A 1,536-well-based kinetic HTS assay for inhibitors of Schistosoma mansoni thioredoxin glutathione reductase.
Abstract: Schistosomiasis is a major neglected tropical disease that currently affects over 200 million people and leads to over 200,000 annual deaths. Schistosoma mansoni parasites survive in humans in part because of a set of antioxidant enzymes that continuously degrade reactive oxygen species produced by the host. A principal component of this defense system has been recently identified as thioredoxin glutathione reductase (TGR), a parasite-specific enzyme that combines the functions of two human counterparts, glutathione reductase and thioredoxin reductase, and as such this enzyme presents an attractive new target for anti-schistosomiasis drug development. Herein, we present the development of a highly miniaturized and robust screening assay for TGR. The 5-mul final volume assay is based on the Ellman reagent [5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)] and utilizes a high-speed absorbance kinetic read to minimize the effect of dust, absorbance interference, and meniscus variation. This assay is further applicable to the testing of other redox enzymes that utilize DTNB as a model substrate.
Lea
Wendy A
WA
NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Jadhav
Ajit
A
Rai
Ganesha
G
Sayed
Ahmed A
AA
Cass
Cynthia L
CL
Inglese
James
J
Williams
David L
DL
Austin
Christopher P
CP
Simeonov
Anton
A
D000818:Animals; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007700:Kinetics; D017382:Reactive Oxygen Species / Q000737:chemistry; D011994:Recombinant Proteins / Q000494:pharmacology; D012550:Schistosoma mansoni / Q000201:enzymology*; D012556:Schistosomicides / Q000494:pharmacology*; D013880:Thioredoxin-Disulfide Reductase / Q000037:antagonists & inhibitors*
D004791:Enzyme Inhibitors; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D012556:Schistosomicides; D004228:Dithionitrobenzoic Acid; D013880:Thioredoxin-Disulfide Reductase