ML323 is a highly potent and selective USP1-UAF1 inhibitor with IC50 of 76 nM. 

ML323 probably exerts its inhibitory effect through an allosteric mechanism. The measured inhibition constants of ML323 for the free enzyme (Ki) and the enzyme-substrate complex (K′i) were 68 nM and 183 nM, respectively, which is in accordance with the IC50 determination. ML323 showed little to no inhibition against the USPs tested, including USP2, USP5, USP7, USP8, USP10, USP11, USP14 and USP21. Notably, ML323 did not inhibit the USP46-UAF1 complex that contains the same UAF1 subunit as the USP1-UAF1 complex and demonstrated little inhibition against USP1 alone (IC50 > 200 μM) in a Ub-Rho assay in which USP1-UAF1 and USP1 demonstrated similar Km values. Furthermore, ML323 displayed little to no inhibition against DUBs in the ubiquitin C-terminal hydrolase (UCH), ovarian tumor protease (OTU) and Machado-Joseph domain (MJD) families and did not inhibit a deSUMOylase, SENP1, and a deneddylase, NEDP1. ML323 inhibits USP1-UAF1 through a mixed inhibition mechanism. inhibition of cellular USP1-UAF1 activity by ML323 compromises Polη-mediated TLS of UV-induced DNA damage. ML323 led to a similar decrease in LD50 in both PD20 and PD20 + D2 cells (to 2.8 and 3.4 J m?2, respectively).