Date Published: January 2, 2008
Publisher: Public Library of Science
Author(s): Anton Simeonov, Ajit Jadhav, Ahmed A. Sayed, Yuhong Wang, Michael E. Nelson, Craig J. Thomas, James Inglese, David L. Williams, Christopher P. Austin, Malcolm Jones
Abstract: Schistosomiasis is a tropical disease associated with high morbidity and mortality, currently affecting over 200 million people worldwide. Praziquantel is the only drug used to treat the disease, and with its increased use the probability of developing drug resistance has grown significantly. The Schistosoma parasites can survive for up to decades in the human host due in part to a unique set of antioxidant enzymes that continuously degrade the reactive oxygen species produced by the host’s innate immune response. Two principal components of this defense system have been recently identified in S. mansoni as thioredoxin/glutathione reductase (TGR) and peroxiredoxin (Prx) and as such these enzymes present attractive new targets for anti-schistosomiasis drug development. Inhibition of TGR/Prx activity was screened in a dual-enzyme format with reducing equivalents being transferred from NADPH to glutathione via a TGR-catalyzed reaction and then to hydrogen peroxide via a Prx-catalyzed step. A fully automated quantitative high-throughput (qHTS) experiment was performed against a collection of 71,028 compounds tested as 7- to 15-point concentration series at 5 µL reaction volume in 1536-well plate format. In order to generate a robust data set and to minimize the effect of compound autofluorescence, apparent reaction rates derived from a kinetic read were utilized instead of end-point measurements. Actives identified from the screen, along with previously untested analogues, were subjected to confirmatory experiments using the screening assay and subsequently against the individual targets in secondary assays. Several novel active series were identified which inhibited TGR at a range of potencies, with IC50s ranging from micromolar to the assay response limit (∼25 nM). This is, to our knowledge, the first report of a large-scale HTS to identify lead compounds for a helminthic disease, and provides a paradigm that can be used to jump-start development of novel therapeutics for other neglected tropical diseases.
Partial Text: Schistosomiasis, also known as bilharzia, a debilitating disease resulting from the infection by the trematode parasite Schistosoma ssp. (S. mansoni, S. mekongi, S. japonicum, S. haematobium, and S. intercalatum) currently affects over 200 million people worldwide, mostly in developing countries . A large percentage of those infected exhibit severe morbidity manifested as growth stunting, lassitude, and cognitive impairment , and an estimated 280,000 people die annually from the disease in sub-Saharan Africa alone . The primary route of infection is via unsafe river and lake water, which is widely used in sub-Saharan Africa and Southeast Asia, among other regions, for irrigation, drinking, cooking, and bathing. Larval parasite forms (residing in and released by snails) can penetrate the skin of people contacting the water. The lifecycle of Schistosoma is exceedingly complex, with the parasite going through a number of stages both outside and inside the human host. Once inside humans, it can survive for years, even decades .
The stability, relatively low cost, and effectiveness of praziquantel has practically created a dependency on this single drug to treat schistosomiasis. Both the success of praziquantel and the general lack of incentives for large pharmaceutical companies to embark on research and development in the area of tropical diseases have led to a fairly dry pipeline for both drugs to treat schistosomiasis and basic research tools to study the lifecycle of this important parasite. To this end, we implemented a highly-miniaturized automated screen of the NCGC small molecule collection in an attempt to identify novel inhibitors of S. mansoni TGR or Prx2, both of which have been recently validated as crucial S. mansoni enzymes and have been proposed as targets for drug development. Prior to HTS adoption, the assay employed monitoring NADPH absorbance. While such a format is very convenient, offering fast access to kinetic data via the use of standard spectrophotometers, measuring absorbance in the UV region in 1536-well density is rarely practical. A significant fraction of organic molecules, as well as dust and buffer components, absorb in the 350 nm range, thereby introducing unacceptably high levels of interference. Additionally, the relatively low extinction coefficient of NADPH coupled with the short optical path length of the plate well significantly reduces the signal available for detection. Because NADPH is naturally fluorescent, emitting at ∼450 nm, while its oxidized counterpart NADP is not, we switched the detection platform for the coupled reaction from absorbance to fluorescence, a step that parallels the application of profluorescent substrates in assays for phosphatases and proteases , with the main difference being the fluorescence change trending from high to low in this reaction.