Discovery andOptimizationof Novel Tricyclic Ubiquitin-SpecificProtease 1 Inhibitors for the Treatment of BRCA-Mutated Breast Cancer

Abstract Ubiquitin-specific protease 1 (USP1) represents an emerging therapeutic target for BRCA-deficient malignancies. Using a scaffold-hopping strategy derived from KSQ-4279, we designed a novel series of USP1 inhibitors featuring a tricyclic core. Among them, two lead compounds 43 and 47 were identified with potent USP1 inhibitory and cellular antiproliferative activity. Further studies in MDA-MB-436 cells demonstrated that compounds 43 and 47 suppressed colony formation and induced S-phase arrest, with time- and concentration-dependently stabilizing ubiquitinated PCNA and amplifying p-H2AX. Importantly, both compounds showed synergistic antiproliferative effects in combination with the PARP inhibitor Olaparib. Supported by its favorable pharmacokinetic properties, compound 43 exhibited significant in vivo anticancer efficacy in an MDA-MB-436 xenograft model, achieving superior tumor growth inhibition both as monotherapy and in combination with Olaparib compared to KSQ-4279. Collectively, compound 43 emerges as a promising preclinical candidate with translational potential for BRCA-mutated breast cancer.