FIGURE 6.

Aurin as a structure-based, geldanamycin-competitive inhibitor of Hsp90; down-regulation of Hsp90 client proteins in aurin-exposed A375 malignant melanoma cells. A, immunoblot detection of Hsp90 client proteins and Hsp90 in A375 cells exposed to aurin (10 μm; ≤24 h). Immunoblot analysis representative of three independent repeats is depicted. B, expression of the AhR target gene CYP1A1 induced by aurin treatment (10 μm, ≤24 h) assessed at the mRNA level (quantitative RT-PCR; mean ± S.D.; n = 3). C and D, Hsp90α inhibition as assessed by fluorescence polarization. A competitive binding assay employing FITC-labeled geldanamycin and purified recombinant Hsp90α was performed comparing inhibitory activity of geldanamycin (panel C) and aurin (panel D). E (inset in D), aurin (<40 μm)- and geldanamycin (40 μm)-induced inhibition of ATPase activity of recombinant human Hsp90α was assessed using the malachite green assay. Data are expressed as % ATPase activity relative to Hsp90α incubated with solvent control (means ± S.D. (n = 3)). F, molecular docking of aurin with HSP90α x-ray structure (ribbon view). Aurin interacts with the HSP90α active site formed by β-sheets at the bottom and α-helices H2, H4, and H7. G, close view of aurin docked with the HSP90α active site. Yellow lines indicate hydrogen bond interactions. Phenolic -OH groups of aurin engage in hydrogen bonding interactions with Asp-93 carboxylate and Asn-106 backbone carbonyl groups. The quinone carbonyl group forms a hydrogen bond with Lys-58. H, close view of geldanamycin docked with the HSP90α active site. Interactions with Asp-93, Lys-58, and Asn-106 are depicted.