Activated Ketones as Inhibitors of Intracellular Ca²⁺-Dependent and Ca²⁺-Independent Phospholipase A₂

Abstract

Studies are reported on two different types of activated ketones as inhibitors of two important intracellular phospholipase A 2 s (PLA 2 ): the group IV 85 kDa Ca 2+ -dependent phospholipase A 2 (cPLA 2 ) and the P388D 1 Ca 2+independent phospholipase A 2 (iPLA 2 ). In a mixed micelle assay, we observed that the reaction progress curve of cPLA 2 in the presence of a trifluoromethyl ketone (TFMK) is linear at pH 7.4, while at pH 9.0 it is nonlinear and slows with time. An investigation of this discrepancy demonstrated that the TFMKs are slow, tight-binding inhibitors of the cPLA 2 at both pH's, that the rate of dissociation of the enzyme-inhibitor complex is the same at both pH's, but that the rate of association of enzyme and inhibitor is slower at pH 7.4 than at pH 9.0. A novel group of activated ketone inhibitors has been synthesized that contain a fatty acyl tricarbonyl. These compounds also inhibit the cPLA 2 in the mixed micelle assay. The inhibition of cPLA 2 by the tricarbonyls is readily reversible upon dilution and does not involve slow binding. For both types of inhibitor, no preference for fatty acid chain was observed as the palmityl analogs inhibited as well as the arachidonoyl analogs, despite the fact that the cPLA 2 shows a strong preference for arachidonoyl-containing phospholipid substrates over palmitoyl-containing substrates. With the iPLA 2 , the inhibition by TFMKs is reversible and does not involve slow or tight binding. The tricarbonyls also inhibited the iPLA 2 , but were less potent than the TFMKs. Unlike the cPLA 2 , the iPLA 2 does exhibit a fatty acid preference as the palmityl analogs of both compounds inhibit better than the arachidonoyl analogs. The palmityl TFMK displayed a 10-fold lower IC 50 at pH 9.0 than at pH 7.5, whereas the potency of the tricarbonyl was unchanged in this range. Thus, the TFMKs inhibit both the cPLA 2 and the iPLA 2 , but the mechanism of inhibition of the two enzymes appears to be quite different. The tricarbonyls also inhibited both enzymes, but in both cases in a reversible manner and as such appear to be poorer inhibitors than the TFMKs.Ketones with enhanced electrophilic reactivity (hydrateforming ketones), such as trifluoromethyl ketones (TFMKs), R-diketones, R-keto esters, and R,β-dioxo esters or vicinal tricarbonyls, 1,2 are a well-established class of inhibitors for proteases, lipases, and other serine esterases. This type of inhibitor has now been used to study phospholipase A 2 s (PLA 2 ), a class of esterases which carry out a reaction similar to that of lipases. Of particular interest are two intracellular PLA 2 s, the Group IV, 85 kDa, Ca 2+ -dependent PLA 2 (cPLA 2 ) 3-5 and the murine P388D 1 80 kDa, Ca 2+ -independent PLA 2 (iPLA 2 ). 6 The mechanisms of action of these two PLA 2 s are not well established, although the cPLA 2 reaction is believed to proceed through an acyl-enzyme intermediate. 7,8 Sharp

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

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“…For ExoU(S142A)-GFP, representative focal planes are shown to demonstrate circumferential plasma membrane (Focal Plane 1) and punctate localization (Focal Plane 2). also called Group IVA PLA 2 ) and Ca 2ϩ -independent PLA 2 s (iPLA 2 , also called Group VIA PLA 2 ) (28,29), blocks 82% of cell killing ( Fig. 2A).…”

Protease activation of calcium-independent phospholipase A2 leads to neutrophil recruitment to coronary artery endothelial cells

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