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.
Highlights
Prostaglandins, which are derived from arachidonic acid, act as potent mediators of pain, fever, and inflammation [1]
The present results demonstrate that the Arg120 residue of hPGHS-1 is essential for the potent inhibitory effects of the acid inhibitors flurbiprofen, indomethacin, diclofenac, ketoprofen, and meclofenamic acid
The decreased or lack of inhibition of prostaglandin G/H synthase (PGHS)-1(Arg120 3 Glu) by acid Nonsteroidal anti-inflammatory drugs (NSAIDs) supports the model proposed by Garavito and his colleagues in which the Arg120 residue of PGHS-1 interacts with the carboxylic acid group of flurbiprofen
Summary
Prostaglandins, which are derived from arachidonic acid, act as potent mediators of pain, fever, and inflammation [1]. At least three types of NSAID-mediated inhibition of PGHS isoforms have been reported. Aspirin has been demonstrated to irreversibly inhibit prostaglandin production of PGHS-1 and stimulate (15R)-hydroxyeicosatetraenoic acid production by PGHS-2 through the acetylation of a specific serine residue in each PGHS isoform [12, 15, 16]. In a second type of NSAIDmediated inhibition, drugs such as indomethacin and flurbiprofen act as time-dependent, irreversible inhibitors of both isoforms without resultant covalent modification [17,18,19]. In a third type of PGHS-mediated inhibition, compounds such as N-(2cyclohexyloxy-4-nitrophenyl)methanesulfonamide (NS398) and 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl)thiophene (DuP697) act as time-independent, reversible inhibitors of PGHS-1 but as time-dependent, irreversible inhibitors of PGHS-2 [17, 18].
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