Fig. 2

NCT-547 targets the C-terminal binding site of HSP90. a-b SKBR3 cells immunostained for HSF-1 (red, a) and HSP70 (green, b) with DAPI (blue) after exposure to NCT-547 (300 nM) and geldanamycin (300 nM) for 24 h. Intensity of nuclear HSF-1 (green) and cytosolic HSP70 (red) is represented in arbitrary units as defined by the software using the intensity profile tool. Gelda; geldanamycin. c No change in protein levels of HSP70 was observed, whereas HSP90 expression was reduced in the presence of NCT-547 (0–10 μM, 72 h). d HSP90α (C-terminal) Inhibitor Screening Assay was used to assess inhibition of the C-terminal binding domain of HSP90 by the inhibitors (novobiocin, gelda and NCT-547). The activity of HSP90 inhibitors was measured with an Alphascreen microplate reader, and the inhibitory effect of each drug was determined at 500 μM. e-h Structural modeling of docking between NCT-547 and hHSP90. e Binding pose of NCT-547 in the C-terminal domain of open state hHSP90 (Surflex-Dock score = 10.228, CScore = 2). Chain A of hHSP90 is rendered as an orange ribbon, and chain B is sky blue. NCT-547 as a ball-and-stick model. Hydrogen bonds and π-cation interactions are represented as yellow and green dashed lines respectively. f View of the entire binding pose of NCT-547 at the dimerization interface. NCT-547 as a space-filling model. g Lipophilicity property surface map (brown color: hydrophobic, blue color: hydrophilic) of the active site. Connolly surface of NCT-547 is shown as pink mesh. h Comparison of the electrostatic complementarity (EC) surface and EC score of ATP with those of NCT-547. Green = perfect electrostatic complementarity (1), grey = both potentials zero, red = perfect electrostatic clash (− 1). i-k NCT-547 degrades HER2 and p95HER2 in HER2- and p95HER2- overexpressing MDA-MB-231 cancer cells. i The expression of HER2, p95HER, phospho-HER2, and phospho-p95HER2 was upregulated in both HER2- and p95HER2-overexpressing MDA-MB-231 (two clones; C1 and C2) compared to the parental MDA-MB-231 cells. j Levels of full-length p185HER2, p95HER2, phospho-HER2 (Tyr1221/1222) and phospho-p95HER2 were detected after treatment of NCT-547 (0–10 μM, 72 h) by immunoblot analysis. k p95HER2-overexpressing MDA-MB-231 cells were co-immunostained for p95HER2 (1:100, green) and ubiquitin (1:100, red) with DAPI (blue) after exposure to NCT-547 (10 μM) for 24 h. Co-localization of ubiquitin and p95HER2 in the plasma membrane is seen as yellow signal (white arrows) at high magnification (× 2000)