Gierse J K, McDonald J J, Hauser S D, Rangwala S H, Koboldt C M, Seibert K
Searle Research and Development, St. Louis, Missouri 63198, USA.
J Biol Chem. 1996 Jun 28;271(26):15810-4. doi: 10.1074/jbc.271.26.15810.
Nonsteroidal anti-inflammatory drugs (NSAIDs) currently available for clinical use inhibit both COX-1 and COX-2. This suggests that clinically useful NSAIDs inhibit pro-inflammatory prostaglandins (PGs) derived from the activity of COX-2, as well as PGs in tissues like the stomach and kidney (via COX-1). A new class of compounds has recently been developed (SC-58125) that have a high degree of selectivity for the inducible form of cyxlooxygenase (COX-2) over the constitutive form (COX-1). This unique class of compounds exhibit a time-dependent irreversible inhibition of COX-2, while reversibly inhibiting COX-1. The molecular basis of this selectivity was probed by site-directed mutagenesis of the active site of COX-2. The sequence differences in the active site were determined by amino acid replacement of the COX-2 sequences based on the known crystal structure of COX-1, which revealed a single amino acid difference in the active site (valine 509 to isoleucine) and a series of differences at the mouth of the active site. Mutants with the single amino acid substitution in the active site and a combination of three changes in the mouth of the active site were made in human COX-2, expressed in insect cells and purified. The single amino acid change of valine 509 to isoleucine confers selectivity of COX-2 inhibitors in the class of SC-58125 and others of the same class (SC-236, NS-398), while commonly used NSAIDs such as indomethacin showed no change in selectivity. Substitutions of COX-1 sequences in COX-2 at the mouth of the active site of COX-2 did not change the selectivity of SC-58125. This indicates that the single amino acid substitution of isoleucine at position 509 for a valine is sufficient to confer COX-2 selectivity in this example of a diaryl-heterocycle COX inhibitor.
目前临床可用的非甾体抗炎药（NSAIDs）会抑制COX-1和COX-2。这表明临床上有用的NSAIDs既能抑制由COX-2活性产生的促炎前列腺素（PGs），也能抑制胃和肾脏等组织中的PGs（通过COX-1）。最近开发了一类新的化合物（SC-58125），它们对诱导型环氧化酶（COX-2）的选择性远高于组成型（COX-1）。这类独特的化合物对COX-2表现出时间依赖性的不可逆抑制，而对COX-1则是可逆抑制。通过对COX-2活性位点进行定点诱变来探究这种选择性的分子基础。基于已知的COX-1晶体结构，通过对COX-2序列进行氨基酸替换来确定活性位点的序列差异，结果显示活性位点存在一个氨基酸差异（缬氨酸509变为异亮氨酸）以及活性位点开口处的一系列差异。在人COX-2中对活性位点进行单氨基酸替换以及在活性位点开口处进行三个变化的组合突变，在昆虫细胞中表达并纯化。缬氨酸509变为异亮氨酸的单氨基酸变化赋予了SC-58125及同一类其他化合物（SC-236、NS-398）对COX-2抑制剂的选择性，而常用的NSAIDs如吲哚美辛的选择性没有变化。在COX-2活性位点开口处将COX-1序列替换到COX-2中并没有改变SC-58125的选择性。这表明在这个二芳基杂环COX抑制剂的例子中，第509位异亮氨酸取代缬氨酸的单氨基酸替换足以赋予COX-2选择性。
