Active site-targeted covalent irreversible inhibitors of USP7 impair the functions of Foxp3+ T-regulatory cells by promoting ubiquitination of Tip60
Fig 5
P217564 regulates the degradation of USP7 substrates and shows durable inhibition of USP7.
(A) HCT116, PC-3, and Jurkat cells were treated with DMSO or P217564 at indicated concentrations for 24 hours; crude cell extracts were subjected to SDS-PAGE electrophoresis, followed by IB with indicated antibodies. (B) P217564 produces long-lasting USP7 inhibition in cells. Jurkat cells were treated with 10μM P217564 for 2 hours, the compound was washed out, and the USP7 activity was measured at various times up to 48 hours using the USP7 IP/activity assay. Cellular levels of USP7 substrates including UHRF1, DNMT1, and HDM2 were detected by Western blot. Anti-Actin blot was used as loading control.