FUNCTIONAL ANALYSIS OF CYCLOOXYGENASE 2

环氧合酶-2（考克斯-2）是花生四烯酸代谢的关键酶，是人类多种急慢性疾病的重要致病因子。考克斯-2抑制剂具有抗炎、镇痛和癌症化学预防作用，但没有困扰传统非甾体抗炎药（NSAID）的严重胃肠道副作用。我们的实验室对考克斯-2的催化和抑制的结构基础感兴趣。我们最近报道了一种荧光猝灭技术，用于监测酶-抑制剂的缔合和解离在真实的时间，并用它来证明，考克斯-2-选择性的二芳基杂环抑制剂是由于他们非常缓慢的解离酶相比，考克斯-1。我们建议使用荧光猝灭，以探索在侧袋的环氧合酶活性位点和收缩，导致环氧合酶活性位点的结合和释放的二芳基杂环从酶的残基的作用。酶-底物相互作用的研究使我们设计了一系列新的共价和非共价考克斯-2抑制剂。将含羧酸的NSAID转化为中性酯或酰胺衍生物显著增加了它们对考克斯-2的选择性。我们建议使用定点突变和X-射线晶体学来阐明考克斯-2与一系列吲哚美辛酰胺和酯相互作用的分子基础。最后，我们对考克斯-2与非甾体抗炎药酯和酰胺相互作用的研究使我们发现了一种新的考克斯-2底物，它可能提供一种新的信号转导途径。我们建议调查代谢的2-花生四烯酸甘油的考克斯-2，其酶的特异性的分子基础，其内过氧化物代谢产物的能力，转化为甘油类花生酸。这些研究将为考克斯-2的功能和抑制提供重要的新见解，COX-2是脂质信号传导和人类疾病的关键酶。

Cyclooxygenase-2 (COX-2) is a key enzyme of arachidonic acid metabolism and an important contributor to a range of acute and chronic human diseases. COX-2 inhibitors are antiinflammatory, analgesic, and cancer chemopreventive but lack the serious gastrointestinal side effects that plague traditional non-steroidal antiinflammatory drugs (NSAIDs). Our laboratory is interested in the structural basis of catalysis and inhibition of COX-2. We recently reported a fluorescence quenching technique for monitoring enzyme-inhibitor association and dissociation in real time and used it to demonstrate that the COX-2-selectivity of diarylheterocycle inhibitors is due to their very slow dissociation from the enzyme compared to COX-1. We propose to use fluorescence quenching to explore the role of residues in a side pocket of the cyclooxygenase active site and in the constriction that leads to the cyclooxygenase active site in the binding and release of diarylheterocycles from the enzyme. Studies of enzyme-substrate interactions led us to design a novel series of covalent and non-covalent COX-2 inhibitors. Conversion of carboxylic acid-containing NSAIDs into neutral ester or amide derivatives dramatically increases their selectivity for COX-2. We propose to use site-directed mutagenesis and X-ray crystallography to elucidate the molecular basis for interaction of COX-2 with a series of indomethacin amides and esters. Finally, our studies of COX-2 interactions with NSAID esters and amides led us to discover a novel substrate for COX-2 that may provide a new pathway of signal transduction. We propose to investigate the metabolism of 2- arachidonylglycerol by COX-2, the molecular basis for its enzyme specificity, and the ability of its endoperoxide metabolite to be converted into glyceryl eicosanoids. These investigations will provide important new insights into the function and inhibition of COX-2, a key enzyme of lipid signalling and human disease.