U.S. patent application number 11/753244 was filed with the patent office on 2007-10-04 for compounds for inhibition of hiv infection by blocking hiv entry.
This patent application is currently assigned to New York Blood Center. Invention is credited to Asim Kumar Debnath, Shibo Jiang.
Application Number | 20070232684 11/753244 |
Document ID | / |
Family ID | 32393342 |
Filed Date | 2007-10-04 |
United States Patent
Application |
20070232684 |
Kind Code |
A1 |
Jiang; Shibo ; et
al. |
October 4, 2007 |
Compounds for inhibition of HIV infection by blocking HIV entry
Abstract
A group of compounds that inhibit HIV replication by blocking
HIV entry was identified. Two representative compounds, designated
NB-2 and NB-64, inhibited HIV replication (p24 production) with
IC.sub.50 values <0.5 .mu.g/ml. It was proved that NB-2 and
NB-64 are HIV entry inhibitors by targeting the HIV gp41 since: 1)
they inhibited HIV-mediated cell fusion; 2) they inhibited HIV
replication only when they were added to the cells less than one
hour after virus addition; 3) they did not block the gp120-CD4
binding; 4) they did not interact with the coreceptor CXCR4 since
they failed to block anti-CXCR4 antibody binding to
CXCR4-expressing cells; 5) they blocked the formation of the gp41
core that is detected by sandwich enzyme linked immunosorbent assay
(ELISA) using a conformation-specific MAb NC-1; 6) they inhibited
the formation of the gp41 six-helix bundle revealed by fluorescence
native-polyacrylamide gel electrophoresis (FN-PAGE); and 7) they
blocked binding of D-peptide to the hydrophobic cavity within gp41
coiled coil domain, modeled by peptide IQN17. These results
suggested that NB-2 and NB-64 may interact with the hydrophobic
cavity and block the formation of the fusion-active gp41 coiled
coil domain, resulting in inhibition of HIV-1 mediated membrane
fusion and virus entry.
Inventors: |
Jiang; Shibo; (Fresh
Meadows, NY) ; Debnath; Asim Kumar; (Fort Lee,
NJ) |
Correspondence
Address: |
LAW OFFICES OF ALBERT WAI-KIT CHAN, LLC
WORLD PLAZA, SUITE 604
141-07 20TH AVENUE
WHITESTONE
NY
11357
US
|
Assignee: |
New York Blood Center
|
Family ID: |
32393342 |
Appl. No.: |
11/753244 |
Filed: |
May 24, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10706027 |
Nov 12, 2003 |
7241803 |
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11753244 |
May 24, 2007 |
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60428055 |
Nov 21, 2002 |
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Current U.S.
Class: |
514/429 ; 514/1;
548/563 |
Current CPC
Class: |
C07D 207/327
20130101 |
Class at
Publication: |
514/429 ;
514/001; 548/563 |
International
Class: |
C07D 207/30 20060101
C07D207/30; A61K 31/00 20060101 A61K031/00; A61K 31/40 20060101
A61K031/40 |
Goverment Interests
[0002] The invention disclosed herein was supported in part by
National Institute of Health Grant RO1 AI46221. Accordingly, the
United States Government may have certain rights in this invention.
Claims
1. A compound of molecular weight from 200 to 1200 Daltons, and
logP of -2.0 to +5.5, capable of interacting with the hydrophobic
cavity and blocking the formulation of the fusion-active gp41
coiled core domain.
2. The compound of claim 1, which is negatively charged.
3. A compound having the following formula: ##STR4## Wherein at
least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6,
R.sub.7, R.sub.8, and R.sub.9 contains COOH or other acidic
groups.
4. The compound of claim 3, wherein X can be C or N; when C or N is
at any X position, the corresponding R group may or may not be
there.
5. The compound of claim 3, wherein X is either O or S; when X is
either O or S, the bond with the next atom, such as C, will be a
single bond and O or S will be unsubstituted.
6. A compound having formula I, wherein X is a carbon or its
pharmaceutically acceptable salts, Wherein: R.sub.1 and R.sub.2 are
independently selected from the group consisting of H, alkyl,
alkenyl, alkynyl, halogen, CN, nitro, OH and OR, where R is alkyl;
R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, and R.sub.9
are each independently selected from the group consisting of H,
alkyl, cycloalkyl, alkenyl, alkynyl, aryl, arylalkyl, heterocyclyl,
tetrazolyl, halogen, OH, CN, NO.sub.2 and OR, where R is alkyl,
NHR, where R is H and alkyl, COOR, where R is H, and alkyl,
SO.sub.3R, where R is H and alkyl, SO.sub.2NHR, where R is H and
alkyl.
7. The compound of claim 4, wherein the group alkyl is substituted
straight or branched alkyl chains carrying 1 to 6 carbon atoms.
8. The compound of claim 5, wherein alkyl is methyl, ethyl,
n-propyl, i-propyl, n-butyl, i-butyl or tert-butyl.
9. The compound of claim 4, wherein alkenyl is substituted straight
or branched alkenyl chains carrying 2 to 6 carbon atoms.
10. The compound of claim 9, wherein the alkenyl is vinyl,
1-propenyl, 2-propenyl, i-propenyl, butenyl or its isomers.
11. The compound of claim 4, wherein alkynyl is substituted
straight or branched alkynyl chains carrying 2 to 6 carbon
atoms.
12. The compound of claim 11, wherein alkynyl group is ethynyl,
propynyl or its isomers, or butynyl or its isomers.
13. The compound of claim 4, wherein suitable substituents of
alkyl, alkenyl and alkynyl can be selected from one or more of
amino, cyano, halogen, hydroxy, alkoxy, aryloxy, aryl,
heterocyclyl, carboxy, nitro, alkyl sulfonyl, aryl sulfonyl, thio,
alkyl thio, or aryl thio.
14. The compound of claim 4, wherein cycloalkyl is substituted
cycloalkyl groups containing 3 to 6 carbon atoms.
15. The compound of claim 14, wherein the cycloalkyl is
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or
adamantyl.
16. The compound of claim 14, wherein the cycloalkyl is benz-fused
to an aromatic cyclic group.
17. The compound of claim 13, wherein the aryl is substituted
phenyl or napthyl.
18. The compound of claim 4, wherein the group heterocyclic is
optionally substituted with saturated, partially saturated, or
aromatic cyclics, which contain one or more heteroatoms selected
from nitrogen, oxygen or sulfur.
19. The compound of claim 18, which is benz-fused to a substituted
aromatic cyclic or heterocyles.
20. The compound of claim 4, wherein the heterocyclic group is
quinolinyl, pyridyl, indolyl, furyl, oxazolyl, thienyl, triazolyl,
pyrazolyl, imidazolyl, benzothiazolyl, benzimidazolyl, piperzinyl,
or benzothiazolyl.
21-38. (canceled)
Description
[0001] This application claims priority of U.S. Ser. No.
60/428,055, filed 21 Nov. 2002, the content of which is
incorporated by reference hereinto this application.
[0003] Throughout this application, various publications are
referenced and full citations for these publications may be found
in the text where they are referenced. Disclosures of these
publications in their entireties are hereby incorporated by
reference into this application to more fully describe the state of
the art to which this invention pertains.
BACKGROUND OF THE INVENTION
[0004] Human immunodeficiency virus type 1 (HIV-1) envelope
glycoprotein (Env) transmembrane subunit gp41 plays a crucial role
in the early steps of viral entry into target cells (38) and may
serve as an important target for development of HIV-1 entry
inhibitors (4, 25). The gp41 molecule consists of three domains,
i.e., cytoplasmic domain, transmembrane domain and extracellular
domain (ectodomain). The ectodomain contains three major functional
regions: the fusion peptide (FP), the N-terminal heptad repeat (NHR
or HR1) and the C-terminal heptad repeat (CHR or HR2). Peptides
derived from the NHR and CHR regions of gp41, designated N- and
C-peptides, have potent antiviral activity against HIV-1 infection
(21, 33, 49, 50). One of the C-peptides, T-20 (previously known as
DP-178 and now as Fuzeon), has shown potent in vivo anti-HIV-1
activity in clinical trials for treatment of patients with HIV-1
infection and AIDS (27, 50) and was recently approved by the US FDA
as the first member of a new class of anti-HIV drugs known as HIV
fusion inhibitors. Discovery of this drug is a great breakthrough
in the development of anti-HIV drugs since it can be used for
treatment of HIV-infected individuals who fail to respond to the
currently available anti-retroviral drugs such as HIV reverse
transcriptase and protease inhibitors (26, 29). However, the future
application of T-20 may be constrained due to its lack of oral
availability and high cost of production. Therefore, it is
essential to develop small molecule anti-HIV-1 compounds with a
mechanism of action similar to that of C-peptides but without the
disadvantages of the peptidic drugs.
[0005] In the study on the mechanism by which C-peptides inhibit
HIV-1 fusion, it has been demonstrated that the gp41 N- and
C-peptides mixed at equimolar concentrations form a stable
.alpha.-helical trimer of antiparallel heterodimers, representing
the fusion-active gp41 core (33, 35). Crystallographic analysis has
revealed that this is a six-stranded .alpha.-helical bundle, in
which three N-helices associate to form the central trimeric
coiled-coil and three C-helices pack obliquely in an antiparallel
manner into the highly conserved hydrophobic grooves on the surface
of this coiled-coil (3, 46, 48). The C-helix interacts with the
N-helix mainly through the hydrophobic residues in the grooves on
the surface of the central coiled-coil trimer. Each of the grooves
on the surface of the N-helix trimer has a deep hydrophobic pocket
that accommodates three conserved hydrophobic residues in the gp41
CHR region (3), suggesting that this pocket is an attractive target
for designing new class of anti-HIV-1 drugs, which may prevent the
early fusion events (2, 3).
[0006] Using the gp41 pocket as the target structure, previously
two small molecule compounds were identified, ADS-J1 (9, 19) and
XTT formazan (51) by applying a computer-aided molecular docking
techniques and a sandwich enzyme linked immunosorbent assay (ELISA)
(23) using a monoclonal antibody (mAb), NC-1, which specifically
recognizes the fusion-active gp41 core structure (20). These
compounds inhibit HIV-1 fusion possibly by docking into the gp41
pocket and interfering with the formation of the gp41 six-helix
bundle formation. However, they may not be good lead compounds for
development of anti-HIV-1 drugs since both are dyes and contain
several reactive groups. Nevertheless, the identification of these
compounds is useful as a proof of concept that a small molecule
organic compound might block the fusion-active gp41 six-helix
bundle formation and inhibit HIV-1 entry. Here it is reported that
the identification of two pyrrole derivatives, designated NB-2 and
NB-64, as novel HIV-1 fusion inhibitors, which may interact with
gp41 at the fusion-intermediate conformation, possibly binding to
the gp41 hydrophobic pocket and surrounding area and block the gp41
six-helix bundle formation, thereby inhibiting the fusion between
the viral and target cell membranes. NB-2 and NB-64 are "drug-like"
compounds and may be used as leads for designing more potent HIV-1
entry inhibitors, which are expected to be developed as a new class
of anti-HIV-1 drugs.
SUMMARY OF THE INVENTION
[0007] It is an object of the present invention to provide
compounds, which are effective against HIV infection.
[0008] It is also an object of the present invention to provide
compounds for design and development of a new class of anti-HIV
drugs by blocking HIV entry.
[0009] It is a further object of the present invention to provide
methods for inhibiting HIV replication or infectivity or treating
HIV infection in a subject without inducing undesirable adverse
effects.
[0010] The present invention comprises compounds of the formula I
or pharmaceutically acceptable salts thereof, ##STR1##
[0011] Wherein at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, R.sub.7, R.sub.8, and R.sub.9 contains COOH or
other acidic groups.
[0012] In an embodiment, X can be C, N, O or S. When X is either O
or S, the bond with the next atom such as C, will be a single bond
and O or S will be unsubstituted. In a further embodiment, X is a
carbon atom, wherein R.sub.1 and R.sub.2 are independently selected
form the group consisting of H, alkyl, alkenyl, alkynyl, halogen,
CN, nitro, OH and OR, where R is alkyl;
[0013] R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, and
R.sub.9 are each independently selected from the group consisting
of H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, arylalkyl,
heterocyclyl, tetrazolyl, halogen, OH, CN, NO.sub.2 and OR, where R
is alkyl, COOR, where R is H and alkyl, SO.sub.3R, where R is H and
alkyl, SO.sub.2NHR, where R is H and alkyl.
[0014] The group alkyl is represented by optionally substituted
straight or branched alkyl chains carrying 1 to 6 carbon atoms and
accordingly preferably stands for methyl, ethyl, n-propyl,
i-propyl, n-butyl, i-butyl and tert-butyl.
[0015] The group alkenyl is represented by optionally substituted
straight or branched alkenyl chains carrying 2 to 6 carbon atoms
and accordingly preferably stands for vinyl, 1-propenyl,
2-propenyl, i-propenyl, and butenyl and its isomers.
[0016] The group alkynyl is represented by optionally substituted
straight or branched alkynyl chains carrying 2 to 6 carbon atoms
and accordingly preferably stands for ethynyl, propynyl and its
isomers, butynyl and its isomers.
[0017] Suitable substituents of alkyl, alkenyl and alkynyl can be
selected from one or more of amino, cyano, halogen, hydroxy,
alkoxy, aryloxy, aryl, heterocyclyl, carboxy, nitro, alkyl
sulfonyl, aryl sulfonyl, thio, alkyl thio, aryl thio.
[0018] The group cycloalkyl is represented by optionally
substituted cycloalkyl groups containing 3 to 6 carbon atoms and
can be selected, e.g., cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, cycloheptyl or adamantyl. All these groups can also be
benz-fused to an aromatic cyclic group, e.g., phenyl.
[0019] The group aryl is represented by optionally substituted
phenyl or napthyl.
[0020] The group heterocyclic stands for optionally substituted
saturated, partially saturated, aromatic cyclics, which contain one
or more heteroatoms selected from nitrogen, oxygen and sulfur and
can also be benz-fused to an optionally substituted aromatic cyclic
or heterocyles.
[0021] Heterocyclic groups can be selected, but not limited to,
from quinolinyl, pyridyl, indolyl, furyl, oxazolyl, thienyl,
triazolyl, pyrazolyl, imidazolyl, benzothiazolyl, benzimidazolyl,
piperzinyl, benzothiazolyl.
[0022] Substituents for aryl and heterocyclyl can be selected from
those mentioned for alkyl.
[0023] The group halogen stands for chloro, bromo, fluoro and
iodo.
[0024] Compounds of formula I, which are acidic in nature can form
pharmaceutically acceptable salts with inorganic and organic bases,
e.g., sodium hydroxide, potassium hydroxide, calcium hydroxide,
barium hydroxide, magnesium hydroxide, N-ethyl piperidine, and
similar other bases. When formula I is basic in nature it can form
pharmaceutically acceptable salts with inorganic and organic acids,
e.g., hydrochloric acid, sulfuric acid, nitric acid, phosphoric
acid, acetic acid, tartaric acid, succinic acid, fumaric acid,
maleic acid, malic acid, citric acid, methane sulfonic acid and
similar other acids. TABLE-US-00001 TABLE 1 I ##STR2## Example#
Code# R.sub.1-R.sub.9 1 NB-1 R.sub.1 = R.sub.2 = CH.sub.3, R.sub.3
= R.sub.4 = R.sub.5 = H, R.sub.6 = COOH, R.sub.7 = CH.sub.3,
R.sub.8 = R.sub.9 = H 2 NB-2 R.sub.1 = R.sub.2 = CH.sub.3, R.sub.3
= H, R.sub.4 = OH, R.sub.5 = COOH, R.sub.6 = R.sub.7 = R.sub.8 =
R.sub.9 = H 3 NB-4 R.sub.1 = R.sub.2 = CH.sub.3, R.sub.3 = R.sub.4
= R.sub.5 = H, R.sub.6 = COOH, R.sub.7 = R.sub.8 = R.sub.9 = H 4
NB-26 R.sub.1 = CH.sub.3, R.sub.2 = C.sub.6H.sub.5, R.sub.3 = H,
R.sub.4 = COOH, R.sub.5 = OH, R.sub.6 = R.sub.7 = R.sub.8 = R.sub.9
= H 5 NB-27 R.sub.1 = CH.sub.3, R.sub.2 = C.sub.6H.sub.5, R.sub.3 =
H, R.sub.4 = COOH, R.sub.5 = Cl, R.sub.6 = R.sub.7 = R.sub.8 =
R.sub.9 = H 6 NB-28 R.sub.1 = CH.sub.3, R.sub.2 = C.sub.6H.sub.5,
R.sub.3 = H, R.sub.4 = COOH, R.sub.5 = R.sub.6 = R.sub.7 = R.sub.8
= R.sub.9 = H 7 NB-29 R.sub.1 = R.sub.2 = CH.sub.3, R.sub.3 =
R.sub.4 = H, R.sub.5 = COOH, R.sub.6 = Cl, R.sub.7 = R.sub.8 =
R.sub.9 = H 8 NB-50 R.sub.1 = R.sub.2 = CH.sub.3, R.sub.3 = R.sub.4
= H, R.sub.5 = COOCH.sub.3, R.sub.6 = R.sub.7 = R.sub.8 = R.sub.9 =
H 9 NB-53 R.sub.1 = R.sub.2 = CH.sub.3, R.sub.3 = R.sub.4 = H,
R.sub.5 = COOH, R.sub.6 = R.sub.7 = R.sub.8 = R.sub.9 = H 10 NB-54
R.sub.1 = R.sub.2 = CH.sub.3, R.sub.3 = R.sub.4 = H, R.sub.5 =
COOH, R.sub.6 = Cl, R.sub.7 = H, R.sub.8 = CHO, R.sub.9 = H 11
NB-56 R.sub.1 = R.sub.2 = CH.sub.3, R.sub.3 = H, R.sub.4 = COOH,
R.sub.5 = OH, R.sub.6 = R.sub.7 = R.sub.8 = R.sub.9 = H 12 NB-63
R.sub.1 = R.sub.2 = R.sub.3 = R.sub.4 = R.sub.5 = H, R.sub.6 =
COOH, R.sub.7 = CH.sub.3, R.sub.8 = R.sub.9 = H 13 NB-64 R.sub.1 =
R.sub.2 = R.sub.3 = H, R.sub.4 = COOH, R.sub.5 = Cl, R.sub.6 =
R.sub.7 = R.sub.8 = R.sub.9 = H 14 NB-67 R.sub.1 = R.sub.2 =
CH.sub.3, R.sub.3 = R.sub.4 = H, R.sub.5 = COOH, R.sub.6 = H,
R.sub.7 = OH, R.sub.8 = R.sub.9 = H 15 NB-68 R.sub.1 = R.sub.2 =
CH.sub.3, R.sub.3 = H, R.sub.4 = COOH, R.sub.5 = H, R.sub.6 = COOH,
R.sub.7 = R.sub.8 = R.sub.9 = H 16 NB-69 R.sub.1 = R.sub.2 =
CH.sub.3, R.sub.3 = H, R.sub.4 = COOH, R.sub.5 = R.sub.6 = H,
R.sub.7 = OH, R.sub.8 = R.sub.9 = H 17 NB-70 R.sub.1 = R.sub.2 =
CH.sub.3, R.sub.3 = R.sub.4 = H, R.sub.5 = Cl, R.sub.6 = COOH,
R.sub.7 = R.sub.8 = R.sub.9 = H 18 NB-71 R.sub.1 = R.sub.2 =
R.sub.3 = CH.sub.3, R.sub.4 = R.sub.5 = H, R.sub.6 = COOH, R.sub.7
= R.sub.8 = R.sub.9 = H 19 NB-72 R.sub.1 = R.sub.2 = CH.sub.3,
R.sub.3 = R.sub.4 = H, R.sub.5 = COOH, R.sub.6 = H, R.sub.7 =
CH.sub.3, R.sub.8 = R.sub.9 = H 20 NB-97 R.sub.1 = R.sub.2 =
R.sub.3 = H, R.sub.4 = COOH, R.sub.5 = R.sub.6 = R.sub.7 = R.sub.8
= R.sub.9 = H 21 NB-99 R.sub.1 = R.sub.2 = CH.sub.3, R.sub.3 =
R.sub.4 = H, R.sub.5 = COOH, R.sub.6 = R.sub.7 = H, R.sub.8 = CHO,
R.sub.9 = H
[0025] A synthetic peptide drug, T-20, has shown potent anti-HIV
activity by blocking HIV entry in clinical trial. However, its
future clinical application will be limited due to lack of oral
availability. A group of organic compounds with low molecular
weight having potent anti-HIV activity were identified by blocking
HIV entry with a mechanism of action similar to that of T-20. Two
representative compounds, designated NB-2 and NB-64, inhibited HIV
replication (p24 production), HIV-mediated cytopathic effect (CPE)
and cell fusion with low IC.sub.50 values (Table 2). It was proved
that NB-2 and NB-64 are HIV entry inhibitors by targeting the HIV
gp41 since: 1) they inhibited HIV-mediated cell fusion; 2) they
inhibited HIV replication only when they were added to the cells
less than two hours after virus addition; 3) they did not block
gp120-CD4 interaction; 4) they did not interact with the coreceptor
CXCR4 since they failed to block anti-CXCR4 antibody binding to
CXCR4-expressing cells; 5) they blocked the formation of the gp41
core detected by sandwich enzyme linked immunosorbent assay (ELISA)
using a conformation-specific MAb NC-1; 6) they inhibited the
formation of the gp41 six-helix bundle revealed by fluorescence
native-polyacrylamide gel electrophoresis (FN-PAGE); and 7) they
blocked binding of D-peptide to the hydrophobic cavity within gp41
coiled coil domain, modeled by peptide IQN17. These results
suggested that NB-2 and NB-64 may interact with the hydrophobic
cavity and block the formation of the fusion-active gp41 coiled
coil domain, resulting in inhibition of HIV-1 mediated membrane
fusion and virus entry.
DETAILED DESCRIPTION OF THE FIGURES
[0026] For the purpose of illustrating the invention, there is
shown in the drawings forms which are presently preferred. It is to
be understood however, that the present invention is not limited to
the precise arrangements and instrumentalities depicted in the
drawings.
[0027] FIG. 1 Chemical structures of NB-2, NB-64, NB-177, and
NB-178. Log P values were calculated using the ClogP software
(Biobyte Corporation, Claremont, Calif.).
[0028] FIG. 2 NB-2 and NB-64 inhibited HIV-1 replication as
indicated by suppression of p24 production. HIV-1.sub.IIIB was used
in this assay. Each sample was tested in triplicate. Error bars
indicate standard deviations.
[0029] FIG. 3 NB-2 and NB-64 inhibited HIV-1 entry. Inhibition of
HIV-1 entry was determined by a time-of-addition assay. NB-2 and
NB-64 were added to MT-2 cells at different intervals
post-infection by HIV-1.sub.IIIB. AZT, a reverse transcriptase
inhibitor, was included as a control. Each sample was tested in
triplicate.
[0030] FIG. 4 NB-2 and NB-64 inhibited HIV-1 mediated membrane
fusion. A) virus-cell fusion. Inhibition of fusion of
HIV-1.sub.NL4-3 pseudotyped viruses expressing envelope
glycoprotein of the HIV-1.sub.SF162 (R5) strain with U87-T4-CCR5
cells was determined by a luciferase assay. Each sample was tested
in triplicate. B) Cell-cell fusion assay. Inhibition of fusion
between HIV-1.sub.IIIB infected H9 cells (H9/HIV-1.sub.IIIB)
labeled with Calcein and MT-2 cells were assessed by a dye transfer
assay as described in the Materials and Methods. Each sample was
tested in quadruplicate.
[0031] FIG. 5 NB-2 and NB-64 did not block gp120-CD4 binding, nor
interact with the coreceptor. A) Binding of gp120 to CD4 in the
presence or absence of the compounds was assessed by ELISA. B)
Binding of anti-CXCR4 antibody, 12G5, to CXCR4-expressing cells,
U373-MAGI-CXCR4.sub.CEM cells, was determined by a cell-based ELISA
(52). Each sample was tested in triplicate.
[0032] FIG. 6 NB-2 and NB-64 inhibited the gp41 six-helix bundle
formation. A) Sandwich ELISA. The compounds NB-2 and NB-64 were
incubated with N36 for 30 min at 37.degree. C. before addition of
C34. Samples were tested in triplicate. B) FN-PAGE. The peptide N36
was incubated with the NB-2 (lane 3) and NB-64 (lane 5) at
37.degree. C. for 30 min before addition of the peptide C34-FITC,
or with C34-FITC at 37.degree. C. for 30 min before addition of the
compounds NB-2 (lane 4) and NB-64 (lane 6), respectively. After
incubation for another 30 min, the mixtures were analyzed by native
PAGE as described in the Materials and Methods.
[0033] FIG. 7 NB-2 and NB-64 disrupted the .alpha.-helical
conformation of the complex formed by N- and C-peptides by CD
spectroscopy. Peptide N36 was incubated with NB-2 and NB-64 at
37.degree. C. for 30 min before addition of C34. After incubation
for additional 30 min, the mixture was measured by a CD
spectrometer.
[0034] FIG. 8 NB-2 and NB-64 blocked a D-peptide binding to the
gp41 pocket. The compounds NB-2 and NB-64, respectively, were
incubated with biotinylated D10-p5-2k peptide at 37.degree. C. for
30 min before addition of the peptide IQN17. Each sample was tested
in triplicate.
DETAILED DESCRIPTION OF THE INVENTION
[0035] This invention provides compounds for inhibition of HIV
infection. Specifically, this invention provides a compound of
molecular weight from 200 to 1200 Daltons, and logP of -2.0 to
+5.5, capable of interacting with the hydrophobic cavity and
blocking the formulation of the fusion-active gp41 coiled core
domain. In a preferred embodiment, the compound is negatively
charged.
[0036] This invention also provides a method for screening
compounds capable of blocking HIV entry. Screening methods of
antiviral compounds targeted to the HIV-1 gp41 core structure were
described in Patent Cooperation Treaty (PCT) application,
PCT/US00/06771, publication no. WO 00/55377, U.S. Pat. No.
6,596,497. The content of this PCT application is hereby
incorporated by reference into this application. For an example,
antiviral compounds may be screened by the following method: [0037]
a) capturing polyclonal antibodies from an animal other than a
mouse, directed against the HIV-1 gp41 trimeric structure
containing three N-peptides of HIV-1 gp41 and three C-peptides of
HIV-1 gp41, onto a solid-phase to form a polyclonal antibody coated
solid-phase; [0038] b) forming a mixture of a compound to be tested
with N-peptides of HIV-1 gp41, and then adding C-peptides of HIV-1
gp41; [0039] c) adding the mixture from step (b) to the polyclonal
antibody coated solid-phase from step (a), then removing unbound
peptides and unbound compound, and then adding a monoclonal
antibody which specifically reacts with the HIV-1 gp41 and three
C-peptides of HIV-1 gp41, but does not react with individual
N-peptides of HIV-1 gp41 and does not react with individual
C-peptides of HIV-1 gp41; and [0040] d) measuring the binding of
said monoclonal antibody.
[0041] In a preferred embodiment, the monoclonal antibody used in
screenings is designated NC-1. In another embodiment, the
monoclonal antibody used is capable of competitively inhibiting
bindings of the NC-1 antibody.
[0042] A biological assay may be used with the above
immunoscreening assay. Said biological assay includes but is not
limited to HIV-mediated cell fusion assay, as described infra.
[0043] The assay may also be fluorescence native polyacrylamide gel
electrophoresis (FN-PAGE).
[0044] This invention also provides the compounds identified by the
screenings. In accordance with the invention, a pharmaceutical
composition for inhibition of HIV infection comprising the
resulting compounds is provided.
[0045] As a result of the screening, this invention provides a
compound having the following formula:
[0046] Wherein at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, R.sub.7, R.sub.8, and R.sub.9 contains COOH or
other acidic groups. ##STR3##
[0047] Wherein, X can be C or N. When N is at any X position, the
corresponding R group may or may not be there. For example, if X is
N at the R.sub.1 site, then there will be no substitution if it is
to maintain a double bond with the X at R.sub.9 site. However, if X
is N at the R.sub.1 site but does not maintain a double bond with
the X at R.sub.9 site, it can then have R.sub.1. In another
embodiment, X is either O or S. When X is either O or S then the
bond with the next atom, such as C, will be a single bond and O or
S will be unsubstituted.
[0048] This invention provides a compound having formula I, wherein
X is a carbon, or its pharmaceutically acceptable salts,
[0049] Wherein:
[0050] R.sub.1 and R.sub.2 are independently selected from the
group consisting of H, alkyl, alkenyl, alkynyl, halogen, CN,
NO.sub.2, OH and OR where R is alkyl;
[0051] R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, and
R.sub.9 are each independently selected from the group consisting
of H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, arylalkyl,
heterocyclyl, tetrazolyl, halogen, OH, CN, NO.sub.2 and OR, where R
is alkyl, NHR, where R is H and alkyl, COOR, where R is H, and
alkyl, SO.sub.3R, where R is H and alkyl, SO.sub.2NHR, where R is H
and alkyl.
[0052] In an embodiment, the group alkyl is substituted with
straight or branched alkyl chains carrying 1 to 6 carbon atoms.
[0053] In another embodiment, alkyl is methyl, ethyl, n-propyl,
i-propyl, n-butyl, i-butyl, or tert-butyl.
[0054] In a separate embodiment, alkenyl is substituted with
straight or branched alkenyl chains carrying 2 to 6 carbon atoms.
The alkenyl includes but is not limited to vinyl, 1-propenyl,
2-propenyl, i-propenyl, butenyl, or its isomers.
[0055] In an embodiment, alkynyl is substituted with straight or
branched alkynyl chains carrying 2 to 6 carbon atoms. The alkynyl
group includes but is not limited to ethynyl, propynyl or its
isomers, or butynyl or its isomers.
[0056] In accordance with this invention, suitable substituents of
alkyl, alkenyl and alkynyl can be selected from one or more of the
following: amino, cyano, halogen, hydroxy, alkoxy, aryloxy, aryl,
heterocyclyl, carboxy, nitro, alkyl sulfonyl, aryl sulfonyl, thio,
alkyl thio, or aryl thio.
[0057] In an embodiment, this invention provides the above
compound, wherein cycloalkyl is substituted with cycloalkyl groups
containing 3 to 6 carbon atoms. The cycloalkyl includes but not
limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl and adamantyl. In a further embodiment, the cycloalkyl
is benz-fused to an aromatic cyclic group.
[0058] In a separate embodiment, the aryl is substituted with
phenyl or napthyl.
[0059] This invention provides the above compound, wherein the
group heterocyclic is optionally substituted with saturated,
partially saturated, or aromatic cyclics, which contain one or more
heteroatoms selected from nitrogen, oxygen or sulfur. In an
embodiment, the compound is benz-fused to a substituted aromatic
cyclic or heterocyles. In a further embodiment, the heterocyclic
group includes but is not limited to quinolinyl, pyridyl, indolyl,
furyl, oxazolyl, thienyl, triazolyl, pyrazolyl, imidazolyl,
benzothiazolyl, benzimidazolyl, piperzinyl, and benzothiazolyl.
[0060] This invention provides the above compound, wherein the
halogen group is chloro, bromo, fluoro, or iodo.
[0061] This invention provides a compound having formula I, which
is acidic and capable of forming pharmaceutically acceptable salts
with inorganic and organic bases. The base includes but is not
limited to sodium hydroxide, potassium hydroxide, calcium
hydroxide, barium hydroxide, magnesium hydroxide, and N-ethyl
piperidine.
[0062] This invention provides the compound having formula I, which
is acidic and capable of forming pharmaceutically acceptable salts
with inorganic and organic acids. The acid includes but is not
limited to hydrochloric acid, sulfuric acid, nitric acid,
phosphoric acid, acetic acid, tartaric acid, succinic acid, fumaric
acid, maleic acid, malic acid, citric acid, and methane sulfonic
acid.
[0063] This invention provides the above compound, wherein X is C;
and R.sub.1 and R.sub.2 are CH.sub.3, respectively; and R.sub.3 is
H; and R.sub.4 is OH; and R.sub.5 is COOH and R.sub.6, R.sub.7,
R.sub.8 and R.sub.9 are each H.
[0064] This invention provides the above compound, wherein R.sub.1,
R.sub.2 and R.sub.3 are H, respectively; and R.sub.4 is COOH,
R.sub.5 is C1 and R.sub.6, R.sub.7, R.sub.8 and R.sub.9 each are H
and X is C.
[0065] This invention provides an antiviral pharmaceutical
composition comprising an effective amount of a compound with
formula I, or a pharmaceutically acceptable salt, and a
pharmaceutically acceptable carrier.
[0066] A "pharmaceutically acceptable carrier" means any of the
standard pharmaceutical carriers. Examples of suitable carriers are
well known in the art and may include but are not limited to any of
the standard pharmaceutical carriers like phosphate buffered saline
solutions, phosphate buffered saline containing Polysorb 80, water,
emulsions such as oil/water emulsion, and various types of wetting
agents. Other carriers may also include sterile solutions, tablets,
coated tablets, and capsules.
[0067] Typically such carriers contain excipients like starch,
milk, sugar, certain types of clay, gelatin, stearic acid or salts
thereof, magnesium or calcium stearate, talc, vegetable fats or
oils, gums, glycols, or other known excipients. Such carriers may
also include flavor and color additives or other ingredients.
Compositions comprising such carriers are formulated by well known
conventional methods.
[0068] This invention provides the above pharmaceutical composition
for treating human immunodeficiency virus (HIV) infection, further
comprising an effective amount of an Acquired Immunodeficiency
Syndrome (AIDS) treatment agent selected from the group consisting
of anti-HIV agents, anti-infective agents, and
immunomodulators.
[0069] This invention provides a method for inhibiting replication
of human immunodeficiency virus in cells comprising of contacting
cells with an effective amount of a compound with formula I to
inhibit the replication of the human immunodeficiency virus.
[0070] This invention provides a method for treating mammals
infected with the human immunodeficiency virus, comprising
administering to said mammals an effective amount of a compound
with formula I, or its pharmaceutically acceptable salts
thereof.
[0071] In an embodiment, the mammal is a human.
[0072] This invention provides a method for preventing
manifestation of Acquired Immunodeficiency Syndrome (AIDS) in a
subject comprising administering to the subject an amount of a
compound with formula I effective to prevent said syndrome in the
subject.
[0073] The invention will be better understood by reference to the
Examples which follow, but those skilled in the art will readily
appreciate that the specific examples are only illustrative and are
not meant to limit the invention as described herein, which is
defined by the claims which follow thereafter.
EXPERIMENTAL DETAILS
Materials and Methods
[0074] Reagents. MT-2 cells, HIV-1.sub.IIIB-infected H9 cells
(H9/HIV-1.sub.IIIB), U87-T4-CXCR4 and U87-T4-CCR5 cells, laboratory
adapted and primary HIV-1 strains, and anti-p24 mAb (183-12H-5C)
were obtained from the NIH AIDS Research and Reference Reagent
Program. Lymphoid cell line CEMx174 5.25M7, kindly provided by C.
Cheng-Mayer, is stably transduced with an HIV-1 long terminal
repeat (LTR)-green fluorescent protein (GFP) reporter and
lusiferase reporter construct. The cells express CD4 and both
coreceptors, CXCR4 and CCR5 (16). These cells were maintained in
RPMI-1640 medium supplemented with 10% FBS, 1 .mu.g/ml puromycin,
200 .mu.g/ml G418. Recombinant soluble CD4 (sCD4) was obtained from
Genentech Inc. (South San Francisco, Calif.). Peptides N36, C34 (3,
35), IQN17 (11), and T22 (39, 40) were synthesized by a standard
solid-phase FMOC method in the MicroChemistry Laboratory of the New
York Blood Center. A biotinylated D-peptide, D10-p5-2K (11), was
also synthesized in-house with D-amino acids and was oxidized as
previously described (11). The peptides were purified to
homogeneity by high-performance liquid chromatography (HPLC). The
identity of the purified peptides was confirmed by laser desorption
mass spectrometry (PerSeptive Biosystems). Rabbit antisera directed
against the mixture of N36/C34 and against IQN17 were prepared as
previously described (20). Mouse mAb NC-1 specific for the gp41
six-helix bundle was prepared and characterized as previously
described (20). Rabbit and mouse IgG were purified using Protein
A/G beads (Pierce, Rockford, Ill.). Mouse mAb 12G5 specific for
CXCR4 was purchased from R&D Systems (Minneapolis, Minn.).
Compounds used for screening were purchased from ChemBridge
Corporation (San Diego, Calif.). NB-177 and NB-178 were purchased
from Maybridge Plc (Trevillett, England). Chloropeptin was a
generous gift from Satoshi Omura and Haruo Tanaka of The Kitasato
Institute, Tokyo, Japan.
[0075] Syncytium-formation assay for screening HIV-1 fusion
inhibitors. HIV-1.sub.IIIB-infected H9 cells (H9/HIV-1.sub.IIIB) at
2.times.10.sup.5/ml were cocultured with MT-2 cells
(2.times.10.sup.6/ml) in the presence of compounds to be screened
(final concentration of compound: 25 .mu.g/ml) in a 96-well plate
at 37.degree. C. for 2 days. HIV-1 induced syncytium formation was
observed under an inverted microscope and scored as "-" (no
syncytium was observed), ".+-." (about 50% syncytia were
inhibited), and "+" (no syncytium formation was inhibited). The
compounds scored with "-" and ".+-." were selected for further
screening by ELISA for inhibitors against the gp41 six-helix bundle
formation.
[0076] ELISA for screening for compounds that inhibit the gp41
six-helix bundle formation. A sandwich ELISA as previously
described (23) was used to screen for compounds that inhibit the
gp41 six-helix bundle formation. Briefly, peptide N36 (2 .mu.M) was
pre-incubated with a test compound at the indicated concentrations
at 37.degree. C. for 30 min, followed by addition of C34 (2 .mu.M).
In the control experiments, N36 was pre-incubated with C34 at
37.degree. C. for 30 min, followed by addition of the test
compound. After incubation at 37.degree. C. for 30 min, the mixture
was added to wells of a 96-well polystyrene plate (Costar, Corning
Inc., Corning, N.Y.) which were precoated with IgG (2 .mu.g/ml)
purified from rabbit antisera directed against the N36/C34 mixture.
Then, the mAb NC-1, biotin-labeled goat-anti-mouse IgG (Sigma
Chemical Co., St. Louis, Mo.), streptavidin-labeled horseradish
peroxidase (SA-HRP) (Zymed, S. San Francisco, Calif.), and the
substrate 3,3',5,5'-tetramethylbenzidine (TMB) (Sigma) were added
sequentially. Absorbance at 450 nm was measured using an ELISA
reader (Ultra 384, Tecan, Research Triangle Park, N.C.). The
percent inhibition by the compounds was calculated as previously
described (18) and the concentration for 50% inhibition (IC.sub.50)
was calculated using the software designated Calcusyn (7), kindly
provided by Dr. T. C. Chou (Sloan-Kettering Cancer Center, New
York, N.Y.).
[0077] Assessment of anti-HIV-1 infectivity. The inhibitory
activity of compounds on infection by laboratory-adapted HIV-1
strains was determined as previously described (18). In brief,
1.times.10.sup.4 MT-2 cells were infected with HIV-1 at 100
TCID.sub.50 (50% tissue culture infective dose) in 200 .mu.l of
RPMI 1640 medium containing 10% FBS in the presence or absence of
compounds at graded concentrations overnight. For the time-of
addition assay, compounds were added at various time
post-infection. Then the culture supernatants were removed and
fresh media were added. On the fourth day post-infection, 100 .mu.l
of culture supernatants were collected from each well, mixed with
equal volumes of 5% Triton X-100 and assayed for p24 antigen, which
was quantitated by ELISA (51). Briefly, the wells of polystyrene
plates (Immulon 1B, Dynex Technology, Chantilly, Va.) were coated
with HIV immunoglobulin (HIVIG), which was prepared from plasma of
HIV-seropositive donors with high neutralizing titers against
HIV-1.sub.IIIB as previously described (44) in 0.085 M
carbonate-bicarbonate buffer (pH 9.6) at 4.degree. C. overnight,
followed by washes with PBS-T buffer (0.01M PBS containing 0.05%
Tween-20) and blocking with PBS containing 1% dry fat-free milk
(Bio-Rad Inc., Hercules, Calif.). Virus lysates were added to the
wells and incubated at 37.degree. C. for 1 h. After extensive
washes, anti-p24 mAb (183-12H-5C), biotin labeled anti-mouse IgG1
(Santa Cruz Biotech., Santa Cruz, Calif.), SA-HRP and TMB were
added sequentially. Reactions were terminated by addition of 1N
H.sub.2SO.sub.4. Absorbance at 450 nm was recorded in an ELISA
reader (Ultra 384, Tecan). Recombinant protein p24 (US Biological,
Swampscott, Mass.) was included for establishing standard dose
response curve.
[0078] Inhibitory activity of compounds on infection by primary
HIV-1 isolates was determined as previously described (41). PBMCs
were isolated from the blood of healthy donors at the New York
Blood Center by standard density gradient centrifugation using
Histopaque-1077 (Sigma). The cells were plated in 75 cm.sup.2
plastic flasks and incubated at 37.degree. C. for 2 hrs. The
nonadherent cells were collected and resuspended at
5.times.10.sup.6 in 10 ml RPMI-1640 medium containing 10% FBS, 5
.mu.g/ml PHA and 100 U/ml IL-2 (Sigma), followed by incubation at
37.degree. C. for 3 days. The PHA-stimulated cells were infected
with corresponding primary HIV-1 isolates at 0.01 multiplicity of
infection (MOI) in the absence or presence of a compound at graded
concentrations. Culture media were changed every 3 days. The
supernatants were collected 7 days post-infection and tested for
p24 antigen by ELISA as described above. The percent inhibition of
p24 production and IC.sub.50 values were calculated as described
above.
[0079] Inhibition of cell-cell fusion. A dye transfer assay was
used for detection of HIV-1 mediated cell fusion as previously
described (21, 24, 34). H9/HIV-1.sub.IIIB cells were labeled with a
fluorescent reagent, Calcein-AM (Molecular Probes, Inc., Eugene,
Oreg.) and then incubated with MT-2 cells (ratio=1:5) in 96-well
plates at 37.degree. C. for 2 hrs in the presence or absence of
compounds tested. The fused and unfused Cacein-labeled
HIV-1-infected cells were counted under an inverted fluorescence
microscope (Zeiss, Germany) with an eyepiece micrometer disc. The
percentage of inhibition of cell fusion and the IC.sub.50 values
were calculated as previously described (21).
[0080] Inhibition of fusion between PBMCs infected by primary HIV-1
strains (X4 and R5 viruses) with CEMx174 5.25 M7 cells, which
express CD4 and both coreceptors, CXCR4 and CCR5, was determined by
a luciferase assay. Briefly, 50 .mu.l of compound at graded
concentration in triplicate was incubated with equal volume of
PHA-stimulated PBMCs (1.times.10.sup.5/ml) infected by
corresponding primary HIV-1 strains, respectively, for 7 days as
described above. After incubation at 37.degree. C. for 30 min, 100
.mu.l of CEMx174 5.25 M7 cells (2.times.10.sup.5) were added and
incubated at 37.degree. C. for three days. The cells were
collected, washed, and lysed with the lysing reagent included in
the luciferase kit (Promega, Corp., Madison, Wis.). Aliquots of
cell lysates were transferred to 96-well flat-bottom luminometer
plates (Costar, Corning Inc., Corning, N.Y.), followed by addition
of luciferase substrate (Promega). The luciferase activity was
measured in the Ultra 384 luminometer (Tecan).
[0081] Inhibition of virus-cell fusion. Inhibition of fusion of
HIV-1.sub.NL4-3 pseudotyped viruses expressing envelope
glycoprotein of the HIV-1.sub.HXB2 (X4) and HIV-1.sub.SF162 (R5)
strains with U87-T4-CXCR4 and U87-T4-CCR5 cells, respectively, was
measured as previously described (15). Briefly, 100 .mu.l of
U87-T4-CXCR4 and U87-T4-CCR5 cells, respectively, at
1.times.10.sup.5 cells/ml were added to the wells of a 96-well
tissue culture plate and cultured at 37.degree. C. overnight. Fifty
.mu.l of a test compound at graded concentrations was mixed with 50
.mu.l of the HIV-1.sub.NL4-3-luc viruses pseudotyped with the
HIV-1.sub.HXB2 and HIV-1.sub.SF162 Env, prepared as described
previously (15), at a final p24 concentration of 0.5 ng/ml in the
presence of Polybrene (10 .mu.g/ml). After incubation at 37.degree.
C. for 30 min, the mixtures were added to the cells and incubated
at 37.degree. C. overnight. The supernatants were removed and fresh
culture medium was added. After incubation at 37.degree. C. for 3
days, the cells were washed twice with PBS and lysed with the
lysing reagent included in a luciferase assay kit (Promega, Corp.,
Madison, Wis.). The cell lysates were transferred to a 96-well
flat-bottom luminometer plate (Costar, Corning Inc., Corning, N.Y.)
and luciferase substrate (Promega) was added. The luciferase
activity was measured immediately on a luminometer (Ultra 384,
Tecan). The % inhibition and IC.sub.50 values were calculated as
described above.
[0082] Detection of in vitro cytotoxicity. The in vitro
cytotoxicity of compounds for MT-2 cells was measured using trypan
blue dye exclusion assay (41). Briefly, 100 .mu.l of MT-2 cells
(2.times.10.sup.6/ml) were incubated with an equal volume of a
compound diluted in culture media at graded concentrations at
37.degree. C. for 2 hr. Ten microliters of cell suspension was
transferred to a well of round-bottom plate and mixed with 10 .mu.l
of 0.04% trypan blue solution. The number of viable cells was
counted using a hemocytometer under a microscope. The CC.sub.50
(the concentration for 50% cytotoxicity) values were calculated
using the software Calcusyn (6).
[0083] Inhibition of gp120 binding to CD4. Wells of polystyrene
plates was coated with 100 .mu.l of sheep anti-gp120 antibody D7324
(Cliniqa, Fallbrook, Calif.) at 2 .mu.g/ml in carbonate buffer (pH
9.6) at 4.degree. C. overnight and blocked with 1% dry fat-free
milk in PBS at 37.degree. C. for 1 h. One hundred microliters of
recombinant gp120 molecule (Immunodiagnostics, Woburn, Mass.) at
0.5 .mu.g/ml in PBS was added and incubated at 37.degree. C. for 1
h, followed by three washes with PBS-T. Soluble CD4 (sCD4) at 0.25
.mu.g/ml was added in the presence of a compound (25 .mu.M) and
incubated at 37.degree. C. for 1 h. After three washes, rabbit
anti-sCD4 IgG (0.25 .mu.g/ml in PBS, 100 .mu.l/well) was added and
incubated at 37.degree. C. for 1 h. Binding of rabbit anti-sCD4 IgG
was determined by sequential addition of biotinylated
goat-anti-rabbit IgG, SA-HRP, and TMB. After the reactions were
terminated, absorbance at 450 nm was recorded in an ELISA reader
(Tecan).
[0084] Inhibition of anti-CXCR4 antibody binding to
CXCR4-expressing cells. The inhibition of the binding of anti-CXCR4
antibody to CXCR4-expressing cells was determined using a
cell-based ELISA as described previously (52). Briefly,
U373-MAGI-CXCR4.sub.CEM cells (1.times.10.sup.5/well) which express
CXCR4 molecules were cultured in a 96-well plate at 37.degree. C.
overnight. The cells were fixed with 5% formaldehyde at room
temperature for 15 min and washed with PBS-T. The cells were
incubated with anti-CXCR4 mAb 12G5 at 37.degree. C. for 1 h in the
presence or absence of compounds tested. Isotype IgG2a was used as
a control. After addition of biotin-labeled goat anti-mouse IgG,
SA-HRP and the substrate TMB sequentially, the absorbance at 450 nm
was measured using an ELISA reader (Tecan). The percentage of
inhibition by the compounds was calculated as described above.
[0085] Circular dichroism (CD) spectroscopy. A test compound at
graded concentrations was incubated with N36 in phosphate buffer
(pH 7.2) at 37.degree. C. for 30 min before addition of C34 in the
same buffer at equimolar concentration. After incubation at
37.degree. C. for 30 min, the mixture was cooled down and CD
spectra of the mixture were measured on the J-715 CD spectrometer
equipped with a thermoelectric temperature controller (Jasco,
Japan). The instrument was calibrated using a two point calibration
method with (+)-10-camphorsulfonic acid. The wavelength dependence
of molar ellipticity (.theta.) was monitored at 0.degree. C. by 5
scans in 1 nm increments with a sampling time of 10 seconds as
previously described (12, 51). The CD signal at 222 nm was measured
as a function of temperature. Thermal melts were performed in
2.degree. C. steps, with 2 min of equilibration at each temperature
and an acquisition time of 0.5 min. The midpoint of the thermal
unfolding transition (T.sub.m) was estimated from the maximum of
the first derivative of the CD signal at 222 nm (1).
[0086] Fluorescence native polyacrylamide gel electrophoresis
(FN-PAGE). FN-PAGE was performed as previously described (32).
Briefly, 18% pre-cast Tris-Glycine gels and Novex X-Cell II Mini
cell (Invitrogen, Carlsbad, Calif.) were used for native-PAGE. The
peptide N36 (40 .mu.M in PBS) was incubated with NB-2 and NB-64 at
200 .mu.g/ml, respectively, at 37.degree. C. for 30 min and the
peptide C34-FITC (40 .mu.M in PBS) was added. In the control, N36
was incubated with C34-FITC at 37.degree. C. for 30 min before
addition of the compounds. After incubation at 37.degree. C. for
another 30 min, the mixture was diluted in Tris-Glycine native
sample buffer, then loaded to 10.times.1.0 mm wells. Gel
electrophorosis was carried out with 125 V constant voltage at room
temperature for about 90 min in Tris-Glycine native running buffer.
Immediately after electrophoresis, image of the fluorescence bands
in the gel was taken by the FluorChem 8800 Imaging System (Alpha
Innotech corp., San Leandro, Calif.) using a transillumination UV
light source with excitation wavelength at 302 nm and a
fluorescence filter with emission wavelength at 520 nm.
[0087] Inhibition of a biotinylated D-peptide binding to IQN17. For
measuring the binding of a D-peptide, D10-p5-2k to IQN17, an ELISA
was established. In brief, wells of a 96-well polystyrene plate
were coated with IgG (10 .mu.g/ml) purified from rabbit antisera
directed against IQN17. The peptide IQN17 (10 .mu.M) was incubated
with a compound at graded concentrations at 37.degree. C. for 30
min before addition of the peptide D10-p5-2K-biotin (5 .mu.M).
After incubation at 37.degree. C. for 30 min, the mixture was added
to the plate, followed by incubation at 37.degree. C. for 30 min.
After extensive washes, D10-p5-2K-biotin bound to the IQN17 was
quantitated by addition of SA-HRP and TMB sequentially. The
absorbance at 450 nm was read using an ELISA reader (Tecan). The
percentage of inhibition by the compounds was calculated as
described above.
Results
Identification of Two Anti-HIV-1 Compounds, NB-2 and NB-64, Through
HTS
[0088] Using syncytium formation assay and sandwich ELISA-based HTS
techniques, a chemical library from ChemBridge Corporation
consisting of 33,040 compounds at a single dose (25 .mu.g/ml) has
been screened. These compounds are "drug-like" molecules which were
rationally pre-selected to form a "universal" library that covers
the maximum pharmacophore diversity with the minimum number of
compounds. Two compounds, termed NB-2 and NB-64, at this
concentration are found to have significantly inhibited HIV-1
mediated syncytium formation and the six-helix bundle formation
between the gp41 N-peptide N36 and C-peptide C34. Both NB-2 and
NB-64 are N-substituted pyrrole derivatives with the molecular
weights of 231 and 222 daltons, and ClogP (a measure of partition
of a drug in water and octanol phase) of 4.15 and 3.15,
respectively (FIG. 1). Another two N-substituted pyrrole
derivatives, NB-177 and NB-178, were obtained from Maybridge as
controls. These two compounds have the same parent structure as
NB-2 and NB-64, respectively, except that they do not have the COOH
group (FIG. 1). But NB-177 and NB-178 had no inhibitory activity in
above two screening assays.
NB-2 and NB-64 have Potent Inhibitory Activity on Infection by
Laboratory-Adapted and Primary HIV-1 Strains
[0089] The inhibitory activity of NB-2 and NB-64 on infection of
MT-2 cells by laboratory-adapted HIV-1 strains and of PBMCs by
primary HIV-1 strains was determined as previously described (18,
41). The in vitro cytotoxicity of NB-2 and NB-64 were determined
using trypan blue exclusion assay (51). As shown in FIG. 2, both
NB-2 and NB-64 significantly inhibited HIV-1.sub.IIIB replication,
while NB-177 and NB-178, have no inhibitory activity at the
concentration up to 80 .mu.g/ml. Both NB-2 and NB-64 had low in
vitro cytotoxicity and high selectivity index (SI>1,000) (Table
2), suggesting NB-2 and NB-64 are potent anti-HIV-1 agents. In
addition to HIV-1.sub.IIIB, NB-2 and NB-64 also inhibited other
laboratory-adapted HIV-1 strains, including one strain resistant to
AZT (AZT-R). Both compounds had potent inhibitory activity on
infection by primary HIV-1 strains with distinct genotypes and
biotypes, although some primary HIV-1 strains (e.g., RU570) were
less sensitive than other strains. These results suggest that NB-2
and NB-64 have potent antiviral activity against a broad spectrum
of HIV-1 strains. TABLE-US-00002 TABLE 2 The in vitro cytotoxicity
of NB-2 and NB-64 and their inhibitory activity on HIV-1.sub.IIIB
replication Compounds NB-2 NB-64 CC.sub.50 (.mu.g/ml) 305.60 .+-.
5.04 >500 IC.sub.50 (.mu.g/ml) 0.24 .+-. 0.05 0.49 .+-. 0.09 SI
(CC.sub.50/IC.sub.50) 1271 >1020
[0090] TABLE-US-00003 TABLE 3 Inhibitory activity of NB-2 and NB-64
on infection by laboratory-adapted and primary HIV-1 strains
IC.sub.50 (.mu.g/ml) for inhibition of p24 production HIV-1 strain
NB-2 NB-64 Laboratory-adapted RF (clade B, X4) 0.45 .+-. 0.04 0.96
.+-. 0.03 SF2 (clade B, X4) 0.44 .+-. 0.03 1.34 .+-. 0.06 AZT-R
(clade B, X4) 1.41 .+-. 0.03 6.25 .+-. 0.25 Primary 94UG103 (clade
A, X4R5) 5.92 .+-. 0.06 7.94 .+-. 0.96 92US657 (clade B, R5) 10.41
.+-. 1.88 6.60 .+-. 1.80 93IN101 (clade C, R5) 1.05 .+-. 0.23 4.38
.+-. 0.19 93TH051 (clade E, X4R5) 1.15 .+-. 0.14 5.24 .+-. 1.80
93BR020 (clade F, X4R5) 2.50 .+-. 0.01 1.05 .+-. 0.18 RU570 (clade
G, R5) 23.12 .+-. 1.23 8.81 .+-. 0.24 BCF02 (Group O, R5) 0.21 .+-.
0.12 1.31 .+-. 0.90
[0091] TABLE-US-00004 TABLE 4 Inhibitory activity of NB-2 and NB-64
on HIV-1 mediated cell-cell fusion IC.sub.50 (.mu.g/ml) for
inhibition of cell-cell fusion Fusion between NB-2 NB-64 MT-2 &
H9/HIV-1.sub.IIIB (clade B, X4) 1.53 .+-. 0.17 2.02 .+-. 0.19
5.25M7 & PBMCs/SF162 (clade B, R5) 4.86 .+-. 0.34 20.86 .+-.
0.20 5.25M7 & PBMCs infected by 92UG029 (clade A, X4) 2.50 .+-.
0.02 2.21 .+-. 0.56 92UG657 (clade B, R5) 6.64 .+-. 1.13 39.00 .+-.
5.27 93IN101 (clade C, R5) 3.27 .+-. 0.18 18.26 .+-. 0.01 93UG065
(clade D, X4R5) 6.07 .+-. 0.07 20.77 .+-. 1.94 CMU02 (clade E, R5)
8.14 .+-. 1.09 4.89 .+-. 1.08 93TH051 (clade E, X4R5) 1.76 .+-.
0.07 12.72 .+-. 0.32 93BR020 (clade F, X4R5) 1.66 .+-. 0.07 2.24
.+-. 0.31 BCF02 (Group O, R5) 0.93 .+-. 0.57 10.30 .+-. 5.90
NB-2 and NB-64 Inhibit HIV-1 Entry by Blocking Membrane Fusion
[0092] A time-of-addition assay was carried out to determine
whether NB-2 and NB-64 are HIV-1 entry inhibitors. MT-2 cells were
incubated with HIV-1.sub.IIIB at 37.degree. C. for 0, 1, 2, 3, 4,
6, and 8 hrs, respectively, before addition of the test compounds
at 10 .mu.g/ml. AZT (2.5 .mu.M) was used as a control. After
culture for another 2 hrs, the cells were washed to remove the free
virus and compounds. The supernatants were collected on day 4
post-infection for measurement of p24 production. NB-2 and NB-64
inhibited HIV-1 replication when they were added to the cells with
virus together, but showed no inhibitory activity if they were
added one hour or longer after virus was added to cells. However,
AZT was still effective in inhibiting HIV-1 replication even it was
added 8 hrs post-infection (FIG. 3).
[0093] Fusion between virus and target cell membranes or between
HIV-infected cells and uninfected cells is the critical steps of
HIV entry into a new target cell. Therefore, it is essential to
determine whether NB-2 and NB-64 inhibit virus-cell and cell-cell
fusion. As shown in FIG. 4A, both NB-2 and NB-64 inhibited fusion
of HIV-1.sub.NL4-3 pseudotyped viruses expressing HIV-1.sub.SF162
(R5) Evn with U87-T4-CCR5 (IC.sub.50 values=3.35.+-.0.32 and
2.79.+-.0.57 .mu.g/ml, respectively), while NB-177 and NB-178 had
no inhibitory activity (FIG. 4B). A similar result was obtained by
using the pseudotyped viruses expressing HIV-1.sub.HXB2 (X4) Env
and U87-T4-CXCR4 cells (data not shown). NB-2 and NB-64 also
significantly inhibited fusion of HIV-1.sub.IIIB infected H9 cells
with uninfected MT-2 cells with IC.sub.50 values of 1.53.+-.0.17
and 2.02.+-.0.19 .mu.g/ml, respectively. However, NB-177 and NB178
had no inhibitory activity on cell-cell fusion (FIG. 4B).
Furthermore, NB-2 and NB-64 inhibited fusion between CEMx174 5.25M7
cells and PBMCs infected by primary HIV-1 strains with distinct
genotypes and phenotypes (Table 4). These results suggest that NB-2
and NB-64 inhibit HIV-1 entry by blocking HIV-1 mediated membrane
fusion.
NB-2 and NB-64 do not Block gp120-CD4 Binding, Nor Interact with
the Coreceptor CXCR4
[0094] The process of HIV-1 entry into a CD4.sup.+ target cell can
be divided into three steps: 1) the virus Env surface subunit gp120
binds to the CD4 molecule; 2) the gp120-CD4 complex interacts with
a coreceptor (CXCR4 or CCR5) on target cells; and 3) the
transmembrane subunit gp41 changes conformation to form the
fusion-active six-helix bundle, resulting in the fusion of viral
envelope with the target cell membranes (45). In the following
experiments, which step of the HIV-1 entry is blocked by NB-2 and
NB-64 was investigated.
[0095] First, it was determined whether NB-2 and NB-64 block
CD4-gp120 interaction. HIV-1 envelope glycoprotein gp120 was
captured by the anti-gp120 antibody coated onto wells of
polystyrene plates. Soluble CD4 (sCD4) was added in the presence or
absence of NB-2 and NB-64. Chloropeptin, a gp120-CD4 binding
inhibitor having a potent anti-HIV-1 activity (36), was used as a
control. After extensive washes, CD4 molecule bound to gp120 was
quantitated by ELISA using anti-CD4 antibody. The results indicated
that chloropeptin at 10 .mu.g/ml markedly inhibited gp120-CD4
binding, while NB-2 and NB-64 at the same concentration did not
significantly inhibited the interaction between CD4 and gp120 (FIG.
5A), suggesting that NB-2 and NB-64 are not targeted to the
gp120-CD4 binding step.
[0096] Then, it was determined whether NB-2 and NB-64 bind to the
HIV-1 coreceptor CXCR4 by a cell-based ELISA as previously
described (52) using a mAb 12G5S, which specifically recognizes
CXCR4 and blocks HIV-1 infection of CXCR4.sup.+ cells (37). T22, a
CXCR4 inhibitory peptide (39), was used as a control. As shown in
FIG. 5B, T22 at 25 .mu.M significantly inhibited 12G5 binding to
U373-MAGI-CXCR4.sub.CEM cells, a CXCR4-expressing cell line.
However, at the same concentration, NB-2 and NB-64 had no
inhibitory activity in this assay, suggesting that NB-2 and NB-64
do not interact with the HIV-1 coreceptor CXCR4 (FIG. 5B).
NB-2 and NB-64 Interfere with the gp41 Six-Helix Bundle
Formation
[0097] Subsequently, a determination was made as the effect of NB-2
and NB-64 on the gp41 six-helix bundle formation, a critical
conformational change during HIV-1 fusion with the target cells. A
model system of the gp41 six-helix bundle was established by mixing
the N- and C-peptides at equal molar concentrations (33) This model
gp41 core structure can be detected by sandwich ELISA using a
conformation-specific mAb, NC-1 (20, 23). Using this system, the
inhibitory activity of NB-2 and NB-64 on the gp41 six-helix bundle
formation was tested. As shown in FIG. 6A, NB-2 and NB-64
significantly inhibited the six-helix bundle formation between N36
and C34 in a dose-dependent manner. Their analogs, NB-177 and
NB-178 had no inhibitory activity on the six-helix bundle formation
at the concentrations up to 80 .mu.g/ml (data not shown). These
results suggest that NB-2 and NB-64 may bind to a component in the
gp41 coiled coil domains and interfere with the association between
the gp41 NHR and CHR regions.
[0098] A convenient biophysical method, FN-PAGE, was previously
developed for revealing the visible bands of gp41 core formed by
N36 and FITC-conjugated C34 (C34-FITC) (32). This method was used
to detect the inhibitory activity of NB-2 and NB-64 on the gp41
core formation. As shown in FIG. 6B, in the absence of N36,
C34-FITC showed a clear band at the lower position (lane 1). When
N36 and C34-FITC was mixed together, two bands were revealed (lane
2). The major band at the upper position corresponds to the gp41
six-helix bundle formed by N36 and C34-FITC as confirmed by Western
blot using the mAb NC-1 (32). The minor band at the lower position
is the isolated C34-FITC. When the compounds NB-2 (lane 3) and
NB-64 (lane 5) were preincubated with N36 before addition of
C34-FITC, the intensity of the upper bands were significantly
decreased while that of the lower bands were increased, suggesting
that the gp41 six-helix bundle formation between N36 and C34 were
inhibited by these two compounds and more isolated C34-FITC was
accumulated. However, if N36 was preincubated with C34-FITC before
addition of NB-2 (lane 4) and NB-64 (lane 6), the six-helix bundle
formation was not inhibited, indicating that NB-2 and NB-64 may
interact with a component in the gp41 NHR region, thus blocking the
formation of the fusion-active gp41 core. However, once the
six-helix bundle is formed, these two compounds cannot disrupt
it.
[0099] NE-2 and NB-64 Interfere with the Conformational Change
During the Interaction Between N- and C-Peptides
[0100] Previous studies demonstrated that the isolated N-peptide
had tendency to aggregate and C-peptide had a random coil structure
in aqueous solution. However, the mixture of the N- and C-peptides
shows a typical .alpha.-helical conformation, as measured by CD
spectroscopy (33), suggesting that the interaction between N- and
C-peptides results in the change of their secondary structure to an
.alpha.-helical coiled-coil conformation. Using a CD
spectrophotometer (Model J-715, Jasco Inc., Japan), it is
demonstrated that NB-2 and NB-64 at 100 .mu.g/ml significantly
disrupted the .alpha.-helicity of the N36/C34 mixture (FIG. 7).
These results confirm that these two compounds interfere with the
conformational change during the interaction between the N- and
C-peptides.
NB-2 and NB-64 Blocked Binding of a D-Peptide to the Pocket
Presented on the gp41 Trimer Modeled by IQN17
[0101] During the process of HIV-1 fusion with the target cell
membrane, the gp41 NHR and CHR regions associate to form fusogenic
core structure. There are three highly conserved symmetrical
hydrophobic grooves on the surface of the internal trimeric
coiled-coil (3, 46, 48) and each of the grooves contains a deep
hydrophobic pocket, which is an attractive target for HIV-1 entry
inhibitors (2, 3). Eckert et al. identified a short circular
anti-HIV-1 peptide consisting of D-amino acids, designated
D10-p5-2K. This peptide specifically binds to the pocket presented
on the IQN17, which mimics the gp41 central trimeric coiled-coil
domain. Similar approach was used to determine whether NB-2 and
NB-64 bind to the pocket and block D10-p5-2K binding to the pocket
on IQN17 trimer. As shown in FIG. 8, both NB-2 and NB-64
significantly inhibited biotinylated D10-p5-2K binding to IQN17.
These results suggest that NB-2 and NB-64 may interact with the
hydrophobic pocket in the gp41 central trimer and block the
interaction between the viral gp41 NHR and CHR regions to form the
six-helix bundle, resulting in inhibition of HIV-1 entry and
replication.
Discussion
[0102] During the past 20 years, one of the greatest progresses in
HIV/AIDS research is the development of anti-HIV drugs (13). So
far, 20 anti-HIV drugs have been approved by the US FDA and more
drug candidates are in the pipelines (13, 43). Most of these drugs
are targeted to the HIV-1 reverse transcriptase and protease. Only
one of them, Fuzeon (T-20), targets the viral envelope glycoprotein
gp41 (5, 17, 29, 50). T-20 (50), like other peptides derived from
the HIV-1 gp41 CHR region, such as SJ-2176 (21, 22) and C34 (35),
inhibits HIV-1 fusion and entry. It has shown great promise against
HIV replication in clinical trials (28, 29). However, it has two
major limitations: lack of oral availability (delivered by
subcutaneous injection) and high cost of production (43). Thus,
development of small molecule HIV-1 fusion inhibitors is urgently
needed.
[0103] It was previously reported that the identification of a
small molecule HIV-1 fusion inhibitor, ADS-J1, through screening
using computer-aided molecular docking techniques and a sandwich
ELISA using a conformation-specific mAb NC-1 (9, 23). However, this
compound is not a good lead compound since it is a dye with azo
bonds and several reactive groups. In addition, it has a molecular
size of 1,087 daltons, larger than most "drug-like" compounds.
Therefore, it was necessary to screen chemical libraries consisting
of "drug-like" compounds using a syncytium formation assay for the
primary screening and the ELISA for the secondary screening.
Although the ELISA is convenient for high throughput screening of
HIV-1 fusion inhibitors targeting gp41 (23), it may miss the
anti-HIV-1 compounds that are targeted to other steps of HIV-1
entry, such as gp120 binding to CD4 and the coreceptors (CXCR4 or
CCR5). Therefore, a syncytium formation assay for the primary
screening was used since this assay can pick up the HIV-1 fusion
inhibitors targeting to any step of HIV-1 entry.
[0104] Using this two-step screening assays, it was identified that
two HIV-1 fusion inhibitors, NB-2 and NB-64, from a chemical
library consisting of 33,040 "drug-like" compounds. These two
compounds have the same parent structure and are N-substituted
pyrrole derivatives. Both are water soluble and "drug-like"
compounds based on the Lipinski's "rule of five" (31), i.e.,
molecular weight<500 daltons, the calculated CLogP<5, H-bond
donors <5 and H-bond acceptors<10. Therefore, these two
compounds may have good permeability and bioavailability.
[0105] NB-2 and NB-64 have potent anti-HIV-1 activity and low
cytotoxicity. They inhibited HIV-1.sub.IIIB replication at
concentration<0.5 .mu.g/ml and selectivity index (SI)>1,000.
The compounds have broad specificity against infection by both
laboratory-adapted and primary HIV-1 strains with distinct
genotypes and phenotypes. The evidence shows that NB-2 and NB-64
interferes with the HIV-1 entry step, more specifically, the
process of gp41-mediated fusion between viral and target cell
membranes as confirmed by a time-of-addition assay (FIG. 3) and
virus-cell and cell-cell fusion assays (FIG. 4). The process of
HIV-1 entry into target cell can be divided into three steps, i.e.,
gp120-CD4 interaction, gp120-CD4 complex binding to a coreceptor
(CXCR4 or CCR5) and gp41 conformational changes. Here it has been
demonstrated that NB-2 and NB-64 cannot block the interaction
between gp120 and CD4 molecules (FIG. 5A), nor interact with CXCR4
(FIG. 5B). However, they do interfere with the gp41 conformational
changes by blocking the formation of the fusion-active gp41
six-helix bundles as demonstrated by several assay systems (FIGS. 6
and 7).
[0106] Although design of small molecule organic compounds to block
protein-protein interaction is a challenging approach for drug
development (8), identification of such inhibitors have been
reported (14, 42, 47). Most recently, a small molecule HIV-1 entry
inhibitor, BMS-378806, was discovered (30). This compound with a
molecular weight of 406.5 is very potent to block interaction
between the viral envelope glycoprotein gp120 and the cellular
receptor CD4. This suggests that a small molecule compound, if
binds to a "hot spot" in a protein, such as a pocket, may
effectively block protein-protein interaction. The deep hydrophobic
pocket in the groove on the surface of the gp41 internal trimer
formed by the NHR domains has been recognized as a "hot spot" since
it may play important roles in the formation and the stability of
the gp41 six-helix bundle (2, 10). NB-2 and NB-64 may bind to the
gp41 pocket since it blocks the binding of D10-p5-2k peptide to the
pocket in the grooves formed by IQN17, a hybrid molecule of GCN4
peptide and the pocket-containing N-peptide portion (FIG. 8).
[0107] To determine the role of the acidic group in NB-2 and NB-64,
Two N-substituted pyrrole derivatives, NB-177 and NB-178 have been
tested, which have the same parent structure as NB-2 and NB-64,
respectively, except that they do not have the COOH group (FIG. 1).
These compounds had no inhibitory activity on HIV-1 replication
(FIG. 2), HIV-1 mediated virus-cell and cell-cell fusion (FIG. 4),
and the gp41 six-helix bundle formation (FIG. 6), suggesting that
the acid group in the NB-2 and NB-64 is critical for their
antiviral activity, consistent with our previous observation on
ADS-J1 which has several acid groups and one of them forms a salt
bridge with the positively charged residue, lysine 574 (K574) in
the gp41 NHR pocket-forming region (19). These results suggest that
NB-2 and NB-64 may interact with a "hot spot" in the gp41 N-helix
coiled coil domain through hydrophobic and ionic interactions and
block the formation of the fusion-active gp41 core, resulting in
inhibition of HIV-1 mediated membrane fusion and virus entry.
[0108] NB-2 and NB-64 have broad anti-HIV-1 activity against
distinct primary HIV-1 strains and specificity to target gp41.
Thus, NB-2 and NB-64 may be used as leads for designing more potent
small molecule HIV-1 entry inhibitors as a new class of anti-HIV-1
drugs.
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