Arginine 120 of Prostaglandin G/H Synthase-1 Is Required for the Inhibition by Nonsteroidal Anti-inflammatory Drugs Containing a Carboxylic Acid Moiety

Abstract

The therapeutic action of nonsteroidal anti-inflammatory drugs (NSAIDs) is exerted through the inhibition of prostaglandin G/H synthase (PGHS), which is expressed as two isoenzymes, termed PGHS-1 and PGHS-2. From the crystal structure of sheep PGHS-1, it has been proposed that the carboxylic acid group of flurbiprofen is located in a favorable position for interacting with the arginine 120 residue of PGHS-1 (Picot, D., Loll, P. J., and Garavito, R. M. (1994) Nature 367, 243-249). Mutation of this Arg120 residue to Glu was performed and expressed in COS-7 cells using a vaccinia virus expression system. Comparison of microsomal enzyme preparations show that the mutation results in a 20-fold reduction in the specific activity of PGHS-1 and in a 100-fold increase in the apparent Km for arachidonic acid. Indomethacin, flurbiprofen, and ketoprofen, inhibitors of PGHS activity containing a free carboxylic acid group, do not exhibit any inhibitory effects against the activity of PGHS-1(Arg120-->Glu). Diclofenac and meclofenamic acid, other NSAIDs containing a free carboxylic acid group, were 50-100-fold less potent inhibitors of the activity of the mutant as compared with the wild type PGHS. In contrast, the nonacid PGHS inhibitors, 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl)thiophene (DuP697) and a desbromo-sulfonamide analogue of DuP697 (L-746,483), were both more potent inhibitors of PGHS-1(Arg120-->Glu) than of the wild tyupe PGHS-1. Inhibition of PGHS-1(Arg120-->Glu) was time-dependent for diclofenac and time-independent for DuP697, as observed for the wild type enzyme, indicating that the mutation does not alter the basic mechanism of inhibition. Aspirin is an acid NSAID that inhibits PGHS-1 through a unique covalent acetylation of the enzyme and also showed a reduced rate of inactivation of the mutated enzyme. These data provide biochemical evidence of the importance of the Arg120 residue in PGHS-1 for interaction with arachidonic acid and NSAIDs containing a free carboxylic acid moiety.

Pre-existent Asymmetry in the Human Cyclooxygenase-2 Sequence Homodimer

“…In contrast, mutations of Arg-120 in E cat do not have major effects on time-dependent inhibition by either flurbiprofen or naproxen. Consistent with previous reports (31,33,46,48,49,64), time-dependent inhibition by celecoxib and related compounds is insensitive to mutations of Arg-120 in either E allo or E cat . Time-dependent inhibition by indomethacin was partially attenuated by mutations of Arg-120 in E cat and unaffected by mutations of Arg-120 in E allo .…”

“…Time-dependent inhibition by indomethacin was partially attenuated by mutations of Arg-120 in E cat and unaffected by mutations of Arg-120 in E allo . Indomethacin exhibits important interactions with Arg-120 in addition to other active site residues, including Val-349, that have been implicated in time-dependent inhibition (31,33,46,50,64).…”

Conversion of Prostaglandin G/H Synthase-1 into an Enzyme Sensitive to PGHS-2-selective Inhibitors by a Double His513→ Arg and Ile523→ Val Mutation

“…Membranes from mock transfected COS-7 cells contained less than 3% of the PGHS activity measured for any of the recombinant PGHS proteins. The time course of PGE 2 production was similar for all recombinant PGHS preparations with a rapid substrate conversion over the first minute of the reaction and a plateau of product formation after a 3-5-min reaction as previously reported (19). The apparent K m values for arachidonic acid were determined and found to be very similar for PGHS-2, PGHS-1, and the mutants of PGHS-1 (0.5-0.9 M) ( Table I).…”

“…The x-ray crystal structure of sheep seminal vesicle PGHS-1 has demonstrated that the cyclooxygenase active site is comprised of a long hy-drophobic channel that is also the site for binding of NSAIDs (18). Several distinctive features of the active site are: 1) an Arg 120 residue at the mouth of the channel that has been demonstrated to be important for binding of arachidonic acid and NSAIDs containing a carboxylic acid residue (19,20); 2) a Tyr 385 residue at the upper portion of the active site that is involved in the formation of a radical at the C-13 position of arachidonic acid (21); and 3) a Ser 530 residue (just below Tyr 385 ) is the residue that is the site of acetylation by aspirin (22). The aspirin acetylated PGHS-2 results in an enzyme form that oxidizes arachidonic acid to 15-HETE (23) with altered sensitivity to inhibition by certain NSAIDs (24).…”

Structural and Functional Basis of Cyclooxygenase Inhibition

“…Site-directed mutation of Arg-120 to alanine in COX-1 reveals that carboxylic acid-containing time-dependent NSAIDs (indomethacin and flurbiprofen) form an ion pair and/or hydrogen bond with Arg-120 and that this interaction is critical for inhibition. 78 The same mutation in COX-2 is not equally effective in eliminating inhibition by indomethacin. 72 Interestingly, a methyl ester derivative of indomethacin exhibits more potent inhibition of the COX-2 R120A mutant than wild-type enzyme, suggesting that interactions with Arg-120 are less important for inhibitor binding and potency with COX-2.…”