Quinazolinone and Phthalazinone Inhibitors of the HDAC6/Ubiquitin Protein–Protein Interaction

Abstract 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). This ZnF-UBD 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 validate previously reported small-molecule inhibitors of the HDAC6 ZnF-UBD/ubiquitin interaction and describe novel N-alkyl moieties based on quinazolinone and phthalazinone scaffolds. Starting from known quinazolinone and phthalazinone scaffolds, a literature and modeling-guided scaffold hop revealed potential for an extended phthalazinone series. Results obtained both in fluorescence polarization (FP) and differential scanning fluorimetry (DSF) confirm this hypothesis. Additionally, late-stage diversification yields compounds with improved predicted physicochemical properties. Finally, machine-learning-based co-folding affinity predictions correlate with experimental IC□□ rank order, highlighting their utility in PPI inhibitor design. These studies continue expanding the chemical space of HDAC6 ZnF-UBD inhibitors and build upon existing foundations for future therapeutic and mechanistic exploration of HDAC6– ubiquitin signaling.