Substrates for Efficient Fluorometric Screening Employing the NAD-Dependent Sirtuin 5 Lysine Deacylase (KDAC) Enzyme

The class III lysine deacylases (KDACs), also known as the sirtuins, have emerged as interesting drug targets for therapeutic intervention in a variety of diseases. To gain a deeper understanding of the processes affected by sirtuins, the development of selective small molecule modulators of individual isozymes has been a longstanding goal. Essential for the discovery of novel modulators, however, are good screening protocols and mechanistic insights with regard to the targets in question. We therefore evaluated the activities of the seven human sirtuin hydrolases against a panel of fluorogenic substrates. Both commonly used, commercially available substrates and novel chemotypes designed to address recent developments in the field of lysine post-translational modification were evaluated. Our investigations led to the discovery of two new fluorogenic ε-N-succinyllysine-containing substrates that enable highly efficient and enzyme-economical screening employing sirtuin 5 (SIRT5). Furthermore, optimized protocols for facile kinetic investigations were developed, which should be valuable for enzyme kinetic investigations. Finally, these protocols were applied to a kinetic analysis of the inhibition of SIRT5 by suramin, a potent sirtuin inhibitor previously shown by X-ray crystallography to bind the substrate pocket of the human SIRT5 KDAC enzyme

Benzodeazaoxaflavins as sirtuin inhibitors with antiproliferative properties in cancer stem cells

Inhibition of sirtuins has recently been proposed as a promising anticancer strategy. Some of the new benzodeazaoxaflavins (2a, 2b, and 2d) here reported as SIRT1/2 inhibitors were endowed with pro-apoptotic properties in human U937 leukemia cells and, most importantly, together with the prototype MC2141 (1) displayed antiproliferative effects in cancer stem cells from patients with colorectal carcinoma and glioblastoma multiforme, known to be highly tumorigenic, resistant to conventional cancer chemotherapy, and responsible, at least in part, for cancer relapse or recurrence. © 2012 American Chemical Society