Mechanism of Polyphenolic Inhibition of NADPH Oxidase

描述（由申请人提供）：多酚是非处方营养制剂中可用的植物提取物中的常见成分，也存在于不同的水果和蔬菜、橄榄油以及红酒和茶等饮料中。基于越来越多的证据，人们认为多酚的化学保护作用涉及抑制活性氧（ROS）的产生，而不仅仅是清除自由基。最近的研究表明，NADPH氧化酶是血管壁的三种主要细胞类型（例如，内皮细胞（EC）、血管平滑肌细胞（VSMC）和外膜成纤维细胞）。人们对血管NADPH氧化酶越来越感兴趣，主要是因为人们认识到氧化应激在血管疾病如动脉粥样硬化的发病机制中起着关键作用。我们小组和其他人进行的初步研究表明，邻甲氧基酚类，如夹竹桃素，在体内被过氧化物酶激活，它们的氧化产物似乎作为NADPH氧化酶组装和激活的抑制剂，尽管氧化产物的确切身份和抑制机制尚未完全阐明.这些信息对于最终开发安全有效的血管NADPH氧化酶抑制剂至关重要。在本申请中，我们提出获得关于夹竹桃素样化合物的活性代谢物和生物化学反应的结构信息，通过所述生物化学反应，夹竹桃素样化合物结合到NADPH氧化酶复合物并抑制NADPH氧化酶复合物的组装和活化。（1）阐明与内皮p47 phox、p67 phox和p22 phox肽序列结合的夹竹桃麻素的活性代谢物的化学结构，并证实这些化合物也与NADPH氧化酶的p47 phox和p67 phox蛋白亚基上的相同结构域结合;（2）确定夹竹桃麻素氧化产物是否破坏参与成熟NADPH氧化酶组装的特异性蛋白质-蛋白质相互作用。这些研究的结果将是一个更详细的了解的化学结构和分子过程的基础，其中夹竹桃素样化合物抑制NADPH氧化酶的组装和激活。

DESCRIPTION (provided by applicant): Polyphenols are common constituents in botanical extracts available in over-the-counter nutraceutical preparations as well as being found in different fruits and vegetables, olive oil, and beverages like red wine and tea. Based on a growing body of evidence it is thought that the chemoprotective effect of polyphenols involves the inhibition of reactive oxygen species (ROS) generation rather than simply scavenging the radicals. Recent studies indicate that NADPH oxidase is the major source of ROS in the three principal cell types of the blood vessel wall (e.g., endothelial cells (EC), vascular smooth muscle cells (VSMC), and adventitial fibroblasts). There has been growing interest in the vascular NADPH oxidases, largely because it is recognized that oxidative stress plays a critical role in the pathogenesis of vascular diseases, such as atherosclerosis. Preliminary studies performed in our group and others have demonstrated that ortho- methoxyphenols, such as apocynin, are activated by peroxidases in vivo and that their oxidation products appear to act as inhibitors of NADPH oxidase assembly and activation, although the exact identity of the oxidation products and the mechanism of inhibition have not been fully elucidated. Such information is critical for the ultimate development of safe and effective inhibitors of vascular NADPH oxidases. In the present application, we propose to obtain structural information on the active metabolites of apocynin-like compounds and the biochemical reactions by which they bind to and inhibit the assembly and activation of the NADPH oxidase complex. Specific goals of this R21 application are to: (1) Elucidate the chemical structures of the active metabolites of apocynin that bind to endothelial p47phox, p67phox, and p22phox peptide sequences and confirm that these compounds also bind to the same domains on the p47phox and p67phox protein subunits of NADPH oxidase; (2) Determine whether apocynin oxidation products disrupt specific protein-protein interactions that are involved in the assembly of mature NADPH oxidase. The outcome of these studies will be the foundation of a more detailed understanding of the chemical structure and molecular process by which apocynin-like compounds inhibit the assembly and activation of NADPH oxidase.