FUNCTIONAL ANALYSIS OF CYCLOOXYGENASE 2

环氧合酶-2 （COX-2）是花生四烯酸代谢的关键酶，是一系列急性和慢性人类疾病的重要贡献者。COX-2抑制剂具有抗炎、镇痛和癌症化学预防作用，但缺乏困扰传统非甾体抗炎药（NSAIDs）的严重胃肠道副作用。我们实验室对COX-2催化和抑制的结构基础很感兴趣。我们最近报道了一种实时监测酶抑制剂结合和解离的荧光猝灭技术，并利用它证明了二芳基杂环抑制剂的cox -2选择性是由于与COX-1相比，它们与酶的解离非常缓慢。我们建议使用荧光猝灭来探索环加氧酶活性位点侧袋中的残基和导致环加氧酶活性位点在二芳基杂环从酶的结合和释放中的收缩的作用。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COX-2抑制剂。将含羧酸的非甾体抗炎药转化为中性酯或酰胺衍生物可显著提高其对COX-2的选择性。我们建议使用定点诱变和x射线晶体学来阐明COX-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COX-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COX-2底物，可能提供了一种新的信号转导途径。我们打算研究COX-2对2-花生四烯基甘油的代谢，其酶特异性的分子基础，以及其内过氧化物代谢产物转化为甘油类二十烷的能力。这些研究将为COX-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.