SIRTUIN ACTIVATORS AND ACTIVATION ASSAYS
Provided are methods and compositions for detecting a compound that activates a sirtuin deacetylase activity on a fluorescent-free activation substrate in vitro. Further provided are sirtuin modulating compounds of the formulas (I)-(XXI), and related compounds (XXXI), (XXXII), (XXXIII), and (XXXIV), including the fluorescent free-substrate SIRT1 activator compounds of formulas (XL), (XI), (XII), and (XIII).
1 . A method of detecting a compound that activates a SIRT1 sirtuin deacetylase activity on a fluorescent-free activation substrate in vitro comprising:
contacting a SIRT1 sirtuin deacetylase with a candidate compound and a fluorescent-free activation substrate comprising an acetylated peptide, polypeptide, or protein substrate that include an activation cofactor moiety;
detecting the level of SIRT1 sirtuin deacetylase activity on the fluorescent-free activation substrate that include an activation cofactor moiety in the presence of the candidate compound; and
comparing the level of SIRT1 sirtuin deacetylase activity on the fluorescent-free activation substrate that include an activation cofactor moiety in the presence of the candidate compound to the level of SIRT1 sirtuin deacetylase activity on the fluorescent-free activation substrate that include an activation cofactor moiety in the absence of the candidate compound,
wherein an increase in the level of SIRT1 sirtuin deacetylase activity on the fluorescent-free activation substrate in the presence of the candidate compound compared to the level of SIRT1 sirtuin deacetylase activity on the fluorescent-free activation substrate in the absence of the candidate compound indicates that the compound is a SIRT1 sirtuin activator.
2 .- 3 . (canceled)
4 . The method of claim 1 , wherein the candidate compound is a SIRT1 activator of an acetylated peptide or polypeptide substrate containing a fluorescent group.
5 . The method of claim 4 , wherein the fluorescent group-containing peptide substrate is the TAMRA-peptide: Ac-EEK (biotin) GQSTSSHSK Ac NleSTEGK (5-TMR) EE-NH 2 .
6 . The method of claim 1 , wherein the sirtuin deacetylase is contacted with a fluorescent-free activation substrate and a candidate compound in the presence of NAD.
7 . The method of claim 1 , wherein the sirtuin deacetylase is contacted with a fluorescent-free activation substrate and a candidate compound in the presence of a hydrolysable NAD analog.
8 . The method of claim 1 , wherein the fluorescent-free activation substrate is a biotinylated polypeptide.
9 . The method of claim 1 , wherein the fluorescent-free activation substrate is the desTAMRA-peptide: Ac-EEK (biotin) GQSTSSHSK Ac NleSTEGKEE-NH 2 .
10 . The method of claim 1 , wherein the fluorescent-free activation substrate is an acetylated peptide or polypeptide free of the fluorescent groups TAMRA (tetramethyl-6-carboxyrhodamine) and AMC (7-amido-4-methyl coumarin).
11 . The method of claim 1 , wherein the fluorescent-free activation substrate is an acetylated peptide selected from the group consisting of Ac-RHKK Ac F-NH 2 and Ac-RHKK Ac W-NH 2 .
12 . The method of claim 1 , wherein the fluorescent-free activation substrate comprises an acetylated peptide, polypeptide, or protein substrate of the sirtuin and an activation cofactor-bearing accessory protein.
13 . The method of claim 12 , wherein the activation cofactor-bearing accessory protein is selected from the group consisting of DBC1 (deleted in breast cancer 1), HIC1 (hypermethylated in cancer 1), AROS (active regulator of SIRT1), and CLOCK.
14 . The method of claim 1 , wherein the acetylated protein is selected from the group consisting of histone H1, histone H3, histone H4, p53, p300, FOXO 1, FOXO 3a, FOXO 4, p65, HIVTat, PGC-1α, PCAF, MyoD, PPARγ, and Ku70.
15 . The method of claim 1 , wherein the level of sirtuin deacetylase activity is detected by measuring the rate of NAD hydrolysis.
16 . The method of claim 1 , wherein the level of sirtuin deacetylase activity is detected by measuring the rate of fluorescent-free activation substrate deacetylation.
17 . The method of claim 1 , wherein the compound is an activator of SIRT1 deacetylation of a fluorescent-free activation substrate.
18 . The method of claim 17 , wherein the compound has the formula:
or a salt thereof, wherein:
R 1 is selected from —(CH 2 ) 3 —CH 3 , and —(CH 2 )CH(CH 3 ) 2 ; and
R 2 is selected from -piperidine and —(CH 2 ) 2 —NH—CH 3 .
19 . The method of claim 18 , wherein the compound, or salt thereof, is selected from the group consisting of:
20 . The method of claim 1 , further comprising:
selecting a candidate compound that is a SIRT1 activator, and
contacting the SIRT1 activator with a test cell and detecting a SIRT1 activation-specific change in the test cell.
21 . The method of claim 20 , wherein the SIRT1 activation-specific change is an increase in FGF21 production.
22 . The method of claim 20 , wherein the SIRT1 activation-specific change is a decrease in LPS-induced TNFα production.
23 . The method of claim 1 , further comprising:
selecting a candidate compound that is a SIRT1 activator, and
administering the SIRT1 activator to a test subject and detecting a SIRT1 activation-specific change in the test subject.
24 . The method of claim 23 , wherein the test subject is a diabetic model mouse and the SIRT1 activation specific change is a lowering of blood glucose.
25 . The method of claim 23 , wherein the test subject is a neurodegenerative disease model mouse and the SIRT1 activation specific change is a decrease in a neurodegenerative disease process or marker.
26 . The method of claim 25 , wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's, Huntington's, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Huntington's disease, and multiple sclerosis (MS).
27 . A fluorescent-free sirtuin substrate having a structural formula selected from the group consisting of Ac-RHKK Ac F-NH 2 and Ac-RHKK Ac W-NH 2 .
28 - 51 . (canceled)
52 . The method of claim 1 , wherein the fluorescent-free acetylated peptide, polypeptide, or protein substrate of SIRT1 comprises an activation cofactor moiety selected from the group consisting of tryptophan or phenylalanine at +1 relative to the position of lysine acetylation.
53 . The method of claim 1 , wherein the fluorescent-free acetylated peptide, polypeptide, or protein substrate of SIRT1 comprises a tryptophan activation cofactor moiety at +1 relative to the position of lysine acetylation.