Human ubiquitin-specific protease 1 (USP1), which is associated with UAF1, has been identified as the deubiquitinase (DUB) responsible for deubiquitinating PCNA, FANCD2 and FANCI in the DNA damage response. ML323 is a potent USP1-UAF1 inhibitor with IC50 values of 76 nM in a ubiquitin-rhodamine (Ub-Rho) assay and 174 nM and 820 nM in orthogonal gel-based assays using K63-linked diubiquitin (di-Ub) and monoubiquitinated PCNA (Ub-PCNA) as substrates, respectively. In vitro, ML323 decreased in the intensity of the labeled USP1 band at concentration ranging in 25 – 100 μM in HEK293T cells. Treatment with 30 μM ML323 increased PCNA and FNACD2 level in H596 cells. Treatment H596 cells with 20 μM ML323 alone did not result in cell cycle delay or arrest. However, treatment of H596 cells with a combination of ML323 (20 μM) and cisplatin (5 μM) increased the percentage of S-phase cell to 65%[3]. Treatment with 5 μM and 10 μM ML323 potently inhibited the induction of caspase-3 and PARP cleavage triggered by elevated glucose, glucose/palmitate, and cytokines in rat β-cell line INS-1E cell[4]. In vivo, administration of ML323 at 5 mg/kg and 10 mg/kg once daily significantly inhibited lung metastatic in mice harboring 4T1 tumors[5]. Treatment with 10 mg/kg ML323before i.p. injection of LPS reduced serum level of IFN-β but did not considerably decrease the amount of TNF-α in C57BL/6J mice. Treatment with 10 mg/kg ML323 inhibited VSV-induced IFN-β expression and enhanced viral replication in C57BL/6J mice[6].
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