title: A specific mechanism for nonspecific activation in reporter-gene assays.
abstract: The importance of bioluminescence in enabling a broad range of high-throughput screening (HTS) assay formats is evidenced by widespread use in industry and academia. Therefore, understanding the mechanisms by which reporter enzyme activity can be modulated by small molecules is critical to the interpretation of HTS data. In this Perspective, we provide evidence for stabilization of luciferase by inhibitors in cell-based luciferase reporter-gene assays resulting in the counterintuitive phenomenon of signal activation. These data were derived from our analysis of luciferase inhibitor compound structures and their prevalence in the Molecular Libraries Small Molecule Repository using 100 HTS experiments available in PubChem. Accordingly, we found an enrichment of luciferase inhibitors in luciferase reporter-gene activation assays but not in assays using other reporters. In addition, for several luciferase inhibitor chemotypes, we measured reporter stabilization and signal activation in cells that paralleled the inhibition determined using purified luciferase to provide further experimental support for these contrasting effects.
authors.affiliation: NIH Chemical Genomics Center, National Institutes of Health, Bethesda, Maryland 20892-3370, USA.
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Auld
authors.forename: Natasha
authors.initials: N
authors.lastname: Thorne
authors.forename: Dac-Trung
authors.initials: DT
authors.lastname: Nguyen
authors.forename: James
authors.initials: J
authors.lastname: Inglese
mesh_terms: D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects*; D017930:Genes, Reporter; D049409:Luciferases, Firefly / Q000037:antagonists & inhibitors / Q000235:genetics; D015394:Molecular Structure; D012680:Sensitivity and Specificity; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship
chemical_list: D004791:Enzyme Inhibitors; D054852:Small Molecule Libraries; D049409:Luciferases, Firefly