini Kit (Qiagen #74106) and reverse
transcribed by the High Capacity cDNA Reverse Transcription Kit
(Applied Biosystems (AB) #4368813). RT-PCR was performed by
ViiA.TM. 7 Real-Time PCR Systems (AB) using TaqMan Fast Advanced
Master Mix (AB #4444964) and TaqMan primer/probe sets in table
below. Gene expression was normalized to 18S (AB #Hs99999901_s1)
and fold change was calculated using the .DELTA..DELTA.Ct method.
For the in vivo samples, the average Ct value+/-SD was determined
for each dose group and fold change compared to vehicle dose group
was calculated using the .DELTA..DELTA.Ct method.
TABLE-US-00018 Gene AB# MYC Hs00153408_m1 EZH2 Hs00172783_m1 PTCH1
Hs00181117_m1 PROM1 Hs01009250_m1 (CD133) GLI1 Hs01110766_m1 DOCK4
Hs00206807_m1 PTPRK Hs00267788_m1 BIN1 Hs00184913_m1
[0496] ELISA:
[0497] Histones were isolated from tumors as previously described
(Daigle et al) and were prepared in equivalent concentrations (0.5
ng/ul for H3 and 4 ng/ul for H3K27Me3) in coating buffer (PBS with
0.05% BSA). Sample or standard (100 .mu.L) was added in duplicate
to two 96-well ELISA plates (Thermo Labsystems, Immulon 4HBX
#3885). Histones isolated from G401 cells that were treated with
DMSO or 10 .mu.mol/L Compound A for 4 days were added to control
wells at the same histone concentration as the tumor histone
samples. The plates were sealed and incubated overnight at
4.degree. C. The following day, plates were washed 3 times with 300
.mu.L/well PBST (PBS with 0.05% Tween 20; 10.times.PBST, KPL
#51-14-02) on a Bio Tek plate washer. Plates were blocked with 300
.mu.L/well of diluent (PBS+2% BSA+0.05% Tween 20), incubated at
room temperature for 2 hours, and washed 3 times with PBST. All
antibodies were diluted in diluent. 100 uL/well of anti-H3K27Me3
(CST #9733, 50% glycerol stock 1:1000) or anti-total H3 (Abcam
#ab1791, 50% glycerol stock 1:10,000) was added to each plate.
Plates were incubated for 90 minutes at room temperature and washed
3 times with PBST. 100 .mu.L/well of anti-Rb-IgG-HRP (Cell
Signaling Technology, 7074) was added 1:2000 to the H3K27Me3 plate
and 1:6000 to the H3 plate and incubated for 90 minutes at room
temperature. Plates were washed 4 times with PB ST. For detection,
100 .mu.L/well of TMB substrate (BioFx Laboratories, #TMBS) was
added and plates incubated in the dark at room temperature for 5
minutes. Reaction was stopped with 100 .mu.L/well 1N H2504.
Absorbance at 450 nm was read on SpectraMax M5 Microplate
reader.
[0498] Xenograft Study:
[0499] All the procedures related to animal handling, care and the
treatment in this study were performed according to the guidelines
approved by the Institutional Animal Care and Use Committee (IACUC)
of Shanghai Chemparner following the guidance of the Association
for Assessment and Accreditation of Laboratory Animal Care
(AAALAC). For the in vivo study, mice were inoculated
subcutaneously at the right flank with G-401 tumor cells
(5.times.10.sup.6/mouse) in 0.2 ml mixture of base media and
Matrigel (McCoy's 5A: Matrigel=1:1) for tumor development. The
treatments were started when the tumor size reached approximately
157 mm3 for the tumor efficacy study (n=16 mice per group).
Compound A or vehicle (0.5% NaCMC+0.1% Tween-80 in water) was
administered orally BID at a dose volume of 10 .mu.L/g for either
21 or 28 days. Animal body weights were measured every day during
the first week, then twice weekly for the remainder of the study.
Tumor size was measured twice weekly in two dimensions using a
caliper, and the volume was expressed in mm.sup.3. For PK/PD
analysis, 8 mice with the largest tumor burden were euthanized for
tumor and blood collection after 21 days of dosing. The remaining
mice continued dosing for one more week, and from day 29, treatment
was stopped and the mice were enrolled in a tumor growth delay
study. Mice were observed as individuals until they reached the
tumor weight endpoint (2000 mm.sup.3) or until day 60 (whichever
came first).
[0500] Pharmacokinetic Analyses:
[0501] Dexamethasone was used as internal standard. An aliquot of
30 .mu.L plasma sample was added with 30 .mu.L IS (Dexamethasone,
1000 ng/mL) and 150 .mu.L ACN. The mixture was vortexed for 5 min
and centrifuged at 14000 rpm for 5 min. An aliquot of 2 .mu.L
supernatant was injected for LC-MS/MS analysis (Q-trap 3200). For
10-fold diluted plasma samples an aliquot of 3 .mu.L plasma sample
was added with 27 .mu.L blank plasma, the dilution factor was 10,
then added with 30 .mu.L IS (Dexamethasone, 1000 ng/mL) and 150
.mu.L ACN. The mixture was vortexed for 5 min and centrifuged at
14000 rpm for 5 min. An aliquot of 2 .mu.L supernatant was injected
for LC-MS/MS analysis. Tumor samples were homogenized on
Beadbeater.RTM. for 30 seconds with 3.times.PBS (w/v) to obtain a
tumor homogenate. An aliquot of 30 .mu.L tumor homogenate sample
was added with 30 .mu.L IS (Dexamethasone, 1000 ng/mL) and 150
.mu.L ACN. The mixture was vortexed for 5 min and centrifuged at
14000 rpm for 5 min. An aliquot of 2 .mu.L supernatant was injected
for LC-MS/MS analysis.
Example 4: General Experimental Procedures
NMR
[0502] .sup.1H-NMR spectra were taken using CDCl.sub.3 unless
otherwise stated and were recorded at 400 or 500 MHz using a Varian
or Oxford instruments magnet (500 MHz) instruments. Multiplicities
indicated are s=singlet, d=doublet, t=triplet, q=quartet,
quint=quintet, sxt=sextet, m=multiplet, dd=doublet of doublets,
dt=doublet of triplets; br indicates a broad signal.
LCMS and HPLC
[0503] Shimadzu LC-Q, Shimadzu LCMS-2010EV or Waters Acquity Ultra
Performance LC. HPLC: Products were analyzed by Shimadzu SPD-20A
with 150.times.4.5 mm YMC ODS-M80 column or 150.times.4.6 mm
YMC-Pack Pro C18 column at 1.0 ml/min.
[0504] Mobile phase was MeCN:H2O=3:2 (containing 0.3% SDS and 0.05%
H.sub.3PO.sub.4),
[0505] 0.05% TFA in water, 0.05% TFA in acetonitrile (gradient
Initial 20%, then 0.05% TFA/MeCN to conc. to 95% in 3 min. holds
for 0.5 min. at 3.51 to 4.50 min then 0.05% TFA/MeCN conc.
20%).
[0506] Alternatively the LCMS, 2 different methods were used; the
one we use the most is the high pH (METCR1600) and the other one
for more standard compounds (METCR1416).
[0507] 0.1% Formic acid in water--Mobile phase "A" 0.1% Formic acid
in acetonitrile -Mobile phase "B" utilizing Waters Atlantis dC18,
2.1 mm.times.100 mm, 3 .mu.m column, with a flow rate=0.6 ml/min
Column temperature=40.degree. C.; Time (mins) % B 0.00 min 5% B.
5.0 mins 100% B, 5.4 mins 100% B and 0.42 mins 5% B
[0508] 3.5 minute method refers to Atlantis dC18, 2.1 mm.times.50
mm, 3 .mu.m column, flow rate of 1 ml/min at 40 C. Mobile phase A
Formic acid (aq.) 0.1% mobile phase B formic acid (MeCN) 0.1%,
injection 3 .mu.L, gradient 0 mins (5% organic), 2.5 min (100%
organic), 2.7 mins (100% organic), 2.71 min (5% organic), 3.5 min
(5% organic)
[0509] 7.0 minute method refers to Atlantis dC18, 2.1 mm.times.100
mm, 3 .mu.m column, flow rate of 0.6 ml/min at 40 C. Mobile phase A
Formic acid (aq.) 0.1% mobile phase B formic acid (MeCN) 0.1%,
injection 3 .mu.L, gradient 0 mins (5% organic), 5 min (100%
organic), 5.4 mins (100% organic), 5.42 min (5% organic), 7 min (5%
organic)
[0510] Both the 3. 5 and 7 minute methods were performed on a MS18
Shimadzu LCMS-2010EV or a MS19 Shimadzu LCMS-2010EV system
utilizing LC-20AB pumps and SPD-M20A PDA detectors.
[0511] Products were purified by HPLC/MS using Waters
AutoPurification System with 3100 Mass Detector.
[0512] HPLC analyses may also be performed on a Shimdazu LC-2010CHT
using an YMC ODS-A, C18, (150.times.4.6.times.5 .mu.m) column at
ambient temperature with a flow Rate of 1.4 ml/min. An injection
volume of 10 .mu.l is utilized and detection occurs via UV/PDA.
Mobile Phase A is 0.05 TFA in water and Mobile Phase B is 0.05 TFA
in acetonitrile with a gradient program of Initial 5% B to 95% B in
8 min, hold for 1.5 min, at 9.51 to 12 min B. conc. 0.5%. The
diluent is the mobile phase Other
[0513] Automated flash column chromatography was performed on a
Biotage Isolera version 4. 10 g SNAP cartridge running at 12 ml/min
or a 25 g SNAP cartridge running at 25 ml/min and detecting at 254
nm and 280 nm.
[0514] Select Nitrile reductions may be performed on a ThalesNano
H-Cube.RTM. according to the conditions described in the
experimental procedure.
[0515] Other related general procedures can also be found in PCT
publication No. WO12/118812, PCT application No. PCT/US2012/033648
and PCT application No. PCT/US2012/033662, each of which is
incorporated herein by reference in its entirety.
Example 5: Synthesis of
N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl
(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-bip-
henyl]-3-carboxamide
##STR00041##
[0516] Step 1: Synthesis of 5-bromo-2-methyl-3-nitrobenzoic
acid
##STR00042##
[0518] To stirred solution of 2-methyl-3-nitrobenzoic acid (100 g,
552 mmol) in conc. H.sub.2SO.sub.4 (400 mL),
1,3-dibromo-5,5-dimethyl-2,4-imidazolidinedione (88 g, 308 mmol)
was added in a portion wise manner at room temperature and the
reaction mixture was then stirred at room temperature for 5 h. The
reaction mixture was poured onto ice cold water, the precipitated
solid was filtered off, washed with water and dried under vacuum to
afford the desired compound as a solid (140 g, 98%). The isolated
compound was taken directly into the next step. .sup.1H NMR
(DMSO-d.sub.6, 400 MHz) .delta. 8.31 (s, 1H), 8.17 (s, 1H), 2.43
(s, 3H).
Step 2: Synthesis of methyl 5-bromo-2-methyl-3-nitrobenzoate
##STR00043##
[0520] To a stirred solution of 5-bromo-2-methyl-3-nitrobenzoic
acid (285 g, 1105 mmol) in DMF (2.8 L) at room temperature was
added sodium carbonate (468 g, 4415 mmol) followed by addition of
methyl iodide (626.6 g, 4415 mmol). The resulting reaction mixture
was heated at 60.degree. C. for 8 h. After completion (monitored by
TLC), the reaction mixture was filtered (to remove sodium
carbonate) and washed with ethyl acetate (1 L.times.3). The
combined filtrate was washed with water (3 L.times.5) and the
aqueous phase was back extracted with ethyl acetate (1 L.times.3).
The combined organic layers were dried over anhydrous sodium
sulfate, filtered and concentrated under reduced pressure to afford
the title compound as a solid (290 g, 97% yield). The isolated
compound was taken directly into the next step. .sup.1H NMR
(CDCl.sub.3, 400 MHz) .delta. 8.17 (s, 1H), 7.91 (s, 1H), 3.96 (s,
3H), 2.59 (s, 3H).
Step 3: Synthesis of methyl 3-amino-5-bromo-2-methylbenzoate
##STR00044##
[0522] To a stirred solution of methyl
5-bromo-2-methyl-3-nitrobenzoate (290 g, 1058 mmol) in ethanol (1.5
L) was added aqueous ammonium chloride (283 g, 5290 mmol dissolved
in 1.5 L water). The resulting mixture was stirred at 80.degree. C.
to which iron powder (472 g, 8451 mmol) was added in a portion wise
manner. The resulting reaction mixture was heated at 80.degree. C.
for 12 h. Upon completion as determined by TLC, the reaction
mixture was hot filtered over Celite.RTM. and the celite bed was
washed with methanol (5 L) followed by washing with 30% MeOH in DCM
(5 L). The combined filtrate was concentrated in-vacuo, the residue
obtained was diluted with aqueous sodium bicarbonate solution (2 L)
and extracted with ethyl acetate (5 L.times.3). The combined
organic layers were dried over anhydrous sodium sulfate, filtered
and concentrated under reduced pressure to afford the title
compound as a solid (220 g, 85%). The compound was taken directly
into the next step. .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 7.37
(s, 1H), 6.92 (s, 1H), 3.94 (s, 3H), 3.80 (bs, 2H), 2.31 (s,
3H).
Step 4: Synthesis of methyl
5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl) amino) benzoate
##STR00045##
[0524] To a stirred solution of methyl
3-amino-5-bromo-2-methylbenzoate(15 g, 61.5 mmol) and
dihydro-2H-pyran-4(3)-one (9.2 g, 92 mmol) in dichloroethane (300
mL) was added acetic acid (22 g, 369 mmol) and the reaction mixture
stirred at room temperature for 15 minutes, then the reaction
mixture was cooled to 0.degree. C. and sodium triacetoxyborohydride
(39 g, 184 mmol) was added. The reaction mixture was stirred
overnight at room temperature. Upon completion of the reaction as
determined by TLC, aqueous sodium bicarbonate solution was added to
the reaction mixture until a pH of 7-8 was obtained. The organic
phase was separated and the aqueous phase was extracted with ethyl
acetate. The combined organic layers were dried over anhydrous
sodium sulfate, filtered and concentrated under reduced pressure.
The crude compound was purified by column chromatography (100-200
mesh silica gel) eluting with ethyl acetate: hexane to afford the
desired compound as a solid (14 g, 69%). .sup.1H NMR (DMSO-d.sub.6,
400 MHz) .delta. 7.01 (s, 1H), 6.98 (s, 1H), 5.00 (d, 1H, J=7.6
Hz), 3.84-3.87 (m, 2H), 3.79 (s, 3H), 3.54-3.56 (m, 1H), 3.43 (t,
2H, J=12 Hz), 2.14 (s, 3H), 1.81-1.84 (m, 2H), 1.47-1.55 (m,
2H).
Step 5: Synthesis of methyl 5-bromo-3-(ethyl
(tetrahydro-2H-pyran-4-yl) amino)-2-methylbenzoate
##STR00046##
[0526] To a stirred solution of methyl
5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl) amino) benzoate (14
g, 42.7 mmol) in dichloroethane (150 mL) was added acetaldehyde
(3.75 g, 85.2 mmol) and acetic acid (15.3 g, 256 mmol). The
resulting reaction mixture was stirred at room temperature for 15
minutes. The mixture was cooled to 0.degree. C. and sodium
triacetoxyborohydride (27 g, 128 mmol) was added. The reaction
mixture was stirred at room temperature for 3 hours. Upon
completion of the reaction as determined by TLC, aqueous sodium
bicarbonate solution was added to the reaction mixture until a pH
7-8 was obtained, the organic phase was separated and the aqueous
phase was extracted with ethyl acetate. The combined organic layers
were dried over anhydrous sodium sulfate, filtered and concentrated
under reduced pressure. The crude compound was purified by column
chromatography (100-200 mesh silica gel) eluting with ethyl
acetate: hexane to afford the desired compound as a viscous liquid
(14 g, 93%). .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta. 7.62 (s,
1H), 7.52 (s, 1H), 3.80 (bs, 5H), 3.31 (t, 2H), 2.97-3.05 (m, 2H),
2.87-2.96 (m, 1H), 2.38 (s, 3H), 1.52-1.61 (m, 2H), 1.37-1.50 (m,
2H), 0.87 (t, 3H, J=6.8 Hz).
Step 6: Synthesis of 5-bromo-N-((4, 6-dimethyl-2-oxo-1,
2-dihydropyridin-3-yl) methyl)-3-(ethyl (tetrahydro-2H-pyran-4-yl)
amino)-2-methylbenzamide
##STR00047##
[0528] To a stirred solution of 5-bromo-3-(ethyl
(tetrahydro-2H-pyran-4-yl) amino)-2-methylbenzoate (14 g, 39.4
mmol) in ethanol (100 mL) was added aqueous NaOH (2.36 g, 59.2 mmol
in 25 mL water) and the resulting mixture was stirred at 60.degree.
C. for 1 h. Upon completion of the reaction as determined by TLC,
the solvent was removed under reduced pressure and the residue
obtained was acidified with 1N HCl until a pH 7 was obtained and
then aqueous citric acid solution was added until a pH 5-6 was
obtained. The aqueous layer was extracted with 10% MeOH in DCM (200
mL.times.3), the combined organic layers were dried over anhydrous
sodium sulfate, filtered and concentrated under reduced pressure to
give the respective acid (14 g, 100%).
[0529] The above acid (14 g, 40.9 mmol) was then dissolved in DMSO
(70 mL) and 3-(amino methyl)-4, 6-dimethylpyridin-2(1H)-one (12.4
g, 81.9 mmol) was added to it. The reaction mixture was stirred at
room temperature for 15 minutes, then PYBOP (31.9 g, 61.4 mmol) was
added and stirring was continued for overnight at room temperature.
Upon completion of the reaction as determined by TLC, the reaction
mixture was poured onto ice-cold water (700 mL), stirred for 30
minutes and the precipitated solid was collected by filtration,
washed with water (500 mL) and air dried. The solid obtained was
stirred with acetonitrile (75 mL.times.2), filtered and air dried.
The solid obtained was again stirred with 5% MeOH in DCM (100 mL),
filtered and dried completely under vacuum to afford the title
compound as a solid (14 g, 74%). .sup.1H NMR (DMSO-d.sub.6, 400
MHz) .delta. 11.47 (s, 1H), 8.23 (t, 1H), 7.30 (s, 1H), 7.08 (s,
1H), 5.85 (s, 1H), 4.23 (d, 2H, J=4.4 Hz), 3.81 (d, 2H, J=10.4 Hz),
3.20-3.26 (m, 2H), 3.00-3.07 (m, 1H), 2.91-2.96 (m, 2H), 2.18 (s,
3H), 2.14 (s, 3H), 2.10 (s, 3H), 1.58-1.60 (m, 2H), 1.45-1.50 (m,
2H), 0.78 (t, 3H, J=6.8 Hz).
Step 7: Synthesis of N-((4, 6-dimethyl-2-oxo-1,
2-dihydropyridin-3-yl) methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)
amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide
##STR00048##
[0531] To a stirred solution of 5-bromo-N-((4, 6-dimethyl-2-oxo-1,
2-dihydropyridin-3-yl) methyl)-3-(ethyl (tetrahydro-2H-pyran-4-yl)
amino)-2-methylbenzamide (14 g, 29.5 mmol) in dioxane/water mixture
(70 mL/14 mL) was added 4-(4-(4, 4, 5, 5-tetramethyl-1, 3,
2-dioxaborolan-2-yl) benzyl) morpholine (13.4 g, 44.2 mmol)
followed by addition of Na.sub.2CO.sub.3 (11.2 g, 106.1 mmol). The
solution was purged with argon for 15 minutes and then Pd
(PPh.sub.3).sub.4 (3.40 g, 2.94 mmol) was added and the solution
was again purged with argon for a further 10 min. The reaction
mixture was heated at 100.degree. C. for 4 h. After completion
(monitored by TLC), the reaction mixture was diluted with water and
extracted with 10% MeOH/DCM. The combined organic layers were dried
over anhydrous sodium sulphate, filtered and concentrated under
reduced pressure. The crude compound was purified by column
chromatography (100-200 mesh silica gel) eluting with methanol: DCM
to the title compound as a solid (12 g, 71%). Analytical Data:
LCMS: 573.35 (M+1).sup.+; HPLC: 99.5% (@ 254 nm) (R.sub.t; 3.999;
Method: Column: YMC ODS-A 150 mm.times.4.6 mm.times.5.mu.; Mobile
Phase: A; 0.05% TFA in water/B; 0.05% TFA in acetonitrile; Inj.
Vol: 10 .mu.L, Col. Temp.: 30.degree. C.; Flow rate: 1.4 mL/min.;
Gradient: 5% B to 95% B in 8 min, Hold for 1.5 min, 9.51-12 min 5%
B); .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta. 11.46 (s, 1H), 8.19
(t, 1H), 7.57 (d, 2H, J=7.2 Hz), 7.36-7.39 (m, 3H), 7.21 (s, 1H),
5.85 (s, 1H), 4.28 (d, 2H, J=2.8 Hz), 3.82 (d, 2H, J=9.6 Hz), 3.57
(bs, 4H), 3.48 (s, 2H), 3.24 (t, 2H, J=10.8 Hz), 3.07-3.09 (m, 2H),
3.01 (m, 1H), 2.36 (m, 4H), 2.24 (s, 3H), 2.20 (s, 3H), 2.10 (s,
3H), 1.64-1.67 (m, 2H), 1.51-1.53 (m, 2H), 0.83 (t, 3H, J=6.4
Hz).
Step 8: Synthesis of
N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl
(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-bip-
henyl]-3-carboxamide trihydrochloride
##STR00049##
[0533] N-((4, 6-dimethyl-2-oxo-1, 2-dihydropyridin-3-yl)
methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)
amino)-4-methyl-4'-(morpholinomethyl)-[1,
1'-biphenyl]-3-carboxamide (12 g, 21.0 mmol) was dissolved in
methanolic HCl (200 mL) and stirred at room temperature for 3 h.
After three hours of stirring, the reaction mixture was
concentrated under reduced pressure. The solid obtained was stirred
with ether (100 mL.times.2) to afford the desired salt as a solid
(11 g, 77%). Analytical Data of the tri-HCl salt: LCMS: 573.40
(M+1).sup.+; HPLC: 99.1% (@ 254 nm) (Rt; 3.961; Method: Column: YMC
ODS-A 150 mm.times.4.6 mm.times.5.mu.; Mobile Phase: A; 0.05% TFA
in water/B; 0.05% TFA in acetonitrile; Inj. Vol: 10 .mu.L, Col.
Temp.: 30.degree. C.; Flow rate: 1.4 mL/min.; Gradient: 5% B to 95%
B in 8 min, Hold for 1.5 min, 9.51-12 min 5% B); .sup.1H NMR
(D.sub.2O 400 MHz) .delta. 7.92 (bs, 1H,) 7.80 (s, 1H), 7.77 (d,
2H, J=8 Hz), 7.63 (s, 1H), 7.61 (s, 1H), 6.30 (s, 1H), 4.48 (s,
2H), 4.42 (s, 2H), 4.09-4.11 (m, 4H), 3.95-3.97 (m, 2H), 3.77 (t,
3H, J=10.4 Hz), 3.44-3.47 (m, 3H), 3.24-3.32 (m, 3H), 2.42 (s, 3H),
2.35 (s, 3H), 2.26 (s, 3H), 2.01 (m, 2H), 1.76 (m, 2H), 1.04 (t,
3H, J=6.8 Hz).
Example 6: N-((4,
6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(((1r,4r)-4-(dimethyla-
mino)cyclohexyl)(ethyl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-bipheny-
l]-3-carboxamide
##STR00050##
[0534] Step 1: 5-bromo-2-methyl-3-nitrobenzoic acid
[0535] To stirred solution of 2-methyl-3-nitrobenzoic acid (100 g,
552.48 mmol) in conc. H.sub.2SO.sub.4 (400 mL),
1,3-dibromo-5,5-dimethyl-2,4-imidazolidinedione (87.98 g, 307.70
mmol) was added in a portion-wise manner at room temperature. The
reaction mixture was then stirred at room temperature for 5 h. The
reaction mixture was poured into ice cold water, the precipitated
solid collected by filtration, washed with water and dried under
vacuum to afford desired 5-bromo-2-methyl-3-nitrobenzoic acid as
off-white solid (140 g, 97.90% yield). .sup.1H NMR (DMSO-d.sub.6,
400 MHz) .delta. 8.31 (s, 1H), 8.17 (s, 1H), 2.43 (s, 3H).
Step 2: methyl 5-bromo-2-methyl-3-nitrobenzoate
[0536] To a stirred solution of 5-bromo-2-methyl-3-nitrobenzoic
acid (285 g, 1104.65 mmol) in DMF (2.8 L) was added sodium
carbonate (468 g, 4415.09 mmol) followed by addition of methyl
iodide (626.63 g, 4415 mmol) at room temperature. The resulting
reaction mixture was stirred at 60.degree. C. for 8 h. The reaction
mixture was then filtered to remove suspended solids which were
washed well with ethyl acetate (3.times.1 L). The combined
filtrates were washed well with water (5.times.3 L) and the aqueous
phase back extracted with ethyl acetate (3.times.1 L). The combined
organic extracts dried over anhydrous sodium sulfate, filtered and
concentrated under reduced pressure to afford methyl
5-bromo-2-methyl-3-nitrobenzoate as an off-white solid (290 g, 97%
yield). .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 8.17 (s, 1H),
7.91 (s, 1H), 3.96 (s, 3H), 2.59 (s, 3H).
Step 3: methyl 3-amino-5-bromo-2-methylbenzoate
[0537] To a stirred solution of methyl
5-bromo-2-methyl-3-nitrobenzoate (290 g, 1058.39 mmol) in ethanol
(1.5 L) was added aqueous ammonium chloride (283 g, 5290 mmol
dissolved in 1.5 L water). The resulting mixture was stirred and
heated at 80.degree. C. followed by addition of iron powder (472 g,
8451 mmol) in portions at 80.degree. C. The resulting reaction
mixture was heated at 80.degree. C. for 12 h. The reaction mixture
was then hot filtered through Celite.RTM. and the Celite.RTM. bed
washed well methanol (5 L) and then with 30% MeOH in DCM (5 L). The
combined filtrates were concentrated in vacuo and the residue
obtained was diluted with aqueous bicarbonate (2 L) and extracted
with ethyl acetate (3.times.5 L). The combined organic layers were
dried over anhydrous sodium sulfate, filtered and concentrated
under reduced pressure to afford methyl
3-amino-5-bromo-2-methylbenzoate as a brown solid (220 g, 89.41%
yield).
[0538] A portion of the product (5 g) was dissolved in hot ethanol
(20 mL), insoluble residue filtered off and mother liquor
concentrated to obtain methyl 3-amino-5-bromo-2-methylbenzoate (3.5
g, 70% yield) with HPLC purity 93.81% as light brown solid. .sup.1H
NMR (CDCl.sub.3, 400 MHz) .delta. 7.37 (s, 1H), 6.92 (s, 1H), 3.94
(s, 3H), 3.80 (bs, 2H), 2.31 (s, 3H).
Step 4: methyl
5-bromo-3-(((1r,4r)-4-((tert-butoxycarbonyl)amino)cyclohexyl)amino)-2-met-
hylbenzoate
[0539] To a stirred solution of methyl
3-amino-5-bromo-2-methylbenzoate(5 g, 20.5 mmol) and tert-butyl
(4-oxocyclohexyl)carbamate (5.69 g, 26.7 mmol) in dichloroethane
(50 mL), acetic acid (7.4 g, 123 mmol) was added and the reaction
was stirred at room temperature for 10 minutes. Sodium
triacetoxyborohydride (13.1 g, 61.7 mmol) was then added at
0.degree. C. and reaction was stirred at room temperature for 16
hours. The reaction was quenched with aqueous sodium bicarbonate,
the organic phase separated and the aqueous phase extracted with
dichloromethane. The combined organic layers were dried over
anhydrous sodium sulfate and concentrated in vacuo. The crude
product was purified by silica gel column chromatography (100-200
mesh size) eluting with 10% ethyl acetate in hexane to afford 3.5 g
of the more polar (trans) isomer, methyl
5-bromo-3-(((1r,4r)-4-((tert-butoxycarbonyl)amino)cyclohexyl)amino)-2-met-
hylbenzoate, as solid (38.46%). .sup.1H NMR (CDCl.sub.3, 400 MHz)
.delta. 7.21 (s, 1H), 6.80 (s, 1H), 4.41 (bs, 1H), 3.85 (s, 3H),
3.60 (m, 1H), 3.45 (m, 1H), 3.20 (m, 1H), 2.22 (s, 3H), 2.15 (bs,
2H), 2.05 (bs, 2H), 1.45 (s, 9H), 1.30 (m, 4H).
Step 5: methyl
5-bromo-3-(((1r,4r)-4-((tert-butoxycarbonyl)amino)cyclohexyl)-(ethyl)amin-
o)-2-methylbenzoate
[0540] To a stirred solution of methyl
5-bromo-3-(((1r,4r)-4-((tert-butoxycarbonyl)amino)-cyclohexyl)(ethyl)amin-
o)-2-methylbenzoate (55 g, 0.124 mol) and acetaldehyde (11 g, 0.25
mol) in dichloroethane (550 mL), acetic acid (44.64 g, 0.744 mol)
was added and the reaction mixture stirred at room temperature for
10 minutes. Sodium triacetoxyborohydride (79 g, 0.372 mol) was then
added at 0.degree. C. and the reaction mixture was stirred at room
temperature for 16 hours. The reaction was quenched with aqueous
sodium bicarbonate, the organic phase separated and the aqueous
phase extracted with dichloromethane. The combined extracts were
dried over anhydrous sodium sulfate and concentrated in-vacuo. The
crude compound was purified by silica gel column chromatography
(100-200 mesh size) eluting with 10% ethyl acetate in hexane to
afford 44 g of methyl
5-bromo-34(1r,4r)-4-((tert-butoxycarbonyl)amino)cyclohexyl)-(ethyl)amino)-
-2-methylbenzoate (75.2%) as solid. .sup.1H NMR (DMSO-d.sub.6, 400
MHz) .delta. 7.55 (s, 1H), 7.45 (s, 1H), 6.65 (d, 1H), 3.80 (s,
3H), 3.15 (bs, 1H), 3.05 (q, 2H), 2.60 (m, 1H), 2.30 (s, 3H), 1.75
(m, 4H), 1.40 (m, 2H), 1.35 (s, 9H), 1.10 (m, 2H), 0.80 (t,
3H).
Step 6: tert-butyl
((1r,4r)-4-((5-bromo-3-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)meth-
yl)carbamoyl)-2-methylphenyl)(ethyl)amino)cyclohexyl)carbamate
[0541] Aqueous NaOH (3.5 g, 0.08 mol in 10 mL H.sub.2O) was added
to a solution of methyl
5-bromo-3-(((1r,4r)-4-((tert-butoxycarbonyl)amino)cyclohexyl)-(ethyl)amin-
o)-2-methylbenzoate (25 g, 0.053 mol) in EtOH (100 mL) and stirred
at 60.degree. C. for 1 h. The ethanol was then removed under
reduced pressure and acidified to pH 8 with dilute HCl and to pH 6
with citric acid. The mixture was extracted with 10% methanol in
DCM (3.times.200 mL). The combined organic layers were dried and
concentrated giving the respective acid (24.2 g, 99.0%). .sup.1H
NMR (DMSO-d.sub.6, 400 MHz) .delta. 13.13 (s, 1H), 7.54 (s, 1H),
7.43 (s, 1H), 6.68 (d, 1H), 3.14 (bs, 1H), 3.03 (q, 2H), 2.56 (m,
1H), 2.33 (s, 3H), 1.80-1.65 (m, 4H), 1.40 (m, 2H), 1.35 (s, 9H),
1.10 (m, 2H), 0.77 (t, 3H).
[0542] The acid (24 g, 0.053 mol) was dissolved in DMSO (100 mL)
and 3-(aminomethyl)-4,6-dimethylpyridin-2(1H)-one (16 g, 0.106 mol)
and triethylamine (5.3 g, 0.053 mol) was added. The reaction
mixture was stirred at room temperature for 15 min before PyBop (41
g, 0.079 mmol) was added and stirring was then continued for
overnight at room temperature. The reaction mixture was poured into
ice water (1 L). The resulting precipitate was collected by
filtration, washed well with water (2.times.1 L) and dried. The
product obtained was further purified by washings with acetonitrile
(3.times.200 mL) and DCM (100 mL) to afford tert-butyl
((1r,4r)-4-((5-bromo-3-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)meth-
yl)carbamoyl)-2-methylphenyl)(ethyl)amino)cyclohexyl)-carbamate (24
g, 77%). .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta. 11.47 (s, 1H),
8.24 (t, 1H), 7.25 (s, 1H), 7.04 (s, 1H), 6.67 (d, 1H), 5.85 (s,
1H), 4.24 (d, 2H), 3.13 (bs, 1H), 3.01 (q, 2H), 2.53 (m, 1H), 2.18
(s, 3H), 2.10 (s, 6H), 1.80-1.65 (m, 4H), 1.40 (m, 2H), 1.35 (s,
9H), 1.10 (m, 2H), 0.77 (t, 3H).
Step 7: tert-butyl
((1r,4r)-4-((5-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carba-
moyl)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-yl)(ethyl)amino)cyc-
lohexyl)carbamate
[0543] To a stirred solution of tert-butyl
((1r,4r)-4-((5-bromo-3-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)meth-
yl)carbamoyl)-2-methylphenyl)(ethyl)amino)cyclohexyl)-carbamate (24
g, 0.041 mol) and
4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)morpholine
(18 g, 0.061 mol) in dioxane/water mixture (160 mL+40 mL),
Na.sub.2CO.sub.3 (15 g, 0.15 mol) was added and solution purged
with argon for 15 min. Pd(PPh.sub.3).sub.4 (4.7 g, 0.041 mol) was
then added and the reaction mixture again purged with argon for 10
min. The reaction mixture was heated at 100.degree. C. for 4 h. The
reaction mixture was then diluted with 10% MeOH/DCM (500 mL) and
filtered. The filtrate was concentrated, diluted with water (500
mL) and extracted with 10% MeOH in DCM (3.times.500 mL). The
combined organic layers were dried over Na.sub.2SO.sub.4 and
solvent removed under reduced pressure. The crude product was
purified by silica gel column chromatography (100-200 mesh) eluting
with 7% MeOH in DCM to afford tert-butyl
((1r,4r)-4-((5-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carba-
moyl)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-yl)(ethyl)amino)cyc-
lohexyl)carbamate (20 g, 71.43%). .sup.1H NMR (DMSO-d.sub.6, 400
MHz) .delta. 11.46 (s, 1H), 8.20 (t, 1H), 7.56 (d, 2H), 7.36 (m,
3H), 7.17 (s, 1H), 6.66 (d, 1H), 5.85 (s, 1H), 4.28 (d, 2H), 3.57
(bs, 4H), 3.48 (s, 2H), 3.20-3.05 (m, 3H), 2.62 (m, 1H), 2.36 (bs,
4H), 2.20 (s, 6H), 2.10 (s, 3H), 1.75 (m, 4H), 1.42 (m, 2H), 1.35
(s, 9H), 1.10 (m, 2H), 0.82 (t, 3H).
Step 8:
5-(((1r,4r)-4-aminocyclohexyl)(ethyl)amino)-N-((4,6-dimethyl-2-oxo-
-1,2-dihydropyridin-3-yl)methyl)-4-methyl-4'-(morpholinomethyl)-[1,1'-biph-
enyl]-3-carboxamide
[0544] To a stirred solution of tert-butyl
((1r,4r)-4-((5-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carba-
moyl)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-yl)(ethyl)amino)cyc-
lohexyl)carbamate (20 g, 0.03 mol) in DCM (200 mL) at 0.degree. C.,
TFA (75 mL) was added and reaction was stirred for 2 h at room
temperature. The reaction mixture was then concentrated to dryness
and the residue basified with aqueous saturated bicarbonate
solution (300 mL) to pH 8. The mixture was extracted with 20%
methanol in DCM (4.times.200 m). The combined extracts were dried
over Na.sub.2SO.sub.4 and the solvent removed under reduced
pressure to afford
5-(((1r,4r)-4-aminocyclohexyl)(ethyl)amino)-N-((4,6-dimethyl-2-oxo-1,2-di-
hydropyridin-3-yl)methyl)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-
-carboxamide (15.5 g, 91%) which was used as is in the next
reaction. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta. 8.18 (bs,
1H), 7.57 (d, 2H), 7.38 (m, 3H), 7.20 (s, 1H), 5.85 (s, 1H), 4.29
(d, 2H), 3.57 (bs, 4H), 3.48 (s, 2H), 3.31 (bs, 2H), 3.10 (m, 2H),
2.91 (m, 1H), 2.67 (m, 1H), 2.36 (bs, 4H), 2.21 (s, 3H), 2.20 (s,
3H), 2.10 (s, 3H), 1.90 (m, 2H), 1.83 (m, 2H), 1.45 (m, 2H), 1.23
(m, 2H), 0.83 (t, 3H).
Step 9:
N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(((1r,4r)-
-4-(dimethylamino)cyclohexyl)(ethyl)amino)-4-methyl-4'-(morpholinomethyl)--
[1,1'-biphenyl]-3-carboxamide
[0545] To a stirred solution of
5-(((1r,4r)-4-aminocyclohexyl)(ethyl)amino)-N-((4,6-dimethyl-2-oxo-1,2-di-
hydropyridin-3-yl)methyl)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-
-carboxamide(14 g, 0.023 mol) in dichloromethane (150 mL) was added
aqueous 35% formaldehyde solution (2.4 g, 0.080 mol) at 0.degree.
C. After stirring for 20 min, Na(OAc).sub.3BH (12.2 g, 0.057 mol)
was added and stirring continued for 2h at 0.degree. C. Water (100
mL) was then added to the reaction mixture and the mixture
extracted with 20% methanol in DCM (3.times.200 mL). The combined
extracts were dried over Na.sub.2SO.sub.4 and the solvent removed
under reduced pressure. The crude product was purified by basic
alumina column chromatography eluting with 6-7% MeOH in DCM to
afford the title compound (10 g, 63.6%). LCMS: 614.65 (M+1).sup.+;
HPLC: 98.88% (@ 210-370 nm) (Rt; 3.724; Method: Column: YMC ODS-A
150 mm.times.4.6 mm.times.5.mu.; Mobile Phase: A; 0.05% TFA in
water/B; 0.05% TFA in acetonitrile; Inj. Vol: 10 .mu.L, Col. Temp.:
30.degree. C.; Flow rate: 1.4 mL/min.; Gradient: 5% B to 95% B in 8
min, Hold for 1.5 min, 9.51-12 min 5% B); .sup.1H NMR
(DMSO-d.sub.6, 400 MHz) .delta. 11.45 (s, 1H), 8.17 (t, 1H), 7.56
(d, 2H, J=8 Hz), 7.36 (m, 3H), 7.17 (s, 1H), 5.85 (s, 1H), 4.29 (d,
2H, J=4.4 Hz), 3.57 (bs, 4H), 3.48 (s, 2H), 3.09 (q, 2H), 2.66 (m,
1H), 2.36 (bs, 4H), 2.21 (s, 3H), 2.20 (s, 3H), 2.11 (s, 9H), 1.79
(m, 4H), 1.36 (m, 2H), 1.11 (m, 2H), 0.82 (t, 3H, J=6.4&6.8
Hz).
Example 7: Bioassay Protocol and General Methods
Protocol for Wild-Type and Mutant PRC2 Enzyme Assays
[0546] General Materials.
[0547] S-adenosylmethionine (SAM), S-adenosylhomocyteine (SAH),
bicine, KCl, Tween20, dimethylsulfoxide (DMSO) and bovine skin
gelatin (BSG) were purchased from Sigma-Aldrich at the highest
level of purity possible. Dithiothreitol (DTT) was purchased from
EMD. .sup.3H-SAM was purchased from American Radiolabeled Chemicals
with a specific activity of 80 Ci/mmol. 384-well streptavidin
Flashplates were purchased from PerkinElmer.
[0548] Substrates.
[0549] Peptides representative of human histone H3 residues 21-44
containing either an unmodified lysine 27 (H3K27me0) or
dimethylated lysine 27 (H3K27me2) were synthesized with a
C-terminal G(K-biotin) linker-affinity tag motif and a C-terminal
amide cap by 21.sup.st Century Biochemicals. The peptides were
high-performance liquid chromatography (HPLC) purified to greater
than 95% purity and confirmed by liquid chromatography mass
spectrometry (LC-MS). The sequences are listed below.
TABLE-US-00019 H3K27me0: (SEQ ID NO: 13)
ATKAARKSAPATGGVKKPHRYRPGGK(biotin)-amide H3K27me2: (SEQ ID NO: 14)
ATKAARK(me2)SAPATGGVKKPHRYRPGGK(biotin)-amide
[0550] Chicken erythrocyte oligonucleosomes were purified from
chicken blood according to established procedures.
[0551] Recombinant PRC2 Complexes.
[0552] Human PRC2 complexes were purified as 4-component enzyme
complexes co-expressed in Spodoptera frugiperda (sf9) cells using a
baculovirus expression system. The subunits expressed were
wild-type EZH2 (NM_004456) or EZH2 Y641F, N, H, S or C mutants
generated from the wild-type EZH2 construct, EED (NM_003797), Suz12
(NM_015355) and RbAp48 (NM_005610). The EED subunit contained an
N-terminal FLAG tag that was used to purify the entire 4-component
complex from sf9 cell lysates. The purity of the complexes met or
exceeded 95% as determined by SDS-PAGE and Agilent Bioanalyzer
analysis. Concentrations of enzyme stock concentrations (generally
0.3-1.0 mg/mL) was determined using a Bradford assay against a
bovine serum albumin (BSA) standard.
[0553] General Procedure for PRC2 Enzyme Assays on Peptide
Substrates.
[0554] The assays were all performed in a buffer consisting of 20
mM bicine (pH=7.6), 0.5 mM DTT, 0.005% BSG and 0.002% Tween20,
prepared on the day of use. Compounds in 100% DMSO (1 .mu.L) were
spotted into polypropylene 384-well V-bottom plates (Greiner) using
a Platemate 2.times.3 outfitted with a 384-channel pipet head
(Thermo). DMSO (1 .mu.L) was added to columns 11, 12, 23, 24, rows
A-H for the maximum signal control, and SAH, a known product and
inhibitor of PRC2 (1 .mu.L) was added to columns 11, 12, 23, 24,
rows I-P for the minimum signal control. A cocktail (40 .mu.L)
containing the wild-type PRC2 enzyme and H3K27me0 peptide or any of
the Y641 mutant enzymes and H3K27me2 peptide was added by Multidrop
Combi (Thermo). The compounds were allowed to incubate with PRC2
for 30 min at 25.degree. C., then a cocktail (10 .mu.L) containing
a mixture of non-radioactive and .sup.3H-SAM was added to initiate
the reaction (final volume=51 .mu.L). In all cases, the final
concentrations were as follows: wild-type or mutant PRC2 enzyme was
4 nM, SAH in the minimum signal control wells was 1 mM and the DMSO
concentration was 1%. The final concentrations of the rest of the
components are indicated in Table 7, below. The assays were stopped
by the addition of non-radioactive SAM (10 .mu.L) to a final
concentration of 600 .mu.M, which dilutes the .sup.3H-SAM to a
level where its incorporation into the peptide substrate is no
longer detectable. 50 .mu.L of the reaction in the 384-well
polypropylene plate was then transferred to a 384-well Flashplate
and the biotinylated peptides were allowed to bind to the
streptavidin surface for at least 1 h before being washed three
times with 0.1% Tween20 in a Biotek ELx405 plate washer. The plates
were then read in a PerkinElmer TopCount platereader to measure the
quantity of .sup.3H-labeled peptide bound to the Flashplate
surface, measured as disintegrations per minute (dpm) or
alternatively, referred to as counts per minute (cpm).
TABLE-US-00020 TABLE 7 Final concentrations of components for each
assay variation based upon EZH2 identity (wild-type or Y641 mutant
EZH2) PRC2 Enzyme (denoted by Non-radioactive SAM EZH2 identity)
Peptide (nM) (nM) .sup.3H-SAM (nM) Wild-type 185 1800 150 Y641F 200
850 150 Y641N 200 850 150 Y641H 200 1750 250 Y641S 200 1300 200
Y641C 200 3750 250
[0555] General Procedure for Wild-Type PRC2 Enzyme Assay on
Oligonucleosome Substrate.
[0556] The assays was performed in a buffer consisting of 20 mM
bicine (pH=7.6), 0.5 mM DTT, 0.005% BSG, 100 mM KCl and 0.002%
Tween20, prepared on the day of use. Compounds in 100% DMSO (1
.mu.L) were spotted into polypropylene 384-well V-bottom plates
(Greiner) using a Platemate 2.times.3 outfitted with a 384-channel
pipet head (Thermo). DMSO (1 .mu.L) was added to columns 11, 12,
23, 24, rows A-H for the maximum signal control, and SAH, a known
product and inhibitor of PRC2 (1 .mu.L) was added to columns 11,
12, 23, 24, rows I-P for the minimum signal control. A cocktail (40
.mu.L) containing the wild-type PRC2 enzyme and chicken erythrocyte
oligonucleosome was added by Multidrop Combi (Thermo). The
compounds were allowed to incubate with PRC2 for 30 min at
25.degree. C., then a cocktail (10 .mu.L) containing a mixture of
non-radioactive and .sup.3H-SAM was added to initiate the reaction
(final volume=51 .mu.L). The final concentrations were as follows:
wild-type PRC2 enzyme was 4 nM, non-radioactive SAM was 430 nM,
.sup.3H-SAM was 120 nM, chicken erythrocyte olignonucleosome was
120 nM, SAH in the minimum signal control wells was 1 mM and the
DMSO concentration was 1%. The assay was stopped by the addition of
non-radioactive SAM (10 .mu.L) to a final concentration of 600
.mu.M, which dilutes the .sup.3H-SAM to a level where its
incorporation into the chicken erythrocyte olignonucleosome
substrate is no longer detectable. 50 .mu.L of the reaction in the
384-well polypropylene plate was then transferred to a 384-well
Flashplate and the chicken erythrocyte nucleosomes were immobilized
to the surface of the plate, which was then washed three times with
0.1% Tween20 in a Biotek ELx405 plate washer. The plates were then
read in a PerkinElmer TopCount platereader to measure the quantity
of .sup.3H-labeled chicken erythrocyte oligonucleosome bound to the
Flashplate surface, measured as disintegrations per minute (dpm) or
alternatively, referred to as counts per minute (cpm).
% Inhibition Calculation ##EQU00001## % inh = 100 - ( d p m cmpd -
d p m min d p m max - d p m min ) .times. 100 ##EQU00001.2##
[0557] Where dpm=disintegrations per minute, cmpd=signal in assay
well, and min and max are the respective minimum and maximum signal
controls.
Four - parameter IC 50 fit ##EQU00002## Y = Bottom + ( Top - Bottom
) 1 + ( X IC 50 ) Hill Coefficient ##EQU00002.2##
[0558] Where top and bottom are the normally allowed to float, but
may be fixed at 100 or 0 respectively in a 3-parameter fit. The
Hill Coefficient normally allowed to float but may also be fixed at
1 in a 3-parameter fit. Y is the % inhibition and X is the compound
concentration.
[0559] IC.sub.50 values for the PRC2 enzyme assays on peptide
substrates (e.g., EZH2 wild type and Y641F) are presented in Table
8 below.
[0560] WSU-DLCL2 Methylation Assay
[0561] WSU-DLCL2 suspension cells were purchased from DSMZ (German
Collection of Microorganisms and Cell Cultures, Braunschweig,
Germany). RPMI/Glutamax Medium, Penicillin-Streptomycin, Heat
Inactivated Fetal Bovine Serum, and D-PBS were purchased from Life
Technologies, Grand Island, N.Y., USA. Extraction Buffer and
Neutralization Buffer (5.times.) were purchased from Active Motif,
Carlsbad, Calif., USA. Rabbit anti-Histone H3 antibody was
purchased from Abcam, Cambridge, Mass., USA. Rabbit anti-H3K27me3
and HRP-conjugated anti-rabbit-IgG were purchased from Cell
Signaling Technology, Danvers, Mass., USA. TMB "Super Sensitive"
substrate was sourced from BioFX Laboratories, Owings Mills, Md.,
USA. IgG-free Bovine Serum Albumin was purchased from Jackson
ImmunoResearch, West Grove, Pa., USA. PBS with Tween
(10.times.PBST) was purchased from KPL, Gaithersburg, Md., USA.
Sulfuric Acid was purchased from Ricca Chemical, Arlington, Tex.,
USA. Immulon ELISA plates were purchased from Thermo, Rochester,
N.Y., USA. V-bottom cell culture plates were purchased from Corning
Inc., Corning, N.Y., USA. V-bottom polypropylene plates were
purchased from Greiner Bio-One, Monroe, N.C., USA.
[0562] WSU-DLCL2 suspension cells were maintained in growth medium
(RPMI 1640 supplemented with 10% v/v heat inactivated fetal bovine
serum and 100 units/mL penicillin-streptomycin) and cultured at
37.degree. C. under 5% CO.sub.2. Under assay conditions, cells were
incubated in Assay Medium (RPMI 1640 supplemented with 20% v/v heat
inactivated fetal bovine serum and 100 units/mL
penicillin-streptomycin) at 37.degree. C. under 5% CO.sub.2 on a
plate shaker.
[0563] WSU-DLCL2 cells were seeded in assay medium at a
concentration of 50,000 cells per mL to a 96-well V-bottom cell
culture plate with 200 .mu.L per well. Compound (1 .mu.L) from 96
well source plates was added directly to V-bottom cell plate.
Plates were incubated on a titer-plate shaker at 37.degree. C., 5%
CO.sub.2 for 96 hours. After four days of incubation, plates were
spun at 241.times.g for five minutes and medium was aspirated
gently from each well of cell plate without disturbing cell pellet.
Pellet was resuspended in 200 .mu.L DPBS and plates were spun again
at 241.times.g for five minutes. The supernatant was aspirated and
cold (4.degree. C.) Extraction buffer (100 .mu.L) was added per
well. Plates were incubated at 4.degree. C. on orbital shaker for
two hours. Plates were spun at 3427.times.g.times.10 minutes.
Supernatant (80 .mu.L per well) was transferred to its respective
well in 96 well V-bottom polypropylene plate. Neutralization Buffer
5.times. (20 per well) was added to V-bottom polypropylene plate
containing supernatant. V-bottom polypropylene plates containing
crude histone preparation (CHP) were incubated on orbital shaker x
five minutes. Crude Histone Preparations were added (24, per well)
to each respective well into duplicate 96 well ELISA plates
containing 100 .mu.L Coating Buffer (1.times.PBS+BSA 0.05% w/v).
Plates were sealed and incubated overnight at 4.degree. C. The
following day, plates were washed three times with 300 .mu.L per
well 1.times.PBST. Wells were blocked for two hours with 300 .mu.L
per well ELISA Diluent ((PBS (1.times.) BSA (2% w/v) and Tween20
(0.05% v/v)). Plates were washed three times with 1.times.PBST. For
the Histone H3 detection plate, 100 per well were added of
anti-Histone-H3 antibody (Abcam, ab1791) diluted 1:10,000 in ELISA
Diluent. For H3K27 trimethylation detection plate, 100 .mu.L per
well were added of anti-H3K27me3 diluted 1:2000 in ELISA diluent.
Plates were incubated for 90 minutes at room temperature. Plates
were washed three times with 300 .mu.L 1.times.PBST per well. For
Histone H3 detection, 100 .mu.L of HRP-conjugated anti-rabbit IgG
antibody diluted to 1:6000 in ELISA diluent was added per well. For
H3K27me3 detection, 100 .mu.L of HRP conjugated anti-rabbit IgG
antibody diluted to 1:4000 in ELISA diluent was added per well.
Plates were incubated at room temperature for 90 minutes. Plates
were washed four times with 1.times.PBST 300 .mu.L per well. TMB
substrate 100 .mu.L was added per well. Histone H3 plates were
incubated for five minutes at room temperature. H3K27me3 plates
were incubated for 10 minutes at room temperature. The reaction was
stopped with sulfuric acid 1N (100 .mu.L per well). Absorbance for
each plate was read at 450 nm.
[0564] First, the ratio for each well was determined by:
( H 3 K 27 me 3 OD 450 value Histone H 3 OD 450 value )
##EQU00003##
[0565] Each plate included eight control wells of DMSO only
treatment (Minimum Inhibition) as well as eight control wells for
maximum inhibition (Background wells).
[0566] The average of the ratio values for each control type was
calculated and used to determine the percent inhibition for each
test well in the plate. Test compound was serially diluted
three-fold in DMSO for a total of ten test concentrations,
beginning at 25 .mu.M. Percent inhibition was determined and
IC.sub.50 curves were generated using duplicate wells per
concentration of compound. IC.sub.50 values for this assay are
presented in Table 8 below.
Percent Inhibition = 100 - ##EQU00004## ( ( ( Individual Test
Sample Ratio ) - ( Background Avg Ratio ) ( Minimum Inhibition
Ratio ) - ( Background Average Ratio ) ) 100 ) ##EQU00004.2##
[0567] Cell Proliferation Analysis
[0568] WSU-DLCL2 suspension cells were purchased from DSMZ (German
Collection of Microorganisms and Cell Cultures, Braunschweig,
Germany). RPMI/Glutamax Medium, Penicillin-Streptomycin, Heat
Inactivated Fetal Bovine Serum were purchased from Life
Technologies, Grand Island, N.Y., USA. V-bottom polypropylene
384-well plates were purchased from Greiner Bio-One, Monroe, N.C.,
USA. Cell culture 384-well white opaque plates were purchased from
Perkin Elmer, Waltham, Mass., USA. Cell-Titer Glo.RTM. was
purchased from Promega Corporation, Madison, Wis., USA. SpectraMax
M5 plate reader was purchased from Molecular Devices LLC,
Sunnyvale, Calif., USA.
[0569] WSU-DLCL2 suspension cells were maintained in growth medium
(RPMI 1640 supplemented with 10% v/v heat inactivated fetal bovine
serum and cultured at 37.degree. C. under 5% CO.sub.2. Under assay
conditions, cells were incubated in Assay Medium (RPMI 1640
supplemented with 20% v/v heat inactivated fetal bovine serum and
100 units/mL penicillin-streptomycin) at 37.degree. C. under 5%
CO.sub.2.
For the assessment of the effect of compounds on the proliferation
of the WSU-DLCL2 cell line, exponentially growing cells were plated
in 384-well white opaque plates at a density of 1250 cell/ml in a
final volume of 50 .mu.l of assay medium. A compound source plate
was prepared by performing triplicate nine-point 3-fold serial
dilutions in DMSO, beginning at 10 mM (final top concentration of
compound in the assay was 20 .mu.M and the DMSO was 0.2%). A 100 nL
aliquot from the compound stock plate was added to its respective
well in the cell plate. The 100% inhibition control consisted of
cells treated with 200 nM final concentration of staurosporine and
the 0% inhibition control consisted of DMSO treated cells. After
addition of compounds, assay plates were incubated for 6 days at
37.degree. C., 5% CO.sub.2, relative humidity >90% for 6 days.
Cell viability was measured by quantization of ATP present in the
cell cultures, adding 35 .mu.l of CellTiter-Glo.RTM..RTM. reagent
to the cell plates. Luminescence was read in the SpectraMax M5. The
concentration inhibiting cell viability by 50% was determined using
a 4-parametric fit of the normalized dose response curves.
INCORPORATION BY REFERENCE
[0570] The entire disclosure of each of the patent documents and
scientific articles referred to herein is incorporated by reference
for all purposes.
[0571] All publications and patent documents cited herein are
incorporated herein by reference as if each such publication or
document was specifically and individually indicated to be
incorporated herein by reference. Citation of publications and
patent documents is not intended as an admission that any is
pertinent prior art, nor does it constitute any admission as to the
contents or date of the same. The invention having now been
described by way of written description, those of skill in the art
will recognize that the invention can be practiced in a variety of
embodiments and that the foregoing description and examples above
are for purposes of illustration and not limitation of the claims
that follow.
EQUIVALENTS
[0572] The invention can be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting on the invention
described herein. Scope of the invention is thus indicated by the
appended claims rather than by the foregoing description, and all
changes that come within the meaning and range of equivalency of
the claims are intended to be embraced therein.
Sequence CWU 1
1
141385PRTHomo sapiens 1Met Met Met Met Ala Leu Ser Lys Thr Phe Gly
Gln Lys Pro Val Lys 1 5 10 15 Phe Gln Leu Glu Asp Asp Gly Glu Phe
Tyr Met Ile Gly Ser Glu Val 20 25 30 Gly Asn Tyr Leu Arg Met Phe
Arg Gly Ser Leu Tyr Lys Arg Tyr Pro 35 40 45 Ser Leu Trp Arg Arg
Leu Ala Thr Val Glu Glu Arg Lys Lys Ile Val 50 55 60 Ala Ser Ser
His Gly Lys Lys Thr Lys Pro Asn Thr Lys Asp His Gly 65 70 75 80 Tyr
Thr Thr Leu Ala Thr Ser Val Thr Leu Leu Lys Ala Ser Glu Val 85 90
95 Glu Glu Ile Leu Asp Gly Asn Asp Glu Lys Tyr Lys Ala Val Ser Ile
100 105 110 Ser Thr Glu Pro Pro Thr Tyr Leu Arg Glu Gln Lys Ala Lys
Arg Asn 115 120 125 Ser Gln Trp Val Pro Thr Leu Pro Asn Ser Ser His
His Leu Asp Ala 130 135 140 Val Pro Cys Ser Thr Thr Ile Asn Arg Asn
Arg Met Gly Arg Asp Lys 145 150 155 160 Lys Arg Thr Phe Pro Leu Cys
Phe Asp Asp His Asp Pro Ala Val Ile 165 170 175 His Glu Asn Ala Ser
Gln Pro Glu Val Leu Val Pro Ile Arg Leu Asp 180 185 190 Met Glu Ile
Asp Gly Gln Lys Leu Arg Asp Ala Phe Thr Trp Asn Met 195 200 205 Asn
Glu Lys Leu Met Thr Pro Glu Met Phe Ser Glu Ile Leu Cys Asp 210 215
220 Asp Leu Asp Leu Asn Pro Leu Thr Phe Val Pro Ala Ile Ala Ser Ala
225 230 235 240 Ile Arg Gln Gln Ile Glu Ser Tyr Pro Thr Asp Ser Ile
Leu Glu Asp 245 250 255 Gln Ser Asp Gln Arg Val Ile Ile Lys Leu Asn
Ile His Val Gly Asn 260 265 270 Ile Ser Leu Val Asp Gln Phe Glu Trp
Asp Met Ser Glu Lys Glu Asn 275 280 285 Ser Pro Glu Lys Phe Ala Leu
Lys Leu Cys Ser Glu Leu Gly Leu Gly 290 295 300 Gly Glu Phe Val Thr
Thr Ile Ala Tyr Ser Ile Arg Gly Gln Leu Ser 305 310 315 320 Trp His
Gln Lys Thr Tyr Ala Phe Ser Glu Asn Pro Leu Pro Thr Val 325 330 335
Glu Ile Ala Ile Arg Asn Thr Gly Asp Ala Asp Gln Trp Cys Pro Leu 340
345 350 Leu Glu Thr Leu Thr Asp Ala Glu Met Glu Lys Lys Ile Arg Asp
Gln 355 360 365 Asp Arg Asn Thr Arg Arg Met Arg Arg Leu Ala Asn Thr
Ala Pro Ala 370 375 380 Trp 385 21717DNAHomo sapiens 2aacgccagcg
cctgcgcact gagggcggcc tggtcgtcgt ctgcggcggc ggcggcggct 60gaggagcccg
gctgaggcgc cagtacccgg cccggtccgc atttcgcctt ccggcttcgg
120tttccctcgg cccagcacgc cccggccccg ccccagccct cctgatccct
cgcagcccgg 180ctccggccgc ccgcctctgc cgccgcaatg atgatgatgg
cgctgagcaa gaccttcggg 240cagaagcccg tgaagttcca gctggaggac
gacggcgagt tctacatgat cggctccgag 300gtgggaaact acctccgtat
gttccgaggt tctctgtaca agagataccc ctcactctgg 360aggcgactag
ccactgtgga agagaggaag aaaatagttg catcgtcaca tggtaaaaaa
420acaaaaccta acactaagga tcacggatac acgactctag ccaccagtgt
gaccctgtta 480aaagcctcgg aagtggaaga gattctggat ggcaacgatg
agaagtacaa ggctgtgtcc 540atcagcacag agccccccac ctacctcagg
gaacagaagg ccaagaggaa cagccagtgg 600gtacccaccc tgcccaacag
ctcccaccac ttagatgccg tgccatgctc cacaaccatc 660aacaggaacc
gcatgggccg agacaagaag agaaccttcc ccctttgctt tgatgaccat
720gacccagctg tgatccatga gaacgcatct cagcccgagg tgctggtccc
catccggctg 780gacatggaga tcgatgggca gaagctgcga gacgccttca
cctggaacat gaatgagaag 840ttgatgacgc ctgagatgtt ttcagaaatc
ctctgtgacg atctggattt gaacccgctg 900acgtttgtgc cagccatcgc
ctctgccatc agacagcaga tcgagtccta ccccacggac 960agcatcctgg
aggaccagtc agaccagcgc gtcatcatca agctgaacat ccatgtggga
1020aacatttccc tggtggacca gtttgagtgg gacatgtcag agaaggagaa
ctcaccagag 1080aagtttgccc tgaagctgtg ctcggagctg gggttgggcg
gggagtttgt caccaccatc 1140gcatacagca tccggggaca gctgagctgg
catcagaaga cctacgcctt cagcgagaac 1200cctctgccca cagtggagat
tgccatccgg aacacgggcg atgcggacca gtggtgccca 1260ctgctggaga
ctctgacaga cgctgagatg gagaagaaga tccgcgacca ggacaggaac
1320acgaggcgga tgaggcgtct tgccaacacg gccccggcct ggtaaccagc
ccatcagcac 1380acggctccca cggagcatct cagaagattg ggccgcctct
cctccatctt ctggcaagga 1440cagaggcgag gggacagccc agcgccatcc
tgaggatcgg gtgggggtgg agtgggggct 1500tccaggtggc ccttcccggc
acacattcca tttgttgagc cccagtcctg ccccccaccc 1560caccctccct
acccctcccc agtctctggg gtcaggaaga aaccttattt taggttgtgt
1620tttgtttttg tataggagcc ccaggcaggg ctagtaacag tttttaaata
aaaggcaaca 1680ggtcatgttc aatttcttca acaaaaaaaa aaaaaaa
17173376PRTHomo sapiens 3Met Met Met Met Ala Leu Ser Lys Thr Phe
Gly Gln Lys Pro Val Lys 1 5 10 15 Phe Gln Leu Glu Asp Asp Gly Glu
Phe Tyr Met Ile Gly Ser Glu Val 20 25 30 Gly Asn Tyr Leu Arg Met
Phe Arg Gly Ser Leu Tyr Lys Arg Tyr Pro 35 40 45 Ser Leu Trp Arg
Arg Leu Ala Thr Val Glu Glu Arg Lys Lys Ile Val 50 55 60 Ala Ser
Ser His Asp His Gly Tyr Thr Thr Leu Ala Thr Ser Val Thr 65 70 75 80
Leu Leu Lys Ala Ser Glu Val Glu Glu Ile Leu Asp Gly Asn Asp Glu 85
90 95 Lys Tyr Lys Ala Val Ser Ile Ser Thr Glu Pro Pro Thr Tyr Leu
Arg 100 105 110 Glu Gln Lys Ala Lys Arg Asn Ser Gln Trp Val Pro Thr
Leu Pro Asn 115 120 125 Ser Ser His His Leu Asp Ala Val Pro Cys Ser
Thr Thr Ile Asn Arg 130 135 140 Asn Arg Met Gly Arg Asp Lys Lys Arg
Thr Phe Pro Leu Cys Phe Asp 145 150 155 160 Asp His Asp Pro Ala Val
Ile His Glu Asn Ala Ser Gln Pro Glu Val 165 170 175 Leu Val Pro Ile
Arg Leu Asp Met Glu Ile Asp Gly Gln Lys Leu Arg 180 185 190 Asp Ala
Phe Thr Trp Asn Met Asn Glu Lys Leu Met Thr Pro Glu Met 195 200 205
Phe Ser Glu Ile Leu Cys Asp Asp Leu Asp Leu Asn Pro Leu Thr Phe 210
215 220 Val Pro Ala Ile Ala Ser Ala Ile Arg Gln Gln Ile Glu Ser Tyr
Pro 225 230 235 240 Thr Asp Ser Ile Leu Glu Asp Gln Ser Asp Gln Arg
Val Ile Ile Lys 245 250 255 Leu Asn Ile His Val Gly Asn Ile Ser Leu
Val Asp Gln Phe Glu Trp 260 265 270 Asp Met Ser Glu Lys Glu Asn Ser
Pro Glu Lys Phe Ala Leu Lys Leu 275 280 285 Cys Ser Glu Leu Gly Leu
Gly Gly Glu Phe Val Thr Thr Ile Ala Tyr 290 295 300 Ser Ile Arg Gly
Gln Leu Ser Trp His Gln Lys Thr Tyr Ala Phe Ser 305 310 315 320 Glu
Asn Pro Leu Pro Thr Val Glu Ile Ala Ile Arg Asn Thr Gly Asp 325 330
335 Ala Asp Gln Trp Cys Pro Leu Leu Glu Thr Leu Thr Asp Ala Glu Met
340 345 350 Glu Lys Lys Ile Arg Asp Gln Asp Arg Asn Thr Arg Arg Met
Arg Arg 355 360 365 Leu Ala Asn Thr Ala Pro Ala Trp 370 375
41690DNAHomo sapiens 4aacgccagcg cctgcgcact gagggcggcc tggtcgtcgt
ctgcggcggc ggcggcggct 60gaggagcccg gctgaggcgc cagtacccgg cccggtccgc
atttcgcctt ccggcttcgg 120tttccctcgg cccagcacgc cccggccccg
ccccagccct cctgatccct cgcagcccgg 180ctccggccgc ccgcctctgc
cgccgcaatg atgatgatgg cgctgagcaa gaccttcggg 240cagaagcccg
tgaagttcca gctggaggac gacggcgagt tctacatgat cggctccgag
300gtgggaaact acctccgtat gttccgaggt tctctgtaca agagataccc
ctcactctgg 360aggcgactag ccactgtgga agagaggaag aaaatagttg
catcgtcaca tgatcacgga 420tacacgactc tagccaccag tgtgaccctg
ttaaaagcct cggaagtgga agagattctg 480gatggcaacg atgagaagta
caaggctgtg tccatcagca cagagccccc cacctacctc 540agggaacaga
aggccaagag gaacagccag tgggtaccca ccctgcccaa cagctcccac
600cacttagatg ccgtgccatg ctccacaacc atcaacagga accgcatggg
ccgagacaag 660aagagaacct tccccctttg ctttgatgac catgacccag
ctgtgatcca tgagaacgca 720tctcagcccg aggtgctggt ccccatccgg
ctggacatgg agatcgatgg gcagaagctg 780cgagacgcct tcacctggaa
catgaatgag aagttgatga cgcctgagat gttttcagaa 840atcctctgtg
acgatctgga tttgaacccg ctgacgtttg tgccagccat cgcctctgcc
900atcagacagc agatcgagtc ctaccccacg gacagcatcc tggaggacca
gtcagaccag 960cgcgtcatca tcaagctgaa catccatgtg ggaaacattt
ccctggtgga ccagtttgag 1020tgggacatgt cagagaagga gaactcacca
gagaagtttg ccctgaagct gtgctcggag 1080ctggggttgg gcggggagtt
tgtcaccacc atcgcataca gcatccgggg acagctgagc 1140tggcatcaga
agacctacgc cttcagcgag aaccctctgc ccacagtgga gattgccatc
1200cggaacacgg gcgatgcgga ccagtggtgc ccactgctgg agactctgac
agacgctgag 1260atggagaaga agatccgcga ccaggacagg aacacgaggc
ggatgaggcg tcttgccaac 1320acggccccgg cctggtaacc agcccatcag
cacacggctc ccacggagca tctcagaaga 1380ttgggccgcc tctcctccat
cttctggcaa ggacagaggc gaggggacag cccagcgcca 1440tcctgaggat
cgggtggggg tggagtgggg gcttccaggt ggcccttccc ggcacacatt
1500ccatttgttg agccccagtc ctgcccccca ccccaccctc cctacccctc
cccagtctct 1560ggggtcagga agaaacctta ttttaggttg tgttttgttt
ttgtatagga gccccaggca 1620gggctagtaa cagtttttaa ataaaaggca
acaggtcatg ttcaatttct tcaacaaaaa 1680aaaaaaaaaa 169052492PRTHomo
sapiens 5Met Thr Ala Glu Pro Met Ser Glu Ser Lys Leu Asn Thr Leu
Val Gln 1 5 10 15 Lys Leu His Asp Phe Leu Ala His Ser Ser Glu Glu
Ser Glu Glu Thr 20 25 30 Ser Ser Pro Pro Arg Leu Ala Met Asn Gln
Asn Thr Asp Lys Ile Ser 35 40 45 Gly Ser Gly Ser Asn Ser Asp Met
Met Glu Asn Ser Lys Glu Glu Gly 50 55 60 Thr Ser Ser Ser Glu Lys
Ser Lys Ser Ser Gly Ser Ser Arg Ser Lys 65 70 75 80 Arg Lys Pro Ser
Ile Val Thr Lys Tyr Val Glu Ser Asp Asp Glu Lys 85 90 95 Pro Leu
Asp Asp Glu Thr Val Asn Glu Asp Ala Ser Asn Glu Asn Ser 100 105 110
Glu Asn Asp Ile Thr Met Gln Ser Leu Pro Lys Gly Thr Val Ile Val 115
120 125 Gln Pro Glu Pro Val Leu Asn Glu Asp Lys Asp Asp Phe Lys Gly
Pro 130 135 140 Glu Phe Arg Ser Arg Ser Lys Met Lys Thr Glu Asn Leu
Lys Lys Arg 145 150 155 160 Gly Glu Asp Gly Leu His Gly Ile Val Ser
Cys Thr Ala Cys Gly Gln 165 170 175 Gln Val Asn His Phe Gln Lys Asp
Ser Ile Tyr Arg His Pro Ser Leu 180 185 190 Gln Val Leu Ile Cys Lys
Asn Cys Phe Lys Tyr Tyr Met Ser Asp Asp 195 200 205 Ile Ser Arg Asp
Ser Asp Gly Met Asp Glu Gln Cys Arg Trp Cys Ala 210 215 220 Glu Gly
Gly Asn Leu Ile Cys Cys Asp Phe Cys His Asn Ala Phe Cys 225 230 235
240 Lys Lys Cys Ile Leu Arg Asn Leu Gly Arg Lys Glu Leu Ser Thr Ile
245 250 255 Met Asp Glu Asn Asn Gln Trp Tyr Cys Tyr Ile Cys His Pro
Glu Pro 260 265 270 Leu Leu Asp Leu Val Thr Ala Cys Asn Ser Val Phe
Glu Asn Leu Glu 275 280 285 Gln Leu Leu Gln Gln Asn Lys Lys Lys Ile
Lys Val Asp Ser Glu Lys 290 295 300 Ser Asn Lys Val Tyr Glu His Thr
Ser Arg Phe Ser Pro Lys Lys Thr 305 310 315 320 Ser Ser Asn Cys Asn
Gly Glu Glu Lys Lys Leu Asp Asp Ser Cys Ser 325 330 335 Gly Ser Val
Thr Tyr Ser Tyr Ser Ala Leu Ile Val Pro Lys Glu Met 340 345 350 Ile
Lys Lys Ala Lys Lys Leu Ile Glu Thr Thr Ala Asn Met Asn Ser 355 360
365 Ser Tyr Val Lys Phe Leu Lys Gln Ala Thr Asp Asn Ser Glu Ile Ser
370 375 380 Ser Ala Thr Lys Leu Arg Gln Leu Lys Ala Phe Lys Ser Val
Leu Ala 385 390 395 400 Asp Ile Lys Lys Ala His Leu Ala Leu Glu Glu
Asp Leu Asn Ser Glu 405 410 415 Phe Arg Ala Met Asp Ala Val Asn Lys
Glu Lys Asn Thr Lys Glu His 420 425 430 Lys Val Ile Asp Ala Lys Phe
Glu Thr Lys Ala Arg Lys Gly Glu Lys 435 440 445 Pro Cys Ala Leu Glu
Lys Lys Asp Ile Ser Lys Ser Glu Ala Lys Leu 450 455 460 Ser Arg Lys
Gln Val Asp Ser Glu His Met His Gln Asn Val Pro Thr 465 470 475 480
Glu Glu Gln Arg Thr Asn Lys Ser Thr Gly Gly Glu His Lys Lys Ser 485
490 495 Asp Arg Lys Glu Glu Pro Gln Tyr Glu Pro Ala Asn Thr Ser Glu
Asp 500 505 510 Leu Asp Met Asp Ile Val Ser Val Pro Ser Ser Val Pro
Glu Asp Ile 515 520 525 Phe Glu Asn Leu Glu Thr Ala Met Glu Val Gln
Ser Ser Val Asp His 530 535 540 Gln Gly Asp Gly Ser Ser Gly Thr Glu
Gln Glu Val Glu Ser Ser Ser 545 550 555 560 Val Lys Leu Asn Ile Ser
Ser Lys Asp Asn Arg Gly Gly Ile Lys Ser 565 570 575 Lys Thr Thr Ala
Lys Val Thr Lys Glu Leu Tyr Val Lys Leu Thr Pro 580 585 590 Val Ser
Leu Ser Asn Ser Pro Ile Lys Gly Ala Asp Cys Gln Glu Val 595 600 605
Pro Gln Asp Lys Asp Gly Tyr Lys Ser Cys Gly Leu Asn Pro Lys Leu 610
615 620 Glu Lys Cys Gly Leu Gly Gln Glu Asn Ser Asp Asn Glu His Leu
Val 625 630 635 640 Glu Asn Glu Val Ser Leu Leu Leu Glu Glu Ser Asp
Leu Arg Arg Ser 645 650 655 Pro Arg Val Lys Thr Thr Pro Leu Arg Arg
Pro Thr Glu Thr Asn Pro 660 665 670 Val Thr Ser Asn Ser Asp Glu Glu
Cys Asn Glu Thr Val Lys Glu Lys 675 680 685 Gln Lys Leu Ser Val Pro
Val Arg Lys Lys Asp Lys Arg Asn Ser Ser 690 695 700 Asp Ser Ala Ile
Asp Asn Pro Lys Pro Asn Lys Leu Pro Lys Ser Lys 705 710 715 720 Gln
Ser Glu Thr Val Asp Gln Asn Ser Asp Ser Asp Glu Met Leu Ala 725 730
735 Ile Leu Lys Glu Val Ser Arg Met Ser His Ser Ser Ser Ser Asp Thr
740 745 750 Asp Ile Asn Glu Ile His Thr Asn His Lys Thr Leu Tyr Asp
Leu Lys 755 760 765 Thr Gln Ala Gly Lys Asp Asp Lys Gly Lys Arg Lys
Arg Lys Ser Ser 770 775 780 Thr Ser Gly Ser Asp Phe Asp Thr Lys Lys
Gly Lys Ser Ala Lys Ser 785 790 795 800 Ser Ile Ile Ser Lys Lys Lys
Arg Gln Thr Gln Ser Glu Ser Ser Asn 805 810 815 Tyr Asp Ser Glu Leu
Glu Lys Glu Ile Lys Ser Met Ser Lys Ile Gly 820 825 830 Ala Ala Arg
Thr Thr Lys Lys Arg Ile Pro Asn Thr Lys Asp Phe Asp 835 840 845 Ser
Ser Glu Asp Glu Lys His Ser Lys Lys Gly Met Asp Asn Gln Gly 850 855
860 His Lys Asn Leu Lys Thr Ser Gln Glu Gly Ser Ser Asp Asp Ala Glu
865 870 875 880 Arg Lys Gln Glu Arg Glu Thr Phe Ser Ser Ala Glu Gly
Thr Val Asp 885 890 895 Lys Asp Thr Thr Ile Met Glu Leu Arg Asp Arg
Leu Pro Lys Lys Gln 900 905 910 Gln Ala Ser Ala Ser Thr Asp Gly Val
Asp Lys Leu Ser Gly Lys Glu 915 920 925 Gln Ser Phe Thr Ser Leu Glu
Val Arg Lys Val Ala Glu Thr Lys Glu 930 935 940 Lys Ser Lys His Leu
Lys Thr Lys Thr Cys Lys Lys Val Gln Asp Gly 945 950 955 960 Leu Ser
Asp Ile Ala Glu Lys Phe Leu Lys Lys Asp Gln Ser Asp Glu 965 970 975
Thr Ser Glu Asp Asp Lys Lys Gln Ser Lys Lys Gly Thr Glu Glu Lys 980
985 990 Lys Lys Pro Ser Asp Phe Lys Lys
Lys Val Ile Lys Met Glu Gln Gln 995 1000 1005 Tyr Glu Ser Ser Ser
Asp Gly Thr Glu Lys Leu Pro Glu Arg Glu 1010 1015 1020 Glu Ile Cys
His Phe Pro Lys Gly Ile Lys Gln Ile Lys Asn Gly 1025 1030 1035 Thr
Thr Asp Gly Glu Lys Lys Ser Lys Lys Ile Arg Asp Lys Thr 1040 1045
1050 Ser Lys Lys Lys Asp Glu Leu Ser Asp Tyr Ala Glu Lys Ser Thr
1055 1060 1065 Gly Lys Gly Asp Ser Cys Asp Ser Ser Glu Asp Lys Lys
Ser Lys 1070 1075 1080 Asn Gly Ala Tyr Gly Arg Glu Lys Lys Arg Cys
Lys Leu Leu Gly 1085 1090 1095 Lys Ser Ser Arg Lys Arg Gln Asp Cys
Ser Ser Ser Asp Thr Glu 1100 1105 1110 Lys Tyr Ser Met Lys Glu Asp
Gly Cys Asn Ser Ser Asp Lys Arg 1115 1120 1125 Leu Lys Arg Ile Glu
Leu Arg Glu Arg Arg Asn Leu Ser Ser Lys 1130 1135 1140 Arg Asn Thr
Lys Glu Ile Gln Ser Gly Ser Ser Ser Ser Asp Ala 1145 1150 1155 Glu
Glu Ser Ser Glu Asp Asn Lys Lys Lys Lys Gln Arg Thr Ser 1160 1165
1170 Ser Lys Lys Lys Ala Val Ile Val Lys Glu Lys Lys Arg Asn Ser
1175 1180 1185 Leu Arg Thr Ser Thr Lys Arg Lys Gln Ala Asp Ile Thr
Ser Ser 1190 1195 1200 Ser Ser Ser Asp Ile Glu Asp Asp Asp Gln Asn
Ser Ile Gly Glu 1205 1210 1215 Gly Ser Ser Asp Glu Gln Lys Ile Lys
Pro Val Thr Glu Asn Leu 1220 1225 1230 Val Leu Ser Ser His Thr Gly
Phe Cys Gln Ser Ser Gly Asp Glu 1235 1240 1245 Ala Leu Ser Lys Ser
Val Pro Val Thr Val Asp Asp Asp Asp Asp 1250 1255 1260 Asp Asn Asp
Pro Glu Asn Arg Ile Ala Lys Lys Met Leu Leu Glu 1265 1270 1275 Glu
Ile Lys Ala Asn Leu Ser Ser Asp Glu Asp Gly Ser Ser Asp 1280 1285
1290 Asp Glu Pro Glu Glu Gly Lys Lys Arg Thr Gly Lys Gln Asn Glu
1295 1300 1305 Glu Asn Pro Gly Asp Glu Glu Ala Lys Asn Gln Val Asn
Ser Glu 1310 1315 1320 Ser Asp Ser Asp Ser Glu Glu Ser Lys Lys Pro
Arg Tyr Arg His 1325 1330 1335 Arg Leu Leu Arg His Lys Leu Thr Val
Ser Asp Gly Glu Ser Gly 1340 1345 1350 Glu Glu Lys Lys Thr Lys Pro
Lys Glu His Lys Glu Val Lys Gly 1355 1360 1365 Arg Asn Arg Arg Lys
Val Ser Ser Glu Asp Ser Glu Asp Ser Asp 1370 1375 1380 Phe Gln Glu
Ser Gly Val Ser Glu Glu Val Ser Glu Ser Glu Asp 1385 1390 1395 Glu
Gln Arg Pro Arg Thr Arg Ser Ala Lys Lys Ala Glu Leu Glu 1400 1405
1410 Glu Asn Gln Arg Ser Tyr Lys Gln Lys Lys Lys Arg Arg Arg Ile
1415 1420 1425 Lys Val Gln Glu Asp Ser Ser Ser Glu Asn Lys Ser Asn
Ser Glu 1430 1435 1440 Glu Glu Glu Glu Glu Lys Glu Glu Glu Glu Glu
Glu Glu Glu Glu 1445 1450 1455 Glu Glu Glu Glu Glu Glu Asp Glu Asn
Asp Asp Ser Lys Ser Pro 1460 1465 1470 Gly Lys Gly Arg Lys Lys Ile
Arg Lys Ile Leu Lys Asp Asp Lys 1475 1480 1485 Leu Arg Thr Glu Thr
Gln Asn Ala Leu Lys Glu Glu Glu Glu Arg 1490 1495 1500 Arg Lys Arg
Ile Ala Glu Arg Glu Arg Glu Arg Glu Lys Leu Arg 1505 1510 1515 Glu
Val Ile Glu Ile Glu Asp Ala Ser Pro Thr Lys Cys Pro Ile 1520 1525
1530 Thr Thr Lys Leu Val Leu Asp Glu Asp Glu Glu Thr Lys Glu Pro
1535 1540 1545 Leu Val Gln Val His Arg Asn Met Val Ile Lys Leu Lys
Pro His 1550 1555 1560 Gln Val Asp Gly Val Gln Phe Met Trp Asp Cys
Cys Cys Glu Ser 1565 1570 1575 Val Lys Lys Thr Lys Lys Ser Pro Gly
Ser Gly Cys Ile Leu Ala 1580 1585 1590 His Cys Met Gly Leu Gly Lys
Thr Leu Gln Val Val Ser Phe Leu 1595 1600 1605 His Thr Val Leu Leu
Cys Asp Lys Leu Asp Phe Ser Thr Ala Leu 1610 1615 1620 Val Val Cys
Pro Leu Asn Thr Ala Leu Asn Trp Met Asn Glu Phe 1625 1630 1635 Glu
Lys Trp Gln Glu Gly Leu Lys Asp Asp Glu Lys Leu Glu Val 1640 1645
1650 Ser Glu Leu Ala Thr Val Lys Arg Pro Gln Glu Arg Ser Tyr Met
1655 1660 1665 Leu Gln Arg Trp Gln Glu Asp Gly Gly Val Met Ile Ile
Gly Tyr 1670 1675 1680 Glu Met Tyr Arg Asn Leu Ala Gln Gly Arg Asn
Val Lys Ser Arg 1685 1690 1695 Lys Leu Lys Glu Ile Phe Asn Lys Ala
Leu Val Asp Pro Gly Pro 1700 1705 1710 Asp Phe Val Val Cys Asp Glu
Gly His Ile Leu Lys Asn Glu Ala 1715 1720 1725 Ser Ala Val Ser Lys
Ala Met Asn Ser Ile Arg Ser Arg Arg Arg 1730 1735 1740 Ile Ile Leu
Thr Gly Thr Pro Leu Gln Asn Asn Leu Ile Glu Tyr 1745 1750 1755 His
Cys Met Val Asn Phe Ile Lys Glu Asn Leu Leu Gly Ser Ile 1760 1765
1770 Lys Glu Phe Arg Asn Arg Phe Ile Asn Pro Ile Gln Asn Gly Gln
1775 1780 1785 Cys Ala Asp Ser Thr Met Val Asp Val Arg Val Met Lys
Lys Arg 1790 1795 1800 Ala His Ile Leu Tyr Glu Met Leu Ala Gly Cys
Val Gln Arg Lys 1805 1810 1815 Asp Tyr Thr Ala Leu Thr Lys Phe Leu
Pro Pro Lys His Glu Tyr 1820 1825 1830 Val Leu Ala Val Arg Met Thr
Ser Ile Gln Cys Lys Leu Tyr Gln 1835 1840 1845 Tyr Tyr Leu Asp His
Leu Thr Gly Val Gly Asn Asn Ser Glu Gly 1850 1855 1860 Gly Arg Gly
Lys Ala Gly Ala Lys Leu Phe Gln Asp Phe Gln Met 1865 1870 1875 Leu
Ser Arg Ile Trp Thr His Pro Trp Cys Leu Gln Leu Asp Tyr 1880 1885
1890 Ile Ser Lys Glu Asn Lys Gly Tyr Phe Asp Glu Asp Ser Met Asp
1895 1900 1905 Glu Phe Ile Ala Ser Asp Ser Asp Glu Thr Ser Met Ser
Leu Ser 1910 1915 1920 Ser Asp Asp Tyr Thr Lys Lys Lys Lys Lys Gly
Lys Lys Gly Lys 1925 1930 1935 Lys Asp Ser Ser Ser Ser Gly Ser Gly
Ser Asp Asn Asp Val Glu 1940 1945 1950 Val Ile Lys Val Trp Asn Ser
Arg Ser Arg Gly Gly Gly Glu Gly 1955 1960 1965 Asn Val Asp Glu Thr
Gly Asn Asn Pro Ser Val Ser Leu Lys Leu 1970 1975 1980 Glu Glu Ser
Lys Ala Thr Ser Ser Ser Asn Pro Ser Ser Pro Ala 1985 1990 1995 Pro
Asp Trp Tyr Lys Asp Phe Val Thr Asp Ala Asp Ala Glu Val 2000 2005
2010 Leu Glu His Ser Gly Lys Met Val Leu Leu Phe Glu Ile Leu Arg
2015 2020 2025 Met Ala Glu Glu Ile Gly Asp Lys Val Leu Val Phe Ser
Gln Ser 2030 2035 2040 Leu Ile Ser Leu Asp Leu Ile Glu Asp Phe Leu
Glu Leu Ala Ser 2045 2050 2055 Arg Glu Lys Thr Glu Asp Lys Asp Lys
Pro Leu Ile Tyr Lys Gly 2060 2065 2070 Glu Gly Lys Trp Leu Arg Asn
Ile Asp Tyr Tyr Arg Leu Asp Gly 2075 2080 2085 Ser Thr Thr Ala Gln
Ser Arg Lys Lys Trp Ala Glu Glu Phe Asn 2090 2095 2100 Asp Glu Thr
Asn Val Arg Gly Arg Leu Phe Ile Ile Ser Thr Lys 2105 2110 2115 Ala
Gly Ser Leu Gly Ile Asn Leu Val Ala Ala Asn Arg Val Ile 2120 2125
2130 Ile Phe Asp Ala Ser Trp Asn Pro Ser Tyr Asp Ile Gln Ser Ile
2135 2140 2145 Phe Arg Val Tyr Arg Phe Gly Gln Thr Lys Pro Val Tyr
Val Tyr 2150 2155 2160 Arg Phe Leu Ala Gln Gly Thr Met Glu Asp Lys
Ile Tyr Asp Arg 2165 2170 2175 Gln Val Thr Lys Gln Ser Leu Ser Phe
Arg Val Val Asp Gln Gln 2180 2185 2190 Gln Val Glu Arg His Phe Thr
Met Asn Glu Leu Thr Glu Leu Tyr 2195 2200 2205 Thr Phe Glu Pro Asp
Leu Leu Asp Asp Pro Asn Ser Glu Lys Lys 2210 2215 2220 Lys Lys Arg
Asp Thr Pro Met Leu Pro Lys Asp Thr Ile Leu Ala 2225 2230 2235 Glu
Leu Leu Gln Ile His Lys Glu His Ile Val Gly Tyr His Glu 2240 2245
2250 His Asp Ser Leu Leu Asp His Lys Glu Glu Glu Glu Leu Thr Glu
2255 2260 2265 Glu Glu Arg Lys Ala Ala Trp Ala Glu Tyr Glu Ala Glu
Lys Lys 2270 2275 2280 Gly Leu Thr Met Arg Phe Asn Ile Pro Thr Gly
Thr Asn Leu Pro 2285 2290 2295 Pro Val Ser Phe Asn Ser Gln Thr Pro
Tyr Ile Pro Phe Asn Leu 2300 2305 2310 Gly Ala Leu Ser Ala Met Ser
Asn Gln Gln Leu Glu Asp Leu Ile 2315 2320 2325 Asn Gln Gly Arg Glu
Lys Val Val Glu Ala Thr Asn Ser Val Thr 2330 2335 2340 Ala Val Arg
Ile Gln Pro Leu Glu Asp Ile Ile Ser Ala Val Trp 2345 2350 2355 Lys
Glu Asn Met Asn Leu Ser Glu Ala Gln Val Gln Ala Leu Ala 2360 2365
2370 Leu Ser Arg Gln Ala Ser Gln Glu Leu Asp Val Lys Arg Arg Glu
2375 2380 2385 Ala Ile Tyr Asn Asp Val Leu Thr Lys Gln Gln Met Leu
Ile Ser 2390 2395 2400 Cys Val Gln Arg Ile Leu Met Asn Arg Arg Leu
Gln Gln Gln Tyr 2405 2410 2415 Asn Gln Gln Gln Gln Gln Gln Met Thr
Tyr Gln Gln Ala Thr Leu 2420 2425 2430 Gly His Leu Met Met Pro Lys
Pro Pro Asn Leu Ile Met Asn Pro 2435 2440 2445 Ser Asn Tyr Gln Gln
Ile Asp Met Arg Gly Met Tyr Gln Pro Val 2450 2455 2460 Ala Gly Gly
Met Gln Pro Pro Pro Leu Gln Arg Ala Pro Pro Pro 2465 2470 2475 Met
Arg Ser Lys Asn Pro Gly Pro Ser Gln Gly Lys Ser Met 2480 2485 2490
611202DNAHomo sapiens 6aattctcctg cctgagcctc ggcccaacaa aatggcggcg
gcagcggtgt cgctttgttt 60ccgcggctcc tgcggcggtg gcagtggtag cggcctttga
gctgtgggga ggttccagca 120gcagctacag tgacgactaa gactccagtg
catttctatc gtaaccgggc gcgggggagc 180gcagatcggc gcccagcaat
cacagaagcc gacaaggcgt tcaagcgaaa acatgaccgc 240tgagcccatg
agtgaaagca agttgaatac attggtgcag aagcttcatg acttccttgc
300acactcatca gaagaatctg aagaaacaag ttctcctcca cgacttgcaa
tgaatcaaaa 360cacagataaa atcagtggtt ctggaagtaa ctctgatatg
atggaaaaca gcaaggaaga 420gggaactagc tcttcagaaa aatccaagtc
ttcaggatcg tcacgatcaa agaggaaacc 480ttcaattgta acaaagtatg
tagaatcaga tgatgaaaaa cctttggatg atgaaactgt 540aaatgaagat
gcgtctaatg aaaattcaga aaatgatatt actatgcaga gcttgccaaa
600aggtacagtg attgtacagc cagagccagt gctgaatgaa gacaaagatg
attttaaagg 660gcctgaattt agaagcagaa gtaaaatgaa aactgaaaat
ctcaaaaaac gcggagaaga 720tgggcttcat gggattgtga gctgcactgc
ttgtggacaa caggtcaatc attttcaaaa 780agattccatt tatagacacc
cttcattgca agttcttatt tgtaagaatt gctttaagta 840ttacatgagt
gatgatatta gccgtgactc agatggaatg gatgaacaat gtaggtggtg
900tgcggaaggt ggaaacttga tttgttgtga cttttgccat aatgctttct
gcaagaaatg 960cattctacgc aaccttggtc gaaaggagtt gtccacaata
atggatgaaa acaaccaatg 1020gtattgctac atttgtcacc cagagccttt
gttggacttg gtcactgcat gtaacagcgt 1080atttgagaat ttagaacagt
tgttgcagca aaataagaag aagataaaag ttgacagtga 1140aaagagtaat
aaagtatatg aacatacatc cagattttct ccaaagaaga ctagttcaaa
1200ttgtaatgga gaagaaaaga aattagatga ttcctgttct ggctctgtaa
cctactctta 1260ttccgcacta attgtgccca aagagatgat taagaaggca
aaaaaactga ttgagaccac 1320agccaacatg aactccagtt atgttaaatt
tttaaagcag gcaacagata attcagaaat 1380cagttctgct acaaaattac
gtcagcttaa ggcttttaag tctgtgttgg ctgatattaa 1440gaaggctcat
cttgcattgg aagaagactt aaattccgag tttcgagcga tggatgctgt
1500aaacaaagag aaaaatacca aagagcataa agtcatagat gctaagtttg
aaacaaaagc 1560acgaaaagga gaaaaacctt gtgctttgga aaagaaggat
atttcaaagt cagaagctaa 1620actttcaaga aaacaggtag atagtgagca
catgcatcag aatgttccaa cagaggaaca 1680aagaacaaat aaaagtaccg
gtggtgaaca taagaaatct gatagaaaag aagaacctca 1740atatgaacct
gccaacactt ctgaagattt agacatggat attgtgtctg ttccttcctc
1800agttccagaa gacatttttg agaatcttga gactgctatg gaagttcaga
gttcagttga 1860tcatcaaggg gatggcagca gtggaactga acaagaagtg
gagagttcat ctgtaaaatt 1920aaatatttct tcaaaagaca acagaggagg
tattaaatca aaaactacag ctaaagtaac 1980aaaagaatta tatgttaaac
tcactcctgt ttccctttct aattccccaa ttaaaggtgc 2040tgattgtcag
gaagttccac aagataaaga tggctataaa agttgtggtc tgaaccccaa
2100gttagagaaa tgtggacttg gacaggaaaa cagtgataat gagcatttgg
ttgaaaatga 2160agtttcatta cttttagagg aatctgatct tcgaagatcc
ccacgtgtaa agactacacc 2220cttgaggcga ccgacagaaa ctaaccctgt
aacatctaat tcagatgaag aatgtaatga 2280aacagttaag gagaaacaaa
aactatcagt tccagtgaga aaaaaggata agcgtaattc 2340ttctgacagt
gctatagata atcctaagcc taataaattg ccaaaatcta agcaatcaga
2400gactgtggat caaaattcag attctgatga aatgctagca atcctcaaag
aggtgagcag 2460gatgagtcac agttcttctt cagatactga tattaatgaa
attcatacaa accataagac 2520tttgtatgat ttaaagactc aggcggggaa
agatgataaa ggaaaaagga aacgaaaaag 2580ttctacatct ggctcagatt
ttgatactaa aaagggcaaa tcagctaaga gctctataat 2640ttctaaaaag
aaacgacaaa cccagtctga gtcttctaat tatgactcag aattagaaaa
2700agagataaag agcatgagta aaattggtgc tgccagaacc accaaaaaaa
gaattccaaa 2760tacaaaagat tttgactctt ctgaagatga gaaacacagc
aaaaaaggaa tggataatca 2820agggcacaaa aatttgaaga cctcacaaga
aggatcatct gatgatgctg aaagaaaaca 2880agagagagag actttctctt
cagcagaagg cacagttgat aaagacacga ccatcatgga 2940attaagagat
cgacttccta agaagcagca agcaagtgct tccactgatg gtgtcgataa
3000gctttctggg aaagagcaga gttttacttc tttggaagtt agaaaagttg
ctgaaactaa 3060agaaaagagc aagcatctca aaaccaaaac atgtaaaaaa
gtacaggatg gcttatctga 3120tattgcagag aaattcctaa agaaagacca
gagcgatgaa acttctgaag atgataaaaa 3180gcagagcaaa aagggaactg
aagaaaaaaa gaaaccttca gactttaaga aaaaagtaat 3240taaaatggaa
caacagtatg aatcttcatc tgatggcact gaaaagttac ctgagcgaga
3300agaaatttgt cattttccta agggcataaa acaaattaag aatggaacaa
ctgatggaga 3360aaagaaaagt aaaaaaataa gagataaaac ttctaaaaag
aaggatgaat tatctgatta 3420tgctgagaag tcaacaggga aaggagatag
ttgtgactct tcagaggata aaaagagtaa 3480gaatggagca tatggtagag
agaagaaaag gtgcaagttg cttggaaaga gttcaaggaa 3540gagacaagat
tgttcatcat ctgatactga gaaatattcc atgaaagaag atggttgtaa
3600ctcttctgat aagagactga aaagaataga attgagggaa agaagaaatt
taagttcaaa 3660gagaaatact aaggaaatac aaagtggctc atcatcatct
gatgctgagg aaagttctga 3720agataataaa aagaagaagc aaagaacttc
atctaaaaag aaggcagtca ttgtcaagga 3780gaaaaagaga aactccctaa
gaacaagcac taaaaggaag caagctgaca ttacatcctc 3840atcttcttct
gatatagaag atgatgatca gaattctata ggtgagggaa gcagcgatga
3900acagaaaatt aagcctgtga ctgaaaattt agtgctgtct tcacatactg
gattttgcca 3960atcttcagga gatgaagcct tatctaaatc agtgcctgtc
acagtggatg atgatgatga 4020cgacaatgat cctgagaata gaattgccaa
gaagatgctt ttagaagaaa ttaaagccaa 4080tctttcctct gatgaggatg
gatcttcaga tgatgagcca gaagaaggga aaaaaagaac 4140tggaaaacaa
aatgaagaaa acccaggaga tgaggaagca aaaaatcaag tcaattctga
4200atcagattca gattctgaag aatctaagaa gccaagatac agacataggc
ttttgcggca 4260caaattgact gtgagtgacg gagaatctgg agaagaaaaa
aagacaaagc ctaaagagca 4320taaagaagtc aaaggcagaa acagaagaaa
ggtgagcagt gaagattcag aagattctga 4380ttttcaggaa tcaggagtta
gtgaagaagt tagtgaatcc gaagatgaac agcggcccag 4440aacaaggtct
gcaaagaaag cagagttgga agaaaatcag cggagctata aacagaaaaa
4500gaaaaggcga cgtattaagg ttcaagaaga ttcatccagt gaaaacaaga
gtaattctga 4560ggaagaagag gaggaaaaag aagaggagga ggaagaggag
gaggaggagg aagaggagga 4620ggaagatgaa aatgatgatt ccaagtctcc
tggaaaaggc agaaagaaaa ttcggaagat 4680tcttaaagat gataaactga
gaacagaaac acaaaatgct cttaaggaag aggaagagag 4740acgaaaacgt
attgctgaga gggagcgtga gcgagaaaaa
ttgagagagg tgatagaaat 4800tgaagatgct tcacccacca agtgtccaat
aacaaccaag ttggttttag atgaagatga 4860agaaaccaaa gaacctttag
tgcaggttca tagaaatatg gttatcaaat tgaaacccca 4920tcaagtagat
ggtgttcagt ttatgtggga ttgctgctgt gagtctgtga aaaaaacaaa
4980gaaatctcca ggttcaggat gcattcttgc ccactgtatg ggccttggta
agactttaca 5040ggtggtaagt tttcttcata cagttctttt gtgtgacaaa
ctggatttca gcacggcgtt 5100agtggtttgt cctcttaata ctgctttgaa
ttggatgaat gaatttgaga agtggcaaga 5160gggattaaaa gatgatgaga
agcttgaggt ttctgaatta gcaactgtga aacgtcctca 5220ggagagaagc
tacatgctgc agaggtggca agaagatggt ggtgttatga tcataggcta
5280tgagatgtat agaaatcttg ctcaaggaag gaatgtgaag agtcggaaac
ttaaagaaat 5340atttaacaaa gctttggttg atccaggccc tgattttgtt
gtttgtgatg aaggccatat 5400tctaaaaaat gaagcatctg ctgtttctaa
agctatgaat tctatacgat caaggaggag 5460gattatttta acaggaacac
cacttcaaaa taacctaatt gagtatcatt gtatggttaa 5520ttttatcaag
gaaaatttac ttggatccat taaggagttc aggaatagat ttataaatcc
5580aattcaaaat ggtcagtgtg cagattctac catggtagat gtcagagtga
tgaaaaaacg 5640tgctcacatt ctctatgaga tgttagctgg atgtgttcag
aggaaagatt atacagcatt 5700aacaaaattc ttgcctccaa aacacgaata
tgtgttagct gtgagaatga cttctattca 5760gtgcaagctc tatcagtact
acttagatca cttaacaggt gtgggcaata atagtgaagg 5820tggaagagga
aaggcaggtg caaagctttt ccaagatttt cagatgttaa gtagaatatg
5880gactcatcct tggtgtttgc agctagacta cattagcaaa gaaaataagg
gttattttga 5940tgaagacagt atggatgaat ttatagcctc agattctgat
gaaacctcca tgagtttaag 6000ctccgatgat tatacaaaaa agaagaaaaa
agggaaaaag gggaaaaaag atagtagctc 6060aagtggaagt ggcagtgaca
atgatgttga agtgattaag gtctggaatt caagatctcg 6120gggaggtggt
gaaggaaatg tggatgaaac aggaaacaat ccttctgttt ctttaaaact
6180ggaagaaagt aaagctactt cttcttctaa tccaagcagc ccagctccag
actggtacaa 6240agattttgtt acagatgctg atgctgaggt tttagagcat
tctgggaaaa tggtacttct 6300ctttgaaatt cttcgaatgg cagaggaaat
tggggataaa gtccttgttt tcagccagtc 6360cctcatatct ctggacttga
ttgaagattt tcttgaatta gctagtaggg agaagacaga 6420agataaagat
aaacccctta tttataaagg tgaggggaag tggcttcgaa acattgacta
6480ttaccgttta gatggttcca ctactgcaca gtcaaggaag aagtgggctg
aagaatttaa 6540tgatgaaact aatgtgagag gacgattatt tatcatttct
actaaagcag gatctctagg 6600aattaatctg gtagctgcta atcgagtaat
tatattcgac gcttcttgga atccatctta 6660tgacatccag agtatattca
gagtttatcg ctttggacaa actaagcctg tttatgtata 6720taggttctta
gctcagggaa ccatggaaga taagatttat gatcggcaag taactaagca
6780gtcactgtct tttcgagttg ttgatcagca gcaggtggag cgtcatttta
ctatgaatga 6840gcttactgaa ctttatactt ttgagccaga cttattagat
gaccctaatt cagaaaagaa 6900gaagaagagg gatactccca tgctgccaaa
ggataccata cttgcagagc tccttcagat 6960acataaagaa cacattgtag
gataccatga acatgattct cttttggacc acaaagaaga 7020agaagagttg
actgaagaag aaagaaaagc agcttgggct gagtatgaag cagagaagaa
7080gggactgacc atgcgtttca acataccaac tgggaccaat ttaccccctg
tcagtttcaa 7140ctctcaaact ccttatattc ctttcaattt gggagccctg
tcagcaatga gtaatcaaca 7200gctggaggac ctcattaatc aaggaagaga
aaaagttgta gaagcaacaa acagtgtgac 7260agcagtgagg attcaacctc
ttgaggatat aatttcagct gtatggaagg agaacatgaa 7320tctctcagag
gcccaagtac aggcgttagc attaagtaga caagccagcc aggagcttga
7380tgttaaacga agagaagcaa tctacaatga tgtattgaca aaacaacaga
tgttaatcag 7440ctgtgttcag cgaatactta tgaacagaag gctccagcag
cagtacaatc agcagcaaca 7500gcaacaaatg acttatcaac aagcaacact
gggtcacctc atgatgccaa agcccccaaa 7560tttgatcatg aatccttcta
actaccagca gattgatatg agaggaatgt atcagccagt 7620ggctggtggt
atgcagccac caccattaca gcgtgcacca cccccaatga gaagcaaaaa
7680tccaggacct tcccaaggga aatcaatgtg attttgcact aaaagcttaa
tggattgtta 7740aaatcataga aagatctttt atttttttag gaatcaatga
cttaacagaa ctcaactgta 7800taaatagttt ggtcccctta aatgccaatc
ttccatatta gttttacttt tttttttttt 7860aaatagggca taccatttct
tcctgacatt tgtcagtgat gttgcctaga atcttcttac 7920acacgctgag
tacagaagat atttcaaatt gttttcagtg aaaacaagtc cttccataat
7980agtaacaact ccacagattt cctctctaaa tttttatgcc tgcttttagc
aaccataaaa 8040ttgtcataaa attaataaat ttaggaaaga ataaagattt
atatattcat tctttacata 8100taaaaacaca cagctgagtt cttagagttg
attcctcaag ttatgaaata cttttgtact 8160taatccattt cttgattaaa
gtgattgaaa tggttttaat gttcttttga ctgaagtctg 8220aaactgggct
cctgctttat tgtctctgtg actgaaagtt agaaactgag ggttatcttt
8280gacacagaat tgtgtgcaat attcttaaat actactgctc taaaagttgg
agaagtcttg 8340cagttatctt agcattgtat aaacagcctt aagtatagcc
taagaagaga attccttttt 8400cttctttagt ccttctgcca ttttttattt
tcagttatat gtgctgaaat aattactggt 8460aaaatttcag ggttgtggat
tatcttccac acatgaattt tctctctcct ggcacgaata 8520taaagcacat
ctcttaactg catggtgcca gtgctaatgc ttcatcctgt tgctggcagt
8580gggatgtgga cttagaaaat caagttctag cattttagta ggttaacact
gaagttgtgg 8640ttgttaggtt cacaccctgt tttataaaca acatcaaaat
ggcagaacca ttgctgactt 8700taggttcaca tgaggaatgt acttttaaca
attcccagta ctatcagtat tgtgaaataa 8760ttcctctgaa agataagaat
cactggcttc tatgcgcttc ttttctctca tcatcatgtt 8820cttttacccc
agtttcctta cattttttta aattgtttca gagtttgttt tttttttagt
8880ttagattgtg aggcaattat taaatcaaaa ttaattcatc caatacccct
ttactagaag 8940ttttactaga aaatgtatta cattttattt tttcttaatc
cagttctgca aaaatgacct 9000ataaatttat tcatgtacaa ttttggttac
ttgaattgtt aaagaaaaca ttgtttttga 9060ctatgggagt caactcaaca
tggcagaacc atttttgaga tgatgataca acaggtagtg 9120aaacagctta
agaattccaa aaaaaaaaaa aaaaaaaaaa aaaagaaaac tgggtttggg
9180ctttgcttta ggtatcactg gattagaatg agtttaacat tagctaaaac
tgctttgagt 9240tgtttggatg attaagagat tgccattttt atcttggaag
aactagtggt aaaacatcca 9300agagcactag gattgtgata cagaatttgt
gaggtttggt ggatccacgc ccctctcccc 9360cactttccca tgatgaaata
tcactaataa atcctgtata tttagatatt atgctagcca 9420tgtaatcaga
tttatttaat tgggtggggc aggtgtgtat ttactttaga aaaaatgaaa
9480aagacaagat ttatgagaaa tatttgaagg cagtacactc tggccaactg
ttaccagttg 9540gtatttctac aagttcagaa tattttaaac ctgatttact
agacctggga attttcaaca 9600tggtctaatt atttactcaa agacatagat
gtgaaaattt taggcaacct tctaaatctt 9660tttcaccatg gatgaaacta
taacttaaag aataatactt agaagggtta attggaaatc 9720agagtttgaa
ataaaacttg gaccactttg tatacactct tctcacttga cattttagct
9780atataatatg tactttgagt ataacatcaa gctttaacaa atatttaaag
acaaaaaaat 9840cacgtcagta aaatactaaa aggctcattt ttatatttgt
tttagatgtt ttaaatagtt 9900gcaatggatt aaaaatgatg atttaaaatg
ttgcttgtaa tacagttttg cctgctaaat 9960tctccacatt ttgtaacctg
ttttatttct ttgggtgtaa agcgtttttg cttagtattg 10020tgatattgta
tatgttttgt cccagttgta tagtaatgtt tcagtccatc atccagcttt
10080ggctgctgaa atcatacagc tgtgaagact tgcctttgtt tctgttagac
tgcttttcag 10140ttctgtattg agtatcttaa gtactgtaga aaagatgtca
cttcttcctt taaggctgtt 10200ttgtaatata tataaggact ggaattgtgt
ttttaaagaa aagcattcaa gtatgacaat 10260atactatctg tgttttcacc
attcaaagtg ctgtttagta gttgaaactt aaactattta 10320atgtcattta
ataaagtgac caaaatgtgt tgtgctcttt attgtatttt cacagctttg
10380aaaatctgtg cacatactgt ttcatagaaa atgtatagct tttgttgtcc
tatataatgg 10440tggttctttt gcacatttag ttatttaata ttgagaggtc
acgaagtttg gttattgaat 10500ctgttatata ctaaattctg taaagggaga
tctctcatct caaaaagaat ttacatacca 10560ggaagtccat gtgtgtttgt
gttagttttg gatgtctttg tgtaatccag ccccatttcc 10620tgtttcccaa
cagctgtaac actcatttta agtcaagcag ggctaccaac ccacacttga
10680tagaaaagct gcttaccatt cagaagcttc cttattacct ggcctccaaa
tgagctgaat 10740attttgtagc cttcccttag ctatgttcat tttccctcca
ttatcataaa atcagatcga 10800tatttatgtg ccccaaacaa aactttaaga
gcagttacat tctgtcccag tagcccttgt 10860ttcctttgag agtagcatgt
tgtgaggcta tagagactta ttctaccagt aaaacaggtc 10920aatcctttta
catgtttatt atactaaaaa ttatgttcag ggtatttact actttatttc
10980accagactca gtctcaagtg acttggctat ctccaaatca gatctaccct
tagagaataa 11040acatttttct accgttattt tttttcaagt ctataatctg
agccagtccc aaaggagtga 11100tcaagtttca gaaatgcttt catcttcaca
acattttata tatactatta tatggggtga 11160ataaagtttt aaatccgaaa
tataaaaaaa aaaaaaaaaa aa 1120272454PRTHomo sapiens 7Met Thr Ala Glu
Pro Met Ser Glu Ser Lys Leu Asn Thr Leu Val Gln 1 5 10 15 Lys Leu
His Asp Phe Leu Ala His Ser Ser Glu Glu Ser Glu Glu Thr 20 25 30
Ser Ser Pro Pro Arg Leu Ala Met Asn Gln Asn Thr Asp Lys Ile Ser 35
40 45 Gly Ser Gly Ser Asn Ser Asp Met Met Glu Asn Ser Lys Glu Glu
Gly 50 55 60 Thr Ser Ser Ser Glu Lys Ser Lys Ser Ser Gly Ser Ser
Arg Ser Lys 65 70 75 80 Arg Lys Pro Ser Ile Val Thr Lys Tyr Val Glu
Ser Asp Asp Glu Lys 85 90 95 Pro Leu Asp Asp Glu Thr Val Asn Glu
Asp Ala Ser Asn Glu Asn Ser 100 105 110 Glu Asn Asp Ile Thr Met Gln
Ser Leu Pro Lys Glu Asp Gly Leu His 115 120 125 Gly Ile Val Ser Cys
Thr Ala Cys Gly Gln Gln Val Asn His Phe Gln 130 135 140 Lys Asp Ser
Ile Tyr Arg His Pro Ser Leu Gln Val Leu Ile Cys Lys 145 150 155 160
Asn Cys Phe Lys Tyr Tyr Met Ser Asp Asp Ile Ser Arg Asp Ser Asp 165
170 175 Gly Met Asp Glu Gln Cys Arg Trp Cys Ala Glu Gly Gly Asn Leu
Ile 180 185 190 Cys Cys Asp Phe Cys His Asn Ala Phe Cys Lys Lys Cys
Ile Leu Arg 195 200 205 Asn Leu Gly Arg Lys Glu Leu Ser Thr Ile Met
Asp Glu Asn Asn Gln 210 215 220 Trp Tyr Cys Tyr Ile Cys His Pro Glu
Pro Leu Leu Asp Leu Val Thr 225 230 235 240 Ala Cys Asn Ser Val Phe
Glu Asn Leu Glu Gln Leu Leu Gln Gln Asn 245 250 255 Lys Lys Lys Ile
Lys Val Asp Ser Glu Lys Ser Asn Lys Val Tyr Glu 260 265 270 His Thr
Ser Arg Phe Ser Pro Lys Lys Thr Ser Ser Asn Cys Asn Gly 275 280 285
Glu Glu Lys Lys Leu Asp Asp Ser Cys Ser Gly Ser Val Thr Tyr Ser 290
295 300 Tyr Ser Ala Leu Ile Val Pro Lys Glu Met Ile Lys Lys Ala Lys
Lys 305 310 315 320 Leu Ile Glu Thr Thr Ala Asn Met Asn Ser Ser Tyr
Val Lys Phe Leu 325 330 335 Lys Gln Ala Thr Asp Asn Ser Glu Ile Ser
Ser Ala Thr Lys Leu Arg 340 345 350 Gln Leu Lys Ala Phe Lys Ser Val
Leu Ala Asp Ile Lys Lys Ala His 355 360 365 Leu Ala Leu Glu Glu Asp
Leu Asn Ser Glu Phe Arg Ala Met Asp Ala 370 375 380 Val Asn Lys Glu
Lys Asn Thr Lys Glu His Lys Val Ile Asp Ala Lys 385 390 395 400 Phe
Glu Thr Lys Ala Arg Lys Gly Glu Lys Pro Cys Ala Leu Glu Lys 405 410
415 Lys Asp Ile Ser Lys Ser Glu Ala Lys Leu Ser Arg Lys Gln Val Asp
420 425 430 Ser Glu His Met His Gln Asn Val Pro Thr Glu Glu Gln Arg
Thr Asn 435 440 445 Lys Ser Thr Gly Gly Glu His Lys Lys Ser Asp Arg
Lys Glu Glu Pro 450 455 460 Gln Tyr Glu Pro Ala Asn Thr Ser Glu Asp
Leu Asp Met Asp Ile Val 465 470 475 480 Ser Val Pro Ser Ser Val Pro
Glu Asp Ile Phe Glu Asn Leu Glu Thr 485 490 495 Ala Met Glu Val Gln
Ser Ser Val Asp His Gln Gly Asp Gly Ser Ser 500 505 510 Gly Thr Glu
Gln Glu Val Glu Ser Ser Ser Val Lys Leu Asn Ile Ser 515 520 525 Ser
Lys Asp Asn Arg Gly Gly Ile Lys Ser Lys Thr Thr Ala Lys Val 530 535
540 Thr Lys Glu Leu Tyr Val Lys Leu Thr Pro Val Ser Leu Ser Asn Ser
545 550 555 560 Pro Ile Lys Gly Ala Asp Cys Gln Glu Val Pro Gln Asp
Lys Asp Gly 565 570 575 Tyr Lys Ser Cys Gly Leu Asn Pro Lys Leu Glu
Lys Cys Gly Leu Gly 580 585 590 Gln Glu Asn Ser Asp Asn Glu His Leu
Val Glu Asn Glu Val Ser Leu 595 600 605 Leu Leu Glu Glu Ser Asp Leu
Arg Arg Ser Pro Arg Val Lys Thr Thr 610 615 620 Pro Leu Arg Arg Pro
Thr Glu Thr Asn Pro Val Thr Ser Asn Ser Asp 625 630 635 640 Glu Glu
Cys Asn Glu Thr Val Lys Glu Lys Gln Lys Leu Ser Val Pro 645 650 655
Val Arg Lys Lys Asp Lys Arg Asn Ser Ser Asp Ser Ala Ile Asp Asn 660
665 670 Pro Lys Pro Asn Lys Leu Pro Lys Ser Lys Gln Ser Glu Thr Val
Asp 675 680 685 Gln Asn Ser Asp Ser Asp Glu Met Leu Ala Ile Leu Lys
Glu Val Ser 690 695 700 Arg Met Ser His Ser Ser Ser Ser Asp Thr Asp
Ile Asn Glu Ile His 705 710 715 720 Thr Asn His Lys Thr Leu Tyr Asp
Leu Lys Thr Gln Ala Gly Lys Asp 725 730 735 Asp Lys Gly Lys Arg Lys
Arg Lys Ser Ser Thr Ser Gly Ser Asp Phe 740 745 750 Asp Thr Lys Lys
Gly Lys Ser Ala Lys Ser Ser Ile Ile Ser Lys Lys 755 760 765 Lys Arg
Gln Thr Gln Ser Glu Ser Ser Asn Tyr Asp Ser Glu Leu Glu 770 775 780
Lys Glu Ile Lys Ser Met Ser Lys Ile Gly Ala Ala Arg Thr Thr Lys 785
790 795 800 Lys Arg Ile Pro Asn Thr Lys Asp Phe Asp Ser Ser Glu Asp
Glu Lys 805 810 815 His Ser Lys Lys Gly Met Asp Asn Gln Gly His Lys
Asn Leu Lys Thr 820 825 830 Ser Gln Glu Gly Ser Ser Asp Asp Ala Glu
Arg Lys Gln Glu Arg Glu 835 840 845 Thr Phe Ser Ser Ala Glu Gly Thr
Val Asp Lys Asp Thr Thr Ile Met 850 855 860 Glu Leu Arg Asp Arg Leu
Pro Lys Lys Gln Gln Ala Ser Ala Ser Thr 865 870 875 880 Asp Gly Val
Asp Lys Leu Ser Gly Lys Glu Gln Ser Phe Thr Ser Leu 885 890 895 Glu
Val Arg Lys Val Ala Glu Thr Lys Glu Lys Ser Lys His Leu Lys 900 905
910 Thr Lys Thr Cys Lys Lys Val Gln Asp Gly Leu Ser Asp Ile Ala Glu
915 920 925 Lys Phe Leu Lys Lys Asp Gln Ser Asp Glu Thr Ser Glu Asp
Asp Lys 930 935 940 Lys Gln Ser Lys Lys Gly Thr Glu Glu Lys Lys Lys
Pro Ser Asp Phe 945 950 955 960 Lys Lys Lys Val Ile Lys Met Glu Gln
Gln Tyr Glu Ser Ser Ser Asp 965 970 975 Gly Thr Glu Lys Leu Pro Glu
Arg Glu Glu Ile Cys His Phe Pro Lys 980 985 990 Gly Ile Lys Gln Ile
Lys Asn Gly Thr Thr Asp Gly Glu Lys Lys Ser 995 1000 1005 Lys Lys
Ile Arg Asp Lys Thr Ser Lys Lys Lys Asp Glu Leu Ser 1010 1015 1020
Asp Tyr Ala Glu Lys Ser Thr Gly Lys Gly Asp Ser Cys Asp Ser 1025
1030 1035 Ser Glu Asp Lys Lys Ser Lys Asn Gly Ala Tyr Gly Arg Glu
Lys 1040 1045 1050 Lys Arg Cys Lys Leu Leu Gly Lys Ser Ser Arg Lys
Arg Gln Asp 1055 1060 1065 Cys Ser Ser Ser Asp Thr Glu Lys Tyr Ser
Met Lys Glu Asp Gly 1070 1075 1080 Cys Asn Ser Ser Asp Lys Arg Leu
Lys Arg Ile Glu Leu Arg Glu 1085 1090 1095 Arg Arg Asn Leu Ser Ser
Lys Arg Asn Thr Lys Glu Ile Gln Ser 1100 1105 1110 Gly Ser Ser Ser
Ser Asp Ala Glu Glu Ser Ser Glu Asp Asn Lys 1115 1120 1125 Lys Lys
Lys Gln Arg Thr Ser Ser Lys Lys Lys Ala Val Ile Val 1130 1135 1140
Lys Glu Lys Lys Arg Asn Ser Leu Arg Thr Ser Thr Lys Arg Lys 1145
1150 1155 Gln Ala Asp Ile Thr Ser Ser Ser Ser Ser Asp Ile Glu Asp
Asp 1160 1165 1170 Asp Gln Asn Ser Ile Gly Glu Gly Ser Ser Asp Glu
Gln Lys Ile 1175 1180 1185 Lys Pro Val Thr Glu Asn Leu Val Leu Ser
Ser His Thr Gly Phe 1190 1195 1200 Cys Gln Ser Ser Gly Asp Glu Ala
Leu Ser Lys Ser Val Pro Val 1205 1210 1215 Thr Val Asp Asp Asp Asp
Asp Asp Asn Asp Pro Glu Asn Arg Ile 1220 1225 1230 Ala Lys Lys Met
Leu Leu Glu Glu Ile Lys Ala Asn Leu Ser Ser 1235 1240 1245 Asp Glu
Asp Gly Ser Ser Asp Asp Glu Pro Glu Glu Gly Lys Lys 1250 1255 1260
Arg Thr Gly Lys Gln Asn Glu Glu Asn Pro Gly Asp Glu Glu Ala 1265
1270 1275 Lys Asn Gln Val Asn Ser Glu Ser Asp Ser Asp Ser Glu Glu
Ser 1280 1285 1290 Lys Lys Pro Arg Tyr Arg His Arg Leu Leu Arg His
Lys Leu Thr 1295
1300 1305 Val Ser Asp Gly Glu Ser Gly Glu Glu Lys Lys Thr Lys Pro
Lys 1310 1315 1320 Glu His Lys Glu Val Lys Gly Arg Asn Arg Arg Lys
Val Ser Ser 1325 1330 1335 Glu Asp Ser Glu Asp Ser Asp Phe Gln Glu
Ser Gly Val Ser Glu 1340 1345 1350 Glu Val Ser Glu Ser Glu Asp Glu
Gln Arg Pro Arg Thr Arg Ser 1355 1360 1365 Ala Lys Lys Ala Glu Leu
Glu Glu Asn Gln Arg Ser Tyr Lys Gln 1370 1375 1380 Lys Lys Lys Arg
Arg Arg Ile Lys Val Gln Glu Asp Ser Ser Ser 1385 1390 1395 Glu Asn
Lys Ser Asn Ser Glu Glu Glu Glu Glu Glu Lys Glu Glu 1400 1405 1410
Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Glu 1415
1420 1425 Asn Asp Asp Ser Lys Ser Pro Gly Lys Gly Arg Lys Lys Ile
Arg 1430 1435 1440 Lys Ile Leu Lys Asp Asp Lys Leu Arg Thr Glu Thr
Gln Asn Ala 1445 1450 1455 Leu Lys Glu Glu Glu Glu Arg Arg Lys Arg
Ile Ala Glu Arg Glu 1460 1465 1470 Arg Glu Arg Glu Lys Leu Arg Glu
Val Ile Glu Ile Glu Asp Ala 1475 1480 1485 Ser Pro Thr Lys Cys Pro
Ile Thr Thr Lys Leu Val Leu Asp Glu 1490 1495 1500 Asp Glu Glu Thr
Lys Glu Pro Leu Val Gln Val His Arg Asn Met 1505 1510 1515 Val Ile
Lys Leu Lys Pro His Gln Val Asp Gly Val Gln Phe Met 1520 1525 1530
Trp Asp Cys Cys Cys Glu Ser Val Lys Lys Thr Lys Lys Ser Pro 1535
1540 1545 Gly Ser Gly Cys Ile Leu Ala His Cys Met Gly Leu Gly Lys
Thr 1550 1555 1560 Leu Gln Val Val Ser Phe Leu His Thr Val Leu Leu
Cys Asp Lys 1565 1570 1575 Leu Asp Phe Ser Thr Ala Leu Val Val Cys
Pro Leu Asn Thr Ala 1580 1585 1590 Leu Asn Trp Met Asn Glu Phe Glu
Lys Trp Gln Glu Gly Leu Lys 1595 1600 1605 Asp Asp Glu Lys Leu Glu
Val Ser Glu Leu Ala Thr Val Lys Arg 1610 1615 1620 Pro Gln Glu Arg
Ser Tyr Met Leu Gln Arg Trp Gln Glu Asp Gly 1625 1630 1635 Gly Val
Met Ile Ile Gly Tyr Glu Met Tyr Arg Asn Leu Ala Gln 1640 1645 1650
Gly Arg Asn Val Lys Ser Arg Lys Leu Lys Glu Ile Phe Asn Lys 1655
1660 1665 Ala Leu Val Asp Pro Gly Pro Asp Phe Val Val Cys Asp Glu
Gly 1670 1675 1680 His Ile Leu Lys Asn Glu Ala Ser Ala Val Ser Lys
Ala Met Asn 1685 1690 1695 Ser Ile Arg Ser Arg Arg Arg Ile Ile Leu
Thr Gly Thr Pro Leu 1700 1705 1710 Gln Asn Asn Leu Ile Glu Tyr His
Cys Met Val Asn Phe Ile Lys 1715 1720 1725 Glu Asn Leu Leu Gly Ser
Ile Lys Glu Phe Arg Asn Arg Phe Ile 1730 1735 1740 Asn Pro Ile Gln
Asn Gly Gln Cys Ala Asp Ser Thr Met Val Asp 1745 1750 1755 Val Arg
Val Met Lys Lys Arg Ala His Ile Leu Tyr Glu Met Leu 1760 1765 1770
Ala Gly Cys Val Gln Arg Lys Asp Tyr Thr Ala Leu Thr Lys Phe 1775
1780 1785 Leu Pro Pro Lys His Glu Tyr Val Leu Ala Val Arg Met Thr
Ser 1790 1795 1800 Ile Gln Cys Lys Leu Tyr Gln Tyr Tyr Leu Asp His
Leu Thr Gly 1805 1810 1815 Val Gly Asn Asn Ser Glu Gly Gly Arg Gly
Lys Ala Gly Ala Lys 1820 1825 1830 Leu Phe Gln Asp Phe Gln Met Leu
Ser Arg Ile Trp Thr His Pro 1835 1840 1845 Trp Cys Leu Gln Leu Asp
Tyr Ile Ser Lys Glu Asn Lys Gly Tyr 1850 1855 1860 Phe Asp Glu Asp
Ser Met Asp Glu Phe Ile Ala Ser Asp Ser Asp 1865 1870 1875 Glu Thr
Ser Met Ser Leu Ser Ser Asp Asp Tyr Thr Lys Lys Lys 1880 1885 1890
Lys Lys Gly Lys Lys Gly Lys Lys Asp Ser Ser Ser Ser Gly Ser 1895
1900 1905 Gly Ser Asp Asn Asp Val Glu Val Ile Lys Val Trp Asn Ser
Arg 1910 1915 1920 Ser Arg Gly Gly Gly Glu Gly Asn Val Asp Glu Thr
Gly Asn Asn 1925 1930 1935 Pro Ser Val Ser Leu Lys Leu Glu Glu Ser
Lys Ala Thr Ser Ser 1940 1945 1950 Ser Asn Pro Ser Ser Pro Ala Pro
Asp Trp Tyr Lys Asp Phe Val 1955 1960 1965 Thr Asp Ala Asp Ala Glu
Val Leu Glu His Ser Gly Lys Met Val 1970 1975 1980 Leu Leu Phe Glu
Ile Leu Arg Met Ala Glu Glu Ile Gly Asp Lys 1985 1990 1995 Val Leu
Val Phe Ser Gln Ser Leu Ile Ser Leu Asp Leu Ile Glu 2000 2005 2010
Asp Phe Leu Glu Leu Ala Ser Arg Glu Lys Thr Glu Asp Lys Asp 2015
2020 2025 Lys Pro Leu Ile Tyr Lys Gly Glu Gly Lys Trp Leu Arg Asn
Ile 2030 2035 2040 Asp Tyr Tyr Arg Leu Asp Gly Ser Thr Thr Ala Gln
Ser Arg Lys 2045 2050 2055 Lys Trp Ala Glu Glu Phe Asn Asp Glu Thr
Asn Val Arg Gly Arg 2060 2065 2070 Leu Phe Ile Ile Ser Thr Lys Ala
Gly Ser Leu Gly Ile Asn Leu 2075 2080 2085 Val Ala Ala Asn Arg Val
Ile Ile Phe Asp Ala Ser Trp Asn Pro 2090 2095 2100 Ser Tyr Asp Ile
Gln Ser Ile Phe Arg Val Tyr Arg Phe Gly Gln 2105 2110 2115 Thr Lys
Pro Val Tyr Val Tyr Arg Phe Leu Ala Gln Gly Thr Met 2120 2125 2130
Glu Asp Lys Ile Tyr Asp Arg Gln Val Thr Lys Gln Ser Leu Ser 2135
2140 2145 Phe Arg Val Val Asp Gln Gln Gln Val Glu Arg His Phe Thr
Met 2150 2155 2160 Asn Glu Leu Thr Glu Leu Tyr Thr Phe Glu Pro Asp
Leu Leu Asp 2165 2170 2175 Asp Pro Asn Ser Glu Lys Lys Lys Lys Arg
Asp Thr Pro Met Leu 2180 2185 2190 Pro Lys Asp Thr Ile Leu Ala Glu
Leu Leu Gln Ile His Lys Glu 2195 2200 2205 His Ile Val Gly Tyr His
Glu His Asp Ser Leu Leu Asp His Lys 2210 2215 2220 Glu Glu Glu Glu
Leu Thr Glu Glu Glu Arg Lys Ala Ala Trp Ala 2225 2230 2235 Glu Tyr
Glu Ala Glu Lys Lys Gly Leu Thr Met Arg Phe Asn Ile 2240 2245 2250
Pro Thr Gly Thr Asn Leu Pro Pro Val Ser Phe Asn Ser Gln Thr 2255
2260 2265 Pro Tyr Ile Pro Phe Asn Leu Gly Ala Leu Ser Ala Met Ser
Asn 2270 2275 2280 Gln Gln Leu Glu Asp Leu Ile Asn Gln Gly Arg Glu
Lys Val Val 2285 2290 2295 Glu Ala Thr Asn Ser Val Thr Ala Val Arg
Ile Gln Pro Leu Glu 2300 2305 2310 Asp Ile Ile Ser Ala Val Trp Lys
Glu Asn Met Asn Leu Ser Glu 2315 2320 2325 Ala Gln Val Gln Ala Leu
Ala Leu Ser Arg Gln Ala Ser Gln Glu 2330 2335 2340 Leu Asp Val Lys
Arg Arg Glu Ala Ile Tyr Asn Asp Val Leu Thr 2345 2350 2355 Lys Gln
Gln Met Leu Ile Ser Cys Val Gln Arg Ile Leu Met Asn 2360 2365 2370
Arg Arg Leu Gln Gln Gln Tyr Asn Gln Gln Gln Gln Gln Gln Met 2375
2380 2385 Thr Tyr Gln Gln Ala Thr Leu Gly His Leu Met Met Pro Lys
Pro 2390 2395 2400 Pro Asn Leu Ile Met Asn Pro Ser Asn Tyr Gln Gln
Ile Asp Met 2405 2410 2415 Arg Gly Met Tyr Gln Pro Val Ala Gly Gly
Met Gln Pro Pro Pro 2420 2425 2430 Leu Gln Arg Ala Pro Pro Pro Met
Arg Ser Lys Asn Pro Gly Pro 2435 2440 2445 Ser Gln Gly Lys Ser Met
2450 811088DNAHomo sapiens 8aattctcctg cctgagcctc ggcccaacaa
aatggcggcg gcagcggtgt cgctttgttt 60ccgcggctcc tgcggcggtg gcagtggtag
cggcctttga gctgtgggga ggttccagca 120gcagctacag tgacgactaa
gactccagtg catttctatc gtaaccgggc gcgggggagc 180gcagatcggc
gcccagcaat cacagaagcc gacaaggcgt tcaagcgaaa acatgaccgc
240tgagcccatg agtgaaagca agttgaatac attggtgcag aagcttcatg
acttccttgc 300acactcatca gaagaatctg aagaaacaag ttctcctcca
cgacttgcaa tgaatcaaaa 360cacagataaa atcagtggtt ctggaagtaa
ctctgatatg atggaaaaca gcaaggaaga 420gggaactagc tcttcagaaa
aatccaagtc ttcaggatcg tcacgatcaa agaggaaacc 480ttcaattgta
acaaagtatg tagaatcaga tgatgaaaaa cctttggatg atgaaactgt
540aaatgaagat gcgtctaatg aaaattcaga aaatgatatt actatgcaga
gcttgccaaa 600agaagatggg cttcatggga ttgtgagctg cactgcttgt
ggacaacagg tcaatcattt 660tcaaaaagat tccatttata gacacccttc
attgcaagtt cttatttgta agaattgctt 720taagtattac atgagtgatg
atattagccg tgactcagat ggaatggatg aacaatgtag 780gtggtgtgcg
gaaggtggaa acttgatttg ttgtgacttt tgccataatg ctttctgcaa
840gaaatgcatt ctacgcaacc ttggtcgaaa ggagttgtcc acaataatgg
atgaaaacaa 900ccaatggtat tgctacattt gtcacccaga gcctttgttg
gacttggtca ctgcatgtaa 960cagcgtattt gagaatttag aacagttgtt
gcagcaaaat aagaagaaga taaaagttga 1020cagtgaaaag agtaataaag
tatatgaaca tacatccaga ttttctccaa agaagactag 1080ttcaaattgt
aatggagaag aaaagaaatt agatgattcc tgttctggct ctgtaaccta
1140ctcttattcc gcactaattg tgcccaaaga gatgattaag aaggcaaaaa
aactgattga 1200gaccacagcc aacatgaact ccagttatgt taaattttta
aagcaggcaa cagataattc 1260agaaatcagt tctgctacaa aattacgtca
gcttaaggct tttaagtctg tgttggctga 1320tattaagaag gctcatcttg
cattggaaga agacttaaat tccgagtttc gagcgatgga 1380tgctgtaaac
aaagagaaaa ataccaaaga gcataaagtc atagatgcta agtttgaaac
1440aaaagcacga aaaggagaaa aaccttgtgc tttggaaaag aaggatattt
caaagtcaga 1500agctaaactt tcaagaaaac aggtagatag tgagcacatg
catcagaatg ttccaacaga 1560ggaacaaaga acaaataaaa gtaccggtgg
tgaacataag aaatctgata gaaaagaaga 1620acctcaatat gaacctgcca
acacttctga agatttagac atggatattg tgtctgttcc 1680ttcctcagtt
ccagaagaca tttttgagaa tcttgagact gctatggaag ttcagagttc
1740agttgatcat caaggggatg gcagcagtgg aactgaacaa gaagtggaga
gttcatctgt 1800aaaattaaat atttcttcaa aagacaacag aggaggtatt
aaatcaaaaa ctacagctaa 1860agtaacaaaa gaattatatg ttaaactcac
tcctgtttcc ctttctaatt ccccaattaa 1920aggtgctgat tgtcaggaag
ttccacaaga taaagatggc tataaaagtt gtggtctgaa 1980ccccaagtta
gagaaatgtg gacttggaca ggaaaacagt gataatgagc atttggttga
2040aaatgaagtt tcattacttt tagaggaatc tgatcttcga agatccccac
gtgtaaagac 2100tacacccttg aggcgaccga cagaaactaa ccctgtaaca
tctaattcag atgaagaatg 2160taatgaaaca gttaaggaga aacaaaaact
atcagttcca gtgagaaaaa aggataagcg 2220taattcttct gacagtgcta
tagataatcc taagcctaat aaattgccaa aatctaagca 2280atcagagact
gtggatcaaa attcagattc tgatgaaatg ctagcaatcc tcaaagaggt
2340gagcaggatg agtcacagtt cttcttcaga tactgatatt aatgaaattc
atacaaacca 2400taagactttg tatgatttaa agactcaggc ggggaaagat
gataaaggaa aaaggaaacg 2460aaaaagttct acatctggct cagattttga
tactaaaaag ggcaaatcag ctaagagctc 2520tataatttct aaaaagaaac
gacaaaccca gtctgagtct tctaattatg actcagaatt 2580agaaaaagag
ataaagagca tgagtaaaat tggtgctgcc agaaccacca aaaaaagaat
2640tccaaataca aaagattttg actcttctga agatgagaaa cacagcaaaa
aaggaatgga 2700taatcaaggg cacaaaaatt tgaagacctc acaagaagga
tcatctgatg atgctgaaag 2760aaaacaagag agagagactt tctcttcagc
agaaggcaca gttgataaag acacgaccat 2820catggaatta agagatcgac
ttcctaagaa gcagcaagca agtgcttcca ctgatggtgt 2880cgataagctt
tctgggaaag agcagagttt tacttctttg gaagttagaa aagttgctga
2940aactaaagaa aagagcaagc atctcaaaac caaaacatgt aaaaaagtac
aggatggctt 3000atctgatatt gcagagaaat tcctaaagaa agaccagagc
gatgaaactt ctgaagatga 3060taaaaagcag agcaaaaagg gaactgaaga
aaaaaagaaa ccttcagact ttaagaaaaa 3120agtaattaaa atggaacaac
agtatgaatc ttcatctgat ggcactgaaa agttacctga 3180gcgagaagaa
atttgtcatt ttcctaaggg cataaaacaa attaagaatg gaacaactga
3240tggagaaaag aaaagtaaaa aaataagaga taaaacttct aaaaagaagg
atgaattatc 3300tgattatgct gagaagtcaa cagggaaagg agatagttgt
gactcttcag aggataaaaa 3360gagtaagaat ggagcatatg gtagagagaa
gaaaaggtgc aagttgcttg gaaagagttc 3420aaggaagaga caagattgtt
catcatctga tactgagaaa tattccatga aagaagatgg 3480ttgtaactct
tctgataaga gactgaaaag aatagaattg agggaaagaa gaaatttaag
3540ttcaaagaga aatactaagg aaatacaaag tggctcatca tcatctgatg
ctgaggaaag 3600ttctgaagat aataaaaaga agaagcaaag aacttcatct
aaaaagaagg cagtcattgt 3660caaggagaaa aagagaaact ccctaagaac
aagcactaaa aggaagcaag ctgacattac 3720atcctcatct tcttctgata
tagaagatga tgatcagaat tctataggtg agggaagcag 3780cgatgaacag
aaaattaagc ctgtgactga aaatttagtg ctgtcttcac atactggatt
3840ttgccaatct tcaggagatg aagccttatc taaatcagtg cctgtcacag
tggatgatga 3900tgatgacgac aatgatcctg agaatagaat tgccaagaag
atgcttttag aagaaattaa 3960agccaatctt tcctctgatg aggatggatc
ttcagatgat gagccagaag aagggaaaaa 4020aagaactgga aaacaaaatg
aagaaaaccc aggagatgag gaagcaaaaa atcaagtcaa 4080ttctgaatca
gattcagatt ctgaagaatc taagaagcca agatacagac ataggctttt
4140gcggcacaaa ttgactgtga gtgacggaga atctggagaa gaaaaaaaga
caaagcctaa 4200agagcataaa gaagtcaaag gcagaaacag aagaaaggtg
agcagtgaag attcagaaga 4260ttctgatttt caggaatcag gagttagtga
agaagttagt gaatccgaag atgaacagcg 4320gcccagaaca aggtctgcaa
agaaagcaga gttggaagaa aatcagcgga gctataaaca 4380gaaaaagaaa
aggcgacgta ttaaggttca agaagattca tccagtgaaa acaagagtaa
4440ttctgaggaa gaagaggagg aaaaagaaga ggaggaggaa gaggaggagg
aggaggaaga 4500ggaggaggaa gatgaaaatg atgattccaa gtctcctgga
aaaggcagaa agaaaattcg 4560gaagattctt aaagatgata aactgagaac
agaaacacaa aatgctctta aggaagagga 4620agagagacga aaacgtattg
ctgagaggga gcgtgagcga gaaaaattga gagaggtgat 4680agaaattgaa
gatgcttcac ccaccaagtg tccaataaca accaagttgg ttttagatga
4740agatgaagaa accaaagaac ctttagtgca ggttcataga aatatggtta
tcaaattgaa 4800accccatcaa gtagatggtg ttcagtttat gtgggattgc
tgctgtgagt ctgtgaaaaa 4860aacaaagaaa tctccaggtt caggatgcat
tcttgcccac tgtatgggcc ttggtaagac 4920tttacaggtg gtaagttttc
ttcatacagt tcttttgtgt gacaaactgg atttcagcac 4980ggcgttagtg
gtttgtcctc ttaatactgc tttgaattgg atgaatgaat ttgagaagtg
5040gcaagaggga ttaaaagatg atgagaagct tgaggtttct gaattagcaa
ctgtgaaacg 5100tcctcaggag agaagctaca tgctgcagag gtggcaagaa
gatggtggtg ttatgatcat 5160aggctatgag atgtatagaa atcttgctca
aggaaggaat gtgaagagtc ggaaacttaa 5220agaaatattt aacaaagctt
tggttgatcc aggccctgat tttgttgttt gtgatgaagg 5280ccatattcta
aaaaatgaag catctgctgt ttctaaagct atgaattcta tacgatcaag
5340gaggaggatt attttaacag gaacaccact tcaaaataac ctaattgagt
atcattgtat 5400ggttaatttt atcaaggaaa atttacttgg atccattaag
gagttcagga atagatttat 5460aaatccaatt caaaatggtc agtgtgcaga
ttctaccatg gtagatgtca gagtgatgaa 5520aaaacgtgct cacattctct
atgagatgtt agctggatgt gttcagagga aagattatac 5580agcattaaca
aaattcttgc ctccaaaaca cgaatatgtg ttagctgtga gaatgacttc
5640tattcagtgc aagctctatc agtactactt agatcactta acaggtgtgg
gcaataatag 5700tgaaggtgga agaggaaagg caggtgcaaa gcttttccaa
gattttcaga tgttaagtag 5760aatatggact catccttggt gtttgcagct
agactacatt agcaaagaaa ataagggtta 5820ttttgatgaa gacagtatgg
atgaatttat agcctcagat tctgatgaaa cctccatgag 5880tttaagctcc
gatgattata caaaaaagaa gaaaaaaggg aaaaagggga aaaaagatag
5940tagctcaagt ggaagtggca gtgacaatga tgttgaagtg attaaggtct
ggaattcaag 6000atctcgggga ggtggtgaag gaaatgtgga tgaaacagga
aacaatcctt ctgtttcttt 6060aaaactggaa gaaagtaaag ctacttcttc
ttctaatcca agcagcccag ctccagactg 6120gtacaaagat tttgttacag
atgctgatgc tgaggtttta gagcattctg ggaaaatggt 6180acttctcttt
gaaattcttc gaatggcaga ggaaattggg gataaagtcc ttgttttcag
6240ccagtccctc atatctctgg acttgattga agattttctt gaattagcta
gtagggagaa 6300gacagaagat aaagataaac cccttattta taaaggtgag
gggaagtggc ttcgaaacat 6360tgactattac cgtttagatg gttccactac
tgcacagtca aggaagaagt gggctgaaga 6420atttaatgat gaaactaatg
tgagaggacg attatttatc atttctacta aagcaggatc 6480tctaggaatt
aatctggtag ctgctaatcg agtaattata ttcgacgctt cttggaatcc
6540atcttatgac atccagagta tattcagagt ttatcgcttt ggacaaacta
agcctgttta 6600tgtatatagg ttcttagctc agggaaccat ggaagataag
atttatgatc ggcaagtaac 6660taagcagtca ctgtcttttc gagttgttga
tcagcagcag gtggagcgtc attttactat 6720gaatgagctt actgaacttt
atacttttga gccagactta ttagatgacc ctaattcaga 6780aaagaagaag
aagagggata ctcccatgct gccaaaggat accatacttg cagagctcct
6840tcagatacat aaagaacaca ttgtaggata ccatgaacat gattctcttt
tggaccacaa 6900agaagaagaa gagttgactg aagaagaaag aaaagcagct
tgggctgagt atgaagcaga 6960gaagaaggga ctgaccatgc gtttcaacat
accaactggg accaatttac cccctgtcag 7020tttcaactct caaactcctt
atattccttt caatttggga gccctgtcag caatgagtaa 7080tcaacagctg
gaggacctca ttaatcaagg aagagaaaaa gttgtagaag caacaaacag
7140tgtgacagca gtgaggattc aacctcttga ggatataatt tcagctgtat
ggaaggagaa 7200catgaatctc tcagaggccc aagtacaggc gttagcatta
agtagacaag ccagccagga 7260gcttgatgtt aaacgaagag aagcaatcta
caatgatgta ttgacaaaac aacagatgtt 7320aatcagctgt gttcagcgaa
tacttatgaa cagaaggctc cagcagcagt acaatcagca 7380gcaacagcaa
caaatgactt atcaacaagc aacactgggt cacctcatga tgccaaagcc
7440cccaaatttg atcatgaatc cttctaacta ccagcagatt gatatgagag
gaatgtatca 7500gccagtggct ggtggtatgc agccaccacc attacagcgt
gcaccacccc caatgagaag 7560caaaaatcca ggaccttccc aagggaaatc
aatgtgattt tgcactaaaa gcttaatgga 7620ttgttaaaat catagaaaga
tcttttattt ttttaggaat caatgactta acagaactca 7680actgtataaa
tagtttggtc cccttaaatg ccaatcttcc atattagttt tacttttttt
7740ttttttaaat agggcatacc atttcttcct gacatttgtc agtgatgttg
cctagaatct 7800tcttacacac gctgagtaca gaagatattt caaattgttt
tcagtgaaaa caagtccttc 7860cataatagta acaactccac agatttcctc
tctaaatttt tatgcctgct tttagcaacc 7920ataaaattgt cataaaatta
ataaatttag gaaagaataa agatttatat attcattctt 7980tacatataaa
aacacacagc tgagttctta gagttgattc ctcaagttat gaaatacttt
8040tgtacttaat ccatttcttg attaaagtga ttgaaatggt tttaatgttc
ttttgactga 8100agtctgaaac tgggctcctg ctttattgtc tctgtgactg
aaagttagaa actgagggtt 8160atctttgaca cagaattgtg tgcaatattc
ttaaatacta ctgctctaaa agttggagaa 8220gtcttgcagt tatcttagca
ttgtataaac agccttaagt atagcctaag aagagaattc 8280ctttttcttc
tttagtcctt ctgccatttt ttattttcag ttatatgtgc tgaaataatt
8340actggtaaaa tttcagggtt gtggattatc ttccacacat gaattttctc
tctcctggca 8400cgaatataaa gcacatctct taactgcatg gtgccagtgc
taatgcttca tcctgttgct 8460ggcagtggga tgtggactta gaaaatcaag
ttctagcatt ttagtaggtt aacactgaag 8520ttgtggttgt taggttcaca
ccctgtttta taaacaacat caaaatggca gaaccattgc 8580tgactttagg
ttcacatgag gaatgtactt ttaacaattc ccagtactat cagtattgtg
8640aaataattcc tctgaaagat aagaatcact ggcttctatg cgcttctttt
ctctcatcat 8700catgttcttt taccccagtt tccttacatt tttttaaatt
gtttcagagt ttgttttttt 8760tttagtttag attgtgaggc aattattaaa
tcaaaattaa ttcatccaat acccctttac 8820tagaagtttt actagaaaat
gtattacatt ttattttttc ttaatccagt tctgcaaaaa 8880tgacctataa
atttattcat gtacaatttt ggttacttga attgttaaag aaaacattgt
8940ttttgactat gggagtcaac tcaacatggc agaaccattt ttgagatgat
gatacaacag 9000gtagtgaaac agcttaagaa ttccaaaaaa aaaaaaaaaa
aaaaaaaaaa gaaaactggg 9060tttgggcttt gctttaggta tcactggatt
agaatgagtt taacattagc taaaactgct 9120ttgagttgtt tggatgatta
agagattgcc atttttatct tggaagaact agtggtaaaa 9180catccaagag
cactaggatt gtgatacaga atttgtgagg tttggtggat ccacgcccct
9240ctcccccact ttcccatgat gaaatatcac taataaatcc tgtatattta
gatattatgc 9300tagccatgta atcagattta tttaattggg tggggcaggt
gtgtatttac tttagaaaaa 9360atgaaaaaga caagatttat gagaaatatt
tgaaggcagt acactctggc caactgttac 9420cagttggtat ttctacaagt
tcagaatatt ttaaacctga tttactagac ctgggaattt 9480tcaacatggt
ctaattattt actcaaagac atagatgtga aaattttagg caaccttcta
9540aatctttttc accatggatg aaactataac ttaaagaata atacttagaa
gggttaattg 9600gaaatcagag tttgaaataa aacttggacc actttgtata
cactcttctc acttgacatt 9660ttagctatat aatatgtact ttgagtataa
catcaagctt taacaaatat ttaaagacaa 9720aaaaatcacg tcagtaaaat
actaaaaggc tcatttttat atttgtttta gatgttttaa 9780atagttgcaa
tggattaaaa atgatgattt aaaatgttgc ttgtaataca gttttgcctg
9840ctaaattctc cacattttgt aacctgtttt atttctttgg gtgtaaagcg
tttttgctta 9900gtattgtgat attgtatatg ttttgtccca gttgtatagt
aatgtttcag tccatcatcc 9960agctttggct gctgaaatca tacagctgtg
aagacttgcc tttgtttctg ttagactgct 10020tttcagttct gtattgagta
tcttaagtac tgtagaaaag atgtcacttc ttcctttaag 10080gctgttttgt
aatatatata aggactggaa ttgtgttttt aaagaaaagc attcaagtat
10140gacaatatac tatctgtgtt ttcaccattc aaagtgctgt ttagtagttg
aaacttaaac 10200tatttaatgt catttaataa agtgaccaaa atgtgttgtg
ctctttattg tattttcaca 10260gctttgaaaa tctgtgcaca tactgtttca
tagaaaatgt atagcttttg ttgtcctata 10320taatggtggt tcttttgcac
atttagttat ttaatattga gaggtcacga agtttggtta 10380ttgaatctgt
tatatactaa attctgtaaa gggagatctc tcatctcaaa aagaatttac
10440ataccaggaa gtccatgtgt gtttgtgtta gttttggatg tctttgtgta
atccagcccc 10500atttcctgtt tcccaacagc tgtaacactc attttaagtc
aagcagggct accaacccac 10560acttgataga aaagctgctt accattcaga
agcttcctta ttacctggcc tccaaatgag 10620ctgaatattt tgtagccttc
ccttagctat gttcattttc cctccattat cataaaatca 10680gatcgatatt
tatgtgcccc aaacaaaact ttaagagcag ttacattctg tcccagtagc
10740ccttgtttcc tttgagagta gcatgttgtg aggctataga gacttattct
accagtaaaa 10800caggtcaatc cttttacatg tttattatac taaaaattat
gttcagggta tttactactt 10860tatttcacca gactcagtct caagtgactt
ggctatctcc aaatcagatc tacccttaga 10920gaataaacat ttttctaccg
ttattttttt tcaagtctat aatctgagcc agtcccaaag 10980gagtgatcaa
gtttcagaaa tgctttcatc ttcacaacat tttatatata ctattatatg
11040gggtgaataa agttttaaat ccgaaatata aaaaaaaaaa aaaaaaaa
1108892285PRTHomo sapiens 9Met Ala Ala Gln Val Ala Pro Ala Ala Ala
Ser Ser Leu Gly Asn Pro 1 5 10 15 Pro Pro Pro Pro Pro Ser Glu Leu
Lys Lys Ala Glu Gln Gln Gln Arg 20 25 30 Glu Glu Ala Gly Gly Glu
Ala Ala Ala Ala Ala Ala Ala Glu Arg Gly 35 40 45 Glu Met Lys Ala
Ala Ala Gly Gln Glu Ser Glu Gly Pro Ala Val Gly 50 55 60 Pro Pro
Gln Pro Leu Gly Lys Glu Leu Gln Asp Gly Ala Glu Ser Asn 65 70 75 80
Gly Gly Gly Gly Gly Gly Gly Ala Gly Ser Gly Gly Gly Pro Gly Ala 85
90 95 Glu Pro Asp Leu Lys Asn Ser Asn Gly Asn Ala Gly Pro Arg Pro
Ala 100 105 110 Leu Asn Asn Asn Leu Thr Glu Pro Pro Gly Gly Gly Gly
Gly Gly Ser 115 120 125 Ser Asp Gly Val Gly Ala Pro Pro His Ser Ala
Ala Ala Ala Leu Pro 130 135 140 Pro Pro Ala Tyr Gly Phe Gly Gln Pro
Tyr Gly Arg Ser Pro Ser Ala 145 150 155 160 Val Ala Ala Ala Ala Ala
Ala Val Phe His Gln Gln His Gly Gly Gln 165 170 175 Gln Ser Pro Gly
Leu Ala Ala Leu Gln Ser Gly Gly Gly Gly Gly Leu 180 185 190 Glu Pro
Tyr Ala Gly Pro Gln Gln Asn Ser His Asp His Gly Phe Pro 195 200 205
Asn His Gln Tyr Asn Ser Tyr Tyr Pro Asn Arg Ser Ala Tyr Pro Pro 210
215 220 Pro Ala Pro Ala Tyr Ala Leu Ser Ser Pro Arg Gly Gly Thr Pro
Gly 225 230 235 240 Ser Gly Ala Ala Ala Ala Ala Gly Ser Lys Pro Pro
Pro Ser Ser Ser 245 250 255 Ala Ser Ala Ser Ser Ser Ser Ser Ser Phe
Ala Gln Gln Arg Phe Gly 260 265 270 Ala Met Gly Gly Gly Gly Pro Ser
Ala Ala Gly Gly Gly Thr Pro Gln 275 280 285 Pro Thr Ala Thr Pro Thr
Leu Asn Gln Leu Leu Thr Ser Pro Ser Ser 290 295 300 Ala Arg Gly Tyr
Gln Gly Tyr Pro Gly Gly Asp Tyr Ser Gly Gly Pro 305 310 315 320 Gln
Asp Gly Gly Ala Gly Lys Gly Pro Ala Asp Met Ala Ser Gln Cys 325 330
335 Trp Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ser Gly Gly
340 345 350 Ala Gln Gln Arg Ser His His Ala Pro Met Ser Pro Gly Ser
Ser Gly 355 360 365 Gly Gly Gly Gln Pro Leu Ala Arg Thr Pro Gln Pro
Ser Ser Pro Met 370 375 380 Asp Gln Met Gly Lys Met Arg Pro Gln Pro
Tyr Gly Gly Thr Asn Pro 385 390 395 400 Tyr Ser Gln Gln Gln Gly Pro
Pro Ser Gly Pro Gln Gln Gly His Gly 405 410 415 Tyr Pro Gly Gln Pro
Tyr Gly Ser Gln Thr Pro Gln Arg Tyr Pro Met 420 425 430 Thr Met Gln
Gly Arg Ala Gln Ser Ala Met Gly Gly Leu Ser Tyr Thr 435 440 445 Gln
Gln Ile Pro Pro Tyr Gly Gln Gln Gly Pro Ser Gly Tyr Gly Gln 450 455
460 Gln Gly Gln Thr Pro Tyr Tyr Asn Gln Gln Ser Pro His Pro Gln Gln
465 470 475 480 Gln Gln Pro Pro Tyr Ser Gln Gln Pro Pro Ser Gln Thr
Pro His Ala 485 490 495 Gln Pro Ser Tyr Gln Gln Gln Pro Gln Ser Gln
Pro Pro Gln Leu Gln 500 505 510 Ser Ser Gln Pro Pro Tyr Ser Gln Gln
Pro Ser Gln Pro Pro His Gln 515 520 525 Gln Ser Pro Ala Pro Tyr Pro
Ser Gln Gln Ser Thr Thr Gln Gln His 530 535 540 Pro Gln Ser Gln Pro
Pro Tyr Ser Gln Pro Gln Ala Gln Ser Pro Tyr 545 550 555 560 Gln Gln
Gln Gln Pro Gln Gln Pro Ala Pro Ser Thr Leu Ser Gln Gln 565 570 575
Ala Ala Tyr Pro Gln Pro Gln Ser Gln Gln Ser Gln Gln Thr Ala Tyr 580
585 590 Ser Gln Gln Arg Phe Pro Pro Pro Gln Glu Leu Ser Gln Asp Ser
Phe 595 600 605 Gly Ser Gln Ala Ser Ser Ala Pro Ser Met Thr Ser Ser
Lys Gly Gly 610 615 620 Gln Glu Asp Met Asn Leu Ser Leu Gln Ser Arg
Pro Ser Ser Leu Pro 625 630 635 640 Asp Leu Ser Gly Ser Ile Asp Asp
Leu Pro Met Gly Thr Glu Gly Ala 645 650 655 Leu Ser Pro Gly Val Ser
Thr Ser Gly Ile Ser Ser Ser Gln Gly Glu 660 665 670 Gln Ser Asn Pro
Ala Gln Ser Pro Phe Ser Pro His Thr Ser Pro His 675 680 685 Leu Pro
Gly Ile Arg Gly Pro Ser Pro Ser Pro Val Gly Ser Pro Ala 690 695 700
Ser Val Ala Gln Ser Arg Ser Gly Pro Leu Ser Pro Ala Ala Val Pro 705
710 715 720 Gly Asn Gln Met Pro Pro Arg Pro Pro Ser Gly Gln Ser Asp
Ser Ile 725 730 735 Met His Pro Ser Met Asn Gln Ser Ser Ile Ala Gln
Asp Arg Gly Tyr 740 745 750 Met Gln Arg Asn Pro Gln Met Pro Gln Tyr
Ser Ser Pro Gln Pro Gly 755 760 765 Ser Ala Leu Ser Pro Arg Gln Pro
Ser Gly Gly Gln Ile His Thr Gly 770 775 780 Met Gly Ser Tyr Gln Gln
Asn Ser Met Gly Ser Tyr Gly Pro Gln Gly 785 790 795 800 Gly Gln Tyr
Gly Pro Gln Gly Gly Tyr Pro Arg Gln Pro Asn Tyr Asn 805 810 815 Ala
Leu Pro Asn Ala Asn Tyr Pro Ser Ala Gly Met Ala Gly Gly Ile 820 825
830 Asn Pro Met Gly Ala Gly Gly Gln Met His Gly Gln Pro Gly Ile Pro
835 840 845 Pro Tyr Gly Thr Leu Pro Pro Gly Arg Met Ser His Ala Ser
Met Gly 850 855 860 Asn Arg Pro Tyr Gly Pro Asn Met Ala Asn Met Pro
Pro Gln Val Gly 865 870 875 880 Ser Gly Met Cys Pro Pro Pro Gly Gly
Met Asn Arg Lys Thr Gln Glu 885 890 895 Thr Ala Val Ala Met His Val
Ala Ala Asn Ser Ile Gln Asn Arg Pro 900 905 910 Pro Gly Tyr Pro Asn
Met Asn Gln Gly Gly Met Met Gly Thr Gly Pro 915 920 925 Pro Tyr Gly
Gln Gly Ile Asn Ser Met Ala Gly Met Ile Asn Pro Gln 930 935 940 Gly
Pro Pro Tyr Ser Met Gly Gly Thr Met Ala Asn Asn Ser Ala Gly 945 950
955 960 Met Ala Ala Ser Pro Glu Met Met Gly Leu Gly Asp Val Lys Leu
Thr 965 970 975 Pro Ala Thr Lys Met Asn Asn Lys Ala Asp Gly Thr Pro
Lys Thr Glu 980 985 990 Ser Lys Ser Lys Lys Ser Ser Ser Ser Thr Thr
Thr Asn Glu Lys Ile 995 1000 1005 Thr Lys Leu Tyr Glu Leu Gly Gly
Glu Pro Glu Arg Lys Met Trp 1010 1015 1020 Val Asp Arg Tyr Leu Ala
Phe Thr Glu Glu Lys Ala Met Gly Met 1025 1030 1035 Thr Asn Leu Pro
Ala Val Gly Arg Lys Pro Leu Asp Leu Tyr Arg 1040 1045 1050 Leu Tyr
Val Ser Val Lys Glu Ile Gly Gly Leu Thr Gln Val Asn 1055 1060 1065
Lys Asn Lys Lys Trp Arg Glu Leu Ala Thr Asn Leu Asn Val Gly 1070
1075 1080 Thr Ser Ser Ser Ala Ala Ser Ser Leu Lys Lys Gln Tyr Ile
Gln 1085 1090 1095 Cys Leu Tyr Ala Phe Glu Cys Lys Ile Glu Arg Gly
Glu Asp Pro 1100 1105 1110 Pro Pro Asp Ile Phe Ala Ala Ala Asp Ser
Lys Lys Ser Gln Pro 1115 1120 1125 Lys Ile Gln Pro Pro Ser Pro Ala
Gly Ser Gly Ser Met Gln Gly 1130 1135 1140 Pro Gln Thr Pro Gln Ser
Thr Ser Ser Ser Met Ala Glu Gly Gly 1145 1150 1155 Asp Leu Lys Pro
Pro Thr Pro Ala Ser Thr Pro His Ser Gln Ile 1160 1165 1170 Pro Pro
Leu Pro Gly Met Ser Arg Ser Asn Ser Val Gly Ile Gln 1175 1180 1185
Asp Ala Phe Asn Asp Gly Ser Asp Ser Thr Phe Gln Lys Arg Asn 1190
1195 1200 Ser Met Thr Pro Asn Pro Gly Tyr Gln Pro Ser Met Asn Thr
Ser 1205 1210 1215 Asp Met Met Gly Arg Met Ser Tyr Glu Pro Asn Lys
Asp Pro Tyr 1220 1225 1230 Gly Ser Met Arg Lys Ala Pro Gly Ser Asp
Pro Phe Met Ser Ser 1235 1240 1245 Gly Gln Gly Pro Asn Gly Gly Met
Gly Asp Pro Tyr Ser Arg Ala 1250 1255 1260 Ala Gly Pro Gly Leu Gly
Asn Val Ala Met Gly Pro Arg Gln His 1265 1270 1275 Tyr Pro Tyr Gly
Gly Pro Tyr Asp Arg Val Arg Thr Glu Pro Gly 1280 1285 1290 Ile Gly
Pro Glu Gly Asn Met Ser Thr Gly Ala Pro Gln Pro Asn 1295 1300 1305
Leu Met Pro Ser Asn Pro Asp Ser Gly Met Tyr Ser Pro Ser Arg 1310
1315 1320 Tyr Pro Pro Gln Gln Gln Gln Gln Gln Gln Gln Arg His Asp
Ser 1325 1330 1335 Tyr Gly Asn Gln Phe Ser Thr Gln Gly Thr Pro Ser
Gly Ser Pro 1340 1345 1350 Phe Pro Ser Gln Gln Thr Thr Met Tyr Gln
Gln Gln Gln Gln Asn 1355 1360 1365 Tyr Lys Arg Pro Met Asp Gly Thr
Tyr Gly Pro Pro Ala Lys Arg 1370 1375 1380 His Glu Gly Glu Met Tyr
Ser Val Pro Tyr Ser Thr Gly Gln Gly 1385 1390 1395 Gln Pro Gln Gln
Gln Gln Leu Pro Pro Ala Gln Pro Gln Pro Ala 1400 1405 1410 Ser Gln
Gln Gln Ala Ala Gln Pro Ser Pro Gln Gln Asp Val Tyr 1415 1420 1425
Asn Gln Tyr Gly Asn Ala Tyr Pro Ala Thr Ala Thr Ala Ala Thr 1430
1435 1440 Glu Arg Arg Pro Ala Gly Gly Pro Gln Asn Gln Phe Pro Phe
Gln 1445 1450 1455 Phe Gly Arg Asp Arg Val Ser Ala Pro Pro Gly Thr
Asn Ala Gln 1460 1465 1470 Gln Asn Met Pro Pro Gln Met Met Gly Gly
Pro Ile Gln Ala Ser 1475 1480 1485 Ala Glu Val Ala Gln Gln Gly Thr
Met Trp Gln Gly Arg Asn Asp 1490 1495 1500 Met Thr Tyr Asn Tyr Ala
Asn Arg Gln Ser Thr Gly Ser Ala Pro 1505 1510 1515 Gln Gly Pro Ala
Tyr His Gly Val Asn Arg Thr Asp Glu Met Leu 1520 1525 1530 His Thr
Asp Gln Arg Ala Asn His Glu Gly Ser Trp Pro Ser His 1535 1540 1545
Gly Thr Arg Gln Pro Pro Tyr Gly Pro Ser Ala Pro Val Pro Pro 1550
1555 1560 Met Thr Arg Pro Pro Pro Ser Asn Tyr Gln Pro Pro Pro Ser
Met 1565 1570 1575 Gln Asn His Ile Pro Gln Val Ser Ser Pro Ala Pro
Leu Pro Arg 1580 1585 1590 Pro Met Glu Asn Arg Thr Ser Pro Ser Lys
Ser Pro Phe Leu His 1595 1600 1605 Ser Gly Met Lys Met Gln Lys Ala
Gly Pro Pro Val Pro Ala Ser 1610 1615 1620 His Ile Ala Pro Ala Pro
Val Gln Pro Pro Met Ile Arg Arg Asp 1625 1630 1635 Ile Thr Phe Pro
Pro Gly Ser Val Glu Ala Thr Gln Pro Val Leu 1640 1645 1650 Lys Gln
Arg Arg Arg Leu Thr Met Lys Asp Ile Gly Thr Pro Glu 1655
1660 1665 Ala Trp Arg Val Met Met Ser Leu Lys Ser Gly Leu Leu Ala
Glu 1670 1675 1680 Ser Thr Trp Ala Leu Asp Thr Ile Asn Ile Leu Leu
Tyr Asp Asp 1685 1690 1695 Asn Ser Ile Met Thr Phe Asn Leu Ser Gln
Leu Pro Gly Leu Leu 1700 1705 1710 Glu Leu Leu Val Glu Tyr Phe Arg
Arg Cys Leu Ile Glu Ile Phe 1715 1720 1725 Gly Ile Leu Lys Glu Tyr
Glu Val Gly Asp Pro Gly Gln Arg Thr 1730 1735 1740 Leu Leu Asp Pro
Gly Arg Phe Ser Lys Val Ser Ser Pro Ala Pro 1745 1750 1755 Met Glu
Gly Gly Glu Glu Glu Glu Glu Leu Leu Gly Pro Lys Leu 1760 1765 1770
Glu Glu Glu Glu Glu Glu Glu Val Val Glu Asn Asp Glu Glu Ile 1775
1780 1785 Ala Phe Ser Gly Lys Asp Lys Pro Ala Ser Glu Asn Ser Glu
Glu 1790 1795 1800 Lys Leu Ile Ser Lys Phe Asp Lys Leu Pro Val Lys
Ile Val Gln 1805 1810 1815 Lys Asn Asp Pro Phe Val Val Asp Cys Ser
Asp Lys Leu Gly Arg 1820 1825 1830 Val Gln Glu Phe Asp Ser Gly Leu
Leu His Trp Arg Ile Gly Gly 1835 1840 1845 Gly Asp Thr Thr Glu His
Ile Gln Thr His Phe Glu Ser Lys Thr 1850 1855 1860 Glu Leu Leu Pro
Ser Arg Pro His Ala Pro Cys Pro Pro Ala Pro 1865 1870 1875 Arg Lys
His Val Thr Thr Ala Glu Gly Thr Pro Gly Thr Thr Asp 1880 1885 1890
Gln Glu Gly Pro Pro Pro Asp Gly Pro Pro Glu Lys Arg Ile Thr 1895
1900 1905 Ala Thr Met Asp Asp Met Leu Ser Thr Arg Ser Ser Thr Leu
Thr 1910 1915 1920 Glu Asp Gly Ala Lys Ser Ser Glu Ala Ile Lys Glu
Ser Ser Lys 1925 1930 1935 Phe Pro Phe Gly Ile Ser Pro Ala Gln Ser
His Arg Asn Ile Lys 1940 1945 1950 Ile Leu Glu Asp Glu Pro His Ser
Lys Asp Glu Thr Pro Leu Cys 1955 1960 1965 Thr Leu Leu Asp Trp Gln
Asp Ser Leu Ala Lys Arg Cys Val Cys 1970 1975 1980 Val Ser Asn Thr
Ile Arg Ser Leu Ser Phe Val Pro Gly Asn Asp 1985 1990 1995 Phe Glu
Met Ser Lys His Pro Gly Leu Leu Leu Ile Leu Gly Lys 2000 2005 2010
Leu Ile Leu Leu His His Lys His Pro Glu Arg Lys Gln Ala Pro 2015
2020 2025 Leu Thr Tyr Glu Lys Glu Glu Glu Gln Asp Gln Gly Val Ser
Cys 2030 2035 2040 Asn Lys Val Glu Trp Trp Trp Asp Cys Leu Glu Met
Leu Arg Glu 2045 2050 2055 Asn Thr Leu Val Thr Leu Ala Asn Ile Ser
Gly Gln Leu Asp Leu 2060 2065 2070 Ser Pro Tyr Pro Glu Ser Ile Cys
Leu Pro Val Leu Asp Gly Leu 2075 2080 2085 Leu His Trp Ala Val Cys
Pro Ser Ala Glu Ala Gln Asp Pro Phe 2090 2095 2100 Ser Thr Leu Gly
Pro Asn Ala Val Leu Ser Pro Gln Arg Leu Val 2105 2110 2115 Leu Glu
Thr Leu Ser Lys Leu Ser Ile Gln Asp Asn Asn Val Asp 2120 2125 2130
Leu Ile Leu Ala Thr Pro Pro Phe Ser Arg Leu Glu Lys Leu Tyr 2135
2140 2145 Ser Thr Met Val Arg Phe Leu Ser Asp Arg Lys Asn Pro Val
Cys 2150 2155 2160 Arg Glu Met Ala Val Val Leu Leu Ala Asn Leu Ala
Gln Gly Asp 2165 2170 2175 Ser Leu Ala Ala Arg Ala Ile Ala Val Gln
Lys Gly Ser Ile Gly 2180 2185 2190 Asn Leu Leu Gly Phe Leu Glu Asp
Ser Leu Ala Ala Thr Gln Phe 2195 2200 2205 Gln Gln Ser Gln Ala Ser
Leu Leu His Met Gln Asn Pro Pro Phe 2210 2215 2220 Glu Pro Thr Ser
Val Asp Met Met Arg Arg Ala Ala Arg Ala Leu 2225 2230 2235 Leu Ala
Leu Ala Lys Val Asp Glu Asn His Ser Glu Phe Thr Leu 2240 2245 2250
Tyr Glu Ser Arg Leu Leu Asp Ile Ser Val Ser Pro Leu Met Asn 2255
2260 2265 Ser Leu Val Ser Gln Val Ile Cys Asp Val Leu Phe Leu Ile
Gly 2270 2275 2280 Gln Ser 2285 108585DNAHomo sapiens 10cagaaagcgg
agagtcacag cggggccagg ccctggggag cggagcctcc accgcccccc 60tcattcccag
gcaagggctt ggggggaatg agccgggaga gccgggtccc gagcctacag
120agccgggagc agctgagccg ccggcgcctc ggccgccgcc gccgcctcct
cctcctccgc 180cgccgccagc ccggagcctg agccggcggg gcggggggga
gaggagcgag cgcagcgcag 240cagcggagcc ccgcgaggcc cgcccgggcg
ggtggggagg gcagcccggg ggactgggcc 300ccggggcggg gtgggagggg
gggagaagac gaagacaggg ccgggtctct ccgcggacga 360gacagcgggg
atcatggccg cgcaggtcgc ccccgccgcc gccagcagcc tgggcaaccc
420gccgccgccg ccgccctcgg agctgaagaa agccgagcag cagcagcggg
aggaggcggg 480gggcgaggcg gcggcggcgg cagcggccga gcgcggggaa
atgaaggcag ccgccgggca 540ggaaagcgag ggccccgccg tggggccgcc
gcagccgctg ggaaaggagc tgcaggacgg 600ggccgagagc aatgggggtg
gcggcggcgg cggagccggc agcggcggcg ggcccggcgc 660ggagccggac
ctgaagaact cgaacgggaa cgcgggccct aggcccgccc tgaacaataa
720cctcacggag ccgcccggcg gcggcggtgg cggcagcagc gatggggtgg
gggcgcctcc 780tcactcagcc gcggccgcct tgccgccccc agcctacggc
ttcgggcaac cctacggccg 840gagcccgtct gccgtcgccg ccgccgcggc
cgccgtcttc caccaacaac atggcggaca 900acaaagccct ggcctggcag
cgctgcagag cggcggcggc gggggcctgg agccctacgc 960ggggccccag
cagaactctc acgaccacgg cttccccaac caccagtaca actcctacta
1020ccccaaccgc agcgcctacc ccccgcccgc cccggcctac gcgctgagct
ccccgagagg 1080tggcactccg ggctccggcg cggcggcggc tgccggctcc
aagccgcctc cctcctccag 1140cgcctccgcc tcctcgtcgt cttcgtcctt
cgctcagcag cgcttcgggg ccatgggggg 1200aggcggcccc tccgcggccg
gcgggggaac tccccagccc accgccaccc ccaccctcaa 1260ccaactgctc
acgtcgccca gctcggcccg gggctaccag ggctaccccg ggggcgacta
1320cagtggcggg ccccaggacg ggggcgccgg caagggcccg gcggacatgg
cctcgcagtg 1380ttggggggct gcggcggcgg cagctgcggc ggcggccgcc
tcgggagggg cccaacaaag 1440gagccaccac gcgcccatga gccccgggag
cagcggcggc ggggggcagc cgctcgcccg 1500gacccctcag ccatccagtc
caatggatca gatgggcaag atgagacctc agccatatgg 1560cgggactaac
ccatactcgc agcaacaggg acctccgtca ggaccgcagc aaggacatgg
1620gtacccaggg cagccatacg ggtcccagac cccgcagcgg tacccgatga
ccatgcaggg 1680ccgggcgcag agtgccatgg gcggcctctc ttatacacag
cagattcctc cttatggaca 1740acaaggcccc agcgggtatg gtcaacaggg
ccagactcca tattacaacc agcaaagtcc 1800tcaccctcag cagcagcagc
caccctactc ccagcaacca ccgtcccaga cccctcatgc 1860ccaaccttcg
tatcagcagc agccacagtc tcaaccacca cagctccagt cctctcagcc
1920tccatactcc cagcagccat cccagcctcc acatcagcag tccccggctc
catacccctc 1980ccagcagtcg acgacacagc agcaccccca gagccagccc
ccctactcac agccacaggc 2040tcagtctcct taccagcagc agcaacctca
gcagccagca ccctcgacgc tctcccagca 2100ggctgcgtat cctcagcccc
agtctcagca gtcccagcaa actgcctatt cccagcagcg 2160cttccctcca
ccgcaggagc tatctcaaga ttcatttggg tctcaggcat cctcagcccc
2220ctcaatgacc tccagtaagg gagggcaaga agatatgaac ctgagccttc
agtcaagacc 2280ctccagcttg cctgatctat ctggttcaat agatgacctc
cccatgggga cagaaggagc 2340tctgagtcct ggagtgagca catcagggat
ttccagcagc caaggagagc agagtaatcc 2400agctcagtct cctttctctc
ctcatacctc ccctcacctg cctggcatcc gaggcccttc 2460cccgtcccct
gttggctctc ccgccagtgt tgctcagtct cgctcaggac cactctcgcc
2520tgctgcagtg ccaggcaacc agatgccacc tcggccaccc agtggccagt
cggacagcat 2580catgcatcct tccatgaacc aatcaagcat tgcccaagat
cgaggttata tgcagaggaa 2640cccccagatg ccccagtaca gttcccccca
gcccggctca gccttatctc cgcgtcagcc 2700ttccggagga cagatacaca
caggcatggg ctcctaccag cagaactcca tggggagcta 2760tggtccccag
gggggtcagt atggcccaca aggtggctac cccaggcagc caaactataa
2820tgccttgccc aatgccaact accccagtgc aggcatggct ggaggcataa
accccatggg 2880tgccggaggt caaatgcatg gacagcctgg catcccacct
tatggcacac tccctccagg 2940gaggatgagt cacgcctcca tgggcaaccg
gccttatggc cctaacatgg ccaatatgcc 3000acctcaggtt gggtcaggga
tgtgtccccc accagggggc atgaaccgga aaacccaaga 3060aactgctgtc
gccatgcatg ttgctgccaa ctctatccaa aacaggccgc caggctaccc
3120caatatgaat caagggggca tgatgggaac tggacctcct tatggacaag
ggattaatag 3180tatggctggc atgatcaacc ctcagggacc cccatattcc
atgggtggaa ccatggccaa 3240caattctgca gggatggcag ccagcccaga
gatgatgggc cttggggatg taaagttaac 3300tccagccacc aaaatgaaca
acaaggcaga tgggacaccc aagacagaat ccaaatccaa 3360gaaatccagt
tcttctacta caaccaatga gaagatcacc aagttgtatg agctgggtgg
3420tgagcctgag aggaagatgt gggtggaccg ttatctggcc ttcactgagg
agaaggccat 3480gggcatgaca aatctgcctg ctgtgggtag gaaacctctg
gacctctatc gcctctatgt 3540gtctgtgaag gagattggtg gattgactca
ggtcaacaag aacaaaaaat ggcgggaact 3600tgcaaccaac ctcaatgtgg
gcacatcaag cagtgctgcc agctccttga aaaagcagta 3660tatccagtgt
ctctatgcct ttgaatgcaa gattgaacgg ggagaagacc ctcccccaga
3720catctttgca gctgctgatt ccaagaagtc ccagcccaag atccagcctc
cctctcctgc 3780gggatcagga tctatgcagg ggccccagac tccccagtca
accagcagtt ccatggcaga 3840aggaggagac ttaaagccac caactccagc
atccacacca cacagtcaga tccccccatt 3900gccaggcatg agcaggagca
attcagttgg gatccaggat gcctttaatg atggaagtga 3960ctccacattc
cagaagcgga attccatgac tccaaaccct gggtatcagc ccagtatgaa
4020tacctctgac atgatggggc gcatgtccta tgagccaaat aaggatcctt
atggcagcat 4080gaggaaagct ccagggagtg atcccttcat gtcctcaggg
cagggcccca acggcgggat 4140gggtgacccc tacagtcgtg ctgccggccc
tgggctagga aatgtggcga tgggaccacg 4200acagcactat ccctatggag
gtccttatga cagagtgagg acggagcctg gaatagggcc 4260tgagggaaac
atgagcactg gggccccaca gccgaatctc atgccttcca acccagactc
4320ggggatgtat tctcctagcc gctacccccc gcagcagcag cagcagcagc
agcaacgaca 4380tgattcctat ggcaatcagt tctccaccca aggcacccct
tctggcagcc ccttccccag 4440ccagcagact acaatgtatc aacagcaaca
gcagaattac aagcggccaa tggatggcac 4500atatggccct cctgccaagc
ggcacgaagg ggagatgtac agcgtgccat acagcactgg 4560gcaggggcag
cctcagcagc agcagttgcc cccagcccag ccccagcctg ccagccagca
4620acaagctgcc cagccttccc ctcagcaaga tgtatacaac cagtatggca
atgcctatcc 4680tgccactgcc acagctgcta ctgagcgccg accagcaggc
ggcccccaga accaatttcc 4740attccagttt ggccgagacc gtgtctctgc
accccctggc accaatgccc agcaaaacat 4800gccaccacaa atgatgggcg
gccccataca ggcatcagct gaggttgctc agcaaggcac 4860catgtggcag
gggcgtaatg acatgaccta taattatgcc aacaggcaga gcacgggctc
4920tgccccccag ggccccgcct atcatggcgt gaaccgaaca gatgaaatgc
tgcacacaga 4980tcagagggcc aaccacgaag gctcgtggcc ttcccatggc
acacgccagc ccccatatgg 5040tccctctgcc cctgtgcccc ccatgacaag
gccccctcca tctaactacc agcccccacc 5100aagcatgcag aatcacattc
ctcaggtatc cagccctgct cccctgcccc ggccaatgga 5160gaaccgcacc
tctcctagca agtctccatt cctgcactct gggatgaaaa tgcagaaggc
5220aggtccccca gtacctgcct cgcacatagc acctgcccct gtgcagcccc
ccatgattcg 5280gcgggatatc accttcccac ctggctctgt tgaagccaca
cagcctgtgt tgaagcagag 5340gaggcggctc acaatgaaag acattggaac
cccggaggca tggcgggtaa tgatgtccct 5400caagtctggt ctcctggcag
agagcacatg ggcattagat accatcaaca tcctgctgta 5460tgatgacaac
agcatcatga ccttcaacct cagtcagctc ccagggttgc tagagctcct
5520tgtagaatat ttccgacgat gcctgattga gatctttggc attttaaagg
agtatgaggt 5580gggtgaccca ggacagagaa cgctactgga tcctgggagg
ttcagcaagg tgtctagtcc 5640agctcccatg gagggtgggg aagaagaaga
agaacttcta ggtcctaaac tagaagagga 5700agaagaagag gaagtagttg
aaaatgatga ggagatagcc ttttcaggca aggacaagcc 5760agcttcagag
aatagtgagg agaagctgat cagtaagttt gacaagcttc cagtaaagat
5820cgtacagaag aatgatccat ttgtggtgga ctgctcagat aagcttgggc
gtgtgcagga 5880gtttgacagt ggcctgctgc actggcggat tggtgggggg
gacaccactg agcatatcca 5940gacccacttc gagagcaaga cagagctgct
gccttcccgg cctcacgcac cctgcccacc 6000agcccctcgg aagcatgtga
caacagcaga gggtacacca gggacaacag accaggaggg 6060gcccccacct
gatggacctc cagaaaaacg gatcacagcc actatggatg acatgttgtc
6120tactcggtct agcaccttga ccgaggatgg agctaagagt tcagaggcca
tcaaggagag 6180cagcaagttt ccatttggca ttagcccagc acagagccac
cggaacatca agatcctaga 6240ggacgaaccc cacagtaagg atgagacccc
actgtgtacc cttctggact ggcaggattc 6300tcttgccaag cgctgcgtct
gtgtgtccaa taccattcga agcctgtcat ttgtgccagg 6360caatgacttt
gagatgtcca aacacccagg gctgctgctc atcctgggca agctgatcct
6420gctgcaccac aagcacccag aacggaagca ggcaccacta acttatgaaa
aggaggagga 6480acaggaccaa ggggtgagct gcaacaaagt ggagtggtgg
tgggactgct tggagatgct 6540ccgggaaaac accttggtta cactcgccaa
catctcgggg cagttggacc tatctccata 6600ccccgagagc atttgcctgc
ctgtcctgga cggactccta cactgggcag tttgcccttc 6660agctgaagcc
caggacccct tttccaccct gggccccaat gccgtccttt ccccgcagag
6720actggtcttg gaaaccctca gcaaactcag catccaggac aacaatgtgg
acctgattct 6780ggccacaccc cccttcagcc gcctggagaa gttgtatagc
actatggtgc gcttcctcag 6840tgaccgaaag aacccggtgt gccgggagat
ggctgtggta ctgctggcca acctggctca 6900gggggacagc ctggcagctc
gtgccattgc agtgcagaag ggcagtatcg gcaacctcct 6960gggcttccta
gaggacagcc ttgccgccac acagttccag cagagccagg ccagcctcct
7020ccacatgcag aacccaccct ttgagccaac tagtgtggac atgatgcggc
gggctgcccg 7080cgcgctgctt gccttggcca aggtggacga gaaccactca
gagtttactc tgtacgaatc 7140acggctgttg gacatctcgg tatcaccgtt
gatgaactca ttggtttcac aagtcatttg 7200tgatgtactg tttttgattg
gccagtcatg acagccgtgg gacacctccc ccccccgtgt 7260gtgtgtgcgt
gtgtggagaa cttagaaact gactgttgcc ctttatttat gcaaaaccac
7320ctcagaatcc agtttaccct gtgctgtcca gcttctccct tgggaaaaag
tctctcctgt 7380ttctctctcc tccttccacc tcccctccct ccatcacctc
acgcctttct gttccttgtc 7440ctcaccttac tcccctcagg accctacccc
accctctttg aaaagacaaa gctctgccta 7500catagaagac tttttttatt
ttaaccaaag ttactgttgt ttacagtgag tttggggaaa 7560aaaaataaaa
taaaaatggc tttcccagtc cttgcatcaa cgggatgcca catttcataa
7620ctgtttttaa tggtaaaaaa aaaaaaaaaa aatacaaaaa aaaattctga
aggacaaaaa 7680aggtgactgc tgaactgtgt gtggtttatt gttgtacatt
cacaatcttg caggagccaa 7740gaagttcgca gttgtgaaca gaccctgttc
actggagagg cctgtgcagt agagtgtaga 7800ccctttcatg tactgtactg
tacacctgat actgtaaaca tactgtaata ataatgtctc 7860acatggaaac
agaaaacgct gggtcagcag caagctgtag tttttaaaaa tgtttttagt
7920taaacgttga ggagaaaaaa aaaaaaggct tttcccccaa agtatcatgt
gtgaacctac 7980aacaccctga cctctttctc tcctccttga ttgtatgaat
aaccctgaga tcacctctta 8040gaactggttt taacctttag ctgcagcggc
tacgctgcca cgtgtgtata tatatgacgt 8100tgtacattgc acataccctt
ggatccccac agtttggtcc tcctcccagc taccccttta 8160tagtatgacg
agttaacaag ttggtgacct gcacaaagcg agacacagct atttaatctc
8220ttgccagata tcgcccctct tggtgcgatg ctgtacaggt ctctgtaaaa
agtccttgct 8280gtctcagcag ccaatcaact tatagtttat ttttttctgg
gtttttgttt tgttttgttt 8340tctttctaat cgaggtgtga aaaagttcta
ggttcagttg aagttctgat gaagaaacac 8400aattgagatt ttttcagtga
taaaatctgc atatttgtat ttcaacaatg tagctaaaac 8460ttgatgtaaa
ttcctccttt ttttcctttt ttggcttaat gaatatcatt tattcagtat
8520gaaatcttta tactatatgt tccacgtgtt aagaataaat gtacattaaa
tcttggtaag 8580acttt 8585112068PRTHomo sapiens 11Met Ala Ala Gln
Val Ala Pro Ala Ala Ala Ser Ser Leu Gly Asn Pro 1 5 10 15 Pro Pro
Pro Pro Pro Ser Glu Leu Lys Lys Ala Glu Gln Gln Gln Arg 20 25 30
Glu Glu Ala Gly Gly Glu Ala Ala Ala Ala Ala Ala Ala Glu Arg Gly 35
40 45 Glu Met Lys Ala Ala Ala Gly Gln Glu Ser Glu Gly Pro Ala Val
Gly 50 55 60 Pro Pro Gln Pro Leu Gly Lys Glu Leu Gln Asp Gly Ala
Glu Ser Asn 65 70 75 80 Gly Gly Gly Gly Gly Gly Gly Ala Gly Ser Gly
Gly Gly Pro Gly Ala 85 90 95 Glu Pro Asp Leu Lys Asn Ser Asn Gly
Asn Ala Gly Pro Arg Pro Ala 100 105 110 Leu Asn Asn Asn Leu Thr Glu
Pro Pro Gly Gly Gly Gly Gly Gly Ser 115 120 125 Ser Asp Gly Val Gly
Ala Pro Pro His Ser Ala Ala Ala Ala Leu Pro 130 135 140 Pro Pro Ala
Tyr Gly Phe Gly Gln Pro Tyr Gly Arg Ser Pro Ser Ala 145 150 155 160
Val Ala Ala Ala Ala Ala Ala Val Phe His Gln Gln His Gly Gly Gln 165
170 175 Gln Ser Pro Gly Leu Ala Ala Leu Gln Ser Gly Gly Gly Gly Gly
Leu 180 185 190 Glu Pro Tyr Ala Gly Pro Gln Gln Asn Ser His Asp His
Gly Phe Pro 195 200 205 Asn His Gln Tyr Asn Ser Tyr Tyr Pro Asn Arg
Ser Ala Tyr Pro Pro 210 215 220 Pro Ala Pro Ala Tyr Ala Leu Ser Ser
Pro Arg Gly Gly Thr Pro Gly 225 230 235 240 Ser Gly Ala Ala Ala Ala
Ala Gly Ser Lys Pro Pro Pro Ser Ser Ser 245 250 255 Ala Ser Ala Ser
Ser Ser Ser Ser Ser Phe Ala Gln Gln Arg Phe Gly 260 265 270 Ala Met
Gly Gly Gly Gly Pro Ser Ala Ala Gly Gly Gly Thr Pro Gln 275 280 285
Pro Thr Ala Thr Pro Thr Leu Asn Gln Leu Leu Thr Ser Pro Ser Ser 290
295 300 Ala Arg Gly Tyr Gln Gly Tyr Pro Gly Gly Asp Tyr Ser Gly Gly
Pro 305 310 315 320 Gln Asp Gly Gly Ala Gly Lys Gly Pro Ala
Asp Met Ala Ser Gln Cys 325 330 335 Trp Gly Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ser Gly Gly 340 345 350 Ala Gln Gln Arg Ser His
His Ala Pro Met Ser Pro Gly Ser Ser Gly 355 360 365 Gly Gly Gly Gln
Pro Leu Ala Arg Thr Pro Gln Pro Ser Ser Pro Met 370 375 380 Asp Gln
Met Gly Lys Met Arg Pro Gln Pro Tyr Gly Gly Thr Asn Pro 385 390 395
400 Tyr Ser Gln Gln Gln Gly Pro Pro Ser Gly Pro Gln Gln Gly His Gly
405 410 415 Tyr Pro Gly Gln Pro Tyr Gly Ser Gln Thr Pro Gln Arg Tyr
Pro Met 420 425 430 Thr Met Gln Gly Arg Ala Gln Ser Ala Met Gly Gly
Leu Ser Tyr Thr 435 440 445 Gln Gln Ile Pro Pro Tyr Gly Gln Gln Gly
Pro Ser Gly Tyr Gly Gln 450 455 460 Gln Gly Gln Thr Pro Tyr Tyr Asn
Gln Gln Ser Pro His Pro Gln Gln 465 470 475 480 Gln Gln Pro Pro Tyr
Ser Gln Gln Pro Pro Ser Gln Thr Pro His Ala 485 490 495 Gln Pro Ser
Tyr Gln Gln Gln Pro Gln Ser Gln Pro Pro Gln Leu Gln 500 505 510 Ser
Ser Gln Pro Pro Tyr Ser Gln Gln Pro Ser Gln Pro Pro His Gln 515 520
525 Gln Ser Pro Ala Pro Tyr Pro Ser Gln Gln Ser Thr Thr Gln Gln His
530 535 540 Pro Gln Ser Gln Pro Pro Tyr Ser Gln Pro Gln Ala Gln Ser
Pro Tyr 545 550 555 560 Gln Gln Gln Gln Pro Gln Gln Pro Ala Pro Ser
Thr Leu Ser Gln Gln 565 570 575 Ala Ala Tyr Pro Gln Pro Gln Ser Gln
Gln Ser Gln Gln Thr Ala Tyr 580 585 590 Ser Gln Gln Arg Phe Pro Pro
Pro Gln Glu Leu Ser Gln Asp Ser Phe 595 600 605 Gly Ser Gln Ala Ser
Ser Ala Pro Ser Met Thr Ser Ser Lys Gly Gly 610 615 620 Gln Glu Asp
Met Asn Leu Ser Leu Gln Ser Arg Pro Ser Ser Leu Pro 625 630 635 640
Asp Leu Ser Gly Ser Ile Asp Asp Leu Pro Met Gly Thr Glu Gly Ala 645
650 655 Leu Ser Pro Gly Val Ser Thr Ser Gly Ile Ser Ser Ser Gln Gly
Glu 660 665 670 Gln Ser Asn Pro Ala Gln Ser Pro Phe Ser Pro His Thr
Ser Pro His 675 680 685 Leu Pro Gly Ile Arg Gly Pro Ser Pro Ser Pro
Val Gly Ser Pro Ala 690 695 700 Ser Val Ala Gln Ser Arg Ser Gly Pro
Leu Ser Pro Ala Ala Val Pro 705 710 715 720 Gly Asn Gln Met Pro Pro
Arg Pro Pro Ser Gly Gln Ser Asp Ser Ile 725 730 735 Met His Pro Ser
Met Asn Gln Ser Ser Ile Ala Gln Asp Arg Gly Tyr 740 745 750 Met Gln
Arg Asn Pro Gln Met Pro Gln Tyr Ser Ser Pro Gln Pro Gly 755 760 765
Ser Ala Leu Ser Pro Arg Gln Pro Ser Gly Gly Gln Ile His Thr Gly 770
775 780 Met Gly Ser Tyr Gln Gln Asn Ser Met Gly Ser Tyr Gly Pro Gln
Gly 785 790 795 800 Gly Gln Tyr Gly Pro Gln Gly Gly Tyr Pro Arg Gln
Pro Asn Tyr Asn 805 810 815 Ala Leu Pro Asn Ala Asn Tyr Pro Ser Ala
Gly Met Ala Gly Gly Ile 820 825 830 Asn Pro Met Gly Ala Gly Gly Gln
Met His Gly Gln Pro Gly Ile Pro 835 840 845 Pro Tyr Gly Thr Leu Pro
Pro Gly Arg Met Ser His Ala Ser Met Gly 850 855 860 Asn Arg Pro Tyr
Gly Pro Asn Met Ala Asn Met Pro Pro Gln Val Gly 865 870 875 880 Ser
Gly Met Cys Pro Pro Pro Gly Gly Met Asn Arg Lys Thr Gln Glu 885 890
895 Thr Ala Val Ala Met His Val Ala Ala Asn Ser Ile Gln Asn Arg Pro
900 905 910 Pro Gly Tyr Pro Asn Met Asn Gln Gly Gly Met Met Gly Thr
Gly Pro 915 920 925 Pro Tyr Gly Gln Gly Ile Asn Ser Met Ala Gly Met
Ile Asn Pro Gln 930 935 940 Gly Pro Pro Tyr Ser Met Gly Gly Thr Met
Ala Asn Asn Ser Ala Gly 945 950 955 960 Met Ala Ala Ser Pro Glu Met
Met Gly Leu Gly Asp Val Lys Leu Thr 965 970 975 Pro Ala Thr Lys Met
Asn Asn Lys Ala Asp Gly Thr Pro Lys Thr Glu 980 985 990 Ser Lys Ser
Lys Lys Ser Ser Ser Ser Thr Thr Thr Asn Glu Lys Ile 995 1000 1005
Thr Lys Leu Tyr Glu Leu Gly Gly Glu Pro Glu Arg Lys Met Trp 1010
1015 1020 Val Asp Arg Tyr Leu Ala Phe Thr Glu Glu Lys Ala Met Gly
Met 1025 1030 1035 Thr Asn Leu Pro Ala Val Gly Arg Lys Pro Leu Asp
Leu Tyr Arg 1040 1045 1050 Leu Tyr Val Ser Val Lys Glu Ile Gly Gly
Leu Thr Gln Val Asn 1055 1060 1065 Lys Asn Lys Lys Trp Arg Glu Leu
Ala Thr Asn Leu Asn Val Gly 1070 1075 1080 Thr Ser Ser Ser Ala Ala
Ser Ser Leu Lys Lys Gln Tyr Ile Gln 1085 1090 1095 Cys Leu Tyr Ala
Phe Glu Cys Lys Ile Glu Arg Gly Glu Asp Pro 1100 1105 1110 Pro Pro
Asp Ile Phe Ala Ala Ala Asp Ser Lys Lys Ser Gln Pro 1115 1120 1125
Lys Ile Gln Pro Pro Ser Pro Ala Gly Ser Gly Ser Met Gln Gly 1130
1135 1140 Pro Gln Thr Pro Gln Ser Thr Ser Ser Ser Met Ala Glu Gly
Gly 1145 1150 1155 Asp Leu Lys Pro Pro Thr Pro Ala Ser Thr Pro His
Ser Gln Ile 1160 1165 1170 Pro Pro Leu Pro Gly Met Ser Arg Ser Asn
Ser Val Gly Ile Gln 1175 1180 1185 Asp Ala Phe Asn Asp Gly Ser Asp
Ser Thr Phe Gln Lys Arg Asn 1190 1195 1200 Ser Met Thr Pro Asn Pro
Gly Tyr Gln Pro Ser Met Asn Thr Ser 1205 1210 1215 Asp Met Met Gly
Arg Met Ser Tyr Glu Pro Asn Lys Asp Pro Tyr 1220 1225 1230 Gly Ser
Met Arg Lys Ala Pro Gly Ser Asp Pro Phe Met Ser Ser 1235 1240 1245
Gly Gln Gly Pro Asn Gly Gly Met Gly Asp Pro Tyr Ser Arg Ala 1250
1255 1260 Ala Gly Pro Gly Leu Gly Asn Val Ala Met Gly Pro Arg Gln
His 1265 1270 1275 Tyr Pro Tyr Gly Gly Pro Tyr Asp Arg Val Arg Thr
Glu Pro Gly 1280 1285 1290 Ile Gly Pro Glu Gly Asn Met Ser Thr Gly
Ala Pro Gln Pro Asn 1295 1300 1305 Leu Met Pro Ser Asn Pro Asp Ser
Gly Met Tyr Ser Pro Ser Arg 1310 1315 1320 Tyr Pro Pro Gln Gln Gln
Gln Gln Gln Gln Gln Arg His Asp Ser 1325 1330 1335 Tyr Gly Asn Gln
Phe Ser Thr Gln Gly Thr Pro Ser Gly Ser Pro 1340 1345 1350 Phe Pro
Ser Gln Gln Thr Thr Met Tyr Gln Gln Gln Gln Gln Val 1355 1360 1365
Ser Ser Pro Ala Pro Leu Pro Arg Pro Met Glu Asn Arg Thr Ser 1370
1375 1380 Pro Ser Lys Ser Pro Phe Leu His Ser Gly Met Lys Met Gln
Lys 1385 1390 1395 Ala Gly Pro Pro Val Pro Ala Ser His Ile Ala Pro
Ala Pro Val 1400 1405 1410 Gln Pro Pro Met Ile Arg Arg Asp Ile Thr
Phe Pro Pro Gly Ser 1415 1420 1425 Val Glu Ala Thr Gln Pro Val Leu
Lys Gln Arg Arg Arg Leu Thr 1430 1435 1440 Met Lys Asp Ile Gly Thr
Pro Glu Ala Trp Arg Val Met Met Ser 1445 1450 1455 Leu Lys Ser Gly
Leu Leu Ala Glu Ser Thr Trp Ala Leu Asp Thr 1460 1465 1470 Ile Asn
Ile Leu Leu Tyr Asp Asp Asn Ser Ile Met Thr Phe Asn 1475 1480 1485
Leu Ser Gln Leu Pro Gly Leu Leu Glu Leu Leu Val Glu Tyr Phe 1490
1495 1500 Arg Arg Cys Leu Ile Glu Ile Phe Gly Ile Leu Lys Glu Tyr
Glu 1505 1510 1515 Val Gly Asp Pro Gly Gln Arg Thr Leu Leu Asp Pro
Gly Arg Phe 1520 1525 1530 Ser Lys Val Ser Ser Pro Ala Pro Met Glu
Gly Gly Glu Glu Glu 1535 1540 1545 Glu Glu Leu Leu Gly Pro Lys Leu
Glu Glu Glu Glu Glu Glu Glu 1550 1555 1560 Val Val Glu Asn Asp Glu
Glu Ile Ala Phe Ser Gly Lys Asp Lys 1565 1570 1575 Pro Ala Ser Glu
Asn Ser Glu Glu Lys Leu Ile Ser Lys Phe Asp 1580 1585 1590 Lys Leu
Pro Val Lys Ile Val Gln Lys Asn Asp Pro Phe Val Val 1595 1600 1605
Asp Cys Ser Asp Lys Leu Gly Arg Val Gln Glu Phe Asp Ser Gly 1610
1615 1620 Leu Leu His Trp Arg Ile Gly Gly Gly Asp Thr Thr Glu His
Ile 1625 1630 1635 Gln Thr His Phe Glu Ser Lys Thr Glu Leu Leu Pro
Ser Arg Pro 1640 1645 1650 His Ala Pro Cys Pro Pro Ala Pro Arg Lys
His Val Thr Thr Ala 1655 1660 1665 Glu Gly Thr Pro Gly Thr Thr Asp
Gln Glu Gly Pro Pro Pro Asp 1670 1675 1680 Gly Pro Pro Glu Lys Arg
Ile Thr Ala Thr Met Asp Asp Met Leu 1685 1690 1695 Ser Thr Arg Ser
Ser Thr Leu Thr Glu Asp Gly Ala Lys Ser Ser 1700 1705 1710 Glu Ala
Ile Lys Glu Ser Ser Lys Phe Pro Phe Gly Ile Ser Pro 1715 1720 1725
Ala Gln Ser His Arg Asn Ile Lys Ile Leu Glu Asp Glu Pro His 1730
1735 1740 Ser Lys Asp Glu Thr Pro Leu Cys Thr Leu Leu Asp Trp Gln
Asp 1745 1750 1755 Ser Leu Ala Lys Arg Cys Val Cys Val Ser Asn Thr
Ile Arg Ser 1760 1765 1770 Leu Ser Phe Val Pro Gly Asn Asp Phe Glu
Met Ser Lys His Pro 1775 1780 1785 Gly Leu Leu Leu Ile Leu Gly Lys
Leu Ile Leu Leu His His Lys 1790 1795 1800 His Pro Glu Arg Lys Gln
Ala Pro Leu Thr Tyr Glu Lys Glu Glu 1805 1810 1815 Glu Gln Asp Gln
Gly Val Ser Cys Asn Lys Val Glu Trp Trp Trp 1820 1825 1830 Asp Cys
Leu Glu Met Leu Arg Glu Asn Thr Leu Val Thr Leu Ala 1835 1840 1845
Asn Ile Ser Gly Gln Leu Asp Leu Ser Pro Tyr Pro Glu Ser Ile 1850
1855 1860 Cys Leu Pro Val Leu Asp Gly Leu Leu His Trp Ala Val Cys
Pro 1865 1870 1875 Ser Ala Glu Ala Gln Asp Pro Phe Ser Thr Leu Gly
Pro Asn Ala 1880 1885 1890 Val Leu Ser Pro Gln Arg Leu Val Leu Glu
Thr Leu Ser Lys Leu 1895 1900 1905 Ser Ile Gln Asp Asn Asn Val Asp
Leu Ile Leu Ala Thr Pro Pro 1910 1915 1920 Phe Ser Arg Leu Glu Lys
Leu Tyr Ser Thr Met Val Arg Phe Leu 1925 1930 1935 Ser Asp Arg Lys
Asn Pro Val Cys Arg Glu Met Ala Val Val Leu 1940 1945 1950 Leu Ala
Asn Leu Ala Gln Gly Asp Ser Leu Ala Ala Arg Ala Ile 1955 1960 1965
Ala Val Gln Lys Gly Ser Ile Gly Asn Leu Leu Gly Phe Leu Glu 1970
1975 1980 Asp Ser Leu Ala Ala Thr Gln Phe Gln Gln Ser Gln Ala Ser
Leu 1985 1990 1995 Leu His Met Gln Asn Pro Pro Phe Glu Pro Thr Ser
Val Asp Met 2000 2005 2010 Met Arg Arg Ala Ala Arg Ala Leu Leu Ala
Leu Ala Lys Val Asp 2015 2020 2025 Glu Asn His Ser Glu Phe Thr Leu
Tyr Glu Ser Arg Leu Leu Asp 2030 2035 2040 Ile Ser Val Ser Pro Leu
Met Asn Ser Leu Val Ser Gln Val Ile 2045 2050 2055 Cys Asp Val Leu
Phe Leu Ile Gly Gln Ser 2060 2065 127934DNAHomo sapiens
12cagaaagcgg agagtcacag cggggccagg ccctggggag cggagcctcc accgcccccc
60tcattcccag gcaagggctt ggggggaatg agccgggaga gccgggtccc gagcctacag
120agccgggagc agctgagccg ccggcgcctc ggccgccgcc gccgcctcct
cctcctccgc 180cgccgccagc ccggagcctg agccggcggg gcggggggga
gaggagcgag cgcagcgcag 240cagcggagcc ccgcgaggcc cgcccgggcg
ggtggggagg gcagcccggg ggactgggcc 300ccggggcggg gtgggagggg
gggagaagac gaagacaggg ccgggtctct ccgcggacga 360gacagcgggg
atcatggccg cgcaggtcgc ccccgccgcc gccagcagcc tgggcaaccc
420gccgccgccg ccgccctcgg agctgaagaa agccgagcag cagcagcggg
aggaggcggg 480gggcgaggcg gcggcggcgg cagcggccga gcgcggggaa
atgaaggcag ccgccgggca 540ggaaagcgag ggccccgccg tggggccgcc
gcagccgctg ggaaaggagc tgcaggacgg 600ggccgagagc aatgggggtg
gcggcggcgg cggagccggc agcggcggcg ggcccggcgc 660ggagccggac
ctgaagaact cgaacgggaa cgcgggccct aggcccgccc tgaacaataa
720cctcacggag ccgcccggcg gcggcggtgg cggcagcagc gatggggtgg
gggcgcctcc 780tcactcagcc gcggccgcct tgccgccccc agcctacggc
ttcgggcaac cctacggccg 840gagcccgtct gccgtcgccg ccgccgcggc
cgccgtcttc caccaacaac atggcggaca 900acaaagccct ggcctggcag
cgctgcagag cggcggcggc gggggcctgg agccctacgc 960ggggccccag
cagaactctc acgaccacgg cttccccaac caccagtaca actcctacta
1020ccccaaccgc agcgcctacc ccccgcccgc cccggcctac gcgctgagct
ccccgagagg 1080tggcactccg ggctccggcg cggcggcggc tgccggctcc
aagccgcctc cctcctccag 1140cgcctccgcc tcctcgtcgt cttcgtcctt
cgctcagcag cgcttcgggg ccatgggggg 1200aggcggcccc tccgcggccg
gcgggggaac tccccagccc accgccaccc ccaccctcaa 1260ccaactgctc
acgtcgccca gctcggcccg gggctaccag ggctaccccg ggggcgacta
1320cagtggcggg ccccaggacg ggggcgccgg caagggcccg gcggacatgg
cctcgcagtg 1380ttggggggct gcggcggcgg cagctgcggc ggcggccgcc
tcgggagggg cccaacaaag 1440gagccaccac gcgcccatga gccccgggag
cagcggcggc ggggggcagc cgctcgcccg 1500gacccctcag ccatccagtc
caatggatca gatgggcaag atgagacctc agccatatgg 1560cgggactaac
ccatactcgc agcaacaggg acctccgtca ggaccgcagc aaggacatgg
1620gtacccaggg cagccatacg ggtcccagac cccgcagcgg tacccgatga
ccatgcaggg 1680ccgggcgcag agtgccatgg gcggcctctc ttatacacag
cagattcctc cttatggaca 1740acaaggcccc agcgggtatg gtcaacaggg
ccagactcca tattacaacc agcaaagtcc 1800tcaccctcag cagcagcagc
caccctactc ccagcaacca ccgtcccaga cccctcatgc 1860ccaaccttcg
tatcagcagc agccacagtc tcaaccacca cagctccagt cctctcagcc
1920tccatactcc cagcagccat cccagcctcc acatcagcag tccccggctc
catacccctc 1980ccagcagtcg acgacacagc agcaccccca gagccagccc
ccctactcac agccacaggc 2040tcagtctcct taccagcagc agcaacctca
gcagccagca ccctcgacgc tctcccagca 2100ggctgcgtat cctcagcccc
agtctcagca gtcccagcaa actgcctatt cccagcagcg 2160cttccctcca
ccgcaggagc tatctcaaga ttcatttggg tctcaggcat cctcagcccc
2220ctcaatgacc tccagtaagg gagggcaaga agatatgaac ctgagccttc
agtcaagacc 2280ctccagcttg cctgatctat ctggttcaat agatgacctc
cccatgggga cagaaggagc 2340tctgagtcct ggagtgagca catcagggat
ttccagcagc caaggagagc agagtaatcc 2400agctcagtct cctttctctc
ctcatacctc ccctcacctg cctggcatcc gaggcccttc 2460cccgtcccct
gttggctctc ccgccagtgt tgctcagtct cgctcaggac cactctcgcc
2520tgctgcagtg ccaggcaacc agatgccacc tcggccaccc agtggccagt
cggacagcat 2580catgcatcct tccatgaacc aatcaagcat tgcccaagat
cgaggttata tgcagaggaa 2640cccccagatg ccccagtaca gttcccccca
gcccggctca gccttatctc cgcgtcagcc 2700ttccggagga cagatacaca
caggcatggg ctcctaccag cagaactcca tggggagcta 2760tggtccccag
gggggtcagt atggcccaca aggtggctac cccaggcagc caaactataa
2820tgccttgccc aatgccaact accccagtgc aggcatggct ggaggcataa
accccatggg 2880tgccggaggt caaatgcatg gacagcctgg catcccacct
tatggcacac tccctccagg 2940gaggatgagt cacgcctcca tgggcaaccg
gccttatggc cctaacatgg ccaatatgcc 3000acctcaggtt gggtcaggga
tgtgtccccc accagggggc atgaaccgga aaacccaaga 3060aactgctgtc
gccatgcatg ttgctgccaa ctctatccaa aacaggccgc caggctaccc
3120caatatgaat caagggggca tgatgggaac tggacctcct tatggacaag
ggattaatag 3180tatggctggc atgatcaacc ctcagggacc cccatattcc
atgggtggaa ccatggccaa 3240caattctgca gggatggcag ccagcccaga
gatgatgggc cttggggatg taaagttaac
3300tccagccacc aaaatgaaca acaaggcaga tgggacaccc aagacagaat
ccaaatccaa 3360gaaatccagt tcttctacta caaccaatga gaagatcacc
aagttgtatg agctgggtgg 3420tgagcctgag aggaagatgt gggtggaccg
ttatctggcc ttcactgagg agaaggccat 3480gggcatgaca aatctgcctg
ctgtgggtag gaaacctctg gacctctatc gcctctatgt 3540gtctgtgaag
gagattggtg gattgactca ggtcaacaag aacaaaaaat ggcgggaact
3600tgcaaccaac ctcaatgtgg gcacatcaag cagtgctgcc agctccttga
aaaagcagta 3660tatccagtgt ctctatgcct ttgaatgcaa gattgaacgg
ggagaagacc ctcccccaga 3720catctttgca gctgctgatt ccaagaagtc
ccagcccaag atccagcctc cctctcctgc 3780gggatcagga tctatgcagg
ggccccagac tccccagtca accagcagtt ccatggcaga 3840aggaggagac
ttaaagccac caactccagc atccacacca cacagtcaga tccccccatt
3900gccaggcatg agcaggagca attcagttgg gatccaggat gcctttaatg
atggaagtga 3960ctccacattc cagaagcgga attccatgac tccaaaccct
gggtatcagc ccagtatgaa 4020tacctctgac atgatggggc gcatgtccta
tgagccaaat aaggatcctt atggcagcat 4080gaggaaagct ccagggagtg
atcccttcat gtcctcaggg cagggcccca acggcgggat 4140gggtgacccc
tacagtcgtg ctgccggccc tgggctagga aatgtggcga tgggaccacg
4200acagcactat ccctatggag gtccttatga cagagtgagg acggagcctg
gaatagggcc 4260tgagggaaac atgagcactg gggccccaca gccgaatctc
atgccttcca acccagactc 4320ggggatgtat tctcctagcc gctacccccc
gcagcagcag cagcagcagc agcaacgaca 4380tgattcctat ggcaatcagt
tctccaccca aggcacccct tctggcagcc ccttccccag 4440ccagcagact
acaatgtatc aacagcaaca gcaggtatcc agccctgctc ccctgccccg
4500gccaatggag aaccgcacct ctcctagcaa gtctccattc ctgcactctg
ggatgaaaat 4560gcagaaggca ggtcccccag tacctgcctc gcacatagca
cctgcccctg tgcagccccc 4620catgattcgg cgggatatca ccttcccacc
tggctctgtt gaagccacac agcctgtgtt 4680gaagcagagg aggcggctca
caatgaaaga cattggaacc ccggaggcat ggcgggtaat 4740gatgtccctc
aagtctggtc tcctggcaga gagcacatgg gcattagata ccatcaacat
4800cctgctgtat gatgacaaca gcatcatgac cttcaacctc agtcagctcc
cagggttgct 4860agagctcctt gtagaatatt tccgacgatg cctgattgag
atctttggca ttttaaagga 4920gtatgaggtg ggtgacccag gacagagaac
gctactggat cctgggaggt tcagcaaggt 4980gtctagtcca gctcccatgg
agggtgggga agaagaagaa gaacttctag gtcctaaact 5040agaagaggaa
gaagaagagg aagtagttga aaatgatgag gagatagcct tttcaggcaa
5100ggacaagcca gcttcagaga atagtgagga gaagctgatc agtaagtttg
acaagcttcc 5160agtaaagatc gtacagaaga atgatccatt tgtggtggac
tgctcagata agcttgggcg 5220tgtgcaggag tttgacagtg gcctgctgca
ctggcggatt ggtggggggg acaccactga 5280gcatatccag acccacttcg
agagcaagac agagctgctg ccttcccggc ctcacgcacc 5340ctgcccacca
gcccctcgga agcatgtgac aacagcagag ggtacaccag ggacaacaga
5400ccaggagggg cccccacctg atggacctcc agaaaaacgg atcacagcca
ctatggatga 5460catgttgtct actcggtcta gcaccttgac cgaggatgga
gctaagagtt cagaggccat 5520caaggagagc agcaagtttc catttggcat
tagcccagca cagagccacc ggaacatcaa 5580gatcctagag gacgaacccc
acagtaagga tgagacccca ctgtgtaccc ttctggactg 5640gcaggattct
cttgccaagc gctgcgtctg tgtgtccaat accattcgaa gcctgtcatt
5700tgtgccaggc aatgactttg agatgtccaa acacccaggg ctgctgctca
tcctgggcaa 5760gctgatcctg ctgcaccaca agcacccaga acggaagcag
gcaccactaa cttatgaaaa 5820ggaggaggaa caggaccaag gggtgagctg
caacaaagtg gagtggtggt gggactgctt 5880ggagatgctc cgggaaaaca
ccttggttac actcgccaac atctcggggc agttggacct 5940atctccatac
cccgagagca tttgcctgcc tgtcctggac ggactcctac actgggcagt
6000ttgcccttca gctgaagccc aggacccctt ttccaccctg ggccccaatg
ccgtcctttc 6060cccgcagaga ctggtcttgg aaaccctcag caaactcagc
atccaggaca acaatgtgga 6120cctgattctg gccacacccc ccttcagccg
cctggagaag ttgtatagca ctatggtgcg 6180cttcctcagt gaccgaaaga
acccggtgtg ccgggagatg gctgtggtac tgctggccaa 6240cctggctcag
ggggacagcc tggcagctcg tgccattgca gtgcagaagg gcagtatcgg
6300caacctcctg ggcttcctag aggacagcct tgccgccaca cagttccagc
agagccaggc 6360cagcctcctc cacatgcaga acccaccctt tgagccaact
agtgtggaca tgatgcggcg 6420ggctgcccgc gcgctgcttg ccttggccaa
ggtggacgag aaccactcag agtttactct 6480gtacgaatca cggctgttgg
acatctcggt atcaccgttg atgaactcat tggtttcaca 6540agtcatttgt
gatgtactgt ttttgattgg ccagtcatga cagccgtggg acacctcccc
6600cccccgtgtg tgtgtgcgtg tgtggagaac ttagaaactg actgttgccc
tttatttatg 6660caaaaccacc tcagaatcca gtttaccctg tgctgtccag
cttctccctt gggaaaaagt 6720ctctcctgtt tctctctcct ccttccacct
cccctccctc catcacctca cgcctttctg 6780ttccttgtcc tcaccttact
cccctcagga ccctacccca ccctctttga aaagacaaag 6840ctctgcctac
atagaagact ttttttattt taaccaaagt tactgttgtt tacagtgagt
6900ttggggaaaa aaaataaaat aaaaatggct ttcccagtcc ttgcatcaac
gggatgccac 6960atttcataac tgtttttaat ggtaaaaaaa aaaaaaaaaa
atacaaaaaa aaattctgaa 7020ggacaaaaaa ggtgactgct gaactgtgtg
tggtttattg ttgtacattc acaatcttgc 7080aggagccaag aagttcgcag
ttgtgaacag accctgttca ctggagaggc ctgtgcagta 7140gagtgtagac
cctttcatgt actgtactgt acacctgata ctgtaaacat actgtaataa
7200taatgtctca catggaaaca gaaaacgctg ggtcagcagc aagctgtagt
ttttaaaaat 7260gtttttagtt aaacgttgag gagaaaaaaa aaaaaggctt
ttcccccaaa gtatcatgtg 7320tgaacctaca acaccctgac ctctttctct
cctccttgat tgtatgaata accctgagat 7380cacctcttag aactggtttt
aacctttagc tgcagcggct acgctgccac gtgtgtatat 7440atatgacgtt
gtacattgca catacccttg gatccccaca gtttggtcct cctcccagct
7500acccctttat agtatgacga gttaacaagt tggtgacctg cacaaagcga
gacacagcta 7560tttaatctct tgccagatat cgcccctctt ggtgcgatgc
tgtacaggtc tctgtaaaaa 7620gtccttgctg tctcagcagc caatcaactt
atagtttatt tttttctggg tttttgtttt 7680gttttgtttt ctttctaatc
gaggtgtgaa aaagttctag gttcagttga agttctgatg 7740aagaaacaca
attgagattt tttcagtgat aaaatctgca tatttgtatt tcaacaatgt
7800agctaaaact tgatgtaaat tcctcctttt tttccttttt tggcttaatg
aatatcattt 7860attcagtatg aaatctttat actatatgtt ccacgtgtta
agaataaatg tacattaaat 7920cttggtaaga cttt 79341326PRTArtificial
SequenceSynthesized peptideMISC_FEATURE(26)..(26)wherein the Lys is
attached to a biotin and an amide 13Ala Thr Lys Ala Ala Arg Lys Ser
Ala Pro Ala Thr Gly Gly Val Lys 1 5 10 15 Lys Pro His Arg Tyr Arg
Pro Gly Gly Lys 20 25 1426PRTArtificial SequenceSynthesized
peptideMISC_FEATURE(7)..(7)wherein the Lys is
trimethylatedMISC_FEATURE(26)..(26)wherein the Lys is attached to a
biotin and an amide 14Ala Thr Lys Ala Ala Arg Lys Ser Ala Pro Ala
Thr Gly Gly Val Lys 1 5 10 15 Lys Pro His Arg Tyr Arg Pro Gly Gly
Lys 20 25
* * * * *
References