U.S. patent application number 11/549482 was filed with the patent office on 2007-06-28 for novel therapeutic target for protozoal diseases.
Invention is credited to Dewal Jani, Rana Nagarkatti, Dharmender Rathore.
Application Number | 20070148185 11/549482 |
Document ID | / |
Family ID | 37948378 |
Filed Date | 2007-06-28 |
United States Patent
Application |
20070148185 |
Kind Code |
A1 |
Rathore; Dharmender ; et
al. |
June 28, 2007 |
Novel therapeutic target for protozoal diseases
Abstract
A novel Hemozoin Detoxification Protein (HDP) from Plasmodium
and related parasites is provided as a target for therapeutic
intervention in diseases caused by the parasites. HDP has been
shown to play a critical role in adhesion to, or invasion into,
host cells by the parasite. Furthermore, HDP catalyzes the
neutralization of heme by the parasite, by promoting its
polymerization into hemozoin. This invention provides methods and
compositions for therapies based on the administration of protein,
DNA or cell-based vaccines and/or antibodies based on HDP, or
antigenic epitopes of HDP, either alone or in combination with
other parasite antigens. Methods for the development and use of
compounds that inhibit the catalytic activity of HDP, and
diagnostic and laboratory methods utilizing HDP are also provided.
HDP is also referred to herein as Fasciclin Related Adhesive
Protein (FRAP).
Inventors: |
Rathore; Dharmender;
(Blacksburg, VA) ; Jani; Dewal; (Blacksburg,
VA) ; Nagarkatti; Rana; (Blacksburg, VA) |
Correspondence
Address: |
WHITHAM, CURTIS & CHRISTOFFERSON & COOK, P.C.
11491 SUNSET HILLS ROAD
SUITE 340
RESTON
VA
20190
US
|
Family ID: |
37948378 |
Appl. No.: |
11/549482 |
Filed: |
October 13, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11249355 |
Oct 14, 2005 |
|
|
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11549482 |
Oct 13, 2006 |
|
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Current U.S.
Class: |
424/191.1 ;
514/217.09; 514/230.5; 514/237.8; 514/25; 514/313; 514/314;
514/320; 514/337; 514/345; 514/367; 514/406; 514/443; 514/731 |
Current CPC
Class: |
A61K 2039/505 20130101;
G01N 33/56905 20130101; Y02A 50/30 20180101; G01N 2333/445
20130101; A61K 39/00 20130101; A61K 2039/53 20130101; C07K 14/445
20130101; A61K 2039/522 20130101 |
Class at
Publication: |
424/191.1 ;
514/367; 514/443; 514/337; 514/230.5; 514/237.8; 514/217.09;
514/025; 514/314; 514/313; 514/731; 514/406; 514/320; 514/345 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61K 31/7024 20060101 A61K031/7024; A61K 31/538
20060101 A61K031/538; A61K 31/5377 20060101 A61K031/5377; A61K
31/4709 20060101 A61K031/4709; A61K 31/4706 20060101
A61K031/4706 |
Claims
1. A method of treating or preventing a disease caused by a
Plasmodium or Theileria parasite in an individual in need thereof,
comprising the step of inhibiting interaction of heme and Heme
Detoxification Protein (HDP) in said individual.
2. The method of claim 1, wherein said step of inhibiting is
carried out by administering to said individual one or more
compounds that inhibit interaction of heme and HDP.
3. The method of claim 2, wherein said one or more compounds bind
to heme.
4. The method of claim 3, wherein said one or more compounds
prevent heme from binding to HDP.
5. The method of claim 3, wherein said one or more compounds allow
the binding of heme to HDP but prevent detoxification of heme by
HDP.
6. The method of claim 2, wherein said one or more compounds bind
to HDP.
7. The method of claim 6, wherein said one or more compounds
prevent binding of heme to HDP.
8. The method of claim 6, wherein said one or more compounds
prevent the production of hemozoin from bound heme.
9. The method of claim 6, wherein said one or more compounds bind
at the active site of HDP.
10. The method of claim 6, wherein said one or more compounds bind
at an allosteric site of HDP.
11. The method of claim 1, wherein said step of inhibiting is
carried out by modification of a cell membrane of said Plasmodium
or Theileria parasite.
12. The method of claim 1, wherein said step of inhibiting is
carried out by inhibiting secretion of HDP from said Plasmodium or
Theileria parasite.
13. The method of claim 2 wherein said disease is malaria.
14. The method of claim 13, wherein said compound is administered
to said individual in combination with one or more of: an
additional antimalarial agent, an agent for reversing antimalarial
resistance, and an adjuvant.
15. The method of claim 14, wherein said compound is administered
prior to, concurrent with, or subsequent to administration of said
additional antimalarial agent or said agent for reversing
antimalarial resistance.
16. The method of claim 14, wherein said additional antimalarial
agent is selected from the group consisting of a) quinolines, b)
folic acid antagonists, c) sulfonamides, and d) antibiotics.
17. The method of claim 14, wherein said agent for reversing
antimalarial resistance is an inhibitor of multidrug
resistance.
18. The method of claim 2, wherein said compound is administered
orally, parenterally, sublingually, rectally, topically or with an
inhalation spray.
19. The method of claim 2 wherein said one or more compounds is
selected from the group consisting of:
(1S)-1-(3,4-dichlorophenyl)-2-(2-imino-1,3-benzothiazol-3(2H)-yl)ethanol;
(2E)-2-[(pyridin-3-ylamino)methylene]-1-benzothiophen-3(2H)-one;
(2E,5E)-5-(5-bromo-3-chloro-2-hydroxybenzylidene)-2-(phenylimino)-1,3-thi-
azolidin-4-one;
(2S)-N-(4-chlorophenyl)-2-methyl-2,3-dihydro-4H-1,4-benzoxazine-4-carboxa-
mide;
(3aR,4R,9bR)-8-chloro-4-(4-chlorophenyl)-9-nitro-3a,4,5,9b-tetrahyd-
ro-3H-cyclopenta[c]quinolin-6-ol;
(3R)-5,7-dichloro-3-hydroxy-3-(2-methylimidazo[1,2-a]pyridin-3-yl)-1,3-di-
hydro-2H-indol-2-one;
[6-hydroxy-2-(4-hydroxyphenyl)-1-benzothien-3-yl][4-(2-piperidin-1-yletho-
xy)phenyl]methanone;
1,2,3,4,6-pentakis-O-(3,4,5-trihydroxybenzoyl)-beta-D-glucopyranose;
1-[(2S)-3-(9H-carbazol-9-yl)-2-hydroxypropyl]-8-hydroxyquinolinium;
1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-1,3-dihydro-2-
H-benzimidazol-2-one;
1-ethyl-6-methoxy-4-methyl-2-[(Z)-(3-methyl-1,3-thiazol-2(3H)-ylidene)met-
hyl]benzo[h]quinolinium; 1H-perimidine-2-carboxylic acid;
2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-4H-chromen-4-one;
2-(4-methoxyphenyl)-4H-1,3-benzoxazin-4-one;
2-(4-methoxyphenyl)pyridin-3-ol;
2-(morpholin-4-ylmethyl)-1-naphthol;
2,2'-buta-1,3-diyne-1,4-diyldiphenol;
2-[(E)-(6-methoxy-1-methylquinolin-2(1H)-ylidene)methyl]-3-methyl-1,3-ben-
zothiazol-3-ium;
2-[(Z)-(3-ethyl-6-methoxy-1,3-benzothiazol-2(3H)-ylidene)methyl]-1,6-dime-
thylquinolinium; 2-[2-(4-hydroxyphenyl)ethyl]-6-methylpyridin-3-ol;
2-[4-(1-benzofuran-2-yl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-5-methoxyph-
enol;
2-{(3R)-1-[4-(2-hydroxyethoxy)benzyl]piperidin-3-yl}-1H-isoindole-1-
,3(2H)-dione;
2-amino-8-(azepan-1-ylmethyl)-3-(1,3-benzothiazol-2-yl)-7-hydroxy-4H-chro-
men-4-one; 2-hydrazino-4-methylquinoline;
2-hydroxy-N-(4-propylbenzoyl)benzamide;
3-(2-hydroxy-5-methoxybenzoyl)-2-(4-methylphenyl)isoindolin-1-one;
3-(3-{[(2-chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile;
3-[(4-{[(2R)-tetrahydrofuran-2-ylmethyl]amino}quinazolin-2-yl)amino]pheno-
l;
3-[4-(9H-fluoren-9-yl)piperazin-1-yl]-N-[3-(trifluoromethyl)phenyl]pro-
panamide;
4-({(2S)-3-[4-(diphenylmethyl)piperazin-1-yl]-2-hydroxypropyl}o-
xy)-1H-indole-2-carbonitrile;
4-(1H-benzimidazol-2-yl)-N,N-dimethylaniline;
4-(6-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;
4-(7-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;
4,4'-(4-phenyl-1H-imidazole-2,5-diyl)diphenol;
4,4'-methylenebis(3-hydroxy-2-naphthoic
acid)-3,3'-[(4-iminocyclohexa-2,5-dien-1-ylidene)methylene]dianiline
(1:1); 4,4'-propane-2,2-diylbis(2-chlorophenol);
4-[(5S)-5-(4-fluorophenyl)-1-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl]-
phenol;
4-[4-(3,4-dihydro-2H-1,5-benzodioxepin-7-yl)-3-methylisoxazol-5-y-
l]benzene-1,3-diol;
4-[5-[5-(4-methylpiperazin-1-yl)-3H-benzoimidazol-2-yl]-1,3-dihydrobenzoi-
midazol-2-ylidene]cyclohexa-2,5-dien-1-one;
4-{[(2-ethylphenyl)amino]methyl}-5-(hydroxymethyl)-2-methylpyridin-3-ol;
4-phenylquinolin-2-amine;
5-(2-hydroxy-5-methylbenzoyl)-1-(4-methylphenyl)-2-oxo-1,2-dihydropyridin-
e-3-carbonitrile;
5-(5-chloro-2-hydroxybenzoyl)-2-oxo-N,1-diphenyl-1,2-dihydropyridine-3-ca-
rboxamide;
5-7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-4H-chromen-4-one;
6-amino-1-ethylbenzo[cd]indol-2(1H)-one;
6-chloro-3-[2-(4-chlorophenyl)ethyl]-3,4-dihydro-2H-1,3-benzoxazine;
7-[(2E)-2-(biphenyl-4-ylmethylene)hydrazino]-N-(2-hydroxyphenyl)-7-oxohep-
tanamide;
7-chloro-N-[2-(dimethylamino)ethyl]-4H-thieno[3,2-c]thiochromen-
e-2-carboxamide; 7-chloro-N-phenylquinolin-4-amine;
7-hydroxy-2-methyl-6-propyl-3-pyridin-2-yl-4H-chromen-4-one;
8-[(2E)-2-(2-bromobenzylidene)hydrazino]-N-(2-hydroxyphenyl)-8-oxooctanam-
ide; CN(C)c1ccc2N=c3cc(C)c(N)cc3=Sc2c1;
COc1cc(O)c-2c(CCc3cc(OC)c(OC)cc32)c1; ethyl
1-benzyl-4-[(dimethylamino)methyl]-5-hydroxy-2-phenyl-1H-indole-3-carboxy-
late; ethyl 2-ethoxy-5-hydroxy-1H-benzo[g]indole-3-carboxylate;
N-(2-ethoxyphenyl)-2-hydroxybenzamide;
N-(2-hydroxybenzoyl)-2-thiophenecarboxamide;
N-(2-hydroxybenzoyl)-3-methoxybenzamide;
N-(3-chloro-4-hydroxy-1-naphthyl)-4-ethoxybenzenesulfonamide;
N-(3-furylcarbonyl)-2-hydroxybenzamide;
N-(4-ethylbenzoyl)-2-hydroxybenzamide;
N-(6-chloro-2-phenyl-4H-chromen-4-ylidene)-1-(2-furyl)methanamine;
N-[(1S)-1-phenylethyl]quinazolin-4-amine;
N-[(4E)-2-(4-methoxyphenyl)-6-methyl-4H-chromen-4-ylidene]-2-phenylethana-
mine; N4-(3,5-dichlorophenyl)-6-methylpyrimidine-2,4-diamine;
N-benzo[g]quinolin-4-yl-N'-isopropylbenzene-1,4-diamine;
O[C@H]1[C@@H](O)[C@@H](COC(.dbd.O)c2cc(O)c(O)c(O)c2)O[C@@H](Oc3ccc(C(.dbd-
.O)CCc4ccc(O)cc4)c(O)c3)[C@@H]1O; and
O[C@H]1[C@H]2[C@H](CC(.dbd.O)O)C(.dbd.O)O[C@@H]3C(COC(.dbd.O)c4cc(O)c(O)c-
(O)c4)O
[C@@H](OC(.dbd.O)c5cc(O)c(O)c(O)c5)C(OC(.dbd.O)c6cc(O)c(O)c(OC1.db-
d.O)c26)[C@@H]3OC (.dbd.O)c7cc(O)c(O)c(O)c7.
20. The method of claim 2 wherein said one or more compounds is
selected from the group consisting of:
(10S)-10-(dimethylamino)-9-methyl-7H,10H-naphtho[1,8-gh]chromen-7-one;
(1E,4E)-1-[4-(dimethylamino)phenyl]-5-(3,4,5-trimethoxyphenyl)penta-1,4-d-
ien-3-one;
(1R,3R)-1-isopropyl-2,3,4,9-tetrahydro-1H-beta-carboline-3-carboxylic
acid;
(1S)-1-(3,4-dichlorophenyl)-2-(2-imino-1,3-benzothiazol-3(2H)-yl)e-
thanol;
(2E)-2-[(pyridin-3-ylamino)methylene]-1-benzothiophen-3(2H)-one;
(2E)-3-(3,4-dimethoxyphenyl)-N-(3,4-dimethylphenyl)acrylamide;
(2E)-6-ethoxy-2-(2-hydroxybenzylidene)-1-benzothiophen-3(2H)-one;
(2E)-N-(2-methyl-1,3-benzothiazol-6-yl)-3-phenylacrylamide;
(2E,5E)-5-(5-bromo-3-chloro-2-hydroxybenzylidene)-2-(phenylimino)-1,3-thi-
azolidin-4-one;
(2E,5Z)-2-[(2-chlorophenyl)imino]-5-(4-hydroxy-3-nitrobenzylidene)-1,3-th-
iazolidin-4-one;
(2R)-1-(benzylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol;
(2R)-2-(2,4-dichlorophenoxy)-N-(5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-3-y-
l)propanamide;
(2R)-2-[(5Z)-5-(4-hydroxy-3,5-dimethoxybenzylidene)-4-oxo-2-thioxo-1,3-th-
iazolidin-3-yl]-3-methylbutanoic acid;
(2R)-2-[(E)-2-(1,3-benzodioxol-5-yl)vinyl]-5,6-dimethyl-2,3-dihydrothieno-
[2,3-d]pyrimidin-4(1H)-one;
(2R,3Z)-6-chloro-3-[(dimethylamino)methylene]-2-methyl-2,3-dihydro-4H-thi-
ochromen-4-one;
(2S)-2-(4-chlorophenyl)-3-oxo-4-phenylbutanenitrile;
(2S)-2-[(5E)-5-(1H-indol-3-ylmethylene)-4-oxo-2-thioxo-1,3-thiazolidin-3--
yl]succinic acid;
(2S)-N-(4-chlorophenyl)-2-methyl-2,3-dihydro-4H-1,4-benzoxazine-4-carboxa-
mide;
(2S,3Z)-3-(1H-indol-3-ylmethylene)-2-phenyl-2,3-dihydro-4H-chromen--
4-one; (2Z)-2-acetamido-N-(3,5-dimethylphenyl)-3-phenylacrylamide;
(2Z,5E)-2-[(3,5-dimethylphenyl)imino]-5-(2-hydroxy-3-methoxybenzylidene)--
1,3-thiazolidin-4-one;
(2Z,5Z)-2-[(2-chlorophenyl)imino]-5-(2-hydroxy-3-nitrobenzylidene)-1,3-th-
iazolidin-4-one;
(3,5-dichloro-2-hydroxyphenyl)(isoxazol-4-yl)methanone;
(3-{(E)-[1-(3-fluorophenyl)-2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene-
]methyl}-1H-indol-1-yl)acetonitrile;
(3aR,4R,9bR)-8-chloro-4-(4-chlorophenyl)-9-nitro-3a,4,5,9b-tetrahydro-3H--
cyclopenta[c]quinolin-6-ol;
(3aS,6aS)-3-benzoyl-1,5-diphenyl-3a,6a-dihydropyrrolo[3,4-c]pyrazole-4,6(-
1H,5H)-dione;
(3R)-3-(2-hydroxy-4-methylphenyl)-N-(2-methoxyphenyl)-3-phenylpropanamide-
;
(3R)-5,7-dichloro-3-hydroxy-3-(2-methylimidazo[1,2-a]pyridin-3-yl)-1,3--
dihydro-2H-indol-2-one;
(3R,3'R,4'S,6'R,8'S,8a'S)-5-(4-hydroxybut-1-yn-1-yl)-6'-[4-(2-hydroxyetho-
xy)phenyl]-1',2-dioxo-3',4'-diphenyl-1,2,3',4',8',8a'-hexahydro-1'H-spiro[-
indole-3,7'-pyrrolo[2, 1-c][1,4]oxazine]-8'-carboxylic acid;
(3S)-3-(2-hydroxy-4-methylbenzoyl)-2-(4-methylphenyl)isoindolin-1-one;
(3S,3aR,6aR)-3-(5-bromo-2-hydroxyphenyl)-5-butyl-2-phenyldihydro-2H-pyrro-
lo[3,4-d]isoxazole-4,6(3H,5H)-dione;
(3S,6S,7R,8aR)-3-(4-acetamidobutyl)-6-(4-hydroxyphenyl)-1,4-dioxooctahydr-
opyrrolo[1,2-a]pyrazine-7-carboxylic acid;
(3Z)-3-(3-hydroxy-4-methoxybenzylidene)-1-methyl-1,3-dihydro-2H-indol-2-o-
ne;
(4E)-2-(4-methoxyphenyl)-4-[(4-methoxyphenyl)imino]-4H-chromen-6-ol;
(4R)-3-(3,4-dichlorophenyl)-4-hydroxy-N-isopropyl-2-oxo-1,2,3,4-tetrahydr-
oquinazoline-4-carboxamide;
(4R)-4-(4-bromophenyl)-3-hydroxy-1-isopropyl-4,8-dihydro-1H-pyrazolo[3,4--
e][1,4]thiazepin-7(6H)-one;
(4R)-4-(4-ethylphenyl)-3-hydroxy-2-phenyl-2,4,6,7,8,9-hexahydro-5H-pyrazo-
lo[3,4-b]quinolin-5-one;
(4R)-5-(2-furylmethyl)-3-(2-hydroxyphenyl)-4-phenyl-4,5-dihydropyrrolo[3,-
4-c]pyrazol-6(1H)-one;
(4R)-N.about.4.about.-(6-chloro-2-methoxyacridin-9-yl)-N.about.1.about.,N-
.about.1.about.-diethylpentane-1,4-diamine;
(4S)-4-(2-bromobenzoyl)-5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one;
(4S)-4-(2-furyl)-3-hydroxy-7,7-dimethyl-2-phenyl-2,4,6,7,8,9-hexahydro-5H-
-pyrazolo[3,4-b]quinolin-5-one;
(4S)-5,7-dihydroxy-4-phenylchroman-2-one;
(4S,5S)-3,5-diphenyl-4,5-dihydro-1H-pyrazol-4-ol;
(4S,7R)-2-amino-4-isobutyl-5-oxo-7-phenyl-5,6,7,8-tetrahydro-4H-chromene--
3-carbonitrile;
(4Z)-2-[2-(4-chlorophenoxy)pyridin-3-yl]-4-[(dimethylamino)methylene]-1,3-
-oxazol-5(4H)-one;
(5E)-1-(4-methylpentyl)-5-(1H-pyrrol-2-ylmethylene)pyrimidine-2,4,6(1H,3H-
,5H)-trione;
(5E)-3-allyl-5-(2-hydroxybenzylidene)-2-thioxo-1,3-thiazolidin-4-one;
(5E)-5-[4-(diethylamino)benzylidene]-3-{[(2-methoxyphenyl)amino]methyl}-1-
,3-thiazolidine-2,4-dione;
(5R)-5-methyl-4-phenyl-1,3,4-thiadiazolidine-2-thione;
(5S,7R)-2,2-dimethyl-5,7-bis(2-phenylethyl)-7,8-dihydro-4H,5H-pyrano[4,3--
d][1,3]dioxine;
(5Z)-5-(4-hydroxybenzylidene)-3-[(2R)-tetrahydrofuran-2-ylmethyl]-2-thiox-
o-1,3-thiazolidin-4-one;
(6E)-5-imino-6-{[1-(2-naphthyl)-1H-pyrrol-2-yl]methylene}-5,6-dihydro-7H--
[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-one;
*c1ceccc1C2C(.dbd.O)N(C)c3ccccc3C2=O;
[(2R)-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]acetic acid;
[(5E)-4-oxo-5-(3-thienylmethylene)-2-thioxo-1,3-thiazolidin-3-yl]acetic
acid;
[(5E)-5-(5-bromo-3-chloro-2-hydroxybenzylidene)-4-oxo-2-thioxo-1,3-
-thiazolidin-3-yl]acetic acid;
[6-hydroxy-2-(4-hydroxyphenyl)-1-benzothien-3-yl][4-(2-piperidin-1-yletho-
xy)phenyl]methanone; {4-[(4-methylphenyl)sulfonyl]phenyl}hydrazine;
1-(2,4-dihydroxy-6-methylphenyl)-2-phenoxyethanone;
1-(2,4-dihydroxyphenyl)-2-(4-isopropylphenoxy)ethanone;
1-(3,4-dihydroxyphenyl)-2-({4-[(3,5-dimethoxyphenyl)amino]quinazolin-2-yl-
}thio)ethanone; 1-(4-chlorophenyl)-1-hydroxy-3-phenylurea;
1-(4-hydroxy-3,5-dimethylphenyl)-2-[(4-methylphenyl)thio]ethanone;
1-(4-iodo-2-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-ol;
1-(5-butyl-2,4-dihydroxyphenyl)-2-pyridin-2-ylethanone;
1-(5-ethyl-2,4-dihydroxyphenyl)-2-(1-methyl-1H-benzimidazol-2-yl)ethanone-
;
1,2,3,4,6-pentakis-O-(3,4,5-trihydroxybenzoyl)-beta-D-glucopyranose;
1-[(2S)-.sup.3-(9H-carbazol-9-yl)-2-hydroxypropyl]-8-hydroxyquinolinium;
1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-1,3-dihydro-2-
H-benzimidazol-2-one;
1-[4-(7-chloroquinolin-4-yl)piperazino]propan-1-one;
1-ethyl-6-methoxy-4-methyl-2-[(Z)-(3-methyl-1,3-thiazol-2(3H)-ylidene)met-
hyl]benzo[h]quinolinium; 1-ethynyl-2-phenoxybenzene;
1H-perimidine-2-carboxylic acid;
2-(1,3-benzodioxol-5-yl)-1-(2,4-dihydroxy-5-propylphenyl)ethanone;
2-(1H-benzimidazol-1-yl)-1-(5-ethyl-2,4-dihydroxyphenyl)ethanone;
2-(2,4-dichlorophenyl)-1H-imidazo[4,5-b]pyridin-1-ol;
2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one;
2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-7-methoxy-4H-chromen-4-one;
2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-4H-chromen-4-one;
2-(4-chlorophenyl)-4H-1,3-benzoxazin-4-one;
2-(4-chlorophenyl)-5-{[(4-pyridin-3-ylpyrimidin-2-yl)thio]methyl}-2,4-dih-
ydro-3H-pyrazol-3-one;
2-(4-fluoro-3-phenoxyphenyl)-3-hydroxy-4H-chromen-4-one;
2-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)-N-(4-methylphenyl)acetamide;
2-(4-methoxyphenyl)-4H-1,3-benzoxazin-4-one;
2-(4-methoxyphenyl)pyridin-3-ol;
2-(4-methylphenyl)-4H-1,3-benzoxazin-4-one;
2-(morpholin-4-ylmethyl)-1-naphthol;
2,2'-buta-1,3-diyne-1,4-diyldiphenol; 2,2'-thiobis(4-chlorophenol);
2,4-dichloro-1-naphthyl[2,2,2-trifluoro-1-methyl-1-(trifluoromethyl)ethyl-
]carbamate; 2-[(2-phenoxyethyl)thio]quinazoline-4-thiol;
2-[(2R)-6,7-dimethyl-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]-N-ethylacet-
amide;
2-[(3-cyano-4-methyl-6-oxo-1,6-dihydropyridin-2-yl)thio]-N-1-napht-
hylacetamide;
2-[(5S)-1-(4-nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl]phenol;
2-[(E)-(6-methoxy-1-methylquinolin-2(1H)-ylidene)methyl]-3-methyl-1,3-ben-
zothiazol-3-ium;
2-[(Z)-(3-ethyl-6-methoxy-1,3-benzothiazol-2(3H)-ylidene)methyl]-1,6-dime-
thylquinolinium; 2-[2-(4-hydroxyphenyl)ethyl]-6-methylpyridin-3-ol;
2-[4-(1-benzofuran-2-yl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-5-methoxyph-
enol; 2-[5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl]phenol;
2-[5-(ethylsulfonyl)-2-hydroxyphenyl]-1H-benzo[de]isoquinoline-1,3(2H)-di-
one;
2-{(1R)-1-[(1-allyl-1H-benzimidazol-2-yl)amino]ethyl}-4-chlorophenol-
;
2-{(3R)-1-[4-(2-hydroxyethoxy)benzyl]piperidin-3-yl}-1H-isoindole-1,3(2-
H)-dione;
2-{[5-chloro-6-methyl-2-(2-pyridinyl)-4-pyrimidinyl]sulfanyl}-1-
-phenyl-1-ethanone;
2-amino-1-(2,4-dimethylphenyl)-1H-pyrrolo[2,3-b]quinoxaline-3-carbonitril-
e;
2-amino-5-butyl-4-(4-hydroxy-3-methoxyphenyl)-6-phenylnicotinonitrile;
2-amino-8-(azepan-1-ylmethyl)-3-(1,3-benzothiazol-2-yl)-7-hydroxy-4H-chr-
omen-4-one; 2-anilino-2-oxoethyl 2-(4-chlorobenzoyl)benzoate;
2-chloro-5-phenyl-3-pyridin-4-yl-4H-1,4-thiazine;
2-chloro-8-hydroxy-10,10-dimethyl-7-phenylpyrido[1,2-a]indol-6(10H)-one;
2-hydrazino-4,6-diphenylpyrimidine; 2-hydrazino-4-methylquinoline;
2-hydroxy-N-(4-methylphenyl)benzamide;
2-hydroxy-N-(4-propylbenzoyl)benzamide;
2-hydroxy-N-[(4-methyl-2-phenyl-1,3-thiazol-5-yl)carbonyl]benzamide;
2-hydroxy-N-[2-(2-methoxyphenyl)acetyl]benzamide;
2-hydroxy-N-{[2-(4-methylphenoxy)-3-pyridinyl]carbonyl}benzamide;
2-hydroxy-N-pyridin-3-ylbenzamide;
2-phenyl-4H-thiochromene-4-thione;
2-phenyl-5-({[5-(trifluoromethyl)pyridin-2-yl]sulfonyl}methyl)-2,4-dihydr-
o-3H-pyrazol-3-one;
2-phenyl-5-(trifluoromethyl)-2,4-dihydro-3H-pyrazol-3-one;
3-(1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)-N-(2-hydroxyphenyl)-N-(-
4-methoxybenzyl)propanamide;
3-(1-acetyl-1H-indol-3-yl)-4-hydroxy-2H-chromen-2-one;
3-(1H-benzimidazol-1-yl)-6-ethyl-7-hydroxy-4H-chromen-4-one;
3-(2-hydroxy-5-methoxybenzoyl)-2-(4-methylphenyl)isoindolin-1-one;
3-(3-{[(2-chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile;
3-(3-{[(4-chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile;
3-(4-bromophenyl)-7-hydroxy-2-methyl-4H-chromen-4-one;
3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-one;
3-(5-{(Z)-[5-(4-methylphenyl)-2-oxofuran-3(2H)-ylidene]methyl}-2-furyl)be-
nzoic acid; 3-(quinazolin-4-ylamino)phenyl thiophene-2-carboxylate;
3,4-dimethoxy-N-(4-methyl-1,3-benzothiazol-2-yl)benzamide;
3,5-dichloro-2-hydroxybenzaldehyde
N-tert-butyl-N'-methylthiosemicarbazone;
3-[(4-{[(2R)-tetrahydrofuran-2-ylmethyl]amino}quinazolin-2-yl)amino]pheno-
l;
3-[2-(4-methoxyphenyl)ethyl]-10-methyl-6-phenyl-3,4-dihydro-2H,8H-chro-
meno[6,7-e][1,3]oxazin-8-one;
3-[4-(9H-fluoren-9-yl)piperazin-1-yl]-N-[3-(trifluoromethyl)phenyl]propan-
amide;
3-{2-[(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-1H-pyrrol--
1-yl}benzoic acid; 3-benzyl-4-hydroxy-1,2-dihydroquinolin-2-one;
3-benzyl-4-hydroxy-1-phenylquinolin-2(1H)-one;
3-benzyl-5,6-bis(4-methoxyphenyl)furo[2,3-d]pyrimidin-4(3H)-imine;
3-benzyl-5-ethyl-4-hydroxy-6-phenyl-1-(1,3-thiazol-2-yl)pyridin-2(1H)-one-
; 3-benzyl-6-ethoxy-4-hydroxyquinolin-2(1H)-one;
3-chloro-N-[2-(methylthio)-1,3-benzothiazol-6-yl]benzamide;
3-hydroxy-N-(2-methylphenyl)-2-naphthamide;
3-methoxy-2-methyl-6-[1'-phenyl-5-(trifluoromethyl)-1H,
1'H-4,4'-bipyrazol-3-yl]phenol;
3-methoxy-N-(3-[1,3]oxazolo[4,5-b]pyridin-2-ylphenyl)benzamide;
3-methyl-1-[3-(trifluoromethyl)phenyl]-1H-pyrazol-5-ol;
3-methyl-1-phenyl-4-(trifluoromethyl)-1,7-dihydro-6H-pyrazolo[3,4-b]pyrid-
in-6-one;
3-methyl-4-[(4-methylphenyl)thio]-1-phenyl-1H-pyrazol-5-yl
methoxyacetate;
3-oxo-3-[3-({[3-(trifluoromethyl)phenyl]thio}methyl)phenyl]propanenitrile-
;
4-({(2S)-3-[4-(diphenylmethyl)piperazin-1-yl]-2-hydroxypropyl}oxy)-1H-i-
ndole-2-carbonitrile; 4-(1H-benzimidazol-2-yl)-N,N-dimethylaniline;
4-(1-propyl-1H-benzimidazol-2-yl)aniline;
4-(2,6-dichlorobenzyl)-3-methyl-1-phenyl-1H-pyrazol-5-ol;
4-(6-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;
4-(7-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;
4,4'-(4-phenyl-1H-imidazole-2,5-diyl)diphenol;
4,4'-[(2,3,5,6-tetrafluoro-1,4-phenylene)bis(oxy)]diphenol;
4,4'-methylenebis(3-hydroxy-2-naphthoic
acid)-3,3'-[(4-iminocyclohexa-2,5-dien-1-ylidene)methylene]dianiline
(1:1); 4,4'-propane-2,2-diylbis(2-chlorophenol);
4-[(3S,6S,7R,8aR)-7-{[2-(4-{(3S,3'S,4'R,6'R,8'R,8a'R)-8'-[(allyloxy)carbo-
nyl]-5-iodo-1',2-dioxo-3',4'-diphenyl-1,2,3',4',8',8a'-hexahydro-1'H-spiro-
[indole-3,7'-pyrrolo[2,
1-c][1,4]oxazin]-6'-yl}phenoxy)ethoxy]carbonyl}-6-(4-hydroxyphenyl)-1,4-d-
ioxooctahydropyrrolo[1,2-a]pyrazin-3-yl]-N,N,N-trimethylbutan-1-aminium;
4-[(4-chlorophenyl)thio]-3-methyl-1-phenyl-1H-pyrazol-5-ol;
4-[(5S)-5-(4-fluorophenyl)-1-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl]-
phenol;
4-[1-(4-hydroxy-3-methoxybenzyl)-1H-benzimidazol-2-yl]-2-methoxyp-
henol;
4-[1'-phenyl-5-(trifluoromethyl)-1H,1'H-4,4'-bipyrazol-3-yl]benzen-
e-1,3-diol;
4-[4-(1,3-benzothiazol-2-yl)-5-methyl-1H-pyrazol-3-yl]benzene-1,3-diol;
4-[4-(3,4-dihydro-2H-1,5-benzodioxepin-7-yl)-3-methylisoxazol-5-yl]benzen-
e-1,3-diol;
4-[4-(4-chlorophenyl)-1,3-thiazol-2-yl]-1-methyl-1H-pyrazol-5-amine;
4-[5-[5-(4-methylpiperazin-1-yl)-3H-benzoimidazol-2-yl]-1,3-dihydrobenzoi-
midazol-2-ylidene]cyclohexa-2,5-dien-1-one;
4-{[(1E)-3-(2-furyl)-3-oxoprop-1-en-1-yl]amino}benzoic acid;
4-{[(2-ethylphenyl)amino]methyl}-5-(hydroxymethyl)-2-methylpyridin-3-ol;
4-{[(2S)-2-ethylpiperidin-1-yl]methyl}-3-hydroxy-1-methyl-6H-benzo[c]chro-
men-6-one;
4-bromo-2-[(E)-(4H-1,2,4-triazol-4-ylimino)methyl]phenol;
4-bromo-2-[5-(2-furyl)-1H-pyrazol-3-yl]phenol;
4-bromo-6-chloro-2-oxo-1,3-benzoxathiol-5-yl ethyl carbonate;
4-ethyl-6-[4-(1-methyl-1H-benzimidazol-2-yl)-1H-pyrazol-3-yl]benzene-1,3--
diol; 4-fluoro-N-[3-(trifluoromethyl)phenyl]benzamide;
4-hydroxy-3-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]-2H-chromen-
-2-one; 4-hydroxy-3-propylquinolin-2(1H)-one;
4-hydroxy-5-phenyl-6H-pyrido[3,2,1-jk]carbazol-6-one;
4-hydroxy-8-methyl-3-{(E)-[(3R)-5-oxo-1,3-diphenylpyrazolidin-4-ylidene]m-
ethyl}quinolin -2(1H)-one; 4-phenylquinolin-2-amine;
5-(2-hydroxy-5-methylbenzoyl)-1-(4-methylphenyl)-2-oxo-1,2-dihydropyridin-
e-3-carbonitrile;
5-(5-bromo-2-hydroxybenzoyl)-1-(2-fluorophenyl)-2-oxo-1,2-dihydropyridine-
-3-carbonitrile;
5-(5-chloro-2-hydroxybenzoyl)-2-oxo-N,1-diphenyl-1,2-dihydropyridine-3-ca-
rboxamide;
5-(benzoylamino)-N,N'-bis(2-hydroxyphenyl)isophthalamide;
5-(diethylamino)-2-{(E)-[(2-phenylethyl)imino]methyl}phenol;
5,7-dihydroxy-4-propyl-2H-chromen-2-one;
5-[(4-methylphenyl)thio]quinazoline-2,4-diamine;
5-{2-[(3,4-dichlorophenyl)thio]ethyl}-2-methylpyridine;
5-benzyl-3-phenyl-5H-pyrazolo[4,3-c]quinolines;
5-benzyl-4-hydroxy-6H-pyrido[3,2,1-jk]carbazol-6-one;
5-chloro-2-hydroxy-N-phenylbenzamide;
5-hydroxy-4-methyl-7-propyl-2H-chromen-2-one;
5-methoxy-2-[3-methyl-4-(1,3-thiazol-4-yl)isoxazol-5-yl]phenol;
5-methyl-2-[5-(2-thienyl)-1H-pyrazol-3-yl]phenol;
6-(4-chlorophenyl)-7-hydroxy-1,3-dimethyl-1H-pyrrolo[3,2-d]pyrimidine-2,4-
(3H,5H)-dione''6,6'-biquinoline;
6-[(S)-[4-(dimethylamino)phenyl](piperidin-1-yl)methyl]-1,3-benzodioxol-5-
-ol;
6-{[(2-ethylphenyl)amino]sulfonyl}-4-oxo-N-[(2S)-tetrahydrofuran-2-y-
lmethyl]-1,4-dihydroquinoline-3-carboxamide;
6-{[(4-chlorophenyl)thio]methyl}-2-phenyl-1H-pyrazolo[3,4-b]pyridine-3,4(-
2H,7H) -dione;
6-{[(4-ethoxyphenyl)(methyl)amino]sulfonyl}-4-oxo-N-[(2S)-tetrahydrofuran-
-2-ylmethyl]-1,4-dihydroquinoline-3-carboxamide;
6-allyl-7-hydroxy-4,8-dimethyl-2H-chromen-2-one;
6-amino-1-ethylbenzo[cd]indol-2(1H)-one;
6-benzyl-7-hydroxy-2,3-dihydro-1H,5H-pyrido[3,2,1-ij]quinolin-5-one;
6-bromo-2-(trifluoromethyl)quinolin-4-ol;
6-butyl-2-(2-furyl)-5-methyl-4,7-dihydropyrazolo[1,5-a]pyrimidin-7-one;
6-chloro-2-(4-chlorophenyl)-1H-benzimidazol-1-ol;
6-chloro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide;
6-chloro-3-(4-methylphenyl)-3,4-dihydro-2H-1,3-benzoxazine;
6-chloro-3-[2-(4-chlorophenyl)ethyl]-3,4-dihydro-2H-1,3-benzoxazine;
6-ethyl-7-hydroxy-3-(1-methyl-1H-benzimidazol-2-yl)-2-(trifluoromethyl)-4-
H-chromen-4-one; 6-fluoro-4-hydroxy-3-phenylquinolin-2(1H)-one;
6-methoxy-N-[(1S)-1-methylpropyl]furo[2,3-b]quinoline-2-carboxamide;
6-phenyl[1,2,3,4]tetraazolo[1,5-b]pyridazin-7-ol;
7-(4-bromophenyl)-5-hydroxy-1,3-benzoxathiol-2-one;
7,8-dihydroxy-2-phenyl-4H-chromen-4-one;
7,8-dihydroxy-4-phenyl-2H-chromen-2-one;
7-[(2E)-2-(4-fluoro-3-phenoxybenzylidene)hydrazino]-N-(2-hydroxyphenyl)-7-
-oxoheptanamide;
7-[(2E)-2-(biphenyl-4-ylmethylene)hydrazino]-N-(2-hydroxyphenyl)-7-oxohep-
tanamide;
7-[2-chloro-5-(trifluoromethyl)phenyl]-5-hydroxy-1,3-benzoxathi-
ol-2-one;
7-{(2E)-2-[(2-fluorobiphenyl-4-yl)methylene]hydrazino}-N-(2-hyd-
roxyphenyl)-7-oxoheptanamide;
7-benzyl-8-hydroxy-10,10-dimethyl-6,10-dihydropyrido[1,2-a]indol-6-one;
7-chloro-4-piperidinoquinoline;
7-chloro-N-(3-fluoro-4-methylphenyl)quinolin-4-amine;
7-chloro-N-[2-(dimethylamino)ethyl]-4H-thieno[3,2-c]thiochromene-2-carbox-
amide; 7-chloro-N-phenylquinolin-4-amine;
7-hydroxy-2-methyl-6-propyl-3-pyridin-2-yl-4H-chromen-4-one;
7-hydroxy-5-methyl-3-(1-phenyl-1H-pyrazol-4-yl)-2-(trifluoromethyl)-4H-ch-
romen-4-one;
7-hydroxy-6-methyl-3-(4-methyl-1,3-thiazol-2-yl)-2-(trifluoromethyl)-4H-c-
hromen-4-one;
8-(trifluoromethoxy)-2-(trifluoromethyl)quinolin-4-ol;
8-[(2E)-2-(2-bromobenzylidene)hydrazino]-N-(2-hydroxyphenyl)-8-oxooctanam-
ide;
8-[(2E)-2-(5-bromo-2-methoxybenzylidene)hydrazino]-N-(2-hydroxypheny-
l)-8-oxooctanamide;
8-{(2E)-2-[(6-bromo-1,3-benzodioxol-5-yl)methylene]hydrazino}-N-(2-hydrox-
yphenyl)-8-oxooctanamide;
8-methoxy-N,N-dimethyl-5H-pyrimido[5,4-b]indol-4-amine;
allyl(3R,3'R,4'S,6'R,8'S,8a'S)-6'-{4-[2-({[(3S,6R,7S,8aS)-3-[4-(dimethyla-
mino)butyl]-6-(4-hydroxyphenyl)-1,4-dioxooctahydropyrrolo[1,2-a]pyrazin-7--
yl]carbonyl}oxy)ethoxy]phenyl}-5-iodo-1',2-dioxo-3',4'-diphenyl-1,2,3',4',-
8',8a'-hexahydro-1'H-spiro[indole-3,7'-pyrrolo[2,1-c][1,4]oxazine]-8'-carb-
oxylate" bis[4-(dimethylamino)phenyl]methanone oxime;
CN(C)c1ccc2N=c3cc(C)c(N)cc3=Sc2c1;
COC(.dbd.O)[C@]1(Cc2ccc(O)c(CC.dbd.C(C)C)c2)OC(.dbd.O)C(.dbd.C1c3ccc(O)cc-
3)O; COc1cc(/C.dbd.C/2\Oc3cc(O)ccc3C2=O)ccc1O;
COc1cc(ccc1O)c2oc3cc(O)cc(O)c3c(.dbd.O)c2O;
COc1cc(O)c-2c(CCc3cc(OC)c(OC)cc32)c1;
ethyl(2E)-3-(2-hydroxy-5-nitrophenyl)acrylate; ethyl
1-benzyl-4-[(dimethylamino)methyl]-5-hydroxy-2-phenyl-1H-indole-3-carboxy-
late; ethyl 2-ethoxy-5-hydroxy-1H-benzo[g]indole-3-carboxylate;
ethyl 4-(benzylamino)-6-ethoxyquinoline-3-carboxylate; ethyl
4-[({[(5R)-5-ethyl-4,6-dioxo-1,4,5,6-tetrahydropyrimidin-2-yl]thio}acetyl-
)amino]benzoate; ethyl
4-[(2-phenylethyl)amino]quinoline-3-carboxylate; ethyl
4-{[(2-anilino-2-oxoethyl)thio]methyl}-5-hydroxy-2-phenyl-1-benzofu-
ran-3-carboxylate; ethyl
4-{[(2E)-3-(2-thienyl)prop-2-enoyl]amino}benzoate; ethyl
6-bromo-4-[(dimethylamino)methyl]-5-hydroxy-1-methyl-2-{[(4-methylphenyl)-
thio]methyl}-1H-indole-3-carboxylate; ethyl
6-ethoxy-4-{[(1R)-1-methylpropyl]amino}quinoline-3-carboxylate;
ethyl
6-methyl-4-[(4-morpholin-4-ylphenyl)amino]quinoline-3-carboxylate;
isopropyl
(2S)-2-{[(2S)-2-{[(2S,3R)-2-{[(2S)-2-amino-3-mercaptopropyl]amino}-3-meth-
ylpentyl]oxy}-3-phenylpropanoyl]amino}-4-(methylsulfonyl)butanoate;
methyl(2Z)-2-(4-hydroxybenzylidene)-3-oxo-2,3-dihydro-1-benzofuran-5-carb-
oxylate; methyl
1-hydroxy-3-methylpyrido[1,2-a]benzimidazole-4-carboxylate; methyl
2,3-bis-O-(biphenyl-2-ylcarbamoyl)-4-O-[(3-ethylphenyl)carbamoyl]-alpha-L-
-idopyranoside; methyl
5-hydroxy-1-[4-(trifluoromethyl)phenyl]-1H-pyrazole-3-carboxylate;
N-(1,3-benzodioxol-5-yl)-7-chloroquinolin-4-amine;
N-(2,3-dihydro-1-benzofuran-5-ylcarbonyl)-2-hydroxybenzamide;
N-(2,5-dimethylphenyl)benzamide;
N-(2-chlorobenzyl)-2-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)acetamide;
N-(2-chlorobenzyl)-2-phenyl-1H-benzimidazole-5-sulfonamide;
N-(2-ethoxyphenyl)-2-hydroxybenzamide;
N-(2-hydroxy-4-methylphenyl)-4-[(methylthio)methyl]benzamide;
N-(2-hydroxybenzoyl)-2-thiophenecarboxamide;
N-(2-hydroxybenzoyl)-3-methoxybenzamide;
N-(2-hydroxybenzoyl)-4-(trifluoromethyl)benzamide;
N-(2-hydroxyphenyl)-8-[(2E)-2-(1-naphthylmethylene)hydrazino]-8-oxooctana-
mide; N-(3-bromo-4-hydroxy-1-naphthyl)-4-chlorobenzenesulfonamide;
N-(3-chloro-4-hydroxy-1-naphthyl)-4-ethoxybenzenesulfonamide;
N-(3-chlorophenyl)-4-(5-hydroxy-1-phenyl-1H-pyrazol-3-yl)piperidine-1-car-
bothioamide; N-(3-furylcarbonyl)-2-hydroxybenzamide;
N-(3-hydroxypyridin-2-yl)-4-phenoxybenzamide;
N-(3-imidazo[1,2-a]pyrimidin-2-ylphenyl)cyclopentanecarboxamide;
N-(4-bromophenyl)-2-[(3-cyano-4-methyl-6-oxo-1,6-dihydropyridin-2-yl)thio-
]acetamide;
N-(4-carbamoylphenyl)-1-phenyl-3-(2-thienyl)-1H-pyrazole-4-carboxamide;
N-(4-ethylbenzoyl)-2-hydroxybenzamide;
N-(4-fluorophenyl)-2-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)acetamide;
N-(5-{[(1S)-1-methylpropyl]thio}-1,3,4-thiadiazol-2-yl)-2-(trifluorometh-
yl)benzamide; N-(5-hydroxy-1-naphthyl)-4-methylbenzenesulfonamide;
N-(6-chloro-2-phenyl-4H-chromen-4-ylidene)-1-(2-furyl)methanamine;
N-(6-methyl-1,3-benzothiazol-2(3H)-ylidene)thiophene-2-carboxamide;
N-(cyclohexylcarbonyl)-2-hydroxybenzamide;
N,2-diphenylquinazolin-4-amine;
N,N,8-trimethyl-5H-pyrimido[5,4-b]indol-4-amine;
N,N'-1H-isoindole-1,3(2H)-diylidenedianiline;
N,N-diethyl-8-methyl-5H-pyrimido[5,4-b]indol-4-amine;
N,N-dimethyl-4-(6-methylimidazo[1,2-a]pyridin-2-yl)benzenesulfonamide;
N-[(1E)-(9-ethyl-9H-carbazol-3-yl)methylene]-4H-1,2,4-triazol-4-amine;
N-[(1E)-1H-indol-3-ylmethylene]-1-propyl-1H-benzimidazol-2-amine;
N-[(1S)-1-benzylpropyl]-6-[(4-methylpiperidin-1-yl)sulfonyl]-4-oxo-1,4-di-
hydroquinoline-3-carboxamide;
N-[(1S)-1-phenylethyl]quinazolin-4-amine;
N-[(3-chloro-1-benzothiophen-2-yl)carbonyl]-2-hydroxybenzamide;
N-[(4E)-2-(4-methoxyphenyl)-6-methyl-4H-chromen-4-ylidene]-2-phenylethana-
mine; N-[2-(1H-benzimidazol-2-yl)phenyl]-2-methylpropanamide;
N-[2-chloro-5-(trifluoromethyl)phenyl]-2-(4,4-dimethyl-2,6-dioxocyclohexy-
l)acetamide;
N-[2-hydroxy-3-(4-oxo-4H-chromen-2-yl)phenyl]acetamide;
N-[3-(1,3-benzothiazol-2-yl)-4-hydroxyphenyl]-2,2-dimethylpropanamide;
N-[3-(1,3-benzothiazol-2-ylthio)-4-hydroxyphenyl]benzenesulfonamide;
N-[3-(1,3-benzothiazol-2-ylthio)-4-hydroxyphenyl]thiophene-2-sulfonamide;
N-[4-(1H-benzimidazol-2-yl)phenyl]-2-(2-methoxyphenyl)acetamide;
N-[4-(ethylsulfonyl)-2-hydroxyphenyl]benzamide;
N-[5-(ethylsulfonyl)-2-hydroxyphenyl]-2-(4-methoxyphenoxy)acetamide;
N4-(3,5-dichlorophenyl)-6-methylpyrimidine-2,4-diamine;
N-benzo[g]quinolin-4-yl-N'-isopropylbenzene-1,4-diamine;
N-benzyl-2-hydroxybenzamide;
N-benzyl-6-{[(3-methoxyphenyl)amino]sulfonyl}-N-methyl-4-oxo-1,4-dihydroq-
uinoline-3-carboxamide;
N-ethyl-3-phenyl-N-(3-phenylpropyl)propan-1-amine;
O[C@H]1[C@@H](O)[C@@H](COC(.dbd.O)c2cc(O)c(O)c(O)c2)O[C@@H](Oc3ccc(C(.dbd-
.O)/C.dbd.C/c4ccccc4)c(O)c3)[C@@H]1O;
O[C@H]1[C@@H](O)[C@@H](COC(.dbd.O)c2cc(O)c(O)c(O)c2)O[C@@H](Oc3ccc(C(.dbd-
.O)CCc4ccc(O)cc4)c(O)c3)[C@@H]1O;
O[C@H]1[C@@H](Oc2c([C@H]3[C@@H](Oc4cc(O)cc(O)c4C3=O)c5ccc(O)cc5)c(O)cc(O)-
c2C1=O)c6ccc(O)cc6;
O[C@H]1[C@H]2[C@H](CC(.dbd.O)O)C(.dbd.O)O[C@@H]3C(COC(.dbd.O)c4cc(O)c(O)c-
(O)c4)
O[C@@H](OC(.dbd.O)c5cc(O)c(O)c(O)c5)C(OC(.dbd.O)c6cc(O)c(O)c(OC1=O)-
c26)[C@@H]3OC(.dbd.O)c7cc(O)c(O)c(O)c7;
OC[C@H]1O[C@@H](OC[C@H]2O[C@@H](Oc3c(oc4cc(O)cc(O)c4c3=O)c5ccc(O)cc5)[
C@H](O)[C@@H](O)[C@@H]2O)[C@H](O)[C@@H](O)[C@@H]1O; and
OC1[C@H](OC(.dbd.O)c2cc(O)c(O)c(O)c2)OC3COC(.dbd.O)c4cc(O)c(O)c(O)c4-c5c(-
O)c(O)c(O)cc5C(.dbd.O)O[C@@H]1[C@@H]3OC(.dbd.O)c6cc(O)c(O)c(O)c6c7c(O)c(O)-
c(O)cc7C(.dbd.O)OOc1ccc(cc1)[C@H]2CC(.dbd.O)c3c(O)cc(O)c([C@H]4[C@@H](Oc5c-
c(O)cc(O)c5C4=O)c6ccc(O)cc6)
c3O2Oc1ccc2C(.dbd.O)/C(.dbd.C/c3ccc(O)c(O)c3)/Oc2c1.
21. A method of inhibiting heme detoxification in a Plasmodium or
Theileria parasite, comprising the step of preventing or
attenuating the production of hemozoin by HDP in said Plasmodium or
Theileria parasite.
22. The method of claim 21 wherein said step of preventing or
attenuating is carried out by a process selected from the group
consisting of: 1) inhibiting interaction of heme and HDP; 2)
preventing an interaction of HDP or heme with cofactors; 3)
preventing dimerization of HDP; and 4) preventing interaction of
HDP or heme with lipids.
23. The method of claim 22, wherein said cofactors are selected
from the group consisting of metal ions, natural ligands or protein
factors.
24. The method of claim 22, wherein said step of preventing or
attenuating is carried out by administering to said individual one
or more compounds selected from the group consisting of:
(1S)-1-(3,4-dichlorophenyl)-2-(2-imino-1,3-benzothiazol-3(2H)-yl)ethanol;
(2E)-2-[(pyridin-3-ylamino)methylene]-1-benzothiophen-3(2H)-one;
(2E,5E)-5-(5-bromo-3-chloro-2-hydroxybenzylidene)-2-(phenylimino)-1,3-thi-
azolidin-4-one;
(2S)-N-(4-chlorophenyl)-2-methyl-2,3-dihydro-4H-1,4-benzoxazine-4-carboxa-
mide;
(3aR,4R,9bR)-8-chloro-4-(4-chlorophenyl)-9-nitro-3a,4,5,9b-tetrahyd-
ro-3H-cyclopenta[c]quinolin-6-ol;
(3R)-5,7-dichloro-3-hydroxy-3-(2-methylimidazo[1,2-a]pyridin-3-yl)-1,3-di-
hydro-2H-indol-2-one;
[6-hydroxy-2-(4-hydroxyphenyl)-1-benzothien-3-yl][4-(2-piperidin-1-yletho-
xy)phenyl]methanone;
1,2,3,4,6-pentakis-O-(3,4,5-trihydroxybenzoyl)-beta-D-glucopyranose;
1-[(2S)-3-(9H-carbazol-9-yl)-2-hydroxypropyl]-8-hydroxyquinolinium;
1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-1,3-dihydro-2-
H-benzimidazol-2-one;
1-ethyl-6-methoxy-4-methyl-2-[(Z)-(3-methyl-1,3-thiazol-2(3H)-ylidene)met-
hyl]benzo[h]quinolinium; 1H-perimidine-2-carboxylic acid;
2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-4H-chromen-4-one;
2-(4-methoxyphenyl)-4H-1,3-benzoxazin-4-one;
2-(4-methoxyphenyl)pyridin-3-ol;
2-(morpholin-4-ylmethyl)-1-naphthol;
2,2'-buta-1,3-diyne-1,4-diyldiphenol;
2-[(E)-(6-methoxy-1-methylquinolin-2(1H)-ylidene)methyl]-3-methyl-1,3-ben-
zothiazol-3-ium;
2-[(Z)-(3-ethyl-6-methoxy-1,3-benzothiazol-2(3H)-ylidene)methyl]-1,6-dime-
thylquinolinium; 2-[2-(4-hydroxyphenyl)ethyl]-6-methylpyridin-3-ol;
2-[4-(1-benzofuran-2-yl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-5-methoxyph-
enol;
2-{(3R)-1-[4-(2-hydroxyethoxy)benzyl]piperidin-3-yl}-1H-isoindole-1-
,3(2H)-dione;
2-amino-8-(azepan-1-ylmethyl)-3-(1,3-benzothiazol-2-yl)-7-hydroxy-4H-chro-
men-4-one; 2-hydrazino-4-methylquinoline;
2-hydroxy-N-(4-propylbenzoyl)benzamide;
3-(2-hydroxy-5-methoxybenzoyl)-2-(4-methylphenyl)isoindolin-1-one;
3-(3-{[(2-chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile;
3-[(4-{[(2R)-tetrahydrofuran-2-ylmethyl]amino}quinazolin-2-yl)amino]pheno-
l;
3-[4-(9H-fluoren-9-yl)piperazin-1-yl]-N-[3-(trifluoromethyl)phenyl]pro-
panamide;
4-({(2S)-3-[4-(diphenylmethyl)piperazin-1-yl]-2-hydroxypropyl}o-
xy)-1H-indole-2-carbonitrile;
4-(1H-benzimidazol-2-yl)-N,N-dimethylaniline;
4-(6-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;
4-(7-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;
4,4'-(4-phenyl-1H-imidazole-2,5-diyl)diphenol;
4,4'-methylenebis(3-hydroxy-2-naphthoic
acid)-3,3'-[(4-iminocyclohexa-2,5-dien-1-ylidene)methylene]dianiline
(1:1); 4,4'-propane-2,2-diylbis(2-chlorophenol);
4-[(5S)-5-(4-fluorophenyl)-1-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl]-
phenol;
4-[4-(3,4-dihydro-2H-1,5-benzodioxepin-7-yl)-3-methylisoxazol-5-y-
l]benzene-1,3-diol;
4-[5-[5-(4-methylpiperazin-1-yl)-3H-benzoimidazol-2-yl]-1,3-dihydrobenzoi-
midazol-2-ylidene]cyclohexa-2,5-dien-1-one;
4-{[(2-ethylphenyl)amino]methyl}-5-(hydroxymethyl)-2-methylpyridin-3-ol;
4-phenylquinolin-2-amine;
5-(2-hydroxy-5-methylbenzoyl)-1-(4-methylphenyl)-2-oxo-1,2-dihydropyridin-
e-3-carbonitrile;
5-(5-chloro-2-hydroxybenzoyl)-2-oxo-N,1-diphenyl-1,2-dihydropyridine-3-ca-
rboxamide;
5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-4H-chromen-4-one;
6-amino-1-ethylbenzo[cd]indol-2(1H)-one;
6-chloro-3-[2-(4-chlorophenyl)ethyl]-3,4-dihydro-2H-1,3-benzoxazine;
7-[(2E)-2-(biphenyl-4-ylmethylene)hydrazino]-N-(2-hydroxyphenyl)-7-oxohep-
tanamide;
7-chloro-N-[2-(dimethylamino)ethyl]-4H-thieno[3,2-c]thiochromen-
e-2-carboxamide; 7-chloro-N-phenylquinolin-4-amine;
7-hydroxy-2-methyl-6-propyl-3-pyridin-2-yl-4H-chromen-4-one;
8-[(2E)-2-(2-bromobenzylidene)hydrazino]-N-(2-hydroxyphenyl)-8-oxooctanam-
ide; CN(C)c1ccc2N=c3cc(C)c(N)cc3=Sc2c1;
COc1cc(O)c-2c(CCc3cc(OC)c(OC)cc32)c1; ethyl
1-benzyl-4-[(dimethylamino)methyl]-5-hydroxy-2-phenyl-1H-indole-3-carboxy-
late; ethyl 2-ethoxy-5-hydroxy-1H-benzo[g]indole-3-carboxylate;
N-(2-ethoxyphenyl)-2-hydroxybenzamide;
N-(2-hydroxybenzoyl)-2-thiophenecarboxamide;
N-(2-hydroxybenzoyl)-3-methoxybenzamide;
N-(3-chloro-4-hydroxy-1-naphthyl)-4-ethoxybenzenesulfonamide;
N-(3-furylcarbonyl)-2-hydroxybenzamide;
N-(4-ethylbenzoyl)-2-hydroxybenzamide;
N-(6-chloro-2-phenyl-4H-chromen-4-ylidene)-1-(2-furyl)methanamine;
N-[(1S)-1-phenylethyl]quinazolin-4-amine;
N-[(4E)-2-(4-methoxyphenyl)-6-methyl-4H-chromen-4-ylidene]-2-phenylethana-
mine; N4-(3,5-dichlorophenyl)-6-methylpyrimidine-2,4-diamine;
N-benzo[g]quinolin-4-yl-N'-isopropylbenzene-1,4-diamine;
O[C@H]1[C@@H](O)[C@@H](COC(.dbd.O)c2cc(O)c(O)c(O)c2)O[C@@H](Oc3ccc(C(.dbd-
.O)CCc4ccc(O)cc4)c(O)c3)[C@@H]1O; and
O[C@H]1[C@H]2[C@H](CC(.dbd.O)O)C(.dbd.O)O[C@@H]3C(COC(.dbd.O)c4cc(O)c(O)c-
(O)c4)
O[C@@H](OC(.dbd.O)c5cc(O)c(O)c(O)c5)C(OC(.dbd.O)c6cc(O)c(O)c(OC1=O)-
c26)[C@@H]3OC(.dbd.O)c7cc(O)c(O)c(O)c7.
25. The method of claim 21 wherein said step of preventing or
attenuating is carried out by administering to said individual one
or more compounds selected from the group consisting of:
(10S)-10-(dimethylamino)-9-methyl-7H,10H-naphtho[1,8-gh]chromen-7-one;
(1E,4E)-1-[4-(dimethylamino)phenyl]-5-(3,4,5-trimethoxyphenyl)penta-1,4-d-
ien-3-one;
(1R,3R)-1-isopropyl-2,3,4,9-tetrahydro-1H-beta-carboline-3-carboxylic
acid;
(1S)-1-(3,4-dichlorophenyl)-2-(2-imino-1,3-benzothiazol-3(2H)-yl)e-
thanol;
(2E)-2-[(pyridin-3-ylamino)methylene]-1-benzothiophen-3(2H)-one;
(2E)-3-(3,4-dimethoxyphenyl)-N-(3,4-dimethylphenyl)acrylamide;
(2E)-6-ethoxy-2-(2-hydroxybenzylidene)-1-benzothiophen-3(2H)-one;
(2E)-N-(2-methyl-1,3-benzothiazol-6-yl)-3-phenylacrylamide;
(2E,5E)-5-(5-bromo-3-chloro-2-hydroxybenzylidene)-2-(phenylimino)-1,3-thi-
azolidin-4-one;
(2E,5Z)-2-[(2-chlorophenyl)imino]-5-(4-hydroxy-3-nitrobenzylidene)-1,3-th-
iazolidin-4-one;
(2R)-1-(benzylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol;
(2R)-2-(2,4-dichlorophenoxy)-N-(5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-3-y-
l)propanamide;
(2R)-2-[(5Z)-5-(4-hydroxy-3,5-dimethoxybenzylidene)-4-oxo-2-thioxo-1,3-th-
iazolidin-3-yl]-3-methylbutanoic acid;
(2R)-2-[(E)-2-(1,3-benzodioxol-5-yl)vinyl]-5,6-dimethyl-2,3-dihydrothieno-
[2,3-d]pyrimidin-4(1H)-one;
(2R,3Z)-6-chloro-3-[(dimethylamino)methylene]-2-methyl-2,3-dihydro-4H-thi-
ochromen-4-one;
(2S)-2-(4-chlorophenyl)-3-oxo-4-phenylbutanenitrile;
(2S)-2-[(5E)-5-(1H-indol-3-ylmethylene)-4-oxo-2-thioxo-1,3-thiazolidin-3--
yl]succinic acid;
(2S)-N-(4-chlorophenyl)-2-methyl-2,3-dihydro-4H-1,4-benzoxazine-4-carboxa-
mide;
(2S,3Z)-3-(1H-indol-3-ylmethylene)-2-phenyl-2,3-dihydro-4H-chromen--
4-one; (2Z)-2-acetamido-N-(3,5-dimethylphenyl)-3-phenylacrylamide;
(2Z,5E)-2-[(3,5-dimethylphenyl)imino]-5-(2-hydroxy-3-methoxybenzylidene)--
1,3-thiazolidin-4-one;
(2Z,5Z)-2-[(2-chlorophenyl)imino]-5-(2-hydroxy-3-nitrobenzylidene)-1,3-th-
iazolidin-4-one;
(3,5-dichloro-2-hydroxyphenyl)(isoxazol-4-yl)methanone;
(3-{(E)-[1-(3-fluorophenyl)-2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene-
]methyl}-1H-indol-1-yl)acetonitrile;
(3aR,4R,9bR)-8-chloro-4-(4-chlorophenyl)-9-nitro-3a,4,5,9b-tetrahydro-3H--
cyclopenta[c]quinolin-6-ol;
(3aS,6aS)-3-benzoyl-1,5-diphenyl-3a,6a-dihydropyrrolo[3,4-c]pyrazole-4,6(-
1H,5H) -dione;
(3R)-3-(2-hydroxy-4-methylphenyl)-N-(2-methoxyphenyl)-3-phenylpropanamide-
;
(3R)-5,7-dichloro-3-hydroxy-3-(2-methylimidazo[1,2-a]pyridin-3-yl)-1,3--
dihydro-2H-indol-2-one;
(3R,3'R,4'S,6'R,8'S,8a'S)-5-(4-hydroxybut-1-yn-1-yl)-6'-[4-(2-hydroxyetho-
xy)phenyl]-1',2-dioxo-3',4'-diphenyl-1,2,3',4',8',8a'-hexahydro-1'H-spiro[-
indole-3,7'-pyrrolo[2,1-c][1,4]oxazine]-8'-carboxylic acid;
(3S)-3-(2-hydroxy-4-methylbenzoyl)-2-(4-methylphenyl)isoindolin-1-one;
(3S,3aR,6aR)-3-(5-bromo-2-hydroxyphenyl)-5-butyl-2-phenyldihydro-2H-pyrro-
lo[3,4-d]isoxazole-4,6(3H,5H)-dione;
(3S,6S,7R,8aR)-3-(4-acetamidobutyl)-6-(4-hydroxyphenyl)-1,4-dioxooctahydr-
opyrrolo[1,2-a]pyrazine-7-carboxylic acid;
(3Z)-3-(3-hydroxy-4-methoxybenzylidene)-1-methyl-1,3-dihydro-2H-indol-2-o-
ne;
(4E)-2-(4-methoxyphenyl)-4-[(4-methoxyphenyl)imino]-4H-chromen-6-ol;
(4R)-3-(3,4-dichlorophenyl)-4-hydroxy-N-isopropyl-2-oxo-1,2,3,4-tetrahydr-
oquinazoline-4-carboxamide;
(4R)-4-(4-bromophenyl)-3-hydroxy-1-isopropyl-4,8-dihydro-1H-pyrazolo[3,4--
e][1,4]thiazepin-7(6H)-one;
(4R)-4-(4-ethylphenyl)-3-hydroxy-2-phenyl-2,4,6,7,8,9-hexahydro-5H-pyrazo-
lo[3,4-b]quinolin-5-one;
(4R)-5-(2-furylmethyl)-3-(2-hydroxyphenyl)-4-phenyl-4,5-dihydropyrrolo[3,-
4-c]pyrazol-6(1H) -one;
(4R)-N.about.4.about.-(6-chloro-2-methoxyacridin-9-yl)-N.about.1.about.,N-
.about.1.about.-diethylpentane-1,4-diamine;
(4S)-4-(2-bromobenzoyl)-5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one;
(4S)-4-(2-furyl)-3-hydroxy-7,7-dimethyl-2-phenyl-2,4,6,7,8,9-hexahydro-5H-
-pyrazolo[3,4-b]quinolin-5-one;
(4S)-5,7-dihydroxy-4-phenylchroman-2-one;
(4S,5S)-3,5-diphenyl-4,5-dihydro-1H-pyrazol-4-ol;
(4S,7R)-2-amino-4-isobutyl-5-oxo-7-phenyl-5,6,7,8-tetrahydro-4H-chromene--
3-carbonitrile;
(4Z)-2-[2-(4-chlorophenoxy)pyridin-3-yl]-4-[(dimethylamino)methylene]-1,3-
-oxazol-5(4H)-one;
(5E)-1-(4-methylpentyl)-5-(1H-pyrrol-2-ylmethylene)pyrimidine-2,4,6(1H,3H-
,5H)-trione;
(5E)-3-allyl-5-(2-hydroxybenzylidene)-2-thioxo-1,3-thiazolidin-4-one;
(5E)-5-[4-(diethylam-ino)benzylidene]-3-{[(2-methoxyphenyl)amino]methyl}1-
,3-thiazolidine-2,4-dione;
(5R)-5-methyl-4-phenyl-1,3,4-thiadiazolidine-2-thione;
(5S,7R)-2,2-dimethyl-5,7-bis(2-phenylethyl)-7,8-dihydro-4H,5H-pyrano[4,3--
d][1,3]dioxine;
(5Z)-5-(4-hydroxybenzylidene)-3-[(2R)-tetrahydrofuran-2-ylmethyl]-2-thiox-
o-1,3-thiazolidin-4-one;
(6E)-5-imino-6-{[1-(2-naphthyl)-1H-pyrrol-2-yl]methylene}-5,6-dihydro-7H--
[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-one;
*cccccc1C2C(.dbd.O)N(C)c3ccccc3C2=O;
[(2R)-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]acetic acid;
[(5E)-4-oxo-5-(3-thienylmethylene)-2-thioxo-1,3-thiazolidin-3-yl]acetic
acid;
[(5E)-5-(5-bromo-3-chloro-2-hydroxybenzylidene)-4-oxo-2-thioxo-1,3-
-thiazolidin-3-yl]acetic acid;
[6-hydroxy-2-(4-hydroxyphenyl)-1-benzothien-3-yl][4-(2-piperidin-1-yletho-
xy)phenyl]methanone; {4-[(4-methylphenyl)sulfonyl]phenyl}hydrazine;
1-(2,4-dihydroxy-6-methylphenyl)-2-phenoxyethanone;
1-(2,4-dihydroxyphenyl)-2-(4-isopropylphenoxy)ethanone;
1-(3,4-dihydroxyphenyl)-2-({4-[(3,5-dimethoxyphenyl)amino]quinazolin-2-yl-
}thio)ethanone; 1-(4-chlorophenyl)-1-hydroxy-3-phenylurea;
1-(4-hydroxy-3,5-dimethylphenyl)-2-[(4-methylphenyl)thio]ethanone;
1-(4-iodo-2-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-ol;
1-(5-butyl-2,4-dihydroxyphenyl)-2-pyridin-2-ylethanone;
1-(5-ethyl-2,4-dihydroxyphenyl)-2-(1-methyl-1H-benzimidazol-2-yl)ethanone-
;
1,2,3,4,6-pentakis-O-(3,4,5-trihydroxybenzoyl)-beta-D-glucopyranose;
1-[(2S)-3-(9H-carbazol-9-yl)-2-hydroxypropyl]-8-hydroxyquinolinium;
1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-1,3-dihydro-2-
H-benzimidazol-2-one;
1-[4-(7-chloroquinolin-4-yl)piperazino]propan-1-one;
1-ethyl-6-methoxy-4-methyl-2-[(Z)-(3-methyl-1,3-thiazol-2(3H)-ylidene)met-
hyl]benzo[h]quinolinium; 1-ethynyl-2-phenoxybenzene;
1H-perimidine-2-carboxylic acid;
2-(1,3-benzodioxol-5-yl)-1-(2,4-dihydroxy-5-propylphenyl)ethanone;
2-(1H-benzimidazol-1-yl)-1-(5-ethyl-2,4-dihydroxyphenyl)ethanone;
2-(2,4-dichlorophenyl)-1H-imidazo[4,5-b]pyridin-1-ol;
2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one;
2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-7-methoxy-4H-chromen-4-one;
2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-4H-chromen-4-one;
2-(4-chlorophenyl)-4H-1,3-benzoxazin-4-one;
2-(4-chlorophenyl)-5-{[(4-pyridin-3-ylpyrimidin-2-yl)thio]methyl}-2,4-dih-
ydro-3H-pyrazol-3-one;
2-(4-fluoro-3-phenoxyphenyl)-3-hydroxy-4H-chromen-4-one;
2-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)-N-(4-methylphenyl)acetamide;
2-(4-methoxyphenyl)-4H-1,3-benzoxazin-4-one;
2-(4-methoxyphenyl)pyridin-3-ol;
2-(4-methylphenyl)-4H-1,3-benzoxazin-4-one;
2-(morpholin-4-ylmethyl)-1-naphthol;
2,2'-buta-1,3-diyne-1,4-diyldiphenol; 2,2'-thiobis(4-chlorophenol);
2,4-dichloro-1-naphthyl[2,2,2-trifluoro-1-methyl-1-(trifluoromethyl)ethyl-
]carbamate; 2-[(2-phenoxyethyl)thio]quinazoline-4-thiol;
2-[(2R)-6,7-dimethyl-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]-N-ethylacet-
amide;
2-[(3-cyano-4-methyl-6-oxo-1,6-dihydropyridin-2-yl)thio]-N-1-napht-
hylacetamide;
2-[(5S)-1-(4-nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl]phenol;
2-[(E)-(6-methoxy-1-methylquinolin-2(1H)-ylidene)methyl]-3-methyl-1,3-ben-
zothiazol-3-ium;
2-[(Z)-(3-ethyl-6-methoxy-1,3-benzothiazol-2(3H)-ylidene)methyl]-1,6-dime-
thylquinolinium; 2-[2-(4-hydroxyphenyl)ethyl]-6-methylpyridin-3-ol;
2-[4-(1-benzofuran-2-yl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-5-methoxyph-
enol; 2-[5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl]phenol;
2-[5-(ethylsulfonyl)-2-hydroxyphenyl]-1H-benzo[de]isoquinoline-1,3(2H)-di-
one;
2-{(1R)-1-[(1-allyl-1H-benzimidazol-2-yl)amino]ethyl}-4-chlorophenol-
;
2-{(3R)-1-[4-(2-hydroxyethoxy)benzyl]piperidin-3-yl}-1H-isoindole-1,3(2-
H)-dione;
2-{[5-chloro-6-methyl-2-(2-pyridinyl)-4-pyrimidinyl]sulfanyl}-1-
-phenyl-1-ethanone;
2-amino-1-(2,4-dimethylphenyl)-1H-pyrrolo[2,3-b]quinoxaline-3-carbonitril-
e;
2-amino-5-butyl-4-(4-hydroxy-3-methoxyphenyl)-6-phenylnicotinonitrile;
2-amino-8-(azepan-1-ylmethyl)-3-(1,3-benzothiazol-2-yl)-7-hydroxy-4H-chr-
omen-4-one; 2-anilino-2-oxoethyl2-(4-chlorobenzoyl)benzoate;
2-chloro-5-phenyl-3-pyridin-4-yl-4H-1,4-thiazine;
2-chloro-8-hydroxy-10,10-dimethyl-7-phenylpyrido[1,2-a]indol-6(10H)-one;
2-hydrazino-4,6-diphenylpyrimidine; 2-hydrazino-4-methylquinoline;
2-hydroxy-N-(4-methylphenyl)benzamide;
2-hydroxy-N-(4-propylbenzoyl)benzamide;
2-hydroxy-N-[(4-methyl-2-phenyl-1,3-thiazol-5-yl)carbonyl]benzamide;
2-hydroxy-N-[2-(2-methoxyphenyl)acetyl]benzamide;
2-hydroxy-N-{[2-(4-methylphenoxy)-3-pyridinyl]carbonyl}benzamide;
2-hydroxy-N-pyridin-3-ylbenzamide;
2-phenyl-4H-thiochromene-4-thione;
2-phenyl-5-({[5-(trifluoromethyl)pyridin-2-yl]sulfonyl}methyl)-2,4-dihydr-
o-3H-pyrazol-3-one;
2-phenyl-5-(trifluoromethyl)-2,4-dihydro-3H-pyrazol-3-one;
3-(1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)-N-(2-hydroxyphenyl)-N-(-
4-methoxybenzyl)propanamide;
3-(1-acetyl-1H-indol-3-yl)-4-hydroxy-2H-chromen-2-one;
3-(1H-benzimidazol-1-yl)-6-ethyl-7-hydroxy-4H-chromen-4-one;
3-(2-hydroxy-5-methoxybenzoyl)-2-(4-methylphenyl)isoindolin-1-one;
3-(3-{[(2-chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile;
3-(3-{[(4-chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile;
3-(4-bromophenyl)-7-hydroxy-2-methyl-4H-chromen-4-one;
3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-one;
3-(5-{(Z)-[5-(4-methylphenyl)-2-oxofuran-3(2H)-ylidene]methyl}-2-furyl)be-
nzoic acid; 3-(quinazolin-4-ylamino)phenyl thiophene-2-carboxylate;
3,4-dimethoxy-N-(4-methyl-1,3-benzothiazol-2-yl)benzamide;
3,5-dichloro-2-hydroxybenzaldehyde
N-tert-butyl-N'-methylthiosemicarbazone;
3-[(4-{[(2R)-tetrahydrofuran-2-ylmethyl]amino}quinazolin-2-yl)amino]pheno-
l;
3-[2-(4-methoxyphenyl)ethyl]-10-methyl-6-phenyl-3,4-dihydro-2H,8H-chro-
meno[6,7-e][1,3]oxazin-8-one;
3-[4-(9H-fluoren-9-yl)piperazin-1-yl]-N-[3-(trifluoromethyl)phenyl]propan-
amide;
3-{2-[(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-1H-pyrrol--
1-yl}benzoic acid; 3-benzyl-4-hydroxy-1,2-dihydroquinolin-2-one;
3-benzyl-4-hydroxy-1-phenylquinolin-2(1H)-one;
3-benzyl-5,6-bis(4-methoxyphenyl)furo[2,3-d]pyrimidin-4(3H)-imine;
3-benzyl-5-ethyl-4-hydroxy-6-phenyl-1-(1,3-thiazol-2-yl)pyridin-2(1H)-one-
; 3-benzyl-6-ethoxy-4-hydroxyquinolin-2(1H)-one;
3-chloro-N-[2-(methylthio)-1,3-benzothiazol-6-yl]benzamide;
3-hydroxy-N-(2-methylphenyl)-2-naphthamide;
3-methoxy-2-methyl-6-[1'-phenyl-5-(trifluoromethyl)-1H,1'H-4,4'-bipyrazol-
-3-yl]phenol;
3-methoxy-N-(3-[1,3]oxazolo[4,5-b]pyridin-2-ylphenyl)benzamide;
3-methyl-1-[3-(trifluoromethyl)phenyl]-1H-pyrazol-5-ol;
3-methyl-1-phenyl-4-(trifluoromethyl)-1,7-dihydro-6H-pyrazolo[3,4-b]pyrid-
in-6-one;
3-methyl-4-[(4-methylphenyl)thio]-1-phenyl-1H-pyrazol-5-yl
methoxyacetate;
3-oxo-3-[3-({[3-(trifluoromethyl)phenyl]thio}methyl)phenyl]propanenitrile-
;
4-({(2S)-3-[4-(diphenylmethyl)piperazin-1-yl]-2-hydroxypropyl}oxy)-1H-i-
ndole-2-carbonitrile; 4-(1H-benzimidazol-2-yl)-N,N-dimethylaniline;
4-(1-propyl-1H-benzimidazol-2-yl)aniline;
4-(2,6-dichlorobenzyl)-3-methyl-1-phenyl-1H-pyrazol-5-ol;
4-(6-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;
4-(7-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;
4,4'-(4-phenyl-1H-imidazole-2,5-diyl)diphenol;
4,4'-[(2,3,5,6-tetrafluoro-1,4-phenylene)bis(oxy)]diphenol;
4,4'-methylenebis(3-hydroxy-2-naphthoic
acid)-3,3'-[(4-iminocyclohexa-2,5-dien-1-ylidene)methylene]dianiline
(1:1); 4,4'-propane-2,2-diylbis(2-chlorophenol);
4-[(3S,6S,7R,8aR)-7-{[2-(4-{(3S,3'S,4'R,6'R,8'R,8a'R)-8'-[(allyloxy)carbo-
nyl]-5-iodo-1',2-dioxo-3',4'-diphenyl-1,2,3',4',8',8a'-hexahydro-1'H-spiro-
[indole-3,7'-pyrrolo[2,
1-c][1,4]oxazin]-6'-yl}phenoxy)ethoxy]carbonyl}-6-(4-hydroxyphenyl)-1,4-d-
ioxooctahydropyrrolo[1,2-a]pyrazin-3-yl]-N,N,N-trimethylbutan-1-aminium;
4-[(4-chlorophenyl)thio]-3-methyl-1-phenyl-1H-pyrazol-5-ol;
4-[(5S)-5-(4-fluorophenyl)-1-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl]-
phenol;
4-[1-(4-hydroxy-3-methoxybenzyl)-1H-benzimidazol-2-yl]-2-methoxyp-
henol;
4-[1'-phenyl-5-(trifluoromethyl)-1H,1'H-4,4'-bipyrazol-3-yl]benzen-
e-1,3-diol;
4-[4-(1,3-benzothiazol-2-yl)-5-methyl-1H-pyrazol-3-yl]benzene-1,3-diol;
4-[4-(3,4-dihydro-2H-1,5-benzodioxepin-7-yl)-3-methylisoxazol-5-yl]benzen-
e-1,3-diol;
4-[4-(4-chlorophenyl)-1,3-thiazol-2-yl]-1-methyl-1H-pyrazol-5-amine;
4-[5-[5-(4-methylpiperazin-1-yl)-3H-benzoimidazol-2-yl]-1,3-dihydrobenzoi-
midazol-2-ylidene]cyclohexa-2,5-dien-1-one;
4-{[(1E)-3-(2-furyl)-3-oxoprop-1-en-1-yl]amino}benzoic acid;
4-{[(2-ethylphenyl)amino]methyl}-5-(hydroxymethyl)-2-methylpyridin-3-ol;
4-{[(2S)-2-ethylpiperidin-1-yl]methyl}-3-hydroxy-1-methyl-6H-benzo[c]chro-
men-6-one;
4-bromo-2-[(E)-(4H-1,2,4-triazol-4-ylimino)methyl]phenol;
4-bromo-2-[5-(2-furyl)-1H-pyrazol-3-yl]phenol;
4-bromo-6-chloro-2-oxo-1,3-benzoxathiol-5-yl ethyl carbonate;
4-ethyl-6-[4-(1-methyl-1H-benzimidazol-2-yl)-1H-pyrazol-3-yl]benzene-1,3--
diol; 4-fluoro-N-[3-(trifluoromethyl)phenyl]benzamide;
4-hydroxy-3-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]-2H-chromen-
-2-one; 4-hydroxy-3-propylquinolin-2(1H)-one;
4-hydroxy-5-phenyl-6H-pyrido[3,2,1-jk]carbazol-6-one;
4-hydroxy-8-methyl-3-{(E)-[(3R)-5-oxo-1,3-diphenylpyrazolidin-4-ylidene]m-
ethyl}quinolin -2(1H)-one; 4-phenylquinolin-2-amine;
5-(2-hydroxy-5-methylbenzoyl)-1-(4-methylphenyl)-2-oxo-1,2-dihydropyridin-
e-3-carbonitrile;
5-(5-bromo-2-hydroxybenzoyl)-1-(2-fluorophenyl)-2-oxo-1,2-dihydropyridine-
-3-carbonitrile;
5-(5-chloro-2-hydroxybenzoyl)-2-oxo-N,1-diphenyl-1,2-dihydropyridine-3-ca-
rboxamide;
5-(benzoylamino)-N,N'-bis(2-hydroxyphenyl)isophthalamide;
5-(diethylamino)-2-{(E)-[(2-phenylethyl)imino]methyl}phenol;
5,7-dihydroxy-4-propyl-2H-chromen-2-one;
5-[(4-methylphenyl)thio]quinazoline-2,4-diamine;
5-{2-[(3,4-dichlorophenyl)thio]ethyl}-2-methylpyridine;
5-benzyl-3-phenyl-5H-pyrazolo[4,3-c]quinolines;
5-benzyl-4-hydroxy-6H-pyrido[3,2,1-jk]carbazol-6-one;
5-chloro-2-hydroxy-N-phenylbenzamide;
5-hydroxy-4-methyl-7-propyl-2H-chromen-2-one;
5-methoxy-2-[3-methyl-4-(1,3-thiazol-4-yl)isoxazol-5-yl]phenol;
5-methyl-2-[5-(2-thienyl)-1H-pyrazol-3-yl]phenol;
6-(4-chlorophenyl)-7-hydroxy-1,3-dimethyl-1H-pyrrolo[3,2-d]pyrimidine-2,4-
(3H,5H)-dione''6,6'-biquinoline;
6-[(S)-[4-(dimethylamino)phenyl](piperidin-1-yl)methyl]-1,3-benzodioxol-5-
-ol;
6-{[(2-ethylphenyl)amino]sulfonyl}-4-oxo-N-[(2S)-tetrahydrofuran-2-y-
lmethyl]-1,4-dihydroquinoline-3-carboxamide;
6-{[(4-chlorophenyl)thio]methyl}-2-phenyl-1H-pyrazolo[3,4-b]pyridine-3,4(-
2H,7H)-dione;
6-{[(4-ethoxyphenyl)(methyl)amino]sulfonyl}-4-oxo-N-[(2S)-tetrahydrofuran-
-2-ylmethyl]-1,4-dihydroquinoline-3-carboxamide;
6-allyl-7-hydroxy-4,8-dimethyl-2H-chromen-2-one;
6-amino-1-ethylbenzo[cd]indol-2(1H)-one;
6-benzyl-7-hydroxy-2,3-dihydro-1H,5H-pyrido[3,2,1-ij]quinolin-5-one;
6-bromo-2-(trifluoromethyl)quinolin-4-ol;
6-butyl-2-(2-furyl)-5-methyl-4,7-dihydropyrazolo[1,5-a]pyrimidin-7-one;
6-chloro-2-(4-chlorophenyl)-1H-benzimidazol-1-ol;
6-chloro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide;
6-chloro-3-(4-methylphenyl)-3,4-dihydro-2H-1,3-benzoxazine;
6-chloro-3-[2-(4-chlorophenyl)ethyl]-3,4-dihydro-2H-1,3-benzoxazine;
6-ethyl-7-hydroxy-3-(1-methyl-1H-benzimidazol-2-yl)-2-(trifluoromethyl)-4-
H-chromen-4-one; 6-fluoro-4-hydroxy-3-phenylquinolin-2(1H)-one;
6-methoxy-N-[(1S)-1-methylpropyl]furo[2,3-b]quinoline-2-carboxamide;
6-phenyl[1,2,3,4]tetraazolo[1,5-b]pyridazin-7-ol;
7-(4-bromophenyl)-5-hydroxy-1,3-benzoxathiol-2-one;
7,8-dihydroxy-2-phenyl-4H-chromen-4-one;
7,8-dihydroxy-4-phenyl-2H-chromen-2-one;
7-[(2E)-2-(4-fluoro-3-phenoxybenzylidene)hydrazino]-N-(2-hydroxyphenyl)-7-
-oxoheptanamide;
7-[(2E)-2-(biphenyl-4-ylmethylene)hydrazino]-N-(2-hydroxyphenyl)-7-oxohep-
tanamide;
7-[2-chloro-5-(trifluoromethyl)phenyl]-5-hydroxy-1,3-benzoxathi-
ol-2-one;
7-{(2E)-2-[(2-fluorobiphenyl-4-yl)methylene]hydrazino}-N-(2-hyd-
roxyphenyl)-7-oxoheptanamide;
7-benzyl-8-hydroxy-10,10-dimethyl-6,10-dihydropyrido[1,2-a]indol-6-one;
7-chloro-4-piperidinoquinoline;
7-chloro-N-(3-fluoro-4-methylphenyl)quinolin-4-amine;
7-chloro-N-[2-(dimethylamino)ethyl]-4H-thieno[3,2-c]thiochromene-2-carbox-
amide; 7-chloro-N-phenylquinolin-4-amine;
7-hydroxy-2-methyl-6-propyl-3-pyridin-2-yl-4H-chromen-4-one;
7-hydroxy-5-methyl-3-(1-phenyl-1H-pyrazol-4-yl)-2-(trifluoromethyl)-4H-ch-
romen-4-one;
7-hydroxy-6-methyl-3-(4-methyl-1,3-thiazol-2-yl)-2-(trifluoromethyl)-4H-c-
hromen-4-one;
8-(trifluoromethoxy)-2-(trifluoromethyl)quinolin-4-ol;
8-[(2E)-2-(2-bromobenzylidene)hydrazino]-N-(2-hydroxyphenyl)-8-oxooctanam-
ide;
8-[(2E)-2-(5-bromo-2-methoxybenzylidene)hydrazino]-N-(2-hydroxypheny-
l)-8-oxooctanamide;
8-{(2E)-2-[(6-bromo-1,3-benzodioxol-5-yl)methylene]hydrazino}-N-(2-hydrox-
yphenyl)-8-oxooctanamide;
8-methoxy-N,N-dimethyl-5H-pyrimido[5,4-b]indol-4-amine;
allyl(3R,3'R,4'S,6'R,8'S,8a'S)-6'-{4-[2-({[(3S,6R,7S,8aS)-3-[4-(dimethyla-
mino)butyl]-6-(4-hydroxyphenyl)-1,4-dioxooctahydropyrrolo[1,2-a]pyrazin-7--
yl]carbonyl}oxy)ethoxy]phenyl}-5-iodo-1',2-dioxo-3',4'-diphenyl-1,2,3',4',-
8',8a'-hexahydro-1'H-spiro[indole-3,7'-pyrrolo[2,1-c][1,4]oxazine]-8'-carb-
oxylate''bis[4-(dimethylamino)phenyl]methanone oxime;
CN(C)c1ccc2N=c3cc(C)c(N)cc3=Sc2c1;
COC(.dbd.O)[C@]1(Cc2ccc(O)c(CC.dbd.C(C)C)c2)OC(.dbd.O)C(.dbd.C1c3ccc(O)cc-
3)O; COc1cc(/C.dbd.C/2\Oc3cc(O)ccc3C2=O)ccc1O;
COc1cc(ccc1O)c2oc3cc(O)cc(O)c3c(.dbd.O)c2O;
COc1cc(O)c-2c(CCc3cc(OC)c(OC)cc32)c1;
ethyl(2E)-3-(2-hydroxy-5-nitrophenyl)acrylate; ethyl
1-benzyl-4-[(dimethylamino)methyl]-5-hydroxy-2-phenyl-1H-indole-3-carboxy-
late; ethyl 2-ethoxy-5-hydroxy-1H-benzo[g]indole-3-carboxylate;
ethyl 4-(benzylamino)-6-ethoxyquinoline-3-carboxylate; ethyl
4-[({[(5R)-5-ethyl-4,6-dioxo-1,4,5,6-tetrahydropyrimidin-2-yl]thio}acetyl-
)amino]benzoate; ethyl
4-[(2-phenylethyl)amino]quinoline-3-carboxylate; ethyl
4-{[(2-anilino-2-oxoethyl)thio]methyl}-5-hydroxy-2-phenyl-1-benzofu-
ran-3-carboxylate; ethyl
4-{[(2E)-3-(2-thienyl)prop-2-enoyl]amino}benzoate; ethyl
6-bromo-4-[(dimethylamino)methyl]-5-hydroxy-1-methyl-2-{[(4-methylphenyl)-
thio]methyl}-1H-indole-3-carboxylate; ethyl
6-ethoxy-4-{[(1R)-1-methylpropyl]amino}quinoline-3-carboxylate;
ethyl
6-methyl-4-[(4-morpholin-4-ylphenyl)amino]quinoline-3-carboxylate;
isopropyl(2S)-2-{[(2S)-2-{[(2S,3R)-2-{[(2S)-2-amino-3-mercaptopropyl]amin-
o}-3-methylpentyl
]oxy}-3-phenylpropanoyl]amino}-4-(methylsulfonyl)butanoate;
methyl(2Z)-2-(4-hydroxybenzylidene)-3-oxo-2,3-dihydro-1-benzofuran-5-carb-
oxylate; methyl
1-hydroxy-3-methylpyrido[1,2-a]benzimidazole-4-carboxylate; methyl
2,3-bis-O-(biphenyl-2-ylcarbamoyl)-4-O-[(3-ethylphenyl)carbamoyl]-alpha-L-
-idopyranoside; methyl
5-hydroxy-1-[4-(trifluoromethyl)phenyl]-1H-pyrazole-3-carboxylate;
N-(1,3-benzodioxol-5-yl)-7-chloroquinolin-4-amine;
N-(2,3-dihydro-1-benzofuran-5-ylcarbonyl)-2-hydroxybenzamide;
N-(2,5-dimethylphenyl)benzamide;
N-(2-chlorobenzyl)-2-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)acetamide;
N-(2-chlorobenzyl)-2-phenyl-1H-benzimidazole-5-sulfonamide;
N-(2-ethoxyphenyl)-2-hydroxybenzamide;
N-(2-hydroxy-4-methylphenyl)-4-[(methylthio)methyl]benzamide;
N-(2-hydroxybenzoyl)-2-thiophenecarboxamide;
N-(2-hydroxybenzoyl)-3-methoxybenzamide;
N-(2-hydroxybenzoyl)-4-(trifluoromethyl)benzamide;
N-(2-hydroxyphenyl)-8-[(2E)-2-(1-naphthylmethylene)hydrazino]-8-oxooctana-
mide; N-(3-bromo-4-hydroxy-1-naphthyl)-4-chlorobenzenesulfonamide;
N-(3-chloro-4-hydroxy-1-naphthyl)-4-ethoxybenzenesulfonamide;
N-(3-chlorophenyl)-4-(5-hydroxy-1-phenyl-1H-pyrazol-3-yl)piperidine-1-car-
bothioamide; N-(3-furylcarbonyl)-2-hydroxybenzamide;
N-(3-hydroxypyridin-2-yl)-4-phenoxybenzamide;
N-(3-imidazo[1,2-a]pyrimidin-2-ylphenyl)cyclopentanecarboxamide;
N-(4-bromophenyl)-2-[(3-cyano-4-methyl-6-oxo-1,6-dihydropyridin-2-yl)thio-
]acetamide;
N-(4-carbamoylphenyl)-1-phenyl-3-(2-thienyl)-1H-pyrazole-4-carboxamide;
N-(4-ethylbenzoyl)-2-hydroxybenzamide;
N-(4-fluorophenyl)-2-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)acetamide;
N-(5-{[(1S)-1-methylpropyl]thio}-1,3,4-thiadiazol-2-yl)-2-(trifluorometh-
yl)benzamide; N-(5-hydroxy-1-naphthyl)-4-methylbenzenesulfonamide;
N-(6-chloro-2-phenyl-4H-chromen-4-ylidene)-1-(2-furyl)methanamine;
N-(6-methyl-1,3-benzothiazol-2(3H)-ylidene)thiophene-2-carboxamide;
N-(cyclohexylcarbonyl)-2-hydroxybenzamide;
N,2-diphenylquinazolin-4-amine;
N,N,8-trimethyl-5H-pyrimido[5,4-b]indol-4-amine;
N,N'-1H-isoindole-1,3(2H)-diylidenedianiline;
N,N-diethyl-8-methyl-5H-pyrimido[5,4-b]indol-4-amine;
N,N-dimethyl-4-(6-methylimidazo[1,2-a]pyridin-2-yl)benzenesulfonamide;
N-[(1E)-(9-ethyl-9H-carbazol-3-yl)methylene]-4H-1,2,4-triazol-4-amine;
N-[(1E)-1H-indol-3-ylmethylene]-1-propyl-1H-benzimidazol-2-amine;
N-[(1S)-1-benzylpropyl]-6-[(4-methylpiperidin-1-yl)sulfonyl]-4-oxo-1,4-di-
hydroquinoline-3-carboxamide;
N-[(1S)-1-phenylethyl]quinazolin-4-amine;
N-[(3-chloro-1-benzothiophen-2-yl)carbonyl]-2-hydroxybenzamide;
N-[(4E)-2-(4-methoxyphenyl)-6-methyl-4H-chromen-4-ylidene]-2-phenylethana-
mine; N-[2-(1H-benzimidazol-2-yl)phenyl]-2-methylpropanamide;
N-[2-chloro-5-(trifluoromethyl)phenyl]-2-(4,4-dimethyl-2,6-dioxocyclohexy-
l)acetamide;
N-[2-hydroxy-3-(4-oxo-4H-chromen-2-yl)phenyl]acetamide;
N-[3-(1,3-benzothiazol-2-yl)-4-hydroxyphenyl]-2,2-dimethylpropanamide;
N-[3-(1,3-benzothiazol-2-ylthio)-4-hydroxyphenyl]benzenesulfonamide;
N-[3-(1,3-benzothiazol-2-ylthio)-4-hydroxyphenyl]thiophene-2-sulfonamide;
N-[4-(1H-benzimidazol-2-yl)phenyl]-2-(2-methoxyphenyl)acetamide;
N-[4-(ethylsulfonyl)-2-hydroxyphenyl]benzamide;
N-[5-(ethylsulfonyl)-2-hydroxyphenyl]-2-(4-methoxyphenoxy)acetamide;
N4-(3,5-dichlorophenyl)-6-methylpyrimidine-2,4-diamine;
N-benzo[g]quinolin-4-yl-N'-isopropylbenzene-1,4-diamine;
N-benzyl-2-hydroxybenzamide;
N-benzyl-6-{[(3-methoxyphenyl)amino]sulfonyl}-N-methyl-4-oxo-1,4-dihydroq-
uinoline-3-carboxamide;
N-ethyl-3-phenyl-N-(3-phenylpropyl)propan-1-amine;
O[C@H]1[C@@H](O)[C@@H](COC(.dbd.O)c2cc(O)c(O)c(O)c2)O[C@@H](Oc3ccc(C(.dbd-
.O)/C.dbd.C/c4ccccc4)c(O)c3)[C@@H]1O;
O[C@H]1[C@@H](O)[C@@H](COC(.dbd.O)c2cc(O)c(O)c(O)c2)O[C@@H](Oc3cCc(C(.dbd-
.O)CCc4ccc(O)cc4)c(O)c3)[C@@H]1O;
O[C@H]1[C@@H](Oc2c([C@H]3[C@@H](Oc4cc(O)cc(O)c4C3=O)c5ccc(O)cc5)c(O)cc(O)-
c2C1=O)c6ccc(O)cc6;
O[C@H]1[C@H]2[C@H](CC(.dbd.O)O)C(.dbd.O)O[C@@H]3C(COC(.dbd.O)c4cc(O)c(O)c-
(O)c4)
O[C@@H](OC(.dbd.O)c5cc(O)c(O)c(O)c5)C(OC(.dbd.O)c6cc(O)c(O)c(OC1=O)-
c26)[C@@H]3OC(.dbd.O)c7cc(O)c(O)c(O)c7; OC[C@H]1O[C@@H](OC
[C@H]2O[C@@H](Oc3c(oc4cc(O)cc(O)c4c3=O)c5cCc(O)cc5)[C@H](O)[C@@H](O)[C@@H-
]2O)[C@H](O)[C@@H](O)[C@@H]1O; and
OC1[C@H](OC(.dbd.O)c2cc(O)c(O)c(O)c2)OC3COC(.dbd.O)c4cc(O)c(O)c(O)c4-c5c(-
O)c(O)c(O)cc5C(.dbd.O)O[C@@H]1[C@@H]3OC(.dbd.O)c6cc(O)c(O)c(O)c6c7c(O)c(O)-
c(O)cc7C(.dbd.O)OOc1ccc(cc1)[C@H]2CC(.dbd.O)c3c(O)cc(O)c([C@H]4[C@@H](Oc5c-
c(O)cc(O)c5C4=O)c6ccc(O)cc6)
c3O2Oc1ccc2C(.dbd.O)/C(.dbd.C/c3ccc(O)c(O)c3)/Oc2c1.
26. The method of claim 21, wherein said method is used to treat or
prevent malaria.
27. A method of treating an individual infected with Plasmodium or
Theileria or who has been or will be exposed to Plasmodium or
Theileria, comprising the step of providing said individual with
one or more compounds that inhibit the ability of HDP to produce
hemozoin from heme.
28. The method of claim 27, wherein said one or more compounds bind
to heme.
29. The method of claim 28, wherein said one or more compounds
prevent heme from binding to HDP.
30. The method of claim 28, wherein said one or more compounds
allow the binding of heme to HDP but prevent detoxification of heme
by HDP.
31. The method of claim 27, wherein said one or more compounds bind
to HDP.
32. The method of claim 31, wherein said one or more compounds
prevent binding of heme to HDP.
33. The method of claim 31, wherein said one or more compounds
prevent the production of hemozoin from bound heme.
34. The method of claim 31, wherein said one or more compound bind
at the active site of HDP.
35. The method of claim 31, wherein said one or more compound bind
at an allosteric site of HDP.
36. The method of claim 27 wherein said one or more compounds is
selected from the group consisting of:
(1S)-1-(3,4-dichlorophenyl)-2-(2-imino-1,3-benzothiazol-3(2H)-yl)ethanol;
(2E)-2-[(pyridin-3-ylamino)methylene]-1-benzothiophen-3(2H)-one;
(2E,5E)-5-(5-bromo-3-chloro-2-hydroxybenzylidene)-2-(phenylimino)-1,3-thi-
azolidin-4-one;
(2S)-N-(4-chlorophenyl)-2-methyl-2,3-dihydro-4H-1,4-benzoxazine-4-carboxa-
mide;
(3aR,4R,9bR)-8-chloro-4-(4-chlorophenyl)-9-nitro-3a,4,5,9b-tetrahyd-
ro-3H-cyclopenta[c]quinolin-6-ol;
(3R)-5,7-dichloro-3-hydroxy-3-(2-methylimidazo[1,2-a]pyridin-3-yl)-1,3-di-
hydro-2H-indol-2-one;
[6-hydroxy-2-(4-hydroxyphenyl)-1-benzothien-3-yl][4-(2-piperidin-1-yletho-
xy)phenyl]methanone;
1,2,3,4,6-pentakis-O-(3,4,5-trihydroxybenzoyl)-beta-D-glucopyranose;
1-[(2S)-3-(9H-carbazol-9-yl)-2-hydroxypropyl]-8-hydroxyquinolinium;
1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-1,3-dihydro-2-
H-benzimidazol-2-one;
1-ethyl-6-methoxy-4-methyl-2-[(Z)-(3-methyl-1,3-thiazol-2(3H)-ylidene)met-
hyl]benzo[h]quinolinium; 1H-perimidine-2-carboxylic acid;
2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-4H-chromen-4-one;
2-(4-methoxyphenyl)-4H-1,3-benzoxazin-4-one;
2-(4-methoxyphenyl)pyridin-3-ol;
2-(morpholin-4-ylmethyl)-1-naphthol;
2,2'-buta-1,3-diyne-1,4-diyldiphenol;
2-[(E)-(6-methoxy-1-methylquinolin-2(1H)-ylidene)methyl]-3-methyl-1,3-ben-
zothiazol-3-ium;
2-[(Z)-(3-ethyl-6-methoxy-1,3-benzothiazol-2(3H)-ylidene)methyl]-1,6-dime-
thylquinolinium; 2-[2-(4-hydroxyphenyl)ethyl]-6-methylpyridin-3-ol;
2-[4-(1-benzofuran-2-yl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-5-methoxyph-
enol;
2-{(3R)-1-[4-(2-hydroxyethoxy)benzyl]piperidin-3-yl}-1H-isoindole-1-
,3(2H)-dione;
2-amino-8-(azepan-1-ylmethyl)-3-(1,3-benzothiazol-2-yl)-7-hydroxy-4H-chro-
men-4-one; 2-hydrazino-4-methylquinoline;
2-hydroxy-N-(4-propylbenzoyl)benzamide;
3-(2-hydroxy-5-methoxybenzoyl)-2-(4-methylphenyl)isoindolin-1-one;
3-(3-{[(2-chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile;
3-[(4-{[(2R)-tetrahydrofiuran-2-ylmethyl]amino}quinazolin-2-yl)amino]phen-
ol;
3-[4-(9H-fluoren-9-yl)piperazin-1-yl]-N-[3-(trifluoromethyl)phenyl]pr-
opanamide;
4-({(2S)-3-[4-(diphenylmethyl)piperazin-1-yl]-2-hydroxypropyl}oxy)-1H-ind-
ole-2-carbonitrile; 4-(1H-benzimidazol-2-yl)-N,N-dimethylaniline;
4-(6-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;
4-(7-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;
4,4'-(4-phenyl-1H-imidazole-2,5-diyl)diphenol;
4,4'-methylenebis(3-hydroxy-2-naphthoic
acid)-3,3'-[(4-iminocyclohexa-2,5-dien-1-ylidene)methylene]dianiline
(1:1); 4,4'-propane-2,2-diylbis(2-chlorophenol);
4-[(5S)-5-(4-fluorophenyl)-1-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl]-
phenol;
4-[4-(3,4-dihydro-2H-1,5-benzodioxepin-7-yl)-3-methylisoxazol-5-y-
l]benzene-1,3-diol;
4-[5-[5-(4-methylpiperazin-1-yl)-3H-benzoimidazol-2-yl]-1,3-dihydrobenzoi-
midazol-2-ylidene]cyclohexa-2,5-dien-1-one;
4-{[(2-ethylphenyl)amino]methyl}-5-(hydroxymethyl)-2-methylpyridin-3-ol;
4-phenylquinolin-2-amine;
5-(2-hydroxy-5-methylbenzoyl)-1-(4-methylphenyl)-2-oxo-1,2-dihydropyridin-
e-3-carbonitrile;
5-(5-chloro-2-hydroxybenzoyl)-2-oxo-N,1-diphenyl-1,2-dihydropyridine-3-ca-
rboxamide;
5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-4H-chromen-4-one;
6-amino-1-ethylbenzo[cd]indol-2(1H)-one;
6-chloro-3-[2-(4-chlorophenyl)ethyl]-3,4-dihydro-2H-1,3-benzoxazine;
7-[(2E)-2-(biphenyl-4-ylmethylene)hydrazino]-N-(2-hydroxyphenyl)-7-oxohep-
tanamide;
7-chloro-N-[2-(dimethylamino)ethyl]-4H-thieno[3,2-c]thiochromen-
e-2-carboxamide; 7-chloro-N-phenylquinolin-4-amine;
7-hydroxy-2-methyl-6-propyl-3-pyridin-2-yl-4H-chromen-4-one;
8-[(2E)-2-(2-bromobenzylidene)hydrazino]-N-(2-hydroxyphenyl)-8-oxooctanam-
ide; CN(C)c1ccc2N=c3cc(C)c(N)cc3=Sc2c1;
COc1cc(O)c-2c(CCc3cc(OC)c(OC)cc32)c1; ethyl
1-benzyl-4-[(dimethylamino)methyl]-5-hydroxy-2-phenyl-1H-indole-3-carboxy-
late; ethyl 2-ethoxy-5-hydroxy-1H-benzo[g]indole-3-carboxylate;
N-(2-ethoxyphenyl)-2-hydroxybenzamide;
N-(2-hydroxybenzoyl)-2-thiophenecarboxamide;
N-(2-hydroxybenzoyl)-3-methoxybenzamide;
N-(3-chloro-4-hydroxy-1-naphthyl)-4-ethoxybenzenesulfonamide;
N-(3-furylcarbonyl)-2-hydroxybenzamide;
N-(4-ethylbenzoyl)-2-hydroxybenzamide;
N-(6-chloro-2-phenyl-4H-chromen-4-ylidene)-1-(2-furyl)methanamine;
N-[(1S)-1-phenylethyl]quinazolin-4-amine;
N-[(4E)-2-(4-methoxyphenyl)-6-methyl-4H-chromen-4-ylidene]-2-phenylethana-
mine; N4-(3,5-dichlorophenyl)-6-methylpyrimidine-2,4-diamine;
N-benzo[g]quinolin-4-yl-N'-isopropylbenzene-1,4-diamine;
O[C@H]1[C@@H](O)[C@@H](COC(.dbd.O)c2cc(O)c(O)c(O)c2)O[C@@H](Oc3ccc(C(.dbd-
.O)CCc4ccc(O)cc4)c(O)c3)[C@@H]1O; and
O[C@H]1[C@H]2[C@H](CC(.dbd.O)O)C(.dbd.O)O[C@@H]3C(COC(.dbd.O)c4cc(O)c(O(O-
)O)c4)
O[C@@H](OC(.dbd.O)c5cc(O)c(O)c(O)c5)C(OC(.dbd.O)c6cc(O)c(O)c(OC1=O)-
c26)[C@@H]3OC (.dbd.O)c7cc(O)c(O)c(O)c7.
37. The method of claim 27 wherein said one or more compounds is
selected from the group consisting of
(10S)-10-(dimethylamino)-9-methyl-7H,10H-naphtho[1,8-gh]chromen-7-one;
(1E,4E)-1-[4-(dimethylamino)phenyl]-5-(3,4,5-trimethoxyphenyl)penta-1,4-d-
ien-3-one;
(1R,3R)-1-isopropyl-2,3,4,9-tetrahydro-1H-beta-carboline-3-carboxylic
acid;
(1S)-1-(3,4-dichlorophenyl)-2-(2-imino-1,3-benzothiazol-3(2H)-yl)e-
thanol;
(2E)-2-[(pyridin-3-ylamino)methylene]-1-benzothiophen-3(2H)-one;
(2E)-3-(3,4-dimethoxyphenyl)-N-(3,4-dimethylphenyl)acrylamide;
(2E)-6-ethoxy-2-(2-hydroxybenzylidene)-1-benzothiophen-3(2H)-one;
(2E)-N-(2-methyl-1,3-benzothiazol-6-yl)-3-phenylacrylamide;
(2E,5E)-5-(5-bromo-3-chloro-2-hydroxybenzylidene)-2-(phenylimino)-1,3-thi-
azolidin-4-one;
(2E,5Z)-2-[(2-chlorophenyl)imino]-5-(4-hydroxy-3-nitrobenzylidene)-1,3-th-
iazolidin-4-one;
(2R)-1-(benzylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol;
(2R)-2-(2,4-dichlorophenoxy)-N-(5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-3-y-
l)propanamide;
(2R)-2-[(5Z)-5-(4-hydroxy-3,5-dimethoxybenzylidene)-4-oxo-2-thioxo-1,3-th-
iazolidin-3-yl]-3-methylbutanoic acid;
(2R)-2-[(E)-2-(1,3-benzodioxol-5-yl)vinyl]-5,6-dimethyl-2,3-dihydrothieno-
[2,3-d]pyrimidin-4(1H)-one;
(2R,3Z)-6-chloro-3-[(dimethylamino)methylene]-2-methyl-2,3-dihydro-4H-thi-
ochromen-4-one;
(2S)-2-(4-chlorophenyl)-3-oxo-4-phenylbutanenitrile;
(2S)-2-[(5E)-5-(1H-indol-3-ylmethylene)-4-oxo-2-thioxo-1,3-thiazolidin-3--
yl]succinic acid;
(2S)-N-(4-chlorophenyl)-2-methyl-2,3-dihydro-4H-1,4-benzoxazine-4-carboxa-
mide;
(2S,3Z)-3-(1H-indol-3-ylmethylene)-2-phenyl-2,3-dihydro-4H-chromen--
4-one; (2Z)-2-acetamido-N-(3,5-dimethylphenyl)-3-phenylacrylamide;
(2Z,5E)-2-[(3,5-dimethylphenyl)imino]-5-(2-hydroxy-3-methoxybenzylidene)--
1,3-thiazolidin-4-one;
(2Z,5Z)-2-[(2-chlorophenyl)imino]-5-(2-hydroxy-3-nitrobenzylidene)-1,3-th-
iazolidin-4-one;
(3,5-dichloro-2-hydroxyphenyl)(isoxazol-4-yl)methanone;
(3-{(E)-[1-(3-fluorophenyl)-2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene-
]methyl}-1H-indol-1-yl) acetonitrile;
(3aR,4R,9bR)-8-chloro-4-(4-chlorophenyl)-9-nitro-3a,4,5,9b-tetrahydro-3H--
cyclopenta[c]quinolin-6-ol;
(3aS,6aS)-3-benzoyl-1,5-diphenyl-3a,6a-dihydropyrrolo[3,4-c]pyrazole-4,6(-
1H,5H)-dione;
(3R)-3-(2-hydroxy-4-methylphenyl)-N-(2-methoxyphenyl)-3-phenylpropanamide-
;
(3R)-5,7-dichloro-3-hydroxy-3-(2-methylimidazo[1,2-a]pyridin-3-yl)-1,3--
dihydro-2H-indol-2-one;
(3R,3'R,4'S,6'R,8'S,8a'S)-5-(4-hydroxybut-1-yn-1-yl)-6'-[4-(2-hydroxyetho-
xy)phenyl]-1',2-dioxo-3',4'-diphenyl-1,2,3',4',8',8a'-hexahydro-1'H-spiro[-
indole-3,7'-pyrrolo[2, 1-c][1,4]oxazine]-8'-carboxylic acid;
(3S)-3-(2-hydroxy-4-methylbenzoyl)-2-(4-methylphenyl)isoindolin-1-one;
(3S,3aR,6aR)-3-(5-bromo-2-hydroxyphenyl)-5-butyl-2-phenyldihydro-2H-pyrro-
lo[3,4-d]isoxazole-4,6(3H,5H)-dione;
(3S,6S,7R,8aR)-3-(4-acetamidobutyl)-6-(4-hydroxyphenyl)-1,4-dioxooctahydr-
opyrrolo[1,2-a]pyrazine-7-carboxylic acid;
(3Z)-3-(3-hydroxy-4-methoxybenzylidene)-1-methyl-1,3-dihydro-2H-indol-2-o-
ne;
(4E)-2-(4-methoxyphenyl)-4-[(4-methoxyphenyl)imino]-4H-chromen-6-ol;
(4R)-3-(3,4-dichlorophenyl)-4-hydroxy-N-isopropyl-2-oxo-1,2,3,4-tetrahydr-
oquinazoline-4-carboxamide;
(4R)-4-(4-bromophenyl)-3-hydroxy-1-isopropyl-4,8-dihydro-1H-pyrazolo[3,4--
e][1,4]thiazepin-7(6H)-one;
(4R)-4-(4-ethylphenyl)-3-hydroxy-2-phenyl-2,4,6,7,8,9-hexahydro-5H-pyrazo-
lo[3,4-b]quinolin-5-one;
(4R)-5-(2-furylmethyl)-3-(2-hydroxyphenyl)-4-phenyl-4,5-dihydropyrrolo[3,-
4-c]pyrazol-6(1H) -one;
(4R)-N.about.4.about.-(6-chloro-2-methoxyacridin-9-yl)-N.about.1.about.,N-
.about.1.about.-diethylpentane-1,4-diamine;
(4S)-4-(2-bromobenzoyl)-5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one;
(4S)-4-(2-furyl)-3-hydroxy-7,7-dimethyl-2-phenyl-2,4,6,7,8,9-hexahydro-5H-
-pyrazolo[3,4-b]quinolin-5-one;
(4S)-5,7-dihydroxy-4-phenylchroman-2-one;
(4S,5S)-3,5-diphenyl-4,5-dihydro-1H-pyrazol-4-ol;
(4S,7R)-2-amino-4-isobutyl-5-oxo-7-phenyl-5,6,7,8-tetrahydro-4H-chromene--
3-carbonitrile;
(4Z)-2-[2-(4-chlorophenoxy)pyridin-3-yl]-4-[(dimethylamino)methylene]-1,3-
-oxazol-5(4H)-one;
(5E)-1-(4-methylpentyl)-5-(1H-pyrrol-2-ylmethylene)pyrimidine-2,4,6(1H,3H-
,5H)-trione;
(5E)-3-allyl-5-(2-hydroxybenzylidene)-2-thioxo-1,3-thiazolidin-4-one;
(5E)-5-[4-(diethylamino)benzylidene]-3-{[(2-methoxyphenyl)amino]methyl}-1-
,3-thiazolidine-2,4-dione;
(5R)-5-methyl-4-phenyl-1,3,4-thiadiazolidine-2-thione;
(5S,7R)-2,2-dimethyl-5,7-bis(2-phenylethyl)-7,8-dihydro-4H,5H-pyrano[4,3--
d][1,3]dioxine;
(5Z)-5-(4-hydroxybenzylidene)-3-[(2R)-tetrahydrofuran-2-ylmethyl]-2-thiox-
o-1,3-thiazolidin-4-one;
(6E)-5-imino-6-{[1-(2-naphthyl)-1H-pyrrol-2-yl]methylene}-5,6-dihydro-7H--
[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-one;
*c1ccccc1C2C(.dbd.O)N(C)c3ccccc3C2=O;
[(2R)-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]acetic acid;
[(5E)-4-oxo-5-(3-thienylmethylene)-2-thioxo-1,3-thiazolidin-3-yl]acetic
acid;
[(5E)-5-(5-bromo-3-chloro-2-hydroxybenzylidene)-4-oxo-2-thioxo-1,3-
-thiazolidin-3-yl]acetic acid;
[6-hydroxy-2-(4-hydroxyphenyl)-1-benzothien-3-yl][4-(2-piperidin-1-yletho-
xy)phenyl]methanone; {4-[(4-methylphenyl)sulfonyl]phenyl}hydrazine;
1-(2,4-dihydroxy-6-methylphenyl)-2-phenoxyethanone;
1-(2,4-dihydroxyphenyl)-2-(4-isopropylphenoxy)ethanone;
1-(3,4-dihydroxyphenyl)-2-({4-[(3,5-dimethoxyphenyl)amino]quinazolin-2-yl-
}thio)ethanone; 1-(4-chlorophenyl)-1-hydroxy-3-phenylurea;
1-(4-hydroxy-3,5-dimethylphenyl)-2-[(4-methylphenyl)thio]ethanone;
1-(4-iodo-2-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-ol;
1-(5-butyl-2,4-dihydroxyphenyl)-2-pyridin-2-ylethanone;
1-(5-ethyl-2,4-dihydroxyphenyl)-2-(1-methyl-1H-benzimidazol-2-yl)ethanone-
;
1,2,3,4,6-pentakis-O-(3,4,5-trihydroxybenzoyl)-beta-D-glucopyranose;
1-[(2S)-3-(9H-carbazol-9-yl)-2-hydroxypropyl]-8-hydroxyquinolinium;
1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-1,3-dihydro-2-
H-benzimidazol-2-one;
1-[4-(7-chloroquinolin-4-yl)piperazino]propan-1-one;
1-ethyl-6-methoxy-4-methyl-2-[(Z)-(3-methyl-1,3-thiazol-2(3H)-ylidene)met-
hyl]benzo[h]quinolinium; 1-ethynyl-2-phenoxybenzene;
1H-perimidine-2-carboxylic acid;
2-(1,3-benzodioxol-5-yl)-1-(2,4-dihydroxy-5-propylphenyl)ethanone;
2-(1H-benzimidazol-1-yl)-1-(5-ethyl-2,4-dihydroxyphenyl)ethanone;
2-(2,4-dichlorophenyl)-1H-imidazo[4,5-b]pyridin-1-ol;
2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one;
2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-7-methoxy-4H-chromen-4-one;
2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-4H-chromen-4-one;
2-(4-chlorophenyl)-4H-1,3-benzoxazin-4-one;
2-(4-chlorophenyl)-5-{[(4-pyridin-3-ylpyrimidin-2-yl)thio]methyl-2,4-dihy-
dro-}H-pyrazol-3-one;
2-(4-fluoro-3-phenoxyphenyl)-3-hydroxy-4H-chromen-4-one;
2-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)-N-(4-methylphenyl)acetamide;
2-(4-methoxyphenyl)-4H-1,3-benzoxazin-4-one;
2-(4-methoxyphenyl)pyridin-3-ol;
2-(4-methylphenyl)-4H-1,3-benzoxazin-4-one;
2-(morpholin-4-ylmethyl)-1-naphthol;
2,2'-buta-1,3-diyne-1,4-diyldiphenol; 2,2'-thiobis(4-chlorophenol);
2,4-dichloro-1-naphthyl[2,2,2-trifluoro-1-methyl-1-(trifluoromethyl)ethyl-
]carbamate; 2-[(2-phenoxyethyl)thio]quinazoline-4-thiol;
2-[(2R)-6,7-dimethyl-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]-N-ethylacet-
amide;
2-[(3-cyano-4-methyl-6-oxo-1,6-dihydropyridin-2-yl)thio]-N-1-napht-
hylacetamide;
2-[(5S)-1-(4-nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl]phenol;
2-[(E)-(6-methoxy-1-methylquinolin-2(1H)-ylidene)methyl]-3-methyl-1,3-ben-
zothiazol-3-ium;
2-[(Z)-(3-ethyl-6-methoxy-1,3-benzothiazol-2(3H)-ylidene)methyl]-1,6-dime-
thylquinolinium; 2-[2-(4-hydroxyphenyl)ethyl]-6-methylpyridin-3-ol;
2-[4-(1-benzofuran-2-yl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-5-methoxyph-
enol; 2-[5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl]phenol;
2-[5-(ethylsulfonyl)-2-hydroxyphenyl]-1H-benzo[de]isoquinoline-1,3(2H)-di-
one;
2-{(1R)-1-[(1-allyl-1H-benzimidazol-2-yl)amino]ethyl}-4-chlorophenol-
;
2-{(3R)-1-[4-(2-hydroxyethoxy)benzyl]piperidin-3-yl}-1H-isoindole-1,3(2-
H)-dione;
2-{[5-chloro-6-methyl-2-(2-pyridinyl)-4-pyrimidinyl]sulfanyl}-1-
-phenyl-1-ethanone;
2-amino-1-(2,4-dimethylphenyl)-1H-pyrrolo[2,3-b]quinoxaline-3-carbonitril-
e;
2-amino-5-butyl-4-(4-hydroxy-3-methoxyphenyl)-6-phenylnicotinonitrile;
2-amino-8-(azepan-1-ylmethyl)-3-(1,3-benzothiazol-2-yl)-7-hydroxy-4H-chr-
omen-4-one; 2-anilino-2-oxoethyl 2-(4-chlorobenzoyl)benzoate;
2-chloro-5-phenyl-3-pyridin-4-yl-4H-1,4-thiazine;
2-chloro-8-hydroxy-10,10-dimethyl-7-phenylpyrido[1,2-a]indol-6(10H)-one;
2-hydrazino-4,6-diphenylpyrimidine; 2-hydrazino-4-methylquinoline;
2-hydroxy-N-(4-methylphenyl)benzamide;
2-hydroxy-N-(4-propylbenzoyl)benzamide;
2-hydroxy-N-[(4-methyl-2-phenyl-1,3-thiazol-5-yl)carbonyl]benzamide;
2-hydroxy-N-[2-(2-methoxyphenyl)acetyl]benzamide;
2-hydroxy-N-{[2-(4-methylphenoxy)-3-pyridinyl]carbonyl}benzamide;
2-hydroxy-N-pyridin-3-ylbenzamide;
2-phenyl-4H-thiochromene-4-thione;
2-phenyl-5-({[5-(trifluoromethyl)pyridin-2-yl]sulfonyl}methyl)-2,4-dihydr-
o-3H-pyrazol-3-one;
2-phenyl-5-(trifluoromethyl)-2,4-dihydro-3H-pyrazol-3-one;
3-(1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)-N-(2-hydroxyphenyl)-N-(-
4-methoxybenzyl)propanamide;
3-(1-acetyl-1H-indol-3-yl)-4-hydroxy-2H-chromen-2-one;
3-(1H-benzimidazol-1-yl)-6-ethyl-7-hydroxy-4H-chromen-4-one;
3-(2-hydroxy-5-methoxybenzoyl)-2-(4-methylphenyl)isoindolin-1-one;
3-(3-{[(2-chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile;
3-(3-{[(4-chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile;
3-(4-bromophenyl)-7-hydroxy-2-methyl-4H-chromen-4-one;
3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-one;
3-(5-{(Z)-[5-(4-methylphenyl)-2-oxofuran-3(2H)-ylidene]methyl}-2-furyl)be-
nzoic acid; 3-(quinazolin-4-ylamino)phenyl thiophene-2-carboxylate;
3,4-dimethoxy-N-(4-methyl-1,3-benzothiazol-2-yl)benzamide;
3,5-dichloro-2-hydroxybenzaldehyde
N-tert-butyl-N'-methylthiosemicarbazone;
3-[(4-{[(2R)-tetrahydrofuran-2-ylmethyl]amino}quinazolin-2-yl)amino]pheno-
l;
3-[2-(4-methoxyphenyl)ethyl]-10-methyl-6-phenyl-3,4-dihydro-2H,8H-chro-
meno[6,7-e][1,3]oxazin-8-one;
3-[4-(9H-fluoren-9-yl)piperazin-1-yl]-N-[3-(trifluoromethyl)phenyl]propan-
amide;
3-{2-[(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-1H-pyrrol--
1-yl}benzoic acid; 3-benzyl-4-hydroxy-1,2-dihydroquinolin-2-one;
3-benzyl-4-hydroxy-1-phenylquinolin-2(1H)-one;
3-benzyl-5,6-bis(4-methoxyphenyl)furo[2,3-d]pyrimidin-4(3H)-imine;
3-benzyl-5-ethyl-4-hydroxy-6-phenyl-1-(1,3-thiazol-2-yl)pyridin-2(1H)-one-
; 3-benzyl-6-ethoxy-4-hydroxyquinolin-2(1H)-one;
3-chloro-N-[2-(methylthio)-1,3-benzothiazol-6-yl]benzamide;
3-hydroxy-N-(2-methylphenyl)-2-naphthamide;
3-methoxy-2-methyl-6-[1'-phenyl-5-(trifluoromethyl)-1H,1'H-4,4'-bipyrazol-
-3-yl]phenol;
3-methoxy-N-(3-[1,3]oxazolo[4,5-b]pyridin-2-ylphenyl)benzamide;
3-methyl-1-[3-(trifluoromethyl)phenyl]-1H-pyrazol-5-ol;
3-methyl-1-phenyl-4-(trifluoromethyl)-1,7-dihydro-6H-pyrazolo[3,4-b]pyrid-
in-6-one;
3-methyl-4-[(4-methylphenyl)thio]-1-phenyl-1H-pyrazol-5-yl
methoxyacetate;
3-oxo-3-[3-({[3-(trifluoromethyl)phenyl]thio}methyl)phenyl]propanenitrile-
;
4-({(2S)-3-[4-(diphenylmethyl)piperazin-1-yl]-2-hydroxypropyl}oxy)-1H-i-
ndole-2-carbonitrile;
4-(1H-benzimidazol-2-yl)-N,N-dimnethylaniline;
4-(1-propyl-1H-benzimidazol-2-yl)aniline;
4-(2,6-dichlorobenzyl)-3-methyl-1-phenyl-1H-pyrazol-5-ol;
4-(6-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;
4-(7-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;
4,4'-(4-phenyl-1H-imidazole-2,5-diyl)diphenol;
4,4'-[(2,3,5,6-tetrafluoro-1,4-phenylene)bis(oxy)]diphenol;
4,4'-methylenebis(3-hydroxy-2-naphthoic
acid)-3,3'-[(4-iminocyclohexa-2,5-dien-1-ylidene)methylene]dianiline
(1:1); 4,4'-propane-2,2-diylbis(2-chlorophenol);
4-[(3S,6S,7R,8aR)-7-{[2-(4-{(3S,3'S,4'R,6'R,8'R,8a'R)-8'-[(allyloxy)carbo-
nyl]-5-iodo-1',2-dioxo-3',4'-diphenyl-1,2,3',4',8',8a'-hexahydro-1'H-spiro-
[indole-3,7'-pyrrolo[2,1-c][1,4]oxazin]-6'-yl}phenoxy)ethoxy]carbonyl}-6-(-
4-hydroxyphenyl)-1,4-dioxooctahydropyrrolo[1,2-a]pyrazin-3-yl]-N,N,N-trime-
thylbutan-1-aminium;
4-[(4-chlorophenyl)thio]-3-methyl-1-phenyl-1H-pyrazol-5-ol;
4-[(5S)-5-(4-fluorophenyl)-1-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl]-
phenol;
4-[1-(4-hydroxy-3-methoxybenzyl)-1H-benzimidazol-2-yl]-2-methoxyp-
henol;
4-[1'-phenyl-5-(trifluoromethyl)-1H,1'H-4,4'-bipyrazol-3-yl]benzen-
e-1,3-diol;
4-[4-(1,3-benzothiazol-2-yl)-5-methyl-1H-pyrazol-3-yl]benzene-1,3-diol;
4-[4-(3,4-dihydro-2H-1,5-benzodioxepin-7-yl)-3-methylisoxazol-5-yl]benzen-
e-1,3-diol;
4-[4-(4-chlorophenyl)-1,3-thiazol-2-yl]-1-methyl-1H-pyrazol-5-amine;
4-[5-[5-(4-methylpiperazin-1-yl)-3H-benzoimidazol-2-yl]-1,3-dihydrobenzoi-
midazol-2-ylidene]cyclohexa-2,5-dien-1-one;
4-{[(1E)-3-(2-furyl)-3-oxoprop-1-en-1-yl]amino}benzoic acid;
4-{[(2-ethylphenyl)amino]methyl}-5-(hydroxymethyl)-2-methylpyridin-3-ol;
4-{[(2S)-2-ethylpiperidin-1-yl]methyl}-3-hydroxy-1-methyl-6H-benzo[c]chro-
men-6-one;
4-bromo-2-[(E)-(4H-1,2,4-triazol-4-ylimino)methyl]phenol;
4-bromo-2-[5-(2-furyl)-1H-pyrazol-3-yl]phenol;
4-bromo-6-chloro-2-oxo-1,3-benzoxathiol-5-yl ethyl carbonate;
4-ethyl-6-[4-(1-methyl-1H-benzimidazol-2-yl)-1H-pyrazol-3-yl]benzene-1,3--
diol; 4-fluoro-N-[3-(trifluoromethyl)phenyl]benzamide;
4-hydroxy-3-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]-2H-chromen-
-2-one; 4-hydroxy-3-propylquinolin-2(1H)-one;
4-hydroxy-5-phenyl-6H-pyrido[3,2,1-jk]carbazol-6-one;
4-hydroxy-8-methyl-3-{(E)-[(3R)-5-oxo-1,3-diphenylpyrazolidin-4-ylidene]m-
ethyl}quinolin -2(1H)-one; 4-phenylquinolin-2-amine;
5-(2-hydroxy-5-methylbenzoyl)-1-(4-methylphenyl)-2-oxo-1,2-dihydropyridin-
e-3-carbonitrile;
5-(5-bromo-2-hydroxybenzoyl)-1-(2-fluorophenyl)-2-oxo-1,2-dihydropyridine-
-3-carbonitrile;
5-(5-chloro-2-hydroxybenzoyl)-2-oxo-N,1-diphenyl-1,2-dihydropyridine-3-ca-
rboxamide;
5-(benzoylamino)-N,N'-bis(2-hydroxyphenyl)isophthalamide;
5-(diethylamino)-2-{(E)-[(2-phenylethyl)imino]methyl}phenol;
5,7-dihydroxy-4-propyl-2H-chromen-2-one;
5-[(4-methylphenyl)thio]quinazoline-2,4-diamine;
5-{2-[(3,4-dichlorophenyl)thio]ethyl}-2-methylpyridine;
5-benzyl-3-phenyl-5H-pyrazolo[4,3-c]quinolines;
5-benzyl-4-hydroxy-6H-pyrido[3,2,1-jk]carbazol-6-one;
5-chloro-2-hydroxy-N-phenylbenzamide;
5-hydroxy-4-methyl-7-propyl-2H-chromen-2-one;
5-methoxy-2-[3-methyl-4-(1,3-thiazol-4-yl)isoxazol-5-yl]phenol;
5-methyl-2-[5-(2-thienyl)-1H-pyrazol-3-yl]phenol;
6-(4-chlorophenyl)-7-hydroxy-1,3-dimethyl-1H-pyrrolo[3,2-d]pyrimidine-2,4-
(3H,5H)-dione''6,6'-biquinoline;
6-[(S)-[4-(dimethylamino)phenyl](piperidin-1-yl)methyl]-1,3-benzodioxol-5-
-ol;
6-{[(2-ethylphenyl)amino]sulfonyl}-4-oxo-N-[(2S)-tetrahydrofuran-2-y-
lmethyl]-1,4-dihydroquinoline-3-carboxamide;
6-{[(4-chlorophenyl)thio]methyl}-2-phenyl-1H-pyrazolo[3,4-b]pyridine-3,4(-
2H,7H)-dione;
6-{[(4-ethoxyphenyl)(methyl)amino]sulfonyl}-4-oxo-N-[(2S)-tetrahydrofuran-
-2-ylmethyl]-1,4-dihydroquinoline-3-carboxamide;
6-allyl-7-hydroxy-4,8-dimethyl-2H-chromen-2-one;
6-amino-1-ethylbenzo[cd]indol-2(1H)-one;
6-benzyl-7-hydroxy-2,3-dihydro-1H,5H-pyrido[3,2,1-ij]quinolin-5-one;
6-bromo-2-(trifluoromethyl)quinolin-4-ol;
6-butyl-2-(2-furyl)-5-methyl-4,7-dihydropyrazolo[1,5-a]pyrimidin-7-one;
6-chloro-2-(4-chlorophenyl)-1H-benzimidazol-1-ol;
6-chloro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide;
6-chloro-3-(4-methylphenyl)-3,4-dihydro-2H-1,3-benzoxazine;
6-chloro-3-[2-(4-chlorophenyl)ethyl]-3,4-dihydro-2H-1,3-benzoxazine;
6-ethyl-7-hydroxy-3-(1-methyl-1H-benzimidazol-2-yl)-2-(trifluoromethyl)-4-
H-chromen-4-one; 6-fluoro-4-hydroxy-3-phenylquinolin-2(1H)-one;
6-methoxy-N-[(1S)-1-methylpropyl]furo[2,3-b]quinoline-2-carboxamide;
6-phenyl[1,2,3,4]tetraazolo[1,5-b]pyridazin-7-ol;
7-(4-bromophenyl)-5-hydroxy-1,3-benzoxathiol-2-one;
7,8-dihydroxy-2-phenyl-4H-chromen-4-one;
7,8-dihydroxy-4-phenyl-2H-chromen-2-one;
7-[(2E)-2-(4-fluoro-3-phenoxybenzylidene)hydrazino]-N-(2-hydroxyphenyl)-7-
-oxoheptanamide;
7-[(2E)-2-(biphenyl-4-ylmethylene)hydrazino]-N-(2-hydroxyphenyl)-7-oxohep-
tanamide;
7-[2-chloro-5-(trifluoromethyl)phenyl]-5-hydroxy-1,3-benzoxathi-
ol-2-one;
7-{(2E)-2-[(2-fluorobiphenyl-4-yl)methylene]hydrazino}-N-(2-hyd-
roxyphenyl)-7-oxoheptanamide;
7-benzyl-8-hydroxy-10,10-dimethyl-6,10-dihydropyrido[1,2-a]indol-6-one;
7-chloro-4-piperidinoquinoline;
7-chloro-N-(3-fluoro-4-methylphenyl)quinolin-4-amine;
7-chloro-N-[2-(dimethylamino)ethyl]-4H-thieno[3,2-c]thiochromene-2-carbox-
amide; 7-chloro-N-phenylquinolin-4-amine;
7-hydroxy-2-methyl-6-propyl-3-pyridin-2-yl-4H-chromen-4-one;
7-hydroxy-5-methyl-3-(1-phenyl-1H-pyrazol-4-yl)-2-(trifluoromethyl)-4H-ch-
romen-4-one;
7-hydroxy-6-methyl-3-(4-methyl-1,3-thiazol-2-yl)-2-(trifluoromethyl)-4H-c-
hromen-4-one;
8-(trifluoromethoxy)-2-(trifluoromethyl)quinolin-4-ol;
8-[(2E)-2-(2-bromobenzylidene)hydrazino]-N-(2-hydroxyphenyl)-8-oxooctanam-
ide;
8-[(2E)-2-(5-bromo-2-methoxybenzylidene)hydrazino]-N-(2-hydroxypheny-
l)-8-oxooctanamide;
8-{(2E)-2-[(6-bromo-1,3-benzodioxol-5-yl)methylene]hydrazino}-N-(2-hydrox-
yphenyl)-8-oxooctanamide;
8-methoxy-N,N-dimethyl-5H-pyrimido[5,4-b]indol-4-amine;
allyl(3R,3'R,4'S,6'R,8'S,8a'S)-6'-{4-[2-({[(3S,6R,7S,8aS)-3-[4-(dimethyla-
mino)butyl]-6-(4-hydroxyphenyl)-1,4-dioxooctahydropyrrolo[1,2-a]pyrazin-7--
yl]carbonyl}oxy)ethoxy]phenyl}-5-iodo-1',2-dioxo-3',4'-diphenyl-1,2,3',4',-
8',8a'-hexahydro-1'H-spiro[indole-3,7'-pyrrolo[2,1-c][1,4]oxazine]-8'-carb-
oxylate''bis[4-(dimethylamino)phenyl]methanone oxime;
CN(C)c1ccc2N=c3cc(C)c(N)cc3=Sc2c1;
COC(.dbd.O)[C@]1(Cc2ccc(O)c(CC.dbd.C(C)C)c2)OC(.dbd.O)C(.dbd.C1c3ccc(O)cc-
3)O; COc1cc(/C.dbd.C/2\Oc3cc(O)ccc3C2=O)ccc1O;
COc1cc(ccc1O)c2oc3cc(O)cc(O)c3c(.dbd.O)c2O;
COc1cc(O)c-2c(CCc3cc(OC)c(OC)cc32)c1;
ethyl(2E)-3-(2-hydroxy-5-nitrophenyl)acrylate; ethyl
1-benzyl-4-[(dimethylamino)methyl]-5-hydroxy-2-phenyl-1H-indole-3-carboxy-
late; ethyl 2-ethoxy-5-hydroxy-1H-benzo[g]indole-3-carboxylate;
ethyl 4-(benzylamino)-6-ethoxyquinoline-3-carboxylate; ethyl
4-[({[(5R)-5-ethyl-4,6-dioxo-1,4,5,6-tetrahydropyrimidin-2-yl]thio}acetyl-
)amino]benzoate; ethyl
4-[(2-phenylethyl)amino]quinoline-3-carboxylate; ethyl
4-{[(2-anilino-2-oxoethyl)thio]methyl}-5-hydroxy-2-phenyl-1-benzofu-
ran-3-carboxylate; ethyl
4-{[(2E)-3-(2-thienyl)prop-2-enoyl]amino}benzoate; ethyl
6-bromo-4-[(dimethylamino)methyl]-5-hydroxy-1-methyl-2-{[(4-methylphenyl)-
thio]methyl}-1H-indole-3-carboxylate; ethyl
6-ethoxy-4-{[(1R)-1-methylpropyl]amino}quinoline-3-carboxylate;
ethyl
6-methyl-4-[(4-morpholin-4-ylphenyl)amino]quinoline-3-carboxylate;
isopropyl
(2S)-2-{[(2S)-2-{[(2S,3R)-2-{[(2S)-2-amino-3-mercaptopropyl]ami-
no}-3-methylpentyl]oxy}-3-phenylpropanoyl]amino}-4-(methylsulfonyl)butanoa-
te;
methyl(2Z)-2-(4-hydroxybenzylidene)-3-oxo-2,3-dihydro-1-benzofuran-5--
carboxylate; methyl
1-hydroxy-3-methylpyrido[1,2-a]benzimidazole-4-carboxylate; methyl
2,3-bis-O-(biphenyl-2-ylcarbamoyl)-4-O-[(3-ethylphenyl)carbamoyl]-alpha-L-
-idopyranoside; methyl
5-hydroxy-1-[4-(trifluoromethyl)phenyl]-1H-pyrazole-3-carboxylate;
N-(1,3-benzodioxol-5-yl)-7-chloroquinolin-4-amine;
N-(2,3-dihydro-1-benzofuran-5-ylcarbonyl)-2-hydroxybenzamide;
N-(2,5-dimethylphenyl)benzamide;
N-(2-chlorobenzyl)-2-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)acetamide;
N-(2-chlorobenzyl)-2-phenyl-1H-benzimidazole-5-sulfonamide;
N-(2-ethoxyphenyl)-2-hydroxybenzamide;
N-(2-hydroxy-4-methylphenyl)-4-[(methylthio)methyl]benzamide;
N-(2-hydroxybenzoyl)-2-thiophenecarboxamide;
N-(2-hydroxybenzoyl)-3-methoxybenzamide;
N-(2-hydroxybenzoyl)-4-(trifluoromethyl)benzamide;
N-(2-hydroxyphenyl)-8-[(2E)-2-(1-naphthylmethylene)hydrazino]-8-oxooctana-
mide; N-(3-bromo-4-hydroxy-1-naphthyl)-4-chlorobenzenesulfonamide;
N-(3-chloro-4-hydroxy-1-naphthyl)-4-ethoxybenzenesulfonamide;
N-(3-chlorophenyl)-4-(5-hydroxy-1-phenyl-1H-pyrazol-3-yl)piperidine-1-car-
bothioamide; N-(3-furylcarbonyl)-2-hydroxybenzamide;
N-(3-hydroxypyridin-2-yl)-4-phenoxybenzamide;
N-(3-imidazo[1,2-a]pyrimidin-2-ylphenyl)cyclopentanecarboxamide;
N-(4-bromophenyl)-2-[(3-cyano-4-methyl-6-oxo-1,6-dihydropyridin-2-yl)thio-
]acetamide;
N-(4-carbamoylphenyl)-1-phenyl-3-(2-thienyl)-1H-pyrazole-4-carboxamide;
N-(4-ethylbenzoyl)-2-hydroxybenzamide;
N-(4-fluorophenyl)-2-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)acetamide;
N-(5-{[(1S)-1-methylpropyl]thio}-1,3,4-thiadiazol-2-yl)-2-(trifluorometh-
yl)benzamide; N-(5-hydroxy-1-naphthyl)-4-methylbenzenesulfonamide;
N-(6-chloro-2-phenyl-4H-chromen-4-ylidene)-1-(2-furyl)methanamine;
N-(6-methyl-1,3-benzothiazol-2(3H)-ylidene)thiophene-2-carboxamide;
N-(cyclohexylcarbonyl)-2-hydroxybenzamide;
N,2-diphenylquinazolin-4-amine;
N,N,8-trimethyl-5H-pyrimido[5,4-b]indol-4-amine;
N,N'-1H-isoindole-1,3(2H)-diylidenedianiline;
N,N-diethyl-8-methyl-5H-pyrimido[5,4-b]indol-4-amine;
N,N-dimethyl-4-(6-methylimidazo[1,2-a]pyridin-2-yl)benzenesulfonamide;
N-[(1E)-(9-ethyl-9H-carbazol-3-yl)methylene]-4H-1,2,4-triazol-4-amine;
N-[(1E)-1H-indol-3-ylmethylene]-1-propyl-1H-benzimidazol-2-amine;
N-[(1S)-1-benzylpropyl]-6-[(4-methylpiperidin-1-yl)sulfonyl]-4-oxo-1,4-di-
hydroquinoline-3-carboxamide;
N-[(1S)-1-phenylethyl]quinazolin-4-amine;
N-[(3-chloro-1-benzothiophen-2-yl)carbonyl]-2-hydroxybenzamide;
N-[(4E)-2-(4-methoxyphenyl)-6-methyl-4H-chromen-4-ylidene]-2-phenylethana-
mine; N-[2-(1H-benzimidazol-2-yl)phenyl]-2-methylpropanamide;
N-[2-chloro-5-(trifluoromethyl)phenyl]-2-(4,4-dimethyl-2,6-dioxocyclohexy-
l)acetamide;
N-[2-hydroxy-3-(4-oxo-4H-chromen-2-yl)phenyl]acetamide;
N-[3-(1,3-benzothiazol-2-yl)-4-hydroxyphenyl]-2,2-dimethylpropanamide;
N-[3-(1,3-benzothiazol-2-ylthio)-4-hydroxyphenyl]benzenesulfonamide;
N-[3-(1,3-benzothiazol-2-ylthio)-4-hydroxyphenyl]thiophene-2-sulfonamide;
N-[4-(1H-benzimidazol-2-yl)phenyl]-2-(2-methoxyphenyl)acetamide;
N-[4-(ethylsulfonyl)-2-hydroxyphenyl]benzamide;
N-[5-(ethylsulfonyl)-2-hydroxyphenyl]-2-(4-methoxyphenoxy)acetamide;
N4-(3,5-dichlorophenyl)-6-methylpyrimidine-2,4-diamine;
N-benzo[g]quinolin-4-yl-N'-isopropylbenzene-1,4-diamine;
N-benzyl-2-hydroxybenzamide;
N-benzyl-6-{[(3-methoxyphenyl)amino]sulfonyl}-N-methyl-4-oxo-1,4-dihydroq-
uinoline-3-carboxamide;
N-ethyl-3-phenyl-N-(3-phenylpropyl)propan-1-amine;
O[C@H]1[C@@H](O)[C@@H](COC(.dbd.O)c2cc(O)c(O)c(O)c2)O[C@@H](Oc3ccc(C(.dbd-
.O)/C.dbd.C/c4ccccc4)c(O)c3)[C@@H]1O;
O[C@H]1[C@@H](O)[C@@H](COC(.dbd.O)c2cc(O)c(O)c(O)c2)O[C@@H](Oc3ccc(C(.dbd-
.O)CCc4ccc(O)cc4)c(O)c3)[C@@H]1O;
O[C@H]1[C@@H](Oc2c([C@H]3[C@@H](Oc4cc(O)cc(O)c4C3=O)c5ccc(O)cc5)c(O)cc(O)-
c2C1=O)c6ccc(O)cc6;
O[C@H]1[C@H]2[C@H](CC(.dbd.O)O)C(.dbd.O)O[C@@H]3C(COC(.dbd.O)c4cc(O)c(O)c-
(O)c4)O[C@@H](OC(.dbd.O)c5cc(O)c(O)c(O)c5)C(OC(.dbd.O)c6cc(O)c(O)c(OC1=O)c-
26)[C@@H]3OC(.dbd.O)c7cc(O)c(O)c(O)c7;
OC[C@H]1O[C@@H](OC[C@H]2O[C@@H](Oc3c(oc4cc(O)cc(O)c4c3=O)c5ccc(O)cc5)[C@H-
](O)[C@@H](O)[C@@H]2O)[C@H](O)[C@@H](O)[C@@H]1O; and
OC1[C@H](OC(.dbd.O)c2cc(O)c(O)c(O)c2)OC3COC(.dbd.O)c4cc(O)c(O)c(O)c4-c5c(-
O)c(O)c(O)cc5C(.dbd.O)O[C@@H]1[C
@@H]3OC(.dbd.O)c6cc(O)c(O)c(O)c6c7c(O)c(O)c(O)cc7C(.dbd.O)OOc1ccc(cc1)[C@-
H]2CC(.dbd.O)c3c(O)cc(O)c([C@H]4[C@@H](Oc5cc(O)cc(O)c5C4=O)c6ccc(O)cc6)
c3020c1ccc2C(.dbd.O)/C(.dbd.C/c3ccc(O)c(O)c3)/Oc2c1.
38. A method for identifying compounds that inhibit HDP expression,
comprising the steps of a) contacting Plasmodium with a test
compound and b) determining whether said Plasmodium expresses
HDP.
39. The method of claim 38, wherein said step of determining is
carried out by measuring mRNA.
40. The method of claim 38, wherein said step of determining is
carried out by measuring HDP.
41. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and an antimalarially effective amount of at
least one compound selected from the group consisting of:
(1S)-1-(3,4-dichlorophenyl)-2-(2-imino-1,3-benzothiazol-3(2H)-yl)ethanol;
(2E)-2-[(pyridin-3-ylamino)methylene]-1-benzothiophen-3(2H)-one;
(2E,5E)-5-(5-bromo-3-chloro-2-hydroxybenzylidene)-2-(phenylimino)-1,3-thi-
azolidin-4-one;
(2S)-N-(4-chlorophenyl)-2-methyl-2,3-dihydro-4H-1,4-benzoxazine-4-carboxa-
mide;
(3aR,4R,9bR)-8-chloro-4-(4-chlorophenyl)-9-nitro-3a,4,5,9b-tetrahyd-
ro-3H-cyclopenta[c]quinolin-6-ol;
(3R)-5,7-dichloro-3-hydroxy-3-(2-methylimidazo[1,2-a]pyridin-3-yl)-1,3-di-
hydro-2H-indol-2-one;
[6-hydroxy-2-(4-hydroxyphenyl)-1-benzothien-3-yl][4-(2-piperidin-1-yletho-
xy)phenyl]methanone;
1,2,3,4,6-pentakis-O-(3,4,5-trihydroxybenzoyl)-beta-D-glucopyranose;
1-[(2S)-3-(9H-carbazol-9-yl)-2-hydroxypropyl]-8-hydroxyquinolinium;
1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-1,3-dihydro-2-
H-benzimidazol-2-one;
1-ethyl-6-methoxy-4-methyl-2-[(Z)-(3-methyl-1,3-thiazol-2(3H)-ylidene)met-
hyl]benzo[h]quinolinium; 1H-perimidine-2-carboxylic acid;
2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-4H-chromen-4-one;
2-(4-methoxyphenyl)-4H-1,3-benzoxazin-4-one;
2-(4-methoxyphenyl)pyridin-3-ol;
2-(morpholin-4-ylmethyl)-1-naphthol;
2,2'-buta-1,3-diyne-1,4-diyldiphenol;
2-[(E)-(6-methoxy-1-methylquinolin-2(1H)-ylidene)methyl]-3-methyl-1,3-ben-
zothiazol-3-ium;
2-[(Z)-(3-ethyl-6-methoxy-1,3-benzothiazol-2(3H)-ylidene)methyl]-1,6-dime-
thylquinolinium; 2-[2-(4-hydroxyphenyl)ethyl]-6-methylpyridin-3-ol;
2-[4-(1-benzofuran-2-yl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-5-methoxyph-
enol;
2-{(3R)-1-[4-(2-hydroxyethoxy)benzyl]piperidin-3-yl-1H-isoindole-1,-
3(2H)-dione;
2-amino-8-(azepan-1-ylmethyl)-3-(1,3-benzothiazol-2-yl)-7-hydroxy-4H-chro-
men-4-one; 2-hydrazino-4-methylquinoline;
2-hydroxy-N-(4-propylbenzoyl)benzamide;
3-(2-hydroxy-5-methoxybenzoyl)-2-(4-methylphenyl)isoindolin-1-one;
3-(3-{[(2-chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile;
3-[(4-{[(2R)-tetrahydrofuran-2-ylmethyl]amino}quinazolin-2-yl)amino]pheno-
l;
3-[4-(9H-fluoren-9-yl)piperazin-1-yl]-N-[3-(trifluoromethyl)phenyl]pro-
panamide;
4-({(2S)-3-[4-(diphenylmethyl)piperazin-1-yl]-2-hydroxypropyl
oxy)-1H-indole-2-carbonitrile;
4-(1H-benzimidazol-2-yl)-N,N-dimethylaniline;
4-(6-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;
4-(7-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;
4,4'-(4-phenyl-1H-imidazole-2,5-diyl)diphenol;
4,4'-methylenebis(3-hydroxy-2-naphthoic
acid)-3,3'-[(4-iminocyclohexa-2,5-dien-1-ylidene)methylene]dianiline
(1:1); 4,4'-propane-2,2-diylbis(2-chlorophenol);
4-[(5S)-5-(4-fluorophenyl)-1-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl]-
phenol;
4-[4-(3,4-dihydro-2H-1,5-benzodioxepin-7-yl)-3-methylisoxazol-5-y-
l]benzene-1,3-diol;
4-[5-[5-(4-methylpiperazin-1-yl)-3H-benzoimidazol-2-yl]-1,3-dihydrobenzoi-
midazol-2-ylidene]cyclohexa-2,5-dien-1-one;
4-{[(2-ethylphenyl)amino]methyl}-5-(hydroxymethyl)-2-methylpyridin-3-ol;
4-phenylquinolin-2-amine;
5-(2-hydroxy-5-methylbenzoyl)-1-(4-methylphenyl)-2-oxo-1,2-dihydropyridin-
e-3-carbonitrile;
5-(5-chloro-2-hydroxybenzoyl)-2-oxo-N,1-diphenyl-1,2-dihydropyridine-3-ca-
rboxamide;
5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-4H-chromen-4-one;
6-amino-1-ethylbenzo[cd]indol-2(1H)-one;
6-chloro-3-[2-(4-chlorophenyl)ethyl]-3,4-dihydro-2H-1,3-benzoxazine;
7-[(2E)-2-(biphenyl-4-ylmethylene)hydrazino]-N-(2-hydroxyphenyl)-7-oxohep-
tanamide;
7-chloro-N-[2-(dimethylarnino)ethyl]-4H-thieno[3,2-c]thiochrome-
ne-2-carboxamide; 7-chloro-N-phenylquinolin-4-amine;
7-hydroxy-2-methyl-6-propyl-3-pyridin-2-yl-4H-chromen-4-one;
8-[(2E)-2-(2-bromobenzylidene)hydrazino]-N-(2-hydroxyphenyl)-8-oxooctanam-
ide; CN(C)c1ccc2N=c3cc(C)c(N)cc3=Sc2c1;
COc1cc(O)c-2c(CCc3cc(OC)c(OC)cc32)c1; ethyl
1-benzyl-4-[(dimethylamino)methyl]-5-hydroxy-2-phenyl-1H-indole-3-carboxy-
late; ethyl 2-ethoxy-5-hydroxy-1H-benzo[g]indole-3-carboxylate;
N-(2-ethoxyphenyl)-2-hydroxybenzarnide;
N-(2-hydroxybenzoyl)-2-thiophenecarboxamide;
N-(2-hydroxybenzoyl)-3-methoxybenzamide;
N-(3-chloro-4-hydroxy-1-naphthyl)-4-ethoxybenzenesulfonamide;
N-(3-furylcarbonyl)-2-hydroxybenzamide;
N-(4-ethylbenzoyl)-2-hydroxybenzamide;
N-(6-chloro-2-phenyl-4H-chromen-4-ylidene)-1-(2-furyl)methanamine;
N-[(1S)-1-phenylethyl]quinazolin-4-amine;
N-[(4E)-2-(4-methoxyphenyl)-6-methyl-4H-chromen-4-ylidene]-2-phenylethana-
mine; N4-(3,5-dichlorophenyl)-6-methylpyrimidine-2,4-diarnine;
N-benzo[g]quinolin-4-yl-N'-isopropylbenzene-1,4-diamine;
O[C@H]1[C@@H](O)[C@@H](COC(.dbd.O)c2cc(O)c(o)c(O)c2)O[C@@H](Oc3ccc(C(.dbd-
.O)CCc4ccc(O)cc4)c(O)c3)[C@@H]1O; and
O[C@H]1[C@H]2[C@H](CC(.dbd.O)O)C(.dbd.O)O[C@@H]3C(COC(.dbd.O)c4cc(O)c(O)c-
(O)c4)
O[C@@H](OC(.dbd.O)c5cc(O)c(O)c(O)c5)C(OC(.dbd.O)c6cc(O)c(O)c(OC1=O)-
c26)[C(@H]3OC(.dbd.O)c7cc(O)c(O)c(O)c7.
42. A pharmaceutical composition comprising a pharmnaceutically
acceptable carrier and an antimalarially effective amount of at
least one compound selected from the group consisting of
(10S)-10-(dimethylamino)-9-methyl-7H,10H-naphtho[1,8-gh]chromen-7-one;
(1E,4E)-1-[4-(dimethylamino)phenyl]-5-(3,4,5-trimethoxyphenyl)penta-1,4-d-
ien-3-one;
(1R,3R)-1-isopropyl-2,3,4,9-tetrahydro-1H-beta-carboline-3-carboxylic
acid;
(1S)-1-(3,4-dichlorophenyl)-2-(2-imino-1,3-benzothiazol-3(2H)-yl)e-
thanol;
(2E)-2-[(pyridin-3-ylamino)methylene]-1-benzothiophen-3(2H)-one;
(2E)-3-(3,4-dimethoxyphenyl)-N-(3,4-dimethylphenyl)acrylamide;
(2E)-6-ethoxy-2-(2-hydroxybenzylidene)-1-benzothiophen-3(2H)-one;
(2E)-N-(2-methyl-1,3-benzothiazol-6-yl)-3-phenylacrylamide;
(2E,5E)-5-(5-bromo-3-chloro-2-hydroxybenzylidene)-2-(phenylimino)-1,3-thi-
azolidin-4-one;
(2E,5Z)-2-[(2-chlorophenyl)imino]-5-(4-hydroxy-3-nitrobenzylidene)-1,3-th-
iazolidin-4-one;
(2R)-1-(benzylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol;
(2R)-2-(2,4-dichlorophenoxy)-N-(5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-3-y-
l)propanamide;
(2R)-2-[(5Z)-5-(4-hydroxy-3,5-dimethoxybenzylidene)-4-oxo-2-thioxo-1,3-th-
iazolidin-3-yl]-3-methylbutanoic acid;
(2R)-2-[(E)-2-(1,3-benzodioxol-5-yl)vinyl]-5,6-dimethyl-2,3-dihydrothieno-
[2,3-d]pyrimidin-4(1H)-one;
(2R,3Z)-6-chloro-3-[(dimethylarnino)methylene]-2-methyl-2,3-dihydro-4H-th-
iochromen-4-one;
(2S)-2-(4-chlorophenyl)-3-oxo-4-phenylbutanenitrile;
(2S)-2-[(5E)-5-(1H-indol-3-ylmethylene)-4-oxo-2-thioxo-1,3-thiazolidin-3--
yl]succinic acid;
(2S)-N-(4-chlorophenyl)-2-methyl-2,3-dihydro-4H-1,4-benzoxazine-4-carboxa-
mide;
(2S,3Z)-3-(1H-indol-3-ylmethylene)-2-phenyl-2,3-dihydro-4H-chromen--
4-one; (2Z)-2-acetamido-N-(3,5-dimethylphenyl)-3-phenylacrylamide;
(2Z,5E)-2-[(3,5-dimethylphenyl)imino]-5-(2-hydroxy-3-methoxybenzylidene)--
1,3-thiazolidin-4-one;
(2Z,5Z)-2-[(2-chlorophenyl)imino]-5-(2-hydroxy-3-nitrobenzylidene)-1,3-th-
iazolidin-4-one;
(3,5-dichloro-2-hydroxyphenyl)(isoxazol-4-yl)methanone;
(3-{(E)-[1-(3-fluorophenyl)-2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene-
]methyl}-1H-indol-1-yl) acetonitrile;
(3aR,4R,9bR)-8-chloro-4-(4-chlorophenyl)-9-nitro-3a,4,5,9b-tetrahydro-3H--
cyclopenta[c]quinolin-6-ol;
(3aS,6aS)-3-benzoyl-1,5-diphenyl-3a,6a-dihydropyrrolo[3,4-c]pyrazole-4,6(-
1H,5H)-dione;
(3R)-3-(2-hydroxy-4-methylphenyl)-N-(2-methoxyphenyl)-3-phenylpropanamide-
;
(3R)-5,7-dichloro-3-hydroxy-3-(2-methylimidazo[1,2-a]pyridin-3-yl)-1,3--
dihydro-2H-indol-2-one;
(3R,3'R,4'S,6'R,8'S,8a'S)-5-(4-hydroxybut-1-yn-1-yl)-6'-[4-(2-hydroxyetho-
xy)phenyl]-1',2-dioxo-3',4'-diphenyl-1,2,3',4',8',8a'-hexahydro-1'H-spiro[-
indole-3,7'-pyrrolo[2, 1-c][1,4]oxazine]-8'-carboxylic acid;
(3S)-3-(2-hydroxy-4-methylbenzoyl)-2-(4-methylphenyl)isoindolin-1-one;
(3S,3aR,6aR)-3-(5-bromo-2-hydroxyphenyl)-5-butyl-2-phenyldihydro-2H-pyrro-
lo[3,4-d]isoxazole-4,6(3H,5H)-dione;
(3S,6S,7R,8aR)-3-(4-acetamidobutyl)-6-(4-hydroxyphenyl)-1,4-dioxooctahydr-
opyrrolo[1,2-a]pyrazine-7-carboxylic acid;
(3Z)-3-(3-hydroxy-4-methoxybenzylidene)-1-methyl-1,3-dihydro-2H-indol-2-o-
ne;
(4E)-2-(4-methoxyphenyl)-4-[(4-methoxyphenyl)imino]-4H-chromen-6-ol;
(4R)-3-(3,4-dichlorophenyl)-4-hydroxy-N-isopropyl-2-oxo-1,2,3,4-tetrahydr-
oquinazoline-4-carboxamide;
(4R)-4-(4-bromophenyl)-3-hydroxy-1-isopropyl-4,8-dihydro-1H-pyrazolo[3,4--
e][1,4]thiazepin-7(6H)-one;
(4R)-4-(4-ethylphenyl)-3-hydroxy-2-phenyl-2,4,6,7,8,9-hexahydro-5H-pyrazo-
lo[3,4-b]quinolin-5-one;
(4R)-5-(2-furylmethyl)-3-(2-hydroxyphenyl)-4-phenyl-4,5-dihydropyrrolo[3,-
4-c]pyrazol-6(1H) -one;
(4R)-N.about.4.about.-(6-chloro-2-methoxyacridin-9-yl)-N.about.1.about.,N-
.about.1.about.-diethylpentane-1,4-diamine;
(4S)-4-(2-bromobenzoyl)-5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one;
(4S)-4-(2-furyl)-3-hydroxy-7,7-dimethyl-2-phenyl-2,4,6,7,8,9-hexahydro-5H-
-pyrazolo[3,4-b]quinolin-5-one;
(4S)-5,7-dihydroxy-4-phenylchroman-2-one;
(4S,5S)-3,5-diphenyl-4,5-dihydro-1H-pyrazol-4-ol;
(4S,7R)-2-amino-4-isobutyl-5-oxo-7-phenyl-5,6,7,8-tetrahydro-4H-chromene--
3-carbonitrile;
(4Z)-2-[2-(4-chlorophenoxy)pyridin-3-yl]-4-[(dimethylamino)methylene]-1,3-
-oxazol-5(4H)-one;
(5E)-1-(4-methylpentyl)-5-(1H-pyrrol-2-ylmethylene)pyrimidine-2,4,6(1H,3H-
,5H)-trione;
(5E)-3-allyl-5-(2-hydroxybenzylidene)-2-thioxo-1,3-thiazolidin-4-one;
(5E)-5-[4-(diethylamino)benzylidene]-3-{[(2-methoxyphenyl)amino]methyl}-1-
,3-thiazolidine-2,4-dione;
(5R)-5-methyl-4-phenyl-1,3,4-thiadiazolidine-2-thione;
(5S,7R)-2,2-dimethyl-5,7-bis(2-phenylethyl)-7,8-dihydro-4H,5H-pyrano[4,3--
d][1,3]dioxine;
(5Z)-5-(4-hydroxybenzylidene)-3-[(2R)-tetrahydrofuran-2-ylmethyl]-2-thiox-
o-1,3-thiazolidin-4-one;
(6E)-5-imino-6-{[1-(2-naphthyl)-1H-pyrrol-2-yl]methylene}-5,6-dihydro-7H--
[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-one;
*c1ccccc1C2C(.dbd.O)N(C)c3ccccc3C2=O;
[(2R)-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]acetic acid;
[(5E)-4-oxo-5-(3-thienylmethylene)-2-thioxo-1,3-thiazolidin-3-yl]acetic
acid;
[(5E)-5-(5-bromo-3-chloro-2-hydroxybenzylidene)-4-oxo-2-thioxo-1,3-
-thiazolidin-3-yl]acetic acid;
[6-hydroxy-2-(4-hydroxyphenyl)-1-benzothien-3-yl][4-(2-piperidin-1-yletho-
xy)phenyl]methanone; {4-[(4-methylphenyl)sulfonyl]phenyl}hydrazine;
1-(2,4-dihydroxy-6-methylphenyl)-2-phenoxyethanone;
1-(2,4-dihydroxyphenyl)-2-(4-isopropylphenoxy)ethanone;
1-(3,4-dihydroxyphenyl)-2-({4-[(3,5-dimethoxyphenyl)amino]quinazolin-2-yl-
}thio)ethanone; 1-(4-chlorophenyl)-1-hydroxy-3-phenylurea;
1-(4-hydroxy-3,5-dimethylphenyl)-2-[(4-methylphenyl)thio]ethanone;
1-(4-iodo-2-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-ol;
1-(5-butyl-2,4-dihydroxyphenyl)-2-pyridin-2-ylethanone;
1-(5-ethyl-2,4-dihydroxyphenyl)-2-(1-methyl-1H-benzimidazol-2-yl)ethanone-
;
1,2,3,4,6-pentakis-O-(3,4,5-trihydroxybenzoyl)-beta-D-glucopyranose;
1-[(2S)-3-(9H-carbazol-9-yl)-2-hydroxypropyl]-8-hydroxyquinolinium;
1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-1,3-dihydro-2-
H-benzimidazol-2-one;
1-[4-(7-chloroquinolin-4-yl)piperazino]propan-1-one;
1-ethyl-6-methoxy-4-methyl-2-[(Z)-(3-methyl-1,3-thiazol-2(3H)-ylidene)met-
hyl]benzo[h]quinolinium; 1-ethynyl-2-phenoxybenzene;
1H-perimidine-2-carboxylic acid;
2-(1,3-benzodioxol-5-yl)-1-(2,4-dihydroxy-5-propylphenyl)ethanone;
2-(1H-benzimidazol-1-yl)-1-(5-ethyl-2,4-dihydroxyphenyl)ethanone;
2-(2,4-dichlorophenyl)-1H-imidazo[4,5-b]pyridin-1-ol;
2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one;
2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-7-methoxy-4H-chromen-4-one;
2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-4H-chromen-4-one;
2-(4-chlorophenyl)-4H-1,3-benzoxazin-4-one;
2-(4-chlorophenyl)-5-{[(4-pyridin-3-ylpyrimidin-2-yl)thio]methyl}-2,4-dih-
ydro-3H-pyrazol-3-one;
2-(4-fluoro-3-phenoxyphenyl)-3-hydroxy-4H-chromen-4-one;
2-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)-N-(4-methylphenyl)acetamide;
2-(4-methoxyphenyl)-4H-1,3-benzoxazin-4-one;
2-(4-methoxyphenyl)pyridin-3-ol;
2-(4-methylphenyl)-4H-1,3-benzoxazin-4-one;
2-(morpholin-4-ylmethyl)-1-naphthol;
2,2'-buta-1,3-diyne-1,4-diyldiphenol; 2,2'-thiobis(4-chlorophenol);
2,4-dichloro-1-naphthyl[2,2,2-trifluoro-1-methyl-1-(trifluoromethyl)ethyl-
]carbamate; 2-[(2-phenoxyethyl)thio]quinazoline-4-thiol;
2-[(2R)-6,7-dimethyl-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]-N-ethylacet-
amide;
2-[(3-cyano-4-methyl-6-oxo-1,6-dihydropyridin-2-yl)thio]-N-1-napht-
hylacetamide;
2-[(5S)-1-(4-nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl]phenol;
2-[(E)-(6-methoxy-1-methylquinolin-2(1H)-ylidene)methyl]-3-methyl-1,3-ben-
zothiazol-3-ium;
2-[(Z)-(3-ethyl-6-methoxy-1,3-benzothiazol-2(3H)-ylidene)methyl]-1,6-dime-
thylquinolinium; 2-[2-(4-hydroxyphenyl)ethyl]-6-methylpyridin-3-ol;
2-[4-(1-benzofuiran-2-yl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-5-methoxyp-
henol; 2-[5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl]phenol;
2-[5-(ethylsulfonyl)-2-hydroxyphenyl]-1H-benzo[de]isoquinoline-1,3(2H)-di-
one;
2-{(1R)-1-[(1-allyl-1H-benzimidazol-2-yl)amino]ethyl}-4-chlorophenol-
;
2-{(3R)-1-[4-(2-hydroxyethoxy)benzyl]piperidin-3-yl}-1H-isoindole-1,3(2-
H)-dione;
2-{[5-chloro-6-methyl-2-(2-pyridinyl)-4-pyrimidinyl]sulfanyl}-1-
-phenyl-1-ethanone;
2-amino-1-(2,4-dimethylphenyl)-1H-pyrrolo[2,3-b]quinoxaline-3-carbonitril-
e;
2-amino-5-butyl-4-(4-hydroxy-3-methoxyphenyl)-6-phenylnicotinonitrile;
2-amino-8-(azepan-1-ylmethyl)-3-(1,3-benzothiazol-2-yl)-7-hydroxy-4H-chr-
omen-4-one; 2-anilino-2-oxoethyl 2-(4-chlorobenzoyl)benzoate;
2-chloro-5-phenyl-3-pyridin-4-yl-4H-1,4-thiazine;
2-chloro-8-hydroxy-10,10-dimethyl-7-phenylpyrido[1,2-a]indol-6(10H)-one;
2-hydrazino-4,6-diphenylpyrimidine; 2-hydrazino-4-methylquinoline;
2-hydroxy-N-(4-methylphenyl)benzamide;
2-hydroxy-N-(4-propylbenzoyl)benzamide;
2-hydroxy-N-[(4-methyl-2-phenyl-1,3-thiazol-5-yl)carbonyl]benzamide;
2-hydroxy-N-[2-(2-methoxyphenyl)acetyl]benzamide;
2-hydroxy-N-{[2-(4-methylphenoxy)-3-pyridinyl]carbonyl}benzamide;
2-hydroxy-N-pyridin-3-ylbenzamide;
2-phenyl-4H-thiochromene-4-thione;
2-phenyl-5-({[5-(trifluoromethyl)pyridin-2-yl]sulfonyl}methyl)-2,4-dihydr-
o-3H-pyrazol-3-one;
2-phenyl-5-(trifluoromethyl)-2,4-dihydro-3H-pyrazol-3-one;
3-(1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)-N-(2-hydroxyphenyl)-N-(-
4-methoxybenzyl)propanamide;
3-(1-acetyl-1H-indol-3-yl)-4-hydroxy-2H-chromen-2-one;
3-(1H-benzimidazol-1-yl)-6-ethyl-7-hydroxy-4H-chromen-4-one;
3-(2-hydroxy-5-methoxybenzoyl)-2-(4-methylphenyl)isoindolin-1-one;
3-(3-{[(2-chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile;
3-(3-{[(4-chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile;
3-(4-bromophenyl)-7-hydroxy-2-methyl-4H-chromen-4-one;
3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-one;
3-(5-{(Z)-[5-(4-methylphenyl)-2-oxofuran-3(2H)-ylidene]methyl}-2-furyl)be-
nzoic acid; 3-(quinazolin-4-ylamino)phenyl thiophene-2-carboxylate;
3,4-dimethoxy-N-(4-methyl-1,3-benzothiazol-2-yl)benzamide;
3,5-dichloro-2-hydroxybenzaldehyde
N-tert-butyl-N'-methylthiosemicarbazone;
3-[(4-{[(2R)-tetrahydrofuran-2-ylmethyl]amino}quinazolin-2-yl)amino]pheno-
l;
3-[2-(4-methoxyphenyl)ethyl]-10-methyl-6-phenyl-3,4-dihydro-2H,8H-chro-
meno[6,7-e][1,3]oxazin-8-one;
3-[4-(9H-fluoren-9-yl)piperazin-1-yl]-N-[3-(trifluoromethyl)phenyl]propan-
amide;
3-{2-[(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-1H-pyrrol--
1-yl}benzoic acid; 3-benzyl-4-hydroxy-1,2-dihydroquinolin-2-one;
3-benzyl-4-hydroxy-1-phenylquinolin-2(1H)-one;
3-benzyl-5,6-bis(4-methoxyphenyl)furo[2,3-d]pyrimidin-4(3H)-imine;
3-benzyl-5-ethyl-4-hydroxy-6-phenyl-1-(1,3-thiazol-2-yl)pyridin-2(1H)-one-
; 3-benzyl-6-ethoxy-4-hydroxyquinolin-2(1H)-one;
3-chloro-N-[2-(methylthio)-1,3-benzothiazol-6-yl]benzamide;
3-hydroxy-N-(2-methylphenyl)-2-naphthamide;
3-methoxy-2-methyl-6-[1'-phenyl-5-(trifluoromethyl)-1H,1'H-4,4'-bipyrazol-
-3-yl]phenol;
3-methoxy-N-(3-[1,3]oxazolo[4,5-b]pyridin-2-ylphenyl)benzamide;
3-methyl-1-[3-(trifluoromethyl)phenyl]-1H-pyrazol-5-ol;
3-methyl-1-phenyl-4-(trifluoromethyl)-1,7-dihydro-6H-pyrazolo[3,4-b]pyrid-
in-6-one;
3-methyl-4-[(4-methylphenyl)thio]-1-phenyl-1H-pyrazol-5-yl
methoxyacetate;
3-oxo-3-[3-({[3-(trifluoromethyl)phenyl]thio}methyl)phenyl]propanenitrile-
;
4-({(2S)-3-[4-(diphenylmethyl)piperazin-1-yl]-2-hydroxypropyl}oxy)-1H-i-
ndole-2-carbonitrile; 4-(1H-benzimidazol-2-yl)-N,N-dimethylaniline;
4-(1-propyl-1H-benzimidazol-2-yl)aniline;
4-(2,6-dichlorobenzyl)-3-methyl-1-phenyl-1H-pyrazol-5-ol;
4-(6-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;
4-(7-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;
4,4'-(4-phenyl-1H-imidazole-2,5-diyl)diphenol;
4,4'-[(2,3,5,6-tetrafluoro-1,4-phenylene)bis(oxy)]diphenol;
4,4'-methylenebis(3-hydroxy-2-naphthoic
acid)-3,3'-[(4-iminocyclohexa-2,5-dien-1-ylidene)methylene]dianiline
(1:1); 4,4'-propane-2,2-diylbis(2-chlorophenol);
4-[(3S,6S,7R,8aR)-7-{[2-(4-{(3S,3'S,4'R,6'R,8'R,8a'R)-8'-[(allyloxy)carbo-
nyl]-5-iodo-1',2-dioxo-3',4'-diphenyl-1,2,3',4',8',8a'-hexahydro-1'H-spiro-
[indole-3,7'-pyrrolo[2,1-c][1,4]oxazin]-6'-yl}phenoxy)ethoxy]carbonyl}-6-(-
4-hydroxyphenyl)-1,4-dioxooctahydropyrrolo[1,2-a]pyrazin-3-yl]-N,N,N-trime-
thylbutan-1-aminium;
4-[(4-chlorophenyl)thio]-3-methyl-1-phenyl-1H-pyrazol-5-ol;
4-[(5S)-5-(4-fluorophenyl)-1-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl]-
phenol;
4-[1-(4-hydroxy-3-methoxybenzyl)-1H-benzimidazol-2-yl]-2-methoxyp-
henol;
4-[1'-phenyl-5-(trifluoromethyl)-1H,1'H-4,4'-bipyrazol-3-yl]benzen-
e-1,3-diol;
4-[4-(1,3-benzothiazol-2-yl)-5-methyl-1H-pyrazol-3-yl]benzene-1,3-diol;
4-[4-(3,4-dihydro-2H-1,5-benzodioxepin-7-yl)-3-methylisoxazol-5-yl]benzen-
e-1,3-diol;
4-[4-(4-chlorophenyl)-1,3-thiazol-2-yl]-1-methyl-1H-pyrazol-5-amine;
4-[5-[5-(4-methylpiperazin-1-yl)-3H-benzoimidazol-2-yl]-1,3-dihydrobenzoi-
midazol-2-ylidene]cyclohexa-2,5-dien-1-one;
4-{[(1E)-3-(2-furyl)-3-oxoprop-1-en-1-yl]amino}benzoic acid;
4-{[(2-ethylphenyl)amino]methyl}-5-(hydroxymethyl)-2-methylpyridin-3-ol;
4-{[(2S)-2-ethylpiperidin-1-yl]methyl}-3-hydroxy-1-methyl-6H-benzo[c]chro-
men-6-one;
4-bromo-2-[(E)-(4H-1,2,4-triazol-4-ylimino)methyl]phenol;
4-bromo-2-[5-(2-furyl)-1H-pyrazol-3-yl]phenol;
4-bromo-6-chloro-2-oxo-1,3-benzoxathiol-5-yl ethyl carbonate;
4-ethyl-6-[4-(1-methyl-H 1H-benzimidazol-2-yl)-H
1H-pyrazol-3-yl]benzene-1,3-diol;
4-fluoro-N-[3-(trifluoromethyl)phenyl]benzamide;
4-hydroxy-3-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]-2H-chromen-
-2-one; 4-hydroxy-3-propylquinolin-2(11)-one;
4-hydroxy-5-phenyl-6H-pyrido[3,2,1-jk]carbazol-6-one;
4-hydroxy-8-methyl-3-{(E)-[(3R)-5-oxo-1,3-diphenylpyrazolidin-4-ylidene]m-
ethyl}quinolin-2(1H)-one; 4-phenylquinolin-2-amine;
5-(2-hydroxy-5-methylbenzoyl)-1-(4-methylphenyl)-2-oxo-1,2-dihydropyridin-
e-3-carbonitrile;
5-(5-bromo-2-hydroxybenzoyl)-1-(2-fluorophenyl)-2-oxo-1,2-dihydropyridine-
-3-carbonitrile;
5-(5-chloro-2-hydroxybenzoyl)-2-oxo-N,1-diphenyl-1,2-dihydropyridine-3-ca-
rboxamide;
5-(benzoylamino)-N,N'-bis(2-hydroxyphenyl)isophthalamide;
5-(diethylamino)-2-{(E)-[(2-phenylethyl)imino]methyl}phenol;
5,7-dihydroxy-4-propyl-2H-chromen-2-one;
5-[(4-methylphenyl)thio]quinazoline-2,4-diamine;
5-{2-[(3,4-dichlorophenyl)thio]ethyl}-2-methylpyridine;
5-benzyl-3-phenyl-5H-pyrazolo[4,3-c]quinolines;
5-benzyl-4-hydroxy-6H-pyrido[3,2,1-jk]carbazol-6-one;
5-chloro-2-hydroxy-N-phenylbenzamide;
5-hydroxy-4-methyl-7-propyl-2H-chromen-2-one;
5-methoxy-2-[3-methyl-4-(1,3-thiazol-4-yl)isoxazol-5-yl]phenol;
5-methyl-2-[5-(2-thienyl)-1H-pyrazol-3-yl]phenol;
6-(4-chlorophenyl)-7-hydroxy-1,3-dimethyl-1H-pyrrolo[3,2-d]pyrimidine-2,4-
(3H,5H)-dione''6,6'-biquinoline;
6-[(S)-[4-(dimethylamino)phenyl](piperidin-1-yl)methyl]-1,3-benzodioxol-5-
-ol;
6-{[(2-ethylphenyl)amino]sulfonyl}-4-oxo-N-[(2S)-tetrahydrofuran-2-y-
lmethyl]-1,4-dihydroquinoline-3-carboxamide;
6-{[(4-chlorophenyl)thio]methyl}-2-phenyl-1H-pyrazolo[3,4-b]pyridine-3,4(-
2H,7H)-dione;
6-{[(4-ethoxyphenyl)(methyl)amino]sulfonyl}-4-oxo-N-[(2S)-tetrahydrofuran-
-2-ylmethyl]-1,4-dihydroquinoline-3-carboxamide;
6-allyl-7-hydroxy-4, 8-dimethyl-2H-chromen-2-one;
6-amino-1-ethylbenzo[cd]indol-2(1H)-one;
6-benzyl-7-hydroxy-2,3-dihydro-1H,5H-pyrido[3,2,1-ij]quinolin-5-one;
6-bromo-2-(trifluoromethyl)quinolin-4-ol;
6-butyl-2-(2-furyl)-5-methyl-4,7-dihydropyrazolo[1,5-a]pyrimidin-7-one;
6-chloro-2-(4-chlorophenyl)-1H-benzimidazol-1-ol;
6-chloro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide;
6-chloro-3-(4-methylphenyl)-3,4-dihydro-2H-1,3-benzoxazine;
6-chloro-3-[2-(4-chlorophenyl)ethyl]-3,4-dihydro-2H-1,3-benzoxazine;
6-ethyl-7-hydroxy-3-(1-methyl-1H-benzimidazol-2-yl)-2-(trifluoromethyl)-4-
H-chromen-4-one; 6-fluoro-4-hydroxy-3-phenylquinolin-2(1H)-one;
6-methoxy-N-[(1S)-1-methylpropyl]furo[2,3-b]quinoline-2-carboxamide;
6-phenyl[1,2,3,4]tetraazolo[1,5-b]pyridazin-7-ol;
7-(4-bromophenyl)-5-hydroxy-1,3-benzoxathiol-2-one;
7,8-dihydroxy-2-phenyl-4H-chromen-4-one;
7,8-dihydroxy-4-phenyl-2H-chromen-2-one;
7-[(2E)-2-(4-fluoro-3-phenoxybenzylidene)hydrazino]-N-(2-hydroxyphenyl)-7-
-oxoheptanamide;
7-[(2E)-2-(biphenyl-4-ylmethylene)hydrazino]-N-(2-hydroxyphenyl)-7-oxohep-
tanamide;
7-[2-chloro-5-(trifluoromethyl)phenyl]-5-hydroxy-1,3-benzoxathi-
ol-2-one;
7-{(2E)-2-[(2-fluorobiphenyl-4-yl)methylene]hydrazino}-N-(2-hyd-
roxyphenyl)-7-oxoheptanamide;
7-benzyl-8-hydroxy-10,10-dimethyl-6,10-dihydropyrido[1,2-a]indol-6-one;
7-chloro-4-piperidinoquinoline;
7-chloro-N-(3-fluoro-4-methylphenyl)quinolin-4-amine;
7-chloro-N-[2-(dimethylamino)ethyl]-4H-thieno[3,2-c]thiochromene-2-carbox-
amide; 7-chloro-N-phenylquinolin-4-amine;
7-hydroxy-2-methyl-6-propyl-3-pyridin-2-yl-4H-chromen-4-one;
7-hydroxy-5-methy1-3-(1-phenyl-1H-pyrazol-4-yl)-2-(trifluoromethyl)-4H-ch-
romen-4-one;
7-hydroxy-6-methyl-3-(4-methyl-1,3-thiazol-2-yl)-2-(trifluoromethyl)-4H-c-
hromen-4-one;
8-(trifluoromethoxy)-2-(trifluoromethyl)quinolin-4-ol;
8-[(2E)-2-(2-bromobenzylidene)hydrazino]-N-(2-hydroxyphenyl)-8-oxooctanam-
ide;
8-[(2E)-2-(5-bromo-2-methoxybenzylidene)hydrazino]-N-(2-hydroxypheny-
l)-8-oxooctanamide;
8-{(2E)-2-[(6-bromo-1,3-benzodioxol-5-yl)methylene]hydrazino}-N-(2-hydrox-
yphenyl)-8-oxooctanamide;
8-methoxy-N,N-dimethyl-5H-pyrimido[5,4-b]indol-4-amine;
allyl(3R,3'R,4'S,6'R,8'S,8a'S)-6'-{4-[2-({[(3S,6R,7S,8aS)-3-[4-(dimethyla-
mino)butyl]-6-(4-hydroxyphenyl)-1,4-dioxooctahydropyrrolo[1,2-a]pyrazin-7--
yl]carbonyl}oxy)ethoxy]phenyl}-5-iodo-1',2-dioxo-3',4'-diphenyl-1,2,3',4',-
8',8a'-hexahydro-1'H-spiro[indole-3,7'-pyrrolo[2,1-c][1,4]oxazine]-8'-carb-
oxylate''bis[4-(dimethylamino)phenyl]methanone oxime;
CN(C)c1ccc2N=c3cc(C)c(N)cc3=Sc2c1;
COC(.dbd.O)[C@]1(Cc2ccc(O)c(CC.dbd.C(C)C)c2)OC(.dbd.O)C(.dbd.C1c3ccc(O)cc-
3)O; COc1cc(/C.dbd.C/2\Oc3cc(O)ccc3C2=O)ccc1O;
COc1cc(ccc1O)c2oc3cc(O)cc(O)c3c(.dbd.O)c2O;
COc1cc(O)c-2c(CCc3cc(OC)c(OC)cc32)c1;
ethyl(2E)-3-(2-hydroxy-5-nitrophenyl)acrylate; ethyl
1-benzyl-4-[(dimethylamino)methyl]-5-hydroxy-2-phenyl-1H-indole-3-carboxy-
late; ethyl 2-ethoxy-5-hydroxy-1H-benzo[g]indole-3-carboxylate;
ethyl 4-(benzylamino)-6-ethoxyquinoline-3-carboxylate; ethyl
4-[({[(5R)-5-ethyl-4,6-dioxo-1,4,5,6-tetrahydropyrimidin-2-yl]thio}acetyl-
)amino]benzoate; ethyl
4-[(2-phenylethyl)amino]quinoline-3-carboxylate; ethyl
4-}[(2-anilino-2-oxoethyl)thio]methyl}-5-hydroxy-2-phenyl-1-benzofu-
ran-3-carboxylate; ethyl
4-{[(2E)-3-(2-thienyl)prop-2-enoyl]amino}benzoate; ethyl
6-bromo-4-[(dimethylamino)methyl]-5-hydroxy-1-methyl-2-{[(4-methylphenyl)-
thio]methyl}-1H-indole-3-carboxylate; ethyl
6-ethoxy-4-{[(1R)-1-methylpropyl]amino}quinoline-3-carboxylate;
ethyl
6-methyl-4-[(4-morpholin-4-ylphenyl)amino]quinoline-3-carboxylate;
isopropyl(2S)-2-{[(2S)-2-{[(2S,3R)-2-{[(2S)-2-amino-3-mercaptopropyl]amin-
o}-3-methylpentyl]oxy}-3-phenylpropanoyl]amino}-4-(methylsulfonyl)butanoat-
e;
methyl(2Z)-2-(4-hydroxybenzylidene)-3-oxo-2,3-dihydro-1-benzofuran-5-c-
arboxylate; methyl
1-hydroxy-3-methylpyrido[1,2-a]benzimidazole-4-carboxylate; methyl
2,3-bis-O-(biphenyl-2-ylcarbamoyl)-4-O-[(3-ethylphenyl)carbamoyl]-alpha-L-
-idopyranoside; methyl
5-hydroxy-1-[4-(trifluoromethyl)phenyl]-1H-pyrazole-3-carboxylate;
N-(1,3-benzodioxol-5-yl)-7-chloroquinolin-4-amine;
N-(2,3-dihydro-1-benzofuran-5-ylcarbonyl)-2-hydroxybenzamide;
N-(2,5-dimethylphenyl)benzamide;
N-(2-chlorobenzyl)-2-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)acetamide;
N-(2-chlorobenzyl)-2-phenyl-1H-benzimidazole-5-sulfonamide;
N-(2-ethoxyphenyl)-2-hydroxybenzamide;
N-(2-hydroxy-4-methylphenyl)-4-[(methylthio)methyl]benzamide;
N-(2-hydroxybenzoyl)-2-thiophenecarboxamide;
N-(2-hydroxybenzoyl)-3-methoxybenzamide;
N-(2-hydroxybenzoyl)-4-(trifluoromethyl)benzamide;
N-(2-hydroxyphenyl)-8-[(2E)-2-(1-naphthylmethylene)hydrazino]-8-oxooctana-
mide; N-(3-bromo-4-hydroxy-1-naphthyl)-4-chlorobenzenesulfonamide;
N-(3-chloro-4-hydroxy-1-naphthyl)-4-ethoxybenzenesulfonamide;
N-(3-chlorophenyl)-4-(5-hydroxy-1-phenyl-1H-pyrazol-3-yl)piperidine-1-car-
bothioamide; N-(3-furylcarbonyl)-2-hydroxybenzamide;
N-(3-hydroxypyridin-2-yl)-4-phenoxybenzamide;
N-(3-imidazo[1,2-a]pyrimidin-2-ylphenyl)cyclopentanecarboxamide;
N-(4-bromophenyl)-2-[(3-cyano-4-methyl-6-oxo-1,6-dihydropyridin-2-yl)thio-
]acetamide;
N-(4-carbamoylphenyl)-1-phenyl-3-(2-thienyl)-1H-pyrazole-4-carboxamide;
N-(4-ethylbenzoyl)-2-hydroxybenzamide;
N-(4-fluorophenyl)-2-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)acetamide;
N-(5-{[(1S)-1-methylpropyl]thio}-1,3,4-thiadiazol-2-yl)-2-(trifluorometh-
yl)benzamide; N-(5-hydroxy-1-naphthyl)-4-methylbenzenesulfonamide;
N-(6-chloro-2-phenyl-4H-chromen-4-ylidene)-1-(2-furyl)methanamine;
N-(6-methyl-1,3-benzothiazol-2(3H)-ylidene)thiophene-2-carboxamide;
N-(cyclohexylcarbonyl)-2-hydroxybenzamide;
N,2-diphenylquinazolin-4-amine;
N,N,8-trimethyl-5H-pyrimido[5,4-b]indol-4-amine;
N,N'-1H-isoindole-1,3(2H)-diylidenedianiline;
N,N-diethyl-8-methyl-5H-pyrimido[5,4-b]indol-4-amine;
N,N-dimethyl-4-(6-methylimidazo[1,2-a]pyridin-2-yl)benzenesulfonamide;
N-[(1E)-(9-ethyl-9H-carbazol-3-yl)methylene]-4H-1,2,4-triazol-4-amine;
N-[(1E)-1H-indol-3-ylmethylene]-1-propyl-1H-benzimidazol-2-amine;
N-[(1S)-1-benzylpropyl]-6-[(4-methylpiperidin-1-yl)sulfonyl]-4-oxo-1,4-di-
hydroquinoline-3-carboxamide;
N-[(1S)-1-phenylethyl]quinazolin-4-amine;
N-[(3-chloro-1-benzothiophen-2-yl)carbonyl]-2-hydroxybenzamide;
N-[(4E)-2-(4-methoxyphenyl)-6-methyl-4H-chromen-4-ylidene]-2-phenylethana-
mine; N-[2-(1H-benzimidazol-2-yl)phenyl]-2-methylpropanamide;
N-[2-chloro-5-(trifluoromethyl)phenyl]-2-(4,4-dimethyl-2,6-dioxocyclohexy-
l)acetamide;
N-[2-hydroxy-3-(4-oxo-4H-chromen-2-yl)phenyl]acetamide;
N-[3-(1,3-benzothiazol-2-yl)-4-hydroxyphenyl]-2,2-dimethylpropanamide;
N-[3-(1,3-benzothiazol-2-ylthio)-4-hydroxyphenyl]benzenesulfonamide;
N-[3-(1,3-benzothiazol-2-ylthio)-4-hydroxyphenyl]thiophene-2-sulfonamide;
N-[4-(1H-benzimidazol-2-yl)phenyl]-2-(2-methoxyphenyl)acetamide;
N-[4-(ethylsulfonyl)-2-hydroxyphenyl]benzamide;
N-[5-(ethylsulfonyl)-2-hydroxyphenyl]-2-(4-methoxyphenoxy)acetamide;
N4-(3,5-dichlorophenyl)-6-methylpyrimidine-2,4-diamine;
N-benzo[g]quinolin-4-yl-N'-isopropylbenzene-1,4-diamine;
N-benzyl-2-hydroxybenzamide;
N-benzyl-6-{[(3-methoxyphenyl)amino]sulfonyl}-N-methyl-4-oxo-1,4-dihydroq-
uinoline-3-carboxamide;
N-ethyl-3-phenyl-N-(3-phenylpropyl)propan-1-amine;
O[C@H]1[C@@H](O)[C@@H](COC(.dbd.O)c2cc(O)c)c(c(O)c2)O[C@@H](Oc3ccc(C(.dbd-
.O)/C.dbd.C/c4ccccc4)c(O)c3)[C@@H]1O;
O[C@H]1[C@@H](O)[C@@H](COC(.dbd.O)c2cc(O)c(O)c(O)c2)O[C@@H](Oc3ccc(C(.dbd-
.O)CCc4ccc(O)cc4)c(O)c3)[C@@H]1O;
O[C@H]1[C@@H](Oc2c([C@H]3[C@@H](Oc4cc(O)cc(O)c4C3=O)c5ccc(O)cc5)c(O)cc(O)-
c2C=O)c6ccc(O)cc6;
O[C@H]1[C@H]2[C@H](CC(.dbd.O)O)C(.dbd.O)O[C@@H]3C(COC(.dbd.O)c4cc(O)c(O)c-
(O)c4)
O[C@@H](OC(.dbd.O)c5cc(O)c(O)c(O)c5)C(OC(.dbd.O)c6cc(O)c(O)c(OC1=O)-
c26)[C@@H]3OC (.dbd.O)c7cc(O)c(O)c(O)c7;
OC[C@H]1O[C@@H](OC[C@H]2O[C@@H](Oc3c(oc4cc(O)cc(O)c4c3=O)c5ccc(O)cc5)[C@H-
](O)[C@@H](O)[C@@H]2O)[C@H](O)[C@@H](O)[C@@H]1O; and
OC1[C@H](OC(.dbd.O)c2cc(O)c(O)c(O)c2)OC3COC(.dbd.O)c4cc(O)c(O)c(O)c4-cc(O-
)c(O)c(O)cc5C(.dbd.O)O[C@@H]1[C@@H]3OC(.dbd.O)c6cc(O)c(O)c(O)c6c7c(O)c(O)c-
(O)cc7C(.dbd.O)OOc1ccc(cc1)[C@H]2CC(.dbd.O)c3c(O)cc(O)c([C@H]4[C@@H](Oc5cc-
(O)cc(O)c5C4=O)c6ccc(O)cc6)
c3O2Oc1ccc2C(.dbd.O)/C(.dbd.C/c3ccc(O)c(O)c3)/Oc2c1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of and claims
benefit of U.S. patent application Ser. No. 11/249,355, the
complete contents of which are hereby incorporated by
reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention generally relates to therapies for the
treatment and prevention of certain parasitic diseases. In
particular, the invention provides method of inhibiting the ability
of Heme Detoxification Protein (HDP) to form hemozoin from heme,
thereby treating or preventing diseases caused by Plasmodium and/or
Theileria species.
[0004] 2. Background of the Invention
[0005] Malaria, a blood-borne infection caused by Plasmodium
parasites, is a major health issue in the tropics, with 300-500
million clinical episodes of this disease occurring each year. A
licensed vaccine against malaria is not available and the parasite
is developing resistance against most of the currently available
antimalarials. There is an urgent need to develop new therapeutics
(drugs and vaccines) against malaria, which will reduce the
morbidity and mortality associated with this disease. The genome of
Plasmodium falciparum has been sequenced and can be exploited to
understand the molecular basis of the onset and sustenance of
infection by these pathogens. Deciphering these mechanisms will
unravel the complex interplay between the troika of host, pathogen
and its environment, which is vital for identifying new targets for
intervention.
[0006] Malaria infection starts with the introduction of Plasmodium
sporozoites into the blood stream of its human host, when it is
bitten by an infected mosquito. Of the four Plasmodium species that
infect humans, P. falciparum is the most virulent--resulting in
severe anemia and cerebral malaria, which can be fatal. Fewer than
200 sporozoites are introduced and even fewer succeed in invading
liver cells, the target organ for the onset of malaria infection in
a host. A successful adhesion and liver cell invasion by the
sporozoite is critical for this onset and is therefore, the
Achilles heel of the parasite. Once inside the liver cell, the
parasite rapidly multiplies and within a few days releases
thousands of parasites, which leads to the clinical pathology of
this disease. Therefore, an ideal approach to control malaria is to
develop a vaccine or therapeutic, which either prevents the
sporozoite from infecting liver cells or destroys the parasite
during liver stages of its life cycle. Such a vaccine is feasible
as animals and human volunteers immunized with Plasmodium
sporozoites that have been attenuated by exposure to X-Ray or gamma
radiation, are protected when subsequently challenged with
infectious sporozoites (Hoffman, et al. (2002) J Infect Dis,
1155-1164; Nussenzweig et al. (1967) Protective immunity produced
by the injection of x-irradiated sporozoites of Plasmodium berghei.
Nature, 216, 160-162.). While this groundbreaking discovery clearly
indicated that it is feasible to make a vaccine against malaria,
the biggest stumbling block for malaria researchers worldwide has
been to decipher the parasite antigens recognized by the host and
to understand the immune mechanisms underlying this protection.
Extensive immunological studies with known sporozoite antigens have
concluded that this protection is not conferred due to a dominant
immune response against a single antigen but is mediated by the
summation of many modest humoral and cell-mediated immune responses
against a large variety of antigens, many of which are currently
not known (Hoffman, S. (1996) Malaria Vaccine Development: A multi
immune response approach. ASM press, Washington, D.C.).
Identification of these antigens is not only the major challenge,
it is vital for the development of a successful vaccine against
malaria.
[0007] Historically, antigen(s) selected as a vaccine candidate in
a given pathosystem are (i) present on the surface of the pathogen,
(ii) are generally involved in host-pathogen interactions and are
therefore, one of the first molecules that are recognized by the
host immune system (Moxon, R. and Rappuoli, R. (2002) Br Med Bull,
62, 45-58). These criteria are also valid for malaria parasite as
the two major vaccine candidates viz., Circumsporozoite protein
(CSP) (Cerami, C. et al. (1992) Cell, 70, 1021-1033) and
Thrombospondin-related anonymous protein (TRAP) (Robson, et al.
(1995) Embo J, 14, 3883-3894) are involved in the invasion of liver
cells by the parasite.
[0008] Upon entering red blood cells, the Plasmodium parasite
undergoes rapid multiplication giving rise to 28-32 parasites in
less than 48 hours. Hemoglobin represents .about.95% of the total
RBC content, and the parasite digests up to 75% of the hemoglobin,
which serves as its source of amino acids. While this process of
hemoglobin digestion provides the parasite with a ready source of
amino acids, it also releases free heme, which in the absence of a
globin moiety, is extremely toxic for the parasite (Gluzman, et al.
(1994) J Clin Invest, 93, 1602-1608.). The parasite survives by
effectively neutralizing toxic heme into a non-toxic and
polymerized product known as hemozoin, which is chemically
identical to .beta.-hematin (Francis,et al. (1997) Annu Rev
Microbiol, 51, 97-123. Most of the currently available
antimalarials have been shown to be binding to free heme, which
inhibits its polymerization, and the toxicity resulting from the
free heme causes the death of the parasite (Slater and Cerami
(1992) Nature, 355, 167-169). Therefore, pathway(s) that lead to
hemozoin formation are extremely attractive drug targets.
Unfortunately, the mechanism(s) in use by the parasite for the
polymerization process is poorly understood. Two parasite proteins
viz., Histidine rich protein II and III have been proposed to be
responsible for this activity (Sullivan, et al. (1996) Science,
271, 219-222.), though parasites lacking either or both of the
proteins make copious amounts of hemozoin without any loss of
activity (Wellems, et al. (1991) Proc Natl Acad Sci USA, 88,
3382-3386). Therefore, an unknown protein(s) has been long thought
to be responsible for this activity.
[0009] The prior art has thus far failed to provide satisfactory
vaccines or drug therapies to combat diseases caused by parasites
such as Plasmodium. There is thus an ongoing need to identify and
characterize potential targets for such therapeutic
intervention.
SUMMARY OF THE INVENTION
[0010] The parasite protein "Fasciclin Related Adhesive Protein"
("FRAP"), which is also referred to as "Heme Detoxification Protein
("HDP") herein, has been discovered, and its use as a target for
therapeutic intervention in parasitic diseases is described herein.
The designations "FRAP" and "HDP" are used interchangeably herein.
FRAP (HDP) is expressed during the infective forms of parasites
such as Plasmodium and Theileria, is intimately involved in the
onset of parasitic infections, and key sequences of the protein are
highly conserved across Plasmodium species and related genera.
Thus, this protein is an ideal target for the treatment and/or
prevention of parasitic diseases by a variety of methods, including
vaccine development. In addition, FRAP (HDP) catalyzes the
neutralization of toxic heme into non-toxic hemozoin. Thus, FRAP
(HDP) is an attractive target for inhibitory drug therapies. Such
therapies may include, for example, the use of compounds that bind
to the HDP protein to either prevent the binding of heme, or to
prevent the conversion of bound heme to hemozoin. Alternatively,
such therapies may involve the use of compounds that bind to heme
to prevent it from binding to HDP, or to prevent its conversion to
hemozoin after binding. The details of these and other mechanisms
of action are described in detail below.
[0011] The present invention provides a composition for eliciting
an immune response to Plasmodium. The composition comprises a
substantially purified synthesized or recombinant protein
comprising an amino acid sequence represented by SEQ ID NO: 1 or
SEQ ID NO: 25; or a substantially purified synthesized or
recombinant protein comprising an amino acid sequence that displays
at least 90% identity to SEQ ID NO: 1 or SEQ ID NO: 25. The
composition may further include at least one of: one or more
additional antigens, and one or more adjuvants. The composition may
further include one or more additional peptides, polypeptides or
proteins each of which is different from said substantially
purified synthesized or recombinant protein.
[0012] The invention also provides a composition for eliciting an
immune response to Plasmodium, which comprises a substantially
purified synthesized or recombinant peptide, polypeptide or protein
comprising an amino acid sequence represented by SEQ ID NO: 37. The
substantially purified synthesized or recombinant peptide,
polypeptide or protein may comprise an amino acid sequence
represented by SEQ ID NO: 24, or an amino acid sequence that
displays at least about 85% identity to SEQ ID NO: 24. The
composition may further include at least one of: one or more
additional antigens, and one or more adjuvants. The composition may
further include one or more additional peptides, polypeptides or
proteins each of which is different from the substantially purified
synthesized or recombinant peptide, polypeptide or protein.
[0013] In addition, the invention provides a vaccine comprising a
substantially purified synthesized or recombinant protein
comprising an amino acid sequence represented by SEQ ID NO: 1 or
SEQ ID NO: 25; or a substantially purified synthesized or
recombinant protein comprising an amino acid sequence that displays
at least 90% identity to SEQ ID NO: 1 or SEQ ID NO: 25. The vaccine
may further include at least one of one or more additional
antigens, and one or more adjuvants.
[0014] In another embodiment, the invention provides a vaccine
comprising a substantially purified synthesized or recombinant
peptide, polypeptide or protein comprising an amino acid sequence
represented by SEQ ID NO: 37. The substantially purified
synthesized or recombinant peptide, polypeptide or protein may
comprise an amino acid sequence represented by SEQ ID NO: 24, or an
amino acid sequence that is at least 85% identical to SEQ ID NO:
24. The vaccine may include at least one of: one or more additional
antigens, and one or more adjuvants. The vaccine may further
include one or more additional peptides, polypeptides or proteins
each of which is different from the substantially purified
synthesized or recombinant peptide, polypeptide or protein.
[0015] In another embodiment, the invention provides a
substantially purified synthesized or recombinantly produced
antibody specific for: a protein with an amino acid sequence
represented by SEQ ID NO: 1 or SEQ ID NO: 25; or a protein with an
amino acid sequence that displays at least 90% identity to SEQ ID
NO: 1 or SEQ ID NO: 25. In some embodiments, the antibody is
chimeric, humanized, or fully human.
[0016] In another embodiment, the invention provides a
substantially purified synthesized or recombinantly produced
antibody specific for: a peptide with an amino acid sequence
represented by SEQ ID NO: 37, or a peptide with an amino acid
sequence represented by SEQ ID NO: 24. In some embodiments, the
antibody is chimeric, humanized, or fully human.
[0017] The invention further provides a transfected cell comprising
expressable recombinant DNA that encodes: one or more of a peptide,
polypeptide or protein which is or includes an amino acid sequence
represented by SEQ ID NO: 1, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ
ID NO: 37; or one or more of a peptide, polypeptide or protein
which is or includes an amino acid sequences that displays at least
90% identity with one or more of SEQ ID NO: 1, SEQ ID NO: 25, or
SEQ ID NO: 37, or at least about 85% identity with SEQ ID NO: 24.
In another embodiment, such transfected cells are used to elicit an
immune response and/or to serve as a vaccine.
[0018] In yet another embodiment, the invention provides a method
of treating or preventing a disease caused by a Plasmodium parasite
in a patient in need thereof. The method comprises the step of
administering to the patient one or more antibodies specific for
one or more amino acid sequences represented by SEQ ID NO: 1, SEQ
ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 37.
The antibody may be synthesized or recombinantly produced.
[0019] In yet another embodiment, the invention provides a method
of eliciting an immune response to a Plasmodium parasite in a
patient in need thereof. The method comprises the step of
administering to the patient one or more peptides, polypeptides or
proteins which comprise one or more amino acid sequences selected
from the group consisting of SEQ ID NO: 1, SEQ ID NO: 24, SEQ ID
NO: 25, SEQ ID NO: 37, and amino acid sequences which display at
least 90% identity with SEQ ID NO: 1, SEQ ID NO: 25, SEQ ID NO: 37,
or at least about 85% identity with SEQ ID NO: 24. The peptides,
polypeptides or proteins may be synthesized or recombinantly
produced.
[0020] In yet another embodiment, the invention provides a method
of treating or preventing a disease caused by a Plasmodium or
Theileria parasite in a patient in need thereof. The method
comprises the step of administering to the patient a compound that
inhibits FRAP protein. In one embodiment, the patient is an animal.
In one embodiment, the compound is an antibody.
[0021] In some instances, the compound interacts with a peptide,
polypeptide protein that comprises an amino acid sequence selected
from the group consisting of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO:
17, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 37.
In addition, the compound may bind to or interact with one or more
of amino acid residues F42, H44 and H122 of FRAP protein encoded by
SEQ ID NOS: 1, 7 and 11, or with one or more equivalent amino acid
residues in other FRAP proteins, i.e. amino acid residues that
fulfill the same or a similar function in another FRAP protein,
such as the proteins encoded by SEQ ID NO: 3, SEQ ID NO: 5, or SEQ
ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID
NO: 19.
[0022] In yet another embodiment, the invention provides a whole
organism vaccine against a parasite. The vaccine comprises an
attenuated parasite which is unable to produce a fully functional
FRAP protein. The attenuated parasite may include one or more
mutations or deletions in a coding region that encodes the fully
functional FRAP protein. One or more mutations may be in a coding
region that encodes the fully functional FRAP protein at a site
which encodes for an amino acid residue selected from the group
consisting of phenylalanine 42, histidine 44, phenylalanine 64,
histidine 79, phenylalanine 90, histidine 122, cysteine 191,
histidine 192 and histidine 197 of FRAP proteins encoded by SEQ ID
NOS: 1, 7 and 11, or the equivalent amino acid residues in other
FRAP proteins, i.e. amino acid residues that fulfill the same or a
similar function in another FRAP protein, such as the proteins
encoded by SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 9, SEQ ID NO:
13, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19. In one
embodiment, the parasite is unable to produce a fully functional
FRAP protein due to RNA silencing. In another embodiment, the
parasite is unable to produce normal levels of a fully functional
FRAP protein due to attenuation of a promoter that is operably
linked to DNA encoding FRAP.
[0023] The invention also provides a method for high throughput
screening for antimalarial agents that inhibit the conversion of
heme to hemozoin. The method comprises the steps of: providing a
potential antimalarial agent; determining a first level of
conversion of heme substrate to hemozoin by FRAP in the presence of
said potential antimalarial agent, and a second level of conversion
of heme substrate to hemozoin by FRAP in the absence of said
potential antimalarial agent; and comparing said first level of
conversion to said second level of conversion, wherein if said
second level of said conversion is higher than said first level of
conversion, said potential antimalarial agent inhibits the
conversion of heme to hemozoin. In some embodiments, FRAP has one
or more amino acid sequences selected from the group consisting of
SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO:
9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and
SEQ ID NO: 19.
[0024] The invention also provides a method for expression and
purification of a recombinant protein. The method comprises the
step of providing a vector that operably encodes the recombinant
protein, wherein said recombinant protein comprises one or more of
SEQ ID NO: 1 or SEQ ID NO: 25. The recombinant protein may be a
fusion protein, and may comprise one or more copies of SEQ ID NO:
24 or SEQ ID NO: 37. The vector may also encode an antigen such as
CSP or TRAP.
[0025] The invention also provides a method for diagnosing prior
exposure to Plasmodium or Theileria. The method comprises the steps
of: obtaining a biological sample from a patient and determining
whether at least one of an amino acid sequence selected from the
group consisting of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 17, SEQ
ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 37, or an
antibody to at least one of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO:
17, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 37
is present in said biological sample.
[0026] The invention also provides a diagnostic assay for
determining exposure to Plasmodium or Theileria, comprising: one or
more substances capable of selectively binding i) at least one
amino acid sequence selected from the group consisting of SEQ ID
NO: 1, SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 24,
SEQ ID NO: 25 and SEQ ID NO: 37; or ii) an antibody to at least one
of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID
NO: 24, SEQ ID NO: 25 and SEQ ID NO: 37; and one or more labels
which are activated upon binding by said one or more
substances.
[0027] The invention also provides a method for identifying
compounds that inhibit heme neutralization by FRAP. The method
comprises the steps of a) contacting FRAP, or an extract containing
FRAP, with a known amount of heme, in the presence or absence of a
known dilution of a test compound; and b) quantitating a percent
inhibition of said heme neutralization by said test compound by
comparing differences in said heme neutralization in the presence
and absence of said test compound. FRAP may have one or more amino
acid sequence selected from the group consisting of SEQ ID NO: 1,
SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:
11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO:
19.
[0028] The invention also provides a method for diagnosing exposure
(prior or ongoing) to Plasmodium or Theileria. The method comprises
the steps of: obtaining a biological sample from a patient and
determining whether at least one of a nucleic acid sequence
selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 8,
SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 26 and SEQ
ID NO: 38, is present in said biological sample. The step of
determining may be performed using polymerase chain reaction.
[0029] The invention also provides a diagnostic kit or assay for
determining exposure (prior or ongoing) to Plasmodium or Theileria.
The kit or assay comprises: one or more nucleic acids which
hybridize to one or more nucleic acid sequence selected from the
group consisting of SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 18, SEQ
ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 38 and SEQ ID NO: 39; and a
mechanism for detecting hybridization. The kit may further comprise
means for quantifying an amount of hybridization, and the one or
more nucleic acids may be bound to a substrate, such as a
biochip.
[0030] The invention further provides a composition for eliciting
an immune response to Plasmodium. The composition comprises a
nucleic acid sequence encoding an amino acid sequence represented
by SEQ ID NO: 1. SEQ ID NO: 7 or SEQ ID NO: 25. The nucleic acid
sequence may be SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 26 or a
sequence that displays at least 90% homology to SEQ ID NO: 2, SEQ
ID NO: 8, or SEQ ID NO: 26. The composition may contain one or more
adjuvants. The composition may contain a nucleic acid encoding one
or more peptides, polypeptides or proteins which are not encoded by
SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 26. In one embodiment,
the nucleic acid sequence is contained in a vector, for example, an
adenoviral vector.
[0031] The invention also provides a composition for eliciting an
immune response to Plasmodium which comprises a nucleic acid
sequence encoding the amino acid sequence represented by SEQ ID NO:
37. In one embodiment, the nucleic acid sequence comprises a
nucleic acid sequence encoding an amino acid sequence represented
by SEQ ID NO: 24. The nucleic acid sequence may be SEQ ID NO: 38 or
SEQ ID NO: 39, or a sequence that displays at least 90% homology to
SEQ ID NO: 38, or a sequence that displays at least 85% homology to
SEQ ID NO: 39. The composition may contain one or more adjuvants,
and may further comprise nucleic acids encoding one or more
peptides, polypeptides or proteins which are not encoded by SEQ ID
NO: 38 or SEQ ID NO: 39. In one embodiment, the nucleic acid
sequence is contained in a vector, for example, an adenoviral
vector.
[0032] The invention also provides a vaccine for eliciting an
immune response to Plasmodium, the vaccine comprising a nucleic
acid sequence encoding an amino acid sequence represented by SEQ ID
NO: 1, SEQ ID NO: 7 or SEQ ID NO: 25. In some embodiments, the
nucleic acid sequence is SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO:
26, or a sequence that displays at least 90% homology to SEQ ID NO:
2, SEQ ID NO: 8, or SEQ ID NO: 26. The composition may contain one
or more adjuvants, and may comprise a nucleic acid encoding one or
more peptides, polypeptides or proteins which are not encoded by
SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 26. In one embodiment,
the nucleic acid sequence is contained in a vector, for example, an
adenoviral vector.
[0033] The invention further provides a vaccine for eliciting an
immune response to Plasmodium, the vaccine comprising a nucleic
acid sequence encoding an amino acid sequence represented by SEQ ID
NO: 37. In one embodiment, the nucleic acid sequence comprises a
nucleic acid sequence encoding an amino acid sequence represented
by SEQ ID NO: 24. The nucleic acid sequence may be SEQ ID NO: 38 or
SEQ ID NO: 39, or a sequence that displays at least 90% homology to
SEQ ID NO: 38, or a sequence that displays at least 85% homology to
SEQ ID NO: 39. The composition may contain one or more adjuvants,
and may comprise nucleic acids encoding one or more peptides,
polypeptides or proteins which are not encoded by SEQ ID NO: 38 or
SEQ ID NO: 39. In one embodiment, the nucleic acid sequence is
contained in a vector, for example, an adenoviral vector.
[0034] The invention further provides a vaccine for eliciting an
immune response to Theileria, the vaccine comprising a nucleic acid
sequence encoding an amino acid sequence represented by SEQ ID NO:
17 or SEQ ID NO: 19. In some embodiments, the nucleic acid sequence
is SEQ ID NO: 18 or SEQ ID NO: 20, or a sequence that displays at
least 90% homology to SEQ ID NO: 18 or SEQ ID NO: 20.
[0035] The invention further provides a method of treating or
preventing a disease caused by a Plasmodium or Theileria parasite
in an individual in need thereof. The method comprises the step of
inhibiting interaction of heme and Heme Detoxification Protein
(HDP) in the individual. Such individuals are typically mammals,
and can be of any species that are susceptible to infection by
Plasmodium or Theileria parasites, e.g. humans, cows, etc.
[0036] In one embodiment of the invention, the step of inhibiting
is carried out by administering to the individual one or more
compounds that inhibit interaction of heme and HDP. In some cases,
the one or more compounds bind to heme and may, for example, 1)
prevent heme from binding to HDP, or 2) allow the binding of heme
to HDP but prevent detoxification of heme by HDP. In other
embodiments, the one or more compounds bind to HDP and may, for
example, 1) prevent binding of heme to HDP, 2) prevent the
production of hemozoin from bound heme, 3) bind at the active site
of HDP, or 4) bind at an allosteric site of HDP. In other
embodiments, the step of inhibiting is carried out by modification
of a cell membrane of the Plasmodium or Theileria parasite. In yet
another embodiment, the step of inhibiting is carried out by
inhibiting secretion of HDP from the Plasmodium or Theileria
parasite.
[0037] In a preferred embodiment of the inveniton, the disease that
is treated or prevented is malaria. In this case, the compound may
be administered to an individual in combination with one or more
of: an additional antimalarial agent, an agent for reversing
antimalarial resistance, and an adjuvant. Administration of the
compound may be prior to, concurrent with, or subsequent to
administration of the additional antimalarial agent or said agent
for reversing antimalarial resistance. Suitable additional
antimalarial agents include a) quinolines, b) folic acid
antagonists, c) sulfonamides, and d) antibiotics. Suitable agents
for reversing antimalarial resistance are, for example, inhibitors
of multidrug resistance. Administration may be accomplished, for
example, orally, parenterally, sublingually, rectally, topically or
with an inhalation spray.
[0038] The invention further provides a method of treating an
individual infected with Plasmodium or Theileria or who has been or
will be exposed to Plasmodium or Theileria, The method comprises
the step of providing the individual with one or more compounds
that inhibit the ability of HDP to produce hemozoin from heme. In
some cases, the one or more compounds bind to heme and may, for
example, 1) prevent heme from binding to HDP, or 2) allow the
binding of heme to HDP but prevent detoxification of heme by HDP.
In other embodiments, the one or more compounds bind to HDP and
may, for example, 1) prevent binding of heme to HDP, 2) prevent the
production of hemozoin from bound heme, 3) bind at the active site
of HDP, or 4) bind at an allosteric site of HDP.
[0039] The invention further provides a method for identifying
compounds that inhibit HDP expression. The method comprises the
steps of a) contacting Plasmodium with a test compound and b)
determining whether the Plasmodium expresses HDP. The step of
determining may be carried out, for example, by measuring mRNA or
by measuring HDP.
[0040] The invention further provides pharmaceutical compositions
comprising a pharmaceutically acceptable carrier and an
antimalarially effective amount of at least one compound selected
Table 11 below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1A-J. This figure shows the amino acid sequences of the
FRAP protein in a variety of organisms as follows: A, Plasmodium
falciparum; B, Plasmodium vivax; C, Plasmodium gallinaceum; D,
Plasmodium knowlesi; E, Plasmodium reichenowi; F, Plasmodium
yoelii; G, Plasmodium berghei; H, Plasmodium chaubaudi; I,
Theileria parva, and J, Theileria annulata.
[0042] FIG. 2A-J. This figure shows the nucleic acid sequences that
encode the FRAP protein in a variety of organisms as follows: A,
Plasmodium falciparum; B, Plasmodium vivax; C, Plasmodium
gallinaceum; D, Plasmodium knowlesi; E, Plasmodium reichenowi; F,
Plasmodium yoelii; G, Plasmodium berghei; H, Plasmodium chaubaudi;
I, Theileria parva, and J, Theileria annulata. The sequences
represent the coding sequence of FRAP from different parasites. The
gene itself is present on three separate exons and the sequence
provided below is intron-free and represents only the coding
sequence of the protein.
[0043] FIG. 3: Multiple sequence alignment of FRAP from Plasmodium
and Theileria parasites. Sequences were aligned using the Clustal W
algorithm. Amino acids in bold (60 total) represent residues that
are conserved across the two genera of phylum apicomplexa. Residues
marked with an asterisk represent amino acid positions that are
identical only in the Plasmodial genus. Overall, the Plasmodial
sequences have 60% sequence identity. FAS1 domain of FRAP has been
aligned with the consensus sequence of FAS1 domain (SEQ ID NO: 21)
and has an e-value of 2e-10. The two conserved motifs have been
underlined.
[0044] FIG. 4. Schematic representation of P. falciparum FRAP gene
organization and the expressed recombinant proteins. (A) FRAP
represents the full length protein encoding 205 amino acids. FRAP 2
represents a truncated version of the full length protein
containing only amino acids 1-87, while FRAP 3 represents amino
acids 88-205, encoding the Fasciclin 1 domain. (B) RT-PCR analysis
of PfFRAP. DNA encoding the coding region of FRAP was amplified by
RT-PCR using total RNA from sporozoite stage of the parasite's
lifecycle. The amplification was performed in the presence (+RT)
and absence of reverse transcriptase (-RT) to rule out the direct
amplification from any contaminating genomic DNA. (C) Recombinant
Expression and Purification of PfFRAP proteins. Full-length FRAP
(lane 1) and its truncated variants, FRAP2 (lane 2) and FRAP3 (lane
3) were purified to homogeneity by a two step chromatography. (D)
Western Blot analysis. Purified proteins were resolved on a 12%
Nu-PAGE gel; transferred onto a nitrocellulose membrane and the
membrane was probed using anti-FRAP2 antibody followed by an
anti-mouse HRP conjugate.
[0045] FIG. 5. Binding analysis of FRAP proteins on HepG2 cells.
Five different concentrations of recombinant proteins were
investigated for their potential to bind to liver cells. Bound
protein was detected using anti-polyhistidine monoclonal followed
by the addition of anti-mouse alkaline phosphatase conjugate and a
fluorescent substrate. Fluorescence was measured using a plate
reader with excitation at 350 nm and emission at 460 nm. Black
bars: CS protein; Hashed bars: FRAP; Grey bars: FRAP2; White bars:
FRAP3.
[0046] FIG. 6. Nature of FRAP receptor on liver cells. Binding
activity of the FRAP proteins was evaluated on liver cells in the
absence or presence of different concentrations of heparin and
Chondroitin sulfate A. Panel A: FRAP; Panel B: FRAP2. Blank and
hashed bars represent inhibition of binding activity in the
presence of different concentrations of heparin and chondroitin
sulfate A, respectively. FIG. 7. Overlap between FRAP-based
peptides. Ten overlapping peptides spanning the FRAP2 sequence were
synthesized and utilized for the identification of regions(s)
recognized by antibodies specific for FRAP.
[0047] FIG. 8. FRAP-mediated neutralization of toxic heme into
non-toxic Hemozoin. 500 pmoles of each of the protein was incubated
with different concentrations of free heme at 37.degree. C. for 16
hours, under acidic conditions (500 mM Sodium acetate pH 5.2).
After 16 hours, free heme was removed by washing and the insoluble
pellet representing hemozoin was solubalized in sodium hydroxide
and estimated using a spectrophotometer. FRAP showed 10-20 fold
more activity in comparison to HRPII, indicating that it could be
the major protein responsible for polymerization of heme in the
parasite.
[0048] FIG. 9. FRAP-mediated hemozoin formation requires intact
protein. Hemozoin formation was investigated with FRAP pretreated
with proteinase K, a nonspecific protease or with buffer alone.
Incubation of FRAP with Proteinase K led to a complete loss of
activity suggesting that the conversion of heme into hemozoin
requires intact FRAP protein.
[0049] FIG. 10. Chemical structure of hemozoin. Dimerization of
heme through a Fe1-O41 linkage leads to the formation of
.beta.-hematin. Oxygen mediated non-covalent interaction between
.beta.-hematin units leads to the stacking and the polymerized
product is known as hemozoin. Adapted from (Pagola et al.,
2000)
[0050] FIG. 11. Spectroscopic verification of FRAP-mediated
polymerized heme as hemozoin. Heme polymerized into hemozoin was
subjected to Fourier transform-Infra Red (FT-IR) spectroscopy to
verify its chemical nature. The insoluble product showed a dramatic
decrease in transmittance at 1664 and 1211 cm.sup.-1, a well
established spectroscopic signature of .beta.-hematin.
[0051] FIG. 12. Time Kinetics of hemozoin formation. FRAP-mediated
hemozoin formation was investigated with respect to time. 500
pmoles of protein was incubated with 300 nmoles of heme for
different times and the amount of heme polymerized was measured as
previously described. Hemozoin formation was found to be
essentially complete by 5 hours.
[0052] FIG. 13. Stoichiometry of FRAP-Heme Interaction.
Stoichiometry of the FRAP-Heme interaction was determined
spectrophotometrically by continuous variation method (Job Plot).
Change in absorbance was measured by using different molar ratios
of FRAP-heme complex. FRAP-Heme have a 1:1 stoichiometry.
[0053] FIG. 14. Inhibition of FRAP-mediated hemozoin formation by
Chloroquine. Hemozoin formation was investigated in the absence or
presence of different concentrations of chloroquine, an
antimalarial drug with high affinity for heme. Chloroquine
inhibited heme polymerization in a dose dependent manner. This
indicates that blocking FRAP-Heme interaction could serve as an
effective antimalarial strategy.
[0054] FIG. 15A and B. A, amino acid and B, nucleic acid encoding
the FRAP2 derivative of FRAP.
[0055] FIG. 16A-F. HDP detoxifies and sequesters heme as Hz. a, HDP
(black bar) mediated Hz production is dose dependent and could be
up to 20 fold higher than HRP II (light grey bar), oleic acid (dark
grey bar) or mono-oleoyl glycerol (white bar). Values are mean
.+-.s.d b, Hz production increases, with increasing amount of HDP
(0-0.5 nmol) in a reaction containing 300 nmol of free heme. c,
Fourier transform infrared spectrum of HDP-derived product showed
absorption peaks at 1660 and 1210 cm-1, which validated it as Hz.
d, HDP-mediated Hz production is restricted to a pH range found
inside the food vacuole. e, Native P. falciparum HDP purified from
intraerythrocytic parasites. Silver stained gel (left panel),
Immunoblot (right panel). f, Native HDP (black bar) can produce Hz.
Hashed bar represents recombinant protein.
[0056] FIG. 17A-B. HDP gene is important for the survival of the
parasite. a, Schematic representation of strategy used for
targeting HDP locus through single cross over recombination. The
anticipated cross-over event at the HDP locus and restriction
enzyme sites Bam HI (B) and Eco RV (E) are shown. b, Lanes a and b
depict Bam HI-linearized pHDPKO (6.3 kb) and Bam HI and Eco RV
digested DNA from wild type P. falciparum parasites containing the
HDP locus (5.3 kb), respectively. Parasites surviving after three
selection cycles (lanes c, d) had an intact HDP locus and an
episomal copy of the pHDPKO plasmid expressing hDHFR. Bar
represents 500 bp.
[0057] FIG. 18A-C. Structural and biochemical analysis of
HDP-mediated Hz formation.a, Heme (100 .mu.M) solution was titrated
into protein (5 .mu.M) and the heat evolved was measured by
Isothermal titration calorimetry. Binding isotherm integrating the
data from the top panel. b, Full length HDP is necessary for Hz
formation as HDP2 (circle) and HDP3 (triangle) alone could not
produce Hz. c, Hz formation activity of P. yoelii HDP (grey bars)
is indistinguishable from its P. falciparum ortholog (black bars).
Values are mean .+-.s.d. with data from at least two independent
experiments.
[0058] FIG. 19A-D. Cloning, expression and purification of HDP
proteins. a, RT-PCR amplification of HDP coding sequence. b,
Schematic representation of HDP gene structure, HDP protein and its
two truncated variants. c, Recombinantly expressed and purified HDP
proteins on a 12% Coomassie stained gel under reducing conditions.
d, Immunoblot of purified proteins with anti-HDP antibodies.
[0059] FIG. 20A-D. Circuitous transport and delivery of HDP to the
food vacuole. a, HDP is secreted into the cytosol of infected
erythrocytes (arrowhead) in early ring stages before any Hz could
be detected inside the parasite. b, After secreting it into the
host cell cytosol, parasite intakes HDP through the cytostome c,
HDP could be found in transport vesicles destined to the food
vacuole. d, Transport vesicles deliver HDP to the food vacuole
where it was present in close proximity of Hz crystals. cyt,
cytostome; fvm, food vacuole membrane; fv, food vacuole; hz,
hemozoin; hdp, heme detoxification protein; hb, hemoglobin; nu,
nucleus; par, parasite; ppm, parasite plasma membrane; pvm,
parasitophorous vacuole membrane; irbc, infected red blood cell;
rbcm, RBC membrane; tv, transport vesicle. Bar, 0.5 .mu.m.
[0060] FIG. 21A-C. HDP is transported to food vacuole along with
hemoglobin. a, HDP(18 nm particles) was found in the cytosol of
infected cells. Inset b, HDP is being internalized along with
hemoglobin (12 nm particles). Inset c, Transport vesicle ready to
deliver both, HDP and hemoglobin to the food vacuole. Bar, 0.5
.mu.m.
[0061] FIG. 22. Comparison of Hemozoin production by HDP protein
from P. vivax and P. falciparum.
[0062] FIG. 23. Results of immunization of mice with either DNA
encoding HDP from P. yoelii or with P. yoelii HDP protein.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE
INVENTION
[0063] The present invention is based on the discovery of several
surprising properties of a previously uncharacterized family of
parasite proteins. The protein family has been designated "FRAP"
for "Fasciclin Related Adhesive Protein". Alternatively, the
protein is denominated "HDP" for "Heme Detoxification Protein".
Herein "FRAP" and "HDP" designate the same entity. This protein is
expressed by Plasmodium and Theileria parasites and is intimately
involved in the onset of parasitic infections. Hence, the FRAP
(HDP) family of proteins, and the nucleic acids that encode them,
are ideal targets for the treatment and/or prevention of certain
parasitic diseases.
[0064] The initial FRAP protein was selected for study based on a
systematic analysis of the genome of Plasmodium falciparum using a
combination of in-silico algorithms, microarray and proteomic
techniques. This process is described in detail in Example 1 of the
Examples section. The study predicted that FRAP should be expressed
on the surface of the P. falciparum sporozoite, and thus would be
involved in early interactions between the sporozoite and host
cells, making it an attractive target for therapeutic intervention.
These predictions have been confirmed. FRAP protein is present in
micronemes, a specialized secretory organelle that transports
proteins to the surface of the Plasmodium sporozoite. FRAP and an
87 amino acid polypeptide derivative, FRAP2 (amino acid sequence,
SEQ ID NO: 25; nucleic acid sequence, SEQ ID NO: 26; FIG. 15) bind
to liver cells, thereby preventing sporozoite invasion. Further,
antibodies specific for FRAP2 also prevent sporozoite invasion of
liver cells. A thirty-two amino acid sequence that is recognized by
these antibodies, encodes the inhibitory epitope and is common to
the FRAP family of proteins (TRSGGLRKPQKVTNDPESINRKVYWCFEHKPV, SEQ
ID NO: 24), has also been discovered. This sequence shows 100%
sequence homology and 87.5% sequence identity within the Plasmodium
genus. In addition, the enzymatic activity of FRAP has been
elucidated. FRAP catalyzes the neutralization of toxic heme into
non-toxic hemozoin, making this protein a highly significant target
for inhibitory drug therapy.
[0065] Herein we describe the application of these discoveries to
the prevention and treatment of parasitic diseases. For example,
FRAP proteins and various derivatives of FRAP proteins, including
the antigenic epitope, and the nucleic acids that encode them, are
useful as vaccine components. In addition, the inhibition of FRAP
proteins or nucleic acids that encode them (e.g. by compounds that
bind to the active site of the protein, or by RNA silencing) also
provides a strategy for therapeutic intervention in parasitic
disease. Further, the invention provides diagnostic tools related
to the detection of parasites harboring either the FRAP protein or
nucleic acids encoding FRAP. Further, the invention provides
methods and compositions for inhibiting the ability of HDP to
detoxify heme, i.e. to convert heme to hemozoin. Thus, the methods
and compositions are useful for the treatment or prevention of
diseases caused by Plasmodium and Theileria parasites. These and
other aspects of the invention are discussed in detail below.
[0066] The FRAP protein that was first identified in (originated
from) P. falciparum and is represented by SEQ ID NO: 1 (see FIG.
1). The protein is encoded by the nucleic acid sequence represented
by SEQ ID NO: 2 (FIG. 2). However, the FRAP family of proteins is
not limited to those originating from P. falciparum. FRAP orthologs
from Plasmodium species other than P. falciparum have been
identified, for example, FRAP orthologs from human (P. vivax)
simian (P. knowlesi, P. reichenowi), avian (P. gallinaceum) and
rodent (P. berghei, P. yoelii and P. chaubaudi) malaria parasites.
Overall, FRAP has extremely high sequence homology across the
Plasmodium genus and the region encoding the inhibitory epitope
identified in P. falciparum protein is very highly conserved in all
known FRAP orthologs. Furthermore, polymerization of human heme
into hemozoin by FRAP from rodent malaria parasite P. yoelii has
been demonstrated. Therefore, FRAP sequences between different
species of the parasites are functionally interchangeable and
transgenic malaria parasites expressing the FRAP sequence from any
member of the Plasmodium genus can be utilized for human malaria
drug and for vaccine development. In addition, FRAP orthologs
present in many related species such as Theileria may also be
utilized for use in drug and vaccine development for the diseases
they cause, e.g. bovine tropical theileriosis (Preston et al.,
Innate and adaptive immune responses co-operate to protect cattle
against Theileria annulata. Parasitol Today. 1999 July;
15(7):268-74). All such orthologs, examples of which are given in
FIG. 1, are encompassed by the present invention. The nucleic acids
that encode some exemplary FRAP proteins are presented in FIG.
2.
[0067] Those of skill in the art will recognize that a FRAP protein
need not have an exact sequence as depicted in FIG. 1 in order to
be suitable for use in the practice of the present invention.
Rather, the invention also encompasses variants (derivatives) of
such proteins. The term "protein" as used herein refers to
sequences of about 100 or more amino acids; and
[0068] the term "polypeptide" refers to sequences of about 100
amino acids or less, although these terms may be used
interchangeably. (Shorter sequences, e.g. about 35 or fewer amino
acids, will generally be referred to as peptides.) Variants or
derivatives of FRAP proteins may be isolated from nature or be
purposefully constructed. The primary sequence of such a variant or
derivative may differ from the original sequence (e.g. as
represented in FIG. 1) in any of several ways, including the
following: conservative amino acid substitutions; non-conservative
amino acid substitutions; truncation by, for example, deletion of
amino acids at the amino or carboxy terminus, or internally within
the molecule; or by addition of amino acids at the amino or carboxy
terminus, or internally within the molecule (e.g. the addition of a
histidine tag for purposes of facilitating protein isolation, the
substitution of residues to alter solubility properties, the
replacement of residues which comprise protease cleavage sites to
eliminate cleavage and increase stability, the replacement of
residues to form a convenient protease cleavage site, the addition
or elimination of glycosylation sites, and the like, for any
reason). Such variants may be naturally occurring (e.g. as the
result of natural variations between species or between
individuals, or as a result of different expression systems used to
produce the amino acid sequence, etc.); or they may be purposefully
introduced (e.g. in a laboratory setting using genetic engineering
techniques). The amino acid sequences may be in a variety of forms,
including a neutral (uncharged) forms, or forms which are salts,
and may contain modifications such as glycosylation, side chain
oxidation or deamidation, phosphorylation and the like. Also
included are amino acid sequences modified by additional
substituents such as glycosyl units, lipids, or inorganic ions such
as phosphates, as well as modifications relating to chemical
conversions or the chains, such as oxidation of sulfhydryl
groups.
[0069] All such variants of the amino acid sequences disclosed
herein are intended to be encompassed by the teachings of the
present invention, provided the variant protein/polypeptide
displays sufficient identity to the original sequence as disclosed
herein, or an amino acid sequence that can be translated from a
nucleic acid sequence disclosed herein. Preferably, amino acid
identity will be in the range of about 50 to 100%, and preferably
about 60 to 100%, or more preferably about 70 to 100%, or even more
preferably about 80 to 100%, or most preferably about 90 to 100%,
or even about 95 to 100%, of the disclosed sequences. The identity
is with reference to the portion of the amino acid sequence that
corresponds to the original amino acid sequence as translated
directly from the nucleic acid sequences disclosed herein, i.e. not
including additional elements that might be added, such as
sequences added to form chimeric proteins, histidine tags, etc.
Those of skill in the art are well acquainted with the methods
available for determining the identity between amino acid
sequences, for example, FASTA, FASTP, the BLAST suite of comparison
software, ClustalW, Lineup, Pileup, or many other alignment
software packages.
[0070] In addition, such protein/polypeptide variants retain at
least about 50 to 100% or more of the activity of the original
polypeptide, and preferably about 60 to 100% or more, or more
preferably about 70 to 100% or more, or even more preferably about
80 to 100% or more, and most preferably about 90 to 100% or more of
the activity of the original sequence. By "activity" we mean the
activity or role of the amino acid sequence in the parasite from
which is was isolated, which may include but is not limited to:
characteristic enzyme activity, activity as a structural component,
role as a membrane component, binding activity, etc.
[0071] The peptides, polypeptides and proteins of the present
invention are generally provided as recombinant molecules, although
the amino acid sequences may also be produced synthetically via
known peptide synthesis techniques. The peptides, polypeptides and
proteins of the present invention are provided in a substantially
purified form, i.e. they are generally free of extraneous materials
(such as other proteins, nucleic acids, lipids, cellular debris,
etc.) and will generally be at least about 75% pure, preferably
about 85% pure, and most preferably at least about 90-95% or more
pure, as would be understood by one of ordinary skill in the
art.
[0072] In general, the proteins and polypeptides of the invention
are produced in recombinant expression systems. In a preferred
embodiment of the present invention, the recombinant system is an
E. coli recombinant system. However, they may also be produced in a
variety of other recombinant expression systems. For example,
yeast, insect cells (using for example, a baculovirus expression
vector), plant cells (e.g. tobacco, potato, corn, etc.), transgenic
animals, or mammalian cell culture systems can be used for
expression of recombinant proteins. Any appropriate expression
system that suitably produces the proteins and polypeptides of the
invention may be used in the practice of the invention. Such
systems and their use for the production of recombinant proteins
are well known to those of ordinary skill in the art.
[0073] The invention also provides antigenic peptides, in
particular an antigenic epitope common to the FRAP family of
proteins. The epitope has the amino acid sequence
TRSGGLRKPQKVTNDPESINRKVYWCFEHKPV (SEQ ID NO: 24). Some modification
of this sequence may be tolerated without compromising the
antigenicity of the sequence. Those of skill in the art will
recognize that peptides may be obtained by several means, including
but not limited to chemical synthesis methods, production using
genetic engineering techniques, enzymatic digestion of larger
polypeptides, etc. The particular source of a peptide is not a
crucial feature of the invention. In a preferred embodiment, the
peptide will be chemically synthesized. In some embodiments of the
invention, the FRAP epitope will be used as an antigen in
combination with at least one other known parasite antigenic
epitope. For example, genetic engineering techniques may be
employed to construct chimeric polypeptides or proteins containing
two or more of such epitopes on the same molecule. Alternatively,
separate preparations of the peptidic epitopes may be prepared and
mixed into a single solution, for example, to be administered as a
vaccine.
[0074] In addition to utilizing FRAP proteins, polypeptides and
peptides, the present invention also encompasses use of the nucleic
acids that encode such amino acid sequences. Exemplary DNA
sequences that encode FRAP proteins are given in FIG. 2A-J. The
nucleic acids may be used as a tool, e.g. to produce a protein.
Alternatively, the nucleic acid sequences themselves may be used in
certain aspects of the invention, e.g. as components of DNA
vaccines, or for gene silencing applications (see below). Those of
skill in the art will recognize that many variants (derivatives) of
such sequences may exist in nature or be constructed which would
still be suitable for use in the practice of the present invention.
For example, with respect to the translation of amino acid
sequences from the nucleic acid sequences, due to the redundancy of
the genetic code, more than one codon may be used to code for an
amino acid. Further, as described above, changes in the amino acid
primary sequence may be desired, and this would necessitate changes
in the encoding nucleic acid sequences. In addition, those of skill
in the art will recognize that many variations of the nucleic acid
sequences may be constructed for purposes related to other aspects
of the invention, for example: for cloning strategies (e.g. the
introduction of restriction enzyme cleavage sites for ease of
manipulation of a sequence for insertion into a vector, for
rendering the sequence compatible with the cloning system vector or
host, for enabling fluorescent or affinity labeling technologies,
etc.), for purposes of modifying transcription (e.g. the
introduction of specific promoter or enhancer sequences, insertion
or deletion of splice signals, for enhancing or negatively
regulating transcription levels, for regulating polyadenylation,
for controlling termination, and the like), or for modification of
active or inactive domains, for elimination or modification of
certain activities or domains, for optimizing expression due to
codon usage or other compositional biases, for addition of
immunologically relevant (enhancing or inhibiting) sequences or for
any other suitable purpose. All such variants of the nucleic acid
sequences encoding the proteins, polypeptides and peptides
disclosed herein are intended to be encompassed by the present
invention, provided the sequences display homology in the range of
about 50 to 100%, and preferably about 60 to 100%, or more
preferably about 70 to 100%, or even more preferably about 80 to
100%, or most preferably about 90 to 100% or about 95 to 100% to
the disclosed sequences. The homology is with reference to the
portion of the nucleic acid sequence that corresponds to the
original sequence, and is not intended to apply to additional
elements such as promoters, vector-derived sequences, restriction
enzyme cleavage sites, etc. derived from other sources. Those of
skill in the art are well-acquainted with methods to determine
nucleic acid similarity or identity using simple software alignment
tools such as FASTA, the BLAST suite of programs, CLUSTAL W,
Lineup, Pileup (GCG), or many others.
[0075] In addition, the nucleic acids are not limited to DNA, but
are intended to encompass other nucleic acids as well, such as
mRNA, RNA-DNA hybrids, and various modified forms of DNA and RNA
known to those of skill in the art. For example, for use in vivo,
nucleic acids may be modified to resist degradation via structural
modification (e.g. by the introduction of secondary structures,
such as stem loops, or via phosphate backbone modifications, etc.).
Alternatively, the nucleic acids may include phosphothioate or
phosphodithioate rather than phosphodiesterase linkages within the
backbone of the molecule, or methylphosphorothiate terminal
linkages. Other variations include but are not limited to:
nontraditional bases such as inosine and queosine; acetyl-, thio-
and similarly modified forms of adenine, cytidine, guanine, thymine
and uridine; stabilized nucleic acid molecules such as nonionic DNA
analogs, alkyl- and aryl phosphonates; nucleic acid molecules which
contain a diol, such as tetrahyleneglycol or hexaethyleneglycol, at
either or both termini; etc. Further, the nucleic acid molecules
may be either single or double stranded, or may comprise segments
of both single and double strand nucleic acid.
[0076] In the course of practicing the invention, FRAP-related
nucleic acid molecules may be cloned into one of many suitable
vectors. In some embodiments, vectors containing nucleic acid
sequences (e.g. DNA) that encode the amino acid sequences of the
invention will encode a single protein, polypeptide, or peptide.
However, this need not always be the case. Such vectors may contain
DNA encoding more than one amino acid sequence, either as separate,
discrete sequences, or combined into a single chimeric sequence.
For example, in the case of an expression vector, two or more
nucleic acids according to the invention may be present in the
vector, and the nucleic acids may be expressed separately,
resulting in the translation of one amino acid sequence for each
nucleic acid. Alternatively, a single polypeptide chain containing
more than one amino acid sequence of the invention, or portions of
more than one amino acid sequence of the invention, may be combined
in tandem. For example, one or more highly antigenic proteins or
regions of proteins of the invention may be expressed as a chimera
from a single DNA sequence. Alternatively, the amino acid sequences
of the invention may be expressed as part of a chimeric protein
comprising amino acid sequences from another source, e.g. antigenic
sequences known to be useful as adjuvants (e.g. PADRE [and other
Pan-DR T helper cell epitope], hepatitis B core antigen, DNA
sequences CPG, other chemokines, CTB or cholera toxin B subunit,
Ricin B and other plant toxin subunits, LPS or lipopolysaccharide,
KLH [key hole limpet hemocyanin], Freund's complete and Freund's
incomplete adjuvant, and many other reagents, etc.), sequences that
permit targeting of the protein to a specific location within the
cell (e.g. nucleus, nucleolus or nuclear membrane,
mitochondrion/mitosome/mitochondria-like organelle, membrane,
endoplasmic reticulum, golgi, rhoptry, dense granules, calcisomes
or acidocalcisomes, and other subcellular organelles compartments,
etc.).
[0077] One application of the present invention is the provision of
vaccines that provide immunity to disease caused by parasites such
as Plasmodium. By "immunity" we mean that administration to an
individual of one or more proteins, polypeptides or peptides of the
invention, or nucleic acids encoding them, either alone or in
combination with other antigenic entities prevents the development
of disease symptoms in that individual after exposure to or
infection by a parasite. Alternatively, the disease symptoms that
develop in the individual may be milder than those that would
otherwise develop in, for example, a matched control individual.
Those of skill in the art are well acquainted with the use and
meaning of "controls" when comparing results of individuals or
populations that have been exposed to different variables (e.g.
vaccinated or not). In particular, the inhibitory epitope peptide
of the invention may be used in combination with one or more other
antigenic epitopes for the production of a multicomponent vaccine.
Such a vaccine addresses previous lackluster vaccine performance by
presenting several highly immunogenic epitopes to the immune system
of a vaccinated individual in a single preparation. This type of
vaccine closely mimics the natural in vivo presentation of antigens
on the surface of a parasite, and thus elicits a robust immune
response.
[0078] According to an embodiment of the invention, the vaccine may
either be prophylactic (i.e. to prevent or attenuate symptoms of
infection) or therapeutic (i.e. to treat disease after infection).
Such vaccines comprise one or more of: immunizing antigen(s),
immunogen(s), polypeptide(s), protein(s) and nucleic acid(s) from
the FRAP family (as described herein), usually in combination with
"pharmaceutically acceptable carriers," which include any carrier
that does not itself induce the production of antibodies harmful to
the individual receiving the composition. Suitable carriers are
typically large, slowly metabolized macromolecules such as
proteins, polysaccharides, polylactic acids, polyglycolic acids,
polymeric amino acids, amino acid copolymers, lipid aggregates
(such as oil droplets or liposomes), and inactive virus particles.
Such carriers are well known to those of ordinary skill in the art.
Additionally, these carriers may function as immunostimulating
agents ("adjuvants"). Furthermore, the antigen or immunogen may be
conjugated to a bacterial toxoid, such as a toxoid from diphtheria,
tetanus, cholera, H. pylori, etc. pathogens. Preferred adjuvants to
enhance effectiveness of the composition include, but are not
limited to: (I) aluminum salts (alum), such as aluminum hydroxide,
aluminum phosphate, aluminum sulfate, etc; (2) oil-in-water
emulsion formulations (with or without other specific
immunostimulating agents such as muramyl peptides (see below) or
bacterial cell wall components), such as for example (a) MF59.TM.
(WO 90/14837; Chapter 10 in Vaccine design: the subunit and
adjuvant approach, eds. Powell & Newman, Plenum Press 1995),
containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally
containing various amounts of MTP-PE (see below), although not
required) formulated into submicron particles using a
microfluidizer such as Model 100Y microfluidizer (Microfluidics,
Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5%
pluronic-blocked polymer L121, and thr-MDP (see below) either
microfluidized into a submicron emulsion or vortexed to generate a
larger particle size emulsion, and (c) Ribi.TM. adjuvant system
(RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene,
0.2% Tween 80, and one or more bacterial cell wall components from
the group consisting of monophosphorylipid A (MPL), trehalose
dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS
(Detox.TM.); (3) saponin adjuvants, such as Stimulon.TM. (Cambridge
Bioscience, Worcester, Mass.) may be used or particles generated
therefrom such as ISCOMs (immunostimulating complexes); (4)
Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant
(IFA); (5) cytokines, such as interleukins (eg. IL-1, IL-2, IL-4,
IL-5, IL-6, IL-7, IL-12, etc.), interferons (eg. gamma interferon),
macrophage colony stimulating factor (M-CSF), tumor necrosis
factor, etc; and (6) other substances that act as immunostimulating
agents to enhance the effectiveness of the composition. Alum and
MF59.TM. are preferred.
[0079] The immunogenic compositions (eg. the immunizing
antigen/immunogen/polypeptide/protein/nucleic acid,
pharmaceutically acceptable carrier, and adjuvant) typically will
contain diluents, such as water, saline, glycerol, ethanol, etc.
Additionally, auxiliary substances, such as wetting or emulsifying
agents, pH buffering substances, and the like, may be present in
such vehicles. Typically, the immunogenic compositions are prepared
as injectables, either as liquid solutions or suspensions; solid
forms suitable for solution in, or suspension in, liquid vehicles
prior to injection may also be prepared. The preparation also may
be emulsified or encapsulated in liposomes for enhanced adjuvant
effect, as discussed above under pharmaceutically acceptable
carriers.
[0080] Immunogenic compositions used as vaccines comprise an
immunologically effective amount of the antigenic or immunogenic
polypeptides, as well as any other of the above-mentioned
components, as needed. By "immunologically effective amount", it is
meant that the administration of that amount to an individual,
either in a single dose or as part of a series, is effective for
eliciting the production of antibodies, for eliciting a cellular
immune response, (or both), and/or for treatment or prevention of
disease. This amount varies depending upon the health and physical
condition of the individual to be treated, the taxonomic group of
individual to be treated (e.g. nonhuman primate, primate, etc.),
the capacity of the individual's immune system to synthesize
antibodies, the degree of protection desired, the formulation of
the vaccine, the treating doctor's assessment of the medical
situation, and other relevant factors. It is expected that the
amount will fall in a relatively broad range that can be determined
through routine trials. The immunogenic compositions are
conventionally administered parenterally, eg. by injection, either
subcutaneously, intramuscularly, intranasally, or
transdermally/transcutaneously. Additional formulations suitable
for other modes of administration include oral and pulmonary
formulations, suppositories, and transdermal applications. Dosage
treatment may be a single dose schedule or a multiple dose
schedule. The vaccine may be administered in conjunction with other
immunoregulatory agents. As an alternative to protein-based
vaccines, DNA vaccination may be employed [eg. Robinson &
Torres (1997) Seminars in Immunology 9:271-283; Donnelly et al.
(1997) Annu Rev Immunol 15:617-648].
[0081] Vaccines can be composed of live, attenuated or killed
organisms, or chemically inactivated toxins (toxoids), against
which the body can raise an effective immune response, leading to
effective protection against the live agent or active toxins
produced during the infection. Combination vaccines make it
possible to immunize individuals against multiple pathogens at a
time. Examples of combination vaccines are DTaP (Diphtheria,
Tetanus, combined with acellular Pertussis) or MMR (Measles, Mumps,
and Rubella). Conjugated vaccines, such as PCV (Pneumococcal
Conjugated Vaccine) provide better immunization of infants. In
conjugated vaccines polysaccharide antigens are chemically linked
to protein antigens which provide a better stimulus for the
immature immune system. Through the use of recombinant DNA
technology it is possible to isolate and express individual genes
or combinations of genes, encoding antigens from pathogens and
produce vaccines by fermentation. Recent advances in genomics and
proteomics of (re-)emerging pathogens will enable entirely new
generations of vaccine based on identification of surface proteins.
Table 1 lists common types of vaccines in current use or in
development, and some important attributes. TABLE-US-00001 TABLE 1
Vaccine types in current use and development Type Vaccine
Advantages Disadvantages Live, attenuated Measles, mumps, Produce a
strong Remote possibility vaccines rubella, polio (Sabin immune
response; that the live microbe vaccine), yellow often give
lifelong could mutate back to fever immunity with one a virulent
form; or two doses must be refrigerated to stay potent Inactivated
or Cholera, flu, Safer and more Produce a weaker "killed" vaccines
hepatitis A, Japanese stable than live immune response
encephalitis, plague, vaccines; don't than live vaccines; polio
(Salk vaccine), require refrigeration; usually require rabies more
easily stored, additional doses transported Toxoid vaccine
Diphtheria, tetanus Teaches immune Protect only against system to
fight off deleterious effect of bacterial toxins; toxin, but do not
often easy to provide protection produce from pathogen Subunit
vaccines Hepatitis B, Targeted to very When developing a pertussis,
specific parts of the new vaccine, pneumonia caused microbe; fewer
identifying the best by Streptococcus antigens, so lower antigens
can be pneumoniae chance of adverse difficult and time reactions
consuming Conjugate vaccines Haemophilus Allow infant influenzae
type B, immune systems to pneumonia caused recognize certain by
Streptococcus antigens pneumoniae DNA vaccines In development
Produce a strong Still in experimental antibody and stages cellular
immune response; relatively easy and inexpensive to produce
Recombinant vector In development Closely mimic a Still in
experimental vaccines natural infection, stages stimulating a
strong immune response
Source: Understanding Vaccines: What they are, how they work. U.S.
DHHS/NIH/NIAID, NIH Publication No. 03-4219, 2003.
[0082] Most vaccines in Table 1 are administered by subcutaneous or
intramuscular injection. The oral route of administration is
occasionally used in case of Oral Polio Vaccine. New vaccine
technology is being developed to produce vaccines that (i) generate
stronger and broader immunity, (ii) meet more stringent safety and
quality requirements, and (iii) that have greater ease of delivery
at lower cost. Therefore, a significant amount of research is
ongoing to develop new delivery methods and adjuvants. For
effective immunization most vaccines are delivered using adjuvants.
Adjuvants are emulsions or formulations, often containing lipids or
aluminum salts, which provide for slow release of the antigen into
the plasma, and also stimulate the immune response in ways that are
not fully understood. Slow release of the antigen is also important
to prevent metabolism and removal from the plasma prior to the
initiation of the immune response. Delivery of antigen to the cells
that participate in antigen presentation, macrophages and dendritic
cells, is also improved by the use of adjuvants. Table 2 lists a
number of commonly used adjuvants and new adjuvant delivery methods
in development. TABLE-US-00002 TABLE 2 Commonly used adjuvants and
new products in development. Adjuvant Category New product or
method Comments/Examples Gel type Aluminium hydroxide/phosphate
Improve delivery to APCs and Calcium phosphate secondary lymphoid
organs Microbial Muramyl dipeptide (MDP) Bacterial exotoxins
Cholera toxin (CT) Endotoxin based adjuvants Escherichia coli heat
labile toxin (LT) Monophosphoryl lipid A (MLA) Particulate
Biodegradable polymer microspheres Immuno-stimulatory complexes
(ISCOMs) Liposomes Oil emulsion/ Freunds incomplete adjuvant Animal
experimental uses only surfactant Microfluidized emulsions MF59
(Squalene), SAF Saponins Qs-21 Synthetic Muramyl peptide
derivatives Murabutide, Threonyl-MDP Non-ionic block co-polymers
L121 Polyphosphazene (PCPP) Cytokines Interleukin-2, -12 Molecules
secreted by GM-CSF macrophages or dendritic cells Interferon gamma
that stimulate the inflammatory and immune response Genetic Genes
encoding cytokines or co- IL-12, IL-2, IFNg, CD40L stimulatory
molecules delivered by plasmids
Sources: Progress in Immunologic Adjuvant Development 1982-2002,
The Jordan Report 2002, US DHHS/NIH/NIAID, and the website located
at www.niaid.nih.gov/daids/vaccine/pdf/compendium.pdf.
[0083] New physical administration methods being developed include
delivery by inhalation, oral delivery, or transdermal delivery.
Inhalation delivery includes intranasal delivery for delivery to
the upper respiratory tract, which is being used in FluMist
(influenza vaccine) or other powder or particle based methods to
deliver immunization to the lower respiratory tract. Oral delivery
includes new formulations to allow antigens to pass through the
stomach and intestinal tract without acid or protease inactivation.
New methods of oral delivery include edible vaccines, where plants
such as potatoes, tomatoes, or bananas are genetically engineered
to express the antigen in parts of the plant that are consumed by
humans. New transdermal delivery methods that avoid injection are
being explored as well. However the large size (high molecular
weight) of the antigen(s) usually is a limitation for this delivery
method. A relatively new delivery method is expression of antigens
in a strain of virus or a bacterium that is not naturally
pathogenic, or is made avirulent either through mutation or genetic
engineering. Attenuated viruses such as polio, or bacteria such as
Vibrio cholerae and Salmonella typhi, are being explored as
delivery vehicles.
[0084] Production methods for vaccines vary with the type of
vaccine. Live, attenuated or killed virus vaccines are produced in
mammalian cell culture. In the latter case virus particles are
killed by chemical inactivation, heat or radiation. A major concern
of mammalian cell culture based production methods is contamination
with other pathogens, specifically retroviruses such as HIV, or
other as of yet uncharacterized mammalian viruses. Influenza
vaccine is produced either through cell culture or growth of virus
in fertilized chicken eggs, followed by purification from the yolk.
Live, attenuated or killed bacterial vaccines are produced by
microbial fermentation. Concerns with this method are contamination
with other micro-organisms (bio-burden), or presence of bacterial
endo- or exo-toxins that can cause anaphylactic shock. Toxoid
vaccines, such as diphtheria or tetanus vaccines, are produced by
microbial fermentation and harvesting of the exo-toxins from the
culture medium. Toxoid vaccines can also be produced with
recombinant DNA technology, followed by purification of the
recombinant protein. Conjugated vaccine components are produced
through multiple methods. The polysaccharide component is harvested
from bacteria grown in culture, and the protein component of the
antigen can be produced through fermentation or recombinant DNA
technology. The conjugation step is done through a chemical
reaction. Subunit vaccines, existing of specific protein antigens
(or combinations) are made through fermentation or recombinant DNA
technology. Other transgenic production methods, such as expression
in the milk of transgenic animals, or production in genetically
engineered plants, are being explored for subunit vaccines as well.
DNA vaccines are produced using recombinant DNA technology. Vector
vaccines are produced through genetic engineering of the vector,
i.e. to produce the antigens of interest, and either microbial
fermentation or mammalian cell culture.
[0085] In particular, with respect to DNA vaccines, U.S. Pat. No.
6,214,804 (Felgner, et al., 2001, the complete contents of which is
hereby incorporated by reference) describes the induction of a
protective immune response in a mammal by injecting a DNA sequence.
Methods for delivering an isolated polynucleotide to the interior
of a cell in a vertebrate are provided. The methods can be used to
deliver a therapeutic polypeptide to the cells of the vertebrate,
to provide an immune response upon in vivo translation of the
polynucleotide, to deliver antisense polynucleotides, to deliver
receptors to the cells of the vertebrate, or to provide transitory
gene therapy.
[0086] In addition, U.S. Pat. No. 6,923,958 (Xiang et al., 2005,
the complete contents of which is hereby incorporated by reference)
describes DNA vaccines encoding carcinoembryonic antigen (CEA) and
a CD40 ligand and methods of their use. The DNA vaccine is
effective for eliciting an immune response against cells that
present a carcinoembryonic antigen, and could be incorporated in a
delivery vector such as an attenuated live bacterium or virus, or a
liposome carrier. Alternatively, the DNA vaccine is administered
orally to a mammal, such as a human, to elicit an immune response
against CEA presenting cells such as colon cancer cells. The mammal
may be further treated with recombinant antibody fusion proteins to
enhance the immune response effectiveness of the vaccine.
[0087] Another embodiment of the invention provides antibodies
specific for FRAP proteins, polypeptides and peptides. As used
herein, the term "antibody" refers to a polypeptide or group of
polypeptides composed of at least one antibody combining site. An
"antibody combining site" is the three-dimensional binding space
with an internal surface shape and charge distribution
complementary to the features of an epitope of an antigen, which
allows binding of the antibody with the antigen. "Antibody"
includes, for example, vertebrate antibodies, hybrid antibodies,
chimeric antibodies, humanised antibodies, fully human antibodies,
altered antibodies, univalent antibodies, Fab proteins and
fragments, and single domain antibodies. Antibodies to the
polypeptides and peptides of the invention, both polyclonal and
monoclonal, may be prepared by conventional methods that are
well-known to those of skill in the art. If desired, the antibodies
(whether polyclonal or monoclonal) may also be labeled using
conventional techniques.
[0088] Antibodies for therapeutic applications for the prevention
or treatment of malarial disease, or diagnostic applications in the
detection of parasite infection, can be made by standard methods.
In most cases the antibodies will be of monoclonal origin, and
either produced in rats or mice.
[0089] Protein for immunization is made by recombinant methods. Any
of the proteins from the group of SEQ ID NOS: 1, 3, 5, 7, 9, 11,
13, 15, 17, and 19, or portions thereof, can be produced by cloning
the corresponding DNA sequences of SEQ ID NOS: 2, 4, 6, 8, 10, 12,
14, 16, 18, and 20, or portions thereof, in recombinant protein
expression vectors. Protein can be produced in this manner in E.
coli, yeast, fungi, plants, mammalian, or insect cells. It is
obvious that the preferred protein used for immunization is from
the Plasmodium species that infect humans, i.e. SEQ ID NOS: 1 and
7. However, in principle SEQ ID NOS: 3, 5, 9, 11, 13 and 15, could
also be used to generate antibodies that are effective as
therapeutics or diagnostic tools. Immunization material for short
peptides and small proteins can also be made through chemical
synthesis.
[0090] For example the 8-mer peptide represented by SEQ ID NO: 37
may be encoded by: ACCAACGACCC AGAAAGTATAAAT (SEQ ID 38), or other
sequences; and the 32-mer peptide represented by SEQ ID NO: 24 may
be encoded by: ACACGAAGTGGCGGTTTAAGAAAACCTCAAAAGG
TAACCAACGACCCAGAAA GT ATAAATAGAAAAGTATATTGGTGTTTTGAACATAA GCCTGTA
(SEQ ID 39), or other sequences. Alternatively, the these peptides
may be chemically synthesized.
[0091] Expressed protein can be purified with standard HPLC and
other chromatographic methods, in quantities and sufficient purity
to be injected in the mice or standard rats. Rats or mice are
injected in the presence of adjuvants, and in a standard schedule
of injections and boosters, in order to generate a vigorous immune
response. In order to make monoclonal antibodies, spleen cells are
harvested from the animals and fused with immortalized cell lines.
Numerous immortalized cell lines are screened for their ability to
secrete antibodies that bind the original antigen used in
immunizations. Positive cell lines are purified and cloned, and
their antibodies are characterized and screened to identify
antibodies that have strong binding characteristics. Upon
identification of such cell lines, the antibody genes are cloned,
sequenced and can be used to engineer mammalian cell culture
strains for high level production.
[0092] In order to avoid a human immune response against the
therapeutic antibody, the sequence of the monoclonal antibody is
modified to most closely resemble the sequence of native human
antibodies. This is done by recombinant DNA methods, through
selective replacement of the significant portions of the munine
antibody light and heavy chain sequences with human sequences
(chimeras), or through replacement of almost all of the
non-variable sections of the murine antibody light and heavy
chains, with those from human antibody chain conserved sequences,
while maintaining the original rat or mouse sequence of the
hyper-variable domain which is responsible for antigen recognition
and binding (`CDR grafting` or `humanization`). For example U.S.
Pat. No. 6,500,931 describes the method of humanizing
antibodies.
[0093] Alternatively, fully human monoclonal antibodies can be made
in mice directly, when these mice are engineered to produce only
human antibody chains. For example the technology practiced by
companies such as Abgenix Inc. [XenoMouse technology, U.S. Pat. No.
6,657,103], Medarex Inc. and GenMab A/S [HuMab Mouse or UltiMAB
technology; WO2005023177] can be used. Purified proteins as
described above are used to immunize such engineered mice.
Monoclonals produced in this manner are produced, screened and
characterized in the standard manner. Fully human antibodies can
also be produced using phage display methods by screening against
human antibody phage display libraries. For example technologies
practiced by companies such as Cambridge Antibody Technology [U.S.
Pat. No. 5,969,108 and U.S. Pat. No. 6,172,197] and others, can be
used to identify fully human antibodies in this manner. Phage
display screening has an added advantage in that the process does
not rely on animal immunization. The genes for fully human
antibodies produced using engineered mice, or identified through
phage display, can be isolated, sequenced and cloned for expression
in mammalian cell lines for high level expression using standard
methods.
[0094] Patents describing this technology in detail are
incorporated herein by reference.
[0095] Such antibodies may be used, for example, for affinity
chromatography, immunoassays, and for distinguishing or identifying
parasite proteins or portions thereof. In a preferred embodiment of
the invention, such antibodies may be used therapeutically, e.g.
for administration to patients suffering from a parasitic disease
such as malaria, or prophylactically in order to prevent a
parasitic disease in patients at risk for developing the
disease.
[0096] In yet another embodiments of the invention cells or cell
lines containing the nucleic acids and/or the amino acid sequences
of the invention as described herein. For example, the cell may be
a host cell that harbors one or more vectors containing nucleic
acid sequences used in the invention (e.g. DNA or RNA) and/or amino
acid sequences of the invention translated from such vectors. Such
cells may contain multiple vectors, and the vectors may be the same
or different. Further, the cells may be either in vitro or in vivo.
The invention also comprehends pharmaceutical compositions and
their use. The pharmaceutical compositions can comprise one or more
proteins, polypeptides, peptides, antibodies, or nucleic acids
according to the invention, or combinations of these. In addition,
the compositions may include compounds that inhibit the interaction
of HDP and heme, thereby preventing or vitiating the ability of HDP
to detoxify heme, e.g. to form hemozoin from heme. The
pharmaceutical compositions comprise a therapeutically effective
amount of such molecules. The term "therapeutically effective
amount" as used herein refers to an amount of a therapeutic agent
that is sufficient to treat, ameliorate, or prevent a disease or
condition, or to exhibit a detectable therapeutic or preventative
effect. The effect can be detected by, for example, chemical
markers or antigen levels. Therapeutic effects also include
reduction of physical symptoms of the parasitic disease. The
precise effective amount for a subject will depend upon several
parameters, including the subject's size, general health, gender,
age, etc., and the therapeutics or combination of therapeutics
selected for administration. Thus, it is not useful to specify an
exact effective amount in advance. However, the effective amount
for a given situation can be determined by routine experimentation
and is within the judgement of those of skill in the art, e.g. a
physician. For purposes of the present invention, an effective dose
will be from about 0.01 mg/kg to 50 mg/kg or about 0.05 mg/kg to
about 10 mg/kg of active, therapeutic agent.
[0097] A pharmaceutical composition may also contain a
pharmaceutically acceptable carrier. The term "pharmaceutically
acceptable carrier" refers to a carrier for administration of a
therapeutic agent, such as antibodies or a polypeptide, genes,
inhibitory compounds, and other therapeutic agents. Suitable
carriers may be large, slowly metabolized macromolecules such as
proteins, polysaccharides, polylactic acids, polyglycolic acids,
polymeric amino acids, amino acid copolymers, and inactive virus
particles. Such carriers are well known to those of ordinary skill
in the art. Pharmaceutically acceptable salts can be used therein,
for example, mineral acid salts such as hydrochlorides,
hydrobromides, phosphates, sulfates, and the like; and the salts of
organic acids such as acetates, propionates, malonates, benzoates,
and the like. A thorough discussion of pharmaceutically acceptable
excipients is available in Remington's Pharmaceutical Sciences
(Mack Pub. Co., N.J. 1991).
[0098] In addition, pharmaceutically acceptable carriers in
therapeutic compositions may contain liquids such as water, saline,
glycerol and ethanol. Additionally, auxiliary substances, such as
wetting or emulsifying agents, pH buffering substances, adjuvants,
and the like, may be present in such vehicles. Typically, the
therapeutic compositions are prepared as injectables, either as
liquid solutions or suspensions; solid forms suitable for solution
in, or suspension in, liquid vehicles prior to injection may also
be prepared. Liposomes are included within the definition of a
pharmaceutically acceptable carrier.
[0099] Once formulated, the compositions of the invention are
administered to the subject. The subjects to be treated may be
animals; in particular, human subjects can be treated. Direct
delivery of the compositions will generally be accomplished by
injection, either subcutaneously, intraperitoneally, intravenously
or intramuscularly or delivered to the interstitial space of a
tissue. Other modes of administration include oral and pulmonary
administration, suppositories, and intranasal, transdermal or
transcutaneous applications (eg. see WO98/20734), needles, and gene
guns or hyposprays. Dosage treatment may be a single dose schedule
or a multiple dose schedule.
[0100] Yet another embodiment of the invention provides tools and
methods for the diagnosis of parasitic infections. Such tools
include primers containing nucleotide sequences that specifically
hybridize to nucleic acid sequences that are unique to FRAP.
Hybridization of the primers to such a unique sequence permits
amplification of the unique sequence (for example, by polymerase
chain reaction (PCR)), thus providing a means to specifically
identify the presence of FRAP in biological samples (blood, feces,
sputum, urine, bronchoaveloar lavage, etc.). Amplification may be
directly from the genome of the organism located in the sample, or
from RNA, e.g. mRNA.
[0101] By "primer" we mean a nucleotide sequence that hybridizes to
another nucleotide sequence of interest, the primer typically being
a relatively short nucleotide sequence (e.g. from about 10 to about
100 base pairs) and the nucleotide sequence of interest typically
being transcribed from the genome of an organism. PCR amplification
techniques are well-known to those of skill in the art. In general,
two primers are selected that target sites that flank the sequence
of interest (e.g. a gene encoding FRAP) for diagnostics or
identification. These primers are designed to recognize only the
target sequence; i.e., they will hybridize only to the target
sequence and to no other sequences. The primers generally range
from 18-nucleotides in length (but can be longer or shorter), have
Tm's (melting temperatures) that are selected to be compatible with
both amplification conditions and with specificity, have little or
no internal structure (stem-loop structures caused by internal
complementarity), little or no ability to dimerize with themselves,
little or no ability to dimerize with the other primer, have few
homopolymeric stretches, etc. Many computer programs (e.g.,
Primer3, Oligo, etc.) are available for primer design. At times, an
internal fluorescent probe is also included for specific use in
even more sensitive and automated tests. The internal probe is
fluorescently labeled such that it is specifically degraded and
therefore fluoresces only if it specifically hybridizes to the
target sequence. Alternately, other fluorescent probes can be
designed that only fluoresce upon binding specifically to an
amplified specific sequence. Thus, several alternative approaches
are available for the generation and detection of specific
sequences amplified by PCR, and any of these can be applied for
diagnostic or identification purposes. (See, for example: Mullis,
K., F. Faloona, S. Scharf, R. Saki, G. Horn, and H. Erlich. (1986)
Specific enzymatic amplication of DNA in vitro: The Polymerase
Chain Reaction. Cold Spring Harbor Symposia on Quantitative Biology
51: 263; Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R.
Higuchi, G. T. Horn, K. B. Mullis, and H. A. Erlich. (1988)
Primer-directed enzymatic amplification of DNA with a thernostable
DNA polymerase. Science 239:487; Schutzbank T E, Stern H J. (1993)
Principles and applications of the polymerase chain reaction. J Int
Fed Clin Chem. 1993 July;5(3):96-105; Erlich H A. (1999) Principles
and applications of the polymerase chain reaction. Rev Immunogenet.
1(2):127-34; Wang, A. M., Doyle, M. V., and D. F. Mark. (1989)
Quantitation of mRNA by the polymerase chain reaction. Proc Natl
Acad Sci USA. 1989 December; 86(24): 9717-9721; Kawasaki, E. S.,
and A. M. Wang. (1989) Detection of gene expression. In: Erlich, H.
A., ed., PCR Technology: Principles and Applications of DNA
Amplification. Stockton Press, Inc., New York, N.Y., pp. 89-97;
Dieter Klein (2002) Quantification using real-time PCR technology:
applications and limitations. Trends in Molecular Medicine,
8(6):257-260; Buck GE. (1996) The polymerase chain reaction: a
revolutionary new procedure for the laboratory diagnosis of
infectious disease. J Ky Med Assoc. Apr; 94(4):148-52.)
[0102] Because the primers are unique to FRAP, a positive
amplification result is indicative of the presence of FRAP in the
biological sample, and thus of infection by a parasite whose genome
encodes FRAP. Similar tests can be carried out with antibodies
specific for FRAP. In this case, a positive result indicates that
the biological sample being tested contains FRAP, and thus, by
inference, the individual from whom the sample was obtained is
infected with a parasite that produced FRAP.
[0103] The invention further provides methods for treating or
preventing a disease caused by a Plasmodium or Theileria parasite
in an individual in need thereof. In one embodiment, this is
accomplished by inhibiting one or more interactions of heme and
Heme Detoxification Protein (HDP). Typically, inhibition is brought
about by the administration of one or more compounds that inhibit
one or more interactions of heme and HDP. In other words, the
ability of HDP to produce hemozoin from heme (i.e. to detoxify
heme) is eliminated or impaired by administration of the compound.
Examples of diseases that can be treated in this manner include but
are not limited to malaria, East Coast Fever caused by Theileria
parasites, etc. Exemplary compounds that may be used in such
methods are listed in Table 11 in the Examples section below. One
or more compounds from one or more of these classes may be
administered, in a quantity sufficient to prevent or ameliorate
disease symptoms.
[0104] Those of skill in the art will recognize that the mechanism
of action of the compounds that are administered can be any of many
known or not yet elucidated types, and that the precise
mechanism(s) will depend on the compound(s) administered. For
example, the compound may bind to the HDP enzyme and prevent the
enzyme from binding to heme. Alternatively, the compound may bind
to HDP and allow heme to also bind to HDP, but prevent further
catalysis and the production of hemozoin. For example, the compound
may bind at the active site or near the active site and sterically
prevent the binding of heme; or the compound may bind at an
allosteric site that influences (e.g. decreases) the activity of
the enzyme; or the compound may cause heme to bind to HDP
irreversibly or with so great an affinity that the ability of HDP
to detoxify heme is eliminated or attenuated. Alternatively, the
compound may bind to heme. In this case, binding of the compound to
heme may prevent the heme from then binding to HDP, or may allow
the heme-compound complex to bind but not be further processed to
hemozoin. Those of skill in the art will recognize that in all
cases, the binding of compounds to HDP or to heme may be reversible
or irreversible, realizing that all binding events involve an
equilibrium distribution of bound and free agents. The criteria for
the use of a compound in the present invention is that the
compound, regardless of its mechanism of action, decrease the
production of hemozoin from heme by at least about 10 to 25%,
preferably from about 25 to 50%, and more preferably from about 50
to 75%, or even from about 75 to 10%.
[0105] Other possible mechanisms of action of the compounds that
are administered include but are not limited to: modification of a
cell membrane of the Plasmodium or Theileria parasite; inhibiting
secretion of HDP from the Plasmodium or Theileria parasite,
inhibiting transport of HDP to the food vacuole, the site of
hemozoin formatin; by binding to free heme (the substrate of HDP)
and preventing its detoxification into Hemozoin; etc.
[0106] The administration of the compound(s) may be carried out by
any suitable means, examples of which include but are not limited
to orally, parenterally, sublingually, rectally, topically or with
an inhalation spray.
[0107] In a preferred embodiment of the invention, the disease that
is prevented or treated is malaria. In this case, the compound that
is administered may be administered in combination with one or more
additional agents such as other antimalarial agents, agents for
reversing antimalarial resistance, and various adjuvants.
Administration of one or more additional antimalarial agents or
agents for reversing antimalarial resistance may occur prior to,
concurrent with, or subsequent to administration of the compound.
Exemplary additional antimalarial agents include but are not
limited to quinolines, folic acid antagonists, sulfonamides, and
antibiotics. An exemplary agent for reversing antimalarial
resistance is an inhibitor of multidrug resistance. Exemplary
adjuvants include but are not limited to those which are suggested
above for use in vaccine preparations, e.g. alum, etc.
[0108] The invention further comprehends pharmaceutical
compositions comprising a pharmaceutically acceptable carrier and
an antimalarially effective amount of at least one compound. By
"antimalarially effective amount" we mean that the compound is
present in the composition in amount that, upon administration to
an individual in need, prevents or lessens the occurrence of
symptoms associated with malaria in the recipient. Such
compositions may include other active agents as well, e.g.
adjuvants, other antimalarial agents (quinolines, etc.), agents
that reverse resistance to malaria, etc.
[0109] The invention also provides method of inhibiting heme
detoxification in a Plasmodium or Theileria parasite by preventing
or attenuating the production of hemozoin by HDP in the parasite.
Those of skill in the art will recognize that various routes of
inhibition may be effective. For example: inhibiting interaction of
heme and HDP; preventing an interaction of HDP or heme with
cofactors; preventing dimerization of HDP; preventing interaction
of HDP or heme with lipids; and others. Exemplary cofactors, the
interaction of which with HDP or heme may be disrupted, include but
are not limited to metal ions, natural ligands and protein
factors.
[0110] The invention also provides methods for identifying
compounds that inhibit HDP expression. The methods include the
steps of a) contacting Plasmodium with a test compound and b)
determining whether HDP is expressed by the Plasmodium. Those of
skill in the art will recognize that there are several suitable
methods to evaluate the outcome of such tests, including but not
limited to measuring mRNA that encodes HDP, measuring HDP protein
production directly (i.e. detecting and measuring the protein
itself), etc.
[0111] The following Examples describe: the discovery and
characterization of the novel FRAP protein family; the expression,
localization and purification of recombinantly expressed FRAP; the
generation of antibodies to FRAP2; experiments demonstrating the
binding of FRAP to liver cells; prevention of sporozoite invasion
of liver cells by FRAP and antibodies to FRAP2; discovery of the
inhibitory epitope of FRAP; FRAP as a drug target; the use of FRAP
in high throughput assays for hemozoin formation for screening
novel antimalarials; siRNA mediated inhibition of FRAP; the
creation of FRAP variant attenuated parasites for use as whole
organism vaccines; and the use of FRAP as a tool for high levels of
expression and purification of recombinant proteins; the screening
of compounds that inhibit HDP; the results of in vivo testing of
DNA that encodes HDP as a vaccine.
[0112] While the invention has been described in terms of its
preferred embodiments, those skilled in the art will recognize that
the invention can be practiced with modification within the spirit
and scope of the appended claims. Accordingly, the present
invention should not be limited to the embodiments as described
above and in the Examples section below, but should further include
all modifications and equivalents thereof within the spirit and
scope of the description provided herein.
EXAMPLES
Example 1
Discovery and Characterization of a Novel Plasmodium falciparum
Protein Involved in Malaria Pathogenesis
[0113] Plasmodium sporozoites adhere to and invade host liver
cells, leading to the onset of malaria. Here we describe a novel,
205 amino acids long, Plasmodium falciparum protein involved in
sporozoite-liver cell interactions. Orthologs of this protein were
identified in seven other Plasmodium species, representing the four
distinct phylogenetic clades, and the protein showed 60% sequence
identity within the genus. Additionally, amino acids 88-205 have a
20% sequence identity to fasciclin 1, an ancient adhesive domain
found in prokaryotes, plants and animal proteins. The DNA encoding
the protein was cloned, expressed in E. coli and the protein was
purified to homogeneity. Immunoelectron microscopy showed that the
protein was localized in the micronemes of the sporozoites. The
protein contributes to sporozoite's adhesion and invasion
activities and antibodies raised against this protein can prevent
>94% of P. falciparum sporozoites from invading liver cells,
thus suggesting a role for this protein in malaria pathogenesis.
Furthermore, we provide evidence that the protein exploits heparan
sulfate proteoglycans expressed on the liver cell surface as its
receptor. Due to its role in host cell adhesion and the presence of
fasciclin 1 domain, we have named this protein as Fasciclin Related
Adhesive Protein or FRAP. Our results show that FRAP is an
excellent target for malaria vaccine development.
[0114] A bite by a parasite-infected mosquito delivers Plasmodium
sporozoites in the blood stream, which is followed by its entry
into the liver cells. A successful adhesion and invasion of liver
cells by the parasite sets the stage for rapid multiplication,
development and subsequent release of parasites in circulation,
leading to the erythrocytic infection and the clinical pathology
associated with malaria. It is widely believed that the host cell
adhesion and invasion is a multistep process involving several
parasitic proteins, many of which are currently not known. Of
these, Circumsporozoite (CS) and Sporozoite Surface
Protein-2/Thrombospondin-Related Anonymous Protein (SSP2/TRAP),
have been extensively investigated (1, 2). Across pathosystems,
proteins involved in host-pathogen interactions are the molecules
of choice for vaccine development. Likewise, CS and SSP2/TRAP have
become major targets for intervention and are being actively
pursued as vaccine candidates (3-6). While the results from these
trials have been encouraging, they have revealed that the
immunological protection against malaria is not conferred due to a
dominant immune response against a single antigen but is mediated
by the summation of many modest humoral and cell-mediated immune
responses against a large variety of known and unknown antigens
(7). Therefore, identification of malarial proteins that are
involved in disease pathogenesis will not only lead to a better
understanding of the disease process, but is also vital for the
development of a successful vaccine against malaria. With the
availability of the genome sequence and proteome analysis of P.
falciparum parasites (8, 9), efforts are now being made to mine
this information for identification and characterization of
proteins that contribute towards pathogenesis (10).
[0115] In recent years, the concept of protein domains and domain
families has risen to greater prominence due to an increasing
realization that by organizing proteins sequences from distinct
organisms into domain families, one can often reliably predict
their molecular functions (11, 12). In case of pathogens,
identification of adhesive domain-containing proteins has played a
pivotal role in deciphering the mechanics of disease pathogenesis.
For example, the Plasmodium genome encodes several proteins that
contain an adhesive thrombospondin type I repeat (TSR) domain, most
of which have now been shown to be involved in host-parasite
interactions (1, 2, 10, 13). Therefore, identification and
characterization of parasite proteins containing adhesive domains
will improve our understanding of the disease process and here we
describe a novel malarial protein that encodes a single fasciclin 1
(FAS1) domain.
[0116] FAS1 is an adhesive domain named due to its initial
discovery in proteins involved in fasciculating axons and growth
cones (14). It is an ancient extracellular adhesive module found in
proteins of prokaryotic, plant and animal origin (15-18). Most of
the FAS1 domain-containing proteins possess multiple copies of the
domain, though proteins encoding only a single copy, have also been
identified (17). A large number of FAS1 domain containing proteins
have been reported in Drosophila and Grasshopper, where they are
involved in neuronal development (19, 20). In contrast, in humans,
FAS1 domains have been found in a large multi-domain scavenger
receptor protein on endothelial cells, involved in the removal of
hyaluronan from blood stream (21), as well as in extracellular
matrix protein, where they mediate corneal epithelial cell adhesion
(22). However, unlike many domains which show a high degree of
sequence conservation, FAS1 domains show huge sequence diversity;
typically have 20% sequence identity in a pairwise alignment (23)
and are recognized by only two short semi-conserved sequence motifs
(underlined in FIG. 3).
[0117] Here we describe a novel P. falciparum FAS1
domain-containing protein and its role in malaria infectivity
during sporozoite stage of the lifecycle. We demonstrate that the
protein contributes towards liver cell adhesion and invasion by the
parasite and have named it as Fasciclin Related Adhesive Protein or
FRAP.
Materials and Methods
[0118] Sequence analysis and identification of FRAP orthologs:
Sequences for P. falciparum (Accession #AAN37059), P. berghei
(Accession #CAH94515) and P. chaubaudi (Accession #CAH77280) FRAP
were obtained from GenBank, where they have been deposited as part
of the parasite genome sequencing projects (8, 24) Using P.
falciparum FRAP sequence, orthologs were identified from
unannotated genome sequences of P. gallinaceum, P. reichenowi, P.
vivax, P. yoelii and P. knowlesi parasites, available at PlasmoDB,
Sanger Center and TIGR web sites (25). FRAP orthologs from
Theileria parva (Accession #EAN32245) and T. annulata (Accession
#CA176887) were from the published genome sequence (26, 27). The
nucleic acid sequences of the genes are provided in FIG. 2A-J. The
amino acid sequences were aligned using Clustal W algorithm (28)
for multiple sequence alignment, using the DNASTAR package. The
amino acid sequences are depicted in FIG. 1, and the alignment is
given in FIG. 3.
[0119] Reverse Transcription, Amplification and Cloning of FRAP
proteins: Total RNA was obtained from highly purified preparations
of P. falciparum (3D7 strain) sporozoites (29). 2.mu.g of total RNA
was reverse transcribed and amplified with the forward 5'
CACCATGAAAAATAGATTTTATTATAATTTG 3' (SEQ ID NO: 22) and reverse 5'
AAAAATGATGGGCTTATCTACTATATG 3' (SEQ ID NO: 23) primers, using
Promega Access RT-PCR kit. The amplified fragment was cloned
inpET101-TOPO (Invitrogen) an E. coli expression vector containing
a C-terminal [His].sub.6 tag, giving rise to plasmid pFRAP. The
forward primers encoded a tetra nucleotide CACC, which facilitated
the directional cloning of amplified fragments in the expression
vector. The authenticity of the clone was verified by DNA
sequencing. Two other FRAP constructs, encoding amino acids 1-87
and 88-205 were generated by PCR-based subcloning using pFRAP as
template, giving rise to plasmid pFRAP2 and pFRAP3 respectively.
Authenticity of these constructs was verified by DNA sequencing.
Sequencing was performed at the core laboratory sequencing facility
of the Virginia Bioinformatics Institute.
[0120] Expression, localization and purification of recombinantly
expressed FRAP protein: For protein expression, E. coli BL21 cells
were transformed with a desired plasmid, grown in super broth, and
at the OD.sub.600=1.0, expression was induced with IPTG, at a final
concentration of 1 mM. Three hours post-induction, the culture was
harvested by centrifugation at 3000 g for 10 minutes. To identify
the intracellular site of accumulation of the protein, pellet was
resuspended in 20% sucrose solution in 20 mM Tris pH 7.5 and
incubated on ice for 10 min. Cells were spun at 5000 g for 20 min
and the pellet was resuspended in chilled water for 10 minutes.
This was followed by centrifugation at 8000 g for 20 minutes to
isolate periplasmic fluid. Spheroplast pellet was further processed
to isolate inclusion bodies, as previously described (30).
Inclusion bodies were solubilized in 1550 mM CAPS buffer containing
0.3% N-lauryl sarkosine and 0.3 M NaCl, pH 11.0 for 30 min and
centrifuged at 10000 g for 30 min at room temperature. The
supernatant was loaded onto a His-Trap High Performance affinity
column (GE Health Care) and bound protein was eluted using an
imidazole gradient in 50 mM CAPS pH 11.0 containing 0.3% N-lauryl
sarkosine and 0.3 M NaCl. Relevant fractions were pooled and
purified to homogeneity by gel filtration chromatography on
Superdex 200 10/300 GL column (GE Health Care). Authenticity of the
purified protein was verified by amino terminal sequencing and
western blotting using anti-polyhistidine tag monoclonal antibody.
For obtaining recombinant CS protein, pCS271IVC a plasmid with a
polyhistidine tag at the carboxyl terminus (1) was expressed in
BL21 E. coli cells and the protein was purified from the periplasm
as previously described (31).
[0121] Generation of anti-FRAP2 antibodies: The protocol for
antibody generation was approved by the animal care committee at
Virginia Tech. 6-8 weeks old female CD1 mice were subcutaneously
immunized with 10 .mu.g of purified FRAP2 emulsified in complete
Freunds adjuvant. Two subsequent booster doses in incomplete
Freunds adjuvant were administered on days 21 and 35, after the
first immunization. Sera were collected two weeks after the last
booster. Antibodies were purified on a Protein G affinity column
using AKTA FPLC chromatography system.
[0122] Confocal analysis: Purified P. falciparum sporozoites were
air dried on a glass slide. The slide was blocked with 5% normal
goat serum in phosphate buffer saline (PBS). Subsequently, the
slide was incubated with doubling dilutions (1:20 to 1:20480) of
anti-FRAP2 or pre immune mouse serum and incubated at room
temperature, in a humidified chamber, for one hour. Unbound
antibodies were removed by washing the slide with TBS containing
0.05% Tween 20 followed by the addition of an anti-mouse FITC
conjugate (1:500 dilution). Confocal imaging was performed using
BioRad Radiance confocal microscope.
[0123] Immunoelectron microscopy: Preparations of Plasmodium
falciparum-infected salivary glands were fixed in 4%
paraformaldehyde (Electron Microscopy Sciences, PA) in 0.25 M HEPES
(pH 7.4) for 1 hr at room temperature, then in 8% paraformaldehyde
in the same buffer overnight at 4.degree. C. They were infiltrated,
frozen and sectioned as previously described (32). The sections
were immunolabeled with mouse anti-FRAP antibodies (1:1000 in
PBS/1% fish skin gelatin), then with anti-mouse IgG antibodies,
followed directly by 10 nm protein A-gold particles (Department of
Cell Biology, Medical School, Utrecht University, the Netherlands)
before examination with a Philips CM120 Electron Microscope
(Eindhoven, the Netherlands) under 80 kV.
[0124] Liver Cell binding assay: The binding of proteins was
assayed on HepG2 cells as described previously (1, 31). Briefly,
cells were plated at a density of 25,000 cells/well, in a 96 well
plate, 36 hours before the start of the experiment. The cells were
fixed with paraformaldehyde, blocked with 1% BSA, followed by the
addition of equimolar concentrations of recombinant proteins. Bound
protein was detected using anti-polyhistidine tag monoclonal
antibody (1:10,000) and anti-mouse antibody conjugated to alkaline
phosphatase (1:2000). Amount of bound protein was detected by using
4-methylumbelliferyl phosphate, a fluorescent substrate, and
measurement of fluorescence using a fluorescent plate reader
(Molecular Devices, CA) with excitation and emission set at 350 nm
and 460 nm respectively. Results are shown as mean.+-.standard
deviation of mean of a representative experiment performed in
triplicate. Binding inhibition assays were performed by combining
the recombinant proteins with increasing amounts of
glycosaminoglycans and incubating at 37.degree. C. for 15 min. For
enzyme treatment, cells were incubated with different
concentrations of Heparinase I or Chondroitinase ABC for 90 minutes
at 37.degree. C. as previously described (31), before the addition
of proteins. The bound protein was assayed as described above.
[0125] All the proteins used in the binding assay possessed a
polyhistidine tag at their carboxyl terminus. Therefore, binding
activity was probed using a polyhistidine tag monoclonal antibody.
This excluded the possibility of misinterpretation of the data due
to differences in antibody affinities.
[0126] Sporozoite Invasion Assay: Invasion assay was performed with
HepG2 (Human hepatoma) cells as previously described (31). Briefly,
HepG2 cells were plated (50,000 cells/0.3 ml) and incubated
overnight at 37.degree. C. in a CO.sub.2 incubator. Next day,
medium was removed and 50 .mu.l of diluted FRAP proteins (final
concentrations: 20 and 10 .mu.g/ml) or anti-FRAP2 antibodies (40
.mu.g/ml final concentration) were added per well. Anti CS
monoclonal antibody NFS1 was used at a final concentration of 100
.mu.g/ml. All protein concentrations and serum dilutions were
evaluated in triplicate. This was immediately followed by the
addition of 20,000 sporozoites in 50 .mu.l of medium to each well.
P. falciparum (strain NF54) sporozoites were obtained from the
salivary glands of An. stephensi mosquitoes as described by Ozaki
(33). The sporozoites were allowed to invade liver cells for three
hours followed by the washing of cells with PBS at pH 7.4.
Subsequently, the cells were fixed with cold methanol. Sporozoites
were visualized by immunostaining using NFS1 as primary antibody
and anti-mouse IgG-peroxidase conjugate. The slides were mounted
with Paramount and only intracellular sporozoites were counted as
described (31). Percentage inhibition of invasion was calculated
with the following formula: [(Control-test)/control].times.100
Results
[0127] Identification and sequence analysis of FRAP: Analysis of
the published DNA sequence of chromosome 14 of P. falciparum (8)
identified a 824 nucleotide sequence (Accession #NP702335)
containing a hypothetical, single copy, three-exon gene, encoding a
205 amino acids long protein (FIG. 1, SEQ ID NO: 1).
Bioinformatical analysis of the predicted protein using the NCBI
conserved domain database (CDD) search tool (34), revealed that the
protein encodes a Fasciclin (FAS1) domain (SMART accession no.
SM00554) from amino acids 88-204 with an e-value of 2e-10. FIG. 1
depicts the FRAP protein sequence and its alignment with the
consensus sequence of FAS1 domain in the database. FAS1 domains are
known for their huge sequence diversity and typically have 20%
sequence identity in a pairwise alignment (23). They are recognized
by only two short semi-conserved sequence motifs (underlined in
FIG. 3). A similar pattern is seen in FRAP as its FAS1 domain has
21% sequence identity with the consensus sequence.
[0128] Using published, unpublished and unannotated sequences in
the databases for pathogens at Sanger, PlasmoDB and TIGR web sites,
P. falciparum FRAP orthologs were identified in all Plasmodial
species that have been sequenced till date or are currently
undergoing sequencing (FIG. 1). Orthologs of P. falciparum FRAP
were found in avian (P. gallinaceum), rodent (P. berghei, P. yoelii
and P. chaubaudi) simian (P. knowlesi and P. reichenowi) and human
(P. vivax) malaria parasites suggesting that the FRAP protein is
most likely present in all the members of Plasmodium genus and,
hence, could be playing an important role in the biology of the
parasite. Within the Plasmodium genus, the protein maintains a 60%
sequence identity (FIG. 3) with 124 out of 205 residues being
identical. Beyond Plasmodium, FRAP homologs were only found in the
two recently sequenced Theileria genomes (26, 27) with an overall
sequence identity of 29% (FIG. 3). In contrast, FRAP homologs could
not be found in the recently sequenced Leishmania (35) and
Trypanosome genomes (36). This selective presence in Plasmodium and
Theileria genomes could point towards a common function of the
protein between otherwise two very different parasites.
[0129] The amino acid sequences for the FRAP proteins discussed
above are depicted in FIG. 1, the nucleic acid sequences that
encode the proteins are depicted in FIG. 2, and the corresponding
SEQ ID NOS: are given in Table 3. TABLE-US-00003 TABLE 3 SEQ ID
NOS: for amino acid and nucleic acid SEQ ID NO: Organism Amino acid
sequence Nucleic acid sequence P. falciparum SEQ ID NO: 1 SEQ ID
NO: 2 P. gallinaceum SEQ ID NO: 3 SEQ ID NO: 4 P. reichenowi SEQ ID
NO: 5 SEQ ID NO: 6 P. vivax SEQ ID NO: 7 SEQ ID NO: 8 P. yoelii SEQ
ID NO: 9 SEQ ID NO: 10 P. knowlesi SEQ ID NO: 11 SEQ ID NO: 12 P.
chaubaudi SEQ ID NO: 13 SEQ ID NO: 14 P. berghei SEQ ID NO: 15 SEQ
ID NO: 16 T. parva SEQ ID NO: 17 SEQ ID NO: 18 T. annulata SEQ ID
NO: 19 SEQ ID NO: 20
Cloning of P. falciparum FRAP: Coding sequence of P. falciparum
FRAP was amplified by RT-PCR using total RNA from the sporozoite
stage of the parasite, giving rise to a 615 bp fragment. This PCR
product was not due to the presence of contaminating genomic DNA in
the RNA preparation, as a parallel reaction performed in the
absence of reverse transcriptase enzyme, showed no amplification.
Also, the size of the amplified fragment, viz. 615 bp, matched the
size of the predicted mature mRNA (FIG. 4b). The amplified fragment
from the sporozoite stage was cloned in a T7 promoter-based E. coli
expression vector, giving rise to plasmid pFRAP. Sequencing of the
cloned DNA fragment authenticated the predicted exon structure and
coding sequence for the FRAP protein (data not shown). To
investigate the role of FAS1 domain in the biology of the protein,
two more plasmid constructs viz., pFRAP2 and pFRAP3, were generated
by sub-cloning, using pFRAP as template. pFRAP2 encoded the DNA
sequence for amino acids 1-87 of the full length protein while
pFRAP3 encoded the FAS1 domain represented by amino acids 88-205
(FIG. 4a). The authenticity of these clones was also verified by
sequencing. Recombinant Expression and Purification of FRAP
proteins: To obtain recombinant FRAP proteins, the desired
construct was transformed in E. coli BL21 cells and the expression
was induced with IPTG. Three hours post induction, the culture was
harvested and the site of accumulation of the recombinant protein
was evaluated by sub-cellular fractionation. For all three FRAP
proteins, the expression was localized in the spheroplast in the
form of insoluble inclusion bodies (data not shown). Spheroplast
pellet was further processed to isolate inclusion bodies, as
previously described (30). Inclusion bodies were solubilized and
the proteins were purified by a combination of affinity and gel
filtration chromatography. The presence of a polyhistidine tag at
the carboxyl terminus of the recombinantly expressed proteins
facilitated the purification and all three proteins were initially
purified on a His-Trap affinity column (data not shown). The
proteins at this stage were 95% pure. Further purification to
apparent homogeneity was done by gel filtration chromatography
(FIG. 4c). Purified FRAP, FRAP2 and FRAP3 had the expected
molecular weights of 27.8, 12.3 and 17.7 kDa respectively and were
recognized by a monoclonal antibody directed against the
polyhistidine tag present at the carboxyl terminus of all the
proteins (FIG. 4d). The first 15 residues of each of the proteins
were also verified by amino terminal sequencing (data not shown).
Together, these results authenticated the recombinant proteins and
suggested that they were structurally intact. FRAP is localized in
the micronemes of the sporozoites: To detect the expression of FRAP
on sporozoites, protein-specific antibodies were raised by
immunizing mice with FRAP2 protein. Anti-FRAP2 antibodies readily
recognized the expression of FRAP protein on the sporozoite (not
shown). The binding was specific as pre-immune serum did not
recognize any expression on the sporozoites. This indicated that
transcription of FRAP mRNA can be correlated to its expression
during the sporozoite stage of the lifecycle. Immunoelectron
microscopy using anti-FRAP2 antibodies revealed that FRAP was
localized in the lumen of micronemes, a specialized secretory
organelle in the cytoplasm (not shown). The protein was present in
the apical micronemes, suggesting that it could be secreted during
the infectivity process. In Plasmodium, micronemes contain several
adhesive domain-containing proteins that are associated with host
cell adhesion and invasion at both, sporozoite and erythrocytic
stages of its lifecycle (13, 37, 38). This suggested that FRAP
could be playing a role in the infectivity process. FRAP is
involved in adhesion of sporozoites to liver cells: FRAP was
investigated for its possible role in host cell adhesion using a
human hepatocyte cell line, HepG2, an established model for
investigating sporozoite-liver cell interactions in malaria (1,
31). FRAP showed a dose dependent binding on liver cells (FIG. 5)
which was comparable to the binding activity of CS protein, a known
parasite protein involved in the adhesion and invasion of liver
cells by the sporozoites (1). This suggested that FRAP could be
serving as one of the parasite ligands in host-parasite
interactions. This host-cell binding activity of FRAP was not due
to the presence of the FAS1 domain alone, as FRAP3, a protein
encoding only the FAS1 domain (amino acids 88-205) did not bind to
liver cells, even at the highest concentration used in the assay
(54). Although FAS 1 domain alone did not show any binding, its
deletion from the full length protein (protein FRAP2) lead to a 50%
loss of activity, in comparison to the full length protein (FIG.
5). This suggested that both, FAS1 domain and the amino terminus
region, contribute to the binding activity of the protein and an
intact FRAP is required for its optimal activity. FRAP binds liver
cells through heparan sulfate proteoglycans: As FRAP showed potent
liver cell binding, the nature of its receptor on liver cells was
investigated by utilizing glycosaminoglycans as competitive
inhibitors. Inhibition of adherence by the addition of soluble
glycosaminoglycans in an assay may suggest that the involved host
receptor is a proteoglycan (31, 39). In the presence of free
heparin, binding activity of FRAP and FRAP2 was reduced by 55 and
60% respectively (FIG. 6). In contrast, chondroitin sulfate A
showed no inhibition at the highest concentration evaluated in the
assay (FIG. 6). This suggested that FRAP utilizes heparan
sulfate-based proteoglycans (HSPG) as a receptor for adhesion.
[0130] The involvement of HSPG as a receptor was further verified
by evaluating the binding of the protein on liver cells that were
pretreated with specific glycosaminoglycan-cleaving enzymes. Cells
were pre-treated with heparinase I or chondroitinase ABC followed
by the evaluation of binding activity of FRAP and FRAP2. Heparinase
I selectively removes heparan sulfate while chondroitinase ABC
cleaves chondroitin sulfate A, B and C type sugars from the liver
cell surface. Both, FRAP and FRAP2 lost 50% of their binding
activity on heparinase I treated cells (Table 4) confirming the
involvement of a heparin-based receptor on the liver cell surface.
CS protein, which binds hepatocytes through HSPG (39) also showed a
similar decrease in binding activity. In contrast, treatment of
liver cells with chondroitinase ABC resulted in no loss of
activity. TABLE-US-00004 TABLE 4 Binding of FRAP proteins to
hepatocytes is inhibited by pretreatment of cells with
glycosaminoglycan cleaving enzyme. Cells were pretreated with
different concentrations of either Heparinase I or Chondroitinase
ABC for 90 minutes followed by the addition of 100 nM of protein.
Inhibition of binding was calculated by comparing the binding of
respective proteins on non-treated HepG2 cells in the same plate.
Inhibition of Binding (%) Enzyme, U/ml FRAP FRAP2 CSP Heparinase I
1.25 39.4 .+-. 4.2 42.3 .+-. 10.4 48.1 .+-. 12.0 2.50 42.1 .+-. 7.6
41.4 .+-. 1.5 57.7 .+-. 7.9 5.00 47.8 .+-. 1.4 49.4 .+-. 9.2 59.1
.+-. 6.5 Chondroitinase ABC 0.01 -- -- -- 0.12 -- -- -- 1.25 -- --
--
[0131] FRAP is involved in liver cell invasion: As FRAP proteins
efficiently bound to HepG2 cells, we investigated the ability of
the two proteins and the anti-FRAP2 antibodies in preventing
invasion of human liver cells by P. falciparum sporozoites in
culture. Both FRAP and FRAP2 could prevent sporozoites from
invading liver cells by 89.5% and 92.4% respectively, at the
highest concentration of the protein used in the assay. This
activity was comparable to the invasion inhibition activity of CSP
protein, which at a similar concentration could also inhibit the
invasion by 92.6%. Anti-FRAP2 antibodies showed extreme potency as
at a concentration of 40 .mu.g/ml, it inhibited sporozoite invasion
by 94.6%, a level comparable to the inhibitory activity of anti-CS
monoclonal antibody NFS1 (Table 5). This indicated that (i) FRAP
not only plays a role in binding, it is also involved in the
invasion process (ii) the protein utilizes its amino terminus
(amino acids 1-87) for its invasion activity and (iii) a potent
antibody response against FRAP2 by the host may play a role in
malaria control. TABLE-US-00005 TABLE 5 FRAP is involved in
invasion of liver cells by P. falciparum sporozoites. Invasion of
HepG2 cells by P. falciparum sporozoites was evaluated in the
presence of different concentrations of free proteins or anti-FRAP2
antibodies and compared with the invasion activity in the presence
of culture medium. % inhibition represents the decrease in the
number of sporozoites that invaded liver cells in comparision to
the invasion level in cells incubated with culture medium.
Concentration Treatment .mu.g/ml % Inhibition Culture Medium --
FRAP 20 89.5 + 1.0 10 80.9 + 1.0 FRAP2 20 92.4 + 3.5 10 88.1 + 4.6
CS Protein 20 92.6 + 2.0 Anti-FRAP2 antibody 40 94.6 + 1.2 Anti-CS
monoclonal 100 97.4 + 0.7
Discussion
[0132] Deciphering the mechanism of infectivity of the malaria
parasite is a major prerequisite for developing intervention
strategies. Key to this process is the unique set of proteins, many
of them currently unknown, expressed by the parasite to bind and
invade host cells. Therefore, a combination of biochemical and
functional studies of malarial genes is required to identify
parasitic proteins involved in pathogenesis.
[0133] We identified P. falciparum FRAP, a new parasite protein and
showed that it is expressed during the sporozoite stage of the
lifecycle. Orthologs of P. falciparum FRAP were identified in
rodent, avian, simian and human malaria species and multiple
sequence alignment revealed that the protein has 60% sequence
identity within the Plasmodium genus (FIG. 3). Its universal
presence and conserved nature suggested that the protein plays an
important role in the biology of the parasite.
[0134] The protein was localized in the sporozoite micronemes by
immunoelectron microscopy. Micronemes are specialized secretory
organelles in Plasmodium and during the sporozoite stage secrete a
wide variety of proteins involved in parasite motility, traversal
and host cell infection. Previously, TRAP/SSP2 and SPECT, two
sporozoite proteins with adhesive Thrombospondin type I repeat
(TSR) domains have been found in the micronemes and have
subsequently been shown to be involved in the infectivity process
(13, 37). As FRAP encoded FAS1, an ancient adhesive domain present
in both prokaryotes and eukaryotes, we therefore investigated the
role of FRAP in host cell adhesion and invasion by the
sporozoites.
[0135] The protein was recombinantly expressed in E. coli and
purified to homogeneity by column chromatography (FIG. 4c). The
purified protein showed robust and dose dependent binding to liver
cells indicating that it is involved in the attachment of
sporozoites to liver cells (FIG. 5). This activity was comparable
to the binding activity of CS protein, considered to be the primary
binding ligand, suggesting that FRAP could be one of the primary
parasite proteins involved in attachment of sporozoites to liver
cells. In .beta.ig-h3, a FAS1 domain-containing human protein
involved in corneal cell adhesion, the adhesion activities of the
protein completely resides in the FAS1 domain (22). To investigate
the role of FAS1 domain in FRAP, we expressed FAS1 domain alone
(amino acids 88-205, protein FRAP3) and evaluated its cell binding
activity on HepG2 cells. The protein did not show any cell binding
activity (FIG. 5), indicating that the deleted segment (amino acids
1-87) of the protein plays an important role in the binding
activity of the protein.
[0136] This was investigated by expressing amino acids 1-87
(protein FRAP2) in E. coli and evaluation of its cell binding
activities on the liver cell line. FRAP2 was capable of binding to
liver cells, albeit at only half the strength of its full length
protein, FRAP. This suggested that amino terminus region of the
protein plays an important role in the host cell binding, however,
an intact FRAP molecule is required for its optimal activity. The
loss of activity seen here could be due to loss of the required
tertiary conformation of the binding domain (due to the absence of
the FAS1 domain) and/or part of the binding motif is present in the
FAS1 domain of the protein. A similar situation exists in the case
of CS protein, where the unique amino terminus region plays an
important role in liver cell binding and invasion activities of the
protein (31).
[0137] FRAP exploited heparan sulfate proteoglycans, expressed on
liver cell surface, as receptor for its biological activities
(Table 4). This was revealed by competition studies with defined
carbohydrates, as well as loss of binding upon enzymatic removal of
host glycans. Heparan sulfate-protein interactions involve
positively charged residues of the protein, which interact with the
negatively charged carboxylate and sulfate ions of the
glycosaminoglycan chain. The amino terminus of FRAP possesses a
disproportionate number of positively charged residues (13 out of
the first 50) some of which are extremely conserved within the
Plasmodium genus (FIG. 3). Their conserved nature suggests that
they could possibly be involved in these interactions. Parallels
exist for such mechanism in other heparin-binding proteins where a
large number of positively charged residues involved in heparin/HS
interaction are present in a close proximity in the protein
(40).
[0138] Entry of sporozoites into the hepatocyte is a multistep
process, where the initial attachment to the hepatocytes is
followed by the invasion of liver cells, by the parasite. To
investigate the role of FRAP in the invasion process, recombinant
FRAP proteins and anti-FRAP2 antibodies were used as competitors in
an in vitro invasion assay. Proteins FRAP, FRAP2 and anti-FRAP2
antibodies inhibited the invasion of liver cells by P. falciparum
sporozoites with extreme competence, showing as high as 94.6%
inhibition (Table 5) in the assay. These levels were comparable to
the inhibitory activity of CSP protein and anti-CSP monoclonal
antibody. These results indicated that FRAP is utilized by
sporozoites for both adhesion and subsequent invasion of liver
cells and the amino terminal region plays an important role in
these processes. It is noteworthy that similar level (>90%) of
inhibition has only been possible by targeting CSP, SSP2/TRAP and
the recently discovered SPATR protein (10). Recently, AMA1 has been
shown to be involved in liver cell invasion but antibodies against
the protein could inhibit the invasion only by about 50% (41). CSP
and SSP2/TRAP are being vigorously pursued as vaccine candidates
and are currently being evaluated in the clinic (4, 5). Involvement
of FRAP in liver cell invasion and its strong inhibition by
antibodies suggest that a potent immunological response against
this protein in vivo could serve as a strategy for intervention and
the immunological competence of FRAP as a vaccine candidate needs
to be investigated.
[0139] Although we have investigated the role of FRAP in the liver
cell adhesion and invasion by the sporozoites, it is noteworthy
that microarray and proteomic studies have revealed that FRAP is
also transcribed and expressed during the erythrocytic stages of
the lifecycle, especially during the schizonts, which is
immediately followed by the release of merozoites and invasion of
red blood cells (9, 42, 43). AMA1 and MAEBL, two micronemal
proteins that are expressed at sporozoites and erythrocytic stages
of the lifecycle, are involved in pathogenesis, both, at
pre-erythrocytic and blood stages, where they play a role in host
cell adhesion and invasion (41, 44-46). With its multistage
expression, it is possible that FRAP could also be involved in
host-parasite interactions during erythrocytic stages of the
lifecycle.
[0140] In conclusion, we have identified and characterized a new
parasite protein involved in malaria pathogenesis at the sporozoite
stage of the lifecycle. It's involvement in pathogenesis indicates
that developing intervention strategies targeting FRAP creates new
treatment options for controlling malaria.
References for Example 1
[0141] 1. Cerami, C., Frevert, U., Sinnis, P., Takacs, B., Clavijo,
P., Santos, M. J. & Nussenzweig, V. (1992) Cell 70, 1021-33.
[0142] 2. Robson, K. J., Frevert, U., Reckmann, I., Cowan, G.,
Beier, J., Scragg, I. G., Takehara, K., Bishop, D. H., Pradel, G.,
Sinden, R. & et al. (1995) Embo J 14, 3883-94. [0143] 3. Wang,
R., Doolan, D. L., Le, T. P., Hedstrom, R. C., Coonan, K. M.,
Charoenvit, Y., Jones, T. R., Hobart, P., Margalith, M., Ng, J.,
Weiss, W. R., Sedegah, M., de Taisne, C., Norman, J. A. &
Hoffman, S. L. (1998) Science 282, 476-80. [0144] 4. Alonso, P. L.,
Sacarlal, J., Aponte, J. J., Leach, A., Macete, E., Milman, J.,
Mandomando, I., Spiessens, B., Guinovart, C., Espasa, M., Bassat,
Q., Aide, P., Ofori-Anyinam, O., Navia, M. M., Corachan, S.,
Ceuppens, M., Dubois, M. C., Demoitie, M. A., Dubovsky, F.,
Menendez, C., Tornieporth, N., Ballou, W. R., Thompson, R. &
Cohen, J. (2004) Lancet 364, 1411-20. [0145] 5. Moorthy, V. S.,
Imoukhuede, E. B., Keating, S., Pinder, M., Webster, D., Skinner,
M. A., Gilbert, S. C., Walraven, G. & Hill, A. V. (2004) J
Infect Dis 189, 2213-9. [0146] 6. Nardin, E. H., Calvo-Calle, J.
M., Oliveira, G. A., Nussenzweig, R. S., Schneider, M., Tiercy, J.
M., Loutan, L., Hochstrasser, D. & Rose, K. (2001) J Immunol
166, 481-9. [0147] 7. Hoffman, S. (1996) Malaria Vaccine
Developmtent: A multi immune response approach (ASM press,
Washington, D.C.). [0148] 8. Gardner, M. J., Hall, N., Fung, E.,
White, O., Berriman, M., Hyman, R. W., Carlton, J. M., Pain, A.,
Nelson, K. E., Bowman, S., Paulsen, I. T., James, K., Eisen, J. A.,
Rutherford, K., Salzberg, S. L., Craig, A., Kyes, S., Chan, M. S.,
Nene, V., Shallom, S. J., Suh, B., Peterson, J., Angiuoli, S.,
Pertea, M., Allen, J., Selengut, J., Haft, D., Mather, M. W.,
Vaidya, A. B., Martin, D. M., Fairlamb, A. H., Fraunholz, M. J.,
Roos, D. S., Ralph, S. A., McFadden, G. I., Cummings, L. M.,
Subramanian, G. M., Mungall, C., Venter, J. C., Carucci, D. J.,
Hoffman, S. L., Newbold, C., Davis, R. W., Fraser, C. M. &
Barrell, B. (2002) Nature 419, 498-511. [0149] 9. Florens, L.,
Washburn, M. P., Raine, J. D., Anthony, R. M., Grainger, M.,
Haynes, J. D., Moch, J. K., Muster, N., Sacci, J. B., Tabb, D. L.,
Witney, A. A., Wolters, D., Wu, Y., Gardner, M. J., Holder, A. A.,
Sinden, R. E., Yates, J. R. & Carucci, D. J. (2002) Nature
5419, 520-6. [0150] 10. Chattopadhyay, R., Rathore, D., Fujioka,
H., Kumar, S., De La Vega, P., Haynes, D., Moch, K., Fryauff, D.,
Wang, R., Carucci, D. J. & Hoffman, S. L. (2003) J Biol Chem
278, 25977-25981. [0151] 11. Bateman, A., Coin, L., Durbin, R.,
Finn, R. D., Hollich, V., Griffiths-Jones, S., Khanna, A.,
Marshall, M., Moxon, S., Sonnhammer, E. L., Studholme, D. J.,
Yeats, C. & Eddy, S. R. (2004) Nucleic Acids Res 32, D138-41.
[0152] 12. Letunic, I., Copley, R. R., Schmidt, S., Ciccarelli, F.
D., Doerks, T., Schultz, J., Ponting, C. P. & Bork, P. (2004)
Nucleic Acids Res 32, D142-4. [0153] 13. Ishino, T., Yano, K.,
Chinzei, Y. & Yuda, M. (2004) PLoS Biol 2, E4. [0154] 14.
Bastiani, M. J., Harrelson, A. L., Snow, P. M. & Goodman, C. S.
(1987) Cell 48, 745-55. [0155] 15. Terasaka, K., Yamaguchi, R.,
Matsuo, K., Yamazaki, A., Nagai, S. & Yamada, T. (1989) FEMS
Microbiol Lett 49, 273-6. [0156] 16. Snow, P. M., Zinn, K.,
Harrelson, A. L., McAllister, L., Schilling, J., Bastiani, M. J.,
Makk, G. & Goodman, C. S. (1988) Proc Natl Acad Sci USA 85,
5291-5. [0157] 17. Johnson, K. L., Jones, B. J., Bacic, A. &
Schultz, C. J. (2003) Plant Physiol 133, 1911-25. [0158] 18. Wang,
W. C., Zinn, K. & Bjorkman, P. J. (1993) J Biol Chem
268,1448-55. [0159] 19. Hu, S., Sonnenfeld, M., Stahl, S. &
Crews, S. T. (1998) J Neurobiol 35, 77-93. [0160] 20. Zinn, K.,
McAllister, L. & Goodman, C. S. (1988) Cell 53, 577-87. [0161]
21. Politz, O., Gratchev, A., McCourt, P. A., Schledzewski, K.,
Guillot, P., Johansson, S., Svineng, G., Franke, P., Kannicht, C.,
Kzhyshkowska, J., Longati, P., Velten, F. W. & Goerdt, S.
(2002) Biochem J 362, 155-64. [0162] 22. Kim, J. E., Kim, S. J.,
Lee, B. H., Park, R. W., Kim, K. S. & Kim, I. S. (2000) J Biol
Chem 275, 30907-15. [0163] 23. Clout, N. J., Tisi, D. &
Hohenester, E. (2003) Structure (Camb) 11, 197-203. [0164] 24.
Hall, N., Karras, M., Raine, J. D., Carlton, J. M., Kooij, T. W.,
Berriman, M., Florens, L., Janssen, C. S., Pain, A., Christophides,
G. K., James, K., Rutherford, K., Harris, B., Harris, D., Churcher,
C., Quail, M. A., Ormond, D., Doggett, J., Trueman, H. E., Mendoza,
J., Bidwell, S. L., Rajandream, M. A., Carucci, D. J., Yates, J.
R., 3rd, Kafatos, F. C., Janse, C. J., Barrell, B., Turner, C. M.,
Waters, A. P. & Sinden, R. E. (2005) Science 307, 82-6. [0165]
25. Bahl, A., Brunk, B., Coppel, R. L., Crabtree, J., Diskin, S.
J., Fraunholz, M. J., Grant, G. R., Gupta, D., Huestis, R. L.,
Kissinger, J. C., Labo, P., Li, L., McWeeney, S. K., Milgram, A.
J., Roos, D. S., Schug, J. & Stoeckert, C. J., Jr. (2002)
Nucleic Acids Res 30, 87-90. [0166] 26. Gardner, M. J., Bishop, R.,
Shah, T., de Villiers, E. P., Carlton, J. M., Hall, N., Ren, Q.,
Paulsen, I. T., Pain, A., Berriman, M., Wilson, R. J., Sato, S.,
Ralph, S. A., Mann, D. J., Xiong, Z., Shallom, S. J., Weidman, J.,
Jiang, L., Lynn, J., Weaver, B., Shoaibi, A., Domingo, A. R.,
Wasawo, D., Crabtree, J., Wortman, J. R., Haas, B., Angiuoli, S.
V., Creasy, T. H., Lu, C., Suh, B., Silva, J. C., Utterback, T. R.,
Feldblyum, T. V., Pertea, M., Allen, J., Nierman, W. C., Taracha,
E. L., Salzberg, S. L., White, O. R., Fitzhugh, H. A., Morzaria,
S., Venter, J. C., Fraser, C. M. & Nene, V. (2005) Science 309,
134-7. [0167] 27. Pain, A., Renauld, H., Berriman, M., Murphy, L.,
Yeats, C. A., Weir, W., Kerhomou, A., Aslett, M., Bishop, R.,
Bouchier, C., Cochet, M., Coulson, R. M., Cronin, A., de Villiers,
E. P., Fraser, A., Fosker, N., Gardner, M., Goble, A.,
Griffiths-Jones, S., Harris, D. E., Katzer, F., Larke, N., Lord,
A., Maser, P., McKellar, S., Mooney, P., Morton, F., Nene, V.,
O'Neil, S., Price, C., Quail, M. A., Rabbinowitsch, E., Rawlings,
N. D., Rutter, S., Saunders, D., Seeger, K., Shah, T., Squares, R.,
Squares, S., Tivey, A., Walker, A. R., Woodward, J., Dobbelaere, D.
A., Langsley, G., Rajandream, M. A., McKeever, D., Shiels, B.,
Tait, A., Barrell, B. & Hall, N. (2005) Science 309, 131-3.
[0168] 28. Thompson, J. D., Higgins, D. G. & Gibson, T. J.
(1994) Nucleic Acids Res 22, 4673-80. [0169] 29. Haynes, J. D.
& Moch, J. K. (2002) Methods Mol Med 72, 489-97. [0170] 30.
Rathore, D., Hrstka, S. C., Sacci, J. B., Jr., De la Vega, P.,
Linhardt, R. J., Kumar, S. & McCutchan, T. F. (2003) J Biol
Chem 278, 40905-10. [0171] 31. Rathore, D., Sacci, J. B., de la
Vega, P. & McCutchan, T. F. (2002) J Biol Chem 277, 7092-8.
[0172] 32. Folsch, H., Pypaert, M., Schu, P. & Mellman, I.
(2001) J Cell Biol 152, 595-606. [0173] 33. Ozaki, L. S., Gwadz, R.
W. & Godson, G. N. (1984) J Parasitol 70, 831-3. [0174] 34.
Marchler-Bauer, A. & Bryant, S. H. (2004) Nucleic Acids Res 32,
W327-31. [0175] 35. Ivens, A. C., Peacock, C. S., Worthey, E. A.,
Murphy, L., Aggarwal, G., Berriman, M., Sisk, E., Rajandream, M.
A., Adlem, E., Aert, R., Anupama, A., Apostolou, Z., Attipoe, P.,
Bason, N., Bauser, C., Beck, A., Beverley, S. M., Bianchettin, G.,
Borzym, K., Bothe, G., Bruschi, C. V., Collins, M., Cadag, E.,
Ciarloni, L., Clayton, C., Coulson, R. M., Cronin, A., Cruz, A. K.,
Davies, R. M., De Gaudenzi, J., Dobson, D. E., Duesterhoeft, A.,
Fazelina, G., Fosker, N., Frasch, A. C., Fraser, A., Fuchs, M.,
Gabel, C., Goble, A., Goffeau, A., Harris, D., Hertz-Fowler, C.,
Hilbert, H., Horn, D., Huang, Y., Klages, S., Knights, A., Kube,
M., Larke, N., Litvin, L., Lord, A., Louie, T., Marra, M., Masuy,
D., Matthews, K., Michaeli, S., Mottram, J. C., Muller-Auer, S.,
Munden, H., Nelson, S., Norbertczak, H., Oliver, K., O'Neil, S.,
Pentony, M., Pohl, T. M., Price, C., Purnelle, B., Quail, M. A.,
Rabbinowitsch, E., Reinhardt, R., Rieger, M., Rinta, J., Robben,
J., Robertson, L., Ruiz, J. C., Rutter, S., Saunders, D., Schafer,
M., Schein, J., Schwartz, D. C., Seeger, K., Seyler, A., Sharp, S.,
Shin, H., Sivam, D., Squares, R., Squares, S., Tosato, V., Vogt,
C., Volckaert, G., Wambutt, R., Warren, T., Wedler, H., Woodward,
J., Zhou, S., Zimmermann, W., Smith, D. F., Blackwell, J. M.,
Stuart, K. D., Barrell, B., et al. (2005) Science 309, 436-42.
[0176] 36. Berriman, M., Ghedin, E., Hertz-Fowler, C., Blandin, G.,
Renauld, H., Bartholomeu, D. C., Lennard, N. J., Caler, E., Hamlin,
N. E., Haas, B., Bohme, U., Hannick, L., Aslett, M. A., Shallom,
J., Marcello, L., Hou, L., Wickstead, B., Alsmark, U. C.,
Arrowsmith, C., Atkin, R. J., Barron, A. J., Bringaud, F., Brooks,
K., Carrington, M., Cherevach, I., Chillingworth, T. J., Churcher,
C., Clark, L. N., Corton, C. H., Cronin, A., Davies, R. M.,
Doggett, J., Djikeng, A., Feldblyum, T., Field, M. C., Fraser, A.,
Goodhead, I., Hance, Z., Harper, D., Harris, B. R., Hauser, H.,
Hostetler, J., Ivens, A., Jagels, K., Johnson, D., Johnson, J.,
Jones, K., Kerhomou, A. X., Koo, H., Larke, N., Landfear, S.,
Larkin, C., Leech, V., Line, A., Lord, A., Macleod, A., Mooney, P.
J., Moule, S., Martin, D. M., Morgan, G. W., Mungall, K.,
Norbertczak, H., Ormond, D., Pai, G., Peacock, C. S., Peterson, J.,
Quail, M. A., Rabbinowitsch, E., Rajandream, M. A., Reitter, C.,
Salzberg, S. L., Sanders, M., Schobel, S., Sharp, S., Simmonds, M.,
Simpson, A. J., Tallon, L., Turner, C. M., Tait, A., Tivey, A. R.,
Van Aken, S., Walker, D., Wanless, D., Wang, S., White, B., White,
O., Whitehead, S., Woodward, J., Wortman, J., Adams, M. D., Embley,
T. M., Gull, K., Ullu, E., Barry, J. D., Fairlamb, A. H.,
Opperdoes, F., Barrell, B. G., Donelson, J. E., Hall, N., Fraser,
C. M., et al. (2005) Science 309, 416-22. [0177] 37. Muller, H. M.,
Reckmann, I., Hollingdale, M. R., Bujard, H., Robson, K. J. &
Crisanti, A. (1993) Embo J 12, 2881-9. [0178] 38. Sim, B. K.,
Chitnis, C. E., Wasniowska, K., Hadley, T. J. & Miller, L. H.
(1994) Science 264, 1941-4. [0179] 39. Frevert, U., Sinnis, P.,
Cerami, C., Shreffler, W., Takacs, B. & Nussenzweig, V. (1993)
J Exp Med 177, 1287-98. [0180] 40. Hileman, R. E., Fromm, J. R.,
Weiler, J. M. & Linhardt, R. J. (1998) Bioessays 20, 156-67.
[0181] 41. Silvie, O., Franetich, J. F., Charrin, S., Mueller, M.
S., Siau, A., Bodescot, M., Rubinstein, E., Hannoun, L.,
Charoenvit, Y., Kocken, C. H., Thomas, A. W., Van Gemert, G. J.,
Sauerwein, R. W., Blackman, M. J., Anders, R. F., Pluschke, G.
& Mazier, D. (2004) J Biol Chem 279, 9490-6. [0182] 42.
Bozdech, Z., Zhu, J., Joachimiak, M. P., Cohen, F. E., Pulliam, B.
& DeRisi, J. L. (2003) Genome Biol 4, R9. [0183] 43. Bozdech,
Z., Llinas, M., Pulliam, B. L., Wong, E. D., Zhu, J. & DeRisi,
J. L. (2003) PLoS Biol 1, E5. [0184] 44. Preiser, P., Renia, L.,
Singh, N., Balu, B., Jarra, W., Voza, T., Kaneko, O., Blair, P.,
Torii, M., Landau, I. & Adams, J. H. (2004) Infect Immun 72,
3604-8. [0185] 45. Kappe, S. H., Noe, A. R., Fraser, T. S., Blair,
P. L. & Adams, J. H. (1998) Proc Natl Acad Sci USA 95, 1230-5.
[0186] 46. Mitchell, G. H., Thomas, A. W., Margos, G., Dluzewski,
A. R. & Bannister, L. H. (2004) Infect Immun 72, 154-8.
Example 2
Inhibitory Epitope in FRAP
[0187] Identification of Inhibitory epitope by peptide mapping As
we have demonstrated that antibodies against FRAP2, an 87 amino
acid polypeptide can prevent invasion, the region of the protein
responsible for this recognition was mapped by developing a set of
overlapping peptides that were utilized for ELISA. A set of 10
overlapping peptides (Table 6) were chemically synthesized and used
as coating antigen to identify the epitope recognized by these
antibodies. Overlapping Peptides HAI-3,4, & 5 were
predominantly recognized by these antibodies, suggesting that a 32
amino acid sequence (TRSGGLRKPQKVTNDPESINRKVYWCFEHKPV, SEQ ID NO:
24), comprised by these peptides is being recognized by the
inhibitory antibodies (Table 7). A sequence comparison of these
peptides reveals that an 8 amino acid sequence (TNDPESIN, SEQ ID
NO: 37) is present in all of them (FIG. 7) suggesting that this
sequence could be an important component of the region recognized
by the anti-protein antibodies. Therefore, the 32 amino acid
sequence or portion thereof can be exploited as part of a
multi-epitope subunit vaccine. The 32 amino acid region has 100%
sequence homology or 87.5% sequence identity within the Plasmodium
genus implying that this region plays a critical role in all the
Plasmodium species and an immune response(s) generated against this
region of the protein in one species could be a factual
representation of immnune responses against other species,
generated by its host. Two other peptides (HAI-7 and HAI-10) were
also recognized by the anti-protein antibodies suggesting that
their recognition is also important in preventing parasites from
initiating an infection. TABLE-US-00006 TABLE 6 Sequence of
peptides chemically synthesized for identification of inhibitory
epitope. Peptide Sequence SEQ ID NO HAI-1 MKNRFYYNLIIKRLYTRSGG SEQ
ID NO: 27 HAI-2 NLIIKRLYTRSGGLRKPQKV SEQ ID NO: 28 HAI-3
TRSGGLRKPQKVTNDPESIN SEQ ID NO: 29 HAI-4 GLRKPQKVTNDPESINRKVY SEQ
ID NO: 30 HAI-5 TNDPESINRKVYWCFEHKPV SEQ ID NO: 31 HAI-6
VYWCFEHKPVKRTIINLIYS SEQ ID NO: 32 HAI-7 KPVKRTIINLIYSHNELKIF SEQ
ID NO: 33 HAI-8 NLIYSHNELKIFSNLLNHPT SEQ ID NO: 34 HAI-9
NELKIFSNLLNHPTVGSSLI SEQ ID NO: 35 HAI-10 NLLNHPTVGSSLIHELSLDG SEQ
ID NO: 36
[0188] TABLE-US-00007 TABLE 7 Recognition of FRAP-derived peptides
by ELISA. Peptides were coated onto the ELISA plate followed by the
addition of log dilutions of antibodies followed by anti-mouse
antibodies conjugated to alkaline phosphatase. Recognition was
measured at 405 nm using an ELISA plate reader. Peptide/ 1:100
1:1000 1:10K Antigen FRAP FRAP2 FRAP FRAP2 FRAP FRAP2 HAI-1 0.010
0.015 -- -- -- -- HAI-2 0.010 0.020 -- -- -- -- HAI-3 0.710 0.530
0.223 0.190 0.026 0.020 HAI-4 0.789 0.710 0.4445 0.630 0.063 0.230
HAI-5 0.660 0.636 0.290 0.465 0.030 0.110 HAI-6 0.005 -- -- -- --
-- HAI-7 0.730 0.065 0.550 0.026 0.165 -- HAI-8 0.020 0.290 --
0.039 -- -- HAI-9 0.030 -- -- -- -- -- HAI-10 0.250 0.300 0.030
0.045 -- -- FRAP* 0.670 0.600 0.650 0.465 0.340 0.130 FRAP** 0.210
0.260 0.070 0.400 -- 0.190 *4 pmol of protein **2 nmol of each
peptide in 50 ul coating buffer
[0189] Optimal recognition of an epitope by the host immune system
requires that the epitope maintains its structural conformation.
While short amino acid sequences can be easily recognized in vitro,
their recognition under in-vivo conditions almost always requires
them to be present as part of a much larger polypeptide. This is
especially important for configurational epitopes present in the
surface antigens of malaria parasite whose recognition requires
that a continuous stretch of amino acids, larger than its
identified epitope, be present for its optimal recognition.
Therefore, a 32 amino acid long region is most likely required for
optimal recognition of FRAP protein by the host immune system and
it could be utilized either alone or in combination with other
known and unknown malarial antigens in a vaccine. [0190] FRAP is
recognized by the host immune system of malaria-infected subjects.
Sera from 17 malaria infected subjects was screened for the
presence of anti-FRAP antibodies, by ELISA.
[0191] 0.5 microgram of purified FRAP protein was coated as antigen
and its recognition was probed with sera at 1:200 dilution. 4 sera
samples from north American volunteers, who have never been exposed
to malaria were used as control. A cutoff value of OD405=0.378,
which represented mean of OD+2 SD was used to determine samples
that were positive. The ELISA results indicated that 10 out of 17
(58.8%) infected subjects had anti-FRAP antibodies (Table 8) with
OD values above the set cutoff TABLE-US-00008 TABLE 8 Recognition
of full length FRAP by sera from infected subjects living in
Bandiagara, a malaria-endemic district in Mali. Sample ID
Absorbance, 405 nm Positive 1A-001 0.79 .+-. 0.07 Y 1A-002 1.03
.+-. 0.05 Y 1A-004 0.47 .+-. 0.02 Y 1A-005 0.23 .+-. 0.01 N 1A-007
0.23 .+-. 0.01 N 1A-008 0.49 .+-. 0.07 Y 1A-010 0.26 .+-. 0.01 N
1A-011 0.21 .+-. 0.01 N 1A-013 0.91 .+-. 0.00 Y 1A-014 0.30 .+-.
0.01 N 1A-016 0.60 .+-. 0.01 Y 1A-017 0.15 .+-. 0.01 N 1A-019 0.43
.+-. 0.00 Y 1A-020 0.56 .+-. 0.01 Y 1A-021 0.40 .+-. 0.00 Y 1A-023
0.41 .+-. 0.02 Y 1A-024 0.20 .+-. 0.00 N
Example 3
FRAP is a Malaria Drug Target
[0192] Once a malaria parasite infects red blood cells, host
hemoglobin serves as its primary source of amino acids required for
its geometric increase in infection. It achieves its goal by
cannibalizing hemoglobin to its constituent amino acids, which it
recycles for its own protein synthesis. While the parasite is
extremely effective in digesting the protein (globin) component of
hemoglobin the heme prosthetic group serves as a challenge to its
survivability. Free heme released from hemoglobin is lethal for the
parasite and to escape its deleterious effects the parasite
enzymatically polymerizes heme into a non-toxic byproduct known as
hemozoin. Therefore, any mechanism by which polymerization of heme
into nontoxic hemozoin can be inhibited will lead to a very
effective therapeutic for malaria.
[0193] We show here that FRAP is responsible for this activity.
FRAP effectively converted toxic heme into inactive hemozoin in a
dose dependent manner (FIG. 8). The hemozoin formation activity was
10-20-fold higher in comparison to histidine rich protein II, the
only known parasite protein capable of making hemozoin. This
activity was specific as it was lost when the protein was
pre-treated with proteinase K (a non specific protease) suggesting
that an intact protein is required for this activity (FIG. 9). The
activity requires the complete protein as two truncated variants of
FRAP (FRAP2 and FRAP3) did not show any hemozoin formation (FIG.
8).
[0194] The authenticity of the polymerized heme as hemozoin was
verified by FT-IR spectroscopy. The IR spectra of hemozoin contains
an intense absorbance at 1664 and 1211 cm.sup.-1, that are absent
in the spectra of free heme (Slater et al., 1991). These are
characteristics of a carboxylate group coordinated to the iron
center of ferriporphyrin (Fe01-O41) arising from stretching of the
localized carbon-oxygen double and single bonds, respectively
(Slater et al., 1991). The chemical structure of .beta.-hematin is
depicted in FIG. 10 (adapted from (Pagola et al., 2000)). The infra
red spectra of the FRAP-generated product showed the characteristic
decrease in transmittance at 1664 and 1211 cm.sup.-1, chemically
validating that the product formed was indeed hemozoin (FIG.
11).
[0195] FRAP residues involved in heme polymerization were
identified by generating 11 variants of FRAP by site-directed
mutagenesis. Evaluation of these mutants for heme
polymerization-activity revealed that three residues viz., F42, H44
& H122 are critically involved in hemozoin formation, as their
conversion to alanine lead to a complete loss of activity (Table
9).
[0196] FRAP protein shows remarkably high amount of sequence
homology between different Plasmodium species. In FRAP, a highly
conserved protein sequence has biological relevance as the residues
shown to be involved in hemozoin formation viz., F42, H44, H122
(Table 9) are not only conserved within the Plasmodium genus, they
are also conserved in Theileria parasites. This indicates that FRAP
protein from a non-human malaria parasite can be used as target for
screening and development of novel inhibitors for FRAP protein of
human malaria parasite.
[0197] This can be achieved by screening a library of small
molecules/inhibitors in vitro in the FRAP-mediated hemozoin
formation assay, which will lead to the identification of a
candidate molecule(s). These molecules can be subsequently
evaluated in an in vitro P. falciparum culture in the laboratory.
Once their efficacy has been proved in vitro, these molecules can
be evaluated in a rodent malaria parasite model. This will be
feasible due to the extremely conserved nature of the protein and
the amino acids residues of FRAP involved in the process of
hemozoin formation (F42, H44, H122), as seen by site-directed
mutagenesis, being identical between all known FRAP proteins (Table
9, FIG. 3).
[0198] Once a small molecule shows efficacy in the mouse malaria
model, it can be directly evaluated in a monkey model without
requiring extensive experimentation as FRAP in P. knowlesi, the
monkey malaria parasite, has the same residues in its active site.
Therefore, it is possible to develop FRAP inhibitors for human
malaria parasite by targeting FRAP sequence from other species of
Plasmodium. TABLE-US-00009 TABLE 9 Identification of FRAP residues
involved in Hemozoin formation. 11 FRAP residues were individually
mutated to alanine by site-directed mutagenesis; proteins were
expressed in E. coli and purified to homogeneity. Polymerization of
heme was investigated with 500 pmoles of each of the proteins and
their activity was compared with the unmutated FRAP. Conversion of
F42, H44 and H122 lead to a complete loss of activity, suggesting a
critical role for these residues in the polymerase activity of the
protein. Heme Polymerized Protein (nmoles) % Decrease FRAP 139.2 --
Y39A 155.2 -- F42A 0.6 99.5 H44A 6.7 95.1 F64A 102.2 26.6 H79A
133.5 4.1 F90A 111.1 20.1 H122A 0.9 99.3 C191A 104.9 24.6 H192A
115.6 16.9 H197A 106.4 23.5
[0199] A time kinetic analysis for hemozoin formation revealed that
the conversion of heme into hemozoin was complete within 5 hours
and was pH dependent where a pH of 5.2 was required for optimal
activity (FIG. 12). Stoichiometric analysis for FRAP-Heme
interaction using continuous variation method (Job's Plot) revealed
that the protein has a 1:1 stoichiometry with heme (FIG. 13).
Hemozoin formation could be effectively inhibited by chloroquine,
an antimalarial that is known to exerts its activity by binding to
free heme and preventing its polymerization into hemozoin (FIG.
14).
[0200] These results clearly demonstrate that (i) FRAP is
responsible for neutralization of heme through a polymerase
activity and (ii) the polymerization can be inhibited by
chloroquine. In addition, the active site residues that are
critical for this activity were identified. Therefore, FRAP is an
efficient drug target for malaria drug development, for example,
for the design of small molecules that bind to the active site and
inhibit the catalytic capability of FRAP.
References for Example 3
[0201] 1. Francis, S. E., Sullivan, D. J., Jr. and Goldberg, D. E.
(1997) Annu Rev Microbiol, 51, 97-123. [0202] 2. Gluzman, I. Y.,
Francis, S. E., Oksman, A., Smith, C. E., Duffin, K. L. and
Goldberg, D. E. (1994) J Clin Invest, 93, 1602-1608. [0203] 3.
Pagola, S., Stephens, P. W., Bohle, D. S., Kosar, A. D. and Madsen,
S. K. (2000) Nature, 404, 307-310. [0204] 4. Slater, A. F. and
Cerami, A. (1992) Nature, 355, 167-169. [0205] 5. Slater, A. F.,
Swiggard, W. J., Orton, B. R., Flitter, W. D., Goldberg, D. E.,
Cerami, A. and Henderson, G. B. (1991) Proc Natl AcadSci USA,
88,325-329. [0206] 6. Sullivan, D. J., Jr., Gluzman, I. Y. and
Goldberg, D. E. (1996) Plasmodium hemozoin formation mediated by
histidine-rich proteins. Science, 271, 219-222. [0207] 7. Wellems,
T. E., Walker-Jonah, A. and Panton, L. J. (1991) Proc Natl Acad Sci
USA, 88, 3382-3386.
Example 4
Use of FRAP in High Through Put Assays for Hemozoin Formation for
Screening Novel Antimalarials
[0208] As described above, the pathway for conversion of heme to
hemozoin is a major drug target. Until now, in vitro screening of
small molecules capable of this blockage has been performed by
evaluating their activity in an assay of hemozoin formation, where
polymerization is being performed using parasite lysate or is
chemically driven requiring extremely high salt concentrations.
These conditions, though yielding hemozoin, are far from perfect as
a typical experiment requires a 16 hour reaction and less than 10%
of the substrate is converted into a product (Tripathi et al.,
2004). Our FRAP-based methodology of hemozoin formation is
extremely superior to the currently available technology, as it
mimics the in vivo process, converts >50% of the initial
substrate into product and can be completed in as little as 5
hours. Therefore, a FRAP-based assay system for the identification
of antimalarials is an assay system of choice for these
processes.
Screening Procedure for Inhibitors of FRAP-mediated Hemozoin
Formation.
[0209] The first assay describes in detail how the hemozoin
formation is investigated. This is the complete detail of the assay
documenting every step of the process. This assay will be used for
studying the role of an inhibitor, as inhibition of FRAP activity
will cause a decrease in hemozoin formation which will b easily
quantifiable by this assay. This assay was used to inhibit hemozoin
formation using chloroquine and has been described as assay 2.
Assay 1: FRAP-mediated Hemozoin formation Assay (all temperatures
in degree C.):
[0210] The standard assay contained in a total volume of 1.0 ml:
500 mM sodium acetate pH 5.2, 300 nmol/ml hemin-Cl (as substrate)
and 500 pmol/ml FRAP, as the source of heme polymerase activity.
The amount of FRAP added was chosen such that 50% of the substrate
was converted into product (insoluble hemozoin) during the assay.
The reaction was initiated by protein addition and allowed to
proceed for 16 hours at 37 degree. The reaction was terminated by
adding 0.01 ml of 10% SDS solution. The reaction tube was
centrifuged at 13,000 rpm for 15 minutes at 23 degrees and the
supernatant was carefully removed. The pellet, which contained the
polymerized and insoluble hemozoin, was resuspended in 1 ml of 0.1M
sodium bicarbonate pH 9.1 containing 2.5% SDS. At this step, any
free heme present in the pellet will go into the solution at it is
soluble in sodium bicarbonate while the hemozoin is insoluble. This
process essentially removes any free heme that could be present in
the pellet. The suspension was spun at 13,000 rpm and the
supernatant, containing unpolymerized substrate was removed. This
process was repeated thrice, followed by washing of the pellet in
pure water. The pellet obtained after final washing was dissolved
in 0.3 ml of 0.1N NaOH and the absorbance of the solution was
measured at 405 nm using a spectrophotometer. Amount of heme
polymerized was calculated utilizing a standard curve, prepared by
dissolving known amounts of commercially available beta-hematin in
0.1N NaOH. Chemically synthesized beta-hematin and biologically
polymerized hemozoin are chemically identical (Pagola et al, 2000
Nature).
[0211] To assure that the heme polymerized was due specifically to
the action of FRAP, a parallel control incubations were performed
which either did not contain any protein or contained bovine serum
albumin, which was used a non-specific protein control.
Furthermore, the hemozoin formation was also evaluated with
truncated variants and point mutants of FRAP to not only describe
its structural requirements, but also pin point the residues that
are involved in the polymerization process.
Assay 2: Inhibition of FRAP-mediated hemozoin formation
[0212] For inhibition studies, the inhibitor under examination was
added to the standard assay cocktail (as described above) at the
desired concentration and the FRAP-mediated hemozoin formation
activity was compared to that found in control (minus inhibitor)
incubations which lacked inhibitor.
[0213] This assay system will be utilized for screening FRAP
inhibitors. A difference in the amount of hemozoin seen in the
presence of an inhibitor with respect to the reaction where the
inhibitor was absent is directly attributable to the activity of
the inhibitor in the reaction.
Example 5
siRNA Mediated Inhibition of FRAP Activities and Genetic Mechanisms
that can Downregulate FRAP Expression Leading to Malaria
Control
[0214] Gene knockout experiments were performed for FRAP to study
its criticality in the life of the parasite. DNA encoding a short
segment of FRAP was cloned into a vector encoding the gene for
Dihydrofolate reductase (DHFR) as a selection marker. The resulting
plasmid vector was transfected into parasites in culture, and the
parasites were then subjected to drug pressure (e.g. Drug WR99210)
to select for parasites that do not encode a functional FRAP gene.
Deletion of FRAP from the genome led to the death of the parasites
indicating that (i) this gene is critical for the survival of the
parasite and (ii) any strategy that can either prevent the
expression of the FRAP gene product or decrease its level of
expression can be exploited for controlling malaria. This result
also gains credence from the biological role of this protein
described by inventors where they have shown that the protein is
involved in the infectivity process and in neutralization of heme,
which is critical for the survival of the parasite. Therefore,
methods that can neutralize the FRAP gene product will
automatically lead to malaria control.
[0215] In the last few years, inhibition of a gene function by
utilizing small inhibitory RNA (siRNA) has been shown to be
feasible for a variety of pathogens. This technology has proved to
be extremely effective in Trypanosome parasites, where it has been
extensively utilized for understanding the role of a particular
gene in the infectivity process and pathogenicity (Best et al.,
2005; Ullu et al., 2002). As deletion of FRAP from the genome is
lethal, and the protein plays an important role in the disease
process, therefore, siRNA mediated gene silencing can be an
effective method for controlling malaria. This is achieved by
designing short segments of sense and anti-sense RNA fragments that
are complementary to the coding sequence of FRAP. These sequences
are delivered to the cytosol of the parasite through a plasmid DNA
construct. Once in the cytosol, transcription of the siRNA occurs
and prevents the expression of FRAP. The result is loss of the
activity of this critical protein, without which the parasite is
not able to survive.
[0216] For example, human Plasmodium parasites can be transformed
with vectors expressing one or more siRNA molecules based on SEQ ID
NOS: 2 or 8. Methods for design of siRNA molecules have been
published by a number of sources. A recent publication by Dharmacon
Inc. (Reynolds, A. et al., Rational design for RNA interference
(2004), Nature Biotechnology 22: 326-330) suggests eight design
criteria optimal for effective siRNA design. The siDESIGN.TM.
Center Program provided by Dharmacon Inc. can be used to design
optimal siRNA molecules based on the SEQIDs 2 or 8, that have one
or more of the following features: have low G/C nucleotide content
(30-52% G/C); three or more A/U nucleotides at the 3'-terminus of
the sense strand (the mRNA coding strand); a lack of internal
repeats that can form secondary structures; and sequence-specific
preferences at the following positions on the sense strand--an A at
position 19, an A at position 3, a U at position 10, and an absence
of a G or C at position 19 and a G at position 13. The resulting
siRNA oligonucleotides can be cloned as a small hairpin RNAs
(shRNA) between a Plasmodium RNA Polymerase III (Pol III) promoter,
which initiates synthesis at a defined distance from the promoter,
and a termination sequence consisting of a string of 4-5 uridines,
or other suitable constitutive promoters can be used as well. When
transfected and co-expressed with a selectable marker into
Plasmodium cells, siRNA expression will reduce the levels of the
endogenous mRNAs corresponding to SEQ ID 2 or 8.
[0217] Several sources are available which give detailed
descriptions of the use of siRNA technology. For example, WO0044895
(Kreutzer and Limmer) specifically covers the use of small dsRNAs
as therapeutics, and specifically to methods and medicaments
involving the use of small dsRNAs formed from two separate strands
and having a region complementary to the target gene.
[0218] US2005026278 (Tuischl et al.) describes a key structural
feature of siRNAs, namely the presence of overhangs at the 3'-end
of each of the two strands and includes data on mammalian cell gene
silencing. U.S. Pat. Nos. 5,898,031 and 6,107,094 (the entire
contents of which are hereby incorporated by reference) describe
degradation of target mRNA mediated by chemically modified
RNAi-like oligonucleotides.
Example 6
A Variant of FRAP that Leads to Attenuated Parasites, which can be
Used as a Whole Organism Vaccine
[0219] We have successfully demonstrated that FRAP performs the
critical neutralization of toxic heme into non-toxic hemozoin. We
have also identified amino acids in FRAP, whose conversion result
in protein variants in which the heme polymerase activity has been
totally lost or has been compromised (Table 7). Developing a
parasite which has been genetically modified in such a way, where
the FRAP gene is present in the genome, but it has been modified by
a genetic modification to a variant copy of the protein, which
encodes a protein that is not fully functional, will give rise to
attenuated parasites. Such a process has been previously
demonstrated in other systems where CSP, a gene encoding a parasite
protein involved in pathogenesis was swapped by genetic
manipulation resulting in attenuated parasites (Tewari et al.,
2005). Attenuated parasites may also be produced using siRNA
vectors as described in the section above.
Example 7
Use of FRAP as a Tool for High Expression of Recombinant Proteins
and Subsequent Purification
[0220] As described in Example 1, expression of DNA encoding FRAP
in E. Coli leads to very high expression and up to 40 mg of
purified protein can be purified from a one liter shaker flask
culture. Obtaining high yields for a recombinant protein and
development of optimal purification strategies has long been
recognized as a major bottleneck for developing therapeutics. In
the field of recombinant protein expression and purification, these
issues have been tackled by expressing a gene of interest fused
with a second gene (commonly called as a tag), which has distinct
binding properties and a high level of expression. The two most
commonly utilized tags for such purposes are DNA encoding for
maltose-binding protein and glutathione S transferase. These tags
not only facilitate purification of protein by exploiting the
distinct binding properties of the tags but also help by enhancing
the expression of the gene of interest.
[0221] The high level of expression of FRAP in its recombinant
expression and its unique capabilities of interaction with heme
makes this protein uniquely fitted to serve as a tag in recombinant
expression vectors. FRAP-based fusions proteins are purified by
affinity chromatography by exploiting its heme-binding properties
in a column chromatography system, where the fusion protein binds
to the column through available heme moiety and is cluted by excess
of free heme. Various fusion proteins of FRAP having epitopes of
CSP and TRAP may be produced by this method for use, e.g. in a
vaccine.
References for Examples 4-7
[0222] Best, A., Handoko, L., Schluter, E. and Goringer, H. U.
(2005) J Biol Chem, 280, 20573-20579. [0223] Tewari, R., Rathore,
D. and Crisanti, A. (2005) Cell Microbiol, 7, 699-707. [0224]
Tripathi, A. K., Khan, S. I., Walker, L. A. and Tekwani, B. L.
(2004) Anal Biochem, 325, 85-91. [0225] Ullu, E., Djikeng, A., Shi,
H. and Tschudi, C. (2002) RNA interference: advances and questions.
Philos Trans R Soc Lond B Biol Sci, 357, 65-70.
Example 8
Production of Fully Human Antibodies
[0226] Fully human monoclonal antibodies against Plasmodium or
Theileria antigens are made in mice directly, when these mice are
engineered to produce only human antibody chains. For example the
technology practiced by companies such as Abgenix Inc. [XenoMouse
technology, U.S. Pat. No. 6,657,103], Medarex Inc. and GenMab A/S
[HuMab Mouse or UltiMAB technology; WO2005023177] can be used.
Purified proteins as described above are used to immunize such
engineered mice. Monoclonals produced in this manner are produced,
screened and characterized in the standard manner. Fully human
antibodies are produced using phage display methods by screening
against human antibody phage display libraries. For example
technologies practiced by companies such as Cambridge Antibody
Technology [U.S. Pat. No. 5,969,108 and U.S. Pat. No. 6,172,197]
and others, can be used to identify fully human antibodies in this
manner. Phage display screening has as an added advantage that the
process does not rely on animal immunization. The genes for fully
human antibodies produced using engineered mice, or identified
through phage display, are isolated, sequenced and cloned for
expression in mammalian cell lines for high level expression using
standard methods.
Example 9
Further Characterization of HDP
[0227] Development of new drugs is urgently needed to replace major
antimalarials that have become ineffective due to increasing drug
resistance. During the intra-erythrocytic stage, malaria parasites
proteolyse globin chains of host hemoglobin1, releasing prosthetic
group heme, which is toxic to the parasite. Heme is immediately
detoxified, primarily by its conversion into a metabolically inert
crystalline material called hemozoin (Hz).sup.2.3, a step essential
for parasite survival and targeted by some of the most effective
antimalarial drugs ever discovered, including chloroquine. These
drugs exert their anti-parasite activity by binding to free
heme.sup.4.5, which prevent its detoxification into Hz. Parasite
factors responsible for heme detoxification are poorly identified
and remain controversial.sup.6.9. In this example, the
identification, genetic characterization and functional activity of
a novel Plasmodium falciparum protein that efficiently converts
free heme into Hz is described. The protein readily converts up to
50% of free heme into Hz, at a rate that is at least an order of
magnitude higher than any of the known parasite factors.sup.6.9
capable of Hz synthesis. Therefore, the polypeptide has been
designated heme detoxification protein or HDP. (Alternatively, the
protein may also be designated "Fasciclin Related Adhesive Protein"
or "FRAP", as is the case in the previous examples. HDP orthologs
have also been identified in rodent, simian and avian Plasmodium
species. HDP is highly conserved within the Plasmodium genus and
appears to be essential as it's gene disruption could not be
achieved in P. falciparum parasites. By immunoelectron microscopy
studies, it has ben demonstrated that after merozoite invasion,
ring form parasites express and secrete this protein into the
erythrocyte cytosol before any detectable amount of Hz is visible
inside the parasite. Subsequently, HDP, accompanied by host
hemoglobin, is delivered to the parasite food vacuole, the site of
Hz formation. Together, these results establish HDP as a key
parasite protein responsible for heme detoxification and therefore,
its targeting could lead to the discovery of novel antimalarial
drugs.
[0228] Major clinical manifestations of malaria are associated with
the development of Plasmodium parasites inside host erythrocytes.
During this stage, heme is detoxified and predominantly sequestered
inside the parasite's food vacuole as Hz, which is chemically and
structurally identical to .beta.-hematin.sup.2.3. The underlying
mechanism, though poorly understood, is believed to be highly
conserved as Hz formation occurs in all the species of Plasmodium
during their intraerythrocytic development, irrespective of the
host species they infect.
[0229] HDP, a single copy, three-exon encoded.sup.10, 205 amino
acid long P. falciparum polypeptide (GenBank Acc#NP.sub.--702335;
FIG. 3) that potently detoxifies heme into Hz (FIG. 4c and 4d) was
identified. The HDP gene was found to be actively transcribed and
expressed during the intraerythrocytic stages, a phase of the
lifecycle where Hz is produced by the parasite (FIG. 4b). The
coding sequence of HDP corresponding to amino acid 1-205 (SEQ ID
NO: 1) was cloned in a T7 promoter-based E. coli expression plasmid
and recombinant HDP was produced and purified to homogeneity (FIG.
4a).
[0230] In a Hz formation assay.sup.6, where heme was present in
several hundred fold molar excess with respect to HDP, it was found
that the protein actively converted heme into Hz, in a dose
dependent manner (FIG. 16a-b). Hz production increased with an
increase in the concentration of either free heme (FIG. 16a) or HDP
(FIG. 16b), converting up to 50% of free heme into Hz, until the
reaction reached equilibrium (FIG. 16a). At the highest heme
concentration tested, HDP produced Hz at a rate of 21 nmol/hr,
which was at least 20 fold higher than that of Histidine Rich
Protein II (HRP II) and unsaturated (oleic acid and mono-oleoyl
glycerol) lipids (FIG. 16a), the only known parasite components
capable of Hz synthesis. This process was HDP-dependent, as in its
absence, Hz production occurred at baseline (0.1-0.2 nmol/hr)
levels. Fourier transform infrared spectroscopy confirmed the
sequestered product as Hz, as it showed characteristic absorption
peaks at 1660 and 1210 cm.sup.-1, a spectroscopic signature.sup.2
of carboxylate side group coordinated to the iron center of
ferriprotoporphyrin IX (FIG. 16c). In vivo, Hz formation occurs in
an acidic (pH 4.5-5.2) milieu.sup.11.12 of the food vacuole and it
was found that HDP had optimal activity in a similar environment
(FIG. 16d), that was indicative of its potential to function in the
food vacuole. It is noteworthy that HRP II (and HRP III), the only
known parasite proteins capable of Hz synthesis.sup.6, are only
found in P. falciparum parasites, where most of the protein
produced is secreted by the parasite.sup.13.14 and Hz production is
unaffected in parasite clones lacking the two proteins.sup.15. This
led to a suggestion that unsaturated membrane lipids could be
producing Hz in the parasite.sup.7.9. However, these results
clearly show that HDP is the most potent parasite factor and could
be the major producer of Hz inside the parasite.
[0231] To investigate whether the heme detoxification activity
demonstrated by recombinant HDP is the true representation of its
role in the parasite, native HDP from erythrocytic stage P.
falciparum parasites was purified. On a SDS-PAGE gel, native HDP
showed an approximate molecular weight of .about.60 KDa, possibly
due to dimerization, and was recognized by anti-HDP antibodies on a
western blot (FIG. 16e). Furthermore, it was found that native HDP
was able to produce Hz at levels comparable to the recombinant
protein (FIG. 16f), which indicated that in vivo, HDP could indeed
be involved in Hz formation.
[0232] Hz formation is an indispensable step in parasite's
lifecycle. As results from our in vitro studies inferred towards a
major role for HDP in this process, its involvement was
investigated in vivo by a genetic knockout experiment in
erythrocytic stage P. falciparum parasites. Disruption of the HDP
locus was attempted by a plasmid-based single cross over
recombination (FIG. 17a). To promote plasmid integration at the
targeted locus, transfected parasites were subjected to three drug
selection cycles over a 12 week period. In two independent
experiments, parasites with a disrupted HDP locus could not be
obtained and the resulting transfectants episomally carried the
pHDPKO plasmid (FIG. 17b) and expressed HDP at levels comparable to
the wild type parasites (not shown), Therefore, it is highly likely
that HDP plays a critical role in Hz formation and its inactivation
may not be possible.
[0233] Inside an infected erythrocyte, up to 75% of the total
hemoglobin is degraded.sup.16 giving rise to large quantities of
free heme, most of which is converted into Hz.sup.7. Having
established the role of HDP in this process, its affinity for heme
was investigated by isothermal titration calorimetry FIG. 18a).
This interaction was studied by measuring the heat change
associated with the binding of heme to HDP, at pH 5.6 where protein
bound heme but did not make any Hz. The interaction revealed a H of
-5.03 kcal/mol, a Kd of 80 nM, and a stoichiometry (n) of 2.7 heme
molecules per HDP polypeptide. This affinity is at least 4 times
higher than HRP II, whose affinity for heme is in 340-940 nM
range.sup.18.19.
[0234] Subsequently HDP sequence were analysed for the presence of
any known heme binding motif using SMART20, a domain identification
tool. While HDP has no homology to any of the known heme-binding
proteins, the analysis revealed that the carboxyl terminus region
(amino acids 88-205) of the protein has homology (e value
3e-.sup.10) to fasciclin-l, an ancient adhesive and highly diverse
domain, present in proteins of prokaryotic.sup.21 and
eukaryotic.sup.22 origin (FIG. 3). To investigate if this domain
alone is responsible for Hz formation, two truncated variants of
HDP were recombinantly produced, one encoding only the fasciclin-1
domain (residues 88-205 of SEQ ID NO: 1; protein HDP3) and the
other encoding residues 1-87 of the full length protein (i.e. of
SEQ ID NO: 1, protein HDP2) (FIG. 19a-d). It was found that neither
fasciclin-1 domain (HDP3) nor the amino terminus region (HDP2)
alone were capable of Hz production (FIG. 18b). Hence, a full
length HDP is required for Hz production.
[0235] As stated earlier, HRP II and HRP III are only found in P.
falciparum parasites but Hz formation occurs in all known species
of Plasmodium. To investigate if HDP is present in all the parasite
species, the genomes of seven other species of Plasmodium.sup.23.24
were examined in silico (FIG. 3). HDP orthologs were found in all
the species with protein showing 60% sequence identity. Evidently,
the protein is functionally conserved as a recombinantly produced
P. yoelii HDP generated Hz at levels indistinguishable from its P.
falciparum ortholog (FIG. 18c). HDP seems to have an ancient
lineage as its homolog was found in Theileria.sup.25 genome (FIG.
3), a hemoprotozoan that sequesters heme into non-toxic aggregates
during the intraerythrocytic stages of its lifecycle.
[0236] As Hz formation occurs inside the food vacuole, to be
functionally relevant, one would anticipate HDP to be present
inside this organelle. Though the protein lacks a classical
N-terminal signal sequence or any known translocation signal that
could predict its possible sorting and transport to its destined
site, the presence of HDP was detected inside the food vacuole
(FIG. 20a-d). Therefore, to comprehend its intracellular
trafficking, intraerythrocytic parasites were analyzed at different
stages of development, for HDP expression. It was discovered that
from the early (ring) stages of infection, HDP is secreted to the
host cell cytosol, before any detectable amount of Hz was visible
inside the parasite (FIG. 20a). The protein accumulated inside the
cytosol of the host cell (FIG. 20b; FIG. 21a-c) and was not
exported out of the infected RBC as it could not be detected in the
concentrated culture supernatant by immunoblot (data not shown).
Subsequently, as parasite development progressed, it was found that
HDP, along with host hemoglobin, is trafficked to the food vacuole,
through the cytostome-mediated pathway (FIG. 20b-d; FIG. 21a-c). By
immunoelectron microscopy, we detected the uptake of HDP through
the cytostome (FIG. 20b, FIG. 21b), its presence in the transport
vesicles (FIG. 20c) and delivery to the food vacuole (FIG. 20d;
FIG. 21c). This novel and circuitous trafficking of HDP is
indicative of a functional convergence in the parasite where host
hemoglobin, HDP and parasite protease26 involved in hemoglobin
proteolysis (and located in the vesicular membrane), are
transported together to the food vacliole.
[0237] This is the first report of a pan-Plasmodium heme
detoxifying protein that is highly efficient in catalyzing the
conversion of heme into Hz. Identification of HDP not only fills an
important gap in our understanding of the mechanism of Hz
production in malaria parasite, but the novel "Outbound-Inbound"
trafficking of HDP also reveals an interesting insight into the
inner workings of the parasite. Due to the rapid emergence of
multi-drig resistant parasites, several major antimalarial drugs
have become ineffective and combination therapy is fast becoming a
mainstay for malaria control.sup.27. This discovery opens new
avenues for designing novel antimalarial drugs that specifically
target HDP and thereby prevent the conversion of heme into Hz.
Methods for Example 9
[0238] Hz formation assay The assay was performed as previously
described.sup.6. Briefly, equimolar amounts (0.5 nmol) of HDP, HRP
II or unsaturated lipids were added to freshly prepared heme
solution in 500 mM sodium acetate buffer pH 5.2, followed by
incubation at 37.degree. C. for 16 hrs. The reaction was stopped by
adding SDS (0.1% final conc.). Unsequestered heme was removed by
repeated washing of the pellet with 2.5% SDS and 0.1 M sodium
bicarbonate (pH 9.1) followed by distilled water till no soluble
heme was visible in the supernatant. Hz pellet was resuspended in
0.1 N NaOH and absorbance was measured at 400 nm. A standard curve
using different concentrations of .beta.-hematin was prepared to
quantitate the amount of heme incorporated into Hz. A reaction
containing buffered heme alone was used as negative control. pH
dependence of HDP was evaluated in 500 MM sodium acetate buffer of
different pH (pH 3.2-6.0). All the Hz formation assays were
performed at least three times in triplicates.
[0239] Purification of native HDP. Anti-HDP antibodies were raised
in rabbits and affinity purified using standard protocols.
Trophozoite stage P. falciparum (3D7 strain) parasites were
isolated from a 20 ml culture using a MACS column (Miltenyi
Biotec), and resuspended in 0.2 ml of solubilization buffer (20 mM
Tris-Cl pH 7.4, 0.5% NP-40, 1.times. Protease Inhibitor Cocktail).
The suspension was subjected to a single freeze-thaw cycle and the
protein extract was clarified by centrifugation at 15,000 g for 15
min at 4.degree. C. Affinity purified anti-HDP antibodies were
coupled to AminoLink.RTM. Plus Coupling Gel using the Seize.RTM.
Primary Immunoprecipitation kit (Pierce Biotechnology), and
utilized for immunoprecipitation of native HDP from the total
protein extract, as per manufacturer's instructions. Purity of the
protein was established by silver staining and the purified protein
was authenticated by an ECL-based immunoblotting system (GE Health
Care).
[0240] Binding affinity. Binding affinity of HDP for heme was
evaluated by Isothermal titration calorimetry where freshly
prepared heme solution was incrementally added to 5 .mu.M HDP (in
50 mM MES, pH 5.6) present inside the ITC cell. Data was collected
at 30.degree. C. at a 420 rpm stir rate using 10 .mu.l injections
of the 100 .mu.M heme into the protein solution. The resulting
measurements delta H vs. molar ratio were fit to a single binding
site model using the MicroCal Origin analysis software.
[0241] Immunoelectron microscopy. P. falciparum infected
erythrocytes were fixed in 4% paraformaldehyde/0.1% glutaraldehyde
in 100 nM PIPES/0.5 mM MgCl.sub.2, pH 7.2 for 1 hr at 4.degree. C.
and used for immunoelectron microscopy as described .sup.26.
Controls omitting the primary antibody were consistently negative
at the concentration of gold-conjugated secondary antibodies used
in these studies.
[0242] Targeted deletion of HDP P. falciparum 3D7 parasites was
cultured in human O+erythrocytes as described previously. Ring
stage parasites at 10% parasitemia were transfected by
electroporation with 100 .mu.g of super coiled pHDPKO, a pHD22Y
based transfection vector containing a 509 bp fragment from the 5'
end of the HDP gene (SEQ ID NO: 2) along with human DHFR selection
cassette, using low voltage/high capacitance conditions .sup.28.
Transfectants were selected in the presence of 10 nM WR99210 (a
gift from Jacobus Pharmaceuticals, Princeton N.J.) and subjected to
three drug selection cycles, each consisting of 21 days of growth
in absence of WR99210 followed by reselection of parasites in the
presence of 10 nM WR99210. Genotypes were analyzed by probing blots
of Eco RV-Bam HI digested total parasite DNA, with a PCR amplified
509 bp fragment of HDP that has been cloned in the transfection
vector. The signal was generated with an Alk Phos direct labelling
and detection kit (GE Healthcare).
[0243] Immunofluorescence Methanol fixed smears of infected RBC at
5% parasitemia were blocked with 2.5% normal goat serum (NGS) for
30 min and incubated with rabbit anti HDP antibodies at 1:200 for 1
h. Bound antibodies were detected using fluorescein isothiocyanate
(FITC)-conjugated goat anti-rabbit IgG diluted to 1:200. Parasite
nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI).
Slides were mounted with the antifade reagent (Vectorshield, KPL)
and images (100.times.magnification) were obtained using Olympus
1.times.70 inverted fluorescence microscope and a Photometrix
cooled charge-coupled device camera (CH350/LCCD) driven by
DELTAVISION software from Applied Precision (Seattle, Wash.).
[0244] Cloning, recombinant expression and purification of HDP
Coding sequence of HDP (SEQ ID NO: 2) was amplified by RT-PCR using
total RNA from the P. falciparum (3D7 strain) erythrocytic stage
parasites. The amplified fragment was cloned in pET101, a V5
epitope and polyhistidine-tag encoding, T7 promoter-based E. coli
expression vector, giving rise to plasmid pHDP. Protein, expressed
in BL21 cells, was localized in inclusion bodies, which were
isolated as described previously.sup.29. Purified inclusion bodies
were solubilized in 50 mM CAPS buffer (pH 11.0) containing 1.5%
N-lauryl sarkosine and 0.3 M NaCl, for 30 min and the solubilized
protein was separated by centrifugation (10,000 g; 30 min). Protein
was purified by affinity chromatography on His-Trap, a high
performance nickel affinity column (GE Health Care) using an
imidazole gradient in 50 mM CAPS pH 11.0 containing 0.3% N-lauryl
sarkosine and 0.3 M NaCl. Protein-containing fractions were pooled
and purified to homogeneity by gel filtration chromatography on
Superdex 200 10/300 GL column (GE Health Care), equilibrated in 25
mM CAPS (pH 11.0) containing 135 mM NaCl. PyHDP (SEQ ID NO:10) was
amplified by RT-PCR using total erythrocytic stage P. yoelii RNA
and cloned in pET101 plasmid. Plasmids encoding protein HDP2 and
HDP3 were generated by sub-cloning using pHDP as template. Their
expression and purification was performed as described above. DNA
encoding P. falciparum Histidine rich protein II was cloned in
pET101 and its expression and purification was performed as
described previously..sup.30
References for Example 9
[0245] 1. Francis, S. E., Sullivan, D. J., Jr. & Goldberg, D.
E. Hemoglobin metabolism in the malaria parasite Plasmodium
falciparum. Annu Rev Microbiol 51, 97-123 (1997). [0246] 2. Slater,
A. F. et al. An iron-carboxylate bond links the heme units of
malaria pigment. Proc Natl Acad Sci USA 88, 325-9 (1991). [0247] 3.
Pagola, S., Stephens, P. W., Bohle, D. S., Kosar, A. D. &
Madsen, S. K. The structure of malaria pigment beta-haematin.
Nature 404, 307-10 (2000). [0248] 4. Sullivan, D. J., Jr., Gluzman,
l. Y., Russell, D. G. & Goldberg, D. E. On the molecular
mechanism of chloroquine's antimalarial action. Proc Natl Acad Sci
USA 93, 11865-70 (1996). [0249] 5. Kannan, R., Sahal, D. &
Chauhan, V. S. Heme-artemisinin adducts are crucial mediators of
the ability of artemisinin to inhibit heme polymerization. Chem
Biol 9, 321-32 (2002). [0250] 6. Sullivan, D. J., Jr., Gluzman, I.
Y. & Goldberg, D. E. Plasmodium hemozoin formation mediated by
histidine-rich proteins. Science 271, 219-22 (1996). [0251] 7.
Bendrat, K., Berger, B. J. & Cerami, A. Haem polymerization in
malaria. Nature 378, 138-9 (1995). [0252] 8. Dorn, A., Stoffel, R.,
Matile, H., Bubendorf, A. & Ridley, R. G. Malarial
haemozoin/beta-haematin supports haem polymerization in the absence
of protein. Nature 10374, 269-71 (1995). [0253] 9. Fitch, C. D.,
Cai, G. Z., Chen, Y. F. & Shoemaker, J. D. Involvement of
lipids in ferriprotoporphyrin IX polymerization in malaria. Biochim
Biophys Acta 1454, 31-7 (1999). [0254] 10. Gardner, M. J. et al.
Genome sequence of the human malaria parasite Plasmodium
falciparum. Nature 419, 498-511 (2002). [0255] 11. Yayon, A.,
Cabantchik, Z. I. & Ginsburg, H. Identification of the acidic
compartment of Plasmodium falciparum-infected human erythrocytes as
the target of the antimalarial drug chloroquine. Embo J 3, 2695-700
(1984). [0256] 12. Hayward, R., Saliba, K. J. & Kirk, K. The pH
of the digestive vacuole of Plasmodium falciparum is not associated
with chloroquine resistance. J Cell Sci 119, 1016-25 (2006). [0257]
13. Akompong, T. et al. Trans expression of a Plasmodium falciparum
histidine-rich protein II (HRPII) reveals sorting of soluble
proteins in the periphery of the host erythrocyte and disrupts
transport to the malarial food vacuole. J Biol Chem 277, 28923-33
(2002). [0258] 14. Howard, R. J. et al. Secretion of a malarial
histidine-rich protein (Pf HRP II) from Plasmodium
falciparum-infected erythrocytes. J Cell Biol 103, 1269-77 (1986).
[0259] 15. Wellems, T. E., Walker-Jonah, A. & Panton, L. J.
Genetic mapping of the chloroquine-resistance locus on Plasmodium
falciparum chromosome 7. Proc Natl Acad Sci USA 88,3382-6(1991).
[0260] 16. Goldberg, D. E., Slater, A. F., Cerami, A. &
Henderson, G. B. Hemoglobin degradation in the malaria parasite
Plasmodium falciparum: an ordered process in a unique organelle.
Proc Natl Acad Sci USA 87, 2931-5 (1990). [0261] 17. Egan, T. J. et
al. Fate of haem iron in the malaria parasite Plasmodium
falciparum. Biochem J 365, 343-7 (2002). [0262] 18. Schneider, E.
L. & Marletta, M. A. Heme binding to the histidine-rich protein
II from Plasmodium falciparum. Biochemistry 44, 979-86 (2005).
[0263] 19. Pandey, A. V. et al. Mechanism of malarial haem
detoxification inhibition by chloroquine. Biochem J 355, 333-8
(2001). [0264] 20. Schultz, J., Milpetz, F., Bork, P. &
Ponting, C. P. SMART, a simple modular architecture research tool:
identification of signaling domains. Proc Natl Acad Sci USA 95,
5857-64 (1998). [0265] 21. Ulstrup, J. C., Jeansson, S., Wiker, H.
G. & Harboe, M. Relationship of secretion pattern and MPB70
homology with osteoblast-specific factor 2 to osteitis following
Mycobacterium bovis BCG vaccination. Infect Immun 63, 672-5 (1995).
[0266] 22. Kim, J. E. et al. Identification of motifs for cell
adhesion within the repeated domains of transforming growth
factor-beta-induced gene, betaig-h3. J Biol Chem 275, 30907-15
(2000). [0267] 23. Carlton, J. M. et al. Genome sequence and
comparative analysis of the model rodent malaria parasite
Plasmodium yoelii yoelii. Nature 419, 512-9 (2002). [0268] 24.
http://www.sanger.ac.uk/Projects/Protozoa/. [0269] 25. Pain, A. et
al. Genome of the host-cell transforming parasite Theileria
annulata compared with T. parva. Science 309, 131-3 (2005). [0270]
26. Klemba, M., Beatty, W., Gluzman, I. & Goldberg, D. E.
Trafficking of plasmepsin II to the food vacuole of the malaria
parasite Plasmodium falciparum. J Cell Biol 164, 47-56 (2004).
[0271] 27. Rathore, D., McCutchan, T. F., Sullivan, M. & Kumar,
S. Antimalarial drugs: current status and new developments. Expert
Opin Investig Drugs 14, 871-83 (2005). [0272] 28. Fidock, D. A.
& Wellems, T. E. Transformation with human dihydrofolate
reductase renders malaria parasites insensitive to WR99210 but does
not affect the intrinsic activity of proguanil. Proc Natl Acad Sci
USA 94, 10931-6 (1997). [0273] 29. Rathore, D. et al. Molecular
mechanism of host specificity in Plasmodium falciparum infection:
role of circumsporozoite protein. J Biol Chem 278, 40905-10 (2003).
[0274] 30. Sullivan, D. J., Jr., Gluzman, I. Y. & Goldberg, D.
E. Plasmodium hemozoin formation mediated by histidine-rich
proteins. Science 271, 219-22 (1996).
Example 10
HDP as an Antimalarial Drug Target for the Two Major Species (P.
falciparum & P. vivax) of Human Malaria
[0275] It has been shown that P. falciparum HDP produces hemozoin
in the parasite. In this example, it is shown that the HDP ortholog
from P. vivax parasites (SEQ ID NO:7) can also produce Hemozoin
(FIG. 22). The experiment was performed as described according to
methods described above for the previous examples. P. vivax is the
second most important human malaria parasite, responsible for
almost 50% of the total malaria cases. Though rarely lethal, it
causes severe morbidity and is a major problem in the southeastern
Asia and Latin America. Therefore, with the demonstration that HDP
from P. vivax (SEQ ID NO:7) also produces Hemozoin, inhibitors of
HDP developed against P. falciparum parasite could also be used to
prevent or treat P. vivax malaria infections.
Example 11
Development of HDP as a Vaccine Candidate
[0276] It has been previously shown that antibodies raised against
HDP can prevent invasion of hepatocytes by P. falciparum parasites,
raising the possibility that HDP could be developed as a vaccine
candidate. This possibility was investigated in a P. yoelii-based
mouse malaria model. Both a protein and a DNA-based approach was
pursued for investigating the potential of HDP in protecting the
host from malaria. Due to differences in haplotype, two different
species of mice were investigated.
Materials and Methods
[0277] Cloning of PyHDP gene from Plasmodium yoelii into a DNA
vaccine plasmid, pVRJ020: RNA from Plasmodium yoelii was used for
amplification of the PyHDP gene. Primers were designed to amplify a
region encoding amino acids 1 to 205 of the PyHDP gene (SEQ ID
NO:9). A BamHI site [bold sequence] was incorporated into the 5'
primer (GGAATTCAGGAGCCCTTCGGATCCAAAAAAAAATTGTAT, SEQ ID NO: 40) and
the 3' primer (CTTCGAATTGAGCTCGGATCCTCAAATTATTGGCTTATCTATGAT SEQ ID
NO: 41). The 3' primer also incorporated a stop codon [underlined
sequence]. The PCR fragment (618 bp) was purified using the PCR
purification kit from Qiagen and digested with BamHI. The base
vector pVR1020.sup.1 containing a kanamycin resistance gene was
also digested with BamHI for 3 hrs at 37.degree. C. During the last
30 min of digestion 1 unit/.mu.l of shrimp alkaline phosphatase was
added to dephosphorylate the ends of the vector. The digested PCR
product and pVR1020 were purified after electrophoresis on a 1%
agarose gel. Ligation was carried out with various vector to insert
ratios. The ligation was performed for 16 hrs at 14.degree. C.
Transformed E coli, DH5.alpha., bacteria were plated on kanamycin
selective media and incubated at 37.degree. C. for 24 hrs. Colonies
contain recombinant plasmids were cultured and plasmids isolated.
The plasmids were sequenced to confirm the insert sequence and
orientation. Plasmid clones were transfected into VM449 cells and
PyHDP expression was confirmed with western blot using 1:200
dilution of anti-PyHDP antibodies raised in mice. Large scale
plasmid DNA from a confirmed PyHDP expressing pVRPyHDP clone was
prepared and purified using an endotoxin-free plasmid purification
Giga kit (QIAGEN Inc., Valencia, Calif.).
[0278] Immunization of Mice: All animal experiments were conducted
in accordance with the guidelines indicated in the National
Institutes of Health Guide to Laboratory Animal Care and were
approved by the Virginia Tech Animal Care and Use Committee. Six
week-old female BALB/c and A/j mice were used for the immunization
and challenge experiment. Three groups of eight mice each were
immunized as indicated in Table 10. This immunization schedule was
repeated twice at intervals of 21 days for all the groups. Groups 1
and 2 were controls for protein and plasmid immunizations, and were
immunized with PBS and base vector pVR1020, respectively. Purified
recombinant PyHDP was used for subcutaneous immunizations at 110
.mu.g/100 .mu.l/mouse (group 3). The first dose was prepared in
complete freunds adjuvant with subsequent doses given in incomplete
freunds adjuvant. For the pVRPyHDP plasmid immunization group
(group 4), DNA was injected intramuscularly (i.m.) into the
gastrocnemius muscle with a 29-gauge needle using 100 .mu.g of DNA
in 100 .mu.l of phosphate-buffered saline (PBS). The last dose of
the DNA vaccine immunization regime was with recombinant PyHDP at
100 .mu.g/100 .mu.l/mouse prepared with incomplete freunds adjuvant
and was administered subcutaneously.
[0279] Sporozoite preparation: Anopheles stephensi mosquitoes were
reared in cages at 27.degree. C. and >80% relative humidity and
were fed with 10% sucrose solution every alternate day [2]. For the
development of the sporozoite stage, mosquitoes starved of sucrose
for 24 hrs, were allowed to blood feed on anesthetized P yoelii
infected mice for 10 minutes. Samples of salivary glands and
stomach were prepared beginning 10 days post feeding to monitor the
development of the mosquito stages of the parasite.
[0280] Sporozoites were isolated using the Ozaki method .sup.3.
Briefly, on the day of challenge (day 0) the mosquitoes were
anesthetized with chloroform and thorax dissected in complete M199
medium. Crushed thorax was loaded on a silanized glass wool column
prepared in Eppendorf tubes, and was centrifuged at 2500 rpm to
collect the flow through. The pellet from 2-3 such tubes were
resuspended and pooled. Sporozoites were counted using a
hemocytometer and resuspended in complete M199 medium at a
concentration of 100 sporozoites per 100 .mu.l. Immunized mice were
challenged with 100 sporozoites injected via the tail vein.
[0281] Monitoring parasitemia: Parasitemia in all mice from all the
groups were monitored on alternate days by conventional Giemsa
staining .sup.4 starting on day 4 after infection. Thin blood films
were prepared by tail bleeding, air dried, and methanol fixed
before staining.
[0282] Parasitemia was monitored for 20 days post infection or till
it reached 40-50%. TABLE-US-00010 TABLE 10 A/J BALB/c Protein
Plasmid Group 1 4 4 1st: Saline + CFA [Control, 2nd: Saline + IFA
Protein] 3rd: Saline + IFA Group 2 4 4 3rd: Saline + IFA 1st:
pVR1020 [Control, 2nd: pVR1020 Plasmid] Group 3 8 8 1st: PyHDP +
CFA [PyHDP] 2nd: PyHDP + IFA 3rd: PyHDP + IFA Group 4 8 8 3rd:
PyHDP + IFA 1st: pVRPyHDP [pVRPyHDP + 2nd: pVRPyHDP PyHDP]
Results: A/J mice immunized with DNA construct encoding PyHDP
showed almost 50% reduction of parasitemia till Day 10 post
sporozoite challenge (FIG. 23). However, animals immunized with
PyHDP protein alone showed a marginal decrease in parasitemia (30%
inhibition). Nevertheless, the initial immune response to the DNA
vaccine was found to be significant. Thus PyHDP is an ideal
candidate for a subunit vaccine.sup.5, and in the presence of other
antigens such as PFTRAP.sup.6 and PFCSP.sup.5 may generate a
protective immune response. Balb/C mice immunized with protein or
the DNA vaccine construct showed no protection.sup.7. and this may
be attributable to strain to strain variations in the immune
system
References for Example 11
[0283] 1. Price, B. M. et al. Protection against Pseudomonas
aeruginosa chronic lung infection in mice by genetic immunization
against outer membrane protein F (OprF) of P. aeruginosa. Infect
Immun 69, 3510-5 (2001). [0284] 2. Porter-Kelley, J. M. et al.
Plasmodium yoelii: axenic development of the parasite mosquito
stages. Exp Parasitol 112, 99-108 (2006). [0285] 3. Ozaki, L. S.,
Gwadz, R. W. & Godson, G. N. Simple centrifugation method for
rapid separation of sporozoites from mosquitoes. J Parasitol 70,
831-3 (1984). [0286] 4. Shute, P. G. & Maryon, M. An Improved
Technique for Staining Malaria Parasites with Giemsa Stain. Arch
Roum Pathol Exp Microbiol 22, 887-94 (1963). [0287] 5. Prieur, E.
et al. A Plasmodium falciparum candidate vaccine based on a
six-antigen polyprotein encoded by recombinant poxviruses. Proc
Natl Acad Sci USA 101, 290-5 (2004). [0288] 6. Schneider, J. et al.
A prime-boost immunisation regimen using DNA followed by
recombinant modified vaccinia virus Ankara induces strong cellular
immune responses against the Plasmodium falciparum TRAP antigen in
chimpanzees. Vaccine 19, 4595-602 (2001). [0289] 7. Belmonte, M. et
al. The infectivity of Plasmodium yoelii in different strains of
mice. J Parasitol 89, 602-3 (2003).
Example 12
HTS Screening of Potential Inhibitors of P. falciparum HDP
[0290] A panel of candidate compounds were tested for their ability
to inhibit HDP from P. falciparum. The protein was prepared as
described above. The testing was carried out as follows:
[0291] HTS screening for identification of HDP inhibitors. HTS
screening was performed at the Chemical Genomics Center of the
Broad Institute of Harvard and MIT (Cambridge, Mass.). A 2.times.
protein stock (10 .mu.M) was prepared in 200 mM Sodium acetate
buffer at pH 5.6. and 35 .mu.l of this solution was dispensed in
each well of a 384 well plate, using an automated dispenser.
Through a robotized transfer mechanism involving steel pins, each
of the protein-containing well (in a 384 well plate) received 300
nl of a compound. After the addition of the compound, the plate was
incubated at room temperature for 60 minutes, followed by an
addition of 35 .mu.l of freshly prepared heme solution at a
concentration of 20 .mu.M. A 1:1 mix of HDP-heme gave rise to the
final concentrations of 5 and 10 .mu.M of protein and heme in the
reaction, respectively. After heme addition, the plate was
incubated in dark for 60 minutes followed by the measurement of
absorbance at 414 nm, utilizing a Synergy plate reader integrated
with a biostack. The reactions were performed in duplicates and
with controls, where the interactions were measured in the absence
of the protein. The readouts were stored and analyzed for the
identification of potential inhibitors of the reaction.
[0292] Data Analysis. Statistical analysis was performed utilizing
a combination of parameters and compounds that showed statistically
significant inhibition were selected. Briefly, the background
absorbance, which can be attributed to heme and compound alone and
measured for each compound utilizing the background plate, was
subtracted from the test reads. Subsequently, the net absorbance
was compared to controls wells, that did not receive the test
compounds and the percent decrease in absorbance was measured by
the following formula: Percent inhibition=[(Absorbance in test
well/Absorbance in control wells).times.100] Activity of HDP
inhibitors on P. falciparum parasites. Selected compounds were
screened for their potential to inhibit the growth of P. falciparum
parasites in culture. Chloroquine sensitive 3D7 strain of the
parasite was utilized for analysis. Briefly, growth of P.
falciparum parasites (1% parasitemia, 1% hematocrit) in RPMI 1640
medium and 0.5% albumax was evaluated in the presence of different
concentrations of the inhibitors and compared with the growth where
parasites were incubated with medium alone. The parasites were
incubated with the inhibitors for 48 hours before the addition of
SYBR Green dye for measuring parasite growth. A recently published
SYBR-Green I based method was utilized for this measurement [9]. As
RBCs are terminally differentiated and lack a nucleus, addition of
SYBR Green to the parasite culture at the end of a desired
incubation time provides a direct measurement of the DNA content of
the parasite. SYBR Green fluorescence was measured using a 384 well
plate spectrofluorometer with an excitation and emission
wavelengths set at 490 and 530 nm, respectively.
Results and Conclusion
[0293] Cell-free HTS for the identification of inhibitors of
HDP-Heme interaction. Using high throughput technology and the
power of combinatorial chemistry, we investigated several thousand
chemical compounds for their potential to inhibit the interactions
between HDP and heme. The screening was facilitated by the
knowledge that HDP, on its interaction with heme, binds to it with
a very strong affinity and gives a Soret peak at 414 nm. This
property of HDP was exploited for designing a simplified assay that
could be utilized for HTS process. A total of 2 grams of HDP was
recombinantly purified from 25 Liters of E. coli culture. The
purified protein was subsequently utilized in the cell-free assay
for the identification of potential inhibitors of HDP-heme
interactions. HTS was performed in 384 well plates where in typical
reaction 5 .mu.M HDP was allowed to interact with 10 .mu.M heme in
the absence (control) or presence of excess of a chemical compound.
The concentration of the chemical compound was in 40-50 .mu.M
range. HDP-heme interaction was measured at 414 nm in the presence
of the compounds and compared with control reactions that only
received the carrier (DMSO). The final concentration of DMSO in the
reaction was 0.4%.
[0294] A total of 110,000 drug-like, diverse heterocyclic chemical
compounds were screened during this process. These compounds were
obtained from several sources including established chemical
vendors (Asinex, Analyticon, Biomol, Bionet, ChemDiv, Enamine,
Maybridge, Spectrum, TimTec) as well as a range of diversity
oriented synthesis compounds that have been generated by academic
research laboratories from around the world. Screening identified
several hundred (300+) compounds (Table 11) that inhibited the
reaction at a statistically significant >30% levels. Successful
events in this initial screen led to the consolidation of select
wells from the original library stock to generate a new second
generation of plate for screening the activity of these compounds
on P. falciparum parasites.
[0295] Antimalarial activity of HDP-inhibitors on P. falciparum
parasites. A total of 327 inhibitors were screened for their
antimalarial activity in a P. falciparum parasite-based cellular
assay. Rescreening of these compounds was performed at 20-40
micromolar final concentration. Parasites were incubated with the
compounds for 60 hours followed by the measurement of parasite DNA
content utilizing a fluorometric assay. The results presented in
Table 11 show the percent inhibition for compounds at the highest
concentration tested in the cell-based antimalaria assay. At the
highest concentration tested, this screen identified 73 compounds
that showed statistically significant >50% inhibition of the
growth of human malaria parasite in culture (Table 11).
[0296] Those of skill in the art will recognize that, while the
particular compounds in Table 11 may be utilized in the invention,
versions of these compounds (i.e. derivatives or analogs thereof)
may also be developed that are optimized for in vivo use, i.e. for
bioactivity. Such optimization may involve, for example,
modifications to increase or decrease the charge of the molecule
(e.g. to increase or decrease solubility, hydrophilicity,
hydrophobicity, affinity for biological membranes, etc.); to
increase toxicity to the parasite; or to decrease toxicity to the
individual being treated. Such modification may also involve the
substitution of charged groups (e.g. carboxyl groups replaced by
sulfates or vice versa); the substitution or replacement of carbon
chains (e.g. increasing or decreasing the number of carbons in an
aliphatic chain, introducing branched carbon chains, double bonds,
triple bonds, etc. or replacing them with unbranched aliphatic
chains), etc. Other modifications may include conjugation of the
molecule to other entities (or to each other) to form chimeric
molecules, e.g. attachment to various targeting moieties (peptides,
etc.); the attachment of lipids or lipophilic moieties; conjugation
to metal ions; and the like. Further, various salts of the
compounds may be utilized in the invention. All such derivatives
and analogs of the compounds in Table 11 are intended to be
encompassed by the present invention, so long as the resulting
derivative/analog has the ability to prevent or inhibit the
interaction of heme with HDP as described herein. Such compounds
will typically be effective in at least the micromolar
concentration range, and preferably in the nanomolar concentration
range when administered in vivo.
[0297] Those of skill in the art will recognize that certain
chemical modification(s) can be introduced as desired into a given
compound to obtain a new derivative with modified biological
properties such as: greater antimalarial potency against a
particular Plasmodium sp., a broader spectrum of antimalarial
activity against diverse Plasmodium sp., enhanced oral
bioavailability, less toxicity in a particular host mammal, more
advantageous pharmacokinetics and/or tissue distribution in a given
host mammal, and the like. Therefore, the present invention
additionally provides methods for obtaining such derivatives by
applying one or more well-known chemical reactions to a given
compound, to provide a derivative wherein one or more phenolic
hydroxyl group(s) may instead be replaced by an ester, sulfonate
ester, or ether group; one or more methyl ether group(s) may
instead be replaced by a phenolic hydroxyl group; one or more
phenolic hydroxyl group(s) may instead be replaced be an aromatic
hydrogen substituent; one or more secondary amine site(s) may
instead be replaced by an amide, sulfonamide, tertiary amine, or
alkyl quaternary ammonium salt; one or more tertiary amine site(s)
may instead by replaced by a secondary amine; and
[0298] one or more aromatic hydrogen substituent(s) may instead be
replaced by a halogen, nitro, amino, hydroxyl, thiol, or cyano
substituent.
[0299] Numerous references describe the process of chemoinformatics
and laboratory-based lead-optimization of pharmaceutical compounds
in general, or antimalarial compounds specifically, and selected
references are incorporated herein. [0300] Oprea, Tudor I. (ed.),
Chemoinformatics in Drug Discovery, Methods and Principles in
Medicinal Chemistry (Volume 23), Edited by Mannhold,
Raimund/Kubinyi, Hugo/Folkers, Gerd. Wiley-VCH, Weinheim, Germany.
[0301] Brown, Nathan; Lewis, Richard A. Exploiting QSAR methods in
lead optimization. Current Opinion in Drug Discovery &
Development (2006), 9(4), 419-424. [0302] Rolf W. Winter, Jane X.
Kelly, Martin J. Smilkstein, Rozalia Dodean, Grover C. Bagby, R.
Keaney Rathbun, Joshua I. Levin, David Hinrichs and Michael K.
Riscoe, Evaluation and lead optimization of anti-malarial
acridones, Experimental Parasitology, Volume 114, Issue 1,
September 2006, Pages 47-56. [0303] Aihua Xie, Prasanna
Sivaprakasam and Robert J. Doerksen, 3D-QSAR analysis of
antimalarial farnesyltransferase inhibitors based on a
2,5-diaminobenzophenone scaffold, Bioorganic & Medicinal
Chemistry, Volume 14, Issue 21, 1 Nov. 2006, Pages 7311-7323.
References for Example 12
[0303] [0304] 1. Francis, S. E., Sullivan, D. J., Jr. &
Goldberg, D. E. (1997) Hemoglobin metabolism in the malaria
parasite Plasmodium falciparum, Annu Rev Microbiol. 51, 97-123.
[0305] 2. Goldberg, D. E., Slater, A. F., Cerami, A. &
Henderson, G. B. (1990) Hemoglobin degradation in the malaria
parasite Plasmodium falciparum: an ordered process in a unique
organelle, Proc Natl Acad Sci USA. 87, 2931-5. [0306] 3. Fitch, C.
D., Chevli, R., Banyal, H. S., Phillips, G., Pfaller, M. A. &
Krogstad, D. J. (1982) Lysis of Plasmodium falciparum by
ferriprotoporphyrin IX and a chloroquine-ferriprotoporphyrin IX
complex, Antimicrob Agents Chemother. 21, 819-22. [0307] 4.
Kikuchi, G., Yoshida, T. & Noguchi, M. (2005) Heme oxygenase
and heme degradation, Biochem Biophys Res Commun. 338, 558-67.
[0308] 5. Leed, A., DuBay, K., Ursos, L. M., Sears, D., De Dios, A.
C. & Roepe, P. D. (2002) Solution structures of antimalarial
drug-heme complexes, Biochemistry. 41, 10245-55. [0309] 6. Pandey,
A. V., Bisht, H., Babbarwal, V. K., Srivastava, J., Pandey, K. C.
& Chauhan, V. S. (2001) Mechanism of malarial haem
detoxification inhibition by chloroquine, Biochem J. 355, 333-8.
[0310] 7. Sullivan, D. J., Jr., Gluzman, I. Y., Russell, D. G.
& Goldberg, D. E. (1996) On the molecular mechanism of
chloroquine's antimalarial action, Proc Natl Acad Sci USA. 93,
11865-70. [0311] 8. Kannan, R., Sahal, D. & Chauhan, V. S.
(2002) Heme-artemisinin adducts are crucial mediators of the
ability of artemisinin to inhibit heme polymerization, Chem Biol.
9, 321-32.
[0312] 9. Bennett, T. N., Paguio, M., Gligorijevic, B., Seudieu,
C., Kosar, A. D., Davidson, E. & Roepe, P. D. (2004) Novel,
rapid, and inexpensive cell-based quantification of antimalarial
drug efficacy, Antimicrob Agents Chemother. 48, 1807-10.
TABLE-US-00011 TABLE 11 % Inhibition HDP- Anti- Heme malarial Plate
Well SMILES Identifier IUPAC Name Interaction activity 2074 F05
CN(C)c1ccc2N.dbd.c3cc(C)c(N) 42.50 100.00 cc3.dbd.Sc2c1 2075 G11
COc1ccc(cc1)c2ncccc2O 2-(4-methoxyphenyl)pyridin-3-ol 61.75 95.80
2069 J20 Cc1ccn2cc(nc2c1)c3ccc(O)
4-(7-methylimidazo[1,2-a]pyridin-2- 47.50 93.70 c(O)c3
yl)benzene-1,2-diol 2020 P06 CCN1/C(.dbd.C/c2ccc3cc(C)
2-[(Z)-(3-ethyl-6-methoxy-1,3- 58.75 93.70
ccc3[n+]2C)/Sc4cc(OC)ccc14 benzothiazol-2(3H)-ylidene)methyl]-
1,6-dimethylquinolinium 2021 B19 CC[n+]1c(/C.dbd.C/2\SC.dbd.CN2C)
1-ethyl-6-methoxy-4-methyl-2-[(Z)- 59.75 93.30
cc(C)c3cc(OC)c4ccccc4c13 (3-methyl-1,3-thiazol-2(3H)-
ylidene)methyl]benzo[h]quinolinium 2046 C02
Oc1ccc(cc1)c2nc(c([nH]2) 4,4'-(4-phenyl-1H-imidazole-2,5- 44.00
92.90 c3ccc(O)cc3)c4ccccc4 diyl)diphenol 2085 H01
Oc1cccc(Nc2nc(NCC3CCCO3) 3-[(4-{[(2R)-tetrahydrofuran-2- 64.50
92.30 c4ccccc4n2)c1 ylmethyl]amino}quinazolin-2- yl)amino]phenol
2105 K04 49.00 91.20 1413 N13 Cc1noc(c1c2ccc3OCCCOc3c2)
4-[4-(3,4-dihydro-2H-1,5- 56.25 90.80 c4ccc(O)cc4O
benzodioxepin-7-yl)-3- methylisoxazol-5-yl]benzene-1,3-diol 2017
B19 Clc1ccc2oc(cc(.dbd.NCc3ccco3)
N-(6-chloro-2-phenyl-4H-chromen-4- 45.25 90.60 c2c1)c4ccccc4
ylidene)-1-(2-furyl)methanamine 2099 E09 Oc1ccc(cc1)c2sc3cc(O)
[6-hydroxy-2-(4-hydroxyphenyl)-1- 49.75 90.10
ccc3c2C(.dbd.O)c4ccc(OCCN5CCCCC5)
benzothien-3-yl][4-(2-piperidin-1- cc4 ylethoxy)phenyl]methanone
2290 H07 Oc1cc(cc(O)c1O)C(.dbd.O) 1,2,3,4,6-pentakis-O-(3,4,5-
41.50 90.10 OC[C@H]2O[C@@H](OC(.dbd.O) trihydroxybenzoyl)-beta-D-
c3cc(O)c(O)c(O)c3)[C@H](OC(.dbd.O) glucopyranose c4cc(O)c(O)c(O)c4)
[C@@H](OC(.dbd.O)c5cc(O)c(O) c(O)c5)[C@@H]2OC(.dbd.O)
c6cc(O)c(O)c(O)c6 2296 J02 O[C@H]1[C@H]2[C@H](CC(.dbd.O) 56.25
90.00 O)C(.dbd.O)O[C@@H]3C(COC(.dbd.O) c4cc(O)c(O)c(O)
c4)O[C@@H](OC(.dbd.O)c5cc(O) c(O)c(O)c5)C(OC(.dbd.O)
c6cc(O)c(O)c(OC1.dbd.O)c26) [C@@H]3OC(.dbd.O)c7cc(O)c(O) c(O)c7
2078 L04 Cc1ccc2nc(cn2c1)c3ccc(O)
4-(6-methylimidazo[1,2-a]pyridin-2- 46.50 89.70 c(O)c3
yl)benzene-1,2-diol 2011 B03 CC(C)Nc1ccc(Nc2ccnc3cc4ccccc4cc23)
N-benzo[g]quinolin-4-yl-N'- 59.00 89.00 cc1
isopropylbenzene-1,4-diamine 2168 C04 OCCOc1ccc(CN2CCC[C@H](C2)
2-{(3R)-1-[4-(2- 37.75 89.00 N3C(.dbd.O)c4ccccc4C3.dbd.O)
hydroxyethoxy)benzyl]piperidin-3-yl}- cc1
1H-isoindole-1,3(2H)-dione 2080 F20 Clc1ccc(CCN2COc3ccc(Cl)
6-chloro-3-[2-(4-chlorophenyl)ethyl]- 57.75 88.90 cc3C2)cc1
3,4-dihydro-2H-1,3-benzoxazine 1446 P02 Cc1cc(Nc2cc(Cl)cc(Cl)c2)
N4-(3,5-dichlorophenyl)-6- 48.00 88.90 nc(N)n1
methylpyrimidine-2,4-diamine 2019 C14 CCOC(.dbd.O)c1c(c2ccccc2)
(ethyl 1-benzyl-4- 49.25 88.70 n(Cc3ccccc3)c4ccc(O)c(CN(C)
[(dimethylamino)methyl]-5-hydroxy- C)c14
2-phenyl-1H-indole-3-carboxylate 2144 I02 48.25 88.70 2016 E14
COc1ccc(cc1)c2c/c(.dbd.NCCc3ccccc3)/ N-[(4E)-2-(4-methoxyphenyl)-6-
45.00 88.50 c4cc(C)ccc4o2 methyl-4H-chromen-4-ylidene]-2-
phenylethanamine 1406 N16 Cc1nc2ccccn2c1C3(O)C(.dbd.O)
(3R)-5,7-dichloro-3-hydroxy-3-(2- 65.00 88.50 Nc4c3cc(Cl)cc4Cl
methylimidazo[1,2-a]pyridin-3-yl)- 1,3-dihydro-2H-indol-2-one 1399
E08 OC(Cn1c(.dbd.N)sc2ccccc12) (1S)-1-(3,4-dichlorophenyl)-2-(2-
50.50 88.30 c3ccc(Cl)c(Cl)c3 imino-1,3-benzothiazol-3(2H)-
yl)ethanol 2021 D19 COc1ccc2N(C)/C(.dbd.C/c3sc4ccccc4[n+]3C)/
2-[(E)-(6-methoxy-1-methylquinolin- 45.25 88.00 C.dbd.Cc2c1
2(1H)-ylidene)methyl]-3-methyl-1,3- benzothiazol-3-ium 1465 J19
Nc1cccc(c1)C(.dbd.C2C.dbd.CC(.dbd.N) 4,4'-methylenebis(3-hydroxy-2-
40.75 88.00 C.dbd.C2)c3cccc(N)c3.OC(.dbd.O) naphthoic
acid)-3,3'-[(4- c1cc2ccccc2c(Cc3c(O) iminocyclohexa-2,5-dien-1-
c(cc4ccccc34)C(.dbd.O)O)c1O ylidene)methylene]dianiline (1:1) 2292
G08 COc1cc(O) 44.00 87.90 c-2c(CCc3cc(OC)c(OC)cc32) c1 2085 L13
CC(Nc1ncnc2ccccc12) N--[(1S)-1-phenylethyl]quinazolin-4- 38.00
87.80 c3ccccc3 amine 2020 K07 Nc1oc2c(CN3CCCCCC3)
2-amino-8-(azepan-1-ylmethyl)-3- 60.75 87.60 c(O)ccc2c(.dbd.O)
(1,3-benzothiazol-2-yl)-7-hydroxy- c1c4nc5ccccc5s4 4H-chromen-4-one
1405 A21 OC(.dbd.O)c1nc2cccc3cccc([nH]1) 1H-perimidine-2-carboxylic
acid 54.25 87.50 c32 1422 M20 CCOc1ccc(cc1)S(.dbd.O)(.dbd.O)
N-(3-chloro-4-hydroxy-1-naphthyl)-4- 57.50 86.50
Nc2cc(Cl)c(O)c3ccccc23 ethoxybenzenesulfonamide 2012 D08
OC(Cn1c2ccccc2c3ccccc13) 1-[(2S)-3-(9H-carbazol-9-yl)-2- 87.25
86.20 C[n+]4cccc5cccc(O)c45 hydroxypropyl]-8-hydroxyquinolinium
1408 C14 Cc1cc(NN)nc2ccccc12 2-hydrazino-4-methylquinoline 57.75
86.00 1471 J20 CN1CCN(CC1)c2ccc3N.dbd.C([NH2]c3c2)
4-[5-[5-(4-methylpiperazin-1-yl)-3H- 49.00 85.80
c4ccc5N.dbd.C([NH2]c5c4)
benzoimidazol-2-yl]-1,3-dihydrobenzoimidazol- c6ccc(O)cc6
2-ylidene]cyclohexa-2,5-dien- 1-one 1415 J19
CCOC(.dbd.O)c1c(OCC)[nH]c2c1cc(O) ethyl 2-ethoxy-5-hydroxy-1H-
50.50 85.20 c3ccccc23 benzo[g]indole-3-carboxylate 1441 F14
Clc1ccccc1SCc2cccc(c2) 3-(3-{[(2- 41.75 85.10 C(.dbd.O)CC#N
chlorophenyl)thio]methyl}phenyl)-3- oxopropanenitrile 2082 C04
Cc1ccc(O)c(CCc2ccc(O)cc2) 2-[2-(4-hydroxyphenyl)ethyl]-6- 33.50
84.80 n1 methylpyridin-3-ol 2296 C09 O[C@H]1[C@@H](O) 43.00 84.50
[C@@H](COC(.dbd.O)c2cc(O)c(O) c(O)c2)O[C@@H](Oc3ccc(C(.dbd.O)
CCc4ccc(O)cc4)c(O)c3) [C@@H]1O 1417 C16 CCN1C(.dbd.O)c2cccc3c(N)
6-amino-1-ethylbenzo[cd]indol- 44.25 82.40 ccc1c23 2(1H)-one 2033
K22 CN(C)CCNC(.dbd.O)c1cc2CSc3cc(Cl)
7-chloro-N-[2-(dimethylamino)ethyl]- 40.75 81.70 ccc3-c2s1
4H-thieno[3,2-c]thiochromene-2- carboxamide 599 O10
O.dbd.C1/C(.dbd.C\Nc2cccnc2)/ (2E)-2-[(pyridin-3- -123.00 80.30
Sc3ccccc13 ylamino)methylene]-1- benzothiophen-3(2H)-one 2041 M08
FC(F)(F)c1cccc(NC(.dbd.O) 3-[4-(9H-fluoren-9-yl)piperazin-1-yl]-
48.75 79.60 CCN2CCN(CC2)C3c4ccccc4-c5ccccc35) N-[3- c1
(trifluoromethyl)phenyl]propanamide 1464 H07
Oc1ccc(cc1n2c(.dbd.O)[nH]c3cc(ccc23) 1-[2-hydroxy-5- 55.50 79.40
C(F)(F)F)C(F)(F)F (trifluoromethyl)phenyl]-5-
(trifluoromethyl)-1,3-dihydro-2H- benzimidazol-2-one 1439 119
Oc1ccccc1C(.dbd.O)NC(.dbd.O) N-(3-furylcarbonyl)-2- 51.00 77.60
c2ccoc2 hydroxybenzamide 2073 I04 Nc1cc(c2ccccc2)c3ccccc3n1
4-phenylquinolin-2-amine 38.25 77.40 2105 E08 41.75 75.80 1438 F17
Oc1ccccc1C(.dbd.O)NC(.dbd.O) N-(2-hydroxybenzoyl)-2- 60.75 75.40
c2cccs2 thiophenecarboxamide 1442 N18 Clc1ccc2c(Nc3ccccc3)
7-chloro-N-phenylquinolin-4-amine 53.25 74.30 ccnc2c1 2160 E04
Oc1cc(O)c2c(.dbd.O)cc(oc2c1) 5,7-dihydroxy-2-(3,4,5- 69.20 74.00
c3cc(O)c(O)c(O)c3 trihydroxyphenyl)-4H-chromen-4- one 1447 D13
COc1ccc(O)c(c1)C(.dbd.O) 3-(2-hydroxy-5-methoxybenzoyl)-2- 81.00
73.60 C2N(C(.dbd.O)c3ccccc23)c4ccc(C)
(4-methylphenyl)isoindolin-1-one cc4 1439 G09
CCCc1ccc(cc1)C(.dbd.O)NC(.dbd.O) 2-hydroxy-N-(4- 43.25 69.80
c2ccccc2O propylbenzoyl)benzamide 1464 J17 OC(COc1cccc2[nH]c(C#N)
4-({(2S)-3-[4- 61.50 68.70 cc12)CN3CCN(CC3)C(c4ccccc4)
(diphenylmethyl)piperazin-1-yl]-2- c5ccccc5
hydroxypropyl}oxy)-1H-indole-2- carbonitrile 1469 D18
Oc1ccc2c(.dbd.O)c(O)c(oc2c1) 2-(3,4-dihydroxyphenyl)-3,7- 46.75
68.50 c3ccc(O)c(O)c3 dihydroxy-4H-chromen-4-one 2144 K02 44.50
68.30 596 G10 Oc1cc(Cl)c([N+](.dbd.O)[O--])
(3aR,4R,9bR)-8-chloro-4-(4- 43.48 67.40 c2C3C.dbd.CCC3C(Nc12)
chlorophenyl)-9-nitro-3a,4,5,9b- c4ccc(Cl)cc4
tetrahydro-3H-cyclopenta[c]quinolin- 6-ol 1409 B15
Oc1ccc(Cl)cc1C(.dbd.O)c2cc(C(.dbd.O)
5-(5-chloro-2-hydroxybenzoyl)-2- 42.00 66.50
Nc3ccccc3)c(.dbd.O)n(c2) oxo-N,1-diphenyl-1,2- c4ccccc4
dihydropyridine-3-carboxamide 599 O09 Oc1c(Cl)cc(Br)
(2E,5E)-5-(5-bromo-3-chloro-2- 43.18 66.30
cc1/C.dbd.C\2/S/C(.dbd.N/c3ccccc3)/NC2.dbd.O hydroxybenzylidene)-2-
(phenylimino)-1,3-thiazolidin-4-one 2131 A18
Oc1ccccc1NC(.dbd.O)CCCCCCC(.dbd.O) 8-[(2E)-2-(2- 42.00 66.10
N/N.dbd.C/c2ccccc2Br bromobenzylidene)hydrazino]-N-(2-
hydroxyphenyl)-8-oxooctanamide 1410 N17 CC(C)(c1ccc(O)c(Cl)c1)
4,4'-propane-2,2-diylbis(2- 46.00 65.30 c2ccc(O)c(Cl)c2
chlorophenol) 1438 J17 COc1cccc(c1)C(.dbd.O)NC(.dbd.O)
N-(2-hydroxybenzoyl)-3- 74.75 64.60 c2ccccc2O methoxybenzamide 587
L11 Oc1ccc(cc1)C2.dbd.NN(C(C2) 4-[(5S)-5-(4-fluorophenyl)-1-(4-
-136.50 64.50 c3ccc(F)cc3)c4ccc(cc4)[N+](.dbd.O)
nitrophenyl)-4,5-dihydro-1H-pyrazol- [O--] 3-yl]phenol 1409 J07
Cc1ccc(cc1)n2cc(cc(C#N) 5-(2-hydroxy-5-methylbenzoyl)-1-(4- 72.75
63.80 c2.dbd.O)C(.dbd.O)c3cc(C)ccc3O methylphenyl)-2-oxo-1,2-
dihydropyridine-3-carbonitrile 2086 H09 Oc1c(CN2CCOCC2)
2-(morpholin-4-ylmethyl)-1-naphthol 80.00 62.90 ccc3ccccc13 1424
H16 CN(C)c1ccc(cc1) 4-(1H-benzimidazol-2-yl)-N,N- 43.75 62.10
c2nc3ccccc3[nH]2 dimethylaniline 1439 I09
CCc1ccc(cc1)C(.dbd.O)NC(.dbd.O) N-(4-ethylbenzoyl)-2- 51.25 60.10
c2ccccc2O hydroxybenzamide 2004 D17 COc1ccc(cc1)c2nc(.dbd.O)
2-(4-methoxyphenyl)-4H-1,3- 51.25 59.50 c3ccccc3o2 benzoxazin-4-one
1413 P10 CCCc1cc2c(.dbd.O)c(c(C)oc2cc1O)
7-hydroxy-2-methyl-6-propyl-3- 37.75 54.70 c3ccccn3
pyridin-2-yl-4H-chromen-4-one 2133 A16
Oc1ccccc1NC(.dbd.O)CCCCCC(.dbd.O) 7-[(2E)-2-(biphenyl-4- 49.50
54.30 N/N.dbd.C/c2ccc(cc2) ylmethylene)hydrazino]-N-(2- c3ccccc3
hydroxyphenyl)-7-oxoheptanamide 2144 E02 44.25 54.20 2047 H22
CCc1ccccc1NCc2c(O)c(C) 4-{[(2-ethylphenyl)amino]methyl}-5- 44.00
53.00 ncc2CO (hydroxymethyl)-2-methylpyridin-3-ol 1408 D13
CCOc1ccccc1NC(.dbd.O) N-(2-ethoxyphenyl)-2- 75.50 52.80 c2ccccc2O
hydroxybenzamide 1463 A19 Oc1ccccc1C#CC#Cc2ccccc2O
2,2'-buta-1,3-diyne-1,4-diyldiphenol -82.25 51.90 2035 C06
CC1CN(C(.dbd.O)Nc2ccc(Cl) (2S)-N-(4-chlorophenyl)-2-methyl- 37.00
51.20 cc2)c3ccccc3O1 2,3-dihydro-4H-1,4-benzoxazine-4- carboxamide
1397 L19 COc1ccc(c2[nH]nc(c2c3cc4ccccc4o3)
2-[4-(1-benzofuran-2-yl)-3- 53.75 50.70 C(F)(F)F)c(O)c1
(trifluoromethyl)-1H-pyrazol-5-yl]-5- methoxyphenol 2131 D05
Oc1ccccc1NC(.dbd.O)CCCCCCC(.dbd.O)
N-(2-hydroxyphenyl)-8-[(2E)-2-(1- 51.25 49.80
N/N.dbd.C/c2cccc3ccccc23 naphthylmethylene)hydrazino]-8-
oxooctanamide 1442 J20 Clc1ccc2c(Nc3ccc4OCOc4c3)
N-(1,3-benzodioxol-5-yl)-7- 59.00 49.50 ccnc2c1
chloroquinolin-4-amine
2015 G08 COc1ccc(/N.dbd.c/2cc(oc3ccc(O)
(4E)-2-(4-methoxyphenyl)-4-[(4- 55.25 49.30 cc23)c4ccc(OC)cc4)cc1
methoxyphenyl)imino]-4H-chromen- 6-ol 2131 C18
Oc1ccccc1NC(.dbd.O)CCCCCCC(.dbd.O) 8-{(2E)-2-[(6-bromo-1,3- 66.25
49.20 N/N.dbd.C/c2cc3OCOc3cc2Br benzodioxol-5-
yl)methylene]hydrazino}-N-(2- hydroxyphenyl)-8-oxooctanamide 2131
C14 COc1ccc(Br)cc1/C.dbd.N/NC(.dbd.O) 8-[(2E)-2-(5-bromo-2- 77.25
48.30 CCCCCCC(.dbd.O) methoxybenzylidene)hydrazino]-N- Nc2ccccc2O
(2-hydroxyphenyl)-8-oxooctanamide 591 D04
Oc1ccc(/C.dbd.C/2\S\C(.dbd.N\c3ccccc3Cl)\
(2E,5Z)-2-[(2-chlorophenyl)imino]-5- 42.88 48.20
NC2.dbd.O)cc1[N+](.dbd.O) (4-hydroxy-3-nitrobenzylidene)-1,3- [O--]
thiazolidin-4-one 2008 J17 CCN(CC)c1ncnc2c3cc(C)
N,N-diethyl-8-methyl-5H- 37.25 47.20 ccc3[nH]c12
pyrimido[5,4-b]indol-4-amine 2086 M07
OC(.dbd.O)CC1Nc2ccccc2NC1.dbd.O [(2R)-3-oxo-1,2,3,4- 73.00 47.10
tetrahydroquinoxalin-2-yl]acetic acid 1453 B20 46.50 46.20 2133 N23
Oc1ccccc1NC(.dbd.O)CCCCCC(.dbd.O) 7-{(2E)-2-[(2-fluorobiphenyl-4-
74.00 45.80 N/N.dbd.C/c2ccc(c(F)c2) yl)methylene]hydrazino}-N-(2-
c3ccccc3 hydroxyphenyl)-7-oxoheptanamide 2027 M05
CCC(C)NC(.dbd.O)c1cc2cc3cc(OC) 6-methoxy-N-[(1S)-1- 42.75 44.90
ccc3nc2o1 methylpropyl]furo[2,3-b]quinoline-2- carboxamide 2003 B09
OC(CNCc1ccccc1)Cn2c3ccc(Cl) (2R)-1-(benzylamino)-3-(3,6- 60.25
44.60 cc3c4cc(Cl)ccc24 dichloro-9H-carbazol-9-yl)propan-2- ol 1439
G19 Cc1nc(sc1C(.dbd.O)NC(.dbd.O)c2ccccc2O)
2-hydroxy-N-[(4-methyl-2-phenyl- 71.25 44.20 c3ccccc3
1,3-thiazol-5-yl)carbonyl]benzamide 1453 F18 46.25 43.30 588 K12
OC(.dbd.O)CN1C(.dbd.O)/C(.dbd.C\c2cc(Br)
[(5E)-5-(5-bromo-3-chloro-2- 49.39 43.10 cc(Cl)c2O)/SC1.dbd.S
hydroxybenzylidene)-4-oxo-2-thioxo- 1,3-thiazolidin-3-yl]acetic
acid 2009 A02 Fc1cccc(c1)N2C(.dbd.O)NC(.dbd.O)/
(3-{(E)-[1-(3-fluorophenyl)-2,4,6- -130.50 42.90
C(.dbd.C\c3cn(CC#N)c4ccccc34)/ trioxotetrahydropyrimidin-5(2H)-
C2.dbd.O ylidene]methyl}-1H-indol-1- yl)acetonitrile 1364 C17
Oc1cc(O)c2C(CC(.dbd.O)Oc2c1) (4S)-5,7-dihydroxy-4- 41.00 42.80
c3ccccc3 phenylchroman-2-one 1408 P11
Cc1ccc(C)c(NC(.dbd.O)c2ccccc2) N-(2,5-dimethylphenyl)benzamide
55.00 41.80 c1 1394 O14 NNc1nc(cc(n1)c2ccccc2)
2-hydrazino-4,6-diphenylpyrimidine 59.25 41.30 c3ccccc3 2100 K09
CN(C)CCCC[C@@H]1NC(.dbd.O) allyl (3R,3'R,4'S,6'R,8'S,8a'S)-6'-{4-
44.00 40.90 [C@@H]2C[C@@H]([C@@H](N2C1.dbd.O)
[2-({[(3S,6R,7S,8aS)-3-[4- c3ccc(O) (dimethylamino)butyl]-6-(4-
cc3)C(.dbd.O)OCCOc4ccc(cc4) hydroxyphenyl)-1,4-
[C@H]5N6[C@@H]([C@@H](C(.dbd.O)
dioxooctahydropyrrolo[1,2-a]pyrazin- OCC.dbd.C)[C@]57C(.dbd.O)
7-yl]carbonyl}oxy)ethoxy]phenyl}-5- Nc8ccc(I)cc87)C(.dbd.O)
iodo-1',2-dioxo-3',4'-diphenyl- O[C@@H]([C@@H]6c9ccccc9)
1,2,3',4',8',8a'-hexahydro-1'H- c % 10ccccc % 10
spiro[indole-3,7'-pyrrolo[2,1- c][1,4]oxazine]-8'-carboxylate 2069
N09 Oc1ccccc1NC(.dbd.O)c2cc(NC(.dbd.O) 5-(benzoylamino)-N,N'-bis(2-
51.25 40.60 c3ccccc3)cc(c2)C(.dbd.O) hydroxyphenyl)isophthalamide
Nc4ccccc4O 2144 G02 52.00 40.50 1412 N21
Oc1ccccc1/C.dbd.C\2/SC(.dbd.S) (5E)-3-allyl-5-(2- 60.25 40.30
N(CC.dbd.C)C2.dbd.O hydroxybenzylidene)-2-thioxo-1,3-
thiazolidin-4-one 2057 C10 CC1(C)c2cc(Cl)
2-chloro-8-hydroxy-10,10-dimethyl- 40.25 38.40
ccc2-n3c1cc(O)c(c4ccccc4)c3.dbd.O
7-phenylpyrido[1,2-a]indol-6(10H)- one 1402 H20
CCc1cc2c(.dbd.O)c(cOc2cc1O) 3-(1H-benzimidazol-1-yl)-6-ethyl-7-
52.00 37.40 n3cnc4ccccc34 hydroxy-4H-chromen-4-one 2001 D13
Cc1ccccc1NC(.dbd.O) 3-hydroxy-N-(2-methylphenyl)-2- 51.25 37.20
c2cc3ccccc3cc2O naphthamide 1364 N13 Clc1ccc(cc1)C(C#N)C(.dbd.O)
(2S)-2-(4-chlorophenyl)-3-oxo-4- 59.25 36.90 Cc2ccccc2
phenylbutanenitrile 1366 O19 Cc1ccc(SCC(.dbd.O)c2cc(C)c(O)
1-(4-hydroxy-3,5-dimethylphenyl)-2- 47.00 35.70 c(C)c2)cc1
[(4-methylphenyl)thio]ethanone 2133 M04
Oc1ccccc1NC(.dbd.O)CCCCCC(.dbd.O) 7-[(2E)-2-(4-fluoro-3- 51.75
35.60 N/N.dbd.C/c2ccc(F)c(Oc3ccccc3)
phenoxybenzylidene)hydrazino]-N- c2 (2-hydroxyphenyl)-7-
oxoheptanamide 2010 M10 Oc1ccc(/C.dbd.C/2\SC(.dbd.S)
(5Z)-5-(4-hydroxybenzylidene)-3- -159.50 34.40
N(CC3CCCO3)C2.dbd.O)cc1 [(2R)-tetrahydrofuran-2-ylmethyl]-2-
thioxo-1,3-thiazolidin-4-one 2034 O10 Oc1ccccc1c2n[nH]c3C(.dbd.O)
(4R)-5-(2-furylmethyl)-3-(2- 50.50 34.40 N(Cc4ccco4)C(c23)c5ccccc5
hydroxyphenyl)-4-phenyl-4,5- dihydropyrrolo[3,4-c]pyrazol-6(1H)-
one 1409 J15 Oc1ccc(Br)cc1C(.dbd.O)c2cc(C#N)
5-(5-bromo-2-hydroxybenzoyl)-1-(2- 74.00 33.60
c(.dbd.O)n(c2)c3ccccc3F fluorophenyl)-2-oxo-1,2-
dihydropyridine-3-carbonitrile 2021 D20 CCOC(.dbd.O)c1cnc2ccc(OCC)
ethyl 6-ethoxy-4-{[(1R)-1- 50.25 32.70 cc2c1NC(C)CC
methylpropyl]amino}quinoline-3- carboxylate 1393 M01
Cc1ccc(NC(.dbd.O)Cc2c(O) 2-(4-hydroxy-2-oxo-1,2- 36.25 32.60
c3ccccc3[nH]c2.dbd.O)cc1 dihydroquinolin-3-yl)-N-(4-
methylphenyl)acetamide 1395 I06 Clc1ccc(cc1)c2nc(.dbd.O)
2-(4-chlorophenyl)-4H-1,3- 46.25 32.50 c3ccccc3o2 benzoxazin-4-one
1414 J16 Cc1[nH]nc(c1c2nc3ccccc3s2) 4-[4-(1,3-benzothiazol-2-yl)-5-
33.25 32.50 c4ccc(O)cc4O methyl-1H-pyrazol-3-yl]benzene-1,3- diol
1398 P07 CC(C)c1ccc(OCC(.dbd.O)c2ccc(O)
1-(2,4-dihydroxyphenyl)-2-(4- 37.00 32.40 cc2O)cc1
isopropylphenoxy)ethanone 1407 N22 OCc1ccc(Br)cc1c2cc([nH]n2)
4-bromo-2-[5-(2-furyl)-1H-pyrazol-3- 55.50 32.00 c3ccco3 yl]phenol
2100 F16 C[N+](C)(C)CCCC[C@@H]1NC(.dbd.O)
4-[(3S,6S,7R,8aR)-7-{[2-(4- 45.25 31.70
[C@H]2C[C@H]([C@H](N2C1.dbd.O) {(3S,3'S,4'R,6'R,8'R,8a'R)-8'-
c3ccc(O)cc3) [(allyloxy)carbonyl]-5-iodo-1',2-dioxo-
C(.dbd.O)OCCOc4ccc(cc4) 3',4'-diphenyl-1,2,3',4',8',8a'-
[C@@H]5N6[C@H]([C@H](C(.dbd.O) hexahydro-1'H-spiro[indole-3,7'-
OCC.dbd.C)[C@@]57C(.dbd.O) pyrrolo[2,1-c][1,4]oxazin]-6'-
Nc8ccc(I)cc87)C(.dbd.O) yl}phenoxy)ethoxy]carbonyl}-6-(4-
O[C@H]([C@H]6c9ccccc9) hydroxyphenyl)-1,4- c % 10cccc % 10
dioxooctahydropyrrolo[1,2-a]pyrazin- 3-yl]-N,N,N-trimethylbutan-1-
aminium 1397 C21 COc1ccc2[nH]c3c(ncnc3c2c1)
8-methoxy-N,N-dimethyl-5H- 43.75 31.60 N(C)C
pyrimido[5,4-b]indol-4-amine 2078 N15
OC(.dbd.O)CN1C(.dbd.O)/C(.dbd.C\c2ccsc2)/
[(5E)-4-oxo-5-(3-thienylmethylene)- 42.25 31.60 SC1.dbd.S
2-thioxo-1,3-thiazolidin-3-yl]acetic acid 1445 F04
Clc1ccc2c(ccnc2c1) 7-chloro-4-piperidinoquinoline 45.50 31.40
N3CCCCC3 2015 A14 COc1cc(/C.dbd.C/C(.dbd.O)\C.dbd.C\c2ccc(cc2)
(1E,4E)-1-[4- -91.00 31.20 N(C)C)cc(OC)
(dimethylamino)phenyl]-5-(3,4,5- c1OC
trimethoxyphenyl)penta-1,4-dien-3- one 2083 D04 CCCCc1c(nc(N)c(C#N)
2-amino-5-butyl-4-(4-hydroxy-3- 40.25 31.20
c1c2ccc(O)c(OC)c2)c3ccccc3 methoxyphenyl)-6- phenylnicotinonitrile
2015 P11 OC(.dbd.O)c1cccc(c1) 3-{2-[(1,3-dioxo-1,3-dihydro-2H-
-100.00 31.20 n2cccc2C.dbd.C3C(.dbd.O)c4ccccc4C3.dbd.O
inden-2-ylidene)methyl]-1H-pyrrol-1- yl}benzoic acid 1398 E06
CCOC(.dbd.O)c1ccc(NC(.dbd.O)/ ethyl 4-{[(2E)-3-(2-thienyl)prop-2-
42.75 31.10 C.dbd.C/c2cccs2)cc1 enoyl]amino}benzoate 1411 A03
Oc1c(Br)cc(NS(.dbd.O)(.dbd.O) N-(3-bromo-4-hydroxy-1-naphthyl)-
38.00 30.90 c2ccc(Cl)cc2)c3ccccc13 4-chlorobenzenesulfonamide 2290
A23 COc1cc(/C.dbd.C/2\Oc3cc(O) 74.50 30.90 ccc3C2.dbd.O)ccc1O 2031
K15 COC(.dbd.O)c1ccc2O/C(.dbd.C\c3ccc(O) methyl (2Z)-2-(4- -91.00
30.90 cc3)/C(.dbd.O)c2c1 hydroxybenzylidene)-3-oxo-2,3-
dihydro-1-benzofuran-5-carboxylate 2009 C17
COc1ccccc1NC(.dbd.O)CC(c2ccccc2) (3R)-3-(2-hydroxy-4-methylphenyl)-
50.75 30.70 c3ccc(C)cc3O N-(2-methoxyphenyl)-3- phenylpropanamide
2069 O12 CCOC(.dbd.O)c1cnc2ccc(OCC) ethyl 4-(benzylamino)-6- 75.50
30.60 cc2c1NCc3ccccc3 ethoxyquinoline-3-carboxylate 1412 F08
ClC1.dbd.C(NC(.dbd.CS1)c2ccccc2)
2-chloro-5-phenyl-3-pyridin-4-yl-4H- 39.75 30.50 c3ccncc3
1,4-thiazine 2003 M11 CCS(.dbd.O)(.dbd.O)c1ccc(O)c(NC(.dbd.O)
N-[5-(ethylsulfonyl)-2- 49.50 30.50 COc2ccc(OC)cc2)c1
hydroxyphenyl]-2-(4- methoxyphenoxy)acetamide 2297 E24
OC[C@H]1O[C@@H](OC[C@H]2O[C@@H](Oc3c(oc4cc(O) 52.25 30.30
cc(O)c4c3.dbd.O)c5ccc(O) cc5)[C@H](O)[C@@H](O)
[C@@H]2O)[C@H](O)[C@@H](O) [C@@H]1O 1463 I17 C#Cc1ccccc1Oc2ccccc2
1-ethynyl-2-phenoxybenzene -123.75 29.80 2099 B17
CC(.dbd.CCC/C(.dbd.C/CC/C(.dbd.C/Cc1c(O)
4-hydroxy-3-[(2E,6E)-3,7,11- 60.00 29.70 c2ccccc2oc1.dbd.O)/C)/
trimethyldodeca-2,6,10-trien-1-yl]- C)C 2H-chromen-2-one 1410 C01
Cc1ccc(cc1)S(.dbd.O)(.dbd.O) {4-[(4- 70.50 29.70 c2ccc(NN)cc2
methylphenyl)sulfonyl]phenyl}hydrazine 2007 C13 CCOC(.dbd.O) ethyl
4-[(2- 73.25 29.60 c1cnc2ccccc2c1NCCc3ccccc3
phenylethyl)amino]quinoline-3- carboxylate 2100 E19
CC(.dbd.O)NCCCC[C@@H]1NC(.dbd.O) (3S,6S,7R,8aR)-3-(4- 42.25 29.60
[C@H]2C[C@H]([C@H](N2C1.dbd.O) acetamidobutyl)-6-(4- c3ccc(O)cc3)
hydroxyphenyl)-1,4- C(.dbd.O)O dioxooctahydropyrrolo[1,2-
a]pyrazine-7-carboxylic acid 2057 E17 Oc1c(Cc2ccccc2)c(.dbd.O)
5-benzyl-4-hydroxy-6H-pyrido[3,2,1- 52.75 29.50
n3c4ccccc4c5cccc1c53 k]carbazol-6-one 2006 P15
Oc1ccc(NS(.dbd.O)(.dbd.O)c2ccccc2)
N-[3-(1,3-benzothiazol-2-ylthio)-4- 54.75 29.40 cc1Sc3nc4ccccc4s3
hydroxyphenyl]benzenesulfonamide 2037 G08 COc1cc(Nc2nc(SCC(.dbd.O)
1-(3,4-dihydroxyphenyl)-2-({4-[(3,5- 44.25 29.20
c3ccc(O)c(O)c3)nc4ccccc24) dimethoxyphenyl)amino]quinazolin-
cc(OC)c1 2-yl}thio)ethanone 1439 M07 Oc1ccccc1C(.dbd.O)NC(.dbd.O)
N-(cyclohexylcarbonyl)-2- 39.75 28.90 C2CCCCC2 hydroxybenzamide
1364 O16 On1c(nc2ccc(Cl)cc12)c3ccc(Cl)
6-chloro-2-(4-chlorophenyl)-1H- 57.50 28.90 cc3 benzimidazol-1-ol
2008 G14 COc1ccc(cc1)c2oc3ncn(Cc4ccccc4) 3-benzyl-5,6-bis(4- 43.50
28.80 c(.dbd.N)c3c2c5ccc(OC) methoxyphenyl)furo[2,3-d]pyrimidin-
cc5 4(3H)-imine 2008 H17 CN(C)c1ncnc2c3cc(C)
N,N,8-trimethyl-5H-pyrimido[5,4- 61.25 28.50 ccc3[nH]c12
b]indol-4-amine 2016 I20 COc1ccc(CN(C(.dbd.O)CCN2C(.dbd.O)
3-(1,1-dioxido-3-oxo-1,2- 44.50 28.20 c3ccccc3S2(.dbd.O).dbd.O)
benzisothiazol-2(3H)-yl)-N-(2- c4ccccc4O)cc1 hydroxyphenyl)-N-(4-
methoxybenzyl)propanamide 2049 N08
FC(F)(F)c1ccc(nc1)S(.dbd.O)(.dbd.O) 2-phenyl-5-({[5- 42.50 28.00
CC2.dbd.NN(C(.dbd.O)C2) (trifluoromethyl)pyridin-2- c3ccccc3
yl]sulfonyl}methyl)-2,4-dihydro-3H- pyrazol-3-one 2043 L02
Clc1ccc(SCc2cc(.dbd.O)c3c(.dbd.O)
6-{[(4-chlorophenyl)thio]methyl}-2- 45.00 27.80
n([nH]c3[nH]2)c4ccccc4) phenyl-1H-pyrazolo[3,4-b]pyridine- cc1
3,4(2H,7H)-dione 2071 D09 Oc1ccc(Br)cc1/C.dbd.N/n2cnnc2
4-bromo-2-[(E)-(4H-1,2,4-triazol-4- 39.75 27.60
ylimino)methyl]phenol 1463 P01 CC1(C)OCC2.dbd.C(CC(CCc3ccccc3)
(5S,7R)-2,2-dimethyl-5,7-bis(2- -85.25 27.40 OC2CCc4ccccc4)O1
phenylethyl)-7,8-dihydro-4H,5H- pyrano[4,3-d][1,3]dioxine 1397 C13
Cc1cc(O)n(n1)c2cccc(c2)C(F) 3-methyl-1-[3- 54.75 27.00 (F)F
(trifluoromethyl)phenyl]-1H-pyrazol- 5-ol 2027 M11
N.dbd.C/1N2N.dbd.CSC2.dbd.NC(.dbd.O)\
(6E)-5-imino-6-{[1-(2-naphthyl)-1H- -109.50 26.90
C1.dbd.C\c3cccn3c4ccc5ccccc5c4
pyrrol-2-yl]methylene}-5,6-dihydro-
7H-[1,3,4]thiadiazolo[3,2-a]pyrimidin- 7-one 2294 A05
OC1[C@H](OC(.dbd.O)c2cc(O) 50.75 26.70 c(O)c(O)c2)OC3COC(.dbd.O)
c4cc(O)c(O)c(O) c4-c5c(O)c(O)c(O)cc5C(.dbd.O)
O[@@H]1[C@@H]3OC(.dbd.O) c6cc(O)c(O)c(O)c6c7c(O)
c(O)c(O)cc7C(.dbd.O)O 1465 K07 CCC(C)[C@@H](CO[C@@H](Cc1ccccc1)
isopropyl (2S)-2-{[(2S)-2-{[(2S,3R)- 37.00 26.70 C(.dbd.O)
2-{[(2S)-2-amino-3- N[C@@H](CCS(.dbd.O)(.dbd.O)C)C(.dbd.O)
mercaptopropyl]amino}-3- OC(C)C)NC[C@@H](N)CS methylpentyl]oxy}-3-
phenylpropanoyl]amino}-4- (methylsulfonyl)butanoate 1410 K11
COc1ccc(/C.dbd.C/2\C(.dbd.O)N(C) (3Z)-3-(3-hydroxy-4- 52.75 26.70
c3ccccc23)cc1O methoxybenzylidene)-1-methyl-1,3-
dihydro-2H-indol-2-one 2004 P09 CCOC(.dbd.O)c1c(oc2ccc(O) ethyl
4-{[(2-anilino-2- 49.75 26.60 c(CSCC(.dbd.O)Nc3ccccc3)c12)
oxoethyl)thio]methyl}-5-hydroxy-2- c4ccccc4
phenyl-1-benzofuran-3-carboxylate 2296 G07 O[C@H]1[C@@H](O) 45.75
25.90 [C@@H](COC(.dbd.O)c2cc(O)c(O)
c(O)c2)O[C@@H](Oc3ccc(C(.dbd.O)/ C.dbd.C/c4ccccc4)c(O)c3) [C@@H]1O
2084 H14 CCOc1ccc2[nH]c(.dbd.O) 3-benzyl-6-ethoxy-4- 71.00 25.90
c(Cc3ccccc3)c(O)c2c1 hydroxyquinolin-2(1H)-one 1442 L20
Cc1ccc(Nc2ccnc3cc(Cl)ccc23) 7-chloro-N-(3-fluoro-4- 70.25 25.90
cc1F methylphenyl)quinolin-4-amine 1465 N16
NS(.dbd.O)(.dbd.O)c1cc2c(N.dbd.CNS2(.dbd.O)
6-chloro-2H-1,2,4-benzothiadiazine- 38.50 25.80 .dbd.O)cc1Cl
7-sulfonamide 1,1-dioxide 2075 O10 CN(C)c1ccc(cc1)C(.dbd.NO) bis[4-
-119.75 25.70 c2ccc(cc2)N(C)C (dimethylamino)phenyl]methanone oxime
2037 B18 CCC(Cc1ccccc1)NC(.dbd.O) N-[(1S)-1-benzylpropyl]-6-[(4-
38.25 25.50 c2c[nH]c3ccc(cc3c2.dbd.O)S(.dbd.O)
methylpiperidin-1-yl)sulfonyl]-4-oxo- (.dbd.O)N4CCC(C)CC4
1,4-dihydroquinoline-3-carboxamide 2042 N03 COc1ccc(CCN2COc3c(C)
3-[2-(4-methoxyphenyl)ethyl]-10- 41.50 25.50
c4oc(.dbd.O)cc(c5ccccc5)c4cc3C2) methyl-6-phenyl-3,4-dihydro-2H,8H-
cc1 chromeno[6,7-e][1,3]oxazin-8-one 597 H21
Oc1ccccc1C2CC(.dbd.NN2c3ccc(cc3)
2-[(5S)-1-(4-nitrophenyl)-3-phenyl- -158.75 25.30
[N+](.dbd.O)[O--]) 4,5-dihydro-1H-pyrazol-5-yl]phenol c4ccccc4 2015
P13 Cc1cc(l)ccc1n2nc(cc2O)C(F) 1-(4-iodo-2-methylphenyl)-3- 41.00
25.30 (F)F (trifluoromethyl)-1H-pyrazol-5-ol 1398 K17
COC(.dbd.O)c1c(C)cc(O) methyl 1-hydroxy-3- 41.25 25.20
n2c3ccccc3nc12 methylpyrido[1,2-a]benzimidazole-4- carboxylate 2098
D22 OCCOc1ccc(cc1) (3R,3'R,4'S,6'R,8'S,8a'S)-5-(4- -84.25 25.10
[C@H]2N3[C@@H]([C@@H](C(.dbd.O) hydroxybut-1-yn-1-yl)-6'-[4-(2-
O)[C@]24C(.dbd.O)Nc5ccc(C#CCCO) hydroxyethoxy)phenyl]-1',2-dioxo-
cc54)C(.dbd.O) 3',4'-diphenyl-1,2,3',4',8',8a'-
O[C@@H]([C@@H]3c6ccccc6) hexahydro-1'H-spiro[indole-3,7'- c7ccccc7
pyrrolo[2,1-c][1,4]oxazine]-8'- carboxylic acid 2290 N23
COc1cc(ccc1O)c2oc3cc(O) 42.50 25.10 cc(O)c3c(.dbd.O)c2O 2094 L02
CCc1cccc(NC(.dbd.O) methyl 2,3-bis-O-(biphenyl-2- -133.75 24.90
O[C@@H]2[C@@H](CO)O[C@H](OC) ylcarbamoyl)-4-O-[(3-
[C@@H](OC(.dbd.O) ethylphenyl)carbamoyl]-alpha-L-
Nc3ccccc3c4ccccc4)[C@H]2OC(.dbd.O) idopyranoside
Nc5ccccc5c6ccccc6)c1 2079 D18 COc1ccccc1c2nnc(o2)
2-[5-(2-methoxyphenyl)-1,3,4- 50.25 24.20 c3ccccc3O
oxadiazol-2-yl]phenol 2043 M16 CC(Nc1nc2ccccc2n1CC.dbd.C)
2-{(1R)-1-[(1-allyl-1H-benzimidazol- 37.50 24.10 c3cc(Cl)ccc3O
2-yl)amino]ethyl}-4-chlorophenol 2086 O22
CC(C)C1NC(Cc2c1[nH]c3ccccc23) (1R,3R)-1-isopropyl-2,3,4,9- 71.00
23.90 C(.dbd.O)O tetrahydro-1H-beta-carboline-3- carboxylic acid
1434 C13 Cn1ncc(c1N)c2nc(cs2) 4-[4-(4-chlorophenyl)-1,3-thiazol-2-
44.50 23.70 c3ccc(Cl)cc3 yl]-1-methyl-1H-pyrazol-5-amine 2018 A06
OC(.dbd.O)c1ccc(N/C.dbd.C/C(.dbd.O)
4-{[(1E)-3-(2-furyl)-3-oxoprop-1-en- 63.50 23.20 c2ccco2)cc1
1-yl]amino}benzoic acid 2040 N04 Oc1c(CC(.dbd.O)NCc2ccccc2Cl)
N-(2-chlorobenzyl)-2-(4-hydroxy-2- 39.50 23.10
c(.dbd.O)[nH]c3ccccc13 oxo-1,2-dihydroquinolin-3- yl)acetamide 1441
N12 FC(F)(F)c1cccc(SCc2cccc(c2) 3-oxo-3-[3-({[3- 48.00 23.00
C(.dbd.O)CC#N)c1 (trifluoromethyl)phenyl]thio}methyl)phenyl]
propanenitrile 2057 E08 CCc1c(O)c(Cc2ccccc2)c(.dbd.O)
3-benzyl-5-ethyl-4-hydroxy-6-phenyl- 46.50 22.80
n(c3nccs3)c1c4ccccc4 1-(1,3-thiazol-2-yl)pyridin-2(1H)-one 2290 K17
Oc1ccc2C(.dbd.O)/C(.dbd.C/c3ccc(O) 49.00 22.80 c(O)c3)/Oc2c1 2026
E05 OC1C(NN.dbd.C1c2ccccc2) (4S,5S)-3,5-diphenyl-4,5-dihydro- 49.25
22.70 c3ccccc3 1H-pyrazol-4-ol 1407 H22 Cc1ccc(c2cc([nH]n2)c3cccs3)
5-methyl-2-[5-(2-thienyl)-1H-pyrazol- 47.25 22.70 c(O)c1
3-yl]phenol 2079 J15 Sc1nc(SCCOc2ccccc2)
2-[(2-phenoxyethyl)thio]quinazoline- 60.50 22.70 nc3ccccc13 4-thiol
1409 C22 Cc1ccc2[nH]c(.dbd.NC(.dbd.O)
N-(6-methyl-1,3-benzothiazol-2(3H)- 40.00 22.60 c3cccs3)sc2c1
ylidene)thiophene-2-carboxamide 588 K09
Oc1c(/C.dbd.C/2\S\C(.dbd.N/c3ccccc3Cl)\
(2Z,5Z)-2-[(2-chlorophenyl)imino]-5- 45.30 22.60
NC2.dbd.O)cccc1[N+](.dbd.O) (2-hydroxy-3-nitrobenzylidene)-1,3-
[O--] thiazolidin-4-one 2005 F12 COc1cc(/C.dbd.C/2\SC(.dbd.S)
(2R)-2-[(5Z)-5-(4-hydroxy-3,5- 52.00 22.20
N(C(C(C)C)C(.dbd.O)O)C2.dbd.O)cc(OC) dimethoxybenzylidene)-4-oxo-2-
c1O thioxo-1,3-thiazolidin-3-yl]-3- methylbutanoic acid 2027 G14
Oc1cc(nn1c2ccccc2) N-(3-chlorophenyl)-4-(5-hydroxy-1- 49.00 22.00
C3CCN(CC3)C(.dbd.S)Nc4ccccc(Cl)c4
phenyl-1H-pyrazol-3-yl)piperidine-1- carbothioamide 1396 O08
CCc1cc(C(.dbd.O)Cc2nc3ccccc3n2C)
1-(5-ethyl-2,4-dihydroxyphenyl)-2-(1- 46.50 22.00 c(O)cc1O
methyl-1H-benzimidazol-2- yl)ethanone 1405 B15
CN(C)S(.dbd.O)(.dbd.O)c1ccc(cc1) N,N-dimethyl-4-(6- 40.50 21.60
c2cn3cc(C)ccc3n2 methylimidazol[1,2-a]pyridin-2-
yl)benzenesulfonamide 2049 D18 CN(C)/C.dbd.C/1\N.dbd.C(OC1.dbd.O)
(4Z)-2-[2-(4-chlorophenoxy)pyridin- 66.75 21.50
c2cccnc2Oc3ccc(Cl)cc3 3-yl]-4-[(dimethylamino)methylene]-
1,3-oxazol-5(4H)-one 1442 I17 Oc1cc(nc2ccc(Br)cc12)C(F)
6-bromo-2-(trifluoromethyl)quinolin- 43.00 21.40 (F)F 4-ol 1409 L21
CC1SC(.dbd.S)NN1c2ccccc2 (5R)-5-methyl-4-phenyl-1,3,4- 114.25 21.30
thiadiazolidine-2-thione 1404 P06 Oc1cccnc1NC(.dbd.O)
N-(3-hydroxypyridin-2-yl)-4- 70.00 21.30 c2ccc(Oc3ccccc3)cc2
phenoxybenzamide 2072 D14 Cc1ccc(CCSc2ccc(Cl)c(Cl)
5-{2-[(3,4-dichlorophenyl)thio]ethyl}- 69.75 21.20 c2)cn1
2-methylpyridine 1449 E20 COC(.dbd.O)c1cc(O)n(n1) methyl
5-hydroxy-1-[4- 44.50 21.20 c2ccc(cc2)C(F)(F)F
(trifluoromethyl)phenyl]-1H-pyrazole- 3-carboxylate 2060 M02
CC/1Sc2ccc(Cl)cc2C(.dbd.O)\ (2R,3Z)-6-chloro-3- 43.00 21.20
C1.dbd.C\N(C)C [(dimethylamino)methylene]-2-
methyl-2,3-dihydro-4H-thiochromen- 4-one 1442 N22
CCC(.dbd.O)N1CCN(CC1) 1-[4-(7-chloroquinolin-4- 41.75 21.20
c2cccnc3cc(Cl)ccc23 yl)piperazino]propan-1-one 1410 G09
CC1(C)CC(.dbd.O)C(CC(.dbd.O) N-[2-chloro-5- 43.25 20.90
Nc2cc(ccc2Cl)C(F)(F)F)C(.dbd.O) (trifluoromethyl)phenyl]-2-(4,4- C1
dimethyl-2,6- dioxocyclohexyl)acetamide 2058 D04
Oc1c(c2ccccc2)c(.dbd.O)[nH]c3ccc(F)
6-fluoro-4-hydroy-3-phenylquinolin- 43.75 20.60 cc13 2(1H)-one 2074
H22 Cc1cc(.dbd.O)[nH]c(SCC(.dbd.O) N-(4-bromophenyl)-2-[(3-cyano-4-
59.00 20.40 Nc2ccc(Br)cc2)c1C#N methyl-6-oxo-1,6-dihydropyridin-2-
yl)thio]aectamide 2027 G19 CCc1ccccc1NS(.dbd.O)(.dbd.O)
6-{[(2-ethylphenyl)amino]sulfonyl}-4- 44.50 20.00
c2ccc3[nH]cc(C(.dbd.O)NCC4CCCO4) oxo-N-[(2S)-tetrahydrofuran-2-
c(50 O)c3c2 ylmethyl]-1,4-dihydroquinoline-3- carboxamide 2022 D10
CC(C)NC(.dbd.O)C1(O)N(C(.dbd.O) (4R)-3-(3,4-dichlorophenyl)-4-
49.00 19.80 Nc2ccccc21)c3ccc(Cl)c(Cl)
hydroxy-N-isopropyl-2-oxo-1,2,3,4- c3 tetrahydroquinazoline-4-
carboxamide 2049 J02 FC(F)(F)C1.dbd.NN(C(.dbd.O)C1)
2-phenyl-5-(trifluoromethyl)-2,4- 41.75 19.30 c2ccccc2
dihydro-3H-pyrazol-3-one 1440 M20 CCCCc1c(C)[nH]c2cc(nn2c1.dbd.O)
6-butyl-2-(2-furyl)-5-methyl-4,7- 44.00 19.20 c3ccco3
dihydropyrazolo[1,5-a]pyrimidin-7- one 1394 F14
CCn1c2ccccc2c3cc(/C.dbd.N/n4cnnc4) N-[(1E)-(9-ethyl-9H-carbazol-3-
46.25 19.00 ccc13 yl)methylene]-4H-1,2,4-triazol-4- amine 1416 K22
Oc1ccc2c(cc(.dbd.O)oc2c1O) 7,8-dihydroxy-4-phenyl-2H- 51.75 19.00
c3ccccc3 chromen-2-one 1413 F17 COc1ccc(c2onc(C)c2c3cscn3)
5-methoxy-2-[3-methyl-4-(1,3- 39.00 18.90 c(O)c1
thiazol-4-yl)isoxazol-5-yl]phenol 2104 I18 73.75 18.40 2006 I09
CCN(CC)c1ccc(/C.dbd.N/CCc2ccccc2) 5-(diethylamino)-2-{(E)-[(2-
65.75 18.00 c(O)c1 phenylethyl)imino]methyl}phenol 2016 H03
ON(C(.dbd.O)Nc1ccccc1)c2ccc(Cl) 1-(4-chlorophenyl)-1-hydroxy-3-
67.50 17.90 cc2 phenylurea 2013 K04 Cc1nn(c(O)c1Sc2ccc(Cl)cc2)
4-[(4-chlorophenyl)thio]-3-methyl-1- 53.00 17.80 c3ccccc3
phenyl-1H-pyrazol-5-ol 2290 N04 Oc1ccc(c(O)c1)c2oc3cc(O)
2-(2,4-dihydroxyphenyl)-3,5,7- 65.00 17.80 cc(O)c3c(.dbd.O)c2O
trihydroxy-4H-chromen-4-one 2058 E18
O.dbd.C1/C(.dbd.C\c2c[nH]c3ccccc23)/
(2S,3Z)-3-(1H-indol-3-ylmethylene)- -109.50 17.50
C(Oc4ccccc14)c5ccccc5 2-phenyl-2,3-dihydro-4H-chromen-4- one 2041
A04 CCOc1ccc(cc1)N(C)S(.dbd.O) 6-{[(4- 40.75 17.30
(.dbd.O)c2ccc3[nH]cc(C(.dbd.O)
ethoxyphenyl)(methyl)amino]sulfonyl}- NCC4CCCO4)c(.dbd.O)c3c2
4-oxo-N-[(2S)-tetrahydrofuran-2- ylmethyl]-1,4-dihydroquinoline-3-
carboxamide 2010 I17 CN(C)C1OC2.dbd.C(C.dbd.C1C)C(.dbd.O)
(10S)-10-(dimethylamino)-9-methyl- 48.00 17.30 c3cccc4cccc2c34
7H,10H-naphtho[1,8-gh]chromen-7- one 2056 P12
Clc1ccc(cc1)N2N.dbd.C(CSc3nccc(n3)
2-(4-chlorophenyl)-5-{[(4-pyridin-3- 61.25 17.20 c4cccnc4)CC2.dbd.O
ylpyrimidin-2-yl)thio]methyl}-2,4- dihydro-3H-pyrazol-3-one 1443
J06 Oc1c(Cc2ccccc2)c(.dbd.O) 3-benzyl-4-hydroxy-1,2- 63.50 16.80
[nH]c3ccccc13 dihydroquinolin-2-one 2084 K01
CC1(C)CC(.dbd.O)C2.dbd.C(C1) (4S)-4-(2-furyl)-3-hydroxy-7,7- 45.00
16.60 Nc3nn(c(O)c3C2c4ccco4) dimethyl-2-phenyl-2,4,6,7,8,9-
c5ccccc5 hexahydro-5H-pyrazolo[3,4- b]quinolin-5-one 1364 E16
On1c(nc2ncccc12)c3ccc(Cl) 2-(2,4-dichlorophenyl)-1H- 61.75 16.50
cc3Cl imidazo[4,5-b]pyridin-1-ol 2030 M08
O.dbd.C(Nc1cccc(c1)c2cn3cccnc3n2) N-(3-imidazo[1,2-a]pyrimidin-2-
45.00 16.40 C4CCCC4 ylphenyl)cyclopentanecarboxamide 2078 J10
Cc1cc(.dbd.O)oc2c(C)c(O)c(CC.dbd.C)
6-allyl-7-hydroxy-4,8-dimethyl-2H- 42.25 16.20 cc12 chromen-2-one
2011 L02 CCOC(.dbd.O)c1cnc2ccc(C) ethyl 6-methyl-4-[(4-morpholin-4-
44.00 16.00 cc2c1Nc3ccc(cc3)N4CCOCC4 ylphenyl)amino]quinoline-3-
carboxylate 2072 J04 Oc1c(oc2ccccc2c1.dbd.O)c3ccc(F)
2-(4-fluoro-3-phenoxyphenyl)-3- 58.50 15.90 c(Oc4ccccc4)c3
hydroxy-4H-chromen-4-one 2014 O13 CCOC(.dbd.O)c1ccc(NC(.dbd.O)
ethyl 4-[({[(5R)-5-ethyl-4,6-dioxo- 49.50 15.90
CSC2.dbd.NC(.dbd.O)C(CC)C(.dbd.O) 1,4,5,6-tetrahydropyrimidin-2-
N2)cc1 yl]thio}acetyl)amino]benzoate 2069 G20
CCS(.dbd.O)(.dbd.O)c1ccc(NC(.dbd.O) N-[4-(ethylsulfonyl)-2- 43.25
15.70 c2ccccc2)c(O)c1 hydroxyphenyl]benzamide
2027 E19 COc1cccc(NS(.dbd.O)(.dbd.O) N-benzyl-6-{[(3- 33.75 15.50
c2ccc3[nH]cc(C(.dbd.O)N(C)Cc4ccccc4)
methoxyphenyl)amino]sulfonyl}-N- c(.dbd.O)c3c2)c1
methyl-4-oxo-1,4-dihydroquinoline- 3-carboxamide 2011 A15
O.dbd.C(Oc1cccc(Nc2ncnc3ccccc23) 3-(quinazolin-4-ylamino)phenyl
48.75 15.40 c1)c4cccs4 thiophene-2-carboxylate 2088 A12
CCOC(.dbd.O)/C.dbd.C/c1cc(ccc1O) ethyl (2E)-3-(2-hydroxy-5- 45.50
15.20 [N+](.dbd.O)[O--] nitrophenyl)acrylate 1431 J17
CSCc1ccc(cc1)C(.dbd.O)Nc2ccc(C) N-(2-hydroxy-4-methylphenyl)-4-
50.50 15.10 cc2O [(methylthio)methyl]benzamide 1415 K11
CCc1cc(c2n[nH]cc2c3nc4ccccc4n3C) 4-ethyl-6-[4-(1-methyl-1H- 46.75
14.90 c(O)cc1O benzimidazol-2-yl)-1H-pyrazol-3- yl]benzene-1,3-diol
1416 N11 CCCc1cc(.dbd.O)oc2cc(O)cc(O)
5,7-dihydroxy-4-propyl-2H-chromen- 62.50 14.90 c12 2-one 2017 K21
CC1.dbd.NN(C(.dbd.O)C1C(.dbd.O) (4S)-4-(2-bromobenzoyl)-5-methyl-
43.25 14.60 c2ccccc2Br)c3ccccc3 2-phenyl-2,4-dihydro-3H-pyrazol-3-
one 1439 C09 Oc1ccccc1C(.dbd.O)NC(.dbd.O)
N-[(3-chloro-1-benzothiophen-2- 73.50 14.50 c2sc3ccccc3c2Cl
yl)carbonyl]-2-hydroxybenzamide 1410 H21
Oc1ccc(Oc2c(F)c(F)c(Oc3ccc(O) 4,4'-[(2,3,5,6-tetrafluoro-1,4- 37.50
14.40 cc3)c(F)c2F)cc1 phenylene)bis(oxy)]diphenol 2012 D07
COc1ccccc1CC(.dbd.O)Nc2ccc(cc2) N-[4-(1H-benzimidazol-2-yl)phenyl]-
74.00 14.20 c3nc4ccccc4[nH]3 2-(2-methoxyphenyl)acetamide 2068 J16
CC(C)CCCN1C(.dbd.O)NC(.dbd.O)/
(5E)-1-(4-methylpentyl)-5-(1H-pyrrol- -112.25 14.00
C(.dbd.C\c2ccc[nH]2)/C1.dbd.O 2-ylmethylene)pyrimidine-
2,4,6(1H,3H,5H)-trione 2293 F13 Oc1ccc(cc1)[C@H]2CC(.dbd.O) 53.25
13.90 c3c(O)cc(O)c([C@H]4[C@@H](Oc5cc(O) cc(O)c5C4.dbd.O)
c6ccc(O)cc6)c3O2 1393 A03 Oc1c(CC(.dbd.O)Nc2ccc(F)cc2)
N-(4-fluorophenyl)-2-(4-hydroxy-2- 37.75 13.80
c(.dbd.O)[nH]c3ccccc13 oxo-1,2-dihydroquinolin-3- yl)acetamide 1439
E21 Cc1ccc(cc1)c2nc(.dbd.O) 2-(4-methylphenyl)-4H-1,3- 52.75 13.70
c3ccccc3o2 benzoxazin-4-one 1439 A09 COc1ccccc1CC(.dbd.O)NC(.dbd.O)
2-hydroxy-N-[2-(2- 37.50 13.60 c2ccccc2O
methoxyphenyl)acetyl]benzamide 2057 M10 Oc1c(Cc2ccccc2)c(.dbd.O)
3-benzyl-4-hydroxy-1- 69.25 13.60 n(c3ccccc3)c4ccccc14
phenylquinolin-2(1H)-one 2016 O15 Oc1cc(c2cc(ccc2Cl)C(F)(F)
7-[2-chloro-5- 41.00 13.60 F)c3oc(.dbd.O)sc3c1
(trifluoromethyl)phenyl]-5-hydroxy- 1,3-benzoxathiol-2-one 1415 K09
CCc1cc2c(.dbd.O)c(c3nc4ccccc4n3C) 6-ethyl-7-hydroxy-3-(1-methyl-1H-
41.75 13.50 c(oc2cc1O)C(F)(F)F benzimidazol-2-yl)-2-
(trifluoromethyl)-4H-chromen-4-one 2059 D01
Cc1ccc(cc1)N2C(C(.dbd.O) (3S)-3-(2-hydroxy-4-methylbenzoyl)- 51.75
13.40 c3ccc(C)cc3O)c4ccccc4C2.dbd.O
2-(4-methylphenyl)isoindolin-1-one 1439 E09
Cc1ccc(Oc2ncccc2C(.dbd.O) 2-hydroxy-N-{[2-(4-methylphenoxy)- 81.00
13.20 NC(.dbd.O)c3ccccc3O)cc1 3-pyridinyl]carbonyl}benzamide 1413
N19 Cc1csc(n1)c2c(oc3cc(O)c(C) 7-hydroxy-6-methyl-3-(4-methyl-1,3-
44.00 13.00 cc3c2.dbd.O)C(F)(F)F
thiazol-2-yl)-2-(trifluoromethyl)-4H- chromen-4-one 2091 D09 -79.00
12.70 1465 D10 CCN(CCCc1ccccc1) N-ethyl-3-phenyl-N-(3- 43.25 12.60
CCCc2ccccc2 phenylpropyl)propan-1-amine 2073 I20
CC(.dbd.O)Nc1cccc(c1O)c2cc(.dbd.O)
N-[2-hydroxy-3-(4-oxo-4H-chromen- 40.50 12.60 c3ccccc3o2
2-yl)phenyl]acetamide 1413 L10 CCCc1cc(C(.dbd.O)Cc2ccc3OCOc3c2)
2-(1,3-benzodioxol-5-yl)-1-(2,4- 55.50 12.50 c(O)cc1O
dihydroxy-5-propylphenyl)ethanone 2067 O04
Cc1sc2NC(NC(.dbd.O)c2c1C)/ (2R)-2-[(E)-2-(1,3-benzodioxol-5- -96.25
12.20 C.dbd.C/c3ccc4OCOc4c3 yl)vinyl]-5,6-dimethyl-2,3-
dihydrothieno[2,3-d]pyrimidin-4(1H)- one 2081 H11
CCCc1cc(O)c2c(C)cc(.dbd.O) 5-hydroxy-4-methyl-7-propyl-2H- 63.25
11.80 oc2c1 chromen-2-one 1406 M17 Oc1cc(c2ccc(Br)cc2)c3oc(.dbd.O)
7-(4-bromophenyl)-5-hydroxy-1,3- 43.50 11.80 sc3c1
benzoxathiol-2-one 1413 D15 Cc1cc(O)cc2oc(c(c3cnn(c3)
7-hydroxy-5-methyl-3-(1-phenyl-1H- 43.00 11.70
c4ccccc4)c(.dbd.O)c12)C(F)(F)F
pyrazol-4-yl)-2-(trifluoromethyl)-4H- chromen-4-one 2014 G14
CC(C)n1nc(O)c2C(SCC(.dbd.O) (4R)-4-(4-bromophenyl)-3-hydroxy- 42.25
11.70 Nc21)c3ccc(Br)cc3 1-isopropyl-4,8-dihydro-1H-
pyrazolo[3,4-e][1,4]thiazepin-7(6H)- one 1425 M21
CCNC(.dbd.O)CC1Nc2cc(C)c(C) 2-[(2R)-6,7-dimethyl-3-oxo-1,2,3,4-
48.50 11.50 cc2NC1.dbd.O tetrahydroquinoxalin-2-yl]-N-
ethylacetamide 2297 M15 COC(.dbd.O)[C@]1(Cc2ccc(O) 49.00 11.20
c(CC.dbd.C(C)C)c2)OC(.dbd.O) C(.dbd.C1c3ccc(O)cc3)O 1412 M06
CC(C)C(.dbd.O) N-[2-(1H-benzimidazol-2-yl)phenyl]- 43.00 11.10
Nc1ccccc1c2nc3ccccc3[nH]2 2-methylpropanamide 1446 D05
CC1(C)c2ccccc2-n3c1cc(O) 7-benzyl-8-hydroxy-10,10-dimethyl- 53.50
11.00 c(Cc4ccccc4)c3.dbd.O 6,10-dihydropyrido[1,2-a]indol-6-one
2081 P14 CCCc1c(O)c2ccccc2[nH]c1.dbd.O
4-hydroxy-3-propylquinolin-2(1H)- 39.00 10.60 one 1425 O02
Oc1ccc(c2n[nH]c(c2c3cnn(c3) 4-[1'-phenyl-5-(trifluoromethyl)- 40.25
10.10 c4ccccc4)C(F)(F)F)c(O)c1 1H,1'H-4,4'-bipyrazol-3-yl]benzene-
1,3-diol 2064 B02 O.dbd.C(C1.dbd.NN(C2C1C(.dbd.O)
(3aS,6aS)-3-benzoyl-1,5-diphenyl- -102.50 9.80
N(C2.dbd.O)c3ccccc3)c4ccccc4) 3a,6a-dihydropyrrolo[3,4-c]pyrazole-
c5ccccc5 4,6(1H,5H)-dione 1412 J10 CSc1nc2ccc(NC(.dbd.O)c3cccc(Cl)
3-chloro-N-[2-(methylthio)-1,3- 45.25 9.80 c3)cc2s1
benzothiazol-6-yl]benzamide 1395 D05 CCCn1c(nc2ccccc12)c3ccc(N)
4-(1-propyl-1H-benzimidazol-2- 39.25 9.60 cc3 yl)aniline 2077 D11
CN(C)c1ccc(cc1)C(N2CCCCC2) 6-[(S)-[4- 46.00 9.20 c3cc4OCOc4cc3O
(dimethylamino)phenyl](piperidin-1- yl)methyl]-1,3-benzodioxol-5-ol
1405 H15 Cc1oc2cc(O)ccc2c(.dbd.O) 3-(4-bromophenyl)-7-hydroxy-2-
51.25 9.20 c1c3ccc(Br)cc3 methyl-4H-chromen-4-one 2073 K12
CC(NC(.dbd.O)Oc1c(Cl)cc(Cl) 2,4-dichloro-1-naphthyl [2,2,2- 53.00
9.20 c2ccccc12)(C(F)(F)F)C(F)(F)F trifluoro-1-methyl-1-
(trifluoromethyl)ethyl]carbamate 2018 O08
Cc1ccc(cc1)C2.dbd.C/C(.dbd.C/c3ccc(o3)
3-(5-{(Z)-[5-(4-methylphenyl)-2- 62.75 8.60 c4cccc(c4)C(.dbd.O)O)/
oxofuran-3(2H)-ylidene]methyl}-2- C(.dbd.O)O2 furyl)benzoic acid
2010 P21 CCOC(.dbd.O)c1c(CSc2ccc(C) ethyl 6-bromo-4- 51.00 8.60
cc2)n(C)c3cc(Br)c(O)c(CN(C) [(dimethylamino)methyl]-5-hydroxy-
C)c13 1-methyl-2-{[(4- methylphenyl)thio]methyl}-1H-indole-
3-carboxylate 1408 L07 Oc1ccccc1C(.dbd.O)Nc2cccnc2
2-hydroxy-N-pyridin-3-ylbenzamide 56.00 8.40 1469 I17
Oc1ccc(CCC(.dbd.O)c2c(O)cc(O) 3-(4-hydroxyphenyl)-1-(2,4,6- 45.50
7.90 cc2O)cc1 trihydroxyphenyl)propan-1-one 1414 B12
CCS(.dbd.O)(.dbd.O)c1ccc(O)c(c1) 2-[5-(ethylsulfonyl)-2- 37.00 7.80
N2C(.dbd.O)c3cccc4cccc(C2.dbd.O) hydroxyphenyl]-1H- c34
benzo[de]isoquinoline-1,3(2H)-dione 1406 I10
Cc1ccc(c(C)c1)n2c(N)c(C#N) 2-amino-1-(2,4-dimethylphenyl)-1H- 46.75
7.50 c3nc4ccccc4nc23 pyrrolo[2,3-b]quinoxaline-3- carbonitrile 1425
A04 COc1ccc(c2n[nH]c(c2c3cnn(c3) 3-methoxy-2-methyl-6-[1'-phenyl-5-
32.75 7.30 c4ccccc4)C(F)(F)F)c(O) (trifluoromethyl)-1H,1'H-4,4'-
c1C bipyrazol-3-yl]phenol 2018 C20 OC(.dbd.O)CC(N1C(.dbd.O)/
(2S)-2-[(5E)-5-(1H-indol-3- 44.50 7.30
C(.dbd.C\c2c[nH]c3ccccc23)/SC1.dbd.S)
ylmethylene)-4-oxo-2-thioxo-1,3- C(.dbd.O)O
thiazolidin-3-yl]succinic acid 1409 P14 Oc1ccccc1C(.dbd.O)
N-benzyl-2-hydroxybenzamide 43.00 7.10 NCc2ccccc2 2058 B10
Cc1nn(c(O)c1Cc2c(Cl) 4-(2,6-dichlorobenzyl)-3-methyl-1- 45.50 6.90
cccc2Cl)c3ccccc3 phenyl-1H-pyrazol-5-ol 2025 O12
Clc1ccccc1CNS(.dbd.O)(.dbd.O) N-(2-chlorobenzyl)-2-phenyl-1H- 45.50
6.80 c2ccc3[nH]c(nc3c2)c4ccccc4 benzimidazole-5-sulfonamide 2072
F12 CCOC(.dbd.O)Oc1c(Cl)cc2oc(.dbd.O) 4-bromo-6-chloro-2-oxo-1,3-
50.00 6.40 sc2c1Br benzoxathiol-5-yl ethyl carbonate 2069 I05
Cc1cccc2c(O)c(/C.dbd.C/3\C(NN(C3.dbd.O)
4-hydroxy-8-methyl-3-{(E)-[(3R)-5- 47.25 6.20 c4ccccc4)c5ccccc5)
oxo-1,3-diphenylpyrazolidin-4- c(.dbd.O)[nH]c12
ylidene]methyl}quinolin-2(1H)-one 1441 L02
Clc1ccc(SCc2cccc(c2)C(.dbd.O) 3-(3-{[(4- 42.50 6.00 CC#N)cc1
chlorophenyl)thio]methyl}phenyl)-3- oxopropanenitrile 2049 B03
CN(/N.dbd.C/c1cc(Cl)cc(Cl)c1O) 3,5-dichloro-2-hydroxybenzaldehyde
56.00 5.90 C(.dbd.S)NC(C)(C)C N-tert-butyl-N'-
methylthiosemicarbazone 2069 I04 Oc1ccc(Cl)cc1Sc2cc(Cl)
2,2'-thiobis(4-chlorophenol) 41.25 5.80 ccc2O 1414 B15
CC(C)CC1C(.dbd.C(N)OC2.dbd.C1C(.dbd.O)
(4S,7R)-2-amino-4-isobutyl-5-oxo-7- 62.25 5.40 CC(C2)c3ccccc3)C#N
phenyl-5,6,7,8-tetrahydro-4H- chromene-3-carbonitrile 1409 M11
Oc1c(Cl)cc(Cl)cc1C(.dbd.O) (3,5-dichloro-2- 67.50 5.40 c2cnoc2
hydroxyphenyl)(isoxazol-4- yl)methanone 1414 J04
CC(C)(C)C(.dbd.O)Nc1ccc(O)c(c1) N-[3-(1,3-benzothiazol-2-yl)-4-
46.75 5.30 c2nc3ccccc3s2 hydroxyphenyl]-2,2- dimethylpropanamide
1416 D17 c1cnc2ccc(cc2c1) 3,6'-biquinoline 38.00 4.70
c3ccc4ncccc4c3 2011 E12 CC(Oc1ccc(Cl)cc1Cl)C(.dbd.O)
(2R)-2-(2,4-dichlorophenoxy)-N-(5- 62.25 4.70
NC2.dbd.NN(C(.dbd.O)C2)c3ccccc3 oxo-1-phenyl-4,5-dihydro-1H-
pyrazol-3-yl)propanamide 2144 J08 45.50 3.90 1426 J16
Oc1ccc(NS(.dbd.O)(.dbd.O)c2cccs2)
N-[3-(1,3-benzothiazol-2-ylthio)-4- 55.75 3.90 cc1Sc3nc4ccccc4s3
hydroxyphenyl]thiophene-2- sulfonamide 588 M05
CCN(CC)c1ccc(/C.dbd.C\2/SC(.dbd.O) (5E)-5-[4- -138.00 3.50
N(CNc3ccccc3OC)C2.dbd.O) (diethylamino)benzylidene]-3-{[(2- cc1
methoxyphenyl)amino]methyl}-1,3- thiazolidine-2,4-dione 2058 E04
Oc1c(c2ccccc2)c(.dbd.O) 4-hydroxy-5-phenyl-6H-pyrido[3,2,1- 43.75
3.10 n3c4ccccc4c5cccc1c53 jk]carbazol-6-one 1443 G08
Cc1ccc(Sc2cccc3nc(N)nc(N) 5-[(4-methylphenyl)thio]quinazoline-
37.50 2.50 c23)cc1 2,4-diamine 2020 K14
CCc1ccc(cc1)C2C3.dbd.C(CCCC3.dbd.O)
(4R)-4-(4-ethylphenyl)-3-hydroxy-2- 61.00 2.30 Nc4nn(c(O)c24)
phenyl-2,4,6,7,8,9-hexahydro-5H- c5ccccc5
pyrazolo[3,4-b]quinolin-5-one 1424 I20 CC(.dbd.O)n1cc(c2c(O)
3-(1-acetyl-1H-indol-3-yl)-4-hydroxy- 51.50 2.00
c3ccccc3oc2.dbd.O)c4ccccc14 2H-chromen-2-one 2081 D13
N(c1ccccc1)c2nc(nc3ccccc23) N,2-diphenylquinazolin-4-amine 50.00
1.30 c4ccccc4 1398 D22 ClC1ccc(cc1)C(.dbd.O) 2-anilino-2-oxoethyl
2-(4- 58.00 0.80 c2ccccc2C(.dbd.O)OCC(.dbd.O)Nc3ccccc3
chlorobenzoyl)benzoate 1394 A01 Fc1ccc(cc1)C(.dbd.O)Nc2cccc(c2)
4-fluoro-N-[3- 28.00 0.00 C(F)(F)F
(trifluoromethyl)phenyl]benzamide 1417 A07 *c1ccccc1C2C(.dbd.O)N(C)
34.75 0.00 c3ccccc3C2.dbd.O 2030 A14
C(c1ccccc1)n2cc3c(nnc3c4ccccc24) 5-benzyl-3-phenyl-5H-pyrazolo[4,3-
51.00 0.00 c5ccccc5 c]quinoline 1413 B06
CCCCc1cc(C(.dbd.O)Cc2ccccn2) 1-(5-butyl-2,4-dihydroxyphenyl)-2-
48.75 0.00 c(O)cc1O pyridin-2-ylethanone 1416 C02
CCOc1ccc2C(.dbd.O)/C(.dbd.C\c3ccccc3O)/ (2E)-6-ethoxy-2-(2- 36.25
0.00 Sc2c1 hydroxybenzylidene)-1- benzothiophen-3(2H)-one 1397 C08
COc1ccc(/C.dbd.C/C(.dbd.O)Nc2ccc(C) (2E)-3-(3,4-dimethoxyphenyl)-N-
50.50 0.00 c(C)c2)cc1OC (3,4-dimethylphenyl)acrylamide 1415 C11
CCc1cc(C(.dbd.O)Cn2cnc3ccccc23)
2-(1H-benzimidazol-1-yl)-1-(5-ethyl- 42.00 0.00 c(O)cc1O
2,4-dihydroxyphenyl)ethanone 1422 C11 COc1cc(Cn2c(nc3ccccc23)
4-[1-(4-hydroxy-3-methoxybenzyl)- 41.75 0.00 c4ccc(O)c(OC)c4)ccc1O
1H-benzimidazol-2-yl]-2- methoxyphenol 1417 C14
CCC(C)Sc1nnc(NC(.dbd.O) N-(5-{[(1S)-1-methylpropyl]thio}- 42.00
0.00 c2ccccc2C(F)(F)F)s1 1,3,4-thiadiazol-2-yl)-2-
(trifluoromethyl)benzamide 1418 D05 COCC(.dbd.O)Oc1c(Sc2ccc(C)
3-methyl-4-[(4-methylphenyl)thio]-1- 40.75 0.00 cc2)c(C)nn1c3ccccc3
phenyl-1H-pyrazol-5-yl methoxyacetate 1469 D07
COc1cc(O)c2c(.dbd.O)c(O)c(oc2c1) 2-(3,4-dihydroxyphenyl)-3,5- 48.50
0.00 c3ccc(O)c(O)c3 dihydroxy-7-methoxy-4H-chromen- 4-one 1446 D07
Oc1cc2nnnn2nc1c3ccccc3 6-phenyl[1,2,3,4]tetraazolo[1,5- 43.50 0.00
b]pyridazin-7-ol 1412 D10 COc1ccc(cc1OC)C(.dbd.O)
3,4-dimethoxy-N-(4-methyl-1,3- 53.25 0.00 Nc2nc3c(C)cccc3s2
benzothiazol-2-yl)benzamide 1432 D13 Oc1c(Cc2ccccc2)c(.dbd.O)
6-benzyl-7-hydroxy-2,3-dihydro- 68.75 0.00 n3CCCc4cccc1c43
1H,5H-pyrido[3,2,1-ij]quinolin-5-one 1408 D15
Cc1ccc(NC(.dbd.O)c2ccccc2O) 2-hydroxy-N-(4- 62.25 0.00 cc1
methylphenyl)benzamide 2080 E05 Cc1ccc(cc1)N2COc3ccc(Cl)
6-chloro-3-(4-methylphenyl)-3,4- 41.50 0.00 cc3C2
dihydro-2H-1,3-benzoxazine 1426 F20
CCCCN1C(.dbd.O)C2ON(C(C2C1.dbd.O) (3S,3aR,6aR)-3-(5-bromo-2- 43.00
0.00 c3cc(Br)ccc3O) hydroxyphenyl)-5-butyl-2- c4ccccc4
phenyldihydro-2H-pyrrolo[3,4- d]isoxazole-4,6(3H,5H)-dione 1442 G19
Oc1cc(nc2c(OC(F)(F)F) 8-(trifluoromethoxy)-2- 52.75 0.00
cccc12)C(F)(F)F (trifluoromethyl)quinolin-4-ol 1410 H10
Cc1nn(c2ccccc2)c3[nH]c(.dbd.O) 3-methyl-1-phenyl-4- 40.50 0.00
cc(c13)C(F)(F)F (trifluoromethyl)-1,7-dihydro-6H-
pyrazolo[3,4-b]pyridin-6-one 1438 H17 Oc1ccccc1C(.dbd.O)NC(.dbd.O)
N-(2-hydroxybenzoyl)-4- 58.00 0.00 c2ccc(cc2)C(F)(F)F
(trifluoromethyl)benzamide 1412 I04
Cc1nc2ccc(NC(.dbd.O)/C.dbd.C/c3ccccc3)
(2E)-N-(2-methyl-1,3-benzothiazol- 62.50 0.00 cc2s1
6-yl)-3-phenylacrylamide 1399 I05 NC(.dbd.O)c1ccc(NC(.dbd.O)
N-(4-carbamoylphenyl)-1-phenyl-3- 39.50 0.00
c2cn(nc2c3cccs3)c4ccccc4)cc1 (2-thienyl)-1H-pyrazole-4- carboxamide
1436 I16 Cc1nc(nc(SCC(.dbd.O)c2ccccc2)
2-{[5-chloro-6-methyl-2-(2-pyridinyl)- 45.75 0.00 c1Cl)c3ccccn3
4-pyrimidinyl]sulfanyl}-1-phenyl-1- ethanone 1409 I20
Cc1ccc(cc1)S(.dbd.O)(.dbd.O) N-(5-hydroxy-1-naphthyl)-4- 44.25 0.00
Nc2cccc3c(O)cccc23 methylbenzenesulfonamide 1394 I20
N/1/C(.dbd.N\c2ccccc2)/ N,N'-1H-isoindole-1,3(2H)- 46.25 0.00
c3ccccc3\C1.dbd.N\c4ccccc4 diylidenedianiline 1410 J05
S.dbd.c1cc(sc2ccccc12) 2-phenyl-4H-thiochromene-4-thione 45.50 0.00
c3ccccc3 1416 J09 CCC1CCCCN1Cc2c(O)cc(C)
4-{[(2S)-2-ethylpiperidin-1-yl]methyl}- 39.00 0.00
c3c4ccccc4c(.dbd.O)oc23 3-hydroxy-1-methyl-6H-
benzo[c]chromen-6-one 1396 J14 CCCn1c(/N.dbd.C/c2c[nH]c3ccccc23)
N-[(1E)-1H-indol-3-ylmethylene]-1- 66.00 0.00 nc4ccccc14
propyl-1H-benzimidazol-2-amine 1439 K19
Oc1ccccc1C(.dbd.O)NC(.dbd.O) N-(2,3-dihydro-1-benzofuran-5- 80.00
0.00 c2ccc3OCCc3c2 ylcarbonyl)-2-hydroxybenzamide 1408 L13
Oc1ccc(Cl)cc1C(.dbd.O) 5-chloro-2-hydroxy-N- 42.25 0.00 Nc2ccccc2
phenylbenzamide 1415 L17 COc1cccc(c1)C(.dbd.O)
3-methoxy-N-(3-[1,3]oxazolo[4,5- 36.00 0.00
Nc2cccc(c2)c3nc4ncccc4o3 b]pyridin-2-ylphenyl)benzamide 2293 L18
O[C@H]1[C@@H](Oc2c([C@H]3[C@@H](Oc4cc(O) 47.25 0.00 cc(O)
c4C3.dbd.O)c5ccc(O)cc5) c(O)cc(O)c2C1.dbd.O)c6ccc(O) cc6 1415 L21
CC(.dbd.O)N/C(.dbd.C\c1ccccc1)/ (2Z)-2-acetamido-N-(3,5- 48.00 0.00
C(.dbd.O)Nc2cc(C)cc(C)c2 dimethylphenyl)-3-phenylacrylamide 1404
M10 Cc1cc(.dbd.O)[nH]c(SCC(.dbd.O) 2-[(3-cyano-4-methyl-6-oxo-1,6-
60.25 0.00 Nc2cccc3ccccc23)c1C#N dihydropyridin-2-yl)thio]-N-1-
naphthylacetamide 593 O09 COc1cccc(/C.dbd.C\2/S/C(.dbd.N\c3cc(C)
(2Z,5E)-2-[(3,5- -1.00 0.00 cc(C)c3)/NC2.dbd.O)c1O
dimethylphenyl)imino]-5-(2-hydroxy- 3-methoxybenzylidene)-1,3-
thiazolidin-4-one 2073 O11 Cn1c(.dbd.O)n(C)c2c(O)c([nH]c2c1.dbd.O)
6-(4-chlorophenyl)-7-hydroxy-1,3- 42.50 0.00 c3ccc(Cl)cc3
dimethyl-1H-pyrrolo[3,2- d]pyrimidine-2,4(3H,5H)-dione 2160 P06
Oc1ccc2c(.dbd.O)cc(oc2c1O) 7,8-dihydroxy-2-phenyl-4H- 54.25 0.00
c3ccccc3 chromen-4-one 1398 P09 Cc1cc(O)cc(O)c1C(.dbd.O)
1-(2,4-dihydroxy-6-methylphenyl)-2- 54.25 0.00 COc2ccccc2
phenoxyethanone
[0313]
Sequence CWU 1
1
53 1 205 PRT Plasmodium falciparum 1 Met Lys Asn Arg Phe Tyr Tyr
Asn Leu Ile Ile Lys Arg Leu Tyr Thr 1 5 10 15 Arg Ser Gly Gly Leu
Arg Lys Pro Gln Lys Val Thr Asn Asp Pro Glu 20 25 30 Ser Ile Asn
Arg Lys Val Tyr Trp Cys Phe Glu His Lys Pro Val Lys 35 40 45 Arg
Thr Ile Ile Asn Leu Ile Tyr Ser His Asn Glu Leu Lys Ile Phe 50 55
60 Ser Asn Leu Leu Asn His Pro Thr Val Gly Ser Ser Leu Ile His Glu
65 70 75 80 Leu Ser Leu Asp Gly Pro Tyr Thr Ala Phe Phe Pro Ser Asn
Glu Ala 85 90 95 Met Gln Leu Ile Asn Ile Glu Ser Phe Asn Lys Leu
Tyr Asn Asp Glu 100 105 110 Asn Lys Leu Ser Glu Phe Val Leu Asn His
Val Thr Lys Glu Tyr Trp 115 120 125 Leu Tyr Arg Asp Leu Tyr Gly Ser
Ser Tyr Gln Pro Trp Leu Met Tyr 130 135 140 Asn Glu Lys Arg Glu Ala
Pro Glu Lys Leu Arg Asn Leu Leu Asn Asn 145 150 155 160 Asp Leu Ile
Val Lys Ile Glu Gly Glu Phe Lys His Cys Asn His Ser 165 170 175 Ile
Tyr Leu Asn Gly Ser Lys Ile Ile Arg Pro Asn Met Lys Cys His 180 185
190 Asn Gly Val Val His Ile Val Asp Lys Pro Ile Ile Phe 195 200 205
2 618 DNA Plasmodium falciparum 2 atgaaaaata gattttatta taatttgata
attaaaagat tatatacacg aagtggcggt 60 ttaagaaaac ctcaaaaggt
aaccaacgac ccagaaagta taaatagaaa agtatattgg 120 tgttttgaac
ataagcctgt aaaaaggaca attattaatt taatatattc acataacgaa 180
ctcaagatat tttctaatct gttaaatcat cctacagttg gcagctcgtt aatacatgaa
240 ttatctctcg atggccctta tactgcattt tttccctcca acgaagccat
gcaattaata 300 aatatagaaa gtttcaataa attgtataac gatgaaaata
aattatcaga atttgtttta 360 aatcacgtta cgaaagaata ttggctgtat
agagatttat atggttcatc ttaccaaccg 420 tggttaatgt acaatgaaaa
aagggaagct ccagaaaaat taagaaattt attgaataat 480 gatttaatag
taaaaattga gggggaattt aaacattgca atcattcgat atatttaaat 540
ggctcaaaaa ttataagacc aaatatgaag tgccacaatg gagttgtgca tatagtagat
600 aagcccatca ttttttaa 618 3 204 PRT Plasmodium gallinaceum 3 Met
Lys Asn Ser Gly Tyr Asn Leu Ile Ile Lys Arg Leu Tyr Thr Arg 1 5 10
15 Ser Gly Gly Leu Arg Lys Pro Gln Lys Val Thr Asn Asp Pro Glu Ser
20 25 30 Ile Asn Arg Lys Val Tyr Trp Cys Phe Glu His Lys Pro Ile
Lys Arg 35 40 45 Thr Ile Val Asn Leu Ile Phe Ser His Lys Glu Leu
Lys Phe Phe Ser 50 55 60 Asn Phe Leu Asn His Pro Asn Val Gly Val
Ser Leu Ile His Glu Leu 65 70 75 80 Ser Leu Glu Gly Pro Phe Thr Gly
Phe Leu Pro Ser Asn Glu Ala Leu 85 90 95 Lys Leu Ile Asn Ser Glu
Cys Leu Asn Lys Leu Tyr Lys Asp Asp Asn 100 105 110 Lys Leu Ser Glu
Phe Val Leu Asn His Phe Thr Lys Asp Phe Trp Leu 115 120 125 Tyr Arg
Asp Leu Tyr Gly Ser Ser Tyr Gln Pro Trp Leu Ile Tyr Asn 130 135 140
Glu Lys Arg Glu Ala Pro Glu Lys Ile Thr Asn Leu Met Asn Asn Asp 145
150 155 160 Leu Ile Val Lys Ile Lys Gly Glu Phe Lys Asn Cys Asp His
Ser Ile 165 170 175 Tyr Leu Asn Glu Ser Lys Ile Ile Arg Pro Asn Met
Lys Cys His Asn 180 185 190 Gly Val Val His Ile Val Asp Lys Pro Ile
Ile Phe 195 200 4 612 DNA Plasmodium gallinaceum 4 atgaaaaata
gtggttataa tttaattatt aaaagactat atactcgtag tggtggatta 60
cgaaaaccac aaaaagtaac taatgatcca gaaagtatta atagaaaagt ttattggtgt
120 tttgaacata aacctattaa aaggacaatt gttaatttaa tattttcaca
taaggaattg 180 aaatttttct ctaatttttt aaaccatcca aatgttggcg
tatcattaat ccatgaatta 240 tctttagagg gaccattcac aggattttta
ccatcaaatg aagcattaaa gttaattaat 300 tcagaatgtt taaataaatt
atataaggat gataataaat tatctgaatt tgttttaaat 360 cattttacaa
aagatttttg gctatataga gatttatatg gatcatcata ccagccttgg 420
ttaatatata atgaaaaaag agaagcacca gaaaaaatca ctaacttaat gaataatgat
480 ttaatagtaa aaataaaagg ggaatttaaa aattgtgatc attcaattta
tttaaacgaa 540 tcaaaaatta tcagacctaa tatgaaatgt cacaatggtg
tagttcatat tgtagataag 600 ccaataatat tt 612 5 204 PRT Plasmodium
reichenowi 5 Met Lys Ile Lys Phe Tyr Asn Leu Ile Ser Lys Arg Leu
Tyr Thr Arg 1 5 10 15 Ser Gly Gly Leu Arg Lys Pro Gln Lys Val Thr
Asn Asp Pro Glu Ser 20 25 30 Ile Asn Arg Lys Val Tyr Trp Cys Phe
Glu His Lys Pro Val Lys Arg 35 40 45 Thr Ile Ile Asn Leu Ile Tyr
Ser His Asn Glu Leu Lys Ile Phe Ser 50 55 60 Asn Leu Leu Asn His
Pro Ile Val Gly Ser Ser Leu Ile His Glu Leu 65 70 75 80 Ser Leu Asp
Gly Pro Tyr Thr Ala Phe Leu Pro Ser Asn Glu Ala Met 85 90 95 Lys
Leu Ile Asn Ile Glu Ser Phe Asn Lys Leu Tyr Asn Asp Glu Asn 100 105
110 Lys Leu Ser Glu Phe Val Leu Asn His Val Thr Lys Glu Tyr Trp Leu
115 120 125 Tyr Arg Asp Leu Tyr Gly Ser Ser Tyr Gln Pro Trp Leu Met
Tyr Asn 130 135 140 Glu Lys Arg Glu Ala Pro Glu Lys Leu Arg Asn Leu
Leu Asn Asn Asp 145 150 155 160 Ile Ile Val Lys Ile Glu Gly Glu Phe
Lys His Cys Asn His Ser Ile 165 170 175 Tyr Leu Asn Gly Ser Lys Ile
Ile Arg Pro Asn Met Lys Cys His Asn 180 185 190 Gly Val Val His Ile
Val Asp Lys Pro Ile Ile Phe 195 200 6 612 DNA Plasmodium reichenowi
6 atgaaaatta aattttataa tttgataagt aaaagattat atactcgaag tggtggttta
60 agaaagcctc aaaaggtaac aaacgaccca gaaagtataa atagaaaagt
atattggtgt 120 tttgaacata agcctgtaaa aaggacaatt attaatttaa
tatattcaca taacgaactc 180 aagatattct ctaatctgtt aaatcatcct
atagttggta gctcgttaat acatgaatta 240 tctctcgatg gcccttatac
tgcatttctt ccctccaacg aagccatgaa attaataaat 300 atagaaagtt
tcaataaatt gtataacgat gaaaataaat tatcagaatt tgttttaaat 360
cacgttacga aagaatattg gctgtataga gatttatatg gttcttctta ccaaccgtgg
420 ttaatgtaca atgaaaaaag ggaagctcca gaaaaattaa gaaatttatt
gaataatgat 480 ataatagtaa aaattgaggg ggaatttaaa cattgcaatc
attcgatata tttaaatggt 540 tcaaaaatta taagaccaaa tatgaagtgc
cacaatggag ttgtgcatat agtagataag 600 cccatcattt tt 612 7 206 PRT
Plasmodium vivax 7 Met Lys Lys Ser Arg Pro Pro Phe Leu Val Ile Lys
Arg Leu Tyr Thr 1 5 10 15 Arg Ser Gly Gly Leu Arg Lys Pro Gln Lys
Val Thr Asn Asp Pro Glu 20 25 30 Ser Ile Asn Arg Lys Thr Tyr Trp
Cys Phe Glu His Lys Pro Ile Lys 35 40 45 Arg Thr Leu Val Asn Leu
Ile Tyr Ser His Asn Glu Leu Lys Leu Phe 50 55 60 Ser Arg Phe Leu
Asn His Pro Asn Val Gly Thr Ser Leu Val His Glu 65 70 75 80 Leu Ser
Leu Glu Gly Pro Tyr Thr Gly Phe Leu Pro Ser Asn Glu Ala 85 90 95
Leu Lys Leu Ile Ser Pro Glu Ser Leu Ala Lys Leu Tyr Glu Glu Gly 100
105 110 Asp Lys Leu Met Glu Phe Val Leu Gly His Phe Ala Lys Asp Phe
Trp 115 120 125 Leu Tyr Arg Asp Leu Tyr Gly Ser Ser Tyr Gln Pro Trp
Leu Val Phe 130 135 140 Asn Glu Arg Arg Asp Ala Pro Glu Lys Ile Thr
Asn Leu Val Asn Arg 145 150 155 160 Asp Leu Leu Val Glu Ile Thr Gly
Glu Phe Lys Asn Cys Asp His Ser 165 170 175 Ile Ser Leu Asn Gly Ala
Lys Ile Ile Arg Pro Asn Met Lys Cys His 180 185 190 Asn Gly Val Val
His Ile Val Asp Arg Pro Ile Ile Gln Arg 195 200 205 8 618 DNA
Plasmodium vivax 8 atgaaaaaga gccgcccacc cttccttgtc attaaaaggc
tatacacacg cagtggcgga 60 ttgaggaaac cgcaaaaagt gacgaacgat
cccgaaagca ttaatcgaaa aacgtactgg 120 tgctttgaac acaaacctat
taagaggacg ttggtcaatt tgatatactc tcataatgaa 180 ttgaaattat
tctcccgttt tcttaatcac cccaatgtgg gtacctccct tgtacacgag 240
ctttccttgg aaggccccta cacggggttc ctgccttcga acgaggctct gaaattgatt
300 agccccgaga gtttagccaa attgtatgaa gaaggagaca agttgatgga
attcgttttg 360 ggccacttcg cgaaggactt ctggctctac agggacctgt
acgggtcgtc ctaccagccc 420 tggctcgtgt tcaacgagag gagggacgcc
cctgagaaaa tcaccaactt agttaacaga 480 gacctacttg tagagataac
aggagagttt aaaaattgcg accactcgat ttccctgaat 540 ggagcgaaga
tcatcagacc gaacatgaag tgccacaacg gagtggtgca cattgtagac 600
aggccgataa tacagagg 618 9 204 PRT Plasmodium yoelii 9 Met Lys Lys
Lys Leu Tyr Asn Leu Val Leu Lys Arg Ser Tyr Thr Arg 1 5 10 15 Ser
Gly Gly Leu Arg Lys Pro Gln Lys Val Thr Asn Asp Pro Glu Ser 20 25
30 Ile Asn Arg Lys Val Tyr Trp Cys Phe Glu His Lys Pro Val Arg Arg
35 40 45 Thr Val Ile Asn Leu Ile Phe Ser His Asn Glu Leu Lys Asn
Phe Ser 50 55 60 Thr Leu Leu Arg Asn Thr Asn Ala Ser Ser Ser Leu
Ile His Glu Leu 65 70 75 80 Ser Leu Glu Gly Pro Tyr Thr Gly Phe Leu
Pro Ser Asp Glu Ala Leu 85 90 95 Asn Leu Leu Ser Thr Asn Ser Leu
Asn Lys Leu Tyr Lys Asp Asp Asn 100 105 110 Lys Met Ser Glu Phe Val
Leu Asn His Phe Thr Lys Gly Leu Trp Met 115 120 125 Tyr Arg Asp Leu
Tyr Gly Ser Ser Tyr Gln Pro Trp Leu Met Tyr Asn 130 135 140 Glu Lys
Arg Glu Ala Pro Glu Lys Ile Gln Thr Leu Val Asn Asn Asp 145 150 155
160 Ile Ile Val Lys Ile Glu Gly Glu Phe Lys Asn Cys Asp His Ser Ile
165 170 175 Tyr Leu Asn Glu Ala Lys Ile Ile Arg Pro Asn Met Lys Cys
His Asn 180 185 190 Gly Ile Ile His Ile Ile Asp Lys Pro Ile Ile Phe
195 200 10 612 DNA Plasmodium yoelii 10 atgaaaaaaa aattgtataa
tttagttctt aaaagaagtt acacacgtag tggcggttta 60 agaaaaccac
aaaaagtaac aaatgatcca gaaagtatta atagaaaggt ttattggtgt 120
tttgaacata aacctgttag gaggactgta attaatttaa tattttccca taatgaatta
180 aaaaactttt caactctttt aagaaataca aatgctagct catcgctaat
tcacgagctg 240 tcattggaag ggccttatac gggatttctt ccatcagacg
aagccttaaa tttattgagt 300 acaaatagtt taaataaatt atataaagat
gataataaaa tgtctgagtt tgttttaaat 360 cattttacta aaggtctgtg
gatgtataga gatttatatg gctcatccta tcagccatgg 420 ctaatgtata
atgaaaaaag agaggcccca gaaaaaatac aaactttagt aaataacgac 480
ataattgtaa aaatagaagg ggaatttaaa aattgtgatc attctatata tttaaatgaa
540 gcaaaaatta taagacccaa tatgaaatgt cataatggca taattcatat
catagataag 600 ccaataattt tt 612 11 206 PRT Plasmodium knowlesi 11
Met Lys Lys Ser His Pro Pro Phe Leu Ile Ile Lys Arg Leu Tyr Thr 1 5
10 15 Arg Ser Gly Gly Leu Arg Lys Pro Gln Lys Val Thr Asn Asp Pro
Glu 20 25 30 Ser Ile Asn Arg Lys Thr Tyr Trp Cys Phe Glu His Lys
Pro Ile Lys 35 40 45 Arg Thr Met Val Asn Leu Ile Tyr Ser His Asn
Glu Leu Lys Leu Phe 50 55 60 Ser Arg Phe Leu Ser His Pro Asn Val
Gly Thr Ser Leu Ile His Glu 65 70 75 80 Leu Ser Leu Glu Gly Pro Tyr
Thr Gly Phe Leu Pro Ser Asn Glu Ala 85 90 95 Leu Lys Leu Ile Ser
Pro Glu Ser Leu Ala Lys Leu Tyr Glu Gln Arg 100 105 110 Asp Lys Leu
Met Glu Phe Val Leu Gly His Phe Thr Lys Asp Phe Trp 115 120 125 Leu
Tyr Arg Asp Leu Tyr Arg Ser Ser Tyr His Pro Trp Leu Val Phe 130 135
140 Asn Glu Lys Arg Glu Ala Pro Glu Lys Ile Thr Asn Leu Val Asn Lys
145 150 155 160 Asp Leu Leu Val Lys Ile Thr Gly Glu Phe Lys Asn Cys
Asp His Ser 165 170 175 Ile Phe Leu Asn Gly Ala Lys Ile Ile Thr Pro
Asn Met Lys Cys His 180 185 190 Asn Gly Val Val His Ile Val Asp Arg
Pro Ile Ile Gln Arg 195 200 205 12 618 DNA Plasmodium knowlesi 12
atgaaaaaga gccacccccc cttccttatc attaaaaggt tatacacacg cagtggagga
60 ttgaggaaac cacaaaaagt gacgaacgat cccgaaagca ttaacagaaa
aacatactgg 120 tgcttcgaac acaaacctat taaaaggacg atggtcaatt
tgatatactc ccacaatgaa 180 ctgaaattat tttcccgctt tctgagtcat
cccaatgtcg gtacctccct catacacgag 240 ctatccttgg aaggccccta
tacrgggttc ctgccttcga acgaagctct gaaattaatt 300 agccccgaaa
gcttagccaa attatatgaa caaagagata aattgatgga atttgttttg 360
gggcacttta cgaaagactt ctggctctac agagatctct acagatcttc ctaccatccc
420 tggctcgtat ttaacgagaa aagggaagcc cctgagaaaa tcaccaactt
agttaacaaa 480 gacctacttg taaaaataac aggagagttt aaaaattgcg
atcactccat tttccttaat 540 ggagcgaaga tcatcacacc aaatatgaag
tgccacaacg gagtggtcca tattgtagac 600 aggccgatta tacagagg 618 13 204
PRT Plasmodium chabaudi 13 Met Lys Lys Lys Leu Tyr Asn Leu Val Leu
Lys Arg Asn Tyr Thr Arg 1 5 10 15 Cys Gly Gly Leu Arg Arg Pro Gln
Lys Val Thr Asn Asp Pro Glu Ser 20 25 30 Ile Asn Arg Lys Val Tyr
Trp Cys Phe Glu His Lys Pro Val Arg Arg 35 40 45 Thr Val Ile Asn
Leu Ile Phe Ser His Asn Glu Leu Lys Asn Phe Ser 50 55 60 Thr Leu
Leu Arg Asn Thr Asn Ala Ser Ser Ser Leu Ile His Glu Leu 65 70 75 80
Ser Leu Glu Gly Pro Tyr Thr Gly Phe Leu Pro Ser Asp Glu Ala Leu 85
90 95 Asn Leu Leu Ser Ala Asn Ser Leu Asn Lys Leu Tyr Asn Asp Asp
Asn 100 105 110 Lys Met Ser Glu Phe Val Leu Asn His Phe Thr Lys Gly
Leu Trp Met 115 120 125 Tyr Arg Asp Leu Tyr Gly Ser Ser Tyr Gln Pro
Trp Leu Met Tyr Asn 130 135 140 Glu Lys Arg Asp Ala Pro Glu Lys Leu
Thr Thr Leu Ile Asn Asn Asp 145 150 155 160 Ile Ile Val Lys Ile Glu
Gly Glu Phe Lys Asn Cys Asp His Ser Ile 165 170 175 Tyr Leu Asn Glu
Ala Lys Ile Ile Arg Pro Asn Met Lys Cys His Asn 180 185 190 Gly Ile
Ile His Ile Ile Asp Lys Pro Ile Ile Phe 195 200 14 612 DNA
Plasmodium chabaudi 14 atgaaaaaaa aattgtataa tttagttctt aaaagaaatt
acacacgctg tggcggttta 60 agaagaccac aaaaagtaac aaatgatcca
gagagtatta atagaaaggt ttattggtgt 120 tttgaacata aacctgttag
gaggactgta attaatttaa tattttccca taatgaatta 180 aaaaactttt
caactctttt aaggaataca aatgctagct catcgctaat tcacgaactg 240
tcattggaag gaccttatac gggatttctt ccttcagacg aggccttaaa tttattgagt
300 gcaaatagct taaataaatt atataatgat gataataaaa tgtctgaatt
cgttttaaat 360 cattttacta aaggtctgtg gatgtacaga gatttatatg
gctcatccta tcagccatgg 420 ctcatgtaca atgaaaaaag agacgcccca
gaaaaattaa caactttaat aaacaacgac 480 ataattgtaa aaatagaagg
agaatttaaa aattgtgatc attccatata tttaaatgaa 540 gcaaaaatta
taaggcccaa tatgaaatgc cacaatggca taattcatat catagataag 600
ccaatcattt tt 612 15 204 PRT Plasmodium berghei 15 Met Lys Lys Lys
Leu Tyr Asn Leu Val Leu Lys Arg Asn Tyr Thr Arg 1 5 10 15 Ser Gly
Gly Leu Arg Lys Pro Gln Lys Val Thr Asn Asp Pro Glu Ser 20 25 30
Ile Asn Arg Lys Val Tyr Trp Cys Phe Glu His Lys Pro Val Arg Arg 35
40 45 Thr Val Ile Asn Leu Ile Phe Ser His Asn Glu Leu Lys Asn Phe
Ser 50 55 60 Thr Leu Leu Lys Asn Thr Asn Ala Ser Ser Ser Leu Ile
His Glu Leu 65 70 75 80 Ser Leu Glu Gly Pro Tyr Thr Gly Phe Leu Pro
Ser Asp Glu Ala Leu 85 90 95 Asn Leu Leu Ser Thr Asn Ser Leu Asn
Lys Leu Tyr Lys Asp Asp Asn 100 105 110 Lys Met Ser Glu Phe Val Leu
Asn His Phe Thr Lys Gly Leu Trp Met 115 120 125 Tyr Arg Asp Leu Tyr
Gly Ser Ser Tyr Gln Pro Trp Leu Met Tyr Asn 130 135 140 Glu Lys Arg
Glu Ala Pro Glu Lys Ile Pro Thr Leu Val Asn Asn Asp 145 150 155 160
Ile Ile Val Lys Ile Glu Gly Glu Phe Lys Asn Cys Asp His Ser Ile 165
170 175 Tyr Leu Asn Glu Ala Lys Ile Ile Arg Pro Asn Met Lys Cys His
Asn 180 185 190
Gly Ile Ile His Ile Ile Asp Lys Pro Ile Ile Phe 195 200 16 612 DNA
Plasmodium berghei 16 atgaaaaaaa aattgtataa tttagttctt aaaagaaatt
acacgcgtag tggcggttta 60 agaaaaccac aaaaagtaac aaatgatcca
gaaagtatta atagaaaggt ttattggtgt 120 tttgagcata aacctgttag
gaggactgta attaatttaa tattttccca taatgaatta 180 aaaaactttt
caactctttt aaaaaataca aatgctagct catcgctaat tcacgaacta 240
tcattggaag ggccttatac gggatttctt ccttcggatg aggccttaaa tttattgagt
300 acaaatagtt taaataaatt atataaagat gataataaaa tgtctgaatt
tgttttaaat 360 cattttacta aaggtctgtg gatgtataga gatttatatg
gctcatccta tcagccatgg 420 ctcatgtaca atgaaaaaag agaggcccca
gaaaaaatac caactttagt aaacaacgac 480 ataattgtaa aaatagaagg
ggaatttaaa aattgtgatc attctatata tttaaatgaa 540 gcaaaaatta
taagacccaa tatgaaatgt cataatggca taattcatat catagataag 600
ccaataattt tt 612 17 199 PRT Theileria parva 17 Met Phe Ile Ser Gln
Ala Leu Leu Trp Arg Ser Asn Phe Gly Gly Leu 1 5 10 15 Lys Lys Leu
Arg Arg Val Thr Lys Asp Pro Asn Val Ile Asn Ser Lys 20 25 30 Val
Tyr Trp Cys Phe Glu His Lys Tyr Ile Arg Arg Thr Val Leu Ser 35 40
45 Phe Cys Asn Asn Asn Pro Phe Thr Arg Ser Phe Ser Ser Leu Ile Asn
50 55 60 Pro Glu Glu Glu Ser Gly Tyr Arg Leu Ser His Glu Leu Ser
Leu Pro 65 70 75 80 Gly Pro Phe Thr Gly Phe Ile Pro Val Asn Glu Gly
Leu Thr Gln Ala 85 90 95 Leu Ser Lys Leu Glu Ala Ser Tyr Lys Asp
Ser Val Val Asp Phe Val 100 105 110 Arg Ser His Phe Thr His Asn Leu
Trp Leu Tyr Arg Asp Ile Leu Gly 115 120 125 Ser Pro Thr Gln Pro Trp
Leu Leu Tyr Asn Lys Thr Arg Lys Phe Pro 130 135 140 Glu Lys Leu Gln
Thr Ile Asn Asn Lys Ser Leu Phe Phe Glu His Thr 145 150 155 160 Gly
Asp Leu Ser Lys Gly Asp Lys Glu Ile Phe Val Asn Gly Ser Lys 165 170
175 Ile Leu Arg Trp Asn Leu Arg Cys His Asn Gly Val Ile His Leu Ile
180 185 190 Asp Lys Pro Leu Phe Asp Ile 195 18 600 DNA Theileria
parva 18 atgtttatct ctcaggccct gttgtggaga tctaattttg gaggcttgaa
aaagttgaga 60 agagtaacaa aggacccgaa cgtcataaat tcaaaggttt
actggtgttt tgaacataaa 120 tatattcgcc gtactgttct ttcattctgt
aataacaacc cctttacgcg ttctttttca 180 agtttaataa atcctgagga
ggaatctggc tataggttat ctcacgagtt atcacttcca 240 gggcctttta
caggctttat tccagtaaat gagggcttaa ctcaggcttt atcaaagcta 300
gaggcttcat acaaggattc tgtcgttgat ttcgtgaggt cccattttac acataactta
360 tggctatatc gtgacatact aggttctcca acccagccct ggttattgta
caataaaact 420 cgaaaatttc cagaaaaact tcaaaccatt aataacaaat
ctttgttctt cgaacacact 480 ggagacttgt caaagggtga taaggaaatc
tttgtaaacg gttcaaagat acttcgctgg 540 aacctgagat gtcataatgg
agttattcac ctgatagata aacctctttt cgatatctaa 600 19 219 PRT
Theileria annulata 19 Met Phe Leu Thr Cys Tyr Phe His Phe Met Met
Phe Thr Ser Lys Ala 1 5 10 15 Leu Ser Trp Arg Ser Asn Phe Gly Gly
Leu Lys Lys Leu Arg Arg Arg 20 25 30 Ser Lys Asp Pro Asn Val Ile
Asn Ser Lys Val Tyr Trp Cys Phe Glu 35 40 45 His Lys Tyr Ile Arg
Arg Thr Val Leu Ser Phe Cys Asn Asn Asn Pro 50 55 60 Phe Thr Arg
Ser Phe Ser Lys Leu Ile Asn Pro Glu Glu Glu Ser Gly 65 70 75 80 Ile
Phe Tyr Phe Leu Ser His Val Leu Gly Tyr Arg Leu Ser His Glu 85 90
95 Leu Ser Leu Pro Gly Pro Phe Thr Gly Phe Ile Pro Val Asn Glu Gly
100 105 110 Leu Thr Gln Ala Leu Pro Lys Leu Glu Ser Ser Tyr Lys Asp
Ala Val 115 120 125 Val Asp Phe Val Arg Ser His Phe Thr His His Leu
Trp Leu His Arg 130 135 140 Asp Leu Leu Gly Ser Pro Thr Gln Pro Trp
Leu Leu Tyr Asn Lys Thr 145 150 155 160 Arg Lys Phe Pro Lys Lys Leu
Gln Thr Leu Asn Asn Lys Ser Leu Phe 165 170 175 Phe Glu His Thr Gly
Asp Leu Ser Lys Gly Asp Lys Glu Ile Phe Val 180 185 190 Asn Gly Ser
Arg Ile Leu Arg Trp Asn Met Arg Cys His Asn Gly Val 195 200 205 Ile
His Leu Ile Asp Lys Pro Leu Phe Asp Ile 210 215 20 660 DNA
Theileria annulata 20 atgtttttaa cttgttattt tcattttatg atgtttactt
ccaaggcctt gtcgtggaga 60 tctaattttg gagggttaaa gaagttaagg
agaagatcaa aggatccaaa cgtcataaat 120 tcaaaggttt attggtgttt
tgagcataaa tatattcgcc gtacagttct ttcattttgt 180 aataataatc
catttacacg ttcattttca aagttaataa atcccgagga agaatcaggt 240
attttttatt ttttaagcca tgttttaggt tatagattat ctcacgagtt atcacttccc
300 gggcctttta cgggcttcat tccagtaaat gaaggcttaa cacaggcctt
accgaagctg 360 gagtcctcat acaaggatgc ggtagttgat ttcgtaaggt
ctcactttac ccatcattta 420 tggctacatc gtgatctgct aggctcacca
acacagccct ggctactgta taacaaaact 480 cgcaaatttc caaaaaaact
acaaaccctt aataacaaat ctttgttctt cgaacacaca 540 ggagatctgt
caaagggtga taaggaaatc tttgtgaatg gatcaaggat acttcgctgg 600
aacatgagat gtcataatgg agttattcac ctgatagata aacccctctt tgatatttag
660 21 87 PRT Artificial consensus sequence 21 Thr Val Phe Ala Pro
Thr Asp Glu Ala Phe Lys Lys Leu Pro Pro Gly 1 5 10 15 Thr Leu Asn
Ser Leu Leu Ala Asp Pro Lys Leu Lys Gln Leu Leu Lys 20 25 30 Tyr
His Ile Val Pro Gly Arg Leu Ser Ser Ala Asp Leu Leu Asn Gly 35 40
45 Gly Thr Leu Pro Thr Leu Ala Gly Ser Lys Leu Arg Val Asn Val Ser
50 55 60 Gly Asn Ser Gly Thr Val Thr Val Asn Gly Ala Arg Ile Val
Glu Ala 65 70 75 80 Asp Ile Ala Ala Thr Asn Gly 85 22 31 DNA
Artificial synthetic oligomucleotide forward primer 22 caccatgaaa
aatagatttt attataattt g 31 23 27 DNA Artificial synthetic
oligonucleotide reverse primer 23 aaaaatgatg ggcttatcta ctatatg 27
24 32 PRT Plasmodium falciparum 24 Thr Arg Ser Gly Gly Leu Arg Lys
Pro Gln Lys Val Thr Asn Asp Pro 1 5 10 15 Glu Ser Ile Asn Arg Lys
Val Tyr Trp Cys Phe Glu His Lys Pro Val 20 25 30 25 87 PRT
Plasmodium falciparum 25 Met Lys Asn Arg Phe Tyr Tyr Asn Leu Ile
Ile Lys Arg Leu Tyr Thr 1 5 10 15 Arg Ser Gly Gly Leu Arg Lys Pro
Gln Lys Val Thr Asn Asp Pro Glu 20 25 30 Ser Ile Asn Arg Lys Val
Tyr Trp Cys Phe Glu His Lys Pro Val Lys 35 40 45 Arg Thr Ile Ile
Asn Leu Ile Tyr Ser His Asn Glu Leu Lys Ile Phe 50 55 60 Ser Asn
Leu Leu Asn His Pro Thr Val Gly Ser Ser Leu Ile His Glu 65 70 75 80
Leu Ser Leu Asp Gly Pro Tyr 85 26 261 DNA Plasmodium falciparum 26
atgaaaaata gattttatta taatttgata attaaaagat tatatacacg aagtggcggt
60 ttaagaaaac ctcaaaaggt aaccaacgac ccagaaagta taaatagaaa
agtatattgg 120 tgttttgaac ataagcctgt aaaaaggaca attattaatt
taatatattc acataacgaa 180 ctcaagatat tttctaatct gttaaatcat
cctacagttg gcagctcgtt aatacatgaa 240 ttatctctcg atggccctta t 261 27
20 PRT Plasmodium falciparum 27 Met Lys Asn Arg Phe Tyr Tyr Asn Leu
Ile Ile Lys Arg Leu Tyr Thr 1 5 10 15 Arg Ser Gly Gly 20 28 20 PRT
Plasmodium falciparum 28 Asn Leu Ile Ile Lys Arg Leu Tyr Thr Arg
Ser Gly Gly Leu Arg Lys 1 5 10 15 Pro Gln Lys Val 20 29 20 PRT
Plasmodium falciparum 29 Thr Arg Ser Gly Gly Leu Arg Lys Pro Gln
Lys Val Thr Asn Asp Pro 1 5 10 15 Glu Ser Ile Asn 20 30 20 PRT
Plasmodium falciparum 30 Gly Leu Arg Lys Pro Gln Lys Val Thr Asn
Asp Pro Glu Ser Ile Asn 1 5 10 15 Arg Lys Val Tyr 20 31 20 PRT
Plasmodium falciparum 31 Thr Asn Asp Pro Glu Ser Ile Asn Arg Lys
Val Tyr Trp Cys Phe Glu 1 5 10 15 His Lys Pro Val 20 32 20 PRT
Plasmodium falciparum 32 Val Tyr Trp Cys Phe Glu His Lys Pro Val
Lys Arg Thr Ile Ile Asn 1 5 10 15 Leu Ile Tyr Ser 20 33 20 PRT
Plasmodium falciparum 33 Lys Pro Val Lys Arg Thr Ile Ile Asn Leu
Ile Tyr Ser His Asn Glu 1 5 10 15 Leu Lys Ile Phe 20 34 20 PRT
Plasmodium falciparum 34 Asn Leu Ile Tyr Ser His Asn Glu Leu Lys
Ile Phe Ser Asn Leu Leu 1 5 10 15 Asn His Pro Thr 20 35 20 PRT
Plasmodium falciparum 35 Asn Glu Leu Lys Ile Phe Ser Asn Leu Leu
Asn His Pro Thr Val Gly 1 5 10 15 Ser Ser Leu Ile 20 36 20 PRT
Plasmodium falciparum 36 Asn Leu Leu Asn His Pro Thr Val Gly Ser
Ser Leu Ile His Glu Leu 1 5 10 15 Ser Leu Asp Gly 20 37 8 PRT
Plasmodium falciparum 37 Thr Asn Asp Pro Glu Ser Ile Asn 1 5 38 24
DNA Plasmodium falciparum 38 accaacgacc cagaaagtat aaat 24 39 96
DNA Plasmodium falciparum 39 acacgaagtg gcggtttaag aaaacctcaa
aaggtaacca acgacccaga aagtataaat 60 agaaaagtat attggtgttt
tgaacataag cctgta 96 40 39 DNA Artificial synthetic oligonucleotide
primer 40 ggaattcagg agcccttcgg atccaaaaaa aaattgtat 39 41 45 DNA
Artificial synthetic oligonucleotide primer 41 cttcgaattg
agctcggatc ctcaaattat tggcttatct atgat 45 42 204 PRT Plasmodium
gallinaceum 42 Met Lys Asn Ser Gly Tyr Asn Leu Ile Ile Lys Arg Leu
Tyr Thr Arg 1 5 10 15 Ser Gly Gly Leu Arg Lys Pro Gln Lys Val Thr
Asn Asp Pro Glu Ser 20 25 30 Ile Asn Arg Lys Val Tyr Trp Cys Phe
Glu His Lys Pro Ile Lys Arg 35 40 45 Thr Ile Val Asn Leu Ile Phe
Ser His Lys Glu Leu Lys Phe Phe Ser 50 55 60 Asn Phe Leu Asn His
Pro Asn Val Gly Val Ser Leu Ile His Glu Leu 65 70 75 80 Ser Leu Glu
Gly Pro Phe Thr Gly Phe Leu Pro Ser Asn Glu Ala Leu 85 90 95 Lys
Leu Ile Asn Ser Glu Cys Leu Asn Lys Leu Tyr Lys Asp Asp Asn 100 105
110 Lys Leu Ser Glu Phe Val Leu Asn His Phe Thr Lys Asp Phe Trp Leu
115 120 125 Tyr Arg Asp Leu Tyr Gly Ser Ser Tyr Gln Pro Trp Leu Ile
Tyr Asn 130 135 140 Glu Lys Arg Glu Ala Pro Glu Lys Ile Thr Asn Leu
Met Asn Asn Asp 145 150 155 160 Leu Ile Val Lys Ile Lys Gly Glu Phe
Lys Asn Cys Asp His Ser Ile 165 170 175 Tyr Leu Asn Glu Ser Lys Ile
Ile Arg Pro Asn Met Lys Cys His Asn 180 185 190 Gly Val Val His Ile
Val Asp Lys Pro Ile Ile Phe 195 200 43 204 PRT Plasmodium
reichenowi 43 Met Lys Ile Lys Phe Tyr Asn Leu Ile Ser Lys Arg Leu
Tyr Thr Arg 1 5 10 15 Ser Gly Gly Leu Arg Lys Pro Gln Lys Val Thr
Asn Asp Pro Glu Ser 20 25 30 Ile Asn Arg Lys Val Tyr Trp Cys Phe
Glu His Lys Pro Val Lys Arg 35 40 45 Thr Ile Ile Asn Leu Ile Tyr
Ser His Asn Glu Leu Lys Ile Phe Ser 50 55 60 Asn Leu Leu Asn His
Pro Ile Val Gly Ser Ser Leu Ile His Glu Leu 65 70 75 80 Ser Leu Asp
Gly Pro Tyr Thr Ala Phe Leu Pro Ser Asn Glu Ala Met 85 90 95 Lys
Leu Ile Asn Ile Glu Ser Phe Asn Lys Leu Tyr Asn Asp Glu Asn 100 105
110 Lys Leu Ser Glu Phe Val Leu Asn His Val Thr Lys Glu Tyr Trp Leu
115 120 125 Tyr Arg Asp Leu Tyr Gly Ser Ser Tyr Gln Pro Trp Leu Met
Tyr Asn 130 135 140 Glu Lys Arg Glu Ala Pro Glu Lys Leu Arg Asn Leu
Leu Asn Asn Asp 145 150 155 160 Ile Ile Val Lys Ile Glu Gly Glu Phe
Lys His Cys Asn His Ser Ile 165 170 175 Tyr Leu Asn Gly Ser Lys Ile
Ile Arg Pro Asn Met Lys Cys His Asn 180 185 190 Gly Val Val His Ile
Val Asp Lys Pro Ile Ile Phe 195 200 44 206 PRT Plasmodium vivax 44
Met Lys Lys Ser Arg Pro Pro Phe Leu Val Ile Lys Arg Leu Tyr Thr 1 5
10 15 Arg Ser Gly Gly Leu Arg Lys Pro Gln Lys Val Thr Asn Asp Pro
Glu 20 25 30 Ser Ile Asn Arg Lys Thr Tyr Trp Cys Phe Glu His Lys
Pro Ile Lys 35 40 45 Arg Thr Leu Val Asn Leu Ile Tyr Ser His Asn
Glu Leu Lys Leu Phe 50 55 60 Ser Arg Phe Leu Asn His Pro Asn Val
Gly Thr Ser Leu Val His Glu 65 70 75 80 Leu Ser Leu Glu Gly Pro Tyr
Thr Gly Phe Leu Pro Ser Asn Glu Ala 85 90 95 Leu Lys Leu Ile Ser
Pro Glu Ser Leu Ala Lys Leu Tyr Glu Glu Gly 100 105 110 Asp Lys Leu
Met Glu Phe Val Leu Gly His Phe Ala Lys Asp Phe Trp 115 120 125 Leu
Tyr Arg Asp Leu Tyr Gly Ser Ser Tyr Gln Pro Trp Leu Val Phe 130 135
140 Asn Glu Arg Arg Asp Ala Pro Glu Lys Ile Thr Asn Leu Val Asn Arg
145 150 155 160 Asp Leu Leu Val Glu Ile Thr Gly Glu Phe Lys Asn Cys
Asp His Ser 165 170 175 Ile Ser Leu Asn Gly Ala Lys Ile Ile Arg Pro
Asn Met Lys Cys His 180 185 190 Asn Gly Val Val His Ile Val Asp Lys
Pro Ile Ile Gln Arg 195 200 205 45 204 PRT Plasmodium yoelii 45 Met
Lys Lys Lys Leu Tyr Asn Leu Val Ile Lys Arg Ser Tyr Thr Arg 1 5 10
15 Ser Gly Gly Leu Arg Lys Pro Gln Lys Val Thr Asn Asp Pro Glu Ser
20 25 30 Ile Asn Arg Lys Val Tyr Trp Cys Phe Glu His Lys Pro Val
Arg Arg 35 40 45 Thr Val Ile Asn Leu Ile Phe Ser His Asn Glu Leu
Lys Asn Phe Ser 50 55 60 Thr Leu Leu Arg Asn Thr Asn Ala Ser Ser
Ser Leu Ile His Glu Leu 65 70 75 80 Ser Leu Glu Gly Pro Tyr Thr Gly
Phe Leu Pro Ser Asp Glu Ala Leu 85 90 95 Asn Leu Leu Ser Thr Asn
Ser Leu Asn Lys Leu Tyr Lys Asp Asp Asn 100 105 110 Lys Met Ser Glu
Phe Val Leu Asn His Phe Thr Lys Gly Leu Trp Met 115 120 125 Tyr Arg
Asp Leu Tyr Gly Ser Ser Tyr Gln Pro Trp Leu Met Tyr Asn 130 135 140
Glu Lys Arg Glu Ala Pro Glu Lys Ile Gln Thr Leu Val Asn Asn Asp 145
150 155 160 Ile Ile Val Lys Ile Glu Gly Glu Phe Lys Asn Cys Asp His
Ser Ile 165 170 175 Tyr Leu Asn Glu Ala Lys Ile Ile Arg Pro Asn Met
Lys Cys His Asn 180 185 190 Gly Ile Ile His Ile Ile Asp Lys Pro Ile
Ile Phe 195 200 46 206 PRT Plasmodium knowlesi 46 Met Lys Lys Ser
His Pro Pro Phe Leu Ile Ile Lys Arg Leu Tyr Thr 1 5 10 15 Arg Ser
Gly Gly Leu Arg Lys Pro Gln Lys Val Thr Asn Asp Pro Glu 20 25 30
Ser Ile Asn Arg Lys Thr Tyr Trp Cys Phe Glu His Lys Pro Ile Lys 35
40 45 Arg Thr Met Val Asn Leu Ile Tyr Ser His Asn Glu Leu Lys Leu
Phe 50 55 60 Ser Arg Phe Leu Ser His Pro Asn Val Gly Thr Ser Leu
Ile His Glu 65 70 75 80 Leu Ser Leu Glu Gly Pro Tyr Thr Gly Phe Leu
Pro Ser Asn Glu Ala 85 90 95 Leu Lys Leu Ile Ser Pro Glu Ser Leu
Ala Lys Leu Tyr Glu Gln Arg 100 105 110 Asp Lys Leu Met Glu Phe Val
Leu Gly His Phe Thr Lys Asp Phe Trp 115 120 125 Leu Tyr Arg Asp Leu
Tyr Arg Ser Ser Tyr His Pro Trp Leu Val Phe 130 135 140 Asn Glu Lys
Arg Glu Ala Pro Glu Lys Ile Thr Asn Leu Val Asn Lys 145 150 155 160
Asp Leu Leu Val Lys Ile Thr Gly Glu Phe Lys Asn Cys Asp His Ser 165
170 175 Ile Phe Leu Asn Gly Ala Lys Ile Ile Thr Pro Asn Met Lys Cys
His 180 185 190 Asn Gly Val
Val His Ile Val Asp Arg Pro Ile Ile Gln Arg 195 200 205 47 204 PRT
Plasmodium chabaudi 47 Met Lys Lys Lys Leu Tyr Asn Leu Val Leu Lys
Arg Asn Tyr Thr Arg 1 5 10 15 Cys Gly Gly Leu Arg Arg Pro Gln Lys
Val Thr Asn Asp Pro Glu Ser 20 25 30 Ile Asn Arg Lys Val Tyr Trp
Cys Phe Glu His Lys Pro Val Arg Arg 35 40 45 Thr Val Ile Asn Leu
Ile Phe Ser His Asn Glu Leu Lys Asn Phe Ser 50 55 60 Thr Leu Leu
Arg Asn Thr Asn Ala Ser Ser Ser Leu Ile His Glu Leu 65 70 75 80 Ser
Leu Glu Gly Pro Tyr Thr Gly Phe Leu Pro Ser Asp Glu Ala Leu 85 90
95 Asn Leu Leu Ser Ala Asn Ser Leu Asn Lys Leu Tyr Asn Asp Asp Asn
100 105 110 Lys Met Ser Glu Phe Val Leu Asn His Phe Thr Lys Gly Leu
Trp Met 115 120 125 Tyr Arg Asp Leu Tyr Gly Ser Ser Tyr Gln Pro Trp
Leu Met Tyr Asn 130 135 140 Glu Lys Arg Asp Ala Pro Glu Lys Leu Thr
Thr Leu Ile Asn Asn Asp 145 150 155 160 Ile Ile Val Lys Ile Glu Gly
Glu Phe Lys Asn Cys Asp His Ser Ile 165 170 175 Tyr Leu Asn Glu Ala
Lys Ile Ile Arg Pro Asn Met Lys Cys His Asn 180 185 190 Gly Ile Ile
His Ile Ile Asp Lys Pro Ile Ile Phe 195 200 48 204 PRT Plasmodium
berghei 48 Met Lys Lys Lys Leu Tyr Asn Leu Val Leu Lys Arg Asn Tyr
Thr Arg 1 5 10 15 Ser Gly Gly Leu Arg Lys Pro Gln Lys Val Thr Asn
Asp Pro Glu Ser 20 25 30 Ile Asn Arg Lys Val Tyr Trp Cys Phe Glu
His Lys Pro Val Arg Arg 35 40 45 Thr Val Ile Asn Leu Ile Phe Ser
His Asn Glu Leu Lys Asn Phe Ser 50 55 60 Thr Leu Leu Lys Asn Thr
Asn Ala Ser Ser Ser Leu Ile His Glu Leu 65 70 75 80 Ser Leu Glu Gly
Pro Tyr Thr Gly Phe Leu Pro Ser Asp Glu Ala Leu 85 90 95 Asn Leu
Leu Ser Thr Asn Ser Leu Asn Lys Leu Tyr Lys Asp Asp Asn 100 105 110
Lys Met Ser Glu Phe Val Leu Asn His Phe Thr Lys Gly Leu Trp Met 115
120 125 Tyr Arg Asp Leu Tyr Gly Ser Ser Tyr Gln Pro Trp Leu Met Tyr
Asn 130 135 140 Glu Lys Arg Glu Ala Pro Glu Lys Ile Pro Thr Leu Val
Asn Asn Asp 145 150 155 160 Ile Ile Val Lys Ile Glu Gly Glu Phe Lys
Asn Cys Asp His Ser Ile 165 170 175 Tyr Leu Asn Glu Ala Lys Ile Ile
Arg Pro Asn Met Lys Cys His Asn 180 185 190 Gly Ile Ile His Ile Ile
Asp Lys Pro Ile Ile Phe 195 200 49 200 PRT Theileria parva 49 Met
Phe Ile Ser Gln Ala Leu Leu Trp Arg Ser Asn Phe Gly Gly Leu 1 5 10
15 Lys Lys Leu Arg Arg Val Thr Lys Asp Pro Asn Val Ile Asn Ser Lys
20 25 30 Val Tyr Trp Cys Phe Glu His Lys Tyr Ile Arg Arg Thr Val
Leu Ser 35 40 45 Phe Cys Asn Asn Asn Pro Phe Thr Arg Ser Phe Ser
Ser Leu Ile Asn 50 55 60 Pro Glu Glu Glu Ser Gly Tyr Arg Leu Ser
His Glu Leu Ser Leu Pro 65 70 75 80 Gly Pro Phe Thr Gly Phe Ile Pro
Val Asn Glu Gly Leu Thr Gln Ala 85 90 95 Ser Leu Ser Lys Leu Glu
Ala Ser Tyr Lys Asp Ser Val Val Asp Phe 100 105 110 Val Arg Ser His
Phe Thr His Asn Leu Trp Leu Tyr Arg Asp Ile Leu 115 120 125 Gly Ser
Pro Thr Gln Pro Trp Leu Leu Tyr Asn Lys Thr Arg Lys Phe 130 135 140
Pro Glu Lys Leu Gln Thr Ile Asn Asn Lys Ser Leu Phe Phe Glu His 145
150 155 160 Thr Gly Asp Leu Ser Lys Gly Asp Lys Glu Ile Phe Val Asn
Gly Ser 165 170 175 Lys Ile Leu Arg Trp Asn Leu Arg Cys His Asn Gly
Val Ile His Leu 180 185 190 Ile Asp Lys Pro Leu Phe Asp Ile 195 200
50 219 PRT Theileria annulata 50 Asn Phe Leu Thr Cys Tyr Phe His
Phe Asn Met Phe Thr Ser Lys Ala 1 5 10 15 Leu Ser Trp Arg Ser Asn
Phe Gly Gly Leu Lys Lys Leu Arg Arg Arg 20 25 30 Ser Lys Asp Pro
Asn Val Ile Asn Ser Lys Val Tyr Trp Cys Phe Glu 35 40 45 His Lys
Tyr Ile Arg Arg Thr Val Leu Ser Phe Cys Asn Asn Asn Pro 50 55 60
Phe Thr Arg Ser Phe Ser Lys Leu Ile Asn Pro Glu Glu Glu Ser Gly 65
70 75 80 Ile Phe Tyr Phe Leu Ser His Val Leu Gly Tyr Arg Leu Ser
His Glu 85 90 95 Leu Ser Leu Pro Gly Pro Phe Thr Gly Phe Ile Pro
Val Asn Glu Gly 100 105 110 Leu Thr Gln Ala Leu Pro Lys Leu Glu Ser
Ser Tyr Lys Asp Ala Val 115 120 125 Val Asp Phe Val Arg Ser His Phe
Thr His His Leu Trp Leu His Arg 130 135 140 Asp Leu Leu Gly Ser Pro
Thr Gln Pro Trp Leu Leu Tyr Asn Lys Thr 145 150 155 160 Arg Lys Phe
Pro Lys Lys Leu Gln Thr Leu Asn Asn Lys Ser Leu Phe 165 170 175 Phe
Glu His Thr Gly Asp Leu Ser Lys Gly Asp Lys Glu Ile Phe Val 180 185
190 Asn Gly Ser Arg Ile Leu Arg Trp Asn Met Arg Cys His Asn Gly Val
195 200 205 Ile His Leu Ile Asp Lys Pro Leu Phe Asp Ile 210 215 51
97 PRT Artificial consensus sequence 51 Thr Val Phe Ala Pro Thr Asp
Glu Ala Phe Lys Lys Leu Pro Pro Gly 1 5 10 15 Thr Leu Asn Ser Leu
Leu Ala Asp Pro Lys Leu Lys Gln Leu Leu Lys 20 25 30 Tyr His Ile
Val Pro Gly Arg Leu Ser Ser Ala Asp Leu Leu Asn Gly 35 40 45 Gly
Thr Leu Pro Thr Leu Ala Gly Ser Lys Leu Arg Val Asn Val Ser 50 55
60 Gly Asn Ser Gly Thr Val Thr Val Asn Gly Ala Arg Ile Val Glu Ala
65 70 75 80 Asp Ile Ala Ala Thr Asn Gly Val Val His Val Ile Asp Arg
Val Leu 85 90 95 Leu 52 261 DNA Artificial nucleic acid encoding
teh FRAP2 derivative of FRAP 52 atgaaaaata gattttatta taatttgata
attaaaagat tatatacacg aagtggcggt 60 ttaagaaaac ctcaaaaggt
aaccaacgac ccagaaagta taaatagaaa agtatattgg 120 tgttttgaac
ataagcctgt aaaaaggaca attattaatt taatatattc acataacgaa 180
ctcaagatat tttctaatct gttaaatcat cctacagttg gcagctcgtt aatacatgaa
240 ttatctctcg atggccctta t 261 53 87 PRT Artificial amino acid
sequence of FRAP2 derivative of FRAP 53 Met Lys Asn Arg Phe Tyr Tyr
Asn Leu Ile Ile Lys Arg Leu Tyr Thr 1 5 10 15 Arg Ser Gly Gly Leu
Arg Lys Pro Gln Lys Val Thr Asn Asp Pro Glu 20 25 30 Ser Ile Asn
Arg Lys Val Tyr Trp Cys Phe Glu His Lys Pro Val Lys 35 40 45 Arg
Thr Ile Ile Asn Leu Ile Tyr Ser His Asn Glu Leu Lys Ile Phe 50 55
60 Ser Asn Leu Leu Asn His Pro Thr Val Gly Ser Ser Leu Ile His Glu
65 70 75 80 Leu Ser Leu Asp Gly Pro Tyr 85
* * * * *
References