The Importance of the “Time Factor” for the Evaluation of Inhibition Mechanisms: The Case of Selected HDAC6 Inhibitors

SIMPLE SUMMARY: Protein lysine acetylation is, with phosphorylation, the most common regulatory post-translational modification of proteins. Histone deacetylases (HDAC) catalyze the removal of acetyl groups from histone and non-histone proteins, participating in the modulation of several pathways. Histone deacetylase 6 is perhaps the most complex among histone deacetylases, comprising two catalytic domains, an N-terminal microtubule-binding domain and a C-terminal ubiquitin–binding domain. Interfering with its catalytic activity by using small synthetic molecules has been shown to be beneficial in the treatment of cancer, and neurological and immunological disorders. Thus, the development of potent and selective inhibitors of HDAC6 is an active field of medicinal chemistry. We shall here discuss the importance of monitoring the kinetics of onset and relief of inhibition to contribute important information on inhibition mechanisms during drug design/development campaigns using selected HDAC6 inhibitors as examples. ABSTRACT: Histone deacetylases (HDACs) participate with histone acetyltransferases in the modulation of the biological activity of a broad array of proteins, besides histones. Histone deacetylase 6 is unique among HDAC as it contains two catalytic domains, an N-terminal microtubule binding region and a C-terminal ubiquitin binding domain. Most of its known biological roles are related to its protein lysine deacetylase activity in the cytoplasm. The design of specific inhibitors is the focus of a large number of medicinal chemistry programs in the academy and industry because lowering HDAC6 activity has been demonstrated to be beneficial for the treatment of several diseases, including cancer, and neurological and immunological disorders. Here, we show how re-evaluation of the mechanism of action of selected HDAC6 inhibitors, by monitoring the time-dependence of the onset and relief of the inhibition, revealed instances of slow-binding/slow-release inhibition. The same approach, in conjunction with X-ray crystallography, in silico modeling and mass spectrometry, helped to propose a model of inhibition of HDAC6 by a novel difluoromethyloxadiazole-based compound that was found to be a slow-binding substrate analog of HDAC6, giving rise to a tightly bound, long-lived inhibitory derivative.