title: Identification of Small-Molecule Frequent Hitters of Glutathione S-Transferase-Glutathione Interaction.
abstract: In high-throughput screening (HTS) campaigns, the binding of glutathione S-transferase (GST) to glutathione (GSH) is used for detection of GST-tagged proteins in protein-protein interactions or enzyme assays. However, many false-positives, so-called frequent hitters (FH), arise that either prevent GST/GSH interaction or interfere with assay signal generation or detection. To identify GST-FH compounds, we analyzed the data of five independent AlphaScreen-based screening campaigns to classify compounds that inhibit the GST/GSH interaction. We identified 53 compounds affecting GST/GSH binding but not influencing His-tag/Ni(2+)-NTA interaction and general AlphaScreen signals. The structures of these 53 experimentally identified GST-FHs were analyzed in chemoinformatic studies to categorize substructural features that promote interference with GST/GSH binding. Here, we confirmed several existing chemoinformatic filters and more importantly extended them as well as added novel filters that specify compounds with anti-GST/GSH activity. Selected compounds were also tested using different antibody-based GST detection technologies and exhibited no interference clearly demonstrating specificity toward their GST/GSH interaction. Thus, these newly described GST-FH will further contribute to the identification of FH compounds containing promiscuous substructures. The developed filters were uploaded to the OCHEM website (http://ochem.eu) and are publicly accessible for analysis of future HTS results.
authors.affiliation: Helmholtz Zentrum München für Gesundheit und Umwelt (GmbH), Assay Development and Screening Platform, Institute of Molecular Toxicology and Pharmacology, Neuherberg, Germany.
authors.forename: Jara K
authors.initials: JK
authors.lastname: Brenke
authors.affiliation: Institute for Organic Chemistry, Technical University Bergakademie Freiberg, Germany.
authors.forename: Elena S
authors.initials: ES
authors.lastname: Salmina
authors.affiliation: Helmholtz Zentrum München für Gesundheit und Umwelt (GmbH), Assay Development and Screening Platform, Institute of Molecular Toxicology and Pharmacology, Neuherberg, Germany.
authors.forename: Larissa
authors.initials: L
authors.lastname: Ringelstetter
authors.affiliation: Helmholtz Zentrum München für Gesundheit und Umwelt (GmbH), Assay Development and Screening Platform, Institute of Molecular Toxicology and Pharmacology, Neuherberg, Germany.
authors.forename: Scarlett
authors.initials: S
authors.lastname: Dornauer
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology, ScreeningPort (Fraunhofer-IME SP), Hamburg, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Kuzikov
authors.affiliation: Helmholtz Zentrum München für Gesundheit und Umwelt (GmbH), Assay Development and Screening Platform, Institute of Molecular Toxicology and Pharmacology, Neuherberg, Germany.
authors.forename: Ina
authors.initials: I
authors.lastname: Rothenaigner
authors.affiliation: Helmholtz Zentrum München für Gesundheit und Umwelt (GmbH), Assay Development and Screening Platform, Institute of Molecular Toxicology and Pharmacology, Neuherberg, Germany.
authors.forename: Kenji
authors.initials: K
authors.lastname: Schorpp
authors.affiliation: Helmholtz Zentrum München für Gesundheit und Umwelt (GmbH), Research Unit Gene Vectors, Munich, Germany German Center for Infection Research (DZIF), Partner Site Munich, Munich, Germany.
authors.forename: Fabian
authors.initials: F
authors.lastname: Giehler
authors.affiliation: Laboratory for Centrosome and Cytoskeleton Biology, CMMC, Cologne, Germany.
authors.forename: Jay
authors.initials: J
authors.lastname: Gopalakrishnan
authors.affiliation: Helmholtz Zentrum München für Gesundheit und Umwelt (GmbH), Research Unit Gene Vectors, Munich, Germany German Center for Infection Research (DZIF), Partner Site Munich, Munich, Germany.
authors.forename: Arnd
authors.initials: A
authors.lastname: Kieser
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology, ScreeningPort (Fraunhofer-IME SP), Hamburg, Germany.
authors.forename: Sheraz
authors.initials: S
authors.lastname: Gul
authors.affiliation: Helmholtz Zentrum München für Gesundheit und Umwelt (GmbH), Institute of Structural Biology, Neuherberg, Germany BigChem GmbH, Ingolstädter Landstrasse 1, Neuherberg, Germany.
authors.forename: Igor V
authors.initials: IV
authors.lastname: Tetko
authors.affiliation: Helmholtz Zentrum München für Gesundheit und Umwelt (GmbH), Assay Development and Screening Platform, Institute of Molecular Toxicology and Pharmacology, Neuherberg, Germany kamyar.hadian@helmholtz-muenchen.de.
authors.forename: Kamyar
authors.initials: K
authors.lastname: Hadian
mesh_terms: D005978:Glutathione / Q000037:antagonists & inhibitors / Q000737:chemistry*; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D060066:Protein Interaction Maps / Q000187:drug effects; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D013379:Substrate Specificity
chemical_list: D054852:Small Molecule Libraries; D005982:Glutathione Transferase; D005978:Glutathione
keywords: AlphaScreen; GST; frequent hitter; glutathione; high-throughput screening