Selective binding of compound 1 (AX) and 2 to the HSP90 C terminus. (A) Scheme of the HSP90 dimerization assay using Autodisplay. HSP90 is displayed on the surface of E coli cells via the Autodisplay technique. The motility of the anchoring domain within the outer membrane of E coli facilitates the dimerization of Hsp90. Dimerized HSP90 on the surface of E coli is capable of binding to fluorescein isothiocyanate (FITC)–labeled p53. This leads to an increase of cellular fluorescence, which can then be detected via flow cytometry. Blocking the dimerization of surface displayed Hsp90 inhibits the binding of FITC-labeled p53 to HSP90 and thus leads to a decrease of cellular fluorescence.³³  (B) Inhibition of dimerization of on E coli cells displayed HSP90 measured via flow cytometry. Experiments were performed 3 times independently (n = 3), and error bars denote the standard deviation. Incubation of E coli BL21 (DE3) cells displaying HSP90 with 1 µM FITC-labeled p53 leads to a high cellular fluorescence, indicating dimerization of HSP90, whereas no cellular fluorescence was detectable in E coli cells without displaying HSP90 (control cells). Preincubation of cells with surface displayed HSP90 with 50 µM of 1 (AX) and 2, respectively, leads to a loss in cellular fluorescence, indicating a lowered binding affinity of FITC-labeled p53 to surface-displayed HSP90. (C) Determination of the apparent K_d value of the NT-647–labeled C-terminal domain of HSP90 and 1 (AX) via MST. A constant amount of the 50 nM–labeled C-terminal domain of HSP90 was used (n = 3). The resulting mean values were determined and used in the K_d fit formula. This yielded an apparent K_d of 27.39 µM for 1 (AX). (D) A cell-based HSP90-dependent luciferase assay was performed on stably expressing K562-luciferase cells. The extent of thermally denatured luciferase refolding (3 minutes at 50°C) in the presence of 1 (AX), NB, and AUY922 was monitored after 180 minutes. (E) Influence of 1 (AX) on the size distribution of HSP90 CTD revealed by sedimentation velocity analysis. 20 μM HSP90 CTD alone (purple), 20 μM HSP90 CTD plus 27.4 μM 1 (AX) (blue), and 20 μM HSP90 CTD plus 54.8 μM 1 (AX) (cyan) were analyzed at 50 000 rpm at 20°C, and the continuous c(s) model was applied to evaluate the data. The s-values were standardized to s_20,w-values. Columns depict the mean of 3 independent experiments (n = 3). Significance analyses of normally distributed data with variance similar between groups used paired, 2-tailed Student t test. *P < .05, **P < .005, ***P < .001.