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Group VIA calcium-independent phospholipase A2 (GVIA iPLA2) has recently emerged as a novel pharmaceutical target. We have now explored the structure-activity relationship between fluoroketones and GVIA iPLA2 inhibition. The presence of a naphthyl group proved to be of paramount importance. 1,1,1-Trifluoro-6-(naphthalen-2-yl)hexan-2-one (FKGK18) is the most potent inhibitor of GVIA iPLA2 (XI(50) 0.0002) ever reported. Being 195 and >455 times more potent for GVIA iPLA2 than for GIVA cPLA2 and GV sPLA2, respectively, makes it a valuable tool to explore the role of GVIA iPLA2 in cells and in vivo models. 1,1,1,2,2,3,3-Heptafluoro-8-(naphthalene-2-yl) octan-4-one inhibited GVIA iPLA2 with a XI(50) value of 0.001, while inhibiting the other intracellular GIVA cPLA2 and GV sPLA2 at least 90-times less potently. Hexa- and octa-fluoro ketones were also found to be potent inhibitors of GVIA iPLA2; however they are not selective.

The 85-kDa GIVA 3 phospholipase A 2 (GIVA PLA 2 ) is a member of the superfamily of phospholipase A 2 enzymes (1, 2) that cleave fatty acids from the sn-2 position of membrane phospholipids. This enzyme was initially isolated from human platelets, and it is specific for phospholipids containing arachidonic acid in the sn-2 position (3, 4). The release of arachidonic acid is the critical first step in the biosynthesis of eicosanoids, which are potent mediators of inflammation and pain (5). There are a number of enzymes and routes by which arachidonic acid can be released from phospholipids, but experiments with GIVA PLA 2 knock-out mice have demonstrated its importance in many inflammatory processes (6 -8), thereby confirming the key role of the GIVA enzyme.The enzyme is composed of two domains, a Ca 2ϩ binding C2 domain and a ␣/␤-hydrolase domain that contains the catalytic site (9). Crystal structures of both the intact enzyme (PDB 1CJY) (10) and the C2 domain alone (11) (PDB 1RLW) have been solved. For the enzyme to be active, it must be sequestered to a phospholipid interface. Binding Ca 2ϩ to the C2 domain accomplishes this as does the binding of two different lipid mediators, e.g. ceramide 1 phosphate (12, 13) and phosphatidylinositol-(4,5) bis-phosphate (14, 15).The Ca 2ϩ binding C2 domain is a conserved domain that is present on many different lipid-binding proteins (16). Ca 2ϩ binding to this domain sequesters the protein to the lipid surface. Extensive studies have been carried out on the C2 domain using a variety of techniques to determine how Ca 2ϩ binding accomplishes lipid surface binding. These studies have included x-ray reflectivity, site-directed mutagenesis, NMR, EPR, and computational methods (17). These studies have shown (18 -23) that lipid binding entails the penetration of Ca 2ϩ binding loops one and three, composed of amino acids 35-39 and 96 -98, into the interface. However, these studies only deal with C2 binding to the membrane, not the intact cPLA 2 enzyme. How the presence of the ␣/␤-hydrolase domain affects surface binding has not been determined in detail due to the difficulties of working with such a large protein.Unlike the C2 domain, the binding of the catalytic domain of the enzyme to the lipid surface has not been extensively studied. Numerous studies using site-directed mutagenesis of the intact protein have localized amino acids that are important for lipid * This work was supported, in whole or in part, by National Institutes of Health Grants GM20501 (to E. A. D.) and CA099835, CA118595, GM037684, AI0220221, and AI022160 (to V. L. W.). This work was also supported by Discovery Grant UC10591 from the University of California Industry.-University Cooperative Research Program (to V. L. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 858-534-7390; E-mail: edennis@ucsd.edu.3 The abbreviations used...

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The GIVA phospholipase A 2 (PLA 2 ) contains two domains: a calcium-binding domain (C2) and a catalytic domain. These domains are linked via a flexible tether. GIVA PLA 2 activity is Ca 2؉ -dependent in that calcium binding promotes protein docking to the phospholipid membrane. In addition, the catalytic domain has a lid that covers the active site, presumably regulating GIVA PLA 2 activity. We now present studies that explore the dynamics and conformational changes of this enzyme in solution utilizing peptide amide hydrogen/deuterium (H/D) exchange coupled with liquid chromatographymass spectrometry (DXMS) to probe the solvent accessibility and backbone flexibility of the C2 domain, the catalytic domain, and the intact GIVA PLA 2 . We also analyzed the changes in H/D exchange of the intact GIVA PLA 2 upon Ca 2؉ binding. The DXMS results showed a fast H/D-exchanging lid and a slow exchanging central core. The C2 domain showed two distinct regions: a fast exchanging region facing away from the catalytic domain and a slow exchanging region present in the "cleft" region between the C2 and catalytic domains. The slow exchanging region of the C2 domain is in tight proximity to the catalytic domain. The effects of Ca 2؉ binding on GIVA PLA 2 are localized in the C2 domain and suggest that binding of two distinct Ca 2؉ ions causes tightening up of the regions that surround the anion hole at the tip of the C2 domain. This conformational change may be the initial step in GIVA PLA 2 activation.The cytosolic group IVA (GIVA) 3 phospholipase A 2 (PLA 2 ), also known as cPLA 2 , was purified and cloned in 1991 (1, 2). It is one of the few phospholipases in the phospholipase A 2 superfamily shown to be important in lipid mediator biosynthesis (3, 4). GIVA PLA 2 hydrolyzes membrane phospholipids at the sn-2 position to release free arachidonic acid, which is the precursor of numerous eicosanoids including the prostaglandins and leukotrienes (5, 6) involved in the inflammatory and pain response (7-9). Understanding the regulation of the catalytic activity of GIVA PLA 2 is crucial for understanding eicosanoid metabolism.The activity of the 85-kDa GIVA PLA 2 has been suggested to be regulated by several factors including the intracellular Ca 2ϩ concentration, its phosphorylation state, and the binding of various activators. Since the GIVA PLA 2 was discovered in 1986, the activity of this enzyme has been known to be Ca 2ϩ -dependent (10, 11). The Ca 2ϩ -dependent lipid-binding domain (C2 domain) at the N terminus, which is linked to a catalytic domain where phospholipid hydrolysis occurs, was later identified (12). Further studies of the GIVA PLA 2 activity showed up-regulation by p38 protein kinase mainly through Ser-505 phosphorylation (13-15). Various membrane-associated activators have been shown to bind to GIVA PLA 2 and upregulate its activity. In particular, we have shown that the membrane-associated phosphatidylinositol 4,5-bisphosphate can bind to the "lysine pocket" of GIVA PLA 2 with high affinity and specificity to ac...

The mechanism of inhibition of group VIA Ca2+-independent phospholipase A2 (iPLA2) by fluoroketone (FK) ligands is examined by a combination of deuterium exchange mass spectrometry (DXMS) and molecular dynamics (MD). Models for iPLA2 were built by homology with the known structure of patatin and equilibrated by extensive MD simulations. Empty pockets were identified during the simulations and studied for their ability to accommodate FK inhibitors. Ligand docking techniques showed that the potent inhibitor 1,1,1,3-tetrafluoro-7-phenylheptan-2-one (PHFK) forms favorable interactions inside an active-site pocket, where it blocks the entrance of phospholipid substrates. The polar fluoroketone headgroup is stabilized by hydrogen bonds with residues Gly486, Gly487, and Ser519. The nonpolar aliphatic chain and aromatic group are stabilized by hydrophobic contacts with Met544, Val548, Phe549, Leu560, and Ala640. The binding mode is supported by DXMS experiments showing an important decrease of deuteration in the contact regions in the presence of the inhibitor. The discovery of the precise binding mode of FK ligands to the iPLA2 should greatly improve our ability to design new inhibitors with higher potency and selectivity.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) plays important roles in both the inhibition and promotion of inflammation in human disease. It catalyzes the hydrolytic inactivation of plasma platelet activating factor (PAF) and is also known as PAF acetylhydrolase. High levels of PAF are implicated in a variety of inflammatory diseases such as asthma, necrotizing enterocolitis and sepsis. Lp-PLA2 also associates with lipoproteins in human plasma where it hydrolyzes oxidized phospholipids to produce pro-inflammatory lipid mediators that can promote inflammation and the development of atherosclerosis. Lp-PLA2 plasma levels have recently been identified as a biomarker of vascular inflammation, atherosclerotic vulnerability, and future cardiovascular events. The enzyme is thus a prominent target for the development of inflammation and atherosclerosis-modulating therapeutics. While the crystallographically-determined structure of the enzyme is known, the enzyme's mechanism of interaction with PAF and the function-modulating lipids in lipoproteins is unknown. We have employed peptide amide hydrogen-deuterium exchange mass spectrometry (DXMS) to characterize the association of Lp-PLA2 with dimyristoyl phosphatidylcholine (DMPC) vesicles, and found that specific residues 113-120 in one of the enzyme's surface-disposed hydrophobic α-helices likely mediate liposome binding.