Identification of deubiquitinase inhibitors via high-throughput screening using a fluorogenic ubiquitin-rhodamine assay

Identification of selective deubiquitinase (DUB) inhibitors is critical for probe development to further understand and explore DUB biological function. Here, we detail the optimization and deployment of an in vitro fluorogenic ubiquitin-rhodamine assay to conduct high-throughput screening of a small molecule library against a panel of DUBs. In screening the compound library against multiple DUBs in parallel, we describe an approach for identifying selective DUB inhibitors and provide a roadmap for enabling selective DUB inhibitor discovery. For complete details on the use and execution of this protocol, please refer to Varca et al. (2021).