Buhrlage, Sara JJaen Maisonet, Isabella2026-03-0420252026-02-272026Jaen Maisonet, Isabella. 2026. Expanding the toolbox for deubiquitylase pharmacology. Doctoral Dissertation, Harvard University Graduate School of Arts and Sciences.32401488https://dash.harvard.edu/handle/1/42731177Ubiquitylation is a posttranslational modification that governs protein cellular fate. Balanced ubiquitin signaling is vital for regulating cell function, replication, and survival, and its dysregulation is implicated in various indications such as neurodegeneration, cancer, autoimmunity, and neurodevelopmental disorders. Ubiquitin is covalently attached to substrates in a stepwise manner by an E1, E2, E3 enzymatic cascade, and is removed by a class of isopeptidases called deubiquitylases (DUBs). DUBs can stabilize both oncogenic drivers and tumor suppressors in cancer. Additionally, both loss-of-function and gain-of-function DUB mutations can cause or drive disease pathologies. Therefore, targeting DUBs with small-molecule tools offers direct and indirect therapeutic approaches for disease intervention. The work in this thesis expands the available chemical toolbox for the interrogation of DUB biology in healthy and disease states, and provides starting points for DUB-targeted therapeutic development. I focus on two DUBs, USP7 and USP48. The potential for USP7-targeted therapeutics in cancer via the regulation of the p53/MDM2 pathway is well-documented. More recently, USP7 has emerged as a critical regulator in neurodevelopment, as mutations in the USP7 gene were identified as causal for the rare neurodevelopmental disorder, Hao-Fountain syndrome (HAFOUS). Our interest in USP48 arose from recent studies by Dana-Farber colleagues that identified USP48 knockout as a novel sensitizer to hypomethylating agents in acute myeloid leukemia and potentially other cancer types. In Chapter 2, I describe the discovery and rigorous mechanistic characterization of MS-8, a first-in-class small-molecule DUB activator. I demonstrate that MS-8 allosterically engages ubiquitin-specific protease 7 (USP7) and induces enzymatic activation, similar to the mechanism of auto-activation by the C-terminal tail of USP7. We assess the therapeutic potential of our activator in vitro and in cells against a panel of HAFOUS patient-derived USP7 variants and identify a subset of mutations sensitive to MS-8 driven activation. Additionally, we provide an initial structure-activity relationship for MS-8 and highlight viable exit vectors on the scaffold for the development of heterobifunctional DUB recruiters. In Chapter 3, I introduce a next generation covalent inhibitor for USP7. Using a structure-guided approach, we elaborate the reported noncovalent Compound 41 into a covalent USP7 inhibitor. Through a focused medicinal chemistry campaign, we optimize our covalent series and validate its useability as a cellular probe in p53-wildtype and p53-mutant cell lines. Lastly, in Chapter 4, I describe the identification and validation of first-in-class selective inhibitors of USP48. I performed a USP48-focused high throughput screen then triaged screening actives in a series of orthogonal assays and counter screens to credential 3 novel scaffolds. We demonstrate the chemical tractability of one of our hits, MD-156, to support further medicinal elaboration. In this chapter, I additionally explore USP48 intramolecular regulation by its C-terminal ubiquitin-like (UBL) domain and define the consequences of UBL domain deletion to USP48 enzymatic activity and substrate-recognition. Ultimately this thesis provides a novel USP7 activator and a framework for future DUB activator discovery, as well as two inhibitors for USP7 and USP48 with pharmacological potential for the treatment of cancers. These agents are important tools for studying the detailed mechanism of pathologies of the DUBs in their respective diseases. Moreover, these compounds and associated knowledgebases set the stage for future DUB-focused biological discovery and targeted therapies.application/pdfendeubiquitylaseDUBsmall moleculesubiquitin signalingBiochemistryChemistryThesis or Dissertation2026-03-040000-0003-4996-3492