Figure 4. Brugia assay identifies species selective Hsp90 inhibitors.

(A–C) Increasing concentrations of indicated inhibitors were added in triplicate to the reaction buffer containing 6 nM of cy3B-GA and Brugia extracts (2 µg/well) (A) or SKBr3 cell lysates (3 µg/well) (B) in a final volume of 100 µL. Free (6 nM cy3B-GA) and bound (6 nM cy3B-GA with 2 µg/well Brugia or 3 µg/well SKBr3 extract) controls were included on each plate. The polarization values were measured after incubation at 4°C for 24 h with the indicated inhibitors to evaluate their Brugia and human tumor Hsp90 affinity. Points, mean; bars, s.d. EC₅₀ values were determined as shown in Fig. 3, and tabulated to indicate the selectivity ratio for the two Hsp90 species (C).