Criteria-guided selection of alternative zinc-binding groups of HDAC3 inhibitors using in silico and in vitro approaches.

Histone deacetylases (HDACs) are epigenetic enzymes linked to several biological functions and diseases. Class I HDACs (HDAC1-3) share high protein sequence and active site similarity. Hydroxamate-based inhibitors, while highly potent, are non-selective with poor pharmacokinetic profile, prompting exploration of alternative zinc-binding groups. The study applied criteria-guided identification of HDAC3 inhibitors with alternative zinc-binding groups following structure-based virtual screening, with a prioritisation of HDAC3-specific binding interactions and bidentate/monodentate interaction with the zinc ion. Metal-focused chemical libraries were screened virtually followed by biochemical assessment of inhibitory activity of the hit compounds and cell cytotoxicity evaluation. Compound a displayed sub-micromolar HDAC3 potency (IC50 = 0.99 μM) but limited selectivity for HDAC3, whereas compound b showed reduced potency (IC50 = 15 μM) yet possessed HDAC3 preference. Molecular docking suggests that the zinc-binding group of the compounds form bidentate/monodentate metal coordination with the zinc ion and hydrogen bond interaction with Asp170. Compound a showed a high cytotoxicity IC50 value of 34.5 μM on neuroblastoma cells (IMR-32 cells) and IC50 on liver (HepG2) cells was >100 μM, while HDAC3 inhibition occurred at sub-micromolar concentrations. Compound b showed HDAC3 inhibitory preference without inducing cytotoxicity on liver and neuroblastoma cells.