Targeting dimerization of bax to modulate bax activity

Abstract

Methods are provided for identifying an agent that directly modulates a Bcl-2- associated x-protein (BAX) by promoting or disrupting dimerization of the BAX. Agents that directly modulate BAX by affecting dimerization are also provided.

1 . A method of identifying an agent as a candidate agent for promoting or inhibiting cell death comprising
 (a) contacting the agent with BCL-2-associated X-protein (BAX) monomers or portions thereof, and/or with BAX dimers, and   (b) measuring if the agent inhibits or promotes dimerization of BAX;   wherein a decrease in binding of BAX monomers to other BAX monomers or to portions of BAX monomers in the presence of the agent compared to in the absence of the agent indicates the agent inhibits dimerization of BAX, wherein an agent that agent inhibits dimerization of BAX is a candidate agent for promoting cell death; and   wherein an increase in binding of BAX monomers or portions thereof to other BAX monomers or portions thereof in the presence of the agent compared to in the absence of the agent indicates that the agent promotes dimerization of BAX, wherein an agent that agent promotes dimerization of BAX is a candidate agent for inhibiting cell death.   
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1 , wherein the agent binds to the N-terminal of BAX at one or more of binding site residues S16, E17, Q18, I19, M20, K21, T22, G23, A24, L25, L26, 127, Q28, G29, F30, I31, Q32, D33, R34, A35, G36, R37, M38, G39, G40, E41, A42, P43, E44, L45, A46, L47, D48, P49, V50, P51, Q52, D53, A54, V129, P130, E131, L132, I133, R134, T135, I136, M137, G138, W139, T140, L141, D142, F143, L144, R145, E146, and R147. 
     
     
         4 . The method of  claim 1 , wherein the agent binds to the C-terminal of BAX at one or more of binding site residues N73, M74, E75, L76, D98, M99, F 100, S101, D102, G103, N104, F105, N106, W107, G108, R109, I152, Q153, D154, Q155, G156, G157, W158, D159, G160, L161, L162, S163, Y164, F165, G166, T167, P168, T169, W170, Q171, T172, V173, T174, i175, F176, V177, A178, G179, V180, L181, T182, A183, S184, L185, T186, I187, W188, K189 K190, M191, and G192. 
     
     
         5 . The method of  claim 1 , wherein measurement of BAX as a dimer or monomer is carried out using one or more of a fluorescent polarization assay, size exclusion chromatography (SEC), polyacrylamide gel electrophoresis, dynamic light scattering and an antibody that specifically binds to either BAX dimer or BAX monomer. 
     
     
         6 . A method for identifying an agent that modulates the activity of BAX comprising
 contacting α9 helix peptide of BAX with N-terminal binding site of BAX in the presence of the agent and in the absence of the agent; and   measuring binding between the α9 helix peptide of BAX and the N-terminal binding site of BAX, wherein decreased binding in the presence of the agent compared to binding in the absence of the agent indicates that the agent is a modulator of the activity of BAX.   
     
     
         7 . The method of  claim 6 , wherein the agent is an inhibitor of BAX. 
     
     
         8 . The method of  claim 6 , wherein the α9 peptide of BAX has the amino acid sequence TWQTVTIFVAGVLTASLTIWKKMG (SEQ ID NO:11). 
     
     
         9 . The method of  claim 6 , wherein the α9 helix peptide of BAX or the N-terminal binding site of BAX is immobilized on a solid substrate. 
     
     
         10 . The method of  claim 6 , wherein binding between the α9 helix peptide of BAX and the N-terminal binding site of BAX is measured using a fluorescence assay. 
     
     
         11 - 14 . (canceled) 
     
     
         15 . A peptide consisting of: 
       
         
           
                 
                 
               
                     
                   a)  
                 
                     
                   (SEQ ID NO: 11) 
                 
                     
                   TWQTVTIFVAGVLTASLTIWKKMG, 
                 
                     
                     
                 
                     
                   b)  
                 
                     
                   (SEQ ID NO: 12) 
                 
                     
                   TWQTVTIFVAGVLTASLT, 
                 
                     
                     
                 
                     
                   c)  
                 
                     
                   (SEQ ID NO: 13) 
                 
                     
                   TWQTVTIFVAGVLTA, 
                 
                     
                     
                 
                     
                   d)  
                 
                     
                   (SEQ ID NO: 14) 
                 
                     
                   TWQTVTIFVAGVL, 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         e) 11-30 amino acid residues comprising the sequence TXQTXXIFXAG (SEQ ID NO:15), where X at any position can independently be any amino acid or unnatural amino acid or non-naturally occurring chemically modified amino acid, 
         f) 15-30 amino acid residues comprising the sequence TXQTXXIFXAGVXTA (SEQ ID NO:16), where X at any position can independently be any amino acid or unnatural amino acid or non-naturally occurring chemically modified amino acid, or 
         g) 11-30 amino acid residues comprising the sequence Y1XY2Y3XXY4Y5XY6Y7 (SEQ ID NO:17), where Y1 is threonine or a conserved amino acid, or unnatural amino acid or non-naturally occurring chemically modified amino acid, Y2 is glutamine or a conserved residue or unnatural amino acid or non-naturally occurring chemically modified amino acid, where Y3 is threonine or a conserved amino acid, or unnatural amino acid or non-naturally occurring chemically modified amino acid, where Y4 is isoleucine or a conserved amino acid, or unnatural amino acid or non-naturally occurring chemically modified amino acid, where Y5 is phenylalanine or a conserved amino acid, or unnatural amino acid or non-naturally occurring chemically modified amino acid, where Y6 is alanine or a conserved amino acid, or unnatural amino acid or non-naturally occurring chemically modified amino acid, where Y7 is glycine or a conserved amino acid, or unnatural amino acid or non-naturally occurring chemically modified amino acid, where X at any position can independently be any amino acid, or natural or non-naturally occurring chemically modified amino acid. 
       
     
     
         16 . The peptide of  claim 15  consisting of the sequence TXQTXXIFXAG (SEQ ID NO:15) or the sequence TXQTXXIFXAGVXTA (SEQ ID NO:16). 
     
     
         17 . A chemically synthesized peptide consisting of any of the peptides of  claim 15 . 
     
     
         18 . A peptide produced by recombinant DNA or cDNA consisting of any of the peptides of  claim 15 . 
     
     
         19 . (canceled) 
     
     
         20 . A method of inhibiting BAX comprising contacting BAX with the peptide of  claim 15 . 
     
     
         21 . The method of  claim 20 , wherein BAX is in a living cell and inhibition of BAX inhibits cell death. 
     
     
         22 . The method of  claim 20 , wherein the BAX is in a subject and the agent is administered to the subject. 
     
     
         23 . (canceled) 
     
     
         24 . The method of  claim 22 , wherein the subject has a disease or disorder associated with premature or unwanted cell death and characterized by abnormal activation, expression or function of BAX. 
     
     
         25 . The method of  claim 24 , wherein the disease or disorder is a cardiovascular disease or disorder, a neurodegenerative or neurological disease or disorder, a liver disease or disorder, a kidney disease or disorder, an immunological disorder, ischemia, infertility, a blood disorder, renal hypoxia, hepatitis, asthma or AIDS. 
     
     
         26 . A labeled peptide comprising the peptide of  claim 15  attached to a fluorescent label or to a radioactive label. 
     
     
         27 . A method of identifying an agent as a candidate agent for inhibiting cell death comprising:
 contacting BCL-2-associated X-protein (BAX) with one or more peptides of  claim 15  in the presence of the agent and in the absence of the agent, and   measuring binding of the one or more peptides to BAX,   wherein decreased binding of the one or more peptides to BAX in the presence of the agent compared to binding of the one or more peptides to BAX in the absence of the agent indicates that the agent is a candidate agent for inhibiting cell death.