However, during SAXS measurements, which were performed on an ensemble of species in solution, we were unable to determine whether 5b-induced oligomers originated from Hsp90α CTD monomeric or dimeric species. In addition, 5b blocks the chaperone function of Hsp90, as determined by TR-FRET assay and in cell-free luciferase refolding assay. Markedly, even though there are differences in the inhibitory concentrations against tested leukemia cells between 5b (IC 50 in submicromolar range) and reference Hsp90 NTD control inhibitors (IC 50 in subnanomolar range), a comparative selectivity profile (in cell-free or cell-based biochemical assays) toward Hsp90 was observed between 5b and Hsp90 NTD reference inhibitors. This data indicate that the conventional Hsp90 NTD inhibitors induce cellular inhibitory effects through their off-target activity, besides targeting Hsp90. Moreover, 5b exhibited potent in vitro anticancer activity against a broad spectrum of therapy-resistant leukemia cell lines (including TKI and proteasome inhibitor-resistant) and primary TKI-resistant (BCR-ABL1+) leukemia patient cells. 5b significantly reduced the leukemia burden in the zebrafish xenotransplantation model and induced apoptosis in TKI-resistant BCR-ABL1 T315I, E255K or M351T mutant cells by destabilizing the BCR-ABL1 expression and, thereby, hampering related downstream signaling cascades without HSR induction. These data collectively established 5b as a first-in-class smallmolecule inhibitor that targets the C-terminal dimerization interface. Expression and purification of recombinant Hsp90: Human Hsp90α CTD (563-732 amino acids) recombinant protein purification was performed as previously described. Human Hsp90α NTD (amino acids 9-236; Addgene #22481) protein was expressed in E. coli BL21-DE3 cells. 76 BL21-DE3 expression strains were grown overnight and used to inoculate LB medium at 37 °C supplemented with 100 μg/mL ampicillin to an (OD 600 = 0.5-0.8), followed by overnight induction of protein expression with 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) at 25 °C. After induction, cells were harvested by centrifugation at 5000 × g and lysed using B-PER bacterial protein extraction reagent (ThermoFisher Scientific, Wesel, Germany). GST-tagged Hsp90 CTD and NTD proteins were affinity purified spin columns (ThermoFisher Scientific) and eluted using glutathione elution buffer. Protein aliquots were made and supplemented with 5% glycerol and stored at -80 °C.