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J Med Chem. 2008 Aug 14;51(15):4724-9. doi: 10.1021/jm8004509. Epub 2008 Jul 23.

False positives in a reporter gene assay: identification and synthesis of substituted N-pyridin-2-ylbenzamides as competitive inhibitors of firefly luciferase.

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DiVision of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden UniVersity, PO Box 9502, 2300 RA Leiden, The Netherlands.


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.

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