1. A high-throughput 1,536-well luminescence assay for glutathione S-transferase activity
- Author
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Adam Yasgar, Hui Wang, Nancy Murphy, Wenhui Zhou, Erika L. Abel, John DiGiovanni, Anton Simeonov, James Inglese, John Shultz, and Fen Huang
- Subjects
Luminescence ,Drug Evaluation, Preclinical ,Drug Resistance ,Isozyme ,Schistosoma japonicum ,law.invention ,Substrate Specificity ,Small Molecule Libraries ,chemistry.chemical_compound ,Mice ,law ,Drug Discovery ,Animals ,Luciferase ,Enzyme Assays ,Glutathione Transferase ,biology ,Glutathione ,Original Articles ,Small molecule ,Luciferin ,Enzyme assay ,Recombinant Proteins ,Isoenzymes ,Biochemistry ,chemistry ,Pharmaceutical Preparations ,Drug Resistance, Neoplasm ,biology.protein ,Recombinant DNA ,Molecular Medicine - Abstract
Glutathione S-transferases (GSTs) constitute a family of detoxification enzymes that catalyze the conjugation of glutathione with a variety of hydrophobic compounds, including drugs and their metabolites, to yield water-soluble derivatives that are excreted in urine or bile. Profiling the effect of small molecules on GST activity is an important component in the characterization of drug candidates and compound libraries. Additionally, specific GST isozymes have been implicated in drug resistance, especially in cancer, and thus represent potential targets for intervention. To date, there are no sensitive miniaturized high-throughput assays available for GST activity detection. A series of GST substrates containing a masked luciferin moiety have been described recently, offering the potential for configuring a sensitive screening assay via coupled luciferase reaction and standard luminescence detection. We report on the optimization and miniaturization of this homogeneous method to 1,536-well format using GSTs from 3 different species: mouse isozyme A4-4, human isozymes A1-1, M1-1, and P1-1, and the major GST from the parasitic worm Schistosoma japonicum.
- Published
- 2010