HIGH THROUGHPUT PROTEIN INTERACTION ASSAY
A method to identify small molecules useful as therapeutics and/or vaccines to prevent, alleviate or ameliorate a pathogenic infection or an autoimmune disorder. The method can be used to screen small molecule test compounds for the ability to disrupt particular antigen-antibody interactions of interest. In one embodiment, the antigen is a pathogen-derived antigen and the antibody decreases or inhibits virulence of the pathogen when bound to the antigen (e.g., a neutralizing antibody, antibody with serum bactericidal activity, etc.). In another embodiment, the antigen is a self-antigen (autoantigen) and the antibody is an autoantibody that is known to be associated with a pathological condition (e.g., autoimmune disorder). Compounds that bind to the antigen or antibody disrupt binding can be used as therapeutics to decrease or inhibit the autoimmune disorder.
1 . A method of identifying a small molecule mimotope of a pathogen-derived antigen comprising:
a) combining the following components
i) an antibody that binds to the antigen;
ii) the pathogen-derived antigen or a fragment or conformational mimetic thereof wherein said fragment or conformational mimetic has a conformation that is substantially similar to that of the antigen such that the antibody binds to the fragment or conformational mimetic; and
iii) a small molecule test compound;
b) incubating the components under conditions which allow components i and ii to bind;
c) measuring the amount of binding between components i and ii, wherein a decrease in binding between components i and ii in the presence of the test compound as compared to in the absence of the test compound indicates the test compound binds to component i or ii and is a candidate compound;
d) determining to which component the candidate compound binds,
wherein binding to the antibody indicates that the candidate compound is a mimotope of the pathogen-derived antigen.
2 . The method of claim 1 , wherein binding of the antibody to the pathogen decreases or inhibits virulence of the pathogen.
3 . The method of claim 1 , wherein the antigen is a fragment or conformational mimetic of the pathogen-derived antigen.
4 . The method of claim 1 , wherein the pathogen-derived antigen is from a pathogen selected from the group consisting of HIV, HCV, and Neisseria meningitidis.
5 . The method of claim 1 , wherein the pathogen-derived antigen is a polypeptide.
6 . The method of claim 5 , wherein the pathogen-derived antigen is a gp41 polypeptide from HIV or a 5-helix polypeptide.
7 . The method of claim 1 , wherein the pathogen-derived antigen is a carbohydrate.
8 . (canceled)
9 . The method of claim 2 , wherein the antibody is a neutralizing antibody.
10 . The method of claim 6 , wherein the antibody is D5.
11 . The method of claim 1 , wherein the antibody binds to polysialic acid on Neisseria meningitides capsules but not to NCAM-associated polysialic acid.
12 . A method of vaccinating an individual against a pathogen comprising administering the mimotope of the pathogen-derived antigen of claim 1 or a derivative thereof to the individual.
13 . A method of vaccinating an individual against HIV comprising administering a mimotope of gp41 or a derivative thereof to the individual, wherein the mimotope is selected from the group consisting of the compounds of any of Tables 2-4.
14 . The method of claim 12 wherein the mimotope is conjugated to an immunogenic carrier protein.
15 . The method of claim 14 , wherein the carrier protein is OMPC.
16 . A method of identifying a small molecule mimotope of a self-antigen or a small molecule antagonist of an autoantibody comprising:
e) combining the following components
i) an autoantibody that binds to the self-antigen, wherein the auto antibody is associated with a pathological condition;
ii) the self-antigen or a fragment or conformational mimetic thereof that has a conformation that is substantially similar to that of the self-antigen such that the autoantibody binds to the fragment or conformational mimetic; and
iv) a small molecule test compound;
f) incubating the components under conditions which allow components i and ii to bind;
g) measuring the amount of binding between components i and ii, wherein a decrease in binding between components i and ii in the presence of the test compound as compared to in the absence of the test compound indicates the test compound binds to component i or ii and is a candidate compound;
h) determining to which component the candidate compound binds,
wherein binding to the autoantibody indicates that the candidate compound is a mimotope of the self-antigen and wherein binding to the self-antigen, fragment or conformational mimetic thereof indicates that the candidate compound is an antagonist of the autoantibody.
17 . The method of claim 16 , wherein the pathological condition is selected from the group consisting of myasthenia gravis, rheumatoid arthritis, lupus erythematosis, diabetes mellitus type 1, and multiple sclerosis.
18 . A method of treating an autoimmune disorder comprising administering to an individual in need thereof the mimetic or the antagonist of claim 16 or a derivative thereof.
19 . The method of claim 18 , wherein the individual is further administered one or more drugs selected from the group consisting of corticosteroid drugs, nonsteroidal anti-inflammatory drugs, and immunosuppressant drugs.
20 . A compound of Formula I
or a pharmaceutically acceptable salt thereof, wherein
R 1 is an optionally substituted aryl or heteroaryl; R 2 is an optionally substituted aryl or heteroaryl;
R 3 is either H or a C 1 -C 6 alkyl;
R 4 is either H or a C 1 -C 6 alkyl;
X is either N or C;
R 5 is selected from the group consisting of:
H,
(C═O)OC 1 -C 6 alkyl,
(C═O)OC 1 -C 6 cyloalkyl,
(C═O)OC 1 -C 6 aryl,
(C═O)OC 1 -C 6 heterocyclyl,
(C═O)OC 1 -C 6 alkyl,
aryl,
C 2 -C 6 alkenyl,
C 2 -C 6 alkynyl,
heterocyclyl,
C 3 -C 6 cycloalkyl,
SO 2 R a , and
(C═O)NR b 2 ,
said alkyl, cycloalkyl, aryl, heterocylyl, alkenyl, and alkynyl is optionally substituted with one or more substituents selected from R 7 ,
R 6 is selected from the group consisting of
H,
C (0-6) alkyl optionally substituted with a heterocyclic ring or aryl,
said alkyl, heterocyclic ring and aryl is optionally substituted with R a ; or
R 5 and R 6 together with X form a monocyclic or bicyclic ring with 5-7 members in each ring and,
when X is C, it optionally contains a 1-4 heteroatoms selected from N, O and S, and
when X is N, it optionally contains 1 to 4 additional heteroatoms selected from N, O and S, said monocylcic or bicyclic ring optionally substituted with one or more substituents selected from R 7 ;
R 7 is selected from the group consisting of:
(C 1 -C 6 )alkyl,
(C═O) r O s (C 1 -C 6 )alkyl,
O r (C 1 -C 3 )perfluoroalkyl,
S(O) m R a ,
SR a ,
(C 1 -C 6 )alkylene-S(O) m R a ,
oxo,
OH,
halo,
CN,
(C 2 -C 6 )alkenyl,
(C 2 -C 6 ) alkynyl,
(C 3 -C 6 )cycloalkyl,
aryl
(C 1 -C 6 )alkylene-aryl,
heterocyclyl,
(C 1 -C 6 )alkylene-heterocyclyl
N(R b ) 2 ,
(C 1 -C 6 )alkylene-N(R b ) 2 ,
C(O) R a ,
CO 2 R a ,
(C 1 -C 6 )alkylene-CO 2 R a ,
C(O)H, and
CO 2 H
(C 1 -C 6 )alkylene-CO 2 H
said alkyl, alkenyl, alkynyl, alkylene, cycloalkyl, aryl, and heterocyclyl is optionally substituted with up to three substituents selected from R b , OH, (C 1 -C 6 )alkoxy, halogen, CO 2 H, CN, O(C═O) (C 1 -C 6 )alkyl, oxo, and N(R b ) 2 ; and wherein each R is independently 0 or 1, each s is independently 0 or 1, and each m is independently 0, 1, or 2
R a is selected from the group consisting of:
(C 1 -C 6 ) alkyl,
(C 3 -C 6 ) cycloalkyl,
aryl,
or heterocyclyl; and
R b is selected from the group consisting of:
H,
(C 1 -C 6 ) alkyl,
aryl,
heterocyclyl,
(C 3 -C 6 ) cycloalkyl,
(C═O)O(C 1 -C 6 )alkyl,
(C═O)(C 1 -C 6 )alkyl or
S(O) 2 R a .
21 . The compound of Formula I or a pharmaceutically acceptable salt thereof, wherein
R 1 is an optionally substituted aryl;
R 2 is an optionally substituted phenyl;
R 3 is either H or a C 1 -C 6 alkyl;
R 4 is either H or a C 1 -C 6 alkyl;
X is either N or C;
R 5 is selected from the group consisting of:
H,
(C═O)OC 1 -C 6 alkyl,
(C═O)OC 1 -C 6 cyloalkyl,
(C═O)OC 1 -C 6 aryl,
(C═O)OC 1 -C 6 heterocyclyl,
(C═O)OC 1 -C 6 alkyl,
aryl,
C 2 -C 6 alkenyl,
C 2 -C 6 alkynyl,
heterocyclyl,
C 3 -C 6 cycloalkyl,
SO 2 R a , and
(C═O)NR b 2 ,
said alkyl, cycloalkyl, aryl, heterocylyl, alkenyl, and alkynyl is optionally substituted with one or more substituents selected from R 7 ,
R 6 is selected from the group consisting of
alkyl substituted with a heterocyclic ring or aryl, and
C (1-6) alkyl substituted with a heterocyclic ring or aryl,
each of which is optionally substituted with R a ; or
R 5 and R 6 together with X form a monocyclic or bicyclic heterocycle with 5-7 members in each ring and,
when X is C, it contains at least one heteroatom selected from N, O and S and optionally containing 1 to 4 additional heteroatoms selected from N, O and S, and
when X is N, it optionally contains 1 to 4 additional heteroatoms selected from N, O and S, said monocylcic or bicyclic heterocycle optionally substituted with one or more substituents selected from R 7 ;
R 7 is selected from the group consisting of:
(C═O) r O s (C 1 -C 6 )alkyl,
O r (C 1 -C 3 )perfluoroalkyl,
S(O) m R a ,
(C 1 -C 6 )alkylene-S(O) m R a ,
oxo,
OH,
halo,
CN,
(C 2 -C 6 )alkenyl,
(C 2 -C 6 ) alkynyl,
(C 3 -C 6 )cycloalkyl,
aryl
(C 1 -C 6 )alkylene-aryl,
heterocyclyl,
(C 1 -C 6 )alkylene-heterocyclyl
N(R b ) 2 ,
(C 1 -C 6 )alkylene-N(R b ) 2 ,
C(O) R a ,
CO 2 R a ,
(C 1 -C 6 )alkylene-CO 2 R a ,
C(O)H, and
CO 2 H
(C 1 -C 6 )alkylene-CO 2 H
said alkyl, alkenyl, alkynyl, alkylene, cycloalkyl, aryl, and heterocyclyl is optionally substituted with up to three substituents selected from R b , OH, (C 1 -C 6 )alkoxy, halogen, CO 2 H, CN, O(C═O) (C 1 -C 6 )alkyl, oxo, and N(R b ) 2 ; and wherein each R is independently 0 or 1, each s is independently 0 or 1, and each m is independently 0, 1, or 2
R a is selected from the group consisting of:
(C 1 -C 6 ) alkyl,
(C 3 -C 6 ) cycloalkyl,
aryl,
or heterocyclyl; and
R b is selected from the group consisting of:
H,
(C 1 -C 6 ) alkyl,
aryl,
heterocyclyl,
(C 3 -C 6 ) cycloalkyl,
(C═O)O(C 1 -C 6 )alkyl,
(C═O)(C 1 -C 6 )alkyl or
S(O) 2 R a .
22 . (canceled)