Specific inhibitors of NFAT activation by calcineurin and their use in treating immune-related diseases

Abstract

Isolated peptide fragments of the conserved regulatory domain of NFAT protein capable of inhibiting protein-protein interaction between calcineurin and NFAT, or a biologically active analog thereof are described. Isolated polynucleotides and gene therapy vectors encoding such peptide fragments are also described. In addition, methods for treating immune-related diseases or conditions and methods for high throughput screening of candidate agents are described. Pharmaceutical compositions are also provided.

What is claimed is:  
     
         1 . A pharmaceutical composition comprising a therapeutically effective amount of an organic molecule capable of inhibiting protein-protein interaction between calcineurin and NFAT, and a pharmaceutically acceptable carrier.  
     
     
         2 . The pharmaceutical composition of  claim 1 , wherein the agent inhibits dephosphorylation of NFAT by calcineurin.  
     
     
         3 . The pharmaceutical composition of  claim 1 , wherein the molecular weight of the organic molecule is less than 2500 Da.  
     
     
         4 . The pharmaceutical composition of  claim 1 , wherein the molecular weight of the organic molecule is between about 100 and 2000 Da.  
     
     
         5 . The pharmaceutical composition of  claim 1 , wherein the molecular weight of the organic molecule is between about 200 and 1500 Da.  
     
     
         6 . The pharmaceutical composition of  claim 1 , wherein the molecular weight of the organic molecule is between about 300 and 1000 Da.  
     
     
         7 . The pharmaceutical composition of  claim 1 , wherein the organic molecule binds calcineurin with an affinity constant of at least about 2×10 4  M −1 .  
     
     
         8 . The pharmaceutical composition of  claim 1 , wherein the organic molecule binds calcineurin with an affinity constant of at least about 10 6  M −1 .  
     
     
         9 . The pharmaceutical composition of  claim 1 , wherein the organic molecule binds calcineurin with an affinity constant of at least about 10 7  M −1 .  
     
     
         10 . The pharmaceutical composition of  claim 1 , wherein the organic molecule binds calcineurin with an affinity constant of at least about 10 8  M −1 .  
     
     
         11 . The pharmaceutical composition of  claim 1 , wherein the organic molecule is a compound selected from the group consisting of: 
 formula (I):                          wherein:    R 1  is hydrogen, C 1 -C 20  alkyl optionally substituted with 1-20 R 6 , C 3 -C 8  cycloalkyl optionally substituted with 1-3 R 6 , aryl optionally substituted with 1-4 R 6 , heterocyclyl optionally substituted with 1-3 R 6 ; heteroaryl optionally substituted with 1-4 R 6 ; C 2 -C 8  alkenyl, or C 2 -C 8  alkynyl, cyano, nitro, carboxy, carbo(C 1 -C 6 )alkoxy, trihalomethyl, halogen, C 1 -C 6  alkoxy, hydroxy, aryloxy, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, or ureido;    R 2  is C 1 -C 20  alkyl optionally substituted with 1-20 R 6 , C 3 -C 8  cycloalkyl optionally substituted with 1-3 R 6 , aryl optionally substituted with 1-4 R 6 , heterocyclyl optionally substituted with 1-3 R 6 , heteroaryl optionally substituted with 1-4 R 6 , C 1 -C 6  alkoxy, or hydroxy;    R 3  is hydrogen or halogen; 
 R 4  is hydrogen, C 1 -C 20  alkyl optionally substituted with 1-20 R 6 , C 3 -C 8  cycloalkyl optionally substituted with 1-3 R 6 , aryl optionally substituted with 1-4 R 6 , heterocyclyl optionally substituted with 1-3 R 6 , heteroaryl optionally substituted with 1-4 R 6 , or halogen;  
 R is NR 7 , O or S;  
 R 6  is halogen, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido; and  
 R 7  is C1-C6 alkyl;  
   formula (II):                          wherein:    R 1  and R 2  are each independently hydrogen, halogen, amino, C 1 -C 6 alkylamino, di(C 1 -C 6 )alkylamino, arylamino, diarylamino, or 4,4-dimethyl-2,6-dioxocyclohexyl;    R 3  is NR 11  or O;    R 4 , R 5  and R 8  are each independently hydrogen, C 1 -C 6  alkyl, halogen, hydroxy, nitro, haloalkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido;    R 6  is hydrogen, halogen, or when taken together with R 7  forms a double bond between the carbon atoms to which they are attached;    R 7  is hydrogen, halogen, or when taken together with R 6  forms a double bond between the carbon atoms to which they are attached;    R 9  is OR 13 , or when taken together with R 10  forms a double bond between the carbon and nitrogen atoms to which they are attached;    R 10  is hydrogen, or when taken together with R 9  forms a double bond between the carbon and nitrogen atoms to which they are attached;    R 11  is SO 2 R 12 ; and    R 12  is aryl optionally substituted with alkyl;    R 13  is alkyl or aryl; and    formula (III):                          wherein,    R 1  and R 4  are each independently O or NR 8 ;    R 2  and R 3  are each independently hydrogen, halogen, or R 2  and R 3  together combine to form aryl optionally substituted with 1-4 R 9 ;    R 5  is hydrogen, halogen, carboxy, acylamino, alkoxycarbonyl, carboxy, alkylcarbonyl, acyloxy, or cyano;    R 6 , R 7  and R 9  are each independently hydrogen, C 1 -C 6  alkyl, halogen, hydroxy, nitro, haloalkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido;    R 8  is SO 2 R 10 ; and    R 10  is aryl optionally substituted with alkyl.    
     
     
         12 . A method for inhibiting protein-protein interaction between calcineurin and NFAT, comprising: 
 providing calcineurin and NFAT;    providing the pharmaceutical composition of  claim 1;  and    contacting the calcineurin, NFAT, and pharmaceutical composition together, such that the protein-protein interaction between calcineurin and NFAT is inhibited.    
     
     
         13 . A method of inhibiting an immune response in an animal, comprising administering to the animal the pharmaceutical composition of  claim 1 .  
     
     
         14 . A method for treating a disease or condition involving hyperactivity or inappropriate activity of the immune system, comprising: 
 identifying an animal suffering from a disease or condition involving hyperactivity or inappropriate activity of the immune system; and    administering to the animal a therapeutically effective amount of the pharmaceutical composition of  claim 1 , to thereby treat the disease or condition involving hyperactivity or inappropriate activity of the immune system.    
     
     
         15 . The method of  claim 14 , wherein the organic molecule is a compound selected from the group consisting of: 
 formula (I):                          wherein:    R 1  is hydrogen, C 1 -C 20  alkyl optionally substituted with 1-20 R 6 , C 3 -C 8  cycloalkyl optionally substituted with 1-3 R 6 , aryl optionally substituted with 1-4 R 6 , heterocyclyl optionally substituted with 1-3 R 6 ; heteroaryl optionally substituted with 1-4 R 6 ; C 2 -C 8  alkenyl, or C 2 -C 8  alkynyl, cyano, nitro, carboxy, carbo(C 1 -C 6 )alkoxy, trihalomethyl, halogen, C 1 -C 6  alkoxy, hydroxy, aryloxy, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, or ureido;    R 2  is C 1 -C 20  alkyl optionally substituted with 1-20 R 6 , C 3 -C 8  cycloalkyl optionally substituted with 1-3 R 6 , aryl optionally substituted with 1-4 R 6 , heterocyclyl optionally substituted with 1-3 R 6 , heteroaryl optionally substituted with 1-4 R 6 , C 1 -C 6  alkoxy, hydroxy;    R 3  is hydrogen or halogen;    R 4  is hydrogen, C 1 -C 20  alkyl optionally substituted with 1-20 R 6 , C 3 -C 8  cycloalkyl optionally substituted with 1-3 R 6 , aryl optionally substituted with 1-4 R 6 , heterocyclyl optionally substituted with 1-3 R 6 , heteroaryl optionally substituted with 1-4 R 6 , or halogen;    R 5  is NR 7 , O or S;    R 6  is halogen, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido; and    R 7  is C1-C6 alkyl;    formula (II):                          wherein:    R 1  and R 2  are each independently hydrogen, halogen, amino, C 1 -C 6 alkylamino, di(C 1 -C 6 )alkylamino, arylamino, diarylamino, or 4,4-dimethyl-2,6-dioxocyclohexyl;    R 3  is NR 11  or O;    R 4 , R 5  and R 8  are each independently hydrogen, C 1 -C 6  alkyl, halogen, hydroxy, nitro, haloalkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido;    R 6  is hydrogen, halogen, or when taken together with R 7  forms a double bond between the carbon atoms to which they are attached;    R 7  is hydrogen, halogen, or when taken together with R 6  forms a double bond between the carbon atoms to which they are attached;    R 9  is OR 13 , or when taken together with R 10  forms a double bond between the carbon and nitrogen atoms to which they are attached;    R 10  is hydrogen, or when taken together with R 9  forms a double bond between the carbon and nitrogen atoms to which they are attached;    R 11  is SO 2 R 12 ; and    R 12  is aryl optionally substituted with alkyl;    R 13  is alkyl or aryl; and    formula (III):                          wherein,    R 1  and R 4  are each independently O or NR 8 ;    R 2  and R 3  are each independently hydrogen, halogen, or R 2  and R 3  together combine to form aryl optionally substituted with 1-4 R 9 ;    R 5  is hydrogen, halogen, carboxy, acylamino, alkoxycarbonyl, carboxy, alkylcarbonyl, acyloxy, or cyano;    R 6 , R 7  and R 9  are each independently hydrogen, C 1 -C 6  alkyl, halogen, hydroxy, nitro, haloalkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido;    R 8  is SO 2 R 10 ; and    R 10  is aryl optionally substituted with alkyl.    
     
     
         16 . The method of  claim 14  wherein the disease or condition involving hyperactivity or inappropriate activity of the immune system is selected from the group consisting of: an acute immune disease, a chronic immune disease, and an autoimmune disease.  
     
     
         17 . A method for treating a disease involving excessive or inappropriate activation of NFAT, or a molecular target thereof, comprising: 
 identifying an animal suffering from a disease involving excessive or inappropriate activation of NFAT or a molecular target thereof, and    administering to the animal a therapeutically effective amount of the pharmaceutical composition of  claim 1 , to thereby treat the disease involving excessive or inappropriate activation of NFAT or molecular target thereof.    
     
     
         18 . A process of making an agent that inhibits protein-protein interaction between calcineurin and NFAT, the process comprising: 
 carrying out a method to identify an agent that inhibits protein-protein interaction between calcineurin and NFAT, wherein the method comprises: 
 providing a first compound selected from the group consisting of calcineurin or a biologically active derivative thereof, and NFAT or a biologically active derivative thereof;  
 providing a second compound selected from the group consisting of calcineurin or a biologically active derivative thereof, and NFAT or a biologically active derivative thereof, wherein the second compound is different from the first compound, and wherein the second compound is labeled;  
 providing a candidate agent;  
 contacting the first compound, the second compound, and the candidate agent with each other; and  
 determining the amount of label bound to the first compound, wherein a reduction in interaction between the first compound and the second compound as assessed by label bound is indicative of usefulness of the candidate agent in inhibiting protein-protein interaction between calcineurin and NFAT; and  
   manufacturing the agent, to thereby make an agent that inhibits protein-protein interaction between calcineurin and NFAT.    
     
     
         19 . The process of  claim 18 , wherein the first compound is calcineurin and the second compound is a biologically active derivative of NFAT.  
     
     
         20 . The process of  claim 18 , wherein the biologically active derivative of NFAT comprises the amino acid sequence GPHPVIVITGPHEE.  
     
     
         21 . A method of manufacturing an agent that inhibits protein-protein interaction between calcineurin and NFAT, the method comprising: 
 providing an organic compound capable of inhibiting protein-protein interaction between calcineurin and NFAT;    providing at least one pharmaceutically acceptable carrier; and    combining the organic compound with the pharmaceutically acceptable carrier, to thereby manufacture an agent that inhibits protein-protein interaction between calcineurin and NFAT.    
     
     
         22 . The method of  claim 21 , further comprising the step of manufacturing the agent into a form suitable for administration to an animal via a route selected from a group consisting of: oral, parenteral, topical, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal.  
     
     
         23 . A method for inhibiting protein-protein interaction between calcineurin and NFAT, comprising: 
 providing calcineurin and NFAT;    providing an organic molecule capable of inhibiting protein-protein interaction between calcineurin and NFAT, wherein the organic molecule is a compound selected from the group consisting of:    formula (I):                          wherein:    R 1  is hydrogen, C 1 -C 20  alkyl optionally substituted with 1-20 R 6 , C 3 -C 8  cycloalkyl optionally substituted with 1-3 R 6 , aryl optionally substituted with 1-4 R 6 , heterocyclyl optionally substituted with 1-3 R 6 ; heteroaryl optionally substituted with 1-4 R 6 ; C 2 -C 8  alkenyl, or C 2 -C 8  alkynyl, cyano, nitro, carboxy, carbo(C 1 -C 6 )alkoxy, trihalomethyl, halogen, C 1 -C 6  alkoxy, hydroxy, aryloxy, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, or ureido;    R 2  is C 1 -C 20  alkyl optionally substituted with 1-20 R 6 , C 3 -C 8  cycloalkyl optionally substituted with 1-3 R 6 , aryl optionally substituted with 1-4 R 6 , heterocyclyl optionally substituted with 1-3 R 6 , heteroaryl optionally substituted with 1-4 R 6 , C 1 -C 6  alkoxy, hydroxy;    R 3  is hydrogen or halogen;    R 4  is hydrogen, C 1 -C 20  alkyl optionally substituted with 1-20 R 6 , C 3 -C 8  cycloalkyl optionally substituted with 1-3 R 6 , aryl optionally substituted with 1-4 R 6 , heterocyclyl optionally substituted with 1-3 R 6 , heteroaryl optionally substituted with 1-4 R 6 , or halogen;    R 5  is NR 7 , O or S;    R 6  is halogen, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido; and    R 7  is C 1 -C 6  alkyl;    formula (II):                          wherein:    R 1  and R 2  are each independently hydrogen, halogen, amino, C 1 -C 6 alkylamino, di(C 1 -C 6 )alkylamino, arylamino, diarylamino, or 4,4-dimethyl-2,6-dioxocyclohexyl;    R 3  is NR 11  or O;    R 4 , R 5  and R 8  are each independently hydrogen, C 1 -C 6  alkyl, halogen, hydroxy, nitro, haloalkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido;    R 6  is hydrogen, halogen, or when taken together with R 7  forms a double bond between the carbon atoms to which they are attached;    R 7  is hydrogen, halogen, or when taken together with R 6  forms a double bond between the carbon atoms to which they are attached;    R 9  is OR 13 , or when taken together with R 10  forms a double bond between the carbon and nitrogen atoms to which they are attached;    R 10  is hydrogen, or when taken together with R 9  forms a double bond between the carbon and nitrogen atoms to which they are attached;    R 11  is SO 2 R 12 ; and    R 12  is aryl optionally substituted with alkyl;    R 13  is alkyl or aryl; and    formula (III):                          wherein,    R 1  and R 4  are each independently O or NR 8 ;    R 2  and R 3  are each independently hydrogen, halogen, or R 2  and R 3  together combine to form aryl optionally substituted with 1-4 R 9 ;    R 5  is hydrogen, halogen, carboxy, acylamino, alkoxycarbonyl, carboxy, alkylcarbonyl, acyloxy, or cyano;    R 6 , R 7  and R 9  are each independently hydrogen, C 1 -C 6  alkyl, halogen, hydroxy, nitro, haloalkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido;    R 8  is SO 2 R 10 ; and    R 10  is aryl optionally substituted with alkyl; and    contacting the calcineurin, NFAT, and organic molecule together such that protein-protein interaction between the calcineurin and the NFAT is inhibited.