一种简单的直接O2消耗法检测COX抑制作用

Q4 Pharmacology, Toxicology and Pharmaceutics
Maria Grazia Perronea, Morena Miciaccia, S. Ferorelli, A. Scilimati
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引用次数: 0

摘要

背景:环氧合酶是一种众所周知的氧化还原酶,它催化花生四烯酸(AA)摄取两摩尔O2,产生氢过氧化物PGG2,然后还原为前列腺素前体前列腺素H2(PGH2)。在这样的反应过程中O2的消耗是环氧合酶活性的量度。所涉及的O2通常通过间接方法测量,在底物AA和/或抑制剂存在的情况下完成。方法:我们使用稳定转染COX-1或COX-2基因的HEK293来源的粘附细胞作为COX酶的来源,开发了一种新的简化且易于实施的O2消耗测量方案。克拉克电极用于测量在酶催化反应过程中O2浓度的变化。结果与讨论:通过测定吲哚美辛、布洛芬、SC560和塞来昔布等已知抑制剂的IC50值,验证了新的测定方法。吲哚美辛和布洛芬是两种传统的非甾体抗炎药。SC560是用于COX-1抑制研究的市售参考化合物。塞来昔布是一种临床使用的COXIB。该方法还用于测定莫非唑酸和P6的动力学和IC50。莫非唑拉是最有效的选择性COX-1抑制剂,也是日本用于治疗类风湿性关节炎的Disopain®的主要活性成分。我们发现的P6与莫非唑酸一起用作“体外”和“体内”COX抑制研究的参考,并用作结构-抑制-活性关系研究的支架。所获得的结果显示了新开发的测定法在抑制剂存在下测量COXs活性以及抑制动力学(即Vmax和Km)的适用性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
A simplified direct O2 consumption-based assay to test COX inhibition
Background: Cyclooxygenase is a well-known oxidoreductase that catalyzes the uptake of two moles of O2 by arachidonic acid (AA) producing the hydroperoxide PGG2, then reduced to the prostaglandin precursor Prostaglandin H2 (PGH2). O2 consumption during such reactions are a measure of cyclooxygenase activity. O2 involved is generally measured by indirect methods, accomplished in the presence of the substrate AA and/or inhibitors. Methods: we developed a new simplified and easy to be carried out protocol for O2 consumption measurement by using disrupted HEK293-derived adherent cells, stably transfected either with COX-1 or COX-2 genes, as a source of the COX enzymes. The Clark electrode is used to measure the O2 concentration variation during the enzyme-catalyzed reactions. Results and Discussion: the novel assay was validated by determining the IC50 values of the known inhibitors such as indomethacin, ibuprofen, SC560 and celecoxib. Indomethacin and ibuprofen are two traditional non-steroidal anti-inflammatory drugs (NSAIDs). SC560 is a commercially available reference compound used for COX-1 inhibition investigations. Celecoxib is a clinically used COXIBs. The assay was also applied to measure the kinetics and IC50 of mofezolac and P6. Mofezolac is the most potent selective COX-1 inhibitor and active principal ingredient of Disopain® used to treat the rheumatoid arthritis in Japan. P6, uncovered by us, is used together with mofezolac as reference in “in vitro” and “in vivo” COX inhibition investigations and as a scaffold for structure-inhibition activity relationship studies. The obtained results showed the suitability of the newly developed assay to measure COXs activity in the presence of inhibitors as well as the kinetics of the inhibition (i.e., Vmax and Km).
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来源期刊
Current Enzyme Inhibition
Current Enzyme Inhibition Pharmacology, Toxicology and Pharmaceutics-Drug Discovery
CiteScore
1.30
自引率
0.00%
发文量
30
期刊介绍: Current Enzyme Inhibition aims to publish all the latest and outstanding developments in enzyme inhibition studies with regards to the mechanisms of inhibitory processes of enzymes, recognition of active sites, and the discovery of agonists and antagonists, leading to the design and development of new drugs of significant therapeutic value. Each issue contains a series of timely, in-depth reviews written by leaders in the field, covering a range of enzymes that can be exploited for drug development. Current Enzyme Inhibition is an essential journal for every pharmaceutical and medicinal chemist who wishes to have up-to-date knowledge about each and every development in the study of enzyme inhibition.
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