All air or moisture sensitive reactions were performed under positive pressure of nitrogen with oven-dried glassware. Therefore, given its favorable biochemical and physicochemical attributes, M元23 can be used by the research community to interrogate USP1 biology in vivo. GW7647 and Pimozide, the first annotated USP1 inhibitors, display fairly promiscuous PubChem activity profile with a 9.9% and 12.0% hit rate respectively while another compound C527 (Publication no. In addition, M元23 addresses the limitations exhibited by the current probes. Subsequent secondary and tertiary assay results also suggest that M元23 inhibitor may be used in combination with known DNA damaging agents, such as cisplatin, to potentiate the cytotoxicity. Ubiquitin-specific Protease 1 (USP1) is a deubiquitinating (DUB) enzyme implicated in DNA damage response through modulation of the ubiquitination levels of key proteins, PCNA and FANCD2, which play important roles in translesion synthesis and Fanconi anemia pathways, respectively. M元23, with its ability to potently and selectively inhibit USP1/UAF1 both in vitro and in a cellular context, enables biologists the evaluation of the USP1/UAF1 deubiquitinase complex as a target for therapeutic development. Recommendations for Scientific Use of the Probe Lastly, M元23 possesses a promising in vitro ADME profile, suggesting its suitability for further testing in PK/PD studies.ġ. In addition, M元23 potentiates the cytotoxicity of cisplatin and increases endogenous monoubiquitination levels of both PCNA and FANCD2, two known cellular targets of USP1/UAF1.
Herein, we describe the discovery and optimization of M元23, a probe molecule that displays reversible, nanomolar inhibitory activity and excellent selectivity toward USP1/UAF1. 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. 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. 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. Advances in pharmacological approaches to target the ubiquitin–proteasome system have revealed several potential new molecular targets within the ubiquitin machinery.