A Single Amino Acid Difference between Cyclooxygenase-1 (COX-1) and −2 (COX-2) Reverses the Selectivity of COX-2 Specific Inhibitors

Abstract

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 timedependent 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.Prostaglandin synthase catalyzes two separate reactions; the first being the cyclooxygenase function, which is the addition of molecular oxygen to arachidonic acid to form the unstable PGG 2 , 1 and the second the further conversion of PGG 2 to the more stable PGH 2 by a peroxidase function. Hence, this "cyclooxygenase" (COX) enzyme performs the critical initial reaction in the arachidonic metabolic cascade leading to the formation of the prostaglandins, thromboxane, and prostacyclin (1).Recently, a second form of the COX enzyme was isolated whose expression is inducible by cytokines and growth factors (COX-2) (2-6). This inducible COX-2 is linked to inflammatory cell types and tissues and is believed to be the target enzyme for the anti-inflammatory activity of nonsteroidal anti-inflammatory drugs (NSAIDs) (7-12). NSAIDs currently available for clinical use inhibit both COX-1 and COX-2 (13, 14). This suggests that clinically useful NSAIDs inhibit pro-inflammatory PGs derived from the activity of COX-2, as well as PGs in tissues like the stomach and kidney (via COX-1). These homeostatic PGs are linked to normal gastric and renal function (15). It is possible that a selective COX-2 inhibitor may eliminate the side effects associated with COX...

Developmental expression of functional cyclooxygenases in zebrafish

“…1). The volume of the arachidonate-binding channel of mammalian COX-1 enzymes is determined by Ile-523, His-513, and Ile-434 (17)(18)(19). These residues are substituted by Val-523, Arg-513, and Val-434 in human COX-2, which opens access to a side pocket in the arachidonate-binding channel for selective COX-2 inhibitors.…”

Structural and Functional Basis of Cyclooxygenase Inhibition

“…Gierse and co-workers probed the molecular basis of the timedependent inhibition of purified human COX-2 by diaryl heterocycles by also constructing a V523I mutant of human COX-2. 97 The V523I mutation abolishes the selectivity of all the inhibitors for COX-2, whereas classical NSAIDs like indomethacin show no change in their selectivity profiles. In addition, a series of mutations were made at the mouth of the active site of human COX-2 (Y115L, S119V, and F357L) to evaluate these residues for their contribution to the selective inhibition of COX-2 by the diaryl heterocycles.…”

“…This is precluded in COX-1 by the extra steric bulk of Ile-523. 96,97 Binding of certain diaryl heterocycles to COX-1 and COX-2 has been directly monitored by fluorescence quenching techniques enabled by the spectral overlap of the fluorescent emission of the inhibitor with the visible absorbance of the heme prosthetic group. 99,100 For example, 4-(2-methyl-4-phenyloxazol-5-yl)benzenesulfonamide 5 (SC-299) is a fluorescent diaryl heterocycle that is a potent, selective inhibitor of COX-2 but a weak, competitive inhibitor of COX-1.…”

“…96,97 Two groups simultaneously studied the role of Val-523 to investigate the structural features of COX-2 that influenced the mechanism of the time-dependent action of COX-2 selective inhibitors. Guo et al created a series of Val-523 mutants in human COX-2 (including V523I) and tested the mutants against compounds 1 and 2 and 5-(4-fluorophenyl)-1-(4-(methylsulfonyl)phenyl)-3-(trifluoromethyl)-1H-pyrazole 3 (SC-58125).…”

Lipopolysaccharide-Induced Increase of Prostaglandin E2 Is Mediated by Inducible Nitric Oxide Synthase Activation of the Constitutive Cyclooxygenase and Induction of Membrane-Associated Prostaglandin E Synthase

“…Its selectivity toward COX-2 has been uniformly proven: it displays IC 50 values of 17 and 0.005 M against human COX-1 and COX-2, respectively. Therefore, the ratio of selectivity IC 50 COX-1/IC 50 COX-2 is of 3400 (25). The specificity is this compound has also been tested in vivo using the rat air pouch-induced inflammation model: SC-236 inhibits COX-2-dependent PGE 2 production with an ED 50 of 0.3 mg/kg when given by gavage, and a dose of 2 mg/kg almost completely (99%) inhibited the COX-2-dependent PGE 2 production, whereas COX-1-dependent PGE 2 production was not affected at doses up to 10 mg/kg (26,27).…”