Resveratrol-like Compounds as SIRT1 Activators

Abstract

The sirtuin 1 (SIRT1) activator resveratrol has emerged as a promising candidate for the prevention of vascular oxidative stress, which is a trigger for endothelial dysfunction. However, its clinical use is limited by low oral bioavailability. In this work, we have applied a previously developed computational protocol to identify the most promising derivatives from our in-house chemical library of resveratrol derivatives. The most promising compounds in terms of SIRT1 activation and oral bioavailability, predicted in silico, were evaluated for their ability to activate the isolated SIRT1 enzyme. Then, we assessed the antioxidant effects of the most effective derivative, compound 3d, in human umbilical vein endothelial cells (HUVECs) injured with H₂O₂ 100 µM. The SIRT1 activator 3d significantly preserved cell viability and prevented an intracellular reactive oxygen species increase in HUVECs exposed to the oxidative stimulus. Such effects were partially reduced in the presence of a sirtuin inhibitor, sirtinol, confirming the potential role of sirtuins in the activity of resveratrol and its derivatives. Although 3d appeared less effective than resveratrol in activating the isolated enzyme, the effects exhibited by both compounds in HUVECs were almost superimposable, suggesting a higher ability of 3d to cross cell membranes and activate the intracellular target SIRT1.

Keywords: SIRT1 activators; computer aided drug discovery; endothelial dysfunction; nature-inspired compounds; oxidative stress; resveratrol; resveratrol-like compounds; sirtuin 1; structure-activity relationship (SAR); vascular endothelium.

Figure 1

Chemical structure of resveratrol and general structure of the library of resveratrol-like compounds.

Figure 2

Chemical structure of the compounds in our in-house chemical library.

Scheme 1

Synthesis of mono- or poly-hydroxylated (E)-benzaldehyde O-benzyl oxime derivatives 1a–e, 2a–e, 3b, 3d–e.

Figure 3

(A) Crystal structure of SIRT1 (grey cartoon representation) in complex with resveratrol (sticks representation) (PDB ID 5BTR); the different binding sites of resveratrol, namely, #site1, #site2, and #site3 are represented in cyan, magenta, and orange, respectively; (B–D) binding mode of compound 3d (dark purple sticks) into SIRT1 with particular focus on #site1 (panel B), #site2 (panel C) and #site3 (panel D). The p53-AMC-peptide is reported as green sticks. Residues in the binding sites are represented by lines while hydrogen bonds are shown as grey dotted lines. Pictures were generated by PyMOL software (The PyMOL Molecular Graphics System, v1.8; Schrödinger, LLC, New York, NY, USA, 2015).

Figure 4

Effects of the screened compounds on isolated SIRT1 enzyme activity. SIRT1 activity is expressed as % of the effect promoted by the reference compound resveratrol (Resv) 100 µM. The sirtuin inhibitor sirtinol (Sirt) 10 µM was used as a negative control. Results are shown as mean ± SEM. * indicates significant difference vs. vehicle (*** p < 0.001).

Figure 5

Graphic representation of SARs resveratrol-like compounds.

Figure 6

Effects of increasing concentrations of resveratrol and the synthetic derivative 3d (10, 30, and 100 µM) on the isolated SIRT1 enzyme. SIRT1 activity is expressed as % of the effect promoted by the reference compound resveratrol (Resv) 100 µM. Results are shown as mean ± SEM. * indicates significant difference vs. vehicle (** p < 0.01; *** p < 0.001).

Figure 7

Preventive effects of resveratrol and compound 3d against H₂O₂-induced reduction in cell viability: A perfect example of a biphasic concentration–response curve. Bars indicate cell viability (%) of HUVECs exposed to H₂O₂ 100 µM for 2 h. Pre-incubation of resveratrol and compound 3d (both 30 µM) for 1 h significantly prevented H₂O₂-induced reduction in cell viability. Conversely, both resveratrol and compound 3d 10 µM and 100 µM did not preserve cell viability in HUVECs exposed to the pro-oxidative stimulus. Data are shown as mean ± SEM. * indicates significant difference vs. vehicle (*** p < 0.001), § indicates significant difference vs. H₂O₂ 100 µM (§§§ p < 0.001) and ^ indicates significant difference vs. Resv 100 µM (^^^ p < 0.001).

Figure 8

Preventive effects of resveratrol and compound 3d against H₂O₂-induced reduction in cell viability: Potential role of sirtuins. Bars indicate cell viability (%) of HUVECs treated with H₂O₂ 100 µM for 2 h. In the absence of the sirtuin inhibitor sirtinol (Sirt 10 µM), both resveratrol (Resv) and compound 3d (30 µM), incubated for 1 h, significantly protected endothelial cells from pro-oxidative cell damage. Conversely, in the presence of Sirt 10 µM, the preventive effects of resveratrol and compound 3d against H₂O₂-induced reduction in cell viability were significantly reduced. Data are shown as mean ± SEM. * indicates significant difference vs. vehicle (*** p < 0.001), § indicates significant difference vs. H₂O₂ 100 µM (§§§ p < 0.001), # indicates significant difference vs. Resv 30 µM + H₂O₂ 100 µM (# p < 0.05) while ^ indicates significant difference vs. 3d 30 µM + H₂O₂ 100 µM (^ p < 0.05).

Figure 9

Preventive effects of resveratrol and 3d against H₂O₂-induced intracellular ROS production in HUVECs. Bars indicate intracellular ROS production (%) in HUVECs exposed to H₂O₂ 100 µM for 2 h. Pre-incubation of resveratrol (Resv) and 3d (10, 30, and 100 µM) for 1 h prevented H₂O₂-induced ROS production in a concentration-dependent manner. Data are shown as mean ± SEM. * indicates significant difference vs. vehicle (*** p < 0.001) while § indicates significant difference vs. H₂O₂ 100 µM (§ p < 0.05; §§§ p < 0.001).

Figure 10

Preventive effects of resveratrol and compound 3d against H₂O₂-induced increase in intracellular ROS production: Potential role of sirtuins. Bars indicate intracellular ROS production (%) in HUVECs treated with H₂O₂ 100 µM for 2 h. In the absence of the sirtuin inhibitor sirtinol (Sirt 10 µM), both resveratrol (Resv) and 3d (30 µM) incubated for 1 h significantly protected endothelial cells from the ROS increase. Conversely, in the presence of Sirt 10 µM, the preventive effects of resveratrol and 3d against H₂O₂-induced increase in ROS were almost abolished. Data are shown as mean ± SEM. * indicates significant difference vs. vehicle (*** p < 0.001), § indicates significant difference vs. H₂O₂ 100 µM (§ p < 0.05; §§ p < 0.01), # indicates significant difference vs. Resv 30 µM + H₂O₂ 100 µM (# p < 0.05) while ^ indicates significant difference vs. 3d 30 µM + H₂O₂ 100 µM (^^ p < 0.01).

Figure 11

Summary of the work. Resveratrol-like compounds from our in-house library were first screened in silico to predict their ability to activate SIRT1 enzyme and bioavailability. Then, six selected compounds were tested in vitro to assess the potential activation of isolated SIRT1 enzyme. The best SIRT1 activator (compound 3d) underwent pharmacological investigation in human endothelial cells (HUVECs). In this final set of experiments, both the reference SIRT1 activator resveratrol and compound 3d prevented H₂O₂-induced cell viability reduction and ROS production, likely via activation of SIRT1.