Inhibition Assay: Different concentrations of TA, TB, TH, or TM were pre-incubated with 1 μM of the sirtuin and 1 mM NAD in 20 mM Tris-HCl buffer (pH 8.0) with 1 mM dithiothreitol (DTT) at 37° C. for 30 minutes. Then 0.05 mM acyl peptides (H3K9Ac for Sirt1, Sirt2 and Sirt3; H3K9Su for Sirt5; H3K9Myr for Sirt6 and Sirt7) were added to initiate the reactions. The total reaction volume iwas 60 mL. Then reactions were incubated at 37° C. for a certain amount of time (2 minutes for Sirt1, 10 minutes for Sirt2 and Sirt5, 30 minutes for Sirt3, and 1 hour for Sirt6 and Sirt7). The reactions were stopped by adding 60 mL of an aqueous solution containing 200 mM HCl and 320 mM acetic acid. After centrifugation to remove precipitated proteins, the supernatant was analyzed by HPLC with a reverse phase C18 column (250×4.6 mm, 90 A, 10 mm, GraceVydac, Southborough, Mass.), with a linear gradient of 0% to 20% B for 10 minutes (1 mL/min).
Inhibition of recombinant human N-terminal His6-tagged SIRT7 (68 to 354 residues) expressed in Escherichia coli Transetta(DE3) using AcIQF as substrate preincubated for 30 mins followed by substrate and NAD+ addition measured after 2 hrs by fluorescence assay
Inhibition of tRNA-activated deacetylation activity of recombinant human full length N-terminal His6-tagged SIRT7 expressed in Escherichia coli using H2N-GGKAPR-[Nepsilon-acetyl-lysine]-QLATKA-CONH2 as substrate measured after 30 mins by HPLC analysis
Inhibition of tRNA-activated His6-tagged human SIRT7 deacetylation activity using acetyl-lysine as substrate in presence of beta-NAD+ after 30 mins by reversed-phase HPLC analysis
Substrate activity at human N-terminal His-tagged SIRT7 (68 to 354 residues) expressed in Escherichia coli at 0.5 uM measured after 2 hrs in presence of NAD+ and trypsin by fluorescence assay
Substrate activity at human N-terminal His-tagged SIRT7 (68 to 354 residues) expressed in Escherichia coli at 5 uM measured after 2 hrs in presence of NAD+ and trypsin by fluorescence assay
Substrate activity at human N-terminal His-tagged SIRT7 (68 to 354 residues) expressed in Escherichia coli at 50 uM measured after 2 hrs in presence of NAD+ and trypsin by fluorescence assay
Binding affinity to SIRT7 (unknown origin) assessed as increase in fluorescence intensity at 2.5 uM measured at 5 mins interval for 1 hr in presence of NAD+ by fluorometric method relative to control
Inhibition of human recombinant SIRT7 at 10 uM incubated for 20 mins followed by mixing and measure after 30 mins by Luminescence assay relative to control