Isoform-selective interaction of cyclooxygenase-2 with indomethacin amides studied by real-time fluorescence, inhibition kinetics, and site-directed mutagenesis.

Abstract

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 interaction with 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 in fluorescence was observed with apoCOX-2 but not with apoCOX-1. The slow, COX-2-specific increase in 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 observed changes 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, and unimolecular in both directions. The forward rate constant for the slow equilibrium determined by fluorescence enhancement [(3.1 +/- 0.6) x 10(-3) s(-1)] corresponded closely to the forward rate constant for inhibition of COX-2 activity [(6.8 +/- 2.3) x 10(-3) s(-1)], suggesting that the slow fluorescence enhancement is associated with selective COX-2 inhibition. Site-directed mutagenesis indicated that residues in the carboxylate-binding region of the COX-2 active site (Arg-120, Tyr-355, and Glu-524) are critical for the binding of the indomethacin conjugates that leads to slow fluorescence enhancement and cyclooxygenase inhibition. The indomethacin conjugates described herein represent powerful tools for the investigation of a novel class of selective inhibitors of COX-2.