SIRT1, 2, and 3 inhibition assay

The continuous assay for comparing isoform specificity of S2P04 was performed using the universal fluorogenic sirtuin substrate described by Schuster et al.⁴¹. Briefly, the inhibition reactions were performed in 50-µL volumes at 37 °C in black, half-area, 96-well plates. The sirtuin enzyme (0.25 µM) was preincubated with different concentrations of the peptide S2P04 (0.0064–50 µM for SIRT1 and SIRT2, 0.5–100 µM for SIRT3) or TB (0.16–150 µM for SIRT1–SIRT3), and 1 mM NAD⁺ in assay buffer (20 mM Tris, pH 7.8, 150 mM NaCl, 5 mM MgCl₂, and 1 mM DTT) for 15 min. Following incubation, the reaction started with the addition of the fluorogenic substrate (5 µM), and product formation was monitored by measuring the fluorescence intensity every 20 s for 5 min (SIRT2) or 30 min (SIRT1 and SIRT3) in a BioTek Synergy H1 microplate reader at λ_ex = 320 nm and λ_em = 408 nm (gain = 130 and read height = 7 mm). Initial rates of product formation were determined by a linear regression of the plot of fluorescence intensity vs. time and normalized relative to 0% (max inhibitor) and 100% (no inhibitor) controls. The assay was repeated to determine the IC₅₀ values of all BuK peptides for SIRT2. IC₅₀ values were determined by nonlinear regression of the plot of the normalized initial rate vs. inhibitor concentration using GraphPad Prism. The determined IC₅₀ values are provided in Supplementary Table ², and inhibition curves can be seen in Supplementary Fig. ²⁰.