False positives in a reporter gene assay:: Identification and synthesis of substituted N-pyridin-2-ylbenzamides as competitive inhibitors of firefly luciferase
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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DOI：
10.1021/jm8004509
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发表时间：
2008-08-14
影响因子：
7.3
通讯作者：
IJzerman, Adriaan P.
中科院分区：
文献类型：
--
作者：
Heitman, Laura H.;van Veldhoven, Jacobus P. D.;IJzerman, Adriaan P.
荧光素酶报告基因检测是高通量筛选活动中常用的技术。在这项研究中，我们报告了一种荧光素酶抑制剂 (1)，它是通过拮抗性 G 蛋白偶联受体荧光素酶报告基因检测筛选而出现的。在体外酶测定中，化合物 I 并未表现出受体活性，而是有效抑制荧光素酶，IC50 值为 1.7 +/- 0.1 μM。此外，1 是底物荧光素的竞争性抑制剂。数据库搜索产生了另一种具有类似 N-吡啶-2-基苯甲酰胺核心的抑制剂 (3)。随后，制备了几种类似物来研究这些荧光素酶抑制剂的构效关系。这产生了该系列中最有效的抑制剂 (6)，IC50 值为 0.069 +/- 0.01 muM。进一步的分子模型研究表明，6 可以容纳在荧光素结合位点中。本文旨在提醒荧光素酶报告基因检测的用户可能出现假阳性结果，包括由于直接荧光素酶抑制而导致的高度“药物样”分子。
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 I was shown to potently inhibit luciferase in an in vitro enzymatic assay with an IC50 value of 1.7 +/- 0.1 mu M. 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 mu M. 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.