Targeting HDAC3 dynamics: Allosteric role of Phe200 in inhibitor binding and breast cancer therapy.

Histone deacetylase 3 (HDAC3) is a key epigenetic regulator implicated in breast cancer progression and represents a promising therapeutic target. Here, we investigated 14 HDAC3-ligand complexes using molecular dynamics (MD) simulations and binding free energy calculations (MM/GBSA) to identify the determinants of inhibitor binding. Key residues consistently engaged across ligands included Gly132, His134-135, Phe144, Asp170, His172, Phe200, Asp259, Leu266, Gly296, Tyr298, and the catalytic Zn ion. Among the compounds, domatinostat and entinostat exhibited the strongest affinities (ΔGbind ≈ -70 kcal/mol), in reasonable agreement with experimental data (r = 0.60). Both ligands also showed small Highest Occupied Molecular Orbital-Lowest Unoccupied Molecular Orbital (HOMO-LUMO) gaps, high softness, and elevated electrophilicity indices, providing chemical cues for the design of next-generation HDAC3 inhibitors. Notably, ligand binding stabilized regions surrounding Phe200 and Asn370, restricting the conformational flexibility required for enzymatic activation. This supports an allosteric inhibition mechanism in which ligands lock HDAC3 into inactive conformations. Collectively, these findings offer mechanistic insights into HDAC3 regulation and highlight structural hot spots for the rational design of selective inhibitors with potential applications in targeted breast cancer therapy.