Article Figures & Data
Figures
- Figure 1.
Identification of HBX 41,108 as a USP7 inhibitor. A, structures of the cyano-indenopyrazine derivatives, HBX 41,108 and HBX 91,490. B, dose-dependent inhibition of USP7 activity by HBX 41,108. Mean ± SD of triplicate values. Representative of four independent experiments. C, HBX 41,108 inhibits USP7 deubiquitination of polyubiquitinated p53. The purified wild-type USP7 enzyme (3 nmol/L final concentration) was incubated for 30 min at room temperature with different doses of HBX 41,108 (lanes 3-6) or ubiquitin vinyl sulfone (UbVS; 0.1 μmol/L; lane 7) or without any inhibitors (lane 2). Purified polyubiquitinated p53 was next added to these different samples (lanes 2-7) or used alone (lane 1) and reactions were incubated at 37°C for 90 min. D, HBX 41,108 acts through a uncompetitive inhibition mechanism. Various concentrations of Ub-AMC (0.15, 0.2, 0.4, 0.6, and 0.8 μmol/L) were added to USP7 (500 pmol/L) in the presence of various concentrations of HBX 41,108 (0.5, 1, 5, 7.5, and 10 μmol/L) for initial rate measurements. Data sets from two independent experiments.
- Figure 2.
HBX 41,108 increases p53 levels in a nongenotoxic manner. A, Western blot analysis of p53, phospho-p53, and p21 protein levels in HCT116 colon cancer cells treated with various doses of HBX 41,108 (1, 3, and 10 μmol/L) for 24 h. B, effect of HBX 41,108 on the relaxation of plasmid DNA by human topoisomerase I in the absence of ethidium bromide during the electrophoresis. Native supercoiled pLAZ (0.13 μg; lane DNA) was incubated with topoisomerase I (4 units) in the absence (lane TopoI) or presence of camptothecin (CPT) and HBX 41,108 at the indicated concentration (μmol/L). Reactions were stopped by adding SDS and proteinase K. DNA samples were separated by electrophoresis in 1% agarose gels. Nck, nicked; Rel, relaxed; Sc, supercoiled. Duplicate experiments.
- Figure 3.
HBX 41,108 inhibits USP7 activity in cells. A, ubiquitinated proteins were precipitated, using Ni-NTA agarose, from HEK293 cells transfected (lanes 2-4) or not (lane 1) with His-tagged ubiquitin and Mdm2 ± wild-type (lane 3) or mutant USP7 (lane 4). Western blots were analyzed for the presence of p53 and USP7. B, experiment was done as in A using HBX 41,108 (10-50 μmol/L). C, HEK293 cells were transfected with wild-type Mdm2, His-tagged ubiquitin, and wild-type or mutant USP7 constructs and treated with HBX 41,108 (1-3 μmol/L). Western blots were analyzed for the presence of Mdm2 and USP7.
- Figure 4.
HBX 41,108 inhibits HCT116 cancer cell growth and induces apoptotic cell death. A, treatment for 24 h with HBX 41,108 decreases HCT116 cell proliferation in a dose-dependent manner (5-bromo-2-deoxyuridine incorporation). B, poly(ADP-ribose) polymerase cleavage following the treatment of HCT116 cells with HBX 41,108 (0.1, 0.3, 1, and 3μmol/L) was detected by Western blotting.
- Figure 5.
HBX 41,108 induces apoptosis in a p53-dependent manner. A, caspase activity was assessed in p53+/+ (HCT116) and p53−/− (PC3) cell lines following HBX 41,108 treatment (0.4, 1.2, and 3.7 μmol/L) using the caspase assay described in Materials and Methods. B,isogenic HCT116 cancer cells (p53+/+, p53−/−) were analyzed for the effects of HBX 41,108 (2, 6, and 18 μmol/L) on caspase-3 cleavage as an indicator of apoptosis induction. C, experiment was done as in B using the genotoxic drug 5-FU.
- Figure 6.
Cooperative effect of HBX 41,108 and 5-FU in inhibiting the growth of HCT116 cells. The IC20 (corresponding to the concentration of HBX 41,108 at which 20% growth inhibition was observed) was used and the growth inhibition obtained at 72 h of drug treatment was determined using the MTS assay. Mean of triplicate experiments.
Tables
- Table 1.
HBX 41,108 specificity on a panel of proteases
Family Name IC50 (μmol/L) Aspartic proteases Renin >10 Cathepsin D >10 Cathepsin E >10 HIV-1 protease >10 Serine proteases Trypsin >10 Factor Xa >10 Thrombin >10 Plasmin >10 Metallo proteases ACE >10 Cysteine proteases Calpain-1 >10 Cathepsin B >1 Cathepsin L >1 Cathepsin S >1 UCH-L1 >1 SENP1 >10 USP7 0.42
Supplementary Data
Files in this Data Supplement:
Volume 8, Issue 8
