Fig 2: Caco-2 cells after cyanidin treatment and expression of SIRT6 protein. Cells were exposed to 0.5% DMSO (control) or various concentration of compound 17 (n = 5) for 24 h. (A) Representative light microscopy images of Caco-2 cells after control (Cnt), 100 µM cyanidin or 200 µM cyanidin treatment. (B) Immunoblotting analysis of SIRT6 protein. SIRT6 protein levels (MW: 39 kDa) were normalized relative to a-tubulin (MW: 50 kDa) and quantification is represented as fold change respect to control. Values are expressed as mean ± SEM of four independent experiment (*p values < 0.05; one way-ANOVA). SIRT6 expression were determined by immunoblotting (B).

Fig 3: SIRT6 and locations of binding sites of activators (light turquoise), inhibitors (yellow), peptide substrates (blue) and for NAD+ (brownish gray). Close-up view of the interactions of best inhibitors, compound 4 (A) and compound 5 (B) and best activator, compound 17 (C). The best activators compound 17 (D) and compound 18 (E) induce changes on the ß6/a6 loop and the orientation of Trp186 and Glu187 similar to known activators oleic acid (F) and linoleic acid (G). Interactions: Yellow dashes indicate hydrogen-bonding, dark green dashes indicate p-p stacking and light purple dash indicates salt bridge (interaction to Asp185). Pink residues and loops indicate the original residue and loop orientation in the protein structure before inhibitor or activator binding.

Fig 4: General scaffold of flavonoids (A). The structures of the most potent SIRT6 modulators are displayed, and additional structures are available in supplementary Tables S1 and S2. The yellow background represents inhibitors, gray represents inhibitors and activators and blue represents activators (B).