Compounds and methods for the treatment of ubiquitin conjugating disorders

Abstract

The present invention provides methods for identifying compounds that selectively bind one or more active sites within an ubiquitin conjugating enzyme. The compounds identified by the methods are useful in the treatment of disorders attributed to dysregulated ubiquitin conjugating enzyme function, specifically in hyperproliferative disorders.

1 . A method of inhibiting an ubiquitin conjugating enzyme with an organic compound comprising, selectively targeting said organic compound to one or more active sites of said ubiquitin conjugating enzyme.  
     
     
         2 . The method of  claim 1 , wherein said active site is within a catalytic site.  
     
     
         3 . The method of  claim 2 , wherein said active site comprises the amino acid residues corresponding to Lys64, Pro66, Lys67, Ile68, Asn84, Ile85, Leu90, Lys91 and Leu120 of SEQ ID NO:1.  
     
     
         4 . The method of  claim 1 , wherein said organic compound interacts with a binding domain comprising the amino acids corresponding to Pro66, Ile85 and Leu90 of SEQ ID NO:1.  
     
     
         5 . The method of  claim 2 , wherein said active site comprises the amino acid residues corresponding to Pro65, Pro66, Lys67, Ile68, Asn84, Leu90, Lys91 and Leu120 of SEQ ID NO:1.  
     
     
         6 . The method of  claim 1 , wherein said organic compound interacts with a binding domain comprising the amino acids corresponding to Pro66, Ile68, Leu90 and Lys91 of SEQ ID NO:1.  
     
     
         7 . The method of  claim 2 , wherein said active site comprises the amino acid residues corresponding to Lys66, Ile67, Ala68, Ser83, Cys85, Leu86, Leu89 and Arg90 of SEQ ID NO:2.  
     
     
         8 . The method of  claim 1 , wherein said organic compound interacts with a binding domain comprising the amino acids corresponding to Ile67 and Leu89 of SEQ ID NO:2.  
     
     
         9 . The method of  claim 2 , wherein said active site comprises the amino acid residues corresponding to Pro64, Pro65, Lys66, Ile67, Ser83, Ile84, Cys85, Leu86, Leu89, Arg90 and Leu119 of SEQ ID NO:2.  
     
     
         10 . The method of  claim 1 , wherein said organic compound interacts with a binding domain comprising the amino acids corresponding to Pro65, Ile67, and Ser 83 of SEQ ID NO:2.  
     
     
         11 . The method of  claim 1 , wherein said organic compound binds to a binding domain in a E1 binding site.  
     
     
         12 . The method of  claim 1 , wherein the ubiquitin conjugating enzyme is selected from the group consisting of: Ubc1, Ubc2, Ubc3, Ubc4, Ubc5, Ubc6, Ubc7, Ubc8, Ubc10, Ubc13.  
     
     
         13 . The method of  claim 12 , wherein said ubiquitin conjugating enzyme is a yeast ubiquitin conjugating enzyme or a mouse, rat, or human homolog.  
     
     
         14 . The method of  claim 2 , wherein said organic compound is a compound of formula (I):  
         Ar—B—NR 1 R 2   (I)  wherein:    Ar is a five or six membered unsubstituted or substituted aromatic ring that is optionally fused to an aromatic or heteroaromatic ring;    B is a bond, CO, SO 2  or (CH 2 ) n  wherein n=1-5; and    R 1  and R 2  are each independently H, alkyl or aryl groups that are optionally substituted, wherein said compound selectively binds to one or more catalytic domains in the ubiquitin conjugating enzyme.    
     
     
         15 . The formula I compound of  claim 14 , wherein Ar is phenyl, pyridyl, napthyl, triazine, triazole, quinoxaline, dibenzofuran, benzimidazole, indene, indeno oxadiazine, indazole or an indole ring; B is a bond, CO or (CH 2 ) n  wherein n=1; and R 1  and R 2  are both H.  
     
     
         16 . The formula I compound of  claim 14 , which is 3-amino-1,2,4-Triazine, 3-amino-1,2,4-triazole, 2-methyl-4-nitroaniline, 2-iodo-4-nitroaniline, 4-amino-3-chloro-5-methylbenzoic acid, 1-(4′Aminophenyl)-1,2,4-triazole, 2-acetamidophenol, 5-chloro-2,3-dihydroxypyridine, 2-methyl-3-(1H-pyrazol-5-yl)imidazo(1,2-a)pyridine, 5-nitro-2,3-dihydro-1H-benzo(d)imidazol-2-one, 4-(methylamino)pyridine, 2-Chloro-4-nitrobenzamide, 2-ethylformanilide, 6-aminoindazole, 2,3-diaminobenzoic acid, 1-(5-chloro-2-methylphenyl)-2-thiourea, 4,5-diiodo-1H-imidazole, 1H-indene-1,3(2H)-dione 1-methylhydrazone, 3-hydroxyindole, 3,4-dihydro-1H-quinoxalin-2-one, 1S,6S,7R,8R,8aR)-1,6,7,8-Tetrahydroxyoctahydroindolizidine, 2-naphthalen-1-yl-2,3-dihydro-1H-pyrimidine, benzo(b)thiophen-3-ylmethylamine, 1-allyl-2-4-dioxo-1-2-3-4-tetrahydro-5-pyrimidinecarbonitrile, Methyl (S)-N-(7-chloro-2,3,4a,5-tetrahydro-4a-(methoxycarbonyl)indeno(1,2-e)(1,3,4)oxadiazin-2-ylcarbonyl)-4′-(trifluoromethoxy)carbanilate (Indoxacarb-MP), 4-acetylpyridine and thioisonicotinamide.  
     
     
         17 . The formula I compound of  claim 14 , which is 4-(Aminomethyl)piperidine.  
     
     
         18 . The formula I compound of  claim 14 , whose molecular weight is less than about 2000 daltons.  
     
     
         19 . The formula I compound of  claim 14 , whose molecular weight is less than about 500 daltons.  
     
     
         20 . The method of  claim 2 , wherein said organic compound is a compound of formula (II):  
         A-(B—NR 1 R 2 ) n   (II)  wherein:    A is a 3-6 membered substituted or unsubstituted cycloaliphatic or a heterocycloaliphatic ring, each of which is optionally fused to an aromatic ring;    B is a bond, CO, SO 2  or (CH 2 ) n  wherein n=1-3; and    R 1  and R 2  are each independently H, alkyl or aryl groups that are optionally substituted, wherein said compound selectively binds to one or more catalytic domains in the ubiquitin conjugating enzyme.    
     
     
         21 . The formula II compound of  claim 20 , wherein A is a heterocycloaliphatic ring comprising, at least one nitrogen atom and optionally, one or more additional heteroatoms selected from the group consisting of: nitrogen (N), oxygen (O) and sulfur (S); B is a bond, CO or (CH 2 ) n  wherein n=1; and R 1  and R 2  are both H.  
     
     
         22 . The formula II compound of  claim 20 , wherein the cycloaliphatic or heterocycloaliphatic ring comprise one or more substituents selected from the group consisting of hydroxyl, halogen, CO and alkyl.  
     
     
         23 . The formula II compound of  claim 20 , which is 1-phenyl-4-methyl-3-pyrazolidone, 4-(aminomethyl)piperidine, N-Phenyl-p-phenylenediamine, 5-(aminomethyl)-3-(2H)-isoxazolone (Muscimol), (R)-2-aminomethylpyrrolidine, 2-pyrrolidinone oxime and 1-cyclopropylethylamine.  
     
     
         24 . The formula II compound of  claim 20 , whose molecular weight is less than about 2000 daltons.  
     
     
         25 . The formula II compound of  claim 20 , whose molecular weight is less than about 500 daltons.  
     
     
         26 . The method of  claim 11 , wherein said organic compound is an aromatic or heteroaromatic compound comprising a piperidinyl, phenyl, quinolinyl and isoquinolinyl ring and having one or more nitrogen containing substitutents selected from the group consisting of NR 3 R 4  (CH 2 ) n N R 3 R 4 , CONH 2 , NH—NH—R 5  and C(S)—NH—R 6 , wherein R 3 , R 4 , R 5  and R 6  are independently H, alkyl, cycloalkyl and aryl, and n is 1-5, and wherein said compound selectively interacts with the E1 binding site of the enzyme.  
     
     
         27 . The method of  claim 26 , wherein the aromatic or heteroaromatic compound is 4-methylcyclohexylamine, 3-(dimethylamino)-1-(5-fluoro-2-hydroxyphenyl)prop-2-en-1-one, 3-ethoxyphenethylamine, 4-fluorobenzenesulfonamide, 2-((4-fluorophenyl)-hydrazono)malononitrile, 3-fluoro-4-hydroxybenzaldehyde and 1-aminoisoquinoline.  
     
     
         28 . The method of  claim 26 , wherein the molecular weight of the aromatic or heteroaromatic compound is less than about 2000 daltons.  
     
     
         29 . The method of  claim 26 , wherein the molecular weight of the aromatic or heteroaromatic compound is less than about 500 daltons.  
     
     
         30 . The method of  claim 11 , wherein said organic compound is a 7 or 8 membered monocyclic or bridged bicyclic compound comprising a cycloheptyl, cyclooctyl, and bicyclo(2,2,1) heptenyl having one or more substitutents selected from the group consisting of NR 3 R 4 , (CH 2 ) n NR 3 R 4 , CONH 2 , NH—NH—R 5  and C(S)—NH—R 6 , wherein R 3 , R 4 , R 5  and R 6  are independently H, alkyl, cycloalkyl and aryl, and n is 1-5, and wherein aid compound selectively interacts with the E1 binding site of the enzyme.  
     
     
         31 . The method of  claim 30 , wherein the 7 or 8-membered monocyclic or bridged bicyclic compound is 6,7-dihydro-5H-dibenzo(a,c)cycloheptene-6-carboxylic acid, N-bicyclo(2.2.1)hept-5-en-2-ylthiourea, N1-cyclooctyl-4-hydroxy-1-piperidinecarbothioamide.  
     
     
         32 . The method of  claim 31 , wherein the molecular weight of the 7 or 8 membered monocyclic or bridged bicyclic compound is less than about 2000 daltons.  
     
     
         33 . The method of  claim 31 , wherein the molecular weight of the 7 or 8 membered monocyclic or bridged bicyclic compound is less than about 500 daltons.  
     
     
         34 . The method of determining a selective interaction between an ubiquitin conjugating enzyme and an organic compound comprising the steps of: 
 a) inputting 3-D co-ordinates of at least a fragment of the ubiquitin conjugating enzyme into an electronic storage medium;    b) determining one or more active sites in said fragment of the ubiquitin conjugating enzyme;    c) simulating a binding interaction between said active sites in said fragments of the ubiquitin conjugating enzyme and a library of organic compounds;    d) identifying compounds in said library of organic compounds that interact with said active sites in said fragments of the ubiquitin conjugating enzyme.    
     
     
         35 . The method of  claim 34 , wherein said ubiquitin conjugating enzyme is selected from the group consisting of: Ubc1, Ubc2, Ubc3, Ubc4, Ubc5, Ubc6, Ubc7, Ubc8, Ubc10, Ubc11, Ubc13.  
     
     
         36 . The method of  claim 35 , wherein said ubiquitin conjugating enzyme is a yeast ubiquitin conjugating enzyme or a mouse, rat, or human homolog.  
     
     
         37 . The method of  claim 34 , wherein said atomic 3-D coordinates are selected from the group consisting of: 1AYZ, 1A3S, 1QCQ, 2UCZ, 1I7K 1J7D, 1JAT, 1JBB and variants thereof.  
     
     
         38 . The method of  claim 34 , wherein step (b) is performed by a molecular topology and charge visualization program.  
     
     
         39 . The method of  claim 34 , wherein said step (c) is performed with LUDI.  
     
     
         40 . The method of  claim 34 , wherein said library of organic compounds is the Available Chemical Directory.  
     
     
         41 . The method of  claim 34 , wherein said active site comprises the amino acid residues corresponding to Lys64, Pro66, Lys67, Ile68, Asn84, Ile85, Leu90, Lys91 and Leu120 of SEQ ID NO:1.  
     
     
         42 . The method of  claim 34 , wherein said active site comprises the amino acid residues corresponding to Pro65, Pro66, Lys67, Ile68, Asn84,Leu90, Lys91 and Leu120 of SEQ ID NO:1.  
     
     
         43 . The method of  claim 34 , wherein said active site comprises the amino acid residues corresponding to Lys66, Ile67, Ala68, Ser83, Cys85, Leu86, Leu89 and Arg90 of SEQ ID NO:2.  
     
     
         44 . The method of  claim 34 , wherein said active site comprises the amino acid residues corresponding to Pro64, Pro65, Lys66, Ile67, Ser83, Ile84, Cys85, Leu86, Leu89, Arg90 and Leu119 of SEQ ID NO:2.  
     
     
         45 . A method of treating or preventing a hyperproliferative disorder by administering an effective amount of a compound of formula (I):  
         Ar—B—NR 1 R 2   (I)  wherein:    Ar is a five or six membered unsubstituted or substituted aromatic ring that is optionally fused to an aromatic or heteroaromatic ring;    B is a bond, CO, SO 2  or (CH 2 ) n  wherein n=1-5; and    R 1  and R 2  are each independently H, alkyl or aryl groups that are optionally substituted. wherein said compound treats or prevents a hyperproliferative disorder.    
     
     
         46 . The formula I compound of  claim 45 , wherein Ar is phenyl, pyridyl, napthyl, triazine, triazole, quinoxaline, dibenzofuran, benzimidazole, indene, indeno oxadiazine, indazole or an indole ring; B is a bond, CO or (CH 2 ) n  wherein n=1; and R 1  and R 2  are both H.  
     
     
         47 . The formula I compound of  claim 45 , which is 3-amino-1,2,4-Triazine, 3-amino-1,2,4-triazole, 2-methyl-4-nitroaniline, 2-iodo-4-nitroaniline, 4-amino-3-chloro-5-methylbenzoic acid, 1-(4′Aminophenyl)-1,2,4-triazole, 2-acetamidophenol, 5-chloro-2,3-dihydroxypyridine, 2-methyl-3-(1H-pyrazol-5-yl)imidazo(1,2-a)pyridine, 5-nitro-2,3-dihydro-1H-benzo(d)imidazol-2-one, 4-(methylamino)pyridine, 2-Chloro-4-nitrobenzamide, 2-ethylformanilide, 6-aminoindazole, 2,3-diaminobenzoic acid, 1-(5-chloro-2-methylphenyl)-2-thiourea, 4,5-diiodo-1H-imidazole, 1H-indene-1,3 (2H)-dione 1-methylhydrazone, 3-hydroxyindole, 3,4-dihydro-1H-quinoxalin-2-one, 1S,6S,7R,8R,8aR)-1,6,7,8-Tetrahydroxyoctahydroindolizidine, 2-naphthalen-1-yl-2,3-dihydro-1H-pyrimidine, benzo(b)thiophen-3-ylmethylamine, 1-allyl-2-4-dioxo-1-2-3-4-tetrahydro-5-pyrimidinecarbonitrile, Methyl (S)-N-(7-chloro-2,3,4a,5-tetrahydro-4a-(methoxycarbonyl)indeno(1,2-e)(1,3,4)oxadiazin-2-ylcarbonyl)-4′-(trifluoromethoxy)carbanilate (Indoxacarb-MP), 4-acetylpyridine and thioisonicotinamide.  
     
     
         48 . The formula I compound of  claim 45 , which is 4-(Aminomethyl)piperidine.  
     
     
         49 . The formula I compound of  claim 45 , whose molecular weight is less than about 2000 daltons.  
     
     
         50 . The formula I compound of  claim 45 , whose molecular weight is less than about 500 daltons.  
     
     
         51 . A method of treating or preventing a hyperproliferative disorder by administering an effective amount of a compound of formula (II):  
         A-(B—NR 1 R 2 ) n   (II)  wherein:    A is a 3-6 membered substituted or unsubstituted cycloaliphatic or a heterocycloaliphatic ring, each of which is optionally fused to an aromatic ring;    B is a bond, CO, SO 2  or (CH 2 ) n  wherein n=1-3; and    R 1  and R 2  are each independently H, alkyl or aryl groups that are optionally substituted.    
     
     
         52 . The formula II compound of  claim 51 , wherein A is a heterocycloaliphatic ring comprising, at least one nitrogen atom and optionally, one or more additional heteroatoms selected from the group consisting of: nitrogen (N), oxygen (O) and sulfur (S); B is a bond, CO or (CH 2 ) n  wherein n=1; and R 1  and R 2  are both H.  
     
     
         53 . The formula II compound of  claim 51 , wherein the cycloaliphatic or heterocycloaliphatic ring comprise one or more substituents selected from the group consisting of hydroxyl, halogen, CO and alkyl.  
     
     
         54 . The formula II compound of  claim 51 , which is 1-phenyl-4-methyl-3-pyrazolidone, 4-(aminomethyl)piperidine, N-Phenyl-p-phenylenediamine, 5-(aminomethyl)-3-(2H)-isoxazolone (Muscimol), (R)-2-aminomethylpyrrolidine, 2-pyrrolidinone oxime and 1-cyclopropylethylamine.  
     
     
         55 . The formula II compound of  claim 51 , whose molecular weight is less than about 2000 daltons.  
     
     
         56 . The formula II compound of  claim 51 , whose molecular weight is less than about 500 daltons.  
     
     
         57 . A method of treating or preventing a hyperproliferative disorder by administering an effective amount of an aromatic or heteroaromatic compound comprising a piperidinyl, phenyl, quinolinyl and isoquinolinyl ring and having one or more nitrogen containing substitutents selected from the group consisting of NR 3 R 4 , (CH 2 ) n N R 3 R 4 , CONH 2 , NH—NH—R 5  and C(S)—NH—R 6 , wherein R 3 , R 4 , R 5  and R 6  are independently H, alkyl, cycloalkyl and aryl, and n is 1-5.  
     
     
         58 . The method of  claim 57 , wherein the aromatic or heteroaromatic compound is 4-methylcyclohexylamine, 3-(dimethylamino)-1-(5-fluoro-2-hydroxyphenyl)prop-2-en-1-one, 3-ethoxyphenethylamine, 4-fluorobenzenesulfonamide, 2-((4-fluorophenyl)-hydrazono)malononitrile, 3-fluoro-4-hydroxybenzaldehyde and 1-aminoisoquinoline.  
     
     
         59 . The method of  claim 57 , wherein the molecular weight of the 7 or 8 membered monocyclic or bridged bicyclic compound is less than about 2000 daltons.  
     
     
         60 . The method of  claim 57 , wherein the molecular weight of the 7 or 8 membered monocyclic or bridged bicyclic compound is less than about 500 daltons.  
     
     
         61 . A method of treating or preventing a hyperproliferative disorder by administering an effective amount of an 7 or 8 membered monocyclic or bridged bicyclic compound comprising a cycloheptyl, cyclooctyl, and bicyclo(2,2,1) heptenyl having one or more substitutents selected from the group consisting of NR 3 R 4 , (CH 2 ) n NR 3 R 4 , CONH 2 , NH—NH—R 5  and C(S)—NH—R 6 , wherein R 3 , R 4 , R 5  and R 6  are independently H, alkyl, cycloalkyl and aryl, and n is 1-5, and wherein aid compound selectively interacts with the E1 binding site of the enzyme.  
     
     
         62 . The method of  claim 61 , wherein the 7 or 8-membered monocyclic or bridged bicyclic compound is 6,7-dihydro-5H-dibenzo(a,c)cycloheptene-6-carboxylic acid, N-bicyclo(2.2.1)hept-5-en-2-ylthiourea, N1-cyclooctyl-4-hydroxy-1-piperidinecarbothioamide.  
     
     
         63 . The method of  claim 61 , wherein the molecular weight of the 7 or 8 membered monocyclic or bridged bicyclic compound is less than about 2000 daltons.  
     
     
         64 . The method of  claim 61 , wherein the molecular weight of the 7 or 8 membered monocyclic or bridged bicyclic compound is less than about 500 daltons.  
     
     
         65 . The method of  claim 45 ,  51 ,  57  or  61 , wherein the hyperproliferative disorder is cancer.  
     
     
         66 . The method of  claim 65 , wherein the cancer is colon cancer.  
     
     
         67 . The method of  claim 65 , wherein the cancer is breast cancer.  
     
     
         68 . A method of treating or preventing a Ubc related disorder by administering an effective amount of a compound of the invention.  
     
     
         69 . The method of  claim 59 , wherein said Ubc related disorder is selected from the group consisting of: hyperproliferative disorders, cystic fibrosis, neurodegenerative disorders and viral disorders.