A continuous sirtuin activity assay without any coupling to enzymatic or chemical reactions.

Sirtuins are NAD(+) dependent lysine deacylases involved in many regulatory processes such as control of metabolic pathways, DNA repair and stress response. Modulators of sirtuin activity are required as tools for uncovering the biological function of these enzymes and as potential therapeutic agents. Systematic discovery of such modulators is hampered by the lack of direct and continuous activity assays. The present study describes a novel continuous assay based on the increase of a fluorescence signal subsequent to sirtuin mediated removal of a fluorescent acyl chain from a modified TNFα-derived peptide. This substrate is well recognized by human sirtuins 1-6 and represents the best sirtuin 2 substrate described so far with a kcat/KM-value of 176 000 M(-1)s(-1). These extraordinary substrate properties allow the first determination of Ki-values for the specific Sirt2 inhibitory peptide S2iL5 (600 nM) and for the quasi-universal sirtuin inhibitor peptide thioxo myristoyl TNFα (80 nM).

The discovery of a highly selective 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one SIRT2 inhibitor that is neuroprotective in an in vitro Parkinson's disease model.

Sirtuins, NAD(+) -dependent histone deacetylases (HDACs), have recently emerged as potential therapeutic targets for the treatment of a variety of diseases. The discovery of potent and isoform-selective inhibitors of this enzyme family should provide chemical tools to help determine the roles of these targets and validate their therapeutic value. Herein, we report the discovery of a novel class of highly selective SIRT2 inhibitors, identified by pharmacophore screening. We report the identification and validation of 3-((2-methoxynaphthalen-1-yl)methyl)-7-((pyridin-3-ylmethyl)amino)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one (ICL-SIRT078), a substrate-competitive SIRT2 inhibitor with a Ki value of 0.62 ± 0.15 µM and more than 50-fold selectivity against SIRT1, 3 and 5. Treatment of MCF-7 breast cancer cells with ICL-SIRT078 results in hyperacetylation of α-tubulin, an established SIRT2 biomarker, at doses comparable with the biochemical IC50 data, while suppressing MCF-7 proliferation at higher concentrations. In concordance with the recent reports that suggest SIRT2 inhibition is a potential strategy for the treatment of Parkinson's disease, we find that compound ICL-SIRT078 has a significant neuroprotective effect in a lactacystin-induced model of Parkinsonian neuronal cell death in the N27 cell line. These results encourage further investigation into the effects of ICL-SIRT078, or an optimised derivative thereof, as a candidate neuroprotective agent in in vivo models of Parkinson's disease.