The evaluation of the binding affinity of compounds towards the ATP pocket of Hsp90 NTD was determined by a competitive binding assay against FITClabeled geldanamycin (GM) using the Hsp90 NTD assay kit (#50293, BPS Bioscience). The inhibitor sample wells were filled with 15 µl 5X Hsp90 assay buffer, 5 µL 40 mM DTT, 5 µL 2 mg/ml BSA, 40 µL H 2 O, 5 µL FITC-labeled GM (100 nM) and 10 µL inhibitor (at indicated concentration). The reaction was initiated by adding 20 µl Hsp90 (17 ng/µl) and incubation at room temperature for 3 hours with slow shaking. Background wells (master mix only); negative controls (FITC-labeled GM, buffer and DMSO) and positive controls (FITC-labeled GM, buffer, DMSO, Hsp90) were also included within the assay plate. Fluorescence was measured at 470 nm excitation wavelength and 525 nm emission wavelength in a microtiter-plate reader (Infinite M1000pro by Tecan). Polarization was calculated using (I II -G(I ┴ )/ (I II + G(I ┴ ))*1000 and a g-factor of 1,187. The percentage of Hsp90-bound FITC labelled GM was calculated using P norm = (P Inhibitor -P neg ) / (P pos -P neg )*100.