title: A high-throughput 1,536-well luminescence assay for glutathione S-transferase activity.
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.
authors.affiliation: NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892-3370, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Yasgar
authors.forename: John
authors.initials: J
authors.lastname: Shultz
authors.forename: Wenhui
authors.initials: W
authors.lastname: Zhou
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.forename: Fen
authors.initials: F
authors.lastname: Huang
authors.forename: Nancy
authors.initials: N
authors.lastname: Murphy
authors.forename: Erika L
authors.initials: EL
authors.lastname: Abel
authors.forename: John
authors.initials: J
authors.lastname: DiGiovanni
authors.forename: James
authors.initials: J
authors.lastname: Inglese
authors.forename: Anton
authors.initials: A
authors.lastname: Simeonov
mesh_terms: D000818:Animals; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods*; D004351:Drug Resistance; D019008:Drug Resistance, Neoplasm; D057075:Enzyme Assays / Q000379:methods*; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D049449:Luminescence; D051379:Mice; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012549:Schistosoma japonicum / Q000187:drug effects / Q000201:enzymology; D054852:Small Molecule Libraries; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D004364:Pharmaceutical Preparations; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; D005982:Glutathione Transferase; D005978:Glutathione