IDENTIFICATION OF INHIBITORS OF TCPC AND TIR NADase ACTIVITY

Abstract

The present invention provides compounds useful as inhibitors of TIR NADase activity, compositions thereof, and methods of using the same. The present invention is useful for inhibition of TIR-domain NADase activity and/or treating microbial infection and/or modulating microbial physiology.

1 . A method of inhibiting TIR NADase activity in a biological sample comprising a TIR NADase, the method comprising, contacting the biological sample with a composition comprising an effective amount of a TIR NADase inhibitor, wherein the TIR NADase inhibitor is one or more of: 
       
         
           
           
               
               
           
         
       
       and derivatives thereof. 
     
     
         2 .- 15 . (canceled) 
     
     
         16 . A method of identifying a TIR NADase inhibitor, comprising:
 a) providing a mixture comprising;
 i) a full length, mutant or fragment of a TIR NADase, 
 ii) NAD+, and; 
 iii) a candidate inhibitor, wherein optionally the mutant or fragment has constitutive NADase activity; 
   b) incubating the mixture;   c) quantifying one or more of NAD+ ADPR, cADPR or nicotinamide in the mixture.   
     
     
         17 . The method of  claim 16 , further comprising:
 a) identifying the candidate inhibitor compound as an NADase inhibitor if one or more of the following is true, the amount of NAD+ is greater or the amount of ADPR, cADPR or nicotinamide is less than that of a control mixture that does not contain the candidate inhibitor.   
     
     
         18 . The method of  claim 16 , further comprising:
 a) determining the molar ratio of NAD+: ADPR; and;   b) identifying the candidate inhibitor compound as an NADase inhibitor if the molar ratio is greater than that of a control mixture that does not contain the candidate inhibitor.   
     
     
         19 . The method of  claim 16 , wherein the quantifying NAD+ and ADPR in the mixture comprises performing an HPLC analysis, performing a chemiluminescence assay, performing liquid chromatography-mass spectroscopy analysis or a combination thereof. 
     
     
         20 . The method of  claim 16 , wherein the mixture comprises a cell lysate from a host cell comprising the full length, mutant or fragment of a TIR NADase. 
     
     
         21 .- 22 . (canceled) 
     
     
         23 . The method of  claim 16 , wherein the full length, mutant or fragment TIR NADase is from TcpC. 
     
     
         24 . The method of  claim 16 , wherein the full length, mutant or fragment TIR NADase is from TirS. 
     
     
         25 .- 27 . (canceled) 
     
     
         28 . A method of treating a microbial infection in a subject in need thereof, wherein the microbe encodes a TIR NADase, the method comprising: administering to said subject a therapeutically effective amount of pharmaceutical composition comprising a TIR NADase inhibitor. 
     
     
         29 . The method of  claim 28 , wherein the TIR NADase inhibitor is Dequalinium 
       
         
           
           
               
               
           
         
       
       or a derivative thereof. 
     
     
         30 . The method of  claim 28 , wherein the TIR NADase inhibitor is Cetylpyridinium 
       
         
           
           
               
               
           
         
       
       or a derivative thereof. 
     
     
         31 . The method of  claim 28 , wherein the TIR NADase inhibitor is Amiloride 
       
         
           
           
               
               
           
         
       
       or a derivative thereof. 
     
     
         32 . The method of  claim 28 , wherein the TIR NADase inhibitor is Acrinol 
       
         
           
           
               
               
           
         
       
       or a derivative thereof. 
     
     
         33 . The method of  claim 28 , wherein the TIR NADase inhibitor is Akrinol 
       
         
           
           
               
               
           
         
       
       or a derivative thereof. 
     
     
         34 . The method of  claim 28 , wherein the infection is caused by a pathogenic bacteria, virus, fungus or protozoa. 
     
     
         35 . The method of  claim 34 , wherein the bacteria is uropathogenic  E. coli.    
     
     
         36 . The method of  claim 35 , wherein the TIR NADase is TcpC. 
     
     
         37 . The method of  claim 34 , wherein the bacteria is  S. aureus.    
     
     
         38 . The method of  claim 37 , wherein the TIR NADase is TirS. 
     
     
         39 . The method of  claim 28 , wherein the TIR NADase inhibitor is administered with one or more additional therapeutically active agents.