Cyanopyrrolidine Inhibitors of Ubiquitin Specific Protease 7 Mediate Desulfhydration of the Active-Site Cysteine

• Published in: ACS chemical biology 2020-06, Vol.15 (6), p.1392-1400
• Main Authors: Bashore, Charlene, Jaishankar, Priyadarshini, Skelton, Nicholas J, Fuhrmann, Jakob, Hearn, Brian R, Liu, Peter S, Renslo, Adam R, Dueber, Erin C
• Format: Article
• Language: English
• Subjects: Catalytic Domain - drug effects; Cysteine - chemistry; Cysteine - metabolism; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - pharmacology; Humans; Molecular Docking Simulation; Peptidomimetics - chemistry; Peptidomimetics - pharmacology; Pyrrolidines - chemistry; Pyrrolidines - pharmacology; Ubiquitin-Specific Peptidase 7 - antagonists & inhibitors; Ubiquitin-Specific Peptidase 7 - chemistry; Ubiquitin-Specific Peptidase 7 - metabolism
• ISSN: 1554-8929; 1554-8937
• DOI: 10.1021/acschembio.0c00031

Ubiquitin specific protease 7 (USP7) regulates the protein stability of key cellular regulators in pathways ranging from apoptosis to neuronal development, making it a promising therapeutic target. Here we used an engineered, activated variant of the USP7 catalytic domain to perform structure-activity studies of electrophilic peptidomimetic inhibitors. Employing this USP7 variant, we found that inhibitors with a cyanopyrrolidine warhead unexpectedly promoted a β-elimination reaction of the initial covalent adducts, thereby converting the active-site cysteine residue to dehydroalanine. We determined that this phenomenon is specific for the USP7 catalytic cysteine and that structural features of the inhibitor and protein microenvironment impact elimination rates. Using comprehensive docking studies, we propose that the characteristic conformational dynamics of USP7 allow access to conformations that promote the ligand-induced elimination. Unlike in conventional reversible-covalent inhibition, the compounds described here irreversibly destroy a catalytic residue while simultaneously converting the inhibitor to a nonelectrophilic byproduct. Accordingly, this unexpected finding expands the scope of covalent inhibitor modalities and offers intriguing insights into enzyme-inhibitor dynamics.