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Small-molecule inhibitor targeting the Hsp90-Cdc37 protein-protein interaction in colorectal cancer

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Science Advances  18 Sep 2019:
Vol. 5, no. 9, eaax2277
DOI: 10.1126/sciadv.aax2277
  • Fig. 1 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. 2 DDO-5936 binds to a critical residue on the Hsp90-Cdc37 PPI interface.

    (A) HSQC spectra (three residues, including R46, E47, and Q133) of 50 μM 15N-labeled Hsp90 N terminus in the absence (magenta) and presence of 250 μM (green) or 500 μM DDO-5936 (blue). (B) Overview of the DDO-5936 binding site on Hsp90. Top: Overall structure with the surface of Hsp90 colored magenta. Inset: Detailed binding region of DDO-5936. Hsp90 is represented as a magenta cartoon, and DDO-5936 is shown as yellow sticks. The residues Arg46, Glu47, and Gln133 are highlighted within the stick model. Hydrogen bonds are displayed as green dotted lines. (C) Binding affinities of WT or mutant Hsp90 proteins for DDO-5936, determined by biolayer interferometry (ForteBio Octet) assay. Data are representative of three independent experiments. (D) Dose-dependent T1ρ NMR spectra for 200 μM DDO-5936 (blue) in the presence of 1 μM Hsp90 (magenta), 5 μM Hsp90 (orange), and 20 μM Hsp90 (green). Data are representative of three independent experiments.

  • Fig. 3 DDO-5936 disrupts the Hsp90-Cdc37 interaction, represses cell proliferation through a strong correlation with the Hsp90-Cdc37 expression level, and selectively down-regulates kinase clients of Hsp90.

    (A) Co-IP in HCT116 cells after treatment with DMSO and increasing concentrations of DDO-5936 (5, 10, and 25 μM) for 24 hours. Western blots were performed with anti-Hsp90, anti-Cdc37, or anti-CDK4 in each experiment. Data are representative of three independent experiments. (B) Correlation between the protein expression level of Hsp90 and Cdc37 in diverse cell lines and antiproliferative activities (IC50 values). The Pearson correlation coefficient (r) was calculated by GraphPad Prism 6.0 software. Data are presented as the means ± SD, n = 6 wells, from three independent experiments. (C) Western blot analysis of Cdc37, p (phosphorylated)–Cdc37, Hsp90, Hsp70, GR (glucocorticoid receptor), p-GR, AKT, p-AKT, ERK1/2 (extracellular signal–regulated kinase 1/2), p-ERK1/2, CDK4, and CDK6 protein expression levels in HCT116 cells after treatment with 0, 1, 5, 10, 20, and 40 μM DDO-5936 or 0, 0.5, 1, and 5 μM AT13387 for 24 hours. β-Actin was used as a loading control. Data are representative of three independent experiments.

  • Fig. 4 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).

  • Fig. 5 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.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/9/eaax2277/DC1

    Supplementary Materials and Methods

    Fig. S1. Searching for critical residues on the Hsp90-Cdc37 binding interface through MD simulation calculation.

    Fig. S2. Identification of critical residues on Hsp90-Cdc37 binding interface.

    Fig. S3. The rational design and discovery of Hsp90-Cdc37 PPI inhibitors.

    Fig. S4. Synthetic scheme and binding site characterization of DDO-5936.

    Fig. S5. DDO-5936 bound to Hsp90 in cells and the antiproliferative activities of DDO-5936 were correlated with the protein expression level of Hsp90 and Cdc37.

    Fig. S6. Quantification of protein expression in Fig. 4 (A and B).

    Fig. S7. Histological morphology of H&E-stained tissues sections of representative nude mice and PK/PD studies of DDO-5936.

    Fig. S8. 1H NMR spectrum and 13C NMR spectrum of DDO-5936.

    Table S1. The change of Gibbs free energy from calculation and experimental results.

    Table S2. ATPase inhibition of DDO-5936 on cell cycle–related kinases by enzyme assays.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • Fig. S1. Searching for critical residues on the Hsp90-Cdc37 binding interface through MD simulation calculation.
    • Fig. S2. Identification of critical residues on Hsp90-Cdc37 binding interface.
    • Fig. S3. The rational design and discovery of Hsp90-Cdc37 PPI inhibitors.
    • Fig. S4. Synthetic scheme and binding site characterization of DDO-5936.
    • Fig. S5. DDO-5936 bound to Hsp90 in cells and the antiproliferative activities of DDO-5936 were correlated with the protein expression level of Hsp90 and Cdc37.
    • Fig. S6. Quantification of protein expression in Fig. 4 (A and B).
    • Fig. S7. Histological morphology of H&E-stained tissues sections of representative nude mice and PK/PD studies of DDO-5936.
    • Fig. S8. 1H NMR spectrum and 13C NMR spectrum of DDO-5936.
    • Table S1. The change of Gibbs free energy from calculation and experimental results.
    • Table S2. ATPase inhibition of DDO-5936 on cell cycle–related kinases by enzyme assays.

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