A single amino acid difference between cyclooxygenase-1 (COX-1) and -2 (COX-2) reverses the selectivity of COX-2 specific inhibitors

被引:348
作者
Gierse, JK
McDonald, JJ
Hauser, SD
Rangwala, SH
Koboldt, CM
Seibert, K
机构
[1] Searle Research and Development, St. Louis
[2] Monsanto/Searle, Chesterfield, MO 63198
关键词
D O I
10.1074/jbc.271.26.15810
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 re versibly 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.
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页码:15810 / 15814
页数:5
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