Discovery of ML323 as a Novel Inhibitor of the USP1/UAF1 Deubiquitinase Complex

Advances in pharmacological approaches to target the ubiquitin–proteasome system have revealed several potential new molecular targets within the ubiquitin machinery. Likewise, the functional consequences of ubiquitination and deubiquitination have recently been linked to a wide variety of critical biological processes well beyond just protein disposal [1]. For example, the deubiquitining enzyme, ubiquitin-specific protease 1 (USP1), in association with its WD40 binding partner, UAF1 (USP1-associated factor 1), is a known regulator of DNA damage response and has been suggested as a promising target to improve the efficacy of the commonly used DNA damaging drugs by modulating the cancer cells' ability to repair or tolerate DNA lesions [2-7]. To further evaluate the therapeutic potential of targeting the USP1/UAF1 deubiquitinase complex, we conducted a quantitative high-throughput screen and a subsequent medicinal chemistry optimization campaign in pursuit of small molecules that inhibit USP1/UAF1. Herein, we describe the discovery and optimization of ML323, a probe molecule that displays reversible, nanomolar inhibitory activity and excellent selectivity toward USP1/UAF1. In addition, ML323 potentiates the cytotoxicity of cisplatin and increases endogenous monoubiquitination levels of both PCNA and FANCD2, two known cellular targets of USP1/UAF1 [8]. Lastly, ML323 possesses a promising in vitro ADME profile, suggesting its suitability for further testing in PK/PD studies.