Characterization of a novel inhibitor of cytosolic phospholipase A2α, pyrrophenone

Abstract

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.

Characterization of prostaglandin E2 generation through the cyclooxygenase (COX)-2 pathway in human neutrophils

“…In addition, mRNA for groups V and X sPLA 2 (GV and GX) have been detected, whereas GIB, GIIA, GIID, GIIE, GIIF, GIII, and GXII sPLA 2 s were not found [19]. Given the demonstrated and prominent role of type IV cPLA 2 in the generation of 5-LO-derived leukotrienes [19], we used an inhibitor displaying highest selectivity for this PLA 2 subtype, termed pyrrophenone [32,33] in order to address its implication in the generation of COX-2-derived PGE 2 . To this end, COX-2 was up-regulated in neutrophils by incubation with a mixture of 1.5 nM GM-CSF and 100 nM TNF-α (GM-CSF/TNF) for 120 min [8] then stimulated with the Ca 2+ -ionophore A23187 for 15 min, in absence or presence of 100 nM pyrrophenone.…”

In Vivo Phospholipase Activity of the Pseudomonas aeruginosa Cytotoxin ExoU and Protection of Mammalian Cells with Phospholipase A2 Inhibitors

“…To assess whether a cellular phospholipase is responsible for cytotoxicity, a specific inhibitor of endogenous cPLA 2 ␣, pyrrophenone (30), and a specific inhibitor of endogenous iPLA 2 , bromoenol lactone (28), were examined. Pyrrophenone is not protective, whereas bromoenol lactone shows only partial protection ( Fig.…”

Roles of ERK and cPLA2 in the Angiotensin II-Mediated Biphasic Regulation of Na+-HCO3 − Transport

“…26 In the presence of AACOCF 3 (10 mol/L), the biphasic regulation of Ang II was lost, and all of the concentrations of Ang II (from 10 Ϫ10 to 10 Ϫ6 mol/L) showed the stimulation of NBC activity ( Figure 3A). In the presence of pyrrophenone (1 mol/L), the virtually identical stimulation of NBC was detected by all of the concentrations of Ang II ( Figure 3B), supporting the role of cPLA 2 in inhibitory effect of Ang II.…”