Design of a novel nucleoside analog as potent inhibitor of the NAD dependent deacetylase, SIRT2

Abstract

Sirtuins (class III histone deacetylase) are evolutionarily conserved NAD(+)-dependent enzymes that catalyze the deacetylation of acetyl-lysine residues of histones and other target proteins. Because of their associations in various pathophysiological conditions, the identification of small molecule modulators has been of significant interest. In the present study, virtual screening was carried out with NCI Diversity Set II using crystal structure of hSIRT2 (PDB ID: 1J8F) as a model for the docking procedure to find potential compounds, which were then subjected to experimental tests for their in vitro SIRT2 inhibitory activity. One of the 40 compounds tested, NSC671136 (IUPAC name: 6-Acetyl-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-5-yl 2,4-dichlorobenzoate) has structurally unique scaffold, showed strong inhibitory activity towards SIRT2 with IC(50) of ~8.7 μM and to a lesser extent on SIRT1 activity. The reported compound is substantially potent compared to the published SIRT2 inhibitors and serves as an excellent base for future lead development.

Keywords: Deacetylase; Inhibitor; SIRT2; Sirtuins; Virtual screening.

Fig. 1

The biologically active hSIRT1 and hSIRT2. a The tertiary structure of the HDAC domain of hSIRT2 (PDB ID: 1J8F), used for docking studies, is drawn as ribbon model, colored from blue (N-terminus) to red (C-terminus). The ligand, NSC671136, shown by sticks, binds at the putative binding site of hSIRT2. The grey sphere represents the Zn²⁺ ion. b An SDS-PAGE profile of the purified HDAC domain of hSIRT1 (left panel) and hSIRT2 (right panel). Lane 1 on each panel represents the molecular weight marker, and lane 2 on each panel indicates hSIRT1 and hSIRT2, respectively. Electrophoresis was done with a 12% SDS polyacrylamide gel, which was stained with Coomassie Brilliant Blue. c Functional assay for hSIRT2. HDAC fluorimetric assay was performed using hSIRT2 protein produced in our lab in the presence of either 100 μM cambinol or 50 μM suramin. Activity of the SIRT2 protein was reduced by half in the presence of 100 μM cambinol and to totally negligible levels in the presence of 50 μM suramin

Fig. 2

Identification of NSC671136 as an inhibitor of hSIRT1 and hSIRT2. a The chemical structure of NSC671136. b A close-up view of a representative docked conformation of NSC671136 binds at the putative binding pocket of hSIRT2. The putative interacting amino acids at the binding pocket region are shown by sticks. c Comparison of NSC671136 mediated inhibition of hSIRT1 and hSIRT2 enzymes. Recombinant His-tagged human SIRT1 and SIRT2 proteins were assayed for deacetylase activity using the HDAC fluorescent activity assay. Results are expressed as the relative activity versus the control whose value is taken as 100% activity. All the values are the average of three independent experiments performed in duplicates

Fig. 3

Dose vs response (IC₅₀) curve of NSC671136 against His-tagged human SIRT2 enzyme. The curve fitting and IC50 determination of NSC671136 was performed using GraphPad Prism 5.0 software. Each data represents the average of triplicate samples