Structure of the Hsp90 dimer (PDB ID: 7L7J), where the NTD is coloured blue, the MD green and the CTD orange. Representative N-terminal inhibitors include geldanamycin, 17-DMAG and TAS-116 – pimitespib, while C-terminal inhibitors include novobiocin and triazole-based inhibitors A, B and C.

Docking binding mode of compound C (in yellow sticks) in the Hsp90β dimer (PDB entry: 5FWK, protomers A and B are coloured grey and green, respectively) C-terminal domain binding site. For clarity, only the amino acids that interact with the inhibitor are shown. Hydrogen bonds are shown as black dashed lines.

Schematic representation of the design workflow following the discovery of compound C, the exploration of its proposed binding mode, the search for the most suitable aromatic substitution pattern and finally, the exploration of potential changes in the basic region of the inhibitor.

Biochemical evaluation of Hsp90 CTD binding using TR-FRET. Residual activities of Hsp90β (A) and Hsp90α (B) CTD when triazole-based inhibitors were applied at 100 µM concentration: compounds C, 5a, 5f, 5p, 5r, 5s, 5t, 5u, 5v, 5w and 5x from Library A are shown in red, compounds 8g, 10g, 10f, and 10i from Library B along with novobiocin at 1000 µM and positive control are presented in blue. The bars represent mean values with SD of at least three replicates. Statistical significance was calculated using one sample t-test (*p<0.05, **p<0.01, ***p<0.001); C) Schematic representation of the mechanism of the TR-FRET assay in which the C-terminal Hsp90 inhibitor prevents the interaction of cyclophilin D and Hsp90 CTD; D) Dose-response curves for compound 5x in the luciferase refolding assay in two biological repetitions performed in triplicates.

1D 1H STD NMR spectra for compound 5x recorded at an Hsp90β:ligand ratio of 1:100. The molecular structure illustrates the proton nomenclature and the colour-coded relative degrees of saturation of individual non-overlapping protons. The STD amplification factors were normalised to the intensity of the signal with the largest STD effect. The reference STD spectrum (top) with proton assignment and the difference STD spectrum (bottom) are shown. The unassigned proton signals between 3.5 and 3.8 ppm belong to protein buffer with glycerol. The spectra are not to scale.

1D 1H STD NMR spectra for compound 10b recorded at an Hsp90β:ligand ratio of 1:100. The molecular structure illustrates the proton nomenclature and the colour-coded relative degrees of saturation of the individual protons. The STD amplification factors were normalised to the intensity of the signal with the largest STD effect. The reference STD spectrum (top) with proton assignment and the difference STD spectrum (bottom) are shown. The low field signal 6’ is missing due to interference from water suppression. Signals 2’, 3’, 4‘, 5‘, and 6’ have insufficient signal-to-noise ratios in the difference STD spectrum. The unassigned proton signals between 3.5 and 3.8 ppm belong to the protein buffer with glycerol. The spectra are not too scale.

A) Docking binding mode of compound 5x (in cyan sticks) in the Hsp90β dimer (PDB entry: 5FWK, protomers A and B are coloured grey and green, respectively) C-terminal domain binding site. For clarity, only the amino acids that interact with the inhibitor are shown. Hydrogen bonds are shown as black dashed lines. B) Schematic representation of the most frequently occurring pharmacophore model during the MD trajectory. Hydrophobic features are shown in yellow, hydrogen bond donor feature in green and positively charged group in blue.

Western blot analysis of effects of compound 5x in MCF-7 cell line on Hsp90 client protein levels (ERα, IGF1R, MEK, ERK 1/2) and representative heat shock proteins (Hsp70, Hsp90) after 24 hours of treatment. A) Representative Western blot images; B) Quantification results normalised on GAPDH levels. The bars represent mean values with SD and Welch’s t-test was used to evaluate statistical significancy (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Full images used for quantification are shown in Supplementary information Figures 11S-14S .

Mechanistic insight into cytostatic behaviour of compound 5x. A) The changes in proportion of annexin V positive cells in dependence of the duration of treatment in MCF-7 cells. B) The extent of different stages of apoptosis at different time points in MCF-7 cells. C) Representative dot plot diagrams for phosphatidylserine exposure and dead cells for all time points in MCF-7 treated with 7 µM compound 5x. D) Dose and time response of phosphatidylserine detection in SK-N-MC cells exposed to 5x. E) Dot plot diagrams for analysis of apoptosis in SK-N-MC cells exposed to 5x for 24 h. F) Cell cycle analysis of MCF-7 cells (2.5 × 105 cells/mL) treated with 7 µM compound 5x for 24 h and 48 h. A representative experiment is shown. For analysis of apoptosis, MCF-7 or SK-N-MC cells were labeled with the combination of SYTOX Blue and R-PE-Annexin V conjugate and analyzed by flow cytometry. The cell histograms showing phases were created by measuring propidium iodide (PI) staining and flow cytometry. Data represents means and SD of at least three independent experiments. Multiple comparison t-tests with Bonferroni correction or Student’s t-test were used (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

A) Graph depicting a statistically significant difference in relative change in tumour size in larvae treated with 1 % DMSO vs. 70 µM of 5x; Error bars represent SEM. Statistical analysis was performed with a Kolmogornov-Smirnov test (**p<0.01) B) Comparison of representative pictures of zebrafish larvae which show an apparent increase in tumour size with 1 % DMSO (1 dpi vs. 3 dpi), while the increase in tumour volume is much smaller when treated with 5x (70 µM).