To assess the selectivity of 5b, biochemical cell-free and cellular assays were performed. First, we evaluated the affinity of 5b against Hsp90 in a cell-free assay, where 5b protected recombinant (fulllength) Hsp90ɑ protein in a dose-dependent fashion from degradation against thermolysin enzyme digestion, an assay commonly used to quantify drug affinity-responsive target stability (DARTS) (Figure ). Next, we performed the cell-free thermal shift assay to determine the potential binding affinity of 5b to recombinant Hsp90α CTD and NTD protein. 5b specifically destabilized the CTD of Hsp90α protein Hsp90 CTD (ΔTm: -8.45 ± 0.55 °C), whereas reference CTD targeting Hsp90i coumermycin A1 (CA1) stabilized Hsp90α CTD protein (Figure , SI Figure and SI Table ).