From the Department of Biophysics and Free Radical Research Center and
From the Department of Biophysics and Free Radical Research Center and.
J Biol Chem. 2014 Jun 6;289(23):16176-89. doi: 10.1074/jbc.M114.548693. Epub 2014 Apr 24.
Recent progress characterizing the reaction mechanism(s) of fluorescent probes with reactive oxygen species has made it possible to rigorously analyze these reactive species in biological systems. We have developed rapid high throughput-compatible assays for monitoring cellular production of superoxide radical anion and hydrogen peroxide using hydropropidine and coumarin boronic acid probes, respectively. Coupling plate reader-based fluorescence measurements with HPLC-based simultaneous monitoring of superoxide radical anion and hydrogen peroxide provides the basis for the screening protocol for NADPH oxidase (Nox) inhibitors. Using this newly developed approach along with the medium-throughput plate reader-based oximetry and EPR spin trapping as confirmatory assays, it is now eminently feasible to rapidly and reliably identify Nox enzyme inhibitors with a markedly lower rate of false positives. These methodological advances provide an opportunity to discover selective inhibitors of Nox isozymes, through enhanced conceptual understanding of their basic mechanisms of action.
近年来，人们对荧光探针与活性氧反应机制的研究取得了进展，这使得在生物系统中严格分析这些活性氧成为可能。我们开发了快速高通量兼容的测定法，分别使用氢丙啶和香豆素硼酸探针监测超氧阴离子自由基和过氧化氢的细胞产生。基于平板阅读器的荧光测量与基于 HPLC 的超氧阴离子自由基和过氧化氢的同时监测相结合，为 NADPH 氧化酶 (Nox) 抑制剂的筛选方案提供了基础。使用这种新开发的方法以及基于中通量平板阅读器的血氧测定法和 EPR 自旋捕获作为确证测定法，现在可以快速可靠地识别 Nox 酶抑制剂，并且假阳性率明显降低。这些方法学的进步为通过增强对其基本作用机制的概念理解，发现 Nox 同工酶的选择性抑制剂提供了机会。
