Determination of Slow-Binding HDAC Inhibitor Potency and Subclass Selectivity

Abstract

Histone deacetylases (HDACs) 1−3 regulate chromatin structure and gene expression. These three enzymes are targets for cancer chemotherapy and have been studied for the treatment of immune disorders and neurodegeneration, but there is a lack of selective pharmacological tool compounds to unravel their individual roles. Potent inhibitors of HDACs 1−3 often display slow-binding kinetics, which causes a delay in inhibitor−enzyme equilibration and may affect assay readout. Here we compare the potencies and selectivities of slow-binding inhibitors measured by discontinuous and continuous assays. We find that entinostat, a clinical candidate, inhibits HDACs 1−3 by a two-step slow-binding mechanism with lower potencies than previously reported. In addition, we show that RGFP966, commercialized as an HDAC3-selective probe, is a slow-binding inhibitor with inhibitor constants of 57, 31, and 13 nM against HDACs 1−3, respectively. These data highlight the need for thorough kinetic investigation in the development of selective HDAC probes.

Tiny Titans: Nanobodies as Powerful Tools for TR‐FRET Assay Development

“…Interestingly, the benzamide RGFP966 showed a relatively fast equilibration rate similar to the hydroxamate‐based ligands. This is consistent with a lower‐affinity binding mode for RGFP966 observed here and in a previous report profiling HDAC1 inhibition by steady‐state enzyme kinetics [31] …”

“…RGFP966 has previously been reported as an HDAC3‐selective inhibitor, lacking HDAC1/2 inhibitory activity at concentrations up to 15 μM [30] . However, more recently Moreno‐Yruela and Olsen have shown that RGFP966 binds with distinct low‐ and high‐affinity binding modes, yielding time‐dependent affinity for HDAC1, consistent with our findings [31] …”

“…This is consistent with a loweraffinity binding mode for RGFP966 observed here and in a previous report profiling HDAC1 inhibition by steady-state enzyme kinetics. [31] Harnessing split-luciferases for TR-FRET applications Thanks to the development of small, bright and stable luciferases such as NanoLuc (Promega), assays based on bioluminescent resonance energy transfer (BRET) have gained increasing popularity in recent years. [32] BRET utilizes light generated by the enzymatic conversion of a luciferase substrate as the energy donor for the excitation of an acceptor fluorophore.…”

“…[30] However, more recently Moreno-Yruela and Olsen have shown that RGFP966 binds with distinct low-and high-affinity binding modes, yielding time-dependent affinity for HDAC1, consistent with our findings. [31] An additional strength of the presented TR-FRET approach is the ability to directly observe binding kinetics. To demonstrate this aspect, we profiled the dose and time-dependent displacement of SAHA-FITC by HDAC inhibitors over the course of 24 h. Representative examples are shown in Figure 6G, confirming the fast-binding kinetics of LBH-589, while Cpd-60 exhibited a > 50-fold increase in binding affinity over 24 h. The time-dependence of HDAC inhibitor binding affinity for the complete reference set is summarized in Figure 6H.…”

Virtual Special Issue: Epigenetics 2022

“…Finally, in a cautionary tale, Moreno-Yruela and Olsen highlight the need for careful analysis of kinetic data when characterizing HDAC inhibitors. The importance of epigenetic targets to drug discovery is also reflected in the patent literature . We capture that in 11 Patent Highlights devoted to novel chemical matter that modulates various epigenetic targets such as protein arginine N -methyl­transferases (PRMT5), bromo­domains, and HDACs, which have potential utility in various disease states such as HIV, Alzheimer’s disease, cancer, fibrosis, inflammation, and cardio­vascular disease. − It is clear that epigenetic regulation will continue to be an important area for medicinal chemistry and drug discovery scientists.…”