T347:Lea: A 1536-well Based Kinetic Assay for Thioredoxin Glutathione Reductase Inhibitor Discovery and Antischistosomal Drug Development

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Author(s) name and affiliation:

Wendy A. Lea1, Ganesha Rai1, Ahmed A. Sayed2, Ajit Jadhav1, Christopher P. Austin1, James Inglese1, Craig J. Thomas1, David L. Williams3, David J. Maloney1 and Anton Simeonov1

1, NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892-3370,

2, Department of Biochemistry, Ain Shams University, Cairo, Egypt

3, Department of Immunology/Microbiology, Rush University Medical Center, 1735 West Harrison Street, Chicago, Illinois 60612

Poster Abstract:

Schistosomiasis is a major neglected disease caused by different species of trematode flatworms of the genus Schistosoma, such as S. mansoni. The World Health Organization estimates that currently over 207 million people are infected with schistosomiasis and another 700 million may be at risk of infection. While extensive vaccine research is ongoing, chemotherapy has remained the only approach and has largely relied on a single drug, praziquantel (PZQ), due to its ready accessibility and efficacy against the adult form of all schistosome species. Despite PZQ’s dramatic effect on infected patients, drug resistance has been reported and novel therapeutic targets and drugs are being sought.
Thioredoxin glutathione reductase (TGR) is a uniquely positioned enzyme that was recently identified as part of S. mansoni’s antioxidant “firewall”. TGR is a multifunctional selenocysteine-containing flavoenzyme that, unlike its human counterparts, catalyzes the interconversion between reduced and oxidized forms of both glutathione and thioredoxins, thus creating a bottleneck in the processing of reactive oxygen species by the parasite. A quantitative high-throughput screen (qHTS) using a TGR-Prx2 (peroxiredoxin, a H2O2 reducing enzyme in S. mansoni) coupled assay against a library of ~71000 small molecules led to the identification of oxadiazole 2-oxides series as novel TGR inhibitor leads that was effective in killing the parasite in both cultured-worm and worm-infected mouse models. With TGR being an attractive antiparasitic target and in order to further characterize the primary hits and assist additional analog optimization and SAR study, a dedicated 1536-well based colorimetric assay targeting specifically TGR was developed and optimized. In this assay, TGR-catalyzed DTNB reduction by NADPH was monitored by measuring the absorbance increase of the reaction product TNB at 412 nm. The assay was implemented in a kinetic mode in order to improve signal strength and to minimize interference from dust, fluidics, or compound quenching. The activity of the previously identified TGR inhibitor furoxan was recapitulated using the assay, with samples obtained from different sources showing consistent single-digit micromolar potency. Compared to the TGR-Prx2 coupled system, this assay addresses TGR as a sole target and obviates the need for post-screen target deconvolution. It was also utilized in an extended mechanistic study where TGR inhibition and parasite injury have been linked to exogenous NO donation. This assay format was subsequently applied in a qHTS screen against a ~370,000 small molecule library in search for novel TGR inhibitors.

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