R&D, Promega Biosciences, San Luis Obispo, CA 93401, USA.
Drug Metab Dispos. 2011 Dec;39(12):2403-10. doi: 10.1124/dmd.111.041541. Epub 2011 Sep 2.
Cytochrome P450 (P450) assays use probe substrates to interrogate the influence of new chemical entities toward P450 enzymes. We report the synthesis and study of a family of bioluminogenic luciferin acetal substrates that are oxidized by P450 enzymes to form luciferase substrates. The luciferin acetals were screened against a panel of purified P450 enzymes. In particular, one proluciferin acetal has demonstrated sensitive and selective CYP3A4-catalyzed oxidation to a luciferin ester-K(m) and k(cat) are 2.88 μM and 5.87 pmol metabolite · min(-1) · pmol enzyme(-1), respectively. The proluciferin acetal was used as a probe substrate to measure IC(50) values of known inhibitors against recombinant CYP3A4 or human liver microsomes. IC(50) values for the known inhibitors correlate strongly with IC(50) values calculated from the traditional high-performance liquid chromatography-based probe substrate testosterone. Luciferin acetals are rapidly oxidized to unstable hemi-orthoesters by CYP3A resulting in luciferin esters and, therefore, are conducive to simple rapid CYP3A bioluminescent assays.
细胞色素 P450（P450）测定法使用探针底物来探究新化学实体对 P450 酶的影响。我们报告了一组生物发光荧光素缩醛底物的合成和研究，这些底物可被 P450 酶氧化形成荧光酶底物。将荧光素缩醛对一组纯化的 P450 酶进行了筛选。特别是，一种前荧光素缩醛已被证明对 CYP3A4 催化的氧化具有灵敏和选择性，生成荧光素酯-K(m)和 k(cat)分别为 2.88 μM 和 5.87 pmol 代谢物·min(-1)·pmol 酶(-1)。该前荧光素缩醛被用作探针底物，以测量已知抑制剂对重组 CYP3A4 或人肝微粒体的 IC(50)值。已知抑制剂的 IC(50)值与基于传统高效液相色谱的探针底物睾酮计算出的 IC(50)值密切相关。荧光素缩醛被 CYP3A 迅速氧化为不稳定的半原甲酸酯，生成荧光素酯，因此有利于简单快速的 CYP3A 生物发光测定法。
