Discovery of novel USP8 inhibitors via Ubiquitin-Rho-110 fluorometric assay based high throughput screening

USP8, one member of deubiquitinating enzymes (DUBs) families, maintains the ubiquitination level of EGFR and regulates the downstream signaling pathways. The deregulation of USP8 has been implicated in many human diseases, especially in cancer. Therefore, USP8 has been identified as a promising target for drug design. Herein, via high throughput screening based on Ubiquitin-rhodamine-110 (Ubiquitin-Rho-110) fluorometric activity assay, we discovered a novel inhibitor DC-U43. By structure optimization, DC-U43-10 reached a half-maximal inhibitory concentration (IC50) value of 2.6 ± 1.1 μM and exhibited 10-fold selectivity against USP7. The binding between DC-U43-10 and USP8 was validated by surface plasmon resonance (SPR) assay with a KD value of 10.5 ± 3.7 μM. It also inhibited the colony formation of H1975 cells. Hence, DC-U43-10 represents a kind of USP8 inhibitors with novel scaffold and has broad prospects for being a probe for USP8-related academic and clinical research.