Fig 1: Pre-incubation and HPLC-based sirtuin assays. (a) Inhibition of SIRT1 (20 nM) and SIRT2 (20 nM)-mediated deacetylation of the H3K9ac residue in a dodecameric peptide (50 µM) determined by HPLC. Error bars represent mean ± SEM of at least 2 independent experiments. See Table S4 (ESI†) for IC50 values against sirtuin mediated H3K9ac deacetylation. (b) Pre-incubation (30 min), with or without NAD+ (500 µM) for selected compounds against SIRT2 (100 nM) demyristoylation using ETDKmyr (50 µM) as substrate. Mean ± SEM based on at least 2 independent experiments performed in duplicate.
Fig 2: Kinetics of SIRT2 inhibition by compounds 25, 26, and 26-D. Sample rate experiment curves for compounds (a) TM, (b) 25, (c) 26, and (d) 26-D, with concentration of inhibitor for each experiment indicated on the right of the curve. (e) Common mechanisms of slow-binding inhibitor kinetics, with associated equilibrium and rate constants. Dependence of the first-order rate constant to reach steady state (kobs) on inhibitor concentration for compounds (f) 25 and (g) 26, following mechanism B, and (h) 26-D, which follows mechanism A. Continuous assays were performed with SIRT2 (20 nM), ETDKmyr (20 µM) as substrate, trypsin (20 ng µL-1), and at designated inhibitor concentrations. See Table S3 (ESI†) for additional details and complete data fitting.
Fig 3: Representative SIRT2 inhibitors. (a) Heterocyclic small molecule inhibitors.30,35,46,47 (b) Chimeric lysine mimic KPM-2.37 (c) Peptide macrocycle S2iL5.34 (d) Mechanism-based inhibitors based on modified lysine.24,32,40 (e) Summary of this study.
Fig 4: Cellular target engagement and SIRT2i effect on perinuclear a-tubulin acetylation. (a) Cellular thermal shift of SIRT2 in HEK293T cells subjected to 1 h treatment with inhibitor at designated concentrations or DMSO (vehicle). Please see Fig. S8 (ESI†) for complete dataset against SIRT1–3 (n = 3). (b–g) Immunofluorescence images (40×) of MCF-7 cells subjected to 6 h treatment with inhibitor [TSA (5 µM), 26 (5 µM), TM (25 µM), 26-D (25 µM)] or DMSO (vehicle). (g) Zoomed image for compound 26. DAPI (blue, nuclear counterstain) and Ac-a-tubulin (green). For additional images (single-filtered and zoomed) for all tested compounds, please see Fig. S9 (ESI†). The data are representative images from two individual experiments.
Fig 5: Structure–activity relationship of SIRT2 inhibitors. Potencies for inhibition of the deacylase activity of recombinant SIRT2 (100 nM) against QPKKac (shown with asterisks**) or ETDKmyr (shown in bold/italics) as substrate (50 µM) are given as mean IC50 values ± standard deviation (SD) or %inhibition. (a) Lead compounds. (b) Amide isosteres. (c) Optimization of position i + 2. (d) Optimization of position + 1. (e) Optimization of N-terminal group. (f) Final inhibitors. Data are based on two individual experiments performed in duplicate. See the ESI† (Scheme S1, Fig. S1–S3 and Table S2) for dose–response curves and selectivity profiling of additional inhibitors (S17–S23) against SIRT1–3. *A single asterisk denotes a stereogenic center.
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