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This ZnF-UBD mediates key protein\u2013protein 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\u25a1\u25a1 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\u2013 ubiquitin signaling.<\/jats:p>","DOI":"10.64898\/2025.12.18.695271","type":"posted-content","created":{"date-parts":[[2025,12,20]],"date-time":"2025-12-20T15:10:11Z","timestamp":1766243411000},"source":"Crossref","is-referenced-by-count":0,"title":["Quinazolinone and Phthalazinone Inhibitors of the HDAC6\/Ubiquitin Protein-Protein Interaction"],"prefix":"10.64898","author":[{"given":"Sydney","family":"Gordon","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Biophysics, University of Pennsylvania, PA 19104"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jordi C.J.","family":"Hintzen","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biophysics, University of Pennsylvania, PA 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