title: An improved beta-lactamase reporter assay: multiplexing with a cytotoxicity readout for enhanced accuracy of hit identification.
abstract: A problem inherent to the use of cellular assays for drug discovery is their sensitivity to cytotoxic compounds, which can result in false hits from certain compound screens. To alleviate the need to follow-up hits from a reporter assay with a separate cytotoxicity assay, the authors have developed a multiplexed assay that combines the readout of a beta-lactamase reporter with that of a homogeneous cytotoxicity indicator. Important aspects to the development of the multiplexed format are addressed, including results that demonstrate that the IC(50) values of 40 select compounds in a beta-lactamase reporter assay for nuclear factor kappa B and SIE pathway antagonists are not affected by the addition of the cytotoxicity indicator. To demonstrate the improvement in hit confirmation, the multiplexed assay was used to perform a small-library screen (7728 compounds) for serotonin 5HT1A receptor antagonists. Hits identified from analysis of the beta-lactamase reporter data alone were compared to those hits determined when the reporter and cytotoxicity data generated from the multiplexed assay were combined. Confirmation rates were determined from compound follow-up using dose-response analysis of the potential antagonist hits identified by the initial screen. In this representative screen, the multiplexed assay approach yielded a 19% reduction in the number of compounds flagged for follow-up, with a 37% decrease in the number of false hits, demonstrating that multiplexing a beta-lactamase reporter assay with a cytotoxicity readout is a highly effective strategy for reducing false hit rates in cell-based compound screening assays.
authors.affiliation: Invitrogen Discovery Sciences, Madison,Wisconsin 53719, USA. tina.hallis@invitrogen.com
authors.forename: Tina M
authors.initials: TM
authors.lastname: Hallis
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Kopp
authors.forename: Jasmin
authors.initials: J
authors.lastname: Gibson
authors.forename: Connie S
authors.initials: CS
authors.lastname: Lebakken
authors.forename: Michael
authors.initials: M
authors.lastname: Hancock
authors.forename: Kaitlyn
authors.initials: K
authors.lastname: Van Den Heuvel-Kramer
authors.forename: Tammy
authors.initials: T
authors.lastname: Turek-Etienne
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D016466:CHO Cells; D002294:Carcinoma, Squamous Cell / Q000473:pathology; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D004353:Drug Evaluation, Preclinical; D005260:Female; D031541:Fluorescence Resonance Energy Transfer; D017930:Genes, Reporter; D006576:Heterocyclic Compounds, 4 or More Rings / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D019169:Jurkat Cells; D008156:Luciferases / Q000378:metabolism; D008954:Models, Biological; D058829:Serotonin 5-HT1 Receptor Antagonists; D013379:Substrate Specificity; D014594:Uterine Neoplasms / Q000473:pathology; D001618:beta-Lactamases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
chemical_list: C524948:CCF4-AM; D006576:Heterocyclic Compounds, 4 or More Rings; D058829:Serotonin 5-HT1 Receptor Antagonists; D008156:Luciferases; D001618:beta-Lactamases