Mycobacterial Disease Detection, Treatment, and Drug Discovery

Abstract

Methods for detecting and treating Mycobacterium -related diseases including reducing Mycobacterial virulence, reducing RV3133c dimerization, and treating a subject with a Mycobacterial infection using identified compounds are disclosed. Examples of compounds useful in the treatment of Mycobacterium -related diseases include N-(4-[(acetylamino)sulfonyl]phenyl)-3-phenylpropanamide; 1-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-(2-imino-3-methyl-2,3-dihydro-1H-benzimidazol-1yl)ethanone hydrochloride; and 1-(1,3-benzoxazol-2-yl)-3-({4-[(2-hydroxyethyl)sulfonyl]phenyl}amino)acrylaldehyde. Further disclosed are methods for identifying agents that interact with a polypeptide in a Mycobacterium cell.

1 .- 66 . (canceled) 
     
     
         67 . A method of reducing Mycobacterial virulence comprising contacting the  Mycobacterium  with a compound of the following formula: 
       
         
           
           
               
               
           
         
         wherein: 
         R 1  is NH—X or C(═O)—X, wherein X is a substituted or unsubstituted C 1 -C 12  alkyl, substituted or unsubstituted C 1 -C 12  alkenyl, substituted or unsubstituted C 1 -C 12  alkynyl, or substituted or unsubstituted aryl; 
         R 4  is OH; SO 2 —Y, wherein Y is a substituted or unsubstituted C 1 -C 12  alkyl, substituted or unsubstituted C 1 -C 12  alkenyl, substituted or unsubstituted C 1 -C 12  alkynyl, or substituted or unsubstituted aryl; or SO 2 —NH—Z, wherein Z is a substituted or unsubstituted C 1 -C 12  alkyl, substituted or unsubstituted C 1 -C 12  alkenyl, substituted or unsubstituted C 1 -C 12  alkynyl, or substituted or unsubstituted aryl; and 
         each of R 2 , R 3 , R 5 , and R 6  is independently H, OH, a substituted or unsubstituted C 1 -C 12  alkyl, substituted or unsubstituted C 1 -C 12  alkenyl, substituted or unsubstituted C 1 -C 12  alkynyl, or substituted or unsubstituted aryl, and pharmaceutically acceptable salts and derivatives thereof, 
         wherein the compound reduces virulence as compared to  Mycobacterium  in the absence of the compound. 
       
     
     
         68 . The method of  claim 67 , wherein contacting the  Mycobacterium  with the compound comprises administering the compound to a subject with a  Mycobacterium  infection. 
     
     
         69 . The method of  claim 67 , wherein X is substituted with a moiety selected from the group consisting of phenyl, carboxyl, benzooxazole, and 1-methyl-1,3-dihydro-benzoimidazol-2-ylideneamine. 
     
     
         70 . The method of  claim 67 , wherein Y is substituted with a hydroxyl moiety. 
     
     
         71 . The method of  claim 67 , wherein Z is substituted with a carbonyl moiety. 
     
     
         72 . The method of  claim 67 , wherein R 1  is selected from the following formulas: 
       
         
           
           
               
               
           
         
       
     
     
         73 . The method of  claim 67 , wherein R 4  is selected from the following formulas: 
       
         
           
           
               
               
           
         
       
     
     
         74 . The method of  claim 67 , wherein the compound is selected from the group consisting of N-(4-[(acetylamino)sulfonyl]phenyl)-3-phenylpropanamide; 1-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-(2-imino-3-methyl-2,3-dihydro-1H-benzimidazol-1 yl)ethanone hydrochloride; and 1-(1,3-benzoxazol-2-yl)-3-({4-[(2-hydroxyethyl)sulfonyl]phenyl}amino)acrylaldehyde; and pharmaceutically acceptable salts and derivatives thereof. 
     
     
         75 . A kit comprising:
 (a) a compound of the following formula:   
       
         
           
           
               
               
           
         
         wherein: 
         R 1  is NH—X or C(═O)—X, wherein X is a substituted or unsubstituted C 1 -C 12  alkyl, substituted or unsubstituted C 1 -C 12  alkenyl, substituted or unsubstituted C 1 -C 12  alkynyl, or substituted or unsubstituted aryl; 
         R 4  is OH; SO 2 —Y, wherein Y is a substituted or unsubstituted C 1 -C 12  alkyl, substituted or unsubstituted C 1 -C 12  alkenyl, substituted or unsubstituted C 1 -C 12  alkynyl, or substituted or unsubstituted aryl; or SO 2 —NH—Z, wherein Z is a substituted or unsubstituted C 1 -C 12  alkyl, substituted or unsubstituted C 1 -C 12  alkenyl, substituted or unsubstituted C 1 -C 12  alkynyl, or substituted or unsubstituted aryl; and 
         each of R 2 , R 3 , R 5 , and R 6  is independently H, OH, a substituted or unsubstituted C 1 -C 12  alkyl, substituted or unsubstituted C 1 -C 12  alkenyl, substituted or unsubstituted C 1 -C 12  alkynyl, or substituted or unsubstituted aryl, and pharmaceutically acceptable salts and derivatives thereof, and 
         (b) instructions for performing the method defined in  claim 67 . 
       
     
     
         76 . A method for detecting biomolecular interactions in  Mycobacterium  comprising:
 (a) combining a first fusion protein and a second fusion protein, wherein the first fusion protein comprises a first fragment of an enzyme reporter molecule and a first Mycobacterial polypeptide, wherein the second fusion protein comprises a second fragment of the enzyme reporter molecule and a second Mycobacterial polypeptide, and wherein association of the first polypeptide with the second polypeptide results in reconstitution of the enzyme reporter activity; and   (b) detecting the enzyme reporter activity, enzyme reporter activity indicating a biomolecular interaction between the first polypeptide and the second polypeptide.   
     
     
         77 . The method of  claim 76 , wherein one or more of the first and second polypeptides are selected from the group consisting of Cfp-10, Esat-6, Rv0686, Rv2151c, Rv2240c, Rv3596c, Rv3800c, and fragments thereof. 
     
     
         78 . The method of  claim 76 , wherein the enzyme reporter molecule comprises DHFR. 
     
     
         79 . The method of  claim 76  further comprising combining an agent with the first fusion protein and second fusion protein, wherein association of the agent with the first polypeptide and with the second polypeptide results in reconstitution of the enzyme reporter activity. 
     
     
         80 . The method of  claim 79 , wherein the agent is linked to a binding molecule, wherein the binding molecule associates with the first polypeptide, and wherein association of the agent with the second polypeptide results in reconstitution of the enzyme reporter activity. 
     
     
         81 . The method of  claim 80 , wherein the binding molecule is rapamycin. 
     
     
         82 . The method of  claim 80 , wherein the first polypeptide is FRB or FKBP. 
     
     
         83 . A kit comprising:
 (a) one or more of a first fusion protein comprising a first Mycobacterial polypeptide and a first enzyme reporter fragment; and   (b) a second enzyme reporter fragment.   
     
     
         84 . The kit of  claim 83 , further comprising a binding molecule, wherein the first fusion protein has affinity for the binding molecule. 
     
     
         85 . A kit as defined in  claim 84 , wherein a cross-linker is attached to the binding molecule. 
     
     
         86 . A kit as defined in  claim 84 , wherein the binding molecule is rapamycin.