Sirtuin Inhibition Assay

A typical reaction contained 800 μM NAD⁺, 500 μM peptide substrate (H3K9Ac for SIRT2, SIRT3 and SIRT6, p53K382Ac for SIRT1 and SIRT5), varying concentrations of trichostatin A (TSA) in 100 mM phosphate buffer pH 7.5. The reactions were initiated by the addition of 10 μM of sirtuin and were incubated at 37°C before being quenched by 8 μL of 10% TFA. The incubation time was controlled so that the conversion of substrate was less than 15%. The samples were then injected on an HPLC fitted to a Macherey-Nagel Nucleosil C18 column. NAD⁺, NAM and AADPR peaks were resolved using a gradient of 0 to 20% methanol in 20 mM ammonium acetate. Chromatograms were analyzed at 260 nm. Reactions are quantified by integrating areas of peaks corresponding to NAD⁺ and AADPR. Rates were plotted as a function of TSA concentration, and points were fitted to the Morrison’s quadratic equation:

Where ν_i is the inhibited turnover, ν₀ is the uninhibited turnover, [E]_T is the total concentration of sirtuin, [I]_T is the total inhibitor concentration, Kiapp is the apparent inhibition constant. This equation has been used to analyze the tight binding inhibition.^35,36 The affinity is determined in terms of free and bound concentrations of enzyme and inhibitor accounting for the effect of binary complex formation on the concentration of free inhibitor.

In addition to TSA, several other small molecules including sodium butyrate, valproic acid and SAHA were also tested against SIRT6. Reactions were carried out in 100 mM phosphate buffer pH 7.5 containing 800 μM NAD⁺, 500 μM H3K9Ac, various concentrations of the small molecules: 0, 0.5, 2 and 5 mM of sodium butyrate or valproic acid; 0, 0.1, 1, 10, 50, 100 and 200 μM of SAHA. The reactions were initiated by the addition of 10 μM of SIRT6 and were incubated at 37°C for 2 h before being quenched by 8 μL of 10% TFA. The samples were then injected on an HPLC fitted to a Macherey-Nagel Nucleosil C18 column. NAD⁺, NAM and AADPR peaks were resolved using a gradient of 0 to 20% methanol in 20 mM ammonium acetate. Chromatograms were analyzed at 260 nm. Reactions are quantified by integrating areas of peaks corresponding to NAD⁺ and AADPR.