(R)-flurbiprofen Substrate-Selective Inhibition of COX-2 and Analog Development

(R)-氟比洛芬 COX-2 底物选择性抑制和类似物开发

• 批准号: 8264004
• 负责人: Daniel Hermanson
• 金额: $ 3.58万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8264004
• 关键词: 2-arachidonylglycerol; Absence of pain sensation; Acids; Adverse effects; Alzheimer' s Disease; Analgesics; Anti-Inflammatory Agents; Anti-inflammatory; Arachidonic Acids; Binding; Binding Sites; Biological Models; Biological Process; Cannabinoids; Carrageenan; Coxibs; Data; Development; Drug Addiction; Drug Delivery Systems; Endocannabinoids; Exhibits; Fatty Acids; General Population; Human; In Vitro; Inflammation; Lead; Libraries; Medical; Mental Depression; Methods; Modeling; Mus; Obesity; Pharmaceutical Preparations; Phospholipase; Play; Process; Prostaglandin-Endoperoxide Synthase; R-flurbiprofen; Regulation; Role; Route; Screening Result; Site-Directed Mutagenesis; Spinal Ganglia; Structure; System; Testing; Work; X-Ray Crystallography; analog; anandamide; base; cyclooxygenase 2; design; enantiomer; ganglion cell; improved; in vitro Assay; in vivo; inflammatory pain; inhibitor/antagonist; insight; interest; macrophage; mutant; novel; painful neuropathy; public health relevance; receptor

DESCRIPTION (provided by applicant): Endocannabinoids are endogenously produced fatty acid derivatives that act at the cannabinoid 1 and 2 receptors. The two predominant endocannabinoids are 2-arachidonoylglycerol (2-AG) and arachidonoylethanolamide (AEA). These molecules are involved in a plethora of biological processes and, in particular, have been shown to modulate analgesic activity. AEA and 2-AG are synthesized on demand by phospholipases and recent work by the Marnett lab has shown that 2-AG and AEA are both substrates of cyclooxygenase-2 (COX-2). Importantly, the inhibition of COX-2 by several non-steroidal anti-inflammatory drugs (NSAIDs), in particular the (S)-2-arylpropionic acid derivative NSAIDs, has been shown to be more potent with respect to the inhibition of endocannabinoid oxygenation as compared to the inhibition of arachidonic acid (AA) oxygenation. The (R)-enantiomers of 2-arylpropionic acid derivative NSAIDs have long been classified as non-inhibitors of COX-2 since they do not inhibit the oxygenation of AA by COX-2. However, our recent work indicates that they are potent inhibitors of endocannabinoid oxygenation by COX-2. Of particular interest, the most potent (R)-enantiomer of a 2- arylpropionic acid derivative NSAID, (R)-flurbiprofen, has been shown to provide analgesia in neuropathic pain models by increasing the levels of AEA. The observed substrate-selective inhibition of COX-2 by (R)-flurbiprofen offers a novel mechanism for achieving this increased AEA level and corresponding analgesia. The mechanism by which (R)-flurbiprofen effects substrate-selective inhibition of COX-2 is presently unknown and will be elucidated using a combination of site-directed mutagenesis and X-ray crystallography. Site-directed mutagenesis will be used to create binding site mutants to probe for residues critical to the substrate-selective inhibition of COX-2 by (R)-flurbiprofen. The mechanism of substrate-selective inhibition by (R)-flurbiprofen will also be defined by solving the crystal structure of (R)-flurbiprofen bound to murine COX-2. The structural and functional insights into the mechanism of substrate-selective inhibition of COX-2 provided by the site-directed mutagenesis and X- ray crystallography will be used to design and synthesize (R)-flurbiprofen analogs with improved potency. The synthesized analogs will be evaluated for substrate-selective inhibition by using an in vitro assay with purified murine or human COX-2 followed by ex vivo testing using RAW 264.7 macrophages and primary dorsal root ganglia as model systems. The most potent substrate-selective inhibitors will then be evaluated and compared to (R)-flurbiprofen in vivo using the mouse carrageenan footpad inflammation model. By defining the mechanism of substrate-selective inhibition of COX-2 by (R)- flurbiprofen and developing and testing novel inhibitors, the project will elucidate a novel mechanism of endocannabinoid regulation and develop lead compounds for the treatment of neuropathic pain. PUBLIC HEALTH RELEVANCE: The development of (R)-flurbiprofen analogs will lead to new anti-inflammatory and pain reducing drugs targeting the endocannabinoid system. These drugs will benefit the general public by providing a new treatment with reduced side effects compared to currently used non-steroidal anti-inflammatory drugs. The drugs may also be applicable to treatment of several prevalent medical conditions that endocannabinoids have been shown to play a role including Alzheimer's disease, drug dependency, obesity, and depression.

描述（由申请人提供）：内源性大麻素是在大麻素1和2受体上作用的内源产生的脂肪酸衍生物。这两个主要的内源性大麻素是2-芳基烯丙基甘油（2-AG）和蛛网膜烯醇乙醇酰胺（AEA）。这些分子参与了许多生物学过程，尤其是已证明可以调节镇痛活性。 AEA和2AG由磷脂酶按需合成，Marnett Lab的最新工作表明2-AG和AEA都是环氧酶-2（COX-2）的底物。重要的是，几种非甾体类抗炎药（NSAIDS）抑制COX-2，特别是（S）-2-芳基丙酸衍生物NSAIDS与抑制抑制AAA的氧化型（AAA）相比，已显示出对抑制内诺甲称型氧气的抑制。长期以来，2-芳基丙酸衍生物NSAIDS的（R） - 替代物已被归类为COX-2的非抑制剂，因为它们不抑制COX-2的AA氧合作用。但是，我们最近的工作表明它们是COX-2对内源性大麻素氧合的有效抑制剂。特别有趣的是，已证明2-芳基丙酸衍生物NSAID（R） - 氟氟芬蛋白的最有效的（R） - 源性体可以通过提高AEA水平来提供神经性疼痛模型中的镇痛作用。通过（R） - 氟叶芬对COX-2对COX-2的底物选择性抑制作用提供了一种新的机制，可实现这一增加的AEA水平和相应的镇痛。目前未知（R） - 氟法效应底物对COX-2的抑制作用的机制尚不清楚，并且将使用位置定向的诱变和X射线晶体学的组合阐明。位置定向的诱变将用于创建结合位点突变体，以探测（R） - 氟吡芬对Cox-2对底物选择性抑制至关重要的残基。 （r） - 氟叶芬的底物选择性抑制的机制也将通过求解与鼠Cox-2结合的（r） - 氟叶芬的晶体结构来定义。对位置定向诱变和X射线晶体学提供的底物选择性抑制机制的结构和功能见解将用于设计和合成（R） - 荧光素（flurbiprofen类似物），具有提高的效力。通过使用纯化的鼠或人Cox-2的体外测定，将评估合成的类似物的底物选择性抑制作用，然后使用RAW 264.7巨噬细胞和原发性背侧神经节作为模型系统进行离体测试。然后，将使用小鼠角叉to虫炎症模型对最有效的底物选择性抑制剂进行评估，并将其与体内（R） - 氟叶芬进行比较。通过定义（R） - 弗拉伯哌芬和开发和测试新型抑制剂对COX-2的底物选择性抑制的机制，该项目将阐明内源性大麻素调节的新型机制，并开发了治疗神经性疼痛的铅化合物。 公共卫生相关性：（R） - 氟法芬类似物的发展将导致针对内源性大麻素系统的新型抗炎和减轻疼痛。与当前使用的非甾体类抗炎药相比，这些药物将通过提供副作用减少的新疗法来使公众受益。这些药物也可能适用于治疗几种普遍的医疗状况，表明内源性大麻素可以发挥作用，包括阿尔茨海默氏病，药物依赖，肥胖和抑郁症。