STRUCTURAL ANALYSIS OF ENZYMES FOR ARCHIDONATE CASCADE FOR DEVELOPING NOVEL DRUGS

非甾体类抗炎药（NSAIDs）的作用是通过抑制环氧合酶（考克斯）的活性来实现的。考克斯有两种同工型，组成型酶考克斯-1和诱导型酶考克斯-2。考克斯-2选择性抑制剂被认为是有希望的NSAID，其副作用低，来源于对考克斯-1活性的抑制。然而，最近的报道显示，考克斯-2选择性抑制剂也有副作用，如增加严重冠心病的风险，这表明越来越需要寻找其他药物靶点。微粒体前列腺素E合成酶（mPGES）是一种新型的靶点，它在炎症部位的诱导选择性比考克斯-2高。另一方面，5-脂氧合酶激活蛋白（FRAP），mPGES的同一家族的成员，和过氧化物酶体增殖物激活受体（PPAR），核受体超家族的成员，被认为是治疗生活方式相关疾病的药物靶标。我们发现，白藜芦醇是红葡萄酒中含有的一种多酚，是过氧化物酶体增殖物激活受体α和γ的双重激动剂，白藜芦醇通过激活过氧化物酶体增殖物激活受体α来保护大脑免受缺血。值得注意的是，MK-886是一种mPGES和FLAP的双重抑制剂，据报道是一种PPARα拮抗剂，表明mPGES抑制剂可能通过其对PPAR的拮抗活性产生副作用。本研究的目的是确定mPGES、FLAP和PPARα的三维结构，为基于其结构的新药开发奠定基础。我们成功地表达和纯化了这些重组蛋白，并仍在研究其良好结晶的条件。我们用新近发展起来的立方相法获得了一种用于FLAP的蛋白晶体。

It is widely accepted that effects of nonsteroidal anti-inflammatory drugs (NSAIDs) result from inhibition of cyclooxygenase (COX) activities. COX has two isoforms, constitutive enzyme COX-1 and inducible enzyme COX-2. COX-2 selective inhibitors are considered as promised NSAIDs with low side effects derived from inhibition of COX-1 activity. However, recent reports show COX-2 selective inhibitors have also side effects such as increase risk of serious coronary heart disease, indicating a growing need for searching other drug targets. As one of the novel targets, microsomal prostaglandin E synthase (mPGES) is noteworthy because it is more selectively induced than COX-2 in inflammatory sites. On the other hand, 5-lipoxygenase activating protein (FRAP), a member of the same family of mPGES, and peroxisome proliferator-activated receptor (PPAR), a member of nuclear receptor superfamily, are recognized as drug targets against lifestyle-related diseases. We found that resveratrol, a polyphenol contained in red wines, is a dual agonist for PPARα and γ, and that resveratrol protects the brain against ischemia by activation of PPARα. Remarkably, MK-886, a dual inhibitor for mPGES and FLAP is reported to be a PPARα antagonist, indicating the possibility of the side effect of mPGES inhibitor by its antagonistic activity for PPAR. The aim of this study is to determine the three-dimensional structures of mPGES, FLAP and PPARα for development of novel drugs based on their structures. We successfully expressed and purified these recombinant proteins, and are still investigating the conditions for their good crystallization. We obtained a protein crystal for FLAP by the lipidic cubic phase method, which is a newly developed.