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. Table of Contents artwork O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/473277v2_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1b1ef16org.highwire.dtl.DTLVardef@c79359org.highwire.dtl.DTLVardef@9ae077org.highwire.dtl.DTLVardef@6ad25_HPS_FORMAT_FIGEXP M_FIG C_FIG