Functional Analysis of Cyclooxygenase-2

描述（由申请人提供）：环氧合酶-2 （COX-2）在花生四烯酸（AA）转化为前列腺素（一类重要的生物活性脂类）中起关键作用。COX-2在炎症组织、癌前病变和癌症中高表达，但在邻近的正常组织中不表达。COX-2抑制剂，无论是异构体选择性还是非选择性，在人体临床试验中表现出抗炎、癌症预防和辅助癌症治疗活性以及心血管副作用。COX-2还氧化AA的酯类和酰胺类衍生物，包括2-花生四烯醇甘油（2-AG）和花生四烯醇乙醇酰胺（AEA）。2-AG和AEA是大麻素受体CB1和CB2的两种最具特征的内源性配体，并通过这些受体发挥多种作用。内源性大麻素生物学的主要焦点一直是神经系统，但越来越多的证据表明，2-AG和AEA具有抗炎和预防癌症的作用。我们假设cox -2依赖性的2-AG和AEA代谢降低了内源性大麻素水平，从而促进了炎症和癌症的发展。我们的实验室研究了COX-2与底物和抑制剂相互作用的结构和功能基础，以便对COX-2的化学生物学产生详细的见解，并利用这些信息合成新的药物来研究或操纵COX-2在体内的功能。我们最近发现，非甾体抗炎药（NSAIDs）是COX-2对AA氧化的弱竞争性抑制剂，是2-AG和AEA氧化的强效非竞争性抑制剂。对于芳基丙酸非甾体抗炎药（profens）的(R)-对映体，如(R)-氟比洛芬、(R)-萘普生和(R)-布洛芬，这是最显著的。虽然这些化合物作为AA氧化抑制剂基本上没有活性，但它们对2-AG和AEA的COX-2氧化具有中等效力的底物选择性抑制剂。这些见解为开发化学探针来研究内源性大麻素的COX-2氧化在体内的重要性提供了机会，并可能解释(R)-profens的抗炎和癌症预防活性。因此，我们建议(1)利用x射线晶体学、定点诱变和酶动力学研究来确定(R)-profen与COX-2结合的结构和功能基础，以便(2)在体内和体外设计和合成更有效和选择性的内源性大麻素氧合抑制剂。优化后的分子将用于(3)验证COX-2降低结肠炎症和癌症模型中内源性大麻素水平的假设，以及COX-2的底物选择性抑制剂可以恢复内源性大麻素水平的假设。这些底物选择性抑制剂可能是候选的癌症化学预防药物，缺乏目前上市的非甾体抗炎药对胃肠道和心血管的副作用。

DESCRIPTION (provided by applicant): Cyclooxygenase-2 (COX-2) plays a key role in the conversion of arachidonic acid (AA) to prostaglandins, which are an important class of bioactive lipids. COX-2 is highly expressed in inflamed tissue, premalignant lesions, and cancers but not in adjacent normal tissue. COX-2 inhibitors, whether isoform-selective or non- selective, exhibit anti-inflammatory, cancer preventive, and adjuvant cancer therapeutic activities as well as cardiovascular side effects in human clinical trials. COX-2 also oxygenates ester and amide derivatives of AA including 2-arachidonoylglycerol (2-AG) and arachidonoylethanolamide (AEA). 2-AG and AEA are the two best characterized endogenous ligands for the cannabinoid receptors - CB1 and CB2 - and exert a plethora of effects through these receptors. The major focus of endocannabinoid biology has been on the nervous system, but increasing evidence indicates that 2-AG and AEA exert anti-inflammatory and cancer preventive effects. We hypothesize that COX-2-dependent metabolism of 2-AG and AEA lowers endocannabinoid levels, thereby contributing to the development of inflammation and cancer. Our laboratory has studied the structural and functional basis of the interaction of COX-2 with substrates and inhibitors in order to generate detailed insights into the chemical biology of COX-2 and to use this information to synthesize novel agents with which to study or manipulate COX-2 function in vivo. We recently discovered that non-steroidal anti-inflammatory drugs (NSAIDs) that are weak, competitive inhibitors of AA oxygenation by COX-2 are potent, non-competitive inhibitors of 2-AG and AEA oxygenation. This is most dramatic for the (R)-enantiomers of arylpropionic acid NSAIDs (profens), such as (R)-flurbiprofen, (R)-naproxen, and (R)-ibuprofen. While essentially inactive as inhibitors of AA oxygenation, these compounds are moderately potent, substrate-selective inhibitors of COX-2 oxygenation of 2-AG and AEA. These insights provide opportunities to develop chemical probes to investigate the in vivo importance of COX-2 oxygenation of endocannabinoids and may explain the reported anti-inflammatory and cancer preventive activities of (R)-profens. Thus, we propose to (1) use X-ray crystallography, site-directed mutagenesis, and enzyme kinetics studies to determine the structural and functional basis for (R)-profen binding to COX-2 in order to (2) design and synthesize more potent and selective inhibitors of endocannabinoid oxygenation in vitro and in vivo. The optimized molecules will be used to (3) test the hypothesis that COX-2 lowers endocannabinoid levels in models of colonic inflammation and cancer and that endocannabinoid levels can be restored by substrate-selective inhibitors of COX-2. These substrate-selective inhibitors may represent candidate cancer chemopreventive agents that lack the gastrointestinal and cardiovascular side effects of currently marketed NSAIDs. PUBLIC HEALTH RELEVANCE: Compounds called endocannabinoids are naturally occurring regulators of inflammation and cancer. Cyclooxygenase-2 metabolizes endocannabinoids and lowers their levels. The development of selective inhibitors of cyclooxygenase-2-dependent endocannabinoid metabolism may produce novel anti-inflammatory and cancer preventive agents.