Introduction. Phospholipase A 2 (PLA 2 ) is a group of lipolytic enzymes that catalyze the hydrolysis of fatty acid ester bonds at the sn-2 position of phospholipids. This enzyme is thought to play an important role in the biosynthesis of eicosanoids via the release of arachidonic acid from biomembranes. Another product from biomembranes, a lysophospholipid, is converted to plateletactivating factor (PAF) known as an inflammatory mediator. PLA 2 s have been generally classified into secretory PLA 2 (sPLA 2 ), cytosolic PLA 2 (cPLA 2 ), and Ca 2+ -independent PLA 2 (iPLA 2 ) by their molecular weights, amino acid sequences, and calcium requirements. 1 cPLA 2 comprises three distinct types of enzymes: R, β, and γ. 2 cPLA 2 R, an 85-kDa protein, contains a calcium-dependent lipid binding domain and a catalytic domain, requires micromolar levels of Ca 2+ for membrane translocation, and has a specificity for arachidonic acid bound to the sn-2 position of phospholipids 3 in contrast with sPLA 2 and iPLA 2 which have broad substrate specificities, suggesting that cPLA 2 R is involved in the production of eicosanoids.

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha), one of the three subtypes of cPLA(2) (alpha, beta and gamma), is thought to be a rate-limiting enzyme in eicosanoid biosynthesis. We developed a novel and potent cPLA(2)alpha inhibitor with an optically active pyrrolidine, termed pyrrophenone, and characterized this compound in detail using enzyme and cellular assay systems. Pyrrophenone, which shows strong inhibition of cPLA(2)alpha activity, is one of the most potent cPLA(2)alpha inhibitors reported to date. Similar inhibitory potencies for cPLA(2)alpha were obtained from three different assays. The inhibitory activity of pyrrophenone is two or three orders of magnitude more potent than arachidonyl trifluoromethyl ketone (AACOCF(3)) under the same assay conditions. Pyrrophenone shows reversible inhibition of cPLA(2)alpha and displays no characteristics of the slow-binding inhibition observed for AACOCF(3). Pyrrophenone also inhibited the esterase and lysophospholipase activities of cPLA(2)alpha. However, the inhibition by pyrrophenone of 14 kDa secretory PLA(2)s, types IB and IIA, was over two orders of magnitude less potent than that for cPLA(2)alpha. Pyrrophenone strongly inhibited arachidonic acid release in calcium ionophore (A23187)-stimulated human monocytic cells (THP-1 cells) in a dose-dependent manner with an IC(50) value of 0.024 microM, followed by suppression of eicosanoid synthesis, and also showed dose-dependent inhibition for interleukin-1-induced prostaglandin E(2) synthesis in human renal mesangial cells with an IC(50) value of 0.0081 microM. The mechanism of inhibition of eicosanoid synthesis in these cell-based assays was due to inhibition of only one step of arachidonic acid release without any effect on cyclo-oxygenase or lipoxygenase pathways. These results suggest that pyrrophenone could be a potential therapeutic agent for inflammatory diseases.

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These results demonstrate that cPLA2 alpha plays an important role in the pathogenesis of collagen-induced arthritis. Oral administration of pyrroxyphene achieved anti-arthritic activity through inhibition of cPLA2 alpha activity, which led to a reduction in eicosanoid levels and suppression of MMP and COX-2 mRNA expression. These results support a potential therapeutic role for cPLA2 alpha inhibitors in the treatment of human rheumatoid arthritis.

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