Isoform-Selective Interaction of Cyclooxygenase-2 with Indomethacin Amides Studied by Real-Time Fluorescence, Inhibition Kinetics, and Site-Directed Mutagenesis

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• 作者：Sergei L. Timofeevski ; Jeffery J. Prusakiewicz ; Carol A. Rouzer ; and Lawrence J. Marnett
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：July 30, 2002
• 年：2002
• 卷：41
• 期：30
• 页码：9654 - 9662
• 全文大小：115K
• 年卷期：v.41,no.30(July 30, 2002)
• ISSN：1520-4995

文摘

Conversion of carboxylate-containing nonsteroidal antiinflammatory drugs, such as indomethacin, to esters or amides provides potent and selective inhibitors of cyclooxygenase-2 (COX-2)[Kalgutkar et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 925-930]. Synthesis of cinnamyl- or coumarinyl-substituted ethanolamide derivatives of indomethacin produced fluorescent probes of inhibitor interactionwith COX-2 and COX-1. Binding of either derivative to apoCOX-2 or apoCOX-1 resulted in a rapid,reversible enhancement of fluorescence. Following this rapid phase, a slow additional increase influorescence was observed with apoCOX-2 but not with apoCOX-1. The slow, COX-2-specific increasein fluorescence was prevented or reversed by addition of the nonfluorescent COX inhibitor (S)-flurbiprofen.Detailed kinetic studies of the interaction of the coumarinyl derivative with COX-2 showed that the observedchanges in fluorescence could be described by two sequential equilibria, the first of which is rapid,reversible, and bimolecular in the forward direction. The second equilibrium is slower, reversible, andunimolecular in both directions. The forward rate constant for the slow equilibrium determined byfluorescence enhancement [(3.1 ± 0.6) × 10^-3 s^-1] corresponded closely to the forward rate constant forinhibition of COX-2 activity [(6.8 ± 2.3) × 10^-3 s^-1], suggesting that the slow fluorescence enhancementis associated with selective COX-2 inhibition. Site-directed mutagenesis indicated that residues in thecarboxylate-binding region of the COX-2 active site (Arg-120, Tyr-355, and Glu-524) are critical for thebinding of the indomethacin conjugates that leads to slow fluorescence enhancement and cyclooxygenaseinhibition. The indomethacin conjugates described herein represent powerful tools for the investigationof a novel class of selective inhibitors of COX-2.