Design and Synthesis of USP1 Inhibitors: Synergistic Antitumor Activity with PARP Inhibitors in Triple-Negative Breast Cancer.

Ubiquitin-specific protease 1 (USP1) regulates the DNA damage response (DDR) by deubiquitinating PCNA, and its inhibition potentiates PARP inhibitor efficacy, highlighting USP1 as a promising therapeutic target in triple-negative breast cancer (TNBC). Guided by USP1-UAF1 structural insights, we optimized the KSQ-4279 scaffold and identified 1,3-dihydro-2H-imidazo[4,5-c]pyridin-2-one as a scaffold-hopping core for proof-of-concept exploration. Representative compound 57 exhibited nanomolar USP1-UAF1 inhibition, antiproliferative activity against MDA-MB-436 cells, favorable human liver microsomal stability, and 76% oral bioavailability in rats. In vitro assays and in vivo xenograft studies demonstrated synergistic antitumor activity between compound 57 and Olaparib, resulting in DNA damage and significantly enhanced tumor growth inhibition compared with Olaparib monotherapy. Transcriptomic analysis and Western blot results further supported enhanced suppression of tumor survival pathways. Collectively, this work establishes compound 57 as a scaffold-hopping, proof-of-concept USP1 inhibitor and provides in vivo validation for USP1-PARP inhibitor combination therapy in TNBC.