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.

Hsp90 Inhibitors as New Leads To Target Parasitic Diarrheal Diseases

“…Hsp90 is also implicated in the development and survival of other intracellular parasites, such as Eimeria tenella (53), Toxoplasma gondii (54), and Trypanosoma cruzi (55). Hsp90 was found to be essential in the filarial nematode Brugia pahangi; exposure of B. pahangi to geldanamycin, a specific inhibitor of Hsp90, killed adult worms and microfilariae in vitro (56,57). Geldanamycin also inhibited P. falciparum Hsp90 (36), T. gondii Hsp90 (54), and T. cruzi Hsp90 (55), leading to the arrest of parasite growth.…”

Fluorescence polarization assays in small molecule screening

“…A large category of diseases where there has been an overall paucity of FP assays is neglected tropical diseases, and a welcome change is a recent work to develop an FP assay targeting Hsp90 in the context of adult filarial worm lysates for development of therapeutics against lymphatic filariasis [109]. Hsp90 has been studied as a target in diseases such as cancer, and FP assays have been applied to evaluate purified Hsp90’s ATPase activity [61], as well as its interaction with other proteins [110].…”

Hsp90 Inhibitors in Parasitic Nematodes: Prospects and Challenges

“…Furthermore, in a fluorescence polarization assay, which also utilized worm lysate, we could detect no significant binding of labeled GA to C. elegans extract, while Hsp90 from Brugia extract bound with similar affinity to that of a breast cancer cell line. In addition, there was no significant difference in GA binding to extracts of wild-type C. elegans, or to worms in which levels of DAF-21 were reduced by ~40% by RNAi [43]. Given the degree of similarity (92%) between the two nematode Hsp90 sequences, these data are hard to explain on the basis of sequence diversity alone.…”

“…A number of other chemical scaffolds including purine-scaffold and isoxazole-based compounds have now been tested against filarial worms with various levels of activity [43,54]. For example, our recent work demonstrated that filarial worms were killed by in vitro exposure to 25.0 nM NVP-AUY922 and showed that this compound was also active in vivo against adult worms [54].…”