Cytosolic phospholipase A2α (cPLA2α), one of the three subtypes of cPLA2 (α, β and γ), is thought to be a rate-limiting enzyme in eicosanoid biosynthesis. We developed a novel and potent cPLA2α 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 cPLA2α activity, is one of the most potent cPLA2α inhibitors reported to date. Similar inhibitory potencies for cPLA2α were obtained from three different assays. The inhibitory activity of pyrrophenone is two or three orders of magnitude more potent than arachidonyl trifluoromethyl ketone (AACOCF3) under the same assay conditions. Pyrrophenone shows reversible inhibition of cPLA2α and displays no characteristics of the slow-binding inhibition observed for AACOCF3. Pyrrophenone also inhibited the esterase and lysophospholipase activities of cPLA2α. However, the inhibition by pyrrophenone of 14kDa secretory PLA2s, types IB and IIA, was over two orders of magnitude less potent than that for cPLA2α. Pyrrophenone strongly inhibited arachidonic acid release in calcium ionophore (A23187)-stimulated human monocytic cells (THP-1 cells) in a dose-dependent manner with an IC50 value of 0.024μM, followed by suppression of eicosanoid synthesis, and also showed dose-dependent inhibition for interleukin-1-induced prostaglandin E2 synthesis in human renal mesangial cells with an IC50 value of 0.0081μM. 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.
- arachidonic acid release
- cPLA2α inhibitor
- cytosolic PLA2α inhibitor
Abbreviations used: PLA2, phospholipase A2; cPLA2, cytosolic PLA2; sPLA2, secretory PLA2; AACOCF3, arachidonyl trifluoromethyl ketone; MAFP, methyl arachidonyl fluorophosphonate; PC, phosphatidylcholine; PAPC, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; [14C]PAPC, 1-palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphocholine; DOG, sn-1,2-dioleoylglycerol; PGE2, prostaglandin E2; LTC4, leukotriene C4; ADAM, 9-anthryldiazomethane; 7-HCA, 7-hydroxycoumarinyl-arachidonate; AA-861, 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone; IL-1β, interleukin-1β; DTT, dithiothreitol; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); FCS, fetal calf serum; DG lipase, diacylglycerol lipase; COX, cyclo-oxygenase; arachidonoyl thio-PC, 1-hexadecyl-2-arachidonoylthio-2-deoxy-sn-glycero-3-phosphocholine; diheptanoyl thio-PC, 1,2-bis(heptanoylthio)-1,2-dideoxy-rac-sn-glycero-3-phosphocholine; LPS, lipopolysaccharide; XI(50), the mol fraction of inhibitor in total lipid that causes 50% inhibition.
- The Biochemical Society, London ©2002