School of Pharmacy, University of Eastern Finland , P.O. Box 1627, FI-70211 Kuopio, Finland.
J Med Chem. 2013 Feb 14;56(3):1064-73. doi: 10.1021/jm301516q. Epub 2013 Jan 17.
Luciferase reporter assays are commonly used in high-throughput screening methods. Here, we report new firefly luciferase (FLuc) inhibitors based on 5-benzyl-3-phenyl-4,5-dihydroisoxazoles and 5-benzyl-3-phenyl-1,4,2-dioxazoles, which showed up as "false positives" in a luciferase reporter gene-based assay for nuclear receptor antagonists. The inhibition was shown to be noncompetitive for both natural enzyme substrates (d-luciferin and ATP) and selective to FLuc and proven to arise from a direct interaction between the enzyme and the inhibitor. Of the 63 evaluated compounds, 28 showed significantly better inhibition potency than the well-known inhibitor resveratrol (IC(50) = 59 nM), with five compounds having distinctly subnanomolar IC(50) values. The most efficient compounds inhibited the luminescence at concentrations lower than (1)/(100) in comparison to resveratrol (lowest IC(50) = 0.26 nM) and can thus be considered to belong to the most potent FLuc inhibitors reported thus far. Overall, the novel inhibitors form a unique molecular library for structure-activity relationship (SAR) analyses.
荧光素酶报告基因检测法常用于高通量筛选方法中。在这里，我们报道了基于 5-苄基-3-苯基-4,5-二氢异恶唑和 5-苄基-3-苯基-1,4,2-二恶唑的新型萤火虫荧光素酶（FLuc）抑制剂，这些抑制剂在基于荧光素酶报告基因的核受体拮抗剂检测中呈现“假阳性”。结果表明，对于天然酶底物（d-荧光素和 ATP）和 FLuc，抑制是非竞争性的，并且对 FLuc 具有选择性，证明酶与抑制剂之间存在直接相互作用。在所评估的 63 种化合物中，有 28 种化合物对酶的抑制活性明显优于众所周知的抑制剂白藜芦醇（IC50 = 59 nM），其中 5 种化合物的 IC50 值明显低于纳摩尔级（IC50 = 0.26 nM）。最有效的化合物在比白藜芦醇（最低 IC50 = 0.26 nM）低 1/100 的浓度下抑制发光，因此可以认为它们属于迄今为止报道的最有效的 FLuc 抑制剂。总的来说，这些新型抑制剂为构效关系（SAR）分析形成了独特的分子文库。
