Gossypol suppresses tumorigenesis through inhibition of multiple deubiquitinating enzymes.

Gossypol is a polyphenolic compound recognized for its role as a Bcl-2 family protein inhibitor, attracting considerable interest mainly due to its antitumor properties. However, its development as a therapeutic agent has faced hurdles from off-target side effects, partially stemming from an incomplete understanding of its target profile. In this study, we identified Gossypol as a multi-deubiquitinating enzyme (DUB) inhibitor, exhibiting preference for the ubiquitin-specific protease (USP) subfamily members. Gossypol demonstrated IC50 values ranging from 0.30 μM to 4.97 μM against USP2 CD, USP7 CD, USP8 CD, USP28 CD and USP9X CD. Mechanistic studies, including 1D NMR, IC50 shift assays, fluorescence quenching experiments, and enzymatic reversibility tests revealed that Gossypol mainly acts as non-covalent inhibitor against multiple DUBs, with binding affinities towards representative USPs between 10 and 30 μM. Cell-based assays confirmed Gossypol's efficacy in inhibiting intracellular DUB activity, leading to increased ubiquitination levels. Moreover, Gossypol exhibited cytotoxic effects on human breast, prostate, and colorectal cancer cell lines, at least partially through downregulating the oncogenic substrates of targeted DUBs such as c-Myc, Mcl-1, MDM2, and Cyclin D1. Collectively, our findings position Gossypol as a promising small-molecule inhibitor targeting DUBs, especially USPs, and provide a rationale for further exploring its therapeutic potential in USP-driven cancers.