High-throughput screening identification of USP7 inhibitors reveals YM155 as a biologically active scaffold with in vivo antitumor efficacy.

Targeting the ubiquitin-proteasome system has emerged as a powerful therapeutic strategy in oncology, particularly through modulation of deubiquitinating enzymes that regulate the stability of oncogenic proteins. Among these, ubiquitin-specific protease 7 (USP7) has garnered considerable attention due to its ability to deubiquitinate and stabilize multiple cancer-relevant substrates, including MDM2, FOXP3, N-Myc, and PTEN. By controlling protein turnover and subcellular localization of substrates, USP7 contributes to tumor progression across diverse types of cancer, positioning it as an attractive therapeutic target. Here, we report the identification of novel small-molecule USP7 inhibitors from a high-throughput screening campaign employing the fluorogenic ubiquitin-rhodamine enzymatic assay. Screening of 2446 compounds led to the identification of five hit compounds with measurable inhibitory activity against recombinant USP7 in vitro. These hits were further evaluated for antiproliferative activity across a panel of human and murine cancer cell lines with distinct molecular backgrounds. Among the identified hits, YM155 displayed the highest activity across all cancer types tested, exhibiting nanomolar IC50 values in the proliferation assays. Importantly, YM155 demonstrated significant tumor growth inhibition in lung and breast cancer mouse models, supporting its in vivo activity and pharmacological tractability in clinically relevant settings. Together, these findings expand the chemical space of USP7 inhibitors and identify YM155 as a hit for further optimization and mechanistic investigation toward USP7-targeted anticancer therapeutics.