U.S. patent application number 17/161505 was filed with the patent office on 2021-12-30 for methods and reagents for analyzing protein-protein interfaces.
The applicant listed for this patent is Revolution Medicines, Inc.. Invention is credited to Neville J. ANTHONY, Seung-Joo LEE, M. James NICHOLS, Uddhav Kumar SHIGDEL, Dylan T. STILES, Sharon A. TOWNSON, Gregory L. VERDINE.
Application Number | 20210405060 17/161505 |
Document ID | / |
Family ID | 1000005830118 |
Filed Date | 2021-12-30 |
United States Patent
Application |
20210405060 |
Kind Code |
A1 |
VERDINE; Gregory L. ; et
al. |
December 30, 2021 |
METHODS AND REAGENTS FOR ANALYZING PROTEIN-PROTEIN INTERFACES
Abstract
The present disclosure provides methods and reagents useful for
analyzing protein-protein interfaces such as interfaces between a
presenter protein (e.g., a member of the FKBP family, a member of
the cyclophilin family, or PIN1) and a target protein. In some
embodiments, the target and/or presenter proteins are intracellular
proteins. In some embodiments, the target and/or presenter proteins
are mammalian proteins.
Inventors: |
VERDINE; Gregory L.;
(Boston, MA) ; NICHOLS; M. James; (Charlestown,
MA) ; TOWNSON; Sharon A.; (Somerville, MA) ;
SHIGDEL; Uddhav Kumar; (East Meadow, NY) ; LEE;
Seung-Joo; (Burlington, MA) ; STILES; Dylan T.;
(Chestnut Hill, MA) ; ANTHONY; Neville J.;
(Northborough, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Revolution Medicines, Inc. |
Redwood City |
CA |
US |
|
|
Family ID: |
1000005830118 |
Appl. No.: |
17/161505 |
Filed: |
January 28, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15974921 |
May 9, 2018 |
10948495 |
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17161505 |
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15282430 |
Sep 30, 2016 |
9989535 |
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15974921 |
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62235896 |
Oct 1, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/6845 20130101;
G01N 2333/82 20130101; G01N 33/566 20130101; G01N 2410/08 20130101;
C07K 5/0215 20130101; C07K 5/0808 20130101; C07K 7/645 20130101;
C07K 1/13 20130101; C07K 1/086 20130101; G01N 2333/90209 20130101;
C07K 5/06034 20130101; C07K 7/64 20130101; C12Q 1/533 20130101;
G01N 2500/02 20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68; C07K 1/13 20060101 C07K001/13; C07K 5/02 20060101
C07K005/02; C07K 7/64 20060101 C07K007/64; C12Q 1/533 20060101
C12Q001/533; G01N 33/566 20060101 G01N033/566; C07K 5/062 20060101
C07K005/062; C07K 1/08 20060101 C07K001/08; C07K 5/083 20060101
C07K005/083 |
Claims
1. A conjugate comprising a presenter protein binding moiety
conjugated to a target protein, (i) wherein the presenter protein
binding moiety is a cyclophilin binding moiety comprising the
structure of Formula III or IV: ##STR00043## wherein Z.sup.3,
Z.sup.4, Z.sup.5, and Z.sup.6 are each, independently, hydroxyl,
optionally substituted C.sub.1-C.sub.6 alkyl, optionally
substituted C.sub.1-C.sub.6 heteroalkyl, or Z.sup.3 and Z.sup.4 or
Z.sup.5 and Z.sup.6 combine to form, with the atoms to which they
are attached, an optionally substituted 10 to 40 member macrocycle;
at least one of Z.sup.3, Z.sup.4, Z.sup.5, Z.sup.6, or R.sup.5
comprises a point of attachment to a cross-linking group; e is 0,
1, 2, 3, or 4; R.sup.5 is optionally substituted C.sub.1-C.sub.6
alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally
substituted C.sub.2-C.sub.6 alkynyl, optionally substituted
C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.2-C.sub.6
heteroalkenyl, optionally substituted C.sub.2-C.sub.6
heteroalkynyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl,
optionally substituted C.sub.6-C.sub.10 aryl, optionally
substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, optionally
substituted C.sub.2-C.sub.9 heterocyclyl, or optionally substituted
C.sub.2-C.sub.9 heterocyclyl C.sub.1-C.sub.6 alkyl; R.sup.6 is
optionally substituted C.sub.1-C.sub.6 alkyl; each R.sup.7 is,
independently, hydroxyl, cyano, optionally substituted amino,
halogen, thiol, optionally substituted C.sub.1-C.sub.6 alkyl,
optionally substituted C.sub.2-C.sub.6 alkenyl, optionally
substituted C.sub.2-C.sub.6 alkynyl, optionally substituted
C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.2-C.sub.6
heteroalkenyl, optionally substituted C.sub.2-C.sub.6
heteroalkynyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl,
optionally substituted C.sub.6-C.sub.10 aryl, optionally
substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, optionally
substituted C.sub.2-C.sub.9 heterocyclyl, or optionally substituted
C.sub.2-C.sub.9 heterocyclyl C.sub.1-C.sub.6 alkyl; and R.sup.8 is
hydrogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally
substituted C.sub.2-C.sub.6 alkenyl, optionally substituted
C.sub.2-C.sub.6 alkynyl, optionally substituted aryl,
C.sub.3-C.sub.7 carbocyclyl, optionally substituted
C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6alkyl, and optionally
substituted C.sub.3-C.sub.7 carbocyclyl C.sub.1-C.sub.6 alkyl; or
(ii) wherein the presenter protein binding moiety is an FKBP
binding moiety comprising the structure of Formula IIa:
##STR00044## wherein Z.sup.1 and Z.sup.2 are each, independently,
optionally substituted C.sub.1-C.sub.6 alkyl, optionally
substituted C.sub.1-C.sub.6 heteroalkyl, or Z.sup.1 and Z.sup.2
combine to form, with the atoms to which they are attached, an
optionally substituted 10 to 40 member macrocycle; at least one of
Z.sup.1 or Z.sup.2 comprises a point of attachment to a
cross-linking group; X.sup.2 is absent, CH.sub.2, O, S, SO,
SO.sub.2, or NR.sup.4; and each R.sup.4 is, independently,
hydrogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally
substituted C.sub.2-C.sub.6 alkenyl, optionally substituted
C.sub.2-C.sub.6 alkynyl, optionally substituted aryl, C.sub.3--C
carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl
C.sub.1-C.sub.6 alkyl, and optionally substituted C.sub.3-C.sub.7
carbocyclyl C.sub.1-C.sub.6 alkyl, wherein the cross-linking group
of the presenter protein binding moiety forms a covalent bond with
the target protein.
2. The conjugate of claim 1, wherein the presenter protein binding
moiety is a cyclophilin binding moiety comprising the structure of
Formula III.
3. The conjugate of claim 2, wherein the cyclophilin binding moiety
is capable of binding PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR,
SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, or
PPWD1.
4. The conjugate of claim 1, wherein the presenter protein binding
moiety is a cyclophilin binding moiety comprising the structure of
Formula IV.
5. The conjugate of claim 4, wherein the cyclophilin binding moiety
is capable of binding PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR,
SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, or
PPWD1.
6. The conjugate of claim 1, wherein the presenter protein binding
moiety is an FKBP binding moiety comprising the structure of
Formula IIa.
7. The conjugate of claim 6, wherein the FKBP binding moiety is
capable of binding FKBP12, FKBP12.6, FKBP13, FKBP25, FKBP51, or
FKBP52.
8. The conjugate of claim 1, wherein the cross-linking group is a
mixed disulfide, maleimide, vinyl sulfone, vinyl ketone, alkyl
halide, isocyanate, isothiocyanate, sulfonyl chloride, acid halide,
active ester, acid anhydride, acylazide, imidoester,
haloheteroaryl, diazo compound, carbodiimide, hydrazide,
alkoxyamine, azide, or alkyne.
9. The conjugate of claim 8, wherein the cross-linking group forms
a covalent bond with the a reactive group of an amino acid side
chain of the target protein.
10. The conjugate of claim 1, wherein the target protein is a
GTPase, GTPase activating protein, Guanine nucleotide-exchange
factor, a heat shock protein, an ion channel, a coiled-coil
protein, a kinase, a phosphatase, a ubiquitin ligase, a
transcription factor, a chromatin modifier/remodeler, or a protein
with classical protein-protein interaction domains and motifs.
11. The conjugate of claim 10, wherein the target protein is a
GTPase.
12. The conjugate of claim 11, wherein the target protein is K-Ras,
H-Ras, or N-Ras.
13. The conjugate of claim 12, wherein the target protein is
K-Ras.
14. The conjugate of claim 13, wherein the target protein is K-Ras
having a G12C mutation.
15. The conjugate of claim 13, wherein the target protein is K-Ras
having a S39C mutation.
16. The conjugate of claim 12, wherein the target protein in
N-Ras.
17. The conjugate of claim 16, wherein the target protein in N-Ras
having a G12C mutation.
Description
REFERENCE TO A SEQUENCE LISTING
[0001] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jan. 28, 2021 is named
50869-010005_Sequence_Listing_1_28_21_ST25 and is 603 bytes in
size.
BACKGROUND
[0002] The vast majority of small molecule drugs act by binding a
functionally important pocket on a target protein, thereby
modulating the activity of that protein. For example, the
cholesterol-lowering drugs statins bind the enzyme active site of
HMG-CoA reductase, thus preventing the enzyme from engaging with
its substrates. The fact that many such drug/target interacting
pairs are known may have misled some into believing that a small
molecule modulator could be discovered for most, if not all,
proteins provided a reasonable amount of time, effort, and
resources. This is far from the case. Current estimates hold that
only about 10% of all human proteins are targetable by small
molecules. The other 90% are currently considered refractory or
intractable toward small molecule drug discovery. Such targets are
commonly referred to as "undruggable." These undruggable targets
include a vast and largely untapped reservoir of medically
important human proteins. Thus, there exists a great deal of
interest in discovering new molecular modalities capable of
modulating the function of such undruggable targets.
SUMMARY
[0003] Small molecules are limited in their targeting ability
because their interactions with the target are driven by adhesive
forces, the strength of which is roughly proportional to contact
surface area. Because of their small size, the only way for a small
molecule to build up enough intermolecular contact surface area to
effectively interact with a target protein is to be literally
engulfed by that protein. Indeed, a large body of both experimental
and computational data supports the view that only those proteins
having a hydrophobic "pocket" on their surface are capable of
binding small molecules. In those cases, binding is enabled by
engulfment.
[0004] Nature has evolved a strategy that allows a small molecule
to interact with target proteins at sites other than hydrophobic
pockets. This strategy is exemplified by naturally occurring
immunosuppressive drugs cyclosporine A, rapamycin, and FK506. The
biological activity of these drugs involves the formation of a
high-affinity complex of the small molecule with a small presenting
protein. The composite surface of the small molecule and the
presenting protein engages the target. Thus, for example, the
binary complex formed between cyclosporin A and cyclophilin A
targets calcineurin with high affinity and specificity, but neither
cyclosporin A or cyclophilin A alone binds calcineurin with
measurable affinity.
[0005] The present inventors have developed compounds and
conjugates useful for identifying presenter protein and target
protein pairs, and probing the interfaces between them for use in
the development of small molecules capable of modulating these
interactions.
[0006] Accordingly, the present disclosure provides methods and
reagents useful for analyzing protein-protein interfaces such as
interfaces between a presenter protein (e.g., a member of the FKBP
family, a member of the cyclophilin family, or PIN1) and a target
protein. Such analysis is useful in aiding the design of small
molecules that are capable of binding simultaneously to both a
presenter protein and a target protein, such that the resulting
small molecule-presenter protein complexes can bind to and modulate
the activity of the target protein. In some embodiments, the target
and/or presenter proteins are intracellular proteins. In some
embodiments, the target and/or presenter proteins are mammalian
proteins.
[0007] In some aspect, the disclosure provides compounds that may
be used as cross-linking substrates. These compounds may include a
protein binding moiety capable of covalent or non-covalent binding
to a protein (e.g., a target protein or a presenter protein) and at
least one cross-linking group capable of a chemoselective reaction
with an amino acid of a different protein than that which binds to
the protein binding moiety. In some embodiments, the compounds
include only one cross-linking group.
[0008] Accordingly, in an aspect, the disclosure provides a
compound including a protein binding moiety (e.g., a presenter
protein binding moiety or a target protein binding moiety) and a
cross-linking group (e.g., a moiety capable of a chemoselective
reaction with an amino acid of a different protein than that which
binds to the protein binding moiety). The protein binding moiety is
capable of binding (covalently or non-covalently) to a protein
(e.g., a presenter protein or target protein, depending upon
whether it is a presenter protein binding moiety or a target
protein binding moiety), while the cross-linking group is capable
of forming a covalent bond with a protein (e.g., a presenter
protein, a target protein, or another compound that is capable of
binding such other protein). In some embodiments, when the compound
includes a presenter protein binding moiety, the compound does not
include a target protein binding moiety. In some embodiments, when
the compound includes a target protein binding moiety, the compound
does not include a presenter protein binding moiety.
[0009] In some embodiments, the cross-linking group is a
sulfhydryl-reactive cross-linking group (e.g., the cross-linking
group includes a mixed disulfide, a maleimide, vinyl sulfone, vinyl
ketone, or an alkyl halide), an amino-reactive cross-linking group,
a carboxyl-reactive cross-linking group, a carbonyl-reactive
cross-linking group, or a triazole-forming cross-linking group.
[0010] In some embodiments, the cross-linking group includes a
mixed disulfide, e.g., the cross-linking group includes the
structure of Formula I:
##STR00001##
[0011] wherein the wavy line illustrates the point of attachment of
the cross-linking group to the remainder of the compound; and
[0012] a is 0, 1, or 2;
[0013] R.sup.A is optionally substituted C.sub.1-C.sub.6 alkyl,
optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally
substituted C.sub.6-C.sub.10 aryl, or optionally substituted
C.sub.2-C.sub.9 heteroaryl.
[0014] In some embodiments, R.sup.A is optionally substituted
C.sub.2-C.sub.9 heteroaryl (e.g., pyridyl). In some embodiments,
the cross-linking group includes the structure:
##STR00002##
[0015] wherein the wavy line illustrates the point of attachment of
the cross-linking group to the remainder of the compound.
[0016] In some embodiments, R.sup.A is optionally substituted
C.sub.1-C.sub.6 alkyl (e.g., methyl). In some embodiments, the
cross-linking group includes the structure:
##STR00003##
[0017] wherein the wavy line illustrates the point of attachment of
the cross-linking group to the remainder of the compound.
[0018] In some embodiments, the cross-linking group includes a
maleimide, e.g., the cross-linking group includes the
structure:
##STR00004##
[0019] wherein the wavy line illustrates the point of attachment of
the cross-linking group to the remainder of the compound.
[0020] In some embodiments, the cross-linking group includes a
vinyl sulfone, e.g., the cross-linking group includes the
structures:
##STR00005##
[0021] wherein the wavy line illustrates the point of attachment of
the cross-linking group to the remainder of the compound.
[0022] In some embodiments, the cross-linking group includes a
vinyl ketone, e.g., the cross-linking group includes the
structures:
##STR00006##
[0023] wherein the wavy line illustrates the point of attachment of
the cross-linking group to the remainder of the compound.
[0024] In some embodiments, the cross-linking group includes an
alkyl halide such as an alkyl chloride, e.g., the cross-linking
group includes the structure:
##STR00007##
[0025] wherein the wavy line illustrates the point of attachment of
the cross-linking group to the remainder of the compound.
[0026] In some embodiments of any of the foregoing compounds, the
protein binding moiety portion is capable of non-covalent
interaction with a protein. In some embodiments of any of the
foregoing compounds, the protein binding moiety portion is capable
of covalent interaction with a protein.
[0027] In some aspects, the disclosure provides a compound
including a presenter protein binding moiety and a cross-linking
group. In some embodiments, the protein binding moiety and the
cross-linking group are attached through a linker.
[0028] In some the disclosure provides a compound having the
structure:
##STR00008## ##STR00009## ##STR00010## ##STR00011##
##STR00012##
[0029] In some aspects, the disclosure provides conjugates, methods
for their synthesis, and uses thereof, including a presenter
protein binding moiety capable of covalent or non-covalent binding
to a presenter protein conjugated to a target protein through a
linker.
[0030] Accordingly, in another aspect, the disclosure provides a
conjugate including a presenter protein binding moiety conjugated
to a target protein. In some embodiments, the presenter protein
binding moiety portion of the conjugate is capable of non-covalent
interaction with a presenter protein. In some embodiments, the
presenter protein binding moiety portion of the conjugate is
capable of covalent interaction with a presenter protein.
[0031] In some aspects, the disclosure provides a method of
producing a conjugate including a presenter protein binding moiety
conjugated to a target protein. This method includes reacting (a) a
compound including a presenter protein binding moiety and a
cross-linking group with (b) a target protein under conditions that
permit production of the conjugate.
[0032] In some aspects, the disclosure provides a method of
producing a conjugate including a presenter protein binding moiety
conjugated to a target protein. This method includes providing (a)
a compound including a presenter protein binding moiety and a
cross-linking group; (b) a target protein; and (c) a presenter
protein; and reacting the compound with the target protein under
conditions that permit production of the conjugate.
[0033] In some aspects, the disclosure provides complexes, methods
for their production, and uses thereof, including a presenter
protein and a conjugate including a presenter protein binding
moiety and a target protein.
[0034] Accordingly, in another aspect, the disclosure provides a
complex including (i) a conjugate including a presenter protein
binding moiety conjugated to a target protein and (ii) a presenter
protein.
[0035] In some aspects, the disclosure provides a method of
producing a complex including (i) a conjugate including a presenter
protein binding moiety conjugated to a target protein and (ii) a
presenter protein. This method includes combining a conjugate
including a presenter protein binding moiety conjugated to a target
protein and a presenter protein under conditions that permit
production of the complex.
[0036] In some aspects, the disclosure provides a method of
producing a complex including (i) a conjugate including a presenter
protein binding moiety conjugated to a target protein and (ii) a
presenter protein. This method includes providing (a) a compound
including a presenter protein binding moiety and a cross-linking
group; (b) a target protein; and (c) a presenter protein; and
reacting the compound with the target protein under conditions that
permit production of the complex.
[0037] In some embodiments of the foregoing methods, the presenter
protein binds to the compound in the absence of the target protein.
In some embodiments of the foregoing methods, the presenter protein
does not substantially bind to the compound in the absence of the
target protein. In some embodiments of the foregoing methods, the
compound and the target protein do not substantially react in the
absence of the presenter protein. In some embodiments of the
foregoing methods, the compound and the target protein react in the
absence of the presenter protein. In some embodiments of the
foregoing methods, the conditions do not include a reducing
reagent. In some embodiments of the foregoing methods, the
conditions include an excess of presenter protein.
[0038] In some embodiments, detectable binding between the compound
and the presenter protein is observed in the absence of the target
protein. In some embodiments, however detectable binding between
the compound and the presenter protein is not observed (e.g., the
presenter protein does not substantially bind to the compound) in
the absence of the target protein. In some embodiments, significant
reaction between the cross-linking group and the target protein
(e.g., significant conjugate formation) is not observed in the
absence of the presenter protein. In some embodiments, however,
significant reaction between the cross-linking group and the target
protein may be observed even in the absence of the presenter
protein. In some embodiments, rate and/or extent of such reaction
(e.g., rate and/or amount of conjugate formation) may differ in a
given assay when presenter protein is present as compared with when
it is absent (e.g., the rate and/or amount of conjugate formation
is 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or 100-fold greater in
the presence of the presenter protein).
[0039] In some embodiments, conjugate production as described
herein is performed under conditions that do not include (e.g., are
substantially free of) a reducing reagent.
[0040] In some embodiments, the present invention provides a
complex comprising (i) a presenter protein; (ii) a compound as
described herein (e.g., compound whose structure includes a
presenter protein binding moiety and a cross-linking group); and
(iii) a target protein. In some embodiments, such complex is
exposed to and/or maintained under conditions that permit reaction
of the cross-linking moiety with the target protein, so that a
cross-link therebetween is formed. In some embodiments, the
cross-link is with a heteroatom in an amino acid (e.g., in an amino
acid side chain) of the target protein. In some embodiments, the
cross-link is with an --S-atom in a cysteine in the target protein.
In some embodiments, the target protein is a variant of a natural
target protein; in some such embodiments, the variant has an amino
acid sequence that shows a high degree (e.g., 80%, 81%, 82%; 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 95%, 96%, 97%,
98%, 99% or higher) with the natural target protein but differs by
substitution or addition of at least one amino acid susceptible to
participation in a cross-link with the cross-linking group (e.g.,
whose amino acid side chain includes a heteroatom that can
participate in such a cross-link).
[0041] In some aspects, the disclosure provides conjugates, methods
for their synthesis, and uses thereof, including a target protein
binding moiety capable of covalent or non-covalent binding to a
target protein conjugated to a presenter protein through a
linker.
[0042] Accordingly, in another aspect, the disclosure provides a
conjugate including a target protein binding moiety conjugated to a
presenter protein. In some embodiments, the target protein binding
moiety portion of the conjugate is capable of non-covalent
interaction with a target protein. In some embodiments, the target
protein binding moiety portion of the conjugate is capable of
non-covalent interaction with target protein. In some embodiments,
the target protein binding moiety and the presenter protein are
conjugated through a linker.
[0043] In some aspects, the disclosure provides a method of
producing a conjugate including a target protein binding moiety
conjugated to a presenter protein. This method includes reacting
(a) a compound including a target protein binding moiety and a
cross-linking group with (b) a presenter protein under conditions
that permit production of the conjugate.
[0044] In some aspects, the disclosure provides a method of
producing a conjugate including a target protein binding moiety
conjugated to a presenter protein. This method includes providing
(a) a compound including a target protein binding moiety and a
cross-linking group; (b) a presenter protein; and (c) a target
protein; and reacting the compound with the presenter protein under
conditions that permit production of the conjugate.
[0045] In some embodiments, detectable binding between the compound
and the target protein is observed absence the presenter protein.
In some embodiments, however detectable binding between the
compound and the target protein is not observed (e.g., the
presenter protein does not substantially bind to the compound) in
the absence of the presenter protein. In some embodiments,
significant reaction between the cross-linking group and the
presenter protein (e.g., significant conjugate formation) is not
observed in the absence of the target protein. In some embodiments,
however, significant reaction between the cross-linking group and
the presenter protein may be observed even in the absence of the
target protein. In some embodiments, the rate and/or extent of such
reaction (e.g., the rate and/or amount of conjugate formation) may
differ in a given assay when presenter protein is present as
compared with when it is absent (e.g., the rate and/or amount of
conjugate formation is 2-fold, 3-fold, 4-fold, 5-fold, 10-fold,
100-fold greater in the presence of the presenter protein).
[0046] In some embodiments, the target protein binds to the
compound in the absence of the presenter protein. In some
embodiments, the target protein does not substantially bind to the
compound in the absence of the presenter protein. In some
embodiments, the presenter protein does not substantially bind to
the compound in the absence of the target protein. In some
embodiments, reaction between the cross-linking group and the
target protein (e.g., conjugate formation) is not observed in the
absence of the presenter protein. In some embodiments, however,
reaction between the cross-linking group and the target protein is
observed even in the absence of the presenter protein. In some
embodiments, conjugate production as described herein is performed
under conditions that do not include (e.g., are substantially free
of) a reducing agent.
[0047] In some embodiments, the present invention provides a
complex comprising (i) a presenter protein; (ii) a compound as
described herein (e.g., compound whose structure includes a
presenter protein binding moiety and a cross-linking group); and
(iii) a target protein. In some embodiments, such complex is
exposed to and/or maintained under conditions that permit reaction
of the cross-linking moiety with the target protein, so that a
cross-link therebetween is formed. In some embodiments, the
cross-link is with a heteroatom in an amino acid (e.g., in an amino
acid side chain) of the target protein. In some embodiments, the
cross-link is with an --S-atom in a cysteine in the target protein.
In some embodiments, the target protein is a variant of a natural
target protein; in some such embodiments, the variant has an amino
acid sequence that shows a high degree (e.g., 80%, 81%, 82%; 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 95%, 96%, 97%,
98%, 99% or higher) with the natural target protein but differs by
substitution or addition of at least one amino acid susceptible to
participation in a cross-link with the cross-linking group (e.g.,
whose amino acid side chain includes a heteroatom that can
participate in such a cross-link).
[0048] In some aspects, the disclosure provides complexes, methods
for their production, and uses thereof, including a target protein
and a conjugate including a target protein binding moiety
conjugated to a presenter protein through a linker.
[0049] In some aspects, the disclosure provides a complex including
(i) a conjugate including a target protein binding moiety
conjugated to a presenter protein; (ii) a target protein; and (iii)
a presenter protein.
[0050] In some embodiments, such complex is exposed to and/or
maintained under conditions that permit reaction of the
cross-linking moiety with the presenter protein, so that a
cross-link therebetween is formed. In some embodiments, the
cross-link is with a heteroatom in an amino acid (e.g., in an amino
acid side chain) of the presenter protein. In some embodiments, the
cross-link is with an --S-atom in a cysteine in the presenter
protein. In some embodiments, the presenter protein is a variant of
a natural presenter protein; in some such embodiments, the variant
has an amino acid sequence that shows a high degree (e.g., 80%,
81%, 82%; 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%,
95%, 96%, 97%, 98%, 99% or higher) with the natural presenter
protein but differs by substitution or addition of at least one
amino acid susceptible to participation in a cross-link with the
cross-linking group (e.g., whose amino acid side chain includes a
heteroatom that can participate in such a cross-link).
[0051] In some aspects, the disclosure provides a method of
producing a complex including (i) a conjugate including a target
protein binding moiety conjugated to a presenter protein and (ii) a
target protein. This method includes combining a conjugate
including a target protein binding moiety conjugated to a presenter
protein and a target protein under conditions that permit
production of the complex.
[0052] In some aspects, the invention features a method of
producing a complex including (i) a conjugate as described herein
(e.g., a conjugate including a target protein binding moiety and a
presenter protein) and (ii) a target protein. In some such
embodiments, a provided method includes combining the conjugate and
target protein under conditions that permit production of the
complex. Alternatively or additionally, in some embodiments, such a
methods includes, for example, (i) combining (a) a compound (e.g.,
a compound whose structure includes a target protein binding moiety
and a cross-linking group); (b) a target protein; and (c) a
presenter protein with one another; and (ii) exposing the
combination to and/or maintaining the combination under conditions
that permit production of the complex. In some such embodiments,
the conditions permit reaction of the cross-linking group with the
presenter protein so that a conjugate is produced.
[0053] In some aspects, the disclosure provides a method of
producing a complex including (i) a conjugate including a target
protein binding moiety conjugated to a presenter protein and (ii) a
target protein. This method includes providing (a) a compound
including a target protein binding moiety and a cross-linking
group; (b) a presenter protein; and (c) a target protein; and
reacting the compound with the presenter protein under conditions
that permit production of the complex.
[0054] In some such embodiments, the conditions are such that the
compound, presenter protein, and/or target protein are
characterized in that detectable binding between the compound and
the target protein is observed in the absence of the presenter
protein. In some embodiments, however, detectable binding between
the compound and the target protein is not observed (e.g., the
target protein does not substantially bind to the compound) under
the conditions in the absence of the presenter protein. In some
embodiments, significant reaction between the cross-linking group
and the presenter protein is not observed in the absence of the
target protein under the conditions. In some embodiments, however,
significant reaction between the cross-linking group and the
presenter protein may be observed even in the absence of the target
protein under the conditions. In some embodiments, the conditions
do not include a reducing reagent. In some embodiments, the
conditions include an excess of presenter protein.
[0055] In some embodiments, the target protein binds to the
compound in the absence of the presenter protein. In some
embodiments, the target protein does not substantially bind to the
compound in the absence of the presenter protein. In some
embodiments, the compound and the presenter protein do not
substantially react in the absence of the target protein. In some
embodiments, the compound and the presenter protein react in the
absence of the target protein. In some embodiments, the conditions
do not include a reducing reagent. In some embodiments, the
conditions include an excess of target protein.
[0056] In some aspects, the disclosure provides compounds including
a presenter protein binding moiety capable on non-covalent
interaction with a presenter protein and a target protein binding
moiety capable of covalent or non-covalent interaction with a
target protein. In some embodiments, the presenter protein binding
moiety and the target protein binding moiety are attached via a
linker.
[0057] Accordingly, in some aspects, the disclosure provides a
compound having the structure of Formula VII:
A-L-B Formula VII [0058] wherein A includes the structure of
Formula VIII:
##STR00013##
[0059] wherein b and c are independently 0, 1, or 2;
[0060] d is 0, 1, 2, 3, 4, 5, 6, or 7;
[0061] X.sup.1 and X.sup.2 are each, independently, absent,
CH.sub.2, O, S, SO, SO.sub.2, or NR.sup.13;
[0062] each R.sup.1 and R.sup.2 are independently hydrogen,
hydroxyl, optionally substituted amino, halogen, thiol, optionally
substituted C.sub.1-C.sub.6 alkyl, optionally substituted
C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6
alkynyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl,
optionally substituted C.sub.2-C.sub.6 heteroalkenyl, optionally
substituted C.sub.2-C.sub.6 heteroalkynyl, optionally substituted
C.sub.3-C.sub.10 carbocyclyl, optionally substituted
C.sub.6-C.sub.10 aryl, optionally substituted C.sub.6-C.sub.10 aryl
C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9
heterocyclyl (e.g., optionally substituted C.sub.2-C.sub.9
heteroaryl), optionally substituted C.sub.2-C.sub.9 heterocyclyl
C.sub.1-C.sub.6 alkyl (e.g., optionally substituted C.sub.2-C.sub.9
heteroaryl C.sub.1-C.sub.6 alkyl), or R.sup.1 and R.sup.2 combine
with the carbon atom to which they are bound to form C.dbd.O or
R.sup.1 and R.sup.2 combine to form an optionally substituted
C.sub.3-C.sub.10 carbocyclyl or optionally substituted
C.sub.2-C.sub.9 heterocyclyl;
[0063] each R.sup.3 is, independently, hydroxyl, optionally
substituted amino, halogen, thiol, optionally substituted
C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6
alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally
substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted
C.sub.2-C.sub.6 heteroalkenyl, optionally substituted
C.sub.2-C.sub.6 heteroalkynyl, optionally substituted
C.sub.3-C.sub.10 carbocyclyl, optionally substituted
C.sub.6-C.sub.10 aryl, optionally substituted C.sub.6-C.sub.10 aryl
C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9
heterocyclyl (e.g., optionally substituted C.sub.2-C.sub.9
heteroaryl), or optionally substituted C.sub.2-C.sub.9 heterocyclyl
C.sub.1-C.sub.6 alkyl (e.g., optionally substituted C.sub.2-C.sub.9
heteroaryl C.sub.1-C.sub.6 alkyl) or two R.sup.8 combine to form an
optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally
substituted C.sub.6-C.sub.10 aryl, optionally substituted
C.sub.2-C.sub.9 heterocyclyl, e.g., optionally substituted
C.sub.2-C.sub.9 heteroaryl;
[0064] R.sup.4 is optionally substituted C.sub.1-C.sub.6 alkyl;
[0065] L is an optional linker; and
[0066] B is a target protein binding moiety.
[0067] In some embodiments of a compound of Formula VII, the target
protein binding moiety, B, is capable of non-covalent interaction
with a target protein. In some embodiments of a compound of Formula
VII, the target protein binding moiety, B, is capable of covalent
interaction with a target protein. In some embodiments of a
compound of Formula VII, the linker, L, is present. In some
embodiments of a compound of Formula VII, the linker, L, is
absent.
[0068] In some aspects, the disclosure provides ternary complexes,
methods for their production, and uses thereof, including a
presenter protein, a target protein, and a compound including a
presenter protein binding moiety and a target protein binding
moiety.
[0069] Accordingly, in another aspect, the disclosure provides a
complex including (i) a compound of Formula VII; (ii) a target
protein; and (iii) a presenter protein.
[0070] In some embodiments, the compounds, conjugates, and
complexes of the present invention may be useful for the
identification of conjugates including a presenter protein binding
moiety and a target protein that are capable of forming complexes
with presenter proteins.
[0071] In some aspects, the invention features a method of
identifying and/or characterizing a conjugate as described herein
(e.g., in which a compound whose structure includes a presenter
protein binding moiety and a cross-linking group, is conjugated to
a target protein) that is capable of forming a complex with a
presenter protein. In some embodiments, such a method includes
steps of: (a) providing (i) such a conjugate (e.g., in which a
compound whose structure includes a presenter protein binding
moiety and a cross-linking group, conjugated to a target protein)
and (ii) a presenter protein; (b) combining the conjugate and the
presenter protein under conditions suitable to permit complex
formation if the conjugate is capable of forming a complex with the
presenter protein; and (c) determining whether a complex comprising
the conjugate and the presenter protein is formed, wherein
formation of the complex indicates that the conjugate is one that
is capable of forming a complex with a presenter protein.
[0072] Accordingly, in some aspects, the disclosure provides a
method of identifying and/or characterizing a conjugate that is
capable of forming a complex with a presenter protein. This method
includes the steps of: (a) providing (i) a conjugate including a
presenter protein binding moiety conjugated to a target protein and
(ii) a presenter protein; (b) combining the conjugate and the
presenter protein under conditions suitable to permit complex
formation if the conjugate is capable of forming a complex with the
presenter protein; and (c) determining whether a complex comprising
the conjugate and the presenter protein is formed, wherein
formation of the complex indicates that the conjugate is one that
is capable of forming a complex with a presenter protein.
[0073] In some embodiments, the compounds, conjugates, and
complexes of the present invention may be useful for the
identification of target proteins capable of forming covalent bonds
to compounds in the presence of a presenter protein.
[0074] Accordingly, in another aspect, the disclosure provides a
method of identifying and/or characterizing a target protein
capable of reacting with a compound in the presence of a presenter
protein, wherein the compound includes a presenter protein binding
moiety and a cross-linking moiety. This method includes the steps
of: (a) providing (i) a compound including a presenter protein
binding moiety and a cross-linking moiety; (ii) a target protein;
and (iii) a presenter protein; (b) combining the compound, the
target protein, and the presenter protein under conditions suitable
for to permit complex formation if the conjugate is capable of
forming a complex with the presenter protein; and (c) determining
whether the target protein and the compound react during formation
of the complex to form a conjugate, wherein if the target protein
and the compound form a conjugate, the target protein is identified
as capable of reacting with the compound in the presence of a
presenter protein.
[0075] In some embodiments, the compounds, conjugates, and
complexes of the invention may be useful for the identification of
target proteins capable of forming complexes with presenter
proteins.
[0076] Accordingly, in another aspect, the disclosure provides a
method of identifying and/or characterizing a target protein which
binds to a presenter protein. This method includes the steps of:
(a) providing (i) a conjugate including a presenter protein binding
moiety conjugated to a target protein and (ii) a presenter protein;
(b) combining the conjugate and the presenter protein under
conditions suitable to permit complex formation if the conjugate is
capable of forming a complex with the presenter protein; and (c)
determining whether the target protein binds to the presenter
protein in the complex, wherein if the target protein binds to the
presenter protein, the target protein is identified as binding to
the presenter protein.
[0077] In some aspects, the disclosure provides a method of
identifying and/or characterizing a target protein which binds to a
presenter protein. This method includes the steps of: (a) providing
(i) a compound including a presenter protein binding moiety and a
cross-linking moiety; (ii) a target protein; and (iii) a presenter
protein; (b) combining the compound, the target protein, and the
presenter protein under conditions suitable for to permit complex
formation if the conjugate is capable of forming a complex with the
presenter protein; and (c) determining whether the target protein
binds to the presenter protein in the complex, wherein if the
target protein binds to the presenter protein, the target protein
is identified as a target protein that binds to a presenter
protein.
[0078] In some aspects, the disclosure provides a method of
identifying and/or characterizing a target protein capable of
forming a complex with a presenter protein. This method includes
the steps of: (a) providing (i) a compound of Formula VII; (ii) a
target protein; and (iii) a presenter protein; (b) combining the
compound, the target protein, and the presenter protein under
conditions suitable to permit complex formation if the conjugate is
capable of forming a complex with the presenter protein; and (c)
determining if the compound, the target protein, and the presenter
protein form a complex, wherein if the compound, the target
protein, and the presenter protein form a complex, the target
protein is identified as a target protein capable of forming a
complex with a presenter protein.
[0079] In some aspects, the disclosure provides a method of
identifying and/or characterizing a target protein which binds to a
presenter protein. This method includes the steps of: (a) providing
(i) a compound of Formula VII; (ii) a target protein; and (iii) a
presenter protein; (b) combining the compound, the target protein,
and the presenter protein under conditions suitable for to permit
complex formation if the compound is capable of forming a complex
with the presenter protein; and (c) determining whether the target
protein binds to the presenter protein in the complex, wherein if
the target protein binds to the presenter protein, the target
protein is identified as a target protein that binds to a presenter
protein.
[0080] In some aspects, the disclosure provides a method of
identifying a target protein capable of forming a complex with a
presenter protein by (a) providing (i) one or more target proteins,
(ii) any of the foregoing compounds; and (iii) a presenter protein
that includes a tag (e.g., an affinity tag); (b) combining the one
or more target proteins, the compound, and the presenter protein
under conditions suitable to permit complex formation if one or
more of the target proteins is capable of forming a complex with
the presenter protein; and (c) determining whether one or more
target proteins form a complex with the compound and the presenter
protein; wherein target proteins that form a complex with the
presenter protein are identified as a target protein capable of
forming a complex with a presenter protein.
[0081] In some embodiments, the determining step comprises
utilizing the tag of said presenter protein to selectively isolate
target proteins which have formed a complex with the presenter
protein (e.g., by use in a pull down experiment). In some
embodiments, the complex includes a target protein, a presenter
protein, and a compound of the invention. In some embodiments, the
complex includes a conjugate including a target protein and a
presenter protein binding moiety (e.g., a conjugate formed by
reaction between a cross-linking group of a compound of the
invention and a reactive amino acid of a target protein) and a
presenter protein. In some embodiments, the method further
comprises (d) identifying the target protein (e.g., determining the
structure of the target protein) in a complex formed between one or
more target proteins, the compound, and the presenter protein. In
some embodiments, the identifying of the structure of the target
protein comprises performing mass spectrometry on the complex. In
some embodiments, determination of whether the target protein and
presenter protein form a complex and/or target protein binds to the
presenter protein in the complex may be carried out using pull down
experiments wherein either the target protein or the presenter
protein is labeled (e.g., wherein a complex may be selectively
pulled down in the presence of target proteins and/or presenter
proteins which are not in a complex).
[0082] In some aspects, the disclosure provides a method of
identifying a target protein capable of forming a complex with a
presenter protein, by (a) providing (i) two or more target
proteins; (ii) any of the foregoing compounds; and (iii) a
presenter protein including an affinity tag; (b) combining the two
or more target proteins, the compound, and the presenter protein
under conditions suitable to permit complex formation if said
target protein is capable of forming a complex with the presenter
protein; (c) selectively isolating one or more complexes of a
target protein, the compound, and the presenter protein formed in
step (b); and (d) identifying the target protein (e.g., determining
the structure of the target protein) in the one or more complexes
isolated in step (c) by mass spectrometry; thereby identifying a
target protein capable of forming a complex with a presenter
protein.
[0083] In some embodiments, the determining step comprises
utilizing the tag of said presenter protein to selectively isolate
target proteins which have formed a complex with the presenter
protein (e.g., by use in a pull down experiment). In some
embodiments, the complex includes a target protein, a presenter
protein, and a compound of the invention. In some embodiments, the
complex includes a conjugate including a target protein and a
presenter protein binding moiety (e.g., a conjugate formed by
reaction between a cross-linking group of a compound of the
invention and a reactive amino acid of a target protein) and a
presenter protein. In some embodiments, determination of whether
the target protein and presenter protein form a complex and/or
target protein binds to the presenter protein in the complex may be
carried out using pull down experiments wherein either the target
protein or the presenter protein is labeled (e.g., wherein a
complex may be selectively pulled down in the presence of target
proteins and/or presenter proteins which are not in a complex).
[0084] In some embodiments, the compounds, conjugates, and
complexes of the present invention may be useful to identify
locations on target proteins to attach presenter protein binding
moieties which result in conjugates capable of forming complexes
with presenter proteins.
[0085] Accordingly, in another aspect, the disclosure provides a
method of identifying and/or characterizing a location on a target
protein to form a conjugate with a presenter protein binding
moiety, which conjugate is capable of forming a complex with a
presenter protein. This method includes the steps of: (a) providing
(i) a conjugate including a presenter protein binding moiety
conjugated to a target protein at a location and (ii) a presenter
protein; (b) combining the conjugate and the presenter protein; (c)
determining if the conjugate and the presenter protein form a
complex; and (d) optionally repeating steps (a) to (c) with the
presenter protein binding moiety conjugated at different locations
on the target protein until a conjugate and the presenter protein
form a complex, wherein a location on a target protein to form a
conjugate with a presenter protein binding moiety, which conjugate
is capable of forming a complex with a presenter protein is
identified if the conjugate and the presenter protein form a
complex. In some embodiments, the presenter protein is a variant of
a naturally occurring target protein.
[0086] In some aspects, the disclosure provides a method of
identifying and/or characterizing a location on a target protein to
form a conjugate with a presenter protein binding moiety, which
conjugate is capable of forming a complex with a presenter protein.
This method includes the steps of: (a) providing (i) a compound
including a presenter protein binding moiety and a cross-linking
group; (ii) a target protein; and (iii) a presenter protein; (b)
combining the compound with the target protein in the presence of
the presenter protein under conditions that permit the formation of
a conjugate including a presenter protein binding moiety conjugated
to a target protein at a location; (c) determining if the conjugate
and the presenter protein form a complex; and (d) optionally
repeating steps (a) to (c) wherein the presenter protein binding
moiety is conjugated at different locations on the target protein
until a conjugate and the presenter protein form a complex; wherein
a location on a target protein to form a conjugate with a presenter
protein binding moiety, which conjugate is capable of forming a
complex with a presenter protein is identified if the conjugate and
the presenter protein form a complex, thereby identifying a
location on a target protein to form a conjugate capable of forming
a complex with a presenter protein. In some embodiments, the target
protein is a variant of a naturally occurring target protein.
[0087] In some embodiments, the compounds, conjugates, and
complexes of the present invention may be useful for identifying
compounds capable of forming covalent bonds to target proteins in
the presence of presenter proteins. In some embodiments, the
compounds identified selectively form covalent bonds with target
proteins in the presence of presenter proteins.
[0088] Accordingly, in another aspect, the disclosure provides a
method of identifying and/or characterizing a compound capable of
covalently binding to a target protein in the presence of a
presenter protein. This method includes the steps of: (a) providing
a sample including (i) a compound including a presenter protein
binding moiety and a cross-linking group; (ii) a target protein;
and (iii) a presenter protein; and (b) determining if the compound
and the target protein form a covalent bond via the cross-linking
group in said compound in the sample, wherein a compound is
identified as covalently binding to a target protein in the
presence of a presenter protein if the compound and the target
protein react in the sample.
[0089] In some aspects, the disclosure provides a method of
identifying and/or characterizing a compound capable of selective
and covalent binding to a target protein in the presence of a
presenter protein. This method includes the steps of: (a) providing
a first sample including (i) a compound including a presenter
protein binding moiety and a cross-linking group; (ii) a target
protein; and (iii) a presenter protein and a second sample
including (i) the same compound including a presenter protein
binding moiety and a cross-linking group as in the first sample and
(ii) the same target protein as in the first sample; and (b)
determining the extent to which the compound and the target protein
react in the first sample compared to the second sample, wherein a
compound is identified as selectively covalently binding to a
target protein in the presence of a presenter protein if the
compound and the target protein reacts in the first sample more
than in the second sample.
[0090] In some embodiments, a compound is identified as selectively
covalently binding to a target protein in the presence of a
presenter protein if the compound and the target protein reacts in
the first sample at least 5-fold more than in the second sample. In
some embodiments, a compound is identified as selectively
covalently binding to a target protein in the presence of a
presenter protein if the compound and the target protein reacts in
the first sample, but does not substantially react in the second
sample.
[0091] In some embodiments, the compounds, conjugates, and
complexes of the present invention may be useful in identifying
conjugates including a target protein and a presenter protein
binding moiety capable of forming complexes with presenter
proteins.
[0092] Accordingly, in another aspect, the disclosure provides a
method of identifying and/or characterizing a conjugate capable of
forming a complex with a presenter protein. This method includes
the steps of: (a) providing (i) a conjugate including a presenter
protein binding moiety conjugated to a target protein and (ii) a
presenter protein; and (b) combining the conjugate and the
presenter protein under conditions suitable for forming a complex;
(c) determining if the conjugate and the presenter protein form a
complex, wherein a conjugate is identified as capable of forming a
complex with a presenter protein if the conjugate and the presenter
protein form a complex, thereby identifying a conjugate capable of
forming a complex with a presenter protein.
[0093] In some embodiments, binding between a conjugate and a
protein may be determined by a method that includes a ternary
time-resolved fluorescence energy transfer assay, a ternary
amplified luminescent proximity homogeneous assay, a isothermal
titration calorimetry, surface plasmon resonance, or nuclear
magnetic resonance.
[0094] In some embodiments, the compounds, conjugates, and
complexes of the present invention may be useful for the
determining the structure of protein-protein interfaces between
presenter proteins and target proteins.
[0095] Accordingly, in another aspect, the disclosure provides a
method of determining the structure of and/or assessing one or more
structural features of an interface in a complex including a
presenter protein and a target protein. This method includes the
steps of: (a) providing (i) a conjugate including a presenter
protein binding moiety conjugated to a target protein and (ii) a
presenter protein; (b) contacting the conjugate with a presenter
protein to form a complex (e.g., in a vial); and (c) determining
the crystal structure of the complex, wherein the structure of the
interface includes at least the portion of the crystal structure
between the presenter protein and the target protein, thereby
determining the structure of an interface in a complex including a
presenter protein and a target protein.
[0096] In some aspects, the disclosure provides a method of
determining the structure of and/or assessing one or more
structural features of an interface in a complex including a
presenter protein and a target protein. This method includes the
steps of: (a) providing (i) a compound including a presenter
protein binding moiety and a cross-linking moiety; (ii) a target
protein; and (iii) a presenter protein; (b) combining the compound,
the target protein, and the presenter protein under conditions
suitable for forming a conjugate between the compound and target
protein and for the formation of a complex between said conjugate
and said presenter protein (e.g., in a vial); and (c) determining
the crystal structure of the complex, wherein the structure of the
interface includes at least the portion of the crystal structure
between the presenter protein and the target protein, thereby
determining the structure of an interface in a complex including a
presenter protein and a target protein.
[0097] In some aspects, the disclosure provides a method of
determining the structure of and/or assessing one or more
structural features of an interface in a complex including a
presenter protein and a target protein. This method includes the
steps of: (a) providing (i) a compound of Formula VII; (ii) a
target protein; and (iii) a presenter protein; (b) forming a
complex including the compound, the target protein, and the
presenter protein (e.g., in a vial); and (c) determining the
crystal structure of the complex, wherein the structure of the
interface includes at least the portion of the crystal structure
between the presenter protein and the target protein, thereby
determining the structure of an interface in a complex including a
presenter protein and a target protein.
[0098] In some aspects, the disclosure provides a method of
determining the structure of and/or assessing one or more
structural features of a protein-protein interface in a complex
including a presenter protein and a target protein. This method
includes the steps of: (a) providing a crystal of any of the
foregoing complexes; and (b) determining the structure of the
crystal, wherein the structure of the interface includes at least
the portion of the crystal structure between the presenter protein
and the target protein, thereby determining the structure of a
protein-protein interface in a complex including a presenter
protein and a target protein.
[0099] In some aspects, the disclosure provides a method of
identifying and/or characterizing compounds capable of modulating
the biological activity of a target protein. This method includes
the steps of: (a) providing the structure of a protein-protein
interface in a complex including a presenter protein and a target
protein (e.g., a structure determined by any of the foregoing
methods); and (b) determining the structure of compounds capable of
binding at the interface, thereby identifying compounds capable of
modulating the biological activity of a target protein. In some
embodiments, the structure of compounds capable of binding at the
interface is determined using computational methods. In some
embodiments, the structure of compounds capable of binding at the
interface is determined by screening of compounds including a
presenter protein binding moiety described herein for complex
formation in the presence of a target protein and a presenter
protein.
[0100] In some aspects, the disclosure provides a method of
obtaining X-ray crystal coordinates for a complex. This method
includes the steps of: (a) providing (i) a conjugate including a
presenter protein binding moiety conjugated to a target protein and
(ii) a presenter protein; (b) combining the conjugate and the
presenter protein under conditions suitable for to permit complex
formation if the conjugate is capable of forming a complex with the
presenter protein; and (c) determining the crystal structure of the
complex, thereby obtaining X-ray crystal coordinates for the
complex.
[0101] In some aspects, the disclosure provides a method of
obtaining X-ray crystal coordinates for a complex. This method
includes the steps of: (a) providing (i) a compound including a
presenter protein binding moiety and a cross-linking moiety; (ii) a
target protein; and (iii) a presenter protein; (b) combining the
compound, the target protein, and the presenter protein under
conditions suitable for to permit complex formation if the compound
is capable of forming a complex with the presenter protein; and (c)
determining the crystal structure of the complex, thereby obtaining
X-ray crystal coordinates for the complex.
[0102] In some aspects, the disclosure provides a method of
obtaining X-ray crystal coordinates for a complex. This method
includes the steps of: (a) providing (i) a compound of the
invention; (ii) a target protein; and (iii) a presenter protein;
(b) combining the compound, the target protein, and the presenter
protein under conditions suitable for to permit complex formation
if the compound is capable of forming a complex with the presenter
protein; and (c) determining the crystal structure of the complex,
thereby obtaining X-ray crystal coordinates for the complex.
[0103] In some aspects, the disclosure provides a method of
determining the residues on a target protein that participate in
binding with a presenter protein. This method includes the steps
of: (a) providing X-ray crystal coordinates of a complex obtained
by a method of the invention; (b) identifying the residues of the
target protein which include an atom within 4 .ANG. of an atom on
the presenter protein; thereby determining the residues on a target
protein that participate in binding with a presenter protein. In
some aspects, the disclosure provides a method of determining
biochemical and/or biophysical properties of any of the presenter
protein/target protein complexes described herein. This method
includes the steps of: (a) providing X-ray crystal coordinates of a
complex described herein obtained by a method described herein; (b)
calculating a biochemical and/or biophysical property of the
complex; thereby determining biochemical and/or biophysical
properties of a presenter protein/target protein complex.
[0104] In some embodiments, the biochemical and/or biophysical
properties include the free energy of binding of a complex, the
K.sub.d of a complex, the K.sub.i of a complex, the K.sub.inact of
a complex, and/or the K.sub.i/K.sub.inact of a complex. In some
embodiments, the biochemical and/or biophysical properties are
determined by isothermal titration calorimetry, surface plasmon
resonance, and/or mass spectrometry.
[0105] In some embodiments, the interface in a complex including a
presenter protein and a target protein is or comprises a binding
pocket.
[0106] In some aspects, the disclosure provides compositions
including any of the foregoing compounds, a target protein, and a
presenter protein in solution.
[0107] In some aspects, the disclosure provides a pharmaceutical
composition including any of the compounds, conjugates, or
complexes of the invention and a pharmaceutically acceptable
excipient. In some embodiments, the pharmaceutical composition is
in unit dosage form.
[0108] In some aspects, the disclosure provides a method of
modulating a target protein (e.g., a eukaryotic target protein such
as a mammalian target protein or a fungal target proteins or a
prokaryotic target protein such as a bacterial target protein). In
some embodiments, such a method includes steps of contacting the
target protein with a modulating (e.g., positive or negative
modulation) amount of any of the compounds (e.g., in the presence
of a presenter protein), conjugates including a target protein
binding moiety, or compositions of the invention.
[0109] In some aspects, the disclosure provides a method of
modulating (e.g., positively or negatively modulating) a target
protein (e.g., a eukaryotic target protein such as a mammalian
target protein or a fungal target proteins or a prokaryotic target
protein such as a bacterial target protein). In some embodiments,
such a method includes steps of contacting a cell expressing the
target protein and a presenter protein with an effective amount of
a compound or composition of the invention under conditions wherein
the compound can form a complex with the presenter protein and the
resulting complex can bind to the target protein, thereby
modulating (e.g., positively or negatively modulating) the target
protein.
[0110] In some aspects, the disclosure provides a method of
modulating (e.g., positively or negatively modulating) a target
protein (e.g., a eukaryotic target protein such as a mammalian
target protein or a fungal target proteins or a prokaryotic target
protein such as a bacterial target protein). In some embodiments,
such a method includes steps of contacting the target protein with
conjugate of the invention including a target protein binding
moiety, thereby modulating the target protein.
[0111] In some aspects, the disclosure provides a method of
inhibiting prolyl isomerase activity. In some embodiments, such a
method includes contacting a cell expressing the prolyl isomerase
with a compound or composition of the invention under conditions
that permit the formation of a complex between the compound and the
prolyl isomerase, thereby inhibiting the prolyl isomerase
activity.
[0112] In some aspects, the disclosure provides a method of forming
a presenter protein/compound complex in a cell. In some
embodiments, such a method includes steps of contacting a cell
expressing the presenter protein with a compound or composition of
the invention under conditions that permit the formation of a
complex between the compound and the presenter protein.
[0113] In some embodiments of any of the foregoing compounds,
conjugates, complexes, compositions, or methods, the presenter
protein binding moiety is capable of binding a protein encoded by
any one of the genes of Table 1. In some embodiments of any of the
foregoing compounds, conjugates, complexes, compositions, or
methods, the presenter protein binding moiety is a prolyl isomerase
binding moiety. In some embodiments of any of the foregoing
compounds, conjugates, complexes, compositions, or methods, the
presenter protein binding moiety is a FKBP binding moiety (e.g.,
the presenter protein binding moiety is capable of binding FKBP12,
FKBP12.6, FKBP13, FKBP25, FKBP51, or FKBP52), a cyclophilin binding
moiety (e.g., the presenter protein binding moiety is capable of
binding PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH,
CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, or PPWD1), or a
PIN1 binding moiety. In some embodiments of any of the foregoing
methods, the presenter protein is known to bind to the presenter
protein binding moiety.
[0114] In some embodiments of any of the foregoing compounds,
conjugates, complexes, compositions, or methods, the presenter
protein binding moiety is a FKBP binding moiety (e.g., a selective
FKBP binding moiety or a non-selective FKBP binding moiety). In
some embodiments of any of the foregoing compounds, conjugates,
complexes, compositions, or methods, the FKBP binding moiety
includes the structure of Formula IIa or IIb:
##STR00014##
[0115] wherein Z.sup.1 and Z.sup.2 are each, independently,
optionally substituted C.sub.1-C.sub.6 alkyl, optionally
substituted C.sub.1-C.sub.6 heteroalkyl, or Z.sup.1 and Z.sup.2
combine to form, with the atoms to which they are attached, an
optionally substituted 10 to 40 member macrocycle; and wherein at
least one of Z.sup.1 or Z.sup.2 includes a point of attachment to
the cross-linking group;
[0116] b and c are independently 0, 1, or 2;
[0117] d is 0, 1, 2, 3, 4, 5, 6, or 7;
[0118] X.sup.1 and X.sup.2 are each, independently, absent,
CH.sub.2, O, S, SO, SO.sub.2, or NR.sup.4;
[0119] each R.sup.1 and R.sup.2 are independently hydrogen,
hydroxyl, optionally substituted amino, halogen, thiol, optionally
substituted C.sub.1-C.sub.6 alkyl, optionally substituted
C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6
alkynyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl,
optionally substituted C.sub.2-C.sub.6 heteroalkenyl, optionally
substituted C.sub.2-C.sub.6 heteroalkynyl, optionally substituted
C.sub.3-C.sub.10 carbocyclyl, optionally substituted
C.sub.6-C.sub.10 aryl, optionally substituted C.sub.6-C.sub.10 aryl
C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9
heterocyclyl (e.g., optionally substituted C.sub.2-C.sub.9
heteroaryl), optionally substituted C.sub.2-C.sub.9 heterocyclyl
C.sub.1-C.sub.6 alkyl (e.g., optionally substituted C.sub.2-C.sub.9
heteroaryl C.sub.1-C.sub.6 alkyl), or R.sup.1 and R.sup.2 combine
with the carbon atom to which they are bound to form C.dbd.O or
R.sup.1 and R.sup.2 combine to form an optionally substituted
C.sub.3-C.sub.10 carbocyclyl or optionally substituted
C.sub.2-C.sub.9 heterocyclyl;
[0120] each R.sup.3 is, independently, hydroxyl, optionally
substituted amino, halogen, thiol, optionally substituted
C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6
alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally
substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted
C.sub.2-C.sub.6 heteroalkenyl, optionally substituted
C.sub.2-C.sub.6 heteroalkynyl, optionally substituted
C.sub.3-C.sub.10 carbocyclyl, optionally substituted
C.sub.6-C.sub.10 aryl, optionally substituted C.sub.6-C.sub.10 aryl
C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9
heterocyclyl (e.g., optionally substituted C.sub.2-C.sub.9
heteroaryl), or optionally substituted C.sub.2-C.sub.9 heterocyclyl
C.sub.1-C.sub.6 alkyl (e.g., optionally substituted C.sub.2-C.sub.9
heteroaryl C.sub.1-C.sub.6 alkyl) or two R.sup.8 combine to form an
optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally
substituted C.sub.6-C.sub.10 aryl, e.g., optionally substituted
C.sub.2-C.sub.9 heteroaryl; and
[0121] each R.sup.4 is, independently, hydrogen, optionally
substituted C.sub.1-C.sub.6 alkyl, optionally substituted
C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6
alkynyl, optionally substituted aryl, C.sub.3--C carbocyclyl,
optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl,
and optionally substituted C.sub.3-C.sub.7 carbocyclyl
C.sub.1-C.sub.6 alkyl.
[0122] In some embodiments of any of the foregoing compounds,
conjugates, complexes, compositions, or methods, the presenter
protein binding moiety includes the structure:
##STR00015## ##STR00016##
[0123] In some embodiments of any of the foregoing compounds,
conjugates, complexes, compositions, or methods, the presenter
protein binding moiety is a cyclophilin binding moiety (e.g., a
selective cyclophilin binding moiety or a non-selective cyclophilin
binding moiety). In some embodiments of any of the foregoing
compounds, conjugates, complexes, compositions, or methods, the
cyclophilin binding moiety includes the structure of Formula III or
IV:
##STR00017##
[0124] wherein Z.sup.3, Z.sup.4, Z.sup.5, and Z.sup.6 are each,
independently, hydroxyl, optionally substituted C.sub.1-C.sub.6
alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, or
Z.sup.3 and Z.sup.4 or Z.sup.5 and Z.sup.6 combine to form, with
the atoms to which they are attached, an optionally substituted 10
to 40 member macrocycle;
[0125] at least one of Z.sup.3, Z.sup.4, Z.sup.5, Z.sup.6, or
R.sup.5 includes a point of attachment to the cross-linking
group;
[0126] e is 0, 1, 2, 3, or 4;
[0127] R.sup.5 is optionally substituted C.sub.1-C.sub.6 alkyl,
optionally substituted C.sub.2-C.sub.6 alkenyl, optionally
substituted C.sub.2-C.sub.6 alkynyl, optionally substituted
C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.2-C.sub.6
heteroalkenyl, optionally substituted C.sub.2-C.sub.6
heteroalkynyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl,
optionally substituted C.sub.6-C.sub.10 aryl, optionally
substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, optionally
substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted
C.sub.2-C.sub.9 heteroaryl C.sub.1-C.sub.6 alkyl, optionally
substituted C.sub.2-C.sub.9 heterocyclyl, or optionally substituted
C.sub.2-C.sub.9 heterocyclyl C.sub.1-C.sub.6 alkyl;
[0128] R.sup.6 is optionally substituted C.sub.1-C.sub.6 alkyl;
[0129] each R.sup.7 is, independently, hydroxyl, cyano, optionally
substituted amino, halogen, thiol, optionally substituted
C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6
alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally
substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted
C.sub.2-C.sub.6 heteroalkenyl, optionally substituted
C.sub.2-C.sub.6 heteroalkynyl, optionally substituted
C.sub.3-C.sub.10 carbocyclyl, optionally substituted
C.sub.6-C.sub.10 aryl, optionally substituted C.sub.6-C.sub.10 aryl
C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9
heterocyclyl (e.g., optionally substituted C.sub.2-C.sub.9
heteroaryl), or optionally substituted C.sub.2-C.sub.9 heterocyclyl
C.sub.1-C.sub.6 alkyl (e.g., optionally substituted C.sub.2-C.sub.9
heteroaryl C.sub.1-C.sub.6 alkyl); and
[0130] R.sup.8 is hydrogen, optionally substituted C.sub.1-C.sub.6
alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally
substituted C.sub.2-C.sub.6 alkynyl, optionally substituted aryl,
C.sub.3--C carbocyclyl, optionally substituted C.sub.6-C.sub.10
aryl C.sub.1-C.sub.6 alkyl, and optionally substituted
C.sub.3-C.sub.7 carbocyclyl C.sub.1-C.sub.6 alkyl.
[0131] In some embodiments of any of the foregoing compounds,
conjugates, complexes, compositions, or methods, the presenter
protein binding moiety includes the structure:
##STR00018##
[0132] In some embodiments of any of the foregoing compounds,
conjugates, complexes, compositions, or methods, the target protein
is a GTPase, GTPase activating protein, Guanine nucleotide-exchange
factor, a heat shock protein, an ion channel, a coiled-coil
protein, a kinase, a phosphatase, a ubiquitin ligase, a
transcription factor, a chromatin modifier/remodeler, or a protein
with classical protein-protein interaction domains and motifs. In
some embodiments of any of the foregoing compounds, conjugates,
complexes, compositions, or methods, the target protein includes an
undruggable surface. In some embodiments of any of the foregoing
compounds, conjugates, complexes, compositions, or methods, the
target protein does not have a traditional binding pocket.
[0133] In some embodiments of any of the foregoing compounds,
conjugates, complexes, compositions, or methods, the amino acid
sequence of the target protein has been modified to substitute at
least one native amino acid with a reactive amino acid (e.g., a
natural amino acid such as a cysteine, lysine, tyrosine, aspartic
acid, glutamic acid, or serine, or a non-natural amino acid). In
some embodiments of any of the foregoing compounds, conjugates,
complexes, compositions, or methods, the amino acid sequence of the
target protein has been modified to substitute at least one native
reactive amino acid (e.g., a cysteine, lysine, tyrosine, aspartic
acid, glutamic acid, or serine) with a non-reactive amino acid
(e.g., a natural amino acid such as a serine, valine, alanine,
isoleucine, threonine, tyrosine, aspartic acid, glutamic acid, or
leucine, or a non-natural amino acid). In some embodiments of any
of the foregoing compounds, conjugates, complexes, compositions, or
methods, the at least one native reactive amino acid is a solvent
exposed amino acid. In some embodiments of any of the foregoing
compounds, conjugates, complexes, compositions, or methods, the
amino acid sequence of the target protein is modified to substitute
all reactive amino acids with a non-reactive amino acid. In some
embodiments of any of the foregoing compounds, conjugates,
complexes, compositions, or methods, the substitution is a
conservative substitution. In some embodiments of any of the
foregoing compounds, conjugates, complexes, compositions, or
methods, the target protein includes only one solvent exposed
reactive amino acid.
[0134] In some embodiments of any of the foregoing compounds,
conjugates, complexes, compositions, or methods, the presenter
protein is a protein encoded by any one of the genes of Table 1. In
some embodiments of any of the foregoing compounds, conjugates,
complexes, compositions, or methods, the presenter protein is a
prolyl isomerase. In some embodiments of any of the foregoing
compounds, conjugates, complexes, compositions, or methods, the
prolyl isomerase is a member of the FKBP family (e.g., FKBP12,
FKBP12.6, FKBP13, FKBP25, FKBP51, or FKBP52), a member of the
cyclophilin family (e.g., PP1A, CYPB, CYPC, CYP40, CYPE, CYPD,
NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2,
or PPWD1), or PIN1.
[0135] In some embodiments of any of the foregoing compounds,
conjugates, complexes, compositions, or methods, the amino acid
sequence of the presenter protein has been modified to substitute
at least one native amino acid with a reactive amino acid (e.g., a
natural amino acid such as a cysteine, lysine, tyrosine, aspartic
acid, glutamic acid, or serine, or a non-natural amino acid). In
some embodiments of any of the foregoing compounds, conjugates,
complexes, compositions, or methods, the amino acid sequence of the
presenter protein has been modified to substitute at least one
native reactive amino acid (e.g., a cysteine, lysine, tyrosine,
aspartic acid, glutamic acid, or serine) with a non-reactive amino
acid (e.g., a natural amino acid such as a serine, valine, alanine,
isoleucine, threonine, tyrosine, aspartic acid, glutamic acid, or
leucine, or a non-natural amino acid). In some embodiments of any
of the foregoing compounds, conjugates, complexes, compositions, or
methods, the at least one native reactive amino acid is a solvent
exposed amino acid. In some embodiments of any of the foregoing
compounds, conjugates, complexes, compositions, or methods, the
amino acid sequence of the presenter protein is modified to
substitute all reactive amino acids with a non-reactive amino acid.
In some embodiments of any of the foregoing compounds, conjugates,
complexes, compositions, or methods, the substitution is a
conservative substitution.
[0136] In some embodiments of any of the foregoing compounds,
conjugates, complexes, compositions, or methods, the linker is 1 to
20 atoms in length. In some embodiments of any of the foregoing
compounds, conjugates, complexes, compositions, or methods, the
linker is 1.5 to 30 angstroms in length.
[0137] In some embodiments of any of the foregoing compounds,
conjugates, complexes, compositions, or methods, the linker has the
structure of Formula V:
A.sup.1-(B.sup.1).sub.f--(C.sup.1).sub.g--(B.sup.2).sub.h-(D)-(B.sup.3).-
sub.i--(C.sup.2).sub.j--(B.sup.4).sub.k-A.sup.2 Formula V
[0138] wherein A.sup.1 is a bond between the linker and protein
binding moiety; A.sup.2 is a bond between the cross-linking group
and the linker; B.sup.1, B.sup.2, B.sup.3, and B.sup.4 each,
independently, is selected from optionally substituted
C.sub.1-C.sub.2 alkyl, optionally substituted C.sub.1-C.sub.3
heteroalkyl, O, S, and NR.sup.N; R.sup.N is hydrogen, optionally
substituted C.sub.1-4 alkyl, optionally substituted C.sub.2-4
alkenyl, optionally substituted C.sub.2-4 alkynyl, optionally
substituted C.sub.2-6 heterocyclyl, optionally substituted
C.sub.6-12 aryl, or optionally substituted C.sub.1-7 heteroalkyl;
C.sub.1 and C.sup.2 are each, independently, selected from
carbonyl, thiocarbonyl, sulphonyl, or phosphoryl; f, g, h, l, j,
and k are each, independently, 0 or 1; and D is optionally
substituted C.sub.1-10 alkyl, optionally substituted C.sub.2-10
alkenyl, optionally substituted C.sub.2-10 alkynyl, optionally
substituted C.sub.2-6 heterocyclyl, optionally substituted
C.sub.6-12 aryl, optionally substituted C.sub.2-C.sub.10
polyethylene glycol, or optionally substituted C.sub.1-10
heteroalkyl, or a chemical bond linking
A.sup.1-(B.sup.1).sub.f--(C.sup.1).sub.g--(B.sup.2).sub.h-- to
--(B.sup.3).sub.i--(C.sup.2).sub.j--(B.sup.4).sub.k-A.sup.2.
[0139] In some embodiments of any of the foregoing compounds,
conjugates, complexes, compositions, or methods, the linker
includes the structure of Formula VI:
##STR00019##
[0140] wherein A.sup.1 is a bond between the linker and protein
binding moiety;
[0141] A.sup.2 is a bond between the cross-linking group and the
linker;
[0142] l is 0, 1, 2, or 3;
[0143] m is 0 or 1;
[0144] n is 0, 1, or 2; and
[0145] X.sup.3, X.sup.4, and X.sup.5 are each, independently,
absent, O, S, --C.dbd.C--, CR.sup.9R.sup.10 or NR.sup.11; and
[0146] each R.sup.9, R.sup.10, and R.sup.11 are, independently,
hydrogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally
substituted C.sub.2-C.sub.6 alkenyl, optionally substituted
C.sub.2-C.sub.6 alkynyl, optionally substituted aryl,
C.sub.3-C.sub.7 carbocyclyl, optionally substituted
C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, and optionally
substituted C.sub.3-C.sub.7 carbocyclyl C.sub.1-C.sub.6 alkyl. In
some embodiments, each R.sup.9, R.sup.10, and R.sup.11 are,
independently, hydrogen, unsubstituted C.sub.1-C.sub.6 alkyl,
unsubstituted C.sub.2-C.sub.6 alkenyl, unsubstituted
C.sub.2-C.sub.6 alkynyl, unsubstituted aryl,
C.sub.3-C.sub.7carbocyclyl, unsubstituted C.sub.6-C.sub.10 aryl
C.sub.1-C.sub.6 alkyl, and unsubstituted C.sub.3-C.sub.7carbocyclyl
C.sub.1-C.sub.6 alkyl.
[0147] In some embodiments of any of the foregoing compounds,
conjugates, complexes, compositions, or methods, the linker
includes the structure:
##STR00020##
Chemical Terms
[0148] Those skilled in the art will appreciate that certain
compounds described herein can exist in one or more different
isomeric (e.g., stereoisomers, geometric isomers, tautomers) and/or
isotopic (e.g., in which one or more atoms has been substituted
with a different isotope of the atom, such as hydrogen substituted
for deuterium) forms. Unless otherwise indicated or clear from
context, a depicted structure can be understood to represent any
such isomeric or isotopic form, individually or in combination.
[0149] Compounds described herein can be asymmetric (e.g., having
one or more stereocenters). All stereoisomers, such as enantiomers
and diastereomers, are intended unless otherwise indicated.
Compounds of the present disclosure that contain asymmetrically
substituted carbon atoms can be isolated in optically active or
racemic forms. Methods on how to prepare optically active forms
from optically active starting materials are known in the art, such
as by resolution of racemic mixtures or by stereoselective
synthesis. Many geometric isomers of olefins, C.dbd.N double bonds,
and the like can also be present in the compounds described herein,
and all such stable isomers are contemplated in the present
disclosure. Cis and trans geometric isomers of the compounds of the
present disclosure are described and may be isolated as a mixture
of isomers or as separated isomeric forms.
[0150] In some embodiments, one or more compounds depicted herein
may exist in different tautomeric forms. As will be clear from
context, unless explicitly excluded, references to such compounds
encompass all such tautomeric forms. In some embodiments,
tautomeric forms result from the swapping of a single bond with an
adjacent double bond and the concomitant migration of a proton. In
certain embodiments, a tautomeric form may be a prototropic
tautomer, which is an isomeric protonation states having the same
empirical formula and total charge as a reference form. Examples of
moieties with prototropic tautomeric forms are ketone--enol pairs,
amide--imidic acid pairs, lactam--lactim pairs, amide--imidic acid
pairs, enamine--imine pairs, and annular forms where a proton can
occupy two or more positions of a heterocyclic system, such as, 1H-
and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and
2H-isoindole, and 1H- and 2H-pyrazole. In some embodiments,
tautomeric forms can be in equilibrium or sterically locked into
one form by appropriate substitution. In certain embodiments,
tautomeric forms result from acetal interconversion, e.g., the
interconversion illustrated in the scheme below:
##STR00021##
[0151] Those skilled in the art will appreciate that, in some
embodiments, isotopes of compounds described herein may be prepared
and/or utilized in accordance with the present invention.
"Isotopes" refers to atoms having the same atomic number but
different mass numbers resulting from a different number of
neutrons in the nuclei. For example, isotopes of hydrogen include
tritium and deuterium. In some embodiments, an isotopic
substitution (e.g., substitution of hydrogen with deuterium) may
alter the physicochemical properties of the molecules, such as
metabolism and/or the rate of racemization of a chiral center.
[0152] As is known in the art, many chemical entities (in
particular many organic molecules and/or many small molecules) can
adopt a variety of different solid forms such as, for example,
amorphous forms and/or crystalline forms (e.g., polymorphs,
hydrates, solvates, etc). In some embodiments, such entities may be
utilized in any form, including in any solid form. In some
embodiments, such entities are utilized in a particular form, for
example in a particular solid form.
[0153] In some embodiments, compounds described and/or depicted
herein may be provided and/or utilized in salt form.
[0154] In certain embodiments, compounds described and/or depicted
herein may be provided and/or utilized in hydrate or solvate
form.
[0155] At various places in the present specification, substituents
of compounds of the present disclosure are disclosed in groups or
in ranges. It is specifically intended that the present disclosure
include each and every individual subcombination of the members of
such groups and ranges. For example, the term "C.sub.1-6 alkyl" is
specifically intended to individually disclose methyl, ethyl,
C.sub.3 alkyl, C.sub.4 alkyl, C.sub.5 alkyl, and C alkyl.
Furthermore, where a compound includes a plurality of positions at
which substitutes are disclosed in groups or in ranges, unless
otherwise indicated, the present disclosure is intended to cover
individual compounds and groups of compounds (e.g., genera and
subgenera) containing each and every individual subcombination of
members at each position.
[0156] Herein a phrase of the form "optionally substituted X"
(e.g., optionally substituted alkyl) is intended to be equivalent
to "X, wherein X is optionally substituted" (e.g., "alkyl, wherein
said alkyl is optionally substituted"). It is not intended to mean
that the feature "X" (e.g. alkyl) per se is optional.
[0157] The term "alkyl," as used herein, refers to saturated
hydrocarbon groups containing from 1 to 20 (e.g., from 1 to 10 or
from 1 to 6) carbons. In some embodiments, an alkyl group is
unbranched (i.e., is linear); in some embodiments, an alkyl group
is branched. Alkyl groups are exemplified by methyl, ethyl, n- and
iso-propyl, n-, sec-, iso- and tert-butyl, neopentyl, and the like,
and may be optionally substituted with one, two, three, or, in the
case of alkyl groups of two carbons or more, four substituents
independently selected from the group consisting of: (1) C.sub.1-6
alkoxy; (2) C.sub.1-6 alkylsulfinyl; (3) amino, as defined herein
(e.g., unsubstituted amino (i.e., --NH.sub.2) or a substituted
amino (i.e., --N(R.sup.N1).sub.2, where R.sup.N1 is as defined for
amino); (4) C.sub.6-10 aryl-C.sub.1-6 alkoxy; (5) azido; (6) halo;
(7) (C.sub.2-9 heterocyclyl)oxy; (8) hydroxyl, optionally
substituted with an O-protecting group; (9) nitro; (10) oxo (e.g.,
carboxyaldehyde or acyl); (11) C.sub.1-7 spirocyclyl; (12)
thioalkoxy; (13) thiol; (14) --CO.sub.2R.sup.A', optionally
substituted with an O-protecting group and where R.sup.A' is
selected from the group consisting of (a) C.sub.1-20 alkyl (e.g.,
C.sub.1-6 alkyl), (b) C.sub.2-20 alkenyl (e.g., C.sub.2-6 alkenyl),
(c) C.sub.6-10 aryl, (d) hydrogen, (e) C.sub.1-6 alk-C.sub.6-10
aryl, (f) amino-C.sub.1-20 alkyl, (g) polyethylene glycol of
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl, and (h)
amino-polyethylene glycol of
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1' wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer
from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1 to 10), and each R.sup.N1 is, independently,
hydrogen or optionally substituted C.sub.1-6 alkyl; (15)
--C(O)NR.sup.B'R.sup.C', where each of R.sup.B' and R.sup.C' is,
independently, selected from the group consisting of (a) hydrogen,
(b) C.sub.1-6 alkyl, (c) C.sub.6-10 aryl, and (d) C.sub.1-6
alk-C.sub.6-10 aryl; (16) --SO.sub.2R.sup.D', where R.sup.D' is
selected from the group consisting of (a) C.sub.1-6 alkyl, (b)
C.sub.6-10 aryl, (c) C.sub.1-6 alk-C.sub.6-10 aryl, and (d)
hydroxyl; (17) --SO.sub.2NR.sup.E'R.sup.F' where each of R.sup.E'
and R.sup.F' is, independently, selected from the group consisting
of (a) hydrogen, (b) C.sub.1-6 alkyl, (c) C.sub.6-10 aryl and (d)
C.sub.1-6 alk-C.sub.6-10 aryl; (18) --C(O)R.sup.G', where R.sup.G'
is selected from the group consisting of (a) C.sub.1-20 alkyl
(e.g., C.sub.1-6 alkyl), (b) C.sub.2-20 alkenyl (e.g., C.sub.2-6
alkenyl), (c) C.sub.6-10 aryl, (d) hydrogen, (e) C.sub.1-6
alk-C.sub.6-10 aryl, (f) amino-C.sub.1-20 alkyl, (g) polyethylene
glycol of
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl, and (h)
amino-polyethylene glycol of
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from to 4), each of s2 and s3, independently, is an integer from
0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6,
or from 1 to 10), and each R.sup.N1 is, independently, hydrogen or
optionally substituted C.sub.1-6 alkyl; (19) --NR.sup.H'C(O)R',
wherein R.sup.H' is selected from the group consisting of (a1)
hydrogen and (b1) C.sub.1-6 alkyl, and R.sup.i' is selected from
the group consisting of (a2) C.sub.1-20 alkyl (e.g., C.sub.1-6
alkyl), (b2) C.sub.2-20 alkenyl (e.g., C.sub.2-6 alkenyl), (c2)
C.sub.6-10 aryl, (d2) hydrogen, (e2) C.sub.1-6 alk-C.sub.6-10 aryl,
(f2) amino-C.sub.1-20 alkyl, (g2) polyethylene glycol of
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3O-
R', wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or
from 1 to 4), each of s2 and s3, independently, is an integer from
0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6,
or from 1 to 10), and R' is H or C.sub.1-20 alkyl, and (h2)
amino-polyethylene glycol of
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 an integer from 1 to 10 (e.g., from 1 to 6 or
from 1 to 4), each of s2 and s3, independently, is an integer from
0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6,
or from 1 to 10), and each R.sup.N1 is, independently, hydrogen or
optionally substituted C.sub.1-6 alkyl; (20)
--NR.sup.J'C(O)OR.sup.K', wherein R.sup.J' is selected from the
group consisting of (a1) hydrogen and (b1) C.sub.1-6 alkyl, and
R.sup.K' is selected from the group consisting of (a2) C.sub.1-20
alkyl (e.g., C.sub.1-6 alkyl), (b2) C.sub.2-20 alkenyl (e.g.,
C.sub.2-6 alkenyl), (c2) C.sub.6-10 aryl, (d2) hydrogen, (e2)
C.sub.1-6 alk-C.sub.6-10 aryl, (f2) amino-C.sub.1-20 alkyl, (g2)
polyethylene glycol of
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl, and (h2)
amino-polyethylene glycol of
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer
from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1 to 10), and each R.sup.N1 is, independently,
hydrogen or optionally substituted C.sub.1-6 alkyl; (21) amidine;
and (22) silyl groups such as trimethylsilyl, t-butyldimethylsilyl,
and tri-isopropylsilyl. In some embodiments, each of these groups
can be further substituted as described herein. For example, the
alkylene group of a C.sub.1-alkaryl can be further substituted with
an oxo group to afford the respective aryloyl substituent.
[0158] The term "alkylene" and the prefix "alk-," as used herein,
represent a saturated divalent hydrocarbon group derived from a
straight or branched chain saturated hydrocarbon by the removal of
two hydrogen atoms, and is exemplified by methylene, ethylene,
isopropylene, and the like. The term "C.sub.x-y alkylene" and the
prefix "C.sub.x-y alk-" represent alkylene groups having between x
and y carbons. Exemplary values for x are 1, 2, 3, 4, 5, and 6, and
exemplary values for y are 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16,
18, or 20 (e.g., C.sub.1-6, C.sub.1-10, C.sub.2-20, C.sub.2-6,
C.sub.2-10, or C.sub.2-20 alkylene). In some embodiments, the
alkylene can be further substituted with 1, 2, 3, or 4 substituent
groups as defined herein for an alkyl group.
[0159] The term "alkenyl," as used herein, represents monovalent
straight or branched chain groups of, unless otherwise specified,
from 2 to 20 carbons (e.g., from 2 to 6 or from 2 to 10 carbons)
containing one or more carbon-carbon double bonds and is
exemplified by ethenyl, 1-propenyl, 2-propenyl,
2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like. Alkenyls
include both cis and trans isomers. Alkenyl groups may be
optionally substituted with 1, 2, 3, or 4 substituent groups that
are selected, independently, from amino, aryl, cycloalkyl, or
heterocyclyl (e.g., heteroaryl), as defined herein, or any of the
exemplary alkyl substituent groups described herein.
[0160] The term "alkynyl," as used herein, represents monovalent
straight or branched chain groups from 2 to 20 carbon atoms (e.g.,
from 2 to 4, from 2 to 6, or from 2 to 10 carbons) containing a
carbon-carbon triple bond and is exemplified by ethynyl,
1-propynyl, and the like. Alkynyl groups may be optionally
substituted with 1, 2, 3, or 4 substituent groups that are
selected, independently, from aryl, cycloalkyl, or heterocyclyl
(e.g., heteroaryl), as defined herein, or any of the exemplary
alkyl substituent groups described herein.
[0161] The term "amino," as used herein, represents
--N(R.sup.N1).sub.2, wherein each R.sup.N1 is, independently, H,
OH, NO.sub.2, N(R.sup.N2).sub.2, SO.sub.2OR.sup.N2,
SO.sub.2R.sup.N2, SOR.sup.N2, an N-protecting group, alkyl,
alkenyl, alkynyl, alkoxy, aryl, alkaryl, cycloalkyl, alkcycloalkyl,
carboxyalkyl (e.g., optionally substituted with an O-protecting
group, such as optionally substituted arylalkoxycarbonyl groups or
any described herein), sulfoalkyl, acyl (e.g., acetyl,
trifluoroacetyl, or others described herein), alkoxycarbonylalkyl
(e.g., optionally substituted with an O-protecting group, such as
optionally substituted arylalkoxycarbonyl groups or any described
herein), heterocyclyl (e.g., heteroaryl), or alkheterocyclyl (e.g.,
alkheteroaryl), wherein each of these recited R.sup.N1 groups can
be optionally substituted, as defined herein for each group; or two
R.sup.N1 combine to form a heterocyclyl or an N-protecting group,
and wherein each R.sup.N2 is, independently, H, alkyl, or aryl. The
amino groups of the invention can be an unsubstituted amino (i.e.,
--NH.sub.2) or a substituted amino (i.e., --N(R.sup.N1).sub.2). In
a preferred embodiment, amino is --NH.sub.2 or --NHR.sup.N1,
wherein R.sup.N1 is, independently, OH, NO.sub.2, NH.sub.2,
NR.sup.N22, SO.sub.2OR.sup.N2, SO.sub.2R.sup.N2, SOR.sup.N2, alkyl,
carboxyalkyl, sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl, or
others described herein), alkoxycarbonylalkyl (e.g.,
t-butoxycarbonylalkyl) or aryl, and each R.sup.N2 can be H,
C.sub.1-20 alkyl (e.g., C.sub.1-6 alkyl), or C.sub.6-10 aryl.
[0162] The term "amino acid," as described herein, refers to a
molecule having a side chain, an amino group, and an acid group
(e.g., a carboxy group of --CO.sub.2H or a sulfo group of
--SO.sub.3H), wherein the amino acid is attached to the parent
molecular group by the side chain, amino group, or acid group
(e.g., the side chain). As used herein, the term "amino acid" in
its broadest sense, refers to any compound and/or substance that
can be incorporated into a polypeptide chain, e.g., through
formation of one or more peptide bonds. In some embodiments, an
amino acid has the general structure H.sub.2N--C(H)(R)--COOH. In
some embodiments, an amino acid is a naturally-occurring amino
acid. In some embodiments, an amino acid is a synthetic amino acid;
in some embodiments, an amino acid is a D-amino acid; in some
embodiments, an amino acid is an L-amino acid. "Standard amino
acid" refers to any of the twenty standard L-amino acids commonly
found in naturally occurring peptides. "Nonstandard amino acid"
refers to any amino acid, other than the standard amino acids,
regardless of whether it is prepared synthetically or obtained from
a natural source. In some embodiments, an amino acid, including a
carboxy- and/or amino-terminal amino acid in a polypeptide, can
contain a structural modification as compared with the general
structure above. For example, in some embodiments, an amino acid
may be modified by methylation, amidation, acetylation, and/or
substitution as compared with the general structure. In some
embodiments, such modification may, for example, alter the
circulating half life of a polypeptide containing the modified
amino acid as compared with one containing an otherwise identical
unmodified amino acid. In some embodiments, such modification does
not significantly alter a relevant activity of a polypeptide
containing the modified amino acid, as compared with one containing
an otherwise identical unmodified amino acid. As will be clear from
context, in some embodiments, the term "amino acid" is used to
refer to a free amino acid; in some embodiments it is used to refer
to an amino acid residue of a polypeptide. In some embodiments, the
amino acid is attached to the parent molecular group by a carbonyl
group, where the side chain or amino group is attached to the
carbonyl group. In some embodiments, the amino acid is an
.alpha.-amino acid. In certain embodiments, the amino acid is a
3-amino acid. In some embodiments, the amino acid is a
.gamma.-amino acid. Exemplary side chains include an optionally
substituted alkyl, aryl, heterocyclyl, alkaryl, alkheterocyclyl,
aminoalkyl, carbamoylalkyl, and carboxyalkyl. Exemplary amino acids
include alanine, arginine, asparagine, aspartic acid, cysteine,
glutamic acid, glutamine, glycine, histidine, hydroxynorvaline,
isoleucine, leucine, lysine, methionine, norvaline, ornithine,
phenylalanine, proline, pyrrolysine, selenocysteine, serine,
taurine, threonine, tryptophan, tyrosine, and valine. Amino acid
groups may be optionally substituted with one, two, three, or, in
the case of amino acid groups of two carbons or more, four
substituents independently selected from the group consisting of:
(1) C.sub.1-6 alkoxy; (2) C.sub.1-6 alkylsulfinyl; (3) amino, as
defined herein (e.g., unsubstituted amino (i.e., --NH.sub.2) or a
substituted amino (i.e., --N(R.sup.N1).sub.2, where R.sup.N1 is as
defined for amino); (4) C.sub.6-10 aryl-C.sub.1-6 alkoxy; (5)
azido; (6) halo; (7) (C.sub.2-9 heterocyclyl)oxy; (8) hydroxyl; (9)
nitro; (10) oxo (e.g., carboxyaldehyde or acyl); (11) C.sub.1-7
spirocyclyl; (12) thioalkoxy; (13) thiol; (14) --CO.sub.2R.sup.A'
where R.sup.A' is selected from the group consisting of (a)
C.sub.1-20 alkyl (e.g., C.sub.1-6 alkyl), (b) C.sub.2-20 alkenyl
(e.g., C.sub.2-6 alkenyl), (c) C.sub.6-10 aryl, (d) hydrogen, (e)
C.sub.1-6 alk-C.sub.6-10 aryl, (f) amino-C.sub.1-20 alkyl, (g)
polyethylene glycol of
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl, and (h)
amino-polyethylene glycol of
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer
from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1 to 10), and each R.sup.N1 is, independently,
hydrogen or optionally substituted C.sub.1-6 alkyl; (15)
--C(O)NR.sup.B'R.sup.C', where each of R.sup.B' and R.sup.C' is,
independently, selected from the group consisting of (a) hydrogen,
(b) C.sub.1-6 alkyl, (c) C.sub.6-10 aryl, and (d) C.sub.1-6
alk-C.sub.6-10 aryl; (16) --SO.sub.2R.sup.D'' where R.sup.D' is
selected from the group consisting of (a) C.sub.1-6 alkyl, (b)
C.sub.6-10 aryl, (c) C.sub.1-6 alk-C.sub.6-10 aryl, and (d)
hydroxyl; (17) --SO.sub.2NR.sup.E'R.sup.F' where each of R.sup.E'
and R.sup.F' is, independently, selected from the group consisting
of (a) hydrogen, (b) C.sub.1-6 alkyl, (c) C.sub.6-10 aryl and (d)
C.sub.1-6 alk-C.sub.6-10 aryl; (18) --C(O)R.sup.G', where R.sup.G'
is selected from the group consisting of (a) C.sub.1-20 alkyl
(e.g., C.sub.1-6 alkyl), (b) C.sub.2-20 alkenyl (e.g., C.sub.2-6
alkenyl), (c) C.sub.6-10 aryl, (d) hydrogen, (e) C.sub.1-6
alk-C.sub.6-10 aryl, (f) amino-C.sub.1-20 alkyl, (g) polyethylene
glycol of
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl, and (h)
amino-polyethylene glycol of
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer
from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1 to 10), and each R.sup.N1 is, independently,
hydrogen or optionally substituted C.sub.1-6 alkyl; (19)
--NR.sup.H'C(O)R', wherein R.sup.H' is selected from the group
consisting of (a1) hydrogen and (b1) C.sub.1-6 alkyl, and R.sup.i'
is selected from the group consisting of (a2) C.sub.1-20 alkyl
(e.g., C.sub.1-6 alkyl), (b2) C.sub.2-20 alkenyl (e.g., C.sub.2-6
alkenyl), (c2) C.sub.6-10 aryl, (d2) hydrogen, (e2) C.sub.1-6
alk-C.sub.6-10 aryl, (f2) amino-C.sub.1-20 alkyl, (g2) polyethylene
glycol of
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3O-
R', wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or
from 1 to 4), each of s2 and s3, independently, is an integer from
0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6,
or from 1 to 10), and R' is H or C.sub.1-20 alkyl, and (h2)
amino-polyethylene glycol of
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 an integer from 1 to 10 (e.g., from 1 to 6 or
from to 4), each of s2 and s3, independently, is an integer from 0
to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or
from 1 to 10), and each R.sup.N1 is, independently, hydrogen or
optionally substituted C.sub.1-6 alkyl; (20)
--NR.sup.J'C(O)OR.sup.K', wherein R.sup.J' is selected from the
group consisting of (a1) hydrogen and (b1) C.sub.1-6 alkyl, and
R.sup.K' is selected from the group consisting of (a2) C.sub.1-20
alkyl (e.g., C.sub.1-6 alkyl), (b2) 02-20 alkenyl (e.g., 02-6
alkenyl), (c2) C.sub.6-10 aryl, (d2) hydrogen, (e2) C.sub.1-6
alk-C.sub.6-10 aryl, (f2) amino-C.sub.1-20 alkyl, (g2) polyethylene
glycol of
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3O-
R', wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or
from 1 to 4), each of s2 and s3, independently, is an integer from
0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6,
or from 1 to 10), and R' is H or C.sub.1-20 alkyl, and (h2)
amino-polyethylene glycol of
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from to 4), each of s2 and s3, independently, is an integer from
0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6,
or from 1 to 10), and each R.sup.N1 is, independently, hydrogen or
optionally substituted C.sub.1-6 alkyl; and (21) amidine. In some
embodiments, each of these groups can be further substituted as
described herein.
[0163] The term "N-alkylated amino acids" as used herein, refers to
amino acids containing an optionally substituted C.sub.1 to C.sub.6
alkyl on the nitrogen of the amino acid that forms the peptidic
bond. N-alkylated amino acids include, but are not limited to,
N-methyl amino acids, such as N-methyl-alanine, N-methyl-threonine,
N-methyl-phenylalanine, N-methyl-aspartic acid, N-methyl-valine,
N-methyl-leucine, N-methyl-glycine, N-methyl-isoleucine,
N(.alpha.)-methyl-lysine, N(.alpha.)-methyl-asparagine, and
N(.alpha.)-methyl-glutamine.
[0164] The term "aryl," as used herein, represents a mono-,
bicyclic, or multicyclic carbocyclic ring system having one or two
aromatic rings and is exemplified by phenyl, naphthyl,
1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, anthracenyl,
phenanthrenyl, fluorenyl, indanyl, indenyl, and the like, and may
be optionally substituted with 1, 2, 3, 4, or 5 substituents
independently selected from the group consisting of: (1) C.sub.1-7
acyl (e.g., carboxyaldehyde); (2) C.sub.1-20 alkyl (e.g., C.sub.1-6
alkyl, C.sub.1-6 alkoxy-C.sub.1-6 alkyl, C.sub.1-6
alkylsulfinyl-C.sub.1-6 alkyl, amino-C.sub.1-6 alkyl,
azido-C.sub.1-6 alkyl, (carboxyaldehyde)-C.sub.1-6 alkyl,
halo-C.sub.1-6 alkyl (e.g., perfluoroalkyl), hydroxy-C.sub.1-6
alkyl, nitro-C.sub.1-6 alkyl, or C.sub.1-6 thioalkoxy-C.sub.1-6
alkyl); (3) C.sub.1-20 alkoxy (e.g., C.sub.1-6 alkoxy, such as
perfluoroalkoxy); (4) C.sub.1-6 alkylsulfinyl; (5) C.sub.6-10 aryl;
(6) amino; (7) C.sub.1-6 alk-C.sub.6-10 aryl; (8) azido; (9)
C.sub.3-8 cycloalkyl; (10) C.sub.1-6 alk-C.sub.3-8 cycloalkyl; (11)
halo; (12) C.sub.1-12 heterocyclyl (e.g., C.sub.1-12 heteroaryl);
(13) (C.sub.1-12 heterocyclyl)oxy; (14) hydroxyl; (15) nitro; (16)
C.sub.1-20 thioalkoxy (e.g., C.sub.1-6 thioalkoxy); (17)
--(CH.sub.2).sub.qCO.sub.2R.sup.A', where q is an integer from zero
to four, and R.sup.A' is selected from the group consisting of (a)
C.sub.1-6 alkyl, (b) C.sub.6-10 aryl, (c) hydrogen, and (d)
C.sub.1-6 alk-C.sub.6-10 aryl; (18)
--(CH.sub.2).sub.qCONR.sup.B'R.sup.C', where q is an integer from
zero to four and where R.sup.B' and R.sup.C' are independently
selected from the group consisting of (a) hydrogen, (b) C.sub.1-6
alkyl, (c) C.sub.6-10 aryl, and (d) C.sub.1-6 alk-C.sub.6-10 aryl;
(19) --(CH.sub.2).sub.qSO.sub.2R.sup.D', where q is an integer from
zero to four and where R.sup.D' is selected from the group
consisting of (a) alkyl, (b) C.sub.6-10 aryl, and (c)
alk-C.sub.6-10 aryl; (20)
--(CH.sub.2).sub.qSO.sub.2NR.sup.E'R.sup.F, where q is an integer
from zero to four and where each of R.sup.E' and R.sup.F' is,
independently, selected from the group consisting of (a) hydrogen,
(b) C.sub.1-6 alkyl, (c) C.sub.6-10 aryl, and (d) C.sub.1-6
alk-C.sub.6-10 aryl; (21) thiol; (22) C.sub.6-10 aryloxy; (23)
C.sub.3-8 cycloalkoxy; (24) C.sub.6-10 aryl-C.sub.1-6 alkoxy; (25)
C.sub.1-6 alk-C.sub.1-12 heterocyclyl (e.g., C.sub.1-6
alk-C.sub.1-12 heteroaryl); (26) C.sub.2-20 alkenyl; and (27)
C.sub.2-20 alkynyl. In some embodiments, each of these groups can
be further substituted as described herein. For example, the
alkylene group of a C.sub.1-alkaryl or a C.sub.1-alkheterocyclyl
can be further substituted with an oxo group to afford the
respective aryloyl and (heterocyclyl)oyl substituent group.
[0165] The "arylalkyl" group, which as used herein, represents an
aryl group, as defined herein, attached to the parent molecular
group through an alkylene group, as defined herein. Exemplary
unsubstituted arylalkyl groups are from 7 to 30 carbons (e.g., from
7 to 16 or from 7 to 20 carbons, such as C.sub.1-6 alk-C.sub.6-10
aryl, C.sub.1-10 alk-C.sub.6-10 aryl, or C.sub.1-20 alk-C.sub.6-10
aryl). In some embodiments, the alkylene and the aryl each can be
further substituted with 1, 2, 3, or 4 substituent groups as
defined herein for the respective groups. Other groups preceded by
the prefix "alk-" are defined in the same manner, where "alk"
refers to a C.sub.1-6 alkylene, unless otherwise noted, and the
attached chemical structure is as defined herein.
[0166] The term "azido" represents an --N.sub.3 group, which can
also be represented as --N.dbd.N.dbd.N.
[0167] The terms "carbocyclic" and "carbocyclyl," as used herein,
refer to an optionally substituted C.sub.3-12 monocyclic, bicyclic,
or tricyclic non-aromatic ring structure in which the rings are
formed by carbon atoms. Carbocyclic structures include cycloalkyl,
cycloalkenyl, and cycloalkynyl groups.
[0168] The "carbocyclylalkyl" group, which as used herein,
represents a carbocyclic group, as defined herein, attached to the
parent molecular group through an alkylene group, as defined
herein. Exemplary unsubstituted carbocyclylalkyl groups are from 7
to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as
C.sub.1-6 alk-C.sub.6-10 carbocyclyl, C.sub.1-10 alk-C.sub.6-10
carbocyclyl, or C.sub.1-20 alk-C.sub.6-10 carbocyclyl). In some
embodiments, the alkylene and the carbocyclyl each can be further
substituted with 1, 2, 3, or 4 substituent groups as defined herein
for the respective groups. Other groups preceded by the prefix
"alk-" are defined in the same manner, where "alk" refers to a
C.sub.1-6 alkylene, unless otherwise noted, and the attached
chemical structure is as defined herein.
[0169] The term "carbonyl," as used herein, represents a C(O)
group, which can also be represented as C.dbd.O.
[0170] The term "carboxy," as used herein, means --CO.sub.2H.
[0171] The term "cyano," as used herein, represents an --CN
group.
[0172] The term "cycloalkyl," as used herein represents a
monovalent saturated or unsaturated non-aromatic cyclic hydrocarbon
group from three to eight carbons, unless otherwise specified, and
is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl, bicycle heptyl, and the like.
[0173] When the cycloalkyl group includes one carbon-carbon double
bond, the cycloalkyl group can be referred to as a "cycloalkenyl"
group. Exemplary cycloalkenyl groups include cyclopentenyl,
cyclohexenyl, and the like. The cycloalkyl groups of this invention
can be optionally substituted with: (1) C.sub.1-7 acyl (e.g.,
carboxyaldehyde); (2) C.sub.1-20 alkyl (e.g., C.sub.1-6 alkyl,
C.sub.1-6 alkoxy-C.sub.1-6 alkyl, C.sub.1-6 alkylsulfinyl-C.sub.1-6
alkyl, amino-C.sub.1-6 alkyl, azido-C.sub.1-6 alkyl,
(carboxyaldehyde)-C.sub.1-6 alkyl, halo-C.sub.1-6 alkyl (e.g.,
perfluoroalkyl), hydroxy-C.sub.1-6 alkyl, nitro-C.sub.1-6 alkyl, or
C.sub.1-6 thioalkoxy-C.sub.1-6 alkyl); (3) C.sub.1-20 alkoxy (e.g.,
C.sub.1-6 alkoxy, such as perfluoroalkoxy); (4) C.sub.1-6
alkylsulfinyl; (5) C.sub.6-10 aryl; (6) amino; (7) C.sub.1-6
alk-C.sub.6-10 aryl; (8) azido; (9) C.sub.3-8 cycloalkyl; (10)
C.sub.1-6 alk-C.sub.3-8 cycloalkyl; (11) halo; (12) C.sub.1-12
heterocyclyl (e.g., C.sub.1-12 heteroaryl); (13) (C.sub.1-12
heterocyclyl)oxy; (14) hydroxyl; (15) nitro; (16) C.sub.1-20
thioalkoxy (e.g., C.sub.1-6 thioalkoxy); (17)
--(CH.sub.2).sub.qCO.sub.2R.sup.A' where q is an integer from zero
to four, and R.sup.A' is selected from the group consisting of (a)
C.sub.1-6 alkyl, (b) C.sub.6-10 aryl, (c) hydrogen, and (d)
C.sub.1-6 alk-C.sub.6-10 aryl; (18)
--(CH.sub.2).sub.qCONR.sup.B'R.sup.C', where q is an integer from
zero to four and where R.sup.B' and R.sup.C' are independently
selected from the group consisting of (a) hydrogen, (b) C.sub.6-10
alkyl, (c) C.sub.6-10 aryl, and (d) C.sub.1-6 alk-C.sub.6-10 aryl;
(19) --(CH.sub.2).sub.qSO.sub.2R.sup.D', where q is an integer from
zero to four and where R.sup.D' is selected from the group
consisting of (a) C.sub.6-10 alkyl, (b) C.sub.6-10 aryl, and (c)
C.sub.1-6 alk-C.sub.6-10 aryl; (20)
--(CH.sub.2).sub.qSO.sub.2NR.sup.E'R.sup.F', where q is an integer
from zero to four and where each of R.sup.E' and R.sup.F' is
independently, selected from the group consisting of (a) hydrogen,
(b) C.sub.6-10 alkyl, (c) C.sub.6-10 aryl, and (d) C.sub.1-6
alk-C.sub.6-10 aryl; (21) thiol; (22) C.sub.6-10 aryloxy; (23)
C.sub.3-8 cycloalkoxy; (24) C.sub.6-10 aryl-C.sub.1-6 alkoxy; (25)
C.sub.1-6 alk-C.sub.1-12 heterocyclyl (e.g., C.sub.1-6
alk-C.sub.1-12 heteroaryl); (26) oxo; (27) C.sub.2-20 alkenyl; and
(28) C.sub.2-20 alkynyl. In some embodiments, each of these groups
can be further substituted as described herein. For example, the
alkylene group of a C.sub.1-alkaryl or a C.sub.1-alkheterocyclyl
can be further substituted with an oxo group to afford the
respective aryloyl and (heterocyclyl)oyl substituent group.
[0174] The "cycloalkylalkyl" group, which as used herein,
represents a cycloalkyl group, as defined herein, attached to the
parent molecular group through an alkylene group, as defined herein
(e.g., an alkylene group of from 1 to 4, from 1 to 6, from 1 to 10,
or form 1 to 20 carbons). In some embodiments, the alkylene and the
cycloalkyl each can be further substituted with 1, 2, 3, or 4
substituent groups as defined herein for the respective group.
[0175] The term "diastereomer," as used herein means stereoisomers
that are not mirror images of one another and are
non-superimposable on one another.
[0176] The term "enantiomer," as used herein, means each individual
optically active form of a compound of the invention, having an
optical purity or enantiomeric excess (as determined by methods
standard in the art) of at least 80% (i.e., at least 90% of one
enantiomer and at most 10% of the other enantiomer), preferably at
least 90% and more preferably at least 98%.
[0177] The term "halo," as used herein, represents a halogen
selected from bromine, chlorine, iodine, or fluorine.
[0178] The term "heteroalkyl," as used herein, refers to an alkyl
group, as defined herein, in which one or two of the constituent
carbon atoms have each been replaced by nitrogen, oxygen, or
sulfur. In some embodiments, the heteroalkyl group can be further
substituted with 1, 2, 3, or 4 substituent groups as described
herein for alkyl groups. The terms "heteroalkenyl" and
heteroalkynyl," as used herein refer to alkenyl and alkynyl groups,
as defined herein, respectively, in which one or two of the
constituent carbon atoms have each been replaced by nitrogen,
oxygen, or sulfur. In some embodiments, the heteroalkenyl and
heteroalkynyl groups can be further substituted with 1, 2, 3, or 4
substituent groups as described herein for alkyl groups.
[0179] The term "heteroaryl," as used herein, represents that
subset of heterocyclyls, as defined herein, which are aromatic:
i.e., they contain 4n+2 pi electrons within the mono- or
multicyclic ring system. Exemplary unsubstituted heteroaryl groups
are of 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2
to 10, or 2 to 9) carbons. In some embodiment, the heteroaryl is
substituted with 1, 2, 3, or 4 substituents groups as defined for a
heterocyclyl group.
[0180] The term "heteroarylalkyl" refers to a heteroaryl group, as
defined herein, attached to the parent molecular group through an
alkylene group, as defined herein. Exemplary unsubstituted
heteroarylalkyl groups are from 2 to 32 carbons (e.g., from 2 to
22, from 2 to 18, from 2 to 17, from 2 to 16, from 3 to 15, from 2
to 14, from 2 to 13, or from 2 to 12 carbons, such as C.sub.1-6
alk-C.sub.1-12 heteroaryl, C.sub.1-10 alk-C.sub.1-12 heteroaryl, or
C.sub.1-20 alk-C.sub.1-12 heteroaryl). In some embodiments, the
alkylene and the heteroaryl each can be further substituted with 1,
2, 3, or 4 substituent groups as defined herein for the respective
group. Heteroarylalkyl groups are a subset of heterocyclylalkyl
groups.
[0181] The term "heterocyclyl," as used herein represents a 5-, 6-
or 7-membered ring, unless otherwise specified, containing one,
two, three, or four heteroatoms independently selected from the
group consisting of nitrogen, oxygen, and sulfur. The 5-membered
ring has zero to two double bonds, and the 6- and 7-membered rings
have zero to three double bonds. Exemplary unsubstituted
heterocyclyl groups are of 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9,
2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. The term
"heterocyclyl" also represents a heterocyclic compound having a
bridged multicyclic structure in which one or more carbons and/or
heteroatoms bridges two non-adjacent members of a monocyclic ring,
e.g., a quinuclidinyl group. The term "heterocyclyl" includes
bicyclic, tricyclic, and tetracyclic groups in which any of the
above heterocyclic rings is fused to one, two, or three carbocyclic
rings, e.g., an aryl ring, a cyclohexane ring, a cyclohexene ring,
a cyclopentane ring, a cyclopentene ring, or another monocyclic
heterocyclic ring, such as indolyl, quinolyl, isoquinolyl,
tetrahydroquinolyl, benzofuryl, benzothienyl and the like. Examples
of fused heterocyclyls include tropanes and
1,2,3,5,8,8a-hexahydroindolizine. Heterocyclics include pyrrolyl,
pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl,
imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, piperidinyl,
homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl,
oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidiniyl, morpholinyl,
thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl,
isothiazolidinyl, indolyl, indazolyl, quinolyl, isoquinolyl,
quinoxalinyl, dihydroquinoxalinyl, quinazolinyl, cinnolinyl,
phthalazinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl,
benzothiadiazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl,
triazolyl, tetrazolyl, oxadiazolyl (e.g., 1,2,3-oxadiazolyl),
purinyl, thiadiazolyl (e.g., 1,2,3-thiadiazolyl),
tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl,
dihydrothienyl, dihydroindolyl, dihydroquinolyl,
tetrahydroquinolyl, tetrahydroisoquinolyl, dihydroisoquinolyl,
pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl,
isobenzofuranyl, benzothienyl, and the like, including dihydro and
tetrahydro forms thereof, where one or more double bonds are
reduced and replaced with hydrogens. Still other exemplary
heterocyclyls include: 2,3,4,5-tetrahydro-2-oxo-oxazolyl;
2,3-dihydro-2-oxo-1H-imidazolyl;
2,3,4,5-tetrahydro-5-oxo-1H-pyrazolyl (e.g.,
2,3,4,5-tetrahydro-2-phenyl-5-oxo-1H-pyrazolyl);
2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (e.g.,
2,3,4,5-tetrahydro-2,4-dioxo-5-methyl-5-phenyl-1H-imidazolyl);
2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (e.g.,
2,3-dihydro-2-thioxo-5-phenyl-1,3,4-oxadiazolyl);
4,5-dihydro-5-oxo-1H-triazolyl (e.g., 4,5-dihydro-3-methyl-4-amino
5-oxo-1H-triazolyl); 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (e.g.,
1,2,3,4-tetrahydro-2,4-dioxo-3,3-diethylpyridinyl);
2,6-dioxo-piperidinyl (e.g.,
2,6-dioxo-3-ethyl-3-phenylpiperidinyl);
1,6-dihydro-6-oxopyridiminyl; 1,6-dihydro-4-oxopyrimidinyl (e.g.,
2-(methylthio)-1,6-dihydro-4-oxo-5-methylpyrimidin-1-yl);
1,2,3,4-tetrahydro-2,4-dioxopyrimidinyl (e.g.,
1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl);
1,6-dihydro-6-oxo-pyridazinyl (e.g.,
1,6-dihydro-6-oxo-3-ethylpyridazinyl);
1,6-dihydro-6-oxo-1,2,4-triazinyl (e.g.,
1,6-dihydro-5-isopropyl-6-oxo-1,2,4-triazinyl);
2,3-dihydro-2-oxo-1H-indolyl (e.g.,
3,3-dimethyl-2,3-dihydro-2-oxo-1H-indolyl and
2,3-dihydro-2-oxo-3,3'-spiropropane-1H-indol-1-yl);
1,3-dihydro-1-oxo-2H-iso-indolyl;
1,3-dihydro-1,3-dioxo-2H-iso-indolyl; 1H-benzopyrazolyl (e.g.,
1-(ethoxycarbonyl)-1H-benzopyrazolyl);
2,3-dihydro-2-oxo-1H-benzimidazolyl (e.g.,
3-ethyl-2,3-dihydro-2-oxo-1H-benzimidazolyl);
2,3-dihydro-2-oxo-benzoxazolyl (e.g.,
5-chloro-2,3-dihydro-2-oxo-benzoxazolyl);
2,3-dihydro-2-oxo-benzoxazolyl; 2-oxo-2H-benzopyranyl;
1,4-benzodioxanyl; 1,3-benzodioxanyl;
2,3-dihydro-3-oxo,4H-1,3-benzothiazinyl;
3,4-dihydro-4-oxo-3H-quinazolinyl (e.g.,
2-methyl-3,4-dihydro-4-oxo-3H-quinazolinyl);
1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (e.g.,
1-ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl);
1,2,3,6-tetrahydro-2,6-dioxo-7H-purinyl (e.g.,
1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-purinyl);
1,2,3,6-tetrahydro-2,6-dioxo-1H-purinyl (e.g.,
1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1H-purinyl);
2-oxobenz[c,d]indolyl; 1,1-dioxo-2H-naphth[1,8-c,d]isothiazolyl;
and 1,8-naphthylenedicarboxamido. Additional heterocyclics include
3,3a,4,5,6,6a-hexahydro-pyrrolo[3,4-b]pyrrol-(2H)-yl, and
2,5-diazabicyclo[2.2.1]heptan-2-yl, homopiperazinyl (or
diazepanyl), tetrahydropyranyl, dithiazolyl, benzofuranyl,
benzothienyl, oxepanyl, thiepanyl, azocanyl, oxecanyl, and
thiocanyl. Heterocyclic groups also include groups of the
formula
##STR00022##
[0182] E' is selected from the group consisting of --N-- and
--CH--; F' is selected from the group consisting of --N.dbd.CH--,
--NH--CH.sub.2--, --NH--C(O)--, --NH--, --CH.dbd.N--,
--CH.sub.2--NH--, --C(O)--NH--, --CH.dbd.CH--, --CH.sub.2--,
--CH.sub.2CH.sub.2--, --CH.sub.2O--, --OCH.sub.2--, --O--, and
--S--; and G' is selected from the group consisting of --CH-- and
--N--. Any of the heterocyclyl groups mentioned herein may be
optionally substituted with one, two, three, four or five
substituents independently selected from the group consisting of:
(1) C.sub.1-7 acyl (e.g., carboxyaldehyde); (2) C.sub.1-20 alkyl
(e.g., C.sub.1-6 alkyl, C.sub.1-6 alkoxy-C.sub.1-6 alkyl, C.sub.1-6
alkylsulfinyl-C.sub.1-6 alkyl, amino-C.sub.1-6 alkyl,
azido-C.sub.1-6 alkyl, (carboxyaldehyde)-C.sub.1-6 alkyl,
halo-C.sub.1-6 alkyl (e.g., perfluoroalkyl), hydroxy-C.sub.1-6
alkyl, nitro-C.sub.1-6 alkyl, or C.sub.1-6 thioalkoxy-C.sub.1-6
alkyl); (3) C.sub.1-20 alkoxy (e.g., C.sub.1-6 alkoxy, such as
perfluoroalkoxy); (4) C.sub.1-6 alkylsulfinyl; (5) C.sub.6-10 aryl;
(6) amino; (7) C.sub.1-6 alk-C.sub.6-10 aryl; (8) azido; (9)
C.sub.3-8 cycloalkyl; (10) C.sub.1-6 alk-C.sub.3-8 cycloalkyl; (11)
halo; (12) C.sub.1-12 heterocyclyl (e.g., C.sub.2-12 heteroaryl);
(13) (C.sub.1-12 heterocyclyl)oxy; (14) hydroxyl; (15) nitro; (16)
C.sub.1-20 thioalkoxy (e.g., C.sub.1-6 thioalkoxy); (17)
--(CH.sub.2).sub.qCO.sub.2R.sup.A', where q is an integer from zero
to four, and R.sup.A' is selected from the group consisting of (a)
C.sub.1-6 alkyl, (b) C.sub.6-10 aryl, (c) hydrogen, and (d)
C.sub.1-6 alk-C.sub.6-10 aryl; (18)
--(CH.sub.2).sub.qCONR.sup.B'R.sup.C', where q is an integer from
zero to four and where R.sup.B' and R.sup.C' are independently
selected from the group consisting of (a) hydrogen, (b) 01-s alkyl,
(c) C.sub.6-10 aryl, and (d) C.sub.1-6 alk-C.sub.6-10 aryl; (19)
--(CH.sub.2).sub.qSO.sub.2R.sup.D', where q is an integer from zero
to four and where R.sup.D' is selected from the group consisting of
(a) C.sub.1-6 alkyl, (b) C.sub.6-10 aryl, and (c) C.sub.1-6
alk-C.sub.6-10 aryl; (20)
--(CH.sub.2).sub.qSO.sub.2NR.sup.E'R.sup.F', where q is an integer
from zero to four and where each of R.sup.E' and R.sup.F' is,
independently, selected from the group consisting of (a) hydrogen,
(b) C.sub.1-6 alkyl, (c) C.sub.6-10 aryl, and (d) C.sub.1-6
alk-C.sub.6-10 aryl; (21) thiol; (22) C.sub.6-10 aryloxy; (23)
C.sub.3-8 cycloalkoxy; (24) arylalkoxy; (25) C.sub.1-6
alk-C.sub.1-12 heterocyclyl (e.g., C.sub.1-6 alk-C.sub.1-12
heteroaryl); (26) oxo; (27) (C.sub.1-12 heterocyclyl)imino; (28)
C.sub.2-20 alkenyl; and (29) C.sub.2-20 alkynyl. In some
embodiments, each of these groups can be further substituted as
described herein. For example, the alkylene group of a
C.sub.1-alkaryl or a C.sub.1-alkheterocyclyl can be further
substituted with an oxo group to afford the respective aryloyl and
(heterocyclyl)oyl substituent group.
[0183] The "heterocyclylalkyl" group, which as used herein,
represents a heterocyclyl group, as defined herein, attached to the
parent molecular group through an alkylene group, as defined
herein. Exemplary unsubstituted heterocyclylalkyl groups are from 2
to 32 carbons (e.g., from 2 to 22, from 2 to 18, from 2 to 17, from
2 to 16, from 3 to 15, from 2 to 14, from 2 to 13, or from 2 to 12
carbons, such as C.sub.1-6 alk-C.sub.1-12 heterocyclyl, C.sub.1-10
alk-C.sub.1-12 heterocyclyl, or C.sub.1-20 alk-C.sub.1-12
heterocyclyl). In some embodiments, the alkylene and the
heterocyclyl each can be further substituted with 1, 2, 3, or 4
substituent groups as defined herein for the respective group.
[0184] The term "hydrocarbon," as used herein, represents a group
consisting only of carbon and hydrogen atoms.
[0185] The term "hydroxyl," as used herein, represents an --OH
group. In some embodiments, the hydroxyl group can be substituted
with 1, 2, 3, or 4 substituent groups (e.g., O-protecting groups)
as defined herein for an alkyl.
[0186] The term "isomer," as used herein, means any tautomer,
stereoisomer, enantiomer, or diastereomer of any compound of the
invention. It is recognized that the compounds of the invention can
have one or more chiral centers and/or double bonds and, therefore,
exist as stereoisomers, such as double-bond isomers (i.e.,
geometric E/Z isomers) or diastereomers (e.g., enantiomers (i.e.,
(+) or (-)) or cis/trans isomers). According to the invention, the
chemical structures depicted herein, and therefore the compounds of
the invention, encompass all of the corresponding stereoisomers,
that is, both the stereomerically pure form (e.g., geometrically
pure, enantiomerically pure, or diastereomerically pure) and
enantiomeric and stereoisomeric mixtures, e.g., racemates.
Enantiomeric and stereoisomeric mixtures of compounds of the
invention can typically be resolved into their component
enantiomers or stereoisomers by well-known methods, such as
chiral-phase gas chromatography, chiral-phase high performance
liquid chromatography, crystallizing the compound as a chiral salt
complex, or crystallizing the compound in a chiral solvent.
Enantiomers and stereoisomers can also be obtained from
stereomerically or enantiomerically pure intermediates, reagents,
and catalysts by well-known asymmetric synthetic methods.
[0187] The term "N-protected amino," as used herein, refers to an
amino group, as defined herein, to which is attached one or two
N-protecting groups, as defined herein.
[0188] The term "N-protecting group," as used herein, represents
those groups intended to protect an amino group against undesirable
reactions during synthetic procedures. Commonly used N-protecting
groups are disclosed in Greene, "Protective Groups in Organic
Synthesis," 3.sup.rd Edition (John Wiley & Sons, New York,
1999), which is incorporated herein by reference. N-protecting
groups include acyl, aryloyl, or carbamyl groups such as formyl,
acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl,
2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl,
o-nitrophenoxyacetyl, .alpha.-chlorobutyryl, benzoyl,
4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral
auxiliaries such as protected or unprotected D, L or D, L-amino
acids such as alanine, leucine, phenylalanine, and the like;
sulfonyl-containing groups such as benzenesulfonyl,
p-toluenesulfonyl, and the like; carbamate forming groups such as
benzyloxycarbonyl, p-chlorobenzyloxycarbonyl,
p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,
2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,
3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl,
2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,
2-nitro-4,5-dimethoxybenzyloxycarbonyl,
3,4,5-trimethoxybenzyloxycarbonyl,
1-(p-biphenylyl)-1-methylethoxycarbonyl,
.alpha.,.alpha.-dimethyl-3,5-dimethoxybenzyloxycarbonyl,
benzhydryloxy carbonyl, t-butyloxycarbonyl,
diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl,
methoxycarbonyl, allyloxycarbonyl, 2,2,2,-trichloroethoxycarbonyl,
phenoxycarbonyl, 4-nitrophenoxy carbonyl,
fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,
adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl,
and the like, alkaryl groups such as benzyl, triphenylmethyl,
benzyloxymethyl, and the like and silyl groups, such as
trimethylsilyl, and the like. Preferred N-protecting groups are
formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl,
phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and
benzyloxycarbonyl (Cbz).
[0189] The term "nitro," as used herein, represents an --NO.sub.2
group.
[0190] The term "O-protecting group," as used herein, represents
those groups intended to protect an oxygen containing (e.g.,
phenol, hydroxyl, or carbonyl) group against undesirable reactions
during synthetic procedures. Commonly used O-protecting groups are
disclosed in Greene, "Protective Groups in Organic Synthesis,"
3.sup.rd Edition (John Wiley & Sons, New York, 1999), which is
incorporated herein by reference. Exemplary O-protecting groups
include acyl, aryloyl, or carbamyl groups, such as formyl, acetyl,
propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl,
trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl,
.alpha.-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl,
t-butyldimethylsilyl, tri-iso-propylsilyloxymethyl,
4,4'-dimethoxytrityl, isobutyryl, phenoxyacetyl,
4-isopropylpehenoxyacetyl, dimethylformamidino, and 4-nitrobenzoyl;
alkylcarbonyl groups, such as acyl, acetyl, propionyl, pivaloyl,
and the like; optionally substituted arylcarbonyl groups, such as
benzoyl; silyl groups, such as trimethylsilyl (TMS),
tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl
(TOM), triisopropylsilyl (TIPS), and the like; ether-forming groups
with the hydroxyl, such methyl, methoxymethyl, tetrahydropyranyl,
benzyl, p-methoxybenzyl, trityl, and the like; alkoxycarbonyls,
such as methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl,
n-isopropoxycarbonyl, n-butyloxycarbonyl, isobutyloxycarbonyl,
sec-butyloxycarbonyl, t-butyloxycarbonyl, 2-ethylhexyloxycarbonyl,
cyclohexyloxycarbonyl, methyloxycarbonyl, and the like;
alkoxyalkoxycarbonyl groups, such as methoxymethoxycarbonyl,
ethoxymethoxycarbonyl, 2-methoxyethoxycarbonyl,
2-ethoxyethoxycarbonyl, 2-butoxyethoxycarbonyl,
2-methoxyethoxymethoxycarbonyl, allyloxycarbonyl,
propargyloxycarbonyl, 2-butenoxycarbonyl,
3-methyl-2-butenoxycarbonyl, and the like; haloalkoxycarbonyls,
such as 2-chloroethoxycarbonyl, 2-chloroethoxycarbonyl,
2,2,2-trichloroethoxycarbonyl, and the like; optionally substituted
arylalkoxycarbonyl groups, such as benzyloxycarbonyl,
p-methylbenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,
p-nitrobenzyloxycarbonyl, 2,4-dinitrobenzyloxycarbonyl,
3,5-dimethylbenzyloxycarbonyl, p-chlorobenzyloxycarbonyl,
p-bromobenzyloxycarbonyl, fluorenylmethyloxycarbonyl, and the like;
and optionally substituted aryloxycarbonyl groups, such as
phenoxycarbonyl, p-nitrophenoxycarbonyl, o-nitrophenoxycarbonyl,
2,4-dinitrophenoxycarbonyl, p-methylphenoxycarbonyl,
m-methylphenoxycarbonyl, o-bromophenoxycarbonyl,
3,5-dimethylphenoxycarbonyl, p-chlorophenoxycarbonyl,
2-chloro-4-nitrophenoxy-carbonyl, and the like); substituted alkyl,
aryl, and alkaryl ethers (e.g., trityl; methylthiomethyl;
methoxymethyl; benzyloxymethyl; siloxymethyl;
2,2,2,-trichloroethoxymethyl; tetrahydropyranyl; tetrahydrofuranyl;
ethoxyethyl; 1-[2-(trimethylsilyl)ethoxy]ethyl;
2-trimethylsilylethyl; t-butyl ether; p-chlorophenyl,
p-methoxyphenyl, p-nitrophenyl, benzyl, p-methoxybenzyl, and
nitrobenzyl); silyl ethers (e.g., trimethylsilyl; triethylsilyl;
triisopropylsilyl; dimethylisopropylsilyl; t-butyldimethylsilyl;
t-butyldiphenylsilyl; tribenzylsilyl; triphenylsilyl; and
diphenymethylsilyl); carbonates (e.g., methyl, methoxymethyl,
9-fluorenylmethyl; ethyl; 2,2,2-trichloroethyl;
2-(trimethylsilyl)ethyl; vinyl, allyl, nitrophenyl; benzyl;
methoxybenzyl; 3,4-dimethoxybenzyl; and nitrobenzyl);
carbonyl-protecting groups (e.g., acetal and ketal groups, such as
dimethyl acetal, 1,3-dioxolane, and the like; acylal groups; and
dithiane groups, such as 1,3-dithianes, 1,3-dithiolane, and the
like); carboxylic acid-protecting groups (e.g., ester groups, such
as methyl ester, benzyl ester, t-butyl ester, orthoesters, and the
like; and oxazoline groups.
[0191] The term "oxo" as used herein, represents .dbd.O.
[0192] The prefix "perfluoro," as used herein, represents anyl
group, as defined herein, where each hydrogen radical bound to the
alkyl group has been replaced by a fluoride radical. For example,
perfluoroalkyl groups are exemplified by trifluoromethyl,
pentafluoroethyl, and the like.
[0193] The term "protected hydroxyl," as used herein, refers to an
oxygen atom bound to an O-protecting group.
[0194] The term "spirocyclyl," as used herein, represents a
C.sub.2-7 alkylene diradical, both ends of which are bonded to the
same carbon atom of the parent group to form a spirocyclic group,
and also a C.sub.1-6 heteroalkylene diradical, both ends of which
are bonded to the same atom. The heteroalkylene radical forming the
spirocyclyl group can containing one, two, three, or four
heteroatoms independently selected from the group consisting of
nitrogen, oxygen, and sulfur. In some embodiments, the spirocyclyl
group includes one to seven carbons, excluding the carbon atom to
which the diradical is attached. The spirocyclyl groups of the
invention may be optionally substituted with 1, 2, 3, or 4
substituents provided herein as optional substituents for
cycloalkyl and/or heterocyclyl groups.
[0195] The term "stereoisomer," as used herein, refers to all
possible different isomeric as well as conformational forms which a
compound may possess (e.g., a compound of any formula described
herein), in particular all possible stereochemically and
conformationally isomeric forms, all diastereomers, enantiomers
and/or conformers of the basic molecular structure. Some compounds
of the present invention may exist in different tautomeric forms,
all of the latter being included within the scope of the present
invention.
[0196] The term "sulfonyl," as used herein, represents an
--S(O).sub.2-- group.
[0197] The term "thiol," as used herein. represents an --SH
group.
Definitions
[0198] In this application, unless otherwise clear from context,
(i) the term "a" may be understood to mean "at least one"; (ii) the
term "or" may be understood to mean "and/or"; (iii) the terms
"comprising" and "including" may be understood to encompass
itemized components or steps whether presented by themselves or
together with one or more additional components or steps; and (iv)
the terms "about" and "approximately" may be understood to permit
standard variation as would be understood by those of ordinary
skill in the art; and (v) where ranges are provided, endpoints are
included.
[0199] As is known in the art, "affinity" is a measure of the
tightness with which a particular ligand binds to its partner.
Affinities can be measured in different ways. In some embodiments,
affinity is measured by a quantitative assay. In some such
embodiments, binding partner concentration may be fixed to be in
excess of ligand concentration so as to mimic physiological
conditions. Alternatively or additionally, in some embodiments,
binding partner concentration and/or ligand concentration may be
varied. In some such embodiments, affinity may be compared to a
reference under comparable conditions (e.g., concentrations).
[0200] As used herein, the terms "approximately" and "about" are
each intended to encompass normal statistical variation as would be
understood by those of ordinary skill in the art as appropriate to
the relevant context. In certain embodiments, the terms
"approximately" or "about" each refer to a range of values that
fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%,
10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either
direction (greater than or less than) of a stated value, unless
otherwise stated or otherwise evident from the context (e.g., where
such number would exceed 100% of a possible value).
[0201] It will be understood that the term "binding" as used
herein, typically refers to association (e.g., non-covalent or
covalent) between or among two or more entities. "Direct" binding
involves physical contact between entities or moieties; indirect
binding involves physical interaction by way of physical contact
with one or more intermediate entities. Binding between two or more
entities can typically be assessed in any of a variety of
contexts--including where interacting entities or moieties are
studied in isolation or in the context of more complex systems
(e.g., while covalently or otherwise associated with a carrier
entity and/or in a biological system or cell).
[0202] The affinity of a molecule X for its partner Y can generally
be represented by the dissociation constant (K.sub.D). Affinity can
be measured by common methods known in the art, including those
described herein. Specific illustrative and exemplary embodiments
for measuring binding affinity are described below. The term
"K.sub.D," as used herein, is intended to refer to the dissociation
equilibrium constant of a particular compound-protein or
complex-protein interaction. Typically, the compounds of the
invention bind to presenter proteins with a dissociation
equilibrium constant (K.sub.D) of less than about 10.sup.-6 M, such
as less than approximately 10.sup.-7 M, 10.sup.-8 M, 10.sup.-9 M,
or 10.sup.-10 M or even lower, e.g., when determined by surface
plasmon resonance (SPR) technology using the presenter protein as
the analyte and the compound as the ligand. The presenter
protein/compound complexes of the invention bind to target proteins
(e.g., a eukaryotic target protein such as a mammalian target
protein or a fungal target protein or a prokaryotic target protein
such as a bacterial target protein) with a dissociation equilibrium
constant (K.sub.D) of less than about 10.sup.-6 M, such as less
than approximately 10.sup.-7 M, 10.sup.-8 M, 10.sup.-9 M, or
10.sup.-10 M or even lower, e.g., when determined by surface
plasmon resonance (SPR) technology using the target protein as the
analyte and the complex as the ligand.
[0203] As used herein, the term "cross-linking group" refers to a
group comprising a reactive functional group capable of chemically
attaching to specific functional groups (e.g., primary amines,
sulfhydryls) on proteins or other molecules. A "moiety capable of a
chemoselective reaction with an amino acid," as used herein refers
to a moiety comprising a reactive functional group capable of
chemically attaching to a functional group of a natural or
non-natural amino acid (e.g., primary and secondary amines,
sulfhydryls, alcohols, carboxyl groups, carbonyls, or triazole
forming functional groups such as azides or alkynes). Examples of
cross-linking groups include sulfhydryl-reactive cross-linking
groups (e.g., groups comprising maleimides, haloacetyls,
pyridyldisulfides, thiosulfonates, or vinylsulfones),
amine-reactive cross-linking groups (e.g., groups comprising esters
such as NHS esters, imidoesters, and pentafluorophenyl esters, or
hydroxymethylphosphine), carboxyl-reactive cross-linking groups
(e.g., groups comprising primary or secondary amines, alcohols, or
thiols), carbonyl-reactive cross-linking groups (e.g., groups
comprising hydrazides or alkoxyamines), and triazole-forming
cross-linking groups (e.g., groups comprising azides or
alkynes).
[0204] As used herein, the term "complex" refers to a group of two
or more compounds and/or proteins which are bound together through
a binding interaction (e.g., a non-covalent interaction, such as a
hydrophobic effect interaction, an electrostatic interaction, a van
der Waals interaction, or .pi.-effect interaction). Examples of
complexes are "presenter protein/conjugate complex" and "target
protein/conjugate complex" which include a conjugate of the
invention bound to a presenter protein or a target protein.
[0205] As used herein, the term "conjugate" refers to a compound
formed by the joining (e.g., via a covalent bond forming reaction)
of two or more chemical compounds (e.g., a compound including a
cross-linking group and a protein such as a target protein or a
presenter protein).
[0206] As used herein, an atom that "participates in binding" is
within 4 .ANG. of the entity to which they bind or connects to an
atom that is with 4 .ANG. of the entity to which they bind.
[0207] The term "presenter protein" refers to a protein that binds
to a small molecule to form a complex that binds to and modulates
the activity of a target protein (e.g., a eukaryotic target protein
such as a mammalian target protein or a fungal target protein or a
prokaryotic target protein such as a bacterial target protein). In
some embodiments, the presenter protein is a relatively abundant
protein (e.g., the presenter protein is sufficiently abundant that
participation in a tripartite complex does not substantially impact
the biological role of the presenter protein in a cell and/or
viability or other attributes of the cell). In certain embodiments,
the presenter protein is a protein that has chaperone activity
within a cell. In some embodiments, the presenter protein is a
protein that has multiple natural interaction partners within a
cell. In certain embodiments, the presenter protein is one which is
known to bind a small molecule to form a binary complex that is
known to or suspected of binding to and modulating the biological
activity of a target protein.
[0208] The term "presenter protein binding moiety" refers to a
group of atoms and the moieties attached thereto (e.g., atoms
within 20 atoms such as, atoms within 15 atoms, atoms within 10,
atoms within 5 atoms) that participate in binding to a presenter
protein such that the compound specifically binds to said presenter
protein, for example, with a K.sub.D of less than 10 .mu.M (e.g.,
less than 5 .mu.M, less than 1 .mu.M, less than 500 nM, less than
200 nM, less than 100 nM, less than 75 nM, less than 50 nM, less
than 25 nM, less than 10 nM) or inhibits the peptidyl-prolyl
isomerase activity of the presenter protein, for example, with an
IC.sub.50 of less than 1 .mu.M (e.g., less than 0.5 .mu.M, less
than 0.1 .mu.M, less than 0.05 .mu.M, less than 0.01 .mu.M). It
will be understood that the presenter protein binding moiety does
not necessarily encompass the entirety of atoms in the compound
that interact with the presenter protein. It will also be
understood that one or more atoms of the presenter protein binding
moiety may be within the target protein binding moiety (e.g.,
eukaryotic target protein binding moiety such as mammalian target
protein binding moiety or fungal target protein binding moiety or
prokaryotic target protein binding moiety such as a bacterial
target protein binding moiety).
[0209] As used herein, "FKBP binding moiety" refers to a presenter
protein binding moiety that is selective for presenter proteins in
the FKBP family of proteins (e.g., FKBP12, FKBP12.6, FKBPP13,
FKBP25, FKBP51, or FKBP52). A "selective FKBP binding moiety," as
used herein, refers to a binding moiety that is specific for one or
more (e.g., two, three, four, five) members of the FKBP family over
all other members of the FKBP family. A "non-selective FKBP binding
moiety," as used herein, refers to a binding moiety that has
comparable affinity (within 2-fold, within 3-fold, within 4-fold,
within 5-fold, within 10-fold) for all members of the FKBP
family.
[0210] The term "protein binding moiety" refers to a group of atoms
and the moieties attached thereto (e.g., atoms within 20 atoms such
as, atoms within 15 atoms, atoms within 10, atoms within 5 atoms)
that participate in binding to a protein (e.g., a presenter protein
or a target protein) such that the compound specifically binds to
said protein, for example, with a K.sub.D of less than 10 .mu.M
(e.g., less than 5 .mu.M, less than 1 .mu.M, less than 500 nM, less
than 200 nM, less than 100 nM, less than 75 nM, less than 50 nM,
less than 25 nM, less than 10 nM) or inhibits the peptidyl-prolyl
isomerase activity of the presenter protein, for example, with an
IC.sub.50 of less than 1 .mu.M (e.g., less than 0.5 .mu.M, less
than 0.1 .mu.M, less than 0.05 .mu.M, less than 0.01 .mu.M). It
will be understood that the protein binding moiety does not
necessarily encompass the entirety of atoms in the compound that
interact with the protein.
[0211] As used herein, the term "react" refers to a process in
which atoms of the same or different elements rearrange themselves
to form a new substance. For example, the formation of a covalent
bond between two atoms such as the reaction between a reactive
amino acid on a protein and a cross-linking group to form a
covalent bond. A reaction may be measured by any method known in
the art, for example, formation of a reaction product can be
determined by LC-MS or NMR.
[0212] As used herein, the term "reactive amino acid" refers to a
natural or non-natural amino acid comprising a functional group
(e.g., a nucleophilic functional group) capable of chemically
attaching to specific functional groups (e.g., a cross-linking
group). Examples of reactive amino acids include cysteine, lysine,
serine, and amino acids having azides on the side chain.
"Non-reactive amino acids" refers to natural or non-natural amino
acids that do not contain a functional group capable of chemically
attaching to specific functional groups. Examples of non-reactive
amino acids include valine, alanine, isoleucine, theronine, and
leucine.
[0213] The term "reference" is often used herein to describe a
standard or control compound, individual, population, sample,
sequence or value against which a compound, individual, population,
sample, sequence or value of interest is compared. In some
embodiments, a reference compound, individual, population, sample,
sequence or value is tested and/or determined substantially
simultaneously with the testing or determination of the compound,
individual, population, sample, sequence or value of interest. In
some embodiments, a reference compound, individual, population,
sample, sequence or value is a historical reference, optionally
embodied in a tangible medium. Typically, as would be understood by
those skilled in the art, a reference compound, individual,
population, sample, sequence or value is determined or
characterized under conditions comparable to those utilized to
determine or characterize the compound, individual, population,
sample, sequence or value of interest.
[0214] As used herein, the term "solvent exposed amino acid" refers
to an amino acid that is accessible to the solvent surrounding the
protein. In some embodiments, a solvent exposed amino acid is an
amino acid that when substituted does not substantially change the
three-dimensional structure of the protein.
[0215] As used herein, the terms "specific binding" or "specific
for" or "specific to" refer to an interaction between a binding
agent and a target entity. As will be understood by those of
ordinary skill, an interaction is considered to be "specific" if it
is favored in the presence of alternative interactions, for
example, binding with a K.sub.D of less than 10 .mu.M (e.g., less
than 5 .mu.M, less than 1 .mu.M, less than 500 nM, less than 200
nM, less than 100 nM, less than 75 nM, less than 50 nM, less than
25 nM, less than 10 nM). In many embodiments, specific interaction
is dependent upon the presence of a particular structural feature
of the target entity (e.g., an epitope, a cleft, a binding site).
It is to be understood that specificity need not be absolute. In
some embodiments, specificity may be evaluated relative to that of
the binding agent for one or more other potential target entities
(e.g., competitors). In some embodiments, specificity is evaluated
relative to that of a reference specific binding agent. In some
embodiments specificity is evaluated relative to that of a
reference non-specific binding agent.
[0216] The term "specific" when used with reference to a compound
having an activity, is understood by those skilled in the art to
mean that the compound discriminates between potential target
entities or states. For example, in some embodiments, a compound is
said to bind "specifically" to its target if it binds
preferentially with that target in the presence of one or more
competing alternative targets. In many embodiments, specific
interaction is dependent upon the presence of a particular
structural feature of the target entity (e.g., an epitope, a cleft,
a binding site). It is to be understood that specificity need not
be absolute. In some embodiments, specificity may be evaluated
relative to that of the binding agent for one or more other
potential target entities (e.g., competitors). In some embodiments,
specificity is evaluated relative to that of a reference specific
binding agent. In some embodiments specificity is evaluated
relative to that of a reference non-specific binding agent. In some
embodiments, the agent or entity does not detectably bind to the
competing alternative target under conditions of binding to its
target entity. In some embodiments, binding agent binds with higher
on-rate, lower off-rate, increased affinity, decreased
dissociation, and/or increased stability to its target entity as
compared with the competing alternative target(s).
[0217] The term "substantially" refers to the qualitative condition
of exhibiting total or near-total extent or degree of a
characteristic or property of interest. One of ordinary skill in
the biological arts will understand that biological and chemical
phenomena rarely, if ever, go to completion and/or proceed to
completeness or achieve or avoid an absolute result. The term
"substantially" is therefore used herein to capture the potential
lack of completeness inherent in many biological and chemical
phenomena.
[0218] The term "does not substantially bind" to a particular
protein as used herein can be exhibited, for example, by a molecule
or portion of a molecule having a K.sub.D for the target of
10.sup.-4 M or greater, alternatively 10.sup.-5 M or greater,
alternatively 10.sup.-6 M or greater, alternatively 10.sup.-7 M or
greater, alternatively 10.sup.-8 M or greater, alternatively
10.sup.-9 M or greater, alternatively 10.sup.-10 M or greater,
alternatively 10.sup.-11 M or greater, alternatively 10.sup.-12 M
or greater, or a K.sub.D in the range of 10.sup.-4 M to 10.sup.-12
M or 10.sup.-6 M to 10.sup.-10 M or 10.sup.-7 M to 10.sup.-9 M.
[0219] The term "target protein" refers to any protein that
participates in a biological pathway associated with a disease,
disorder or condition. In some embodiments, the target protein is
not mTOR or calcineurin. In some embodiments, the target protein is
capable of forming a tripartite complex with a presenter protein
and a small molecule. In some embodiments, a target protein is a
naturally-occurring protein; in some such embodiments, a target
protein is naturally found in certain mammalian cells (e.g., a
mammalian target protein), fungal cells (e.g., a fungal target
protein), bacterial cells (e.g., a bacterial target protein) or
plant cells (e.g., a plant target protein). In some embodiments, a
target protein is characterized by natural interaction with one or
more natural presenter protein/natural small molecule complexes. In
some embodiments, a target protein is characterized by natural
interactions with a plurality of different natural presenter
protein/natural small molecule complexes; in some such embodiments
some or all of the complexes utilize the same presenter protein
(and different small molecules). In some embodiments, a target
protein does not substantially bind to a complex of cyclosporin,
rapamycin, or FK506 and a presenter protein (e.g., FKBP). Target
proteins can be naturally occurring, e.g., wild type.
Alternatively, the target protein can vary from the wild type
protein but still retain biological function, e.g., as an allelic
variant, a splice mutant or a biologically active fragment.
Exemplary mammalian target proteins are GTPases, GTPase activating
protein, Guanine nucleotide-exchange factor, heat shock proteins,
ion channels, coiled-coil proteins, kinases, phosphatases,
ubiquitin ligases, transcription factors, chromatin
modifier/remodelers, proteins with classical protein-protein
interaction domains and motifs, or any other proteins that
participate in a biological pathway associated with a disease,
disorder or condition.
[0220] In some embodiments, the target protein is a modified target
protein. A modified target protein can include an amino acid
insertion, deletion, or substitution, either conservative or
non-conservative (e.g., D-amino acids, desamino acids) in the
protein sequence (e.g., where such changes do not substantially
alter the biological activity of the polypeptide). In particular,
the addition of one or more cysteine residues to the amino or
carboxy terminus of any of the polypeptides of the invention can
facilitate conjugation of these proteins by, e.g., disulfide
bonding. In some embodiments, one or more reactive amino acid
residues (e.g., cysteines) are removed to decrease the number of
possible conjugation sites on the protein. Amino acid substitutions
can be conservative (i.e., wherein a residue is replaced by another
of the same general type or group) or non-conservative (i.e.,
wherein a residue is replaced by an amino acid of another type). In
addition, a naturally occurring amino acid can be substituted for a
non-naturally occurring amino acid (i.e., non-naturally occurring
conservative amino acid substitution or a non-naturally occurring
non-conservative amino acid substitution).
[0221] The term "target protein binding moiety" refers to a group
of ring atoms and the moieties attached thereto (e.g., atoms within
20 atoms such as, atoms within 15 atoms, atoms within 10 atoms,
within 5 atoms) that participate in binding to a target protein
(e.g., a eukaryotic target protein such as a mammalian target
protein or a fungal target protein or a prokaryotic target protein
such as a bacterial target protein) when the compound is in a
complex with a presenter protein. It will be understood that the
target protein binding moiety does not necessarily encompass the
entirety of atoms in the compound that interact with the target
protein. It will also be understood that one or more atoms of the
presenter protein binding moiety may also be present in the target
protein binding moiety.
[0222] The term "traditional binding pocket" refers to cavities or
pockets on a protein structure with physiochemical and/or geometric
properties comparable to proteins whose activity has been modulated
by one or more small molecules. In some embodiments, a traditional
binding pocket is a well-defined pocket with a volume greater than
1000 A.sup.3. Those of ordinary skill in the art are familiar with
the concept of a traditional binding pocket and, moreover are aware
of its relationship to "druggability". In certain embodiments, a
protein is considered to not have a traditional binding pocket if
it is undruggable, as defined herein.
[0223] The term "undruggable target" refers to proteins that are
not members of a protein family which is known to be targeted by
drugs and/or does not possess a binding site that is suitable for
high-affinity binding to a small molecule. Methods for determining
whether a target protein is undruggable are known in the art. For
example, whether a target protein is undruggable may be determined
using an structure-based algorithim, such as those used by the
program DOGSITESCORER.RTM. (Universitat Hamburg, Hamburg, Germany)
that assesses druggability based on parameters computed for binding
pockets on a protein including volume, surface area, lipophilic
surface area, depth, and/or hydrophobic ratio.
BRIEF DESCRIPTION OF THE DRAWINGS
[0224] FIG. 1 is an image illustrating SDS-PAGE analysis of
KRAS.sub.GTP/S39C lite/C2-FK506 conjugates. Lane 1:
KRAS.sub.GTP/S39C lite; Lane 2: KRAS.sub.GTP/S39C lite/C2-FK506
reaction mixture; Lane 3: KRAS.sub.GTP/S39C lite/C2-FK506 reaction
mixture+100 mM DTT.
[0225] FIG. 2 is an image illustrating SDS-PAGE analysis of
KRAS.sub.GTP/G12C lite/SFAX9DS conjugates.
[0226] FIGS. 3A and 3B are images illustrating SEC and SDS-PAGE
Analysis of KRAS.sub.GTP/S39C lite/C.sub.2-Holt/FKBP12 Complex
Formation. FIG. 3A) SEC purification profile. Dashed blue lines
indicate the peak corresponding to elution of the KRAS.sub.GTP/S39C
lite/C2-Holt/FKBP12 ternary complex; FIG. 3B) SDS-PAGE analysis of
SEC elution peaks. Dashed blue lines correspond with the fractions
collected for the KRAS.sub.GTP/S39C lite/C2-Holt/FKBP12 elution
peak.
[0227] FIG. 4 is an image illustrating SEC profile and SDS-PAGE
analysis of the elution peaks confirm the formation of
KRAS.sub.GDP/S39C lite/SFAC4DS/CypA.sub.C52S complex.
[0228] FIGS. 5A and 5B are images illustrating the SEC profile and
SDS-PAGE analysis of free PTP1B.sub.S187C lite and FKBP12 proteins
and the PTP1B.sub.S187C lite/C3-SLF/FKBP12 complex.
[0229] FIG. 6 is an image illustrating crosslinking efficiency of
C3- and C4-SLF by SDS-PAGE.
[0230] FIGS. 7A and 7B is an image illustrating the crystal
structure of FKBP12-Compound 1-KRAS.sub.GTP/S39C complex. FIG. 7A)
Ribbon representation showing FKBP12, KRAS.sub.GTP/S39C and the
ligand. Fo-Fc electron density at 3 a shown is shown for the ligand
in the close-up view. FIG. 7B) Surface representation of the
complex with atoms within 4 .ANG. proximity to either ligand or
partner protein colored in red.
[0231] FIG. 8 is an image illustrating the crystal structure of
CypA.sub.C52S-SFAC4DS-KRAS.sub.GDP/S39C.
[0232] FIGS. 9A and 9B are images illustrating the crystal
structure of FKBP12-C3SLF-PTP1B.sub.S187C. FIG. 9A illustrates that
the crystal contains two complex molecules of
FKBP12-C3SLF-PTP1B.sub.S187C in the asymmetric unit. FIG. 9B
illustrates that the buried surface area of PTP1B.sub.S187C is 427
.ANG..sup.2 and the buried surface area of C3-SLF is 615
.ANG..sup.2.
[0233] FIG. 10 is an image illustrating the crystal structure of
MCL1.sub.S245C/C3SLF/FKBP52.
[0234] FIG. 11 is an image illustrating the binding curve of W21487
dependent complex formation of CYPA-W21487-KRAS.sub.G12C-GTP
ternary complex.
[0235] FIG. 12 is an image illustrating the binding curve of W21487
dependent complex formation of CYPA-W21487-KRAS.sub.G12C-GTP
ternary complex.
[0236] FIG. 13 is an image illustrating ITC measurements for the
binding of FKBP12-Compound 1 and FKBP12-Compound 2 binary complexes
to CEP250.
[0237] FIG. 14 is an image illustrating SPR sensorgrams for the
binding of FKBP12/Compound 1 to CEP250.sub.11.4 and
CEP250.sub.29.2.
[0238] FIG. 15 is an image illustrating sensogram and steady state
fitting curves for the binding of CYPA/Compound 3 to
KRAS.sub.G12C-GTP.
[0239] FIG. 16 is an image illustrating fluorescence polarization
curves for CypA:C3DS:KRAS complex formation.
[0240] FIGS. 17A-17C are images illustrating the 2D 1H-15N
TROSY-HSQC spectrum of KRAS.sub.G12C-GTP (FIG. 17A), the addition
of a stoichiometric amount of CYPA (FIG. 17B), and KRAS and CYPA
alone (FIG. 17C).
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0241] Small molecules are limited in their targeting ability
because their interactions with the target are driven by adhesive
forces, the strength of which is roughly proportional to contact
surface area. Because of their small size, the only way for a small
molecule to build up enough intermolecular contact surface area to
effectively interact with a target protein is to be literally
engulfed by that protein. Indeed, a large body of both experimental
and computational data supports the view that only those proteins
having a hydrophobic "pocket" on their surface are capable of
binding small molecules. In those cases, binding is enabled by
engulfment.
[0242] Nature has evolved a strategy that allows a small molecule
to interact with target proteins at sites other than hydrophobic
pockets. This strategy is exemplified by naturally occurring
immunosuppressive drugs cyclosporine A, rapamycin, and FK506. The
biological activity of these drugs involves the formation of a
high-affinity complex of the small molecule with a small presenting
protein. The composite surface of the small molecule and the
presenting protein engages the target. Thus, for example, the
binary complex formed between cyclosporin A and cyclophilin A
targets calcineurin with high affinity and specificity, but neither
cyclosporin A or cyclophilin A alone binds calcineurin with
measurable affinity.
[0243] Many important therapeutic targets exert their function by
complexation with other proteins. The protein/protein interaction
surfaces in many of these systems contain an inner core of
hydrophobic side chains surrounded by a wide ring of polar
residues. The hydrophobic residues contribute nearly all of the
energetically favorable contacts, and hence this cluster has been
designated as a "hotspot" for engagement in protein-protein
interactions. Importantly, in the aforementioned complexes of
naturally occurring small molecules with small presenting proteins,
the small molecule provides a cluster of hydrophobic functionality
akin to a hotspot, and the protein provides the ring of mostly
polar residues. In other words, presented small molecule systems
mimic the surface architecture employed widely in natural
protein/protein interaction systems.
[0244] Nature has demonstrated the ability to reprogram the target
specificity of presented small molecules--portable hotspots-through
evolutionary diversification. In the best characterized example,
the complex formed between FK506 binding protein (FKBP) and FK506
targets calcineurin. However, FKBP can also form a complex with the
related molecule rapamycin, and that complex interacts with a
completely different target, TorC1. To date, no methodology has
been developed to reprogram the binding and modulating ability of
presenter protein/ligand interfaces so that they can interact with
and modulate other target proteins that have previously been
considered undruggable.
[0245] In addition, it is well established that some drug
candidates fail because they modulate the activity of both the
intended target and other non-intended proteins as well. The
problem is particularly daunting when the drug binding site of the
target protein is similar to binding sites in non-target proteins.
The insulin like growth factor receptor (IGF-1R), whose ATP binding
pocket is structurally similar to the binding pocket of the
non-target insulin receptor (IR), is one such example. Small
molecule development candidates that were designed to target IGF-1R
typically have the unacceptable side effect of also modulating the
insulin receptor. However, structural dissimilarities do exist
between these two proteins in the regions surrounding the ATP
binding pocket. Despite such knowledge, no methodology exists to
date to take advantage of those differences and develop drugs that
are specific to IGF-1R over IR.
[0246] The present disclosure provides methods and reagents useful
for analyzing protein-protein interfaces such as the interface
between a presenter protein (e.g., a member of the FKBP family, a
member of the cyclophilin family, or PIN1) and a target protein. In
some embodiments, the target and/or presenter proteins are
intracellular proteins. In some embodiments, the target and/or
presenter proteins are mammalian proteins. In some embodiments,
these methods and reagents may be useful for identifying target
proteins amenable to inhibition or activation by forming a complex
with a presenter protein and a small molecule. In some embodiments,
these methods and reagents may be useful in identifying compounds
capable of inhibiting or activating target proteins by forming a
complex with a presenter protein and the target protein.
Compounds and Conjugates
[0247] The disclosure provides compounds including a protein
binding moiety (e.g., a presenter protein binding moiety or target
protein binding moiety) and a cross-linking group. The invention
also features conjugates including a protein binding moiety
conjugated to a protein, e.g., a presenter protein binding moiety
conjugated to a target protein or a target protein binding moiety
conjugated to a presenter protein.
[0248] The invention also features compounds of Formula VII:
A-L-B Formula VII
[0249] wherein A comprises the structure of Formula VIII:
##STR00023##
[0250] In some embodiments, the compound of the invention is:
##STR00024## ##STR00025## ##STR00026## ##STR00027##
##STR00028##
[0251] Cross-Linking Groups
[0252] In some embodiments, compounds of the invention include a
cross-linking group. A cross-linking group refers to a group
comprising a reactive functional group capable of chemically
attaching to specific functional groups (e.g., primary amines,
sulfhydryls) on proteins or other molecules. Examples of
cross-linking groups include sulfhydryl-reactive cross-linking
groups (e.g., groups comprising maleimides, haloacetyls,
pyridyldisulfides, thiosulfonates, or vinylsulfones),
amine-reactive cross-linking groups (e.g., groups comprising esters
such as NHS esters, imidoesters, and pentafluorophenyl esters, or
hydroxymethylphosphine), carboxyl-reactive cross-linking groups
(e.g., groups comprising primary or secondary amines, alcohols,
orthiols), carbonyl-reactive cross-linking groups (e.g., groups
comprising hydrazides or alkoxyamines), and triazole-forming
cross-linking groups (e.g., groups comprising azides or
alkynes).
[0253] Exemplary cross-linking groups include 2'-pyridyldisulfide,
4'-pyridyldisulfide iodoacetyl, maleimide, thioesters,
alkyldisulfides, alkylamine disulfides, nitrobenzoic acid
disulfide, anhydrides, NHS esters, aldehydes, alkyl chlorides,
alkynes, and azides.
[0254] Presenter Protein Binding Moieties
[0255] In some embodiments, compounds of the invention include a
presenter protein binding moiety. In some embodiments, a presenter
protein binding moiety includes a group of atoms (e.g., 5 to 20
atoms, 5 to 10 atoms, 10 to 20 atoms) and may include any moieties
attached thereto (e.g., atoms within 20 atoms, atoms within 15
atoms, atoms within 10 atoms, atoms within 5 atoms) that
participate in binding to a presenter protein such that a provided
compound specifically binds to said presenter protein, for example,
with a K.sub.D of less than 10 .mu.M (e.g., less than 5 .mu.M, less
than 1 .mu.M, less than 500 nM, less than 200 nM, less than 100 nM,
less than 75 nM, less than 50 nM, less than 25 nM, less than 10 nM)
or inhibits the peptidyl-prolyl isomerase activity of the presenter
protein, for example, with an IC.sub.50 of less than 1 .mu.M (e.g.,
less than 0.5 .mu.M, less than 0.1 .mu.M, less than 0.05 .mu.M,
less than 0.01 .mu.M). In some embodiments, the presenter protein
binding moiety does not encompass the entirety of atoms in a
provided compound that interact with the presenter protein. In
certain embodiments, one or more atoms of the presenter protein
binding moiety do not interact with the presenter protein.
[0256] In some embodiments, a presenter protein binding moiety
includes a N-acyl proline moiety, a N-acyl-pipecolic acid moiety, a
N-acyl 3-morpholino-carboxylic acid moiety, and/or a N-acyl
piperzic acid moiety (e.g., with acylation on either nitrogen atom.
In certain embodiments, a presenter protein binding moiety includes
a N-acyl-pipecolic acid moiety. In some embodiments, a presenter
protein binding moiety includes a N-acyl proline moiety. In certain
embodiments, a presenter protein binding moiety includes a N-acyl
3-morpholino-carboxylic acid moiety. In some embodiments, a
presenter protein binding moiety includes a N-acyl piperzic acid
moiety.
[0257] In some embodiments, at least one atom of a presenter
protein binding moiety participates in binding with one or more
(e.g., two, three, four, five, six, seven, eight, nine, ten,
eleven, twelve, thirteen, fourteen, or fifteen) of Tyr 27, Phe 37,
Asp 38, Arg 41, Phe 47, Gln 54, Glu 55, Val 56, Ile 57, Trp 60, Ala
82, Try 83, His 88, Ile 92, and/or Phe 100 of FKBP12. In some
embodiments, at least one at of a presenter protein binding moiety
participates in binding with at least one (e.g., two, three, or
four) of Arg 41, Gln 54, Glu 55, and/or Ala 82 of FKBP12.
[0258] In some embodiments, a presenter protein binding moiety has
a structure according to Formula II-IV:
##STR00029##
[0259] In some embodiments, a presenter protein binding moiety
includes or consists of the structure:
##STR00030## ##STR00031## ##STR00032## ##STR00033## ##STR00034##
##STR00035## ##STR00036## ##STR00037##
[0260] or a stereoisomer thereof.
[0261] A presenter protein can bind to an atom in a presenter
protein binding moiety. Alternatively or additionally, a presenter
protein can bind to two or more atoms in a presenter protein
binding moiety. In another alternative, a presenter protein bind
can to a substituent attached to one or more atoms in a presenter
protein binding moiety. Furthermore, in some embodiments, a
presenter protein can bind to an atom in a presenter protein
binding moiety and to a substituent attached to one or more atoms
in a presenter protein binding moiety. In some embodiments, a
presenter protein binds to a group that mimics a natural ligand of
a presenter protein and wherein the group that mimics a natural
ligand of a presenter protein is attached to a presenter protein
binding moiety. In some embodiments, a presenter protein binds to a
presenter protein and affinity of a presenter protein for a
presenter protein in the binary complex is increased relative to
the affinity of a presenter protein for a presenter protein in the
absence of the complex. Binding in such examples is typically
through, but not limited to non-covalent interactions of a
presenter protein to a presenter protein binding moiety.
[0262] Target Protein Binding Moieties
[0263] In some embodiments, compounds of the invention include a
target protein binding moiety (e.g., a eukaryotic target protein
binding moiety such as a mammalian target protein binding moiety or
a fungal target protein binding moiety or a prokaryotic target
protein binding moiety such as a bacterial target protein binding
moiety). In some embodiments, the target protein binding moiety
includes a group of atoms (e.g., 5 to 20 atoms, 5 to 10 atoms, 10
to 20 atoms) and may include any moieties attached thereto (e.g.,
atoms within 20 atoms, atoms within 15 atoms, atoms within 10
atoms, atoms within 5 atoms) that specifically bind to a target
protein. In some embodiments, a target protein binding moiety
comprises a plurality of the atoms in the compound that interact
with the target protein. In certain embodiments, one or more atoms
of a target protein binding moiety do not interact with the target
protein.
[0264] A target protein can bind to an atom in a target protein
binding moiety. Alternatively or additionally, a target protein can
bind to two or more atoms in a target protein binding moiety. In
another alternative, a target protein bind can to a substituent
attached to one or more atoms in a target protein binding moiety.
In another alternative, a target protein can bind to an atom in a
target protein binding moiety and to a substituent attached to one
or more atoms in a target protein binding moiety. In another
alternative, a target protein binds to a group that mimics a
natural ligand of a target protein and wherein the group that
mimics a natural ligand of a target protein is attached to a target
protein binding moiety. In yet another alternative, a target
protein binds to a presenter protein and the affinity of a target
protein for a presenter protein in the binary complex is increased
relative to the affinity of a target protein for a presenter
protein in the absence of the complex. Binding in these examples is
typically through, but not limited to non-covalent interactions of
a target protein to a target protein binding moiety.
[0265] Linkers
[0266] The compounds of the invention include a linker (e.g.,
moiety linker joining a protein binding moiety (e.g., a presenter
protein binding moiety or a target protein binding moiety) to a
cross-linking group or a linker joining a protein binding moiety to
a protein (e.g., a presenter protein or target protein). The linker
component of the invention is, at its simplest, a bond, but may
also provide a linear, cyclic, or branched molecular skeleton
having pendant groups covalently linking two moieties.
[0267] In some embodiments, at least one atom of a linker
participates in binding to the presenter protein and/or the target
protein. In certain embodiments, at least one atom of a linker does
not participate in binding to the presenter protein and/or the
target protein.
[0268] Thus, a linker, when included in a compound and/or conjugate
as described herein, achieves linking of two (or more) moieties by
covalent means, involving bond formation with one or more
functional groups located on either moiety. Examples of chemically
reactive functional groups which may be employed for this purpose
include, without limitation, amino, hydroxyl, sulfhydryl, carboxyl,
carbonyl, carbohydrate groups, vicinal diols, thioethers,
2-aminoalcohols, 2-aminothiols, guanidinyl, imidazolyl, and
phenolic groups.
[0269] In some embodiments, such covalent linking of two (or more)
moieties may be effected using a linker that contains reactive
moieties capable of reaction with such functional groups present in
either moiety. For example, an amine group of a moiety may react
with a carboxyl group of the linker, or an activated derivative
thereof, resulting in the formation of an amide linking the
two.
[0270] Examples of moieties capable of reaction with sulfhydryl
groups include .alpha.-haloacetyl compounds of the type
XCH.sub.2CO-- (where X.dbd.Br, Cl, or I), which show particular
reactivity for sulfhydryl groups, but which can also be used to
modify imidazolyl, thioether, phenol, and amino groups as described
by Gurd, Methods Enzymol. 11:532 (1967). N-Maleimide derivatives
are also considered selective towards sulfhydryl groups, but may
additionally be useful in coupling to amino groups under certain
conditions. Reagents such as 2-iminothiolane (Traut et al.,
Biochemistry 12:3266 (1973)), which introduce a thiol group through
conversion of an amino group, may be considered as sulfhydryl
reagents if linking occurs through the formation of disulfide
bridges.
[0271] Examples of reactive moieties capable of reaction with amino
groups include, for example, alkylating and acylating agents.
Representative alkylating agents include:
[0272] (i) .alpha.-haloacetyl compounds, which show specificity
towards amino groups in the absence of reactive thiol groups and
are of the type XCH.sub.2CO-- (where X.dbd.Br, Cl, or I), for
example, as described by Wong Biochemistry 24:5337 (1979);
[0273] (ii) N-maleimide derivatives, which may react with amino
groups either through a Michael type reaction or through acylation
by addition to the ring carbonyl group, for example, as described
by Smyth et al., J. Am. Chem. Soc. 82:4600 (1960) and Biochem. J.
91:589 (1964);
[0274] (iii) aryl halides such as reactive nitrohaloaromatic
compounds;
[0275] (iv) alkyl halides, as described, for example, by McKenzie
et al., J. Protein Chem. 7:581 (1988);
[0276] (v) aldehydes and ketones capable of Schiff's base formation
with amino groups, the adducts formed usually being stabilized
through reduction to give a stable amine;
[0277] (vi) epoxide derivatives such as epichlorohydrin and
bisoxiranes, which may react with amino, sulfhydryl, or phenolic
hydroxyl groups;
[0278] (vii) chlorine-containing derivatives of s-triazines, which
are very reactive towards nucleophiles such as amino, sufhydryl,
and hydroxyl groups;
[0279] (viii) aziridines based on s-triazine compounds detailed
above, e.g., as described by Ross, J. Adv. Cancer Res. 2:1 (1954),
which react with nucleophiles such as amino groups by ring
opening;
[0280] (ix) squaric acid diethyl esters as described by Tietze,
Chem. Ber. 124:1215 (1991); and
[0281] (x) .alpha.-haloalkyl ethers, which are more reactive
alkylating agents than normal alkyl halides because of the
activation caused by the ether oxygen atom, as described by
Benneche et al., Eur. J. Med. Chem. 28:463 (1993).
[0282] Representative amino-reactive acylating agents include:
[0283] (i) isocyanates and isothiocyanates, particularly aromatic
derivatives, which form stable urea and thiourea derivatives
respectively;
[0284] (ii) sulfonyl chlorides, which have been described by Herzig
et al., Biopolymers 2:349 (1964);
[0285] (iii) acid halides;
[0286] (iv) active esters such as nitrophenylesters or
N-hydroxysuccinimidyl esters;
[0287] (v) acid anhydrides such as mixed, symmetrical, or
N-carboxyanhydrides;
[0288] (vi) other useful reagents for amide bond formation, for
example, as described by M. Bodansky, Principles of Peptide
Synthesis, Springer-Verlag, 1984;
[0289] (vii) acylazides, e.g., wherein the azide group is generated
from a preformed hydrazide derivative using sodium nitrite, as
described by Wetz et al., Anal. Biochem. 58:347 (1974);
[0290] (viii) imidoesters, which form stable amidines on reaction
with amino groups, for example, as described by Hunter and Ludwig,
J. Am. Chem. Soc. 84:3491 (1962); and
[0291] (ix) haloheteroaryl groups such as halopyridine or
halopyrimidine.
[0292] Aldehydes and ketones may be reacted with amines to form
Schiff's bases, which may advantageously be stabilized through
reductive amination. Alkoxylamino moieties readily react with
ketones and aldehydes to produce stable alkoxamines, for example,
as described by Webb et al., in Bioconjugate Chem. 1:96 (1990).
[0293] Examples of reactive moieties capable of reaction with
carboxyl groups include diazo compounds such as diazoacetate esters
and diazoacetamides, which react with high specificity to generate
ester groups, for example, as described by Herriot, Adv. Protein
Chem. 3:169 (1947). Carboxyl modifying reagents such as
carbodiimides, which react through O-acylurea formation followed by
amide bond formation, may also be employed.
[0294] It will be appreciated that functional groups in either
moiety may, if desired, be converted to other functional groups
prior to reaction, for example, to confer additional reactivity or
selectivity. Examples of methods useful for this purpose include
conversion of amines to carboxyls using reagents such as
dicarboxylic anhydrides; conversion of amines to thiols using
reagents such as N-acetylhomocysteine thiolactone,
S-acetylmercaptosuccinic anhydride, 2-iminothiolane, or
thiol-containing succinimidyl derivatives; conversion of thiols to
carboxyls using reagents such as .alpha.-haloacetates; conversion
of thiols to amines using reagents such as ethylenimine or
2-bromoethylamine; conversion of carboxyls to amines using reagents
such as carbodiimides followed by diamines; and conversion of
alcohols to thiols using reagents such as tosyl chloride followed
by transesterification with thioacetate and hydrolysis to the thiol
with sodium acetate.
[0295] So-called zero-length linkers, involving direct covalent
joining of a reactive chemical group of one moiety with a reactive
chemical group of the other without introducing additional linking
material may, if desired, be used in accordance with the
invention.
[0296] More commonly, however, the linker includes two or more
reactive moieties, as described above, connected by a spacer
element. The presence of such a spacer permits bifunctional linkers
to react with specific functional groups within either moiety,
resulting in a covalent linkage between the two. The reactive
moieties in a linker may be the same (homobifunctional linker) or
different (heterobifunctional linker, or, where several dissimilar
reactive moieties are present, heteromultifunctional linker),
providing a diversity of potential reagents that may bring about
covalent attachment between the two moieties.
[0297] Spacer elements in the linker typically consist of linear or
branched chains and may include a C.sub.1-10 alkyl, C.sub.2-10
alkenyl, C.sub.2-10 alkynyl, C.sub.2-6 heterocyclyl, C.sub.6-12
aryl, C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl,
C.sub.2-C.sub.100 polyethylene glycol, or C.sub.1-10
heteroalkyl.
[0298] In some instances, the linker is described by Formula V.
[0299] Examples of homobifunctional linkers useful in the
preparation of conjugates of the invention include, without
limitation, diamines and diols selected from ethylenediamine,
propylenediamine and hexamethylenediamine, ethylene glycol,
diethylene glycol, propylene glycol, 1,4-butanediol,
1,6-hexanediol, cyclohexanediol, and polycaprolactone diol.
[0300] In some embodiments, the linker is a bond or a linear chain
of up to 10 atoms, independently selected from carbon, nitrogen,
oxygen, sulfur or phosphorous atoms, wherein each atom in the chain
is optionally substituted with one or more substituents
independently selected from alkyl, alkenyl, alkynyl, aryl,
heteroaryl, chloro, iodo, bromo, fluoro, hydroxyl, alkoxy, aryloxy,
carboxy, amino, alkylamino, dialkylamino, acylamino, carboxamido,
cyano, oxo, thio, alkylthio, arylthio, acylthio, alkylsulfonate,
arylsulfonate, phosphoryl, and sulfonyl, and wherein any two atoms
in the chain may be taken together with the substituents bound
thereto to form a ring, wherein the ring may be further substituted
and/or fused to one or more optionally substituted carbocyclic,
heterocyclic, aryl, or heteroaryl rings.
[0301] In some embodiments, a linker has the structure of Formula
XIX:
A.sup.1-(B.sup.1).sub.a--(C.sup.1).sub.b--(B.sup.2).sub.c-(D)-(B.sup.3).-
sub.d--(C.sup.2).sub.e--(B.sup.4).sub.f-A.sup.2 Formula XIX
where A.sup.1 is a bond between the linker and presenter protein
binding moiety; A.sup.2 is a bond between the mammalian target
interacting moiety and the linker; B.sup.1, B.sup.2, B.sup.3, and
B.sup.4 each, independently, is selected from optionally
substituted C.sub.1-C.sub.2 alkyl, optionally substituted
C.sub.1-C.sub.3 heteroalkyl, O, S, and NR.sup.N; R.sup.N is
hydrogen, optionally substituted C.sub.1-4 alkyl, optionally
substituted C.sub.2-4 alkenyl, optionally substituted C.sub.2-4
alkynyl, optionally substituted C.sub.2-6 heterocyclyl, optionally
substituted C.sub.6-12 aryl, or optionally substituted C.sub.1-7
heteroalkyl; C.sup.1 and C.sup.2 are each, independently, selected
from carbonyl, thiocarbonyl, sulphonyl, or phosphoryl; a, b, c, d,
e, and f are each, independently, 0 or 1; and D is optionally
substituted C.sub.1-10 alkyl, optionally substituted C.sub.2-10
alkenyl, optionally substituted C.sub.2-10 alkynyl, optionally
substituted C.sub.2-6 heterocyclyl, optionally substituted
C.sub.6-12 aryl, optionally substituted C.sub.2-C.sub.10
polyethylene glycol, or optionally substituted C.sub.1-10
heteroalkyl, or a chemical bond linking
A.sup.1-(B.sup.1).sub.a--(C.sup.1).sub.b--(B.sup.2).sub.c-- to
--(B.sup.3).sub.d--(C.sup.2).sub.e--(B.sup.4).sub.f-A.sup.2.
Proteins
[0302] Presenter Proteins
[0303] Presenter proteins can bind a small molecule to form a
complex, which can bind to and modulate the activity of a target
protein (e.g., a eukaryotic target protein such as a mammalian
target protein or a fungal target protein or a prokaryotic target
protein such as a bacterial target protein). In some embodiments,
the presenter protein is a mammalian presenter protein (e.g., a
human presenter protein). In some embodiments, the presenter
protein is a fungal presenter protein. In certain embodiments, the
presenter protein is a bacterial presenter protein. In some
embodiments, the presenter protein is a plant presenter protein. In
some embodiments, the presenter protein is a relatively abundant
protein (e.g., the presenter protein is sufficiently abundant that
participation in a tripartite complex does not materially
negatively impact the biological role of the presenter protein in a
cell and/or viability or other attributes of the cell). In some
embodiments, the presenter protein is more abundant than the target
protein. In certain embodiments, the presenter protein is a protein
that has chaperone activity within a cell. In some embodiments, the
presenter protein has multiple natural interaction partners within
a cell. In certain embodiments, the presenter protein is one which
is known to bind a small molecule to form a binary complex that is
known to or suspected of binding to and modulating the biological
activity of a target protein. Immunophilins are a class of
presenter proteins which are known to have these functions and
include FKBPs and cyclophilins. In some embodiments, a reference
presenter protein exhibits peptidyl prolyl isomerase activity; in
some embodiments, a presenter protein shows comparable activity to
the reference presenter protein. In certain embodiments, the
presenter protein is a member of the FKBP family (e.g., FKBP12,
FKBP12.6, FKBP13, FKBP19, FKBP22, FKBP23, FKBP25, FKBP36, FKBP38,
FKBP51, FKBP52, FKBP60, FKBP65, and FKBP133), a member of the
cyclophilin family (e.g., PP1A, CYPB, CYPC, CYP40, CYPE, CYPD,
NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2,
PPWD1, PPIAL4A, PPIAL4B, PPIAL4C, PPIAL4D, or PPIAL4G), or PIN1.
The "FKBP family" is a family of proteins that have prolyl
isomerase activity and function as protein folding chaperones for
proteins containing proline residues. Genes that encode proteins in
this family include AIP, AIPL1, FKBP1A, FKBP1B, FKBP2, FKBP3,
FKBP4, FKBP5, FKBP6, FKBP7, FKBP8, FKBP9, FKBP9L, FKBP10, FKBP11,
FKBP14, FKBP15, and LOC541473.
[0304] The "cyclophilin family" is a family of proteins that bind
to cyclosporine. Genes that encode proteins in this family include
PPIA, PPIB, PPIC, PPID, PPIE, PPIF, PPIG, PPIH, SDCCAG-10, PPIL1,
PPIL2, PPIL3, PPIL4, P270, PPWD1, and COAS-2. Exemplary
cyclophilins include PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR,
SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2,
PPWD1, PPIAL4A, PPIAL4B, PPIAL4C, PPIAL4D, and PPIAL4G.
[0305] In some embodiments, a presenter protein is a chaperone
protein such as GRP78/BiP, GRP94, GRP170, calnexin, calreticulin,
HSP47, ERp29, Protein disulfide isomerase (PDI), and ERp57.
[0306] In some embodiments, a presenter protein is an allelic
variant or splice variant of a FKBP or cyclophilin disclosed
herein.
[0307] In some embodiments, a presenter protein is a polypeptide
whose amino acid sequence i) shows significant identity with that
of a reference presenter protein; ii) includes a portion that shows
significant identity with a corresponding portion of a reference
presenter protein; and/or iii) includes at least one characteristic
sequence found in presenter protein. In many embodiments, identity
is considered "significant" for the purposes of defining an
presenter protein if it is above 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
higher. In some embodiments, the portion showing significant
identity has a length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300,
350, 450, 500, 550, 600 amino acids or more.
[0308] Representative presenter proteins are encoded by the genes
or homologs thereof listed in Table 1; in some embodiments, a
reference presenter protein is encoded by a gene set forth in Table
1. Also, those of ordinary skill in the art, referring to Table 1,
can readily identify sequences that are characteristic of presenter
proteins generally, and/or of particular subsets of presenter
proteins.
TABLE-US-00001 TABLE 1 Genes that Encode Selected Presenter
Proteins Uniprot Accession Gene Name Number AIP O00170 AIPL1 Q9NZN9
FKBP1A P62942 FKBP1B P68106 FKBP2 P26885 FKBP3 Q00688 FKBP4 Q02790
FKBP5 Q13451 FKBP6 O75344 FKBP7 Q9Y680 FKBP8 Q14318 FKBP9 O95302
FKBP9L Q75LS8 FKBP10 Q96AY3 FKBP11 Q9NYL4 FKBP14 Q9NWM8 FKBP15
Q5T1M5 LOC541473 -- PPIA Q567Q0 PPIB P23284 PPIC P45877 PPID Q08752
PPIE Q9UNP9 PPIG Q13427 PPIH O43447 PPIL1 Q9Y3C6 PPIL2 Q13356 PPIL3
Q9H2H8 PPIL4 Q8WUA2 PPIL5 Q32Q17 PPIL6 Q8IXY8 PPWD1 Q96BP3
[0309] Target Proteins
[0310] A target protein (e.g., a eukaryotic target protein such as
a mammalian target protein or a fungal target protein or a
prokaryotic target protein such as a bacterial target protein) is a
protein which mediates a disease condition or a symptom of a
disease condition. As such, a desirable therapeutic effect can be
achieved by modulating (inhibiting or increasing) its activity.
Target proteins useful in the complexes and methods of the
invention include those which do not naturally associate with a
presenter protein, e.g., those which have an affinity for a
presenter protein in the absence of a binary complex with a
compound of the invention of greater than 1 .mu.M, preferably
greater than 5 .mu.M, and more preferably greater than 10 .mu.M.
Alternatively, target proteins which do not naturally associate
with a presenter protein are those which have an affinity for a
compound of the invention in the absence of a binary complex
greater than 1 .mu.M, preferably greater than 5 .mu.M, and more
preferably greater than 10 .mu.M. In another alternative, target
proteins which do not naturally associate with a presenter protein
are those which have an affinity for a binary complex of
cyclosporine, rapamycin, or FK506 and a presenter protein (e.g.,
FKBP) of greater than 1 .mu.M, preferably greater than 5 .mu.M, and
more preferably greater than 10 .mu.M. In yet another alternative,
target proteins that do not naturally associate with a presenter
protein are those which are other than calcineurin or mTOR. The
selection of suitable target proteins for the complexes and methods
of the invention may depend on the presenter protein. For example,
target proteins that have low affinity for a cyclophilin may have
high affinity for an FKBP and would not be used together with the
latter.
[0311] Target proteins can be naturally occurring, e.g., wild type.
Alternatively, a target protein can vary from the wild type protein
but still retain biological function, e.g., as an allelic variant,
a splice mutant or a biologically active fragment.
[0312] In some embodiments, a target protein is a transmembrane
protein. In some embodiments, a target protein has a coiled coil
structure. In certain embodiments, a target protein is one protein
of a dimeric complex.
[0313] In some embodiments, a target protein of the invention
includes one or more surface sites (e.g., a flat surface site)
characterized in that, in the absence of forming a presenter
protein/compound complex, small molecules typically demonstrate low
or undetectable binding to the site(s). In some embodiments, a
target protein includes one or more surface sites (e.g., a flat
surface site) to which, in the absence of forming a presenter
protein/compound complex, a particular small molecule (e.g., the
compound) shows low or undetectable binding (e.g., binding at least
2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 fold or more lower
than that observed with a presenter protein/compound complex
involving the same compound). In some embodiments, a target protein
has a surface characterized by one or more sites (and, in some
embodiments, an entire surface) that lack(s) any traditional
binding pocket, for example, a cavity or pocket on the protein
structure with physiochemical and/or geometric properties
comparable to proteins whose activity has been modulated by one or
more small molecules. In certain embodiments, a target protein has
a traditional binding pocket and a site for a protein-protein
interaction. In some embodiments, a target protein is an
undruggable target, for example, a target protein is not a member
of a protein family which is known to be targeted by drugs and/or
does not possess a binding site that is expected (e.g., according
to art-accepted understanding, as discussed herein) to be suitable
for binding to a small molecule. In some embodiments, the protein
includes at least one reactive cysteine.
[0314] In some embodiments, the target protein is a GTPase such as
DIRAS1, DIRAS2, DIRAS3, ERAS, GEM, HRAS, KRAS, MRAS, NKIRAS1,
NKIRAS2, NRAS, RALA, RALB, RAP1A, RAP1B, RAP2A, RAP2B, RAP2C,
RASD1, RASD2, RASL10A, RASL10B, RASL11A, RASL11B, RASL12, REM1,
REM2, RERG, RERGL, RRAD, RRAS, RRAS2, RHOA, RHOB, RHOBTB1, RHOBTB2,
RHOBTB3, RHOC, RHOD, RHOF, RHOG, RHOH, RHOJ, RHOQ, RHOU, RHOV,
RND1, RND2, RND3, RAC1, RAC2, RAC3, CDCl42, RAB1A, RAB1B, RAB2,
RAB3A, RAB3B, RAB3C, RAB3D, RAB4A, RAB4B, RABSA, RAB5B, RABSC,
RAB6A, RAB6B, RAB6C, RAB7A, RAB7B, RAB7L1, RAB8A, RAB8B, RAB9,
RAB9B, RABL2A, RABL2B, RABL4, RAB10, RAB11A, RAB11B, RAB12, RAB13,
RAB14, RAB15, RAB17, RAB18, RAB19, RAB20, RAB21, RAB22A, RAB23,
RAB24, RAB25, RAB26, RAB27A, RAB27B, RAB2B, RAB2B, RAB30, RAB31,
RAB32, RAB33A, RAB33B, RAB34, RAB35, RAB36, RAB37, RAB3B, RAB39,
RAB39B, RAB40A, RAB40AL, RAB40B, RAB40C, RAB41, RAB42, RAB43,
RAP1A, RAP1B, RAP2A, RAP2B, RAP2C, ARF1, ARF3, ARF4, ARF5, ARF6,
ARL1, ARL2, ARL3, ARL4, ARL5, ARL5C, ARL6, ARL7, ARL8, ARL9,
ARL10A, ARL10B, ARL10C, ARL11, ARL13A, ARL13B, ARL14, ARL15, ARL16,
ARL17, TRIM23, ARL4D, ARFRP1, ARL13B, RAN, RHEB, RHEBL1, RRAD, GEM,
REM, REM2, RIT1, RIT2, RHOT1, or RHOT2. In some embodiments, the
target protein is a GTPase activating protein such as NF1, IQGAP1,
PLEXIN-B1, RASAL1, RASAL2, ARHGAP5, ARHGAP8, ARHGAP12, ARHGAP22,
ARHGAP25, BCR, DLC1, DLC2, DLC3, GRAF, RALBP1, RAP1GAP, SIPA1,
TSC2, AGAP2, ASAP1, or ASAP3. In some embodiments, the target
protein is a Guanine nucleotide-exchange factor such as CNRASGEF,
RASGEF1A, RASGRF2, RASGRP1, RASGRP4, SOS1, RALGDS, RGL1, RGL2, RGR,
ARHGEF10, ASEF/ARHGEF4, ASEF2, DBS, ECT2, GEF-H1, LARG, NET1,
OBSCURIN, P-REX1, P-REX2, PDZ-RHOGEF, TEM4, TIAM1, TRIO, VAV1,
VAV2, VAV3, DOCK1, DOCK2, DOCK3, DOCK4, DOCK8, DOCK10, C3G,
BIG2/ARFGEF2, EFA6, FBX8, or GEP100. In certain embodiments, the
target protein is a protein with a protein-protein interaction
domain such as ARM; BAR; BEACH; BH; BIR; BRCT; BROMO; BTB; C1; C2;
CARD; CC; CALM; CH; CHROMO; CUE; DEATH; DED; DEP; DH; EF-hand; EH;
ENTH; EVH1; F-box; FERM; FF; FH2; FHA; FYVE; GAT; GEL; GLUE; GRAM;
GRIP; GYF; HEAT; HECT; IQ; LRR; MBT; MH1; MH2; MIU; NZF; PAS; PB1;
PDZ; PH; POLO-Box; PTB; PUF; PWWP; PX; RGS; RING; SAM; SC; SH2;
SH3; SOCS; SPRY; START; SWIRM; TIR; TPR; TRAF; SNARE; TUBBY; TUDOR;
UBA; UEV; UIM; VHL; VHS; WD40; WW; SH2; SH3; TRAF; Bromodomain; or
TPR. In some embodiments, the target protein is a heat shock
protein such as Hsp20, Hsp27, Hsp70, Hsp84, alpha B crystalline,
TRAP-1, hsf1, or Hsp90. In certain embodiments, the target protein
is an ion channel such as Cav2.2, Cav3.2, IKACh, Kv1.5, TRPA1,
NAv1.7, Nav1.8, Nav1.9, P2X3, or P2X4. In some embodiments, the
target protein is a coiled-coil protein such as geminin, SPAG4,
VAV1, MAD1, ROCK1, RNF31, NEDP1, HCCM, EEA1, Vimentin, ATF4, Nemo,
SNAP25, Syntaxin 1a, FYCO1, or CEP250. In certain embodiments, the
target protein is a kinase such as Cyclin D1, ABL, ALK, AXL, BTK,
EGFR, FMS, FAK, FGFR1, 2, 3, 4, FLT3, HER2/ErbB2, HER3/ErbB3,
HER4/ErbB4, IGF1R, INSR, JAK1, JAK2, JAK3, KIT, MET, PDGFRA,
PDGFRB, RET RON, ROR1, ROR2, ROS, SRC, SYK, TIE1, TIE2, TRKA, TRKB,
KDR, AKT1, AKT2, AKT3, PDK1, PKC, RHO, ROCK1, RSK1, RKS2, RKS3,
ATM, ATR, CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9,
CDK10, ERK1, ERK2, ERK3, ERK4, GSK3A, GSK3B, JNK1, JNK2, JNK3,
AurA, AurB, PLK1, PLK2, PLK3, PLK4, IKK, KIN1, cRaf, PKN3, c-Src,
Fak, PyK2, or AMPK. In some embodiments, the target protein is a
phosphatase such as WIP1, SHP2, SHP1, PRL-3, PTP1B, or STEP. In
certain embodiments the target protein is a ubiquitin or
ubiquitin-like protein (such as NEDD8, ATG8 proteins, SUMO
proteins, ISG15), activating enzyme (E1's such as UBA1, UBA2, UBA3,
UBA5, UBA6, UBA7, ATG7, NAE1, SAE1), conjugation enzyme (E2's such
as UBE proteins, ATG3, BIRC6), ligation enzyme (E3's such as BMI-1,
MDM2, NEDD4-1, Beta-TRCP, SKP2, E6AP, CBL-B, orAPC/C), and
ubiquitin or ubiquitin-like protein protease. In some embodiments,
the target protein is a chromatin modifier/remodeler such as a
chromatin modifier/remodeler encoded by the gene BRG1, BRM, ATRX,
PRDM3, ASH1L, CBP, KAT6A, KAT6B, MLL, NSD1, SETD2, EP300, KAT2A, or
CREBBP. In some embodiments, the target protein is a transcription
factor such as a transcription factor encoded by the gene EHF,
ELF1, ELF3, ELF4, ELF5, ELK1, ELK3, ELK4, ERF, ERG, ETS1, ETV1,
ETV2, ETV3, ETV4, ETV5, ETV6, FEV, FLI1, GAVPA, SPDEF, SP11, SPIC,
SPIB, E2F1, E2F2, E2F3, E2F4, E2F7, E2F8, ARNTL, BHLHA15, BHLHB2,
BHLBHB3, BHLHE22, BHLHE23, BHLHE41, CLOCK, FIGLA, HAS5, HES7, HEY1,
HEY2, ID4, MAX, MESP1, MLX, MLXIPL, MNT, MSC, MYF6, NEUROD2,
NEUROG2, NHLH1, OLIG1, OLIG2, OLIG3, SREBF2, TCF3, TCF4, TFAP4,
TFE3, TFEB, TFEC, USF1, ARF4, ATF7, BATF3, CEBPB, CEBPD, CEBPG,
CREB3, CREB3L1, DBP, HLF, JDP2, MAFF, MAFG, MAFK, NRL, NFE2, NFIL3,
TEF, XBP1, PROX1, TEAD1, TEAD3, TEAD4, ONECUT3, ALX3, ALX4, ARX,
BARHL2, BARX, BSX, CART1, CDX1, CDX2, DLX1, DLX2, DLX3, DLX4, DLX5,
DLX6, DMBX1, DPRX, DRGX, DUXA, EMX1, EMX2, EN1, EN2, ESX1, EVX1,
EVX2, GBX1, GBX2, GSC, GSC2, GSX1, GSX2, HESX1, HMX1, HMX2, HMX3,
HNF1A, HNF1B, HOMEZ, HOXA1, HOXA10, HOXA13, HOXA2, HOXAB13, HOXB2,
HOXB3, HOXB5, HOXC10, HOXC11, HOXC12, HOXC13, HOXD11, HOXD12,
HOXD13, HOXD8, IRX2, IRX5, ISL2, ISX, LBX2, LHX2, LHX6, LHX9,
LMX1A, LMX1B, MEIS1, MEIS2, MEIS3, MEOX1, MEOX2, MIXL1, MNX1, MSX1,
MSX2, NKX2-3, NKX2-8, NKX3-1, NKX3-2, NKX6-1, NKX6-2, NOTO,
ONECUT1, ONECUT2, OTX1, OTX2, PDX1, PHOX2A, PHOX2B, PITX1, PITX3,
PKNOX1, PROP1, PRRX1, PRRX2, RAX, RAXL1, RHOXF1, SHOX, SHOX2,
TGIF1, TGIF2, TGIF2LX, UNCX, VAX1, VAX2, VENTX, VSX1, VSX2, CUX1,
CUX2, POU1F1, POU2F1, POU2F2, POU2F3, POU3F1, POU3F2, POU3F3,
POU3F4, POU4F1, POU4F2, POU4F3, POU5F1P1, POU6F2, RFX2, RFX3, RFX4,
RFX5, TFAP2A, TFAP2B, TFAP2C, GRHL1, TFCP2, NFIA, NFIB, NFIX, GCM1,
GCM2, HSF1, HSF2, HSF4, HSFY2, EBF1, IRF3, IRF4, IRF5, IRF7, IRF8,
IRF9, MEF2A, MEF2B, MEF2D, SRF, NRF1, CPEB1, GMEB2, MYBL1, MYBL2,
SMAD3, CENPB, PAX1, PAX2, PAX9, PAX3, PAX4, PAX5, PAX6, PAX7,
BCL6B, EGR1, EGR2, EGR3, EGR4, GLIS1, GLIS2, GLI2, GLIS3, HIC2,
HINFP1, KLF13, KLF14, KLF16, MTF1, PRDM1, PRDM4, SCRT1, SCRT2,
SNAI2, SP1, SP3, SP4, SP8, YY1, YY2, ZBED1, ZBTB7A, ZBTB7B, ZBTB7C,
ZIC1, ZIC3, ZIC4, ZNF143, ZNF232, ZNF238, ZNF282, ZNF306, ZNF410,
ZNF435, ZBTB49, ZNF524, ZNF713, ZNF740, ZNF75A, ZNF784, ZSCAN4,
CTCF, LEF1, SOX10, SOX14, SOX15, SOX18, SOX2, SOX21, SOX4, SOX7,
SOX8, SOX9, SRY, TCF7L1, FOXO3, FOXB1, FOXC1, FOXC2, FOXD2, FOXD3,
FOXG1, FOXI1, FOXJ2, FOXJ3, FOXK1, FOXL1, FOXO1, FOXO4, FOXO6,
FOXP3, EOMES, MGA, NFAT5, NFATC1, NFKB1, NFKB2, TP63, RUNX2, RUNX3,
T, TBR1, TBX1, TBX15, TBX19, TBX2, TBX20, TBX21, TBX4, TBX5, AR,
ESR1, ESRRA, ESRRB, ESRRG, HNF4A, NR2C2, NR2E1, NR2F1, NR2F6,
NR3C1, NR3C2, NR4A2, RARA, RARB, RARG, RORA, RXRA, RXRB, RXRG,
THRA, THRB, VDR, GATA3, GATA4, or GATA5; or C-myc, Max, Stat3,
Stat4, Stat6, androgen receptor, C-Jun, C-Fox, N-Myc, L-Myc, MITF,
Hif-1alpha, Hif-2alpha, Bc16, E2F1, NF-kappaB, Stat5, or ER(coact).
In certain embodiments, the target protein is TrkA, P2Y14, mPEGS,
ASK1, ALK, Bcl-2, BCL-XL, mSIN1, ROR.gamma.t, IL17RA, eIF4E, TLR7R,
PCSK9, IgE R, CD40, CD40L, Shn-3, TNFR1, TNFR2, IL31RA, OSMR,
IL12beta1,2, Tau, FASN, KCTD 6, KCTD 9, Raptor, Rictor, RALGAPA,
RALGAPB, Annexin family members, BCOR, NCOR, beta catenin, AAC 11,
PLD1, PLD2, Frizzled7, RaLP, MLL-1, Myb, Ezh2, RhoGD12, EGFR,
CTLA4R, GCGC (coact), Adiponectin R2, GPR 81, IMPDH2, IL-4R,
IL-13R, IL-1R, IL2-R, IL-6R, IL-22R, TNF-R, TLR4, MyD88, Keap1, or
Nrlp3.
[0315] Protein Variants
[0316] A protein or polypeptide variant, as described herein,
generally has an amino acid sequence that shows significant (e.g.,
80% or more, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)
identity with that of a reference polypeptide (e.g., a presenter
protein or target protein as described herein such as for example a
mammalian presenter protein or target protein) but includes a
limited number of particular amino acid changes (e.g., insertions,
deletions, or substitutions, either conservative or
non-conservative and/or including one or more amino acid variants
or analogs (e.g., D-amino acids, desamino acids) relative to the
reference polypeptide. In certain embodiments, a variant shares a
relevant biological activity (e.g., binding to a particular
compound or moiety thereof) with the reference polypeptide; in some
such embodiments, the variant displays such activity at a level
that is not less than about 50% of that of the reference
polypeptide and/or is not less than about 0.5 fold below that of
the reference polypeptide.
[0317] In some embodiments, a variant polypeptide has an amino acid
sequence that differs from that of a reference polypeptide at least
(or only) in that the variant has a larger number of cysteine
residues and/or has one or more cysteine residues at a position
corresponding to a non-cysteine residue in the reference
polypeptide. For example, in some embodiments, addition of one or
more cysteine residues to the amino or carboxy terminus of any of a
polypeptide (e.g., of a presenter protein and/or of a target
protein) as described herein can facilitate conjugation of such
polypeptide by, e.g., disulfide bonding. In some embodiments, amino
acid substitutions can be conservative (i.e., wherein a residue is
replaced by another of the same general type or group) or
non-conservative (i.e., wherein a residue is replaced by an amino
acid of another type). In some embodiments, a naturally occurring
amino acid can be substituted for a non-naturally occurring amino
acid (i.e., non-naturally occurring conservative amino acid
substitution or a non-naturally occurring non-conservative amino
acid substitution), or vice versa.
[0318] Polypeptides made synthetically can include substitutions of
amino acids not naturally encoded by DNA (e.g., non-naturally
occurring or unnatural amino acid). Examples of non-naturally
occurring amino acids include D-amino acids, an amino acid having
an azide-containing side chain, an amino acid having an
acetylaminomethyl group attached to a sulfur atom of a cysteine, a
pegylated amino acid, the omega amino acids of the formula
NH.sub.2(CH.sub.2).sub.nCOOH wherein n is 2-6, neutral nonpolar
amino acids, such as sarcosine, t-butyl alanine, t-butyl glycine,
N-methyl isoleucine, and norleucine. Phenylglycine may substitute
for Trp, Tyr, or Phe; citrulline and methionine sulfoxide are
neutral nonpolar, cysteic acid is acidic, and ornithine is basic.
Proline may be substituted with hydroxyproline and retain the
conformation conferring properties.
[0319] Analogs may be generated by substitutional mutagenesis and
retain the structure (e.g., a local structure or global structure)
of the original protein. Examples of substitutions identified as
"conservative substitutions" are shown in Table 2. If such
substitutions result in a change not desired, then other type of
substitutions, denominated "exemplary substitutions" in Table 2, or
as further described herein in reference to amino acid classes, are
introduced and the products screened.
[0320] Substantial modifications in function or immunological
identity are accomplished by selecting substitutions that differ
significantly in their effect on maintaining (a) the structure of
the protein backbone in the area of the substitution, for example,
as a sheet or helical conformation. (b) the charge or
hydrophobicity of the molecule at the target site, or (c) the bulk
of the side chain. Naturally occurring residues are divided into
groups based on common side chain properties:
(1) hydrophobic: norleucine, methionine (Met), Alanine (Ala),
Valine (Val), Leucine (Leu), Isoleucine (Ile), Histidine (His),
Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe), (2) neutral
hydrophilic: Cysteine (Cys), Serine (Ser), Threonine (Thr) (3)
acidic/negatively charged: Aspartic acid (Asp), Glutamic acid (Glu)
(4) basic: Asparagine (Asn), Glutamine (Gln), Histidine (His),
Lysine (Lys), Arginine (Arg) (5) residues that influence chain
orientation: Glycine (Gly), Proline (Pro); (6) aromatic: Tryptophan
(Trp), Tyrosine (Tyr), Phenylalanine (Phe), Histidine (His), (7)
polar: Ser, Thr, Asn, Gin (8) basic positively charged: Arg, Lys,
His, and; (9) charged: Asp, Glu, Arg, Lys, His Other amino acid
substitutions are listed in Table 2.
TABLE-US-00002 TABLE 2 Amino acid substitutions Conservative
Original residue Exemplary substitution substitution Ala (A) Val,
Leu, Ile Val Arg (R) Lys, Gln, Asn Lys Asn (N) Gln, His, Lys, Arg
Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp
Gly (G) Pro Pro His (H) Asn, Gln, Lys, Arg Arg Ile (I) Leu, Val,
Met, Ala, Phe, norleucine Leu Leu (L) Norleucine, Ile, Val, Met,
Ala, Phe Ile Lys (K) Arg, Gln, Asn Arg Met (M) Leu, Phe, Ile Leu
Phe (F) Leu, Val, Ile, Ala Leu Pro (P) Gly Gly Ser (S) Thr Thr
Thr(T) Ser Ser Trp (W) Tyr Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val
(V) Ile, Leu, Met, Phe, Ala, norleucine Leu
[0321] Protein Variants with Altered Reactive Amino Acid
Profiles
[0322] In some embodiments, a protein or polypeptide variant may
include the addition of one or more reactive amino acid residues
(e.g., cysteines) to a protein (e.g., at the amino or carboxy
terminus of any of the proteins described herein) can facilitate
conjugation of these proteins by, e.g., disulfide bonding. In some
embodiments, one or more reactive amino acids (e.g., cysteines) may
be removed to decrease the number of possible conjugation sites on
a protein. Amino acid substitutions can be conservative (i.e.,
wherein a residue is replaced by another of the same general type
or group) or non-conservative (i.e., wherein a residue is replaced
by an amino acid of another type). In addition, a naturally
occurring amino acid can be substituted for a non-naturally
occurring amino acid (i.e., non-naturally occurring conservative
amino acid substitution or a non-naturally occurring
non-conservative amino acid substitution).
[0323] As is known in the art, e.g., as described in Chin, J. W.,
Expanding and Reprogramming the Genetic Code of Cells and Animals,
Annual Review of Biochemistry, Vol. 83: 379-408, unnatural amino
acids may be incorporated into proteins made in vitro. For example,
in one system, UAG amber (stop) codons have been used to
incorporate pyrrolysine via an archeal tRNA synthetase and tRNA and
these can also be used to incorporate azides and alkynes via
feeding. Other side chains on unnatural amino acids that have been
demonstrated in the art include cyclopropene, trans-cyclooctene,
bicyclo[6.1.0]nonyne-lysine, coumarins, p-azidophenylalanine,
N6-[(2-propynloxy)carbonyl]-L-Lysine,
bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN),
N-5-norbornene-2-yloxycarbonyl-1-lysine,
N-tert-butyloxycarbonyl-1-lysine,
N-2-azidoethyloxycarbonyl-1-lysine, N-L-thiaprolyl-L-lysine,
N-D-cysteinyl-L-lysine, N-L-cysteinyl-L-lysine,
N6-[(2-propynyloxy)carbonyl]-L-lysine,
N6-[(2-azidoethoxy)carbonyl]-L-lysine, benzophenone,
4-(6-methyl-s-tetrazin-3-yl)aminophenylalanine, and
cyclooctynes.
Complexes
[0324] In naturally occurring protein-protein interactions, binding
events are typically driven largely by hydrophobic residues on flat
surface sites of the interacting proteins, in contrast to many
small molecule-protein interactions which are driven by
interactions between the small molecule in a cavity or pocket on
the protein. Commonly, hydrophobic residues on a protein's flat
surface site form a hydrophobic hot spot wherein most of the
binding interactions between or among interacting proteins are van
der Waals interactions. In some situations, a small molecule may
provide a "portable hotspot" (or portion thereof) in that it
participates in or generates such as a hydrophobic interaction site
on a protein (e.g., a presenter proteins) where such does not exist
absent the small molecule; aspects of the present disclosure are
particularly applicable to such situations. For example, in some
embodiments, a compound (and/or a tagged form thereof) as described
herein forms a complex with a protein (e.g., a presenter
protein/compound complex) and participates in pseudo
protein-protein interactions (e.g., forming a tripartite complex
with a target protein).
[0325] Many mammalian proteins are able to bind to any of a
plurality of different partners; in some cases, such alternative
binding interactions contribute to biological activity of the
proteins. Many of these proteins adapt the inherent variability of
the hot spot protein regions to present the same residues in
different structural contexts. More specifically, the
protein-protein interactions can be mediated by a class of natural
products produced by a select group of fungal and bacterial
species. These molecules exhibit both a common structural
organization and resultant functionality that provides the ability
to modulate protein-protein interaction. These molecules contain a
presenter protein binding moiety that is highly conserved and a
target protein interacting moiety that exhibits a high degree of
variability among the different natural products. The presenter
protein binding moiety confers specificity for the presenter
protein and allows the molecule to bind to the presenter protein to
form a complex; the mammalian target protein binding moiety confers
specificity for the target protein and allows the binary complex to
bind to the target protein, typically modulating (e.g., positively
or negatively modulating) its activity. In the present invention, a
binary complex (e.g., between a compound and presenter protein or a
compound and a target protein) is mimicked by conjugating a
presenter protein binding moiety to a target protein or a target
protein binding moiety to presenter protein. The resulting
conjugates of the invention may then bind to a presenter protein or
target protein forming a complex that mimics the tripartite
complex. These complexes may be used, e.g., to determine the
structure of the interface between the presenter protein and the
target protein. Furthermore, by simplifying the formation of the
complex, e.g., by conjugated a presenter protein binding moiety to
a target protein, the compounds of the invention may be used, e.g.,
to identify target proteins capable of binding to presenter
proteins.
Uses
Identification of Target Proteins
[0326] In some embodiments, the compounds, conjugates, complexes,
compositions, and/or methods of the present invention may be useful
to identify target proteins capable of forming complexes with
presenter proteins (e.g., in the presence of a small molecule). The
target proteins may be identified by formation of conjugates
including a presenter protein binding moiety conjugated to a target
moiety and determining if the conjugate forms a complex with a
presenter protein.
[0327] Most target proteins known in the art to form ternary
complexes with presenter proteins and small molecules were
identified fortuitously during determination of the mechanism of
action of the small molecule. The present methods allow for the
rational identification of target proteins capable of forming
complexes with presenter proteins in the presence of small
molecules by covalently conjugating a presenter protein binding
moiety to the target molecule, allowing formation of a complex
prior to identification of a compound capable of binding both the
presenter protein and target protein simultaneously.
[0328] Screening of small molecules for their ability to facilitate
complex formation between the presenter protein and identified
target protein could then be carried out to identify potential
therapeutics capable of modulating the biological activity of the
target protein.
[0329] In some embodiments, the compounds of the invention may be
used to identify target proteins capable of forming complexes with
presenter proteins. For example, target proteins may be identified
by combining one or more target proteins with a labeled presenter
protein (e.g., labeled with biotin) in the presence of a compound
of the invention under conditions suitable to allow for formation
of a presenter protein/target protein complex. The target proteins
which do not form complexes with presenter proteins may then be
removed (e.g., washed out) and the target proteins which form
complexes may then be pulled down using the label on the presenter
protein and analyzed. In some embodiments, the pulled down target
proteins may be analyzed by mass spectrometry to determine their
identity.
Compound Design
[0330] In some embodiments, the compounds, conjugates, complexes,
compositions, and/or methods of the present invention may be useful
for the design of compounds capable of modulating the biological
activity of target proteins for use in the treatment of
disease.
[0331] For example, formation of complexes of presenter proteins
and conjugates of the invention can facilitate determination of the
structure of the protein-protein interface between a presenter
protein and a target protein by crystallization and crystal
structure determination of the complex. Once the crystal structure
of a complex of the invention is determined, methods known in the
art for rational drug design may be used to develop small molecules
capable of facilitating complex formation between the presenter
protein and the target protein such as computational chemistry
methods to build structures de novo and/or fragment based drug
design using methods such as fragment soaking the crystals of
complexes of the invention and determining the resulting
structure.
[0332] The compounds designed as described above may then be
screened to determine their ability to modulate the biological
activity of the target protein and modified using medicinal
chemistry techniques, as necessary, to produce therapeutically
useful compounds.
Identification of Covalent Small Molecule Therapeutics
[0333] In some embodiments, the compounds, conjugates, complexes,
compositions, and/or methods of the present invention may be useful
for identifying compounds capable of modulating the biological
activity of target proteins through covalent interaction.
[0334] For example, the compounds of the inventions may be screened
for their ability to covalently bind to target proteins in the
presence and absence of presenter proteins to identify compounds
capable of selectively binding to target proteins only in the
presence of a presenter protein. These compounds may then be tested
for their ability to modulate biological activity of the target
protein and modified using medicinal chemistry techniques, as
necessary, to produce therapeutically useful compounds.
Determination of Biochemical and/or Biophysical Properties
[0335] In some embodiments, the compounds, conjugates, complexes,
compositions, and/or methods of the invention may be useful for
determining biochemical and/or biophysical properties of a protein
or complex.
[0336] For example, the free energy of binding between a conjugate
including a presenter protein binding moiety and a target protein
and a presenter protein may be determined, e.g., by isothermal
titration calorimetry. The K.sub.d of a conjugate including a
presenter protein binding moiety and a target protein for a
presenter protein may be determined, e.g., by surface plasmon
resonance. The K.sub.i, K.sub.inact, and/or K.sub.i/K.sub.inact for
a compound and a presenter protein for a target protein may be
determined, e.g., by mass spectrometry.
Treatment of Diseases or Disorders
[0337] Compounds, conjugates, and complexes described herein may be
useful in the methods of treating diseases or disorders related to
the target proteins described herein, and, while not bound by
theory, are believed to exert their desirable effects through their
ability to modulate (e.g., positively or negatively modulate) the
activity of a target protein (e.g., a eukaryotic target protein
such as a mammalian target protein or a fungal target protein or a
prokaryotic target protein such as a bacterial target protein),
through interaction with presenter proteins and the target
protein.
[0338] Kits
[0339] In some embodiments, the present invention relates to a kit
for conveniently and effectively carrying out the methods in
accordance with the present invention. In general, the
pharmaceutical pack or kit comprises one or more containers filled
with one or more of the ingredients of the pharmaceutical
compositions of the invention. Such kits are especially suited for
the delivery of solid oral forms such as tablets or capsules. Such
a kit preferably includes a number of unit dosages, and may also
include a card having the dosages oriented in the order of their
intended use. If desired, for instance if the subject suffers from
Alzheimer's disease, a memory aid can be provided, for example in
the form of numbers, letters, or other markings or with a calendar
insert, designating the days in the treatment schedule in which the
dosages can be administered. Alternatively, placebo dosages, or
calcium dietary supplements, either in a form similar to or
distinct from the dosages of the pharmaceutical compositions, can
be included to provide a kit in which a dosage is taken every day.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceutical products, which notice
reflects approval by the agency of manufacture, use or sale for
human administration.
[0340] Pharmaceutical Compositions
[0341] For use as treatment of human and animal subjects, the
compounds and conjugates of the invention can be formulated as
pharmaceutical or veterinary compositions. Depending on the subject
to be treated, the mode of administration, and the type of
treatment desired--e.g., prevention, prophylaxis, or therapy--the
compounds are formulated in ways consonant with these parameters. A
summary of such techniques is found in Remington: The Science and
Practice of Pharmacy, 21.sup.st Edition, Lippincott Williams &
Wilkins, (2005); and Encyclopedia of Pharmaceutical Technology,
eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New
York, each of which is incorporated herein by reference.
[0342] Compounds described herein may be present in amounts
totaling 1-95% by weight of the total weight of the composition.
The composition may be provided in a dosage form that is suitable
for intraarticular, oral, parenteral (e.g., intravenous,
intramuscular), rectal, cutaneous, subcutaneous, topical,
transdermal, sublingual, nasal, vaginal, intravesicular,
intraurethral, intrathecal, epidural, aural, or ocular
administration, or by injection, inhalation, or direct contact with
the nasal, genitourinary, reproductive or oral mucosa. Thus, the
pharmaceutical composition may be in the form of, e.g., tablets,
capsules, pills, powders, granulates, suspensions, emulsions,
solutions, gels including hydrogels, pastes, ointments, creams,
plasters, drenches, osmotic delivery devices, suppositories,
enemas, injectables, implants, sprays, preparations suitable for
iontophoretic delivery, or aerosols. The compositions may be
formulated according to conventional pharmaceutical practice.
[0343] In general, for use in treatment, compounds described herein
may be used alone, or in combination with one or more other active
agents. An example of other pharmaceuticals to combine with the
compounds described herein would include pharmaceuticals for the
treatment of the same indication. Another example of a potential
pharmaceutical to combine with compounds described herein would
include pharmaceuticals for the treatment of different yet
associated or related symptoms or indications. Depending on the
mode of administration, compounds are formulated into suitable
compositions to permit facile delivery. Each compound of a
combination therapy may be formulated in a variety of ways that are
known in the art. For example, the first and second agents of the
combination therapy may be formulated together or separately.
Desirably, the first and second agents are formulated together for
the simultaneous or near simultaneous administration of the
agents.
[0344] Compounds of the invention may be prepared and used as
pharmaceutical compositions comprising an effective amount of a
compound described herein and a pharmaceutically acceptable carrier
or excipient, as is well known in the art. In some embodiments, a
composition includes at least two different pharmaceutically
acceptable excipients or carriers.
[0345] Formulations may be prepared in a manner suitable for
systemic administration or topical or local administration.
Systemic formulations include those designed for injection (e.g.,
intramuscular, intravenous or subcutaneous injection) or may be
prepared for transdermal, transmucosal, or oral administration. A
formulation generally include diluents as well as, in some cases,
adjuvants, buffers, preservatives and the like. Compounds can be
administered also in liposomal compositions or as
microemulsions.
[0346] For injection, formulations can be prepared in conventional
forms as liquid solutions or suspensions or as solid forms suitable
for solution or suspension in liquid prior to injection or as
emulsions. Suitable excipients include, for example, water, saline,
dextrose, glycerol and the like. Such compositions may also contain
amounts of nontoxic auxiliary substances such as wetting or
emulsifying agents, pH buffering agents and the like, such as, for
example, sodium acetate, sorbitan monolaurate, and so forth.
[0347] Various sustained release systems for drugs have also been
devised. See, for example, U.S. Pat. No. 5,624,677, which is herein
incorporated by reference.
[0348] Systemic administration may also include relatively
noninvasive methods such as the use of suppositories, transdermal
patches, transmucosal delivery and intranasal administration. Oral
administration is also suitable for compounds of the invention.
Suitable forms include syrups, capsules, and tablets, as is
understood in the art.
[0349] Each compound of a combination therapy, as described herein,
may be formulated in a variety of ways that are known in the art.
For example, the first and second agents of the combination therapy
may be formulated together or separately.
[0350] The individually or separately formulated agents can be
packaged together as a kit. Non-limiting examples include, but are
not limited to, kits that contain, e.g., two pills, a pill and a
powder, a suppository and a liquid in a vial, two topical creams,
etc. The kit can include optional components that aid in the
administration of the unit dose to subjects, such as vials for
reconstituting powder forms, syringes for injection, customized IV
delivery systems, inhalers, etc. Additionally, the unit dose kit
can contain instructions for preparation and administration of the
compositions. The kit may be manufactured as a single use unit dose
for one subject, multiple uses for a particular subject (at a
constant dose or in which the individual compounds may vary in
potency as therapy progresses); or the kit may contain multiple
doses suitable for administration to multiple subjects ("bulk
packaging"). The kit components may be assembled in cartons,
blister packs, bottles, tubes, and the like.
[0351] Formulations for oral use include tablets containing the
active ingredient(s) in a mixture with nontoxic pharmaceutically
acceptable excipients. These excipients may be, for example, inert
diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol,
microcrystalline cellulose, starches including potato starch,
calcium carbonate, sodium chloride, lactose, calcium phosphate,
calcium sulfate, or sodium phosphate); granulating and
disintegrating agents (e.g., cellulose derivatives including
microcrystalline cellulose, starches including potato starch,
croscarmellose sodium, alginates, or alginic acid); binding agents
(e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium
alginate, gelatin, starch, pregelatinized starch, microcrystalline
cellulose, magnesium aluminum silicate, carboxymethylcellulose
sodium, methylcellulose, hydroxypropyl methylcellulose,
ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and
lubricating agents, glidants, and antiadhesives (e.g., magnesium
stearate, zinc stearate, stearic acid, silicas, hydrogenated
vegetable oils, or talc). Other pharmaceutically acceptable
excipients can be colorants, flavoring agents, plasticizers,
humectants, buffering agents, and the like.
[0352] Two or more compounds may be mixed together in a tablet,
capsule, or other vehicle, or may be partitioned. In one example,
the first compound is contained on the inside of the tablet, and
the second compound is on the outside, such that a substantial
portion of the second compound is released prior to the release of
the first compound.
[0353] Formulations for oral use may also be provided as chewable
tablets, or as hard gelatin capsules wherein the active ingredient
is mixed with an inert solid diluents (e.g., potato starch,
lactose, microcrystalline cellulose, calcium carbonate, calcium
phosphate or kaolin), or as soft gelatin capsules wherein the
active ingredient is mixed with water or an oil medium, for
example, peanut oil, liquid paraffin, or olive oil. Powders,
granulates, and pellets may be prepared using the ingredients
mentioned above under tablets and capsules in a conventional manner
using, e.g., a mixer, a fluid bed apparatus or a spray drying
equipment.
[0354] Dissolution or diffusion controlled release can be achieved
by appropriate coating of a tablet, capsule, pellet, or granulate
formulation of compounds, or by incorporating the compound into an
appropriate matrix. A controlled release coating may include one or
more of the coating substances mentioned above and/or, e.g.,
shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl
alcohol, glyceryl monostearate, glyceryl distearate, glycerol
palmitostearate, ethylcellulose, acrylic resins, dl-polylactic
acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl
acetate, vinyl pyrrolidone, polyethylene, polymethacrylate,
methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels,
1,3 butylene glycol, ethylene glycol methacrylate, and/or
polyethylene glycols. In a controlled release matrix formulation,
the matrix material may also include, e.g., hydrated
methylcellulose, carnauba wax and stearyl alcohol, carbopol 934,
silicone, glyceryl tristearate, methyl acrylate-methyl
methacrylate, polyvinyl chloride, polyethylene, and/or halogenated
fluorocarbon.
[0355] The liquid forms in which the compounds and compositions of
the present invention can be incorporated for administration orally
include aqueous solutions, suitably flavored syrups, aqueous or oil
suspensions, and flavored emulsions with edible oils such as
cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as
elixirs and similar pharmaceutical vehicles.
[0356] Generally, when administered to a human, the oral dosage of
any of the compounds of the combination of the invention depends on
the nature of the compound, and can readily be determined by one
skilled in the art. Typically, such dosage is normally about 0.001
mg to 2000 mg per day, desirably about 1 mg to 1000 mg per day, and
more desirably about 5 mg to 500 mg per day. Dosages up to 200 mg
per day may be necessary.
[0357] Administration of each drug in a combination therapy, as
described herein, can, independently, be one to four times daily
for one day to one year, and may even be for the life of the
subject. Chronic, long-term administration may be indicated.
EXAMPLES
Example 1: Synthesis of Certain Cross-Linking Reagents
Synthesis of
(R)-3-(3,4-dimethoxyphenyl)-1-(3-(3-(pyridin-2-yldisulfanyl)propanamido)p-
henyl)propyl
(S)-1-(3,3-dimethyl-2-oxopentanoyl)piperidine-2-carboxylate
(C3-SLF)
##STR00038##
[0359] To a solution of Aniline 1 (90 mg, 172 .mu.mol, 1 eq),
disulfide 2 (74 mg, 343 .mu.mol, 2 eq) and diisopropylethylamine
(149 .mu.L, 111 mg, 858 .mu.mol, 5 eq) in DNF (3 mL) was added HATU
(130 mg, 343 .mu.mol, 2 eq) and the reaction was stirred at room
temperature for 24 h. The reaction mixture was diluted with water
and extracted with ethyl acetate (3.times.). The organic extracts
were washed with water, saturated sodium chloride, dried over
magnesium sulfate and evaporated. The residue was purified on
Silica gel gradient elution (20% ethyl acetate: 80%
heptane.fwdarw.100% ethyl acetate) to provide the tittle compound
C3-SLF (50 mg, 40%). MS (ESI) calc=722.3 (M+H), obs=722.3.
Synthesis of
(R)-3-(3,4-dimethoxyphenyl)-1-(3-(4-(pyridin-2-yldisulfanyl)butanamido)ph-
enyl)propyl
(S)-1-(3,3-dimethyl-2-oxopentanoyl)piperidine-2-carboxylate
(C4-SLF)
##STR00039##
[0361] To a solution of Aniline 1 (90 mg, 172 .mu.mol, 1 eq),
disulfide 2 (79 mg, 343 .mu.mol, 2 eq) and diisopropylethylamine
(149 .mu.L, 111 mg, 858 .mu.mol, 5 eq) in DMF (3 mL) was added HATU
(130 mg, 343 .mu.mol, 2 eq) and the reaction mixture was stirred at
room temperature for 24 h. The reaction mixture was diluted with
water and extracted with ethyl acetate (3.times.). The organic
extracts were washed with water, saturated sodium chloride, dried
over magnesium sulfate and evaporated. The residue was purified on
Silica gel gradient elution (20% ethyl acetate: 80%
heptane.fwdarw.100% ethyl acetate) to provide the tittle compound
C4-SLF (98 mg, 77%). MS (ESI) calc=736.3 (M+H), obs=736.3.
Synthesis of methyl
(S)-1-((S)-3-(3-hydroxyphenyl)-2-((S)-3-methyl-2-(4-(pyridin-2-yldisulfan-
yl)butanamido)butanamido)propanoyl)hexahydropyridazine-3-carboxylate
(SFAC4DS)
##STR00040##
[0363] Amine 1 was prepared according to Paquette et al., JACS
2002(124), 4257-4270. To a solution of amine 1 (20 mg, 49.2
.mu.mol) in 1 mL of acetonitrile was added triethylamine (16.5
.mu.L, 118 .mu.mol, 2.4 eq), followed by acid chloride 2 (13.8 mg,
59.2 .mu.mol, 1.2 eq). The reaction mixture was stirred at room
temperature for 14 h, then concentrated, and the residue was
purified by preparative TLC (dichloromethane:MeOH:NH.sub.4OH,
20:1:0.1) to afford 14.0 mg (47%) of the product as a colorless
foam. R.sub.f=0.59 (dichloromethane:MeOH:NH.sub.4OH, 10:1:0.1). MS
(ESI) calc=618.2 (M+H), obs=618.2.
Synthesis of methyl
(S)-1-((S)-3-(3-hydroxyphenyl)-2-((S)-3-methyl-2-(3-(2-(pyridin-2-yldisul-
fanyl)ethoxy)propanamido)butanamido)propanoyl)hexahydropyridazine-3-carbox-
ylate (SFAX6)
##STR00041##
[0365] Carboxylic acid 2 (70 mg, 0.270 mmol) and HBTU (204 mg,
0.540 mmol, 2.00 eq) were mixed in 3 mL of acetonitrile, and the
resulting suspension was stirred at room temperature for 15 min.
Following this period, amine 1 (110 mg, 0.270 mmol, 1.00 eq) was
added followed by triethylamine (113 .mu.L, 0.810 mmol, 3.00 eq)
and the mixture was stirred at room temperature for 18 h. The
mixture was then treated with 20 mL of saturated sodium bicarbonate
and extracted with 2.times.30 mL portions of ethyl acetate. The
pooled organic extracts were washed with 2.times.20 mL portions of
brine, dried over saturated sodium sulfate, filtered and
concentrated under vacuum. The residue was purified using silica
gel chromatography, eluting with dichloromethane:MeOH, 100:1 to
50:1, affording 70 mg (40%) of the product as a colorless oil.
R.sub.f=0.31 (dichloromethane:MeOH, 20:1). MS (ESI) calc=648.2
(M+H), obs=648.2.
Synthesis of
N-(4-((2S,11R,14S,17S,20S,23S,26S)-26-ethyl-23-((1R,2R,E)-1-hydroxy-2-met-
hylhex-4-en-1-yl)-14,17-diisobutyl-20-isopropyl-4,11,13,16,19,22,28,31-oct-
amethyl-3,6,9,12,15,18,21,24,27,30,33-undecaoxo-1,4,7,10,13,16,19,22,25,28-
,31-undecaazacyclotritriacontan-2-yl)butyl)-4-(pyridin-2-yldisulfanyl)buta-
namide (CsA3)
##STR00042##
[0367] To a solution of amine 1 (100 mg, 90.5 .mu.mol) and
carboxylic acid 2 (31 mg, 135.2 .mu.mol, 1.5 eq) in 6 mL of NMP was
added HATU (51 mg, 134.1 .mu.mol, 1.48 eq) and DIPEA (70 .mu.L,
401.9 .mu.mol, 4.4 eq). The reaction was stirred for 1 h at room
temperature, then diluted with water and extracted with 3.times.30
mL portions of ethyl acetate. The organic extracts were washed with
saturated sodium chloride solution and concentrated under vacuum.
The crude material was purified by reversed phase chromatography on
C.sub.18 media, eluting with gradient of 15% acetonitrile:85% water
(both containing 0.1% formic acid) to 100% acetonitrile (containing
0.1% formic acid). MS (ESI) calc=658.9 (M+2H), obs=659.0.
Example 2: Synthesis of Certain Conjugates
[0368] General Protocol: This protocol describes a method for the
formation of target protein-compound conjugates.
[0369] Reagents: Compound in 100% DMSO (in-house) and mammalian
target protein (in-house)
[0370] Equipment: Mini-PROTEAN TGX Gel (Bio-Rad)
[0371] Experimental Protocol: A 1:2 molar ratio of target protein
and compound are mixed together in 12.5 mM HEPES pH 7.4, 75 mM NaCl
buffer containing 2% DMSO. The reaction is incubated at 37.degree.
C. for 30 min, followed by overnight incubation at room
temperature. Cross-linking efficiency is assessed by SDS-PAGE gel.
Conjugates migrate slower than non-cross-linked target protein. For
thiol reactive compounds, the Cys specific attachment of the
compound to the target protein can be further confirmed by SDS-PAGE
after the addition of 100 mM DTT to the reaction mixture, which
reduces the conjugate back into its components.
A. Formation of KRAS.sub.GTP/S39C Lite/C2-FK506 Conjugates
[0372] Reagents: C2-FK506 in 100% DMSO (in-house),
KRAS.sub.GTP/S39C lite (in house; residues 1-169 containing
G12V/S39C/C51S/C80L/C118S).
[0373] Equipment: Mini-PROTEAN TGX Gel (Bio-Rad)
[0374] Experimental Protocol: A 1:2 molar ratio of
KRAS.sub.GTP/S39C lite and C2-FK506 are mixed together in 12.5 mM
HEPES pH 7.4, 75 mM NaCl buffer containing 2% DMSO. The reaction is
incubated at 37.degree. C. for 30 min, followed by overnight
incubation at room temperature. Cross-linking efficiency is
assessed by SDS-PAGE gel. Attachment of C2-FK506 to Cysteine 39 on
KRAS.sub.GTP/S39C lite is also assessed by incubation of the
reaction mixture with 100 mM DTT.
[0375] Results: C2-FK506 cross-links efficiently with
KRAS.sub.GTP/S39C lite and is specific for Cysteine 39 (FIG.
1).
B. Formation of KRAS.sub.GTP/G12C Lite/SFAX9DS Conjugates
[0376] Reagents: SFAX9DS in 100% DMSO (in-house), KRAS.sub.GTP/G12C
lite (in house; residues 1-169 containing
G12C/C51S/C80L/C118S).
[0377] Equipment: Mini-PROTEAN TGX Gel (Bio-Rad)
[0378] Experimental Protocol: A 1:2 molar ratio of
KRAS.sub.GTP/G12C lite and SFAX9DS are mixed together in 12.5 mM
HEPES pH 7.4, 75 mM NaCl buffer containing 2% DMSO. The reaction is
incubated at 37.degree. C. for 30 min, followed by overnight
incubation at room temperature. Cross-linking efficiency is
assessed by SDS-PAGE gel. Wild-type CypA also cross-linked with the
compound. Cysteine 52 as a reactive Cysteine on CypA and was
mutated to Serine to abrogate presenter cross-linking.
[0379] Results: SFAX9DS cross-links efficiently with
KRAS.sub.GTP/G12C lite protein and CypA.sub.C52S does not
cross-link to SFAX9DS (FIG. 2).
Example 3: Formation of Certain Complexes
[0380] General Protocol: This protocol describes two methods for
the formation and isolation of complexes comprised of presenter
protein, compound, and mammalian target protein.
[0381] Reagents: Compound in 100% DMSO (in-house), presenter
protein (in-house) and mammalian target protein (in-house)
[0382] Equipment: Mini-PROTEAN TGX Gel (Bio-Rad), Superdex 75 (GE
Healthcare, CV 120 mL)
[0383] Experimental Protocol A: Pre-Conjugated Compound and
Protein
[0384] A 1:2 molar ratio of conjugate and presenter protein are
mixed together in 12.5 mM HEPES pH 7.4, 75 mM NaCl buffer
containing 2% DMSO. The reaction is incubated at 37.degree. C. for
30 min, followed by overnight incubation at room temperature. Pure
complex is isolated by Size Exclusion Chromatography (SEC)
purification. The reaction mixture is directly injected on a
Superdex 75 column (CV 120 mL) pre-equilibrated with buffer
containing 12.5 mM HEPES pH 7.4, 75 mM NaCl. Complex elutes at a
higher molecular weight than unreacted target protein and presenter
protein. To confirm presence of complex in the elution peak,
samples are assessed by SDS-PAGE.
[0385] Experimental Protocol B: Cross-Linking Reagent, Presenter
Protein and Target Protein
[0386] A 1:2:2 molar ratio of compound, presenter protein, and
target protein are mixed together in 12.5 mM HEPES pH 7.4, 75 mM
NaCl buffer containing 2% DMSO. The reaction is incubated at
37.degree. C. for 30 min, followed by overnight incubation at room
temperature. Pure complex is isolated by Size Exclusion
Chromatography (SEC) purification. The reaction mixture is directly
injected on a Superdex 75 column (CV 120 mL) pre-equilibrated with
buffer containing 12.5 mM HEPES pH 7.4, 75 mM NaCl. Complex elutes
at a higher molecular weight than unreacted target protein and
presenter protein. To confirm presence of complex in the elution
peak, samples are assessed by SDS-PAGE.
A. Formation of KRAS.sub.GTP/S39C lite/C2-Holt/FKBP12 ternary
complex
[0387] Reagents: C2-Holt in 100% DMSO (in-house), KRAS.sub.GTP/S39C
lite (in house; residues 1-169 containing
G12V/S39C/C51S/C80L/C118S), and FKBP12 (in-house).
[0388] Equipment: Mini-PROTEAN TGX Gel (Bio-Rad), Superdex 75 (GE
Healthcare, CV 120 mL)
[0389] Experimental Protocol: A 1:2:2 molar ratio of C2-Holt,
FKBP12, and KRAS.sub.GTP/S39C lite are mixed together in 12.5 mM
HEPES pH 7.4, 75 mM NaCl buffer containing 2% DMSO. The reaction is
incubated at 37.degree. C. for 30 min, followed by overnight
incubation at room temperature. Pure complex is isolated by Size
Exclusion Chromatography (SEC) purification. The reaction mixture
is directly injected on a Superdex 75 column (CV 120 mL)
pre-equilibrated with buffer containing 12.5 mM HEPES pH 7.4, 75 mM
NaCl. Complex elutes at around 69 mL post injection and unreacted
KRAS.sub.GTP/S39C lite and FKBP12 elutes at around 75 mL and 87 mL
post injection respectively. To confirm the presence of
KRAS.sub.GTP/S39C lite and FKBP12 in the elution peak, samples are
also assessed by SDS-PAGE.
[0390] Results: The SEC profile and SDS-PAGE analysis of the
elution peaks confirm the formation of KRAS.sub.GTP/S39C
lite/C2-Holt/FKBP12 complex (FIGS. 3A and 3B).
B. Formation of KRAS.sub.GDP/S39C Lite/SFAC4DS/CypA.sub.C52S
Ternary Complex
[0391] Reagents: SFAC4DS in 100% DMSO (in-house), KRAS.sub.GDP/S39C
lite (in house; residues 1-169 containing
G12V/S39C/C51S/C80L/C118S), and CypA.sub.C52S (in-house).
[0392] Equipment: Mini-PROTEAN TGX Gel (Bio-Rad), Superdex 75 (GE
Healthcare, CV 120 mL)
[0393] Experimental Protocol: A 1:2:2 molar ratio of SFAC4DS,
CypA.sub.C52S, and KRAS.sub.GDP/S39C lite are mixed together in
12.5 mM HEPES pH 7.4, 75 mM NaCl buffer containing 2% DMSO. The
reaction is incubated at 37.degree. C. for 30 min, followed by
overnight incubation at room temperature. Pure complex is isolated
by Size Exclusion Chromatography (SEC) purification. The reaction
mixture is directly injected on a Superdex 75 column (CV 120 mL)
pre-equilibrated with buffer containing 12.5 mM HEPES pH 7.4, 75 mM
NaCl. Complex elutes at around 69 mL post injection and unreacted
KRAS.sub.GDP/S39C lite and CypA.sub.C52S elutes at around 75 mL and
80 mL post injection respectively. To confirm the presence of
KRAS.sub.GDP/S39C lite and CypA.sub.C52S in the elution peak,
samples are also assessed by SDS-PAGE.
[0394] Results: The SEC profile and SDS-PAGE analysis of the
elution peaks confirm the formation of KRAS.sub.GDP/S39C
lite/SFAC4DS/CypA.sub.C52S complex (FIG. 4).
C. Formation of PTP1B.sub.S187C Lite/C3-SLF/FKBP12 Ternary
Complex
[0395] Reagents: C3-SLF in 100% DMSO (in-house), PTP1BE186C lite
(in house; residues 1-293 containing C32S/C92V/C121S/S187C), and
FKBP12 (in-house).
[0396] Equipment: Mini-PROTEAN TGX Gel (Bio-Rad), Superdex 75 (GE
Healthcare, CV 120 mL)
[0397] Experimental Protocol: A 1:3:3 molar ratio of C3-SLF,
FKBP12, and PTP1B.sub.S187C lite are mixed together in 12.5 mM
HEPES pH 7.4, 75 mM NaCl buffer containing 4% DMSO. The reaction is
incubated at 37.degree. C. for 30 min, followed by overnight
incubation at room temperature. Pure complex is isolated by Size
Exclusion Chromatography (SEC) purification. The reaction mixture
is directly injected on a Superdex 75 column (CV 120 mL)
pre-equilibrated with buffer containing 12.5 mM HEPES pH 7.4, 75 mM
NaCl. Complex elutes at around 62 mL post injection and unreacted
FKBP12 elutes at around 75 mL (Dimer) and 90 mL (Monomer) post
injection respectively. To confirm the presence of PTP1B.sub.S187C
lite and FKBP12 in the elution peak, samples are also assessed by
SDS-PAGE. Free PTP1B.sub.S187C lite and FKBP12 mixture are
subjected to a Superdex 75 column under the same condition to
determine their elution time.
[0398] Results: The SEC profile and SDS-PAGE analysis of free
PTP1B.sub.S187C lite and FKBP12 proteins (FIG. 5A) confirming the
free PTP1B.sub.S187C lite elutes at around 64-65 ml. The SEC
profile and SDS-PAGE analysis of the elution peaks confirm the
formation of PTP1B.sub.S187C lite/C3-SLF/FKBP12 complex, which
elutes at around 61 ml (FIG. 5B).
Example 4: Conjugate Formation when Presenter Protein is Present,
but not when it is Absent
[0399] This protocol describes methods to analyzing cross-linking
efficiency using mass spectrometry and gel shift assay in effort to
assess the presenter dependency of conjugate formation.
[0400] Reagents: Compound in 100% DMSO (in-house), FKBP12
(in-house), KRAS.sub.GTP/G12C (in-house, residues 1-169).
[0401] Experimental protocol: In order to follow the kinetics of
disulfide crosslinking reactions, Agilent 6230 TOF-LC/MS and
Agilent 1260 HPLC instruments employing a AdvanceBio RP-mAb C4
column (2.1.times.100 mm, 3.5 .mu.m) and equipped with an
auto-sampler were used. HPLC grade acetonitrile and water (each
containing 1.0 mM ammonium formate and 1% formic acid by volume)
were used as mobile phase with the following ramp: 0.6 ml/min flow
rate, water:acetonitrile=95:5 from 0.0 to 13.0 min ramping to
water:acetonitrile=5:95 from 13.0 to 17.0 min. Total time=17.0
min.
[0402] All crosslinking reactions were performed in 1.5 mL amber
colored glass vials equipped with 0.5 mL glass inserts. A
water-soluble peptide (SEQ ID NO: 1: YQNLLVGRNRGEEILD) was employed
as an internal standard. Although the actual sequence of the
internal standard is inconsequential, choice of amino acid residues
were critical to avoid interference in the crosslinking assays.
Hence, proline (interfering with FKBP12) and cysteine (interfering
with disulfide bond formation) residues were excluded. All
reactions and standard solutions were prepared in HEPES (pH 7.4,
1.0 mM MgCl.sub.2) buffer.
[0403] Prior to every reaction, a standard curve was obtained for
the individual components using a series of standard solutions (an
example of standard curve analysis for FKBP12 is shown in Table 3
below). Using the data from standard curve, .mu.mol of protein
samples were plotted against the ratio of areas (sample:std) and a
linear fit (y=mx+c) was employed to obtain the slope and intercept.
The value of slope and intercept involved in these standard curves
were accounted for during evaluation of the substrate and product
concentration before and during the course of the reaction. For
every substrate/product, an initial injection was followed up by a
blank injection to verify presence of any residual
protein/reagents. Based on this analysis the sequence of
auto-sampler can be adjusted to included appropriate number of
blank injections for removal of residual components, if any. The MS
spectra were analyzed using Agilent MassHunter v B.07.0
software.
TABLE-US-00003 TABLE 3 Standard curve for FKBP12. Slope = 6.53,
Intercept = -0.64, R.sup.2 = 0.999 Concen- Area under Area under
tration internal standard FKBP-12 Ratio of Solution (mM) (R.sub.t =
7.1 min) (R.sub.t = 8.9 min) Sample:Std 1 100 2615940.1 40314760.1
15.41 2 10 5244417.4 8718886.5 1.66 3 1 5868006.9 1745344.5 0.30 4
0.5 5407354.8 910748.2 0.17 5 0.1 5626447.0 237074.5 0.04
[0404] In a representative experiment to assess the presenter
dependency of ligand cross-linking on the target protein,
KRAS.sub.GTP/G12C and either C3- or C4-SLF ligand were incubated in
the presence or absence of FKBP12 at concentrations of 2 .mu.M
KRAS, 10 .mu.M FKBP12, 10 .mu.M C3- or C4-SLF for 4 hours at room
temperature in 12.5 mM HEPES pH 7.4, 75 mM NaCl, 1 mM MgCl.sub.2,
3% DMSO. Analysis of the amount of KRAS undergoing disulfide
cross-linking with the ligand using the method above. As shown in
Table 4, 5- to 10-fold increased cross-linking efficiency was
observed in the presence of the presenter:
TABLE-US-00004 TABLE 4 Crosslinking efficiency of C3- and C4-SLF
analyzed MS C3-SLF C4-SLF FKBP12 - + - + % xlink to KRAS 9.6 47.5
7.0 69.8
[0405] In parallel to mass spectrometry analysis, cross-linking
reactions with C3- or C4-SLF were subjected to gel shift assay
using 12% SDS-PAGE in the presence or absence of FKBP12 at the same
experimental condition described above except that cross-linking
reactions were set up at higher concentrations (60 .mu.M KRAS, 180
.mu.M FKBP12, and 180 .mu.M C3- or C4-SLF) and they were quenched
by MMTS to terminate the reaction. Similar to the MS data, the
ligand cross-linking efficiency was boosted significantly in the
presence of FKBP12, which is more pronounced for C4-SLF (FIG.
6).
Example 5: Determination of Presenter Protein/Target Protein
Interface Structure by X-Ray Analysis
[0406] This protocol describes the crystallization and structure
determination method for the crystal structure of a ternary complex
of FKBP12-C2Holt-KRAS.sub.GTP/S39C.
A. Crystal Structure Determination of
FKBP12-C2Holt-KRAS.sub.GTP/S39C Ternary Complex
[0407] Reagents: Ligand (C2Holt) in 100% DMSO (in-house), FKBP12
(in-house), KRAS.sub.GTP/S39C lite (in-house, residues 1-169
containing G12V/S39C/C51S/C80L/C118S).
[0408] Equipment: Superdex 75 (GE Healthcare)
[0409] Experimental Protocol: C Holt and FKBP12 were added to
KRAS.sub.GTP/S39C lite at 3:1 and 1.5:1 molar excess in 12.5 mM
HEPES pH 7.4, 75 mM NaCl buffer, 1 mM MgCl.sub.2, 2% DMSO, and
incubated overnight at 20.degree. C. or 36-72 hours at 4.degree. C.
Pure complex was isolated by size exclusion chromatography on a
Superdex 75 column in 12.5 mM HEPES pH 7.4, 75 mM NaCl, and 1 mM
MgCl.sub.2. Purified complex (at 15-20 mg/ml) was subjected to
crystallization screening at 20.degree. C. using sitting drop vapor
diffusion method. Crystals were grown in a well solution containing
0.1 M MES pH 6.5, 20-22% PEG 20,000. For data collection crystals
were transferred to a solution containing mother liquor
supplemented with 15% glycerol, and then frozen in liquid nitrogen.
Diffraction datasets were collected at the Advanced Photon Source
(APS) and processed with the HKL program. Molecular replacement
solutions were obtained using the program PHASER in the CCP4 suite,
using the published structure of FKBP12 (PDB-ID 1FKD) and KRAS
(PDB-ID 3GFT) as search models. Subsequent model building and
refinement were performed according to standard protocols with the
software packages CCP4 and COOT.
[0410] Results: Overall structure of
FKBP12-C2Holt-KRAS.sub.GTP/S39C: The crystal contains one
heterodimer of FKBP12 and KRAS.sub.GTP/S39C in the asymmetric unit
(FIG. 7). The model comprised residues Met1 to Glu108 of FKBP12 and
Met1 to Lys169 of KRAS.sub.GTP/S39C. The resulting electron density
shows unambiguous binding mode, including the orientation and
conformation of the ligand. The continuous electron density was
observed for the disulfide generated from the cysteine of the
protein and the sulfur from the ligand.
[0411] The KRAS.sub.GTP/S39C residues involved in binding C2Holt (4
.ANG. distance cut-off) are Glu37, Cys39, Leu56, and Met67. The
KRAS.sub.GTP/S39C residues involved in binding to FKBP12 are Glu3,
Lys5, Ile36, Cys39, Tyr40, Arg41, Asp54, Glu63, Tyr64, Met67, and
Arg73. The FKBP12 residues involved in binding KRAS.sub.GTP/S39C
are Arg43, Lys53, Gln54, Glu55, Thr86, Pro89, Gly90, and Ile92. The
FKBP12 residues involved in binding C2Holt are Tyr27, Phe37, Asp38,
Phe47, Glu55, Val56, Ile57, Trp60, Tyr83, His88, Ile91, Ile92, and
Phe100.
[0412] The total buried surface area of the complex is 1,947
.ANG..sup.2. The buried surface area of KRAS.sub.GTP/S39C is 600
.ANG..sup.2 of which 501 .ANG..sup.2 is contributed by FKBP12
(83%), and 99 .ANG..sup.2 contributed by C2Holt (17%). The buried
surface area of FKBP12 is 762 .ANG..sup.2 of which 500 .ANG..sup.2
contributed by KRAS.sub.GTP/S39C (66%) and 262 .ANG..sup.2
contributed by C2Holt (34%). The buried surface area of C2 Holt is
584 .ANG..sup.2 of which 132 .ANG..sup.2 contributed by
KRAS.sub.GTP/S39C (23%) and 452 .ANG..sup.2 contributed by FKBP12
(77%). The protein-protein interface between KRAS.sub.GTP/S39C and
FKBP12 is formed by both hydrophobic and polar interactions,
including three intermolecular H-bonds. The binding interface
between C2 Holt and FKBP12 is largely contributed by hydrophobic
interactions, but also contributed by three H-bonds between three
carbonyl groups of the ligand and Tyr27, Ile57, and Tyr83 of
FKBP12. C2 Holt forms minimal contact with KRAS.sub.GTP/S39C by
design (99 .ANG..sup.2) but forms one H-bond with Glu37 of
KRAS.sub.GTP/S39C. Data collection and refinement statistics of the
final structure are listed in Table 5 below.
B. Crystal structure determination of
KRAS.sub.GDP/S39C/SFAC4DS/CypA.sub.C52S ternary complex
[0413] Reagents: Ligand (SFAC4DS) in 100% DMSO (in-house),
CypA.sub.C52S (in-house), KRAS.sub.GDP/S39C lite (in-house,
residues 1-169 containing G12V/S39C/C51S/C80L/C118S).
[0414] Equipment: Superdex 75 (GE Healthcare)
[0415] Experimental Protocol: SFAC4DS and CypA.sub.C52S were added
to KRAS.sub.GDP/S39C lite at 2:1 and 2:1 molar excess in 12.5 mM
HEPES pH 7.4, 75 mM NaCl buffer, 1 mM MgCl.sub.2, 2% DMSO, and
incubated overnight at 20.degree. C. Pure complex was isolated by
size exclusion chromatography on a Superdex 75 column in 12.5 mM
HEPES pH 7.4, 75 mM NaCl, and 1 mM MgCl.sub.2. Purified complex (at
15 mg/ml) was subjected to crystallization screening at 20.degree.
C. using sitting drop vapor diffusion method. Crystals were grown
in a well solution containing 0.1 M Bis-Tris pH 6.5, 25% PEG 3350.
For data collection, crystals were transferred to a solution
containing mother liquor supplemented with extra PEG 3350 to make
it 40% of PEG, and then frozen in liquid nitrogen. Diffraction
datasets were collected at the Advanced Light Source (ALS) and
processed with the HKL program. Molecular replacement solutions
were obtained using the program PHASER in the CCP4 suite, using the
published structure of CypA (PDB-ID 1CWA) and KRAS (PDB-ID 3GFT) as
search models. Subsequent model building and refinement were
performed according to standard protocols with the software
packages CCP4 and COOT.
[0416] Results: Overall structure of
CypA.sub.C52S-SFAC4DS-KRAS.sub.GDP/S39C: The crystal contains one
heterodimer of CypA.sub.C52S and KRAS.sub.GDP/S39C in the
asymmetric unit (FIG. 8). The model comprised residues Met1 to
Glu165 of CypA and Met1 to Lys169 of KRAS.sub.GDP/S39C. The
resulting electron density shows unambiguous binding mode,
including the orientation and conformation of the ligand. The
continuous electron density was observed for the disulfide
generated from the cysteine of the protein and the sulfur from the
ligand.
[0417] The KRAS.sub.GDP/S39C residues involved in binding SFAC4DS
(4 .ANG. distance cut-off) are Glu3, Lys5, Cys39, Arg41, Leu52,
Asp54, Ile55 and Leu56. The KRAS.sub.GDP/S39C residues involved in
binding to CypA.sub.C52S are Glu37, Asp38, Cys39, Arg41, Gln43,
Leu56, Ala66, Met67, Gln70, and Thr74. The CypA.sub.C52S residues
involved in binding KRAS.sub.GDP/S39C are Arg55, Ile57, Arg69,
Asn71, Thr73, Ala81, Ala103, Arg148, and Asn149. The CypA.sub.C52S
residues involved in binding SFAC4DS are Arg55, Phe60, Met61,
Gln63, Gly72, Ala101, Asn102, Gln111, Phe113, and His126.
[0418] The total buried surface area of this complex cannot be
calculated due to partial structural disorder at the
protein-protein interface. Excluding the disordered region for
calculation, the buried surface area at the protein-protein
interface is greater than 1,350 .ANG..sup.2, of which over 30% is
contributed by SFAC4DS (443 .ANG..sup.2). The protein-protein
interface between KRAS.sub.GDP/S39C and CypA.sub.C52S is formed by
both hydrophobic and polar interactions, including two
intermolecular H-bonds. The binding interface between SFAC4DS and
CypA is contributed both by hydrophobic and polar interactions.
There are six H-bonds between carbonyl and N--H groups of the
ligand and residues Arg55, Gln63, Asn102, and His126 of
CypA.sub.C52S. SFAC4DS forms minimal direct contact with
KRAS.sub.GDP/S39C but forms one H-bond with Arg41 of
KRAS.sub.GDP/S39C. Data collection and refinement statistics of the
final structure are listed in Table 5 below.
C. Crystal Structure Determination of PTP1B.sub.S187C/C3SLF/FKBP12
Ternary Complex
[0419] Reagents: Ligand (C3-SLF) in 100% DMSO (in-house), FKBP12
(in-house), PTP1B.sub.S187C lite (in-house, residues 1-169
containing C32S/C92V/C121S/S187C).
[0420] Equipment: Superdex 75 (GE Healthcare), Gryphon (Art Robbins
Instruments)
[0421] Experimental Protocol: C3SLF and FKBP12 were added to
PTP1B.sub.S187C lite at 3:1 molar excess in 12.5 mM HEPES pH 7.4,
75 mM NaCl buffer, 4% DMSO, and incubated 36-72 hours at 4.degree.
C. Pure complex was isolated by size exclusion chromatography on a
Superdex 75 column in 12.5 mM HEPES pH 7.4 and 75 mM NaCl. Purified
complex (at 15 mg/ml) was subjected to crystallization screening at
20.degree. C. using sitting drop vapor diffusion method. Crystals
were grown in a well solution containing 0.2 M Magnesium Acetate,
20% w/v PEG 3350. For data collection, crystals were transferred to
a solution containing mother liquor supplemented with 25% PEG400,
and then frozen in liquid nitrogen. Diffraction datasets were
collected at the Advanced Photon Source (APS) and processed with
the XDS program. Molecular replacement solutions were obtained
using the program PHASER in the CCP4 suite, using the published
structure of FKBP12 (PDB-ID 2PPN) and PTP1B (PDB-ID 2NT7) as search
models. Subsequent model building and refinement were performed
according to standard protocols with the software packages CCP4 and
COOT.
[0422] Results: Overall structure of FKBP12-C3SLF-PTP1B.sub.S187C:
The crystal contains two complex molecules of
FKBP12-C3SLF-PTP1B.sub.S187C in the asymmetric unit (FIG. 9A). The
model comprised residues Gly2 to Glu108 of FKBP12 and Glu6 to
Phe280 of PTP1B.sub.S187C. The resulting electron density shows
unambiguous binding mode, including the orientation and
conformation of the ligand. The continuous electron density was
observed for the disulfide generated from the cysteine of the
protein and the sulfur from the ligand.
[0423] The total buried surface area of the complex is 1,042
.ANG..sup.2. The buried surface area of PTP1B.sub.S187C is 427
.ANG..sup.2. The buried surface area of C3-SLF is 615 .ANG..sup.2
(FIG. 9B). The protein-protein interface between PTP1B.sub.S187C
and FKBP12 is formed by both hydrophobic and polar
interactions.
D. Crystal Structure Determination of MCL1.sub.S245C/C3SLF/FKBP52
Ternary Complex
[0424] Reagents: Ligand (C3-SLF) in 100% DMSO (in-house), FKBP52
(in-house, residues 1-140), MCL1.sub.S245C lite (in-house, residues
172-327 containing S245C/C286S).
[0425] Equipment: Superdex 75 (GE Healthcare), Gryphon (Art Robbins
Instruments)
[0426] Experimental Protocol: C3SLF and FKBP52 were mixed with
MCL1.sub.S245C lite at 3:1 molar excess in 12.5 mM HEPES pH 7.4, 75
mM NaCl buffer, 2% DMSO, and incubated 24-48 hours at 4.degree. C.
Pure complex was isolated by size exclusion chromatography on a
Superdex 75 column in 12.5 mM HEPES pH 7.4 and 75 mM NaCl. Purified
complex (at 15 mg/ml) was subjected to crystallization screening at
20.degree. C. using sitting drop vapor diffusion method. Crystals
were grown in a well solution containing 2.1 M Malic acid. For data
collection, crystals were transferred to a solution containing
mother liquor supplemented with 20% glycerol, and then flash-frozen
in liquid nitrogen. 3.0 .ANG. resolution diffraction dataset was
measured at the Advanced Photon Source (APS) and processed with the
XDS program. Molecular replacement solutions were obtained using
the program PHASER in the CCP4 suite, using the published structure
of FKBP52 (PDB-ID 1 N1A) and PTP1B (PDB-ID 3MK8) as search models.
Subsequent model building and refinement were performed according
to standard protocols with the software packages CCP4 and COOT.
[0427] Results: The crystal contains one complex molecule of
MCL1.sub.S245C/C3SLF/FKBP52 in the asymmetric unit (FIG. 10). The
resulting electron density revealed unambiguous binding between two
proteins, including the orientation and conformation of the ligand.
The continuous electron density was observed for the disulfide
generated from the cysteine of the protein and the sulfur from the
ligand. The total buried surface area of the complex is 1,410
.ANG..sup.2, of which approximately 60% is contributed by FKBP52
(804 .ANG..sup.2), and approximately 40% by C3-SLF (606
.ANG..sup.2). Due to the limited resolution, the detailed analysis
in the protein-protein and protein-ligand interaction was not
feasible.
TABLE-US-00005 TABLE 5 Data collection and refinement statistics
Structure A Structure B Structure C Structure D Resolution [.ANG.]
47.8-1.4 70.1-1.6 49.0-2.4 65.9-3.0 Number of reflections
(working/test) 50,557/2,708 34,377/1,790 30,175/1,543 5,343/245
R.sub.cryst [%] 18.9 17.9 23.0 33.2 R.sub.free[%].sup.1 21.7 21.2
28.1 37.8 Total number of atoms: Protein 2,202 2,528 6,192 2,035
Water 268 171 38 0 Ligands 69 63 43 43 Ions 1 1 0 0 Deviation from
ideal geometry: .sup.2 Bond lengths [.ANG.] 0.007 0.011 0.004 0.010
Bond angles [.degree.] 1.33 1.49 0.87 1.30 Ramachandran plot:
.sup.3 Most favoured regions [%] 93.8 96.5 93.3 95.2 Allowed
regions [%] 6.2 3.1 4.9 4.4 Disallowed region [%] 0.0 0.3 1.8 0.4
.sup.1Test-set contains 5% of measured reflections .sup.2 Root mean
square deviations from geometric target values .sup.3 Calculated
with RAMPAGE
Example 6: Determination of Complex Formation by TR-FRET
[0428] TR-FRET technology (LANCE, Perkin Elmer) is a standard
method to detect the binary association of two fusion-tagged
proteins, e.g., protein 1/tag A and protein 2/tag B, where A and B
can be any of glutathione-S-transferase (GST), hexahistidine
(His.sub.6), FLAG, biotin-avi, Myc, and Hemagglutinin (HA). In this
example, the technology is used to measure the compound-facilitated
association of a presenter protein with a target protein. A mixture
of a presenter protein/tag A and a target protein/tag B are added
to a 384-well assay plate containing compounds of the invention and
incubated for 15 minutes. A mixture of anti-fusion tag A or B
Europium-chelate donor and anti-fusion tag A or B allophycocyanin
acceptor or Ulight acceptor reagents are added and the reactions
are incubated for 240 minutes. The TR-FRET signal is read on an
Envision microplate reader (Perkin Elmer) using excitation=320 nm,
emission=665/615 nm. Compounds that facilitate ternary complex
formation are identified as those eliciting an increase in the
TR-FRET ratio relative to DMSO control wells.
Determination of CYPA-Compound 3-KRAS.sub.G12C-GTP Complex
Formation by TR-FRET Avi-tagged Cyclophilin A and His-tagged KRAS
G.sub.12C-GTP were mixed with increasing concentration of ligand
(Compound 3) and incubated at room temperature for 15 minutes to
allow formation of ternary complex. A pre-mixture of Anti-His
Eu-W1024 and Streptavidin APC were then added and incubated for 60
minutes. TR-FRET signal is read on an EnVision microplate reader
(Perkin Elmer, Ex 320 nm, Em 665/615 nm). A counter screen without
presenter and target protein is also run to rule out the
contribution of compounds alone.
[0429] Reagents and Instrument [0430] His6-KRAS.sub.G12C-GTP (in
house; residues 1-169); 1.2 mM in PBS buffer, pH 7.4 [0431]
Avi-CYPA (in house; residues 1-165); 556 .mu.M in PBS buffer, pH
7.4 [0432] Anti-His Eu-W1024 (Perkin Elmer) [0433] Streptavidin APC
(Perkin Elmer) [0434] Ligand (W21487), 10 mM in 100% DMSO [0435]
EnVision (Perkin Elmer) [0436] Combi Mutidrop liquid dispenser with
8-channel small volume cassette [0437] 384-w ProxiPlate (black)
[0438] Experimental Protocol [0439] 1. Use Mosquito to dispense 100
nL/well of compounds (varying concentration in DMSO) into 384-w
black ProxiPlate to make assay-ready-plate (ARP). [0440] 2. Make
2.times. assay buffer containing 40 mM Hepes pH 8.0, 200 mM NaCl, 2
mM MgCl.sub.2, 0.1% BSA and 0.004% Tween-20. [0441] 3. Make
2.times.PRE-MIX A: 100 nM of His6-KRas G12C-GTP (1-169) and 1000 nM
of Avi-CypA (1-165) in 1.times. assay buffer. [0442] 4. Use
MutiDrop Combi to dispense 2.times.PRE-MIX A into ARP, 5
.mu.l/well. Incubate 15 min at RT. [0443] 5. Make 2.times.PRE-MIX
B: 10 nM of anti-His Eu-W1024 and 40 nM of SA APC. [0444] 6. Use
MutiDrop Combi to dispense 2.times.PRE-MIX B into ARP, 5
.mu.l/well. Shake briefly on Combi and incubate 60 min at RT.
[0445] 7. Read on EnVision (Ex: 320 nm; Em1:615 nm; Em2: 665 nm).
[0446] 8. Data is processed using Dotmatics. Curves are fit using a
4 parameter non-linear fit to determine the EC50 value for
formation of the ternary complex.
[0447] Results: The binding curve (FIG. 11) demonstrates Compound 3
dependent complex formation of CYPA-Compound 3-KRAS.sub.G12C-GTP
ternary complex, with a calculated EC50 value of 2.1 .mu.M
Example 7: Determination of Complex Formation by Amplified
Luminescent Proximity Homogeneous Assay
[0448] AlphaScreen technology (Perkin Elmer) is a standard method
to detect the binary association of two fusion-tagged proteins,
e.g., protein 1/tag A and protein 2/tag B, where A and B can be any
of glutathione-S-transferase (GST), hexahistidine (His.sub.6),
FLAG, biotin-avi, Myc, and Hemagglutinin (HA). In this example, the
technology is used to measure the compound-facilitated association
of a presenter protein with a target protein. A mixture of
presenter protein/tag A and target protein/tag B are added to a
384-well assay plate containing compounds of the invention and
incubated for 15 minutes. A mixture of anti-fusion tag A or B
AlphaScreen donor beads and anti-fusion tag A or B AlphaScreen
acceptor beads are added and the reactions are incubated for 240
minutes. The AlphaScreen signal is read on an Envision microplate
reader (Perkin Elmer) using excitation=680 nm, emission=585 nm.
Compounds that facilitate ternary complex formation are identified
as those eliciting an increase in the AlphaScreen signal relative
to DMSO control wells.
Determination of CYPA-Compound 3-KRAS.sub.G12C-GTP Complex
Formation by alpha LISA Avi-tagged Cyclophilin A and His-tagged
KRAS.sub.G12C-GTP were mixed with increasing concentration of
ligand (Compound 3) and incubated at room temperature for 60
minutes to allow formation of ternary complex. A pre-mixture of
Nickel chelate donor beads and Streptavidin acceptor beads were
then added and incubated for 60 minutes. AlphaLISA signal is read
on an EnVision microplate reader (Perkin Elmer, Ex 680 nm, Em 615
nm). A counter screen without presenter and target protein is also
run to rule out the contribution of compounds alone.
[0449] Reagents and Instrument: [0450] His6-KRAS.sub.G12C-GTP (in
house; residues 1-169); 1.2 mM in PBS buffer, pH 7.4 [0451]
Avi-CYPA (in house; residues 1-165); 556 uM in PBS buffer, pH 7.4
[0452] Nickel chelate donor beads (Perkin Elmer) [0453]
Streptavidin acceptor beads (Perkin Elmer) [0454] Ligand (W21487),
10 mM in 100% DMSO [0455] EnVision (Perkin Elmer) [0456] Combi
Mutidrop liquid dispenser with 8-channel small volume cassette
[0457] alphaPlate-384 plate (white)
[0458] Experimental Protocol: [0459] 1. Use Mosquito to dispense
100 nL/well of compounds (varying concentration in DMSO) into
384-well black ProxiPlate to make assay-ready-plate (ARP). [0460]
2. Make 2.times. assay buffer containing 40 mM Hepes pH 8.0, 200 mM
NaCl, 2 mM MgCl.sub.2 and 0.004% Tween-20. [0461] 3. Make
2.times.PRE-MIX A: 300 nM of His6-KRas G12C-GTP (1-169) and 300 nM
of Avi-CypA (1-165) in 1.times. assay buffer. [0462] 4. Use
MutiDrop Combi to dispense 2.times.PRE-MIX A into ARP, 5
.mu.l/well. Incubate 60 min at RT. [0463] 5. Make 2.times.PRE-MIX
B: 30 .mu.g/ml of streptavidin acceptor beads and 30 .mu.g/ml of
Nickel chelate donor beads. [0464] 6. Use MutiDrop Combi to
dispense 2.times.PRE-MIX B into ARP, 5 .mu.l/well. Shake briefly on
Combi and incubate 60 min at RT. [0465] 7. Read on EnVision (Ex:
680 nm; Em1:615 nm). [0466] 8. Data is processed using Dotmatics.
Curves are fit using a 4 parameter non-linear fit to determine the
EC50 value for formation of the ternary complex.
[0467] Results: The binding curve (FIG. 12) demonstrates Compound 3
dependent complex formation of CYPA-Compound 3-KRAS.sub.G12C-GTP
ternary complex, with a calculated EC50 value of 0.99 .mu.M.
Example 8: Determination of Complex Formation by Isothermal
Titration Calorimetry
[0468] Isothermal Titration Calorimetry (ITC) is an established
biophysical technique used to directly measure the heat change
associated with the binary interaction of two proteins or protein
to a ligand. Measurement of the heat change allows accurate
determination of association constants (K.sub.a), reaction
stoichiometry (N), and the change in binding enthalpy (.DELTA.H).
Gibbs energy changes (.DELTA.G) and entropy changes (.DELTA.S) can
also be determined using the relationship:
.DELTA.G=-RTInK.sub.a=.DELTA.H-T.DELTA.S (where R is the gas
constant and Tis the absolute temperature). In this example, the
method is used to measure binding (e.g., non-covalent or covalent
binding) of a compound or conjugate of the invention to a presenter
protein.
Determination of Kinetics and Thermodynamics of Binding Between
FKBP12-Compound 1 and CEP250 by ITC
[0469] Reagents: Compound 1 and Compound 2 in 100% DMSO (in-house),
Protein Buffer (10 mM HEPES, pH 7.5, 75 mM NaCl, 0.5 mM TCEP),
assay buffer (protein buffer+1% DMSO), FKBP12 (in-house),
CEP250.sub.29.4 (in-house, residues 1982-2231) and CEP250.sub.11.4
(in-house, residues 2134-2231).
[0470] Equipment: MicroCal.TM. ITC200 (GE Healthcare). Instrument
parameters are shown in Table 6.
TABLE-US-00006 TABLE 6 Isothermal Titration Calorimetry instrument
parameters Experimental device MicroCal .TM. ITC.sub.200 (GE
Healthcare) Sample cell volume (.mu.l) 270 Injector volume (.mu.l)
40 Experimental parameters Total number of Injections 19 Cell
Temperature (.degree. C.) 25 Reference Power (.mu.Cal/s) 5 Initial
Delay (s) 200 Stirring Speed (rpm) 750 Injection parameters Volume
(.mu.l) 2 Duration (s) 4 Spacing (s) 170-200 Filter Period (s) 5
Feedback Mode/Gain High
[0471] Experimental Protocol: FKBP12 stock solution is diluted to
10 .mu.M in assay buffer (1% DMSO final). Compound is added to
FKBP12 to 20 .mu.M (1% DMSO final), and binary complex is filled
into the reaction cell of the ITC device after 5-10 min
pre-incubation time. CEP250 protein stocks are diluted to 50 .mu.M
in assay buffer and supplemented with 20 .mu.M compound (1% DMSO
final) before being filled into the injection syringe. A control
experiment in the absence of compound is also run to determine the
heat associated with operational artifacts and the dilution of
titrant as it is injected from the syringe into the reaction cell.
More detailed experimental parameters are shown in Table 7.
TABLE-US-00007 TABLE 7 Final protein and ligand concentrations DMSO
conc. Experiment Cell content Syringe content Ligand (%) 3 FKBP12,
10 .mu.M CEP250.sub.29.2, 50 .mu.M None 1.0 4 FKBP12, 10 .mu.M
CEP250.sub.11.4, 50 .mu.M None 1.0 5 FKBP12, 10 .mu.M
CEP250.sub.29.2, 118 .mu.M Compound 1b, 20 .mu.M 1.0 6 FKBP12, 10
.mu.M CEP250.sub.29.2, 118 .mu.M Compound 2, 20 .mu.M 1.0 7 FKBP12,
10 .mu.M CEP250.sub.11.4, 68 .mu.M Compound 1b, 20 .mu.M 1.0 8
FKBP12, 10 .mu.M CEP250.sub.11.4, 68 .mu.M Compound 2, 20 .mu.M
1.0
[0472] Data Fitting: Data were fitted with the Origin ITC200
software according to the following procedure: [0473] 1) Read raw
data [0474] 2) In "mRawITC": adjust integration peaks and baseline,
integrate all peaks [0475] 3) In "Delta H"--data control: remove
bad data (injection #1 and other artifacts), subtract straight line
(background subtraction) [0476] 4) In "Delta H"--model fitting:
select one set of sites model, perform fitting with
Levenberg-Marquardt algorithm until Chi Square is not reduced
further, finish with "done" (parameters N, K.sub.a and .DELTA.H are
calculated based on fitting)
[0477] Results: ITC measurements for the binding of FKBP12-Compound
1 and FKBP12-Compound 2 binary complexes to CEP250 are summarized
in Table 8 and FIG. 13. Overall, the data for FKBP12-Compound 1 and
FKBP12-Compound 2 binary complexes binding to CEP250.sub.11.4 and
CEP250.sub.29.4 show similar interaction parameters. K.sub.d values
were similar for all combinations. All interactions show an almost
identical thermodynamic profile in which binding is characterized
by a purely enthalpic binding mode (-T*.DELTA.S term is positive
and does not contribute to the Gibbs free energy). Binding
stoichiometries for all interactions were N=0.5-0.6 and support a
1:2 binding ratio for 1 CEP250 homodimer binding to 2 FKBP12
molecules, as evidenced in the crystal structure of
CEP250.sub.11.4/Compound 1/FKBP12.
TABLE-US-00008 TABLE 8 Determination of FKBP12-Compound 1-CEP250
ternary complex formation by ITC Kd .DELTA.H -T*.DELTA.S .DELTA.H
Experiment T (K) N (.mu.M)* (kJ*mol.sup.-1)** (kJ*mol.sup.-1)***
(kJ*mol-1)**** 3 298 N.D. N.D. N.D. N.D. N.D. 4 298 N.D. N.D. N.D.
N.D. N.D. 5 298 0.50 0.19 -52.21 13.80 -38.41 6 298 0.57 0.36
-58.48 21.73 -36.74 7 298 0.56 0.07 -49.37 8.62 -40.75 8 298 0.54
0.08 -47.78 7.41 -40.36 *K.sub.d (calculated from K.sub.a =
1/K.sub.d **.DELTA.H ***T*.DELTA.S (calculated from - T.DELTA.S =
.DELTA.G - .DELTA.H) ****.DELTA.G = -RT In K.sub.a = RT In
K.sub.d
Example 9: Determination of Kinetics of Binding Between Conjugates
and Proteins by Surface Plasmon Resonance
[0478] Surface Plasmon Resonance (SPR) is a biophysical technique
used to measure the kinetics associated with the binary interaction
of either two proteins or a protein to a ligand. Typically, one
component of the binary interacting pair is immobilized on a flow
cell of an activated sensor chip via a fusion tag. Increasing
concentrations of the second component (the analyte) are then
injected over the active surface for a fixed time. An increase in
SPR signal (expressed in resonance units, RU) during the
association phase and decrease in SPR signal during the
dissociation phase is indicative of an interaction and can be fit
to a binding model to determine associated K.sub.D, K.sub.a,
K.sub.d values. In this example, the method is used to measure
kinetics for the binding of a conjugate of the invention to a
presenter protein, in which either (i) the conjugate is immobilized
on the chip via fusion tag and a presenter protein is injected over
the surface, or (ii) a presenter protein is immobilized on the chip
via a fusion tag and a conjugate is injected over the surface.
Determination of Kinetics of Binding Between FKBP12-Compound 1 and
CEP250 by SPR
[0479] This protocol utilizes Surface Plasmon Resonance (SPR) as a
method to determine kinetics (K.sub.D, K.sub.a, K.sub.d) for the
binding of CEP250 (analyte) to immobilized FKBP12-Compound 1 binary
complex (ligand).
[0480] Reagents: Compound 1 in 100% DMSO (in-house),
10.times.HBS-P+ buffer (GE Healthcare BR-1006-71), Assay buffer
(1.times.HBS-P+ buffer, 1% DMSO, 1 .mu.M Compound 1), 12.times.HIS
tagged FKBP12 (in-house), CEP250.sub.29.2 (residues 1982-2231) and
CEP250.sub.11.4 (residues 2134-2231) (in-house).
[0481] Equipment: BIACORE.TM. X100 (GE Healthcare)
[0482] Supplies: NTA Sensor chip (GE Healthcare BR-1000-34)
[0483] Experimental Protocol: Experiments are performed at
25.degree. C. Stock solution of 12.times.HIS tagged FKBP12 is
diluted to 100 nM in assay buffer containing 1 .mu.M Compound 1 (1%
DMSO final). Approximately 200-400 RU of FKBP12 is immobilized on
one of two flow cells of an activated NTA chip. The second flow
cell is not activated as a reference for non-specific interaction
of the analyte to the sensor chip. Various concentrations of CEP250
(1 nM-1 .mu.M range), serially diluted into the same assay buffer
containing 1 .mu.M Compound 1 (1% DMSO final), are injected onto
the FKBP12 surface and reference surface at a flow rate of 10
.mu.l/min. The surface is regenerated between analyte injections
with 350 mM EDTA.
[0484] Data Fitting: The BiaEvaluation software program is used for
data fitting. All data is reference subtracted against both the
reference flow cell and a buffer injection. For kinetic analyses,
data is locally fit to a 1:1 interaction model.
[0485] Results: SPR sensorgrams and are shown in FIG. 14.
Dissociation constants (K.sub.D) of 5.4 nM and 0.29 nM were
determined for the binding of FKBP12/Compound 1 to CEP250114 and
CEP250.sub.29.2, respectively.
Example 10: Determination of Kinetics of Binding Between Conjugates
and Proteins by Biolayer Interferometry
[0486] Biolayer Inferometry (BLI) is a biophysical technique used
to measure the kinetics associated with the binary interaction of
either two proteins or a protein to a ligand. Typically, one
component of the binary interacting pair is immobilized on a
biosensor tip via a fusion tag. Increasing concentrations of the
second component (the analyte) are then injected over the biosensor
tip for a fixed time. An increase in BLI signal (expressed in
optical thickness, nm) during the association phase and decrease in
BLI signal during the dissociation phase is indicative of an
interaction and can be fit to a binding model to determine
associated K.sub.D, K.sub.a, K.sub.d values. In this example, the
method is used to measure kinetics for the binding of a conjugate
of the invention to a presenter protein, in which either (i) the
conjugate is immobilized on the tip via fusion tag and a presenter
protein is injected over the surface, or (ii) a presenter protein
is immobilized on the tip via a fusion tag and a conjugate is
injected over the surface.
Determination of Kinetics of Binding Between CYPA-Compound 3 and
KRAS.sub.G12C-GTP by BLI
[0487] This protocol utilizes Biolayer Interferometry (BLI) as a
method to determine the dissociation constant (K.sub.D) for the
binding of KRAS.sub.G12C-GTP (analyte) to immobilized CYPA-Compound
3 binary complex (ligand).
[0488] Reagents: Compound 3 in 100% DMSO (in-house), ForteBio
Kinetic Buffer (ForteBio Inc., Menlo Park, Calif.), Assay Buffer
(Kinetic Buffer, 1% DMSO, 2 .mu.M Compound 3), Avi-tagged CYPA
(in-house), KRAS.sub.G12C-GTP (residues 1-169) (in-house).
[0489] Equipment: Octet Red 96 instrument (ForteBio Inc., Menlo
Park, Calif.)
[0490] Supplies: Streptavidin (SA) biosensors (ForteBio)
[0491] Experimental Protocol: Streptavidin (SA) biosensors were
coated in a solution containing 10 .mu.M Avi-CYPA protein at
25.degree. C. to a loading signal of 0.6 nm. The loading of the
protein showed stability over time and an absence of baseline
drift. The formation of the ternary complex was evaluated in
dose-response experiments with KRAS.sub.G12C-GTP protein
concentrations starting from 200 .mu.M in a 1:2 dilution series.
For negative control, sensors coated with Avi-CYPA protein were
dipped into wells containing only the screening buffer
(supplemented with 2 .mu.M Compound 3). Corrected binding response
sensograms were recorded and analyzed.
[0492] Data Fitting: Analysis on the ForteBio Octet RED instrument
was performed using the ForteBio software. The analysis accounts
for non-specific binding, background, and signal drift and
minimizes well based and sensor variability. Dose-dependent
formation of the ternary complex was observed and the corresponding
equilibrium dissociation constants (K.sub.D) were determined.
[0493] Results: Sensogram and steady state fitting curves are shown
in FIG. 15. A dissociation constant (K.sub.D) of 44 .mu.M was
determined for the binding of CYPA/Compound 3 to
KRAS.sub.G12C-GTP.
Example 11: Proteomic Identification of FKBP12 Bound Target
Proteins for Cross-Linking Reagents
[0494] Reagents: Compound in 100% DMSO (in-house), N-terminal
biotin-FKBP12 (in-house), HEK293T cell lysate (in-house).
[0495] Experimental protocol: HEK293T cell lysate was prepared
using a lysis buffer consisting of 40 mM HEPES, pH 7.3, 120 mM
NaCl, 2 mM MgCl.sub.2, 2 mM CaCl.sub.2), 0.5% octyl-b-glucoside,
and EDTA free protease inhibitor cocktail (Roche) using sonication
(4, 10 second pulses at 20% power) on ice. The lysate was first
cleared via centrifugation and the resulting supernatant is passed
through a 0.2 mm syringe filter on ice. N-terminal biotin labeled
FKBP12 was added to 500 ml of the lysate to a final concentration
of 4 mM, mixed via pipetting, and then compound was added to a
final concentration of 10 mM with the reaction being mixed via
pipetting. 60 mL of 50% slurry agarose-Streptavidin resin
(pre-equilibrated in the lysis buffer) was added and the reaction
is allowed to proceed at 4.degree. C. with gentle rocking for 1
hour. After incubation, the resin was gently pelleted, washed 4
times with 1 mL of lysis buffer on ice via addition,
centrifugation, and aspiration and then washed another 4 times with
1 mL of lysis buffer without detergent in the same physical manner.
Retained proteins were eluted from the resin using 8M Urea, pH 8.0
in HEPES buffer, diluted to 7M Urea with 100 mM HEPES, pH 8.0 and
Endoproteinase Lys-C added for protein digestion at 37.degree. C.
for 2 hours. Next, the sample was diluted to 0.8M Urea using 100 mM
HEPES, pH8.0, Trypsin was added, and the sample digested for an
additional 16 hours at 37.degree. C. After digestion was complete,
the sample was prepared for LC-MS/MS analysis using a C18 SPE
filter onto which the sample was loaded, washed, eluted, desiccated
in a speed-vac, and finally suspended in 10 ml of 5% acetonitrile,
5% formic acid buffer for LC-MS/MS analysis. LC-MS/MS analysis was
performed on a Thermo-Fisher LTQ-Velos-Pro OrbiTrap mass
spectrometer using a top 20 data dependent acquisition method and
8-35% acetonitrile gradient for the HPLC. Peptide sequences were
assigned using the Sequest algorithm and identified proteins are
compared to control samples (DMSO only) in order to identify
candidate target proteins.
[0496] Results: Using the above protocol, >100 target proteins
have been identified as being capable of binding to a presenter
protein in the presence of a cross-linking compound. The identified
target proteins include kinases, phosphatases, ubiquitin ligases,
DNA binding proteins, heat shock proteins, DNA helicases, GTPase
activating proteins, nucleotide binding proteins, and miscellaneous
protein binding proteins.
Example 12: Determination of Binding Between Conjugates and
Proteins by Fluorescence Polarization
[0497] The technique of fluorescence polarization (FP) is based on
the observation that when a fluorescently labeled molecule is
excited by polarized light, it emits light with a degree of
polarization that is inversely proportional to the rate of
molecular rotation. Small molecules rotate quickly during the
excited state, and upon emission, have low polarization values.
Large complexes, formed by binding of a labeled molecule to a
second molecule, rotate little during the excited state, and
therefore have high polarization values. This property of
fluorescence can be used to measure the interaction of a labeled
ligand with a larger protein and provides a basis for direct and
competition binding assays. In this example, the method is used to
measure the binding of compound or conjugate of the invention to
the presenter protein and establish ternary complex formation with
a target protein.
Determination of CypA:C3DS:KRAS Complex Formation by FP
[0498] Reagents: C3DS in 100% DMSO (in-house), Protein Buffer (12.5
mM HEPES pH=7.4, 1 mM MgCl.sub.2), assay buffer (25 mM HEPES, pH
7.3, 0.002% Tween 20, 0.1% BSA, 10 mM NaCl, 1 mM MgCl.sub.2), CYPA
(in-house), Mant-GMP-PNP loaded KRAS (1-169 residues).
[0499] Equipment: SpectraMax
[0500] Experimental Protocol: KRAS stock solution is loaded to
final concentration of 0.8 .mu.M in assay buffer (1% DMSO final).
Compound (C3DS) is added to a final concentration of 10 .mu.M and
the reaction mixture is dispensed to a 384 well Costar black plate.
CYPA is serially diluted into the wells of the plate and allowed to
incubate for 15 mins at room temperature. A control experiment in
the absence of compound is also run to determine the association of
the CYPA to KRAS in the absence of compound. The reaction mixtures
are excited at 355 nm and the emission signal is recorded at 455
nm. The signals are measured at perpendicular and parallel planes
and the polarization is recorded using the following equation.
FP(polarization units.times.10{circumflex over (
)}-3)=Signal(Parallel)-Signal(Perpendicular)/[Signal(Parallel)+Signal(Per-
pendicular)]
[0501] Results: A representative curve and is shown in FIG. 16 and
a table listing the EC50 (concentration require to enhance the FP
signal of the KRAS by 50%) is listed below. The curves were fit to
a four-parameter equation and the EC50s obtained indicate the
effect of the ligand C3DS towards enhancing the binding between
CYPA and KRAS.
TABLE-US-00009 CypA:Kras CypA:C3DS:Kras EC50 30 .mu.M 2.3 .mu.M
Example 13: Determination of Binding Between Conjugates and
Proteins by Nuclear Magnetic Resonance
[0502] Nuclear Magnetic Resonance (NMR) spectroscopy is a technique
used to solve three-dimensional structures and study the dynamics
of proteins and protein-ligand complexes. In addition, it can be
used to identify the ligand binding site in protein-ligand
interaction. Out of several available NMR approaches, protein
structure based ligand screening (highly sensitive 2D
.sup.1H-.sup.15N TROSY-HSQC spectrum) and identification of
critical residues involved in ligand (drug) binding is the most
sensitive method for such studies. Addition of sequentially
increasing ligand concentration into protein's NMR sample and
collection of 2D .sup.1H-.sup.15N TROSY-HSQC provides atomic level
highly resolved residue perturbation information, called chemical
shift perturbation (CSP), that directly provides more accurate
information on identification of ligand binding site not possible
by any other biophysical techniques available. With this approach
weak, intermediate, and strong affinity of ligand binding to a
protein or binary protein complex can be studied, and this
information can be directly linked to existing structural, dynamic,
and kinetic information. In this example, the method is used to
demonstrate binding (e.g. non-covalent or covalent) of a compound
(drug) or conjugate of the invention to a presenter protein.
Determination of KRAS(G12C)-Cyclophilin-Compound 3 Binding in
Ternary Complex by Solution NMR Spectroscopy
[0503] Reagents: Compound 3 in 100% DMSO (in-house), Protein Buffer
(50 mM TRIS-d.sub.11, 50 mM NaCl, pH 7.0, 1 mM TCEP-d.sub.16, 1 mM
MgCl.sub.2), Additives in NMR sample of KRAS (100 .mu.M DSS in 93%
H.sub.2O and 7% D.sub.2O), assay buffer (protein buffer+increasing
equivalents of drug in DMSO (<5%)), GMP-15N-KRAS(G12C)-16
(N-His, residues 1-169, in-house), unlabeled (UL) Cyclophilin
(CYPA; residues 1-165) (in-house).
[0504] Equipment: BrukerAvance 800 MHz Spectrometer equipped with 5
mm CPTCl .sup.1H-.sup.13C/.sup.15N/D Z-GRD Z44909/0026 cryoprobe
(Bruker). High precision 5 mm NMR tubes are used in these
experiments.
[0505] NMR Data processing and analysis: Linux computers running
Topspin v3.1 and NMRPipe/NMRDraw for processing, and CCPNMR
"analysis" program for data analysis.
[0506] Experimental protocol: GMP-.sup.15N-KRAS(G12C) -16 stock
solution of 0.72 mM in protein buffer was used to prepare 0.18 mM
NMR sample in 600 .mu.l (including NMR additives). DSS was used as
internal standard (.sup.1H peak at 0.0 ppm) for chemical shift
referencing. 2D .sup.1H-.sup.15N TROSY-HSQC spectrum of .sup.15N
KRAS was collected (data size 2048.times.128). One equivalent (0.18
mM) of CYPA in protein buffer was added (from stock solution of 0.4
mM) into NMR sample and agitated for 10 minutes. Final NMR sample
volume was maintained to 600 .mu.l. 2D .sup.1H-.sup.15N TROSY-HSQC
spectrum of binary complex (.sup.15N-KRAS+UL-CYPA) was collected
(data size 2048.times.128) keeping other acquisition parameters the
same (only KRAS .sup.1H-.sup.15N correlation crosspeaks are visible
in the spectrum). A stock solution of 20 mM Compound 3 in 100% DMSO
was used for NMR titrations. Compound 3 was sequentially added into
NMR sample to obtain its 0.5, 1.0, 2.5, and 5.0 equivalents (to
that of 15N-KRAS concentration) in the NMR sample of binary complex
(.sup.15N-KRAS+UL-CYPA). At each stages sample volume of 600 .mu.l
was maintained while keeping the acquisition parameters the same.
At each stages of Compound 3 addition, 2D .sup.1H-.sup.15N
TROSY-HSQC spectrum was acquired to investigate chemical shift
perturbation (CSP) of KRAS residues. All spectra were superimposed
to each other. Effective CSP at each Compound 3 titration point is
determined using the difference of chemical shifts of each residue
of KRAS in ternary complex (KRAS+CYPA+Compound 3) versus the binary
complex (KRAS+CYPA). Subsequently, weighted average chemical shift
(.DELTA..delta..sub.weighted) of each KRAS residue are determined
using the below formula:
.DELTA..delta..sub.weighted=[(.DELTA..sup.1H).sup.2+(.DELTA..sup.15N/5).-
sup.2].sup.1/2
[0507] Residues eliciting .DELTA..delta..sub.weighted greater than
one standard deviation from overall average are considered
significantly perturbed and used in the binding site mapping. In
separate titration experiments, we have collected 2D
.sup.1H-.sup.15N TROSY-HSQC spectra on binary complex (KRAS+CYPA)
by sequentially adding DMSO equivalents (to meet equivalent solvent
concentration as in above experiment) to subtract contribution from
DMSO addition. In second control experiment, we have collected
series of 2D .sup.1H-.sup.15N TROSY-HSQC spectra of 15N-KRAS
titrated with Compound 3 at different equivalents (in absence of
CYPA).
[0508] The effective CSP is tabulated and analyzed. Drug binding
residues of KRAS (in presence of CYPA) is mapped onto the protein
surface. Dissociation constant, K.sub.D, is determined.
[0509] Experimental and Processing Parameters:
Spectrum data size: 2048 (1H dimension).times.128 (15N-dimension)
Number of scans: 4
Temperature: 298 K
Quadrature Detection Mode: DQD (1H) and Echo-AntiEcho (15N)
[0510] Data sizes were extended by applying forward-backward linear
prediction within the indirect dimension. Data sets were
extrapolated by zero filling once in each dimension prior to
Fourier transformation.
[0511] Results: 2D 1H-15N TROSY-HSQC spectrum of KRAS.sub.G12C-GTP
is shown in FIG. 17A. Adding a stoichiometric amount of CYPA has no
effect on KRAS amide backbone crosspeaks (FIG. 17B), indicating
that KRAS and CYPA do not interact directly. Titration of W21487
into a 1:1 sample of CYPA:KRAS elicits distinct chemical shifts
(FIG. 17C), indicative of a direct interaction with KRAS.
Example 14. Determination of Binding Between Conjugates and
Proteins by Microscale Thermophoresis
[0512] Microscale Thermophoresis (MST) is a technique for
characterization of biomolecular interactions by correlating any
changes in the molecular properties of the molecule such as size,
conformation to its mobility in a directed temperature gradient.
The generation of the gradient is induced by an infrared laser. The
movement of the biomolecule is frequently characterized by labeling
the molecule using covalently attached fluorophores or even
intrinsic fluorescence. In this example, the method is used to
measure the binding of compound or conjugate of the invention to
the presenter protein and establish ternary complex formation with
a target protein, in which either (i) the conjugate is labelled
with a fluorophore and a presenter protein is titrated in, or (ii)
a presenter protein is labelled with a fluorophore and a conjugate
is titrated in.
Example 15. Determination of Binding Between Conjugates and
Proteins by Second Harmonic Generation Technology
[0513] Second Harmonic Generation (SHG) is an optical phenomenon
that can be used to measure conformational changes in aqueous
solution in real time. SHG signal intensity is sensitive to average
angular orientation of dye labeled to a protein tethered to a
surface and magnitude of signal change directly correlates to
amount of angular change. Different conformations can be classified
by magnitude of signal change upon binding, signal relative to
baseline (more vertical orientations relative to surface produce
positive signal changes and vice versa) and kinetics. In this
example, the method is used to measure the binding of compound or
conjugate of the invention to the presenter protein and establish
ternary complex formation with a target protein, in which either
(i) a dye labelled conjugate is immobilized on the surface via a
fusion tag and a presenter protein is injected over the surface, or
(ii) a dye labelled presenter protein is immobilized on the surface
via a fusion tag and a conjugate is injected over the surface.
Example 16. Determination of Binding Between Conjugates and
Proteins by Differential Scanning Fluorimetry
[0514] Differential Scanning Fluorimetry (DSF) is a solution based
biophysical technique used to measure the melting temperature
(T.sub.m) of a protein. In a typical experiment, protein of
interest is subject to increasing heat (typically from 4.degree.
C.-95.degree. C.) in the presence of a fluorescent dye (e.g. SYPRO
orange). Fluorescent intensities are plotted as a function of
temperature and the T.sub.m is calculated from the negative
derivative minimum of the fluorescence signal. For a target
protein, the thermal shift (.DELTA.T.sub.m) in the presence of a
small molecule can be measured to assess whether the small molecule
binds to and stabilizes the protein. In this example, the method is
used to measure the thermal shift (e.g., non-covalent or covalent
binding) of a compound or conjugate of the invention to a presenter
protein, in which either (i) the conjugate is labelled with a
fluorescent dye and a presenter protein is titrated in, or (ii) a
presenter protein is labelled with a fluorescent dye and a
conjugate is titrated in.
Example 17. Determination of Binding Between Conjugates and
Proteins by NanoDSF
[0515] NanoDSF is an advanced DSF method for measuring the T.sub.m
of a protein using intrinsic tryptophan or tyrosine fluorescence.
In a typical experiment, protein of interest is subject to
increasing heat (typically from 4.degree. C.-95.degree. C.) and
fluorescent intensities of intrinsic tryptophan or tyrosine
residues are monitored as a function of temperature. T.sub.m can be
calculated from the changes in tryptophan fluorescence intensity,
or from the ratio of tryptophan emission at 330 and 350 nm, which
describes the shift of tryptophan emission upon unfolding. For a
target protein, .DELTA.T.sub.m in the presence of a small molecule
can be measured to assess whether the small molecule binds to and
stabilizes the protein. In this example, the method is used to
measure the thermal shift (e.g., non-covalent or covalent binding)
of a compound or conjugate of the invention to a presenter protein,
in which either (i) the fluorescence of the conjugate is measured
and a presenter protein is titrated in, or (ii) the fluorescence of
a presenter protein is monitored and a conjugate is titrated
in.
Example 18. Determination of Complex Formation by Differential
Light Scattering
[0516] Dynamic light scattering (DLS) is an established biophysics
method used to measures time-dependent fluctuations in the
scattering intensity arising from particles undergoing random
Brownian motion. Diffusion coefficient and particle size
information can be obtained from the analysis of these
fluctuations. More specifically, the method provides the ability to
measure size characteristics including radius and molecular weight
of proteins in aqueous solution. In this example, the method is
used to measure change in radius or molecular weight in either (i)
the presenter protein upon binding of conjugate of the invention or
(ii) the conjugate of the invention upon binding to a presenter
protein.
Example 19. Determination of Binding Between Conjugates and
Proteins by Sonic Wave Acoustic Technology
[0517] Surface Acoustic Wave (SAW) technology is a biophysical
method used for the real-time detection of binding-induced
conformational changes through monitoring the shift in the phase of
surface acoustic waves that travel along the biosensor. It can be
used to measure the kinetics associated with the binary interaction
of either two proteins or a protein to a ligand. Typically, one
component of the binary interacting pair is immobilized on the
biosensor via a fusion tag. Increasing concentrations of the second
component (the analyte) are then injected over the biosensor for a
fixed time. An increase in signal (measured either through a change
in wave phase or amplitude) during the association phase and
decrease in signal during the dissociation phase is indicative of
an interaction and can be fit to a binding model to determine
associated K.sub.D, K.sub.a, K.sub.d values. In this example, the
method is used to measure kinetics for the binding of a conjugate
of the invention to a presenter protein, in which either (i) the
conjugate is immobilized on the biosensor chip via fusion tag and a
presenter protein is injected over the surface, or (ii) a presenter
protein.
Example 20: Determination of Complex Formation by Small-Angle X-Ray
Scattering
[0518] Small-Angle X-Ray Scattering (SAXS) is a solution based
method used to determine the structure of a protein in terms of
average particle size and shape. It is capable of delivering
structural information in the resolution range between 1 and 25 nm,
and of repeat distances in partially ordered systems of up to 150
nm in size. Ultra small-angle scattering (USAS) can resolve even
larger dimensions. In a typical scattering experiment, a solution
of protein or protein complex are exposed to X-rays (with
wavelength A typically around 0.15 nm). The scattered intensity
I(s) is recorded as a function of momentum transfer s (s=4.pi. sin
.theta./.lamda., where 2.theta. is the angle between the incident
and scattered radiation). From the intensity of the solution the
scattering from only the solvent is subtracted. An X-ray scattering
curve (intensity versus scattering angle) is then used to create a
low-resolution model of a protein or protein complex. In this
example, the method is used to identify existence of a ternary
complex (e.g., non-covalent or covalent binding) of a compound or
conjugate of the invention to a presenter protein.
OTHER EMBODIMENTS
[0519] It is to be understood that while the present disclosure has
been described in conjunction with the detailed description
thereof, the foregoing description is intended to illustrate and
not limit the scope of the present disclosure, which is defined by
the scope of the appended claims. Other aspects, advantages, and
alterations are within the scope of the following claims.
[0520] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments in accordance with the
invention described herein. The scope of the present invention is
not intended to be limited to the above Description, but rather is
as set forth in the appended claims.
[0521] In the claims, articles such as "a," "an," and "the" may
mean one or more than one unless indicated to the contrary or
otherwise evident from the context. Claims or descriptions that
include "or" between one or more members of a group are considered
satisfied if one, more than one, or all of the group members are
present in, employed in, or otherwise relevant to a given product
or process unless indicated to the contrary or otherwise evident
from the context. The invention includes embodiments in which
exactly one member of the group is present in, employed in, or
otherwise relevant to a given product or process. The invention
includes embodiments in which more than one, or all of the group
members are present in, employed in, or otherwise relevant to a
given product or process.
[0522] It is also noted that the term "comprising" is intended to
be open and permits but does not require the inclusion of
additional elements or steps. When the term "comprising" is used
herein, the term "consisting of" is thus also encompassed and
disclosed.
[0523] Where ranges are given, endpoints are included. Furthermore,
it is to be understood that unless otherwise indicated or otherwise
evident from the context and understanding of one of ordinary skill
in the art, values that are expressed as ranges can assume any
specific value or subrange within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower
limit of the range, unless the context clearly dictates
otherwise.
[0524] In addition, it is to be understood that any particular
embodiment of the present invention that falls within the prior art
may be explicitly excluded from any one or more of the claims.
Since such embodiments are deemed to be known to one of ordinary
skill in the art, they may be excluded even if the exclusion is not
set forth explicitly herein. Any particular embodiment of the
compositions of the invention (e.g., any polynucleotide or protein
encoded thereby; any method of production; any method of use) can
be excluded from any one or more claims, for any reason, whether or
not related to the existence of prior art.
Sequence CWU 1
1
1116PRTArtificial SequenceSynthetic construct 1Tyr Gln Asn Leu Leu
Val Gly Arg Asn Arg Gly Glu Glu Ile Leu Asp1 5 10 15
* * * * *