Virtual Screening Combined with Enzymatic Assays to Guide the Discovery of Novel SIRT2 Inhibitors

Abstract

Sirtuin isoform 2 (SIRT2) is one of the seven sirtuin isoforms present in humans, being classified as class III histone deacetylases (HDACs). Based on the high sequence similarity among SIRTs, the identification of isoform selective modulators represents a challenging task, especially for the high conservation observed in the catalytic site. Efforts in rationalizing selectivity based on key residues belonging to the SIRT2 enzyme were accompanied in 2015 by the publication of the first X-ray crystallographic structure of the potent and selective SIRT2 inhibitor SirReal2. The subsequent studies led to different experimental data regarding this protein in complex with further different chemo-types as SIRT2 inhibitors. Herein, we reported preliminary Structure-Based Virtual Screening (SBVS) studies using a commercially available library of compounds to identify novel scaffolds for the design of new SIRT2 inhibitors. Biochemical assays involving five selected compounds allowed us to highlight the most effective chemical features supporting the observed SIRT2 inhibitory ability. This information guided the following in silico evaluation and in vitro testing of further compounds from in-house libraries of pyrazolo-pyrimidine derivatives towards novel SIRT2 inhibitors (1–5). The final results indicated the effectiveness of this scaffold for the design of promising and selective SIRT2 inhibitors, featuring the highest inhibition among the tested compounds, and validating the applied strategy.

Novel Thiazole-Based SIRT2 Inhibitors Discovered via Molecular Modelling Studies and Enzymatic Assays

“…This piece of information revealed structural differences in the positioning featured by the residues of the SIRT2 binding site, when in presence of different chemo-types, via induced-fit events. Notably, this kind of result was recently described by us when comparing eight X-ray crystallographic data of SIRT2 [ 23 ]. Conversely, the protein domains outside the inhibitor binding cavity were properly superposed, as shown by the alignment and superimposition of the two PDB codes in Figure S5 .…”

“…To ascertain the reliability of the procedure so as to identify novel putative SIRT2 ligands, the known SIRT2 inhibitor AGK2 [ 42 ] was also evaluated via molecular docking calculations. Notably, this procedure was recently applied by us with success to identify novel scaffolds for the design of new SIRT2 inhibitors [ 23 ]. According to our results, all of the compounds occupied SIRT2 pockets A and B with the terminal aromatic ring.…”

“…The synthesis of peptides H3K9Ac and H3K9Palm and the evaluation of the SIRT2 deacetylase activity and of the SIRT6 deacetylase and depalmitoylase activity were carried out as previously described [ 23 ]. The IC 50 of compound 1a was determined as in [ 43 ].…”

“…Indeed, efforts to explore SIRT2 selectivity were achieved in 2015 through the publication of the first X-ray crystallographic structure of the protein in complex with the potent and selective inhibitor SirReal2 (PDB code = 4RMG) [ 22 ]. Subsequent studies led to further different experimental data regarding SIRT2 in the presence of further different chemo-types as SIRT2 inhibitors [ 23 ].…”

Recent Advances in the Discovery of SIRT1/2 Inhibitors via Computational Methods: A Perspective

“…Regarding the non-reaction mechanism related to SIRT2Is, most of the reported Xcrystallographic data contain an SIRT2 isoform-selective ligand. Indeed, most of the chemical diversity thus far explored is limited to the SirReal2 series, as previously reported by us [66] and confirmed by the abundance of X-ray SIRT2 structures complexes containing SirReal2 or its derivatives (see Table 3).…”