U.S. patent application number 15/477966 was filed with the patent office on 2017-10-05 for protein-protein interaction inducing technology.
The applicant listed for this patent is Arvinas, Inc.. Invention is credited to Andrew P. Crew, Hanqing Dong, Brian Hamman, Taavi Neklesa, Jing Wang.
Application Number | 20170281784 15/477966 |
Document ID | / |
Family ID | 59958990 |
Filed Date | 2017-10-05 |
United States Patent
Application |
20170281784 |
Kind Code |
A1 |
Wang; Jing ; et al. |
October 5, 2017 |
PROTEIN-PROTEIN INTERACTION INDUCING TECHNOLOGY
Abstract
The present disclosure is based on the surprising and unexpected
discovery that a ligand molecule with certain characteristics is
able to bind to two protein molecules simultaneously and recruit
them to form a transient or stable protein-protein interaction
complex. The protein-protein interaction and other cross-domain
interactions gained in this process contribute additional
stabilization energy to the complex beyond the combination of the
binary binding energies, and therefore, largely increase the
binding potency of the ligand. Accordingly, the present disclosure
provides a Protein-Protein Interaction Inducing Technology (PPIIT),
which includes a method to design and identify the tripartite or
bifunctional compounds and use such compounds to induce
protein-protein interactions in various contexts. The present
disclosure also provides a composition for the purpose of inducing
protein-protein interactions.
Inventors: |
Wang; Jing; (Milford,
CT) ; Crew; Andrew P.; (Guilford, CT) ; Dong;
Hanqing; (Madison, CT) ; Neklesa; Taavi;
(Orange, CT) ; Hamman; Brian; (Orange,
CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Arvinas, Inc. |
New Haven |
CT |
US |
|
|
Family ID: |
59958990 |
Appl. No.: |
15/477966 |
Filed: |
April 3, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62318630 |
Apr 5, 2016 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/4166 20130101;
A61K 31/277 20130101; A61K 38/05 20130101; G16B 20/00 20190201;
A61K 31/551 20130101; A61K 47/55 20170801; G16B 15/00 20190201;
G16C 20/50 20190201; G01N 33/6845 20130101 |
International
Class: |
A61K 47/48 20060101
A61K047/48; G06F 19/18 20060101 G06F019/18; G06F 19/16 20060101
G06F019/16; A61K 31/277 20060101 A61K031/277; C40B 30/04 20060101
C40B030/04; A61K 31/551 20060101 A61K031/551; A61K 31/4166 20060101
A61K031/4166; G01N 33/68 20060101 G01N033/68; A61K 38/05 20060101
A61K038/05 |
Claims
1. A method of designing a bifunctional compound capable of
effectuating protein-protein interactions between a first protein
molecule (A) and a second protein molecule (B), the method
comprising: (a) providing a bifunctional ligand (L) of structure
WA-C.sub.n-WB, wherein WA is a warhead targeting the first protein
A, WB is a warhead targeting the second protein B, and C is a
connector with length or number of atoms n, covalently linked to WA
and WB; (b) measuring the ternary binding potency and the binary
binding potencies of the ligand L with respect to the first protein
A and the second protein B; and (c) determining the capability of
the ligand to induce an interaction between the first protein A and
the second protein B.
2. The method of claim 1, wherein at least one of the warhead WA,
warhead WB, and connector C is a chemical group or moiety.
3. The method of claim 2, wherein the warheads WA and WB are
derived from compounds known to bind to proteins A and B,
respectively.
4. The method of claim 1, wherein the connector C is a linear chain
of carbon atoms or a linear chain of alternating carbon atoms and
heteroatoms.
5. The method of claim 4, wherein any two heteroatoms are separated
by at least two carbon atoms.
6. The method of claim 1, wherein the method of designing the
bifunctional ligand further comprises a step of modifying the chain
length or the number of atoms of the connector C to determine the
appropriate chain length or number of atoms for inducing
protein-protein interactions.
7. The method of claim 6, wherein the method includes the steps of
determining comprising: (a) synthesizing a set of compounds with
the number of atoms in C varying n between 0 and 30 while keeping
the warheads WA and WB constant; (b) measuring the binding of each
compound to determine which compounds have a superior ternary
binding potency relative to a corresponding binary binding potency
for proteins A and B; and (c) determining the n values that give
rise to the potencies indicative of the existence of
protein-protein interactions.
8. The method of claim 7, wherein determining the chain length or
the number of atoms of the connector C (n) further comprises: (d)
changing the attachment points on WA and WB that are used to link
the warheads to the connector C, and repeat steps (a) through (c)
to find additional compounds with protein-protein interactions.
9. The method of claim 1, wherein the connector C is a chain with
branched groups and/or contains rings.
10. The method of claim 1, wherein the measuring step further
comprises: measuring the influence of the first protein or the
second protein on a binding constant of another protein toward the
ligand to evaluate the capability of the ligand L to induce the
protein-protein interaction.
11. The method of claim 10, wherein the method comprises a step of:
performing molecular dynamics simulations to demonstrate
protein-protein interactions and other cross-domain interactions in
ternary systems composed of the first protein A, the second protein
B, and the ligand L to evaluate the capability of the ligand L to
induce the protein-protein interaction.
12. The method of claim 11, wherein the first protein A and the
second protein B are the same protein
13. The method of claim 11, wherein the first protein A and the
second protein B are different proteins.
14. The method of claim 1, further comprising selecting a ligand
with a ternary complex that results in surface area burial greater
than the sum of the surface area burial of the corresponding
warhead monomers with the first and second proteins.
15. A compound resulting from the method of claim 1.
16. A method of treating or preventing a disease or disorder, the
method comprising: administering an effective amount of a compound
of claim 15.
17. A method of designing a tripartite or bifunctional ligand that
induces protein-protein interaction(s) between a first protein
molecule (A) and a second protein molecule (B), the method
comprising: designing, preparing, and/or synthesizing a plurality
of tripartite and/or bifunctional compounds with the general
structure WA-C-WB or WA-WB, wherein WA is a warhead that associates
with the first protein, WB is a warhead that associates with the
second protein, and C is a connector covalently linked or bound to
WA and WB; designing, preparing, and/or synthesizing control
compounds; quantifying induced protein-protein interactions with at
least one of biochemical assays, cellular assays, and molecular
dynamics simulations; and selecting the tripartite or bifunctional
compound/ligand that induces protein-protein interactions and/or
other cross-domain interactions in the ternary complex.
18. The method of claim 17, wherein designing, preparing, and/or
synthesizing includes varying a length of the connector between 0
atoms to 30 atoms while maintaining the same warheads and
connection points between the connector and the warheads.
19. The method of claim 18, wherein the length of the connector is
varied by an increment of 1 to 3 atoms.
20. The method of claim 17, wherein the covalent link between the
connector and WA and/or WB is at a solvent-exposed point.
21. The method claim 17, wherein the plurality of tripartite and/or
bifunctional compounds comprises subsets of compounds having a
unique covalent link between warhead WA and warhead WB or a unique
series of covalent links between warhead WA, the connector, and
warhead WB, relative to the other subsets.
22. The method of claim 17, wherein designing, preparing, and/or
synthesizing control compounds comprises modifying either warhead
WA or WB such that substantially all of its association/binding
ability to protein A or protein B is removed.
23. The method of claim 17, wherein quantifying protein-protein
interactions using biochemical assays comprises determining whether
(i) the tripartite or bifunctional compound binding/associating
with protein A and protein B produce synergism, or (ii) the
tripartite or bifunctional compound/ligand induces ternary binding
potency.
24. The method of claim 17, wherein selecting the tripartite or
bifunctional compound/ligand that induces protein-protein
interactions in the ternary complex comprises: selecting at least
one tripartite or bifunctional compound/ligands that have a ratio
.alpha. that is greater than about 1, wherein the ratio .alpha. is
IC.sub.50.sup.A over IC.sub.50.sup.A/B or IC.sub.50.sup.B over
IC.sub.50.sup.B/A; and/or selecting at least one tripartite or
bifunctional compounds/ligands that have a ratio .alpha.T that is
greater than about 1, wherein the ratio .alpha.T is a ratio of the
lower of IC.sub.50.sup.A and IC.sub.50.sup.B over
IC.sub.50.sup.T.
25. The method of claim 17, wherein quantifying induced
protein-protein interactions comprises performing molecular
dynamics simulations on tripartite and/or bifunctional compounds
that are determined to induce protein-protein interaction(s) by
either biochemical assays or cellular assays.
26. The method of claim 17, wherein the protein-protein
interactions for a particular conformation determined by molecular
dynamics simulations are examined by calculating at least one of
atom distances, surface area burial, and interaction energies for
the ternary complex formation and a binary complex formation.
27. The method claim 17, wherein the protein-protein interactions
for a particular conformation determined by molecular dynamics
simulations are examined along the simulation trajectory and the
critical distances related to the interactions and the
intermolecular energies between critical groups can be calculated
along the simulation time.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of earlier filed U.S.
Provisional Patent Application Ser. No. 62/318,630 titled:
"Protein-Protein Interaction Inducing Technology", filed on Apr. 5,
2016, which is hereby incorporated herein by reference in its
entirety.
BACKGROUND
1. Field of the Discovery
[0002] The present disclosure relates to the field of designing
compounds to modify protein activity or behavior, such as for
example, by inducing protein-protein interactions, antagonizing or
agonizing protein function, and/or impacting their ability to
associate with partners, modifying their conformational stability,
effectuating ubiquitination and degradation, or causing them to
undergo other post-translational modifications including
phosphorylation, dephosphorylation and other modifications.
Modification of protein activity or behavior can lead to changes
in, e.g., transcriptional activities, cell proliferation and/or
differentiation, apoptosis.
2. Background
[0003] Designing small molecule compounds or ligands to bind to a
protein and thus block the function of that protein is a highly
desired approach for developing drugs for pharmaceutical
applications. However, many proteins are considered undruggable
because they lack binding pockets deep enough to allow development
of high affinity compounds, and high affinity is a prerequisite for
in vivo efficacy and specificity in many cases. The particular
examples include, but not limited to, receptor proteins and
scaffolding proteins, which do not have enzymatic active sites and
only have interaction surfaces for associating with protein or
non-protein partners.
[0004] There exists an ongoing need for a technology that
facilitates drug design. In particular, it is desirable to design
compounds in which one ligand molecule is used to simultaneously
target two protein molecules (of the same protein or different
proteins), the ligand molecule induces protein-protein interaction,
and the protein-protein interaction energy gained in this process
enhances the potency of the ligand.
SUMMARY
[0005] The present disclosure provides a Protein-Protein
Interaction Inducing Technology (PPIIT), which allows one to design
potent ligands for "undruggable" targets, or proteins with only
shallow binding sites. The present disclosure is based on the
surprising and unexpected discovery that a ligand molecule with
certain characteristics is able to bind to two protein molecules
simultaneously and recruit them to form a transient or stable
protein-protein interaction complex. The protein-protein
interaction and other cross-domain interactions gained in this
process contribute additional stabilization energy to the complex
beyond the combination of the binary binding energies, and
therefore, largely increase the binding potency of the ligand (FIG.
1). That is, a ligand molecule with certain characteristics
provides a protein-protein interaction between two protein
molecules that is greater than one would expect given the ligands
binding efficiency for either protein individually. Accordingly,
the present disclosure provides a method for discovering, designing
or deriving a ligand for forming a transient or stable interaction
between two of the same proteins or two different proteins. The
present disclosure also provides a composition that induces
protein-protein interactions between, e.g., two of the same
proteins or two different proteins, and the use thereof.
[0006] An aspect of the disclosure provides a ligand L able to
induce protein-protein interactions between protein A (a first
protein) and protein B (a second protein). In an embodiment, the
ligand has a generic chemical structure of WA-C-WB, wherein the WA
is a warhead (e.g., a chemical group or moiety) targeting protein
A, the WB is a warhead targeting protein B, and C is a connector
(e.g., a chemical group or moiety) providing appropriate spacing
between WA and WB. C is covalently linked to WA and WB. The
distance or the range of distance between WA and WB in the
tripartite molecule WA-C-WB is critical for the protein-protein
interaction between the protein A and protein B to occur, and the
lengths of C allowing optimal or desirable protein-protein
interactions depend upon the structures of protein A and protein B
involved. In an embodiment, C has a chain length or number of atoms
in a range of 0 to 30 atoms, e.g., the C chain length can be 1, 2,
3, 4, 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, or 30 atoms. In certain
embodiments, the chain is substituted or unsubstituted. That is, in
an embodiment, the connector is 0 atoms and the ligand L has a
general structure of WA-WB. Therefore, the present disclosure also
relates a method to determine a length and other structural
features of the connector C so that the tripartite ligand WA-C-WB,
after it binds protein A and protein B with the respective
warheads, allows protein A and protein B to interact with each
other favorably and optimally (i.e., the connector provides the
ligand with a binding efficiency greater than the warheads WA and
WB provide individually).
[0007] In an additional aspect, the disclosure provides a method
for determining whether or not a ligand L induces a protein-protein
interaction between protein A and protein B. In an embodiment, the
method includes quantifying and comparing a ternary binding potency
(A.L.B) and a corresponding binary binding potency. In a particular
embodiment, the ternary binding potency is the IC.sub.50 or a total
concentration of the ligand at which the concentration of a ternary
complex A.L.B is half of its maximum value, determined by varying
the concentration of the ligand systematically. In another
embodiment, a binary binding potency for A.L is the IC.sub.50 or
the total concentration of the ligand at which the concentration of
the binary complex A.L is half of its maximum value, determined by
varying the concentration of the ligand in the absence of a
contribution of protein B. In an additional embodiment, a binary
binding potency for B.L is the IC.sub.50 or the total concentration
of the ligand at which the concentration of the binary complex B.L
is half of its maximum value, determined by varying the
concentration of the ligand in the absence of the contribution of
protein A. The protein concentrations for protein A and protein B
are kept constant in the determination of the above three
IC.sub.50s for the ligand. According to an embodiment, if the
ternary IC.sub.50 is smaller than both of the corresponding binary
IC.sub.50s, protein-protein interactions should exist.
[0008] In another aspect of the disclosure, the protein-protein
interactions are determined by measuring a synergism of binding
affinities. The binding constant between protein A and ligand L can
be measured in the absence or in the presence of the contribution
of protein B. In an embodiment, if the binding constant between
protein A and ligand L is smaller (i.e., has a higher affinity) in
the presence of the contribution of protein B than in the absence
of the contribution of B, the existence of protein-protein
interactions between protein A and protein B can be assumed.
Similarly, a binding constant between protein B and ligand L can be
measured in the absence or in the presence of the contribution of
protein A. In another embodiment, observing a strengthening of
binding of protein B and ligand L by protein A signals the
existence of protein-protein interactions.
[0009] In an aspect of the disclosure, the existence of
protein-protein interactions between protein A and protein B
mediated by ligand L can be demonstrated by performing molecular
dynamics (MD) simulation. That is, in an embodiment, the method
comprises performing an MD simulation. In a particular embodiment,
the MD simulation is performed on a starting conformation of a
ternary model A.L.B, wherein the warheads of the ligand L occupy
the corresponding binding sites of protein A and protein B and the
connector of ligand L assumes an extended or arbitrary
conformation. The MD simulation with explicit water can lead to a
complex in which protein A physically interacts with protein B
and/or the B-binding warhead of the ligand L or protein B
physically interacts with protein A and/or the A-binding warhead of
the ligand L.
[0010] In additional embodiments, the protein-protein interaction
of the present disclosure includes at least one of ion-pair,
hydrogen bonding, and hydrophobic interactions, and/or the
formation of hydrophobic clusters contributed by the non-polar
groups of different proteins and ligand molecules. In another
embodiment, the A-L-B ternary system adopts an arrangement that
includes interactions between at least one of: the two warheads WA
and WB of a single ligand, protein A and WB, protein B and WA, and
the proteins and the connector C Like the pure protein-protein
interaction, these cross-domain interactions involving parts of a
ligand can also contribute extra binding energy beyond the
combination of the binary binding energies (i.e., stemming from the
docking of the warheads to the respective binding sites). As used
herein, the term "protein-protein interaction" can refer to, unless
explicitly stated otherwise or by the context of its use, these
cross-domain interactions as well as pure protein-protein
interactions.
[0011] In one embodiment, the present disclosure provides a method
to find the connector C for a tripartite ligand L of chemical
structure WA-C-WB to induce protein-protein interaction between A
and B. In an embodiment, WA and WB are chemical moieties known to
bind to A and B, respectively, and C is a linear chain of carbon
atoms or a linear chain of alternating carbon atoms and
heteroatoms. In a particular embodiment, any two heteroatoms are
separated by at least two carbon atoms, thereby producing a
chemically stable compound. In some embodiments, the method
includes synthesizing a set of compounds in which the length of the
connector C is systematically varied by changing the number of
atoms constituting C, and WA and WB remain constant. In an
embodiment, the length of connector C is varied within a range
between 0 and 30 atoms, wherein the incremental differences in
length can be between 1 to 3 atoms. However, other ranges and
increments of exploration are possible. In certain embodiments, the
method comprising synthesizing ligands with different attachment
points on WA and WB to link the connector C. In another embodiment,
once attachment points on WA and WB to link the connector C is
determined, the length of the connector C can be examined, as
discussed above, with the new attachment points. This process of
changing attachment points and exploring the length of connector C
can be repeated to find ligands that produce greater
protein-protein interactions. Each compound in the process is
measured to determine and compare its ternary binding potency and
binary binding potencies. Embodiments of the disclosure can
determine which ranges of connector length and which attachment
points correspond to best ternary binding potencies, and whether or
not any of the ternary binding potency values are more potent than
the corresponding binary binding potency values, thereby indicating
the existence of the protein-protein interaction. Whether or not
significantly favorable protein-protein interactions can be found
for a given protein or protein pair using this process depends on
the nature of the proteins involved, but embodiments of the
disclosure provides a system to efficiently determine ligands that
can provide favorable protein-protein interactions.
[0012] Embodiments of the method can result in a ligand L
(compound) wherein the connector C contains no atoms. That is, the
compound(s) can have a bi-functional chemical structure of WA-WB,
wherein the two warheads are directly linked with a covalent bond
or fused through a common bond. Some protein pairs can adopt such
an interaction mode that allows close approach between WA and WB so
that compounds like WA-WB are suitable to mediate the interactions
between protein A and protein B.
[0013] The protein pair A and B between which the interactions are
sought can be different proteins or the same protein. In
embodiments where proteins A and B are identical (i.e., the same
proteins), the two warheads WA and WB of the ligand of type WA-C-WB
or WA-WB can be identical or different as long as they are able to
bind to the corresponding targets. The method to measure the
protein-protein interaction can be the same as described previously
or can be inferred directly from the previous description for the
case where protein A and protein B represent different
proteins.
[0014] The present disclosure provides compounds that can induce
protein-protein interactions between protein pairs that do not
naturally interact. The present PPIIT process was applied to the
following protein pairs and induced favorable protein-protein
interactions between each of the pairs: androgen receptor (AR) with
von Hippel-Lindau protein (VHL), AR with cereblon, estrogen
receptor (ER) with VHL, bromodomain-containing protein 4 (BRD4)
with VHL, and BRD4 with cereblon. These pairs are not known to
interact with each other naturally. Embodiments of the present
disclosure can also be used to strengthen the interactions between
protein pairs that do interact with each other naturally.
[0015] In an embodiment, the present disclosure provides a process
to optimize a ligand L which has measurable capability of inducing
protein-protein interaction between protein A and protein B. The
ligand can have recognizable warheads WA and WB for targeting
protein A and protein B, respectively. In particular embodiments,
the generic chemical structure of the ligand is WA-C-WB in which C
is a connector covalently linked to WA and WB, or WA-WB in which WA
and WB are covalently linked. Some embodiments of the present
disclosure include modifying the chemical structure of the ligand
to change its ability to induce protein-protein interactions and/or
other characteristics related to its chemical structure. In an
embodiment, the process of optimization comprises building or
synthesizing an initial molecular model for the ternary complex
A.L.B in which the warheads of the ligand L occupy the
corresponding binding sites of protein A and protein B,
respectively. The crystal structures or validated models of
complexes of A.L and B.L can be useful input to determine how the
warheads dock onto protein A and protein B. In a particular
embodiment, the method includes performing MD simulation with
explicit water on the initial model of ternary complex and a
representative conformation is derived from the simulation
trajectory. The representative conformation can be used to redesign
the connector and/or modify the warheads.
[0016] The present description also provides a composition useful
for inducing protein-protein interactions. The composition
comprises a compound or composition that is derived from the
compound exploration or design processes described above. In an
embodiment, the composition has a generic chemical structure of
WA-C-WB in which WA and WB are warheads targeting protein A and
protein B, respectively, and C is a connector covalently linked to
WA and WB. WA and WB can be known ligands of protein A and protein
B or they can also be derived using established technologies in the
art such as high-throughput screening and/or structure-based drug
design. In one embodiment, C is a linear chain of carbon atoms or a
linear chain of alternating carbon atoms and heteroatoms and the
length of the connector C as well as the attachment points on WA
and WB are derived using the exploration process described
previously. In another embodiment, C contains branched groups,
saturated rings, and/or non-saturated rings, each with or without
heteroatoms. In yet another embodiment, the connector C is reduced
to zero so that the composition has a generic structure of WA-WB in
which the two warheads are directly linked together through a
covalent bond, sharing a common atom, or sharing a common bond
(i.e., fusion).
[0017] In additional embodiments, the description provides methods
for treating or ameliorating a disease, disorder or symptom thereof
in a subject or a patient, e.g., an animal such as a human,
comprising administering to a subject in need thereof a composition
comprising an effective amount, e.g., a therapeutically effective
amount, of a compound as described herein or salt form thereof, and
a pharmaceutically acceptable excipient, carrier, adjuvant, another
bioactive agent or combination thereof, wherein the composition is
effective for treating or ameliorating the disease or disorder or
symptom thereof in the subject.
BRIEF DESCRIPTION OF THE FIGURES
[0018] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0019] FIG. 1A and FIG. 1B illustrate the general concept of the
Protein-Protein Interaction Inducing Technology (PPIIT). (FIG. 1A):
Protein A has a shallow binding site so that only weakly
associating small molecule binders can be found or designed to
associate with protein A. (FIG. 1B): A tripartite or bi-functional
compound can be designed to recruit two protein molecules together
to form a transient or stable complex. The protein-protein
interaction and other cross-domain interactions gained in this
process contribute additional stabilization energy to the complex
beyond the combination of the binary binding energies, and thus
largely increase the potency of the compound (i.e., the ability of
the compound to bind protein A and protein B). This technology can
work to induce either homodimer (A and B are the same protein) or
heterodimer (A and B are different proteins) depending upon the
design of the warheads of the tripartite or bi-functional compound.
The connector length of the tripartite compound needs to be in the
correct range in order for a favorable protein-protein docking pose
to be found.
[0020] FIG. 2 is an illustration of a general concept of examining
connectors of differing lengths and different connection points (or
links) with WA and WB to optimize the tripartite compound's ability
to induce protein-protein interactions or other cross-domain
interactions. A similar technique can be used to optimize a
bifunctional compound/ligand.
[0021] FIG. 3 is an overview of the representative conformation of
the AR.L.VHL ternary complex derived from a MD simulation. Elongins
B (EB) and Elongins C (EC) are included as part of VHL in the
simulation. AR is rendered as purple ribbons and wires; VHL/EB/EC
is rendered as dark green ribbons and wires; and the ligand is
rendered as tubes with yellow-green color for carbon, red for
oxygen and blue for nitrogen. The protein atoms within 10 .ANG.
radius of the ligand are covered with molecular surface in which
the AR part is in pink and the VHL part is in grey-blue. One can
see that the molecular surfaces of AR and VHL merge into a
contiguous surface and form a collective binding site for the
ligand.
[0022] FIG. 4 is an amplified view of the AR.L.VHL ternary complex
focused on the ligand and the protein-protein interface. The ligand
is represented as tubes with orange for carbon, red for oxygen and
blue for nitrogen. AR is in purple ribbons and wires, and VHL is in
dark green ribbons and wires. Some of the interfacial residues are
in tubes.
[0023] FIG. 5 is an overview of the representative conformation of
the BRD4.L'.VHL ternary complex derived from a MD simulation. BRD4
is rendered as purple ribbons and wires; VHL is rendered as dark
green ribbons and wires; and the ligand is rendered as tubes with
yellow-green color for carbon, red for oxygen and blue for
nitrogen. The protein atoms within 10 .ANG. radius of the ligand
are covered with molecular surface in which the BRD4 part is in
pink and the VHL part is in grey-blue. One can see that the
molecular surfaces of BRD4 and VHL merge into a contiguous surface
and form a collective binding site for the ligand.
[0024] FIG. 6 is an amplified view of the BRD4.L'.VHL ternary
complex focused on the ligand and the protein-protein interface.
The ligand is represented as tubes with orange for carbon, red for
oxygen and blue for nitrogen. BRD4 is in purple ribbons and wires
and VHL is in dark green ribbons and wires. Some of the interfacial
residues are in tubes.
[0025] FIG. 7 visualizes the trimer of AR, VHL and a tripartite
ligand where AR and VHL dock together, and the ligand connector
interacts with the proteins while allowing the warheads to plug
into the respective binding sites. This suggests that the length
and the chemical composition of the connector are important for the
potency of the compound.
[0026] FIG. 8 visualizes the trimer of BRD4, cereblon and a
tripartite ligand where BRD4 and cereblon dock together, and the
ligand connector adopts a conformation to allow the warheads to
dock into the respective binding sites and also dock against each
other at the same time. This further suggests that both the length
and the chemical composition of the connector are important for the
potency of the compound.
[0027] FIG. 9 visualizes the trimer of BRD4, VHL and the most
potent ligand from the connector length exploration. BRD4 is
covered by pink surface. VHL is covered by grey-blue surface. The
ligand is rendered as tubes with the warheads' carbon atoms in
orange and the connector's carbon atoms in yellow-green.
DETAILED DESCRIPTION
[0028] The following is a detailed description provided to aid
those skilled in the art in practicing the present disclosure.
Those of ordinary skill in the art may make modifications and
variations in the embodiments described herein without departing
from the spirit or scope of the present disclosure. All
publications, patent applications, patents, figures and other
references mentioned herein are expressly incorporated by reference
in their entirety.
[0029] The present disclosure relates to a method of designing
compounds (compositions) that induce protein-protein interactions
between a given pair of protein molecules A (i.e. protein A) and B
(i.e. protein B), wherein protein A and protein B can be two
different proteins or two of the same proteins. The present
disclosure also provides a composition or the characteristics of a
composition capable of inducing said protein-protein
interaction.
[0030] As such, in certain aspects the description provides methods
of designing a bifunctional compound capable of effectuating
protein-protein interactions between a first protein molecule (A)
and a second protein molecule (B), the method comprising: (a)
providing a bifunctional ligand (L) of structure WA-C.sub.n-WB,
wherein WA is a warhead targeting the first protein A, WB is a
warhead targeting the second protein B, and C is a connector with
length or number of atoms n, covalently linked to WA and WB; (b)
measuring the ternary binding potency and the binary binding
potencies of the ligand L with respect to the first protein A and
the second protein B; and (c) determining the capability of the
ligand to induce an interaction between the first protein A and the
second protein B.
[0031] In any of the aspects or embodiments described herein, at
least one of the warhead WA, warhead WB, and connector C is a
chemical group or moiety. In any of the aspects or embodiments
described herein, the warheads WA and WB are derived from compounds
known to bind to proteins A and B, respectively.
[0032] In any of the aspects or embodiments described herein, the
connector C is a linear chain of carbon atoms or a linear chain of
alternating carbon atoms and heteroatoms. In any of the aspects or
embodiments described herein, any two heteroatoms are separated by
at least two carbon atoms.
[0033] In any of the aspects or embodiments described herein, the
method of designing the bi-functional ligand further comprises a
step of modifying the chain length or the number of atoms of the
connector C to determine the appropriate chain length or number of
atoms for inducing protein-protein interactions, determining
comprising.
[0034] In any of the aspects or embodiments described herein, the
method includes the steps of (a) synthesizing a set of compounds
with the number of atoms in C varying n between 0 and 30 while
keeping the warheads WA and WB constant; (b) measuring the binding
of each compound to determine which compounds have a superior
ternary binding potency relative to a corresponding binary binding
potency for proteins A and B; and (c) determining the n values that
give rise to the potencies indicative of the existence of
protein-protein interactions.
[0035] In any of the aspects or embodiments described herein,
determining the chain length or the number of atoms of the
connector C (n) further comprises the step of changing the
attachment points on WA and WB that are used to link the warheads
to the connector C, and repeat steps (a) through (c) to find
additional compounds with protein-protein interactions.
[0036] In any of the aspects or embodiments described herein,
connector C is a chain with branched groups and/or contains
rings.
[0037] In any of the aspects or embodiments described herein, the
measuring step further comprises: measuring the influence of the
first protein or the second protein on a binding constant of
another protein toward the ligand to evaluate the capability of the
ligand L to induce the protein-protein interaction.
[0038] In any of the aspects or embodiments described herein, the
method comprises a step of: performing molecular dynamics
simulations to demonstrate protein-protein interactions and other
cross-domain interactions in ternary systems composed of the first
protein A, the second protein B, and the ligand L to evaluate the
capability of the ligand L to induce the protein-protein
interaction.
[0039] In any of the aspects or embodiments described herein, the
first protein A and the second protein B are the same protein. In
any of the aspects or embodiments described herein, the first
protein A and the second protein B are different proteins.
[0040] In any of the aspects or embodiments described herein, the
method further comprises selecting a ligand with a ternary complex
that results in surface area burial greater than the sum of the
surface area burial of the corresponding warhead monomers with the
first and second proteins.
[0041] In another aspect, the description provides a compound
resulting from performing a method as described herein.
[0042] In another aspect, the description provides a compound for
use in treating or preventing a disease or disorder, administering
an effective amount of a compound as described herein to a person
in need thereof.
[0043] In another aspect, the description provides a method of
designing a tripartite or bifunctional ligand that induces
protein-protein interaction(s) between a first protein molecule (A)
and a second protein molecule (B), the method comprising:
designing, preparing, and/or synthesizing a plurality of tripartite
and/or bifunctional compounds with the general structure WA-C-WB or
WA-WB, wherein WA is a warhead that associates with the first
protein, WB is a warhead that associates with the second protein,
and C is a connector covalently linked or bound to WA and WB;
designing, preparing, and/or synthesizing control compounds;
quantifying induced protein-protein interactions with at least one
of biochemical assays, cellular assays, and molecular dynamics
simulations; and selecting the tripartite or bifunctional
compound/ligand that induces protein-protein interactions and/or
other cross-domain interactions in the ternary complex.
[0044] In any of the aspects or embodiments described herein,
designing, preparing, and/or synthesizing includes varying a length
of the connector between 0 atoms to 30 atoms while maintaining the
same warheads and connection points between the connector and the
warheads. In any of the aspects or embodiments described herein,
the length of the connector is varied by an increment of 1 to 3
atoms. In any of the aspects or embodiments described herein, the
covalent link between the connector and WA and/or WB is at a
solvent-exposed point.
[0045] In any of the aspects or embodiments described herein, the
plurality of tripartite and/or bifunctional compounds comprises
subsets of compounds having a unique covalent link between warhead
WA and warhead WB or a unique series of covalent links between
warhead WA, the connector, and warhead WB, relative to the other
subsets.
[0046] In any of the aspects or embodiments described herein,
designing, preparing, and/or synthesizing control compounds
comprises modifying either warhead WA or WB such that substantially
all of its association/binding ability to protein A or protein B is
removed.
[0047] In any of the aspects or embodiments described herein,
quantifying protein-protein interactions using biochemical assays
comprises determining whether (i) the tripartite or bifunctional
compound binding/associating with protein A and protein B produce
synergism, or (ii) the tripartite or bifunctional compound/ligand
induces ternary binding potency.
[0048] In any of the aspects or embodiments described herein,
selecting the tripartite or bifunctional compound/ligand that
induces protein-protein interactions in the ternary complex
comprises: selecting at least one tripartite or bifunctional
compound/ligands that have a ratio .alpha. that is greater than
about 1, wherein the ratio .alpha. is IC.sub.50.sup.A over
IC.sub.50.sup.A/B or IC.sub.50.sup.B over IC.sub.50.sup.B/A; and/or
selecting at least one tripartite or bifunctional compounds/ligands
that have a ratio .alpha.T that is greater than about 1, wherein
the ratio .alpha.T is a ratio of the lower of IC.sub.50.sup.A and
IC.sub.50.sup.B over IC.sub.50.sup.T.
[0049] In any of the aspects or embodiments described herein,
quantifying induced protein-protein interactions comprises
performing molecular dynamics simulations on tripartite and/or
bifunctional compounds that are determined to induce
protein-protein interaction(s) by either biochemical assays or
cellular assays.
[0050] In any of the aspects or embodiments described herein, the
protein-protein interactions for a particular conformation
determined by molecular dynamics simulations are examined by
calculating at least one of atom distances, surface area burial,
and interaction energies for the ternary complex formation and a
binary complex formation.
[0051] In any of the aspects or embodiments described herein, the
protein-protein interactions for a particular conformation
determined by molecular dynamics simulations are examined along the
simulation trajectory and the critical distances related to the
interactions and the intermolecular energies between critical
groups can be calculated along the simulation time.
[0052] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. The
terminology used in the description is for describing particular
embodiments only and is not intended to be limiting of the
invention.
[0053] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise (such as in the case
of a group containing a number of carbon atoms in which case each
carbon atom number falling within the range is provided), between
the upper and lower limit of that range and any other stated or
intervening value in that stated range is encompassed within the
invention. The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either both of those included limits
are also included in the invention.
[0054] The following terms are used to describe the present
invention. In instances where a term is not specifically defined
herein, that term is given an art-recognized meaning by those of
ordinary skill applying that term in context to its use in
describing the present invention.
[0055] The articles "a" and "an" as used herein and in the appended
claims are used herein to refer to one or to more than one (i.e.,
to at least one) of the grammatical object of the article unless
the context clearly indicates otherwise. By way of example, "an
element" means one element or more than one element.
[0056] The phrase "and/or," as used herein in the specification and
in the claims, should be understood to mean "either or both" of the
elements so conjoined, i.e., elements that are conjunctively
present in some cases and disjunctively present in other cases.
Multiple elements listed with "and/or" should be construed in the
same fashion, i.e., "one or more" of the elements so conjoined.
Other elements may optionally be present other than the elements
specifically identified by the "and/or" clause, whether related or
unrelated to those elements specifically identified. Thus, as a
non-limiting example, a reference to "A and/or B", when used in
conjunction with open-ended language such as "comprising" can
refer, in one embodiment, to A only (optionally including elements
other than B); in another embodiment, to B only (optionally
including elements other than A); in yet another embodiment, to
both A and B (optionally including other elements); etc.
[0057] As used herein in the specification and in the claims, "or"
should be understood to have the same meaning as "and/or" as
defined above. For example, when separating items in a list, "or"
or "and/or" shall be interpreted as being inclusive, i.e., the
inclusion of at least one, but also including more than one, of a
number or list of elements, and, optionally, additional unlisted
items. Only terms clearly indicated to the contrary, such as "only
one of" or "exactly one of," or, when used in the claims,
"consisting of," will refer to the inclusion of exactly one element
of a number or list of elements. In general, the term "or" as used
herein shall only be interpreted as indicating exclusive
alternatives (i.e., "one or the other but not both") when preceded
by terms of exclusivity, such as "either," "one of," "only one of,"
or "exactly one of."
[0058] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," "composed of," and
the like are to be understood to be open-ended, i.e., to mean
including but not limited to. Only the transitional phrases
"consisting of" and "consisting essentially of" shall be closed or
semi-closed transitional phrases, respectively, as set forth in the
United States Patent Office Manual of Patent Examining Procedures,
Section 2111.03.
[0059] As used herein in the specification and in the claims, the
phrase "at least one," in reference to a list of one or more
elements, should be understood to mean at least one element
selected from anyone or more of the elements in the list of
elements, but not necessarily including at least one of each and
every element specifically listed within the list of elements and
not excluding any combinations of elements in the list of elements.
This definition also allows that elements may optionally be present
other than the elements specifically identified within the list of
elements to which the phrase "at least one" refers, whether related
or unrelated to those elements specifically identified. Thus, as a
nonlimiting example, "at least one of A and B" (or, equivalently,
"at least one of A or B," or, equivalently "at least one of A
and/or B") can refer, in one embodiment, to at least one,
optionally including more than one, A, with no B present (and
optionally including elements other than B); in another embodiment,
to at least one, optionally including more than one, B, with no A
present (and optionally including elements other than A); in yet
another embodiment, to at least one, optionally including more than
one, A, and at least one, optionally including more than one, B
(and optionally including other elements); etc.
[0060] It should also be understood that, in certain methods
described herein that include more than one step or act, the order
of the steps or acts of the method is not necessarily limited to
the order in which the steps or acts of the method are recited
unless the context indicates otherwise.
[0061] The terms "co-administration" and "co-administering" or
"combination therapy" refer to both concurrent administration
(administration of two or more therapeutic agents at the same time)
and time varied administration (administration of one or more
therapeutic agents at a time different from that of the
administration of an additional therapeutic agent or agents), as
long as the therapeutic agents are present in the patient to some
extent, preferably at effective amounts, at the same time. In
certain preferred aspects, one or more of the present compounds
described herein, are coadministered in combination with at least
one additional bioactive agent, especially including an anticancer
agent. In particularly preferred aspects, the co-administration of
compounds results in synergistic activity and/or therapy, including
anticancer activity.
[0062] The term "compound", as used herein, unless otherwise
indicated, refers to any specific chemical compound disclosed
herein and includes tautomers, regioisomers, geometric isomers, and
where applicable, stereoisomers, including optical isomers
(enantiomers) and other steroisomers (diastereomers) thereof, as
well as pharmaceutically acceptable salts and derivatives
(including prodrug forms) thereof where applicable, in context.
Within its use in context, the term compound generally refers to a
single compound, but also may include other compounds such as
stereoisomers, regioisomers and/or optical isomers (including
racemic mixtures) as well as specific enantiomers or
enantiomerically enriched mixtures of disclosed compounds. The term
also refers, in context to prodrug forms of compounds which have
been modified to facilitate the administration and delivery of
compounds to a site of activity. It is noted that in describing the
present compounds, numerous substituents and variables associated
with same, among others, are described. It is understood by those
of ordinary skill that molecules which are described herein are
stable compounds as generally described hereunder. When the bond is
shown, both a double bond and single bond are represented within
the context of the compound shown.
[0063] The term "patient" or "subject" is used throughout the
specification to describe an animal, preferably a human or a
domesticated animal, to whom treatment, including prophylactic
treatment, with the compositions according to the present
disclosure is provided. For treatment of those infections,
conditions or disease states which are specific for a specific
animal such as a human patient, the term patient refers to that
specific animal, including a domesticated animal such as a dog or
cat or a farm animal such as a horse, cow, sheep, etc. In general,
in the present disclosure, the term patient refers to a human
patient unless otherwise stated or implied from the context of the
use of the term.
[0064] The term "effective" is used to describe an amount of a
compound, composition or component which, when used within the
context of its intended use, effects an intended result. The term
effective subsumes all other effective amount or effective
concentration terms, which are otherwise described or used in the
present application.
[0065] FIG. 1A and FIG. 1B illustrates a ligand of the present
disclosure. The ligand is able to induce protein-protein
interactions between two protein molecules and has a generic
chemical structure of WA-C-WB. WA is a warhead that targets protein
molecule A (e.g., binds or interacts with protein molecule A). WB
is a warhead that targets protein molecule B (e.g., binds or
interacts with protein molecule B). C is a connector that provides
appropriate spacing between warhead WA and warhead WB. In a
particular embodiment, the connector C is covalently linked to
warhead WA and warhead WB.
[0066] As shown below, the distance or the range of the distance
between warhead WA and warhead WB in the tripartite molecule
WA-C-WB is critical for the protein-protein interaction between
protein A and protein B to occur, and the lengths of the connector
C associated with optimal or desirable protein-protein interactions
depend upon the structures of protein A and protein B involved
(FIG. 2). Therefore, a major part of the present disclosure relates
to a process of chemical exploration to identify an appropriate
connector C. In an embodiment, the warhead WA and/or warhead WB is
a known ligand of protein A and/or protein B. In another
embodiment, warhead WA and/or warhead WB is derived using
established technologies in the art, for example, a high-through
screening and/or structure-based drug design may be utilized to
discover/derive a previously unknown ligand for protein A and/or
protein B. In some embodiments, for certain proteins A and B, a
ligand of generic chemical structure of WA-WB is appropriate to
induce the corresponding protein-protein interaction, wherein no
connector is needed and the warheads are linked directly through a
covalent bond, a common atom, or fused together through a common
bond.
[0067] In an aspect of the present disclosure, a method of
designing tripartite or bifunctional compounds is provided. The
method comprises: designing, preparing, and/or synthesizing a
plurality of tripartite and/or bifunctional compounds (i.e.,
ligands) with the general structure WA-C-WB or WA-WB; designing,
preparing, and/or synthesizing control compounds; and quantifying
induced protein-protein interactions with at least one of
biochemical assays, cellular assays (i.e. in a cellular context),
and molecular dynamics simulations; and selecting the tripartite or
bifunctional compound/ligand that induces protein-protein
interactions and/or other cross-domain interactions in the ternary
complex.
[0068] WA selectively binds protein A and WB selectively binds
protein B. The warheads WA and WB can be independently either known
association/binding partners of protein A and protein B,
respectively, or they can be selected through a conventional high
throughput screening and/or structure-based drug design. Proteins A
and B can be the same protein or be two different proteins. In an
embodiment, WA and WB are linked to connector C via covalent bonds.
In another embodiment, WA and WB are directly linked through a
common bond, a common atom, or fused together through a common
bond.
[0069] In certain embodiments, designing, preparing, and/or
synthesizing includes varying a length of the connector. That is,
the connectors of the plurality of tripartite and/or bifunctional
compounds vary in length, i.e. the number of atoms along its length
(n) are different, while the connectors of each of the plurality of
tripartite and/or bifunctional compounds are linked in the same
fashion to the same WA and WB. This allows for determination of the
optimal connector length to induce protein-protein interactions. In
an embodiment, the length of the connector is varied between 0 to
30 atoms. In a particular embodiment, the length of the connector
is varied by an increment of 1-6 atoms, 1-5 atoms, 1-4 atoms, 1-3
atoms, 1-2 atoms, 2-6 atoms, 2-5 atoms, 2-4 atoms, 2-3 atoms, 3-6
atoms, 3-5 atoms, 3-4 atoms, 4-6 atoms, 4-5 atoms, or 5-6 atoms. In
an embodiment, the connector is varied by an increment of 1, 2, 3,
4, 5, 6, 7, 8, 9, or 10 atoms. In certain embodiments, the
connector chain is 1, 2, 3, 4, 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, or 30
atoms, and may be substituted or unsubstituted.
[0070] In other embodiments, the connector is linked to WA and/or
WB via a solvent-exposed point. In some embodiments, the plurality
of tripartite and/or bifunctional compounds comprises subsets of
compound in which each subset of compounds has a unique link
relative to the other subsets. That is, each subset of compounds
has a unique set of attachment points between WA, WB and the
connector, and/or WA and WB. Furthermore, each subset with a unique
link comprises a plurality of tripartite and/or bifunctional
compounds, each having a unique connector for the subset of
compounds. In an embodiment, each of the compounds of the subset
has a connector of a different length within the range of 0 to 30
atoms.
[0071] In particular embodiments, designing, preparing, and/or
synthesizing control compounds comprises modifying either WA or WB
such that substantially all of its association/binding ability to
protein A or protein B, respectively, is removed. For example, WA
would retain its ability to associate with or bind protein A, while
WB would be modified such that WB* substantially lacks the ability
to associate with or bind protein B, or vise versa (please note
that an asterisk denotes a WA or WB wish substantially diminished
or eliminated ability to bind or associate with protein A or
protein B). The ability for WA or WB to associate/bind with its
respective protein can be diminished/eliminated by, for example,
adding a methyl group to cause clashes with the association/binding
pocket of the warhead or inverting at least one chiral center that
is critical for the association/binding. Production of the control
compounds allow for the examination for induced protein-protein
interactions while accounting for membrane permeability difference
that would exist if WA or WB were removed from the compound. As
discussed below, these modified tripartite or bifunctional
compounds allow for measurement of binary binding potencies, which
can be compared with measured ternary binding potency of the parent
tripartite or bifunctional compound in cellular assays to determine
whether the parent tripartite or bifunctional compound induced
protein-protein interactions.
[0072] In some embodiment, quantifying protein-protein interactions
using biochemical assays comprises determining whether (i) the
tripartite or bifunctional compound binding/associating with
protein A and protein B produce synergism (i.e. whether the binding
potency of the compound/ligand to protein A or protein B is
strengthened by the presence of the association/binding of the
other protein (protein B or protein A, respectively) to the
compound/ligand) or (ii) the tripartite or bifunctional
compound/ligand induces ternary binding potency (i.e. the
tripartite or bifunctional compound/ligands ability to produce
ternary species).
[0073] In an embodiment, determining whether the compound/ligand
produces synergism includes comparing binary binding potencies of
binary control compounds, i.e. WA-C and C-WB (for tripartite
compounds/ligands) or WA and WB (for bifunctional
compounds/ligands), with the binding potency of its respective
tripartite or bifunctional compound ligand (i.e., WA-C-WB or
WA-WB). The binary binding potencies of binary control compounds
are a measure of the binding potency between each warheads and its
associated protein. The binary binding potency of a tripartite or
bifunctional compound with respect to A, or the binary binding
IC.sub.50.sup.A, can be determined by titrating the corresponding
mono-functional compound WA-C-WB* or WA-WB* (as discussed above, an
asterisk denotes a warhead WA or WB with substantially diminished
or eliminated capacity/ability to bind to protein A or protein B)
into a solution of A. A dose-response curve reflective of the
complex formation between compound WA-C-WB* or WA-WB* and the
protein A is measured as a function of the compound concentration.
In an embodiment, the IC.sub.50.sup.A is the total compound
concentration at which the response reaches a half height of its
maximum.
[0074] In a similar fashion, the binary binding potency of a
tripartite or bifunctional compound with respect to B, or the
binary binding IC.sub.50.sup.B, can be determined by titrating the
corresponding mono-functional compound WA*-C-WB or WA*-WB into a
solution of B and measuring the dose-response curve reflective of
the complex formation between protein B and compound WA*-C-WB or
WA*-WB. In an embodiment, the IC.sub.50.sup.B is the total compound
concentration at which the response reaches a half height of its
maximum. When protein A and protein B are the same protein, and
warheads WA and WB are identical, IC.sub.50.sup.A and
IC.sub.50.sup.B are the same. When protein A and protein B are
different proteins, IC.sub.50.sup.A can be determined by titrating
the compound WA-C-WB or WA-WB into the solution of A without
protein B, and/or IC.sub.50.sup.B can be determined by titrating
the compound WA-C-WB or WA-WB into the solution of B without
protein A.
[0075] The binding potency of the tripartite or bifunctional
compound/ligand can be examined relative to protein A or protein B.
For example, the binding potency of the tripartite or bifunctional
compound/ligand can be measured by titrating the compound into a
solution of mixture of protein A and protein B. A dose-response
curve reflective of the complex formation between the compound and
protein A can be measured as a function of the compound
concentration. IC.sub.50.sup.A/B is the total compound
concentration at which the response between the compound and
protein A reaches a half height of its maximum, and which includes
the contributions of the binary complex A-L and ternary complex
A-L-B. Similarly, a dose-response curve reflective of the complex
formation between the compound and protein B can be measured as a
function of the compound concentration. IC.sub.50.sup.B/A is the
total compound concentration at which the response between the
compound and protein B reaches a half height of its maximum, and
which includes the contributions of the binary complex B-L and
ternary complex A-L-B. If A and B are identical (i.e., the same
protein), IC.sub.50.sup.A/B=IC.sub.50.sup.B/A and can be determined
by titrating the compound into the solution of the proteins.
[0076] In an embodiment, comparing binary binding potencies of
binary control compounds with the binding potency of its respective
tripartite or bifunctional compound ligand comprises producing a
ratio .alpha. of the binary IC.sub.50.sup.A over IC.sub.50.sup.A/B.
In another embodiment, the ratio .alpha. is of the binary
IC.sub.50.sup.B over IC.sub.50.sup.B/A. When the same protein
concentrations are used to determine the ratio .alpha.: there is
substantially no interaction between protein A and protein B when
.alpha. is about 1, there is a favorable interaction between
protein A and protein B (i.e. a favorable protein-protein
interaction) when .alpha. is greater than about 1, and there is an
unfavorable interaction between protein A and B (i.e., an
unfavorable protein-protein interaction) when .alpha. is less than
about 1.
[0077] In an embodiment, determining whether the tripartite or
bifunctional compound/ligand induces ternary binding potency
comprises comparing binary binding potencies of binary control
compounds, i.e. WA-C and C-WB (for tripartite compounds/ligands) or
WA and WB (for bifunctional compounds/ligands), with a ternary
binding potency (i.e., WA-C-WB or WA-WB). The binary binding
potencies can be determined as described above. The ternary binding
potency can be determined by titrating the tripartite or
bifunctional compound/ligand into a solution of a mixture of
proteins A and B, and producing a dose-response curve reflective of
the ternary complex formation A-L-B measured as a function of the
compound concentration. Ternary binding IC.sub.50.sup.T is the
total compound concentration at which the response first reaches a
half height of its maximum value (i.e. the earliest occasion). In
another embodiment, comparing binary binding potencies of binary
control compounds with a ternary binding potency comprises
producing a ratio .alpha..sup.T of the lower value of binary
binding IC.sub.50.sup.A and IC.sub.50.sup.B over the ternary
binding IC.sub.50.sup.T. When the same protein concentrations are
used to determine IC.sub.50.sup.T and IC.sub.50.sup.A or
IC.sub.50.sup.B: there is a favorable interaction between protein A
and protein B (i.e. a favorable protein-protein interaction) when
.alpha..sup.T is greater than about 1, there is an unfavorable
interaction between protein A and B (i.e., an unfavorable
protein-protein interaction) when .alpha..sup.T is less than or
equal to about 0.1; and there is a moderate or no protein-protein
interaction between protein A and protein B when .alpha..sup.T is
in a range of about 0.1 to about 1.
[0078] Quantifying the induced protein-protein interactions with
cellular assays (i.e. in a cellular context) can comprise
determining whether (i) the tripartite or bifunctional compound
binding/associating with protein A and protein B produce synergism,
or (ii) the tripartite or bifunctional compound/ligand induces
ternary binding potency. In an embodiment, when examining
synergism, a binary binding potency and a ternary binding potency
are determined in a cellular assay. In a particular embodiment,
comparing the binary binding potency with the ternary binding
potency includes producing a ratio .alpha. selected from:
IC.sub.50.sup.A/IC.sub.50.sup.A/B and
IC.sub.50.sup.B/IC.sub.50.sup.B/A.
[0079] The ratios can be determined as follows. Binary binding
IC.sub.50.sup.A between a tripartite or bifunctional compound and
protein A can be determined by, for example, titrating a tripartite
or bifunctional compound substantially lacking the ability to
bind/associate with protein B (e.g., WA-WB* or WA-C-WB*) into a
medium with cells that express protein A and protein B, and
producing a dose-response curve reflective of the complex formation
between the compound and protein A in the cells, measured as a
function of the compound concentration. As such, IC.sub.50.sup.A is
the total compound concentration at which the response reaches a
half height of its maximum. The binary binding IC.sub.50.sup.B
between a tripartite or bifunctional compound and protein B can be
determined by, for example, titrating a tripartite or bifunctional
compound substantially lacking the ability to bind/associate with
protein A (e.g., WA*-WB or WA*-C-WB) into a medium with cells that
express protein A and protein B, and producing a dose-response
curve reflective of the complex formation between the compound and
protein B in the cells, measured as a function of the compound
concentration. As such, IC.sub.50.sup.B is the total compound
concentration at which the response reaches a half height of its
maximum. When protein A and protein B are the same protein and the
two warheads WA and WB are identical, IC.sub.50.sup.A and
IC.sub.50.sup.B are not differentiable and the two determinations
become one.
[0080] The binding potency of the tripartite or bifunctional
compound to protein A in the presence of the contribution of
protein B, i.e. IC.sub.50.sup.A/B, can be determined by, for
example, titrating the tripartite or bifunctional compound/ligand
(i.e. WA-C-WB or WA-WB) into a medium with cells that express
protein A and protein B, and producing a dose-response curve
reflective of the complex formation between the compound/ligand and
protein A in the cells, measured as a function of the compound
concentration (the contributions are from binary species A-L and
ternary species A-L-B). As such, the IC.sub.50.sup.A/B is the total
compound/ligand concentration at which the response reaches half
the height of its maximum. The binding potency of the tripartite or
bifunctional compound and protein B in the presence of the
contribution of protein A, i.e., IC.sub.50.sup.B/A, can be
determined by, for example, titrating the tripartite or
bifunctional compound ligand into a medium with cells that express
protein A and protein B, and producing a dose-response curve
reflective of the complex formation between the compound/ligand and
protein B in the cells, measured as a function of the compound
concentration (the contributions are from binary species B-L and
ternary species A-L-B). As such, the IC.sub.50.sup.B/A is the total
compound/ligand concentration at which the response reaches half
the height of its maximum. When protein A and protein B are the
same protein and the two warheads WA and WB are identical,
IC.sub.50.sup.A/B and IC.sub.50.sup.B/A are not differentiable and
the two determinations become one.
[0081] The ratios of IC.sub.50.sup.A/IC.sub.50.sup.A/B and
IC.sub.50.sup.B/IC.sub.50.sup.B/A should converge so either may be
used as the ratio .alpha.. When the membrane permeability of the
control compounds (e.g., WA*-C-WB, WA-C-WB*, WA*-WB, or WA-WB*) are
the same or similar to that of the tripartite or bifunctional
compound, the ratio .alpha. is independent of the membrane
permeability of the compound and is a measure of the interactions
between proteins A and B in the ternary complex with the compound.
As such, the ligand induces: a favorable protein-protein
interaction between protein A and protein B when .alpha. is greater
than about 1; an unfavorable protein-protein interaction between
protein A and protein B when .alpha. is less than about 1; and no
protein-protein interaction between protein A and protein B when
.alpha. is about 1.
[0082] The ternary binding potency of the tripartite or
bifunctional compound to protein A and protein B can be determined
by titrating the compound into medium with cells expressing protein
A and protein B, and producing a dose-response curve reflective of
ternary species formation, such as A-L-B. Ternary binding
IC.sub.50.sup.T can be the total compound concentration at which
the response first reaches half the height of its maximum (i.e. the
first occasion it reaches half the height of its maximum). In an
embodiment, .alpha..sup.T is a ratio of the smaller binary binding
IC.sub.50.sup.A and IC.sub.50.sup.B over the ternary binding
IC.sub.50.sup.T. The value .alpha..sup.T is independent of membrane
permeability of the compounds because of the cancelation of the
effect by the parent tripartite or bifunctional compound and the
control compounds. As such, the ligand induces: a favorable
protein-protein interaction between protein A and protein B when
.alpha..sup.T is greater than about 1; an unfavorable
protein-protein interaction between protein A and protein B when
.alpha..sup.T is less than or equal to about 0.1; and moderate or
no protein-protein interaction between protein A and protein B when
.alpha. is in a range of about 0.1 to about 1.
[0083] In an embodiment, quantifying the induced protein-protein
interactions with molecular dynamics simulations comprises:
building an initial model of a ternary complex of the tripartite or
bifunctional compound/ligand with protein A and protein B (i.e.,
A-L-B, A-WA-WB-B, A-WA-C-WB-B, etc.) by docking protein A with WA
and protein B with WB. In particular embodiment, docking of protein
A and protein B is guided by existing crystal structures for binary
complexes (e.g., WA-A or WB-B). In another embodiment, docking of
protein A and protein B is performed by a docking program. In an
embodiment, at least one of the following characteristics is used:
(i) a connector conformation is set to an extended conformation
arbitrarily; (ii) the system is solvated with explicit waters and
counter ions in about 0.1 M sodium chloride; (iii) the molecular
dynamics simulation is performed on the system for a 40 nanoseconds
production time; (iv) coordinate frames from the last 10
nanoseconds of simulation are subject to clustering analysis; and
(v) a frame closest to the center of a largest cluster is
considered as the most populated conformation and used as a
representative conformation of the system. In some embodiments, at
least two, three, four, or all five of the characteristics are
used.
[0084] In an embodiment, protein-protein interactions and other
cross-domains interactions for a particular conformation are
examined by calculating at least one of atom distances, surface
area burial, and interaction energies (e.g. on a group-to-group
basis) for the ternary complex formation and a binary complex
formation. In another embodiment, protein-protein interactions and
other cross-domain interactions are monitored along the simulation
trajectory and the critical distances related to the interactions
and the intermolecular energies between critical groups can be
calculated along the simulation time.
[0085] As such, selecting the tripartite or bifunctional
compound/ligand that induces protein-protein interactions and/or
other cross-domain interactions in the ternary complex can be
achieved by selecting the compound that demonstrates synergism
and/or ternary binding potency that is greater/strengthened
relative to the value predicted by binary binding potency, as
described above.
[0086] Furthermore, in an embodiment, any compound with an .alpha.
value or .alpha..sup.T value that demonstrates there is a
protein-protein interaction or other cross-domain interactions is
subject to molecular dynamics simulation, e.g. as described above.
The simulation can therefore reveal how the ligand, protein A, and
protein B interact with each other to induce the cross-domain
interactions. In some embodiment, the representative conformation
of the ternary complex from the molecular dynamics simulations have
a trimer conformation (i.e., ternary conformation) in which protein
molecules A and B form a complex that has a collective binding site
for the compound/ligand. In an embodiment, part or all of the
connector interacts with the rest of the solute system and
contributes to the stability of the ternary complex. The
representative conformation of the ternary complex is used to guide
optimization of the compound.
[0087] In additional embodiments, designing, preparing, and/or
synthesizing a plurality of tripartite and/or bifunctional
compounds can include making other modifications to the
compound/ligand including: (a) adding branches to a linear
connector; (b) replacing at least one flexible portion of the
connector with a rigid group; (c) constraining parts of the
connector by cyclization; (d) replacing at least one atom of the
connector with atoms or groups of different nature (e.g. carbon to
oxygen replacement, oxygen to carbon replacement, etc.); (e) adding
or removing atoms or groups to or from the connector to lengthen or
shorten the connector, respectively; and (f) adding, removing, or
changing groups within at least one of the warheads.
[0088] The modification of the tripartite or bifunctional
compound/ligand is performed to find a tripartite or bifunctional
compound/ligand with increased potency, improved physicochemical
properties, and/or improved metabolic stability. The modifications
are guided by the trimer conformation such that the trimer complex
is stabilized or at least not destabilized too much to lose the
corresponding potency. In an embodiment, the length of a modified
connector allows the placement of the warheads WA and WB into the
respective binding sites of protein A and protein B without change
of binding modes. In another embodiment, the selected tripartite or
bifunctional compound/ligand that has enhanced ability to induce
protein-protein interactions or other cross-domain interactions
relative to a parent tripartite or bifunctional compound comprises
at least one of: (a) a more rigid connector than the parent
compound that adopts the same conformation as the parent in its
ternary conformation, (b) fills more hydrophobic cavities than the
parent compound in the ternary conformation; and (c) forms
additional hydrogen bonds or ion-pair interactions than the parent
compound in the ternary conformation.
[0089] In an additional embodiment, a modification is considered as
a good/favorable modification when the ternary conformation is
maintained relative to the parent compound. In another embodiment,
a modification is considered a favorable/good modification when a
surface area burial for ternary conformation for the modified
compound is about equal to or greater than that of the parent
compound. Conversely, a decrease in burial surface relative to a
parent compound indicates that the modification was unfavorable or
bad.
[0090] The modified compounds that are judged reasonable by the
modeling studies above are synthesized and tested with the assay
methods described in the previous section. If one seeks to optimize
the physicochemical properties or metabolic stability, the
corresponding parameters for the compound are measured also.
Several cycles of modeling, modification, synthesis and testing may
be needed to achieve the desired objectives of optimization.
[0091] In an embodiment, the capability of a compound to induce a
protein-protein interaction is determined through biochemical
experiments in which the ternary IC.sub.50 and binary IC.sub.50s
are measured in an aqueous solution or buffer containing related
proteins and compound. In another embodiment, the capability of a
compound to induce a protein-protein interaction is determined
through cell-based assays in which a compound is added to a medium
containing a cell line expressing the related proteins. The related
proteins can be intracellular proteins or membrane-bound proteins.
The ternary and binary IC.sub.50s are the total compound
concentrations in the respective conditions. In a particular
embodiment, the IC.sub.50s are determined by measuring specific
cellular responses reflective of, and proportional to, the
concentrations of the corresponding ternary species and binary
species formed by the added compound with the corresponding
proteins. In another embodiment, the binary IC.sub.50s is
determined by using decoy compounds wherein one of the warheads is
altered in a way that its binding to the corresponding target is
disabled. Comparing a ternary binding IC.sub.50 and the
corresponding binary binding IC.sub.50s can provide a measurement
of the protein-protein interaction. In a cell-based assay, the
individual IC.sub.50s are affected by the membrane permeability of
the compound, however, the ratio between the ternary binding
IC.sub.50 and the corresponding binary binding IC.sub.50s is
independent of the membrane permeability because the factor of the
membrane permeability is canceled out.
[0092] In another embodiment, the chemical matter of the present
disclosure (i.e., the ligand) can be administered to animals for in
vivo experiments. After administration, the pharmacokinetic
parameters of the compound can be measured to examine the effects
of the compound in different tissues and blood components. The in
vivo functional effects the compound has on, e.g. Tumor Growth
Inhibition (TGI), Prostate-Specific Antigens (PSA) as well as
others, can be examined/measured.
[0093] The composition of the present disclosure may be
administered to a subject, e.g., a human, in any medically
acceptable way, and which may depend on the disease condition or
injury being treated. Possible administration routes include e.g.,
injections, by parenteral routes such as intravascular,
intravenous, intraepidural or others, as well as oral, nasal,
ophthalmic, rectal, topical, or pulmonary, e.g. by inhalation.
Administration is discussed in greater detail below.
[0094] In one embodiment, the method comprises performing molecular
dynamics simulations with explicit water and counter-ions on
ternary complexes A-L-B with protein A and protein B as target
proteins and the ligand L binding to the proteins A and B
simultaneously. In an embodiment, the method includes determining
the most populated conformation (representative conformation)
during a simulation trajectory. In an additional embodiment, the
method comprises examining the protein-protein interactions in the
simulated structures. As such, the ligand can be redesigned to have
a better fit to ternary complexes based on the simulated
structures.
[0095] In another embodiment, the method comprises determining
crystal structures of at least one ternary complex A-L-B with x-ray
crystallography or NMR spectroscopy. The protein-protein
interactions in the ternary complexes can be examined, and the
ligands can be redesigned based on the complex structures. In
another embodiment, the method comprises monitoring the
protein-protein interactions by detecting spectroscopic signals of
the relevant groups of the molecules. In yet another embodiment,
the method comprises inferring the protein-protein interactions
from heat absorption or release of the ternary binding and binary
binding processes using calorimetry-based methods.
Therapeutic Compositions
[0096] Pharmaceutical compositions comprising combinations of an
effective amount of at least one bifunctional compound as described
herein, and one or more of the compounds otherwise described
herein, all in effective amounts, in combination with a
pharmaceutically effective amount of a carrier, additive or
excipient, represents a further aspect of the present
disclosure.
[0097] The present disclosure includes, where applicable, the
compositions comprising the pharmaceutically acceptable salts, in
particular, acid or base addition salts of compounds as described
herein. The acids which are used to prepare the pharmaceutically
acceptable acid addition salts of the aforementioned base compounds
useful according to this aspect are those which form non-toxic acid
addition salts, i.e., salts containing pharmacologically acceptable
anions, such as the hydrochloride, hydrobromide, hydroiodide,
nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate,
lactate, citrate, acid citrate, tartrate, bitartrate, succinate,
maleate, fumarate, gluconate, saccharate, benzoate,
methanesulfonate, ethanesulfonate, benzenesulfonate,
p-toluenesulfonate and pamoate [i.e.,
1,1'-methylene-bis-(2-hydroxy-3 naphthoate)]salts, among numerous
others.
[0098] Pharmaceutically acceptable base addition salts may also be
used to produce pharmaceutically acceptable salt forms of the
compounds or derivatives according to the present disclosure. The
chemical bases that may be used as reagents to prepare
pharmaceutically acceptable base salts of the present compounds
that are acidic in nature are those that form non-toxic base salts
with such compounds. Such non-toxic base salts include, but are not
limited to those derived from such pharmacologically acceptable
cations such as alkali metal cations (eg., potassium and sodium)
and alkaline earth metal cations (eg, calcium, zinc and magnesium),
ammonium or water-soluble amine addition salts such as
N-methylglucamine-(meglumine), and the lower alkanolammonium and
other base salts of pharmaceutically acceptable organic amines,
among others.
[0099] The compounds as described herein may, in accordance with
the disclosure, be administered in single or divided doses by the
oral, parenteral or topical routes. Administration of the active
compound may range from continuous (intravenous drip) to several
oral administrations per day (for example, Q.I.D.) and may include
oral, topical, parenteral, intramuscular, intravenous,
sub-cutaneous, transdermal (which may include a penetration
enhancement agent), buccal, sublingual and suppository
administration, among other routes of administration. Enteric
coated oral tablets may also be used to enhance bioavailability of
the compounds from an oral route of administration. The most
effective dosage form will depend upon the pharmacokinetics of the
particular agent chosen as well as the severity of disease in the
patient. Administration of compounds according to the present
disclosure as sprays, mists, or aerosols for intra-nasal,
intra-tracheal or pulmonary administration may also be used. The
present disclosure therefore also is directed to pharmaceutical
compositions comprising an effective amount of compound as
described herein, optionally in combination with a pharmaceutically
acceptable carrier, additive or excipient. Compounds according to
the present disclosureion may be administered in immediate release,
intermediate release or sustained or controlled release forms.
Sustained or controlled release forms are preferably administered
orally, but also in suppository and transdermal or other topical
forms. Intramuscular injections in liposomal form may also be used
to control or sustain the release of compound at an injection
site.
[0100] The compositions as described herein may be formulated in a
conventional manner using one or more pharmaceutically acceptable
carriers and may also be administered in controlled-release
formulations. Pharmaceutically acceptable carriers that may be used
in these pharmaceutical compositions include, but are not limited
to, ion exchangers, alumina, aluminum stearate, lecithin, serum
proteins, such as human serum albumin, buffer substances such as
phosphates, glycine, sorbic acid, potassium sorbate, partial
glyceride mixtures of saturated vegetable fatty acids, water, salts
or electrolytes, such as prolamine sulfate, disodium hydrogen
phosphate, potassium hydrogen phosphate, sodium chloride, zinc
salts, colloidal silica, magnesium trisilicate, polyvinyl
pyrrolidone, cellulose-based substances, polyethylene glycol,
sodium carboxymethylcellulose, polyacrylates, waxes,
polyethylene-polyoxypropylene-block polymers, polyethylene glycol
and wool fat.
[0101] The compositions as described herein may be administered
orally, parenterally, by inhalation spray, topically, rectally,
nasally, buccally, vaginally or via an implanted reservoir. The
term "parenteral" as used herein includes subcutaneous,
intravenous, intramuscular, intra-articular, intra-synovial,
intrasternal, intrathecal, intrahepatic, intralesional and
intracranial injection or infusion techniques. Preferably, the
compositions are administered orally, intraperitoneally or
intravenously.
[0102] Sterile injectable forms of the compositions as described
herein may be aqueous or oleaginous suspension. These suspensions
may be formulated according to techniques known in the art using
suitable dispersing or wetting agents and suspending agents. The
sterile injectable preparation may also be a sterile injectable
solution or suspension in a non-toxic parenterally-acceptable
diluent or solvent, for example as a solution in 1, 3-butanediol.
Among the acceptable vehicles and solvents that may be employed are
water, Ringer's solution and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally employed as a
solvent or suspending medium. For this purpose, any bland fixed oil
may be employed including synthetic mono- or di-glycerides. Fatty
acids, such as oleic acid and its glyceride derivatives are useful
in the preparation of injectables, as are natural
pharmaceutically-acceptable oils, such as olive oil or castor oil,
especially in their polyoxyethylated versions. These oil solutions
or suspensions may also contain a long-chain alcohol diluent or
dispersant, such as Ph. Helv or similar alcohol.
[0103] The pharmaceutical compositions as described herein may be
orally administered in any orally acceptable dosage form including,
but not limited to, capsules, tablets, aqueous suspensions or
solutions. In the case of tablets for oral use, carriers which are
commonly used include lactose and corn starch. Lubricating agents,
such as magnesium stearate, are also typically added. For oral
administration in a capsule form, useful diluents include lactose
and dried corn starch. When aqueous suspensions are required for
oral use, the active ingredient is combined with emulsifying and
suspending agents. If desired, certain sweetening, flavoring or
coloring agents may also be added.
[0104] Alternatively, the pharmaceutical compositions as described
herein may be administered in the form of suppositories for rectal
administration. These can be prepared by mixing the agent with a
suitable non-irritating excipient, which is solid at room
temperature but liquid at rectal temperature and therefore will
melt in the rectum to release the drug. Such materials include
cocoa butter, beeswax and polyethylene glycols.
[0105] The pharmaceutical compositions as described herein may also
be administered topically. Suitable topical formulations are
readily prepared for each of these areas or organs. Topical
application for the lower intestinal tract can be effected in a
rectal suppository formulation (see above) or in a suitable enema
formulation. Topically-acceptable transdermal patches may also be
used.
[0106] For topical applications, the pharmaceutical compositions
may be formulated in a suitable ointment containing the active
component suspended or dissolved in one or more carriers. Carriers
for topical administration of the compounds of this disclosure
include, but are not limited to, mineral oil, liquid petrolatum,
white petrolatum, propylene glycol, polyoxyethylene,
polyoxypropylene compound, emulsifying wax and water. In certain
preferred aspects of the disclosure, the compounds may be coated
onto a stent which is to be surgically implanted into a patient in
order to inhibit or reduce the likelihood of occlusion occurring in
the stent in the patient.
[0107] Alternatively, the pharmaceutical compositions can be
formulated in a suitable lotion or cream containing the active
components suspended or dissolved in one or more pharmaceutically
acceptable carriers. Suitable carriers include, but are not limited
to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl
esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and
water.
[0108] For ophthalmic use, the pharmaceutical compositions may be
formulated as micronized suspensions in isotonic, pH adjusted
sterile saline, or, preferably, as solutions in isotonic, pH
adjusted sterile saline, either with our without a preservative
such as benzylalkonium chloride. Alternatively, for ophthalmic
uses, the pharmaceutical compositions may be formulated in an
ointment such as petrolatum.
[0109] The pharmaceutical compositions as described herein may also
be administered by nasal aerosol or inhalation. Such compositions
are prepared according to techniques well-known in the art of
pharmaceutical formulation and may be prepared as solutions in
saline, employing benzyl alcohol or other suitable preservatives,
absorption promoters to enhance bioavailability, fluorocarbons,
and/or other conventional solubilizing or dispersing agents.
[0110] The amount of compound in a pharmaceutical composition as
described herein that may be combined with the carrier materials to
produce a single dosage form will vary depending upon the host and
disease treated, the particular mode of administration. Preferably,
the compositions should be formulated to contain between about 0.05
milligram to about 750 milligrams or more, more preferably about 1
milligram to about 600 milligrams, and even more preferably about
10 milligrams to about 500 milligrams of active ingredient, alone
or in combination with at least one other compound according to the
present disclosure.
[0111] It should also be understood that a specific dosage and
treatment regimen for any particular patient will depend upon a
variety of factors, including the activity of the specific compound
employed, the age, body weight, general health, sex, diet, time of
administration, rate of excretion, drug combination, and the
judgment of the treating physician and the severity of the
particular disease or condition being treated.
[0112] A patient or subject in need of therapy using compounds
according to the methods described herein can be treated by
administering to the patient (subject) an effective amount of the
compound according to the present disclosure including
pharmaceutically acceptable salts, solvates or polymorphs, thereof
optionally in a pharmaceutically acceptable carrier or diluent,
either alone, or in combination with other known erythopoiesis
stimulating agents as otherwise identified herein.
[0113] These compounds can be administered by any appropriate
route, for example, orally, parenterally, intravenously,
intradermally, subcutaneously, or topically, including
transdermally, in liquid, cream, gel, or solid form, or by aerosol
form.
[0114] The active compound is included in the pharmaceutically
acceptable carrier or diluent in an amount sufficient to deliver to
a patient a therapeutically effective amount for the desired
indication, without causing serious toxic effects in the patient
treated. A preferred dose of the active compound for all of the
herein-mentioned conditions is in the range from about 10 ng/kg to
300 mg/kg, preferably 0.1 to 100 mg/kg per day, more generally 0.5
to about 25 mg per kilogram body weight of the recipient/patient
per day. A typical topical dosage will range from 0.01-5% wt/wt in
a suitable carrier.
[0115] The compound is conveniently administered in any suitable
unit dosage form, including but not limited to one containing less
than 1 mg, 1 mg to 3000 mg, preferably 5 to 500 mg of active
ingredient per unit dosage form. An oral dosage of about 25-250 mg
is often convenient.
[0116] The active ingredient is preferably administered to achieve
peak plasma concentrations of the active compound of about
0.00001-30 mM, preferably about 0.1-30 .mu.M. This may be achieved,
for example, by the intravenous injection of a solution or
formulation of the active ingredient, optionally in saline, or an
aqueous medium or administered as a bolus of the active ingredient.
Oral administration is also appropriate to generate effective
plasma concentrations of active agent.
[0117] The concentration of active compound in the drug composition
will depend on absorption, distribution, inactivation, and
excretion rates of the drug as well as other factors known to those
of skill in the art. It is to be noted that dosage values will also
vary with the severity of the condition to be alleviated. It is to
be further understood that for any particular subject, specific
dosage regimens should be adjusted over time according to the
individual need and the professional judgment of the person
administering or supervising the administration of the
compositions, and that the concentration ranges set forth herein
are exemplary only and are not intended to limit the scope or
practice of the claimed composition. The active ingredient may be
administered at once, or may be divided into a number of smaller
doses to be administered at varying intervals of time.
[0118] Oral compositions will generally include an inert diluent or
an edible carrier. They may be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound or its prodrug derivative can
be incorporated with excipients and used in the form of tablets,
troches, or capsules. Pharmaceutically compatible binding agents,
and/or adjuvant materials can be included as part of the
composition.
[0119] The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth
or gelatin; an excipient such as starch or lactose, a dispersing
agent such as alginic acid, Primogel, or corn starch; a lubricant
such as magnesium stearate or Sterotes; a glidant such as colloidal
silicon dioxide; a sweetening agent such as sucrose or saccharin;
or a flavoring agent such as peppermint, methyl salicylate, or
orange flavoring. When the dosage unit form is a capsule, it can
contain, in addition to material of the above type, a liquid
carrier such as a fatty oil. In addition, dosage unit forms can
contain various other materials which modify the physical form of
the dosage unit, for example, coatings of sugar, shellac, or
enteric agents.
[0120] The active compound or pharmaceutically acceptable salt
thereof can be administered as a component of an elixir,
suspension, syrup, wafer, chewing gum or the like. A syrup may
contain, in addition to the active compounds, sucrose as a
sweetening agent and certain preservatives, dyes and colorings and
flavors.
[0121] The active compound or pharmaceutically acceptable salts
thereof can also be mixed with other active materials that do not
impair the desired action, or with materials that supplement the
desired action, such as erythropoietin stimulating agents,
including EPO and darbapoietin alfa, among others. In certain
preferred aspects of the disclosure, one or more compounds
according to the present disclosure are co-administered with
another bioactive agent, such as an erythropoietin stimulating
agent or would healing agent, including an antibiotic, as otherwise
described herein.
[0122] Solutions or suspensions used for parenteral, intradermal,
subcutaneous, or topical application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose. The parental preparation can be
enclosed in ampoules, disposable syringes or multiple dose vials
made of glass or plastic.
[0123] If administered intravenously, preferred carriers are
physiological saline or phosphate buffered saline (PBS).
[0124] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art.
[0125] Liposomal suspensions may also be pharmaceutically
acceptable carriers. These may be prepared according to methods
known to those skilled in the art, for example, as described in
U.S. Pat. No. 4,522,811 (which is incorporated herein by reference
in its entirety). For example, liposome formulations may be
prepared by dissolving appropriate lipid(s) (such as stearoyl
phosphatidyl ethanolamine, stearoyl phosphatidyl choline,
arachadoyl phosphatidyl choline, and cholesterol) in an inorganic
solvent that is then evaporated, leaving behind a thin film of
dried lipid on the surface of the container. An aqueous solution of
the active compound are then introduced into the container. The
container is then swirled by hand to free lipid material from the
sides of the container and to disperse lipid aggregates, thereby
forming the liposomal suspension.
Therapeutic Methods
[0126] In an additional aspect, the description provides
therapeutic compositions comprising an effective amount of a
compound as described herein or salt form thereof, and a
pharmaceutically acceptable carrier. The therapeutic compositions
modulate protein degradation in a patient or subject, for example,
an animal such as a human, and can be used for treating or
ameliorating disease states or conditions which are modulated
through the degraded protein.
[0127] The terms "treat", "treating", and "treatment", etc., as
used herein, refer to any action providing a benefit to a patient
for which the present compounds may be administered, including the
treatment of any disease state or condition which is modulated
through the protein to which the present compounds bind. Disease
states or conditions, including cancer, which may be treated using
compounds according to the present disclosure are set forth
hereinabove.
[0128] The description provides therapeutic compositions as
described herein for effectuating the degradation of proteins of
interest for the treatment or amelioration of a disease, e.g.,
cancer. In certain additional embodiments, the disease is multiple
myeloma. As such, in another aspect, the description provides a
method of ubiquitinating/degrading a target protein in a cell. In
certain embodiments, the method comprises administering a
bifunctional compound as described herein comprising, e.g., a ILM
and a PTM, preferably linked through a linker moiety, as otherwise
described herein, wherein the ILM is coupled to the PTM and wherein
the ILM recognizes a ubiquitin pathway protein (e.g., an ubiquitin
ligase, preferably an E3 ubiquitin ligase such as, e.g., cereblon)
and the PTM recognizes the target protein such that degradation of
the target protein will occur when the target protein is placed in
proximity to the ubiquitin ligase, thus resulting in
degradation/inhibition of the effects of the target protein and the
control of protein levels. The control of protein levels afforded
by the present disclosure provides treatment of a disease state or
condition, which is modulated through the target protein by
lowering the level of that protein in the cell, e.g., cell of a
patient. In certain embodiments, the method comprises administering
an effective amount of a compound as described herein, optionally
including a pharamaceutically acceptable excipient, carrier,
adjuvant, another bioactive agent or combination thereof.
[0129] In additional embodiments, the description provides methods
for treating or emeliorating a disease, disorder or symptom thereof
in a subject or a patient, e.g., an animal such as a human,
comprising administering to a subject in need thereof a composition
comprising an effective amount, e.g., a therapeutically effective
amount, of a compound as described herein or salt form thereof, and
a pharmaceutically acceptable excipient, carrier, adjuvant, another
bioactive agent or combination thereof, wherein the composition is
effective for treating or ameliorating the disease or disorder or
symptom thereof in the subject.
[0130] In another aspect, the description provides methods for
identifying the effects of the degradation of proteins of interest
in a biological system using compounds according to the present
disclosure.
[0131] In another embodiment, the present disclosure is directed to
a method of treating a human patient in need for a disease state or
condition modulated through a protein where the degradation of that
protein will produce a therapeutic effect in that patient, the
method comprising administering to a patient in need an effective
amount of a compound according to the present disclosure,
optionally in combination with another bioactive agent. The disease
state or condition may be a disease caused by a microbial agent or
other exogenous agent such as a virus, bacteria, fungus, protozoa
or other microbe or may be a disease state, which is caused by
overexpression of a protein, which leads to a disease state and/or
condition
[0132] The term "disease state or condition" is used to describe
any disease state or condition wherein protein dysregulation (i.e.,
the amount of protein expressed in a patient is elevated) occurs
and where degradation of one or more proteins in a patient may
provide beneficial therapy or relief of symptoms to a patient in
need thereof. In certain instances, the disease state or condition
may be cured.
[0133] Disease states of conditions which may be treated using
compounds according to the present disclosure include, for example,
asthma, autoimmune diseases such as multiple sclerosis, various
cancers, ciliopathies, cleft palate, diabetes, heart disease,
hypertension, inflammatory bowel disease, mental retardation, mood
disorder, obesity, refractive error, infertility, Angelman
syndrome, Canavan disease, Coeliac disease, Charcot-Marie-Tooth
disease, Cystic fibrosis, Duchenne muscular dystrophy,
Haemochromatosis, Haemophilia, Klinefelter's syndrome,
Neurofibromatosis, Phenylketonuria, Polycystic kidney disease,
(PKD1) or 4 (PKD2) Prader-Willi syndrome, Sickle-cell disease,
Tay-Sachs disease, Turner syndrome.
[0134] Further disease states or conditions which may be treated by
compounds according to the present disclosure include Alzheimer's
disease, Amyotrophic lateral sclerosis (Lou Gehrig's disease),
Anorexia nervosa, Anxiety disorder, Atherosclerosis, Attention
deficit hyperactivity disorder, Autism, Bipolar disorder, Chronic
fatigue syndrome, Chronic obstructive pulmonary disease, Crohn's
disease, Coronary heart disease, Dementia, Depression, Diabetes
mellitus type 1, Diabetes mellitus type 2, Epilepsy, Guillain-Barre
syndrome, Irritable bowel syndrome, Lupus, Metabolic syndrome,
Multiple sclerosis, Myocardial infarction, Obesity,
Obsessive-compulsive disorder, Panic disorder, Parkinson's disease,
Psoriasis, Rheumatoid arthritis, Sarcoidosis, Schizophrenia,
Stroke, Thromboangiitis obliterans, Tourette syndrome,
Vasculitis.
[0135] Still additional disease states or conditions which can be
treated by compounds according to the present disclosure include
aceruloplasminemia, Achondrogenesis type II, achondroplasia,
Acrocephaly, Gaucher disease type 2, acute intermittent porphyria,
Canavan disease, Adenomatous Polyposis Coli, ALA dehydratase
deficiency, adenylosuccinate lyase deficiency, Adrenogenital
syndrome, Adrenoleukodystrophy, ALA-D porphyria, ALA dehydratase
deficiency, Alkaptonuria, Alexander disease, Alkaptonuric
ochronosis, alpha 1-antitrypsin deficiency, alpha-1 proteinase
inhibitor, emphysema, amyotrophic lateral sclerosis Alstrom
syndrome, Alexander disease, Amelogenesis imperfecta, ALA
dehydratase deficiency, Anderson-Fabry disease, androgen
insensitivity syndrome, Anemia Angiokeratoma Corporis Diffusum,
Angiomatosis retinae (von Hippel-Lindau disease) Apert syndrome,
Arachnodactyly (Marfan syndrome), Stickler syndrome, Arthrochalasis
multiplex congenital (Ehlers-Danlos syndrome#arthrochalasia type)
ataxia telangiectasia, Rett syndrome, primary pulmonary
hypertension, Sandhoff disease, neurofibromatosis type II,
Beare-Stevenson cutis gyrata syndrome, Mediterranean fever,
familial, Benjamin syndrome, beta-thalassemia, Bilateral Acoustic
Neurofibromatosis (neurofibromatosis type II), factor V Leiden
thrombophilia, Bloch-Sulzberger syndrome (incontinentia pigmenti),
Bloom syndrome, X-linked sideroblastic anemia, Bonnevie-Ullrich
syndrome (Turner syndrome), Bourneville disease (tuberous
sclerosis), prion disease, Birt-Hogg-Dube syndrome, Brittle bone
disease (osteogenesis imperfecta), Broad Thumb-Hallux syndrome
(Rubinstein-Taybi syndrome), Bronze Diabetes/Bronzed Cirrhosis
(hemochromatosis), Bulbospinal muscular atrophy (Kennedy's
disease), Burger-Grutz syndrome (lipoprotein lipase deficiency),
CGD Chronic granulomatous disorder, Campomelic dysplasia,
biotinidase deficiency, Cardiomyopathy (Noonan syndrome), Cri du
chat, CAVD (congenital absence of the vas deferens), Caylor
cardiofacial syndrome (CBAVD), CEP (congenital erythropoietic
porphyria), cystic fibrosis, congenital hypothyroidism,
Chondrodystrophy syndrome (achondroplasia),
otospondylomegaepiphyseal dysplasia, Lesch-Nyhan syndrome,
galactosemia, Ehlers-Danlos syndrome, Thanatophoric dysplasia,
Coffin-Lowry syndrome, Cockayne syndrome, (familial adenomatous
polyposis), Congenital erythropoietic porphyria, Congenital heart
disease, Methemoglobinemia/Congenital methaemoglobinaemia,
achondroplasia, X-linked sideroblastic anemia, Connective tissue
disease, Conotruncal anomaly face syndrome, Cooley's Anemia
(beta-thalassemia), Copper storage disease (Wilson's disease),
Copper transport disease (Menkes disease), hereditary
coproporphyria, Cowden syndrome, Craniofacial dysarthrosis (Crouzon
syndrome), Creutzfeldt-Jakob disease (prion disease), Cockayne
syndrome, Cowden syndrome, Curschmann-Batten-Steinert syndrome
(myotonic dystrophy), Beare-Stevenson cutis gyrata syndrome,
primary hyperoxaluria, spondyloepimetaphyseal dysplasia (Strudwick
type), muscular dystrophy, Duchenne and Becker types (DBMD), Usher
syndrome, Degenerative nerve diseases including de Grouchy syndrome
and Dejerine-Sottas syndrome, developmental disabilities, distal
spinal muscular atrophy, type V, androgen insensitivity syndrome,
Diffuse Globoid Body Sclerosis (Krabbe disease), Di George's
syndrome, Dihydrotestosterone receptor deficiency, androgen
insensitivity syndrome, Down syndrome, Dwarfism, erythropoietic
protoporphyria Erythroid 5-aminolevulinate synthetase deficiency,
Erythropoietic porphyria, erythropoietic protoporphyria,
erythropoietic uroporphyria, Friedreich's ataxia, familial
paroxysmal polyserositis, porphyria cutanea tarda, familial
pressure sensitive neuropathy, primary pulmonary hypertension
(PPH), Fibrocystic disease of the pancreas, fragile X syndrome,
galactosemia, genetic brain disorders, Giant cell hepatitis
(Neonatal hemochromatosis), Gronblad-Strandberg syndrome
(pseudoxanthoma elasticum), Gunther disease (congenital
erythropoietic porphyria), haemochromatosis, Hallgren syndrome,
sickle cell anemia, hemophilia, hepatoerythropoietic porphyria
(HEP), Hippel-Lindau disease (von Hippel-Lindau disease),
Huntington's disease, Hutchinson-Gilford progeria syndrome
(progeria), Hyperandrogenism, Hypochondroplasia, Hypochromic
anemia, Immune system disorders, including X-linked severe combined
immunodeficiency, Insley-Astley syndrome, Jackson-Weiss syndrome,
Joubert syndrome, Lesch-Nyhan syndrome, Jackson-Weiss syndrome,
Kidney diseases, including hyperoxaluria, Klinefelter's syndrome,
Kniest dysplasia, Lacunar dementia, Langer-Saldino achondrogenesis,
ataxia telangiectasia, Lynch syndrome, Lysyl-hydroxylase
deficiency, Machado-Joseph disease, Metabolic disorders, including
Kniest dysplasia, Marfan syndrome, Movement disorders, Mowat-Wilson
syndrome, cystic fibrosis, Muenke syndrome, Multiple
neurofibromatosis, Nance-Insley syndrome, Nance-Sweeney
chondrodysplasia, Niemann-Pick disease, Noack syndrome (Pfeiffer
syndrome), Osler-Weber-Rendu disease, Peutz-Jeghers syndrome,
Polycystic kidney disease, polyostotic fibrous dysplasia
(McCune-Albright syndrome), Peutz-Jeghers syndrome,
Prader-Labhart-Willi syndrome, hemochromatosis, primary
hyperuricemia syndrome (Lesch-Nyhan syndrome), primary pulmonary
hypertension, primary senile degenerative dementia, prion disease,
progeria (Hutchinson Gilford Progeria Syndrome), progressive
chorea, chronic hereditary (Huntington) (Huntington's disease),
progressive muscular atrophy, spinal muscular atrophy, propionic
acidemia, protoporphyria, proximal myotonic dystrophy, pulmonary
arterial hypertension, PXE (pseudoxanthoma elasticum), Rb
(retinoblastoma), Recklinghausen disease (neurofibromatosis type
I), Recurrent polyserositis, Retinal disorders, Retinoblastoma,
Rett syndrome, RFALS type 3, Ricker syndrome, Riley-Day syndrome,
Roussy-Levy syndrome, severe achondroplasia with developmental
delay and acanthosis nigricans (SADDAN), Li-Fraumeni syndrome,
sarcoma, breast, leukemia, and adrenal gland (SBLA) syndrome,
sclerosis tuberose (tuberous sclerosis), SDAT, SED congenital
(spondyloepiphyseal dysplasia congenita), SED Strudwick
(spondyloepimetaphyseal dysplasia, Strudwick type), SEDc
(spondyloepiphyseal dysplasia congenita) SEMD, Strudwick type
(spondyloepimetaphyseal dysplasia, Strudwick type), Shprintzen
syndrome, Skin pigmentation disorders, Smith-Lemli-Opitz syndrome,
South-African genetic porphyria (variegate porphyria),
infantile-onset ascending hereditary spastic paralysis, Speech and
communication disorders, sphingolipidosis, Tay-Sachs disease,
spinocerebellar ataxia, Stickler syndrome, stroke, androgen
insensitivity syndrome, tetrahydrobiopterin deficiency,
beta-thalassemia, Thyroid disease, Tomaculous neuropathy
(hereditary neuropathy with liability to pressure palsies),
Treacher Collins syndrome, Triplo X syndrome (triple X syndrome),
Trisomy 21 (Down syndrome), Trisomy X, VHL syndrome (von
Hippel-Lindau disease), Vision impairment and blindness (Alstrom
syndrome), Vrolik disease, Waardenburg syndrome, Warburg Sjo
Fledelius Syndrome, Weis senbacher-Zweymuller syndrome,
Wolf-Hirschhorn syndrome, Wolff Periodic disease, Weis
senbacher-Zweymuller syndrome and Xeroderma pigmentosum, among
others.
[0136] The term "neoplasia" or "cancer" is used throughout the
specification to refer to the pathological process that results in
the formation and growth of a cancerous or malignant neoplasm,
i.e., abnormal tissue that grows by cellular proliferation, often
more rapidly than normal and continues to grow after the stimuli
that initiated the new growth cease. Malignant neoplasms show
partial or complete lack of structural organization and functional
coordination with the normal tissue and most invade surrounding
tissues, metastasize to several sites, and are likely to recur
after attempted removal and to cause the death of the patient
unless adequately treated. As used herein, the term neoplasia is
used to describe all cancerous disease states and embraces or
encompasses the pathological process associated with malignant
hematogenous, ascitic and solid tumors. Exemplary cancers which may
be treated by the present compounds either alone or in combination
with at least one additional anti-cancer agent include
squamous-cell carcinoma, basal cell carcinoma, adenocarcinoma,
hepatocellular carcinomas, and renal cell carcinomas, cancer of the
bladder, bowel, breast, cervix, colon, esophagus, head, kidney,
liver, lung, neck, ovary, pancreas, prostate, and stomach;
leukemias; benign and malignant lymphomas, particularly Burkitt's
lymphoma and Non-Hodgkin's lymphoma; benign and malignant
melanomas; myeloproliferative diseases; sarcomas, including Ewing's
sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma,
myosarcomas, peripheral neuroepithelioma, synovial sarcoma,
gliomas, astrocytomas, oligodendrogliomas, ependymomas,
gliobastomas, neuroblastomas, ganglioneuromas, gangliogliomas,
medulloblastomas, pineal cell tumors, meningiomas, meningeal
sarcomas, neurofibromas, and Schwannomas; bowel cancer, breast
cancer, prostate cancer, cervical cancer, uterine cancer, lung
cancer, ovarian cancer, testicular cancer, thyroid cancer,
astrocytoma, esophageal cancer, pancreatic cancer, stomach cancer,
liver cancer, colon cancer, melanoma; carcinosarcoma, Hodgkin's
disease, Wilms' tumor and teratocarcinomas. Additional cancers
which may be treated using compounds according to the present
disclosure include, for example, T-lineage Acute lymphoblastic
Leukemia (T-ALL), T-lineage lymphoblastic Lymphoma (T-LL),
Peripheral T-cell lymphoma, Adult T-cell Leukemia, Pre-B ALL, Pre-B
Lymphomas, Large B-cell Lymphoma, Burkitts Lymphoma, B-cell ALL,
Philadelphia chromosome positive ALL and Philadelphia chromosome
positive CML.
[0137] The term "bioactive agent" is used to describe an agent,
other than a compound according to the present disclosure, which is
used in combination with the present compounds as an agent with
biological activity to assist in effecting an intended therapy,
inhibition and/or prevention/prophylaxis for which the present
compounds are used. Preferred bioactive agents for use herein
include those agents which have pharmacological activity similar to
that for which the present compounds are used or administered and
include for example, anti-cancer agents, antiviral agents,
especially including anti-HIV agents and anti-HCV agents,
antimicrobial agents, antifungal agents, etc.
[0138] The term "additional anti-cancer agent" is used to describe
an anti-cancer agent, which may be combined with compounds
according to the present disclosure to treat cancer. These agents
include, for example, everolimus, trabectedin, abraxane, TLK 286,
AV-299, DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244
(ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin,
vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263,
a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an
aurora kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an
HDAC inhbitor, a c-MET inhibitor, a PARP inhibitor, a Cdk
inhibitor, an EGFR TK inhibitor, an IGFR-TK inhibitor, an anti-HGF
antibody, a PI3 kinase inhibitor, an AKT inhibitor, an mTORC1/2
inhibitor, a JAK/STAT inhibitor, a checkpoint-1 or 2 inhibitor, a
focal adhesion kinase inhibitor, a Map kinase kinase (mek)
inhibitor, a VEGF trap antibody, pemetrexed, erlotinib, dasatanib,
nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Lep-etu,
nolatrexed, azd2171, batabulin, ofatumumab, zanolimumab,
edotecarin, tetrandrine, rubitecan, tesmilifene, oblimersen,
ticilimumab, ipilimumab, gossypol, Bio 111, 131-I-TM-601, ALT-110,
BIO 140, CC 8490, cilengitide, gimatecan, IL13-PE38QQR, INO 1001,
IPdR.sub.1 KRX-0402, lucanthone, LY317615, neuradiab, vitespan, Rta
744, Sdx 102, talampanel, atrasentan, Xr 311, romidepsin,
ADS-100380, sunitinib, 5-fluorouracil, vorinostat, etoposide,
gemcitabine, doxorubicin, liposomal doxorubicin,
5'-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709,
seliciclib; PD0325901, AZD-6244, capecitabine, L-Glutamic acid,
N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-
benzoyl]-, disodium salt, heptahydrate, camptothecin, PEG-labeled
irinotecan, tamoxifen, toremifene citrate, anastrazole, exemestane,
letrozole, DES(diethylstilbestrol), estradiol, estrogen, conjugated
estrogen, bevacizumab, IMC-1C11, CHIR-258);
3-[5-(methylsulfonylpiperadinemethyl)-indolyl-quinolone, vatalanib,
AG-013736, AVE-0005, goserelin acetate, leuprolide acetate,
triptorelin pamoate, medroxyprogesterone acetate,
hydroxyprogesterone caproate, megestrol acetate, raloxifene,
bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714;
TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF
antibody, erbitux, EKB-569, PKI-166, GW-572016, Ionafarnib,
BMS-214662, tipifarnib; amifostine, NVP-LAQ824, suberoyl analide
hydroxamic acid, valproic acid, trichostatin A, FK-228, SU11248,
sorafenib, KRN951, aminoglutethimide, arnsacrine, anagrelide,
L-asparaginase, Bacillus Calmette-Guerin (BCG) vaccine, adriamycin,
bleomycin, buserelin, busulfan, carboplatin, carmustine,
chlorambucil, cisplatin, cladribine, clodronate, cyproterone,
cytarabine, dacarbazine, dactinomycin, daunorubicin,
diethylstilbestrol, epirubicin, fludarabine, fludrocortisone,
fluoxymesterone, flutamide, gleevec, gemcitabine, hydroxyurea,
idarubicin, ifosfamide, imatinib, leuprolide, levamisole,
lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesna,
methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide,
octreotide, oxaliplatin, pamidronate, pentostatin, plicamycin,
porfimer, procarbazine, raltitrexed, rituximab, streptozocin,
teniposide, testosterone, thalidomide, thioguanine, thiotepa,
tretinoin, vindesine, 13-cis-retinoic acid, phenylalanine mustard,
uracil mustard, estramustine, altretamine, floxuridine,
5-deooxyuridine, cytosine arabinoside, 6-mecaptopurine,
deoxycoformycin, calcitriol, valrubicin, mithramycin, vinblastine,
vinorelbine, topotecan, razoxin, marimastat, COL-3, neovastat,
BMS-275291, squalamine, endostatin, SU5416, SU6668, EMD121974,
interleukin-12, IM862, angiostatin, vitaxin, droloxifene,
idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab,
denileukin diftitox, gefitinib, bortezimib, paclitaxel,
cremophor-free paclitaxel, docetaxel, epithilone B, BMS-247550,
BMS-310705, droloxifene, 4-hydroxytamoxifen, pipendoxifene,
ERA-923, arzoxifene, fulvestrant, acolbifene, lasofoxifene,
idoxifene, TSE-424, HMR-3339, ZK186619, topotecan, PTK787/ZK
222584, VX-745, PD 184352, rapamycin,
40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001,
ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646,
wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin,
erythropoietin, granulocyte colony-stimulating factor,
zolendronate, prednisone, cetuximab, granulocyte macrophage
colony-stimulating factor, histrelin, pegylated interferon alfa-2a,
interferon alfa-2a, pegylated interferon alfa-2b, interferon
alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab,
hydrocortisone, interleukin-11, dexrazoxane, alemtuzumab,
all-transretinoic acid, ketoconazole, interleukin-2, megestrol,
immune globulin, nitrogen mustard, methylprednisolone, ibritgumomab
tiuxetan, androgens, decitabine, hexamethylmelamine, bexarotene,
tositumomab, arsenic trioxide, cortisone, editronate, mitotane,
cyclosporine, liposomal daunorubicin, Edwina-asparaginase,
strontium 89, casopitant, netupitant, an NK-1 receptor antagonist,
palonosetron, aprepitant, diphenhydramine, hydroxyzine,
metoclopramide, lorazepam, alprazolam, haloperidol, droperidol,
dronabinol, dexamethasone, methylprednisolone, prochlorperazine,
granisetron, ondansetron, dolasetron, tropisetron, pegfilgrastim,
erythropoietin, epoetin alfa, darbepoetin alfa and mixtures
thereof.
[0139] The term "anti-HIV agent" or "additional anti-HIV agent"
includes, for example, nucleoside reverse transcriptase inhibitors
(NRTI), other non-nucloeoside reverse transcriptase inhibitors
(i.e., those which are not representative of the present
disclosure), protease inhibitors, fusion inhibitors, among others,
exemplary compounds of which may include, for example, 3TC
(Lamivudine), AZT (Zidovudine), (-)-FTC, ddI (Didanosine), ddC
(zalcitabine), abacavir (ABC), tenofovir (PMPA), D-D4FC (Reverset),
D4T (Stavudine), Racivir, L-FddC, L-FD4C, NVP (Nevirapine), DLV
(Delavirdine), EFV (Efavirenz), SQVM (Saquinavir mesylate), RTV
(Ritonavir), IDV (Indinavir), SQV (Saquinavir), NFV (Nelfinavir),
APV (Amprenavir), LPV (Lopinavir), fusion inhibitors such as T20,
among others, fuseon and mixtures thereof, including anti-HIV
compounds presently in clinical trials or in development.
[0140] Other anti-HIV agents which may be used in coadministration
with compounds according to the present disclosure include, for
example, other NNRTI's (i.e., other than the NNRTI's according to
the present disclosure) may be selected from the group consisting
of nevirapine (BI-R6-587), delavirdine (U-90152S/T), efavirenz
(DMP-266), UC-781
(N-[4-chloro-3-(3-methyl-2-butenyloxy)phenyl]-2methyl3-furancarbothiamide-
), etravirine (TMC 125), Trovirdine (Ly300046.HCl), MKC-442
(emivirine, coactinon), HI-236, HI-240, HI-280, HI-281, rilpivirine
(TMC-278), MSC-127, HBY 097, DMP266, Baicalin (TJN-151) ADAM-II
(Methyl
3',3'-dichloro-4',4''-dimethoxy-5',5''-bis(methoxycarbonyl)-6,6-diphenylh-
exenoate), Methyl
3-Bromo-5-(1-5-bromo-4-methoxy-3-(methoxycarbonyl)phenyl)hept-1-enyl)-2-m-
ethoxybenzoate (Alkenyldiarylmethane analog, Adam analog),
(5-chloro-3-(phenylsulfinyl)-2'-indolecarboxamide), AAP-BHAP
(U-104489 or PNU-104489), Capravirine (AG-1549, S-1153), atevirdine
(U-87201E), aurin tricarboxylic acid (SD-095345),
1-[(6-cyano-2-indolyl)carbonyl]-4-[3-(isopropylamino)-2-pyridinyl]piperaz-
ine,
1-[5-[[N-(methyl)methylsulfonylamino]-2-indolylcarbonyl-4-[3-(isoprop-
ylamino)-2-pyridinyl]piperazine,
1-[3-(Ethylamino)-2-[pyridinyl]-4-[(5-hydroxy-2-indolyl)carbonyl]piperazi-
ne,
1-[(6-Formyl-2-indolyl)carbonyl]-4-[3-(isopropylamino)-2-pyridinyl]pip-
erazine,
1-[[5-(Methylsulfonyloxy)-2-indoyly)carbonyl]-4-[3-(isopropylamin-
o)-2-pyridinyl]piperazine, U88204E, Bis(2-nitrophenyl)sulfone (NSC
633001), Calanolide A (NSC675451), Calanolide B,
6-Benzyl-5-methyl-2-(cyclohexyloxy)pyrimidin-4-one (DABO-546), DPC
961, E-EBU, E-EBU-dm, E-EPSeU, E-EPU, Foscarnet (Foscavir), HEPT
(1-[(2-Hydroxyethoxy)methyl]-6-(phenylthio)thymine), HEPT-M
(1-[(2-Hydroxyethoxy)methyl]-6-(3-methylphenyl)thio)thymine),
HEPT-S(1-[(2-Hydroxyethoxy)methyl]-6-(phenylthio)-2-thiothymine),
Inophyllum P, L-737,126, Michellamine A (NSC650898), Michellamine B
(NSC649324), Michellamine F,
6-(3,5-Dimethylbenzyl)-1-[(2-hydroxyethoxy)methyl]-5-isopropyluracil,
6-(3,5-Dimethylbenzyl)-1-(ethyoxymethyl)-5-isopropyluracil, NPPS,
E-BPTU (NSC 648400), Oltipraz
(4-Methyl-5-(pyrazinyl)-3H-1,2-dithiole-3-thione),
N-{2-(2-Chloro-6-fluorophenethyl]-N'-(2-thiazolyl)thiourea (PETT
Cl, F derivative),
N-{2-(2,6-Difluorophenethyl]-N'-[2-(5-bromopyridyl)]thiourea {PETT
derivative),
N-{2-(2,6-Difluorophenethyl]-N'-[2-(5-methylpyridyl)]thiourea {PETT
Pyridyl derivative),
N-[2-(3-Fluorofuranyl)ethyl]-N'-[2-(5-chloropyridyl)]thiourea,
N-[2-(2-Fluoro-6-ethoxyphenethyl)]-N'-[2-(5-bromopyridyl)]thiourea,
N-(2-Phenethyl)-N'-(2-thiazolyl)thiourea (LY-73497), L-697,639,
L-697,593, L-697,661,
342-(4,7-Difluorobenzoxazol-2-yl)ethyl}-5-ethyl-6-methyl(pypridin-2(1H)-t-
hione (2-Pyridinone Derivative),
3-[[(2-Methoxy-5,6-dimethyl-3-pyridyl)methyl]amine]-5-ethyl-6-methyl(pypr-
idin-2(1H)-thione, R82150, R82913, R87232, R88703, R89439
(Loviride), R90385, S-2720, Suramin Sodium, TBZ
(Thiazolobenzimidazole, NSC 625487), Thiazoloisoindol-5-one,
(+)(R)-9b-(3,5-Dimethylphenyl-2,3-dihydrothiazolo
[2,3-a]isoindol-5(9bH)-one, Tivirapine (R86183), UC-38 and UC-84,
among others.
[0141] The term "pharmaceutically acceptable salt" is used
throughout the specification to describe, where applicable, a salt
form of one or more of the compounds described herein which are
presented to increase the solubility of the compound in the gastic
juices of the patient's gastrointestinal tract in order to promote
dissolution and the bioavailability of the compounds.
Pharmaceutically acceptable salts include those derived from
pharmaceutically acceptable inorganic or organic bases and acids,
where applicable. Suitable salts include those derived from alkali
metals such as potassium and sodium, alkaline earth metals such as
calcium, magnesium and ammonium salts, among numerous other acids
and bases well known in the pharmaceutical art. Sodium and
potassium salts are particularly preferred as neutralization salts
of the phosphates according to the present disclosure.
[0142] The term "pharmaceutically acceptable derivative" is used
throughout the specification to describe any pharmaceutically
acceptable prodrug form (such as an ester, amide other prodrug
group), which, upon administration to a patient, provides directly
or indirectly the present compound or an active metabolite of the
present compound.
EXAMPLES
[0143] Embodiments of the present disclosure generate compounds
likely to induce protein-protein interactions inside cells for the
following protein pairs: AR with VHL, AR with cereblon, ER with
VHL, BRD4 with VHL, and BRD4 with cereblon. Molecular dynamics
simulations of the corresponding ternary systems and measurements
of compound potencies in the related cell-based assays, as
exemplified by the embodiments below, were performed and led to the
surprising and unexpected discovery of the method of the
disclosure. The following examples are intended to illustrate but
not to limit the disclosure in any manner, shape or form, either
explicitly or implicitly. While they are typical of those that
might be used, other procedures, methodologies, or techniques known
to those skilled in the art may alternatively be used.
[0144] Examination of Tripartite Ligand AR-C-VHL.
[0145] A tripartite ligand L with the following structure was
examined:
##STR00001##
[0146] It is composed of an AR-binding warhead which is connected
to a VHL-binding warhead by a linear connector. This compound was
incubated with VCaP cells which have significant expression of AR
and VHL E3 ligase system. The experimental outcome indicated that
the compound caused AR degradation with an IC.sub.50 of 0.688 nM.
The compound's (i.e., the ligand's) potency was much greater than
the estimated binary binding potencies. The binary binding potency
of the ligand L to AR was 342 nM based on a separate cell-based
assay measuring displacement of a known AR ligand R1881 by the
compound. The binary binding potency of the ligand L to VHL was
estimated to be weaker than 3960 nM in the cells because the
corresponding IC.sub.50 in a biochemical binding assay was 3960 nM
and the membrane permeability of the compound was shown to be very
low. The ratio between the smaller binary binding IC.sub.50s and
the ternary binding IC.sub.50 was about 497-fold. MD simulation of
a ternary system composed of AR ligand-binding domain, a ligand and
the complex of VHL with elongin B (EB) and elongin C (EC) in the
presence of explicit water was performed. The ligand in the
simulation is a close analog of the ligand L above and only differs
from the ligand L above by having a methyl substitution at the
4-position of the thiazole group. The simulation led to a dynamic
trajectory with substantial protein-protein interactions. The
protein-protein interactions add extra binding energy on top of the
amounts that stem from the ligand warheads in binding to the
respective targets, and thus contribute to the superior/enhanced
protein degradation potency relative to the binary binding
potencies. This example reveals a way to induce protein-protein
interactions via a tripartite or bi-functional ligand and achieve
superior or enhanced binding potency and therefore, protein
degradation.
[0147] The representative conformation derived from the MD
simulation of the ternary complex of the AR ligand-binding domain,
the ligand and the complex of VHL with EB and EC revealed that the
molecular surface of AR merged with that of VHL so that the two
proteins formed a collective binding site for the ligand. See FIG.
3. Such a ligand-induced trimer formation of AR.L.VHL caused a
large portion of the surface area, i.e. 1716.4 .ANG..sup.2, to be
buried relative to the monomeric state, while the formation of a
binary complex of AR.L buried a surface area of about 694.7
.ANG..sup.2 and the formation of a binary complex of VHL.L buried a
surface area of about 547 .ANG..sup.2. The amount of the surface
area buried due to the trimer AR.L.VHL was more than the combined
amounts of the surface area buried due to the separate dimers AR.L
and L.VHL by about 474.7 .ANG..sup.2. The complex of VHL, EB and EC
is considered as one entity and collectively called "VHL" in the
descriptions concerning MD simulation in this and other examples.
The cross-domain interactions stabilizing the trimer complex, shown
in FIG. 4, included an ion-pair between Asp879 of AR and Arg108 of
VHL, a hydrophobic cluster composed of Phe697, Ala698 and Leu880 of
AR with Pro71, His110, Tyr112 and the nonpolar part of Arg69 of
VHL, and a second hydrophobic cluster composed of Ile882 of AR,
Tyr98 and Pro99 of VHL and the phenyl-thiazole group of the ligand.
The C-terminal end of a a-helix of AR docked onto the
phenyl-thiazole part of the ligand. In addition, a water molecule
formed stable bridging interactions by forming a trifurcating
hydrogen bond with the backbone carbonyl of Leu880 of AR, the side
chain phenol of Tyr112 of VHL and the backbone carbonyl of the
t-butyl glycine of the ligand. The connector of the ligand
interacted with both proteins. A hydrogen bond between the backbone
amide of Ala698 of AR and the backbone carbonyl of Arg69 of VHL was
seen in the representative conformation. However, this hydrogen
bond underwent disappearing/reappearing cycles during the dynamics
trajectory, which could be defined as a transient hydrogen
bond.
[0148] Examination of Tripartite Ligand BRD4-C-VHL.
[0149] A tripartite ligand L with the following structure was
examined:
##STR00002##
[0150] It is composed of a BRD4-binding warhead which is connected
to a VHL-binding warhead by a linear connector. This compound was
incubated with 22RV1 cells, which have significant expression of
BRD4 and VHL E3 ligase system. Either the inhibition of BRD4 (by
binding and blocking the active site of BRD4) or the degradation of
BRD4 leads to the down-regulation of the expression level of a
downstream protein c-Myc. The experimental outcome in an ELISA
assay demonstrated that the compound down-regulated the c-Myc
expression with an IC.sub.50 of 0.305 nM. A Western blot assay
confirmed that the degradation potency DC.sub.50 of this compound
with respect to BRD4 was less than 1 nM. A decoy compound which
differs from the ligand L above by having inverse chirality at c1
and c2 positions, and thus lack VHL-binding capability, showed an
IC.sub.50 greater than 1000 nM in c-Myc expression. Since the decoy
compound has no VHL-binding capability while retaining the
BRD4-binding capability, the effect produced by the decoy compound
should be attributed to the binary binding to BRD4. Therefore, it
can be inferred that the binary binding potency of ligand L to BRD4
is greater than 1000 nM. The binary binding potency of the L to VHL
was 3960 nM in a biochemical assay suggesting that the
corresponding binary IC.sub.50 in the cell assay should be greater
than this value given the poor membrane permeability of this type
of compounds. The ratio between the smaller binary binding
IC.sub.50 and the ternary binding IC.sub.50 is greater than
3279-fold. MD simulation of a ternary system composed of BRD4
bromodomain 1, the ligand L and the complex of VHL with EB and EC
in the presence of explicit water was performed. The simulation
resulted in a dynamic trajectory with substantial protein-protein
interactions. The protein-protein interactions provided extra
stabilization energy to the ternary complex, and therefore,
contribute to the superior/enhanced protein degradation potency
relative to the binary binding potencies. As such, this is another
example that a tripartite or bi-functional ligand can induce
protein-protein interactions and achieve superior potency (e.g.,
binding and/or protein degradation).
[0151] The representative conformation derived from the MD
simulation of the ternary complex of the BRD4 bromodomain 1, the
ligand L' and VHL revealed that the molecular surface of BRD4
merged with that of VHL so that the two proteins formed a
collective binding site for the ligand. (Ligand L' differs from
Ligand L by not having a methyl group on the benzylic position of
the VHL ligand.) See FIG. 5. Such a ligand-induced trimer formation
of BRD4.L'.VHL caused a large portion of the surface area to be
buried, i.e. about 1760.9 .ANG..sup.2, relative to the monomeric
state, while the formation of a binary complex of BRD4.L' buried a
surface area of about 572.4 .ANG..sup.2 and the formation of a
binary complex of VHL.L' buried a surface area of about 660
.ANG..sup.2. The amount of the surface area buried due to the
trimer BRD4.L'.VHL was more than the combined amounts of the
surface area buried due to the separate dimers BRD4.L' and L'.VHL
by about 528.5 .ANG..sup.2. The cross-domain interactions
stabilizing the trimer complex (FIG. 6) included a hydrophobic
cluster contributed by Trp81, Val87, Leu92 and the dimethylthienyl
part of the BRD4-binding warhead from the BRD4 side and Pro71,
Tyr112 and His110 of VHL, and an ion-pair interaction between
Asp145 of BRD4 and Arg69 of VHL. The cross-domain interactions also
included the contributions from the connector which interacted with
both BRD4 and VHL.
[0152] Examination of the Length n of the Connector Linking an
AR-Binding Warhead and a VHL-Binding Warhead.
[0153] The connector linking the AR-binding warhead and the
VHL-binding warhead was systematically probed from 4 to 23 atoms.
The results are shown in Table 1. The AR degradation potency
DC.sub.50 and the maximum percentage of degradation D.sub.max of AR
in LNCaP cells were measured for each of these compounds. While
DC.sub.50 is the primary measure of the degradation potency,
D.sub.max provides a second measure in that a compound is
considered to be weak if its D.sub.max is not sufficiently high
(<60%) with respect to its DC.sub.50. The binary binding potency
with respect to AR was also measured for some of the compounds by
the cell-based ligand (R1881) displacement assay. The ratio .alpha.
between the AR-binding potency and the degradation potency are
calculated when it is possible. The results demonstrate that when n
ranges between 11 and 14 atom, the degradation potency reaches the
optimum and exhibits a superior binding/degradation potency over
the binary binding potencies. When n is gradually shortened from 11
atoms, the degradation potency declines until 8 atoms, where
degradation potency drops off. These results are consistent with
the notion of a stable trimer in which the connector needs to be
sufficiently long to span a defined distance. Otherwise, clashes
between the proteins or dislocation of the warhead(s) are expected.
When n is larger than the optimum range, the degradation potency
also decreased. Ligands with a larger n required that one protein
molecule to explore a larger volume of space translationally and
rotationally with respect to another protein molecule in order to
find the specific interaction surface(s), and as a result, the
formation of a protein-protein interaction complex is more
difficult due to the entropic effect. MD simulation on compounds 8
and 9 showed that these compounds induced similar trimer complexes
(complexes with protein-protein interactions). The representative
conformation of the trimer for compound 9 is shown in FIG. 7,
indicating that a connector of a length of 12 atoms is compatible
with the trimer conformation. The connector adopts a near-extended
conformation with few twists, consistent with the fact that
shortening the connector to 11 atoms long (compound 8) is
well-tolerated, but further shortening starts to destabilize the
trimer. The connector is not totally solvent-exposed, but binds to
the induced pocket formed by the two protein molecules, suggesting
that changing the connector length will change its interaction
energy with the protein molecules as well as its conformational
energy stemming from the chain twisting, in addition to the
entropic effect mentioned above.
##STR00003##
TABLE-US-00001 TABLE 1 Exploration of Connector Length n Linking an
AR-Binding Warhead and a VHL-Binding Warhead, Measurement of AR
Degradation Potency DC.sub.50, Maximum AR Degradation D.sub.max,
Binary Binding Potency IC.sub.50 to AR in cells and Calculation of
the Ratio .alpha. between DC.sub.50 and IC.sub.50. DC.sub.50
D.sub.max IC.sub.50.sup.AR ID Connector n (.mu.M) (%) (.mu.M)
.alpha. 1 ##STR00004## 4 >3 35 0.97 <1 2 ##STR00005## 5 1.7
51 2.14 <1 3 ##STR00006## 6 9.3 56 4 ##STR00007## 7 >3 18 5
##STR00008## 8 >3 38 2.08 <1 6 ##STR00009## 9 0.072 54 7
##STR00010## 10 0.39 56 8 ##STR00011## 11 0.0262 62 2.14 81.7 9
##STR00012## 12 0.0344 83 0.81 23.5 10 ##STR00013## 13 0.060 77 11
##STR00014## 14 0.011 60 1.15 105 12 ##STR00015## 16 >3 42 1.15
<1 13 ##STR00016## 17 >3 24 5.29 <1 14 ##STR00017## 18
0.22 52 4.95 15 ##STR00018## 19 0.029 58 3.48 16 ##STR00019## 20
3.0 90 1.75 0.58 17 ##STR00020## 21 >3 43 1.42 <1 18
##STR00021## 23 >3 47 0.96 <1
[0154] Examination of the Length n of the Connector Linking a
BRD4-Binding Warhead and a Cereblon-Binding Warhead.
[0155] The connector linking the BRD-4 binding warhead and the
cereblon-binding warhead was systematically probed from 2 to 8
atoms. The data is shown in Table 2. For comparison, the parent
inhibitor of BRD4 (compound JQ1) was also prepared. Each compound
was incubated with 22RV1 cells which have significant expression of
BRD4 and the cereblon E3 ligase system. The expression level of a
downstream protein c-Myc was measured. In this assay, either the
inhibition of BRD4 (by binding and blocking the active site of
BRD4) or the degradation of BRD4 leads to the down-regulation of
the expression level of c-Myc. The results indicated that a
connector length of 5 or 7 atoms led to the most potent IC.sub.50
(0.682 or 0.049 nM). Shortening the length to 4 atoms or less
decreases the compound potency to a stationary level around 120 to
250 nM, while extending the length to 8 atoms decreases the
compound potency to about 13 nM. The BRD4 inhibitor JQ1 exhibited
an IC.sub.50 of 101 nM, similar to those with shorter connectors.
It is likely that the compounds with connector lengths less than 5
atoms act like pure BRD4 inhibitors, which bind and inhibit BRD4
without causing degradation. The c-Myc expression inhibition
IC.sub.50 of these compounds reflect the binary binding potency to
BRD4. The compounds with greater connector lengths induce ternary
complex formation which recruits BRD4 and cereblon together and
causes ubiquitination of BRD4 by the cereblon E3 ligase system and
the subsequent degradation of BRD4. The degradation potencies
substantially surpass the binary inhibitory potency due to the
binding to BRD4. They also surpass the binary binding potency of
the cereblon-binding warhead (pomalidomide), since pomalidomide has
a K.sub.d of 157 nM based on the literature (Fischer et al. Nature
2014). The MD simulations on compounds 23 and 24 showed that these
compounds induced similar trimer complexes in which cereblon
directly interacted with the bromodomain 1 of BRD4. The
representative conformation of the trimer for compound 24 is given
in FIG. 8, and which demonstrates that a connector length of 7
atoms is compatible with the trimer conformation. The connector
lengths shorter than 5 atoms are too short to span the distance
required to allow the predicted trimer, consistent with the
observed lower potencies. The difference in potency between
compound 22 and compound 23, which differ by having an
oxygen-to-carbon replacement in the middle of the connector, can be
rationalized by the fact that the oxygen atom facilitates the
gauche conformation of the next adjacent bond. In addition to the
interactions between BRD4 and cereblon, the BRD4-binding warhead
and the cereblon-binding warhead also interact between each other.
These cross-domain interactions largely contribute to the stability
of the ternary complexes, and thus to the potency of the
corresponding compounds.
##STR00022##
TABLE-US-00002 TABLE 2 Exploration of Connector Length n Linking a
BRD4-Binding Warhead and a Cereblon-Binding Warhead and Measurement
of c-Myc Expression Inhibition IC.sub.50 and Maximum Inhibition
I.sub.max in 22RV1 cells. I.sub.max ID Connector n IC.sub.50
(.mu.M) (%) 19 ##STR00023## 2 0.187 96 20 ##STR00024## 3 0.123 93
21 ##STR00025## 4 0.250 88 22 ##STR00026## 5 0.00379 100 23
##STR00027## 5 0.000682 100 24 ##STR00028## 7 0.000049 98 25
##STR00029## 8 0.013 100 JQ1 0.101 93
[0156] Examination of the Length n of the Connector Linking a
BRD4-Binding Warhead and a VHL-Binding Warhead.
[0157] The connector linking the BRD4 warhead and the VHL-binding
warhead was systematically probed from 5 to 19 atoms. The results
are shown in Table 3. Each compound was incubated with 22RV1 cells
which have significant expression of BRD4 and the VHL E3 ligase
system. The expression level of a downstream protein c-Myc was
measured. In this assay, either the inhibition of BRD4 (e.g., by
binding and blocking the active site of BRD4) or the degradation of
BRD4 results in the down-regulation of the expression level of
c-Myc. The results indicate that the connector length range between
8 and 11 atoms gave rise to the optimal range of IC.sub.50 (0.16 to
19 nM). Shortening the length of the connector to 5-7 atoms
decreased the compound potency to a stationary level around 78 to
95 nM. Extending the length of the connector from 11 to 19 atoms
gradually decreased the compound potency to about 700 nM. The
degradation potencies corresponding to the optimal connector length
range substantially surpass the binary inhibitory potencies due to
the binding to BRD4 or VHL. The MD simulations on compound 29
showed that this compound induced a trimer in which VHL directly
interacted with the bromodomain 1 of BRD4. The representative
conformation of the trimer is shown in FIG. 9, indicating that a
connector length of 8 atoms is compatible with the trimer
conformation. The connector lengths of 5 to 7 atoms disturb the
trimer conformation, consistent with the observed lower potencies.
Extending the connector length longer than 8 atoms is tolerated
because the connector in this case is significantly exposed to
solvent. A longer connector can adopt a loop conformation with the
middle part projecting to the solvent space. However, long
connectors will suffer entropic penalties as previously stated.
[0158] In order to study the roles of the oxygen atoms in the
connector of compound 29, these oxygen atoms were replaced by
carbon atoms, sulfur atom or sulfonic group. The results are shown
in Table 4. It turned out that the oxygen atom closer to the BRD4
side was important for the potency while the other oxygen atom
seems important in a lesser extent. As shown in FIG. 9, the
predicted trimer conformation showed the first oxygen atom
facilitated the gauche conformation between this atom and the amide
group. Replacing it with a carbon atom, a sulfur atom or a sulfonic
group destabilizes the conformation and thus leads to the decrease
of potency. The sulfonic group would be particularly disturbing due
to the conformational restraints and desolvation cost. These
results demonstrate that although the length is the primary
requirement for a connector to allow trimer formation, the chemical
composition of the connector is also important since the connector
needs to adopt specific conformations in certain regions and it has
certain interactions with the targets.
[0159] Demonstration of the Induction of the Protein-Protein
Interactions Between VHL and ER-.alpha. by Two Tripartite Compounds
Using Surface Plasmon Resonance (SPR) Method.
[0160] The SPR experiments were conducted on a Biacore3000 (GE
Healthcare). His-tagged VHL protein was immobilized on a
carboxymethylated dextran surface with nitriloacetic acid, taking
advantage of NTA/Ni.sup.2+ chelation. The dissociation constant
(K.sub.d) of a compound to VHL was determined in a twelve
point-concentration assay, and the K.sub.d of the equimolar mixture
of a same compound and ER-.alpha. to VHL was determined in a same
way, which allows the comparison of the two K.sub.d values. This
comparative experiment was done for two tripartite compounds
respectively. Each of the two tripartite compounds contains an
ER-binding warhead and a VHL-binding warhead with a connector
covalently linking the two warheads. The two compounds only differ
in their ER-binding warheads. This set of experiments indicated
that the K.sub.d of the first compound to VHL in the absence of
ER-.alpha. was about 1.00 .mu.M while the K.sub.d to VHL was
changed to 0.010 .mu.M in the presence of ER-.alpha., corresponding
to a potency increase of about 100-fold. Similarly, the K.sub.d of
the second compound to VHL in the absence of ER-.alpha. was about
0.700 .mu.M while the K.sub.d to VHL was changed to 0.0045 .mu.M in
the presence of ER-.alpha., corresponding to a potency increase of
about 140-fold. These large increases in potency show that the
designed tripartite compounds induce the protein-protein
interactions and/or cross-domain interactions for the corresponding
proteins.
[0161] Demonstration of the Induction of the Protein-Protein
Interactions Between VHL and ER-.alpha. by a Tripartite Compound
Using AlphaLisa Assay.
[0162] The AlphaLisa assay was performed with GST-ER-.alpha.,
VHL-His and a tripartite compound composed of an ER-binding warhead
and a VHL-binding warhead and a connector. After a ten-minute
pre-incubation of the compound with equimolar mixture of
GST-ER-.alpha. and VHL-His, 7.5 .mu.L of anti-His conjugated
AlphaLISA acceptor beads diluted 100.times. in Buffer A was added
to each well of the assay plate, followed by another 5 minute
incubation in the dark. Finally, 7.5 .mu.L of
glutathione-conjugated AlphaLISA Donor beads diluted 100.times. in
Buffer B was added, followed by another 5 minute incubation at room
temperature in the dark. A series of concentrations of the
compounds was tested. The percentage of the ternary complex
formation at each concentration was calculated from the signal and
the dissociation constant K.sub.d of the ternary complex formation
was determined. It indicated a K.sub.d of 1.5 nM for the ternary
complex formation to compare with the binary binding constant of
209 nM between the compound and ER-.alpha. and the binary binding
constant of 228 nM between the compound and VHL. Thus, the ternary
binding affinity was about at least 139-fold more potent than the
binary binding affinities, suggesting that the designed tripartite
compound induced the protein-protein interactions for the
corresponding proteins. (The binary binding constant between
ER-.alpha. and the compound was determined using fluorescent
polarization assay and the binary binding constant between VHL and
the compound was determined using AlphaLisa assay.)
##STR00030##
TABLE-US-00003 TABLE 3 Exploration of Connector Length n Linking a
BRD4-Binding Warhead and a VHL-Binding Warhead and Measurement of
c-Myc Expression Inhibition IC.sub.50 and Maximum Inhibition
I.sub.max in 22RV1 cells. ID Connector n IC.sub.50 (.mu.M)
I.sub.max (%) 26 ##STR00031## 5 0.095 98 27 ##STR00032## 6 0.078
100 28 ##STR00033## 7 0.0805 94 29 ##STR00034## 8 0.00016 97 30
##STR00035## 9 0.0194 100 31 ##STR00036## 10 0.00122 99.3 32
##STR00037## 11 0.00086 98 33 ##STR00038## 12 0.005 97 34
##STR00039## 13 0.034 94 35 ##STR00040## 16 0.206 93 36
##STR00041## 17 0.126 96 37 ##STR00042## 18 0.202 96.4 38
##STR00043## 19 0.707 62.2
TABLE-US-00004 TABLE 4 Changes of Chemical Composition of the Best
Connector from the Set in Table 3 and Measurement of c-Myc
Expression Inhibition IC.sub.50 and Maximum Inhibition IC.sub.50
I.sub.max ID Connector n (.mu.M) (%) 29 ##STR00044## 8 0.00016 97
39 ##STR00045## 8 0.0102 95.4 40 ##STR00046## 8 0.0209 99 41
##STR00047## 8 0.0257 100 42 ##STR00048## 8 0.122 88.3
Methods of the Examples
[0163] Molecular Modeling and Molecular Dynamics Simulation.
[0164] The computer programs mentioned below are the 2013, 2014 and
2015 releases distributed by Schrodinger Inc. headquartered in New
York. The crystal structures of the complexes of VHL/EB/EC with
ligands, AR with ligands (PDB codes: 3V4A and 2YLO), BRD4 with
ligand (PDB code: 3MXF) and cereblon with ligand (PDB codes: 4TZ4
and 4CI2) were retrieved and imported to Maestro98 and Maestro102.
The hydrogen atoms and missing side chains were added using Protein
Preparation Wizard. For VHL, BRD4 and cereblon complexes, the
ligands were modified into the corresponding warheads mentioned in
the disclosure. Two crystal structures of cereblon were merged to
generate a model of human cereblon. For AR, the crystal structures
have the closed conformation concerning H12 helix and adjacent
loops which is not compatible with the desired warheads. A homology
model of an open conformation was built by combining these crystal
structures and the open conformation structures of ER (PDB codes:
1ERR, 3ERT and 2YJA). The H12 helix and the loops of AR were
replaced by the corresponding open conformation pieces of ER and
the side chains were mutated to the corresponding AR amino acids.
The ligand in AR was modified into the desired warheads. Molecular
dynamics simulation was performed for each homology model. A
representative conformation was generated for each model from the
molecular dynamics trajectory. A connector was built to link
between each AR-binding or BRD4-binding warhead and each
VHL-binding or cereblon-binding warhead while keeping the
interactions between each warhead and the corresponding protein
counterpart unchanged, using Maestro modeling tools. Each ternary
complex obtained in this way was subjected to molecular dynamics
simulation and a representative conformation was derived from the
trajectory generated.
[0165] Each molecular dynamics simulation was done with a general
protocol as follows. A system was solvated with explicit water and
0.15 M sodium chloride within a rectangle box of which each face is
10 .ANG. away from the nearest atom of the solute system. The box
had such an orientation relative to the solute system that the box
volume is minimized. The box was confined using the periodic
boundary condition. Additional sodium or chloride ions were added
to bring the total charge to zero. The program Desmond was used for
simulation with OPLS2.1 force-field. Each simulation underwent
seven stages: minimization with restraints on solute, minimization
without any restraints, simulation with Berendsen NVT at
temperature of 10 K with small time-steps with restraints on solute
heavy atoms, simulation with Berendsen NPT at temperature of 10 K
with restraints on solute heavy atoms, simulation with Berendsen
NPT at temperature of 310 K with restraints on solute heavy atoms,
simulation with Berendsen NPT at temperature of 310 K without
restraints, and the production stage with RESPA integrator at
temperature of 310 K and pressure of 1.01325 bar. The Coulombic
interaction was treated with Smooth Particle Mesh Edward method
with Ewald tolerance at 1.times.e.sup.-9. The energies were saved
at every 1.2 picosecond and the trajectory was saved at every 4.8
picosecond. The production stage was typically run for 20
nanoseconds, and when the starting point was a rough model, another
20 nanoseconds follow.
[0166] After each molecular dynamics simulation, a cluster analysis
was performed on the frames of the last 20 nanosecond trajectory
using one of the supporting scripts of Desmond. Different frames
were aligned by minimizing the Root-Mean-Square Deviation (RMSD) of
the corresponding solute heavy atoms between frames. The RMSD of
the aligned frames were used as the distances for clustering using
hierarchical method. The entire population of frames was clustered
into two clusters. The frame closest to the center of the largest
cluster was selected as the representative conformation from the
simulation which can be nominally considered as the most populated
conformation.
[0167] The molecular surface areas were calculated using the
Molecular Surface program of Maestro suite with a resolution at 0.3
.ANG. and probe radius of 1.4 .ANG.. The surface burial due to a
binary complex formation is defined as the difference between the
surface area of the whole binary complex and the sum of the surface
areas of the corresponding monomers in isolation. The surface
burial due to a ternary complex formation is defined as the
difference between the surface area of the whole ternary complex
and the sum of the surface areas of the corresponding monomers in
isolation. Thus, the surface burials were calculated by first
calculating the surface areas of the individual systems and then
calculating the differences accordingly.
Chemical Synthesis: General Scheme and Examples
[0168] A tripartite compound of WA-C-WB, or their pharmaceutically
acceptable salts, polymorphic forms, prodrugs, solvate forms and
isotope containing derivatives thereof, may be prepared by the
general approaches described below (scheme 1), together with
synthetic methods known in the art of organic chemistry, or
modifications and derivatizations that are familiar to those of
ordinary skill in the art.
##STR00049##
TABLE-US-00005 TABLE 5 Exemplary Compounds. Ex # Structure Compound
name and Analytical data E1 ##STR00050##
(2S,4R)-1-[(2S)-3,3-dimethyl-2-(2-{[5-
(4-{[trans-3-(3-chloro-4-cyanophenyl)-
2,2,4,4,-tetramethylcyclobutyl] carbamoyl}phenoxy)pentyl]oxy}
actamido)butanoyl]-4-hydroxy-N-{[4-
(1,3-thiazol-5-yl)phenyl]methyl} pyrrolidine-2-carboxamide .sup.1H
NMR (400 MHz, CDCl3): .delta. 0.95 (s, 9H), 1.22 (s, 6H), 1.27 (s,
6H), 1.56- 1.58 (m, 2H), 1.68-1.70 (m, 2H), 1.83- 1.86 (m, 2H),
2.11-2.12 (m, 1H), 2.54 (br, 1H), 3.52-3.63 (m, 3H), 3.91-4.16 (m,
7H), 4.28-4.54 (m, 5H), 4.70-4.71 (m, 1H), 6.19 (d, J = 6.8 Hz,
1H), 6.80-6.97 (m, 4H), 7.17 (d, J = 8.4 Hz, 1H), 7.32 (d, J = 6.8
Hz, 2H), 7.48- 7.58 (m, 3H), 7.72-7.74 (m, 2H), 8.03- 8.10 (m, 2H),
8.78 (br, 1H); LC-MS: (ES.sup.+): m/z 941.20 [M + H.sup.+] E2
##STR00051## (2S,4R)-1-((S)-2-(2-(3-(5-(4-(3-(4-
cyano-3-(trifluoromethyl)phenyl)-5,5-
dimethyl-4-oxo-2-thioximidazolidin-1- yl)phenoxy)pentyloxy)propoxy)
acetamido)-3,3-dimethylbutanoyl)-4-
hydroxy-N-(4-(4-methylthiazol-5-
yl)benzyl)pyrrolidine-2-carboxamide 1H NMR (400 MHz, CDCl3):
.delta. 7.96 (s, 2H), 7.86 (d, J = 8.6 Hz, 1H), 7.19 (d, J = 8.8
Hz, 2H), 7.02 (d, J = 8.6 Hz, 2H), 4.50 (s, 2H), 4.30 (t, J = 6.4
Hz, 2H), 4.02 (t, J = 6.4 Hz, 2H), 3.53 (m, 2H), 3.44 (m, 2H),
1.96-1.80 (m, 4H), 1.69-1.53 (m, 2H), 1.49 (s, 6H), 1.48 (s, 9H),
1.44-1.22 (m, 2H); Mass (ES+): m/z 686.35 [MNa+]
Synthesis of Example E1
##STR00052##
[0169] Step 1: Synthesis of methyl
4-{[5-({[(2S)-1-[(2S,4R)-4-hydroxy-2-({[4-(1,3-thiazol-5-yl)phenyl]methyl-
}carbamoyl)pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]carbamoyl}methoxy-
)pentyl]oxy}benzoate
##STR00053##
[0171] To a stirred solution of
2-({5-[4-(methoxycarbonyl)phenoxy]pentyl}oxy)acetic acid (200 mg),
(2S,4R)-1-[(2S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-{[4-(1,3-thiazo-
l-5-yl)phenyl]methyl}pyrrolidine-2-carboxamide hydrogen chloride
salt (149 mg, 0.32 mmol), N-ethyl-N-isopropylpropan-2-amine (185
mg, 1.44 mmol) in anhydrous N,N-dimethylformamide (5 mL) was added
HATU (2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate) (203 mg, 0.54 mmol) at 0.degree. C. The
resulting mixture was allowed to warm up to rt and stirred at rt
for 20 min. TLC and LC-MS showed formation of the desired product.
The mixture was partitioned between ethyl acetate (100 mL) and
water (50 mL). The organic layer was collected, washed with brine
(50 mL), dried over anhydrous sodium sulfate, and concentrated
under reduced pressure to give a crude residue which was purified
by silica gel flash chromatography (eluent 2% methanol in methylene
dichloride) to afford the titled product (yield 25%, 2 steps) as a
white solid. Mass: (ES.sup.+): m/z 695.30 [M+H.sup.+].
Step 2: Synthesis of
4-{[5-({[(2S)-1-[(2S,4R)-4-hydroxy-2-({[4-(1,3-thiazol-5-yl)phenyl]methyl-
}carbamoyl)pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]carbamoyl}methoxy-
)pentyl]oxy}benzoic acid
##STR00054##
[0173] To a stirred solution of methyl
4-{[5-({[(2S)-1-[(2S,4R)-4-hydroxy-2-({[4-(1,3-thiazol-5-yl)phenyl]methyl-
}carbamoyl)pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]carbamoyl}methoxy-
)pentyl]oxy}benzoate (150 mg, 0.22 mmol) in a mixed solvents of
tetrahydrofuran (4 mL)-water (2 mL)-methanol (1 ml) was added
lithium hydroxide monohydrate (36 mg, 0.86 mmol) at rt. The
resulting mixture was stirred at 35.degree. C. overnight. TLC and
LC-MS showed formation of the desired product. The reaction mixture
was acidified with aqueous HCl (3N) to pH=3-4 and extracted with
methylene dichloride (50 mL.times.2). The organic layers were
combined, washed with brine, dried over Na.sub.2SO.sub.4 and
concentrated to afford the titled product (110 mg, crude) as a
white solid which was used for next step without further
purification. Mass: (ES.sup.+): m/z 681.20 [M+H.sup.+].
Step 3: Synthesis of Example E1
##STR00055##
[0175] To a stirred mixture of
4-{[5-({[(2S)-1-[(2S,4R)-4-hydroxy-2-({[4-(1,3-thiazol-5-yl)phenyl]methyl-
}carbamoyl)pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]carbamoyl}methoxy-
)pentyl]oxy}benzoic acid (110 mg, 0.16 mmol),
2-chloro-4-[trans-3-amino-2,2,4,4-tetramethylcyclobutoxy]benzonitrile
hydrogen chloride salt (50 mg, 0.16 mmol),
N-ethyl-N-isopropylpropan-2-amine (77 mg, 0.64 mmol) in anhydrous
N,N-dimethylformamide (4 mL) was added HATU
((2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate)) (68 mg, 0.18 mmol) at 0.degree. C. The
resulting mixture was allowed to warm up to rt and stirred at rt
for 20 min. TLC and LC-MS showed formation of the desired product.
The reaction mixture was partitioned between ethyl acetate (100 mL)
and water (40 mL). The organic phase was separated, washed with
brine (50 mL), dried over anhydrous sodium sulfate, and
concentrated under reduced pressure to give a crude residue which
was purified by preparative TLC (eluent: 5% methanol in methylene
dichloride) to afford the titled product (yield 25%, 2 steps) as a
white solid.
Synthesis of 2-({5-[4-(methoxycarbonyl)phenoxy]pentyl}oxy)acetic
acid
##STR00056##
[0176] Step 1: Synthesis of tert-butyl
2-{[5-(benzyloxy)pentyl]oxy}acetate
##STR00057##
[0178] To a stirred mixture of 5-(benzyloxy)pentan-1-ol (10 g, 51.5
mmol), tert-butyl 2-bromoacetate (40.2 g, 206 mmol) and tetrabutyl
ammonium chloride (14.2 g, 51.5 mmol) in methylene dichloride (60
mL) was added sodium hydroxide (40 ml, 35% in water) at rt, and the
resulting mixture was stirred at rt for 16 h. The reaction mixture
was then partitioned between methylene dichloride (200 mL) and
water (100 mL). The organic layer was collected and washed with
brine (50 mL), dried over anhydrous sodium sulfate, and
concentrated under reduced pressure to give a crude residue which
was purified by silica gel flash chromatography (eluent: 5% ethyl
acetate in hexane) to afford tert-butyl
2-{[5-(benzyloxy)pentyl]oxy}acetate (yield 31.6%) as light yellow
oil. LC-MS: (ES.sup.+): m/z 331.10 [M+Na.sup.+], .sup.1H NMR (400
MHz, CDCl.sub.3): .delta. 1.48 (s, 9H), 1.63-1.67 (m, 6H),
3.46-3.53 (m, 4H), 4.10 (s, 2H), 4.50 (s, 2H), 7.28-7.34 (m,
5H).
Step 2: Synthesis of tert-butyl 2-[(5-hydroxypentyl)oxy]acetate
##STR00058##
[0180] To a stirred solution of tert-butyl
2-{[5-(benzyloxy)pentyl]oxy}acetate (5 g, 16.2 mmol) in ethanol
(100 ml) under a nitrogen atmosphere was added palladium on carbon
(10%, 600 mg) at rt. The resulting mixture was stirred at
50.degree. C. overnight under hydrogen atmosphere (hydrogen
balloon). TLC showed formation of desired product. Palladium on
carbon was removed through filtration and washed with ethyl acetate
(50 mL). The filtrate was concentrated under reduced pressure to
afford tert-butyl 2-[(5-hydroxypentyl)oxy]acetate (2.5 g, crude) as
colorless oil which was used in next step without further
purification.
Step 3: Synthesis of tert-butyl
2-({5-[(4-methylbenzenesulfonyl)oxy]pentyl}oxy)acetate
##STR00059##
[0182] To a stirred solution of tert-butyl
2-[(5-hydroxypentyl)oxy]acetate (2.5 g, crude) and triethylamine
(3.5 g, 34.5 mmol) in anhydrous methylene dichloride (50 mL) was
added a solution of 4-toluenesulfonyl chloride (2.7 g, 13.8 mmol)
in anhydrous methylene dichloride (8 mL) drop wise at 0.degree. C.
The reaction mixture was allowed to warm up to rt and stirred at rt
overnight. TLC showed formation of desired product. The mixture was
quenched with aqueous solution of potassium carbonate (1N, 50 mL)
at rt and extracted with ethyl acetate (50 mL.times.3). The organic
layers were combined, washed with brine (50 mL), dried over
anhydrous sodium sulfate, and concentrated under reduced pressure
to give a crude residue which was purified by silica gel flash
chromatography (eluent: 1% methanol in methylene dichloride) to
afford tert-butyl
2-({5-[(4-methylbenzenesulfonyl)oxy]pentyl}oxy)acetate (yield
35.1%) as colorless oil. Mass: (ES.sup.+): m/z 395.10
[MNa.sup.+].
Step 4: Synthesis of methyl
4-({5-[2-(tert-butoxy)-2-oxoethoxy]pentyl}oxy)benzoate
##STR00060##
[0184] To a stirred mixture of tert-butyl
2-({5-[(4-methylbenzenesulfonyl)oxy]pentyl}oxy)acetate (1.0 g, 2.7
mmol) and potassium carbonate (266 mg, 1.6 mmol) in acetonitrile
(15 mL) was added methyl 4-hydroxybenzoate (500 mg, 3.29 mmol) at
rt. The resulting mixture was refluxed overnight. TLC showed
formation of desired product. The reaction mixture was cooled to
rt, and partitioned between ethyl acetate (150 mL) and water (50
mL). The organic layer was washed with washed with brine (50 mL),
dried over anhydrous sodium sulfate, and concentrated under reduced
pressure to give a crude residue which was purified by silica gel
flash chromatography (eluent 10% ethyl acetate in hexane) to afford
methyl 4-({5-[2-(tert-butoxy)-2-oxoethoxy]pentyl}oxy)benzoate
(yield 33%) as colorless oil. Mass (ES.sup.+): m/z 353.10
[M+Na.sup.+]; .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 1.48 (s,
9H), 1.55-1.61 (m, 2H), 1.68-1.72 (m, 2H), 1.80-1.87 (m, 2H), 3.55
(t, J=6.4 Hz, 2H), 3.88 (s, 3H), 3.96 (s, 2H), 4.02 (t, J=6.4 Hz,
2H), 6.89 (d, J=9.2 Hz, 2H), 7.97 (d, J=9.2 Hz, 2H).
Step 5: Synthesis of
2-({5-[4-(methoxycarbonyl)phenoxy]pentyl}oxy)acetic acid
##STR00061##
[0186] To a stirred solution of methyl
4-({5-[2-(tert-butoxy)-2-oxoethoxy]pentyl}oxy)benzoate (300 mg,
0.85 mmol) in DCM (4 mL) was added and TFA (2 ml) at rt, the
resulting solution was stirred at room temperature for 1 h. TLC
showed formation of the desired product. The solvent was evaporated
to afford 2-({5-[4-(methoxycarbonyl)phenoxy]pentyl}oxy)acetic acid
(200 mg, crude) as yellow oil which was used in next step without
further purification.
Synthesis of an AR-Binding Warhead
[0187] WA:
2-chloro-4-(3-(4-hydroxyphenyl)-4,4-dimethyl-5-oxo-2-thioxoimid-
azolidin-1-yl)benzonitrile
##STR00062##
Step 1: Synthesis of 2-trifluoromethyl-4-isothiocyanatobenzonitrile
(B)
[0188] To a stirred solution of 4-amino-2-trifluomethylbenzonitrile
(A, 1 g, 6.55 mmol) in dichloromethane (9 mL) was added sodium
bicarbonate (2.21 g, 26.31 mmol) and water (9 mL). The resulting
mixture was cooled to 0.degree. C., to which thiophosgene (817 mg,
7.11 mmol) was added in drop wise in 30 min at 0.degree. C. The
resulting mixture was then warmed up to rt and stirred at rt for 1
h. The reaction mixture was diluted with dichloromethane (200 mL),
washed with brine (50 mL.times.2), dried over anhydrous sodium
sulfate and then concentrated under reduced pressure to give a
crude residue. The residue was purified by flash silica gel
chromatography (eluent: ethyl acetate/petroleum ether (v:v=1:30))
to give desired product (yield: 71%) .sup.1HNMR (400 MHz,
CDCl.sub.3): .delta. 7.69 (d, J=8.0 Hz, 1H), 7.38 (s, 1H), 7.28 (m,
1H);
Step 2: Synthesis of
2-trifluoromethyl-4-[3-(4-hydroxyphenyl)-5-imino-4,
4-dimethyl-2-sulfanylideneimidazolidin-1-yl]benzonitrile (C)
[0189] To a stirred solution of
2-trifluoromethyl-4-isothiocyanatobenzonitrile (B, 399 mg, 2.05
mmol) in toluene (5 mL) was added
2-[(4-hydroxyphenyl)amino]-2-methylpropanenitrile (C, 300 mg, 1.70
mmol) and 4-dimethylaminopyridine (312 mg, 2.55 mmol). The
resulting solution was then heated in an oil bath to 100.degree. C.
and stirred at the same temperature for 16 h. LC-MS indicated
formation of the desired product. The reaction mixture was
concentrated under vacuum to give a crude reside which was purified
by flash silica gel chromatography (eluent: ethyl acetate/petroleum
ether (v:v=1:1)) to give desired product (yield: 48%) as a brown
solid. LC-MS (ES.sup.+): m/z 370.95 [MH.sup.+], t.sub.R=0.74 min
(2.0 minute run);
Step 3: Synthesis of
2-trifluoromethyl-4-[3-(4-hydroxyphenyl)-4,4-dimethyl-5-oxo-2-sulfanylide-
neimidazolidin-1-yl]benzonitrile (WA)
[0190] To a stirred solution of
2-trifluomethyl-4-[3-(4-hydroxyphenyl)-5-imino-4,
4-dimethyl-2-sulfanylideneimidazolidin-1-yl]benzonitrile (C, 300
mg, 0.81 mmol) in methanol (6 mL) was added aqueous hydrogen
chloride (2N, 3.0 mL). The resulting solution was then heated in an
oil bath to 100.degree. C. and stirred at the same temperature for
2 h. The reaction mixture was diluted with water (30 mL), extracted
with ethyl acetate (60 mL.times.3), washed with water (50 mL),
dried over anhydrous sodium sulfate and concentrated under vacuum
to give titled product (yield: 93%) as a yellow solid, which was
used for the next step without any further purifications. LC-MS
(ES.sup.+): m/z 372.00 [MH.sup.+], t.sub.R=0.97 min (2.0 minute
run).
Synthesis of VHL-Binding Warheads
ULM-1:
(2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-meth-
ylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide
##STR00063## ##STR00064##
[0191] Step 1: Synthesis of
4-(4-methyl-1,3-thiazol-5-yl)benzonitrile (G)
[0192] To a stirred solution of 4-bromobenzonitrile (E, 20 g,
109.88 mmol) in DMA (250 mL) under a nitrogen atmosphere was added
4-methyl-1,3-thiazole (F, 21.88 g, 220.67 mmol), palladium (II)
acetate (743 mg, 3.31 mmol) and potassium acetate (21.66 g, 220.71
mmol) at rt. The resulting solution was heated to 150.degree. C.
and stirred at this temperature for 5 h, LC-MS indicated formation
of the desired product. The reaction was cooled to rt, diluted with
1 L of water and extracted with ethyl acetate (300 mL.times.3). The
organic layers were combined, washed with saturated aqueous
solution of sodium chloride (200 mL), dried over anhydrous sodium
sulfate and then concentrated under reduced pressure to give a
crude residue, which was purified by flash silica gel
chromatography (eluent: ethyl acetate/petroleum ether, v:v=1:5) to
give the G (yield: 91%) as a white solid.
Step 2: Synthesis of
[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methanamine (H)
[0193] To a stirred solution of
4-(4-methyl-1,3-thiazol-5-yl)benzonitrile (G, 35.0 g, 174.8 mmol)
in tetrahydrofuran (1000 mL) was added LiAlH.sub.4 (20.0 g, 526.3
mmol) in portions at 0.degree. C. in 10 min under a nitrogen
atmosphere. The resulting solution was then stirred at 60.degree.
C. for 3 h. LC-MS indicated formation of the desired product. The
reaction was then cooled to 0.degree. C., quenched by the addition
water (20 mL, added slowly), aq. solution of NaOH(15%, 20 mL) and
water (60 mL). The resulting mixture was then extracted with ethyl
acetate (300 mL.times.2). The organic layers were combined, washed
with saturated aqueous solution of sodium chloride (100 mL), dried
over anhydrous sodium sulfate and then concentrated under reduced
pressure to give a crude residue, which was purified by flash
silica gel chromatography (eluent: dichloromethane/methanol
(v:v=10:1)) to give H (yield: 56%) as a yellow oil.
Step 3: synthesis of tert-butyl
(2S,4R)-4-hydroxy-2-({[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}carbamo-
yl)pyrrolidine-1-carboxylate (J)
[0194] To a stirred solution of
(2S,4R)-1-[(tert-butoxy)carbonyl]-4-hydroxypyrrolidine-2-carboxylic
acid (I, 2.7 g, 11.7 mmol) in N,N-dimethylformamide (20 mL) was
added DIEA (2.52 g, 19.50 mmol), HATU (4.47 g, 11.76 mmol) and
[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methanamine (H, 2.0 g, 9.79
mmol) at rt. The resulting mixture was stirred at rt overnight,
LC-MS indicated formation of the desired product. The reaction
mixture was diluted with water (20 mL) and extracted with ethyl
acetate (50 mL.times.3). The organic layers were combined, washed
with saturated aqueous solution of sodium chloride (50 mL), dried
over anhydrous sodium sulfate and then concentrated under reduced
pressure to give a crude residue, which was purified by flash
silica gel chromatography (eluent: dichloromethane/methanol
(v:v=20:1)) to give J (yield: 56%) as a yellow solid.
Step 4: Synthesis of
(2S,4R)-4-hydroxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}pyrrolid-
ine-2-carboxamide hydrochloride (K)
[0195] To a stirred solution of tert-butyl
(2S,4R)-4-hydroxy-2-({[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}carbamo-
yl)pyrrolidine-1-carboxylate (J, 45 g, 107.78 mmol), was added a
solution of hydrogen chloride in dioxane (4N, 300 mL). The
resulting solution was stirred at 20.degree. C. for 2 h. The solids
were collected by filtration to give K (yield: 98%) as a yellow
solid, which was used for the next step without any further
purification.
Step 5: Synthesis of tert-butyl
N-[(2S)-1-[(2S,4R)-4-hydroxy-2-({[4-(4-methyl-1,3-thiazol-5-yl)phenyl]met-
hyl}carbamoyl)pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]carbamate
(M)
[0196] To a stirred solution of
(2S)-2-{[(tert-butoxy)carbonyl]amino}-3,3-dimethylbutanoic acid (L,
15.7 g, 68.0 mmol) in N,N-dimethylformamide (500 mL) was added DIEA
(29.2 g, 225.9 mmol), HATU (25.9 g, 68.1 mmol) and
(2S,4R)-4-hydroxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}pyrrolid-
ine-2-carboxamide hydrochloride (K, 20.0 g, 56.5 mmol) at rt. The
resulting solution was stirred at rt for 16 h, LC-MS indicated
formation of the desired product. The reaction mixture was diluted
by water (200 mL) and extracted with ethyl acetate (200
mL.times.3). The organic layers were combined, washed with
saturated aqueous solution of sodium chloride (50 mL.times.2),
dried over anhydrous sodium sulfate and then concentrated under
reduced pressure to give a crude residue, which was purified by
flash silica gel chromatography (eluent: ethyl acetate/petroleum
ether (v:v=2:1)) to give M (yield: 51%) as a yellow solid.
Step 6: Synthesis of
(2S,4R)-1-[(2S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-{[4-(4-methyl-1-
,3-thiazol-5-yl)phenyl]methyl}pyrrolidine-2-carboxamide
hydrochloride (ULM-1)
[0197] To a stirred solution of tert-butyl
N-[(2S)-1-[(2S,4R)-4-hydroxy-2-({[4-(4-methyl-1,3-thiazol-5-yl)phenyl]met-
hyl}carbamoyl)pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]carbamate
(M, 12 g, 22.61 mmol) in dioxane (20 mL) was added a solution of
hydrogen chloride in dioxane (4N, 80 mL) at rt. The resulting
solution was stirred at rt for 2 h, LC-MS indicated formation of
the desired product. Precipitated solids were collected by
filtration to give ULM-1 (yield: 48%) as a yellow solid. .sup.1HNMR
(400 MHz, CD.sub.3OD): .delta. 9.84-9.82 (s, 1H), 7.58-7.54 (m,
4H), 4.71-4.41 (m, 4H), 4.13-4.08 (m, 1H), 3.86-3.71 (m, 2H), 3.36
(s, 1H), 2.60-2.58 (s, 3H), 2.35-2.07 (m, 2H), 1.19-1.12 (m, 9H).
LC-MS (ES.sup.+): m/z 431.11 [MH.sup.+], t.sub.R=0.73 min (2.0
minute run).
ULM-2:
(2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(thiazo-
l-5-yl)benzyl)pyrrolidine-2-carboxamide
##STR00065##
[0199] ULM-2 was synthesized according to similar procedure
described above for the synthesis of ULM-1, utilizing
4-bromobenzonitrile and 1,3-thiazole as starting materials. LC-MS
(ES.sup.+): m/z 417.10 [MH.sup.+], t.sub.R=0.51 min (2.0 minute
run).
ULM-3:
(2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N--((S)-1-(4-
-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide
##STR00066##
[0200] Step 1: Synthesis of tert-butyl
N-[(1S)-1-(4-bromophenyl)ethyl]carbamate (O)
[0201] To a stirred mixture of (1S)-1-(4-bromophenyl)ethan-1-amine
(N, 10.0 g, 49.98 mmol) in dichloromethane (100 mL) was added
Et.sub.3N (10.0 g, 99.01 mmol) and (Boc).sub.2O (13.0 g, 59.63
mmol). The resulting mixture was stirred at rt for 2 h. The bulk of
solvent was then removed under reduced pressure to give a crude
residue, which was purified by flash silica gel chromatography
(eluent: ethyl acetate/petroleum ether, v:v=1:10) to give O (yield:
99%) as a white solid.
Step 2: Synthesis of tert-butyl
N-[(1S)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]carbamate
(P)
[0202] To a stirred solution of tert-butyl
N-[(1S)-1-(4-bromophenyl)ethyl]carbamate (0, 15.0 g, 49.97 mmol) in
DMA (100 mL), under an atmosphere of nitrogen, was added
4-methyl-1,3-thiazole (9.9 g, 99.84 mmol), potassium acetate (9.8
g, 99.86 mmol) and Pd(OAc).sub.2 (112.5 mg, 0.50 mmol) at rt. The
resulting mixture was then stirred at 120.degree. C. for 2 h. The
reaction mixture was then cooled to rt, diluted by water (120 mL),
and extracted with ethyl acetate (200 mL.times.3). The organic
layers were combined, dried over anhydrous sodium sulfate and then
concentrated under reduced pressure to give a crude residue, which
was purified by flash silica gel chromatography (eluent: ethyl
acetate/petroleum ether, v:v=1:5) to give P (yield: 47%) as a white
solid. LC-MS (ES.sup.+): m/z 319.13 [MH.sup.+], t.sub.R=0.97 min
(2.0 minute run).
Step 3. Synthesis of
(1S)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethan-1-amine
hydrochloride (Q)
[0203] To a stirred solution of tert-butyl
N-[(1S)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]carbamate (P,
7.5 g, 23.55 mmol) in methanol (20 mL) was bubbled in hydrogen
chloride (gas) at rt for 2 h. Then the resulting mixture was
concentrated under vacuum to give Q (yield: 86%) as a white solid,
which was used in the next step without any further
purifications.
[0204] Intermediate Q was converted to ULM-3 in a similar manner as
described for the conversion of H to ULM-1. .sup.1H NMR (300 MHz,
DMSO): .delta. 8.99 (s, 1H), 8.57-8.55 (d, J=7.8 Hz, 1H), 8.01 (br.
s, 3H), 7.46-7.43 (d, J=8.4 Hz, 2H), 7.39-7.37 (d, J=8.4 Hz, 2H),
4.98-4.90 (m, 1H), 4.57-4.51 (m, 1H), 4.34 (br. s, 1H), 3.94-3.92
(m, 1H), 3.69-3.66 (m, 1H), 3.53-3.49 (m, 1H), 2.52 (s, 3H),
2.10-2.07 (m, 1H), 1.83-1.81 (m, 1H), 1.40-1.30 (m, 3H), 1.03 (s,
9H). LC-MS (ES.sup.+): m/z 445.05 [MH.sup.+], t.sub.R=0.53 min (2.0
minute run).
Synthesis of Example E2
E2:
(2S,4R)-1-((S)-2-(2-(3-(5-(4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,-
5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)phenoxy)pentyloxy)propoxy)aceta-
mido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)py-
rrolidine-2-carboxamide
##STR00067##
[0205] Step 1: Synthesis of tert-butyl
2-(3-{[5-(4-{3-[4-cyano-3-(trifluoromethyl)phenyl]-5,5-dimethyl-4-oxo-2-s-
ulfanylideneimidazolidin-1-yl}phenoxy)pentyl]oxy}propoxy)acetate
(BG)
[0206] To a stirred solution of tert-butyl
2-[3-[(5-[[(4-methylbenzene)sulfonyl]oxy]pentyl)oxy]propoxy]acetate
(AB, 150 mg, 0.35 mmol) in acetonitrile (10 mL) was added
4-[3-(4-hydroxyphenyl)-4,4-dimethyl-5-oxo-2-sulfanylideneimidazolidin-1-y-
l]-2-(trifluoromethyl)benzonitrile (ABM-3, 141 mg, 0.35 mmol) and
potassium carbonate (144 mg, 1.04 mmol). The resulting mixture was
stirred overnight at 80.degree. C. in an oil bath. LC-MS indicated
formation of the desired product. The reaction mixture was then
extracted with ethyl acetate (20 mL.times.2). The organic layers
were combined, washed with saturated aqueous solution of sodium
chloride (20 mL), dried over anhydrous sodium sulfate and then
concentrated under reduced pressure to give a crude residue, which
was purified by flash silica gel chromatography (eluent: ethyl
acetate/petroleum ether, v:v=1:1) to give 0.22 g of BG as a yellow
oil. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 7.96 (s, 2H), 7.86
(d, J=8.6 Hz, 1H), 7.19 (d, J=8.8 Hz, 2H), 7.02 (d, J=8.6 Hz, 2H),
4.50 (s, 2H), 4.30 (t, J=6.4 Hz, 2H), 4.02 (t, J=6.4 Hz, 2H), 3.53
(m, 2H), 3.44 (m, 2H), 1.96-1.80 (m, 4H), 1.69-1.53 (m, 2H), 1.49
(s, 6H), 1.48 (s, 9H), 1.44-1.22 (m, 2H); Mass (ES.sup.+): m/z
686.35 [MNa.sup.+].
Step 2: Synthesis of
2-(3-[[5-(4-[3-[4-cyano-3-(trifluoromethyl)phenyl]-5,5-dimethyl-4-oxo-2-s-
ulfanylideneimidazolidin-1-yl]phenoxy)pentyl]oxy]propoxy)acetic
acid (BH)
[0207] To a stirred solution of tert-butyl
2-(3-{[5-(4-{3-[4-cyano-3-(trifluoromethyl)phenyl]-5,5-dimethyl-4-oxo-2-s-
ulfanylideneimidazolidin-1-yl}phenoxy)pentyl]oxy}propoxy)acetate
(BG, 220 mg, 0.33 mmol) in dioxane (4.0 mL) was added hydrogen
chloride (2N in water, 1.0 mL). The resulting mixture was stirred
at 80.degree. C. for 2 h. LC-MS indicated formation of the desired
product. The resulting mixture was concentrated under reduced
pressure to provide 200 mg of BH as light yellow oil. Mass
(ES.sup.+): m/z 608.25 [MH.sup.+].
Step 3: Synthesis of E2
[0208] To a stirred solution of
2-(3-[[5-(4-[3-[4-cyano-3-(trifluoromethyl)phenyl]-5,5-dimethyl-4-oxo-2-s-
ulfanylideneimidazolidin-1-yl]phenoxy)pentyl]oxy]propoxy)acetic
acid (BH, 160 mg, 0.26 mmol) in N,N-dimethylformamide (5 mL) was
added
(2S,4R)-1-[(2S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-{[4-(4-methyl-1-
,3-thiazol-5-yl)phenyl]methyl}pyrrolidine-2-carboxamide
hydrochloride (ULM-1, 182 mg, 0.39 mmol), DIPEA (151 mg, 1.17
mmol), EDCI (101 mg, 0.53 mmol) and HOBt (70 mg, 0.52 mmol). The
resulting mixture was stirred at rt for 5 h and LC-MS indicated
formation of the desired product. Water (20 mL) was added to the
reaction, the resulting mixture was extracted with ethyl acetate
(20 mL.times.2). The organic layers were combined, washed with
saturated aqueous solution of sodium chloride (20 mL), dried over
anhydrous sodium sulfate and then concentrated under reduced
pressure to give a crude residue. The residue was purified by
Prep-HPLC to give 60 mg of Example 1 as a white solid. .sup.1H NMR
(400 MHz, CD.sub.3OD): .delta. 8.88 (s, 1H), 8.16 (d, J=8.0 Hz,
2H), 8.00 (s, 1H), 7.49-7.42 (m, 4H), 7.28 (d, J=8.8 Hz, 2H), 7.06
(m, 2H), 4.87 (s, 1H), 4.59 (m, 3H), 4.37 (m, 1H), 4.05 (m, 4H),
3.88 (m, 2H), 3.65 (m, 2H), 3.58 (m, 2H), 3.50 (m, 2H), 2.48 (s,
3H), 2.25 (m, 1H), 2.10 (m, 1H), 1.90 (m, 2H), 1.80 (m, 2H), 1.66
(m, 2H), 1.56 (s, 8H), 1.04 (s, 9H); LC-MS (ES.sup.+): m/z 1020.20
[MH.sup.+], t.sub.R=2.28 min (3.6 minute run).
[0209] Androgen Receptor ELISA Assay
[0210] Compounds have been evaluated in this assay in LNCaP and/or
VCaP cells utilizing similar protocols. The protocols used with
VCaP cells are described below. The androgen receptor ELISA assay
was performed using PathScan AR ELISA (Cell Signaling
Catalog#12850) according to the following assay steps:
[0211] VCaP cells are seeded at 30,000 cells/well at a volume of
200 .mu.L/well in VCaP assay medium [Phenol red free RPMI (Gibco
Cat#11835-030); 5% Charcoal Stripped (Dextran treated) FBS (Omega
Scientific, Cat#FB-04); Pen/Strep Life Technologies (Gibco Cat#:
10378-016); 0.1 nM R1881 (Sigma, Cat# R0908) is added upon the
start of the assay, not during initial plating of the cells] in
Corning 3904 plates. The cells are grown for a minimum of 3
days.
[0212] First, cells are dosed with compounds diluted in 0.1%
DMSO--use a polypropylene plate according to the following
protocol: (1)(i) make 1000.times. stock plate in DMSO; (ii) 20 mM
stock diluted 1/6.7 with DMSO (5 .mu.L+28.3 .mu.L DMSO)=3 mM into
row H; (iii) perform serial dilutions in 1/2 log doses (10 .mu.L of
compound+20 .mu.L DMSO) from row H towards row B. Reserve row A for
DMSO; (iv) 7 doses total (final concentration in this 1000.times.
plate will be 3 mM, 1 mM, 333 .mu.M, 111 .mu.M, etc). (2)(i) Make
10.times. stock plate in media; (ii) transfer 2.5 .mu.L of the
1000.times. stock to a new 10.times. stock plate (use 12 channel
pipet, start at A, DMSO control, work thru H). When 247.5 .mu.L of
media is added to this plate, it will serve as a 10.times. stock;
(iii) make media+1 nM R1881 for making 10.times. stock plate; (iv)
add 247.5 .mu.L of media with 1 nM R1881 to each well of the
10.times. stock plate, mix.
[0213] Then 22 .mu.L of 10.times. stock is added to cells and
incubated for 24 h. 1.times. Cell Signaling Cell lysis buffer is
made (Catalogue #9803; comes with the kit)--prepare for 50
.mu.L/well. Keep on ice. Media is aspirated, and 50 .mu.L 1.times.
cell lysis buffer/well is added. The cells are placed on ice for 10
minutes. The solution is mixed and transferred to PCR plate, and
centrifuged at 4 C for 10 minutes at 4000 rpm.
[0214] 5 .mu.L is transferred to fresh plate (use immediately or
freeze -80 C); 115 .mu.L ELISA Dilutant is added (0.15 ug/ml-0.075
ug/ml; comes with the PathScan ELISA).
[0215] Add 100 .mu.L/well AR Elisa; cover and shake, 37 C for 2
hrs; dump, tap, wash 4.times. 200 .mu.L ELISA wash buffer; add 100
.mu.L/well mouse AR detection Ab; cover and shake, 37 C for 1 hr;
dump, tap, wash 4.times. 200 .mu.L ELISA wash buffer; add 100
.mu.L/well anti-mouse--HRP conjugated Ab (comes with the kit);
cover and shake, 37 C for 30 min; allow TMB reagent to come to RT;
dump, tap, wash 4.times. 200 .mu.L Elisa wash buffer; tap; add 100
.mu.L TMB, shake 5 min--while watching color. Add the stop reagent
when light blue color develops. Add 100 .mu.L Stop solution; shake
and read at 450 nM.
[0216] Ligand Competition Assay
[0217] The experimental protocol is based on reference Gustafson et
al. (Angew. Chem. Int. Ed., 54: 9659-9662). HEK293 cells were
transiently transfected using Fugene 6 to overexpress wildtype
Androgen Receptor. During binding assay, transfected cells were
grown in DMEM supplemented with 10% charcoal-stripped FBS. Cells
were treated for two hours with the indicated competitor compound
in the presence of 0.1 nM .sup.3H-R1881. Cells were washed in PBS,
lysed in 200 ul lysis buffer (2% SDS, 10% Glycerol, 10 mM Tris-HCl
[pH 6.8]), and cleared with the addition of 300 ul of 10 mM
Tris-HCl (pH 8.0). 300 ul of these lysates were added to 3 mL of
Cytoscint (MP Biomedicals), and scintillation counting was
performed on a Beckman LS 6000SC instrument. Counts (cpm) were
normalized to total protein amount in each sample, as determined
via the Pierce BCA Protein Assay Kit (Thermo Scientific) per the
manufacturer's instructions.
[0218] Surface Plasmon Resonance (SPR) Method.
[0219] The SPR experiments were conducted on a Biacore3000 (GE
Healthcare). His-tagged VHL protein was immobilized on a
carboxymethylated dextran surface with nitriloacetic acid (NTA),
taking advantage of NTA/Ni.sup.2+ chelation. The prepared surface
equilibrated over three hours in running buffer (10 mM HEPES buffer
@ pH 7.4, 150 mM NaCl, 0.4 mg/mL BSA, 0.005% P20, 2% DMSO). All
compounds were prepared in 100% DMSO stock plates with a top
concentration of 500 mM in a 3.times. serial dilution. Compounds
were transferred from the stock plate to the assay plate and
diluted into running buffer (no DMSO) to measure direct binding.
Equimolar concentration of ER-.alpha. was added to corresponding
compound wells to measure cooperative binding. All compounds were
run as a twelve point-concentration series with a final assay top
concentration of 1 mM. Data analysis was performed in Scrubber 2
(BioLogic software, Campbell, Australia). Blanks were subtracted
and data was corrected for DMSO against a standard DMSO curve. All
reported K.sub.d values represent an average of at least N=2 and
were obtained by fitting to a minimum of five concentrations using
a 1:1 fitting algorithm.
[0220] AlphaLISA Assay Method for Measuring K.sub.d Values of a
Trimeric Complex.
[0221] Compounds in 10% DMSO were serially diluted in 3-fold
increments in an intermediate plate and then 3 uL was transferred
to 384-well OptiPlates (Perkin Elmer, #6007290). Next, equimolar
His-tagged VHL (made at Arvinas) and GST-tagged ER-.alpha. (Thermo
Fisher, #A15677) were mixed to a final concentration of 14 nM each
in Buffer A (50 mM HEPES, pH 7.5, 50 mM NaCl, 69 uM Brij, 0.1 mg/mL
BSA) and then 13 uL of this mixture was transferred to each well of
the 384-well assay plate containing compounds. After a ten-minute
pre-incubation of the compound/protein mixture, 7.5 uL of anti-His
conjugated AlphaLISA acceptor beads (PerkinElmer, # AL128M) diluted
100.times. in Buffer A was added to each well of the assay plate,
followed by another 5 minute incubation in the dark. Finally, 7.5
uL of glutathione-conjugated AlphaLISA Donor beads (PerkinElmer,
#6765301) diluted 100.times. in Buffer B was added, followed by
another 5 minute incubation at room temperature in the dark. Assay
plates were then read using a Synergy2 Multi-Mode plate reader
(BioTek, Winooski, Vt.) after excitation thru a 680/30 nm
excitation filter and collection of emission thru a 615/16 nm
filter. A zero compound control was used to estimate 615 nm
emission signal at 0% trimer formation, whereas, the max 615 nm
emission value obtained at optimal compound concentration was used
to estimate 100% trimer formation. Percent trimer formation was
then calculated from 615 nm emission values using the following
equation: ((615 nm observed-615 nm 0% control)/(615 nm 100%
control-615 nm 0% control))*100%. All data points were then fit
using a non-linear regression analysis in GraphPad Prism.
[0222] AlphaLISA Competitive Binding Assay for VHL.
[0223] Six microliters of 40 uM VHL-his stock (made at Arvinas) and
60 ul of 10 uM biotinylated VHL ligand probe (made at Arvinas) were
added into 8 ml of Buffer A (50 mM HEPES, pH 7.5, 50 mM NaCl, 69 uM
Brij, 0.1 mg/mL BSA). Final concentrations were 30 nM VHL and 75 nM
probe and this mixture was incubated at room temperature for 10
minutes. Meanwhile, compounds in 10% DMSO were serially diluted in
3-fold increments in an intermediate plate and then 3 uL was
transferred to 384-well OptiPlates (Perkin Elmer, #6007290). Next,
12 ul of the VHL-his/probe mix was added to each well of the assay
plate and incubated 15 minutes at room temperature. After a
ten-minute pre-incubation of the compound/protein mixture, 7.5 uL
of anti-His conjugated AlphaLISA acceptor beads (Perkin Elmer,
#AL128M) diluted 100.times. in Buffer A was added to each well of
the assay plate, followed by another 5 minute incubation in the
dark. Finally, 7.5 uL of streptavidin-conjugated AlphaLISA Donor
beads (PerkinElmer, #6760002) diluted 100.times. in Buffer A was
added followed by another 5 minutes incubation at room temperature
in the dark. Assay plates were then read using a Synergy2
Multi-Mode plate reader (BioTek, Winooski, Vt.) after excitation
thru a 680/30 nm excitation filter and collection of emission thru
a 615/16 nm filter. A zero compound control was used to estimate
615 nm emission signal at 0% binding, whereas, signal after
addition of 100.times. excess ligand (without biotin) was used to
estimate 615 nm emission at 100% binding. Percent binding of
compound was then calculated from 615 nm emission values using the
following equation: (1-(615 nm compound-615 nm @ 100% binding)/(615
nm @ 0% binding-615 nm @ 100% binding))*100%. All data were then
fit using a non-linear regression analysis in GraphPad Prism. Since
the K.sub.d of the biotinylated VHL probe is 7.5 uM (data not
shown), K.sub.d values were easily calculated from IC.sub.50 values
using the well-established Cheng-Prusoff equation.
[0224] Western Blot Analysis
[0225] Cultured cells were collected in lysis buffer containing 40
mM HEPES (pH 7.4), 140 mM NaCl, 2.5 mM EDTA, 1% NP-40, 0.1% SDS,
and protease inhibitor cocktail. After 10 min of centrifugation
(14,000 rpm), supernatant was collected for protein concentration
determination by the bicinchoninic acid method and subjected for
immunoblotting by standard protocol. Western blot results were
visualized using Bio-Rad Clarity ECL Western Blotting Substrate on
a Bio-Rad ChemiDoc MP imaging system.
[0226] c-Myc ELISA Assay Protocol
[0227] 22RV-1 cells were purchased from ATCC and cultured in RPMI
+10% FBS media. Cells were harvested using trypsin (Gibco
#25200-114), counted and seeded at 30,000 cells/well at a volume of
75 .mu.L/well in RPMI +10% FBS media in 96-well plates. The cells
were dosed with compounds diluted in 0.1% DMSO, incubated for 18 h
then washed and lysed in 50 uL RIPA buffer (50 mM Tris pH8, 150 mM
NaCl, 1% Tx-100, 0.1% SDS, 0.5% sodium deoxycholate) supplemented
with protease and phosphatase inhibitors. The lysates were
clarified at 4000 rpm at 4.degree. C. for 10 minutes then aliquots
were added into a 96-well ELISA plate of Novex Human c-Myc ELISA
kit from Life Technologies Catalog #KH02041. 50 .quadrature.L of
c-Myc Detection antibody was added into every well, the plates
incubated at room temperature for 3 hrs, then washed with ELISA
wash buffer. 100 .mu.L of the anti-rabbit IgG-HRP secondary
antibody was added to each well and incubated at room temperature
for 30 minutes. The plates were washed with ELISA wash buffer, 100
.mu.L TMB added to each well, and then monitored every 5 minutes
for a color change. 100 .mu.L of stop solution is added and the
plates read at 450 nm.
[0228] VHL Fluorescence Polarization (FP) Assay
[0229] VHL ligands were dissolved in DMSO (20 mM), and then serial
diluted with 3-fold increments in DMSO. This serial dilution was
diluted 10-fold with Assay Buffer (50 mM Tris pH 7.5, 200 mM NaCl,
2 mM DTT). Two .mu.L of the 10% DMSO serial dilution series was
pipetted into 384-well Corning 3575 plate. Purified VHL was diluted
in Assay Buffer to 250 nM and 8 .mu.L was transferred to each well.
The plate was incubated for 1 hr. 5-FAM-DEALA[HYP]YIPMDDDFQLRSF
peptide (synthesized by BIOMATIK) was diluted in Assay Buffer to 40
nM and 10 .mu.L was pipetted to each well. The final concentration
of the labeled peptide and VHL protein in the assay is 20 nM and
100 nM, respectively. The plate was incubated for 2 hrs, after
which Fluorescence Polarization was determined with BioTek Cytation
plate reader using the following BioTek filters: EX 485/20 EM
528/20. The percent inhibition was determined by normalizing to
maximum and minimum polarization, and graphed against the log
[ligand]. IC.sub.50 values were determined using Prism 6.
[0230] In summary, the present disclosure provides a method and
materials, including the theoretical understanding, to develop
ligands that cause protein-protein interactions between targeted
protein molecules. Accordingly, the present disclosure also defines
the generic chemical structures for the compounds that have the
ability to induce protein-protein interactions between any given
targets.
[0231] While preferred embodiments of the disclosure have been
shown and described herein, it will be understood that such
embodiments are provided by way of example only. Numerous
variations, changes and substitutions will occur to those skilled
in the art without departing from the spirit of the disclosure.
Accordingly, it is intended that the appended claims cover all such
variations as fall within the spirit and scope of the
disclosure.
[0232] The contents of all references, patents, pending patent
applications and published patents, cited throughout this
application are hereby expressly incorporated by reference.
[0233] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the disclosure described
herein. Such equivalents are intended to be encompassed by the
following claims. It is understood that the detailed examples and
embodiments described herein are given by way of example for
illustrative purposes only, and are in no way considered to be
limiting to the invention. Various modifications or changes in
light thereof will be suggested to persons skilled in the art and
are included within the spirit and purview of this application and
are considered within the scope of the appended claims. For
example, the relative quantities of the ingredients may be varied
to optimize the desired effects, additional ingredients may be
added, and/or similar ingredients may be substituted for one or
more of the ingredients described. Additional advantageous features
and functionalities associated with the systems, methods, and
processes of the present disclosure will be apparent from the
appended claims. Moreover, those skilled in the art will recognize,
or be able to ascertain using no more than routine experimentation,
many equivalents to the specific embodiments of the disclosure
described herein. Such equivalents are intended to be encompassed
by the following claims.
* * * * *