Tsotsou Georgia E, Di Nardo Giovanna, Sadeghi Sheila J, Fruttero Roberta, Lazzarato Loretta, Bertinaria Massimo, Gilardi Gianfranco
Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy.
J Biomol Screen. 2013 Feb;18(2):211-8. doi: 10.1177/1087057112459351. Epub 2012 Sep 13.
This work presents the validation of a rapid screening procedure for the catalysis of cytochrome P450 on new chemical entities. The assay is tested on the prototypical, catalytically self-sufficient and soluble cytochrome P450 BM3 from Bacillus megaterium that shares a high degree of homology with mammalian counterparts. The so-called alkali assay developed in our laboratory is validated here also by product formation and molecular modeling on a number of derivatives sharing the molecular scaffold of the 1,2,5-oxadiazole ring, a class of molecules very different from the long-chain fatty acids known to be oxidized by cytochrome P450 BM3. The alkali assay reveals the ability of this cytochrome to oxidize NADPH in the presence of nine out of thirteen 1,2,5-oxadiazole derivatives tested. The enzyme shows high affinity and coupling efficiencies when incubated with four 1,2,5-oxadiazole derivatives. The presence of oxidation products deriving from catalysis was also confirmed by high-performance liquid chromatography (HPLC). Molecular docking suggests that a key factor for the 1,2,5-oxadiazole derivatives to enter the active site and induce catalysis is the presence of the -SO(2) moiety bridging the 1,2,5-oxadiazole and phenyl rings. These data indicate that the alkali assay is able to quickly and cheaply detect the recognition of new substrates by cytochrome P450. The assay is not intended to substitute HPLC-mass spectrometry analysis, but it is a preliminary screening that allows elimination of obvious nonsubstrates from the start.
这项工作展示了一种针对新化学实体的细胞色素P450催化作用的快速筛选程序的验证。该测定法在来自巨大芽孢杆菌的典型、具有催化自足性且可溶的细胞色素P450 BM3上进行测试，它与哺乳动物的对应物具有高度同源性。我们实验室开发的所谓碱测定法，在此也通过产物形成以及对一系列共享1,2,5 - 恶二唑环分子支架的衍生物进行分子建模来验证，这是一类与已知被细胞色素P450 BM3氧化的长链脂肪酸非常不同的分子。碱测定法揭示了这种细胞色素在测试的十三种1,2,5 - 恶二唑衍生物中的九种存在时氧化NADPH的能力。当与四种1,2,5 - 恶二唑衍生物孵育时，该酶显示出高亲和力和偶联效率。高效液相色谱（HPLC）也证实了催化产生氧化产物的存在。分子对接表明，1,2,5 - 恶二唑衍生物进入活性位点并诱导催化的一个关键因素是存在连接1,2,5 - 恶二唑和苯环的 -SO(2) 部分。这些数据表明碱测定法能够快速且廉价地检测细胞色素P450对新底物的识别。该测定法并非旨在替代HPLC - 质谱分析，而是一种初步筛选，能够从一开始就排除明显的非底物。
