Somvanshi Rishi K, Kumar Ashwini, Kant Shashi, Gupta Deepti, Singh S Bhaskar, Das Utpal, Srinivasan Alagiri, Singh Tej P, Dey Sharmistha
Department of Biophysics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India.
Biochem Biophys Res Commun. 2007 Sep 14;361(1):37-42. doi: 10.1016/j.bbrc.2007.06.122. Epub 2007 Jul 5.
Cyclooxygenase (COX) is a key enzyme in the biosynthetic pathway leading to the formation of prostaglandins, which are mediators of inflammation [D.L. Dewitt, W.L. Smith, Primary structure of prostaglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence, Proc. Natl. Acad. Sci. USA 85 (1988) 1412-1416, 1]. It exists mainly in two isoforms COX-1 and COX-2 [A. Raz, A. Wyche, N. Siegel, P. Needleman, Regulation of fibroblast cyclooxygenase synthesis by interleukin-1, J. Biol. Chem. 263 (1988) 3022-3028, 2]. The conventional non-steroidal anti-inflammatory drugs (NSAIDs) have adverse gastrointestinal side-effects, because they inhibit both isoforms [T.D. Warner, F. Guiliano, I. Vojnovic, A. Bukasa, J.A. Mitchell, J.P. Vane, Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated with human gastrointestinal toxicity: a full in vitro analysis, Proc. Natl. Acad. Sci. USA 96 (1999) 7563-7568, 3; L.J. Marnett, A.S. Kalgutkar, Cyclooxygenase 2 inhibitors: discovery, selectivity and the future, Trends Pharmacol. Sci. 20 (1999) 465-469, 4; J.R. Vane, NSAIDs, Cox-2 inhibitors, and the gut, Lancet 346 (1995) 1105-1106, 5]. Therefore drugs which selectively inhibit COX-2, known as coxibs were developed. Recent reports on the harmful cardiovascular and renovascular side-effects of the anti-inflammatory drugs have led to the quest for a novel class of COX-2 selective inhibitors. Keeping this in mind, we have used the X-ray crystal structures of the complexes of the COX-1 and COX-2 with the known inhibitors for a rational, structure based approach to design a small peptide, which is potent inhibitor for COX-2. The peptides have been checked experimentally by in-vitro kinetic studies using surface plasmon resonance (SPR) and other biochemical methods. We have identified a tripeptide inhibitor which is a potential lead for a new class of COX-2 inhibitor. The dissociation constant (K(D)) determined for COX-2 with peptide WCS is 1.90x10(-10)M, the kinetic constant (K(i)) determined by spectrophotometry is 4.85x10(-9)M and the IC(50) value is 1.5x10(-8)M by ELISA test.
环氧化酶（COX）是导致前列腺素形成的生物合成途径中的关键酶，前列腺素是炎症介质[D.L. 德维特，W.L. 史密斯，从互补DNA序列确定的绵羊精囊前列腺素G/H合酶的一级结构，《美国国家科学院院刊》85 (1988) 1412 - 1416，1]。它主要以两种同工型COX - 1和COX - 2的形式存在[A. 拉兹，A. 怀奇，N. 西格尔，P. 尼德曼，白细胞介素 - 1对成纤维细胞环氧化酶合成的调节，《生物化学杂志》263 (1988) 3022 - 3028，2]。传统的非甾体抗炎药（NSAIDs）具有不良的胃肠道副作用，因为它们会抑制这两种同工型[T.D. 华纳，F. 朱利亚诺，I. 沃伊诺维奇，A. 布卡萨，J.A. 米切尔，J.P. 韦恩，环氧化酶 - 1而非环氧化酶 - 2的非甾体药物选择性与人胃肠道毒性相关：全面的体外分析，《美国国家科学院院刊》96 (1999) 7563 - 7568，3；L.J. 马内特，A.S. 卡尔古特卡尔，环氧化酶2抑制剂：发现、选择性及未来，《药理学趋势》20 (1999) 465 - 469，4；J.R. 韦恩，非甾体抗炎药、Cox - 2抑制剂与肠道，《柳叶刀》346 (1995) 1105 - 1106，5]。因此，开发了选择性抑制COX - 2的药物，即昔布类药物。近期关于抗炎药物有害心血管和肾血管副作用的报道促使人们寻求一类新型的COX - 2选择性抑制剂。考虑到这一点，我们利用COX - 1和COX - 2与已知抑制剂的复合物的X射线晶体结构，采用基于结构的合理方法设计一种小肽，它是COX - 2的有效抑制剂。这些肽已通过使用表面等离子体共振（SPR）的体外动力学研究和其他生化方法进行了实验验证。我们鉴定出一种三肽抑制剂，它是一类新型COX - 2抑制剂的潜在先导化合物。用肽WCS测定的COX - 2的解离常数（K(D)）为1.90×10(-10)M，通过分光光度法测定的动力学常数（K(i)）为4.85×10(-9)M，通过ELISA试验测定的IC(50)值为1.5×10(-8)M。
