(D) Ovarian cancer tissues from non-neoplastic, serous carcinoma, mucinous adenocarcinoma and endometrioid carcinoma were stained with the isotype control or anti-USP7 antibodies. speculated that CDDO-Me may target USP7 in ovarian cancer cells. We demonstrated that Cryaa ovarian cancer cells have higher USP7 expression than their normal counterparts. Knockdown of USP7 inhibits the proliferation of ovarian cancer cells both and gel-based assay. The IC50 of CDDO-Me for USP7 inhibition was 14.08 M (Figure ?(Figure1C).1C). USP7 belongs to cysteine protease, which including palpain-like proteases (such as cathepsin B), caspase-like enzymes and deubiquitinating enzymes. To see whether CDDO-Me affects other cysteine protease, we measured its effect on cathepsin β-Apo-13-carotenone D3 B and cathepsin D. Even at a concentration of 100 M, CDDO-Me could not significantly inhibit the activity of cathepsin B and cathepsin D (Figure 1D, 1E). By contrast, E64 and pepstatin A, which are known inhibitors of cathepsin B and cathepsin D, markedly inhibited the activities of cathepsin B and cathepsin D (Figure 1D, 1E). Moreover, we examined the effect of CDDO-Me on other deubiquitiating enzymes with the similar structure to USP7. Interestingly, CDDO-Me also has inhibitory activity against USP2 with IC50 at 22.33 M (Supplementary Figure S1). Together, β-Apo-13-carotenone D3 these data show that CDDO-Me could inhibit USP7 β-Apo-13-carotenone D3 activity gel-based USP7 activity assay, various concentrations of CDDO-Me were pre-incubated with 80 nM USP7 before GST-UBA52 was added. After incubation, the reactions were stopped, and the products were separated by 12% SDS-PAGE and visualized by Coomassie brilliant blue (G250), and the IC50 is 14.08 M (C). (DCE) The effect of 50 and 100 M CDDO-Me on the activity of cathepsin B (D) and cathepsin D (E) were determined as described in the Materials and Methods section; 50 M E64 (inhibitor of cathepsin B) and 50 M pepstatin A (inhibitor of cathepsin D) were used as positive controls. All experiments were performed at least three times with the same results. CDDO-Me inhibits USP7 activity independent of the Michael acceptor in the A ring We next tried to determine the mode of action of CDDO-Me on USP7. CDDO-Me has two electrophilic Michael acceptor sites in the A and C rings. CDDO-Me can interact with proteins containing structurally available redox-sensitive cysteine residues such as IKK, STAT3 . Given that USP7 is a cysteine protein, we hypothesized that CDDO-Me may covalently bind to USP7 and inhibit its activity in an irreversible manner. Unexpectedly, our results showed that CDDO-Me inhibited USP7 activity in a reversible manner (Figure ?(Figure2A).2A). Therefore, we suspected that the two Michael acceptor sites may not be necessary for the inhibitory effect of CDDO-Me. To address this, we attempted to reduce the double bonds in the A and C rings of CDDO-Me. However, we could only reduce the double bond in the A ring could be (CDDO-MeR) (Figure ?(Figure2B).2B). Interestingly, CDDO-MeR inhibited the USP7 activity at concentrations similar to that of CDDO-Me (Figure ?(Figure2C).2C). Moreover, preincubation with dithiothreitol (DTT) at higher concentrations (40C80 mM) abrogated the activity of CDDO-Me but not that of CDDO-MeR (Figure ?(Figure2D).2D). These data suggest that CDDO-Me inhibits USP7 activity via a mechanism independent of the presence of the Michael acceptor site in the A ring. Open in a separate window Figure 2 Reduced CDDO-Me inhibits USP7(A) Time course of the inhibitory effect of CDDO-Me on USP7. β-Apo-13-carotenone D3 USP7 was pre-incubated for different time periods with DMSO or CDDO-Me before initiating the enzymatic reaction by adding the Ub-AMC substrate (300 nM), and the activity of USP7 was measured. (B) Chemical structure of reduced CDDO-Me (CDDO-MeR). (C) The inhibitory effect of CDDO-MeR on USP7 activity was assessed by a gel-based assay and IC50 was determined. (D) CDDO-Me (Me) and CDDO-MeR (MeR) were β-Apo-13-carotenone D3 pre-incubated with different concentrations of DTT, after which their inhibitory effect on USP7 was determined by a gel-based assay. All experiments were performed at least three times with the same results. The binding mode between USP7 and CDDO-Me was further explored by molecular docking..