U.S. patent application number 12/047789 was filed with the patent office on 2008-09-11 for antiviral inhibition of capsid proteins.
Invention is credited to Michael F. SUMMERS, Chun TANG.
Application Number | 20080221215 12/047789 |
Document ID | / |
Family ID | 29255357 |
Filed Date | 2008-09-11 |
United States Patent
Application |
20080221215 |
Kind Code |
A1 |
SUMMERS; Michael F. ; et
al. |
September 11, 2008 |
ANTIVIRAL INHIBITION OF CAPSID PROTEINS
Abstract
Methods for evaluating the antiviral activity of test compounds
are provided. Further aspects of the methods involve the retroviral
capsid protein of HIV-1. In another aspect, methods of reducing
mortality associated with AIDS with a compound that binds to the
apical cleft near the C-terminal end of the N-terminal domain of
the HIV-1 capsid protein are provided. Derivatives of CAP-1, CAP-2,
CAP-3, CAP-4, CAP-5, CAP-6 and CAP-7 are described that bind to the
apical cleft of the N-terminal domain of the HIV-1 capsid protein
and inhibit proper assembly of the core particle.
Inventors: |
SUMMERS; Michael F.;
(Ellicott City, MD) ; TANG; Chun; (Baltimore,
MD) |
Correspondence
Address: |
WILMERHALE/BOSTON
60 STATE STREET
BOSTON
MA
02109
US
|
Family ID: |
29255357 |
Appl. No.: |
12/047789 |
Filed: |
March 13, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10420438 |
Apr 22, 2003 |
7361459 |
|
|
12047789 |
|
|
|
|
60374557 |
Apr 22, 2002 |
|
|
|
60375852 |
Apr 25, 2002 |
|
|
|
60404043 |
Aug 16, 2002 |
|
|
|
Current U.S.
Class: |
514/595 ; 435/5;
564/47 |
Current CPC
Class: |
Y02A 90/26 20180101;
A61K 31/341 20130101; G16B 15/00 20190201; G16C 20/60 20190201;
A61P 31/18 20180101; C07C 275/32 20130101; C07C 275/30 20130101;
G01N 2500/04 20130101; A61K 31/277 20130101; C07C 281/06 20130101;
C12Q 1/18 20130101; G16B 35/00 20190201; A61P 43/00 20180101; A61K
31/17 20130101; C07K 14/005 20130101; C12N 2740/16222 20130101;
C07C 275/40 20130101; C07C 275/42 20130101; Y02A 90/10 20180101;
C07D 307/52 20130101; G16C 20/50 20190201; A61K 31/00 20130101 |
Class at
Publication: |
514/595 ; 564/47;
435/5 |
International
Class: |
A61K 31/17 20060101
A61K031/17; C07C 273/00 20060101 C07C273/00; C12Q 1/70 20060101
C12Q001/70; A61P 31/18 20060101 A61P031/18 |
Goverment Interests
GOVERNMENT LICENSE RIGHTS
[0002] The U.S. Government has a paid-up license in this invention
and the right in limited circumstances to require the patent owner
to license others on reasonable terms as provided for by the terms
of grant no. A130917 awarded by the National Institutes of Health.
Claims
1.-8. (canceled)
9. A method of reducing mortality associated with AIDS comprising
the step of administering a therapeutically effective amount of a
compound that binds to the apical cleft near the C-terminal end of
the N-terminal domain of the HIV capsid protein to a human
suffering from AIDS.
10. The method of claim 9 wherein the capsid protein is an HIV-1
capsid protein.
11. The method of claim 9 wherein the compound is selected form the
group consisting of
N-(3-chloro-4-methylphenyl)-N'-[2-thioethyl-2'-[5-(dimethylaminomethyl)]--
2-methylfuryl]urea (CAP-1),
N-(4-N-acetamidophenyl)-N'-(3-nitro-4-methyl phenyl)urea (CAP-2),
N-(2-propyl)-N'-(3-nitro-4-methyl phenyl)urea (CAP-3),
N-(3-chloro-4-methyl phenyl)-N'-(4-cyanophenyl)urea (CAP-4),
N-(3-chloro-4-methyl
phenyl)-N'-[4-(1,1,1-trichloromethyl)phenyl]urea (CAP-5),
N-(3-nitro-4-fluorophenyl)-N'-[3-(1,1,1-trifluoromethyl)phenyl]u-
rea (CAP-6), N-[(3-chloro-4-methyl phenyl)-N',N'-propyl]urea
(CAP-7).
12. The method of claim 11 wherein the compound is
N-(3-chloro-4-methylphenyl)-N'-[2-thioethyl-2'-[5-(dimethylaminomethyl)]--
2-methylfuryl]urea (CAP-1).
13. The method of claim 11 wherein the compound is
N-(4-N-acetamidophenyl)-N'-(3-nitro-4-methyl phenyl)urea
(CAP-2).
14. The method of claim 11 wherein the compound is
N-(2-propyl)-N'-(3-nitro-4-methyl phenyl)urea (CAP-3).
15. The method of claim 11 wherein the compound is
N-(3-chloro-4-methyl phenyl)-N'-(4-cyanophenyl)urea (CAP-4).
16. The method of claim 11 wherein the compound is
N-(3-chloro-4-methyl
phenyl)-N'-[4-(1,1,1-trichloromethyl)phenyl]urea (CAP-5).
17. The method of claim 11 wherein the compound is
N-(3-nitro-4-fluorophenyl)-N'-[3-(1,1,1-trifluoromethyl)phenyl]urea
(CAP-6).
18. The method of claim 11 wherein the compound is
N-[(3-chloro-4-methyl phenyl)-N',N'-propyl]urea (CAP-7).
19. A method of treating a human suffering from AIDS comprising the
step of administering a compound that binds to the apical cleft
near the C-terminal end of the N-terminal domain of the HIV capsid
protein in an amount effective to reduce the number and severity of
morbidities.
20. The method of claim 19 wherein the HIV capsid protein is an
HIV-1 capsid protein.
21. The method of claim 19 wherein the compound is selected form
the group consisting of
N-(3-chloro-4-methylphenyl)-N'-[2-thioethyl-2'-[5-(dimethylaminomethyl)]--
2-methylfuryl]urea (CAP-1),
N-(4-N-acetamidophenyl)-N'-(3-nitro-4-methyl phenyl)urea (CAP-2),
N-(2-propyl)-N'-(3-nitro-4-methyl phenyl)urea (CAP-3),
N-(3-chloro-4-methyl phenyl)-N'-(4-cyanophenyl)urea (CAP-4),
N-(3-chloro-4-methyl
phenyl)-N'-[4-(1,1,1-trichloromethyl)phenyl]urea (CAP-5),
N-(3-nitro-4-fluorophenyl)-N'-[3-(1,1,1-trifluoromethyl)phenyl]u-
rea (CAP-6), N-[(3-chloro-4-methyl phenyl)-N',N'-propyl]urea
(CAP-7).
22. The method of claim 21 wherein the compound is
N-(3-chloro-4-methylphenyl)-N'-[2-thioethyl-2'-[5-(dimethylaminomethyl)]--
2-methylfuryl]urea (CAP-1).
23. The method of claim 21 wherein the compound is
N-(4-N-acetamidophenyl)-N'-(3-nitro-4-methyl phenyl)urea
(CAP-2).
24. The method of claim 21 wherein the compound is
N-(2-propyl)-N'-(3-nitro-4-methyl phenyl)urea (CAP-3).
25. The method of claim 21 wherein the compound is
N-(3-chloro-4-methyl phenyl)-N'-(4-cyanophenyl)urea (CAP-4).
26. The method of claim 21 wherein the compound is
N-(3-chloro-4-methyl
phenyl)-N'-[4-(1,1,1-trichloromethyl)phenyl]urea (CAP-5).
27. The method of claim 21 wherein the compound is
N-(3-nitro-4-fluorophenyl)-N'-[3-(1,1,1-trifluoromethyl)phenyl]urea
(CAP-6).
28. The method of claim 21 wherein the compound is),
N-[(3-chloro-4-methyl phenyl)-N',N'-propyl]urea (CAP-7).
29. A method of evaluating a compound for the ability to inhibit
.beta.-hairpin formation of Gag comprising: a) contacting said
compound with Gag.sup.283 or a fragment thereof, and b) determining
the ability of said compound to interfere with .beta.-hairpin
formation of Gag.sup.283.
30. A method of screening a candidate compound for the ability to
inhibit .beta.-hairpin formation of Gag.sup.283 comprising: a)
contacting said compound with Gag.sup.283 or a fragment thereof,
and b) determining the ability of said compound to interfere with
.beta.-hairpin formation of Gag.sup.283.
31. A method of identifying a compound for the ability to inhibit
.beta.-hairpin formation of Gag.sup.283 comprising: a) generating a
3D computer model of Gag.sup.283 using Gag.sup.283 molecular
coordinates, and b) using said model to identify a compound that
binds to Gag.sup.283.
32. A method of identifying a compound that binds to the apical
cleft near the C-terminal end of the N-terminal domain of a viral
capsid protein comprising: a) generating a 3D computer model of
Gag.sup.283 using Gag.sup.283 molecular coordinates, and b) using
said model to identify a compound that binds to said apical
cleft.
33. The method of claim 32 wherein the capsid protein is an HIV-1
capsid protein.
34. A method of inhibiting capsid assembly with a compound that
binds to the apical cleft near the C-terminal end of the N-terminal
domain of a capsid protein.
35. The method of claim 34 wherein the capsid protein is selected
from the group consisting of viral capsid proteins and retroviral
capsid proteins.
36. The method of claim 35 wherein the capsid protein is an HIV-1
capsid protein.
37. The method of claim 34 wherein said compound is selected from
the group consisting of
N-(3-chloro-4-methylphenyl)-N'-[2-thioethyl-2'-[5-(dimethylaminomethyl)]--
2-methylfuryl]urea (CAP-1),
N-(4-N-acetamidophenyl)-N'-(3-nitro-4-methyl phenyl)urea (CAP-2),
N-(2-propyl)-N'-(3-nitro-4-methyl phenyl)urea (CAP-3),
N-(3-chloro-4-methylphenyl)-N'-(4-cyanophenyl)urea (CAP-4),
N-(3-chloro-4-methyl
phenyl)-N'-[4-(1,1,1-trichloromethyl)phenyl]urea (CAP-5),
N-(3-nitro-4-fluorophenyl)-N'-[3-(1,1,1-trifluoromethyl)phenyl]u-
rea (CAP-6), N-[(3-chloro-4-methyl phenyl)-N',N'-propyl]urea
(CAP-7).
38. The method of claim 36 wherein said compound is selected from
the group consisting of
N-(3-chloro-4-methylphenyl)-N'-[2-thioethyl-2'-[5-(dimethylaminomethyl)]--
2-methylfuryl]urea (CAP-1),
N-(4-N-acetamidophenyl)-N'-(3-nitro-4-methyl phenyl)urea (CAP-2),
N-(2-propyl)-N'-(3-nitro-4-methyl phenyl)urea (CAP-3),
N-(3-chloro-4-methyl phenyl)-N'-(4-cyanophenyl)urea (CAP-4),
N-(3-chloro-4-methyl
phenyl)-N'-[4-(1,1,1-trichloromethyl)phenyl]urea (CAP-5),
N-(3-nitro-4-fluorophenyl)-N'-[3-(1,1,1-trifluoromethyl)phenyl]u-
rea (CAP-6), N-[(3-chloro-4-methyl phenyl)-N',N'-propyl]urea
(CAP-7).
39. A method of inhibiting capsid disassembly during infectivity
with a compound that binds to the apical cleft near the C-terminal
end of the N-terminal domain of a capsid protein.
40. The method of claim 39 wherein the capsid protein is selected
from the group consisting of viral capsid proteins and retroviral
capsid proteins.
41. The method of claim 39 wherein the capsid protein is an HIV-1
capsid protein.
42. The method of claim 39 wherein said compound is selected from
the group consisting of
N-(3-chloro-4-methylphenyl)-N'-[2-thioethyl-2'-[5-(dimethylaminomethyl)]--
2-methylfuryl]urea (CAP-1),
N-(4-N-acetamidophenyl)-N'-(3-nitro-4-methyl phenyl)urea (CAP-2),
N-(2-propyl)-N'-(3-nitro-4-methyl phenyl)urea (CAP-3),
N-(3-chloro-4-methyl phenyl)-N'-(4-cyanophenyl)urea (CAP-4),
N-(3-chloro-4-methyl
phenyl)-N'-[4-(1,1,1-trichloromethyl)phenyl]urea (CAP-5),
N-(3-nitro-4-fluorophenyl)-N'-[3-(1,1,1-trifluoromethyl)phenyl]u-
rea (CAP-6), N-[(3-chloro-4-methyl phenyl)-N',N'-propyl]urea
(CAP-7).
43. The method of claim 41 wherein said compound is selected from
the group consisting of
N-(3-chloro-4-methylphenyl)-N'-[2-thioethyl-2'-[5-(dimethylaminomethyl)]--
2-methylfuryl]urea (CAP-1),
N-(4-N-acetamidophenyl)-N'-(3-nitro-4-methyl phenyl)urea (CAP-2),
N-(2-propyl)-N'-(3-nitro-4-methyl phenyl)urea (CAP-3),
N-(3-chloro-4-methyl phenyl)-N'-(4-cyanophenyl)urea (CAP-4),
N-(3-chloro-4-methyl
phenyl)-N'-[4-(1,1,1-trichloromethyl)phenyl]urea (CAP-5),
N-(3-nitro-4-fluorophenyl)-N'-[3-(1,1,1-trifluoromethyl)phenyl]u-
rea (CAP-6), N-[(3-chloro-4-methyl phenyl)-N',N'-propyl]urea
(CAP-7).
44. A compound or its pharmaceutically acceptable salt having the
formula I: ##STR00003## wherein: (a) R.sub.1 represents hydrogen,
halogen, cyano, trifluoromethyl, trichloromethyl, nitro,
--OR.sub.8, --SR.sub.8, --NHR.sub.8, --NR.sub.8R.sub.9, --NHCOOH,
--NHCH.sub.2COOH, --NHCHR.sub.8COOH, --NHCR.sub.8R.sub.9COOH,
--NR.sub.8COOR.sub.9, --COOR.sub.8 or a hydrocarbon group
comprising a straight chained, branched or cyclic group each
containing up to 9 carbon atoms; (b) R.sub.2 represents hydrogen,
halogen, cyano, trifluoromethyl, trichloromethyl, nitro,
--OR.sub.8, --SR.sub.8, --NHR.sub.8, --NR.sub.8R.sub.9, --NHCOOH,
--NHCH.sub.2COOH, --NHCHR.sub.8COOH, --NHCR.sub.8R.sub.9COOH,
--NR.sub.8COOR.sub.9, --COOR.sub.8 or a hydrocarbon group
comprising a straight chained, branched or cyclic group each
containing up to 9 carbon atoms; (c) R.sub.3 represents hydrogen,
halogen, cyano, trifluoromethyl, trichloromethyl, nitro,
--OR.sub.8, --SR.sub.8, --NHR.sub.8, --NR.sub.8R.sub.9, --NHCOOH,
--NHCH.sub.2COOH, --NHCHR.sub.8COOH, --NHCR.sub.8R.sub.9COOH,
--NR.sub.8COOR.sub.9, --COOR.sub.8 or a hydrocarbon group
comprising a straight chained, branched or cyclic group each
containing up to 9 carbon atoms; (d) R.sub.4 represents hydrogen,
halogen, cyano, trifluoromethyl, trichloromethyl, nitro,
--OR.sub.8, --SR.sub.8, --NHR.sub.8, --NR.sub.8R.sub.9, --NHCOOH,
--NHCH.sub.2COOH, --NHCHR.sub.8COOH, --NHCR.sub.8R.sub.9COOH,
--NR.sub.8COOR.sub.9, --COOR.sub.8 or a hydrocarbon group
comprising a straight chained, branched or cyclic group each
containing up to 9 carbon atoms; (e) R.sub.5 represents hydrogen,
halogen, cyano, trifluoromethyl, trichloromethyl, nitro,
--OR.sub.8, --SR.sub.8, --NHR.sub.8, --NR.sub.8R.sub.9, --NHCOOH,
--NHCH.sub.2COOH, --NHCHR.sub.8COOH, --NHCR.sub.8R.sub.9COOH,
--NR.sub.8COOR.sub.9, --COOR.sub.8 or a hydrocarbon group
comprising a straight chained, branched or cyclic group each
containing up to 9 carbon atoms; (f) Halogen is limited to fluoro,
chloro, bromo, and iodo; (g) R.sub.8 and R.sub.9 are independently
methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl,
t-butyl, pentyl, hexyl, neo-pentyl, cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cyclopropylmethyl, cyclopropylethyl,
cyclobutylmethyl, cyclobutylethyl, cyclopentylmethyl,
cyclohexylmethyl, or cyclohexylethyl; (h) The letters n, m, and p
represent independently any integer from 1 to 6; (i) R.sub.6 and
R.sub.7 are independently selected from the group consisting of
hydrogen or a hydrocarbon group comprising a straight chained,
branched or cyclic group each containing up to 6 carbon atoms; (j)
X is selected from the group consisting of O or S; (k) Y is
heterocyclic, carbocyclic, or optionally substituted phenyl; (l)
Heterocyclic is selected from any stable 5, 6, or 7-membered
monocyclic or bicyclic or 7, 8, 9, or 10-membered bicyclic
heterocyclic ring which is saturated, partially unsaturated or
unsaturated (aromatic), and which consists of carbon atoms and 1,
2, 3, or 4 heteroatoms independently selected from the group
consisting of N, NH, O and S and including any bicyclic group in
which any of the above-defined heterocyclic rings is fused to a
benzene ring. The nitrogen and sulfur heteroatoms may optionally be
oxidized. The heterocyclic ring may be attached to its pendant
group at any heteroatom or carbon atom which results in a stable
structure. The heterocyclic rings described herein may be
substituted on carbon or on a nitrogen atom if the resulting
compound is stable. If specifically noted, a nitrogen in the
heterocycle may optionally be quaternized. It is preferred that
when the total number of S and O atoms in the heterocycle exceeds
1, then these heteroatoms are not adjacent to one another. As used
herein, the term "aromatic heterocyclic system" is intended to mean
a stable 5- to 7-membered monocyclic or bicyclic or 7- to
10-membered bicyclic heterocyclic aromatic ring which consists of
carbon atoms and from 1 to 4 heteroatoms independently selected
from the group consisting of N, O and S. Examples of heterocycles
include, but are not limited to, 1H-indazole, 2-pyrrolidonyl,
2H,6H-1,5,2-dithiazinyl, 2H-pyrrolyl, 3H-indolyl, 4-piperidonyl,
4aH-carbazole, 4H-quinolizinyl, 6H-1,2,5-thiadiazinyl, acridinyl,
azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl,
benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl,
benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazalonyl,
carbazolyl, 4aH-carbazolyl, .beta.-carbolinyl, chromanyl,
chromenyl, cinnolinyl, decahydroquinolinyl,
2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran,
furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl,
1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl,
isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl,
isoindolyl, isoquinolinyl (benzimidazolyl), isothiazolyl,
isoxazolyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl,
oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl,
1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl,
oxazolidinylperimidinyl, phenanthridinyl, phenanthrolinyl,
phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl,
phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, pteridinyl,
piperidonyl, 4-piperidonyl, pteridinyl, purinyl, pyranyl,
pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl,
pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl,
pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl,
quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,
carbolinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,
tetrahydroquinolinyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,
1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,
thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,
thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl,
1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, tetrazolyl, and
xanthenyl, Preferred heterocycles include, but are not limited to,
pyridinyl, thiophenyl, furanyl, indazolyl, benzothiazolyl,
benzimidazolyl, benzothiaphenyl, benzofuranyl, benzoxazolyl,
benzisoxazolyl, quinolinyl, isoquinolinyl, imidazolyl, indolyl,
isoidolyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,
pyrrazolyl, 1,2,4-triazolyl, 1,2,3-triazolyl, tetrazolyl,
thiazolyl, oxazolyl, pyrazinyl, and pyrimidinyl, and fused ring and
spiro compounds containing the above heterocycles; (m) carbocyclic
is intended to mean any stable 3, 4, 5, 6, or 7-membered monocyclic
or bicyclic or 7, 8, 9, 10, 11, 12, or 13-membered bicyclic or
tricyclic, any of which may be saturated, partially unsaturated, or
aromatic. Examples of such carbocycles include, but are not limited
to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
adamantyl, cyclooctyl, [3.3.0] bicyclooctane, [4.3.0]bicyclononane,
[4.4.0]bicyclodecane (decalin), [2.2.2]bicyclooctane, fluorenyl,
phenyl, naphthyl, indanyl, adamantyl, or tetrahydronaphthyl
(tetralin); (n) Substituted phenyl is defined by Structure II
##STR00004## wherein: (o) R.sub.10 represents hydrogen, halogen,
cyano, trifluoromethyl, trichloromethyl, nitro, --OR.sub.8,
--SR.sub.8, --NHR.sub.8, --NR.sub.8R.sub.9, --NHCOOH,
--NHCH.sub.2COOH, --NR.sub.8COOR.sub.9, --COOR.sub.8 or a
hydrocarbon group comprising a straight chained, branched or cyclic
group each containing up to 9 carbon atoms; (p) R.sub.11
independently represents hydrogen, halogen, cyano, trifluoromethyl,
trichloromethyl, nitro, --OR.sub.8, --SR.sub.8, --NHR.sub.8,
--NR.sub.8R.sub.9, --NHCOOH, --NHCH.sub.2COOH,
--NR.sub.8COOR.sub.9, --COOR.sub.8 or a hydrocarbon group
comprising a straight chained, branched or cyclic group each
containing up to 9 carbon atoms; (q) R.sub.12 independently
represents hydrogen, halogen, cyano, trifluoromethyl,
trichloromethyl, nitro, --OR.sub.8, --SR.sub.8, --NHR.sub.8,
--NR.sub.8R.sub.9, --NHCOOH, --NHCH.sub.2COOH,
--NR.sub.8COOR.sub.9, --COOR.sub.8 or a hydrocarbon group
comprising a straight chained, branched or cyclic group each
containing up to 9 carbon atoms; (r) R.sub.13 independently
represents hydrogen, halogen, cyano, trifluoromethyl,
trichloromethyl, nitro, --OR.sub.8, --SR.sub.8, --NHR.sub.8,
--NR.sub.8R.sub.9, --NHCOOH, --NHCH.sub.2COOH,
--NR.sub.8COOR.sub.9, --COOR.sub.8 or a hydrocarbon group
comprising a straight chained, branched or cyclic group each
containing up to 9 carbon atoms; (s) R.sub.14 independently
represents hydrogen, halogen, cyano, trifluoromethyl,
trichloromethyl, nitro, --OR.sub.8, --SR.sub.8, --NHR.sub.8,
--NR.sub.8R.sub.9, --NHCOOH, --NHCH.sub.2COOH,
--NR.sub.8COOR.sub.9, --COOR.sub.8 or a hydrocarbon group
comprising a straight chained, branched or cyclic group each
containing up to 9 carbon atoms; (t) R.sub.8 and R.sub.9 are
independently methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl,
sec-butyl, t-butyl, pentyl, hexyl, neo-pentyl, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cyclopropylmethyl,
cyclopropylethyl, cyclobutylmethyl, cyclobutylethyl,
cyclopentylmethyl, cyclohexylmethyl, or cyclohexylethyl; (u)
Halogen is limited to fluoro, chloro, bromo, and iodo.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional
application No. 60/374,557 filed Apr. 22, 2002, U.S. provisional
application No. 60/375,852 filed Apr. 25, 2002 and U.S. provisional
application No. 60/404,043 filed Aug. 16, 2002.
FIELD OF THE INVENTION
[0003] The invention relates to treatment of Acquired
Immunodeficiency Syndrome (AIDS). In particular, the invention
relates to treatment of AIDS by inhibition of the Human
immunodeficiency virus type 1 (HIV-1) capsid protein.
BACKGROUND OF THE INVENTION
[0004] Viruses are noncellular infective agents that are capable of
reproducing only in an appropriate host cell. Viruses are typically
smaller than bacteria, and can infect animal, plant, or bacterial
cells. Many viruses are important agents of disease. The infective
particle (virion) consists of a core of nucleic acid surrounded by
a proteinaceous capsid and, in some cases, an outer envelope.
[0005] Retroviruses are enveloped single-stranded RNA viruses
infecting animals. The family consists of three groups: the
spumaviruses such as the human foamy virus; the lentiviruses, such
as the human immunodeficiency virus types 1 and 2, as well as visna
virus of sheep; and the oncoviruses.
[0006] The retrovirus particle is composed of two identical RNA
molecules. Each genome is a positive sense, single-stranded RNA
molecule, which is capped at the 5' end and polyadenylated at the
3' tail. The prototype C-type oncoviral RNA genome contains three
open reading frames call gag, pol and env, bounded by regions that
contain signals essential for expression of the viral genes. The
gag region encodes the structural proteins of the viral capsid. The
pol region encodes a viral proteinase as well as the proteins for
genome processing, including reverse transcriptase, ribonuclease H
and endonuclease enzymatic activities. The env region specifies the
glycoproteins of the viral envelope. In addition to these three
open reading frames, the more complex genomes of the lentiviruses
and the spumaviruses carry additional open reading frames which
encode regulatory proteins involved in the control of genome
expression.
[0007] There are substantial homologies between the capsid proteins
of both viruses and retroviruses. One of skill in the art can
easily identify these homologies by searching any of the standard
biotechnology search engines.
[0008] AIDS is a retroviral disease characterized by profound
immunosuppression that leads to opportunistic infections, secondary
neoplasms and neurologic manifestations. The magnitude of this
modern plague is truly staggering. In the United States, AIDS is
the leading cause of death in men between 25 and 44 years of age,
and it is the third leading cause of death of women in this age
group. Although initially recognized and reported in the United
States, it has now been reported from more than 193 countries
around the world. The pool of HIV-infected persons in Africa and
Asia is large and rapidly expanding. Transmission of HIV occurs
under conditions that facilitate exchange of blood or body fluids
containing the virus or virus-infected cells. Hence, the three
major routes of transmission are sexual contact, parenteral
inoculation, and passage of the virus from infected mothers to
their newborns. Because of the almost uniformly fatal outcome of
AIDS, finding effective treatments for the disease remains a
serious medical problem.
[0009] There is little doubt that AIDS is caused by HIV, a
nontransforming human retrovirus belonging to the lentivirus
family. O'Brien, et al., HIV causes AIDS: Koch's postulates
fulfilled, Curr Opin Immunol 8:613 (1996). Two genetically
different but related forms of HIV, called HIV-1 and HIV-2, have
been isolated from patients with AIDS. HIV-1 is the most common
type associated with AIDS in the United States, Europe, and Central
Africa, whereas HIV-2 causes similar disease principally in West
Africa.
[0010] Similar to most retroviruses, the HIV-1 virion is spherical
and contains an electron-dense, cone shaped core surrounded by a
lipid envelope derived from the host cell membrane. The virus core
contains (1) the major capsid protein p24 (CA), (2) nucleocapsid
protein p7/p9, (3) two copies of genomic RNA, and (4) the three
viral enzymes (protease (PR), reverse transcriptase (RT), and
integrase). The viral core is surrounded by a matrix protein called
p17, which lies underneath the virion envelope. Studding the viral
envelope are two viral glycoproteins, gp 120 and gp 41, which are
critical for HIV infection of cells.
[0011] As with other retroviruses, the HIV proviral genome contains
the gag, pol, and env genes, which code for various viral proteins.
The products of the gag and pol genes are translated initially into
large precursor proteins that must be cleaved by the viral protease
to yield the mature proteins.
[0012] The CA is initially synthesized as a domain within a 55 kDa
Gag precursor polyprotein. Approximately 4,000 copies of Gag
assemble at the plasma membrane and bud to form an immature virus
particle. Subsequent to budding, the CA is liberated by proteolytic
cleavage of Gag, which triggers a conformational change that
promotes assembly of the capsid particle. Gitti, et al., Structure
of the amino-terminal core domain of the HIV-1 capsid protein,
Science, 273: 231-35 (1996); von Schwedler, et al., Proteolytic
refolding of the HIV-1 capsid protein amino-terminus facilitates
viral core assembly, EMBO J., 17: 1555-68 (1998); Gross, et al.,
N-terminal extension of human immunodeficiency virus capsid protein
converts the in vitro assembly phenotype from tubular to spherical
particles, J. Virol., 72: 4798-4810 (1998). Two copies of the viral
genome and enzymes essential for infectivity become encapsidated in
the central, cone shaped capsid of the mature virion.
[0013] Several recent studies have shown that proper capsid
assembly is critical for viral infectivity. Mutations in CA that
inhibit assembly are lethal and mutations that alter capsid
stability severely attenuate replication making the CA an
attractive potential antiviral target. Tang, et al., Human
immunodeficiency virus type 1 N-terminal capsid mutants that
exhibit aberrant core morphology are blocked in initiation of
reverse transcription in infected cells, J. Virol. 75: 9357-66
(2001); Reicin, et al., The role of Gag in human immunodeficiency
virus type 1 virion morphogenesis and early steps of the viral life
cycle, J. Virol., 70: 8645-52 (1996); and Forshey, et al.,
Formation of a human immunodeficiency virus type 1 core of optimal
stability is crucial for viral replication, J. Virol. 76: 5667-5677
(2002).
[0014] Although antiviral agents have been developed that bind to
the capsid protein of picornaviruses and suppress infectivity by
inhibiting disassembly of the capsid shell, Smith, et al., The site
of attachment in human rhinovirus 14 for antiviral agents that
inhibit uncoating, Science, 233: 1286-93 (1986), inhibitors of HIV
capsid assembly or disassembly have not yet been identified.
Currently available drugs for the treatment of HIV infection target
the RT and PR enzymes, two of fifteen proteins encoded by the viral
genome. These drugs are marginally effective when administered
independently due to the rapid emergence of resistant strains that
are selected under conditions of incomplete viral suppression.
Richman, D. D., HIV chemotherapy, Nature 410: 995-1001 (2001).
Although sustained reductions in viral load can be achieved when
inhibitors are used in appropriate combinations (highly affective
anti-retroviral therapy, HAART), Richman, D. D., HIV chemotherapy,
Nature 410: 995-1001 (2001); Pillay, et al, Incidence and impact of
resistance against approved antiretroviral drugs, Rev Med Virol,
10: 231-53 (2000), inadequate suppression due to poor compliance,
resistance, and interactions with other drugs or diet is a
significant problem that limits the effectiveness of HAART therapy
for many patients and can lead to the spread of drug-resistant
strains. Mansky, et al, Combination of drugs an drug-resistant
reverse transcriptase results in a multiplicative increase of human
immunodeficiency virus type 1 mutant frequencies, J. Virol., 76:
9253-59 (2002); Coffin, J., HIV population dynamic in vivo:
implications for genetic variation, pathogenesis, and therapy,
Science, 267: 483-89 (1995); Kuritzkes, D. R., Clinical
significance of drug resistance in HIV-1 infection, AIDS, 10:
S27-33 (1996).
[0015] In spite of the availability of HAART therapy, the mortality
and morbidities associated with AIDS remain significant and
unresolved by current therapies. New therapeutic compounds and
methods are needed that could reduce or ameliorate the adverse
events and improve the clinical outcome of AIDS, including, for
example, reducing mortality and improving the quality of life of
those suffering from the disease.
SUMMARY OF THE INVENTION
[0016] The invention provides novel methods for evaluating the
antiviral activity of test compounds. The method includes (a)
contacting a test compound with Gag.sup.283 or a fragment thereof,
(b) determining the ability of the test compound to bind to the
apical cleft near the C-terminal end of the N-terminal domain of
the capsid protein, and (c) evaluating the antiviral effect of the
test compound. The capsid protein can be a viral capsid protein or
a retroviral capsid protein. The retroviral capsid protein
includes, but is not limited to HIV-1 and HIV-2. The capsid protein
can be immature or mature. In addition, the antiviral effect
includes, but is not limited to, inhibition of capsid assembly
during viral maturation and inhibition of disassembly during
infectivity.
[0017] The invention also provides a method of reducing mortality
associated with AIDS. The method includes administering a
therapeutically effective amount of a compound that binds to the
apical cleft near the C-terminal end of the N-terminal domain of
the HIV capsid protein to a human suffering from AIDS. Compounds of
the invention include, but are not limited to,
N-(3-chloro-4-methylphenyl)-N'-[2-thioethyl-2'-[5-(dimethylaminomethyl)]--
2-methylfuryl]urea (CAP-1),
N-(4-N-acetamidophenyl)-N'-(3-nitro-4-methyl phenyl)urea (CAP-2),
N-(2-propyl)-N'-(3-nitro-4-methyl phenyl)urea (CAP-3),
N-(3-chloro-4-methyl phenyl)-N'-(4-cyanophenyl)urea (CAP-4),
N-(3-chloro-4-methyl
phenyl)-N'-[4-(1,1,1-trichloromethyl)phenyl]urea (CAP-5),
N-(3-nitro-4-fluorophenyl)-N'-[3-(1,1,1-trifluoromethyl)phenyl]u-
rea (CAP-6), N-[(3-chloro-4-methyl phenyl)-N',N'-propyl]urea
(CAP-7).
[0018] The invention also provides a method for treating a human
suffering from AIDS. The method includes administering a compound
that binds the apical cleft near the C-terminal end of the
N-terminal domain of the HIV capsid protein in an amount effective
to reduce the number and severity of morbidities associated with
AIDS. Compounds of the invention include, but are not limited to,
N-(3-chloro-4-methylphenyl)-N'-[2-thioethyl-2'-[5-(dimethylaminomethyl)]--
2-methylfuryl]urea (CAP-1),
N-(4-N-acetamidophenyl)-N'-(3-nitro-4-methyl phenyl)urea (CAP-2),
N-(2-propyl)-N'-(3-nitro-4-methyl phenyl)urea (CAP-3),
N-(3-chloro-4-methyl phenyl)-N'-(4-cyanophenyl)urea (CAP-4),
N-(3-chloro-4-methyl
phenyl)-N'-[4-(1,1,1-trichloromethyl)phenyl]urea (CAP-5),
N-(3-nitro-4-fluorophenyl)-N'-[3-(1,1,1-trifluoromethyl)phenyl]u-
rea (CAP-6), N-[(3-chloro-4-methyl phenyl)-N',N'-propyl]urea
(CAP-7).
[0019] The invention also provides a method of evaluating a test
compound for the ability to inhibit .beta.-hairpin formation of
Gag.sup.283. The method includes (a) contacting a test compound
with Gag.sup.283 or a fragment thereof, and (b) determining the
ability of the compound to interfere with .beta.-hairpin formation
of Gag.sup.283.
[0020] The invention also provides a method of screening a test
compound for the ability to inhibit .beta.-hairpin formation of
Gag.sup.283. The method includes (a) contacting the test compound
with Gag.sup.283 or a fragment thereof, and (b) determining the
ability of the compound to interfere with .beta.-hairpin formation
of Gag.sup.283.
[0021] The invention further provides a method of identifying a
test compound for the ability to inhibit .beta.-hairpin formation
of Gag.sup.283. The method includes (a) generating a 3D computer
model of Gag.sup.283 using Gag.sup.283 molecular coordinates, and
(b) using the model to identify a test compound that binds to
Gag.sup.283.
[0022] The invention further provides a method of identifying a
test compound that binds to the apical cleft near the C-terminal
end of the N-terminal domain of a capsid protein. The method
includes (a) generating a 3D computer model of Gag.sup.283 using
Gag.sup.283 molecular coordinates, and (b) using the model to
identify a test compound that binds to the apical cleft. The capsid
protein can be a viral capsid protein or a retroviral capsid
protein. The retroviral capsid protein includes, but is not limited
to HIV-1 and HIV-2.
[0023] The invention also provides for any derivative or
pharmaceutically acceptable salts of CAP-1, CAP-2, CAP-2, CAP-3,
CAP-4, CAP-5, CAP-6 or CAP-7 having the formula I described below
that is an antiviral compound that binds to the apical site of the
N-terminal domain of the HIV-1 capsid protein and inhibits proper
assembly of the core particle. In particular, the invention
provides derivatives or pharmaceutically acceptable salts of CAP-1
or its related molecules that contain a urea group that contain a
substituted aromatic substituent on one nitrogen and a flexible
tether attached to a second aromatic group on the other
nitrogen.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 is a representation of diffusion tensors determined
independently for the MA and CA.sup.N domains of Gag.sup.283.
D.sub.zz denotes the principle component of the axially symmetric
diffusion tensor.
[0025] FIG. 2 is a comparison of the X-ray and NMR structures
determined to date for the CA.sup.N domain of the HIV-1 capsid
protein.
[0026] FIG. 3 is a representation of CAP-1 docked in the apical
cleft of the HIV-1 capsid protein.
[0027] FIG. 4 is a representation of the short-range interactions
involved in the binding of CAP-1 to the apical cleft of the HIV-1
capsid protein.
[0028] FIG. 5 represents a resolution enhanced .sup.1H-.sup.15N
HSQC spectrum obtained for a .sup.2H, .sup.15N-labeled Gag.sup.283
sample.
[0029] FIG. 6 is a representation of NMR relaxation and chemical
shift data that identifies regions of structure and mobility.
[0030] FIG. 7 is a tabular representation of structural statistics
for the 20 lowest energy Gag.sup.283 conformers.
[0031] FIG. 8 is a representation of backbone .sup.15N
R.sub.2/R.sub.1 values determined for residues that exhibit neither
significant internal mobility nor conformational exchange.
[0032] FIG. 9 is a comparison of the NMR structures determined for
CA.sup.N and Gag.sup.283 showing conformational differences
associated with helix 6.
[0033] FIG. 10 is a representation of the shift of helix 6 and the
concomitant change in the position of the CypA binding loop.
[0034] FIG. 11 is a representation of the chemical shift
differences observed for the backbone atoms of the immature and
mature CA.sup.N domains.
[0035] FIG. 12 is a representation of an overlay of 2D
.sup.1H-.sup.15N HSQC spectra obtained for the HIV-1 capsid protein
N-terminal domain upon titration with CAP-1.
[0036] FIG. 13 is a representation of an overlay of 2D .sup.1H-15N
HSQC spectra obtained for the HIV-1 capsid protein N-terminal
domain upon titration with CAP-1, CAP-2, CAP-3 and CAP-4.
[0037] FIG. 14 is a representation of an overlay of 2D
.sup.1H-.sup.15N HSQC spectra obtained for the HIV-1 capsid protein
N-terminal domain upon titration with CAP-5, CAP-6 and CAP-7.
[0038] FIG. 15 is a representation of .sup.15N NMR chemical shift
titration data for residues Ser 33 (squares), Val 59 (diamonds) and
Gly 60 (stars) and fit to 1:1 binding isotherms
(K.sub.d=0.82.+-.0.18 mM).
[0039] FIG. 16 is a graphical representation of turbidity assay
results showing the effects of CA-binding compounds on in vitro
capsid assembly.
[0040] FIG. 17 is a graphical representation of viral infectivity
(diamonds), cell viability (squares) and virus production
(triangles) from latent infected U1 cultures as a function of added
CAP-1.
[0041] FIG. 18 is a graphical representation of cell viability,
virus particle associated RT and CA levels and infectious units
obtained upon incubation of infected U1 and MAGI cells for 72 hours
with 100 .mu.M CAP-1.
[0042] FIG. 19 is a representation of capsid protein Western blot
assays showing relative concentrations of intra- and extracellular
viral CA in the free, Gag polyprotein and partially processed
states as a function of added CAP-1.
DETAILED DESCRIPTION OF THE INVENTION
[0043] The CA of the mature HIV-1 contains an N-terminal
.beta.-hairpin that is essential for formation of the capsid core
particle. CA is generated by proteolytic cleavage of the Gag
precursor polyprotein during viral maturation. To date,
high-resolution structural studies have focused on the proteins of
the mature virus. The mature HIV-1 CA protein consists of
N-terminal core (CA.sup.N) and C-terminal dimerization (CA.sup.C)
domains that are folded independently and connected by a flexible
linker. The CA.sup.N core domain contains a 13 residue N-terminal
.beta.-hairpin that is stabilized in part by a salt bridge between
the terminal NH.sub.2.sup.+ group of Pro 1 and the side chain
carboxyl group of Asp 51. These residues are highly conserved and
it is likely that all other retroviruses except the spumaviruses
contain a similar N-terminal .beta.-hairpin. Mutagenesis studies
indicate that the .beta.-hairpin is required for the formation of
HIV-1 capsid core particle and that it probably functions by
participating directly in intermolecular CA-CA interactions.
[0044] The CA domain of Gag is also responsible for packaging about
200 copies of the host protein, CypA, which is a prolyl isomerase
and a chaperone protein that is essential for HIV-1 infectivity.
Although the precise function of CypA is not known, it is suspected
that the protein facilitates disassembly of the capsid core during
infectivity.
[0045] Cryo-electron microscopic (EM) methods have been used to
study the structure of the Gag polyprotein in immature virions and
in virus-like particles. At low resolution, the assembled Gag
proteins appear as an electron dense layer associated with the
viral membrane. More recent studies have shown that the electron
dense layer is actually composed of several spherical shells of
density, including an outer shell associated directly with the
lipid bilayer that corresponds to the MA domains of the Gag
proteins, two shells corresponding to the CA.sup.N and CA.sup.C
domains, and a fourth innermost shell that corresponds to the NC
domain of Gag and the associated RNA genome. The thicknesses of MA
and CA.sup.N shells correspond closely to the lengths (measured
from N- to C-terminus) of the folded domains (FIG. 1).
Interestingly, the separation between the MA and CA.sup.N shells is
only 40 .ANG., even though the inventors' indicated that these
domains can be separated by over 80 .ANG. in extended forms of
Gag.sup.283. The separation could be even greater if the flexible
C-terminal helix of MA is partially unfolded. This indicates that
factors other than the length of the flexible linker define the
separation of the shells in the immature particles and demonstrates
that the MA and CA domains form ordered shells with defined
protein-protein interactions.
[0046] Upon proteolytic maturation, the spherical CA shells of the
immature virion condense to form the characteristic cone-shaped
capsid core particle. The N-terminal .beta.-hairpin is required for
the formation of the capsid core particle. Mutations designed to
inhibit .beta.-hairpin formation in vivo, including Pro 133 to Leu
and Asp 183 to Ala, result in the formation of virus particles that
are unable to form normal capsid cores and are non-infectious. In
addition, whereas native CA molecules can assemble into tubes that
have some features resembling those of the native capsid core,
addition of residues at the N-terminus of CA, or mutation of Asp
183 to Ala, lead to the formation of a heterogeneous mixture of
structures that are generally spherical and resemble the capsid
shells of immature virions. The .beta.-hairpin makes extensive
lattice contacts in crystal structures of both the intact CA
protein bound to an antibody fragment, and to the CA.sup.N domain
bound to CypA, suggesting that the .beta.-hairpin promotes capsid
assembly by participating directly in intermolecular CA-CA
interactions.
[0047] The .beta.-hairpin is unfolded in the immature protein and
the .beta.-hairpin formation occurs subsequent to proteolytic
cleavage of Gag, triggering capsid assembly. Hairpin formation is
stabilized by the salt bridge that can subsequently form between
Pro 133-NH.sub.2.sup.+ and the COO-- side chain of conserved Asp
183, as well as by numerous additional interactions that are not
present in the immature protein. These additional interactions
involve not only inter-.beta.-strand hydrogen bonding and packing,
but also interactions between the hairpin and the rest of CA.sup.N
domain, including the new hydrogen bond between the backbone NH of
Ile 134 and the carbonyl of Gly 178 and hydrophobic packing between
the side chains of Ile 134 (.beta.-hairpin), Thr 180 (helix 3) and
Ile 247 and Met 250 (helix 6). Thus, the capsid assembly mechanism
has features that appear very similar to those employed for enzyme
activation by the trypsin family of serine proteases, in which
proteolytic cleavage of the inactive zymogen results in a new
N-terminal NH.sub.3.sup.+ group that forms a salt bridge with a
buried carboxyl group and activates the enzyme.
[0048] The process of uncoating is dependent on the presence of
CypA and CypA packaging by HIV-1 is essential for infectivity.
Virions containing mutations in CA that abolish CypA binding are
able to assemble and mature and appear normal in cryo-EM images.
However, although these particles can fuse and penetrate target
cells, they are unable to reverse-transcribe their genomes,
indicating that CypA is necessary for an early event in the
replication cycle that most likely occurs after virus maturation
and membrane fusion. Interestingly, mutant virions that do not
efficiently package CypA are infectious in cell lines that contain
abnormally high intrinsic concentrations of CypA, and revertant
mutants that arise spontaneously in the presence of cyclosporine
analogs are dependent on these CypA inhibitors for infectivity.
Taken together, these findings indicate that capsid assembly and
disassembly processes are finely tuned and that relatively minor
mutations or alterations in cellular conditions can affect the
stability of the capsid core.
[0049] The NMR data described below indicates that .beta.-hairpin
formation induces an approximately 2 .ANG. displacement of helix-6
and a concomitant shift of the CypA binding site. In view of the
fact that (i) the .beta.-hairpin is clearly important for capsid
assembly, and (ii) CypA is likely involved in disassembly,
conformational coupling between these sites is relevant to events
associated with CypA-mediated uncoating. Thus, the binding of CypA
to the exposed loop shifts the position of helix-6, resulting in
either the destabilization or repositioning of the .beta.-hairpin
in a manner that destabilizes the capsid and promotes uncoating.
NMR chemical shift mapping experiments revealed substantial shifts
for the backbone amide signals of helix-6 upon CypA binding to the
mature CA.sup.N domain. Although the .beta.-hairpin of mature
CA.sup.N is relatively mobile, its position is well defined in the
NMR structure of the free domain.
[0050] In contrast, in the X-ray crystal structure of the
CypA-CA.sup.N dimer, well-defined electron density was observed for
only one of the two .beta.-hairpins. The missing electron density
in the X-ray structure could therefore be at least partially due to
the binding of CypA. Furthermore, comparison of the five X-ray and
NMR structures that have been determined thus far for HIV-1
CA.sup.N domain revealed that the position of helix-6 can vary,
depending on the conditions employed for structural studies (FIG.
2). Temperature factors reported for the backbone atoms of helix-6
are generally greater than those of the other helices in the
high-resolution X-ray structure (2.36 .ANG.) of the mature
CA.sup.N:CypA complex. Taken together, these data indicate that
helix-6 is relatively mobile and that its position is sensitive to
CypA binding and .beta.-hairpin formation. Finally, the affinity of
CypA for Gag is 1000-fold greater than its affinity for the mature
capsid protein. These differences in affinity can be explained by
the conformational coupling of the .beta.-hairpin and CypA binding
sites.
[0051] The three dimensional structure of the N-terminal half
(residues 2-283) of the HIV-1 Gag polyprotein was determined using
the following general methods. DNA encoding the N-terminal 283
residues of HIV-1 Gag precursor protein and an appended C-terminal
His.sub.6 tag was amplified from pNL4-3 plasmid using the
polymerase chain reaction (PCR). Plasmid carrying Gag.sup.283 was
transformed into BL21 codon plus RIL cell line (Stratagene, La
Jolla, Calif.). The transformed cells were grown on M9 minimum
medium containing .sup.15NH.sub.4Cl and/or UL-[.sup.13C]glucose
(Cambridge Isotope Laboratories, Andover, Mass.) as its sole
nitrogen and/or carbon source either in H.sub.2O or in 99%
.sup.2H.sub.2O (Martek, Columbia, Md.). Protein was purified with
cobalt affinity resin (Clontech, Palo Alto, Calif.) in a single
step. Typically, one liter of growth medium yields 20 mg
Gag.sup.283. Its molecular weight was confirmed by ESI-MS.
[0052] NMR data were collected with 1 mM protein samples containing
50 mM sodium acetate buffer at pH 5.0, 100 mM NaCl, 5 mM
.beta.Mercaptoethanol and 1.times. protease inhibitor cocktail
(Calbiochem, San Diego, Calif.). All NMR studies were carried out
with a 600 MHZ Bruker AVANCE DRX spectrometer equipped with
xyz-gradients triple resonance probe; T=30.degree. C., 2D .sup.15N
HSQC, 3D constant time HNHA, HN(CO)CA, HNCO, and 4D
.sup.15N/.sup.15N-edited NOESY (4DNN) data were used for backbone
assignments. The 4DNN data were obtained for a U-.sup.2H/.sup.15N
labeled sample; T.sub.mix=200 ms. Other NOE data collected on
U-.sup.13C/.sup.15N labeled sample include 4D
.sup.13C/.sup.15N-edited NOESY (T.sub.mix=120 ms) and 4D
.sup.13C/.sup.13C-edited NOESY (T.sub.mix=100 ms recorded in
2H.sub.2O). NMR data was processed with NMRPipe and analyzed in
NMRView. Signals were assigned using standard assignment
strategies.
[0053] The NOE cross-peaks were quantitatively categorized as
strong, medium and weak and used to assign upper distance limits of
2.7, 3.2 and 5.0 .ANG., respectively. Distance involving methyl
groups, degenerate germinal methylene groups, degenerate aromatic
protons and non-stereo-specifically assigned methyl groups of
leucine or valine were compensated by adding 0.5, 0.8, 2.3 and 1.5
.ANG., respectively. Backbone hydrogen bond restraints (1.8-2.7
.ANG. for H--O distances and 2.4-3.2 .ANG. for N--O distances) were
implemented to reinforce canonical secondary structures based on
characteristic NOE patterns and chemical shift indices.
[0054] Backbone dihedral angle restraints were obtained by analysis
of .sup.13C.sup..alpha., .sup.13C.sup..beta., .sup.1H.sup..alpha.,
.sup.13C and .sup.15N chemical shifts using the program TALOS.
Structure calculations performed with DYANA were initially carried
out using only distance restraints derived from the NOE data.
Hydrogen bond and dihedral angle restraints were subsequently
incorporated and target functions were further minimized. The
quality of the generated structures was assessed with Procheck-NMR.
Images were generated with Chimera and MolScript and rendered with
Raster 3D.
[0055] .sup.15N relaxation data were for U-.sup.2H/.sup.15N labeled
Gag.sup.283 samples prepared under conditions described above.
{.sup.1H}-.sup.15N steady state heteronuclear NOE (XNOE) for
backbone .sup.15N nuclei were measured with inverse detected water
flip-back pulse sequence. Longitudinal relaxation rates R.sub.1 and
transverse relaxation rates R.sub.2 were measured by collecting
eight two-dimensional spectra at different delays in an interleaved
mode. The eight delays for .sup.15N T.sub.1 (=1/R.sub.1) are 10.04,
120.49, 512.09, 763.12, 1004.11, 1506.16, 2008.21, 2510.26 ms and
for .sup.15N T.sub.2 (=1/R.sub.2) are 21.10, 36.70, 52.30, 67.89,
83.49, 99.09, 114.69, 130.29 ms; 4 s recovery delay.
[0056] The XNOE value of a given residue was derived from the
intensity ratio (I/I.sub.0) of .sup.15N/.sup.1H correlation peak in
the presence of proton saturation (I) and in the absence of proton
saturation (I.sub.0). Errors were estimated from the baseline
noise. Relaxation rates for each well-resolved peak were obtained
by fitting peak intensities of eight spectra into two parameter
(R.sub.2) or three-parameter exponential (R.sub.1) decay using
commercial software Orgin 6.0 (microcal, Northampton, Mass.).
Reported errors are calculated during the filling of the relaxation
data. NH groups exhibiting significant internal motions at ps-ns
timescale (XNOE<0.70) or chemical exchange on .mu.s-ms timescale
(significant increase of R.sub.2 without concomitant increase in
R.sub.1) were excluded from rotational diffusion calculations, and
the remaining relaxation data were analyzed with Quadric Diffusion
1.12 (A.G. Palmer, Columbia University).
[0057] NMR chemical shift assignments for Gag.sup.283 have been
deposited with the BioMagResBank (http://bmrb.wisc.edu), accession
number 5316. Coordinates for the 20 conformers with lowest target
functions, and the associated restraint list, have been deposited
in the Protein Data Bank (http://www.rcsb.org/pdb), accession
number 1L6N. The restraint list and coordinates for the refined
structure of the mature CA.sup.N domain have also been deposited,
accession number (1GWP).
[0058] The three dimensional structure of the N-terminal half
(residues 2-283) of the HIV-1 Gag polyprotein revealed a surface
cleft that is exposed on the CA domain of the immature protein but
becomes filled by residues of an N-terminal .beta.-hairpin of the
CA protein upon proteolytic maturation. Compounds that bind to the
.beta.-hairpin cleft inhibit viral maturation and possess antiviral
activity.
[0059] Further studies revealed an addition binding site (apical
cleft) near the C-terminal end of the N-terminal domain of CA.
Unlike the .beta.-hairpin pocket, the apical cleft is present on
both the mature and immature forms of the N-terminal domain of CA.
Compounds that bind to this apical cleft inhibit capsid assembly
and have antiviral properties. Residues of CA with backbone amide
signals that are most significantly perturbed by binding of
inhibitors to the apical cleft are either strictly conserved (Glu
35, Val 36, Val 59, Gly 60, His 62, Gln 63, Ala 65, Tyr 145) or
rarely and conservatively substituted (number of occurrences in
parentheses: E29D (2) K30R (1), A31G (16), A31N (1), F32L (1), SeeN
(13), G61E (1), M144T (1)) among the 93 genome sequences in the HIV
Sequence Compendium. Most of the conserved residues are exposed on
the surface of the N-terminal domain suggesting a possible
macromolecular interactive function. Residues of the apical cleft
of the N-terminal domain participate in an intermolecular CA
(N-terminal domain)-CA (C-terminal domain) interface upon in vitro
capsid formation. This, in combination with the other disclosures
contained herein, provides compelling evidence that the inhibitor
compounds function mechanistically by inhibiting intermolecular
CA-CA interactions necessary for proper capsid assembly.
[0060] Residues Trp 23 and Val 59 exhibit significant chemical
shift changes upon inhibitor ligand binding despite the fact that
they are buried between helices 1, 2 and 3 of the CA monomer. It
is, therefore, likely that the assembly inhibitors alter the local
structure of the capsid protein and may thereby either
competitively inhibit CA-CA interactions or promote the formation
of a structurally distorted capsid shell.
[0061] Inhibition of capsid assembly does not require ligands with
exceptionally high affinity for CA. This is likely due to the high
local concentration of Gag molecules in assembled virions (14 mM),
which favors binding by ligands with even modest affinities, e.g.,
N-(3-chloro-4-methylphenyl)-N'-[2-thioethyl-2'-[5-(dimethylaminomethyl)]--
2-methylfuryl]urea (CAP-1). Thus conservatively assuming that
cytosolic drug concentrations in the budding virus and cells are
equal (100 .mu.M), the percentage of viral CAP-1 molecules bound to
CA can be estimated by standard mass action calculations which
affords a value for the concentration of bound CAP-1 ([CA:CAP-1])
of 94 .mu.M. This indicates that 94% of the CAP-1 molecules in
immature virions (100 .mu.M dose) should be bound to Gag, and that
binding to as few as approximately 25 molecules of Gag per virion
is sufficient to inhibit core assembly during viral maturation.
[0062] FIG. 3 shows the small molecule ligand CAP-1 docked in the
structure of the capsid protein. The experimentally determined NMR
structure of the capsid protein is utilized to generate the Connoly
surface. The wireframe model of the ligand is illustrated in green.
The ligand structure is minimized with appropriate ab initio
methods. The capsid structure is colored to represent
hydrophobicity (blue, most hydrophobic; red, most hydrophilic;
white, intermediate hydrophobicity).
[0063] The experimentally determined important interactions are
shown in FIG. 4. Notable is the interaction between the aromatic
ring of Tyr 145 and the furan of CAP-1; the chlorine of the
aromatic ring in CAP 1 and the hydrophobic sidechain of Ile 37; the
interaction of the sulfur in the disulfide component of the
molecule and the hydroxyl group of Ser 146; and the hydroxyl
sidechain of Ser 33 and the nitrogen of the dimethylaminomethyl
substituent on the 5-position of the furan of CAP 1.
[0064] Other ureas including all of those described in the present
application are experimentally determined to possess functionally
very similar interactions with the capsid protein. This is evident
from the two-dimensional NMR data presented in FIGS. 13-14,
below.
[0065] The data from the examination of the multidimensional NMR
spectra of the capsid protein in the presence of the ligands CAP-1
and its structural homologues demonstrate the existence of a
potentially high affinity binding site for ligands. A multiplicity
of specific and selective interactions were unexpectedly found
which not only demonstrate the existence of such a site, but help
to suggest how very high affinity ligands might be designed for the
site.
[0066] It is observed that several structural features represent
requirements for the ligand. It is necessary to have a substituted
phenyl or other aryl group on one side of the molecule, which
contains a relatively bulky substituent. In the present situation,
the chlorine atom serves this purpose. However, the experimental
data combined with model fitting suggest that the use of a Br- or
even I-atom might increase the affinity of binding. Other
substituents on this aromatic ring are not as clearly indicated in
terms of position, but the binding site model would be positively
effected, in our view, with the inclusion of a substituent such as
cyano or dialkylamino ortho- to the halogen. The 3,4-dichlorophenyl
or 3-chloro-4-bromo phenyl moieties also would be active.
[0067] The relative position of the sulfur atom in CAP-1 is highly
significant. It is probable that using a different oxidation state
of the sulfur would not lend itself to activity in the present
system. This is true for a sulfone or sulfonamide. The furan could
equally be replaced with a variety of heterocyclic groups with
various substituents on the sidechain. In addition to the
dimethylaminomethyl, replacement of this group with groups such as
guanidine, N-cyanoguanidine, N-nitroguanidine, and such would
probably yield active compounds.
[0068] Compounds with higher affinity for cytosolic Gag will be
concentrated in assembling viruses, and compounds with greater
affinity for CA are more potent inhibitors of in vitro assembly. It
is therefore possible to rationally design agents with increased
potency as antiviral inhibitors of HIV-1 capsid assembly.
[0069] Derivatives of CAP-1, CAP-2, CAP-2, CAP-3, CAP-4, CAP-5,
CAP-6 or CAP-7 that are antiviral compounds that bind to the apical
site of the N-terminal domain of the HIV-1 capsid protein and
inhibit proper assembly of the core particle are also disclosed. In
particular, the invention provides derivatives of CAP-1 or its
related molecules that contain a urea group that contains a
substituted aromatic substituent on one nitrogen and a flexible
tether attached to a second aromatic group on the other nitrogen.
For example, such derivatives are compounds or pharmaceutically
acceptable salts having formula I:
##STR00001##
wherein: [0070] (a) R.sub.1 represents hydrogen, halogen, cyano,
trifluoromethyl, trichloromethyl, nitro, --OR.sub.8, --SR.sub.8,
--NHR.sub.8, --NR.sub.8R.sub.9, --NHCOOH, --NHCH.sub.2COOH,
--NHCHR.sub.8COOH, --NHCR.sub.8R.sub.9COOH, --NR.sub.8COOR.sub.9,
--COOR.sub.8 or a hydrocarbon group comprising a straight chained,
branched or cyclic group each containing up to 9 carbon atoms;
[0071] (b) R.sub.2 represents hydrogen, halogen, cyano,
trifluoromethyl, trichloromethyl, nitro, --OR.sub.8, --SR.sub.8,
--NHR.sub.8, --NR.sub.8R.sub.9, --NHCOOH, --NHCH.sub.2COOH,
--NHCHR.sub.8COOH, --NHCR.sub.8R.sub.9COOH, --NR.sub.8COOR.sub.9,
--COOR.sub.8 or a hydrocarbon group comprising a straight chained,
branched or cyclic group each containing up to 9 carbon atoms;
[0072] (c) R.sub.3 represents hydrogen, halogen, cyano,
trifluoromethyl, trichloromethyl, nitro, --OR.sub.8, --SR.sub.8,
--NHR.sub.8, --NR.sub.8R.sub.9, --NHCOOH, --NHCH.sub.2COOH,
--NHCHR.sub.8COOH, --NHCR.sub.8R.sub.9COOH, --NR.sub.8COOR.sub.9,
--COOR.sub.8 or a hydrocarbon group comprising a straight chained,
branched or cyclic group each containing up to 9 carbon atoms;
[0073] (d) R.sub.4 represents hydrogen, halogen, cyano,
trifluoromethyl, trichloromethyl, nitro, --OR.sub.8, --SR.sub.8,
--NHR.sub.8, --NR.sub.8R.sub.9, --NHCOOH, --NHCH.sub.2COOH,
--NHCHR.sub.8COOH, --NHCR.sub.8R.sub.9COOH, --NR.sub.8COOR.sub.9,
--COOR.sub.8 or a hydrocarbon group comprising a straight chained,
branched or cyclic group each containing up to 9 carbon atoms;
[0074] (e) R.sub.5 represents hydrogen, halogen, cyano,
trifluoromethyl, trichloromethyl, nitro, --OR.sub.8, --SR.sub.8,
--NHR.sub.8, --NR.sub.8R.sub.9, --NHCOOH, --NHCH.sub.2COOH,
--NHCHR.sub.8COOH, --NHCR.sub.8R.sub.9COOH, --NR.sub.8COOR.sub.9,
--COOR.sub.8 or a hydrocarbon group comprising a straight chained,
branched or cyclic group each containing up to 9 carbon atoms;
[0075] (f) Halogen is limited to fluoro, chloro, bromo, and iodo
[0076] (g) R.sub.8 and R.sub.9 are independently methyl, ethyl,
n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, pentyl,
hexyl, neo-pentyl, cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl,
cyclobutylethyl, cyclopentylmethyl, cyclohexylmethyl, or
cyclohexylethyl [0077] (h) The letters n, m, and p represent
independently any integer from 1 to 6 [0078] (i) R.sub.6 and
R.sub.7 are independently selected from the group consisting of
hydrogen or a hydrocarbon group comprising a straight chained,
branched or cyclic group each containing up to 6 carbon atoms
[0079] (j) X is selected from the group consisting of O or S [0080]
(k) Y is heterocyclic, carbocyclic, or optionally substituted
phenyl. [0081] (l) Heterocyclic is selected from any stable 5, 6,
or 7-membered monocyclic or bicyclic or 7, 8, 9, or 10-membered
bicyclic heterocyclic ring which is saturated, partially
unsaturated or unsaturated (aromatic), and which consists of carbon
atoms and 1, 2, 3, or 4 heteroatoms independently selected from the
group consisting of N, NH, O and S and including any bicyclic group
in which any of the above-defined heterocyclic rings is fused to a
benzene ring. The nitrogen and sulfur heteroatoms may optionally be
oxidized. The heterocyclic ring may be attached to its pendant
group at any heteroatom or carbon atom, which results in a stable
structure. The heterocyclic rings described herein may be
substituted on carbon or on a nitrogen atom if the resulting
compound is stable. If specifically noted, a nitrogen in the
heterocycle may optionally be quaternized. It is preferred that
when the total number of S and O atoms in the heterocycle exceeds
1, then these heteroatoms are not adjacent to one another. As used
herein, the term "aromatic heterocyclic system" is intended to mean
a stable 5- to 7-membered monocyclic or bicyclic or 7- to
10-membered bicyclic heterocyclic aromatic ring which consists of
carbon atoms and from 1 to 4 heteroatoms independently selected
from the group consisting of N, O and S. Examples of heterocycles
include, but are not limited to, 1H-indazole, 2-pyrrolidonyl,
2H,6H-1,5,2-dithiazinyl, 2H-pyrrolyl, 3H-indolyl, 4-piperidonyl,
4aH-carbazole, 4H-quinolizinyl, 6H-1,2,5-thiadiazinyl, acridinyl,
azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl,
benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl,
benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazalonyl,
carbazolyl, 4aH-carbazolyl, .beta.-carbolinyl, chromanyl,
chromenyl, cinnolinyl, decahydroquinolinyl,
2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran,
furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl,
1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl,
isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl,
isoindolyl, isoquinolinyl (benzimidazolyl), isothiazolyl,
isoxazolyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl,
oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl,
1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl,
oxazolidinylperimidinyl, phenanthridinyl, phenanthrolinyl,
phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl,
phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, pteridinyl,
piperidonyl, 4-piperidonyl, pteridinyl, purinyl, pyranyl,
pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl,
pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl,
pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl,
quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,
carbolinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,
tetrahydroquinolinyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,
1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,
thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,
thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl,
1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, tetrazolyl, and
xanthenyl. Preferred heterocycles include, but are not limited to,
pyridinyl, thiophenyl, furanyl, indazolyl, benzothiazolyl,
benzimidazolyl, benzothiaphenyl, benzofuranyl, benzoxazolyl,
benzisoxazolyl, quinolinyl, isoquinolinyl, imidazolyl, indolyl,
isoidolyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,
pyrrazolyl, 1,2,4-triazolyl, 1,2,3-triazolyl, tetrazolyl,
thiazolyl, oxazolyl, pyrazinyl, and pyrimidinyl, and fused ring and
spiro compounds containing the above heterocycles. [0082] (m)
carbocyclic is intended to mean any stable 3, 4, 5, 6, or
7-membered monocyclic or bicyclic or 7, 8, 9, 10, 11, 12, or
13-membered bicyclic or tricyclic, any of which may be saturated,
partially unsaturated, or aromatic. Examples of such carbocycles
include, but are not limited to, cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclooctyl,
[3.3.0] bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane
(decalin), [2.2.2]bicyclooctane, fluorenyl, phenyl, naphthyl,
indanyl, adamantyl, or tetrahydronaphthyl (tetralin). [0083] (n)
Substituted phenyl is defined by formula II
##STR00002##
[0083] wherein: [0084] (o) R.sub.10 represents hydrogen, halogen,
cyano, trifluoromethyl, trichloromethyl, nitro, --OR.sub.8,
--SR.sub.8, --NHR.sub.8, --NR.sub.8R.sub.9, --NHCOOH,
--NHCH.sub.2COOH, --NR.sub.8COOR.sub.9, --COOR.sub.8 or a
hydrocarbon group comprising a straight chained, branched or cyclic
group each containing up to 9 carbon atoms; [0085] (p) R.sub.11
independently represents hydrogen, halogen, cyano, trifluoromethyl,
trichloromethyl, nitro, --OR.sub.8, --SR.sub.8, --NHR.sub.8,
--NR.sub.8R.sub.9, --NHCOOH, --NHCH.sub.2COOH,
--NR.sub.8COOR.sub.9, --COOR.sub.8 or a hydrocarbon group
comprising a straight chained, branched or cyclic group each
containing up to 9 carbon atoms; [0086] (q) R.sub.12 independently
represents hydrogen, halogen, cyano, trifluoromethyl,
trichloromethyl, nitro, --OR.sub.8, --SR.sub.8, --NHR.sub.8,
--NR.sub.8R.sub.9, --NHCOOH, --NHCH.sub.2COOH,
--NR.sub.8COOR.sub.9, --COOR.sub.8 or a hydrocarbon group
comprising a straight chained, branched or cyclic group each
containing up to 9 carbon atoms; [0087] (r) R.sub.13 independently
represents hydrogen, halogen, cyano, trifluoromethyl,
trichloromethyl, nitro, --OR.sub.8, --SR.sub.8, --NHR.sub.8,
--NR.sub.8R.sub.9, --NHCOOH, --NHCH.sub.2COOH,
--NR.sub.8COOR.sub.9, --COOR.sub.8 or a hydrocarbon group
comprising a straight chained, branched or cyclic group each
containing up to 9 carbon atoms; [0088] (s) R.sub.14 independently
represents hydrogen, halogen, cyano, trifluoromethyl,
trichloromethyl, nitro, --OR.sub.8, --SR.sub.8, --NHR.sub.8,
--NR.sub.8R.sub.9, --NHCOOH, --NHCH.sub.2COOH,
--NR.sub.8COOR.sub.9, --COOR.sub.8 or a hydrocarbon group
comprising a straight chained, branched or cyclic group each
containing up to 9 carbon atoms; [0089] (t) R.sub.8 and R.sub.9 are
independently methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl,
sec-butyl, t-butyl, pentyl, hexyl, neo-pentyl, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cyclopropylmethyl,
cyclopropylethyl, cyclobutylmethyl, cyclobutylethyl,
cyclopentylmethyl, cyclohexylmethyl, or cyclohexylethyl [0090] (u)
Halogen is limited to fluoro, chloro, bromo, and iodo.
[0091] Therapeutic compositions comprising compounds that bind to
the apical cleft of HIV-1 capsid protein may be administered
systemically or topically. Systemic routes of administration
include oral, intravenous, intramuscular or subcutaneous injection
(including into a depot for long-term release), intraocular and
retrobulbar, intrathecal, intraperitoneal (e.g., by intraperitoneal
lavage), intrapulmonary using aerosolized or nebulized drug, or
transdermal. Topical routes include administration in the form of
salves, ophthalmic drops, eardrops, irrigation fluids (for, e.g.,
irrigation of wounds) or medicated shampoos. Those skilled in the
art can readily optimize effective dosages and administration
regimens for therapeutic compositions comprising compounds that
bind to the apical cleft of HIV-1 capsid protein, as determined by
good medical practice and the clinical condition of the individual
patient.
[0092] The administration of compounds that bind to the apical
cleft of HIV-1 capsid protein is preferably accomplished with a
pharmaceutical composition comprising such a compound and a
pharmaceutically acceptable diluent, adjuvant or carrier. The
compound may be administered without or in conjunction with known
surfactants, other chemotherapeutic agents or additional known
antiviral agents.
[0093] Other aspects and advantages of the invention will be
understood upon consideration of the following illustrative
examples. Example 1 describes the overall structure and dynamics of
Gag.sup.283. Example 2 describes the structure of the MA domain of
Gag.sup.283. Example 3 describes the structure of the CA.sup.N
domain of Gag.sup.283. Example 4 describes the identification of
the ligand binding site on the N-terminal domain of CA. Example 5
describes the in vitro inhibition of capsid assembly by compounds
that bind to the apical cleft of HIV-1 capsid protein. Example 6
describes the inhibition of viral infectivity by compounds that
bind to the apical cleft of HIV-1 capsid protein. Example 7
describes the in vivo inhibition of capsid assembly.
EXAMPLE 1
The Overall Structure and Dynamics of Gag.sup.283
[0094] A 32.2 kD recombinant polypeptide (Gag.sup.283)
corresponding to the 283 N-terminal residues of HIV-1.sub.pNL4-3
(plus an additional C-terminal hexahistidine tag) was cloned and
prepared for NMR studies (Mr.sub.calc=32216.6 Daltons,
Mr.sub.exp=32216.1.+-.0.8 Daltons. High quality NMR spectra were
obtained (FIG. 5), enabling nearly complete .sup.1H, .sup.15N and
.sup.13C NMR signals assignments. NMR chemical shifts of the
backbone atoms are indicative of a highly helical structure (FIG.
6).
[0095] Residues Gly 2-Ser 6, Gly 123-Val 143 and Pro 279-Leu 283
exhibit relatively low {.sup.1H}-.sup.15N heteronuclear NOE (XNOE)
and T.sub.2 relaxation values (FIG. 6), as well as few or no medium
range .sup.1H-.sup.1H NOEs, indicating that these residues are
conformationally labile. A total of 2,376 experimentally derived
restraints (2046 distance restraints and 332 torsion angle
restraints, corresponding to 17.7 restraints per restrained
residue) were employed for the relatively non-labile residues Val
7-Thr 122 and His 144-Ser 278. Twenty refined structures with
target functions of 1.50.+-.0.24 .ANG..sup.2 and good structural
statistics (FIG. 7) were generated with DYANA. Residues VAL 7-Thr
122 of MA and His 144-Ser 278 of CA.sup.N exhibit good convergence,
with pairwise RMS deviations for backbone heavy atoms of the
helices of 0.41.+-.0.09 and 0.72.+-.0.14 .ANG., respectively. Since
no restraints were employed for the mobile residues that connect
the folded domains (Gly 123-Val 143), and no NOEs were observed
between the domains, the relative orientation of the MA and
CA.sup.N domains is not defined. The calculations indicate that the
two domains can be separated by up to 80 .ANG. when residues Gly
123-Val 143 are fully extended.
[0096] The T.sub.1/T.sub.2 ratios obtained for the backbone NH
groups indicate that the MA and CA.sup.N domains tumble with
different rotational correlation times. For both domains
T.sub.1/T.sub.2 (=R.sub.2/R.sub.1) ratios for residues of the
different helices were generally clustered, which is indicative of
anisotropic rotational diffusion, (FIG. 8). Errors associated with
the T.sub.1/T.sub.2 ratios of the CA.sup.N domain are somewhat
larger than those of the MA domain due to the larger line widths of
the CA.sup.N signals (and corresponding smaller signal-to-noise
ratio) (FIG. 6). The relatively poor clustering observed for some
helices of the CA.sup.N domain (especially helix 7) is likely due
to the presence of weak CA.sup.N-CA.sup.N intermolecular
interactions. Indeed the scatter in T.sub.1/T.sub.2 ratios for a
given helix decreased as sample concentrations were reduced from
1.0 to 0.25 mM, but the average T.sub.1/T.sub.2 ratios were
essentially unaffected. The rotational diffusion properties of the
MA and CA.sup.N domains were calculated independently. Best
statistical fits were obtained using prolate axially symmetric
diffusion models, which afforded rotational correlation times of
10.0.+-.0.1 ns and 13.2.+-.0.2 ns for the MA and CA.sup.N domains,
respectively. The principal axes of the diffusion tensors (i.e.,
the axes about which rotational diffusion is fastest) are nearly
coincident with vectors that connect the N- and C-terminal residues
of the folded domains (FIG. 1).
EXAMPLE 2
Structure of the MA Domain of Gag.sup.283
[0097] The structure of the MA domain of Gag.sup.283 was
essentially identical to that observed for the isolated, mature
protein. Superposition of the backbone heavy atoms of rigid
residues of the mature and immature MA NMR structures afforded
pairwise RMS deviations of 1.27.+-.0.005 .ANG.. The structure was
in better agreement with the X-ray structure of the mature MA
trimer (1.14.+-.0.09 .ANG.) due to the use of more modern
methodologies in the current NMR study (i.e., chemical shift-based
restraints and the use of 4D NMR data). The fit improved to
0.90.+-.0.11 .ANG. when a 3.sub.10 helix that undergoes
conformational changes upon trimerization (Pro 66-Gly 71) was
removed from the fitting with the X-ray structure. The
.sup.1H-.sup.1H NOE data indicated that the C-terminal helix
extends beyond the globular portion of the domain to residue Thr
122. However, the C.sub..alpha. chemical shift indices
progressively decreased from helical to random coil values for
residue Ile 104-Gln 117, and together with the relaxation data
indicated a progressive shift from predominantly .alpha.-helical to
predominantly random coil conformations (FIG. 4). This finding was
consistent with X-ray structural data reported for the mature MA
trimer, in which electron density for these residues varied
substantially among the six different molecules of the unit
cell.
EXAMPLE 3
Structure of the CA.sup.N Domain of Gag.sup.283
[0098] The overall structure of the CA.sup.N domain of Gag.sup.283
is very similar to that observed for the mature CA.sup.N domain. To
facilitate comparisons, the amino acid numbering scheme of immature
CA.sup.N was also used for the mature domain (i.e., Pro 133 is the
N-terminal residue of mature CA.sup.N). Residues Ser 148-Lys 162,
Ser 165-Ser 176, Thr 180-Val 191, His 194-His 216, Arg 232-Ala 237,
Thr 242-Thr 251, Val 258-Ser 278 form seven .alpha.-helices (helix
1-7, respectively) that are packed together in a flat and
triangular shape, and residues Pro 217-Pro 231 (which include the
CypA binding site) form a conformationally flexible loop.
Superposition of the backbone heavy atoms of the helical residues
of the mature and immature forms of CA.sup.N NMR structures afford
pairwise RMS deviations of 1.32.+-.0.13 .ANG..
[0099] In contrast, the conformation of residues Pro 133-Val 143 of
Gag.sup.213 is substantially different from that observed in the
mature CA.sup.N protein. No intermediate of long-range
.sup.1H-.sup.1H NOEs were observed for these residues, and the
chemical shift index data indicated that they exist in a random
coil conformation (FIG. 6). Also, the .sup.15N NMR relaxation
properties of these residues indicated a high degree of
conformational mobility (FIG. 6). In the mature CA.sup.N domain,
residues Pro 133-Asn 137 pair with residues Gln 141-Gln 145 to form
anti-parallel strands of a .beta.-hairpin that packs against
helix-6, and the backbone NH.sub.2.sup.+ group of Pro 133 forms a
salt bridge with the partially buried side chain of Asp 183. In
addition, although residues His 144-Ile 147 interact with globular
portion of the CA.sup.N domain in both the immature and mature
structures, differences in the NOE data indicated subtle but
significant structural differences. For example, in the mature
CA.sup.N domain, the His 144-H.beta. protons exhibited moderate
intensity NOEs with the side chain methyl protons of Ile 247 (helix
6), whereas these groups give rise to very weak NOEs in the mature
domain.
[0100] In addition, helix 6 was shifted by approximately 2 .ANG.
relative to its position in the mature protein (FIGS. 10 and 11).
In the immature domain, the side chain of Thr 180, which caps helix
3, packs within a hydrophobic cluster formed by the side chains of
Leu 243, Ile 247 and Met 250 of helix 6 (FIG. 9), and very strong
.sup.1H-.sup.1H NOEs were observed between the Thr 180 and Ile 247
methyl groups. However, in the mature protein, the side chain of
Ile 134 is inserted into this hydrophobic pocket, resulting in an
approximately 2 .ANG. separation of the Thr 180 and Ile 247 side
chains and a substantial reduction in the intensities of the
associated inter-residue NOEs.
[0101] Furthermore, the .chi..sub.2-angle of the Leu 243 side chain
differs by an approximately 90.degree. rotation, and the methyl
group of Met 250 is reoriented in the immature domain in a manner
to partially fill the space occupied by the Ile 134 side chain of
the mature domain (FIG. 9). Helix 6 also makes contact with the
CypA binding loop. Although the loop is flexible, a qualitative
comparison of the two ensembles of NMR structures indicated that
Pro 222, which binds to the active site of CypA, was shifted by
several angstroms relative to its position in the mature CA.sup.N
domain (FIG. 10). Significant .sup.1H, .sup.15N and .sup.13C
chemical shift differences were observed for residues Pro 133-Ile
147, which were extended in the immature protein but form a
.beta.-hairpin in the mature domain (FIG. 11). Significant shifts
were also observed for Ala 179, Thr 180, Ile 243, Ile 247, Met 250
and Leu 243, which interact with the .beta.-sheet in the mature
CA.sup.N domain. These shift differences are fully consistent with
the structural changes described above.
[0102] Finally, signal doubling and R.sub.2 exchange broadening was
observed for the backbone amides of Glu 177 and Gly 178 (FIGS. 5
and 6). There are no bulky aromatic side chains in the vicinity of
these residues that might cause these effects. Instead, the signal
doubling was due to heterogeneity in the .PSI. and .PHI. torsion
angles associated with the Glu 177-Gly 178-Ala 179 peptide bonds.
Signal doubling was not observed for these residues in the mature
protein in which the carbonyl of Gly 178 forms a hydrogen bond with
the Ile 134 backbone NH of the .beta.-hairpin.
EXAMPLE 4
Identification of a Ligand Binding Site of the N-Terminal Domain of
CA
[0103] DNA encoding the CA N-terminal domain (residues 1-151) was
amplified from HIV-1 cDNA plasmid pNL-4-3 and an oligonucleotide
encoding a C-terminal hexahistidine tag was appended to the gene.
The DNA was inserted into a p11a expression vector (Novagen,
Madison, Wis.), and the protein product was purified by cobalt
affinity chromatography (Clontech, Palo Alto, Calif.);
MW.sub.calc=17523.0 daltons, MW.sub.obs=17523.10.+-.0.44 daltons
(electrospray Mass Spectrometry). The plasmid for the full length,
native capsid protein was kindly provided by Dr. W. I. Sundquist
(University of Utah, Salt Lake City, Utah), and the protein was
purified as described. NMR spectra were assigned using conventional
triple resonance methods. Binding isotherms from .sup.1H-.sup.15N
NMR HSQC titration experiments were calculated with ORIGIN 7.0
software (MicroCal, Northampton, Mass.).
[0104] To identify compounds that inhibit functions of the capsid
protein, public domain chemical libraries were screened for
compounds that might bind to clefts on the surface of the capsid
protein and tested for binding using NMR titration spectroscopy.
Screening efforts focused mainly on a .beta.-hairpin cleft that is
exposed on the surface of the N-terminal domain of the immature
capsid protein but becomes occupied by residues of a .beta.-hairpin
that forms after proteolytic cleavage of Gag. Compounds from public
domain chemical libraries were screened using DOCK-4.0, and 40
compounds with good theoretical binding properties (binding energy
<-26 kCal/mol, Contact Score <-40) were experimentally tested
for binding to the intact CA protein, the N-terminal domain of CA,
and a 283 residue fragment of the immature Gag polyprotein
(Gag.sup.283). Several compounds that bind to the capsid protein at
a site that appears to be important for capsid assembly were
identified. Exemplary compounds include, but are not limited to,
N-(3-chloro-4-methylphenyl)-N'-[2-thioethyl-2'-[5-(dimethylaminomethyl)]--
2-methylfuryl]urea (CAP-1),
N-(4-N-acetamidophenyl)-N'-(3-nitro-4-methyl phenyl)urea (CAP-2),
N-(2-propyl)-N'-(3-nitro-4-methyl phenyl)urea (CAP-3),
N-(3-chloro-4-methyl phenyl)-N'-(4-cyanophenyl)urea (CAP-4),
N-(3-chloro-4-methyl
phenyl)-N'-[4-(1,1,1-trichloromethyl)phenyl]urea (CAP-5),
N-(3-nitro-4-fluorophenyl)-N'-[3-(1,1,1-trifluoromethyl)phenyl]u-
rea (CAP-6), N-[(3-chloro-4-methyl phenyl)-N',N'-propyl]urea
(CAP-7). One compound,
N-(3-chloro-4-methylphenyl)-N'-[2-thioethyl-2'-[5-(dimethylamin-
omethyl)]-2-methylfuryl]urea (CAP-1) was particularly
well-tolerated in cell cultures enabling the in vivo antiviral and
mechanistic studies described below.
[0105] The mature N-terminal domain was titrated with
N-(3-chloro-4-methylphenyl)-N'-[2-thioethyl-2'-[5-(dimethylaminomethyl)]--
2-methylfuryl]urea (CAP-1). Representative .sup.1H-15N HSQC NMR
data obtained upon titration of the mature NTD with CAP-1 is shown
in FIG. 12. Although most signals were unaffected by the
titrations, a subset of signals shifted as a function of increasing
CAP-1 concentration, indicating site-specific binding.
[0106] Similarly, other compounds that bind to the apical cleft of
the N-terminal domain of the HIV-1 capsid protein were titrated. In
FIG. 13, the superposition of .sup.1H-.sup.15N HSQC NMR data
obtained upon titration of compounds CAP-1, CAP-2, CAP-3, and CAP-4
with the HIV-1 capsid NTD is provided. Compounds CAP-1 and CAP-2
bind to the apical site of the protein, as evidenced by specific
chemical shift changes as a function of added compound. Slight
perturbations and signal broadening observed upon addition of CAP-3
and CAP-4 indicate very weak binding. In FIG. 14, the superposition
of .sup.1H-15N HSQC NMR data obtained in the presence and absence
of capsid binding compounds CAP-5, CAP-6, and CAP-7 with the HIV-1
capsid NTD is provided. Signals perturbed by binding are shown in
circles. For comparison, results obtained with two structurally
related compounds that do not bind to the capsid protein are also
shown in FIG. 14.
[0107] The chemical shift changes fit to 1:1 binding isotherms and
afforded an equilibrium dissociation constant (K.sub.d) of
0.82.+-.0.18 mM at 35.degree. C. (FIG. 15). Significantly tighter
binding was observed for a second compound,
1-(4-(N-methyl-acetamido)phenyl)-3-(4-methyl-3-nitrophenyl)urea
(CAP-2), K.sub.d=52.+-.27 .mu.M. In both cases, the CA residues
perturbed by binding (.sup.1H.sub.N .DELTA..delta.>0.1 ppm;
.sup.15N .DELTA..delta.>0.5 ppm; Glu29, Lys30, Ala31, Phe32,
Ser33, Glu35, Val36, Val59, Gly60, Gly61, His62, Gln63, Ala65,
Met144 and Tyr145) are located at or near the apex of a helical
bundle (helices 1, 2, 3, 4 and 7). Essentially identical results
were obtained for titrations with Gag.sup.283 and intact CA,
indicating that the binding site remains accessible in Gag-like
constructs containing the native N-terminal matrix (MA) and
C-terminal capsid (CTD) domains, and that binding is insensitive to
the maturation state of the protein.
EXAMPLE 5
In vitro Inhibition of Capsid Assembly
[0108] Turbidity assays were performed at 21.degree. C. using a
Beckman DU650 spectrophotometer operating at 350 nm wavelength.
Concentrated ligand in DMSO (0.2 .mu.l) was added to a 250 .mu.l
aqueous solution containing the capsid protein ([CA]=60 .mu.M;
[NaH.sub.2PO.sub.4]=50 mM; pH 8.0). Particulates were removed by
centrifugation, and capsid assembly was initiated by addition of a
concentrated NaCl solution (5 M, 250 .mu.l). Spectral measurements
were made every 10 s, following a short initial delay to allow
sample equilibration. Relative assembly rates were estimated from
initial slopes of the plots of absorbance vs. time.
[0109] In the absence of other viral constituents, HIV-1 CA can
assemble into tubes with structural features that resemble mature
cores. Tube formation leads to increases in sample turbidity that
can be monitored spectrophotometrically, and this assay was used to
probe for potential inhibitory effects of the CAP compounds on in
vitro capsid assembly. As shown in FIG. 17, dissolution of native
HIV-1 CA into assembly buffer (50 mM phosphate buffer, pH 8.0, 2.5
M NaCl, 0.04% v/v DMSO) led to an increase in absorbance at an
initial rate of 204.+-.36 mOD/min (determined from the initial
slope and reported as the mean .+-.standard deviation from three
experiments). As expected, compounds tested that do not bind CA did
not affect the rate of assembly. However, the assembly rate was
diminished in a dose-dependent manner by both CAP-1 and CAP-2, with
the more tightly binding CAP-2 having a more pronounced effect. As
shown in FIG. 16, the initial assembly rates in the presence of
CAP-1 decreased to 93.+-.3 and 67.+-.16 mOD/min at CAP-1: CA ratios
of 1:1 and 2:1, respectively. For comparison, assembly rates in the
presence of the more tightly binding CAP-2 decreased to 81.+-.2 and
39.+-.11 mOD/min at CAP-2: CA ratios of 0.5:1 and 1:1,
respectively. These data confirm that CA-binding compounds can
inhibit capsid assembly in vitro, and that the relative efficacy of
assembly inhibition is dependent on the affinity of the ligands for
the CA protein.
EXAMPLE 6
Inhibition of Viral Infectivity
[0110] U1 cells (5.times.10.sup.5 cells/ml) were mixed with
TNF-.alpha. (10 ng/ml, Sigma) for activation of HIV virion
production and treated with CAP-1 at different concentrations.
Cultures were harvested 72 hours after treatment. Cell viability
was measured using the MTS cell proliferation assay (CellTiter 96
Aqueous One Solution Cell Proliferation Assay, Promega, Madison,
Wis.). Supernatants were collected, the cell debris removed by low
speed centrifugation, and the particles in the supernatants
pelleted by microcentrifugation. Infectious units associated with
the particles were measured as described in Kimpton, et al.,
Detection of replication-competent and pseudotyped HIV with a
sensitive cell line on the basis of activation of an integrated
.beta.-galactosidase gene, J. Virol. 66: 2232-39, 1992, except that
.beta.-gal activities were measured using a Tropix Gal-Screen
detector system (Applied Biosystems, Foster City, Calif.).
Particle-associated RT activities were determined as described in
Huang, et al., p6Gag is required for particle production from full
length human immunodeficiency virus type 1 molecular clones
expressing protease, J. Virol., 96: 6810-18, 1995. Cell lysates and
pelleted particles were subjected to SDS-PAGE analysis using AIDS
patient sera (AIDS Research and Reference Regent Program, NIAID,
NIH). Quantitative p24 (CA) assays were performed with the HIV-1
p24 Antigen Capture ELISA kit (AIDS vaccine program,
FCRDC/SAIC/NCI, Frederick, Md.). MAGI cells were washed after viral
adsorption (HIV-1.sub.RF) with PBS and were fed fresh media
containing CAP-1 at various concentrations. 72 hours
post-infection, the culture supernatants were harvested and
pre-cleared. Virus particles present on the supernatants were
collected by microcentrifugation and particle-associated RT
activity and infectivity were subsequently measured.
[0111] The CAP compounds were tested for toxicity and antiviral
activity using HIV-1 producing, latent infected U1 cells. This
assay allows assessment of antiviral effects on late phase
replication events. Although CAP-2 was too cytotoxic for in vivo
evaluations, CAP-1 was non-toxic under the conditions employed, and
its application led to dose dependent reductions in supernatant
infectivity, FIG. 17. At 100 .mu.M CAP-1, the U1 cells were fully
viable, but infectivity was reduced by ca. 95% relative to
untreated samples, FIGS. 18 and 19.
[0112] To determine if CAP-1 affects viral gene expression and
particle production, reverse transcriptase (RT) activity and CA
(p24) levels were measured for the supernatants after pelleting and
removal of the cells. As shown in FIG. 18, both the CA levels and
RT activities were unaffected by CAP-1, indicating that antiviral
activity is not due to inhibited virus production. In addition, the
p24 (CA) levels observed in Western data obtained for treated and
untreated samples were very similar, FIG. 19, indicating that CAP-1
does not significantly affect proteolytic processing of Gag.
Consistent with this finding, CAP-1 did not affect in vitro
protease activity. The p24 Western data also indicated a reduction
in the intracellular levels of Gag (p55) as a function of
increasing levels of CAP-1, whereas the levels of the Gag cleavage
products p24 and p41 remained relatively unaffected. These findings
demonstrate that CAP-1 promotes the intracellular degradation of
the full length Gag polyprotein. Quantitation of gp120 was also
obtained for the supernatant using antibodies against gp120. No
differences were observed between the treated and untreated
samples, indicating that CAP-1 does not inhibit the synthesis or
viral incorporation of the envelope glycoprotein.
[0113] Antiviral activity was also tested in a second cellular
assay using MAGI cell cultures. As observed in the UI assay,
treatment of infected, virus producing MAGI cells with CAP-1 led to
dose dependent reductions in virus particle infectivity, with
infectious units dropping by nearly two log units to less than 2%
of the untreated levels at 100 .mu.M CAP-1, FIG. 18. No reductions
in viral RT activity were observed, providing further evidence that
antiviral activity is not due to inhibited virus production. Virus
production was also unaffected by pre-incubation of either MAGI
cells or virus particles with CAP-1, indicating that CAP-1 is not
virucidal and does not directly inhibit early phase events. These
data collectively indicate that the antiviral activity of CAP-1 is
due to the inhibition of a late phase viral event that is different
from events targeted by other anti-HIV agents currently under
investigation or in clinical use.
EXAMPLE 7
In Vivo Inhibition of Capsid Assembly
[0114] Treated (CAP-1) and untreated virus-producing U1 cells were
pelleted, washed in phosphate-buffered saline (PBS) and resuspended
in at least ten cell pellet volumes of fixative (100 mM sodium
cacodylate, pH 7.2, 2.5% glutaraldehyde, 1.6% paraformaldehyde,
0.5% picric acid). Cells were fixed for 24-48 hours, after which
fixative was removed and cells were washed twice in PBS and
pelleted in eppendorf centrifuge tubes. Washed cell pellets were
post-fixed one hour in 1% osmium tetroxide plus 0.8% potassium
ferricyanide in 100 mM sodium cacodylate, pH 7.2. After thorough
rinsing in water, cells were pre-stained in 4% uranyl acetate one
hour, thoroughly rinsed, dehydrated, infiltrated overnight in 1:1
acetone:Epon 812, infiltrated one hour with 100% Epon 812 resin,
and embedded in the resin. After polymerization, 60-80 nm thin
sections were cut on a Reichert untramicrotome, stained 5 minutes
in lead citrate, rinsed, post-stained 30 minutes in uranyl acetate,
rinsed and dried. EM was performed at 60 kV on a Philips
CMi20/Biotwin equipped with a 1024.times.1024 Gatan multiscan CCD,
and images were collected at original magnifications of
25,880.times.-36,960.times., corresponding t resolutions of 8.9 and
6.5 .ANG./pixel, respectively. For each sample, 4 separate EM grids
were viewed, and at least 47 were collected, corresponding to a
minimum total area of 35 micron.sup.2.
[0115] Since CAP-1 did not inhibit virus production, it was
possible to examine the virons produced form treated cells for
morphological defects by EM. The capsids of mature virions
generally appear as central, conical (approximately 30% of the
particles) or spherical (approximately 40%) structures, depending
on the orientation of the cone during thin section sample
preparation, and about 30% of virions in EM preparations typically
exhibit an immature phenotype characterized by the lack of central
electron density. Virions from untreated U1 cell cultures generated
these typical results. However, particles generated from the
treated cells exhibited greater size heterogeneity, which is
indicative of a Gag assembly defect. In addition, only 35% of the
particles from the treated cells contained a dense, centrally
located core, compared to 70% from the untreated cells. Most
strikingly, none of the particles from the treated cells exhibited
a cone-shaped core that is the hallmark of HIV, indicating that
CAP-1 inhibits assembly of the mature core. Essentially identical
phenotypes were observed previously for virions generated with CA
mutations designed to disrupt intermolecular CA-CA interactions.
Thus, this EM data indicates that CAP-1 inhibits capsid assembly
during viral maturation and interferes to some extent with normal
Gag-Gag interactions during assembly of the immature particle.
[0116] Numerous modifications and variations of the above-described
invention are expected to occur to those of skill in the art.
Accordingly, only such limitations as appear in the appended claims
should be placed thereon.
* * * * *
References