4.3. Cell-Free Assay on Isolated SIRT1 Enzyme

SIRT1 activity was measured using a SIRT1 direct fluorescent screening assay kit (Cayman Chemical, Ann Arbor, MI, USA), according to the manufacturer’s protocol. Briefly, potential SIRT1 activators were freshly dissolved in DMSO (10⁻² M) and diluted in the assay buffer up to a final concentration of 100 µM. Then, the reconstituted SIRT1 human recombinant enzyme, assay buffer, and selected compounds or vehicle (DMSO 1%) were added in triplicate to a 96-well plate. The SIRT1 activator resveratrol 100 µM [43] and the sirtuin inhibitor sirtinol 10 µM were used as positive and negative controls, respectively. The multi-well plate was incubated in the dark for 45 min at room temperature on a shaker. Then, 50 µL of a stop/developing solution was added to each well and incubated for 30 min at room temperature to allow the generation of a fluorescent reaction product. The fluorescence intensity was measured with the microplate reader EnSpire (PerkinElmer, Waltham, MA, USA) at λex = 350 nm and λem = 450 nm. The possible interference of compounds with the developer and/or fluorophore was evaluated before performing the assay. In a separate set of experiments, resveratrol and the most effective SIRT1 activator compound 3d were tested also at lower concentrations (10 and 30 µM). Once the fluorescence values of the background (DMSO 1%) were subtracted, results were normalized considering the vehicle as 0% of SIRT1 activity and resveratrol 100 µM as 100%. Data are shown as the mean ± standard error of the mean (SEM). Statistical analysis included a one-way ANOVA followed by Bonferroni’s post hoc test, and statistical significance was set at p < 0.05. Analysis was performed with the software GraphPad Prism (version 5.0).