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 step of:
(c) 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. The method described in claim 1 wherein the transcription factor is a p53 protein or an active portion thereof.
21. The method described in claim 15 wherein the transcription factor is a p53 protein or an active portion thereof.
22. A method of screening a compound, comprising the steps of:
(a) providing a reaction mixture comprising Sir2, a transcription factor that binds a specific DNA site, and the compound; and
(b) determining if the compound modulates Sir2 interaction with the transcription factor,
thereby screening the compound.
23. A method of screening a compound, comprising the steps of:
(a) providing a reaction mixture comprising Sir2, a transcription factor that binds a specific DNA site, and the compound; and
(b) determining if the compound modulates Sir2-mediated deacetylation of the transcription factor,
thereby screening the compound.