Fig 2: DDO-5936 dose-dependently impairs the growth of xenografted HCT116 cells in nude mice.(A) Body weights of nude mice treated with either vehicle or DDO-5936 are shown. Body weight is plotted as the means ± SEM (n = 6 mice for each group). (B) Volume measurements of HCT116 xenograft tumors treated with vehicle, DDO-5936 (50 mg/kg per day), or DDO-5936 (100 mg/kg) for 21 days. DDO-5936 was administered by intraperitoneal injection once a day. Tumor volume is plotted as the means ± SEM (n = 6 mice for each group) (**P < 0.01, ***P < 0.001, paired Student’s t test, two-tailed). (C) Volume distribution of HCT116 xenograft tumors treated with vehicle or DDO-5936 at 21 days. (D) Western blot analysis of Hsp90, Hsp70, GR, p-GR, AKT, p-AKT, ERK1/2, p-ERK1/2, CDK4, and CDK6 in three representative xenograft tumor tissue samples from each group. β-Actin was used as a loading control. Data are representative of three technical replicates. (E) Schematic model of the process in which DDO-5936 modulates the Hsp90-Cdc37 chaperone cycle. DDO-5936 inhibited the Hsp90-Cdc37 PPI through binding to a previously unknown pocket on Hsp90 involving E47, a binding determinant of the Hsp90-Cdc37 complex, inhibiting the maturation of the kinase client to implement the antiproliferation effects of HCT116 cells.

Fig 3: Discovery of DDO-5936, an Hsp90-Cdc37 PPI inhibitor without ATPase inhibition, based on a site screening strategy involving critical residues identified at the binding interface.(A) Binding affinities of the WT Hsp90-Cdc37 complex and complexes with key residues mutated, determined by ITC. Hydrogen bonds are presented as red dotted lines. Data are from three independent experiments. (B) Chemical structures of compound 11 and DDO-5936. (C) Dose-dependent inhibition of the Hsp90-Cdc37 interaction by compound 11, DDO-5936, and AT13387. Twofold diluted compounds with seven concentrations were simultaneously tested by an Hsp90-Cdc37 homogeneous time-resolved fluorescence (HTRF) assay. The results are shown as the means ± SD, n = 3 wells, from three independent experiments. P values were calculated by pairwise comparisons to the dimethyl sulfoxide (DMSO) control (**P<0.01, ***P<0.001, Student’s t test). ns, not significant. (D) Thermostability of Hsp90 treated with 0, 10, 50, 100, and 200 μM DDO-5936, determined by a standard thermo shift assay. ΔTm results were obtained from the mean values of three independent assays (n = 6 wells). (E) Dose-dependent competitive binding of compound 11, DDO-5936, and AT13387 to the Hsp90 ATP binding site, determined by fluorescence polarization (FP) assays using a fluorescein isothiocyanate–geldanamycin probe. Data are presented as the means ± SD, n = 3 wells, from three independent experiments. N/A, not applicable. (F) Dose dependence of compound 11, DDO-5936, and AT13387 inhibition of Hsp90 ATPase activity. Data are presented as the means ± SD, n = 3 wells, from three independent experiments. (G) 1H saturation transfer difference (STD) NMR spectrum of 100 μM DDO-5936 binding to 5 μM Hsp90. ppm, parts per million.

Fig 4: ITCH may function as an E3 ubiquitin ligase targeting CDK4 for degradation. (A) Relative CDK4 mRNA expression in HCT116 (Vector/HA-CCDC68) and RKO (Vector/HA-CCDC68) cells. Student′s t-test, ns, not significant. (B) CDK4 protein degradation was monitored in HCT116 (CCDC68) and HCT116 (Vector) cells pretreated with MG132 for 5 h followed by CHX for various times. (C) CDK4 protein degradation rates in HCT116 (CCDC68) and HCT116 (Vector) cells. Three independent degradation experiments were performed, and the results were analyzed using ImageJ software and analyzed by Student′s t-test. *P < 0.05. (D) Molecules potentially involved in the regulation of CDK4 protein degradation were predicted by the UbiBrowser database. (E) Venn diagram screening the E3 enzymes involved in the regulation of CDK4 protein degradation. (F) Expression of CDK4, ITCH, BTRC, CDC37, FBXO4, and SOCS7 in HCT116 (Vector/HA-CCDC68) and RKO (Vector/HA-CCDC68) cells.

Fig 5: DDO-5936 arrests the cell cycle in HCT116 cells.(A) Western blot analysis of Cdc37, Hsp90, GR, p-GR, AKT, p-AKT, ERK1/2, and p-ERK1/2 protein expression levels in Cdc37-WT HCT116 cells and corresponding Cdc37-KO HCT116 cells after treatment with 0, 10, or 25 μM DDO-5936 for 24 hours. β-Actin was used as a loading control. Data are representative of three independent experiments. (B) Western blot analysis of Cdc37, CDK2, CDK4, CDK6, p21, p27, cyclin D1, and cyclin D3 protein expression levels in Cdc37-WT HCT116 cells and corresponding Cdc37-KO HCT116 cells after treatment with 0, 10, or 25 μM DDO-5936 for 24 hours. β-Actin was used as a loading control. Data are representative of three independent experiments. (C) Top: Cell cycle distribution measured by propidium iodide (PI) staining of Cdc37-WT HCT116 cells and corresponding Cdc37-KO cells after treatment with 0, 10, or 25 μM DDO-5936 for 24 hours. Bottom: The cell cycle distribution is represented as a graphic histogram. Data are representative of three independent experiments as the means ± SD (***P < 0.001).