Sir2 products and activities
View Patent ↗A novel compound, 2′/3′-O-acetyl-ADP-ribose, is provided. The compound is a mixture of the 2′ and 3′ regioisomers of O-acetyl-ADP ribose, and is formed nonenzymatically from 2′-O-acetyl-ADP-ribose, which is the newly discovered product of the reaction of Sir2 enzymes with acetylated peptides and NAD + . Analogs of 2′/3′-O-acetyl-ADP-ribose are also provided. Additionally, methods of preparing 2′/3′-O-acetyl-ADP-ribose, methods of determining whether a test compound is an inhibitor of a Sir2 enzyme, methods of detecting Sir2 activity in a composition, methods of deacetylating an acetylated peptide, and methods of inhibiting the deacetylation of an acetylated peptide are provided. Prodrugs of 2′/3′-O-acetyl-ADP-ribose are also provided.
1. A method of determining whether a test compound is an inhibitor of a Sir2 enzyme, the method comprising
combining the test compound with the Sir2 enzyme, NAD + and an acetylated peptide substrate of Sir2 in a reaction mixture under conditions and for a time sufficient to deacetylate the peptide in the absence of the test compound;
quantifying 2′/3′-O-acetyl-ADP-ribose produced from the acetylated peptide; then
comparing the quantity of 2′/3′-O-acetyl-ADP-ribose produced with a quantity of 2′/3′-O-acetyl-ADP-ribose produced under the same conditions but without the test compound;
wherein the presence of less 2′-O-acetyl-ADP-ribose and/or 3′-O-acetyl-ADP-ribose than without the test compound indicates that the test compound is an inhibitor of the Sir2 enzyme.
2. The method of claim 1 , wherein the 2′/3′-O-acetyl-ADP-ribose is quantified after subjecting the reaction mixture to chromatography.
3. The method of claim 2 , wherein the chromatography is HPLC.
4. The method of claim 2 , wherein the chromatography is on a Sephadex-DEAE column.
5. The method of claim 1 , wherein the NAD + is radiolabeled such that the 2′/3′-O-acetyl-ADP-ribose is radiolabeled after the peptide is deacetylated.
6. The method of claim 5 , wherein the NAD + is radiolabeled at the 5′ position with 14 C.
7. The method of claim 1 , wherein the acetyl group on the acetylated peptide is radiolabeled.
8. The method of claim 1 , wherein the acetylated peptide substrate consists at least two amino acids, wherein at least one of the amino acids comprise a lysine residue that is acetylated at the ε-amino moiety.
9. The method of claim 8 , wherein the acetylated peptide substrate consists of at least three amino acid residues.
10. The method of claim 8 , wherein the acetylated peptide substrate consists of at least five amino acid residues.
11. The method of claim 8 , wherein the acetylated peptide substrate consists of at least ten amino acid residues.
12. The method of claim 8 , wherein the acetylated peptide substrate consists of at least fifteen amino acid residues.
13. The method of claim 8 , wherein the acetylated peptide substrate consists of at least eighteen amino acid residues.
14. The method of claim 1 , wherein the acetylated peptide substrate is homologous to an acetylated region of a regulatory domain of a p53.
15. The method of claim 14 , wherein the acetylated peptide comprises JB11 or JB12.
16. The method of claim 15 , wherein the acetylated peptide substrate is an acetylated p53.
17. The method of claim 1 , wherein the acetylated peptide substrate is homologous to an acetylated region of a histone.
18. The method of claim 17 , wherein the acetylated peptide is an acetylated histone.
19. The method of claim 1 , wherein the Sir2 enzyme is derived from a prokaryote.
20. The method of claim 19 , wherein the Sir2 enzyme is derived from an archaeal bacterium.
21. The method of claim 19 , wherein the Sir2 enzyme is derived from a eubacterium.
22. The method of claim 1 , wherein the Sir2 enzyme is from a eukaryote.
23. The method of claim 22 , wherein the Sir2 enzyme is from a yeast.
24. The method of claim 22 , wherein the Sir2 enzyme is from a human.