title: False positives in a reporter gene assay: identification and synthesis of substituted N-pyridin-2-ylbenzamides as competitive inhibitors of firefly luciferase.
abstract: Luciferase reporter-gene assays are a commonly used technique in high-throughput screening campaigns. In this study, we report on a luciferase inhibitor (1), which emerged from an antagonistic G protein-coupled receptor luciferase reporter-gene assay screen. Instead of displaying receptor activity, compound 1 was shown to potently inhibit luciferase in an in vitro enzymatic assay with an IC50 value of 1.7 +/- 0.1 microM. In addition, 1 was a competitive inhibitor with respect to the substrate luciferin. A database search yielded another inhibitor (3) with a similar N-pyridin-2-ylbenzamide core. Subsequently, several analogues were prepared to investigate the structure-activity relationships of these luciferase inhibitors. This yielded the most potent inhibitor of this series (6) with an IC50 value of 0.069 +/- 0.01 microM. Further molecular modeling studies suggested that 6 can be accommodated in the luciferin binding site. This paper is meant to alert users of luciferase reporter-gene assays for possible false positive hits including highly "druglike" molecules due to direct luciferase inhibition.
authors.affiliation: DiVision of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden UniVersity, PO Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: Laura H
authors.initials: LH
authors.lastname: Heitman
authors.forename: Jacobus P D
authors.initials: JP
authors.lastname: van Veldhoven
authors.forename: Annelien M
authors.initials: AM
authors.lastname: Zweemer
authors.forename: Kai
authors.initials: K
authors.lastname: Ye
authors.forename: Johannes
authors.initials: J
authors.lastname: Brussee
authors.forename: Adriaan P
authors.initials: AP
authors.lastname: IJzerman
mesh_terms: D001549:Benzamides / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D002384:Catalysis; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D005189:False Positive Reactions; D017930:Genes, Reporter / Q000235:genetics*; D049409:Luciferases, Firefly / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D011725:Pyridines / Q000737:chemistry*; D013329:Structure-Activity Relationship
chemical_list: D001549:Benzamides; D004791:Enzyme Inhibitors; D011725:Pyridines; D049409:Luciferases, Firefly