FUNCTIONAL ANALYSIS OF CYCLOOXYGENASE 2

环氧合酶-2(COX-2)是花生四烯酸代谢的关键酶，是人类一系列急、慢性疾病的重要致病因子。COX-2抑制剂具有抗炎、止痛和癌症化学预防作用，但缺乏困扰传统非类固醇抗炎药(NSAIDs)的严重胃肠道副作用。我们实验室对COX-2的催化和抑制的结构基础感兴趣。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，并用它来证明二芳基杂环抑制剂的COX-2选择性是因为它们与COX-1相比从酶上解离得非常慢。我们建议使用荧光猝灭来探索环氧合酶活性部位侧袋中的残基在环氧合酶活性部位的收缩中的作用，以及在二芳基杂环从酶中结合和释放中的作用。酶-底物相互作用的研究引导我们设计了一系列新的共价和非共价COX-2抑制剂。将含有羧酸的非甾体抗炎药转化为中性的酯或酰胺类化合物，大大提高了它们对COX-2的选择性。我们建议使用定点突变和X射线结晶学来阐明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NSAID酯和酰胺的相互作用的研究使我们发现了COX-2的新底物，这可能提供一种新的信号转导途径。我们建议研究COX-2对2-花生四烯基甘油的代谢，其酶专一性的分子基础，以及其内过氧化代谢产物转化为甘油二十烷基类化合物的能力。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.