SIR2 Activity

Abstract

This invention relates to methods of screening compounds that modulate cellular and organismal processes by modification of the activity of SIR2 and/or transcription factors, e.g., p53, particularly methods of screening for compounds that modify lifespan and/or metabolism of a cell or an organism by modulation of the activity of SIR2 and/or transcription factors, e.g., p53, and more particularly to methods of screening for compounds that modulate the activity of Sir2 and/or transcription factors, e.g., p53. In particular, the present invention relates to a method for screening a compound, by providing a test mixture comprising a transcription factor, Sir2, and a Sir2 cofactor with the compound, and evaluating an activity of a component of the test mixture in the presence of the compound. The invention further relates to therapeutic uses of said compounds. The invention further relates to a method of modifying the acetylation status of a transcription factor binding site on histone or DNA by raising local concentrations of Sir2.

1 . A method of screening a compound, comprising the steps of:
 (a) providing a reaction mixture comprising Sir2, a transcription factor, and the compound; and   (b) determining if the compound modulates Sir2 interaction with the transcription factor, thereby screening the compound.   
     
     
         2 . The method of  claim 1 , wherein the Sir2 interaction with the transcription factor is direct binding, covalent modification in one or both of the Sir2 or transcription factor, a change in cellular location of the test compound, Sir2, or the transcription factor, or an alteration in activity, stability, or structure. 
     
     
         3 . The method of  claim 2 , wherein the determining includes comparing the binding of Sir2 to the transcription factor at a first concentration of the compound and at a second concentration of the compound. 
     
     
         4 . The method of  claim 3 , wherein the first or second concentration of the compound is zero. 
     
     
         5 . The method of  claim 1 , wherein the reaction mixture further comprises a Sir2 cofactor. 
     
     
         6 . The method of  claim 5 , wherein the Sir2 cofactor is NAD or an NAD analog. 
     
     
         7 . The method of  claim 1  wherein the Sir2 is a Sir2 variant that has reduced deacetylase activity. 
     
     
         8 . The methods of  claim 1 , wherein the Sir2 is human. 
     
     
         9 . The method of  claim 8 , wherein the Sir2 is human SIRT1. 
     
     
         10 . The method of  claim 1 , wherein the Sir2 is murine. 
     
     
         11 . The method of  claim 10 , wherein the Sir2 is murine Sir2α. 
     
     
         12 . The method of  claim 1 , wherein the Sir2 is exogenous and expressed from a heterologous nucleic acid. 
     
     
         13 . The method of  claim 1 , wherein the transcription factor is exogenous and expressed from a heterologous nucleic acid. 
     
     
         14 . The method of  claim 1 , further comprising the steps of:
 (c) repeating steps (a) and (b) to confirm a modulatory effect of the compound on Sir2 interaction with the transcription factor, and   (d) contacting or administering the compound with or to a cell or animal to evaluate the effect of the compound on the cell or animal.   
     
     
         15 . A method of screening a compound, comprising the steps of:
 (a) providing a reaction mixture comprising Sir2, a transcription factor, and the compound; and   (b) determining if the compound modulates Sir2-mediated deacetylation of the transcription factor,   thereby screening the compound.   
     
     
         16 . The method of  claim 15 , wherein the determining includes comparing the acetylation status of the transcription factor, at a first concentration of the compound and at a second concentration of the compound. 
     
     
         17 . The method of  claim 16 , wherein the first or second concentration of the compound is zero. 
     
     
         18 . The method of  claim 17 , wherein the reaction mixture further comprises a Sir2 cofactor. 
     
     
         19 . The method of  claim 18 , wherein the Sir2 cofactor is NAD or an NAD analog. 
     
     
         20 . A method of screening a compound, comprising the steps of:
 (a) providing a compound that modulates interaction of an Sir2 with a transcription factor;   (b) contacting the compound with a cell or a system comprising an Sir2 or an active portion thereof and a transcription factor or an active portion thereof; and   (c) determining if the compound modulates transcription of a gene whose expression is regulated by the transcription factor,   thereby screening the compound.   
     
     
         21 . The method of  claim 20 , wherein the Sir2 interaction with the transcription factor is direct binding, covalent modification in one or both of the Sir2 or transcription factor, a change in cellular location of the test compound, Sir2, or the transcription factor, or an alteration in activity, stability, or structure. 
     
     
         22 . The isolated complex described in  claim 20  wherein the transcription factor binds a specific DNA site. 
     
     
         23 . The method of  claim 20  wherein the determining includes comparing the binding of Sir2 to the transcription factor at a first concentration of the compound and at a second concentration of the compound. 
     
     
         24 . The method of  claim 23 , wherein the first or second concentration of the compound is zero. 
     
     
         25 . The method of  claim 20 , wherein the cell or system further comprises a Sir2 cofactor. 
     
     
         26 . The method of  claim 25 , wherein the Sir2 cofactor is NAD or an NAD analog. 
     
     
         27 . The method of  claim 20  wherein the Sir2 is a Sir2 variant that has reduced deacetylase activity. 
     
     
         28 . The method of  claim 20 , wherein the Sir2 is human. 
     
     
         29 . The method of  claim 28 , wherein the Sir2 is human SIRT1. 
     
     
         30 . The method of  claim 20 , wherein the Sir2 is murine. 
     
     
         31 . The method of  claim 30 , wherein the Sir2 is murine Sir2α. 
     
     
         32 . The method of  claim 20 , wherein the Sir2 is exogenous and expressed from a heterologous nucleic acid. 
     
     
         33 . The method of  claim 20 , wherein the transcription factor is exogenous and expressed from a heterologous nucleic acid. 
     
     
         34 . The method of  claim 20 , further comprising, after step (c), the steps of:
 (d) contacting or administering the compound with or to a cell or animal; and   (e) evaluating the effect of the compound on the cell or animal.