Functional Analysis of Cyclooxygenase-2

描述（由申请人提供）：环氧合酶-2（考克斯-2）在花生四烯酸（AA）转化为胡萝卜素（一种重要的生物活性脂质）中起关键作用。考克斯-2在炎症组织、癌前病变和癌症中高度表达，但在邻近的正常组织中不表达。考克斯-2抑制剂，无论是同种型选择性的还是非选择性的，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。考克斯-2还氧化AA的酯和酰胺衍生物，包括2-花生四烯酰甘油（2-AG）和花生四烯酰乙醇酰胺（AEA）。2-AG和AEA是大麻素受体CB1和CB2的两种最佳表征的内源性配体，并通过这些受体发挥过多的作用。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发挥抗炎和癌症预防作用。我们推测2-AG和AEA的考克斯-2依赖性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。我们的实验室已经研究了考克斯-2与底物和抑制剂相互作用的结构和功能基础，以产生对考克斯-2的化学生物学的详细了解，并利用这些信息合成新的药物，以研究或操纵考克斯-2在体内的功能。我们最近发现，非甾体抗炎药（NSAID）是AA通过考克斯-2氧化的弱的竞争性抑制剂，是2-AG和AEA氧化的有效的非竞争性抑制剂。这对于芳基丙酸NSAID（profens）的（R）-对映异构体如（R）-氟比洛芬、（R）-萘普生和（R）-布洛芬是最显著的。虽然作为AA氧合的抑制剂基本上无活性，但这些化合物是2-AG和AEA的考克斯-2氧合的中等有效的底物选择性抑制剂。这些见解提供了机会，开发化学探针，调查内源性大麻素的考克斯-2氧化的体内重要性，并可能解释所报道的抗炎和癌症预防活动的（R）-profens。因此，我们建议（1）使用X-射线晶体学，定点诱变，和酶动力学研究，以确定（R）-洛芬结合考克斯-2的结构和功能基础，以便（2）设计和合成更有效的和选择性的内源性大麻素氧化抑制剂在体外和体内。优化的分子将用于（3）检验以下假设：考克斯-2降低结肠炎症和癌症模型中的内源性大麻素水平，并且内源性大麻素水平可以通过考克斯-2的底物选择性抑制剂恢复。这些底物选择性抑制剂可能代表候选癌症化学预防剂，其缺乏目前市售NSAID的胃肠道和心血管副作用。 公共卫生相关性：被称为内源性大麻素的化合物是炎症和癌症的天然调节剂。环氧合酶-2代谢内源性大麻素并降低其水平。环氧化酶-2依赖性内源性大麻素代谢的选择性抑制剂的发展可能会产生新的抗炎和癌症预防剂。

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.