Edward A. Dennis

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Name: Dennis, Edward

Organization: University of California , USA

Department: Department of Chemistry and Biochemistry

Title: Professor(PhD)

Chemicals
References

Borrelia burgdorferi infection induces lipid mediator production during Lyme arthritis

Co-reporter:Charles R. Brown, Edward A. Dennis

Biochimie 2017 Volume 141(Volume 141) pp:

Publication Date(Web):1 October 2017

DOI:10.1016/j.biochi.2017.06.010

•C3H mice infected with Borrelia burgdorferi develop an inflammatory arthritis.•Select bioactive lipids from infected ankles were quantified using lipidomics.•COX-2 inhibition or deletion results in non-resolution of arthritis.•Lipidomics revealed that COX-2 deletion leads to loss of some 5-LOX metabolites.•5-LOX inhibition or deletion also resulted in non-resolution of arthritis.Experimental Lyme arthritis provides a mouse model for exploring the development of pathology following infection of C3H mice with Borrelia burgdorferi. Infected mice develop a reliable inflammatory arthritis of the ankle joint with severity that typically peaks around two to three weeks post-infection and then undergoes spontaneous resolution. This makes experimental Lyme arthritis an excellent model for investigating the mechanisms that drive both the development and resolution phases of inflammatory disease. Eicosanoids are powerful lipid mediators of inflammation and are known to regulate multiple aspects of inflammatory processes. While much is known about the role of eicosanoids in regulating immune responses during autoimmune disease and cancer, relatively little is known about their role during bacterial infection. In this review, we discuss the role of eicosanoid biosynthetic pathways in mediating inflammatory responses during bacterial infection using experimental Lyme arthritis as a model system. We point out the critical role eicosanoids play in disease development and highlight surprising differences between sterile autoimmune responses and those occurring in response to bacterial infection. These differences should be kept in mind when designing therapies and treatments for inflammatory diseases.

Development of Potent and Selective Inhibitors for Group VIA Calcium-Independent Phospholipase A2 Guided by Molecular Dynamics and Structure–Activity Relationships

Co-reporter:Varnavas D. Mouchlis; Dimitris Limnios; Maroula G. Kokotou; Efrosini Barbayianni; George Kokotos; J. Andrew McCammon

Journal of Medicinal Chemistry 2016 Volume 59(Issue 9) pp:4403-4414

Publication Date(Web):April 17, 2016

DOI:10.1021/acs.jmedchem.6b00377

The development of inhibitors for phospholipase A2 (PLA2) is important in elucidating the enzymes implication in various biological pathways. PLA2 enzymes are an important pharmacological target implicated in various inflammatory diseases. Computational chemistry, organic synthesis, and in vitro assays were employed to develop potent and selective inhibitors for group VIA calcium-independent PLA2. A set of fluoroketone inhibitors was studied for their binding mode with two human cytosolic PLA2 enzymes: group IVA cPLA2 and group VIA iPLA2. New compounds were synthesized and assayed toward three major PLA2s. This study led to the development of four potent and selective thioether fluoroketone inhibitors as well as a thioether keto-1,2,4-oxadiazole inhibitor for GVIA iPLA2, which will serve as lead compounds for future development and studies. The keto-1,2,4-oxadiazole functionality with a thioether is a novel structure, and it will be used as a lead to develop inhibitors with higher potency and selectivity toward GVIA iPLA2.

Computer-aided drug design guided by hydrogen/deuterium exchange mass spectrometry: A powerful combination for the development of potent and selective inhibitors of Group VIA calcium-independent phospholipase A2

Co-reporter:Varnavas D. Mouchlis, Christophe Morisseau, Bruce D. Hammock, Sheng Li, J. Andrew McCammon, Edward A. Dennis

Bioorganic & Medicinal Chemistry 2016 Volume 24(Issue 20) pp:4801-4811

Publication Date(Web):15 October 2016

DOI:10.1016/j.bmc.2016.05.009

Potent and selective inhibitors for phospholipases A2 (PLA2) are useful for studying their intracellular functions. PLA2 enzymes liberate arachidonic acid from phospholipids activating eicosanoid pathways that involve cyclooxygenase (COX) and lipoxygenase (LOX) leading to inflammation. Anti-inflammatory drugs target COX and LOX; thus, PLA2 can also be targeted to diminish inflammation at an earlier stage in the process. This paper describes the employment of enzymatic assays, hydrogen/deuterium exchange mass spectrometry (DXMS) and computational chemistry to develop PLA2 inhibitors. Beta-thioether trifluoromethylketones (TFKs) were screened against human GVIA calcium-independent, GIVA cytosolic and GV secreted PLA2s. These compounds exhibited inhibition toward Group VIA calcium-independent PLA2 (GVIA iPLA2), with the most potent and selective inhibitor 3 (OTFP) obtaining an XI(50) of 0.0002 mole fraction (IC50 of 110 nM). DXMS binding experiments in the presence of OTFP revealed the peptide regions of GVIA iPLA2 that interact with the inhibitor. Molecular docking and dynamics simulations in the presence of a membrane were guided by the DXMS data in order to identify the binding mode of OTFP. Clustering analysis showed the binding mode of OTFP that occupied 70% of the binding modes occurring during the simulation. The resulted 3D complex was used for docking studies and a structure–activity relationship (SAR) was established. This paper describes a novel multidisciplinary approach in which a 3D complex of GVIA iPLA2 with an inhibitor is reported and validated by experimental data. The SAR showed that the sulfur atom is vital for the potency of beta-thioether analogues, while the hydrophobic chain is important for selectivity. This work constitutes the foundation for further design, synthesis and inhibition studies in order to develop new beta-thioether analogues that are potent and selective for GVIA iPLA2 exclusively.Figure optionsDownload full-size imageDownload as PowerPoint slide

2-Oxoamide inhibitors of cytosolic group IVA phospholipase A2 with reduced lipophilicity

Co-reporter:Georgia Antonopoulou, Victoria Magrioti, Maroula G. Kokotou, Aikaterini Nikolaou, Efrosini Barbayianni, Varnavas D. Mouchlis, Edward A. Dennis, George Kokotos

Bioorganic & Medicinal Chemistry 2016 Volume 24(Issue 19) pp:4544-4554

Publication Date(Web):1 October 2016

DOI:10.1016/j.bmc.2016.07.057

Cytosolic GIVA phospholipase A2 (GIVA cPLA2) initiates the eicosanoid pathway of inflammation and thus inhibitors of this enzyme constitute novel potential agents for the treatment of inflammatory diseases. Traditionally, GIVA cPLA2 inhibitors have suffered systemically from high lipophilicity. We have developed a variety of long chain 2-oxoamides as inhibitors of GIVA PLA2. Among them, AX048 was found to produce a potent analgesic effect. We have now reduced the lipophilicity of AX048 by replacing the long aliphatic chain with a chain containing an ether linked aromatic ring with in vitro inhibitory activities similar to AX048.

Computational Modeling of Competitive Metabolism between ω3- and ω6-Polyunsaturated Fatty Acids in Inflammatory Macrophages

Co-reporter:Shakti Gupta, Yasuyuki Kihara, Mano R. Maurya, Paul C. Norris, Edward A. Dennis, and Shankar Subramaniam

The Journal of Physical Chemistry B 2016 Volume 120(Issue 33) pp:8346-8353

Publication Date(Web):April 11, 2016

DOI:10.1021/acs.jpcb.6b02036

Arachidonic acid (AA), a representative ω6-polyunsaturated fatty acid (PUFA), is a precursor of 2-series prostaglandins (PGs) that play important roles in inflammation, pain, fever, and related disorders including cardiovascular diseases. Eating fish or supplementation with the ω3-PUFAs such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is widely assumed to be beneficial in preventing cardiovascular diseases. A proposed mechanism for a cardio-protective role of ω3-PUFAs assumes competition between AA and ω3-PUFAs for cyclooxygenases (COX), leading to reduced production of 2-series PGs. In this study, we have used a systems biology approach to integrate existing knowledge and novel high-throughput data that facilitates a quantitative understanding of the molecular mechanism of ω3- and ω6-PUFA metabolism in mammalian cells. We have developed a quantitative computational model of the competitive metabolism of AA and EPA via the COX pathway through a two-step matrix-based approach to estimate the rate constants. This model was developed by using lipidomic data sets that were experimentally obtained from EPA-supplemented ATP-stimulated RAW264.7 macrophages. The resulting model fits the experimental data well for all metabolites and demonstrates that the integrated metabolic and signaling networks and the experimental data are consistent with one another. The robustness of the model was validated through parametric sensitivity and uncertainty analysis. We also validated the model by predicting the results from other independent experiments involving AA- and DHA-supplemented ATP-stimulated RAW264.7 cells using the parameters estimated with EPA. Furthermore, we showed that the higher affinity of EPA binding to COX compared with AA was able to inhibit AA metabolism effectively. Thus, our model captures the essential features of competitive metabolism of ω3- and ω6-PUFAs.

Membranes serve as allosteric activators of phospholipase A2, enabling it to extract, bind, and hydrolyze phospholipid substrates

Co-reporter:Varnavas D. Mouchlis;Denis Bucher;J. Andrew McCammon

PNAS 2015 Volume 112 (Issue 6 ) pp:E516-E525

Publication Date(Web):2015-02-10

DOI:10.1073/pnas.1424651112

Defining the molecular details and consequences of the association of water-soluble proteins with membranes is fundamental to understanding protein–lipid interactions and membrane functioning. Phospholipase A2 (PLA2) enzymes, which catalyze the hydrolysis of phospholipid substrates that compose the membrane bilayers, provide the ideal system for studying protein–lipid interactions. Our study focuses on understanding the catalytic cycle of two different human PLA2s: the cytosolic Group IVA cPLA2 and calcium-independent Group VIA iPLA2. Computer-aided techniques guided by deuterium exchange mass spectrometry data, were used to create structural complexes of each enzyme with a single phospholipid substrate molecule, whereas the substrate extraction process was studied using steered molecular dynamics simulations. Molecular dynamic simulations of the enzyme–substrate–membrane systems revealed important information about the mechanisms by which these enzymes associate with the membrane and then extract and bind their phospholipid substrate. Our data support the hypothesis that the membrane acts as an allosteric ligand that binds at the allosteric site of the enzyme’s interfacial surface, shifting its conformation from a closed (inactive) state in water to an open (active) state at the membrane interface.

Comprehensive ultra-performance liquid chromatographic separation and mass spectrometric analysis of eicosanoid metabolites in human samples

Co-reporter:Yan Wang, Aaron M. Armando, Oswald Quehenberger, Chao Yan, Edward A. Dennis

Journal of Chromatography A 2014 Volume 1359() pp:60-69

Publication Date(Web):12 September 2014

DOI:10.1016/j.chroma.2014.07.006

•Describe an UPLC-QTRAP/MS/MS method to detect eicosanoids.•Monitor a total of 184 eicosanoids in a single 5 min UPLC run.•The method was validated by using a human plasma sample.•The method was shown to be fast, sensitive and reliable.Over the past decade, the number of known eicosanoids has expanded immensely and we have now developed an ultra-performance liquid chromatography–electrospray ionization triple quadrupole mass spectrometric (UPLC-QTRAP/MS/MS) method to monitor and quantify numerous eicosanoids. The UPLC-QTRAP/MS/MS approach utilizes scheduled multiple reaction monitoring (MRM) to optimize sensitivity, number of metabolites that can be analyzed and the time requirement of the analysis. A total of 184 eicosanoids including 26 deuterated internal standards can be separated and monitored in a single 5 min UPLC run. To demonstrate a practical application, human plasma samples were analyzed following solid-phase extraction (SPE) and the recovery rate and matrix effects were determined for the 26 deuterated internal standards added to the plasma. The method was validated and shown to be sensitive with the limit of quantitation at pg levels for most compounds, accurate with recovery rates of 70–120%, and precise with a CV < 30 for all compounds. Also, the method showed a linear response over a range spanning several orders of magnitude. In a QC human plasma sample, we identified and rigorously quantified over 120 eicosanoids.

Phospholipase A2 regulates eicosanoid class switching during inflammasome activation

Co-reporter:Paul C. Norris;David Gosselin;Donna Reichart;Christopher K. Glass;

Proceedings of the National Academy of Sciences 2014 111(35) pp:12746-12751

Publication Date(Web):August 19, 2014

DOI:10.1073/pnas.1404372111

Initiation and resolution of inflammation are considered to be tightly connected processes. Lipoxins (LX) are proresolution lipid mediators that inhibit phlogistic neutrophil recruitment and promote wound-healing macrophage recruitment in humans via potent and specific signaling through the LXA₄ receptor (ALX). One model of lipoxin biosynthesis involves sequential metabolism of arachidonic acid by two cell types expressing a combined transcellular metabolon. It is currently unclear how lipoxins are efficiently formed from precursors or if they are directly generated after receptor-mediated inflammatory commitment. Here, we provide evidence for a pathway by which lipoxins are generated in macrophages as a consequence of sequential activation of toll-like receptor 4 (TLR4), a receptor for endotoxin, and P2X₇, a purinergic receptor for extracellular ATP. Initial activation of TLR4 results in accumulation of the cyclooxygenase-2–derived lipoxin precursor 15-hydroxyeicosatetraenoic acid (15-HETE) in esterified form within membrane phospholipids, which can be enhanced by aspirin (ASA) treatment. Subsequent activation of P2X₇ results in efficient hydrolysis of 15-HETE from membrane phospholipids by group IVA cytosolic phospholipase A₂, and its conversion to bioactive lipoxins by 5-lipoxygenase. Our results demonstrate how a single immune cell can store a proresolving lipid precursor and then release it for bioactive maturation and secretion, conceptually similar to the production and inflammasome-dependent maturation of the proinflammatory IL-1 family cytokines. These findings provide evidence for receptor-specific and combinatorial control of pro- and anti-inflammatory eicosanoid biosynthesis, and potential avenues to modulate inflammatory indices without inhibiting downstream eicosanoid pathways.

New potent and selective polyfluoroalkyl ketone inhibitors of GVIA calcium-independent phospholipase A2

Co-reporter:Victoria Magrioti, Aikaterini Nikolaou, Annetta Smyrniotou, Ishita Shah, Violetta Constantinou-Kokotou, Edward A. Dennis, George Kokotos

Bioorganic & Medicinal Chemistry 2013 Volume 21(Issue 18) pp:5823-5829

Publication Date(Web):15 September 2013

DOI:10.1016/j.bmc.2013.07.010

Group VIA calcium-independent phospholipase A2 (GVIA iPLA2) has recently emerged as an important pharmaceutical target. Selective and potent GVIA iPLA2 inhibitors can be used to study its role in various neurological disorders. In the current work, we explore the significance of the introduction of a substituent in previously reported potent GVIA iPLA2 inhibitors. 1,1,1,2,2-Pentafluoro-7-(4-methoxyphenyl)heptan-3-one (GK187) is the most potent and selective GVIA iPLA2 inhibitor ever reported with a XI(50) value of 0.0001, and with no significant inhibition against GIVA cPLA2 or GV sPLA2. We also compare the inhibition of two difluoromethyl ketones on GVIA iPLA2, GIVA cPLA2, and GV sPLA2.

Fluoroketone Inhibition of Ca2+-Independent Phospholipase A2 through Binding Pocket Association Defined by Hydrogen/Deuterium Exchange and Molecular Dynamics

Co-reporter:Yuan-Hao Hsu ; Denis Bucher ; Jian Cao ; Sheng Li ; Sheng-Wei Yang ; George Kokotos ▽; Virgil L. Woods ; Jr; J. Andrew McCammon

Journal of the American Chemical Society 2012 Volume 135(Issue 4) pp:1330-1337

Publication Date(Web):December 20, 2012

DOI:10.1021/ja306490g

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.

Omega-3 fatty acids cause dramatic changes in TLR4 and purinergic eicosanoid signaling

Co-reporter:Paul C. Norris

PNAS 2012 Volume 109 (Issue 22 ) pp:8517-8522

Publication Date(Web):2012-05-29

DOI:10.1073/pnas.1200189109

Dietary fish oil containing ω3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), elicit cardioprotective and anti-inflammatory effects through unresolved mechanisms that may involve competition and inhibition at multiple levels. Here, we report the effects of arachidonic acid (AA), EPA, and DHA supplementation on membrane incorporation, phospholipase A2 catalyzed release, and eicosanoid production in RAW264.7 macrophages. Using a targeted lipidomics approach, we observed that Toll-like receptor 4 and purinergic receptor activation of supplemented cells leads to the release of 22-carbon fatty acids that potently inhibit cyclooxygenase pathways. This inhibition was able to shunt metabolism of AA to lipoxygenase pathways, augmenting leukotriene and other lipoxygenase mediator synthesis. In resident peritoneal macrophages, docosapentaenoic acid (DPA) was responsible for cyclooxygenase inhibition after EPA supplementation, offering fresh insights into how EPA exerts anti-inflammatory effects indirectly through elongation to 22-carbon DPA.

Spinal 12-lipoxygenase-derived hepoxilin A3 contributes to inflammatory hyperalgesia via activation of TRPV1 and TRPA1 receptors

Co-reporter:Ann M. Gregus;Suzanne Doolen;Darren S. Dumlao;Matthew W. Buczynski;Toshifumi Takasusuki;Bethany L. Fitzsimmons;Xiao-Ying Hua;Bradley K. Taylor;Tony L. Yaksh

PNAS 2012 109 (17 ) pp:6721-6726

Publication Date(Web):2012-04-24

DOI:10.1073/pnas.1110460109

Peripheral inflammation initiates changes in spinal nociceptive processing leading to hyperalgesia. Previously, we demonstrated that among 102 lipid species detected by LC-MS/MS analysis in rat spinal cord, the most notable increases that occur after intraplantar carrageenan are metabolites of 12-lipoxygenases (12-LOX), particularly hepoxilins (HXA3 and HXB3). Thus, we examined involvement of spinal LOX enzymes in inflammatory hyperalgesia. In the current work, we found that intrathecal (IT) delivery of the LOX inhibitor nordihydroguaiaretic acid prevented the carrageenan-evoked increase in spinal HXB3 at doses that attenuated the associated hyperalgesia. Furthermore, IT delivery of inhibitors targeting 12-LOX (CDC, Baicalein), but not 5-LOX (Zileuton) dose-dependently attenuated tactile allodynia. Similarly, IT delivery of 12-LOX metabolites of arachidonic acid 12(S)-HpETE, 12(S)-HETE, HXA3, or HXB3 evoked profound, persistent tactile allodynia, but 12(S)-HpETE and HXA3 produced relatively modest, transient heat hyperalgesia. The pronociceptive effect of HXA3 correlated with enhanced release of Substance P from primary sensory afferents. Importantly, HXA3 triggered sustained mobilization of calcium in cells stably overexpressing TRPV1 or TRPA1 receptors and in acutely dissociated rodent sensory neurons. Constitutive deletion or antagonists of TRPV1 (AMG9810) or TRPA1 (HC030031) attenuated this action. Furthermore, pretreatment with antihyperalgesic doses of AMG9810 or HC030031 reduced spinal HXA3-evoked allodynia. These data indicate that spinal HXA3 is increased by peripheral inflammation and promotes initiation of facilitated nociceptive processing through direct activation of TRPV1 and TRPA1 at central terminals.

Phospholipase A2 Enzymes: Physical Structure, Biological Function, Disease Implication, Chemical Inhibition, and Therapeutic Intervention

Co-reporter:Edward A. Dennis, Jian Cao, Yuan-Hao Hsu, Victoria Magrioti, and George Kokotos

Chemical Reviews 2011 Volume 111(Issue 10) pp:6130

Publication Date(Web):September 12, 2011

DOI:10.1021/cr200085w

Lipoprotein-Associated Phospholipase A2 Interacts with Phospholipid Vesicles via a Surface-Disposed Hydrophobic α-Helix

Co-reporter:Jian Cao, Yuan-Hao Hsu, Sheng Li, Virgil L. Woods Jr., and Edward A. Dennis

Biochemistry 2011 Volume 50(Issue 23) pp:

Publication Date(Web):May 9, 2011

DOI:10.1021/bi101916w

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 dimyristoylphosphatidylcholine (DMPC) vesicles and found that specific residues 113–120 in one of the enzyme’s surface-disposed hydrophobic α-helices likely mediate liposome binding.

Potent and Selective Fluoroketone Inhibitors of Group VIA Calcium-Independent Phospholipase A2

Co-reporter:George Kokotos ; Yuan-Hao Hsu ; John E. Burke ; Constantinos Baskakis ; Christoforos G. Kokotos ; Victoria Magrioti

Journal of Medicinal Chemistry 2010 Volume 53(Issue 9) pp:3602-3610

Publication Date(Web):April 6, 2010

DOI:10.1021/jm901872v

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 octafluoro ketones were also found to be potent inhibitors of GVIA iPLA2; however, they are not selective.

Inflammatory hyperalgesia induces essential bioactive lipid production in the spinal cord

Co-reporter:Matthew W. Buczynski;Camilla I. Svensson;Darren S. Dumlao;Bethany L. Fitzsimmons;Jae-Hang Shim;Thomas J. Scherbart;Faith E. Jacobsen;Xiao-Ying Hua;Tony L. Yaksh

Journal of Neurochemistry 2010 Volume 114( Issue 4) pp:981-993

Publication Date(Web):

DOI:10.1111/j.1471-4159.2010.06815.x

J. Neurochem. (2010) 114, 981–993.

Abstract

Lipid molecules play an important role in regulating the sensitivity of sensory neurons and enhancing pain perception, and growing evidence indicates that the effect occurs both at the site of injury and in the spinal cord. Using high-throughput mass spectrometry methodology, we sought to determine the contribution of spinal bioactive lipid species to inflammation-induced hyperalgesia in rats. Quantitative analysis of CSF and spinal cord tissue for eicosanoids, ethanolamides and fatty acids revealed the presence of 102 distinct lipid species. After induction of peripheral inflammation by intra-plantar injection of carrageenan to the ipsilateral hind paw, lipid changes in cyclooxygenase (COX) and 12-lipoxygenase (12-LOX) signaling pathways peaked at 4 h in the CSF. In contrast, changes occurred in a temporally disparate manner in the spinal cord with LOX-derived hepoxilins followed by COX-derived prostaglandin E₂, and subsequently the ethanolamine anandamide. Systemic treatment with the mu opioid agonist morphine, the COX inhibitor ketorolac, or the LOX inhibitor nordihydroguaiaretic acid significantly reduced tactile allodynia, while their effects on the lipid metabolites were different. Morphine did not alter the lipid profile in the presence or absence of carrageenan inflammation. Ketorolac caused a global reduction in eicosanoid metabolism in naïve animals that remained suppressed following injection of carrageenan. Nordihydroguaiaretic acid-treated animals also displayed reduced basal levels of COX and 12-LOX metabolites, but only 12-LOX metabolites remained decreased after carrageenan treatment. These findings suggest that both COX and 12-LOX play an important role in the induction of carrageenan-mediated hyperalgesia through these pathways.

2-Oxoamide inhibitors of phospholipase A2 activity and cellular arachidonate release based on dipeptides and pseudodipeptides

Co-reporter:Efrosini Barbayianni, Daren Stephens, Andrej Grkovich, Victoria Magrioti, Yuan-Hao Hsu, Panagiotis Dolatzas, Dimitrios Kalogiannidis, Edward A. Dennis, George Kokotos

Bioorganic & Medicinal Chemistry 2009 Volume 17(Issue 13) pp:4833-4843

Publication Date(Web):1 July 2009

DOI:10.1016/j.bmc.2009.03.069

A series of 2-oxoamides based on dipeptides and pseudodipeptides were synthesized and their activities towards two human intracellular phospholipases A2 (GIVA cPLA2 and GVIA iPLA2) and one human secretory phospholipase A2 (GV sPLA2) were evaluated. Derivatives containing a free carboxyl group are selective GIVA cPLA2 inhibitors. A derivative based on the ethyl ester of an ether pseudodipeptide is the first 2-oxoamide, which preferentially inhibits GVIA iPLA2. The effect of 2-oxoamides on the generation of arachidonic acid from RAW 264.7 macrophages was also studied and it was found that selective GIVA cPLA2 inhibitors preferentially inhibited cellular arachidonic acid release; one pseudodipeptide gave an IC50 value of 2 μM.

Phospholipase A2 Biochemistry

Co-reporter:John E. Burke;Edward A. Dennis

Cardiovascular Drugs and Therapy 2009 Volume 23( Issue 1) pp:49-59

Publication Date(Web):2009/02/01

DOI:10.1007/s10557-008-6132-9

The phospholipase A2 (PLA2) superfamily consists of many different groups of enzymes that catalyze the hydrolysis of the sn-2 ester bond in a variety of different phospholipids. The products of this reaction, a free fatty acid, and lysophospholipid have many different important physiological roles. There are five main types of PLA2: the secreted sPLA2’s, the cytosolic cPLA2’s, the Ca2+independent iPLA2’s, the PAF acetylhydrolases, and the lysosomal PLA2’s. This review focuses on the superfamily of PLA2 enzymes, and then uses three specific examples of these enzymes to examine the differing biochemistry of the three main types of these enzymes. These three examples are the GIA cobra venom PLA2, the GIVA cytosolic cPLA2, and the GVIA Ca2+-independent iPLA2.

Lipidomics joins the omics evolution

Co-reporter:Edward A. Dennis

PNAS 2009 106 (7 ) pp:2089-2090

Publication Date(Web):2009-02-17

DOI:10.1073/pnas.0812636106

Synthesis of Polyfluoro Ketones for Selective Inhibition of Human Phospholipase A2 Enzymes

Co-reporter:Constantinos Baskakis ; Victoria Magrioti ; Naomi Cotton ; Daren Stephens ; Violetta Constantinou-Kokotou ; Edward A. Dennis ;George Kokotos

Journal of Medicinal Chemistry 2008 Volume 51(Issue 24) pp:8027-8037

Publication Date(Web):November 24, 2008

DOI:10.1021/jm800649q

The development of selective inhibitors for individual PLA2 enzymes is necessary in order to target PLA2-specific signaling pathways, but it is challenging due to the observed promiscuity of known PLA2 inhibitors. In the current work, we present the development and application of a variety of synthetic routes to produce pentafluoro, tetrafluoro, and trifluoro derivatives of activated carbonyl groups in order to screen for selective inhibitors and characterize the chemical properties that can lead to selective inhibition. Our results demonstrate that the pentafluoroethyl ketone functionality favors selective inhibition of the GVIA iPLA2, a very important enzyme for which specific, potent, reversible inhibitors are needed. We find that 1,1,1,2,2-pentafluoro-7-phenyl-heptan-3-one (FKGK11) is a selective inhibitor of GVIA iPLA2 (XI(50) = 0.0073). Furthermore, we conclude that the introduction of an additional fluorine atom at the α′ position of a trifluoromethyl ketone constitutes an important strategy for the development of new potent GVIA iPLA2 inhibitors.

Structure–activity relationships of natural and non-natural amino acid-based amide and 2-oxoamide inhibitors of human phospholipase A2 enzymes

Co-reporter:Georgia Antonopoulou, Efrosini Barbayianni, Victoria Magrioti, Naomi Cotton, Daren Stephens, Violetta Constantinou-Kokotou, Edward A. Dennis, George Kokotos

Bioorganic & Medicinal Chemistry 2008 Volume 16(Issue 24) pp:10257-10269

Publication Date(Web):15 December 2008

DOI:10.1016/j.bmc.2008.10.046

A variety of 2-oxoamides and related amides based on natural and non-natural amino acids were synthesized. Their activity on two human intracellular phospholipases (GIVA cPLA2 and GVIA iPLA2) and one human secretory phospholipase (GV sPLA2) was evaluated. We show that an amide based on (R)-γ-norleucine is a highly selective inhibitor of GV sPLA2.

Interaction of Group IA Phospholipase A2 with Metal Ions and Phospholipid Vesicles Probed with Deuterium Exchange Mass Spectrometry

Co-reporter:John E. Burke, Mark J. Karbarz, Raymond A. Deems, Sheng Li, Virgil L. Woods Jr. and Edward A. Dennis

Biochemistry 2008 Volume 47(Issue 24) pp:

Publication Date(Web):May 24, 2008

DOI:10.1021/bi8000962

Deuterium exchange mass spectrometric evaluation of the cobra venom (Naja naja naja) group IA phospholipase A2 (GIA PLA2) was carried out in the presence of metal ions Ca2+ and Ba2+ and phospholipid vesicles. Novel conditions for digesting highly disulfide bonded proteins and a methodology for studying protein−lipid interactions using deuterium exchange have been developed. The enzyme exhibits unexpectedly slow rates of exchange in the two large α-helices of residues 43−53 and 89−101, which suggests that these α-helices are highly rigidified by the four disulfide bonds in this region. The binding of Ca2+ or Ba2+ ions decreased the deuterium exchange rates for five regions of the protein (residues 24−27, 29−40, 43−53, 103−110, and 111−114). The magnitude of the changes was the same for both ions with the exception of regions of residues 24−27 and 103−110 which showed greater changes for Ca2+. The crystal structure of the N. naja naja GIA PLA2 contains a single Ca2+ bound in the catalytic site, but the crystal structures of related PLA2s contain a second Ca2+ binding site. The deuterium exchange studies reported here clearly show that in solution the GIA PLA2 does in fact bind two Ca2+ ions. With dimyristoylphosphatidylcholine (DMPC) phospholipid vesicles with 100 μM Ca2+ present at 0 °C, significant areas on the i-face of the enzyme showed decreases in the rate of exchange. These areas included regions of residues 3−8, 18−21, and 56−64 which include Tyr-3, Trp-61, Tyr-63, and Phe-64 proposed to penetrate the membrane surface. These regions also contained Phe-5 and Trp-19, proposed to bind the fatty acyl tails of substrate.

A Macrophage Cell Model for Selective Metalloproteinase Inhibitor Design

Co-reporter:Faith E. Jacobsen ;Matthew W. Buczynski ;Seth M. Cohen

ChemBioChem 2008 Volume 9( Issue 13) pp:2087-2095

Publication Date(Web):

DOI:10.1002/cbic.200800148

Abstract

The desire to inhibit zinc-dependent matrix metalloproteinases (MMPs) has, over the course of the last 30 years, led to the development of a plethora of MMP inhibitors that bind directly to the active-site metal. With one exception, all of these drugs have failed in clinical trials, due to many factors, including an apparent lack of specificity for MMPs. To address the question of whether these inhibitors are selective for MMPs in a biological setting, a cell-based screening method is presented to compare the relative activities of zinc, heme iron, and non-heme iron enzymes in the presence of these compounds using the RAW264.7 macrophage cell line. We screened nine different zinc-binding groups (ZBGs), four established MMP inhibitors (MMPis), and two novel MMP inhibitors developed in our laboratory to determine their selectivities against five different metalloenzymes. Using this model, we identified two nitrogen donor compounds—2,2′-dipyridylamine (DPA) and triazacyclononane (TACN)—as the most selective ZBGs for zinc metalloenzyme inhibitor development. We also demonstrated that the model could predict known nonspecific interactions of some of the most commonly used MMPis, and could also give cross-reactivity information for newly developed MMPis. This work demonstrates the utility of cell-based assays in both the design and the screening of novel metalloenzyme inhibitors.

Spinal phospholipase A2 in inflammatory hyperalgesia: role of Group IVA cPLA2

Co-reporter:Karin Killermann Lucas;Camilla I Svensson;Xiao-Ying Hua;Tony L Yaksh;Edward A Dennis

British Journal of Pharmacology 2005 Volume 144(Issue 7) pp:

Publication Date(Web):29 JAN 2009

DOI:10.1038/sj.bjp.0706116

1
Current work has shown the importance of spinal cyclooxygenase (COX) products in facilitatory processes leading to tissue injury induced hyperalgesia. This cascade must originate with free arachidonic acid (AA) released by the activity of spinal phospholipase A₂'s (PLA₂). In the present work, we studied the role of PLA₂'s in spinal sensitization.
2
We first demonstrate the presence of constitutive mRNA in the spinal cord for PLA₂ Groups IB, IIA, IIC, IVA, V and VI by reverse transcription–polymerase chain reaction (RT–PCR) and sequencing. Using quantitative-PCR, we found that Group IVA cPLA₂ and Group VI iPLA₂ are the predominant PLA₂ messages in the spinal cord. Western blotting and activity assays specific for Group IVA cPLA₂ and Group VI iPLA₂ verified the presence of these enzymes. PLA₂ activity in spinal cord homogenates was suppressed by methyl arachidonyl fluorophosphonate (MAFP) and arachidonyl trifluoromethylketone (AACOCF₃), mixed inhibitors of Group IVA cPLA₂ and Group VI iPLA₂ as well as by bromoenol lactone (BEL), a Group VI iPLA₂ inhibitor. The spinal expression of PLA₂ mRNA or protein was not altered in the face of peripheral inflammation. Secondly, we showed that intrathecal (i.t.) administration of MAFP and AACOCF₃, but not BEL, dose-dependently prevented thermal hyperalgesia induced by intraplantar carrageenan as well as formalin-induced flinching. Finally, i.t. injection of AACOCF₃, at antihyperalgesic doses, decreased the release of prostaglandin E₂ (PGE₂) into spinal dialysate evoked by i.t. NMDA, while i.t. injection of BEL had no effect.
3
Taken together, this work points to a role for constitutive Group IVA cPLA₂ in spinal nociceptive processing.

British Journal of Pharmacology (2005) 144, 940–952. doi:10.1038/sj.bjp.0706116

A comprehensive classification system for lipids

Co-reporter:Eoin Fahy;Shankar Subramaniam;H. Alex Brown;Christopher K. Glass;Alfred H. Merrill Jr.;Robert C. Murphy;Christian R. H. Raetz;David W. Russell;Yousuke Seyama;Walter Shaw;Takao Shimizu;Friedrich Spener;Gerrit van Meer;Michael S. VanNieuwenhze;Stephen H. White;Joseph L. Witztum

European Journal of Lipid Science and Technology 2005 Volume 107(Issue 5) pp:

Publication Date(Web):27 MAY 2005

DOI:10.1002/ejlt.200405001

Lipids are produced, transported, and recognized by the concerted actions of numerous enzymes, binding proteins, and receptors. A comprehensive analysis of lipid molecules, “lipidomics,” in the context of genomics and proteomics is crucial to understanding cellular physiology and pathology; consequently, lipid biology has become a major research target of the postgenomic revolution and systems biology. To facilitate international communication about lipids, a comprehensive classification of lipids with a common platform that is compatible with informatics requirements has been developed to deal with the massive amounts of data that will be generated by our lipid community. As an initial step in this development, we divide lipids into eight categories (fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, and polyketides) containing distinct classes and subclasses of molecules, devise a common manner of representing the chemical structures of individual lipids and their derivatives, and provide a 12 digit identifier for each unique lipid molecule. The lipid classification scheme is chemically based and driven by the distinct hydrophobic and hydrophilic elements that compose the lipid. This structured vocabulary will facilitate the systematization of lipid biology and enable the cataloging of lipids and their properties in a way that is compatible with other macromolecular databases.[Reprinted with copyright permission from the Journal of Lipid Research. 2005. 46: 839–861.]

Lipidomics analysis of essential fatty acids in macrophages

Co-reporter:Oswald Quehenberger, Aaron Armando, Darren Dumlao, Daren L. Stephens, Edward A. Dennis

Prostaglandins, Leukotrienes and Essential Fatty Acids (PLEFA) (September–November 2008) Volume 79(Issues 3–5) pp:123-129

Publication Date(Web):1 September 2008

DOI:10.1016/j.plefa.2008.09.021

The Lipid Metabolites and Pathway Strategy (LIPID MAPS) Consortium is a nationwide initiative that has taken on the task of employing lipidomics to advance our understanding of lipid metabolism at the molecular and mechanistic level in living organisms. An important step toward this goal is to craft enabling analytical procedures to comprehensively measure all lipid species, to establish the precise structural identity of the lipid molecules analyzed, and to generate accurate quantitative information. The LIPID MAPS Consortium has succeeded in the implementation of a complete infrastructure that now provides tools for analysis of the global lipidome in cultured and primary cells. Here we illustrate the advancement of a gas chromatography mass spectrometry (GC/MS) procedure for the analysis of essential fatty acids in RAW 264.7 cells. Our method allows for the specific identification and quantification of over 30 fatty acids present in cells in their free form in a single analytical GC/MS run. Free fatty acids are selectively extracted in the presence of deuterated internal standards, which permit subsequent estimation of extraction efficiencies and quantification with high accuracy. Mass spectrometer conditions were optimized for single-ion monitoring, which provides an extremely sensitive technology to measure fatty acids from biological samples in trace amounts. These methods will be presented in the context of our broader effort to analyze all fatty acids as well as their metabolites in inflammatory cells.

High-throughput lipidomic analysis of fatty acid derived eicosanoids and N-acylethanolamines

Co-reporter:Darren S. Dumlao, Matthew W. Buczynski, Paul C. Norris, Richard Harkewicz, Edward A. Dennis

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids (November 2011) Volume 1811(Issue 11) pp:

Publication Date(Web):November 2011

DOI:10.1016/j.bbalip.2011.06.005

Fatty acid-derived eicosanoids and N-acylethanolamines (NAE) are important bioactive lipid mediators involved in numerous biological processes including cell signaling and disease progression. To facilitate research on these lipid mediators, we have developed a targeted high-throughput mass spectrometric based methodology to monitor and quantitate both eicosanoids and NAEs, and can be analyzed separately or together in series. Each methodology utilizes scheduled multiple reaction monitoring (sMRM) pairs in conjunction with a 25 min reverse-phase HPLC separation. The eicosanoid methodology monitors 141 unique metabolites and quantitative amounts can be determined for over 100 of these metabolites against standards. The analysis covers eicosanoids generated from cycloxygenase, lipoxygenase, cytochrome P450 enzymes, and those generated from non-enzymatic pathways. The NAE analysis monitors 36 metabolites and quantitative amounts can be determined for 33 of these metabolites against standards. The NAE method contains metabolites derived from saturated fatty acids, unsaturated fatty acids, and eicosanoids. The lower limit of detection for eicosanoids ranges from 0.1 pg to 1 pg, while NAEs ranges from 0.1 pg to 1000 pg. The rationale and design of the methodology is discussed. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.Research highlights► We present a high-throughput methodology for eicosanoids and N-acylethanolamines. ► The rationale behind the method design is addressed. ► Quantitation for 100 eicosanoid and 33 N-acylethanolamine species. ► Limits of detections are determined. ► Biological application of the methodology is demonstrated.

High sensitivity quantitative lipidomics analysis of fatty acids in biological samples by gas chromatography–mass spectrometry

Co-reporter:Oswald Quehenberger, Aaron M. Armando, Edward A. Dennis

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids (November 2011) Volume 1811(Issue 11) pp:

Publication Date(Web):November 2011

DOI:10.1016/j.bbalip.2011.07.006

Historically considered to be simple membrane components serving as structural elements and energy storing entities, fatty acids are now increasingly recognized as potent signaling molecules involved in many metabolic processes. Quantitative determination of fatty acids and exploration of fatty acid profiles have become common place in lipid analysis. We present here a reliable and sensitive method for comprehensive analysis of free fatty acids and fatty acid composition of complex lipids in biological material. The separation and quantitation of fatty acids are achieved by capillary gas chromatography. The analytical method uses pentafluorobenzyl bromide derivatization and negative chemical ionization gas chromatography–mass spectrometry. The chromatographic procedure provides base line separation between saturated and unsaturated fatty acids of different chain lengths as well as between most positional isomers. Fatty acids are extracted in the presence of isotope-labeled internal standards for high quantitation accuracy. Mass spectrometer conditions are optimized for broad detection capacity and sensitivity capable of measuring trace amounts of fatty acids in complex biological samples. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.Highlights► We review quantitative analyses of fatty acids in biological material. ► Chromatographic separation of fatty acids is achieved by capillary GC. ► Mass spectrometry using soft ionization techniques enables sensitive detection. ► Isotope dilution method facilitates accurate and precise quantitation.

A lipidomic perspective on inflammatory macrophage eicosanoid signaling

Co-reporter:Paul C. Norris, Edward A. Dennis

Advances in Biological Regulation (January 2014) Volume 54() pp:

Publication Date(Web):1 January 2014

DOI:10.1016/j.jbior.2013.09.009

Macrophages are central to essential physiological processes including the regulation of innate and adaptive immunity, but they are also central to a number of inflammatory disease states. These immune cells also possess remarkable plasticity and display various shades of functionalities based on changes in the surrounding molecular environment. Macrophage biology has defined various phenotypes and roles in inflammation based primarily on cytokine and chemokine profiles of cells in different activation states. Importantly, macrophages are elite producers of eicosanoids and other related lipid mediators during inflammation, but specific roles of these molecules have not generally been incorporated into the larger context of macrophage biology. In this review, we discuss the current classification of macrophage types and their roles in inflammation and disease, along with the practical challenges of studying biologically relevant phenotypes ex vivo. Using the latest advances in eicosanoid lipidomics, we highlight several key studies from our laboratory that provide a comprehensive understanding of how eicosanoid metabolism differs between macrophage phenotypes, along with how this metabolism is altered by changes in membrane fatty acid distribution and varied durations of Toll-like receptor (TLR) priming. In conclusion, we summarize several examples of the benefit of macrophage plasticity to develop accurate cellular mechanisms of lipid metabolism, and insights from lipidomic analyses about the differences in eicosanoid pathway enzyme activity in vitro vs. in cells ex vivo. Examples of new techniques to further understand the role of macrophage eicosanoid signaling in vivo are also discussed.

Phosphatidic acid phosphohydrolase in the regulation of inflammatory signaling

Co-reporter:Andrej Grkovich, Edward A. Dennis

Advances in Enzyme Regulation (2009) Volume 49(Issue 1) pp:

Publication Date(Web):1 January 2009

DOI:10.1016/j.advenzreg.2009.01.005

2-Oxoamides based on dipeptides as selective calcium-independent phospholipase A2 inhibitors

Co-reporter:Anneta Smyrniotou, Maroula G. Kokotou, Varnavas D. Mouchlis, Efrosini Barbayianni, George Kokotos, Edward A. Dennis, Violetta Constantinou-Kokotou

Bioorganic & Medicinal Chemistry (1 February 2017) Volume 25(Issue 3) pp:

Publication Date(Web):1 February 2017

DOI:10.1016/j.bmc.2016.12.007

Calcium-independent phospholipase A2 (GVIA iPLA2) has recently attracted interest as a medicinal target. The number of known GVIA iPLA2 inhibitors is limited to a handful of synthetic compounds (bromoenol lactone and polyfluoroketones). To expand the chemical diversity, a variety of 2-oxoamides based on dipeptides and ether dipeptides were synthesized and studied for their in vitro inhibitory activity on human GVIA iPLA2 and their selectivity over the other major intracellular GIVA cPLA2 and the secreted GV sPLA2. Structure-activity relationship studies revealed the first 2-oxoamide derivative (GK317), which presents potent inhibition of GVIA iPLA2 (XI(50) value of 0.007) and at the same time significant selectivity over GIVA cPLA2 and GV sPLA2.