Structural requirements for time-dependent inhibition of prostaglandin biosynthesis by anti-inflammatory drugs.

Abstract

Several anti-inflammatory drugs have been examined for their ability to cause a time-dependent destruction of the fatty acid oxygenase that produces prostaglandins. All of the time-dependent inhibitors contained carboxylic acid moieties, and in addition all but one of the drugs contained a halogen atom. Structural analogs (of the timedependent inhibitors) lacking halogen atoms were unable to cause a time-dependent destruction of the enzyme.The timedependent property of an inhibitor was totally eliminated after methylation of the carboxylate group. Methylation did not, however, alter the ability of the inhibitors to competitively inhibit the oxygenase.Thus, the reversible binding of the agents at the active site was not appreciably dependent upon the free carboxyl group, whereas the subsequent irreversible process was. Since the first report by Vane (1) on the inhibition of the biosynthesis Qf prostaglandins by aspirin, a wide variety of anti-inflammatory drugs has been shown to inhibit the synthesis of prostaglandins, including pyrazolones, arylacetic acids, fenamates, and salicylates (2). The concept that inflammation could be mediated by controlling prostaglandin synthesis has intensified the search for effective inhibitors of the formation of prostaglandins. Many inhibitors investigated contain hydrophobic portions likely to compete with substrate acid for binding to a hydrophobic site on the fatty acid oxygenase that produces prostaglandins. Data recently summarized (3, 4) support the concept of such a site.Time-dependent inhibition of the fatty acid oxygenase by aspirin and indomethacin was first reported in 1971 (5). Awareness of this mode of inhibition has added another dimension to our understanding of the control of prostaglandin production, since the potency of an inhibitor may not depend solely on its affinity for binding the oxygenase, but also on the amount of time the drug remains in contact with the enzyme. The mechanism of the time-dependent loss of activity is not well understood, although recent evidence suggests that aspirin may exert its time-dependent effect by acylation of the fatty acid oxygenase (6). The current results make it unlikely that acylation is a general mechanism for all time-dependent inhibitions of anti-inflammatory agents since several time-dependent inhibitors lack an activated acyl group (see Fig. 2).We initiated the present study to examine common structural features that might be associated with the time-dependent action of the irreversible agents. The enzyme (see ref. 7 for preparation) was routinely homogenized in 0.1 M Tris-HCl (pH 8.5), containing 0.67 mM phenol, to give a concentration of 20-60 mg/ml in buffer. This was allowed to stand for 30 min at 250 to increase its activity (8). MATERIALS AND METHODSDetermination of Fatty Acid Oxygenase Activity. Oxygen consumption was measured at 30 i 0.50 on a Yellow Springs Instrument Co. model 53 Oxygen Monitor. Continuous recordings were made on a dual channel recorder with one channel recording the oxygen electrod...

Structural and Functional Basis of Cyclooxygenase Inhibition

“…The tight binding of indomethacin to COX-1 requires the carboxylic acid group because its methyl ester lacks the time-dependent step but retains the fast-reversible (timeindependent) step. 43 In contrast, esterification of indomethacin does not abolish time-dependent inhibition of COX-2, although it does reduce its tightness of binding to the enzyme. 72,73 Indomethacin is recovered intact after prolonged incubation with either enzyme, suggesting that the time-dependent inhibition of COX likely involves a conformational change rather than a covalent interaction with the protein 71 as is seen for aspirin inhibition.…”

“…43 In this study, ibuprofen (2-[4-(2-methylpropyl)phenyl]propanoic acid) and mefenamic acid (2-(2,3-dimethylphenyl)aminobenzoic acid) ( Figure 7) display competitive and rapidly reversible inhibition of the COX activity of sheep seminal vesicle preparations that is characterized by a single-step mechanism (one forward and one reverse rate constant to describe enzymeinhibitor association and dissociation, respectively). On the other hand, flurbiprofen (2-(3-fluoro-4-phenylphenyl)propanoic acid) and meclofenamic acid (2-(2,6-dichloro-3-methylphenyl)aminobenzoic acid) exhibit time-dependent, functionally irreversible inhibition of COX activity that displays a two-step mechanism.…”

“…Aspirin ( Figure 7) inhibits cyclooxygenase activity in a time-dependent fashion, although it is the least potent of the time-dependent COX inhibitors as reflected in its unusually high K I value for initial association with the enzyme. 43 Reaction of COX with aspirin containing a radiolabeled acetyl group leads to incorporation of radioactivity into the protein. 44,45 Arachidonic acid inhibits acetylation, suggesting that aspirin acetylates a residue in the COX active site channel.…”

“…The phenylpropionic acid inhibitors ibuprofen and mefenamic acid ( Figure 7) are competitive and rapidly reversible inhibitors of COX that inhibit with a single-step kinetic mechanism, whereas structural analogues of each inhibitor (flurbiprofen and meclofenamic acid) are time-dependent, functionally irreversible inhibitors that follow a two-step mode for COX inhibition. 43 The original kinetic model developed by Rome and Lands explains the kinetic differences between these two classes of inhibitors (eq 1). 43 Ibuprofen and mefenamic acid associate with Figure 8.…”

“…43 The original kinetic model developed by Rome and Lands explains the kinetic differences between these two classes of inhibitors (eq 1). 43 Ibuprofen and mefenamic acid associate with Figure 8. Crystal structure of an aspirin analogue in the active site of oCOX-1.…”