Stabilizing HDAC11 with SAHA to assay slow-binding benzamide inhibitors

Abstract

Among 18 human histone deacetylases (HDAC), HDAC11 is least studied. MS275, a benzamide HDAC inhibitor (HDACi), was stereotypically considered to selectively target Class I HDACs. We verified this slow-binding inhibitor also targeted HDAC11. In a traditional enzyme based assay, MS275 at low concentrations surprisingly behaved as an agonist. This was attributed to the poor stability of HDAC11 which lost 40% activity in 3h at 37°C. By adding 0.2μM SAHA, HDAC11 activity was stabilized during the 3-h assay period. Since 0.2μM SAHA inhibited 50% HDAC11 activity, the apparent IC₅₀' of MS275 was adjusted to the true IC₅₀=0.65μM. Finally, the new method demonstrated its superiority in one-dose-screening assays by decreasing false negative results. This work highlighted an optimized strategy to assay slow-binding inhibitors of unstable proteins with known fast-binding inhibitors. It should be especially useful in a hit-discovery stage to find moderate potent compounds.

Keywords: Chaperone molecule; HDAC11; Inhibitor screening; Slow-binding; Unstable protein.