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

描述(由申请人提供)：内源性大麻素是作用于大麻素1和2受体的内源性脂肪酸衍生物。两种主要的内源性大麻素是2-花生四烯基甘油(2-AG)和花生四烯基乙醇胺(AEA)。这些分子参与了过多的生物过程，特别是已经被证明可以调节止痛活性。AEA和2-AG是由磷脂酶根据需要合成的，Marnett实验室最近的工作表明，2-AG和AEA都是环氧合酶-2(COX-2)的底物。重要的是，与抑制花生四烯酸(AA)氧化相比，几种非甾体抗炎药(NSAIDs)，特别是(S)-2-芳基丙酸衍生物NSAIDs对COX-2的抑制作用被证明是更有效的。2-芳基丙酸衍生物的(R)-对映体长期以来一直被归类为COX-2的非抑制剂，因为它们不抑制COX-2对AA的氧化。然而，我们最近的工作表明，它们是环氧合酶-2对内源性大麻素氧合的有效抑制物。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，已被证明通过增加AEA的水平在神经病理性疼痛模型中提供镇痛。(R)-氟比洛芬对COX-2的底物选择性抑制为实现这种升高的AEA水平和相应的镇痛提供了新的机制。(R)-氟比洛芬影响底物选择性抑制COX-2的机制目前尚不清楚，将结合定点突变和X射线结晶学来阐明。定点突变将被用于创建结合位点突变体，以探测对(R)-氟比洛芬对COX-2的底物选择性抑制至关重要的残基。(R)-氟比洛芬对底物选择性抑制的机制也将通过解算与小鼠COX-2结合的(R)-氟比洛芬的晶体结构来确定。通过定点突变和X射线结晶学对底物选择性抑制COX-2机制的结构和功能的洞察，将被用于设计和合成效力更高的(R)-氟比洛芬类似物。合成的类似物将通过使用纯化的小鼠或人COX-2的体外实验和以RAW 264.7巨噬细胞和初级背根神经节为模型系统的体外测试来评估底物选择性抑制。然后将使用小鼠角叉菜胶足垫炎症模型在体内评估最有效的底物选择性抑制剂并与(R)-氟比洛芬进行比较。通过确定(R)-氟比洛芬对COX-2底物选择性抑制的机制以及开发和测试新的抑制剂，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病理性疼痛的先导化合物。

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.