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

描述（由申请人提供）：内源性大麻素是内源性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-花生四烯醇甘油（2-AG）和花生四烯醇乙醇酰胺（AEA）。这些分子参与了大量的生物过程，特别是，已被证明可以调节镇痛活性。AEA和2-AG是由磷脂酶根据需要合成的，Marnett实验室最近的研究表明，2-AG和AEA都是环氧化酶-2 （COX-2）的底物。重要的是，几种非甾体抗炎药（NSAIDs）对COX-2的抑制作用，特别是(S)-2-芳基丙酸衍生物NSAIDs，已被证明在抑制内源性大麻素氧合方面比抑制花生四烯酸（AA）氧合更有效。2-芳基丙酸衍生物nsaid的(R)-对映体长期以来被归类为COX-2的非抑制剂，因为它们不抑制COX-2对AA的氧化作用。然而，我们最近的工作表明它们是内源性大麻素COX-2氧化的有效抑制剂。特别有趣的是，2-芳基丙酸衍生物NSAID的最有效(R)-对映体(R)-氟比洛芬，已被证明通过增加AEA水平来提供神经性疼痛模型的镇痛。(R)-氟比洛芬对COX-2的底物选择性抑制为实现AEA水平的提高和相应的镇痛提供了一种新的机制。(R)-氟比洛芬作用底物选择性抑制COX-2的机制目前尚不清楚，将通过位点定向诱变和x射线晶体学的结合来阐明。位点定向诱变将用于创建结合位点突变体，以探测(R)-氟比洛芬对COX-2的底物选择性抑制至关重要的残基。通过求解(R)-氟比洛芬与小鼠COX-2结合的晶体结构，确定(R)-氟比洛芬对底物选择性抑制的机制。位点定向诱变和X射线晶体学提供的对底物选择性抑制COX-2机制的结构和功能见解将用于设计和合成具有更高效力的(R)-氟比洛芬类似物。合成的类似物将通过纯化的小鼠或人COX-2进行体外实验，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，以评估底物选择性抑制作用。然后使用小鼠卡拉胶足垫炎症模型评估最有效的底物选择性抑制剂，并将其与(R)-氟比洛芬在体内进行比较。通过明确(R)-氟比洛芬对COX-2的底物选择性抑制机制，并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，并开发用于神经性疼痛治疗的先导化合物。

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.