Determination of Slow-binding HDAC Inhibitor Potency and Subclass Selectivity

Histone deacetylases (HDACs) 1-3 regulate chromatin structure and gene expression. These three enzymes are targets for cancer chemotherapy and are 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 potency and selectivity 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 HDAC3-selective probe, is a slow-binding inhibitor with inhibitor constants of 57 nM, 31 nM, and 13 nM against HDACs 1-3, respectively. These data highlight a need for thorough kinetic investigation in the development of selective HDAC probes.

Kinetic and Interaction Studies of Adenosine-5′-Triphosphate (ATP) Hydrolysis with Polyoxovanadates

The reactivity of polyoxovanadates towards adenosine-5′-triphosphate (ATP) hydrolysis at pH 2, 4, 6 and 7 is reported. Detailed kinetic investigation of ATP hydrolysis in the presence of polyoxovanadates was performed through multinuclear nuclear magnetic resonance (NMR) spectroscopy. In general, rate acceleration of up to five orders of magnitude was observed in the presence of vanadates compared to spontaneous ATP hydrolysis, with the greatest acceleration observed for reactions carried out at pH 2. Interestingly, the effectiveness of vanadates in promoting ATP hydrolysis decreased as the pH of the reaction solution increased; nevertheless, at pH = 7, the rate increase of one order of magnitude in comparison to blank reactions was still observed. Interactions between vanadate species in solution and ATP were investigated by means of 31P and 51V NMR spectroscopy, and this pointed towards the preferential interaction of vanadium with the phosphate groups rather than other regions of the ATP molecule.