title: 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.
authors.affiliation: Department of Molecular Pharmacology and Chemistry, Sloan Kettering Institute, New York, New York, United States of America.
authors.forename: Tony
authors.initials: T
authors.lastname: Taldone
authors.forename: Victoria
authors.initials: V
authors.lastname: Gillan
authors.forename: Weilin
authors.initials: W
authors.lastname: Sun
authors.forename: Anna
authors.initials: A
authors.lastname: Rodina
authors.forename: Pallav
authors.initials: P
authors.lastname: Patel
authors.forename: Kirsty
authors.initials: K
authors.lastname: Maitland
authors.forename: Kerry
authors.initials: K
authors.lastname: O'Neill
authors.forename: Gabriela
authors.initials: G
authors.lastname: Chiosis
authors.forename: Eileen
authors.initials: E
authors.lastname: Devaney
mesh_terms: D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology*; D001665:Binding Sites; D017179:Brugia pahangi / Q000187:drug effects* / Q000378:metabolism; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery / Q000379:methods*; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D005260:Female; D005368:Filariasis / Q000188:drug therapy / Q000469:parasitology; D005454:Fluorescence Polarization; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D047029:Lactams, Macrocyclic / Q000494:pharmacology*; D011485:Protein Binding; D015203:Reproducibility of Results
chemical_list: D016227:Benzoquinones; D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; C001277:geldanamycin