Fig 1: V517F mutation translocates the outside side chain of Y514 into the compound binding cleft of the catalytic domain, resulting in a decreased ability of USP7i binding.a The association constant (Kd) for the interaction between USP7CD, V517FCD, USP7-797, or FT671 was determined using an isothermal titration calorimetry (ITC) assay. C value was calculated as the ratio of the protein analyte concentration to Kd. b The structural superposition between the AF_USP7_V517FCD (purple) and USP7CD_FT671 (PDB ID: 5NGE) (green). A zoomed-in view of the boxed region from the lower right inset, showing clashes between the mutant USP7 Y514 residues and the FT671 compound (orange) in wild-type USP7CD as indicated by a red circle. Lower right inset, atomic models of the catalytic domain of the USP7_V517F mutant (purple) and the wild-type USP7 (green) bound with FT671. c Structural superposition among USP4 (2Y6E), USP8 (2GFO), USP9 (5WCH), USP12 (5K16), USP15 (6GHA), USP34 (7W3R) and USP7CD (1NB8). A zoomed-in view of the boxed region from the lower right inset, showing the rotamer conformations of Y514 or its counterparts in different USP members. Lower right inset, atomic models of the catalytic domain of the USP members. d The association constant (Kd) for the interaction between V517G/ V517A/ V517I/ V517Y, and FT671 was determined using an isothermal titration calorimetry (ITC) assay.
Fig 2: Resistant clones contain a heterozygous mutation in USP7.a The mRNA and protein levels of USP7 were assessed in parental and resistant cells using quantitative RT-PCR and Western blotting. The mRNA level are presented as mean ± SD (n = 3), and blot is representative of three independent experiments. b Representative western blot of three experiments showing the levels of p53, p21, Rad18, DNMT1, and UHRF1 in parental and V517F mutant cells after treatment with USP7i for 24 h. c Schematic representation of location of USP7 mutations detected in resistant clones. Sanger sequencing confirmed the presence of the heterozygous NM_003470.3:c.G2170>T (V517F) mutation in all six resistant clones. d The enzyme activity of wild-type (WT) and V517F mutant was assessed by measuring the cleavage activity of the AMC fluorophore from ubiquitin-AMC. Left panel: the catalytic domains of both the wild-type USP7 (WT_CD) and the V517F mutation (V517F_CD) were confirmed through Coomassie Brilliant Blue fast staining. Right panel: the fluorescence intensity was calculated as the mean ± SD (n = 3 technical replicates; 3 independent experiments). e The inhibition of WT or V517F mutant by USP7-797 or FT671 was assessed to determine the enzyme activity inhibition. Data shown are the mean ± SD (n = 3 technical replicates; 3 independent experiments). f The IC50 values of USP7-797, FT671, and GNE6640 in WT and V517F mutants were determined. Data are presented as mean ± SD (n = 3 technical replicates; 3 independent experiments).
Fig 3: Cell lines containing the V517F knock-in mutation and overexpression demonstrate resistance to USP7i.a Schematic representation of generation of V517F knock-in (KI) cell lines in CHP-212 cells using CRISRP/Cas9 technology. The nucleotide sequences of the USP7 gene at the target site in the V517F KI monoclonal cells are shown in the right panel. b, c The CHP-212 V517F KI and LNCaP V517F KI clones exhibit resistance to USP7i and a reduction in USP7 pathway inhibition. Following treatment with the specified compounds for 5 days, cell viability was assessed using the SRB assay for CHP-212, and CCK8 assay for LNCaP cells. Western blotting analysis was conducted to assess the indicated protein levels following treatment with 1 μM USP7-797 or FT671 for 24 h. Data shown are mean ± SD (n = 3 technical replicates; 3 independent experiments). d Exogenous expression of V517F mutant in Capan-1 cells resulted in reduced sensitivity to USP7i. Following stable transfection with full-length USP7 WT or V517F mutant, subsequent treatment with USP7-797 for indicated days was analyzed by SRB assays or western blotting (lower panel). Data shown are mean ± SD (n = 3 technical replicates; 3 independent experiments). e Exogenous expression of V517F mutant in CHP-212 and LNCaP cells decreased their sensitivity to USP7i. Cells were treated with USP7-797 or FT671 for 5 days and then subjected to SRB or CCK8 assays. Data shown are mean ± SD (n = 3 technical replicates; 3 independent experiments).
Supplier Page from Sino Biological, Inc. for Human USP7/HAUSP Gene ORF cDNA clone in cloning vector