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). 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 ₅₀ 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.