The evaluation of the Hsp90 binding affinity to PPID (Cyclophilin D) was performed using the Hsp90 CTD TR-FRET assay kit (#50289, BPS Bioscience, San Diego, CA, USA). Terbium (Tb) labeled donor and dye-labeled acceptor were diluted 100-fold in (1X) Hsp90 assay buffer 2. 10 µL diluted tb-labeled donor and 10 µl diluted dye-labeled acceptor were mixed with 4 µL (indicated concentration) inhibitor, 10 µL of 3 ng/µl PPID-GST-tag (BPS Bioscience) and 6 µL of 2 ng/µl biotin labeled CTD of Hsp90 (BPS Biosciences). For the positive control, the inhibitor was substituted for DMSO and for the negative control PPID-GST-tag was substituted for 1X Hsp90 assay buffer. Samples were incubated for 2 hours at RT protected from light and measured with a microplate-reader (SPARK10M, Tecan). Fluorescence was measured using a time resolved reading mode with two subsequent measurement: First measurement was performed using 340 nm / 620 nm (excitation/emission) wavelength with a lag time of 60 µs and integration time of 500 µs. Second measurement was performed using 340 nm / 665 nm (excitation/emission) wavelength with a lag time of 60 µs and integration time of 500 µs. Data analysis is performed using the TR-FRET ratio (665 nm emission / 620 nm emission). The TR-FRET ratios are normalized to % activity by setting the negative control as zero percent activity and the positive control as one hundred percent activity [(FRETsample -FRET neg )/(FRET pos -FRET neg ) * 100%].