Figure 2.

Selection of 5b as a lead candidate. (a) Schematic view of the Hsp90 dimerization assay using Autodisplay. (b) Flow cytometry measurements of the inhibition of dimerized Hsp90α displayed on E. coli cells.³⁶E. coli BL21 (DE3) cells displaying Hsp90α incubated with 1 μM FITC-labeled p53 lead to a high cellular fluorescence indicating dimerization of Hsp90α. The value obtained was set as 0% inhibition. In contrast, E. coli cells without displaying Hsp90α (control cells) show no cellular fluorescence. The value obtained here was set as 100% inhibition. Preincubation of E. coli cells with surface-displayed Hsp90α with 50 μM of the respective substance leads to a lowered cellular fluorescence intensity indicating a lowered binding affinity of FITC-labeled p53 to surface-displayed Hsp90α. These values were set in relation to obtain the relative inhibition of dimerization. (c) Apparent K_D values of the purified CTD of Hsp90α and the respective substance measured via the MST method. A constant amount of 50 nM labeled CTD of Hsp90 was used, and three independent measurements were performed. The resulting mean values were determined and used in the K_D fit formula. (d) Cellular viability assessment of a leukemic cell line (K562) measured by incubating with the indicated inhibitors for 72 h, followed by a viability measurement using an ATP-based Celltiter Glo assay. (e) Selection of 5b as a lead candidate on the basis of high inhibition of Hsp90α dimerization, low apparent K_D, and low IC₅₀ (μM) in a tested leukemic cell line.