Next, to assess the ability of 5b to inhibit Hsp90 chaperone function, a cell-free luciferase-refolding assay was performed using rabbit reticulocyte lysates as a source of Hsp90. Exposure of 5b decreased the luciferase refolding capacity in a dose-dependent manner by blocking the chaperone function of Hsp90 (Figure ). The known Hsp90 NTD inhibitors geldanamycin (GM) and TM served as positive controls. Besides, to assess the specific effect of 5b in obstructing Hsp90 CTD-interacting cochaperones, a time-resolved fluorescence resonance energy transfer (TR-FRET) assay was conducted. 45 5b blocked the binding of PPID (or cyclophillin D, an Hsp90 CTD-interacting chaperone) to recombinant Hsp90α or Hsp90β CTD protein comparable to CA1 treatment, whereas PU-H71, TM, and GM served as negative controls (Figure , Table ). To rule out the possible interaction of 5b with the NTD of Hsp90α, a fluorescence polarization (FP) competitive assay was carried out using FITClabelled GM (Figure , SI Table ). As expected, 5b did not show any interaction with the NTD of Hsp90, whereas unlabeled Hsp90 NTD targeting inhibitors GM, Ganetespib (GP), TM, and PU-H71 served as positive controls. Hsp90α (1 µg) was incubated with 5b at indicated concentrations, followed by digestion with thermolysin. Treated protein samples were electrophoresed (SDS-PAGE) and immunoblotted with anti-Hsp90α for detecting protection of Hsp90α protein by 5b (the upper band is protected from proteolysis). b) Cell-free thermal shift assay was performed by incubating recombinant Hsp90α CTD protein with 5b, at an increasing temperature (up to 95 °C). Melting temperature (T m ) without inhibitors (DMSO) was used as a control. c) The dose-dependent intracellular (K562 cells) thermal stabilization (CETSA ITDRF ) of Hsp90 after 5b incubation (24 h) at its increasing concentration (1.25 µM -5 µM). d) 5b inhibits Hsp90α chaperone function, comparable to TM and GM, in cell-free luciferase refolding assay, where the incubation of the inhibitors prevented the rabbit reticulocyte lysate (a source of Hsp90) assisted refolding of denatured luciferase. e) Incubation of 5b blocked the binding of Hsp90 CTD-interacting cochaperone (PPID) in TR-FRET measurements. f) 5b did not reduce the amount of Hsp90-bound FITC-labelled GM and, therefore, does not compete for the GM binding pocket of full-length Hsp90α. Unlabelled GM, GP, PUH71, and TM served as positive controls and NB and CA1 as negative controls.