NTD targeting Hsp90i Tanespimycin (TM) and PUH-71 served as positive (Hsp90α NTD) or negative control (Hsp90α CTD) in this assay. The thermostabilizing effect of 5b to its target (total Hsp90) was also assessed in a cellular setup, termed cellular thermal shift assay (CETSA) , a biophysical method based on the ligand-induced thermal stabilization of the protein to directly probe the target engagement in the living cells (SI Table , SI Figure ). The protein quantification for CETSA was performed using a digital western blotter for sensitive and quantitative evaluation of the ligand-protected intracellular Hsp90, whereas TM and PU-H71 served as controls. Next, the thermal stability of intracellular Hsp90 in an increasing concentration of 5b (at a fixed temperature) was determined, a method termed isothermal dose-response fingerprint ITDRF CETSA . 40 5b induced thermal stability of Hsp90 in a dose-dependent fashion, confirming its intracellular and specific target engagement (Figure , SI Table ).