The design, synthesis and biological evaluation of novel small-molecule HDAC inhibitors
Creators: name: Di Fruscia, Paolo
Publisher: name: Imperial College London
Year: 2012

description: NAD+-Dependent histone deacetylases, also called sirtuins, are a family of enzymes that catalyse the cleavage of acetyl groups from lysine amino acid residues in histones and a variety of nonhistone proteins, playing a critical role in numerous biological processes. They have been found to be deregulated in several age-related diseases such as cancer and neurological disorders. Consequently, their role as therapeutic targets is widely explored. To date, seven different sirtuins (SIRT1-7) have been found in mammals, with SIRT1 and SIRT2 being the most studied. Nicotinamide (1), EX-527 (2) and 2-anilinobenzamide (3) have been reported as SIRT inhibitors (Figure 1). In an effort to discover alternative and selective nicotinamide pocket (C-pocket) sirtuin inhibitors, we decided to prepare and screen a small-molecule compound library based around the 10,11-dihydro-5H-dibenzo[b,f]azepine scaffold (4), structurally related to the above known SIRT inhibitors. Successfully, this approach led us to the identification of novel, highly selective low-micromolar SIRT2 inhibitors. In vitro screening assays revealed our best hit (5) to have an IC50 of 18 !M against SIRT2 and high selectivity over the SIRT1 isoform (Figure 2). Cellular screening assays, performed on MCF-7 cells, confirmed the in vitro selectivity and showed compound 5 to have antiproliferative activity at a concentration of 30 !M. Finally, docking studies with compound 5 were performed to predict its binding mode at the SIRT2 catalytic site and rationalise the observed isozyme Successfully, this approach led us to the identification of novel, highly selective low-micromolar SIRT2 inhibitors. In vitro screening assays revealed our best hit (5) to have an IC50 of 18 !M against SIRT2 and high selectivity over the SIRT1 isoform (Figure 2). Cellular screening assays, performed on MCF-7 cells, confirmed the in vitro selectivity and showed compound 5 to have antiproliferative activity at a concentration of 30 !M. Finally, docking studies with compound 5 were performed to predict its binding mode at the SIRT2 catalytic site and rationalise the observed isozyme selectivity. Further biological evaluation of hit molecule 5 is currently underway. 2) Towards the Discovery of Novel HDAC4 Inhibitors The class IIa HDAC4 enzyme is under extensive investigation, since its cellular functions have potential implications in human disease. Recently, it has been found that HDAC4 levels are significantly increased in platinum-resistant ovarian cancer cells and silencing of HDAC4 in those cell lines restores platinum sensitivity. Inhibition of this HDAC isozyme with pharmacological intervention could therefore potentially reverse clinically acquired platinum resistance in cancer cells. Hence, selective HDAC4 inhibitors have the potential to represent a novel class of anticancer agents, useful in combination therapy with other HDAC inhibitors or conventional chemotherapeutic agents. To date, only a limited number of HDAC4 inhibitors have been discovered and they all show dose-limiting toxicity, poor pharmacokinetics and metabolic instability, mainly due to the presence of reactive zinc-binding groups, such as the trifluoromethylketone and the hydroxamic acid moieties (Figure 3). In order to identify novel, non-toxic and selective HDAC4 inhibitors, we considered a novel molecular framework, bearing an alternative, drug-like zinc-binding group. A small compound library was synthesised and evaluated for its potential to inhibit the HDAC4 isoform, and restore platinum sensitivity in two different types of platinum-resistant ovarian cancer cell lines: PEO4 and SKOV3. Disappointingly, functional screens showed no HDAC4 inhibitory activity for the newly designed compounds, however a few molecules were found to be active in cellular assays. Remarkably, compounds 10 and 11 (Figure 4) were able to resensitise PEO4 and SKOV3 platinum-resistant ovarian cancer cell lines at low-micromolar concentrations, without showing any general cytotoxicity, even at concentrations up to 100 μM. Additional biological investigation is planned, with the aim to gain insights into the molecular mechanisms underlying this novel class of compounds.