Pharmacophore-based discovery of a novel cytosolic phospholipase A(2)? inhibitor.
ABSTRACT: The release of arachidonic acid, a precursor in the production of prostaglandins and leukotrienes, is achieved by activity of the cytosolic phospholipase A(2)? (cPLA(2)?). Signaling mediated by this class of bioactive lipids, which are collectively referred to as eicosanoids, has numerous effects in physiological and pathological processes. Herein, we report the development of a ligand-based pharmacophore model and pharmacophore-based virtual screening of the National Cancer Institute (NCI) database, leading to the identification of 4-(hexadecyloxy)-3-(2-(hydroxyimino)-3-oxobutanamido)benzoic acid (NSC 119957) as cPLA(2)? inhibitor in cell-free and cell-based in vitro assays.
Project description:Resident tissue macrophages are activated by the fungal pathogen Candida albicans to release eicosanoids, which are important modulators of inflammation and immune responses. Our objective was to identify the macrophage receptors engaged by C. albicans that mediate activation of group IVA cytosolic phospholipase A(2) (cPLA(2)?), a regulatory enzyme that releases arachidonic acid (AA) for production of prostaglandins and leukotrienes. A comparison of peritoneal macrophages from wild type and knock-out mice demonstrates that the ?-glucan receptor Dectin-1 and MyD88 regulate early release of AA and eicosanoids in response to C. albicans. However, cyclooxygenase 2 (COX2) expression and later phase eicosanoid production are defective in MyD88(-/-) but not Dectin-1(-/-) macrophages. Furthermore, C. albicans-stimulated activation of MAPK and phosphorylation of cPLA(2)? on Ser-505 are regulated by MyD88 and not Dectin-1. In contrast, Dectin-1 mediates MAPK activation, cPLA(2)? phosphorylation, and COX2 expression in response to particulate ?-glucan suggesting that other receptors engaged by C. albicans preferentially mediate these responses. Results also implicate the mannan-binding receptor Dectin-2 in regulating cPLA(2)?. C. albicans-stimulated MAPK activation and AA release are blocked by d-mannose and Dectin-2-specific antibody, and overexpression of Dectin-2 in RAW264.7 macrophages enhances C. albicans-stimulated MAPK activation, AA release, and COX2 expression. In addition, calcium mobilization is enhanced in RAW264.7 macrophages overexpressing Dectin-1 or -2. The results demonstrate that C. albicans engages both ?-glucan and mannan-binding receptors on macrophages that act with MyD88 to regulate the activation of cPLA(2)? and eicosanoid production.
Project description:Cytosolic phospholipase A(2)? (cPLA(2)?) is a rate-limiting key enzyme controlling the release of arachidonic acid (AA) substrate for the synthesis of prostaglandins and leukotrienes. This study was designed to explore the role of hepatocyte cPLA(2)? in Fas-mediated liver injury, in vivo.Transgenic mice with targeted expression of cPLA(2)? under control of the albumin-promoter enhancer and wild-type mice were injected intraperitoneally with anti-Fas antibody Jo2 or lipopolysaccharide plus d-galactosamine and monitored for liver injury and survival at various time points.The cPLA(2)? Tg mice resist Fas-induced liver failure, as reflected by the lower serum transaminase levels, fewer apoptotic hepatocytes, reduced caspase activation, and reduced PARP cleavage when compared to the matched wild type mice. Inhibition of cPLA(2)? by its pharmacological inhibitor, pyrrolidine, enhanced Jo2-induced liver injury in both cPLA(2)? Tg and wild type mice. Hepatic overexpression of cPLA(2)? increases the expression of EGFR in the liver and the EGFR inhibitor, AG1478, exacerbated Jo2-mediated liver injury. The cPLA(2)? transgenic mice develop more prominent liver tissue damage than wild-type mice after LPS/d-galactosamine injection.Hepatocyte cPLA(2)? protects against Fas-induced liver injury and this effect is mediated at least in part through the upregulation of EGFR.
Project description:Phospholipase A(2) enzymes hydrolyze phospholipids to liberate arachidonic acid for the biosynthesis of prostaglandins and leukotrienes. In the vascular endothelium, group IV phospholipase A(2)? (cPLA(2)?) enzyme activity is regulated by reversible association with the Golgi apparatus. Here we provide evidence for a plasma membrane cell adhesion complex that regulates endothelial cell confluence and simultaneously controls cPLA(2)? localization and enzymatic activity. Confluent endothelial cells display pronounced accumulation of vascular endothelial cadherin (VE-cadherin) at cell-cell junctions, and mechanical wounding of the monolayer stimulates VE-cadherin complex disassembly and cPLA(2)? release from the Golgi apparatus. VE-cadherin depletion inhibits both recruitment of cPLA(2)? to the Golgi and formation of tubules by endothelial cells. Perturbing VE-cadherin and increasing the soluble cPLA(2)? fraction also stimulated arachidonic acid and prostaglandin production. Of importance, reverse genetics shows that ?-catenin and ?-catenin, but not ?-catenin, regulates cPLA(2)? Golgi localization linked to cell confluence. Furthermore, cPLA(2)? Golgi localization also required partitioning defective protein 3 (PAR3) and annexin A1. Disruption of F-actin internalizes VE-cadherin and releases cPLA(2)? from the adhesion complex and Golgi apparatus. Finally, depletion of either PAR3 or ?-catenin promotes cPLA(2)?-dependent endothelial tubule formation. Thus a VE-cadherin-PAR3-?-catenin adhesion complex regulates cPLA(2)? recruitment to the Golgi apparatus, with functional consequences for vascular physiology.
Project description:Group IVA cytosolic phospholipase A(2) (cPLA(2)?) catalyzes the first step in the arachidonic acid cascade leading to the synthesis of important lipid mediators, the prostaglandins and leukotrienes. We previously described a patient deficient in cPLA(2)? activity, which was associated with mutations in both alleles encoding the enzyme. In this paper, we describe the biochemical characterization of each of these mutations. Using saturating concentrations of calcium, we showed that the R485H mutant was nearly devoid of any catalytic activity, that the S111P mutation did not affect the enzyme activity, and that the known K651R polymorphism was associated with activity slightly higher than that of the wild type. Using MDCK cells, we showed that translocation to the Golgi in response to serum activation was impaired for the S111P mutant but not for the other mutants. Using immortalized mouse lung fibroblasts lacking endogenous cPLA(2)? activity, we showed that both mutations S111P and R485H/K651R caused a profound defect in the enzyme catalytic activity in response to cell stimulation with serum. Taken together, our results show that the S111P mutation hampers calcium binding and membrane translocation without affecting the catalytic activity, and that the mutation R485H does not affect membrane translocation but blocks catalytic activity that leads to inactivation of the enzyme. Interestingly, our results show that the common K651R polymorphism confers slightly higher activity to the enzyme, suggesting a role of this residue in favoring a catalytically active conformation of cPLA(2)?. Our results define how the mutations negatively influence cPLA(2)? function and explain the inability of the proband to release arachidonic acid for eicosanoid production.
Project description:Proinflammatory eicosanoids (prostaglandins and leukotrienes) and specialized pro-resolving mediators (SPM) are temporally regulated during infections. Here we show that human macrophage phenotypes biosynthesize unique lipid mediator signatures when exposed to pathogenic bacteria. E. coli and S. aureus each stimulate predominantly proinflammatory 5-lipoxygenase (LOX) and cyclooxygenase pathways (i.e., leukotriene B4 and prostaglandin E2) in M1 macrophages. These pathogens stimulate M2 macrophages to produce SPMs including resolvin D2 (RvD2), RvD5, and maresin-1. E. coli activates M2 macrophages to translocate 5-LOX and 15-LOX-1 to different subcellular locales in a Ca2+-dependent manner. Neither attenuated nor non-pathogenic E. coli mobilize Ca2+ or activate LOXs, rather these bacteria stimulate prostaglandin production. RvD5 is more potent than leukotriene B4 at enhancing macrophage phagocytosis. These results indicate that M1 and M2 macrophages respond to pathogenic bacteria differently, producing either leukotrienes or resolvins that further distinguish inflammatory or pro-resolving phenotypes.
Project description:Lipid mediators including classical arachidonic acid-derived eicosanoids (e.g. prostaglandins and leukotrienes) and more recently identified specialized pro-resolving-mediator metabolites of the omega-3 fatty acids play essential roles in initiation, self-limitation, and active resolution of acute inflammatory responses. In this study, we examined the bioactive lipid mediator profile of human skeletal muscle at rest and following acute resistance exercise. Twelve male subjects completed a single bout of maximal isokinetic unilateral knee extension exercise and muscle biopsies were taken from the m.vastus lateralis before and at 2, 4, and 24 h of recovery. Muscle tissue lipid mediator profile was analyzed via liquid chromatography-mass spectrometry (LC-MS)-based targeted lipidomics. At 2 h postexercise, there was an increased intramuscular abundance of cyclooxygenase (COX)-derived thromboxanes (TXB<sub>2</sub> : 3.33 fold) and prostaglandins (PGE<sub>2</sub> : 2.52 fold and PGF<sub>2?</sub> : 1.77 fold). Resistance exercise also transiently increased muscle concentrations of lipoxygenase (LOX) pathway-derived leukotrienes (12-Oxo LTB<sub>4</sub> : 1.49 fold and 20-COOH LTB<sub>4</sub> : 2.91 fold), monohydroxy-eicosatetraenoic acids (5-HETE: 2.66 fold, 12-HETE: 2.83 fold, and 15-HETE: 1.69 fold) and monohydroxy-docosahexaenoic acids (4-HDoHE: 1.69 fold, 7-HDoHE: 1.58 fold and 14-HDoHE: 2.35 fold). Furthermore, the abundance of CYP pathway-derived epoxy- and dihydroxy-eicosatrienoic acids was increased in 2 h postexercise biopsies (5,6-EpETrE: 2.48 fold, 11,12-DiHETrE: 1.66 fold and 14,15-DiHETrE: 2.23 fold). These data reveal a range of bioactive lipid mediators as present within human skeletal muscle tissue and demonstrate that acute resistance exercise transiently stimulates the local production of both proinflammatory eicosanoids and pathway markers in specialized proresolving mediator biosynthesis circuits.
Project description:Neuroinflammation is involved in various central nervous system (CNS) disorders, including brain infections, ischemia, trauma, stroke, and degenerative CNS diseases. In the CNS inflammation, secretory phospholipase A?-IIA (sPLA?-IIA) acts as a mediator, resulting in the generation of the precursors of pro-inflammatory lipid mediators, such as prostaglandins (PGs) and leukotrienes (LTs). However, the role of sPLA?-IIA in neuroinflammation is more complicated and remains unclear yet. In the present study, we investigated the effect of sPLA?-IIA inhibition by specific inhibitor SC-215 on the inflammation in LPS-induced mice cerebral cortex and primary astrocytes. Our results showed that the inhibition of sPLA?-IIA alleviated the release of PGE? by suppressing the activation of ERK1/2, cPLA??, COX-2 and mPGES-1. These findings demonstrated that sPLA?-IIA showed the potential to regulate the neuroinflammation in vivo and in vitro, indicating that sPLA?-IIA might be a novel target for the treatment of acute neuroinflammation.
Project description:Mast cells play an important role in both innate and acquired immunity as well as several pathological conditions including allergy, arthritis and neoplasia. They influence these processes by producing a variety of mediators including cytokines, chemokines and eicosanoids. Very little is currently known about the spectrum of inflammatory mediators, particularly eicosanoids (prostaglandins and leukotrienes), produced by canine mast cells. This is important since modulating mast cell derived eicosanoids may help in the treatment of autoimmune and inflammatory disorders. The purpose of this study was to investigate the spectrum of eicosanoids produced by normal canine mast cells and to evaluate the effects of cytokines and non-steroidal anti-inflammatory mediators (NSAIDS) on eicosanoid production and release. Canine bone marrow derived cultured mast cells (cBMCMCs) expressed COX-1, COX-2, and 5-LOX and synthesized and released PGD2, PGE2, LTB4, and LTC4 following activation by a variety of stimuli. The selective COX-2 NSAIDs carprofen (Rimadyl) and deracoxib (Deramaxx) inhibited PGD2 and PGE2 production but only slightly inhibited LTB4 and LTC4. The mixed COX-1/COX-2 inhibitor piroxicam blocked PGD2 and PGE2 production, but upregulated LTC4 following treatment while tepoxilan (Zubrin), a pan COX/LOX inhibitor, markedly reduced the production of all eicosanoids. The LOX inhibitor nordihydroguaiaretic acid (NDGA) prevented LTB4/LTC4 release and BMBMC degranulation. Pre-incubation of cBMCMCs with IL-4 and SCF sensitized these cells to degranulation in response to substance P. In conclusion, canine BMCMCs produce an array of eicosanoids similar to those produced by mast cells from other species. Tepoxilan appeared to be the most effective NSAID for blocking eicosanoid production and thus may be useful for modulating mast cell mediated responses in dogs.
Project description:Detection of microbial products by host inflammasomes is an important mechanism of innate immune surveillance. Inflammasomes activate the caspase-1 (CASP1) protease, which processes the cytokines interleukin (IL)-1? and IL-18, and initiates a lytic host cell death called pyroptosis. To identify novel CASP1 functions in vivo, we devised a strategy for cytosolic delivery of bacterial flagellin, a specific ligand for the NAIP5 (NLR family, apoptosis inhibitory protein 5)/NLRC4 (NLR family, CARD-domain-containing 4) inflammasome. Here we show that systemic inflammasome activation by flagellin leads to a loss of vascular fluid into the intestine and peritoneal cavity, resulting in rapid (less than 30?min) death in mice. This unexpected response depends on the inflammasome components NAIP5, NLRC4 and CASP1, but is independent of the production of IL-1? or IL-18. Instead, inflammasome activation results, within minutes, in an 'eicosanoid storm'--a pathological release of signalling lipids, including prostaglandins and leukotrienes, that rapidly initiate inflammation and vascular fluid loss. Mice deficient in cyclooxygenase-1, a critical enzyme in prostaglandin biosynthesis, are resistant to these rapid pathological effects of systemic inflammasome activation by either flagellin or anthrax lethal toxin. Inflammasome-dependent biosynthesis of eicosanoids is mediated by the activation of cytosolic phospholipase?A(2) in resident peritoneal macrophages, which are specifically primed for the production of eicosanoids by high expression of eicosanoid biosynthetic enzymes. Our results therefore identify eicosanoids as a previously unrecognized cell-type-specific signalling output of the inflammasome with marked physiological consequences in vivo.
Project description:ideR, an essential gene of Mycobacterium tuberculosis, is an attractive drug target as its conditional knockout displayed attenuated growth phenotype in vitro and in vivo. To the best of our knowledge, no inhibitors of IdeR are identified. We carried out virtual screening of NCI database against the IdeR DNA binding domain followed by inhibition studies using EMSA. Nine compounds exhibited potent inhibition with NSC 281033 (I-20) and NSC 12453 (I-42) exhibiting IC50 values of 2?µg/ml and 1?µg/ml, respectively. We then attempted to optimize the leads firstly by structure based similarity search resulting in a class of inhibitors based on I-42 containing benzene sulfonic acid, 4-hydroxy-3-[(2-hydroxy-1-naphthalenyl) azo] scaffold with 4 molecules exhibiting IC50???10?µg/ml. Secondly, optimization included development of energy based pharmacophore and screening of ZINC database followed by docking studies, yielding a molecule with IC50 of 60?µg/ml. More importantly, a five-point pharmacophore model provided insight into the features essential for IdeR inhibition. Five molecules with promising IC50 values also inhibited M. tuberculosis growth in broth culture with MIC90 ranging from 17.5?µg/ml to 100?µg/ml and negligible cytotoxicity in various cell lines. We believe our work opens up avenues for further optimization studies.