Quinazolinone and Phthalazinone Inhibitors of the HDAC6/Ubiquitin Protein-Protein Interaction.

Histone deacetylase 6 (HDAC6) is a class IIb histone deacetylase that regulates diverse cytosolic acetylation through its two catalytic deacetylase domains and a C-terminal zinc finger ubiquitin-binding domain (ZnF-UBD) which mediates key protein-protein interactions (PPIs) that couple deacetylation and ubiquitin-dependent degradation. While most HDAC6 inhibitors target the catalytic domains, the ZnF-UBD represents an underexplored target. Here, we report the development of small-molecule inhibitors of the HDAC6 ZnF-UBD/ubiquitin interaction based on quinazolinone and phthalazinone scaffolds. Starting from known quinazolinone inhibitors, a modeling-guided scaffold hop revealed a novel phthalazinone series and late-stage diversification yield compounds with improved predicted physicochemical properties. Furthermore, machine-learning-based co-folding affinity predictions reproduced experimental IC rank order, highlighting their utility in PPI inhibitor design. These studies expand the chemical space of HDAC6 ZnF-UBD inhibitors and provide a foundation for future therapeutic and mechanistic exploration of HDAC6-ubiquitin signaling.