U.S. patent application number 16/819130 was filed with the patent office on 2020-10-29 for camptothecin derivatives as anti-hiv agents and methods of identifying agents that disrupt vif self-association.
This patent application is currently assigned to OYAGEN, INC.. The applicant listed for this patent is OYAGEN, INC.. Invention is credited to Ryan P. BENNETT, Harold C. SMITH.
Application Number | 20200338067 16/819130 |
Document ID | / |
Family ID | 1000004944566 |
Filed Date | 2020-10-29 |
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United States Patent
Application |
20200338067 |
Kind Code |
A1 |
SMITH; Harold C. ; et
al. |
October 29, 2020 |
CAMPTOTHECIN DERIVATIVES AS ANTI-HIV AGENTS AND METHODS OF
IDENTIFYING AGENTS THAT DISRUPT VIF SELF-ASSOCIATION
Abstract
The present invention relates to the use of camptothecin
derivatives as anti-HIV agents that disrupt self-association of the
viral infectivity factor (Vif) found in HIV and other retroviruses.
The present invention also relates to methods of identifying agents
that disrupt VIf self-association and methods of using these
agents, including methods of treating or preventing HIV
infection.
Inventors: |
SMITH; Harold C.;
(Rochester, NY) ; BENNETT; Ryan P.; (Clifton
Springs, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OYAGEN, INC. |
Rochester |
NY |
US |
|
|
Assignee: |
OYAGEN, INC.
Rochester
NY
|
Family ID: |
1000004944566 |
Appl. No.: |
16/819130 |
Filed: |
March 15, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14900666 |
Dec 22, 2015 |
10588902 |
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PCT/US2014/043974 |
Jun 24, 2014 |
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16819130 |
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61838574 |
Jun 24, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/6845 20130101;
G01N 2333/15 20130101; G01N 2500/02 20130101; G01N 2333/163
20130101; G01N 2500/10 20130101; A61K 31/4745 20130101; A61K 45/06
20130101 |
International
Class: |
A61K 31/4745 20060101
A61K031/4745; A61K 45/06 20060101 A61K045/06; G01N 33/68 20060101
G01N033/68 |
Goverment Interests
GOVERNMENT RIGHTS STATEMENT
[0002] The present invention was made with U.S. Government support
under
[0003] National Institutes of Health Grant No. R21NS067671-01. The
U.S. Government has certain rights in the invention.
Claims
1. A method for treating or preventing HIV infection or AIDS in a
patient, said method comprising: administering to a patient in need
of such treatment or prevention a therapeutically effective amount
of a compound of Formula (I): ##STR00004## or a pharmaceutically
acceptable salt thereof, wherein: Q is selected from NH, O, and S;
R.sup.20a and R.sup.20b are individually selected from hydrogen,
hydroxy, and C.sub.1-6alkyl; R.sup.21 is selected from hydrogen,
--NHC(.dbd.O)(CH.sub.2).sub.pNR.sup.23R.sup.24, and
--(CH.sub.2).sup.pNR.sup.23R.sup.24; p is 0, 1, 2, 3, or 4;
R.sup.22 is selected from hydrogen and hydroxyl; R.sup.23 and
R.sup.24 are individually selected from hydrogen and
C.sub.1-6alkyl; and R.sup.25 and R.sup.26 are individually selected
from hydrogen and --NO.sub.2.
2-11. (canceled)
12. The method according to claim 1, wherein the compound of
Formula (I) or pharmaceutically acceptable salt thereof is
administered with a pharmaceutically acceptable carrier.
13. The method according to claim 1, further comprising:
administering a therapeutically effective amount of at least one
other agent for treating HIV selected from the group consisting of
HIV reverse transcriptase inhibitors, non-nucleoside HIV reverse
transcriptase inhibitors, HIV protease inhibitors, HIV fusion
inhibitors, HIV attachment inhibitors, CCR5 inhibitors, CXCR4
inhibitors, HIV budding or maturation inhibitors, and HIV integrase
inhibitors.
14. The method according to claim 17, wherein said contacting step
is effective to inhibit A method for inhibiting infectivity of a
lentivirus in the cell.
15. (canceled)
16. The method according to claim 14, wherein the lentivirus is
selected from the group consisting of HIV-1 and HIV-2.
17. A method for inhibiting Vif self-association in a cell, said
method comprising: contacting a cell with an inhibitory-effective
amount of a compound of Formula (I) or a pharmaceutically
acceptable salt thereof, wherein said compound of Formula (I)
comprises: ##STR00005## or a pharmaceutically acceptable salt
thereof, wherein: Q is selected from NH, O, and S; R.sup.20a and
R.sup.20b are individually selected from hydrogen, hydroxy, and
C.sub.1-6alkyl; R.sup.21 is selected from hydrogen,
--NHC(.dbd.O)(CH.sub.2).sup.pNR.sup.23R.sup.24, and
--(CH.sub.2).sup.pNR.sup.23R.sup.24 p is 0, 1, 2, 3, or 4; R.sup.22
is selected from hydrogen and hydroxyl; R.sup.23 and R.sup.24 are
individually selected from hydrogen and C.sub.1-6alkyl; and
R.sup.25 and R.sup.26 are individually selected from hydrogen and
--NO.sub.2.
18. The method according to claim 17, wherein said compound of
Formula (I) or pharmaceutically acceptable salt thereof is
administered with a pharmaceutically acceptable carrier, wherein
said compound of Formula (I) or pharmaceutically acceptable salt
thereof is effective to disrupt or inhibit multimerization of Vif
in the cell, thereby inhibiting Vif self-association in the
cell.
19. (canceled)
20. A method of identifying an agent that disrupts Vif
self-association, said method comprising: providing a Vif:Vif
complex comprising a first Vif protein or fragment associated with
a second Vif protein or fragment; contacting the Vif:Vif complex
with a test agent under conditions effective to generate a
detectable signal when the Vif:Vif complex is disrupted; and
detecting the detectable signal to determine whether or not the
test agent disrupts the Vif:Vif complex, wherein disruption of the
Vif:Vif complex by the test agent identifies an agent that disrupts
Vif self-association.
21. (canceled)
22. The method according to claim 20, wherein the test agent is
from a library of small molecule compounds.
23. The method according to claim 20, wherein the contacting step
comprises incubating the Vif:Vif complex with one type of test
agent or more than one type of test agent.
24. (canceled)
25. The method according to claim 20, wherein the detectable signal
is detected using a detection technique selected from the group
consisting of fluorimetry, microscopy, spectrophotometry,
computer-aided visualization, and the like, or combinations
thereof.
26. The method according to claim 25, wherein the detectable signal
is selected from the group consisting of a fluorescent signal, a
phosphorescent signal, a luminescent signal, an absorbent signal,
and a chromogenic signal.
27. The method according to claim 26, wherein the fluorescent
signal is detectable by its fluorescence properties selected from
the group consisting of fluorescence resonance energy transfer
(FRET), fluorescence emission intensity, and fluorescence lifetime
(FL).
28. The method according to claim 20, wherein the Vif:Vif complex
is provided with a first detection moiety attached to the first Vif
protein or fragment and a second detection moiety attached to the
second Vif protein or fragment wherein the first detection moiety
and the second detection moiety comprise a fluorescence resonance
energy transfer (FRET) pair, wherein the first detection moiety is
a FRET donor and the second detection moiety is a FRET
acceptor.
29. (canceled)
30. (canceled)
31. The method according to claim 28, wherein the FRET donor and
the FRET acceptor comprise a fluorophore pair selected from the
group consisting of EGFP-REACh2, GFP-YFP, EGFP-YFP, EGFP-REACh2,
CFP-YFP, CFP-dsRED, BFP-GFP, GFP or YFP-dsRED, Cy3-Cy5,
Alexa488-Alexa555, Alexa488-Cy3, FITC-Rhodamine (TRITC), YFP-TRITC
or Cy3, and the like.
32. The method according to claim 20, wherein the Vif:Vif complex
is provided in a host cell co-transfected with a first plasmid
encoding the first Vif protein or fragment and a second plasmid
encoding the second Vif protein or fragment.
33. (canceled)
34. (canceled)
35. The method according to claim 32, wherein the host cell is
stably or transiently co-transfected with the first and second
plasmids.
36-50. (canceled)
51. A method for treating or preventing HIV infection or AIDS in a
patient, said method comprising: identifying an agent that disrupts
Vif self-association by performing the method according to claim
20; and administering to a patient in need of such treatment or
prevention a therapeutically effective amount of the agent.
52-55. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/900,666, filed Dec. 22, 2015, now U.S. Pat.
No. 10,588,902, issued Mar. 17, 2020, which is a U.S. National
Phase filing under 35 U.S.C. .sctn. 371 of International
Application No. PCT/US2014/043974, filed Jun. 24, 2014, and
published as WO 2014/210082-A2 on Dec. 31, 2014, which claims
priority benefit of U.S. Provisional Patent Application Ser. No.
61/838,574, filed Jun. 24, 2013. The entire contents of each of the
prior applications are incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0004] The present invention relates to the use of camptothecin
derivatives as anti-HIV agents that disrupt self-association of the
viral infectivity factor (Vif) found in HIV and other retroviruses.
The present invention also relates to methods of identifying agents
that disrupt VIf self-association and methods of using these
agents, including methods of treating or preventing HIV
infection.
BACKGROUND OF THE INVENTION
[0005] HIV-1 is the causative agent of AIDS and presently infects
approximately 33 million persons worldwide with approximately 1.9
million infected persons in North America alone. Recent studies
have shown that HIV/AIDS has become a global epidemic that is not
under control in developing nations. The rapid emergence of
drug-resistant strains of HIV throughout the world has placed a
priority on innovative approaches for the identification of novel
drug targets that may lead to a new class of anti-retroviral
therapies.
[0006] The virus contains a 10-kb single-stranded RNA genome that
encodes three major classes of gene products that include: (i)
structural proteins (Gag, Pol and Env); (ii) essential trans-acting
proteins (Tat, Rev); and (iii) "auxiliary" proteins that are not
required for efficient virus replication in permissive cells (Vpr,
Vif, Vpu, Nef) [reviewed in (1)]. There has been a heightened
interest in Vif as an antiviral target because of the discovery
that the primary function of Vif is to overcome the action of a
cellular antiviral protein known as APOBEC3G or A3G (2).
[0007] In 1984, it was determined that HIV was the virus that
causes AIDS and researchers declared that a vaccine would be
available within two years. Nearly three decades later, there is
still no vaccine available and the primary focus remains on
developing therapeutics for those already infected. Currently, the
primary HIV preventative is the combination treatment known as
STRIBILD.TM.. This medication contains 3 components of the highly
active anti-retroviral therapy (HAART) regimen (i.e., one integrase
(IN) and two reverse transcriptase (RT) inhibitors). However, HIV
strains with resistance to some or all of these components had
already emerged prior to the availability of STRIBILD.TM., thus
rendering it ineffective against such strains. Furthermore, not
only has HIV developed resistance to STRIBILD.TM. components, but
it also has developed resistance to all HAART medications to date,
including inhibitors of all HIV enzymatic and viral entry targets.
In fact, it is common to see drug resistance even among
treatment-naive individuals worldwide, emphasizing that at least
some of the current drugs have limited efficacy in a subset of
untreated, infected individuals. The barrier to developing
resistance to HIV drugs is low and often a single codon change in
the targeted protein is sufficient to cause resistance to more than
one inhibitor of the same class (i.e., M461/L/V in the HIV protease
confers resistance to 7 out of 8 inhibitors). The ever-present
problem of drug resistance together with the lack of success in
developing a vaccine accentuate the need for novel HIV prevention
and treatment strategies that are unlikely to develop
resistance.
[0008] The present invention is directed toward overcoming these
and other deficiencies in the art.
SUMMARY OF THE INVENTION
[0009] The present invention is based, in part, on the discovery
that identifying agents that disrupt Vif self-association can lead
to the identification of novel agents for use as anti-HIV
therapeutics.
[0010] In one aspect, the present invention provides compounds that
are effective as inhibitors or disruptors of Vif self-association.
The present invention further relates to various uses of these
compounds.
[0011] The present invention also provides a high throughput
primary screen for small molecules and other agents that have Vif
multimerization antagonist activity. In one embodiment, this HTS
primary screen is based on a live cell quenched fluorescence
resonance energy transfer (FRET) assay.
[0012] In a more particular embodiment, the present invention
provides a homogeneous assay based on the expression of fluorescent
protein chimeras of Vif in HEK 293T cells to achieve
distance-dependent quenching through FRET mediated by Vif
multimerization. Compounds that disrupt Vif multimerization will
yield an enhanced fluorescence signal. Hits from the primary screen
can then be subjected to an orthogonal secondary screen (e.g., in
Escherichia coli). Hits from the secondary screen can then be
validated for their (1) antiviral activity through infectivity
assays; (2) ability to inhibit co-immunoprecipitation of
differentially epitope tagged Vif; and (3) ability to protect
APOBEC3G from Vif-dependent degradation.
[0013] Compounds identified using the assays of the present
invention can be used as lead compounds to address a mandate for
novel therapeutics and also provide new research reagents to study
the structure and function of Vif.
[0014] The present invention also provides a method of treating or
preventing HIV infection or AIDS in a patient using anti-HIV agents
identified using the assay of the present invention. Further
aspects and embodiments are described in more detail herein
below.
[0015] In one aspect, the present invention addresses the
deficiency in the art of effective assays for identifying small
molecules that disrupt Vif dimerization and, therefore, have
anti-HIV activity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] For the purpose of illustrating the invention, there are
depicted in the drawings certain embodiments of the invention.
However, the invention is not limited to the precise arrangements
and instrumentalities of the embodiments depicted in the
drawings.
[0017] The patent or application file may contain at least one
drawing executed in color. Copies of this patent or patent
application publication with color drawings, if any, will be
provided by the U.S. Patent and Trademark Office upon request and
payment of the necessary fee.
[0018] FIG. 1 is a schematic illustrating how A3G can integrate
with viral particles and inhibit viral replication when Vif is
disabled.
[0019] FIG. 2 is a schematic illustrating how HIV evades A3G twice
during infection.
[0020] FIG. 3 is a graph illustrating that Vif dimerization
antagonist peptides suppress HIV-1 infectivity. MT2 cells grown in
microtiter dishes were infected with live HIV-1 virus at 0.01 MOI
and treated every other day with either AZT (1 .mu.M), Control
peptide (50 .mu.M), Peptide 1 (50 .mu.M) or Peptide 2 (50 .mu.M) or
left untreated (viral control). At the indicated days
post-infection, cells were harvested for cell lysate preparation
and reverse transcriptase quantification. Lysates were prepared
from parallel cultures of uninfected and untreated cells (cell
control) as controls for the reverse transcriptase assays.
[0021] FIG. 4 are schematics illustrating an antiviral strategy
that involves disrupting Vif dimerization. The left schematic
illustrates HIV life cycle enabled with Vif dimer formed. The right
schematic illustrates A3G host-defense enabled without Vif
dimer.
[0022] FIG. 5A is a schematic showing the qFRET assay for use in
identifying small molecules that interfere with Vif
self-association.
[0023] FIG. 5B are photographs (top panels) and Western blots
(bottom panels) illustrating in-cell high throughput screen for Vif
dimerization antagonists.
[0024] FIG. 6 is a graph showing hit rate from screening a small
library of compounds using one embodiment of an assay according to
the present invention.
[0025] FIG. 7 is a flowchart illustrating one embodiment of a
critical path to finding an initial hit for a Vif inhibitor small
molecule (e.g., camptothecin, CPT).
[0026] FIG. 8 illustrates aspects of a medicinal chemistry strategy
for modifying an initial hit from a Vif inhibitor assay of the
present invention for pharmaceutical use.
[0027] FIGS. 9A and 9B illustrate results of an in vitro drug
screening assay for toxicity of the OYA002-16 small molecule of the
present invention.
[0028] FIG. 10 illustrates in-cell secondary screen fro
Vif-dependent A3G degradation.
[0029] FIG. 11 are graphs showing results from a primary screen
(left side) and a secondary screen (right side) of the OYA002-16
small molecule scaffold.
[0030] FIG. 12A is a schematic showing aspects of a single cycle
infectivity assay of the present disclosure.
[0031] FIG. 12B are graphs illustrating that OYA002-16 maintains an
A3G and Vif-dependent antiviral effect in single cycle HIV
infectivity experiments. The left graph illustrates pseudotyped
single cycle HIV infections. The right graph shows Vif and
A3G-dependent effect on pseudotyped HIV infectivity expressed as %
of control.
[0032] FIG. 13 illustrates results of experiments showing that
OYA002-16 increases A3G in viral particles.
[0033] FIG. 14 is an illustration relating to a Next Gen Sequencing
for A3G signature mutation profiles.
[0034] FIG. 15 illustrates graphs showing results relating to
efficacy studies of lead scaffold OYA002-16.
[0035] FIGS. 16A-1, 16A-2, 16A-3, 16A-4 are graphs showing results
of efficacy studies with regard to OYA002-16.
[0036] FIGS. 16B-1 and 16B-2 are graphs and illustrations showing
results of efficacy studies with regard to OYA002-16 and
OYA004-06.
[0037] FIG. 17 is a graph showing results of toxicity studies
relating to lead scaffold OYA002-16.
[0038] FIG. 18 is a graph showing results of stability and
solubility studies relating to lead scaffold OYA002-16.
[0039] FIGS. 19A and 19B are illustrations showing that unbiased
computer docking predicts OYA002-16 scaffold favorably binds to
solvent exposed surface of Vif.
[0040] FIGS. 20A-1 and 20A-2 illustrate lead optimization goals for
improving solubility and decreasing toxicity with respect to a Vif
inhibitor of the present disclosure.
[0041] FIG. 20B illustrate lead optimization goals for improving
solubility and decreasing toxicity with respect to a Vif inhibitor
of the present disclosure.
[0042] FIG. 21A illustrates derivatives of one small molecule lead
scaffold of the present disclosure.
[0043] FIG. 21B are schematics of the synthetic pathways of various
embodiments of the Vif inhibitor for use in the present invention,
including deoxycamptothecin lactam (denoted as formula 2 in the
figure) (corresponds to Formula (I-c)), 9-glycinamido camptothecin
lactam (denoted as formula 5 in the figure) (corresponds to Formula
(I-d)),9-glycinamido deoxycamptothecin lactam (denoted as formula 7
in the figure) (corresponds to Formula (I-e)), topotecan lactam
(denoted as formula 9 in the figure) (corresponds to Formula
(I-f)), and deoxytopotecan lactam (denoted as formula 10 in the
figure) (corresponds to Formula (I-g)).
DETAILED DESCRIPTION OF THE INVENTION
[0044] The present invention is based, in part, on the discovery
that disrupting self-association of the HIV viral infectivity
factor (Vif) can be a mechanism for use in identifying agents that
can be used as anti-HIV agents.
[0045] Vif binds to and induces the destruction of APOBEC3G (also
referred to herein as "A3G"), which is a broad antiviral
host-defense factor. Therefore, Vif is essential for HIV infection.
Vif subunits interact to form multimers and this property has been
shown to be necessary for HIV infectivity. The segment of Vif that
mediates subunit interaction was previously determined to be
proline-proline-leucine-proline (PPLP). However, to date, there has
not been an effective high throughput screening (HTS) assay to
identify agents that disrupt Vif self-association. The present
invention is effective to address this need.
[0046] As shown in FIG. 1, A3G can integrate with viral particles
and inhibit viral replication when Vif is disabled. For example, as
shown in FIG. 1: Part 1: In the absence of a functional Vif A3G is
incorporated into viral particles and is bound to viral RNA upon
release of the viral proteins into the target cell. Part 2: During
reverse transcription of the viral RNA into proviral DNA A3G causes
dC to dU hypermutations (X) on the viral minus strand that is
single-stranded in the small window of time before the positive
strand is synthesized. Part 3: Mutated proviral DNA is either
degraded by DNA repair machinery recognizing dU in DNA or
incorporated into the host genome with dG to dA mutations in the
positive strand since reverse transcriptase reads the dU mutations
as needing a dA complementary nucleotide instead of a dG. Part 4:
The virus uses the host cell machinery to make mutated viral RNA
and proteins; the mutations cause missense and stop codons that are
catastrophic for viral function. Part 5: Thus, the progeny virions
are defective and non-infective.
[0047] As shown in FIG. 2, HIV evades A3G twice during infection.
For example, as shown in FIG. 2, HIV Ssuppresses A3G host defense
activity in permissive cells by: (1) Early Block: HIV infection
induces A3G inactivation through complex formation with host cell
RNAs. (2) Late Block: Vif is expressed and Vif dimers bind to A3G
to direct its destruction before A3G can be packaged with
virions.
[0048] Inhibitors of Vif Self-Association
[0049] In one aspect, the present invention provides small molecule
compounds that are inhibitors of Vif self-association (also
referred to generally herein as "Vif inhibitors").
[0050] Vif binds to and induces the destruction of APOBEC3G (also
referred to herein as "A3G"), which is a broad antiviral
host-defense factor. Therefore, Vif is essential for HIV infection.
Vif subunits interact to form multimers and this property has been
shown to be necessary for HIV infectivity. The segment of Vif that
mediates subunit interaction was previously determined to be
proline-proline-leucine-proline (PPLP) (see FIG. 3). As shown in
FIG. 3, its has been confirmed and established that the antiviral
activity of VDA peptides were dependent on Vif and A3G for highest
efficacy (J H Miller et al., Retrovirology 2007).
[0051] In various embodiments, small molecule compounds or other
agents that disrupt Vif self-association (also referred to herein
as "Vif dimerization" or "Vif multimerization") are suitable as Vif
inhibitors in accordance with the present invention. As shown in
FIG. 4, as provided by the present disclosure, one antiviral
strategy aginst HIV is to disrupt Vif dimerization.
[0052] In one embodiment, the Vif inhibitor of the present
invention is effective to inhibit Vif dimerization by direct or
indirect inhibition of binding of Vif dimers at the Vif
dimerization domain, said Vif dimerization domain comprising the
amino acid sequence of proline-proline-leucine-proline (PPLP). In
another embodiment, the Vif inhibitor of the present invention is
effective to inhibit Vif from binding to A3G. In another
embodiment, the
[0053] Vif inhibitor of the present invention is effective to
inhibit Vif-dependent degradation of A3G. In another embodiment,
the Vif inhibitor of the present invention is effective to inhibit
Vif-dependent degradation of A3G by inhibiting interaction of Vif
with one or more enzymes selected from the group consisting of
Cullin 5, Elongin B, and Elongin C, thereby inhibiting
ubiquitination of A3G.
[0054] Vif inhibitors for use in the methods of the present
disclosure are as disclosed herein.
[0055] In certain embodiments, the present invention provides
camptothecin derivatives as small molecule compounds that were
identified using the screening assay of the present invention. The
small molecule compounds are effective as inhibitors of Vif
self-association.
[0056] In certain embodiments, the Vif inhibitor of the present
invention can include, without limitation, camptotechin, topotecan,
irinotecan, and analogs thereof and those having a related chemical
scaffold (chemotype) thereof. Certain camptothecin derivatives for
use as Vif inhibitors of the present disclosure are illustrated by
chemical structure in FIGS. 7, 8, 16B-2, 20A-1, 20B, 21A, and 21B
hereof.
[0057] In one aspect, the present invention provides a compound of
formula (I):
##STR00001##
or a pharmaceutically acceptable salt thereof, wherein:
[0058] Q is selected from NH, O, and S;
[0059] R.sup.20a and R.sup.20b are individually selected from
hydrogen, hydroxy, and C.sub.1-6alkyl;
[0060] R.sup.21 is selected from hydrogen,
--NHC(.dbd.O)(CH.sub.2).sub.pNR.sup.23R.sup.24 and
(CH.sub.2).sub.pNR.sup.23R.sup.24;
[0061] p is 0, 1, 2, 3, or 4;
[0062] R.sup.22 is selected from hydrogen and hydroxyl;
[0063] R.sup.23 and R.sup.24 are individually selected from
hydrogen and C.sub.1-6alkyl; and
[0064] R.sup.25 and R.sup.26 are individually selected from
hydrogen and --NO.sub.2.
[0065] In one sub-group of compounds, Q is O.
[0066] In one sub-group of compounds, Q is selected from NH and
S.
[0067] In a particular sub-group of compounds, Q is NH.
[0068] In another particular sub-group of compounds, Q is S.
[0069] In one sub-group of compounds, R.sup.20a is
C.sub.1-6alkyl.
[0070] In one sub-group of compounds, R.sup.20a and R.sup.20b are
individually selected from hydrogen, hydroxy, methyl, and
ethyl.
[0071] In one sub-group of compounds R.sup.20a is selected from
hydrogen and hydroxy.
[0072] In a particular sub-group of compounds, R.sup.20a is
hydrogen.
[0073] In another particular sub-group of compounds, R.sup.20a is
hydroxy.
[0074] In one sub-group of compounds R.sup.20b is
C.sub.1-6alkyl.
[0075] In one sub-group of compounds R.sup.20b is hydrogen.
[0076] In a particular sub-group of compounds, R.sup.20b is
ethyl.
[0077] In one sub-group of compounds, R.sup.20a is hydrogen or
hydroxy and R.sup.20b is ethyl.
[0078] In a particular sub-group of compounds, R.sup.20a is
hydrogen and R.sup.20b is ethyl.
[0079] In a particular sub-group of compounds, R.sup.20a is hydroxy
and R.sup.20b is ethyl.
[0080] In one sub-group of compounds, R.sup.21 is hydrogen.
[0081] In another sub-group of compounds, R.sup.21 is
--NHC(.dbd.O)(CH.sub.2).sub.pNR.sup.23R.sup.24 (e.g.,
--NHC(.dbd.O)CH.sub.2NR.sup.23R.sup.24, such as
--NHC(.dbd.O)CH.sub.2NH.sub.2)
[0082] In another sub-group of compounds, R.sub.21 is
--NHC(CH.sub.2).sub.pNR.sup.23R.sup.24 (e.g.,
--CH.sub.2N(CH.sub.3).sub.2).
[0083] In one subgroup of compounds, p is 1, 2, 3, or 4. In a
particular subgroup, p is 1 or 2. In a more particular subgroup of
compounds, p is 1.
[0084] In one subgroup of compounds, R.sup.22 is hydrogen.
[0085] In one subgroup of compounds, R.sup.22 is hydroxyl.
[0086] In one subgroup, R.sup.23 and R.sup.24 are individually
selected from hydrogen and methyl.
[0087] In one subgroup of compounds, R.sup.25 and R.sup.26 are both
hydrogen.
[0088] In one subgroup of compounds, R.sup.25 and R.sup.26 are both
--NO.sub.2.
[0089] In particular embodiments, a suitable Vif inhibitor of
formula (I) is a compound selected from the group consisting of
##STR00002## ##STR00003##
[0090] The compound of formula (I-a) is also referred to herein as
"OYA-002-16" or "OYA002-16." The compound of formula (I-b) is also
referred to herein as "OYA-004-006" or "OYA004-006." The compound
of formula (I-c) is also referred to herein as deoxycamptothecin
lactam (denoted as formula 2 in the figure). The compound of
formula (I-d) is also referred to herein as 9-glycinamido
camptothecin lactam (denoted as formula 5 in the figure). The
compound of formula (I-e) is also referred to herein as
9-glycinamido deoxycamptothecin lactam (denoted as formula 7 in the
figure). The compound of formula (I-f) is also referred to herein
as topotecan lactam (denoted as formula 9 in the figure). The
compound of formula (I-g) is also referred to herein as and
deoxytopotecan lactam (denoted as formula 10 in the figure).
[0091] The Vif inhibitor compounds for use in the methods of the
present invention can include functional derivatives of any of the
Vif inhibitor compounds disclosed herein, and pharmaceutically
acceptable salts thereof.
[0092] In another aspect, the present invention provides
pharmaceutical compositions comprising a compound of Formula
(I).
[0093] As used herein, the following definitions shall apply unless
otherwise indicated. For purposes of this invention, the chemical
elements are identified in accordance with the Periodic Table of
the Elements, CAS version, Handbook of Chemistry and Physics, 75th
Ed. Additionally, general principles of organic chemistry are
described in "Organic Chemistry", Thomas Sorrell, University
Science Books, Sausalito: 1999, and "March's Advanced Organic
Chemistry", 5th Ed., Ed.: Smith, M. B. and March, J., John Wiley
& Sons, New York: 2001, which are herein incorporated by
reference in their entirety.
[0094] Unless otherwise specified, alkyl is intended to include
linear, branched, or cyclic hydrocarbon structures and combinations
thereof. A combination would be, for example, cyclopropylmethyl.
Lower alkyl refers to alkyl groups of from 1 to 6 carbon atoms.
Examples of lower alkyl groups include methyl, ethyl, propyl,
isopropyl, butyl, s-and t-butyl and the like. Preferred alkyl
groups are those of C.sub.20 or below. Cycloalkyl is a subset of
alkyl and includes cyclic hydrocarbon groups of from 3 to 8 carbon
atoms. Examples of cycloalkyl groups include c-propyl, c-butyl,
c-pentyl, norbornyl and the like.
[0095] C.sub.1 to C.sub.20 hydrocarbon includes alkyl, cycloalkyl,
polycycloalkyl, alkenyl, alkynyl, aryl and combinations thereof.
Examples include benzyl, phenethyl, cyclohexylmethyl, camphoryl and
naphthylethyl. Hydrocarbon refers to any substituent comprised of
hydrogen and carbon as the only elemental constituents.
[0096] An "alkenyl" group refers to an unsaturated hydrocarbon
group containing at least one carbon-carbon double bond, including
straight-chain, branched-chain, and cyclic groups. Preferably, the
alkenyl group has 1 to 12 carbons. More perferably it is a lower
alkenyl of from 1 to 7 carbons. The alkenyl group may be
substituted or unsubstituted. When substituted, the substituted
group(s) is preferably hydroxyl, cyano, alkoxy, .dbd.O, .dbd.S,
NO.sub.2, N(CH.sub.3).sub.2, halogen, amino, or SH.
[0097] An "alkynyl" group refers to an unsaturated hydrocarbon
group containing at least one carbon-carbon triple bond, including
straight-chain, branched chain, and cyclic groups. Preferably, the
alkynyl group has 1 to 12 carbons. More perferably it is a lower
alkynyl of from 1 to 7 carbons. The alkynyl group may be
substituted or unsubstituted. When substituted, the substituted
group(s) is preferably hydroxyl, cyano, alkoxy, .dbd.O, .dbd.S,
NO.sub.2, N(CH.sub.3).sub.2, amino, or SH.
[0098] "Alkylene" means a linear saturated divalent hydrocarbon
radical of one to six carbon atoms or a branched saturated divalent
hydrocarbon radical of three to six carbon atoms, e.g., methylene,
ethylene, 2,2-dimethylethylene, propylene, 2-methylpropylene,
butylene, pentylene, and the like.
[0099] "Alkoxyalkyl" means a moiety of the formula
R.sup.a--O--R.sup.b--, where R.sup.a is alkyl and R.sup.b is
alkylene as defined herein. Exemplary alkoxyalkyl groups include,
by way of example, 2-methoxyethyl, 3-methoxypropyl,
1-methyl-2-methoxyethyl, 1-(2-methoxyethyl)-3-methoxypropyl, and
1-(2-methoxyethyl)-3-methoxypropyl.
[0100] The abbreviations Me, Et, Ph, Tf, Ts and Ms represent
methyl, ethyl, phenyl, trifluoromethanesulfonyl, toluenesulfonyl
and methanesulfonyl, respectively. A comprehensive list of
abbreviations utilized by organic chemists (i.e., persons of
ordinary skill in the art) appears in the first issue of each
volume of the Journal of Organic Chemistry. The list, which is
typically presented in a table entitled "Standard List of
Abbreviations" is incorporated herein by reference.
[0101] Unless otherwise specified, the term "carbocycle" is
intended to include ring systems in which the ring atoms are all
carbon but of any oxidation state. Thus (C.sub.3-C.sub.10)
carbocycle refers to both non-aromatic and aromatic systems,
including such systems as cyclopropane, benzene and cyclohexene;
(C.sub.8-C.sub.12) carbopolycycle refers to such systems as
norbornane, decalin, indane and naphthalene. Carbocycle, if not
otherwise limited, refers to monocycles, bicycles and
polycycles.
[0102] Alkoxy or alkoxyl refers to groups of from 1 to 8 carbon
atoms of a straight, branched or cyclic configuration and
combinations thereof attached to the parent structure through an
oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy,
cyclopropyloxy, cyclohexyloxy and the like. Lower-alkoxy refers to
groups containing one to four carbons. For the purpose of this
application, alkoxy and lower alkoxy include methylenedioxy and
ethylenedioxy. A particular subgroup of alkoxy is C.sub.1-6alkoxy,
which refers to alkoxy having 1, 2, 3, 4, 5, or 6 carbon atoms.
[0103] Oxaalkyl refers to alkyl residues in which one or more
carbons (and their associated hydrogens) have been replaced by
oxygen. Examples include methoxypropoxy, 3,6,9-trioxadecyl and the
like. The term oxaalkyl is intended as it is understood in the art
[see Naming and Indexing of Chemical Substances for Chemical
Abstracts, published by the American Chemical Society, 196, but
without the restriction of 127(a)], i.e. it refers to compounds in
which the oxygen is bonded via a single bond to its adjacent atoms
(forming ether bonds); it does not refer to doubly bonded oxygen,
as would be found in carbonyl groups. Similarly, thiaalkyl and
azaalkyl refer to alkyl residues in which one or more carbons has
been replaced by sulfur or nitrogen, respectively. Examples include
ethylaminoethyl and methylthiopropyl.
[0104] Unless otherwise specified, acyl refers to formyl and to
groups of 1, 2, 3, 4, 5, 6, 7 and 8 carbon atoms of a straight,
branched, cyclic configuration, saturated, unsaturated and aromatic
and combinations thereof, attached to the parent structure through
a carbonyl functionality. One or more carbons in the acyl residue
may be replaced by nitrogen, oxygen or sulfur as long as the point
of attachment to the parent remains at the carbonyl. Examples
include acetyl, benzoyl, propionyl, isobutyryl, t-butoxycarbonyl,
benzyloxycarbonyl and the like. Lower-acyl refers to groups
containing one to four carbons. The double bonded oxygen, when
referred to as a substituent itself is called "oxo".
[0105] Aryl and heteroaryl mean (i) a phenyl group (or benzene) or
a monocyclic 5- or 6-membered heteroaromatic ring containing 1-4
heteroatoms selected from O, N, or S; (ii) a bicyclic 9- or
10-membered aromatic or heteroaromatic ring system containing 0-4
heteroatoms selected from O, N, or S; or (iii) a tricyclic 13- or
14-membered aromatic or heteroaromatic ring system containing 0-5
heteroatoms selected from O, N, or S. The aromatic 6- to
14-membered carbocyclic rings include, e.g., benzene, naphthalene,
indane, tetralin, and fluorene and the 5- to 10-membered aromatic
heterocyclic rings include, e.g., imidazole, pyridine, indole,
thiophene, benzopyranone, thiazole, furan, benzimidazole,
quinoline, isoquinoline, quinoxaline, pyrimidine, pyrazine,
tetrazole and pyrazole. As used herein aryl and heteroaryl refer to
residues in which one or more rings are aromatic, but not all need
be.
[0106] Arylalkyl refers to a substituent in which an aryl residue
is attached to the parent structure through alkyl. Examples are
benzyl, phenethyl and the like. Heteroarylalkyl refers to a
substituent in which a heteroaryl residue is attached to the parent
structure through alkyl. In one embodiment, the alkyl group of an
arylalkyl or a heteroarylalkyl is an alkyl group of from 1 to 6
carbons. Examples include, e.g., pyridinylmethyl, pyrimidinylethyl
and the like.
[0107] Heterocycle means a cycloalkyl or aryl carbocycle residue in
which from one to three carbons is replaced by a heteroatom
selected from the group consisting of N, O and S. The nitrogen and
sulfur heteroatoms may optionally be oxidized, and the nitrogen
heteroatom may optionally be quaternized. Unless otherwise
specified, a heterocycle may be non-aromatic or aromatic. Examples
of heterocycles that fall within the scope of the invention include
pyrrolidine, pyrazole, pyrrole, indole, quinoline, isoquinoline,
tetrahydroisoquinoline, benzofuran, benzodioxan, benzodioxole
(commonly referred to as methylenedioxyphenyl, when occurring as a
substituent), tetrazole, morpholine, thiazole, pyridine,
pyridazine, pyrimidine, thiophene, furan, oxazole, oxazoline,
isoxazole, dioxane, tetrahydrofuran and the like. It is to be noted
that heteroaryl is a subset of heterocycle in which the heterocycle
is aromatic. Examples of heterocyclyl residues additionally include
piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl,
2-oxo-pyrrolidinyl, 2-oxoazepinyl, azepinyl, 4-piperidinyl,
pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyrazinyl,
oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolyl,
quinuclidinyl, isothiazolidinyl, benzimidazolyl, thiadiazolyl,
benzopyranyl, benzothiazolyl, tetrahydrofuryl, tetrahydropyranyl,
thienyl, benzothienyl, thiamorpholinyl, thiamorpholinylsulfoxide,
thiamorpholinylsulfone, oxadiazolyl, triazolyl and
tetrahydroquinolinyl.
[0108] As used herein, the term "optionally substituted" may be
used interchangeably with "unsubstituted or substituted". The term
"substituted" refers to the replacement of one or more hydrogen
atoms in a specified group with a specified radical. For example,
substituted alkyl, aryl, cycloalkyl, heterocyclyl etc. refer to
alkyl, aryl, cycloalkyl, or heterocyclyl wherein one or more H
atoms in each residue are replaced with halogen, haloalkyl, alkyl,
acyl, alkoxyalkyl, hydroxyloweralkyl, carbonyl, phenyl, heteroaryl,
benzenesulfonyl, hydroxy, loweralkoxy, haloalkoxy, oxaalkyl,
carboxy, alkoxycarbonyl [-C(.dbd.O)O-alkyl], alkoxycarbonylamino
[HNC(.dbd.O)O-alkyl], carboxamido [--C(.dbd.O)NH.sub.2],
alkylaminocarbonyl [--C(.dbd.O)NH-alkyl], cyano, acetoxy, nitro,
amino, alkylamino, dialkylamino, (alkyl)(aryl)aminoalkyl,
alkylaminoalkyl (including cycloalkylaminoalkyl),
dialkylaminoalkyl, dialkylaminoalkoxy, heterocyclylalkoxy,
mercapto, alkylthio, sulfoxide, sulfone, sulfonylamino,
alkylsulfinyl, alkylsulfonyl, acylaminoalkyl, acylaminoalkoxy,
acylamino, amidino, aryl, benzyl, heterocyclyl, heterocyclylalkyl,
phenoxy, benzyloxy, heteroaryloxy, hydroxyimino, alkoxyimino,
oxaalkyl, aminosulfonyl, trityl, amidino, guanidino, ureido,
benzyloxyphenyl, and benzyloxy. "Oxo" is also included among the
substituents referred to in "optionally substituted"; it will be
appreciated by persons of skill in the art that, because oxo is a
divalent radical, there are circumstances in which it will not be
appropriate as a substituent (e.g. on phenyl). In one embodiment,
1, 2 or 3 hydrogen atoms are replaced with a specified radical. In
the case of alkyl and cycloalkyl, more than three hydrogen atoms
can be replaced by fluorine; indeed, all available hydrogen atoms
could be replaced by fluorine.
[0109] The terms "haloalkyl" and "haloalkoxy" mean alkyl or alkoxy,
respectively, substituted with one or more halogen atoms. The terms
"alkylcarbonyl" and "alkoxycarbonyl" mean --C(.dbd.O)alkyl or
--C(O)alkoxy, respectively.
[0110] The term "halogen" means fluorine, chlorine, bromine or
iodine. In one embodiment, halogen may be fluorine or chlorine.
[0111] The term "heterocyclic group" includes within its scope
aromatic, non-aromatic, unsaturated, partially saturated and fully
saturated heterocyclic ring systems. In general, such groups may be
monocyclic or bicyclic and may contain, for example, 3 to 12 ring
members, more usually 5 to 10 ring members. Examples of monocyclic
groups are groups containing 3, 4, 5, 6, 7, and 8 ring members,
more usually 3 to 7, and preferably 5 or 6 ring members. A
particular non-limiting example is a morpholinyl group.
[0112] It is understood that any alkyl, alkenyl, alkynyl,
cycloalkyl and cycloalkenyl moiety described herein can also be an
aliphatic group, an alicyclic group or a heterocyclic group. An
"aliphatic group" is non-aromatic moiety that may contain any
combination of carbon atoms, hydrogen atoms, halogen atoms, oxygen,
nitrogen or other atoms, and optionally contain one or more units
of unsaturation, e.g., double and/or triple bonds. An aliphatic
group may be straight chained, branched or cyclic and preferably
contains between about 1 and about 24 carbon atoms, more typically
between about 1 and about 12 carbon atoms. In addition to aliphatic
hydrocarbon groups, aliphatic groups include, for example,
polyalkoxyalkyls, such as polyalkylene glycols, polyamines, and
polyimines, for example. Such aliphatic groups may be further
substituted. It is understood that aliphatic groups may be used in
place of the alkyl, alkenyl, alkynyl, alkylene, alkenylene, and
alkynylene groups described herein.
[0113] Substituents R.sup.n are generally defined when introduced
and retain that definition throughout the specification and in all
independent claims.
[0114] As used herein, and as would be understood by the person of
skill in the art, the recitation of "a compound"--unless expressly
further limited--is intended to include salts, solvates and
inclusion complexes of that compound. Unless otherwise stated or
depicted, structures depicted herein are also meant to include all
stereoisomeric (e.g., enantiomeric, diastereomeric, and cis-trans
isomeric) forms of the structure; for example, the R and S
configurations for each asymmetric center, (Z) and (E) double bond
isomers, and (Z) and (E) conformational isomers. Therefore, single
stereochemical isomers as well as enantiomeric, diastereomeric, and
cis-trans isomeric (or conformational) mixtures of the present
compounds are within the scope of the invention. Unless otherwise
stated, all tautomeric forms of the compounds of the invention are
within the scope of the invention. Additionally, unless otherwise
stated, structures depicted herein are also meant to include
compounds that differ only in the presence of one or more
isotopically enriched atoms. For example, compounds having the
present structures except for the replacement of hydrogen by
deuterium or tritium, or the replacement of a carbon by a
.sup.13C-- or .sup.14C-enriched carbon are within the scope of this
invention. Such compounds are useful, for example, as analytical
tools or probes in biological assays. The term "solvate" refers to
a compound of Formula I in the solid state, wherein molecules of a
suitable solvent are incorporated in the crystal lattice. A
suitable solvent for therapeutic administration is physiologically
tolerable at the dosage administered. Examples of suitable solvents
for therapeutic administration are ethanol and water. When water is
the solvent, the solvate is referred to as a hydrate. In general,
solvates are formed by dissolving the compound in the appropriate
solvent and isolating the solvate by cooling or using an
antisolvent. The solvate is typically dried or azeotroped under
ambient conditions. Inclusion complexes are described in Remington:
The Science and Practice of Pharmacy 19.sup.th Ed. (1995) volume 1,
page 176-177, which is incorporated herein by reference. The most
commonly employed inclusion complexes are those with cyclodextrins,
and all cyclodextrin complexes, natural and synthetic, are
specifically encompassed within the claims.
[0115] The term "pharmaceutically acceptable salt" refers to salts
prepared from pharmaceutically acceptable non-toxic acids or bases
including inorganic acids and bases and organic acids and bases.
When the compounds of the present invention are basic, salts may be
prepared from pharmaceutically acceptable non-toxic acids including
inorganic and organic acids. Suitable pharmaceutically acceptable
acid addition salts for the compounds of the present invention
include acetic, adipic, alginic, ascorbic, aspartic,
benzenesulfonic (besylate), benzoic, boric, butyric, camphoric,
camphorsulfonic, carbonic, citric, ethanedisulfonic,
ethanesulfonic, ethylenediaminetetraacetic, formic, fumaric,
glucoheptonic, gluconic, glutamic, hydrobromic, hydrochloric,
hydroiodic, hydroxynaphthoic, isethionic, lactic, lactobionic,
laurylsulfonic, maleic, malic, mandelic, methanesulfonic, mucic,
naphthylenesulfonic, nitric, oleic, pamoic, pantothenic,
phosphoric, pivalic, polygalacturonic, salicylic, stearic,
succinic, sulfuric, tannic, tartaric acid, teoclatic,
p-toluenesulfonic, and the like. When the compounds contain an
acidic side chain, suitable pharmaceutically acceptable base
addition salts for the compounds of the present invention include,
but are not limited to, metallic salts made from aluminum, calcium,
lithium, magnesium, potassium, sodium and zinc or organic salts
made from lysine, arginine, N,N'-dibenzylethylenediamine,
chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine
(N-methylglucamine) and procaine. Further pharmaceutically
acceptable salts include, when appropriate, nontoxic ammonium
cations and carboxylate, sulfonate and phosphonate anions attached
to alkyl having from 1 to 20 carbon atoms.
[0116] While it may be possible for the compounds of the invention
to be administered as the raw chemical, it is preferable to present
them as a pharmaceutical composition. According to a further
aspect, the present invention provides a pharmaceutical composition
comprising a compound of the invention or a pharmaceutically
acceptable salt or solvate thereof, together with one or more
pharmaceutical carriers thereof and optionally one or more other
therapeutic ingredients. The carrier(s) must be "acceptable" in the
sense of being compatible with the other ingredients of the
formulation and not deleterious to the recipient thereof.
[0117] As used herein, the term "physiologically functional
derivative" refers to any pharmaceutically acceptable derivative of
a compound of the present invention that, upon administration to a
mammal, is capable of providing (directly or indirectly) a compound
of the present invention or an active metabolite thereof. Such
derivatives, for example, esters and amides, will be clear to those
skilled in the art, without undue experimentation. Reference may be
made to the teaching of Burger's Medicinal Chemistry And Drug
Discovery, 5.sup.th Edition, Vol 1: Principles and Practice, which
is incorporated herein by reference to the extent that it teaches
physiologically functional derivatives.
[0118] As used herein, the term "effective amount" means that
amount of a drug or pharmaceutical agent that will elicit the
biological or medical response of a tissue, system, animal, or
human that is being sought, for instance, by a researcher or
clinician. The term "therapeutically effective amount" means any
amount which, as compared to a corresponding subject who has not
received such amount, results in improved treatment, healing,
prevention, or amelioration of a disease, disorder, or side effect,
or a decrease in the rate of advancement of a disease or disorder.
The term also includes within its scope amounts effective to
enhance normal physiological function. For use in therapy,
therapeutically effective amounts of a compound of the present
invention, as well as salts, solvates, and physiological functional
derivatives thereof, may be administered as the raw chemical.
Additionally, the active ingredient may be presented as a
pharmaceutical composition.
[0119] Pharmaceutical compositions of the present invention
comprise an effective amount of one or more compound of the present
invention, or additional agent dissolved or dispersed in a
pharmaceutically acceptable carrier. The phrases "pharmaceutical or
pharmacologically acceptable" refers to molecular entities and
compositions that do not produce an adverse, allergic or other
untoward reaction when administered to an animal, such as, for
example, a human, as appropriate. The preparation of a
pharmaceutical composition that contains at least one compound of
the present invention, or additional active ingredient will be
known to those of skill in the art in light of the present
disclosure, as exemplified by Remington's Pharmaceutical Sciences,
18th Ed. Mack Printing Company, 1990, incorporated herein by
reference. Moreover, for animal (e.g., human) administration, it
will be understood that preparations should meet sterility,
pyrogenicity, general safety and purity standards as required by
FDA Office of Biological Standards.
[0120] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
surfactants, antioxidants, preservatives (e.g., antibacterial
agents, antifungal agents), isotonic agents, absorption delaying
agents, salts, preservatives, drugs, drug stabilizers, gels,
binders, excipients, disintegration agents, lubricants, sweetening
agents, flavoring agents, dyes, such like materials and
combinations thereof, as would be known to one of ordinary skill in
the art (see, for example, Remington's Pharmaceutical Sciences,
18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated
herein by reference). Except insofar as any conventional carrier is
incompatible with the active ingredient, its use in the
pharmaceutical compositions is contemplated.
[0121] The term "lentivirus" as used herein may be any of a variety
of members of this genus of viruses. The lentivirus may be, e.g.,
one that infects a mammal, such as a sheep, goat, horse, cow or
primate, including human. Typical such viruses include, e.g., Vizna
virus (which infects sheep); simian immunodeficiency virus (SIV),
bovine immunodeficiency virus (BIV), chimeric simian/human
immunodeficiency virus (SHIV), feline immunodeficiency virus (FIV)
and human immunodeficiency virus (HIV). "HIV," as used herein,
refers to both HIV-1 and HIV-2. Much of the discussion herein is
directed to HIV or HIV-1; however, it is to be understood that
other suitable lentiviruses are also included.
[0122] The term "mammal" as used herein refers to any non-human
mammal. Such mammals are, for example, rodents, non-human primates,
sheep, dogs, cows, and pigs. The preferred non-human mammals are
selected from the rodent family including rat and mouse, more
preferably mouse. The preferred mammal is a human.
[0123] As used herein, the terms "peptide," "polypeptide," and
"protein" are used interchangeably, and refer to a compound
comprised of amino acid residues covalently linked by peptide
bonds. A protein or peptide must contain at least two amino acids,
and no limitation is placed on the maximum number of amino acids
which can comprise a protein's or peptide's sequence. Polypeptides
include any peptide or protein comprising two or more amino acids
joined to each other by peptide bonds. As used herein, the term
refers to both short chains, which also commonly are referred to in
the art as peptides, oligopeptides and oligomers, for example, and
to longer chains, which generally are referred to in the art as
proteins, of which there are many types. "Polypeptides" include,
for example, biologically active fragments, substantially
homologous polypeptides, oligopeptide, homodimers, heterodimers,
variants of polypeptides, modified polypeptides, derivatives,
analogs, fusion proteins, among others. The polypeptides include
natural peptides, recombinant peptides, synthetic peptides, or a
combination thereof.
[0124] "Pharmaceutically acceptable" means physiologically
tolerable, for either human or veterinary applications. In
addition, "pharmaceutically acceptable" is meant a material that is
not biologically or otherwise undesirable, i.e., the material may
be administered to a subject without causing any undesirable
biological effects or interacting in a deleterious manner with any
of the other components of the pharmaceutical composition in which
it is contained. Essentially, the pharmaceutically acceptable
material is nontoxic to the recipient. The carrier would naturally
be selected to minimize any degradation of the active ingredient
and to minimize any adverse side effects in the subject, as would
be well known to one of skill in the art. For a discussion of
pharmaceutically acceptable carriers and other components of
pharmaceutical compositions, see, e.g., Remington's Pharmaceutical
Sciences, 18th ed., Mack Publishing Company, 1990.
[0125] As used herein, "pharmaceutical compositions" include
formulations for human and veterinary use.
[0126] As used herein, the terms "prevent," "preventing,"
"prevention," "prophylactic treatment" and the like refer to
reducing the probability of developing a disorder or condition in a
subject, who does not have, but is at risk of or susceptible to
developing a disorder or condition.
[0127] "Test agents" or otherwise "test compounds" as used herein
refers to an agent or compound that is to be screened in one or
more of the assays described herein. Test agents include compounds
of a variety of general types including, but not limited to, small
organic molecules, known pharmaceuticals, polypeptides;
carbohydrates such as oligosaccharides and polysaccharides;
polynucleotides; lipids or phospholipids; fatty acids; steroids; or
amino acid analogs. Test agents can be obtained from libraries,
such as natural product libraries and combinatorial libraries. In
addition, methods of automating assays are known that permit
screening of several thousands of compounds in a short period.
[0128] As used herein, the terms "treat," "treating," "treatment,"
and the like refer to reducing or ameliorating a disorder and/or
symptoms associated therewith. It will be appreciated that,
although not precluded, treating a disorder or condition does not
require that the disorder, condition or symptoms associated
therewith be completely eliminated.
[0129] "Variant" as the term is used herein, is a nucleic acid
sequence or a peptide sequence that differs in sequence from a
reference nucleic acid sequence or peptide sequence respectively,
but retains essential properties of the reference molecule. Changes
in the sequence of a nucleic acid variant may not alter the amino
acid sequence of a peptide encoded by the reference nucleic acid,
or may result in amino acid substitutions, additions, deletions,
fusions and truncations. Changes in the sequence of peptide
variants are typically limited or conservative, so that the
sequences of the reference peptide and the variant are closely
similar overall and, in many regions, identical. A variant and
reference peptide can differ in amino acid sequence by one or more
substitutions, additions, deletions in any combination. A variant
of a nucleic acid or peptide can be a naturally occurring such as
an allelic variant, or can be a variant that is not known to occur
naturally. Non-naturally occurring variants of nucleic acids and
peptides may be made by mutagenesis techniques or by direct
synthesis.
[0130] "Viral infectivity" as that term is used herein means any of
the infection of a cell, the replication of a virus therein, and
the production of progeny virions therefrom.
[0131] A "virion" is a complete viral particle; nucleic acid and
capsid, further including and a lipid envelope in the case of some
viruses.
[0132] Methods of Using the Inhibitors of Vif Self-Association
[0133] The inhibitors of Vif self-association described herein can
be used for various uses.
[0134] In one embodiment, the inhibitors of Vif self-association
described herein can be used in a method for treating or preventing
HIV infection or AIDS in a patient. This method involves
administering to a patient in need of such treatment or prevention
a therapeutically effective amount of a compound of described
herein, or a pharmaceutically acceptable salt thereof. The method
can further include administering a therapeutically effective
amount of at least one other agent for treating HIV selected from
the group consisting of HIV reverse transcriptase inhibitors,
non-nucleoside HIV reverse transcriptase inhibitors, HIV protease
inhibitors, HIV fusion inhibitors, HIV attachment inhibitors, CCRS
inhibitors, CXCR4 inhibitors, HIV budding or maturation inhibitors,
and HIV integrase inhibitors.
[0135] In one embodiment, the inhibitors of Vif self-association
described herein can be used in a method for inhibiting infectivity
of a lentivirus in a cell. This method involves contacting a cell
with an antiviral-effective amount of a compound described herein,
or a pharmaceutically acceptable salt thereof.
[0136] In one embodiment, the inhibitors of Vif self-association
described herein can be used in a method for inhibiting Vif
self-association in a cell. This method involves contacting a cell
with an inhibitory-effective amount of a compound described herein,
or a pharmaceutically acceptable salt thereof.
[0137] The present invention further provides various methods of
using the Vif self-association inhibitors, where the first step
involves conducting the screening assay of the present invention to
identify the agents as being inhibitors of Vif self-association.
Such methods are described below.
[0138] In one embodiment, the present invention provides a method
for inhibiting infectivity of a lentivirus. This method involves
identifying an agent that disrupts Vif self-association by
performing the screening method of the present invention, and
contacting a cell with an antiviral-effective amount of said agent
under conditions effective to disrupt or inhibit multimerization of
Vif in the cell, thereby inhibiting infectivity of the lentivirus.
In one embodiment, the agent is effective to inhibit dimerization
by direct or indirect inhibition of binding of Vif dimmers at the
Vif dimerization domain, said Vif dimerization domain comprising
the amino acid sequence of proline-proline-leucine-proline
(PPLP).
[0139] In one embodiment, the present invention provides a method
for inhibiting Vif self-association in a cell. This method involves
identifying an agent that disrupts Vif self-association by
performing the screening method of the present invention, and then
contacting a cell with an inhibitory-effective amount of said agent
under conditions effective to disrupt or inhibit multimerization of
Vif in the cell, thereby inhibiting Vif self-association in the
cell.
[0140] In one embodiment, the present invention provides a method
for treating or preventing HIV infection or AIDS in a patient. This
method involves identifying an agent that disrupts Vif
self-association by performing the screening method of the present
invention, and then administering to a patient in need of such
treatment or prevention a therapeutically effective amount of the
agent.
[0141] Methods of Treatment
[0142] In one embodiment, the present invention provides methods of
treating a disease, disorder, or condition associated with a viral
infection. Preferably, the viral infection is HIV. The method
comprises administering to a subject, such as a mammal, preferably
a human, a therapeutically effective amount of a pharmaceutical
composition that inhibits Vif self-association.
[0143] The invention includes compounds identified using the
screening methods discussed elsewhere herein. Such a compound can
be used as a therapeutic to treat an HIV infection or otherwise a
disorder associated with the inability to dissociate Vif:Vif
complexes.
[0144] The ability for a compound to inhibit Vif self-association
can provide a therapeutic to protect or otherwise prevent viral
infection, for example HIV infection.
[0145] Thus, the invention includes pharmaceutical compositions.
Pharmaceutically acceptable carriers that are useful include, but
are not limited to, glycerol, water, saline, ethanol and other
pharmaceutically acceptable salt solutions such as phosphates and
salts of organic acids. Examples of these and other
pharmaceutically acceptable carriers are described in Remington's
Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey),
the disclosure of which is incorporated by reference as if set
forth in its entirety herein.
[0146] The pharmaceutical compositions may be prepared, packaged,
or sold in the form of a sterile injectable aqueous or oily
suspension or solution. This suspension or solution may be
formulated according to the known art, and may comprise, in
addition to the active ingredient, additional ingredients such as
the dispersing agents, wetting agents, or suspending agents
described herein. Such sterile injectable formulations may be
prepared using a non-toxic peritoneally-acceptable diluent or
solvent, such as water or 1,3-butane diol, for example. Other
acceptable diluents and solvents include, but are not limited to,
Ringer's solution, isotonic sodium chloride solution, and fixed
oils such as synthetic mono- or di-glycerides.
[0147] Pharmaceutical compositions that are useful in the methods
of the invention may be administered, prepared, packaged, and/or
sold in formulations suitable for oral, rectal, vaginal,
peritoneal, topical, pulmonary, intranasal, buccal, ophthalmic, or
another route of administration. Other contemplated formulations
include projected nanoparticles, liposomal preparations, resealed
erythrocytes containing the active ingredient, and
immunologically-based formulations.
[0148] The compositions of the invention may be administered via
numerous routes, including, but not limited to, oral, rectal,
vaginal, peritoneal, topical, pulmonary, intranasal, buccal, or
ophthalmic administration routes. The route(s) of administration
will be readily apparent to the skilled artisan and will depend
upon any number of factors including the type and severity of the
disease being treated, the type and age of the veterinary or human
patient being treated, and the like.
[0149] As used herein, "peritoneal administration" of a
pharmaceutical composition includes any route of administration
characterized by physical breaching of a tissue of a subject and
administration of the pharmaceutical composition through the breach
in the tissue. Peritoneal administration thus includes, but is not
limited to, administration of a pharmaceutical composition by
injection of the composition, by application of the composition
through a surgical incision, by application of the composition
through a tissue-penetrating non-surgical wound, and the like. In
particular, peritoneal administration is contemplated to include,
but is not limited to, subcutaneous, intraperitoneal,
intramuscular, intrastemal injection, and kidney dialytic infusion
techniques.
[0150] A pharmaceutical composition can consist of the active
ingredient alone, in a form suitable for administration to a
subject, or the pharmaceutical composition may comprise the active
ingredient and one or more pharmaceutically acceptable carriers,
one or more additional ingredients, or some combination of these.
The active ingredient may be present in the pharmaceutical
composition in the form of a physiologically acceptable ester or
salt, such as in combination with a physiologically acceptable
cation or anion, as is well known in the art.
[0151] The formulations of the pharmaceutical compositions
described herein may be prepared by any method known or hereafter
developed in the art of pharmacology. In general, such preparatory
methods include the step of bringing the active ingredient into
association with a carrier or one or more other accessory
ingredients, and then, if necessary or desirable, shaping or
packaging the product into a desired single- or multi-dose
unit.
[0152] Although the descriptions of pharmaceutical compositions
provided herein are principally directed to pharmaceutical
compositions that are suitable for ethical administration to
humans, it will be understood by the skilled artisan that such
compositions are generally suitable for administration to animals
of all sorts. Modification of pharmaceutical compositions suitable
for administration to humans in order to render the compositions
suitable for administration to various animals is well understood,
and the ordinarily skilled veterinary pharmacologist can design and
perform such modification with merely ordinary, if any,
experimentation. Subjects to which administration of the
pharmaceutical compositions of the invention is contemplated
include, but are not limited to, humans and other primates, mammals
including commercially relevant mammals such as cattle, pigs,
horses, sheep, cats, and dogs.
[0153] Controlled- or sustained-release formulations of a
pharmaceutical composition of the invention may be made using
conventional technology.
[0154] Formulations of a pharmaceutical composition suitable for
peritoneal administration comprise the active ingredient combined
with a pharmaceutically acceptable carrier, such as sterile water
or sterile isotonic saline. Such formulations may be prepared,
packaged, or sold in a form suitable for bolus administration or
for continuous administration. Injectable formulations may be
prepared, packaged, or sold in unit dosage form, such as in ampules
or in multi-dose containers containing a preservative. Formulations
for peritoneal administration include, but are not limited to,
suspensions, solutions, emulsions in oily or aqueous vehicles,
pastes, and implantable sustained-release or biodegradable
formulations. Such formulations may further comprise one or more
additional ingredients including, but not limited to, suspending,
stabilizing, or dispersing agents. In one embodiment of a
formulation for peritoneal administration, the active ingredient is
provided in dry (i.e., powder or granular) form for reconstitution
with a suitable vehicle (e.g., sterile pyrogen-free water) prior to
peritoneal administration of the reconstituted composition.
[0155] The pharmaceutical compositions may be prepared, packaged,
or sold in the form of a sterile injectable aqueous or oily
suspension or solution. This suspension or solution may be
formulated according to the known art, and may comprise, in
addition to the active ingredient, additional ingredients such as
the dispersing agents, wetting agents, or suspending agents
described herein. Such sterile injectable formulations may be
prepared using a non-toxic peritoneally-acceptable diluent or
solvent, such as water or 1,3-butane diol, for example. Other
acceptable diluents and solvents include, but are not limited to,
Ringer's solution, isotonic sodium chloride solution, and fixed
oils such as synthetic mono- or di-glycerides. Other
parentally-administrable formulations which are useful include
those which comprise the active ingredient in microcrystalline
form, in a liposomal preparation, or as a component of a
biodegradable polymer systems. Compositions for sustained release
or implantation may comprise pharmaceutically acceptable polymeric
or hydrophobic materials such as an emulsion, an ion exchange
resin, a sparingly soluble polymer, or a sparingly soluble
salt.
[0156] Formulations suitable for topical administration include,
but are not limited to, liquid or semi-liquid preparations such as
liniments, lotions, oil-in-water or water-in-oil emulsions such as
creams, ointments or pastes, and solutions or suspensions.
Topically-administrable formulations may, for example, comprise
from about 1% to about 10% (w/w) active ingredient, although the
concentration of the active ingredient may be as high as the
solubility limit of the active ingredient in the solvent.
Formulations for topical administration may further comprise one or
more of the additional ingredients described herein.
[0157] Typically, dosages of the compound of the invention which
may be administered to an animal, preferably a human, will vary
depending upon any number of factors, including but not limited to,
the type of animal and type of disease state being treated, the age
of the animal and the route of administration.
[0158] The compound can be administered to an animal as frequently
as several times daily, or it may be administered less frequently,
such as once a day, once a week, once every two weeks, once a
month, or even less frequently, such as once every several months
or even once a year or less. The frequency of the dose will be
readily apparent to the skilled artisan and will depend upon any
number of factors, such as, but not limited to, the type and
severity of the disease being treated, the type and age of the
animal, and the like. Preferably, the compound is, but need not be,
administered as a bolus injection that provides lasting effects for
at least one day following injection. The bolus injection can be
provided intraperitoneally.
[0159] Method of Screening
[0160] The current invention relates to a method of screening for
an agent (e.g., a small molecule compound) that disrupts Vif
self-association (also referred to herein as Vif dimerization and
Vif multimerization).
[0161] In one aspect, the present invention provides a method of
identifying an agent that disrupts Vif self-association. This
method involves (i) providing a Vif:Vif complex comprising a first
Vif protein or fragment associated with a second Vif protein or
fragment; (ii) contacting the Vif:Vif complex with a test agent
under conditions effective to generate a detectable signal when the
Vif if complex is disrupted; and (iii) detecting the detectable
signal to determine whether or not the test agent disrupts the
Vif:Vif complex, wherein disruption of the Vif if complex by the
test agent identifies an agent that disrupts Vif
self-association.
[0162] A suitable test agent can include a small molecule, a
peptide, a polypeptide, an oligosaccharide, a polysaccharide, a
polynucleotide, a lipid, a phospholipid, a fatty acid, a steroid,
an amino acid analog, and the like. In one embodiment, the test
agent is from a library of small molecule compounds.
[0163] In one embodiment, the contacting step comprises incubating
the Vif:Vif complex with one type of test agent or more than one
type of test agent.
[0164] In another embodiment, the contacting step comprises
associating the test agent with the Vif:Vif complex either directly
or indirectly.
[0165] The detactable signal may be detected using a detection
technique selected from the group consisting of fluorimetry,
microscopy, spectrophotometry, computer-aided visualization, and
the like, or combinations thereof.
[0166] The detectable signal may be selected from the group
consisting of a fluorescent signal, a phosphorescent signal, a
luminescent signal, an absorbent signal, and a chromogenic
signal.
[0167] In one embodiment, the fluorescent signal is detectable by
its fluorescence properties selected from the group consisting of
fluorescence resonance energy transfer
[0168] (FRET), fluorescence emission intensity, and fluorescence
lifetime (FL).
[0169] In one embodiment, the Vif:Vif complex is provided with a
first detection moiety attached to the first Vif protein or
fragment and a second detection moiety attached to the second Vif
protein or fragment.
[0170] In one embodiment, the first detection moiety and the second
detection moiety generate a detectable signal in a
distance-dependent manner, so that disruption of the Vif:Vif
complex is sufficient to separate the first detection moiety and
the second detection moiety a distance effective to generate the
detectable signal.
[0171] In one embodiment, the first detection moiety and the second
detection moiety comprise a fluorescence resonance energy transfer
(FRET) pair, wherein the first detection moiety is a FRET donor and
the second detection moiety is a FRET acceptor. The FRET donor and
the FRET acceptor can comprise a fluorophore pair selected from the
group consisting of EGFP-REACh2, GFP-YFP, EGFP-YFP, GFP-REACh2,
CFP-YFP, CFP-dsRED, BFP-GFP, GFP or YFP-dsRED, Cy3-Cy5,
Alexa488-Alexa555, Alexa488-Cy3, FITC-Rhodamine (TRITC), YFP-TRITC
or Cy3, and the like.
[0172] In one embodiment, the Vif:Vif complex is provided in a host
cell co-transfected with a first plasmid encoding the first Vif
protein or fragment and a second plasmid encoding the second Vif
protein or fragment.
[0173] In one embodiment, the ratio of the first plasmid to the
second plasmid is effective to optimize the generation of the
detectable signal when the Vif:Vif complex is disrupted. The
optimized ratio of the first plasmid to the second plasmid may be
about 1:4, wherein the first plasmid further comprises a signal
donor moiety and the second plasmid further comprises a signal
quencher moiety.
[0174] In one embodiment, the host cell is stably or transiently
co-transfected with the first and second plasmids.
[0175] In one embodiment, the host cell is selected from the group
consisting of a mammalian cell, an insect cell, a bacterial cell,
and a fungal cell. A suitable mammalian cell can include a human
cell.
[0176] In one embodiment, the host cell is a cell culture
comprising a cell line that is stably co-transfected with the first
and second plasmids.
[0177] The method of identifying an agent that disrupts Vif
self-association of the present invention can be configured as a
high throughput screening assay. The high throughput screening
assay can have a Z'-factor of between about 0.5 and about 1.0.
[0178] The method of identifying an agent that disrupts Vif
self-association of the present invention can further involve (i)
quantitating the detectable signal; (ii) amplifying the detectable
signal; and (iii) attaching a first epitope tag to the first Vif
protein or fragment and attaching a second epitope tag to the
second Vif protein or fragment, wherein said first and second
epitope tags are different from one another.
[0179] In one embodiment, the first and second epitope tags are
selected from the group consisting of AU1 epitope tags, AU5 epitope
tags, Beta-galactosidase epitope tags, c-Myc epitope tags, ECS
epitope tags, GST epitope tags, Histidine epitope tags, V5 epitope
tags, GFP epitope tags, HA epitope tags, and the like.
[0180] The method of identifying an agent that disrupts Vif
self-association of the present invention can further involve
subjecting the test agent identified as disrupting the Vif:Vif
complex to a validation assay effective to confirm disruption of
Vif self-association by the test agents.
[0181] The method of identifying an agent that disrupts Vif
self-association of the present invention can further involve
subjecting the test agent identified as disrupting the Vif:Vif
complex to toxicity, permeability, and/or solubility assays.
[0182] Other methods, as well as variation of the methods disclosed
herein will be apparent from the description of this invention. For
example, the test compound may be either fixed or increased, a
plurality of compounds or proteins may be tested at a single
time.
[0183] Based on the disclosure presented herein, the screening
method of the invention is applicable to a robust Forster quenched
resonance energy transfer (FgRET) assay for high-throughput
compound library screening in microtiter plates. The assay is based
on selective placement of chromoproteins or chromophores that allow
reporting on Vif:Vif complex disruption. For example, an
appropriately positioned FRET donor and FRET quencher will results
in a "dark" signal when the quaternary complex is formed between
Vif dimers, and a "light" signal when the Vif:Vif complex is
disrupted.
[0184] The skilled artisan would also appreciate, in view of the
disclosure provided herein, that standard binding assays known in
the art, or those to be developed in the future, can be used to
assess the disruption of Vif self-assocation in the presence or
absence of the test compound to identify a useful compound. Thus,
the invention includes any compound identified using this
method.
[0185] The screening method includes contacting a mixture
comprising recombinant Vif dimers with a test compound and
detecting the presence of the Vif:Vif complex, where a decrease in
the level of Vif:Vif complex compared to the amount in the absence
of the test compound or a control indicates that the test compound
is able to inhibit Vif self-association. In certain embodiments,
the control is the same assay performed with the test compound at a
different concentration (e.g. a lower concentration), or in the
absence of the test agent, etc.
[0186] Determining the ability of the test compound to interfere
with the formation of the Vif:Vif complex, can be accomplished, for
example, by coupling the Vif dimers with a tag, radioisotope, or
enzymatic label such that the Vif:Vif complex can be measured by
detecting the labeled component in the complex. For example, a
component of the complex (e.g., a single Vif protein) can be
labeled with .sup.32P, .sup.125I, .sup.35S, .sup.14C, or .sup.3H,
either directly or indirectly, and the radioisotope detected by
direct counting of radioemission or by scintillation counting.
Alternatively, a component of the complex can be enzymatically
labeled with, for example, horseradish peroxidase, alkaline
phosphatase, or luciferase, and the enzymatic label is then
detected by determination of conversion of an appropriate substrate
to product.
[0187] Determining the ability of the test compound to interfere
with the Vif self-association can also be accomplished using
technology such as real-time Biomolecular Interaction Analysis
(BIA) as described in Sjolander et al., 1991, Anal. Chem.
63:2338-2345 and Szabo et al., 1995, Curr. Opin. Struct. Biol.
5:699-705. BIA is a technology for studying biospecific
interactions in real time, without labeling any of the interactants
(e.g., BIAcore, BIAcore International AB, Uppsala, Sweden). Changes
in the optical phenomenon of surface plasmon resonance (SPR) can be
used as an indication of real-time reactions between biological
molecules.
[0188] In more than one embodiment of the methods of the present
invention, it may be desirable to immobilize particular Vif dimers
to facilitate separation of complexed from uncomplexed forms of one
or both of the molecules, as well as to accommodate automation of
the assay. The effect of a test compound on the Vif:Vif complex,
can be accomplished using any vessel suitable for containing the
reactants. Examples of such vessels include microtiter plates, test
tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided which adds a domain that allows one or both
of the proteins to be bound to a matrix. For example,
glutathione-S-transferase/target fusion proteins can be adsorbed
onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.)
or glutathione-derivatized micrometer plates, which are then
combined with the other corresponding component of the Vif:Vif
complex in the presence of the test compound. The mixture is
incubated under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads or microtiter plate wells are washed to remove any
unbound material, the matrix is immobilized in the case of beads,
and the formation of the complex is determined either directly or
indirectly, for example, as described above.
[0189] The test compounds can be obtained using any of the numerous
approaches in combinatorial library methods known in the art,
including: biological libraries; spatially addressable parallel
solid phase or solution phase libraries; synthetic library methods
requiring deconvolution; the "one-bead one-compound" library
method; and synthetic library methods using affinity chromatography
selection. The biological library approach is limited to peptide
libraries, while the other four approaches are applicable to
peptide, non-peptide oligomer or small molecule libraries of
compounds (Lam et al., 1997, Anticancer Drug Des. 12:45).
[0190] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example, in: DeWitt et al., 1993,
Proc. Natl. Acad. USA 90:6909; Erb et al., 1994, Proc. Natl. Acad.
Sci. USA 91:11422; Zuckermann et al., 1994, J. Med. Chem. 37:2678;
Cho et al., 1993, Science 261:1303; Carrell et al., 1994, Angew.
Chem. Int. Ed. Engl. 33:2059; Carell et al., 1994, Angew. Chem.
Int. Ed. Engl. 33:2061; and Gallop et al., 1994, J. Med. Chem.
37:1233.
[0191] Libraries of compounds may be presented in solution (e.g.,
Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam, 1991,
Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556),
bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat.
No. '409), plasmids (Cull et al., 1992, Proc. Natl. Acad. Sci. USA
89:1865-1869) or on phage (Scott and Smith, 1990, Science
249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al.,
1990, Proc. Natl. Acad. Sci. USA 87:6378-6382; Felici, 1991, J.
Mol. Biol. 222:301-310; and Ladner supra).
[0192] In situations where "high-throughput" modalities are
preferred, it is typical to that new chemical entities with useful
properties are generated by identifying a chemical compound (called
a "lead compound") with some desirable property or activity,
creating variants of the lead compound, and evaluating the property
and activity of those variant compounds. The current trend is to
shorten the time scale for all aspects of drug discovery.
[0193] In one embodiment, high throughput screening methods involve
providing a library containing a large number of compounds
(candidate compounds) potentially having the desired activity. Such
"combinatorial chemical libraries" are then screened in one or more
assays, as described herein, to identify those library members
(particular chemical species or subclasses) that display a desired
characteristic activity. The compounds thus identified can serve as
conventional "lead compounds" or can themselves be used as
potential or actual therapeutics.
[0194] Unless defined otherwise, 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. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the described
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and described the methods and/or materials in
connection with which the publications are cited.
[0195] It must be noted that as used herein and in the appended
claims, the singular forms "a," "and," and "the" include plural
references unless the context clearly dictates otherwise. All
technical and scientific terms used herein have the same
meaning.
[0196] Publications discussed herein are provided solely for their
disclosure prior to the filing date of the described application.
Nothing herein is to be construed as an admission that the present
invention is not entitled to antedate such publication by virtue of
prior invention. Further, the dates of publication provided may be
different from the actual publication dates which may need to be
independently confirmed.
EXAMPLES
[0197] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the described invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g., amounts, temperature, etc.), but some experimental
errors and deviations should be accounted for. Unless indicated
otherwise, parts are parts by weight, molecular weight is weight
average molecular weight, temperature is in degrees Centigrade, and
pressure is at or near atmospheric.
Example 1
Assay Development for High Throughput Molecular Screening
[0198] I. Specific Aims
[0199] The proposed research seeks to develop a novel high
throughput screen based on quenched FRET to identify small
molecules that bind to the HIV protein known as Viral Infectivity
Factor (Vif) and disrupt its self-association. The primary function
of Vif is to bind to the host-defense factor known as APOBEC3G
(A3G) and induce A3G degradation through a
polyubiquitination-dependent proteosomal pathway. Although Vif was
discovered more than a decade ago, its requirement was only known
as `being essential for infection of non-permissive cells`. The
function of Vif was revealed in the discovery of A3G as a
host-defense factor. A3G binds to single-stranded replicating HIV
DNA and introduces multiple dC to dU mutations in the negative
strand that templates dG to dA mutations in the protein-coding
strand of HIV in the absence of Vif. During the late phase of HIV
infection, A3G can become packaged with virions such that it is in
position to interact with nascent DNA during viral replication upon
infection. Vif prevents A3G viral packaging while also reducing the
cellular abundance of A3G thereby promoting viral infectivity.
[0200] Research by our lab and others revealed that multimerization
of Vif through a small C-terminal motif, .sup.161PPLP.sup.164, was
required for the interaction of Vif with A3G. The critical
importance of Vif self-association through this motif was
demonstrated with Vif multimerization antagonist peptides that also
contained the HIV TAT membrane transduction motif in order to
penetrate cells. This peptide prevented co-immunoprecipitation of
Vif, markedly reduced Vif-dependent A3G destruction and restored
A3G antiviral activity in the presence of Vif. Ultimately small
molecules with Vif multimerization antagonistic activity are of
greater long-term value in the drug industry. Given the antiviral
capacity of the peptide in living cells we believe Vif
multimerization is an accessible target in vivo with significance
equal to the A3G-Vif interaction. In fact, the C-terminal
self-association motif is relatively small and does not overlap
with any of the other Vif or A3G interaction domains making it
perhaps a more attractive target than the relatively large A3G-Vif
interaction domain (residues 40-44 and 52-72) in the N-terminus of
Vif.
[0201] We seek to develop a primary and secondary screen and apply
`hit` validation assays for small molecules that disrupt Vif s
ability to multimerize (directly or allosterically) in order to
protect A3G antiviral activity from Vif mediated inhibition. Given
the increasing preponderance of HIV strains that are resistant to
the current antiviral drugs on the market, a therapeutic against a
novel target such as Vif multimerization would have a significant
impact on the worldwide epidemic of HIV/AIDS.
[0202] Specific Aim 1. Optimize a primary high throughput screen in
384-well format that is based on Vif multimerization and quenched
FRET. EGFP-VS-Vif (the fluorescence donor) and Vif-HA-REACh2 (the
acceptor and non-fluorescent YFP variant that quenches EGFP
fluorescence) will be co-expressed in HEK 293T cells. Compounds
that dissociate Vif multimers will induce EGFP fluorescence making
this a positive screen for small molecules that disrupt Vif
self-association.
[0203] Specific Aim 2. Develop and optimize a secondary screen in
microtiter well format to validate `hits` from the primary screen.
In this E. coli-based assay, one Vif is linked to the periplasmic
transporter signal peptide ssTorA and another Vif is linked to
.beta.-lactamas e (Bla). In order for cells to survive under
ampicillin selection the Vif linked to ssTorA must multimerize with
Vif linked to Bla thereby enabling transport of Bla to the
periplasm where it neutralizes ampicillin. In the presence of small
molecules that disrupt Vif self-association the bacteria will not
grow in the presence of ampicillin.
[0204] Specific Aim 3. Perform `hit` validation assays to confirm
that small molecules selected by the primary and secondary screens
have antiviral activity through their antagonism of Vif
self-association. Antiviral activity will be validated for each
compound with a luciferase viral infectivity reporter assay using
infected TZM-bl cells in microtiter plate format. Each compound's
ability to inhibit Vif-Vif interaction will be evaluated by
co-immunoprecipitation. Western blot analysis of whole cell
extracts and purified viral particles from cells transfected with
viral DNA and A3G will demonstrate the efficacy of compounds in
protecting A3G from Vif-dependent degradation, thereby enabling A3G
packaging within virions.
[0205] II. Background and Significance
[0206] The virus contains a 10-kb single-stranded RNA genome that
encodes three major classes of gene products that include: (i)
structural proteins (Gag, Pol and Env); (ii) essential trans-acting
proteins (Tat, Rev); and (iii) "auxiliary" proteins that are not
required for efficient virus replication in permissive cells (Vpr,
Vif, Vpu, Nef) [reviewed in (1)]. There has been a heightened
interest in Vif as an antiviral target because of the discovery
that the primary function of Vif is to overcome the action of a
cellular antiviral protein known as APOBEC3G or A3G (2). In
permissive cells (e.g. 293T, SUPT1 and CEM-SS T cell lines)
vif-deleted HIV-1 clones replicate with an efficiency that is
essentially identical to that of wild-type virus. However in
non-permissive cells (e.g. primary T cells, macrophages, or CEM, H9
and HUT78 T cell lines), vif-deleted HIV-1 clones replicate with
100- to 1000-fold reduced efficiency (3-8). The failure of
Vif-deficient HIV-1 mutants to accumulate reverse transcripts and
generate integrating provirus in the non-permissive cells is due to
the ability of A3G to interact with viral replication complexes and
impair their progression as well as A3G mutagenic activity on
nascent proviral single-stranded DNA (2,9-11).
[0207] Discovery of A3G. The function of A3G (formerly named CEM15)
as an antiviral host factor was discovered in 2002 in experiments
designed to identify host cell factors in non-permissive cells that
would necessitate the expression of Vif (2). Heterokaryons
consisting of non-permissive and permissive cells retained the
non-permissive phenotype for Vif-deficient virus, demonstrating
expression of a dominant neutralizing factor in non-permissive
cells (3,4). Subtractive transcriptome analysis identified a cDNA
encoding A3G (2) as a member of the APOBEC family of cytidine
deaminases active on single-stranded nucleic acids (12,13).
Transfection of permissive cells with A3G cDNA was necessary and
sufficient for conversion to the non-permissive phenotype for
Vif-deficient HIV-1 infectivity (2).
[0208] A3G antiviral mechanism. Multiple labs have characterized a
deaminase-dependent antiviral function of A3G and its packaging
into HIV virions (9-11). Sequencing of proviral genomes revealed
that cells infected with virions containing A3G had dG to dA
hypermutations throughout the protein encoding positive strand
(9-11), consistent with A3G dC to dU mutation of the negative
strand during reverse transcription (11). Furthermore, A3G acts
processively 3' to 5' along the minus-strand HIV DNA template
(14,15) with mutations occurring in regions where the HIV DNA is
single-stranded for the longest period of time during HIV reverse
transcription (16,17). The hypermutations introduce multiple
premature stop codons and codon sense changes that negatively
affect the virus (9-11). The dU mutations in minus-strand viral DNA
can trigger the uracil base excision pathway mediated by uracil DNA
glycosylase (UDG) that is recruited into virions (18,19), leading
to cleavage of viral DNA before integration into host DNA (10).
Reduction in proviral DNA can also occur through what has been
proposed to be a physical block to reverse transcription by A3G
(5,6,20-22).
[0209] Vif-dependent inhibition of A3G antiviral activity.
Vif-expressing viruses overcome A3G by suppressing viral packaging
of A3G and targeting it for proteosomal degradation (23-26). Vif
promotes A3G degradation through its ability to bind to the
ubiquitination machinery. A consensus SOCS (suppressor of cytokine
signaling)-box in the C-terminus of Vif (residues 144-SLQYLA-149)
binds to the Elongin C subunit of the E3 ubiquitin ligase complex
that also contains Cullin 5 and Elongin B (26). Vif also contains a
zinc binding HCCH motif (residues
108-HX.sub.5-CX.sub.18CX.sub.5H-138) that confers an interaction
with Cullin 5 (27). Vif serves as a bridge for A3G to Elongin C and
Cullin 5 in the E3 ubiquitin ligase complex, leading to
polyubiquitination of both Vif and A3G (25-27). Recent studies have
shown that only polyubiquitination of Vif on one or more of its 16
lysine residues is required for proteosomal degradation of A3G and
Vif (28). Site-directed mutagenesis demonstrated that alteration of
a single amino acid within A3G could affect Vif interaction
(29-31). An aspartic acid at position 128 in A3G is required for
HIV-1 Vif to degrade human A3G whereas a lysine at position 128 is
required for simian immunodeficiency virus from African green
monkey (SlVagm) Vif to degrade agmA3G (29-31). Alanine scanning
mutation analysis of A3G revealed that residues adjacent to D128
are also crucial for Vif interaction with A3G, specifically proline
129 and aspartic acid 130 (32). On the other hand, relatively large
regions within the N-terminus of Vif are involved in its
interaction with A3G. Deletion and point mutation analyses of Vif
identified residues 40-YRHHY-44 and 52-72 as being critical regions
within Vif responsible for A3G interaction and degradation
(33-36).
[0210] Vif self-association. An analysis of Vif deletion mutants in
the Zhang lab at Thomas Jefferson University in 2001 revealed that
residues 151-164 were critical for Vif multimerization, an
interaction that was required for infectivity of non-permissive
cells (37). Subsequent phage display revealed that peptides with a
PXP motif bound to PPLP within Vif (residues 161-164), and in doing
so blocked Vif multimerization in vitro (38). Upon linkage of a
cell transducing peptide to PPLP containing peptides of Vif, both
the Zhang lab (using antennapedia homeodomain, RQIKIWFQNRRMKWKK)
and our lab (using HIV TAT transduction domain, YGRKKRRQRRRG)
revealed that these peptide chimeras transduced cells and blocked
live HIV infectivity (38,39). A3G incorporation into viral
particles was enhanced in the presence of the peptide resulting in
marked suppression of HIV infectivity (39). Donahue et al.
demonstrated that mutating the PPLP motif to AAAP enabled A3G
antiviral activity. More importantly, they showed through
co-immunoprecipitation analysis that the Vif multimerization mutant
had significantly reduced interaction with A3G. On the other hand,
the Vif mutant retained interactions with Elongin C and Cullin 5 in
a manner equivalent to wild-type Vif (40). The data reveal that Vif
self-association is essential for both viral infectivity and Vif
interaction with A3G. Moreover, the Vif multimerization domain can
be disrupted in vivo, demonstrating its potential as a drug
target.
[0211] Advantage of targeting Vif self-association. To date, four
characteristics of Vif-A3G interaction have been studied in enough
detail to make them of potential interest as drug targets. These
are: (i) Vif self-association, (ii) the Vif surface and (iii) the
A3G surface that contribute to the interface of Vif-A3G complexes,
and (iv) Vif polyubiquitination.
[0212] Vif polyubiquitination may be the most difficult
functionality of Vif to selectively target, because there are 16
lysines on Vif that are capable of being polyubiquitinated (28).
Small molecules that affect ubiquitination of Vif are likely to be
toxic given that ubiquitin-mediated degradation is an essential
part of the cell and `hits` on this target are likely to have
off-target effects leading to toxicity. Moreover, Vif bound to A3G
that is not degraded would likely still prevent A3G viral
packaging.
[0213] There has been some promising work involving the Vif-A3G
interface. The Gabuzda lab evaluated 15-mer peptides of Vif regions
for their ability to antagonize the Vif-A3G interaction. A peptide
containing amino acids 57-71 of Vif was identified that blocked
Vif-A3G interaction in vitro (41). However the efficacy of this
peptide as an antiviral in vivo is yet to be determined. The Rana
lab has identified a small molecule that is capable of blocking
Vif-dependent degradation of A3G in HEK 293T cells through HTS
based on Vif-dependent degradation of a fluorescently tagged A3G.
The molecular target of the small molecule and its mechanism of
action are unclear (42).
[0214] Considering A3G as a drug target, the major caveat to
targeting the N-terminal region of A3G involved in Vif binding is
the fact that the same region of A3G is also involved in crucial
interactions for its cellular and antiviral activity. Deletion
analysis revealed that residues 104-156 of A3G were crucial for HIV
Gag binding and viral packaging (43,44). Also, scanning alanine
mutagenesis demonstrated that amino acids 124-YYFW-127 were
especially important for viral packaging (32). The Smith lab
recently showed that there is a cytoplasmic retention signal in
residues 113-128 of A3G that interacts with an as-of-yet
unidentified cytoplasmic partner that prevents A3G from entering
the nucleus (45). The related proteins, APOBEC1 and AID, must
traffic to the nucleus but their nuclear import and access to
genomic DNA are strictly regulated (46) to prevent their potential
genotoxicity due to unregulated DNA deaminase activity (47-53).
Therefore, small molecules that prevent A3G binding to Vif at
residues 128-130 of A3G (32) have the potential negative outcome of
affecting A3G viral packaging or enabling A3G access to the
genome.
[0215] We propose that the Vif multimerization domain is an
attractive target for drug development. Blocking the Vif
self-association has proven to be an accessible target in vivo and
disrupting Vif self-association prevents Vif-A3G interaction in a
manner that will prevent the degradation of A3G and preserve its
antiviral activity (38,39). Preliminary data will demonstrate the
practicality of using Vif for the development of HTS that are
biased for Vif multimerization.
[0216] Based on these considerations, the goal of this proposal is
to develop a human cell-based homogenous assay as a primary HTS and
an orthogonal secondary screen in E. coli for small molecules that
antagonize Vif self-association. Viral infectivity assays,
co-immunoprecipitation of differentially tagged Vif subunits and
whole cell A3G quantification and A3G viral encapsidation will
serve as functional endpoints to validate hits obtained from a
preliminary library screening.
[0217] III. Preliminary Results
[0218] Vif self-association is an accessible target. Our studies
with a peptide containing the Vif multimerization motif and the HIV
TAT transduction motif demonstrated that Vif self-association is
accessible in vivo. The peptide prevented live HIV viral infection
of H9 and MT-2 T cell lines that endogenously express A3G. After
twenty days of infection the peptide blocked viral infectivity,
reducing reverse transcriptase (RT) activity in cell supernatants
to levels that were on par with those from no virus cell control or
cells treated with the potent antiviral AZT. The reduction in
infectivity was dependent on the presence of Vif and A3G (39) and
the peptide specifically allowed 2.6-fold more A3G to enter viral
particles as evident when the A3G western blot signals of (+) and
(-) peptide were normalized for p24 gag recovery. This demonstrated
that targeting Vif self-association alleviated the Vif-dependent
inhibition of A3G viral packaging.
[0219] Development of the quenched FRET primary screen. EGFP is a
FRET donor and REACh2 (Resonance Energy Accepting Chromoprotein 2)
is a non-fluorescent FRET acceptor (54). The non-fluorescent REACh2
is able to quench EGFP signal in a distance-dependent manner when
they are linked to interacting domains. However, if there is no
interaction, EGFP and REACh2 are not proximal and quenching will
not occur. This is an ideal system for HTS in which the default
condition is quenched signal due to interacting Vif molecules
linked to the FRET pair. A small molecule `hit` will produce a
positive fluorescent signal by interfering with Vif
self-association and alleviating the quench.
[0220] We tested various combinations of N- and C-terminally tagged
Vif constructs and determined that EGFP-VS-Vif and Vif-HA-REACh2
yielded the most significant quench. The system employs the use of
HEK 293T cells due to their high transfection efficiency (up to 90%
with FUGENE 6 or HD.RTM. lipofection reagent) and Vif s established
functionality in these cells demonstrated by many investigators
(24,29,32,42). Transient transfection allows for high expression of
the protein, which is important for robust FRET signals. In
addition, transiently transfected cells have the ability to
maintain an expression level of REACh2-HA-Vif that is higher than
EGFP-VS-Vif to ensure maximum amount of quenched protein in the
cell. In fact stable cell lines expressing the FRET pair have been
established but these proved to have lower levels of Vif expression
than transiently transfected cells and consequently produced very
low signals.
[0221] DNA ratios greater than or equal to 4:1 REACh2 to EGFP
maintained quenched signal in the vast majority of cells.
EGFP-V5-Vif alone has a strong baseline fluorescence. When
EGFP-VS-Vif and Vif-HA-REACh2 are co-expressed there is a
significant reduction in fluorescence intensity due to REACh2
quenching of EGFP signal. Addition of the Vif multimerization
antagonist peptide (described above) at 50 .mu.M liberates
EGFP-V5-Vif and relieves the quench. Cells treated with the peptide
antagonist will serve as a positive control condition in the
assay.
[0222] There was no quench with the multimerization-deficient
4A-Vif mutant (161-PPLP-164 to AAAA) in the equivalent conditions
to wild-type Vif. As expected the addition of peptide to cells
expressing mutant 4A-Vif did not promote additional fluorescence.
Westerns for HA and V5 demonstrated consistent expression of the
transfected constructs confirming that the lack of fluorescence is
not due to less expression of the EGFP-VS-Vif, but is in fact due
to quenched FRET.
[0223] Adapting the Quenched FRET Assay to 96-well and 384-Well
Format
[0224] Experimentals relating to adapting the quenched FRET assay
to 96-well and 384-well format are set forth below: [0225]
Description of reagents and readouts: We are currently capable of
screening small libraries in 96-well format, and have optimized
transfections for 384-well format. The assay is cell based
transient transfection of two plasmids. One plasmid contains
EGFP-VS-Vif (EVV) and the other contains Vif-HA-REACh2 (VHR).
REACh2 is a non-fluorescent YFP variant that quenches EGFP through
FRET, so in the default state Vif dimerizes and the EGFP signal is
quenched, a compound that affects the interaction will cause an
increase in fluorescence due to lack of FRET from interacting
proteins (aka "releasing of the quench"). Our read out is
fluorescence at GFP's excitation and emission in a PE Victor 3
plate reader. We have to express the REACh2 protein 4 times higher
than the EGFP in order to ensure good quench and we could not
recapitulate that in stable cell lines at the consistency, ratio
and expression level we can achieve with transient transfection.
[0226] Data confirming assay protocol: We have gone through a
significant amount of troubleshooting to obtain Z'-factors and CVs
that are optimal for HTS. We have also worked out a background
correction to account for variability within plates and between
plates. Using this optimized protocol the Z'-factors are always
above 0.5 in our hands. We have a peptide that we have tested as a
positive control that registers as a dose dependent "hit" with a
Z-score <3. We also have some promising small molecules from the
NCC library that passed secondary validation by counterscreening
for toxicity and antiviral activity. [0227] Signal of sufficient
intensity: Using the GFP/FITC excitation and emission of 485 and
535, respectively, in the PE Victor 3 Multilabel Plate Reader
quenched signal is typically >20,000 RFU above background and
the positive control is >100,000 RFU above the quenched
condition. These values can vary depending on exposure time for the
plate read and aperture size, but this is a typical signal range
for a one second reads using a normal aperture size setting. [0228]
CVs and Z'-factors: [0229] 96-well format numbers from pilot
screen: [0230] CV quench=2.4 % [0231] CV positive control=3.6%
[0232] Z'-factor=0.51 [0233] 384-well format numbers: [0234] CV
quench=1.4% [0235] CV positive control=3.3% [0236] Z'-factor=0.66
[0237] Oya001 peptide "hit" control. [0238] Standard Deviation of
980=1 Z-score [0239] This experiment involved three test wells for
each concentration of Oya001 peptide and 15 controls for quenched
and positive signal. Plate reads were performed before adding
peptide and 1.5 hours after peptide addition. The differentials
between these two reads were used in the analysis (.DELTA.RFU).
[0240] CV quench=1.7 % [0241] CV positive control=1.9% [0242]
Z'-factor=0.63 [0243] We have published data showing that this
peptide directly affects our target (39). The data in FIG. 6 shows
a clear dose dependence with the peptide in the HTS assay revealing
z-scores of 1.36, 1.96, 3.01, and 4.37 that relate to the
91.2.sup.th, 97.4.sup.th, 99.9.sup.th and >99.9.sup.th
percentile for 12.5, 25, 50, and 75 uM of Oya001 peptide,
respectively. [0244] Knowledge of control parameters [0245] DMSO
tolerance [0246] The assay tolerates DMSO very well at 0.1-1%, See
the toxicity test reported in FIGS. 11A-11B, in which the SMVDAs or
DMSO alone were added at 1%. Moreover, all pilot screens were
performed at .sup..about.0.1% DMSO and SMVDAs or DMSO alone
(controls) were added to cells anywhere between 0.1-0.5% in the HIV
infectivity counterscreens. [0247] Plate-to-Plate variation
(384-well plates with 40.+-.samples per plate) [0248] Plate 1:
[0249] CV quench=1.4 % [0250] CV positive control=5.2% [0251]
Z'-factor=0.63 [0252] Plate 2: [0253] CV quench=1.6 % [0254] CV
positive control=4.7% [0255] Z'-factor=0.66 [0256] Plate 3: [0257]
CV quench=2.0 % [0258] CV positive control=5.9% [0259]
Z'-factor=0.59 [0260] CVs for Average RFU values from Plates 1-3
[0261] CV quench=0.9 [0262] CV positive control=2.5% [0263]
Background Correction [0264] Z-Score Normalization allows for cross
plate comparison of experimental data points by making all plate
means and standard deviations equal via the plate variability
correction procedures shown in equation 1 and 4. Further
calculating the systematic variability (equation 2) and applying
the correction (equation 3) controls for variability due to error
in plating, cell growth or other systematic error. Finally, Z-Score
transformation allows data to be fit against a normal distribution.
This takes the arbitrary nature of `Relative Fluorescent Units` and
frames the data in the context of a Z-Score, or deviation. HTS hits
are generally selected as a function of deviation from the sample
population, thus framing the data in an easily interpreted context
through this normalization procedure. [0265] Equations: [0266]
initial Plate Normalization
[0266] x i ' = x i - .mu. .sigma. ( 1 ) ##EQU00001## [0267]
Normalizes data (x.sub.f)so plate mean (p) and plate standard
deviation (.sigma.) are 0 and 1, respectively, [0268] Well
Background Calculation
[0268] z i = 1 N j = 1 N x i , j ' ( 2 ) ##EQU00002## [0269]
Calculates systematic background z.sub.i from the mean of data
points x' of well i access plates j of plate set 1, 2, . . . N. All
data points x'.gtoreq.3 are excluded when N15.100. [0270] Well
Background Correction
[0270] x.sup.n.sub.i=x.sup.t.sub.i-z.sub.i (3) [0271] Subtracts
systematic background z.sub.i from normalized data point x'
yielding background corrected data point x'' [0272]
Re-normalization Post-Background Correction
[0272] x i ''' = x i - .mu. '' .sigma. ( 4 ) ##EQU00003## [0273] A
final re-normailzation using corrected data x.sup.x, subtracting
plate mean, .mu., and dividing by plate standard deviation,
.sigma.. This corrects plate .mu. and .sigma. back to 0 and 1 for
cross plate comparison,
[0274] Development of an orthogonal E. coli based secondary screen.
The secondary screen takes advantage of the Twin-arginine
translocation (Tat) pathway found in plants and bacteria (55). The
Tat pore is in the cytoplasmic membrane of E. coli and is made up
of three proteins, Tat A, B and C. A small N-terminal signaling
domain (i.e. ssTorA) is recognized and cleaved in transport. Folded
proteins and protein complexes of two or more intact proteins,
where only one protein carries a Tat signal, can be exported by the
Tat pathway in a process termed the "hitchhiker" mechanism (55,56).
.beta.-lactamase must be transported to the periplasm to breakdown
ampicillin (Amp), thus mediating growth under Amp selection. This
assay has been successfully adapted to study protein-protein
interactions and used as a screen for compounds that affect these
interactions (57,58).
[0275] OyaGen, Inc has obtained a license to use this system from
Vybion, Inc (www.vybion.com/?page=tech_procode). We have
re-engineered the assay to co-express ssTorA-Vif and
.beta.-lactamase (Bla)-Vif under L-arabinose (Ara) induction in E.
coli. Multimerization of Vif links ssTorA-Vif to Bla-Vif, allowing
Tat-mediated transport of Bla-Vif to the periplasm. In the presence
of small molecules that prevent Vif multimerization, Bla-Vif cannot
be transported to the periplasm and cells will not survive.
[0276] The constructs pBAD18-Cm-ssTorA-VS-Vif and
pBAD33-Kan-Bla-HA-Vif have been cloned, and found to express well
in the MC4100 E. coli strain as confirmed by western blotting.
pBAD18 is a high copy plasmid whereas pBAD33 is a low copy plasmid.
Bla-Vif was put in the low copy plasmid to avoid Amp resistance due
to an overabundance of Bla-Vif in the media that can leach out of
dead or dying cells. Initial tests in 96-well format carried out as
five 200 .mu.l cultures revealed no differential growth +/-Ara in
the absence of Amp. When Amp was added (30 .mu.g/mL) to cultures
simultaneous with Ara, antibiotic resistant growth became apparent
by 2 hours following Ara induction. The difference in growth
between cultures +/-Amp continued to increase up to the termination
of the study at 8 hours. Data in FIG. 9 are the average of three
repeats of the experiment. At 8 hours, the difference was 0.419
average OD 600 readings +/-Ara induction. The standard deviations
were low at 0.021 for +Ara and 0.005 for -Ara, suggesting a
possible Z'-factor favorable for HTS at 0.807.
[0277] We will further evaluate media conditions that effect
bacterial growth rate (10, 50 LB, supplemented with minimal media
(M9) salts and `super` broths) in an effort to improve the
differential between +/-Amp. This is important to achieve good
differential growth as early as possible and obtain the best
coefficient of variance, signal to noise ratio and Z'-factor.
Dependence of this HTS secondary screen on the Vif multimerization
domain and DMSO tolerance also will be determined.
Example 2
Screening, Validating, and Vetting Vif Dimerization Disruptors
[0278] Part 1. Validating the Assay. HTS analysis of Vif-Vif
multimerization through quenched FRET utilizes Vif-HA-REACh2
(quencher) and EGFP-VS-Vif (fluorophore) at an optimized ratio of
plasmids transiently transfected into 293T cells. The interaction
of Vif molecules enables quenching of EGFP signal by REACh2.
Control experiments with either peptides that mimic this domain
prevented Vif-Vif interaction or mutations within the PPLP domain
crucial for Vif-Vif interaction prevent quenching and have stronger
fluorescence signals. The legend describes the abbreviations used.
Western blotting of extracts from transfected cells showed
equivalent expression of the donor/quencher pair mutant constructs
and donor/quencher pair in peptide treated cells when compared to
control.
[0279] Part 2. Screening a Small Library. The screen has been
optimized to yield CVs less than 3% and a Z' factor of 0.61 in
96-well format. To date two libraries have been screened totaling
2446 compounds at 5.mu.M, with a smaller subset tested by qHTS at
50, 25, and 5.mu.M concentrations. In these libraries eight small
molecules had to be ruled out due to auto-fluorescence. After
background correction and normalizing of values for plate position
variability in the screen, 26 small molecules were determined to be
hits (SMVDA1-26 for Small Molecules Vif Dimerization Antagonists
1-26). The hit rate was .about.1%.
[0280] Hits were selected based on three criteria: 1) High hit
(Z-score .gtoreq.1.8, .about.97% and above the normal
distribution), 2) Multiple hits (two or more Z-score values
.gtoreq.0.9, .about.82% and above the normal distribution in the
three concentrations tested), and 3) Dose dependence. All small
molecules with at least one of these criteria were assessed and 24
of the top 26 had at least two of the three criteria. Two
exceptions were SMVDA2 and SMVDA17 which only met one criteria
(SMVDA2 was a high hit at the lowest concentration tested and
SMVDA17 had a Z score of 1.4 for both of the lowest concentrations
tested (so relatively close to the high hit cut off of 1.8).
[0281] Part 3. Vetting the Hits for Toxicity. We next analyzed hits
for toxicity. We focused on hits that showed dose dependence or
were `high hits` at multiple concentrations. We analyzed toxicity
of the compounds at 50, 25, and 5.mu.M using Promega's Cell-titer
Glo, a luciferase based assay that determines ATP concentration.
10,000 cell/well of 293T cells were plated into 96-well format and
dosed in triplicate with the small molecules and analyzed with the
Cell-titer Glo kit 24 hours later. The data showed that SMVDA1-14
had low to no toxicity at all doses, while SMVDA15-17 were toxic at
50 and 25 .mu.M.
[0282] Some hits were not evaluated for toxicity because they were
inconsistent hits in the HTS assay and these included: SMVDA20-22
which were `high hits` in the HTS assay at 50 .mu.M but showed no
dose dependence; SMVDA23-25 which were `medium hits` at 50 .mu.M,
high at 25 .mu.M and low at 5 .mu.M and SMVDA26 which was a `high
hit` at 50 and 5 .mu.M but low at 25 .mu.M.
[0283] Part 4. Vetting the Hits for Antiviral Activity. The
antiviral activity of the hits in a single round infection with
psuedotyped HIV was assessed. The assay is conducted using producer
cells that do or do not express A3G and viruses that do or do not
express Vif.
[0284] The wildtype HIV proviral vector codes for all HIV genes
except nef (replaced with EGFP) and env. The delta Vif proviral
vector is identical to wildtype except that it contains a stop
codon early within the vif gene. Delta Vif +A3G is a strong
positive control for this assay because without Vif present, A3G is
able to be encapsidated into viral particles and have a strong
antiviral effect. Alternatively, in the absence of A3G, both wild
type and Delta Vif viruses should have good infectivity.
[0285] Virus was made by transfecting these vectors with VSV-G coat
protein from a separate vector, as well as V5-APOBEC3G (A3G) in the
+A3G conditions. Transfecting the coat protein on a separate
vector, allows for only a single round of infection. The ratio of
proviral DNA:VSV-G:A3G was set to 1:0.5:0.05, which established
levels of A3G that were comparable to endogenous A3G. Cells were
dosed with chemistries 5 hours after transfection and viral
particles were harvested from the media 24 hours after transfecting
by filtering through a 0.45-micron syringe filter. Viral load was
normalized with a p24 ELISA Kit (Zeptometrix, Buffalo, N.Y.). Equal
viral loads were then added in triplicate to TZM-bl reporter cells
that express luciferase from the HIV-LTR promoter. 48 hours after
infection luciferase levels were assessed with Steady-Glo reagent
(Promega).
[0286] The first chemistries tested showed dose dependence and were
high hits at high compound concentrations in HTS. SMVDA1,
SMVDA11-15, and SMVDA18-19 were tested at 50 and 25 .mu.M with A3G
present in the first infectivity assay. The criteria for a compound
as having antiviral activity were based on % infectivity relative
to DMSO only control. Hits that inhibited infectivity to less than
60% of control were considered to have antiviral activity. Only
SMVDA1, 18, and 19 were able to show a significant decrease in
infectivity at both concentrations, but SMVDA18 and 19 have not
been evaluated further because, although they were not toxic, they
also were not positive hits at 5.mu.M in the HTS assay.
[0287] Chemistries that had antiviral activity at lower doses and
SMVDA1 were tested in the infectivity assay at 5 .mu.M. Although
levels of infectivity were not affected as much as they were for
the higher doses tested SMVDA1-6 were able to decrease infectivity
to less than 60% of control. SMVDA7-10 had minimal effects on
infectivity at 5 .mu.M and were eliminated from further
consideration.
[0288] Since SMVDA1 seemed to be the best candidate so far, we
looked closer at the structure and noticed that a related chemistry
was also in the initial screen but had been filtered out because it
had a strong auto-fluorescence signal (named SMVDA1.1). Given its
close relationship to SMVDA1 we tested SMVDA1.1 further in the
infectivity assay side-by-side with SMVDA1 at 5, 1 and 0.5 .mu.M.
While SMVDA1 had a strong effect at 50 and 25 .mu.M its antiviral
activity at lower doses was not as strong, being somewhat effective
at 5 and 1 .mu.M by knocking down infectivity by .about.50%, yet
having minimal effect on infectivity at 0.5 .mu.M. On the other
hand, SMVDA1.1 was able to reduce infectivity to less than 30% of
control at all three concentrations tested.
[0289] At this stage we had 7 compounds with antiviral activity
based on +A3G infectivity assays. All seven hits were tested
further for their ability to show a differential in infectivity
between +Vif & A3G and -Vif & A3G. The rationale here is
that these compounds should show a Vif-selective response if they
are truly acting as antagonist of Vif dimerization and sparing A3G.
Along these lines, SMVDA4-6 did not have any significant
differential between +/-Vif & A3G, thus they were eliminated
from further consideration. This left SMVDA1, 1.1, 2 and 3, which
all showed some differential between the two conditions. This
suggested a certain level of target specificity. The most
significant differentials were at 5 .mu.M for SMVDA1 and 0.5 .mu.M
for SMVDA1.1.
[0290] Part 5. Vetting the Hits for A3G Viral Particle Content.
Another way to observe target specificity is by looking at the
amount of A3G that is encapsulated into the viral particle. Since
Vif blocks A3G from getting into the virus, more A3G should be
present in viral particles isolated from cells dosed with a small
molecule that disables Vif s function. This was observed in the
case of SMVDA1 and 1.1 and, as seen with the infectivity data,
SMVDA1.1 worked better at lower doses and seemed to have the most
A3G in the virus at 5 .mu.M. Although it must be noted that more
volume was required to normalize the p24 load with 11 and 5.mu.M
SMVDA1.1 compared to other small molecules suggesting that higher
doses might be cytotoxic, resulting in lower yield of virus. The
fact that even the lowest dose of SMVDA1.1 was effective suggested
a true effect on the Vif. Supporting this conclusion was the
finding that very little A3G was present in viral particles dosed
with SMVDA2 and 3 over a larger range of doses. This suggests that
their antiviral activity was not selective for Vif.
[0291] Our complete analysis of the hits from the initial screen
left us with two related compounds (SMVDA1 and 1.1) that passed all
our tests. Given the close relationship between these compounds our
selection of these compounds suggest that one chemotype or chemical
scaffold has been identified that SAR may optimize for nanomolar
target selectivity and lower cell toxicity. Moreover the low
micromolar efficacy of these compounds suggests that medicinal
chemistry, may be able to identify compounds with nanomolar
antiviral IC50 and IC95.
Example 3
Studies and Assays Relating to Small Molecule Vif Inhibitors of the
Present Disclosure
[0292] This example relates to various studies and assays with
respect to the identification, testing, and modification of small
molecule Vif inhibitors of the present disclosure.
[0293] As shown inFIG. 5A, a qFRET assay for use in identifying
small molecules that interfere with Vif self-association.
[0294] As shown in FIG. 5B, an in-cell high throughput screen for
Vif dimerization antagonists was conducted. HTS analysis of Vif-Vif
multimerization through quenched FRET utilizes transiently
transfected Vif-HA-REACh2 (quencher) and EGFP-VS-Vif (fluorophore)
at an optimized ratio in 293T cells. The interaction of Vif
molecules enables quenching of EGFP signal by REACh2. Control
experiments with either mutations within the PPLP domain crucial
for Vif-Vif interaction (4A mutant) or peptides that mimic this
domain prevented Vif-Vif interaction and consequently resulted in a
stronger fluorescence signal (a hit produces a positive signal).
The positive control peptide confirmed that Vif-Vif multimerization
was responsible for the quenched FRET along with western blotting
showing equivalent expression of mutants or peptide treated cells
when compared to control. These confirmations established that the
assay was optimized for HTS. The screen has been optimized to yield
CVs less than 3% and a Z' factor greater than 0.6 in 96-well and
384-well formats. Transient transfection is necessary for EGFP-Vif
since over expression of Vif inhibits the cell-cycle and stable
cell lines are difficult to establish.
[0295] As shown in FIG. 6, hit rate from screening a small library
of compounds was determined and a hit zone was identified. In this
study, a sampling of 446 out of 2446 chemistries was tested.
[0296] As shown in FIG. 7, one embodiment of a path to finding an
initial hit for a Vif inhibitor small molecule (e.g., camptothecin,
CPT) was developed. As shown in FIG. 8, after identifying an
initial hit, a medicinal chemistry strategy for modifying the
initial hit from a Vif inhibitor assay of the present invention for
pharmaceutical use was conducted. For example, one hit related to
camptothecin. Camptothecin (CPT) is a known inhibitor of
Topoisomerase I (Topol), which causes toxicity to actively
replicating cells. Therefore a CPT derivative that is known to not
inhibit Topol activity was determined to be a good way to test if
CPT derivatives with Vif inhibiting activity could be uncoupled
from Topol inhibition and toxicity.
[0297] FIGS. 9A and 9B illustrate results of an in vitro drug
screening assay for toxicity of the OYA002-16 small molecule of the
present invention. As shown in FIG. 9A, in vitro Topol drug
screening assay (Topogen) shows that increasing amounts of CPT trap
plasmid DNA into open nicked circles (increase in top band) while
OYA002-16 shows no increase in open nicked circles through Topol
poisoning compared to the 0 chem control (blue arrow). As shown in
FIG. 9B, another test of a compound's effect on toxicity and Topol
activity is a cell cycle test. After 24 hours of compound treatment
cells were fixed in methanol and treated with propidium iodide (PI)
to stain the cellular DNA in A3.01 cells (a CEM derived T-cell
line). Flow cytometry of the cells can detect if the cells are in
the G1 S or G2 phase of mitosis based on amount of PI staining in
each cell. Topol inhibition causes a strong G2 block as seen with
0.2 .mu.M CPT but 0.2 and 0.4 .mu.M of OYA002-16 had a minimal
amount of G2 block (22% in G2 compared to the DMSO control at
17%).
[0298] FIG. 10 illustrates in-cell secondary screen fro
Vif-dependent A3G degradation. As shown in FIG. 10, A3G-mCherry is
stably expressed in 293T cells under puromycin selection. 50 ng of
Vif was transiently transfected into the cells in 384-well format
with Turbofect. 4 hours after transfection the chemistries were
added to cells. 24 hours after chemistries were added the mCherry
signal was read on a Biotek Synergy 4 plate reader. The signal from
plated cells not transfected with Vif was averaged and set at 100%
(left image), and cells transfected with Vif and treated with DMSO
only were averaged and set at 0% (right image). A chemistry that
inhibits Vif s ability to chaperone A3G to the proteasomal
degradation pathway would result in an increased mCherry signal
compared to the DMSO only control, and any signal that is much
higher than the no-Vif positive control is likely to be due to
autofluorescence from the chemistry itself
[0299] FIG. 11 shows results from a primary screen (left side) and
a secondary screen (right side) of the OYA002-16 small molecule
scaffold. As shown in FIG. 11, OYA002-16 shows dose dependence in
both the primary screen described in FIG. 5A and the secondary
screen described in FIG. 10. The primary screen is represented as a
change in RFU over the quenched control (ARFU). The secondary
screen is compared to the +Vif (0%) and -Vif (100%) controls as
described in FIG. 10.
[0300] As shown in FIGS. 12A and 12B, a single cycle infectivity
assay was used to study OYA002-16. FIG. 12A is a visual
representation of how the single cycle infectivity experiments were
done. They are in 6-well format in order to obtain enough virus to
do viral particle purifications for western blot detection of A3G
in the viral particle (FIG. 13). The antiviral activity of the hits
in a single-round infection with pseudotyped HIV were conducted
using HEK293T producer cells +/-A3G and viruses that are +/-Vif The
wild type HIV proviral vector codes for all HIV genes except nef
(replaced with EGFP) and env. The DVif proviral vector is identical
to wild type except that it contains a stop codon early within the
Vif gene. DVif +A3G is a strong positive control for this assay
because without Vif present, A3G is able to be encapsidated within
viral particles and have strong antiviral activity. Alternatively,
in the absence of A3G, both wild type and DVif viruses should have
high infectivity.
[0301] Single-round infectivity assays utilized transient
co-transfection of the viral vectors with VSV-G coat protein vector
and V5-A3G in the +A3G conditions using Fugene HD (Promega).
Proviral DNA:VSV-G:A3G were added to cells with a ratio of
1:0.5:0.08 which establishes levels of A3G that are comparable to
endogenous A3G. These virus producer cells were dosed with
compounds four hours after transfection and viral particles were
harvested from the media 24 hours after transfecting by filtering
through a 0.45-micron syringe filter. Viral load was then
normalized with a p24 ELISA (Perkin Elmer).
[0302] The infections utilized TZM-bl reporter cells that contain
stably integrated luciferase that is driven by the HIV-LTR
promoter, therefore luciferase is expressed upon successful HIV
infection. Triplicate infections in 96-well plates at 10,000
cells/well with 500 pg p24/well proceeded for 48 hours before the
addition of SteadyGlo.TM. Reagent (Promega) to each well for 30
minutes. Luminescence was measured as a quantitative metric for
changes in infectivity with each compound as compared to controls,
in which relative luminescence units (RLU) with no compounds are
set to 100%.
[0303] The gold standard for a Vif dimerization antagonist is
antiviral activity that is Vif- and A3G-dependent. This is best
represented as a differential between infectivity in the presence
of Vif and A3G vs the absence of Vif and A3G.
[0304] As shown in FIG. 12B, OYA002-16 showed a dose dependent
antiviral effect only when Vif and A3G were expressed (left). The
highest concentration of 33.3 04 is shown as a % of controls in the
absence of OYA002-16 (right).
[0305] FIG. 13 illustrates results of experiments showing that
OYA002-16 increases A3G in viral particles. As shown in FIG. 13,
+Vif virus +A3G produced in 293T cells in the presence of
increasing amounts of OYA002-16 (1.67 04 to 33.3 04) were purified
through a 20% sucrose cushion and western blotted for p24 and V5
tagged A3G (same virus was used in infectivity experiments in FIGS.
12A and 12B).
[0306] FIG. 14 is an illustration relating to a Next Gen Sequencing
for A3G signature mutation profiles. Next Gen Sequencing with the
Ion Torrent platform was used to sequence over 27 million bases for
each sample. An 885 bp region of Pol was nested PCR amplified from
genomic DNA isolated from TZM-bl cells infected in the previous
Figures with -Vif virus +A3G (Positive Control) and +Vif virus +A3G
& OYA002-16 (OYA002-16) and -Vif -A3G & OYA002-16 (Negative
Control). There were 60 possible A3G "GG" signature mutation sites
(A3G mediated single-stranded DNA mutations occur preferentially at
the 3' C of CC dinucleotide pairs) in the region sequenced. 34
sites were mutated at a rate of 15- to 339-fold over the background
mutation rate for the Positive Control. 34 sites were mutated at a
rate of 48 to 200-fold over background for the OYA002-16 sample. 21
sites overlapped between the two.
[0307] As shown in FIG. 15, efficacy studies of lead scaffold
OYA002-16 were conducted. OYA002-16 was evaluated in the low MOI
acute infection assay in CEM-SS (low A3G CEM derived T-cells) and
A3.01 cells (high A3G CEM derived T-cells, 7-fold more than CEM-SS
by qRT-PCR) to determine the relative antiviral activity with
compound added every other day for 14 days. Each of the test
materials was added to the uninfected cells at three concentrations
immediately prior to infection with HIV-1IIIB. Virus replication
was monitored by reverse transcriptase (RT) activity on a daily
basis over the course of 14 days. DMSO solvent alone was evaluated
in parallel. The cytotoxicity of the test materials to uninfected
cells treated with the same compound concentrations was evaluated
on days 5 and 14 using XTT tetrazolium salt.
[0308] As shown in FIGS. 16A-1, 16A-2, 16A-3, 16A-4, 16B-1, and
16B-2, efficacy studies were conducted on OYA002-16 and OYA004-06.
These compounds were tested in a standard PBMC cell-based
microtiter anti-HIV assay against HIV-1 isolates representing
different viral subtypes, and co-receptor tropisms. Test drug
dilutions were prepared at a 2.times. concentration in microtiter
tubes and 100 .mu.L of each concentration was placed in appropriate
wells using the standard format. 50 .mu.L of a predetermined
dilution of virus stock was placed in each test well (final
MOI=0.1). Separate plates were prepared identically without virus
for drug cytotoxicity studies using an MTS assay system (Tox). The
PBMC cultures were maintained for seven days following infection at
37.degree. C., 5% CO2. After this period, cell-free supernatant
samples were collected for analysis of reverse transcriptase
activity, and compound cytotoxicity was measured by addition of MTS
to the separate cytotoxicity plates for determination of cell
viability. The PBMC data analysis includes the calculation of IC50
(50% inhibition of virus replication), IC90 (90% inhibition of
virus replication), TC50 (50% cytotoxicity), and therapeutic index
values (TI=TC/IC; also referred to as Antiviral Index or AI).
[0309] As shown in FIG. 17, toxicity studies were conducted with
regard to lead scaffold OYA002-16. All cells are plated on 384-well
collagen-coated, tissue culture-treated black walled clear bottomed
polystyrene plates. Rat hepatocytes are seeded and dosed on the day
they are harvested. Dosing takes place after attachment to the
plate. They are treated with test compound at a 10 point dose range
of concentrations and incubated for 24 and 48 hrs. HepG2 cells are
seeded on the day prior to dosing and are treated with test
compound at a 10 point dose range of concentrations and incubated
for 24 and 72 hours. At the end of the incubation period, the cells
are loaded with the relevant dye/antibody for each cell health
marker. The plates are then scanned using an automated High Content
Imager, ArrayScan.RTM. VTI (Thermo Fisher Cellomics). The GSH, ROS,
and 5 day toxicity assays were run at 1 time point, 18 hours
post-dosing for GSH, 4 hr post-dose for ROS, and 5 days post-drug
treatment for the 5 day assay. The largest effect of OYA002-16 is
on Cytostasis in HepG2 cells but at antiviral doses in A3.01, there
are minimal cell cycle effects (FIG. 15). Moreover, there seems to
be minimal effect in Stress and Apoptosis assays along with
Viability tests in all tested cell types.
[0310] As shown in FIG. 18, stability and solubility studies were
conducted on lead scaffold OYA002-16. Plasma Protein Binding (PPB):
Test agent is added to plasma. This mixture is dialyzed in a RED
Device (Pierce) per the manufacturers' instructions against PBS and
incubated on an orbital shaker. After incubation, aliquots from
both the plasma and PBS sides are collected, an equal amount of PBS
is added to the plasma sample, and an equal volume of plasma is
added to the PBS sample. A methanol-containing internal standard
(three volumes) is added to precipitate the proteins and release
the test agents. After centrifugation, the supernatant is
transferred to a new plate and analyzed by LC/MS/MS.
[0311] Solubility: 0.7 mg of OYA002-16 was suspended in 100.8 -82 l
Lac-Buffer pH 4.5 or pH 7.4, vortexed for 10 min and warmed-up at
37.degree. C. for 10 min, sonicated for 5 min and vortexed again
for 10 min and centrifuged for 10 min. Supernatant was directly
injected for concentration measurement. pH 7.4 Solubility: 65-72
.mu.M by UV, 58-63 .mu.M by CLND. pH 4.5 Solubility: 74-92 .mu.M by
UV, 74 .mu.M by CLND.
[0312] OYA002-16 was found to be 100% stable after 48 h in PBS and
after 3 freeze/thaw cycles.
[0313] Standard Protocol for Stability Test in PBS Buffer: For each
compound, a 0.250 uM stock solution in DMSO was prepared and
sonicated until clear. Then, for each stability test, the cpd was
formulated by adding 10 uL of the stock solution to 990 uL of PBS
buffer, pH 7.0 and then a 200 uL aliquot was incubated 37.degree.
C. for 24 or 48 hrs.
[0314] Standard Protocol for Stability Test after 3 Feeze/Thaw
Cycles: For each compound, an aliquot of the stock solution (250 uM
in DMSO) was frozen for 24 hrs at -80.degree. C. and thawed for 1
hr, 3 times, then diluted 10 times in 40% CAN and analyzed by
HPLC.
[0315] Standard Analytical Protocol: HPLC conditions used for the
experiments were as follows: Column: Luna C-18, 150x4.6 from
Phenomenex. Eluent: 42% aqueous CAN with 0.75 mM NH4OAc, pH=6.8.
Detection: UV at 254 nm. Temperature: 35.degree. C. Flow: 1.5
mL/min. Injection: 20 uL (solubility, freeze/thaw) or 200 uL
(stability).
[0316] As shown in FIGS. 19A and 19B, unbiased computer docking
predicted that the OYA002-16 scaffold favorably binds to the
solvent exposed surface of Vif. As shown in FIG. 19A, a single
pentameric complex of Vif, Elo B, Elo C, CBFb & CulS was
extracted from the original crystal structure (Guo, et. al, Nature
2014) and saved as a pdb file. Autodock Vina was used to read this
file, added polar hydrogen atoms to the pentamer, and created a
rectangular grid covering all of the exposed surface to interrogate
exclusively this area of the pentameric complex. The 2D structure
of OYA002-16 was drawn and used open babel to add missing
hydrogens, calculate atomic charges, introduce or subtract
hydrogens based on the expected protonated state at physiological
pH 7.4, and minimize the molecular structure to obtain their 3D
coordinates for in silico docking. Autodock Vina was used to read
the surface grid file of Vif along with the files containing the
coordinates of OYA002-16. The software automatically docked the
compound at every point of the surface grid and was instructed to
dock a compound 300 times at every detected binding site with
favorable .DELTA.G binding energy outputting a file with the 3D
coordinates of the compound according to the interactions at the
most favorable binding sites. As shown in FIG. 19B, this unbiased
surface probing identified the above binding site where OYA002-16
selectively interacted with a -.DELTA.G value of -13.9 kcal/mol for
the lowest energy binding site. Close examination of the crystal
structure confirmed that the compounds docks near Vif dimerization
residues 161-PPLP-164.
[0317] As shown in FIGS. 20A-1, 20A-2, and 20B, lead optimization
goals for improving solubility and decreasing toxicity with respect
to a Vif inhibitor of the present disclosure were followed. As
shown in FIG. 20B, deoxy-CPT was active in 4 of 68 Anti-Cancer
Screens at NCI under PubChem BioAssays. Similar to the lactam in
OYA002-16, the deoxy at the 20 position eliminates a crucial
hydrogen bond with the Topol active site. 9-glycineamido-20(S)-CPT
was found to be active in 37 of 76 Anti-Cancer Screens at NCI under
PubChem BioAssays. The peptide bond off of the 9 position enables
solubilizing capabilities with peptide R-groups. Topotecan was
found to be active in most Anti-Cancer Screens at NCI under PubChem
BioAssays as this is currently an approved chemotherapy drug. The
advantage of Topotecan is that is has been optimized for treatment
in people with favorable ADME profiles. With the exception of
Deoxy-CPT, these CPT derivatives are likely to be too toxic to
pursue for an AIDS therapeutic, however, they can be modified with
lead attributes for the Vif target (i.e. Lactam-CPT aka OYA002-16).
The ultimate goal will be to synthesize and evaluate 5 new
compounds for improved toxicity and solubility and favorable DMPK
& ADMET.
[0318] FIG. 21A illustrates derivatives of CPT and topotecan that
may be useful as Vif inhibitors in accordance with the present
invention. As shown in FIG. 21B, various synthetic pathways of
various embodiments of the Vif inhibitors for use in the present
invention are set forth, including those for deoxycamptothecin
lactam (denoted as formula 2 in the figure) (corresponds to Formula
(I-c)), 9-glycinamido camptothecin lactam (denoted as formula 5 in
the figure) (corresponds to Formula (I-d)),9-glycinamido
deoxycamptothecin lactam (denoted as formula 7 in the figure)
(corresponds to Formula (I-e)), topotecan lactam (denoted as
formula 9 in the figure) (corresponds to Formula (I-f)), and
deoxytopotecan lactam (denoted as formula 10 in the figure)
(corresponds to Formula (I-g)).
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[0382] While the present invention has been described with
reference to the specific embodiments thereof it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adopt a particular situation,
material, composition of matter, process, process step or steps, to
the objective spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims
appended hereto.
* * * * *