Metabolic stability screening of 5b in human liver microsomes revealed 91 % stability after a 40 min incubation at 37 C (Supplementary Note 2). Propanolol, a reference drug with medium to high metabolic stability, showed 74 % of the parent compound remaining, and therefore demonstrated slightly decreased stability compared with 5b. Calculated results for the intrinsic clearance suggest that 5b (6 µL/min/mg) is a low-clearance compound with estimated long half-life (n = 2). CTD was incubated with 63 µM BS 3 crosslinker, with (at indicated concentration) or without 5b, followed by immunoblotting with anti-Hsp90 (AC88) antibody. b) Scattering data of Hsp90α CTD is shown in black dots, with grey error bars. The ab-initio DAMMIF model fit is shown as a red line. The intensity is displayed as a function of momentum transfer s. c) The volumetric envelope, calculated from the scattering data using DAMMIF , is shown as a blue surface. The monomers of the predicted Hsp90 CTD dimer model are shown in green and cyan. Superimposing was done using SUPCOMB. 57 d) The Radius of gyration (Rg) of the different Hsp90α CTD protein samples was calculated using the Guinier approximation. The theoretical Rg of the tetramer was calculated using CRYSOL based on the structure PDB ID 3q6m. 59 e) Native Hsp90 complexes in K-562 (24h administration of 5b) was identified by running Blue Native (BN) gels followed by immunoblotting analysis. The cytotoxic concentration of 5b resulted in the potent disruption of Hsp90ɑ, Hsp90β, Hsp40, and Hsp27 complexes and monomers/dimers. AUY922 exposure elevated the expression of HSR associated protein complexes and monomers/dimers (Hsp40 and Hsp27), whereas Hsp60 served as loading controls.