Structural-based inhibitors of the glutathione binding site in aldose reductase, methods of screening therefor and methods of use

Abstract

Provided herein is a crystallized ternary structure of aldose reductase (AR) bound to NADPH and γ-glutamyl-S-(1,2-dicarboxyethyl)cysteinylglycine (DCEG). Also provided are specific inhibitors of glutathione-aldehyde binding to aldose reductase which are designed via at least computer modeling of the ternary AR:NADPH:DCEG structure and methods of designing and of screening the inhibitors for inhibition of glutathione-aldehyde binding to aldose reductase. In addition methods of treating a pathophysiological state or symptoms thereof resulting from aldose reductase-mediated signaling in a cytotoxic pathway using a small interfering RNA (siRNA) or the designed inhibitors.

1 . A method of treating a pathophysiological state or symptoms thereof resulting from aldose reductase-mediated signaling in a cytotoxic pathway in a subject, comprising: 
 administering a pharmacologically effective amount of an inhibitor of aldose reductase to the subject thereby preventing aldose reductase mediated signaling.    
     
     
         2 . The method of  claim 1 , wherein the inhibitor is a small interfering RNA (siRNA) or an antisense molecule.  
     
     
         3 . The method of  claim 2 , wherein said siRNA comprises a vector effective to transfect a cell characteristic of said pathophysiological state.  
     
     
         4 . The method of  claim 3 , wherein said cell is a colon cancer cell.  
     
     
         5 . The method of  claim 1 , wherein the siRNA has the sequence shown in SEQ ID NO: 1.  
     
     
         6 . The method of  claim 1 , wherein the inhibitor is effective to inhibit reduction of a glutathione-aldehyde conjugate by aldose reductase.  
     
     
         7 . The method of  claim 6 , wherein said inhibitor interacts with a glutathione binding domain, but does not block a carbonyl binding site, in an active pocket of an aldose reductase having a three-dimensional conformation determined by DCEG binding to AR:NADPH.  
     
     
         8 . The method of  claim 7 , wherein said glutathione-binding domain comprises residues Trp-20, Trp-79, Trp-111, Trp-219, Phe-122, Val-47, Cys-298, Ala-299, Ser-302, Leu-300, and Leu-301.  
     
     
         9 . The method of  claim 7 , wherein said active pocket comprises three flexible loops A, B, and C, said inhibitor interacting with at least said C loop.  
     
     
         10 . The method of  claim 9 , wherein C loop comprises residues Ser-302, Ala-299, Leu-300, and Leu-301.  
     
     
         11 . The method of  claim 10 , wherein residues Ser-302, Ala-299, Leu-300, and Leu-301 interact with said inhibitor via a network of water molecules within the C loop.  
     
     
         12 . The method of  claim 7 , wherein said carbonyl binding site comprises residues Tyr-48, His-110, and Trp-111 and NADPH.  
     
     
         13 . The method of  claim 7 , wherein said inhibitor has a γ-glutamylcysteinylglycine backbone with an S-cysteinyl-substituted moiety.  
     
     
         14 . The method of  claim 1 , wherein said pathophysiological state is a cancer.  
     
     
         15 . The method of  claim 14 , wherein said cancer is colon cancer, breast cancer, prostate cancer and lung cancer.  
     
     
         16 . The method of  claim 1 , wherein said pathophysiological state is characterized by inflammation.  
     
     
         17 . The method of  claim 16 , wherein said inflammation is induced by lipopolysaccharide (LPS) and/or is related to sepsis.  
     
     
         18 . The method of  claim 1 , wherein said cytotoxic pathway is a PLC/PKC/NF-κB pathway.  
     
     
         19 . The method of  claim 18 , wherein signaling by NF-□B, prostaglandin E2 (PGE2), or cyclooxygenase (Cox-2) or a combination thereof is inhibited.  
     
     
         20 . A method of treating colon cancer in a subject, comprising: 
 administering a pharmacologically effective amount of an aldose reductase small interfering RNA (siRNA) to the subject to inhibit colon cancer cell proliferation thereby treating the colon cancer.    
     
     
         21 . The method of  claim 20 , wherein said siRNA comprises a vector effective to transfect a colon cancer cell.  
     
     
         22 . The method of  claim 20 , wherein the siRNA has the sequence shown in SEQ ID NO: 1.