Blobaum Anna L, Marnett Lawrence J
A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Department of Biochemistry, Vanderbilt Institute for Chemical Biology, Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
J Biol Chem. 2007 Jun 1;282(22):16379-90. doi: 10.1074/jbc.M609883200. Epub 2007 Apr 12.
Lumiracoxib is the first example of a marketed COX-2 inhibitor of the arylacetic acid class, and it is reported to be the most selective COXIB in vivo. However, the molecular basis of its COX-2 inhibition has not been completely defined. Using standard assays, lumiracoxib was found to be a poor inhibitor of purified ovine COX-1 and a relatively weak inhibitor of purified human COX-2. The extent of COX-2 inhibition plateaued at around 50% and suggested that the inhibitor may be reversibly bound to the enzyme. Kinetic studies with lumiracoxib demonstrated that it was a time-dependent and slowly reversible inhibitor of human COX-2 that exhibited at least two binding steps during inhibition. Derivatives of lumiracoxib were synthesized with or without the methyl group on the phenylacetic acid ring and with various substitutions on the lower aniline ring. Inhibition studies demonstrated that the methyl group on the phenylacetic acid ring is required for COX-2 selectivity. The chemical identity and position of the substituents on the lower aniline ring were important in determining the potency and extent of COX inhibition as well as COX-2 selectivity. Mutation of Ser-530 to Ala or Val-349 to Ala or Leu abolished the potent inhibition observed with wild-type human COX-2 and key lumiracoxib analogs. Interestingly, a Val-349 to Ile mutant was inhibited with equal potency to human COX-2 with 2,6-dichloro-, 2,6-dimethyl-, or 2-chloro-6-methyl-substituted inhibitors and, in the case of lumiracoxib, actually showed an increase in potency. Taken together with a recent crystal structure of a lumiracoxib-COX-2 complex, the kinetic analyses presented herein of the inhibition of mutant COX-2s by lumiracoxib allows the definition of the molecular basis of COX-2 inhibition.
鲁米昔布是上市的芳基乙酸类COX-2抑制剂中的首个实例，据报道它是体内最具选择性的环氧化酶抑制剂（COXIB）。然而，其抑制COX-2的分子基础尚未完全明确。使用标准测定方法发现，鲁米昔布是纯化绵羊COX-1的低效抑制剂，也是纯化人COX-2的相对较弱抑制剂。COX-2的抑制程度在约50%时趋于平稳，这表明该抑制剂可能与酶可逆结合。对鲁米昔布的动力学研究表明，它是一种时间依赖性且缓慢可逆的人COX-2抑制剂，在抑制过程中表现出至少两个结合步骤。合成了在苯乙酸环上有或没有甲基以及在下位苯胺环上有各种取代基的鲁米昔布衍生物。抑制研究表明，苯乙酸环上的甲基是COX-2选择性所必需的。下位苯胺环上取代基的化学特性和位置对于确定COX抑制的效力和程度以及COX-2选择性很重要。将Ser-530突变为Ala或Val-349突变为Ala或Leu消除了野生型人COX-2和关键鲁米昔布类似物所观察到的强效抑制作用。有趣的是，Val-349突变为Ile的突变体对2,6-二氯-、2,6-二甲基-或2-氯-6-甲基取代的抑制剂抑制人COX-2的效力相同，就鲁米昔布而言，实际上效力有所增加。结合最近鲁米昔布-COX-2复合物的晶体结构，本文对鲁米昔布抑制突变型COX-2的动力学分析使得能够定义COX-2抑制的分子基础。
