Figure 3. Brugia assay validation.

(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) in a final volume of 100 µL. Free (6 nM cy3B-GA) and bound (6 nM cy3B-GA with 2 µg/well Brugia extract) controls were included on each plate. The polarization values were measured after incubation at 4°C for the indicated times to evaluate assay stability (A) or for 24 h with the indicated inhibitors to evaluate their affinity for Brugia Hsp90 (B, C). The competitive effect was expressed as percentage of control and was calculated by dividing the millipolarization (mP; subtracting free cy3B-GA) value from inhibitor wells by the average mP (subtracting free cy3B-GA) from controls (cy3B-GA and cell lysate with vehicle DMSO) in each plate. Ligand binding was plotted against the log₁₀ inhibitor concentration, and EC₅₀ values were calculated using a nonlinear least-square curve-fitting program in Prism 4.0. Points, mean; bars, s.d. (D) Six adult female B. pahangi were incubated individually in 2.0 ml of tissue culture medium containing GA at 1.0 µM, PU-H71 at 10, 5 or 2.5 µM, PU-DZ8 at 10 or 5 µM, DMSO or medium alone. Graphs show mean and SD of Mf output over a three-day period from six female worms per group. Data combined from two separate experiments. *** P<0.005 for all drug concentrations vs DMSO except for PU-H71 at 2.5 µM where P = 0.0260 (**).