Assay strategies for the discovery and validation of therapeutics targeting Brugia pahangi Hsp90

Abstract

The chemotherapy of lymphatic filariasis relies upon drugs such as diethylcarbamazine and ivermectin that largely target the microfilarial stages of the parasite, necessitating continued treatment over the long reproductive life span of the adult worm. The identification of compounds that target adult worms has been a long-term goal of WHO. Here we describe a fluorescence polarization assay for the identification of compounds that target Hsp90 in adult filarial worms. The assay was originally developed to identify inhibitors of Hsp90 in tumor cells, and relies upon the ability of small molecules to inhibit the binding of fluorescently labelled geldanamycin to Hsp90. We demonstrate that the assay works well with soluble extracts of Brugia, while extracts of the free-living nematode C. elegans fail to bind the probe, in agreement with data from other experiments. The assay was validated using known inhibitors of Hsp90 that compete with geldanamycin for binding to Hsp90, including members of the synthetic purine-scaffold series of compounds. The efficacy of some of these compounds against adult worms was confirmed in vitro. Moreover, the assay is sufficiently sensitive to differentiate between binding of purine-scaffold compounds to human and Brugia Hsp90. The assay is suitable for high-throughput screening and provides the first example of a format with the potential to identify novel inhibitors of Hsp90 in filarial worms and in other parasitic species where Hsp90 may be a target.

Figure 1. Brugia assay development.

(A–C) Dose-response curve for the binding of 6 nM cy3B-GA to Hsp90 present in the adult Brugia pahangi worm extract (A), SKBr3 cell lysate (B) and C. elegans extract (-RNAi) or to a C. elegans extract in which hsp90 had been depleted by RNAi by approximately 40% (+RNAi) (C) Various amounts of total lysate protein dissolved in binding buffer (0–20 µg/well) were incubated in triplicate wells with the ligand at 4°C, and the response was measured at the indicated time intervals. Fluorescence polarization was read with an Analyst GT instrument. Values obtained at several time intervals were plotted against the amount of added total protein. The assay window data were obtained by subtracting free tracer values from values recorded in the presence of specified protein concentrations. Data were analyzed and plotted in Prism 4.0. Points, mean; bars, s.d. (D) Overlay of GA-bound homology models (derived using Prime software of Schrodinger L.L.C, NY) of B. pahangi (orange, Accession number AJ005784) and C. elegans (green, Accession number Z75530) and the X-ray crystal structure of human Hsp90α (blue, PDB ID: 1YET).

Figure 2. Analysis of Brugia assay performance.

(A–C) Data collected at equilibrium in the binding experiment described in Fig1A were transformed and analyzed using a nonlinear regression method in Prism 4.0, and Hill plots were constructed. Specific binding represents the contribution of bound ligand to total recorded values. (D) Two 96-well plates each containing 48 free tracer control wells (6 nM cy3B-GA) and 48 bound tracer control wells (6 nM cy3B–GA with added lysate, 2 µg/well) were used to determine the suitability of the assay for high-throughput screening. The millipolarization value for each well was recorded, and average values corresponding to each plate were plotted. The signal-to-noise ratios and the Z' factors were calculated as indicated in Methods.

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 (**).

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).