U.S. patent application number 13/723101 was filed with the patent office on 2013-08-08 for compositions and methods for modulating interaction between polypeptides.
This patent application is currently assigned to Massachusetts Institute of Technology. The applicant listed for this patent is The General Hospital Corporation, Massachusetts Institute of Technology. Invention is credited to Steven M. Hersch, David E. Housman, Aleksey G. Kazantsev, Anne B. Young.
Application Number | 20130203733 13/723101 |
Document ID | / |
Family ID | 34975307 |
Filed Date | 2013-08-08 |
United States Patent
Application |
20130203733 |
Kind Code |
A1 |
Kazantsev; Aleksey G. ; et
al. |
August 8, 2013 |
COMPOSITIONS AND METHODS FOR MODULATING INTERACTION BETWEEN
POLYPEPTIDES
Abstract
The present invention is based, in part, on assays we conducted
that revealed compounds that may be used to treat or prevent
diseases characterized by an abnormal or undesirable association of
one protein with another.
Inventors: |
Kazantsev; Aleksey G.;
(Brookline, MA) ; Young; Anne B.; (Boston, MA)
; Housman; David E.; (Newton, MA) ; Hersch; Steven
M.; (Jamaica Plain, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The General Hospital Corporation;
Massachusetts Institute of Technology; |
Boston
Cambridge |
MA
MA |
US
US |
|
|
Assignee: |
Massachusetts Institute of
Technology
Cambridge
MA
The General Hospital Corporation
Boston
MA
|
Family ID: |
34975307 |
Appl. No.: |
13/723101 |
Filed: |
December 20, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11076093 |
Mar 7, 2005 |
8404747 |
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13723101 |
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60550748 |
Mar 5, 2004 |
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60630252 |
Nov 22, 2004 |
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60630264 |
Nov 22, 2004 |
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60630231 |
Nov 22, 2004 |
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60630221 |
Nov 22, 2004 |
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60630262 |
Nov 22, 2004 |
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60630230 |
Nov 22, 2004 |
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60633487 |
Dec 6, 2004 |
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Current U.S.
Class: |
514/212.01 ;
514/603; 540/604; 544/126; 544/361; 544/363; 546/171; 546/99;
548/437; 549/69; 564/86 |
Current CPC
Class: |
C07D 215/42 20130101;
A61K 31/21 20130101; C07C 2601/16 20170501; C07C 311/46 20130101;
A61K 31/277 20130101; C07D 333/38 20130101; C07D 401/06 20130101;
C07D 209/56 20130101; C07D 295/20 20130101; C07D 217/24 20130101;
C07C 311/44 20130101; C07D 215/40 20130101; A61K 31/4015 20130101;
C07D 333/36 20130101; A61K 31/445 20130101; C07D 209/80 20130101;
C07D 223/02 20130101; C07D 295/26 20130101; C07D 221/06
20130101 |
Class at
Publication: |
514/212.01 ;
514/603; 540/604; 544/126; 544/361; 544/363; 546/99; 546/171;
548/437; 549/69; 564/86 |
International
Class: |
C07D 223/02 20060101
C07D223/02; C07D 217/24 20060101 C07D217/24; C07C 311/44 20060101
C07C311/44; C07D 209/80 20060101 C07D209/80; C07D 333/36 20060101
C07D333/36; C07D 295/20 20060101 C07D295/20; C07D 215/40 20060101
C07D215/40 |
Claims
1. A pharmaceutical composition comprising a compound of Formula
II: ##STR00094## wherein, Z is O or S; Y is O, NR.sup.25 or
CR.sup.26R.sup.27; each R.sup.21 and R.sup.22 is, independently,
halo, hydroxy, nitro, cyano, amino, amido, or alkyl; R.sup.23 is
alkyl, cyclyl, aryl, heteroaryl, cyclylalkyl, arylalkyl, or
heteroarylalkyl, or when taken together with R.sup.24 and the
nitrogen to which it is attached forms a ring; wherein R.sup.23 is
optionally substituted with 1-3 R.sup.28. R.sup.24 is H, alkyl, or
when taken together with R.sup.23 and the nitrogen to which it is
attached forms a ring; wherein R.sup.24 is optionally substituted
with 1-3 R.sup.28. R.sup.25 is H or alkyl; each R.sup.26 and
R.sup.27 is independently H or alkyl; each R.sup.28 is
independently halo, hydroxy, nitro, cyano, amino, amido, or alkyl;
each p and q are independently an integer from 0-4.
2. The composition of claim 1, wherein Z is O.
3. The composition of claim 1, wherein Y is NR.sup.25.
4. The composition of claim 1, wherein R.sup.25 is H.
5. The composition of claim 1, wherein Y is CR.sup.26R.sup.27.
6. The composition of claim 1, wherein each R.sup.21 and R.sup.22
is independently halo or hydroxy.
7-9. (canceled)
10. The composition of claim 1, wherein R.sup.22 is halo; and q is
1.
11. The composition of claim 10, wherein p is 0.
12. The composition of claim 10, wherein R.sup.22 is bromo.
13-24. (canceled)
25. A pharmaceutical composition comprising:
4-Bromo-N-(4-bromo-phenyl)-3-cyclohexylsulfamoyl-benzamide;
N-(4-Bromo-phenyl)-3-(4-bromo-phenylsulfamoyl)-benzamide;
3-(4-Bromo-phenylsulfamoyl)-N-phenyl-benzamide;
4-Bromo-3-cyclohexylsulfamoyl-N-phenyl-benzamide;
N-(4-Bromo-phenyl)-3-cyclohexylsulfamoyl-benzamide; or
1-[3-(Azepane-1-sulfonyl)-2-bromo-phenyl]-2-(3-hydroxy-phenyl)-ethanone.
26-28. (canceled)
29. A method of treating a subject who has been diagnosed as
having, or who is at risk of developing, a disorder characterized
by an undesirable association of proteins, the method comprising
identifying the subject and administering to the subject a
therapeutically effective amount of the pharmaceutical composition
of claim 1.
30. The method of claim 29, wherein the subject has been diagnosed
as having, or is at risk of developing, Huntington's disease.
31. The method of claim 29, wherein the subject has been diagnosed
as having, or is at risk of developing, Parkinson's disease.
32. The method of claim 29, wherein the subject has been diagnosed
as having, or is at risk of developing, spinal and bulbar muscular
atrophy, dentatorubral-pallidoluysian atrophy, spinocerebellar
ataxia type 1 (SCA1), SCA2, SCA6, SCA7, Machado-Joseph disease
(MJD/SCA3), breast cancer, amyloidosis, myeloma, Creutzfeldt-Jakob
disease, kuru, cystic fibrosis, neuroblastoma, carcinoma, or
alpha-1-antitrypsin deficiency disease.
33. A pharmaceutical composition comprising: (a) a compound Formula
I: ##STR00095## X is C(O) or a bond; each R.sup.11 and R.sup.12 is,
independently, halo, nitro, amino, hydroxy, alkoxy, alkyl, alkenyl,
alkynyl, cyclyl, heterocyclyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, NR.sup.15C(O)R.sup.14, or C(O)NR.sup.15R.sup.16,
each of which is optionally substituted with 1-4 R.sup.17; R.sup.13
is H, alkyl, alkenyl, alkynyl, amino, hydroxy, aryl, arylalkyl,
arylamino, heterocyclyl, heterocyclylalkyl, heteroaryl,
heteroarylalkyl, cyclyl, cyclylalkyl, aminoalkyl, hydroxyalkyl, or
alkoxyalkyl, each of which is optionally substituted with 1-4
R.sup.18; R.sup.14 is H, alkyl, alkenyl, alkynyl, cyclyl,
heterocyclyl, aryl, or heteroaryl; each of R.sup.15 and R.sup.16
is, independently, H, hydroxy, alkoxy, alkyl, alkenyl, alkynyl,
cyclyl, heterocyclyl, aryl, arylalkyl, heteroaryl, or
heteroarylalkyl; each R.sup.17 is, independently, halo, alkyl,
alkoxy, or hydroxy; each R.sup.18 is, independently, halo, alkyl,
amino, hydroxy, C(O)NR.sup.15R.sup.16, NR.sup.15C(O)R.sup.14, or
hydroxyalkyl; and m and n are each, independently, an integer from
0 to 3; (b) a compound of Formula III: ##STR00096## A is N or
CR.sup.32; B is N or CH; R.sup.31 is H or NR.sup.33R.sup.34,
wherein R.sup.31 is optionally substituted with 1-3 R.sup.35;
R.sup.32 is H or NR.sup.33R.sup.34, wherein R.sup.32 is optionally
substituted with 1-3 R.sup.35; R.sup.33 is H, alkyl, or taken
together with R.sup.34 and the nitrogen to which it is attached
forms a heterocyclyl ring; R.sup.34 is H, alkyl, arylalkyl,
heteroarylalkyl, cyclylalkyl, heterocyclylalkyl or taken together
with R.sup.33 and the nitrogen to which it is attached to form a
heterocyclyl ring; each R.sup.35 is, independently, halo, hydroxy,
amino, nitro, alkyl, aryl, arylacyl, arylalkyl, heteroaryl,
heteroarylacyl, heteroarylalkyl, cyclylacyl, heterocyclylacyl, or
alkylacyl; R.sup.35 is optionally substituted with 1-4 R.sup.36;
and each R.sup.36 is, independently, halo, alkyl, nitro, amino or
hydroxy; or (c) a compound of Formula IV: ##STR00097## D is O, S,
or NH; E is O or NH; R.sup.41 is halo, alkyl, amino, hydroxy, or
alkoxy; R.sup.42 is alkyl, arylalkyl, cyclyl, or cyclylalkyl;
R.sup.43 is alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, cyclyl, cyclylalkyl, heterocyclyl, or
heterocyclylalkyl, where R.sup.43 is optionally substituted with
1-4 R.sup.45; R.sup.44 is alkyl, cyclyl, cyclylalkyl, aryl,
arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, or
heterocyclylalkyl, where R.sup.44 is optionally substituted with
1-4 R.sup.46; each R.sup.45 is independently halo, alkyl, amino,
amido, hydroxy, alkoxy, nitro, cyano, thio, alkylthio, sulfonyl, or
sulfonamidyl; and each R.sup.46 is independently halo, alkyl,
amino, amido, hydroxy, alkoxy, nitro, cyano, thio, alkylthio,
sulfonyl, or sulfonamidyl.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/076,093, filed Mar. 7, 2005, which claims priority to U.S.
Application Ser. No. 60/550,748, filed on Mar. 5, 2004; to U.S.
Ser. No. 60/630,252, filed on Nov. 22, 2004; to U.S. Ser. No.
60/630,264, filed on Nov. 22, 2004; to U.S. Ser. No. 60/630,231,
filed on Nov. 22, 2004; to U.S. Ser. No. 60/630,221, filed on Nov.
22, 2004; to U.S. Ser. No. 60/630,262, filed on Nov. 22, 2004; to
U.S. Ser. No. 60/630,230, filed on Nov. 22, 2004; and to U.S. Ser.
No. 60/633,487, filed on Dec. 6, 2004, each of which are hereby
incorporated by reference in the present application.
TECHNICAL FIELD
[0002] This invention relates to compositions and methods for
modulating the interaction between polypeptides. We describe
exemplary compounds, which may be contained in pharmaceutical
compositions, the screening methods by which they were discovered,
and their use as therapeutic or prophylactic agents.
BACKGROUND
[0003] At least eight progressive neurodegenerative disorders are
caused by an expansion of the naturally occurring CAG tract that
encodes a polyglutamine (polyQ) repeat within the corresponding
protein. These diseases include Huntington's disease (HD), spinal
and bulbar muscular atrophy (SBMA; also known as Kennedy's
disease), dentatorubral-pallidoluysian atrophy, spinocerebellar
ataxia type 1 (SCA1), SCA2, SCA6, SCA7, and Machado-Joseph disease
(MJD/SCA3)(Reddy et al., Trends Neurosci. 22:248-255, 1999). With
the exception of SCA6 (CACNL1A4; Zhuchenko et al., Nature 15:62-69,
1997), which is characterized by a minimal repeat expansion,
affected individuals typically show a similar range of repeat
expansion above .about.35 repeats (Kakizuka et al., Trends Genet.
14:396-402, 1998).
[0004] Of the diseases listed above, HD has arguably been studied
the most intensely, and one of the tools extensively used in those
studies is a transgenic mouse model. The neurons within mice
transgenic for exon 1 of huntingtin are marked by intranuclear
inclusions that contain huntingtin and ubiquitin (Bates et al.,
Brain Pathol. 8:699-714, 1998; and Paulson et al., Am. J. Hum.
Genet. 64:339-345, 1999). These inclusions indicate that protein
misfolding and aggregation mediate neuronal pathogenesis (Davies et
al., Cell 90:537-548, 1997). Moreover, nuclear inclusions have been
observed in the affected regions of brains of patients diagnosed as
having a polyQ-associated disease (Kakizuka et al., Trends Genet.
14:396-402, 1998; DiFiglia et al., Science 277:1990-1993, 1997;
Bates et al., Brain Pathol. 8:699-714, 1998; and Paulson et al.,
Am. J. Hum. Genet. 64:339-345, 1999).
SUMMARY
[0005] The present invention is based, in part, on our discovery of
compounds that can be used to treat or prevent diseases that are
believed to be caused by an aberrant association of proteins within
a cell. The compounds can, for example, be used in the treatment or
prevention of neurological disorders in which polypeptides form
aggregates or other complexes within cells. The compounds were
identified in our screening assays based on their ability to
inhibit or facilitate the association of one protein with another.
While these compounds may mediate the undesirable association that
occurs between polypeptides in the course of certain diseases,
whether by modulating that association or an upstream or downstream
event, the invention is not limited to compounds that exert their
effect on the disease process by any particular mechanism. While we
tend to use the term "compound(s)", we may also use terms like
"agent(s)" to refer to the molecules described herein.
[0006] We have placed each of the compounds we identified into one
of five categories. The compounds in the first four categories are
represented by Formulas I-IV. The compounds in the fifth category
are represented by Formulas V(a)-V(u). The invention encompasses
these compounds in, for example, a substantially pure form, as well
as various compositions containing one or more of them (e.g.,
pharmaceutical formulations) and methods of using them.
[0007] Formula I is:
##STR00001##
[0008] In Formula I, X can be C(O) or a bond; each R.sup.11 and
R.sup.12 can be, independently, halo (e.g., bromo), nitro, amino,
hydroxy, alkoxy, alkyl, alkenyl, alkynyl, cyclyl, heterocyclyl,
aryl, arylalkyl, heteroaryl, or heteroarylalkyl,
NR.sup.15C(O)R.sup.14, or C(O)NR.sup.15R.sup.16; each of which can
be optionally substituted with 1-4 R.sup.17; R.sup.13 can be H,
alkyl, alkenyl, alkynyl, amino, hydroxy, aryl, arylalkyl,
arylamino, heterocyclyl, heterocyclylalkyl, heteroaryl,
heteroarylalkyl, cyclyl, cyclylalkyl, aminoalkyl, hydroxyalkyl, or
alkoxyalkyl; each of which can be optionally substituted with 1-4
R.sup.18; R.sup.14 can be H, alkyl, alkenyl, alkynyl, cyclyl,
heterocyclyl, aryl, or heteroaryl; each of R.sup.15 and R.sup.16
can be, independently, H, hydroxy, alkoxy, alkyl, alkenyl, alkynyl,
cyclyl, heterocyclyl, aryl, arylalkyl, heteroaryl, or
heteroarylalkyl; each R.sup.17 can be, independently, halo (e.g.,
bromo), alkyl, alkoxy, or hydroxy; each R.sup.18 can be,
independently, halo (e.g., bromo), alkyl, amino, hydroxy,
C(O)NR.sup.15R.sup.16, NR.sup.15C(O)R.sup.14, or hydroxyalkyl; and
m and n are each, independently, an integer from 0 to 3. The
compositions of the invention can include a compound of Formula I,
with the proviso that the compound is not Scriptaid
(6-(1,3-Dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxyhexanamide),
which is a histone deacetylase (HDAc) inhibitor; Mitonafide
(5-nitro-2-(2-dimethylaminoethyl)-benzo(de)isoquinoline-1,3-dione),
which is an intercalating agent; or Amonafide
(1H-Benz[de]isoquinoline-1,3(2H)-dione,
5-amino-2-[2-(dimethylamino)ethyl]-(9CI), which is also an
intercalating agent. The proviso can extend to
5-amino-2-(2-diethylamino-ethyl)-benzo[de]isoquinoline-1,3-dione
(see Table 1). The excluded compounds may be encompassed by the
invention when newly formulated in a particular manner (e.g., as
one of the pharmaceutical formulations set out below) or as part of
a kit or as packaged for storage, shipment, or sale. Of course,
their use in treating or preventing a disease characterized by an
unwanted association of proteins is also new. Specific compounds
that conform to Formula I are shown in Table 1.
[0009] Formula II is:
##STR00002##
[0010] In Formula II, Z can be O or S; Y can be O, NR.sup.25 or
CR.sup.26R.sup.27; each of R.sup.21 and R.sup.22 can be
independently halo (e.g., bromo), hydroxy, nitro, cyano, amino,
amido, or alkyl; R.sup.23 can be alkyl, cyclyl, aryl, heteroaryl,
cyclylalkyl, arylalkyl, or heteroarylalkyl, or can be taken
together with R.sup.24 and the nitrogen to which it is attached to
form a ring where R.sup.23 is optionally substituted with 1-3
R.sup.28. R.sup.24 can be H or alkyl, or can be taken together with
R.sup.23 and the nitrogen to which it is attached to form a ring
where R.sup.24 is optionally substituted with 1-3 R.sup.28;
R.sup.25 can be H or alkyl; each of R.sup.26 and R.sup.27 can be,
independently, H or alkyl; each R.sup.28 can be independently halo
(e.g., bromo), hydroxy, nitro, cyano, amino, amido, or alkyl; and
each p and q can be, independently, an integer from 0-4. Specific
compounds that conform to Formula II are shown in Table II.
[0011] Formula III is:
##STR00003##
[0012] In Formula III, A can be N or CR.sup.32; B can be N or CH;
R.sup.31 can be H or NR.sup.33R.sup.34; R.sup.31 can be optionally
substituted with 1-3 R.sup.35; R.sup.32 can be H or
NR.sup.33R.sup.34; R.sup.32 can be optionally substituted with 1-3
R.sup.35; R.sup.33 can be H, alkyl, or taken together with R.sup.34
and the nitrogen to which it is attached forms a heterocyclyl ring;
R.sup.34 can be H, alkyl, arylalkyl, heteroarylalkyl, cyclylalkyl,
heterocyclylalkyl or taken together with R.sup.33 and the nitrogen
to which it is attached to form a heterocyclyl ring; each R.sup.35
can be, independently, halo (e.g., bromo), hydroxy, amino, nitro,
alkyl, aryl, arylacyl, arylalkyl, heteroaryl, heteroarylacyl,
heteroarylalkyl; cyclylacyl; heterocyclylacyl; or alkylacyl;
R.sup.35 can be optionally substituted with 1-4 R.sup.36; each
R.sup.36 can be, independently, halo (e.g., bromo), alkyl, nitro,
amino or hydroxy. Specific compounds that conform to Formula III
are shown in Table 3.
[0013] Formula IV is:
##STR00004##
[0014] In Formula IV, D can be O, S, or NH; E can be O or NH;
R.sup.41 can be halo (e.g., bromo), alkyl, amino, hydroxy, alkoxy;
R.sup.42 can be alkyl, arylalkyl, cyclyl, or cyclylalkyl; R.sup.43
can be alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, cyclyl, cyclylalkyl, heterocyclyl, or
heterocyclylalkyl, where R.sup.43 is optionally substituted with
1-4 R.sup.45; R.sup.44 can be alkyl, cyclyl, cyclylalkyl, aryl,
arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, or
heterocyclylalkyl, where R.sup.44 is optionally substituted with
1-4 R.sup.46; each R.sup.45 can be independently halo (e.g.,
bromo), alkyl, amino, amido, hydroxy, alkoxy, nitro, cyano, thio,
alkylthio, sulfonyl, or sulfonamidyl; and each R.sup.46 can be
independently halo (e.g., bromo), alkyl, amino, amido, hydroxy,
alkoxy, nitro, cyano, thio, alkylthio, sulfonyl, or sulfonamidyl.
Specific compounds that conform to Formula IV are shown in Table
4.
[0015] Each of the variables designated by, for example, R, X, Y,
m, and n in any of the formulas disclosed herein can be selected
independently. While we tend to use the term "compound(s)", we may
also use terms like "agent(s)" to refer to the molecules described
herein.
[0016] The compounds of Formulas V(a) through V(u) are shown in
Table 5.
[0017] The invention also encompasses pharmaceutically acceptable
salts or solvates of a compound of any of Formulas I-IV or
V(a)-V(u), and prodrugs, metabolites, structural analogs, and other
pharmaceutically useful variants thereof. These other variants may
be, for example, a complex containing the compound and a targeting
moiety, as described further below, or a detectable marker (e.g.,
the compound may be joined to a fluorescent compound or may
incorporate a radioactive isotope). When in the form of a prodrug,
a compound may be modified in vivo (e.g., intracellularly) after
being administered to a patient or to a cell in culture. The
modified compound (i.e., the processed prodrug) may be identical to
a compound described herein and will be biologically active or have
enough activity to be clinically beneficial. The same is true of a
metabolite; a given compound may be modified within a cell and yet
retain sufficient biological activity to be clinically useful.
[0018] Packaged products (e.g., sterile containers containing one
or more of the compounds described herein and packaged for storage,
shipment, or sale) and kits, including at least one compound of the
invention and instructions for use, are also within the scope of
the invention.
[0019] In one aspect, the invention features substantially pure
preparations of the compounds described herein or combinations
thereof. A naturally occurring compound is substantially pure when
it is separated to some degree from the compound(s) or other
entities (e.g., proteins, fats, or minerals) it is associated with
in nature. For example, a naturally occurring compound described
herein is substantially pure when it has been separated from 50%,
60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of
the compound(s) or other moieties it is associated with in nature.
While the compounds of the invention may be naturally occurring and
may be purified using conventional techniques, they may also be
non-naturally occurring and may be synthesized (naturally occurring
compounds can be synthesized as well). Compounds prepared by
chemical synthesis are substantially pure, as are compounds that
have been separated from a library of chemical compounds. A
substantially pure compound may be one that is separated from all
the other members of the compound library or it may be one that has
been separated to a limited extent (e.g., it may remain associated
with a limited number (e.g., 1, 2, 3, 4, or 5-10) of other members
of the library). A compound library is not a pharmaceutical or
therapeutic composition.
[0020] Regardless of their original source or the manner in which
they are obtained, the compounds of the invention can be formulated
in accordance with their use. For example, the compounds can be
formulated within compositions for application to cells in tissue
culture or for administration to a patient. For example, the
compounds can be mixed with a sterile, pharmaceutically acceptable
diluent (such as normal saline). As noted below, and as known in
the art, the type of diluent can vary depending upon the intended
route of administration. The resulting compositions can include
additional agents, such as preservatives. The compounds may also be
applied to a surface of a device (e.g., a catheter) or contained
within a pump, patch, or other drug delivery device.
[0021] Whether in cell culture or in vivo, when proteins associate
(either on the cell surface, within the cell, or following
secretion from the cell), they may do so in a variety of ways. When
a compound of the invention interferes with the way one protein
would otherwise interact with another (i.e., the way proteins would
associate in the absence of the compound), the compound may
mediate, for example, aggregation, dimerization, multimerization,
accumulation or participation within complexes, or any other
physiologically significant association between proteins.
[0022] We may refer to the protein that is affected by the compound
as a target protein. The target protein may be the protein most
directly involved with, or associated with, the disease process.
For example, the target protein can be an A.beta. protein found in
the plaques associated with Alzheimer's disease, a tau protein
within a neurofibrillary tangle, the Huntingtin protein that
aggregates in Huntington's disease, or oncogenic proteins such as
fos and jun. We may refer to these target proteins as primary
target proteins. Alternatively, the target protein can be a protein
that is active upstream or downstream in a biochemical pathway in
which the primary target protein is active. We may refer to these
target proteins as secondary target proteins. For example, the
secondary target protein can be a transcription factor that
facilitates expression of a gene encoding a primary target protein.
The secondary target protein could also be a protein whose activity
changes upon interacting with the primary target protein. For
example, where the primary target protein is an enzyme, the
secondary target protein can be that enzyme's substrate.
Alternatively, the secondary target protein could be a kinase that
activates the primary target protein by phosphorylating it. These
scenarios are meant to describe the manner in which the compounds
of the invention may exert their effect on protein-protein
interaction within a cell, but the invention is not so limited. The
invention encompasses compounds according to the formulas described
herein, compositions containing them (e.g., pharmaceutical
formulations), and methods of using them regardless of the
mechanism by which they work. The target proteins may contain
stretches of consecutive glutamine residues or glutamine-rich
regions (e.g., regions containing a sufficient number of glutamine
residues that protein behavior is adversely affected), but the
compounds of the invention may mediate or modulate association
between other polypeptides as well.
[0023] The target proteins, whether primary or secondary, can be
identical or non-identical (e.g., a compound of the invention can
facilitate or inhibit the dimerization of proteins in a homodimer
or heterodimer or may facilitate or inhibit the aggregation of one
Huntingtin protein to another). Some of the target proteins
affected by the compounds of the present invention may contain
stretches of consecutive glutamine residues or glutamine-rich
regions, but the invention is not so limited; the compounds of the
invention may mediate association between other proteins (e.g., the
secondary target proteins described above) or between primary and
secondary proteins. The compounds of the invention may be useful in
the treatment of aggregation-associated diseases by affecting the
disease mechanism in another way (e.g., by facilitating degradation
of a target protein).
[0024] "PolyQ-containing" polypeptides include a number of
consecutive glutamine residues, which may be described in the art
as homopolymeric polyQ regions, while "glutamine-rich" polypeptides
include other (non-glutamine) amino acid residues interspersed
within glutamine residues. The transcriptional factor CBP, the
yeast prion proteins RNQ1 and Sup35, Sp1, and the TAFII130 subunit
of the transcription factor TFIID are examples of proteins that
include glutamine-rich regions. While the number of consecutive
glutamine residues may be quite low (e.g., as few as 3-10 (e.g.,
five)), polyQ-containing polypeptides typically have about 26 or
more consecutive amino acid residues (e.g., 28, 30, 33, 34, 35, 36,
37, 40, 42, 47, 50, 52, 60, 65, 70, 72, 75, 80, 85, 95, 100, 103,
104, 110, 119, 120, 130, 140, 144, 151, 160, 170, 180, 190, 191,
195, 200, 210, 230, 250, 270 or 300 consecutive glutamine
residues). For example, a glutamine-rich polypeptide can have at
least 32 consecutive glutamine residues. Polypeptides having such a
region of consecutive glutamine residues may also be referred to as
having an "extended" polyglutamine region. PolyQ-containing or
glutamine-rich polypeptides can be naturally occurring polypeptides
such as the huntingtin protein, atrophin-1, ataxin-1, ataxin-2,
ataxin-3, the .alpha.1a-voltage dependent calcium channel,
ataxin-7, the androgen receptor, alpha-, beta-, and
gamma-synucleins, polypeptides involved in amyloidosis, such as
those containing immunoglobulin light chains, amyloid-associated
proteins (e.g., alpha1-antichymotrypsin, apolipoprotein E (apoE),
SP-40, and ubiquitin), mutant transthyretin, beta2 microglobulin,
beta2 amyloid protein, and the prion proteins. Other proteins that
may be affected by the compounds of the present invention include
those that form complexes with cellular receptors (e.g., a cell
surface or nuclear receptor) or that participate in dimers or
multimers (e.g., transcription factors). Accordingly, the invention
features compounds that inhibit the aggregation of (or other
undesirable association between) any one or more of the
aforementioned polypeptides, and methods of treating a subject in
which any one of those polypeptides associate, or fail to
associate, to an extent that cellular function is disrupted and a
disease state results (e.g., a subject having immunoglobulin light
chain amyloidosis, HD, Parkinson's disease, adult-onset diabetes,
cirrhosis, emphysema, or a prion disease, such as Creutzfeldt-Jakob
disease). For example, one or more of the compounds of the
invention may block nuclear aggregation of androgen receptors.
Accordingly, the invention features compounds that inhibit the
aggregation of (or other undesirable association between) any one
of the aforementioned polypeptides and methods of treating a
subject in which any one of those polypeptides associate, or fail
to associate, to an extent that cellular function is disrupted and
a disease state results.
[0025] In addition to determining the effect of a compound on
polypeptide association (and, in animal models or clinical trials,
the effect of a compound on the signs and symptoms of a disease),
the assays or screens can include a step in which one determines
cellular toxicity. One can also generate a dose response profile of
putative assay hits and record the results in a screening database
(which is also within the scope of the present invention).
[0026] In specific embodiments, the compositions of the present
invention can be administered to a subject having immunoglobulin
light chain amyloidosis, HD, Parkinson's disease, adult-onset
diabetes, cirrhosis (e.g., cirrhosis of the liver), emphysema, or a
prion disease, such as Creutzfeldt-Jakob disease. Other conditions
that can be treated or prevented with one or more of the compounds
of the present invention include amyotrophic lateral sclerosis,
dentatorubral pallidoluysian atrophy, spinal bulbar muscular
atrophy (SBMA; also known as Kennedy's disease), any of the several
types of spinocerebellar ataxias (e.g., SCA1, SCA2, SCA6, SCA7 and
Machado-Joseph disease (MJD/SCA3)), dentatorubral-pallidoluysian
atrophy, and disorders in which polyglutamine-containing
transcription factors or coactivators are undesirably active (e.g.,
disorders associated with homodimerization of jun or hexamerization
of p53). For example, a subject may have been diagnosed as having,
or at risk for developing, a carcinoma (e.g., breast cancer),
amyloidosis, a myeloma, kuru, a neuroblastoma, cystic fibrosis, an
alpha-1-antitrypsin deficiency disease, or a disorder with a
similar underlying cellular basis (i.e., an association with
undesirable (e.g., excessive or insufficient) protein-protein
aggregation, dimerization, or other interaction).
[0027] Therapeutic methods featured in the invention can include
the step of identifying a subject in need of treatment. The subject
can be identified by, for example, a health care professional
(e.g., a physician) on the basis of subjective or objective
information (e.g., based on comments from the subject, a physical
examination, and/or on measurable parameters (i.e., diagnostic
tests)). Subjects who are treated with the compounds featured in
the invention may have been diagnosed with any disease
characterized by aberrant or undesirable association between
proteins, whether that association occurs to a greater or lesser
extent than is normal (in, e.g., a healthy patient) or desirable.
Alternatively, the subject may be at risk for developing these
disorders. For example, a subject may have a family history or a
genetic mutation or element (e.g., an expanded trinucleotide
repeat) that contributes to the development of disease. Human
subjects, in consult with their physicians and/or other health care
professionals, can decide whether their risk is great enough to
undergo preventative care (as is the case for any prophylactic
treatment or procedure). While the subjects of the preventative
and/or therapeutic regimes described herein may be human, the
compounds and compositions of the invention can also be
administered to non-human subjects (e.g., domesticated animals
(such as a dog or cat), livestock (e.g., a cow, pig, sheep, goat,
or horse), or animals kept in captivity (e.g., any of the large
cats, non-human primates, zebra, giraffes, elephants, and the like
kept in zoos, parks, or preserves)).
[0028] The prophylactic and therapeutic methods can be carried out
by administering to the subject a pharmaceutical composition
containing a therapeutically effective amount of one or more of the
compounds described herein. While a single compound may be
effective, the invention is not so limited. A subject can be
treated with multiple compounds, administered simultaneously or
sequentially (i.e., before or after a compound of the present
invention). For example, a subject can be treated with one or more
of the compounds described herein and, optionally, a
chemotherapeutic agent, an analgesic, a bronchodilator, levodopa or
a similar medication, haloperidol, or risperdone. In other
embodiments, the "second" agent can be a vitamin, mineral, nucleic
acid (e.g., an antisense oligonucleotide or siRNA), a therapeutic
protein (e.g., a peptide), including therapeutic antibodies or
antigen-binding portions thereof, or an anti-inflammatory agent.
Compositions containing a compound of the invention and a second
agent, as described herein, are also within the scope of the
present invention.
[0029] The combination therapy will, of course, depend on the
disorder being treated. Where a compound of the invention is
administered to treat a patient with a cancer, it may be combined
with a known chemotherapeutic agent used to treat that type of
cancer; where a compound of the invention is administered to treat
a patient with Parkinson's disease, it may be combined with a
medication to increase dopamine levels in the brain; and so
forth.
[0030] Compounds that mediate association between proteins can also
be used to diagnose diseases characterized by protein aggregation
(or, as noted above, other undesirable interaction (e.g.,
dimerization or complex formation)). These methods can be carried
out by providing a biological sample from a patient suspected of
having a disease associated with an abnormal or undesirable
association between proteins; exposing the sample to a compound of
the invention; and determining whether the compound modulates the
association of proteins within the sample. The compound can be one
that is known to interact directly with a primary target or one
that modulates protein-protein interaction by acting upstream or
downstream from the primary target. The compound can also be one
that is known to interact with proteins in the context of the
suspected disease. For example, a compound that is known to inhibit
the aggregation of Huntingtin can be used to diagnose a patient
suspected of having HD. The sample will be exposed to the compound
for a time and under conditions (e.g., physiological conditions of
temperature and pH) sufficient to permit the compound to affect
proteins within the sample (e.g., Huntingtin, tau, or A.beta.
proteins within cells within the sample). The diagnostic methods
can be carried out before, after, or in conjunction with other
diagnostic tests, and their results can inform the subject's
treatment regime. For example, where a compound is found to
modulate the aggregation of Huntingtin proteins in a sample
obtained from a patient suspected of having HD, that compound may
then be used to treat the patient.
[0031] With respect to "aggregation associated" diseases, the
predominant theory is that the protein rich aggregates that form
within cells are deleterious. However, a contrary theory holds that
aggregation is a cellular defense mechanism; harmful proteins
aggregate, forming large inclusions that are targeted by, and
slowly degraded by, cellular enzymes. If the latter theory proves
true to any extent, compounds that facilitate aggregation will be
efficacious therapeutic agents. If, instead, the former theory
emerges, compounds that inhibit protein-protein aggregation will be
efficacious therapeutic agents. In either event, all of the present
compounds can be formulated for use in cell culture and/or in vivo
administration and supplied as reagents for research, as described
herein. For example, the compounds can be used to generate cellular
or animal models of the diseases described above, and the cellular
or animal models can include a step of determining a dose response
profile and cellular toxicity.
[0032] Compounds that can mediate association between proteins
(e.g., polyglutamine-containing polypeptides) can be identified by
the screening methods of the invention. As noted above, these
compounds may modulate interaction between proteins in different
ways; the screening methods of the invention are not limited to
those that identify compounds that work by any particular
mechanism, nor are the compounds so limited. In some embodiments,
the compounds may bind to polypeptides prone to aggregate. In other
embodiments, the compounds may act as transcriptional repressors or
enhancers (in this scenario, a compound stimulates or inhibits
transcription of a gene encoding a polypeptide that aggregates or
that is prone to aberrant aggregation; by virtue of, respectively,
increasing or decreasing the amount of the protein within the cell,
the protein becomes more or less likely to aggregate). The
compounds of the invention may also (or may alternatively) affect
protein or RNA stability, thereby affecting polypeptide
accumulation within a cell (more stable RNAs or proteins being more
prone to associate to an undesirable extent). The compounds may
also modulate the post-translational processing of a protein
(improperly processed proteins being less prone to associate to an
undesirable extent). For example, a compound may interact with a
kinase, phosphatase, methyl transferase, ubiquitinase, protease
(e.g., an aspartyl protease such as cathepsin D or BACE-1 or
BACE-2), polymerase (e.g., PARP-1), or other modifying enzyme.
Interruption of post-translational processing events may alter the
ability of a protein to aggregate, or may alter the stability of a
protein, which in turn may affect the accumulation of polypeptide
aggregates or other complexes as discussed above. In yet other
embodiments, a compound may interact with (e.g., bind to) a protein
that mediates protein folding, such as a chaperone protein (e.g., a
stress protein such as a heat shock protein (hsp; e.g., a human hsp
or an hsp expressed within a human cell)). Interaction of the
compound with the chaperone can stabilize, or otherwise modify, its
activity, thereby modifying protein folding in the cell and the
tendency of proteins to aggregate. Yet other compounds may modulate
aggregation by rescuing proteasome dysfunction. Representative
compounds are shown in Tables 1-5.
[0033] Other features and advantages of the invention will be
apparent from the accompanying drawings and description, and from
the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIGS. 1A-1C. FIG. 1A is a bar graph showing luciferase
expression in Sp1-luc/111Q striatum double knock-in cells following
treatment with different compounds, as indicated. Fluorescence was
measured 24 hours after addition of compound. FIG. 1B is a bar
graph as described in FIG. 1A. Fluorescence was measured 48 hours
after addition of compound. FIG. 1C is a bar graph as described in
FIG. 1A. Fluorescence was measured 72 hours after addition of
compound.
DETAILED DESCRIPTION
[0035] Small molecule-based therapeutics have provided the means to
successfully treat many diseases, and the identification of
pharmacological agents that can reverse, block, or delay
disease-linked processes in model systems is critical to the
development of effective treatments for the diseases described
herein. Our assays employ in vitro model systems that recapitulate
key features of disease pathology and that are adaptable to high
throughput screening against a large collection of chemical
compounds.
[0036] Using our assays and screens, we have identified compounds
we believe are capable of modulating (either directly or
indirectly) the association of polypeptides including those that,
when abnormally expressed or associated, cause pathological
disorders such as Parkinson's disease, Huntington's disease, and
the other diseases referred to herein (we tend to use the term
"disease" to refer to any disorder, unwanted condition, or
syndrome). The compounds described herein can be used to modulate
(e.g., inhibit) the aggregation of polypeptides, such as
polyQ-containing polypeptides that are associated with pathological
disorders, as well as non-naturally occurring polypeptides (e.g.,
polyQ-containing polypeptides that are used in disease models, such
as models of HD). Before describing exemplary compounds, we provide
exemplary assays that can be used to test (or further test) those
compounds as well as to identify other compounds or moieties, such
as proteins (e.g., antibodies) and nucleic acids (e.g.,
oligonucleotides or molecules that mediate RNAi (e.g., siRNAs or
shRNAs)) useful in the diagnosis, prevention, or treatment of a
disease characterized by an abnormal association of one protein
with another.
[0037] Assays:
[0038] A variety of assays are available to identify, test and/or
monitor the effect of a compound or other moiety on protein
association (e.g., the aggregation of polyglutamine
repeat-containing polypeptides). In one assay, for example, a cell
expresses a fusion protein that contains a detectable label such as
a fluorescent or luminescent polypeptide (e.g., a fusion protein
that contains a detectable label and a glutamine-rich polypeptide).
The polypeptide can be one that naturally fluoresces or a
non-fluorescent polypeptide that is labeled with a tag (e.g., an
enzyme, fluorescent, luminescent (e.g., bioluminescent), or
otherwise detectable tag). Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase,
and acetylcholinesterase; examples of suitable fluorescent
materials include umbelliferone, fluorescein, fluorescein
isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein,
dansyl chloride, phycoerythrin, green fluorescent protein, and blue
fluorescent protein; an example of a luminescent material is
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin; and examples of suitable radioactive
materials include .sup.125I, .sup.131I, .sup.35S, .sup.32P, and
.sup.3H. These labels and tags may be used not only to label
substrates useful in the assays, but also to label the compounds
identified therein. The coupling of a label to the
aggregation-disposed polypeptide or a compound that affects such a
peptide can be carried out by chemical methods known in the
art.
[0039] In an exemplary assay, the cell is exposed to a compound
(e.g., incubated with the compound), and the signal from the
detectable label can be evaluated. If a fluorescent tag (e.g., GFP,
EGFP, BFP, etc. . . . ) is used, the fluorescent aggregates can be
detected and quantified by fluorescent microscopy.
Aggregate-positive cells and the effects of compounds on protein
aggregation can be detected by any method known in the art (e.g.,
using a fluorometer or by fluorescence-activated cell sorting). To
facilitate the reading, cells containing aggregates and soluble
fluorescent polypeptides can be incubated for an extended time.
Prolonged incubation promotes cellular degradation of soluble
polyQ-containing proteins, leaving intact aggregates that are
resistant to degradation. Cells typically degrade soluble
polypeptides rapidly. For example, PC12 cells usually clear soluble
polyQ in about 24 hours, whereas intracellular aggregates are
retained much longer. An increase in the intensity of the
detectable label (e.g., an increase in fluorescence) following
incubation of the cell with the compound indicates that the
compound promotes interaction (e.g., aggregation) between
polypeptides (e.g., glutamine-rich polypeptides). Conversely, a
decrease in the intensity of the detectable label indicates that
the compound inhibits protein interaction.
[0040] A detectable label can be a label detected by indirect
methods, as by an antibody detection assay. For example, the
aggregation-disposed protein can be fused to an N-terminal amino
acid sequence consisting of 5-35 (e.g., 5, 8, 10, 12, 15, 18, 20,
23, 27, 29, 31, or 35) amino acid residues from any existing
protein or of any random sequence. The N-terminal amino acid
sequence can also be the FLAG-tag sequence (MYKDDDDK (SEQ ID
NO:1)). Alternatively, the tag can be a histidine (His) tag,
influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag, or
thioredoxin (Trx) tag. Other detectable protein fusion tags include
beta-galactosidase, beta-glucuronidase, glutathione-S-transferase
(GST), luciferase, horseradish peroxidase (HRP) and chloramphenicol
acetyltransferase (CAT). When a non-fluorescent tag is used,
association of aggregation-disposed polypeptides can be assessed by
exposing the cell to antibodies that specifically bind the
non-fluorescent polypeptide. Of course, the antibodies that
specifically bind the non-fluorescent polypeptide can be
fluorescently labeled. In any event, while fluorescence can be
measured with a device, such as a fluorimeter, it is also possible
to detect changes in fluorescence by viewing a labeled cell
directly under the microscope. Changes in the size of protein
aggregates can be readily apparent to the eye and can be detected
by automated systems.
[0041] To most accurately assess a compound, the signal generated
by the detectable label (e.g., fluorescent polypeptide) can be
assessed just after the cell has been exposed to the compound
(i.e., before any significant incubation has occurred) as well as
after the period of incubation. When the method is carried out in
this way, the first reading will more accurately reflect the
background signal intensity by taking into account any fluorescence
emitted by the compound per se. Of course, less accurate
measurements can be obtained in other ways (e.g., by assessing
background signal before the compound is added to the cell). In
this method, as well as the others described herein, the compound
can be virtually any substance (e.g., the compound can be a
biological molecule, such as a polypeptide expressed in the cell, a
chemical compound, or a small molecule). Libraries that encode or
contain candidate compounds are available to those of ordinary
skill in the art through charitable sources (e.g., ChemBridge
Corporation (San Diego, Calif.) (which provides useful information
about chemical libraries on the worldwide web)) and commercial
suppliers.
[0042] The cells that can be used in the methods and assays
described herein can be mammalian cells (e.g., the cell of a
rodent, non-human primate, or human) or yeast cells (of any
strain). Regardless of the cell type used, the recombinant proteins
(e.g., fusion proteins) they express can be placed under the
control of an inducible promoter. Many useful inducible promoters
are known in the art. For example, in the event yeast cells are
employed, the fusion protein can be placed under the control of a
Gal1 promoter.
[0043] In one assay, a compound that modulates (e.g., inhibits or
promotes) the interaction (e.g., aggregation) of polypeptides, such
as glutamine-rich polypeptides, can be identified by obtaining a
cell that expresses a fusion protein that includes the polypeptide
(e.g., a glutamine-rich polypeptide, whether naturally or
non-naturally occurring), exposing the cell to the compound, and
assessing the growth rate of the cell. An increase in the growth
rate of the cell can indicate that the compound favorably modulates
(e.g., inhibits) the interaction of the polypeptides. Conversely,
suppression of growth can indicate that the compound stimulates or
promotes the interaction of the polypeptides and that interaction
is deleterious to the cell. The significance of the results may
differ depending upon the disease model (e.g., a model of cancer;
or an assay conducted with tumor cells in culture or in vivo). The
polypeptides whose aggregation is in question can be identical to
one another or they may differ from one another.
[0044] Assays can be used to identify a gene product that mediates
interaction of aggregation-disposed polypeptides or other target
proteins (i.e., a gene product that, possibly in concert with other
gene products, functions to either promote or inhibit the
association of polypeptides, including glutamine-rich
polypeptides). Gene products, which serve as targets for
therapeutic agents, can be identified in assays in which
fluorescence, cell growth, or both, are assessed. For example, a
gene product that mediates the interaction between
aggregation-disposed polypeptides can be identified by obtaining a
mutant yeast cell that expresses an aggregation-disposed
polypeptide and assessing the rate of growth of the cell. An
increase in the rate of growth, relative to that of a wild type
yeast cell that expresses the aggregation-disposed polypeptide
indicates that the gene product that is mutant in the yeast cell is
a gene product that mediates aggregation of the
aggregation-disposed polypeptides. Alternatively, where a
fluorescence-based assay is used, a gene product that mediates
interaction of aggregation-disposed polypeptides can be identified
by obtaining a mutant yeast cell that expresses fusion protein that
includes an aggregation-disposed polypeptide and a fluorescent
polypeptide, exposing the cell to the compound, incubating the cell
with the compound, and assessing the fluorescence emitted by the
fluorescent polypeptide. A decrease in fluorescence, relative to
that of a wild type yeast cell that expresses the
aggregation-disposed polypeptide, indicates that the gene product
that is mutant in the yeast cell is a gene product that mediates
aggregation of glutamine-rich polypeptides.
[0045] Another method that can be used to identify a target for a
therapeutic agent is carried out by obtaining cells that express a
fusion protein that includes a polypeptide (e.g., a polypeptide
prone to aggregation (e.g., a glutamine-rich polypeptide, perhaps
in the context of a disease process) or other target protein),
transfecting the cells with an expression library of mammalian
genes, and assessing the growth of the cells. An alteration in the
growth of a cell (among those transfected and relative to
non-transfected cells) indicates that that cell has been
transfected with a mammalian gene that mediates aggregation of the
polypeptide (e.g., the glutamine-rich polypeptide). Therefore, the
gene, or the gene's product, is a target for a therapeutic agent
that mediates the aggregation of glutamine-rich polypeptides. Here
again, the method can be fluorescence-based, in which case the cell
would express a fusion protein that includes a glutamine-rich
polypeptide and a fluorescent polypeptide, and fluorescent
emission, rather than cell growth, would be assessed.
[0046] Cultured cells, whether labeled or exposed to a
detectably-labeled compound or not, can also be used to carry out
toxicity studies of the compounds described herein and others
(e.g., others identified by the assays of the invention). Using
such studies, we determined that none of compounds C1-C8 are
cytotoxic. Compounds that undesirably block interactions between
proteins (e.g., transcription factors) are very likely to affect
cell viability.
[0047] For use in the screening methods, both naturally occurring
and non-naturally occurring aggregation-disposed polypeptides can
be produced recombinantly. Recombinant methods can be used to fuse
other proteins (e.g., heterologous proteins) to the
aggregation-disposed polypeptides. For example, a glutamine-rich
polypeptide such as huntingtin, can be fused to an antigenic tag,
such as c-myc or FLAG-tag, or a proteinaceous label such as a green
fluorescent protein (GFP, which term includes enhanced GFP, or
"EGFP"). Such fusion proteins, nucleic acid sequences encoding
them, and expression vectors useful in mediating expression are
also within the scope of the present invention. The cell in which
the recombinant polypeptide is produced can be used directly in the
methods of the invention, or the recombinant polypeptide can be
purified from the culture medium or from a lysate of the cells.
Cells that include an association-disposed polypeptide and,
optionally, a compound disclosed herein (e.g., a compound of any of
Tables 1-5 or a salt, solvate, biologically active variant or other
analog thereof) are within the scope of the present invention, as
are arrays of such cells.
[0048] Variants of the aggregation-disposed polypeptides can also
be targets of the compounds of the invention, or used in the assays
described herein. Variants can be prepared by substituting selected
amino acid residues in the polypeptides. A variant of an
aggregation-disposed polypeptide includes a polypeptide that has
high sequence identity (e.g., 60, 70, 80, 90, 95, 96, 97, 98, or
99%) to an aggregation-disposed polypeptide and retains the ability
to aggregate.
[0049] Isolated nucleic acid molecules that encode naturally
occurring, aggregation-disposed polypeptides, variants thereof, or
non-naturally occurring aggregation-disposed polypeptides are
useful in the methods of the invention and in the assays described
herein. Naturally occurring nucleic acid sequences that encode
aggregation-disposed polypeptides are well known in the art and can
be obtained, for example, from GENBANK.TM.. The nucleic acid
triplet that encodes the amino acid glutamine can be either CAA or
CAG. The CAA or CAG codons need not be present in equal numbers and
need not form a repeating pattern.
[0050] Typically, expressing an aggregation-disposed polypeptide in
a cell involves inserting an aggregation-disposed polypeptide
coding sequence into a vector, where it is operably linked to one
or more expression control sequences. The need for, and identity
of, expression control sequences will vary according to the type of
cell in which the aggregation-disposed polypeptide sequence is to
be expressed. Examples of expression control sequences include
transcriptional promoters, enhancers, suitable mRNA ribosomal
binding sites, and sequences that terminate transcription and
translation.
[0051] Suitable expression control sequences can be selected by one
of ordinary skill in the art. Standard methods can be used by the
skilled person to construct expression vectors. See, generally,
Sambrook et al., 1989, Cloning--A Laboratory Manual (2.sup.nd Ed),
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
[0052] Useful vectors include plasmid vectors and viral vectors.
Viral vectors can be, for example, those derived from retroviruses,
adenoviruses, adeno-associated virus, SV40 virus, pox viruses, or
herpes viruses. Once introduced into a host cell (e.g., a bacterial
cell, a yeast cell, an insect cell, an avian cell, or a mammalian
cell), the vector can remain episomal, or be incorporated into the
genome of the host cell. Useful vectors include vectors that can be
purchased commercially, e.g., pcDNA 3.1-based vectors can be
purchased from Invitrogen (Carlsbad, Calif.).
[0053] Compounds:
[0054] Using assays such as those described above, we have
identified certain compounds, which are categorized according to
one of Formulas I-IV or one of Formulas V(a)-V(u). The invention
encompasses these compounds in, for example, a substantially pure
form, as well as various compositions containing one or more of
them (e.g., pharmaceutical formulations, packaged products, and
kits) and methods of using them.
##STR00005##
[0055] Compounds that can be used in practicing the invention,
having the general formula of Formula I can contain a
benzo[de]isoquinoline-1,3-dione or 1H-benzo[cd]indol-2-one core.
The core can be substituted, for example at the nitrogen, or on one
or both of the phenyl moieties.
[0056] Any ring carbon atom can be substituted, for example with
one or more R.sup.1 and R.sup.2 as defined above. For example,
R.sup.1 and R.sup.2 can include, without limitation, halo, nitro,
amino, hydroxy, alkoxy, alkyl, alkenyl, alkynyl, cyclyl,
heterocyclyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, or
amido, each of which can be further substituted with substituents.
Examples of substituents include, but are not limited to halo,
alkyl, alkoxy, or hydroxy.
[0057] The nitrogen is bound to R.sup.3. Examples of R.sup.3
include, but are not limited to H, alkyl, alkenyl, alkynyl, amino,
hydroxy, aryl, arylalkyl, arylamino, heterocyclyl,
heterocyclylalkyl, heteroaryl, heteroarylalkyl, cyclyl,
cyclylalkyl, aminoalkyl, hydroxyalkyl, or alkoxyalkyl each of which
can be further substituted with substituents. Examples of
substituents include, but are not limited to halo, alkyl, amino,
hydroxy, amido, or hydroxyalkyl.
[0058] In specific embodiments, the invention features a purified
or substantially pure compound of Formula I and compositions
comprising such compounds (e.g., pharmaceutical or physiologically
acceptable compositions). Referring to Formula I, X can be C(O) or
a bond; each R.sup.1 and R.sup.2 can be, independently halo, nitro,
amino, hydroxy, alkoxy, alkyl, alkenyl, alkynyl, cyclyl,
heterocyclyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl,
NR.sup.5C(O)R.sup.4, or C(O)NR.sup.5R.sup.6; each of which can be
optionally substituted with 1-4 R.sup.7; R.sup.3 can be H, alkyl,
alkenyl, alkynyl, amino, hydroxy, aryl, arylalkyl, arylamino,
heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl,
cyclyl, cyclylalkyl, aminoalkyl, hydroxyalkyl, or alkoxyalkyl; each
of which can be optionally substituted with 1-4 R.sup.8; R.sup.4
can be H, alkyl, alkenyl, alkynyl, cyclyl, heterocyclyl, aryl, or
heteroaryl; each R.sup.5 and R.sup.6 can be, independently, H,
hydroxy, alkoxy, alkyl, alkenyl, alkynyl, cyclyl, heterocyclyl,
aryl, arylalkyl, heteroaryl, or heteroarylalkyl; each R.sup.7 can
be, independently, halo, alkyl, alkoxy, or hydroxy; each R.sup.8
can be, independently, halo, alkyl, amino, hydroxy,
C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.4, or hydroxyalkyl; and m
and n can each be, independently, an integer from 0 to 3. In
specific embodiments, R.sup.1 can be heterocylcyl (e.g., in this
embodiment and others, R.sup.1 can be a nitrogen-containing
heterocyclyl), nitro or amino; m can be 1; and n can be 0. In other
embodiments, R.sup.1 can be morpholinyl, piperidinyl, or
piperazinyl. Each R.sup.1 and R.sup.2 can be, independently halo,
and each of m and n can be, independently, 1. Either or both of
R.sup.1 and R.sup.2 can be chloro. Either or both of m and n can be
0.
[0059] R.sup.3 can be H, alkyl, amino, aryl, arylamino, arylalkyl,
heterocyclylalkyl (e.g., morpholinyl, piperidinyl or piperazinyl
that includes a C.sub.2-C.sub.4 alkyl) aminoalkyl, hydroxyalkyl, or
alkoxyalkyl. When R.sup.3 is an aminoalkyl, it can be optionally
substituted with alkyl, hydroxyalkyl or C(O)NR.sup.5R.sup.6.
[0060] In specific embodiments, X is C(O); R.sup.1 is heterocylcyl
(e.g., a nitrogen-containing heterocyclyl), nitro or amino; R.sup.3
is alkyl, amino, aryl, arylamino, arylalkyl, heterocyclylalkyl,
aminoalkyl, hydroxyalkyl, or alkoxyalkyl; m is 1; and n is 0. More
specifically, R.sup.1 can be morpholinyl, piperidinyl, or
piperazinyl. Where R.sup.3 is aminoalkyl, it can be optionally
substituted with hydroxyalkyl or C(O)NR.sup.5R.sup.6.
[0061] In another embodiment, X is C(O); R.sup.1 and R.sup.2 are
each independently halo; R.sup.3 is alkyl, amino, aryl, arylamino,
arylalkyl, heterocyclylalkyl, aminoalkyl, hydroxyalkyl, or
alkoxyalkyl; and m and n are each independently 1. For example,
R.sup.3 can be heterocyclylalkyl or aminoalkyl, and where R.sup.3
is aminoalkyl, it can be optionally substituted with hydroxyalkyl
or C(O)NR.sup.5R.sup.6.
[0062] In another embodiment, X is C(O); R.sup.3 is alkyl, amino,
aryl, arylamino, arylalkyl, heterocyclylalkyl, aminoalkyl,
hydroxyalkyl, or alkoxyalkyl; and m and n are each independently 0.
For example, R.sup.3 can be heterocyclylalkyl or aminoalkyl, and
where R.sup.3 is aminoalkyl, it can be optionally substituted with
alkyl, hydroxyalkyl or C(O)NR.sup.5R.sup.6. In this or other
embodiments, where an R group (e.g., R.sup.3) is heterocyclylalkyl,
the heterocyclylalkyl can include a heterocyclyl of morpholinyl,
piperidinyl and piperazinyl, and the heterocyclylalkyl can include
a C.sub.2-C.sub.4 alkyl.
[0063] In another embodiment, X is a bond, and R.sup.3 is H or
alkyl. R.sup.2 can be amino, nitro, or NR.sup.5C(O)R.sup.4, and n
can be 1. Alternatively, m and n can be 0.
[0064] The compounds and compositions of the invention can be, or
can include: 2-Phenylamino-benzo[de]isoquinoline-1,3-dione;
2-Benzyl-6-morpholin-4-yl-benzo[de]isoquinoline-1,3-dione;
6-(4-Methyl-piperazin-1-yl)-2-phenyl-benzo[de]isoquinoline-1,3-dione;
2-(2-Morpholin-4-yl-ethyl)-benzo[de]isoquinoline-1,3-dione;
6-(1,3-Dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-hexanoic acid
hydroxyamide;
2-[3-(Hydroxymethyl-amino)-propyl]-benzo[de]isoquinoline-1,3-dione;
2-[2-(Hydroxymethyl-amino)-ethyl]-benzo[de]isoquinoline-1,3-dione;
2-(3-Methoxy-propyl)-benzo[de]isoquinoline-1,3-dione;
2-(3-Dimethylamino-propyl)-benzo[de]isoquinoline-1,3-dione;
2-(3-Morpholin-4-yl-propyl)-benzo[de]isoquinoline-1,3-dione;
2-(2-Piperidin-4-yl-ethyl)-benzo[de]isoquinoline-1,3-dione;
2-(3-Piperidin-1-yl-propyl)-benzo[de]isoquinoline-1,3-dione;
2-Dimethylamino-benzo[de]isoquinoline-1,3-dione;
2-(2-Dimethylamino-ethyl)-benzo[de]isoquinoline-1,3-dione;
2-(3-Methyl-butyl)-benzo[de] isoquinoline-1,3-dione;
6,7-Dichloro-2-(2-dimethylamino-ethyl)-benzo[de]isoquinoline-1,3-dione;
2-(2-Piperidin-1-yl-ethyl)-benzo[de]isoquinoline-1,3-dione;
2-(2-piperazin-1-yl-ethyl)-benzo[de]isoquinoline-1,3-dione;
2-(2-Morpholin-4-yl-ethyl)-benzo[de]isoquinoline-1,3-dione;
2-(2-Diethylamino-ethyl)-benzo[de]isoquinoline-1,3-dione;
6-Amino-1H-benzo[cd]indol-2-one; 1-Ethyl-1H-benzo[cd]indol-2-one;
6-Amino-1-ethyl-1H-benzo[cd]indol-2-one;
N-(1-Ethyl-2-oxo-1,2-dihydro-benzo[cd]indol-6-yl)-acetamide;
N-Ethyl-2-(2-oxo-2H-benzo[cd]indol-1-yl)-acetamide;
2-Butyl-benzo[de]isoquinoline-1,3-dione;
2-(2-Methoxy-ethyl)-benzo[de]isoquinoline-1,3-dione;
2-Hydroxymethyl-6-piperidin-1-yl-benzo[de]isoquinoline-1,3-dione;
6-(4-Methyl-piperazin-1-yl)-2-phenyl-benzo[de]isoquinoline-1,3-dione;
and/or
2-[3-(Hydroxymethyl-amino)-propyl]-benzo[de]isoquinoline-1,3-dione-
. For example, the compounds and compositions of the invention can
be, or can include a compound shown in Table 1.
TABLE-US-00001 TABLE 1 GI ##STR00006## Formula I A1 ##STR00007##
2-Phenylamino- benzo[de]isoquinoline-1,3-dione A2 aka C7 aka C4
##STR00008## 2-Benzyl-6-morpholin-4-yl-
benzo[de]isoquinoline-1,3-dione A3 aka C4-34 ##STR00009##
6-(4-Methyl-piperazin-1-yl)-2- phenyl-benzo[de]isoquinoline-1,3-
dione A4 ##STR00010## 2-(2-Morpholin-4-yl-ethyl)-
benzo[de]isoquinoline-1,3-dione A5 Scriptaid ##STR00011##
6-(1,3-Dioxo-1H,3H- benzo[de]isoquinolin-2-yl)- hexanoic acid
hydroxyamide A6 ##STR00012## 2-[3-(Hydroxymethyl-amino)-
propyl]-benzo[de]isoquinoline-1,3- dione A7 aka C9-3 ##STR00013##
2-[2-(Hydroxymethyl-amino)- ethyl]-benzo[de]isoquinoline-1,3- dione
A8 aka C9-2 ##STR00014## 2-(3-Methoxy-propyl)-
benzo[de]isoquinoline-1,3-dione A9 aka C9-1 aka C91 ##STR00015##
2-(3-Dimethylamino-propyl)- benzo[de]isoquinoline-1,3-dione A10
##STR00016## 2-(3-Morpholin-4-yl-propyl)-
benzo[de]isoquinoline-1,3-dione A11 aka C9-7 ##STR00017##
2-(2-Piperidin-4-yl-ethyl)- benzo[de]isoquinoline-1,3-dione A12 aka
C9-4A ##STR00018## 2-(3-Piperidin-1-yl-propyl)-
benzo[de]isoquinoline-1,3-dione A13 ##STR00019## 2-Dimethylamino-
benzo[de]isoquinoline-1,3-dione A14 aka C9-1B ##STR00020##
2-(2-Dimethylamino-ethyl)- benzo[de]isoquinoline-1,3-dione A15 aka
C9-1A ##STR00021## 2-(3-Methyl-butyl)-
benzo[de]isoquinoline-1,3-dione A16 aka C9-1 aka C1 ##STR00022##
6,7-Dichloro-2-(2-dimethylamino- ethyl)-benzo[de]isoquinoline-1,3-
dione A17 ##STR00023## 2-(2-Dimethylamino-ethyl)-5-nitro-
benzo[de]isoquinoline-1,3-dione A18 ##STR00024##
5-Amino-2-(2-dimethylamino- ethyl)-benzo[de]isoquinoline-1,3- dione
A19 aka C9-4 ##STR00025## 2-(2-Piperidin-1-yl-ethyl)-
benzo[de]isoquinoline-1,3-dione A20 aka C9-5 ##STR00026##
2-(2-Piperazin-1-yl-ethyl)- benzo[de]isoquinoline-1,3 -dione A21
##STR00027## 2-(2-Morpholin-4-yl-ethyl)-
benzo[de]isoquinoline-1,3-dione A22 aka C96C ##STR00028##
5-Amino-2-(2-diethylamino-ethyl)- benzo[de]isoquinoline-1,3-dione
A23 aka C9-6B ##STR00029## 2-(2-Diethylamino-ethyl)-
benzo[de]isoquinoline-1,3-dione A24 ##STR00030##
6-Amino-1H-benzo[cd]indol-2-one A25 ##STR00031##
1-Ethyl-1H-benzo[cd]indol-2-one A26 ##STR00032##
6-Amino-1-ethyl-1H- benzo[cd]indol-2-one A27 aka CG4 ##STR00033##
N-(1-Ethyl-2-oxo-1,2-dihydro- benzo[cd]indol-6-yl)-acetamide A28
##STR00034## N-Ethyl-2-(2-oxo-2H- benzo[cd]indol-1-yl)-acetamide
A29 ##STR00035## 2-Butyl-benzo[de]isoquinoline-1,3- dione A30
##STR00036## 2-(2-Methoxy-ethyl)- benzo[de]isoquinoline-1,3-dione
A31 aka C4-7 ##STR00037## 2-Hydroxymethyl-6-piperidin-1-yl-
benzo[de]isoquinoline-1,3-dione A33 aka C9 aka C4-DAK ##STR00038##
2-[3-(Hydroxymethyl-amino)- propyl]-benzo[de]isoquinoline-1,3-
dione A34 ##STR00039## A35 ##STR00040## N-(1-ethyl-2-oxo-1,2-
dihydrobenzo[cd]indol-7- yl)acetamide
[0065] While pharmaceutical formulations are described further
below, we note here, that the compounds of the invention, including
those just described, can be formulated for oral or parenteral
administration to a patient. Likewise, while methods are described
further elsewhere herein, we note that the invention encompasses
methods of treating a subject who has, who has been diagnosed as
having, or who is at risk of developing, a disorder characterized
by an undesirable association of proteins. The methods can include
the step of identifying the subject (or patient) and administering
to the subject a therapeutically effective amount of a
pharmaceutical composition that includes any of the compounds
described herein (e.g., a compound conforming to Formula I). The
subject may have been diagnosed as having, or at risk of
developing, Huntington's disease, Parkinson's disease, spinal and
bulbar muscular atrophy, dentatorubral-pallidoluysian atrophy,
spinocerebellar ataxia type 1 (SCA1), SCA2, SCA6, SCA7,
Machado-Joseph disease (MJD/SCA3), a carcinoma associated with
oncoprotein association (e.g., dimerization) (e.g., breast cancer),
amyloidosis, myeloma, Creutzfeldt-Jakob disease, kuru, cystic
fibrosis, neuroblastoma, or alpha-1-antitrypsin deficiency
disease.
[0066] With respect to C9, which conforms to Formula I, our initial
studies revealed that this compound increases protein-protein
aggregation (as described further below, C9 facilitated aggregation
of polyglutamine-containing proteins in a cell-free
membrane-trapping assay and in neurons in cultured brain slices at
concentrations higher than 10 .mu.M). Based on this information, we
readily identified two compounds that are variants of C9 that also
facilitate protein-protein aggregation (C9-1 and C9-2, shown
below). Accordingly, C9, C9-1 and C9-2 are within the scope of the
present invention and can be formulated in pharmaceutically or
physiologically acceptable compositions and applied to cells to, we
believe, facilitate association between polypeptides. Our present
results lead us to conclude that C9 interferes with transcriptional
machinery, possibly by intercalating into the DNA's double helix
and forming high affinity hydrogen bonds with GC rich motifs.
##STR00041##
[0067] In Formula II, Z can be O or S; Y can be O, NR.sup.25 or
CR.sup.26R.sup.27; each of R.sup.21 and R.sup.22 can be
independently halo (e.g., bromo), hydroxy, nitro, cyano, amino,
amido, or alkyl; R.sup.23 can be alkyl, cyclyl, aryl, heteroaryl,
cyclylalkyl, arylalkyl, or heteroarylalkyl, or can be taken
together with R.sup.24 and the nitrogen to which it is attached to
form a ring where R.sup.23 is optionally substituted with 1-3
R.sup.28. R.sup.24 can be H or alkyl, or can be taken together with
R.sup.23 and the nitrogen to which it is attached to form a ring
where R.sup.24 is optionally substituted with 1-3 R.sup.28;
R.sup.25 can be H or alkyl; each of R.sup.26 and R.sup.27 can be,
independently, H or alkyl; each R.sup.28 can be independently halo
(e.g., bromo), hydroxy, nitro, cyano, amino, amido, or alkyl; and
each p and q can be, independently, an integer from 0-4. Specific
compounds that conform to Formula II are shown in Table 2.
[0068] In specific embodiments, the invention features a purified
or substantially pure compound of Formula II and compositions
comprising such compounds (e.g., pharmaceutical or physiologically
acceptable compositions). Referring to Formula II, Z can be O or S;
Y can O, NR.sup.25 or CR.sup.26R.sup.27; each of R.sup.21 and
R.sup.22 can be, independently, halo, hydroxy, nitro, cyano, amino,
amido, or alkyl; R.sup.23 can be alkyl, cyclyl, aryl, heteroaryl,
cyclylalkyl, arylalkyl, or heteroarylalkyl, or when taken together
with R.sup.24 and the nitrogen to which it is attached can form a
ring. For example, R.sup.23 can optionally be substituted with 1-3
R.sup.28. R.sup.24 can be H, alkyl, or when taken together with
R.sup.23 and the nitrogen to which it is attached can form a ring.
For example, R.sup.24 can be optionally substituted with 1-3
R.sup.28. R.sup.25 can be H or alkyl; each of R.sup.26 and R.sup.27
can be, independently, H or alkyl; each R.sup.28 can be,
independently, halo, hydroxy, nitro, cyano, amino, amido, or alkyl;
each p can be an integer from 0-5, inclusive; and each q can be,
independent of p, an integer from 0-4, inclusive (i.e., 0, 1, 2, 3,
or 4).
[0069] In specific embodiments, Z is 0, and Y is NR.sup.25. In this
embodiment and others, R.sup.25 can be H. Alternatively, Y can be
CR.sup.26R.sup.27. Each R.sup.21 and R.sup.22 can be independently
halo or hydroxy. In some embodiments, R.sup.21 is halo and p is 1.
In that instance, q can be, for example, 0. In connection with
Formula II or any other of the formulas presented herein, the
halogen can be any radical of fluorine, chlorine, bromine or
iodine. Thus, in specific embodiments, R.sup.22 can be halo (e.g.,
bromo) and q can be 1. In that instance, p can be, for example, 0.
In some embodiments, p and q are 0.
[0070] R.sup.23 can be cyclyl or aryl, and the aryl can be
substituted with bromo. R.sup.24 can be H or alkyl, and R.sup.23
and R.sup.24, taken together with the nitrogen to which they are
attached, can form a ring.
[0071] In another embodiment, Z is O; Y is NR.sup.25; each of
R.sup.21 and R.sup.22 are, independently, halo (e.g., bromo),
hydroxy or alkyl; R.sup.23 is cyclyl or aryl; R.sup.24 is H or
alkyl; and each p and q is 0 or 1.
[0072] In another embodiment, Z is O; Y is CR.sup.26R.sup.27; each
R.sup.21 and R.sup.22 is independently halo (e.g., bromo, chloro,
fluoro, or iodo), hydroxy, or alkyl; R.sup.23 and R.sup.24, taken
together with the nitrogen to which they are attached, can form a
ring; and each p and q is 0 or 1. For example, R.sup.21 can be
hydroxy, p can be 1, and q can be 0.
[0073] The compounds and compositions of the invention can be, or
can include:
4-Bromo-N-(4-Bromo-phenyl)-3-(4-bromo-phenylsulfamoyl)-benzamide- ;
3-(4-Bromo-phenylsulfamoyl)-N-phenyl-benzamide;
4-Bromo-3-cyclohexylsulfamoyl-N-phenyl-benzamide;
N-(4-Bromo-phenyl)-3-cyclohexylsulfamoyl-benzamide; and/or
1-[3-(Azepane-1-sulfonyl)-2-bromo-phenyl]-2-(3-hydroxy-phenyl)-ethanone.
For example, the compounds and compositions of the invention can
be, or can include a compound shown in Table 2.
TABLE-US-00002 TABLE 2 GII ##STR00042## Formula II B1 ##STR00043##
4-Bromo-N-(4-bromo-phenyl)-3- cyclohexylsulfamoyl-benzamide B2 aka
C2-8 aka C8 ##STR00044## N-(4-Bromo-phenyl)-3-(4-bromo-
phenylsulfamoyl)-benzamide B3 ##STR00045##
3-(4-Bromo-phenylsulfamoyl)-N- phenyl-benzamide B4 ##STR00046##
4-Bromo-3-cyclohexylsulfamoyl- N-phenyl-benzamide B5 ##STR00047##
N-(4-Bromo-phenyl)-3- cyclohexylsulfamoyl-benzamide B6 aka C2-10
##STR00048## 1-[3-(Azepane-1-sulfonyl)-2-
bromo-phenyl]-2-(3-hydroxy- phenyl)-ethanone B7 aka C2-11
##STR00049## 3-(N-(4- methoxyphenyl)sulfamoyl)-4-
methyl-N-o-tolylbenzamide B8 ##STR00050##
3-(azepan-1-ylsulfonyl)-N-(3- hydroxyphenyl)-4- methylbenzamide B9
##STR00051## 5-(azepan-1-ylsulfonyl)-2-chloro-
N-(3-hydroxyphenyl)benzamide
[0074] While pharmaceutical formulations are described further
below, we note here, that the compounds of the invention, including
those just described, can be formulated for oral or parenteral
administration to a patient. Likewise, while methods are described
further elsewhere herein, we note that the invention encompasses
methods of treating a subject who has, who has been diagnosed as
having, or who is at risk of developing, a disorder characterized
by an undesirable association of proteins. The methods can include
the step of identifying the subject (or patient) and administering
to the subject a therapeutically effective amount of a
pharmaceutical composition that includes any of the compounds
described herein (e.g., a compound conforming to Formula II). The
subject may have been diagnosed as having, or at risk of
developing, Huntington's disease, Parkinson's disease, spinal and
bulbar muscular atrophy, dentatorubral-pallidoluysian atrophy,
spinocerebellar ataxia type 1 (SCA1), SCA2, SCA6, SCA7,
Machado-Joseph disease (MJD/SCA3), a carcinoma associated with
oncoprotein association (e.g., dimerization) (e.g., breast cancer),
amyloidosis, myeloma, Creutzfeldt-Jakob disease, kuru, cystic
fibrosis, neuroblastoma, or alpha-1-antitrypsin deficiency
disease.
##STR00052##
[0075] In Formula III, A can be N or CR.sup.32; B can be N or CH;
R.sup.31 can be H or NR.sup.33R.sup.34; R.sup.31 can be optionally
substituted with 1-3 R.sup.35; R.sup.32 can be H or
NR.sup.33R.sup.34; R.sup.32 can be optionally substituted with 1-3
R.sup.35; R.sup.33 can be H, alkyl, or taken together with R.sup.34
and the nitrogen to which it is attached forms a heterocyclyl ring;
R.sup.34 can be H, alkyl, arylalkyl, heteroarylalkyl, cyclylalkyl,
heterocyclylalkyl or taken together with R.sup.33 and the nitrogen
to which it is attached to form a heterocyclyl ring; each R.sup.35
can be, independently, halo (e.g., bromo), hydroxy, amino, nitro,
alkyl, aryl, arylacyl, arylalkyl, heteroaryl, heteroarylacyl,
heteroarylalkyl; cyclylacyl; heterocyclylacyl; or alkylacyl;
R.sup.35 can be optionally substituted with 1-4 R.sup.36; each
R.sup.36 can be, independently, halo (e.g., bromo), alkyl, nitro,
amino or hydroxy. Specific compounds that conform to Formula III
are shown in Table 3.
[0076] In specific embodiments, the invention features a purified
or substantially pure compound of Formula III and compositions
comprising such compounds (e.g., pharmaceutical or physiologically
acceptable compositions). Referring to Formula III, A can be N or
CR.sup.32; B can be N or CH; R.sup.31 can be H or
NR.sup.33R.sup.34; wherein R.sup.31 is optionally substituted with
1-3 R.sup.35; R.sup.32 can be H or NR.sup.33R.sup.34; wherein
R.sup.32 optionally substituted with 1-3 R.sup.35; R.sup.33 can be
H, alkyl, or taken together with R.sup.34 and the nitrogen to which
it is attached, can form a heterocyclyl ring; R.sup.34 can be H,
alkyl, arylalkyl, heteroarylalkyl, cyclylalkyl, heterocyclylalkyl
or, taken together with R.sup.33 and the nitrogen to which it is
attached, can form a heterocyclyl ring; each R.sup.35 can be,
independently halo, hydroxy, amino, nitro, alkyl, aryl, arylacyl,
arylalkyl, heteroaryl, heteroarylacyl, heteroarylalkyl, cyclylacyl,
heterocyclylacyl, or alkylacyl, wherein R.sup.35 can be,
optionally, substituted with 1-4 R.sup.36; and each R.sup.36 can
be, independently, halo, alkyl, nitro, amino or hydroxy.
[0077] For example, A can be CR.sup.32; B can be N; and R.sup.32
can be H. R.sup.31 can be NR.sup.33R.sup.34 and R.sup.33 and
R.sup.34, together with the nitrogen to which they are attached,
can form a heterocyclyl ring. For example, R.sup.33 and R.sup.34,
together with the nitrogen to which they are attached, can form a
ring (e.g., a piperazinyl, piperidinyl, or morpholinyl ring). The
heterocyclyl ring can be substituted with R.sup.35, which can be an
alkyl, hydroxyl, or arylacyl group. Where R.sup.35 is arylacyl, it
can be substituted with 1-3 halo.
[0078] In another embodiment, A is N, B is CH, and R.sup.31 is
NR.sup.33R.sup.34. In this embodiment and others, R.sup.33 and
R.sup.34, together with the nitrogen to which they are attached,
can form a heterocyclyl ring (e.g., a piperazinyl, piperidinyl, or
morpholinyl ring). The heterocyclyl ring can be substituted with
R.sup.35, which can be an alkyl, hydroxyl, or arylacyl. The
araylacyl can be substituted with 1-3 halo.
[0079] In another embodiment, A is CR.sup.32, B is N, R.sup.31 is
H, and R.sup.32 is NR.sup.33R.sup.34. In this embodiment and
others, R.sup.33 and R.sup.34, together with the nitrogen to which
they are attached, can form a heterocyclyl ring (e.g., a
piperazinyl or piperidinyl ring). The heterocyclyl ring can be
substituted with R.sup.35, which can be, for example, alkyl or
arylacyl.
[0080] Accordingly, the compounds and compositions of the invention
can be, or can include:
(4-Chloro-phenyl)-[4-(8-nitro-quinolin-5-yl)-piperazin-1-yl]-methanone;
(2-Chloro-4,5-difluoro-phenyl)-[4-(5-nitro-quinolin-8-yl)-piperazin-1-yl]-
-methanone;
(2-Chloro-4,5-difluoro-phenyl)-[4-(8-nitro-quinolin-5-yl)-piperazin-1-yl]-
-methanone;
(2-Fluoro-phenyl)-[4-(8-nitro-quinolin-5-yl)-piperazin-1-yl]-methanone;
(3,4-Dichloro-phenyl)-[4-(5-nitro-quinolin-8-yl)-piperazin-1-yl]-methanon-
e; 1-(8-Nitro-quinolin-5-yl)-piperidin-3-ol;
(2-Fluoro-phenyl)-[4-(5-nitro-quinolin-8-yl)-piperazin-1-yl]-methanone;
8-Nitro-4-piperidin-1-yl-quinoline;
4-(4-Methyl-piperazin-1-yl)-8-nitro-quinoline;
5-Morpholin-4-yl-8-nitro-quinoline;
(3-Morpholin-4-yl-propyl)-(8-nitro-quinolin-5-yl)-amine;
8-Nitro-5-piperazin-1-yl-quinoline;
8-(4-Methyl-piperazin-1-yl)-5-nitro-quinoline; and/or
5-Nitro-8-(4-phenyl-piperazin-1-yl)-quinoline. For example, the
compounds and compositions of the invention can be, or can include
a compound shown in Table 3.
TABLE-US-00003 TABLE 3 GIII ##STR00053## Formula III E1
##STR00054## (4-Chloro-phenyl)-[4-(8-nitro-quinolin-
5-yl)-piperazin-1-yl]-methanone E2 ##STR00055##
(2-Chloro-4,5-difluoro-phenyl)-[4-(5-
nitro-quinolin-8-yl)-piperazin-1-yl]- methanone E3 ##STR00056##
(2-Chloro-4,5-difluoro-phenyl)-[4-(8-
nitro-quinolin-5-yl)-piperazin-1-yl]- methanone E4 ##STR00057##
(2-Fluoro-phenyl)-[4-(8-nitro-quinolin-
5-yl)-piperazin-1-yl]-methanone E5 ##STR00058##
(3,4-Dichloro-phenyl)-[4-(5-nitro- quinolin-8-yl)-piperazin-1-yl]-
methanone E7 ##STR00059## (2-Fluoro-phenyl)-[4-(5-nitro-quinolin-
8-yl)-piperazin-1-yl]-methanone E8 ##STR00060##
8-Nitro-4-piperidin-1-yl-quinoline E9 ##STR00061##
4-(4-Methyl-piperazin-1-yl)-8-nitro- quinoline E10 ##STR00062##
5-Morpholin-4-yl-8-nitro-quinoline E11 ##STR00063##
(3-Morpholin-4-yl-propyl)-(8-nitro- quinolin-5-yl)-amine E12
##STR00064## 8-Nitro-5-piperazin-1-yl-quinoline E13 ##STR00065##
8-(4-Methyl-piperazin-1-yl)-5-nitro- quinoline E14 ##STR00066##
5-Nitro-8-(4-phenyl-piperazin-1-yl)- quinoline
[0081] While pharmaceutical formulations are described further
below, we note here, that the compounds of the invention, including
those just described, can be formulated for oral or parenteral
administration to a patient. Likewise, while methods are described
further elsewhere herein, we note that the invention encompasses
methods of treating a subject who has, who has been diagnosed as
having, or who is at risk of developing, a disorder characterized
by an undesirable association of proteins. The methods can include
the step of identifying the subject (or patient) and administering
to the subject a therapeutically effective amount of a
pharmaceutical composition that includes any of the compounds
described herein (e.g., a compound conforming to Formula III). The
subject may have been diagnosed as having, or at risk of
developing, Huntington's disease, Parkinson's disease, spinal and
bulbar muscular atrophy, dentatorubral-pallidoluysian atrophy,
spinocerebellar ataxia type 1 (SCA1), SCA2, SCA6, SCA7,
Machado-Joseph disease (MJD/SCA3), a carcinoma associated with
oncoprotein association (e.g., dimerization) (e.g., breast cancer),
amyloidosis, myeloma, Creutzfeldt-Jakob disease, kuru, cystic
fibrosis, neuroblastoma, or alpha-1-antitrypsin deficiency
disease.
##STR00067##
[0082] In Formula IV, D can be O, S, or NH; E can be O or NH;
R.sup.41 can be halo (e.g., bromo), alkyl, amino, hydroxy, alkoxy;
R.sup.42 can be alkyl, arylalkyl, cyclyl, or cyclylalkyl; R.sup.43
can be alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, cyclyl, cyclylalkyl, heterocyclyl, or
heterocyclylalkyl, where R.sup.43 is optionally substituted with
1-4 R.sup.45; R.sup.44 can be alkyl, cyclyl, cyclylalkyl, aryl,
arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, or
heterocyclylalkyl, where R.sup.44 is optionally substituted with
1-4 R.sup.46; each R.sup.45 can be independently halo (e.g.,
bromo), alkyl, amino, amido, hydroxy, alkoxy, nitro, cyano, thio,
alkylthio, sulfonyl, or sulfonamidyl; and each R.sup.46 can be
independently halo (e.g., bromo), alkyl, amino, amido, hydroxy,
alkoxy, nitro, cyano, thio, alkylthio, sulfonyl, or sulfonamidyl.
Specific compounds that conform to Formula IV are shown in Table
4.
[0083] In specific embodiments, the invention features a purified
or substantially pure compound of Formula IV and compositions
comprising such compounds (e.g., pharmaceutical or physiologically
acceptable compositions). Referring to Formula IV, D can be O, S,
or NH; E can be O or NH; R.sup.41 can be halo, alkyl, amino,
hydroxy, alkoxy; R.sup.42 can be alkyl, arylalkyl, cyclyl, or
cyclylalkyl; R.sup.43 can be alkyl, alkenyl, alkynyl, aryl,
arylalkyl, heteroaryl, heteroarylalkyl, cyclyl, R.sup.44
cyclylalkyl, heterocyclyl, heterocyclylalkyl, wherein R.sup.43 is
optionally substituted with 1-4 R.sup.45; can be alkyl, cyclyl,
cyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl,
heterocyclyl, or heterocyclylalkyl, wherein R.sup.44 is optionally
substituted with 1-4 R.sup.46; each R.sup.45 can be, independently,
halo (e.g., bromo), alkyl, amino, amido, hydroxy, alkoxy, nitro,
cyano, thio, alkylthio, sulfonyl, or sulfonamidyl; and each
R.sup.46 can be, independently, halo, alkyl, amino, amido, hydroxy,
alkoxy, nitro, cyano, thio, alkylthio, sulfonyl, or sulfonamidyl.
For example, D can be S, R.sup.41 can be alkyl or methyl, E can be
NH, R.sup.44 can be alkyl or aryl (e.g., aryl substituted with
alkoxy).
[0084] In other embodiments, E can be O, and R.sup.42 and R.sup.44
can be alkyl (e.g., C.sub.2-3 alkyl). In this or other embodiments,
R.sup.42 can be ethyl or isopropyl. In this or other embodiments,
R.sup.43 can be aryl or arylalkyl (e.g., R.sup.43 can be aryl
substituted with 1-4 halo, alkyl, or sulfonamidyl groups or
arylalkyl substituted with alkoxy).
[0085] In some embodiments, D can be S; E can be NH; R.sup.41 can
be alkyl; and R.sup.42 can be alkyl. In this or other embodiments,
R.sup.43 can be aryl or arylalkyl (e.g., R.sup.43 can be aryl
substituted with 1-4 halo, alkyl, or sulfonamidyl groups or
arylalkyl substituted with alkoxy). R.sup.44 can be alkyl or aryl
(e.g., aryl substituted with alkoxy).
[0086] In another embodiment, D can be S; E can be O; R.sup.41 can
be alkyl; and R.sup.42 can be alkyl. Further, R.sup.43 can be aryl,
and R.sup.44 can be alkyl. R.sup.43 can aryl substituted with two
chloro and one sulfonamidyl.
[0087] Accordingly, the compounds and compositions of the invention
can be, or can include:
2-[2-(4-Methoxy-phenyl)-acetylamino]-5-(2-methoxy-phenylcarbamoyl)-4-meth-
yl-thiophene-3-carboxylic acid ethyl ester;
5-(2-Methoxy-phenylcarbamoyl)-4-methyl-2-(2-methyl-benzoylamino)-thiophen-
e-3-carboxylic acid ethyl ester;
5-Diethylcarbamoyl-2-(4-fluoro-benzoylamino)-4-methyl-thiophene-3-carboxy-
lic acid isopropyl ester; or
5-(2,4-Dichloro-5-sulfamoyl-benzoylamino)-3-methyl-thiophene-2,4-dicarbox-
ylic acid diethyl ester. The invention encompasses the compounds
shown in Table 4 and compositions containing them.
TABLE-US-00004 TABLE 4 GIV ##STR00068## Formula IV D1 ##STR00069##
2-[2-(4-Methoxy-phenyl)- acetylamino]-5-(2-methoxy-
phenylcarbamoyl)-4-methyl- thiophene-3-carboxylic acid ethyl ester
D2 ##STR00070## 5-(2-Methoxy-phenylcarbamoyl)-4-
methyl-2-(2-methyl-benzoylamino)- thiophene-3-carboxylic acid ethyl
ester D3 ##STR00071## 5-Diethylcarbamoyl-2-(4-fluoro-
benzoylamino)-4-methyl-thiophene- 3-carboxylic acid isopropyl ester
D4 ##STR00072## 5-(2,4-Dichloro-5-sulfamoyl-
benzoylamino)-3-methyl-thiophene- 2,4-dicarboxylic acid diethyl
ester
[0088] Further with respect to Formula IV, and in some embodiments,
R.sup.41 is alkyl, for example a lower alkyl, such as methyl,
ethyl, propyl, or butyl. Preferred embodiments may include those
where R.sup.41 is methyl.
[0089] The core can also be substituted with an ester, for example,
as depicted above as C(O)OR.sup.42. Examples of suitable R.sup.42
moieties include, but are not limited to alkyl, arylalkyl, cyclyl,
or cyclylalkyl. The core can also be substituted with at least one
amide positioned adjacent to the heteroatom. For example, as can be
seen above, the core is substituted with NHC(O)R.sup.43. Examples
of R.sup.43 include, but are not limited to alkyl, alkenyl,
alkynyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cyclyl,
cyclylalkyl, heterocyclyl, heterocyclylalkyl. In some instances,
R.sup.43 is further substituted with one or more substituents,
including for example, halo, alkyl, amino, amido, hydroxy, alkoxy,
nitro, cyano, thio, alkylthio, sulfonyl, or sulfonamidyl.
[0090] The core can be substituted with an additional carbonyl, for
example an ester or an amide. In preferred embodiment, the core is
substituted as above with C(O)NHR.sup.44. Examples of R.sup.44
include, but are not limited to alkyl, cyclyl, cyclylalkyl, aryl,
arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, or
heterocyclylalkyl. In some instances, R.sup.44 is further
substituted with one or more substituents, including for example,
halo, alkyl, amino, amido, hydroxy, alkoxy, nitro, cyano, thio,
alkylthio, sulfonyl, or sulfonamidyl.
[0091] While pharmaceutical formulations are described further
below, we note here, that the compounds of the invention, including
those just described, can be formulated for oral or parenteral
administration to a patient. Likewise, while methods are described
further elsewhere herein, we note that the invention encompasses
methods of treating a subject who has, who has been diagnosed as
having, or who is at risk of developing, a disorder characterized
by an undesirable association of proteins. The methods can include
the step of identifying the subject (or patient) and administering
to the subject a therapeutically effective amount of a
pharmaceutical composition that includes any of the compounds
described herein (e.g., a compound conforming to Formula IV). The
subject may have been diagnosed as having, or at risk of
developing, Huntington's disease, Parkinson's disease, spinal and
bulbar muscular atrophy, dentatorubral-pallidoluysian atrophy,
spinocerebellar ataxia type 1 (SCA1), SCA2, SCA6, SCA7,
Machado-Joseph disease (MJD/SCA3), a carcinoma associated with
oncoprotein association (e.g., dimerization) (e.g., breast cancer),
amyloidosis, myeloma, Creutzfeldt-Jakob disease, kuru, cystic
fibrosis, neuroblastoma, or alpha-1-antitrypsin deficiency
disease.
[0092] The compounds of Formulas V(a) through V(u) are shown in
Table 5.
TABLE-US-00005 TABLE 5 Compound M1 Formula V(a) ##STR00073##
3-Amino-4,6-dimethyl-thieno[2,3-b]pyridine-2-carboxylic acid amide
Compound M2 Formula V(b) ##STR00074##
N-(4-Phenylamino-phenyl)-2-m-tolyloxy-acetamide Compound M3 Formula
V(c) ##STR00075## N-(4-(phenylamino)phenyl)-2-(o-tolyloxy)acetamide
Compound M4 Formula V(d) ##STR00076##
5-Bromo-N-(4-sulfamoyl-phenyl)-nicotinamide Compound M5 Formula
V(e) ##STR00077##
2-[N'-(3-Aminomethoxy-4-hydroxy-benzylidene)-hydrazino]-N,N-diethyl-2-oxo-
-acetamide Compound M6 Formula V(f) ##STR00078##
N-(2,4-Dimethoxy-benzylidene)-N'-(3-methyl-3H-benzothiazol-2-ylidene)-hyd-
razine Compound M7 Formula V(g) ##STR00079##
{2-[(5-Nitro-furan-2-carbonyl)-amino]-thiazol-4-yl}- acetic acid
ethyl ester Compound M8 Formula V(h) ##STR00080##
[2-(6-Ethoxy-4-methyl-quinazolin-2-ylamino)-5-oxo-1,5-dihydro-imidazol-4--
ylidene]-acetic acid Compound M9 Formula V(i) ##STR00081##
3-[2-(2,4-Dimethyl-thiazol-5-yl)-2-oxo-ethylidene]-3,4-dihydro-benzo[1,4]-
oxazin-2-one Compound M10 Formula V(j) ##STR00082##
(E)-2,4-dichloro-6-((2-hydroxy-3,5-dimethylphenylimino)methyl)phenol
Compound M11 Formula V(k) ##STR00083##
N,N'-(ethane-1,2-diyl)bis(N-benzylfuran-2-carboxamide) Compound M14
Formula V(l) aka C3 aka C6 ##STR00084##
3-[2-(2-Methylsulfanyl-but-1-enyl)-naphtho[1,2-d]thiazol-1-yl]-propane-1--
sulfonic acid anion Compound M15 Formula V(m) aka C5 ##STR00085##
2-Hydroxy-benzoic acid 2-(4-ethoxy-phenyl)-2-oxo-ethyl ester
Compound M16 Formula V(n) ##STR00086## Compound M17 Formula V(o)
aka C3-6 ##STR00087## 2-Phenyl-thiazolo[4,5-c]quinoline Compound
M18 Formula V(p) ##STR00088##
8,9-Dimethoxy-5,5-dimethyl-5,6-dihydro-2H-1,2,4-triaza-benzo[e]azulen-3-o-
ne Compound M19 Formula V(q) ##STR00089##
4-[4-(3,4-Dimethoxy-phenyl)-thiazol-2-ylamino]-phenol Compound M20
Formula V(r) ##STR00090##
2-[1-(2-Hydroxy-phenylamino)-propylidene]-5-phenyl-cyclohexane-1,3-dione
Compound M21 Formula V(s) ##STR00091##
2-(4-Nitro-thiophen-2-yl)-2,3-dihydro-1H-benzoimidazole Compound
M22 Formula V(t) aka C3-5 ##STR00092##
2-(4-chlorophenyl)thiazolo[4,5-c]quinoline Compound M23 Formula
V(u) ##STR00093## 3-butyl-2-methyl-2,3-dihydrobenzo[d]thiazole
[0093] Each of the variables designated by, for example, R, X, Y,
m, and n in any of the formulas disclosed herein can be selected
independently. While we tend to use the term "compound(s)", we may
also use terms like "agent(s)" to refer to the molecules described
herein.
DEFINITIONS
[0094] The following definitions apply to the terms used in
connection with any of the formulas herein. The term "halo" or
"halogen" refers to any radical of fluorine, chlorine, bromine or
iodine. The terms "cyclylalkyl" and "cycloalkyl" refer to saturated
monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon
groups. Any atom can be substituted by, for example, one or more
substituents. Cycloalkyl groups can contain fused rings, which
share a common carbon atom. Cycloalkyl moieties can include, for
example, cyclopropyl, cyclohexyl, methylcyclohexyl (the point of
attachment to another moiety can be either the methyl group or a
cyclohexyl ring carbon), adamantyl, and norbornyl.
[0095] The term "alkenyl" refers to a straight or branched
hydrocarbon chain containing 2-20 carbon atoms and having one or
more double bonds. Any atom can be substituted by one or more
substituents. Alkenyl groups can include, for example, allyl,
propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups. One of the
double bond carbons can optionally be the point of attachment of
the alkenyl substituent. The term "alkynyl" refers to a straight or
branched hydrocarbon chain containing 2-20 carbon atoms and having
one or more triple bonds. Any atom can be substituted by one or
more substituents. Alkynyl groups can include, for example,
ethynyl, propargyl, and 3-hexynyl. One of the triple bond carbons
can optionally be the point of attachment of the alkynyl
substituent.
[0096] The term "alkoxy" refers to an --O-alkyl radical. The term
"heterocyclyl" refers to a monocyclic, bicyclic, tricyclic or other
polycyclic ring system having: 1-4 heteroatoms if monocyclic; 1-8
heteroatoms if bicyclic; or 1-10 heteroatoms if tricyclic. The
heteroatoms can be O, N, or S (e.g., carbon atoms and 1-4, 1-8, or
1-10 heteroatoms of N, O, or S if monocyclic, bicyclic, or
tricyclic, respectively). The heteroatom can optionally be the
point of attachment of the heterocyclyl substituent. Any atom can
be substituted, by, for example, one or more substituents. The
heterocyclyl groups can contain fused rings, which share a common
carbon atom. Heterocyclyl groups can include, for example,
tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino,
pyrrolinyl, and pyrrolidinyl.
[0097] The term "heteroaryl" refers to an aromatic monocyclic,
bicyclic, tricyclic, or other polycyclic hydrocarbon groups having:
1-4 heteroatoms if monocyclic; 1-8 heteroatoms if bicyclic; or 1-10
heteroatoms if tricyclic. The heteroatoms can be O, N, or S (e.g.,
carbon atoms and 1-4, 1-8, or 1-10 heteroatoms of N, O, or S if
monocyclic, bicyclic, or tricyclic, respectively). Any atom can be
substituted by, for example, one or more substituents. Heteroaryl
groups can contain fused rings, which share a common carbon atom.
Heteroaryl groups include pyridyl, thienyl, furanyl, imidazolyl,
and pyrrolyl.
[0098] The term "oxo" refers to an oxygen atom, which forms a
carbonyl when attached to carbon, an N-oxide when attached to
nitrogen, and a sulfoxide or sulfone when attached to sulfur.
[0099] The term "substituents" refers to a group "substituted" on,
for example, an alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl,
heteroaralkyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl,
aryl, or heteroaryl group at any atom of that group. In one aspect,
the substituents on a group are independently any one single, or
any subset of, the aforementioned substituents. In another aspect,
a substituent may itself be substituted with any one of the above
substituents.
[0100] Salts, Solvates, and Other Variants:
[0101] The invention also encompasses pharmaceutically acceptable
salts or solvates of a compound of any of Formulas I-IV or
V(a)-V(u), and prodrugs, metabolites, structural analogs, and other
pharmaceutically useful variants thereof. These other variants may
be, for example, a complex containing the compound and a targeting
moiety, as described further below, a second therapeutic agent or a
detectable marker (e.g., the compound may incorporate a radioactive
isotope or be joined to a fluorescent compound). When in the form
of a prodrug, a compound may be modified in vivo (e.g.,
intracellularly) after being administered to a patient or to a cell
in culture. The modified compound (i.e., the processed prodrug) may
be identical to a compound described herein and will be
biologically active or have enough activity to be clinically
beneficial. The same is true of a metabolite; a given compound may
be modified within a cell and yet retain sufficient biological
activity to be clinically useful.
[0102] A salt, for example, can be formed between an anion and a
positively charged substituent (e.g., amino) on a compound
described herein. Suitable anions include chloride, bromide,
iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate,
trifluoroacetate, and acetate. Likewise, a salt can also be formed
between a cation and a negatively charged substituent (e.g.,
carboxylate) on a compound described herein. Suitable cations
include sodium ion, potassium ion, magnesium ion, calcium ion, and
an ammonium cation such as tetramethylammonium ion.
[0103] Examples of prodrugs include esters and other
pharmaceutically acceptable derivatives, which, upon administration
to a subject, are capable of providing active compounds. A
"prodrug" may be any pharmaceutically acceptable salt, ester, salt
of an ester, or other derivative of a compound of this invention
(for example an imidate ester of an amide), which, upon
administration to a recipient, is capable of providing (directly or
indirectly) a compound of this invention. Particularly favored
derivatives and prodrugs are those that increase the
bioavailability of the compounds of this invention when such
compounds are administered to a mammal (e.g., by allowing an orally
administered compound to be more readily absorbed into the blood)
or which enhance delivery of the parent compound to a biological
compartment (e.g., the brain or lymphatic system) relative to the
parent species. Preferred prodrugs include derivatives where a
group which enhances aqueous solubility or active transport through
the gut membrane is appended to the structure of formulae described
herein.
[0104] The compounds of this invention may be modified by appending
appropriate functionalities to enhance selected biological
properties (e.g., targeting to a particular tissue). Such
modifications are known in the art and include those which increase
biological penetration into a given biological compartment (e.g.,
blood, lymphatic system, central nervous system), increase oral
availability, increase solubility to allow administration by
injection, alter metabolism and alter rate of excretion.
[0105] The compounds of the invention may contain one or more
asymmetric centers and thus occur as racemates and racemic
mixtures, single enantiomers, individual diastereomers and
diastereomeric mixtures. All such isomeric forms of these compounds
are expressly included in the present invention. The compounds of
this invention may also contain linkages (e.g., carbon-carbon
bonds) wherein bond rotation is restricted about that particular
linkage (e.g., restriction resulting from the presence of a ring or
double bond). Accordingly, all cis/trans and E/Z isomers are
expressly included in the present invention. The compounds of this
invention may also be represented in multiple tautomeric forms, in
such instances, the invention expressly includes all tautomeric
forms of the compounds described herein, even though only a single
tautomeric form may be represented (e.g., alkylation of a ring
system may result in alkylation at multiple sites, the invention
expressly includes all such reaction products). All such isomeric
forms of such compounds are expressly included in the present
invention. All crystal forms of the compounds described herein are
expressly included in the present invention.
[0106] As noted, the compounds of the invention may be mixed with
or joined to a detectable marker or tag, to another therapeutic
agent, or to a moiety that facilitates passage across the
blood-brain barrier (see below).
[0107] Packaged Products:
[0108] The compounds described herein can be packaged in suitable
containers labeled, for example, for use as a therapy to treat a
disease or disorder characterized by protein aggregation or another
form of undesirable association. The containers can include the
compound (i.e., the diagnostic/prophylactic/therapeutic agent) and
one or more of a suitable stabilizer, carrier molecule, flavoring,
and/or the like, as appropriate for the intended use. Accordingly,
packaged products (e.g., sterile containers containing one or more
of the compounds described herein and packaged for storage,
shipment, or sale at concentrated or ready-to-use concentrations)
and kits, including at least one compound of the invention and
instructions for use, are also within the scope of the invention. A
product can include a container (e.g., a vial, jar, bottle, bag, or
the like) containing one or more compounds of the invention and a
legend (e.g., a printed label or insert or other medium describing
the product's use (e.g., an audio- or videotape)). The legend can
be associated with the container (e.g., affixed to the container)
and can describe the manner in which the compound therein should be
administered (e.g., the frequency and route of administration),
indications therefore, and other uses. The compounds can be ready
for administration (e.g., present in dose-appropriate units), and
may include a pharmaceutically acceptable adjuvant, carrier or
other diluent and/or an additional therapeutic agent.
Alternatively, the compounds can be provided in a concentrated form
with a diluent and instructions for dilution.
[0109] Stability:
[0110] Combinations of substituents and variables envisioned by
this invention are only those that result in the formation of
stable compounds. The term "stable," as used herein, refers to
compounds that are stable enough to allow manufacture and that
maintain their integrity for a sufficient period of time to be
useful for the purposes detailed herein (e.g., therapeutic or
prophylactic administration to a subject).
[0111] Purity:
[0112] In one aspect, the invention features substantially pure
preparations of the compounds described herein or combinations
thereof. A naturally occurring compound is substantially pure when
it is separated to some degree from the compound(s) or other
entities (e.g., proteins, fats, or minerals) it is associated with
in nature. For example, a naturally occurring compound described
herein is substantially pure when it has been separated from 50%,
60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of
the compound(s) or other moieties it is associated with in nature.
These degrees of purity are not limiting, however, the compounds of
the invention need be only as pure as necessary to cause a
beneficial clinical result and to conform with good manufacturing
practices. While the compounds of the invention may be naturally
occurring and may be purified using conventional techniques, they
may also be non-naturally occurring and may be synthesized
(naturally occurring compounds can be synthesized as well; see
below). Compounds prepared by chemical synthesis are substantially
pure, as are compounds that have been separated from a library of
chemical compounds. A substantially pure compound may be one that
is separated from all the other members of the compound library or
it may be one that has been separated to a limited extent (e.g., it
may remain associated with a limited number (e.g., 1, 2, 3, 4, or
5-10) of other members of the library. As noted, while more than
one of the agents described herein can be formulated within the
same composition, and while the compositions can also include a
second therapeutic agent (as described herein), the pharmaceutical
compositions of the invention expressly exclude extremely
heterogeneous mixtures, such as libraries (e.g., combinatorial or
compound libraries, including those that contain synthetic and/or
natural products, and custom analog libraries, which may contain
compounds based on a common scaffold). Such libraries can include
hundreds or thousands of distinct compounds or random pools
thereof. Whether or not commercially available, such libraries are
excluded from the meaning of a pharmaceutical composition.
[0113] Formulations:
[0114] Regardless of their original source or the manner in which
they are obtained, the compounds of the invention can be formulated
in accordance with their use. For example, the compounds can be
formulated within compositions for application to cells in tissue
culture or for administration to a patient. For example, the
compounds can be mixed with a sterile, pharmaceutically acceptable
diluent (such as normal saline). As noted below, and as known in
the art, the type of diluent can vary depending upon the intended
route of administration. The resulting compositions can include
additional agents, such as preservatives. The compounds may also be
applied to a surface of a device (e.g., a catheter) or contained
within a pump, patch, or other drug delivery device. The
therapeutic agents of the invention can be administered alone, or
in a mixture, in the presence of a pharmaceutically acceptable
excipient or carrier (e.g., physiological saline). The excipient or
carrier is selected on the basis of the mode and route of
administration. Suitable pharmaceutical carriers, as well as
pharmaceutical necessities for use in pharmaceutical formulations,
are described in Remington's Pharmaceutical Sciences (E. W.
Martin), a well-known reference text in this field, and in the
USP/NF (United States Pharmacopeia and the National
Formularly).
[0115] A pharmaceutical composition (e.g., a composition containing
a therapeutic agent or the DNA molecule encoding it) is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include oral, rectal, and
parenteral, for example, intravenous, intradermal, and
subcutaneous, transdermal (topical), and transmucosal
administration. Variants of the compounds described herein,
formulated to cross the blood-brain barrier, are described
below.
[0116] Diagnostic, Prophylactic and Therapeutic Use:
[0117] The compounds identified by the methods described herein
(which may also be referred to herein as "therapeutic agents") may
be used to treat a variety of disorders, including Huntington's
disease. For example, the compounds described herein can be
included as therapeutic agents in pharmaceutical compositions to
treat HD and other conditions described herein that are mediated by
(or characterized by) protein-protein association. The therapeutic
agents of the invention can be used to treat any disease, disorder,
or condition that results from an abnormal or undesirable
association between two polypeptides (like or unlike). For example,
the therapeutic agents of the invention can be used to treat
neurodegenerative disorders (e.g., Huntington's Disease can be
inhibited by inhibiting the association of huntingtin proteins) and
disorders in which polyglutamine-containing transcription factors
or coactivators are undesirably active (e.g., disorders (e.g.,
cancers) associated with homodimerization of jun or hexamerization
of p53).
[0118] Treating a subject can encompass administration of a
therapeutic agent as a prophylactic measure to prevent the
occurrence of disease or to lessen the severity or duration of the
symptoms associated with the disease. Physicians and others of
ordinary skill in the art routinely make determinations as to the
success or failure of a treatment. Treatment can be deemed
successful despite the fact that not every symptom of the disease
is totally eradicated. Treatment can also be deemed successful
despite side-effects.
[0119] It is usual in the course of developing a therapeutic agent
that tests of that agent in vitro or in cell culture are followed
by tests in animal models of human disease, and further, by
clinical trials for safety and efficacy in humans. Accepted animal
models for many diseases are now known to those of ordinary skill
in the art. For example, therapeutic agents of the present
invention can be screened in a Drosophila model of
neurodegeneration as well as in more evolutionarily advanced
animals.
[0120] Mammalian models for Huntington's disease are available. To
generate similar animal models, a homolog of the
aggregation-disposed polypeptide is first cloned from the genome of
the selected mammal using standard techniques. For example, the
sequence can be amplified by PCR or obtained by screening an
appropriate library under conditions of low stringency (as
described, e.g., in Sambrook et al. supra.). Subsequently,
trinucleotide repeats can be introduced into the gene by molecular
cloning and mutagenesis techniques. For example, in a HD model, CAG
repeats can be introduced in the HD gene. The site for insertion of
the repeat sequence can be located by alignment of the cDNA from
the desired mammal with the human cDNA for the aggregation-disposed
protein. The modified gene with artificially expanded repeats can
be reintroduced into the mammal using standard methods for
transgenesis.
[0121] Methods for generating transgenic mice are routine in the
art (See, e.g., Hogan et al., Manipulating the Mouse Embryo, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, New York
(1994)). As an example, a mouse bearing a transgene comprising the
HD gene and expanded CAG repeats has symptoms similar to the human
disease. Murine symptoms can include hyperactivity, circling,
abnormal gait, tremors, learning deficits, hypoactivity, and
hypokinesis. Neuropathological symptoms include general brain
atrophy, progressive striatal atrophy, neuropil aggregates,
inclusions in the striatum, reduced dendritic spines, cell loss in
the cortex, and striatum.
[0122] Any of these behavioral or physiological deficits can be
assessed in order to determine the efficacy of a given therapeutic
agent of the invention. For example, the agent can be administered
to a transgenic mouse model, generated as described above. The
symptoms of a treated mouse can be compared to untreated mice at
various times during and after treatment. In addition, treated and
untreated mice can be sacrificed at various intervals after
treatment, and the neuropathology of the brain can be analyzed.
Thus, the efficacy of the treatment can be evaluated readily by
comparing the behavioral symptoms, neuropathological symptoms, and
clinical symptoms of treated and untreated mice.
[0123] In specific embodiments, the compositions of the present
invention can be administered to a subject having any disease
mediated by (or characterized by) an abnormal or unwanted
association of one protein with another. Examples include
immunoglobulin light chain amyloidosis, HD, Parkinson's disease,
adult-onset diabetes, cirrhosis (e.g., cirrhosis of the liver),
emphysema, or a prion disease, such as Creutzfeldt-Jakob disease.
Other conditions that can be treated or prevented with one or more
of the compounds of the present invention include amyotrophic
lateral sclerosis, dentatorubral pallidoluysian atrophy, spinal
bulbar muscular atrophy (SBMA; also known as Kennedy's disease),
any of the several types of spinocerebellar ataxias (e.g., SCA1,
SCA2, SCA6, SCA7 and Machado-Joseph disease (MJD/SCA3)),
dentatorubral-pallidoluysian atrophy, and disorders in which
polyglutamine-containing transcription factors or coactivators are
undesirably active (e.g., disorders associated with
homodimerization of jun or hexamerization of p53). For example, a
subject may have been diagnosed as having, or at risk for
developing, a carcinoma (e.g., breast cancer), amyloidosis, a
myeloma, kuru, a neuroblastoma, cystic fibrosis, an
alpha-1-antitrypsin deficiency disease, or a disorder with a
similar underlying cellular basis (i.e., an association with
undesirable (e.g., excessive or insufficient) protein-protein
aggregation, dimerization, or other interaction).
[0124] The synuclein proteins (alpha, beta and gamma synuclein)
have been implicated in Parkinson's disease and breast cancer, and
are targets for the compositions of the invention. As targets,
these proteins can be incorporated in the screening assays
described herein. Proteins such as amyloid light chains and
amyloid-associated proteins, which are associated with amyloidosis,
can also be targeted by the compositions and methods of the
invention. Other aggregation- or association-disposed polypeptides
include: mutant transthyretin, which is associated with familial
amyloid polyneuropathies; beta2 microglobulin, aggregation of which
causes complications during chronic renal dialysis; immunoglobulin
light chain, which is associated with multiple myelomas and various
other B-cell proliferations; prion proteins, which cause spongiform
encephalopathies such as Creutzfeldt-Jakob disease and kuru in
humans; cystic fibrosis transmembrane conductance regulator (CFTR),
which is a hallmark of cystic fibrosis; p53, which has been
observed to aggregate in some neuroblastomas, carcinomas, and
myelomas; and alpha-1-antitrypsin, which aggregates in patients
with alpha-1-antitrypsin deficiency disease.
[0125] Subjects who are treated with the compounds of the invention
may have been diagnosed with any disease characterized by aberrant
or undesirable association between proteins, whether that
association occurs to a greater or lesser extent than is normal
(in, e.g., a healthy patient) or desirable. Alternatively, the
subject may be at risk for developing these disorders. For example,
a subject may have a family history or a genetic mutation or
element (e.g., an expanded trinucleotide repeat) that contributes
to the development of disease. Human subjects, in consult with
their physicians and/or other health care professionals, can decide
whether their risk is great enough to undergo preventative care (as
is the case for any prophylactic treatment or procedure). While the
subjects of the preventative and/or therapeutic regimes described
herein may be human, the compounds and compositions of the
invention can also be administered to non-human subjects.
[0126] The prophylactic and therapeutic methods can be carried out
by administering to the subject a pharmaceutical composition
containing a therapeutically effective amount of one or more of the
compounds described herein. While a single compound may be
effective, the invention is not so limited. A subject can be
treated with multiple compounds, administered simultaneously or
sequentially. For example, a subject can be treated with one or
more of the compounds described herein and, optionally, a
chemotherapeutic agent, an analgesic, a bronchodilator, levodopa or
a similar medication. The combination therapy will, of course,
depend on the disorder being treated. Where a compound of the
invention is administered to treat a patient with a cancer, it may
be combined with a known chemotherapeutic agents used to treat that
type of cancer; where a compound of the invention is administered
to treat a patient with Parkinson's disease, it may be combined
with a medication to increase dopamine levels in the brain; and so
forth.
[0127] Compounds that mediate association between proteins can also
be used to diagnose diseases characterized by protein aggregation
(or, as noted above, other undesirable interaction (e.g.,
dimerization or complex formation)). These methods can be carried
out by providing a biological sample from a patient suspected of
having a disease associated with an abnormal or undesirable
association between proteins; exposing the sample to a compound of
the invention; and determining whether the compound modulates the
association of proteins within the sample. The compound can be one
that is known to interact directly with a primary target or one
that modulates protein-protein interaction by acting upstream from
the primary target. The compound can also be one that is known to
interact with proteins in the context of the suspected disease. For
example, a compound that is known to inhibit the aggregation of
Huntingtin can be used to diagnose a patient suspected of having
HD. The sample will be exposed to the compound for a time and under
conditions (e.g., physiological conditions of temperature and pH)
sufficient to permit the compound to affect proteins within the
sample (e.g., Huntingtin, tau, or A.beta. proteins within cells
within the sample). The diagnostic methods can be carried out
before, after, or in conjunction with other diagnostic tests, and
their results can inform the subject's treatment regime. For
example, where a compound is found to modulate the aggregation of
Huntingtin proteins in a sample obtained from a patient suspected
of having HD, that compound may then be used to treat the
patient.
[0128] The blood-brain barrier is an obstacle for the delivery of
drugs from circulation in the bloodstream to the brain. The
endothelial cells of brain capillaries are connected by tight
intercellular junctions, which inhibit the passive movement of
compounds out of the blood plasma into the brain. These cells also
have reduced pinocytic vesicles in order to restrict the
indiscriminate transport of materials intracellularly. These
features of the brain regulate the exchange of materials between
plasma and the central nervous system. Both active and passive
transport mechanisms operate to exclude certain molecules from
traversing the barrier. For example, lipophilic compounds are more
permeable to the barrier than hydrophilic compounds (Goldstein et
al., Scientific American 255:74-83, 1996; Pardridge et al.,
Endocrin. Rev. 7:314-330, 1996).
[0129] However, the blood-brain barrier must also allow for the
selective transport of desired materials into the brain in order to
nourish the central nervous system and to remove waste products.
The mechanisms by which this is accomplished can provide the means
for supplying the therapeutic agents described herein.
[0130] The compositions of the invention can be delivered to the
CNS following conjugation with other compounds as follows (and as
described further in, for example, U.S. Pat. No. 5,994,392). In one
instance, polar groups on a compound are masked to generate a
derivative with enhanced lipophilic qualities. For example,
norepinephrine and dopamine have been modified with diacetyl and
triacetyl esters to mask hydroxyl groups. An implementation of this
strategy has been previously used to create a pro-drug derivative
of dopamine (see U.S. Pat. No. 5,994,392). The modified drugs are
generally referred to as pro-drugs, and the compounds of the
invention encompass those described herein in which polar groups
are masked. This method may have the additional advantage of
providing an inactive species of the compound in the general
circulation. After crossing the blood-brain barrier, enzymes
present in the central nervous system are able to hydrolyze the
linkages (e.g., ester linkages), thereby unmasking the compound and
liberating the active drug. Thus, compounds of the invention can be
chemically modified to create pro-drugs by, e.g., conjugation to a
lipophilic moiety or carrier. A compound or a variant thereof
having at least one free hydroxyl or amino group can be coupled to
a desired carrier (e.g., a fatty acid, a steroid, or another
lipophilic moiety).
[0131] More specifically, and for example, the hydroxyl groups can
first be protected with acetonide. The protected agent is then
reacted with the desired carrier in the presence of a
water-extracting compound (e.g., dicyclohexyl carbodiiamide), in a
solvent (e.g., dioxane, tetrahydrofurane), or N,N dimethylformamide
at room temperature. The solvent is then removed, and the product
is extracted using methods routinely used by those of ordinary
skill in the art. Amine groups can be coupled to a carboxyl group
in the desired carrier. An amide bond is formed with an acid
chloride or low carbon ester derivative of the carrier. Bond
formation is accompanied by HCl and alcohol liberation. Alcohol
groups on the compound can be coupled to a desired carrier using
ester bonds by forming an anhydride derivative, i.e. the acid
chloride derivative, of the carrier. One of ordinary skill in the
art of chemistry will recognize that phosphoramide, sulfate,
sulfonate, phosphate, and urethane couplings are also useful for
coupling a therapeutic agent (e.g., a compound described herein) to
a desired carrier. A useful and adaptable method for lipidation of
antibodies is described by Cruikshank et al. (J. Acquired Immune
Deficiency Syndromes and Human Retrovirology 14:193, 1997).
[0132] Procedures for delivering therapeutic agents (or
"compounds") of the invention to the CNS can also be carried out
using the transferrin receptor as described, for example, in U.S.
Pat. No. 6,015,555. To implement this procedure, the agents are
conjugated to a molecule that specifically binds to the transferrin
receptor (e.g., an antibody or antigen-binding fragment thereof, or
transferrin). Methods for obtaining antibodies against the
transferrin receptor and for coupling the antibodies to a desired
compound are also described in U.S. Pat. No. 6,015,555.
[0133] Monoclonal antibodies that specifically bind to the
transferrin receptor include OX-26, T58/30, and B3/25 (Omary et
al., Nature 286:888-891, 1980), T56/14 (Gatter et al., J. Clin.
Path. 36:539-545, 1983), OKT-9 (Sutherland et al., Proc. Natl.
Acad. Sci. USA 78:4515-4519, 1981), L5.1 (Rovera, Blood 59:671-678,
1982) and 5E-9 (Haynes et al., J. Immunol. 127:347-351, 1981). In
one embodiment, the monoclonal antibody OX-26 is used. The antibody
of choice can be an Fab fragment, a F(ab').sub.2 fragment, a
humanized antibody, a chimeric antibody, or a single chain
antibody.
[0134] The antibody to the transferrin receptor is conjugated to a
desired compound with either a cleavable or non-cleavable linker.
The preferred type of linker can be determined without undue
experimentation by making cleavable and non-cleavable conjugates
and assaying their activity in, for example, an in vitro or cell
culture assay described herein. The conjugates can be further
tested in vivo (e.g., in a animal model of a disease of interest).
Examples of chemical systems for generating non-cleavable linkers
include the carbodiimmide, periodate, sulfhydryl-maleimide, and
N-succinimidyl-3-(2-puridyldithio) propionate (SPDP) systems.
Carbodiimide activates carboxylic acid groups, which then react
with an amino group to generate a noncleavable amide bond. This
reaction may be especially useful for coupling two proteins.
Periodate is used to activate an aldehyde on an oligosaccharide
group such that it can react with an amino group to generate a
stable conjugate. Alternatively, a hydrazide derivative of the
desired compound can be reacted with the antibody oxidized with
periodate. Sulfhydryl-maleimide and SDPD use sulfhydryl chemistry
to generate non-cleavable bonds. SDPD is a heterobifunctional
crosslinker that introduces thiol-reactive groups. In the
sulfhydryl-maleimide system, an NHS ester (e.g.,
gamma-maleimidobutyric acid NHS ester) is used to generate
maleimide derivative, for example, of a protein drug or antibody.
The maleimide derivative can react with a free sulfhydryl group on
the other molecule.
[0135] Cleavable linkers are also useful. Cleavable linkers include
acid labile linkers such as cis-aconitic acid, cis-carboxylic
alkadienes, cis-carboxylic alkatrienes, and polypeptide-maleic
anhydrides (see U.S. Pat. No. 5,144,011).
[0136] In one embodiment, the compound is a compound having one of
the structures shown in Tables 1-5. Such a compound can be
covalently attached to an antibody specific for the transferrin
receptor. In one embodiment, use of a single chain antibody is
preferred in order to facilitate covalent fusion with the
therapeutic agent.
[0137] The targeting antibody can be linked covalently to the
therapeutic agent (or "compound") of the invention. A protease
recognition site can be included in the linker if cleavage of the
antibody is required after delivery.
[0138] The efficacy of strategies to deliver a desired compound
across the blood-brain barrier can, of course, be monitored. The
desired compound, conjugated for delivery across the blood-brain
barrier, is administered to a test mammal (e.g., a rat, a mouse, a
non-human primate, a cow, a dog, a rabbit, a cat, or a sheep). One
of ordinary skill in the art will, however, recognize that the
permeability of the blood-brain barrier varies from species to
species, with the human blood-brain barrier being the least
permeable. The mode of administration can be the same as the
desired mode of treatment (e.g., intravenous). For a comprehensive
analysis, a set of test mammals is used. The test mammals are
sacrificed at various times after the agent is administered and are
then perfused through the heart with, e.g., Dulbecco's
phosphate-buffered saline (DPBS) to clear the blood from all
organs. The brain is removed, frozen in liquid nitrogen, and
subsequently sectioned in a cryostat. The sections are placed on
glass microscope slides. The presence of the desired agent is then
detected in the section, for example with an antibody, or by having
administered a radiolabeled or otherwise tagged compound (such
labeled therapeutic compounds as described above). Detection is
indicative of the compound having successfully traversed the
blood-brain barrier. If a method of enhancing the compounds
permeability to the blood-brain barrier is being assessed, then the
amount of the agent detected in a brain section can be compared to
the amount detected in a brain section from an animal treated with
the same compound without the enhancing method.
[0139] The terms "blood-brain barrier permeant" or "blood-brain
barrier permeable" are qualities of a compound for which the ratio
of a compound's distribution at equilibrium in the cerebrospinal
fluid (CSF) relative to its distribution in the plasma (CSF/plasma
ratio) is greater than at least (or about) 0.01, 0.02, 0.05, or
0.1. While lower ratios are generally preferred, any ratio that
allows a compound to be used clinically is acceptable.
[0140] To facilitate targeting to a polypeptide of interest (e.g.,
to a Huntingtin or jun protein), the compound (e.g., a compound
conforming to any of Formulas I IV or V(a) V(u)) can include a
moiety that specifically binds to the target protein. For example,
a compound conforming to Formula I can be joined to an antibody or
an antigen-binding portion thereof (e.g., a single chain antibody)
that specifically binds the target protein (e.g., Huntingtin or
jun). Targeting moieties are described further below.
[0141] A therapeutic vector can be administered to a subject, for
example, by intravenous injection, by local administration (see
U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g.,
Chen et al., Proc. Natl. Acad. Sci. USA 91:3054-3057, 1994). The
compound can be further formulated, for example, to delay or
prolong the release of the active agent by means of a slow release
matrix.
[0142] Regardless of whether or not the compound is to cross the
blood-brain barrier, it can be conjugated to a targeting agent that
facilitates interaction with a target protein (e.g., Huntingtin or
jun). As noted, the compound can be directly or indirectly joined
to an antibody (e.g., a single chain antibody) or an
antigen-binding fragment thereof that specifically binds the target
protein.
[0143] An appropriate dosage of the therapeutic agents of the
invention must be determined. An effective amount of a therapeutic
compound is the amount or dose required to ameliorate a symptom of
a disorder associated with protein aggregation, such as a disorder
characterized by a trinucleotide repeat expansion. Determining the
amount required to treat a subject is routine to one of ordinary
skill in the art (e.g., a physician, pharmacist, or researcher).
First, the toxicity and therapeutic efficacy of an agent (i.e. a
tri-domain molecule) is determined. Routine protocols are available
for determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population) in non-human animals. The therapeutic
index is measured as the ratio of the LD.sub.50/ED.sub.50.
Compounds, formulations, and methods of administration with high
therapeutic indices are preferable as such treatments have little
toxicity at dosages that provide high efficacy. Compounds with
toxic or undesirable side effects can be used, if means are
available to deliver the compound to the affected tissue, while
minimizing damage to unaffected tissue.
[0144] In formulating a dosage range for use in humans, the
effective dose of a therapeutic agent can be estimated from in
vitro cell studies and in vivo studies with animal models. If an
effective dose is determined for ameliorating a symptom in cell
culture, a dose can be formulated in an animal in order to achieve
a circulating plasma concentration of sodium butyrate that falls in
this range. An exemplary dose produces a plasma concentration that
exceeds the IC.sub.50 (i.e., the concentration of the test compound
which achieves a half-maximal inhibition of symptoms) as determined
in cell culture assays. The circulating plasma concentration can be
determined, for example, by administering a labeled therapeutic
composition to the test animal, obtaining a blood sample, and
quantitating the amount of labeled compound present at various
times after administration.
[0145] An appropriate daily dose of a therapeutic agent can be
between about 0.1 mg/kg of body weight to about 500 mg/kg, or
between about 1 mg/kg to about 100 mg/kg. The dose can be adjusted
in accordance with the blood-brain barrier permeability of the
compound. For example, a therapeutic compound can be administered
at a dosage of 50 mg/kg to 100 mg/kg in order to treat the brain.
The dose for a patient can be optimized while the patient is under
care of a physician, pharmacist, or researcher. For example, a
relatively low dose of a tri-domain therapeutic can be administered
initially. The patient can be monitored for symptoms of the
disorder being treated (e.g., HD). The dose can be increased until
an appropriate response is obtained. In addition, the specific dose
level for any particular subject can vary depending on the age,
body weight, general health, gender, and diet of the subject, the
time of administration, the route of administration, the rate of
excretion, and other drugs provided in combination.
[0146] As occurs in the course of all drug development, optimal
treatment regimes will emerge through further modeling and clinical
trials. It may be, for example, that a patient will receive a
combination of compounds that act synergistically to inhibit
polypeptide association by the same or different mechanisms of
action. Combination therapies may also rely on administration of a
compound that interferes with gene transcription (e.g., a small
molecule or a nucleic acid that mediates RNAi) and a compound that
facilitates degradation of any remaining unwanted
polypeptide-containing complexes.
[0147] The efficacy of a dose of any therapeutic agent can be
determined in a subject. For example, the subject can be monitored
for clinical symptoms, for example, a symptom of a trinucleotide
repeat disease, such as a symptom of HD. Behavioral symptoms of HD
include irritability, apathy, lethargy, depression, hostile
outbursts, loss of memory and/or judgment, loss of ability to
concentrate, anxiety, slurred speech, difficulty swallowing and/or
eating, and inability to recognize persons. Clinical symptoms of HD
include loss of coordination, loss of balance, inability to walk,
uncontrolled movements of the fingers, feet, face, and/or trunk,
rapid twitching, tremors, chorea, rigidity, and akinesia (severe
rigidity).
[0148] Methods of Making:
[0149] The compounds of the invention or biologically active
variants thereof (e.g., salts) may be synthesized in vitro,
produced in vivo (e.g., produced within the body (e.g.,
intracellularly) following administration to a patient), or
produced following application to a cell in culture. Accordingly,
the present invention features methods of making the compounds and
compositions of the present invention. The compounds can be
synthesized using routine techniques known to one of ordinary skill
in the art. For example, the compounds can be made by providing a
starting compound or intermediate and reacting the compound or
intermediate with one or more chemical reagents in one or more
steps to produce a compound described herein (e.g., a compound of
any of Formulas I-IV or V(a)-V(u)).
[0150] Some of the compounds described herein can be obtained from
commercial sources. As noted, others can be synthesized by
conventional methods using commercially available starting
materials and reagents. The compounds described herein can be
separated from a reaction mixture and further purified by a method
such as column chromatography, high-pressure liquid chromatography,
or recrystallization. As can be appreciated by one of ordinary
skill in the art, further methods of synthesizing the compounds of
the formulae herein are available. Additionally, the various
synthetic steps may be performed in an alternate sequence or order
to give the desired compounds. Synthetic chemistry transformations
and protecting group methodologies (protection and deprotection)
useful in synthesizing the compounds described herein are known in
the art and include, for example, those such as described in R.
Larock, Comprehensive Organic Transformations, VCH Publishers
(1989); T. W. Greene and P. G. M. Wuts, Protective Groups in
Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); 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), and
subsequent editions thereof. Techniques useful for the separation
of isomers, for example, stereoisomers are within skill of the art
and are described in Eliel, E. L.; Wilen, S. H.; Mander, L. N.
Stereochemistry of Organic Compounds, Wiley Interscience, NY, 1994.
For example compounds can be resolved via formation of
diasteromeric salts, for example, with a chiral base, for example,
(+) or (-) a-methylbenzylamine, or via high performance liquid
chromatography using a chiral column.
[0151] Platform and Scaffold Use:
[0152] In an alternate embodiment, the compounds described herein
may be used as platforms or scaffolds that may be utilized in
combinatorial chemistry techniques for preparation of derivatives
and/or chemical libraries of compounds. Such derivatives and
libraries of compounds have biological activity and are useful for
identifying and designing compounds possessing a particular
activity. Combinatorial techniques suitable for utilizing the
compounds described herein are known in the art as exemplified by
Obrecht, D. and Villalgrodo, J. M., "Solid-Supported Combinatorial
and Parallel Synthesis of Small-Molecular-Weight Compound
Libraries", Pergamon-Elsevier Science Limited (1998), and include
those such as the "split and pool" or "parallel" synthesis
techniques, solid-phase and solution-phase techniques, and encoding
techniques (see, for example, Czarnik, Curr. Opin. Chem. Bio. 1:60,
1997). Thus, one embodiment relates to methods of using the
compounds described herein for generating derivatives or chemical
libraries. The methods can be carried out by performing these, and
optionally additional, steps: (1) providing a body comprising a
plurality of wells; (2) providing one or more compounds identified
by methods described herein in each well (e.g., any of the
compounds of Formulas I-IV or V(a)-V(u); (3) providing an
additional one or more chemicals in each well, where the compound,
upon exposure to the chemical(s) may produce one or more products;
and (4) isolating the resulting one or more products from each
well. We may refer to the original compound as the "first" compound
and to the chemical as the "second" compound. The order in which
the first and second compounds are added to the wells can vary, and
the methods can be carried out in vitro or in cell culture. Lead
derivatives can be further tested in animal models.
[0153] In alternate embodiments, the methods of using the compounds
described herein for generating derivatives or chemical libraries
can be carried out using a solid support. These methods can be
carried out by, for example: (1) providing one or more of the
compounds described herein attached to a solid support; (2)
treating the one or more compounds identified by methods described
herein attached to a solid support with one or more additional
compounds or chemicals; (3) isolating the resulting one or more
products from the solid support. In these methods, "tags" or
identifier or labeling moieties may be attached to and/or detached
from the compounds described herein or their derivatives, to
facilitate tracking, identification or isolation of the desired
products or their intermediates. Such moieties are known in the art
and exemplary tags are noted above. The chemicals (or "second"
compound(s)) used in the aforementioned methods may include, for
example, solvents, reagents, catalysts, protecting group and
deprotecting group reagents, and the like. Examples of such
chemicals are those that appear in the various synthetic and
protecting group chemistry texts and treatises which are known in
the art and may be referenced herein.
[0154] Databases:
[0155] In one aspect, the invention includes cell-based and in
vitro assays (e.g., high throughput screens) that can be used with
essentially any compound collection. Following an assay, the result
can be recorded in a database, and such databases are also within
the scope of the present invention. For example, the invention
features a computer-readable database that includes a plurality of
records. Each record includes (a) a first field that includes
information reflecting the identity of an agent (e.g., an agent
within one of the types of libraries described herein) and (b) a
second field that includes information concerning the impact of the
agent on polypeptide association. Additional fields may include the
results of toxicity tests, dose-response tests, and the like. The
information contained with the fields can be obtained in any order
(e.g., the information reflecting protein association can be
obtained first). However, to help ensure the integrity of the
database, the information should be obtained independently (or
"blindly"). The database can also include a field comparing the
agent to a clinical outcome (e.g., an improvement in a sign or
symptom associated with Parkinson's disease, Huntington's disease,
cancer, or any of the other disorders described herein). The number
of records can be, but is not necessarily, great. For example, a
useful database can include at least 10, 25, 50, 100, 250, 500,
1000, 1500, 1800, 2000, or 2500 records.
[0156] The invention is further illustrated by the following
examples, which should not be construed as further limiting.
EXAMPLES
Example 1
Effect of Expanded Polyglutamine (polyQ) on Cell Growth and Protein
Aggregation
[0157] We expressed sequences encoding fragments of the huntingtin
gene having either an expanded region of glutamine residues (104Q)
or a normal length (25Q) in the yeast S. cerevisiae. We fused the
genes for 104Q and 25Q to the DNA sequence encoding green
fluorescent protein to allow microscopic detection of the
corresponding polypeptide in vivo. Both genes also contained a
short sequence corresponding to the FLAG-tag at the N-terminus. We
expressed the 104Q and 25Q transgenes in yeast under the regulation
of a Gall promoter. Upon transfer of the cells to a
galactose-containing medium, 104Q formed multiple aggregates (10-20
per cell) in the cytoplasm within 4-6 hours, while 25Q was soluble.
While 25Q-GFP had no effect on the growth rate of the cells, 104Q
expression caused a general cessation of growth. With time,
expression of 104Q strongly declined and yeast growth was partially
restored, but at a very low rate. The presence of FLAG-tag in the
104Q sequence may be critical for growth cessation.
Example 2
A Screen for Genes that Modulate the Toxicity of polyQ-Containing
Polypeptides and Formation of Inclusion Bodies
[0158] We performed a screen for yeast genes involved in polyQ
aggregation by identifying suppressors of the polyQ-dependent
growth defect. A number of mutant clones expressing 104Q formed
large colonies (i.e., clones in which the growth defect was
suppressed) and were selected for further analysis. In some of
these colonies, aggregation of 104Q was strongly inhibited, while
in others, aggregation was normal. Three clones that demonstrated
suppressed aggregation were analyzed. About 85% of the cells in
these clones did not have inclusion bodies at all, while about 15%
of the cells had one large aggregate. Notably, expression levels of
104Q in these mutants were much higher than in the parental wild
type, and reached the levels of expression of 25Q. Increased
expression of 104Q in the mutants closely correlated with increased
fluorescence. We identified mutations in these clones by
complementation analysis, which showed the mutations to be in the
HSP104 gene. Precise deletion of the gene hsp104 led to suppression
of the growth defect caused by 104Q, prevented aggregation of this
polypeptide, and allowed its high expression. Mutations in ssa1,
ssa2, and ydj 1 genes also affected 104Q aggregation but caused
very different phenotypes. Many more inclusion bodies were formed
in these mutants, but they were much smaller than in the wild type,
and the fraction of 104Q in these inclusion bodies was very low.
Similar to the hsp104 mutation, ssa1, ssa2, and ydj 1 mutations
cause a reduced formation of inclusion bodies and also suppressed
the growth defect caused by the extended polyQ domain. Based on
these data, we established a simple screen to identify mammalian
genes that inhibit formation of aggregates when overexpressed. An
expression library of mammalian genes from HeLa cells was
transfected into 104Q-GFP-expressing yeast cells and large clones
(i.e., those in which growth defect was suppressed), were selected.
Out of about 30,000 colonies screened, 21 colonies demonstrated
suppression of the growth defect. Many of the plasmids were found
to be "false positives" since they were unable to suppress the
growth defect after isolation and re-transforming into a different
104Q-expressing clone. Two plasmids (each present in several
selected clones) encoding mammalian genes partially suppressed 104Q
aggregation. These plasmids caused phenotypes similar to those of
cells carrying mutations in the Hsp70 or DnaJ genes. Sequencing of
these genes revealed that one encoded the chaperonin TCP1.alpha.,
and another encoded an unknown ORF. This genetic approach can
provide insight to the mechanisms driving formation of inclusion
bodies, and can identify potential targets for design of drugs that
affect aggregation of proteins, such as the polyQ-containing
proteins described above.
Example 3
Screening of Chemical Compound Libraries
[0159] We designed a screen for chemical compounds that can affect
protein aggregation. The screen was designed based on the
phenotypical differences between cells expressing 104Q and 25Q. The
phenotypes included difference in growth rate and in fluorescence
levels. We used microtiter plates to screen chemical libraries by
the described method. We grew yeast transformed with either the
104Q-, or the 25Q-encoding plasmids separately in liquid medium in
the presence of glucose, which inhibits polyQ expression. In the
mid-log phase of growth, we washed the cells to remove glucose and
resuspended them in a medium containing galactose, which induces
polyQ expression. We placed the cell suspensions in 96- or 348-well
plates and incubated them with aeration at 30.degree. C. for 20
hours. We then measured cell density and fluorescence using a plate
reader. Very little increase in the culture density and
fluorescence was seen with cells expressing 104Q, but both
parameters increased strongly for cells expressing 25Q. In general,
an approximately 3-5 fold difference in cell number, and an
approximately 20-30 fold difference in fluorescence was observed in
104Q and 25Q cultures. Compounds from a chemical library were added
to 104Q culture right after plating into the microtiter plates.
Compounds that enhanced cell growth or fluorescence were selected.
These compounds were then tested for the ability to reduce
aggregation of 104Q. Thirteen compounds were found to either
enhance growth or reduce aggregation of cells expressing 104Q, or
cause both effects. These compounds were then tested for the
ability to reduce aggregation of 104Q in cells of the
neuron-derived PC12 cell line. The compounds identified by this
method are leads for development of drugs for treatment of diseases
related to expansion of polyQ.
Example 4
Screening Method for Using an Epitope Tagged polyQ Peptide in a
Screen for Compounds that Inhibit Protein Aggregation
[0160] An epitope-tagged polyQ peptide can be used to screen for,
and thereby identify, compounds that modulate (e.g., inhibit)
protein aggregation or another type of association between
proteins. For example, cells can be plated on UV-treated coverslips
in a 2 cm 6-well plate and incubated overnight in 2 ml 10% serum
containing DME media. The next day, the cells are transiently
transfected with 1-2 .mu.g of DNA plasmid encoding a myc-tagged
polyQ polypeptide using the lipofection reagent, Transfectam.RTM.
(Promega, Madison, Wis.), following the Promega standard protocol.
After 48 hours, the cells are washed twice in PBS, fixed in 2%
formaldehyde for 10 minutes, treated for five minutes with 0.1%
Triton-X (to permeabilize the cells), then incubated in a
humidified chamber with PBS with 10% goat serum and 0.2% Tween-20.
Following incubation, the cells can be visualized with an anti-myc
fluorescent antibody (Cy3, rhodamine and FITC are commonly used
fluorophores, FLAG, myc and HA are common epitope tags); and
analyzed for inhibition of aggregation using high magnification
fluorescent microscopy. The examination, whether the assay is
configured as described here or as described elsewhere herein, can
be carried out by eye or automated. For example, a microscope stage
can be automatically moved (by, for example, a robotic device) so
that each well appears beneath the lens and is photographed. A
computer attached to the microscope can receive and analyze the
data.
Example 5
Screen to Identify Transcriptional Repressors and Activators, and
Compounds Facilitating Degradation of Extended polyQ
[0161] High sensitivity of R2/6 cell lines to chemical treatment
was used as a basis for a high-throughput screen of 30,000
compounds. The purpose of the screen was to identify
transcriptional repressors, such as cystamine and C9 (A33 in Table
1), transcriptional activators, and compounds facilitating
degradation of extended polyQ.
[0162] The compound C9 (aka C4-DAK) down-regulated expression of a
mutant Huntingtin transgene (htt) in ecdyson-based inducible PC12
cells and down-regulated expression of htt and SOD1 transgenes
(implicated in ALS) in transiently transfected HeLa cells. The
compound C9 (A33 in Table 1) has structural similarity to the
histone deacetylase (HDAc) inhibitor, scriptaid
(6-(1,3-Dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxyhexanam-
ide) (Su et al., Cancer Res. 60:3137, 2000; A5 in Table 1). We
observed the most dramatic effect on expression level by C9 and
scriptaid in the PC12 cell line 14A2/6 expressing the extended
polyQ mutant htt(104Q). The effect of C9 on the down-regulation of
polyQ expression, was similar to cystamine, a drug that has shown
efficacy in a mouse model for Huntington's Disease (HD). Cystamine
blocked protein aggregation and prolonged the survival of R2/6 HD
mice by an unknown mechanism. Both C9 and cystamine reversed the
stimulatory effect of the HDAc inhibitor, scriptaid, on expression
and aggregation in PC12 cells. 14A2/6 cells were at least 5-fold
more responsive to chemical treatment than other cell lines tested.
2.5 .mu.M of Scriptaid in a media caused dramatic overexpression of
a transgene, aggregation in 100% of the cells, and cell death of
14A2/6 cells. In other cell lines, the same concentration of
Scriptaid only modestly stimulated the expression of the transgene.
Both cystamine and C9 rescued PC12 cells from scriptaid-dependent
synthetic lethality, whereas the other blockers of aggregation
failed to reverse the effect of scriptaid on transgene expression
or PC12 cell death.
Example 6
A Screen for Small Molecule Inhibitors of Protein Aggregation
[0163] In the study that follows, we identified and characterized
small chemical molecules that inhibit protein association by
screening a library of 16,000 such molecules. We used a yeast-based
assay for aggregation, and selected nine structurally diverse
inhibitors. We found that four of these molecules suppressed
protein association well in mammalian cells in vivo but not in
vitro. This suggests that the molecules either depend on metabolic
conversion to become inhibitors or inhibit aggregation indirectly,
targeting cellular pathway(s) of protein aggregation that are
active in whole animals. In view of these four compounds, we
synthesized more than 100 structural analogs and tested them in
cell culture to select the compounds most effective in inhibiting
aggregation. We studied the effects of the most potent compounds on
polyQ aggregation in neurons in brain slices isolated from HD
transgenic mice and maintained in vitro.
[0164] For our high throughput screen, we developed a novel yeast
model of polyglutamine aggregation using an amino-terminal fragment
of mutant HD protein (Htt) containing extended polyglutamines
(103Q). This mutant efficiently aggregates in cells and causes
cytotoxicity.
[0165] For the compound screen, we engineered the Erg6 yeast strain
to express the "103Q" polypeptide tagged with EGFP under the
control of a GAL1 promoter. This strain, engineered as described
here, is within the scope of the present invention. The erg6
mutation inhibits ergosterol biosynthesis, which enhances membrane
fluidity and results in increased membrane permeability to a
variety of chemical compounds. The yeast culture was grown to
mid-logarithmic phase, shifted to galactose medium to induce 103Q
expression, placed in 96-well plates, and supplemented with
compounds. We used the optical density at 600 nm to monitor yeast
growth and fluorescence in FITC channel to monitor expression
levels of 103Q-GFP fusion polypeptides. From the screened
compounds, we selected chemical compounds that caused an increase
in growth and/or an increase in fluorescence. The ability of
pre-selected compounds to suppress 103Q aggregation was examined
microscopically, and we identified nine inhibitors of aggregation
from the library screened (from Diverse Set collection, ChemBridge
Corp., San Diego, Calif.).
[0166] To study the inhibitors we identified using the yeast screen
in mammalian cells, we employed ecdyson-inducible rat
phaeochromocytoma (PC12) cells. Compounds were tested in two
clones, 14A2/5 and 14A2/6, with highest aggregation rate. We
incubated undifferentiated PC12 cells with muristerone A to induce
103Q expression, and we exposed the cells to compounds at
concentrations ranging from 1 to 10 .mu.M. Four compounds,
including Cl (A16 in Table 1), C2 (B1 in Table 2), C3 (M14 in Table
5), and C4 (A2 in Table 1), showed significant inhibitory effects
on aggregation of 103Q in the two PC12 clones tested. Under the
test conditions, the IC.sub.50 for compound C1 was 10 .mu.M; for
compounds C2 and C3, it was 5 .mu.M; and for compound C4 it was 2.5
.mu.M. All compounds were non-toxic except C2, which was toxic at
concentrations higher than 5 .mu.M.
[0167] The inhibitory activity we observed with C1-C4 in the PC12
cell model described above was confirmed in Cos1 cells transiently
transfected with a CMV promoter-based DNA construct that encodes an
Htt exon I fragment of Huntington's disease protein (Htt)) having
51 consecutive glutamine residues (HD Q51). When expressed in Cos1
cells, HD 51Q polypeptides readily form aggregates. Transfected
Cos1 cells were incubated with C.sub.1-C.sub.4 compounds in culture
medium and lysed. The lysates were then heat-denatured in SDS and
passed through a cellulose acetate membrane, which captures
aggregated, but not soluble polypeptides. Polyglutamine aggregates
trapped on membranes were detected by immunostaining with antibody
specific to extended polyQ. Pre-incubation of cells with compounds
C1, C3, and C4 decreased the amount of retained polyQ, indicating
inhibitory effects on polyQ aggregation. The IC.sub.50 for compound
Cl was 10 .mu.M and the IC.sub.50 for compounds C3 and C4 was 5
.mu.M. As C2 was toxic for Cos1 cells, its effect on aggregation
was not determined.
[0168] The protein levels of Htt fragments containing 103Q in PC12
cells were not affected by incubation with compounds at the
concentrations tested. Furthermore, proteolytic capacity of cells
treated with compound C2 appeared to be normal, since levels of a
highly unstable endogenous p53 protein in PC12 cells were not
changed. Together, these data suggest that inhibition of aggregate
formation by C2 was not related to general inhibition of cell
viability, general cessation of metabolism or an increase in the
cells' general capacity to degrade or refold abnormal proteins.
[0169] To determine whether C2 could directly interfere with polyQ
aggregation, we reproduced this process in vitro and assayed the
inhibitory effects of the compounds in a cell-free trap assay.
Recombinant HD Q51 polypeptide, purified from bacteria, was
incubated in the presence or in the absence of C2 for a time
sufficient to allow aggregation. Then, upon heat-denaturation in
SDS buffer, samples were filtered through a cellulose acetate
membrane. Harsh denaturation conditions separated unaggregated
soluble peptides, which passed through the membrane, and insoluble
aggregates, which were retained on the membrane. The polyQ
aggregates trapped on the membrane were subsequently immunostained
and quantified. The compound failed to block aggregation of pure
polyglutamines in vitro even at high concentrations (up to 100 mM).
These data suggest that the molecular targets of these compounds
were not soluble or aggregated polyQ.
[0170] Suppression of aggregation of 103Q could be related to
potential induction of heat shock proteins that facilitate folding
and degradation of abnormal polypeptides. However, the compounds
failed to affect expression of the major inducible heat shock
protein, Hsp72, indicating that expression of Hsps is not regulated
by the tested compounds in PC12 cells.
[0171] To devise more potent inhibitors of polyQ aggregation, we
assembled a focus library consisting of chemical compounds having
more than 70% structural similarity to C1-C4. We tested these
analogs at concentrations ranging from 0.025 .mu.M to 5.0 .mu.M for
their effects on polyQ aggregation in PC12 cells. Among 24 analogs
of C1; 28 analogs of C2; 24 analogs of C3, and 53 analogs of C4, we
identified several potent inhibitors of polyQ aggregation. From our
C2 focus library, we isolated compound C2-8 (B2 in Table 2), which
inhibits polyQ aggregation with an IC.sub.50 value 50 nM. We also
identified C2-10 (B6 in Table 2) and C2-11 (M16 (formula V(n)) in
Table 5), and B7, B8 and B9 in Table 2. From our C3 focus library,
we isolated compound C3-5 (M22 (formula V(t)) in Table 5) (having
an IC.sub.50 value of 100 nM) and compound C3-6 (M17 (formula V(o))
in Table 5) (having an IC.sub.50 value of 5 .mu.M). From our C4
library, we isolated compound C4-7 (A31 in Table 1), which has an
IC.sub.50 value 100 nM. We also identified C4-34 (A3 in Table
1).
[0172] The compounds demonstrated no effects on expression levels
of 103Q polypeptides and no toxicity at the concentrations used to
inhibit aggregation. In the C1 library, no inhibitors of polyQ
aggregation with high potency were found. The original "hit"
compounds C1-C4 and their structural analogs displayed specificity
in inhibiting aggregation. Although these compounds were potent
inhibitors of polyQ aggregation, they failed to inhibit
alpha-synuclein aggregation in preliminary tests in a cellular
model of Parkinson's disease.
[0173] The effects of the selected compounds on polyQ aggregation
in neuronal tissues were assessed in brain slice cultures from the
transgenic mouse R6/2 model of HD. R6/2 mice ubiquitously express
human Huntingtin exon I containing 150Q (HD Q150), which causes
neuropathology resembling key neurological changes in HD patients.
Formation of neuronal polyQ aggregates precedes pathological
behavioural changes in R6/2 mice. An organotypic slice culture
assay has been developed in order to establish an ex vivo system
that closely models the process of aggregation occurring in R6/2
mouse brains. The early appearance of polyQ aggregates in the R6/2
mouse hippocampus makes it an ideal model to test aggregation
inhibitors in an ex vivo system: aggregates appear in neurons in
the slice cultures at the same time they appear in neurons in
intact brains of transgenic mice. The potency of any aggregate
inhibitor can be assessed directly in neurons in brain slices (thus
bypassing the blood brain barrier, the major obstacle to test
compounds in neuronal tissues).
[0174] Aggregation in brain slice cultures was assessed during four
weeks using three parameters: (1) the number of aggregates per
square millimeter, (2) the density of individual aggregates, and
(3) the size of the aggregates. These parameters can be assessed in
any of the assays described herein. Brain slices maintained in
culture for 2, 3 or 4 weeks were fixed and immunostained using an
anti-Htt-antibody. Fluorescent images of aggregates were captured
as Z stacks using a Confocal Microscope, capturing aggregates
throughout the entire thickness of the slice. To obtain
statistically significant data, 20 sections for each sample were
quantified with a macro computer program (Paul Wetton at Image
Associates, UK), which measured aggregate count, aggregate density
and aggregate size.
[0175] The test compounds were added to the slice cultures from Day
1 in regular media, which were changed twice a week. The four
hit-compounds, C1-C4, were tested at concentrations of 0.1 mM, 1
mM, 10 mM and 100 mM. The five structural analogues, C5-C9 (C5 is
in M15 in Table 5; C6 is M14 in Table 5; C7 is A2 in Table 1; C8 is
B2 in Table 2; C9 is A33 in Table 1), were tested at concentrations
0.001 mM, 0.01 mM, 0.1 mM, 1 mM and 10 mM.
[0176] The inhibitory effects on aggregation of primary
hit-compounds were restricted to 2-3 weeks in brain slice culture
at 0.1-10 .mu.M concentrations. At higher concentrations of 10-100
.mu.M, these compounds were toxic for neurons.
[0177] To date, the most successful inhibitor identified by this
screen was C2-8 (B2 in Table 2). At the 3-week time point,
aggregates intensity and area was inhibited only by the highest
tested concentration of 10 mM. After 4 weeks of incubation with
C2-8, inhibition occurred at concentrations ranging from 0.1-10 mM
for every parameter assayed, but 10 mM showed the strongest
effect.
[0178] Compound C2-8 was also tested in a cell-free trap assay of
purified polyQ, and showed inhibitory effects on aggregation,
albeit at very high concentrations (IC.sub.50 25 .mu.M). Congo Red,
which is structurally unrelated to C2-8, was used as a control. Our
data suggested that either C2-8 is metabolically converted by cells
into a highly potent inhibitor or that it affected cellular factors
involved in regulation of protein aggregation.
Example 7
Naphthylamine Scaffold Compounds
[0179] Previous studies for drugs to treat HD identified
naphthylamine scaffold compounds. Compounds from this chemical
class were identified as inhibitors of polyglutamine aggregation
and transcriptional regulators. The compounds include HDAc
inhibitors such as scriptaid, DAc inhibitors, DNA intercalation
agents, inhibitors of topoisomerase II, and the anticancer drugs
amonofide and mitonafide.
[0180] A variety of animal models of Huntington disease have been
developed. Examination of the degeneration of photoreceptor neurons
facilitated by extended polyQ expression is one such model (Jackson
et al., Neuron 21:633-642, 1998).
[0181] We used the photoreceptor model in Drosophila to test a
variety of different compounds, including naphthylamine
derivatives, for an effect on neurodegeneration. One naphthylamine
analog, C9-2 (A8 in Table 1), showed dramatic neuroprotection. C9-2
is 2-(3-methoxypropyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione.
[0182] Biochemical activity associated with C9-2 is not well
understood. The compound is structurally similar to scriptaid, but
unlike scriptaid, C9-2 does not appear to inhibit HDAc activity.
Further, no transcriptional activation or repression was observed
when C9-2 was tested in the striatum of 11Q/111Q cells and PC12
cells, nor has DNA binding activity been detected. C9-2 also does
not appear to suppress aggregation of polyQ-containing
polypeptides. Future studies include testing the compound for
anti-apoptotic activity, and transcriptional profiling using a
striatum cell line derived from a double knock-out mutant htt
mouse. The C9-2 analogs C9-2B (A30 in Table 1) and C9-2A (control)
(A29 in Table 1) are being tested for an effect on photoreceptor
neurodegeneration in the Drosophila HD model.
Example 8
Transcriptional Dysregulation
[0183] Transcriptional dysregulation is a hallmark of HD.
Regulators of global transcription, including HDAc inhibitors, have
been shown to ameliorate disease pathology. We found that the
compound C9 could repress transcription in PC12 cells expressing a
polypeptide with an extended polyQ (103Q) N-terminal fragment.
Expression of the polypeptide was under control of an inducible
ecdysone receptor (EcR)-based promoter.
[0184] We then tested C9 in chromatin immunoprecipitation (ChIP)
assays. Such assays are useful for measuring binding of
transcriptional factors and histones to DNA. It was previously
demonstrated that cells derived from striatum of double knock-in
mutant (111Q) full-length htt mice are deficient in binding of the
transcription factor SP 1 to dopamine receptor (D2) DNA. The
compound C9 was found to restore binding of SP 1 and increase
binding of histones (and acetylated histones in particular) to D2
DNA. The compound also demonstrated unspecific toxicity to cells at
concentrations higher than 10 .mu.M.
[0185] C9 was tested in the HD Drosophila model and showed modest
rescue of neurodegeneration of photoreceptor neurons.
[0186] Mouse trials were also conducted with C9, and while the
compound was found to be bioavailable, an effect was not observed,
presumably due to low potency of the compound. Scriptaid was toxic
to flies.
[0187] We conducted structure activity relationship (SAR) studies
to optimize transcription activation. We developed a novel
transcription-based assay for this purpose. A luciferase reporter
gene was stably integrated into 111Q/111Q striatum double knock-in
cells and constitutively expressed under control of a promoter
containing six copies of the SP 1 trancription factor binding motif
(6XSP1). In this system, C9 was determined to have an EC.sub.200 of
7.5 .mu.M. This result is similar to the transcription activation
efficacy observed for scriptaid. C9 was also found to activate
transcription in the neuronal cell line H4.
[0188] A series of C9 derivatives were designed, synthesized, and
tested in the ChIP and transcription-based assays described above.
The analog C91 (A9 in Table 1) was more potent (4 .mu.M) than C9,
and exhibited a similar result in ChIP experiments. C9-2 and C9-3
(A7 in Table 1) did not effectively activate transcription.
Compound C9-1B (A14 in Table 1) was even more potent than C9 and
C91, with an EC.sub.200 of 50 nM. This compound also demonstrated
some weak nonspecific toxicity in the range of 1.25-5.0 .mu.M. The
compounds tested generally demonstrated the best response 72 hours
after exposing the cells to the chemicals. This result contrasts
with that of the HDAc inhibitors which demonstrate a peak
transcriptional response in the transcription assay at 24 hours
following exposure of cells to compounds. The delay in the response
in the C9 compounds suggests that the mechanism of transcriptional
activation is different than that of the HDAc inhibitors. The
delayed response also indicates that the compounds are generally
stable in the cellular environment.
[0189] In the course of the SAR study, we also identified
structures that had previously been developed as cancer drugs, such
as the naphthalimides, mitonafide and amonafide (C91CN and C91C,
respectively). These compounds are known to be DNA intercalators
with antitumor activity. Testing of the DNA-binding activity of the
C9 compounds revealed no correlation between constant DNA binding
and transcriptional response. This is in contrast to the effect of
mithramycin which binds DNA and was shown to be highly efficacious
in a mouse HD model (Ferrante et al., Soc Neurosci Abstr 28: 725,
2002b). Mithramycin potently activated transcription in our assay.
Similar to many anti-tumor compounds, mithramycin is toxic to
cells. The C9 series lead compounds, however, do not show a similar
toxicity.
[0190] Compounds of the C9 series did not directly inhibit HDAc
activity, but C9-4 (A19 in Table 1) demonstrated indirect HDAc
inhibitory activity at high concentrations, targeting
down-regulation of HDAc 5 specifically. Thus, the C9 compounds may
modulate transcriptional activity by an indirect effect on the HDAc
pathway.
[0191] C9-1B and C9-6B (C9-1B is A14 in Table 1; C9-6B is A23 in
Table 1) will be tested in a mouse model of HD.
[0192] A series of rationally-designed scaffold-type compounds,
called the CG series of compounds, was tested in the transcription
assay described above. CG4 (A27 in Table 1) was the most potent
transcriptional activator, but was less potent than C9-4.
Example 9
Screening of Chemical Compound Libraries
[0193] A PC12 cell line was engineered to express extended polyQ
under the control of an ecdysone-inducible promoter. In the assay
cell line, an N-terminal fragment of htt containing an extended
polyQ tract was stably expressed as a fusion with EGFP
(htt103Q-EGFP), from the pIND vector. Expression of extended polyQ
in these cells was controlled by addition of an ecdysone analog, in
this case muristerone A. In this assay, EGFP fluorescence directly
correlated with htt103Q-EGFP expression levels. This system was
designed to assess the effects of specific compounds on overall
levels of extended polyglutamines in cells, using a simple
fluorescent-based read-out.
[0194] Using this assay, several compounds were identified as
facilitators of protein aggregation. These compounds are D1, D2,
and D3, shown in Table 4. PC12 cells expressing the extended polyQ
polypeptide exhibited proteasomal dysfunction, and compounds D1 and
D2 relieved this phenotype. The compound D4 was found to inhibit
protein aggregation.
Other Embodiments
[0195] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
* * * * *