(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）：内源性大麻素是内源产生的脂肪酸衍生物，作用于大麻素 1 和 2 受体。两种主要的内源性大麻素是 2-花生四烯酰甘油 (2-AG) 和花生四烯酰乙醇酰胺 (AEA)。这些分子参与多种生物过程，特别是已被证明可以调节镇痛活性。 AEA 和 2-AG 由磷脂酶按需合成，Marnett 实验室最近的工作表明，2-AG 和 AEA 都是环氧合酶-2 (COX-2) 的底物。重要的是，几种非甾体抗炎药 (NSAID)，特别是 (S)-2-芳基丙酸衍生物 NSAID，对 COX-2 的抑制作用已被证明在抑制内源性大麻素氧合方面更有效与花生四烯酸（AA）氧化的抑制相比。 2-芳基丙酸衍生物 NSAID 的 (R)-对映体长期以来一直被归类为 COX-2 的非抑制剂，因为它们不会抑制 COX-2 对 AA 的氧化作用。然而，我们最近的工作表明它们是 COX-2 内源性大麻素氧化的有效抑制剂。特别令人感兴趣的是，2-芳基丙酸衍生物 NSAID 的最有效的 (R)-对映体，(R)-氟比洛芬，已被证明可以通过增加 AEA 的水平在神经性疼痛模型中提供镇痛作用。观察到的 (R)-氟比洛芬对 COX-2 的底物选择性抑制为实现 AEA 水平的增加和相应的镇痛提供了一种新的机制。 (R)-氟比洛芬对 COX-2 进行底物选择性抑制的机制目前尚不清楚，将通过结合定点诱变和 X 射线晶体学来阐明。定点诱变将用于创建结合位点突变体，以探测对 (R)-氟比洛芬底物选择性抑制 COX-2 至关重要的残基。 (R)-氟比洛芬的底物选择性抑制机制也将通过解析与鼠 COX-2 结合的 (R)-氟比洛芬的晶体结构来确定。通过定点诱变和 X 射线晶体学提供的对 COX-2 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具有改进效力的 (R)-氟比洛芬类似物。将通过使用纯化的小鼠或人 COX-2 进行体外测定，然后使用 RAW 264.7 巨噬细胞和初级背根神经节作为模型系统进行离体测试，评估合成类似物的底物选择性抑制。然后将使用小鼠角叉菜胶足垫炎症模型对最有效的底物选择性抑制剂进行评估，并在体内与 (R)-氟比洛芬进行比较。通过定义(R)-氟比洛芬底物选择性抑制COX-2的机制以及开发和测试新型抑制剂，该项目将阐明内源性大麻素调节的新机制并开发用于治疗神经性疼痛的先导化合物。 公共健康相关性：(R)-氟比洛芬类似物的开发将带来针对内源性大麻素系统的新型抗炎和止痛药物。与目前使用的非甾体抗炎药相比，这些药物将提供一种副作用减少的新治疗方法，从而使广大公众受益。这些药物还可用于治疗内源性大麻素已被证明发挥作用的几种常见疾病，包括阿尔茨海默病、药物依赖、肥胖和抑郁症。