BACKGROUND AND PURPOSEw3-polyunsaturated fatty acids (w3-PUFAs) are known to exert anti-inflammatory effects in various disease models although their direct targets are only poorly characterized. EXPERIMENTAL APPROACHHere we report on two new cPLA2 inhibitors, the w3-derivatives AVX001 and AVX002, and their effects on inflammatory PGE2 production in cultures of renal mesangial cells. KEY RESULTSAVX001 and AVX002 dose-dependently inhibited the group IVA cytosolic phospholipase A2 (cPLA2) in an in vitro activity assay with similar IC50 values for AVX001 and AVX002, whereas the known cPLA2 inhibitor AACOCF3 was less potent and docosahexaenoic acid (DHA) was inactive. In renal mesangial cells, AVX001 and AVX002 suppressed IL-1b-induced PGE2 synthesis. Mechanistically, this effect occurred by a down-regulation of IL-1b-induced group IIA-sPLA2 protein expression, mRNA expression and promoter activity. A similar but less potent effect was seen with AACOCF3 and no effect was seen with DHA. As gene expression of sPLA2 is known to be regulated by the transcription factor NF-kB, we further investigated NF-kB activation. Both compounds prevented NF-kB activation by blocking degradation of the inhibitor of kB. CONCLUSIONS AND IMPLICATIONSThese data show for the first time that the novel cPLA2 inhibitors AVX001 and AVX002 exert an anti-inflammatory effect in cultures of renal mesangial cells and reduce the pro-inflammatory mediator PGE2 through an inhibitory effect on NF-kB activation. Therefore, these compounds may represent promising novel drugs for the treatment of inflammatory disorders. AbbreviationsAACOCF3, ATK, arachidonyl-trifluoromethyl ketone; AVX001, 1-octadeca-2,6,9,12,15-pentaenylsulfanyl-propan-2-one; AVX002, 1-octadeca-3,6,9,12,15-pentaenylsulfanyl-propan-2-one; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; IkB, inhibitor of kB; MAFP, methyl-arachidonyl fluorophosphonate; PAF-AH, PAF acetylhydrolase; PUFA, polyunsaturated fatty acid; RT-PCR, reverse transcriptase-PCR BJP British Journal of Pharmacology DOI:10.1111DOI:10. /j.1476DOI:10. -5381.2012 British Journal of Pharmacology (2012) IntroductionMesangial cells are specialized smooth muscle-like cells located in the renal glomerulus and are not only involved in the regulation of the glomerular filtration rate and in the preservation of the structural integrity of the glomerulus, but also play a central role in most pathological processes of the renal glomerulus (Kashgarian and Sterzel, 1992;Pfeilschifter, 1994;Gómez-Guerrero et al., 2005). Upon activation by a variety of pro-inflammatory cytokines, mesangial cells respond with three prominent reactions which are all hallmarks of many forms of glomerulonephritis: (i) increased proliferation; (ii) increased mediator production, including cytokines, chemokines, NO, superoxide radicals and PGs; and (iii) increased extracellular matrix production (Kashgarian and Sterzel, 1992;Pfeilschifter, 1994). The detailed mechanisms underlying these cellular responses are still not completely understood. One importan...

Group IVA cytosolic phospholipase A2 (GIVA cPLA2) is the rate-limiting provider of pro-inflammatory mediators in many tissues and is thus an attractive target for the development of novel anti-inflammatory agents. In this work, we present the synthesis of new thiazolyl ketones and the study of their activities in vitro, in cells, and in vivo. Within this series of compounds, methyl 2-(2-(4-octylphenoxy)acetyl)thiazole-4-carboxylate (GK470) was found to be the most potent inhibitor of GIVA cPLA2, exhibiting an XI(50) value of 0.011 mole fraction in a mixed micelle assay and an IC50 of 300 nM in a vesicle assay. In a cellular assay using SW982 fibroblast-like synoviocytes, it suppressed the release of arachidonic acid with an IC50 value of 0.6 μM. In a prophylactic collagen-induced arthritis model, it exhibited an anti-inflammatory effect comparable to the reference drug methotrexate, whereas in a therapeutic model, it showed results comparable to those of the reference drug Enbrel. In both models, it significantly reduced plasma PGE2 levels.

BackgroundGroup IVA cytosolic phospholipase A2 (cPLA2α) plays an important role in tumorigenesis and angiogenesis. It is overexpressed in basal-like breast cancer (BLBC), which is aggressive and usually triple-negative, making it unresponsive to current targeted therapies. Here, we evaluated the anti-angiogenic effects of a specific cPLA2α inhibitor, AVX235, in a patient-derived triple-negative BLBC model.MethodsMice bearing orthotopic xenografts received i.p. injections of AVX235 or DMSO vehicle daily for 1 week and then every other day for up to 19 days. Six treated and six control mice were terminated after 2 days of treatment, and the tumors excised for high resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS) and prostaglandin E2 (PGE2) enzyme immunoassay (EIA) analysis. A 1-week imaging study was performed on a separate cohort of mice. Longitudinal dynamic contrast enhanced (DCE)-MRI was performed before, after 4 days, and after 1 week of treatment. The mice were then perfused with a radiopaque vascular casting agent, and the tumors excised for micro-CT angiography. Subsequently, tumors were sectioned and stained with lectin and for Ki67 or α-smooth muscle actin to quantify endothelial cell proliferation and vessel maturity, respectively. Partial least squares discriminant analysis was performed on the multivariate HR MAS MRS data, and non-parametric univariate analyses using Mann–Whitney U tests (α = 0.05) were performed on all other data.ResultsGlycerophosphocholine and PGE2 levels, measured by HR MAS MRS and EIA, respectively, were lower in treated tumors after 2 days of treatment. These molecular changes are expected downstream effects of cPLA2α inhibition and were followed by significant tumor growth inhibition after 8 days of treatment. DCE-MRI revealed that AVX235 treatment caused a decrease in tumor perfusion. Concordantly, micro-CT angiography showed that vessel volume fraction, density, and caliber were reduced in treated tumors. Moreover, histology showed decreased endothelial cell proliferation and fewer immature vessels in treated tumors.ConclusionsThese results demonstrate that cPLA2α inhibition with AVX235 resulted in decreased vascularization and perfusion and subsequent inhibition of tumor growth. Thus, cPLA2α inhibition may be a potential new therapeutic option for triple-negative basal-like breast cancer.

IntroductionRheumatoid arthritis (RA) is an inflammatory disease of the joint characterized by chronic synovitis causing pain, swelling and loss of function due to destruction of cartilage and bone. The complex series of pathological events occurring in RA is largely regulated via excessive production of pro-inflammatory cytokines, the most prominent being tumor necrosis factor (TNF). The objective of this work was to elucidate possible involvement of group IVA cytosolic phospholipase A2 (cPLA2α) in TNF-induced regulation of synovitis and joint destructive effectors in RA, to evaluate the potential of cPLA2α as a future therapeutic target.MethodsThe involvement of cPLA2α in tumor necrosis factor (TNF)-induced intracellular signaling cascades in synoviocytes (synovial fibroblast-like cells) was analyzed by arachidonic acid (AA) release assay, synoviocyte enzyme activity assay, gene expression analysis by real-time PCR and ELISA immunoassay for the detection of prostaglandin E2 (PGE2), interleukin 8 (IL8) and stromelysin-1 (MMP3), respectively. ResultsInhibitors of cPLA2α enzyme activity (AVX002, ATK) significantly reduced TNF-induced cellular release of AA, PGE2, IL8 and MMP3. This reduction was evident both at transcriptional, protein or metabolite levels. Interestingly, cPLA2α inhibition affected several key points of the arachidonyl cascade; AA-release, cyclooxygenase-2 (COX2) expression and PGE2 production. Furthermore, the results suggest that cPLA2α is subject to transcriptional auto-regulation as inhibition of cPLA2α resulted in reduced PLA2G4A gene expression in TNF-stimulated synoviocytes. ConclusionscPLA2α appears to be an important regulator of central effectors of inflammation and joint destruction, namely MMP3, IL8, COX2, and PGE2. Decreased transcription of the PLA2G4A and COX2 genes in response to cPLA2α enzyme inhibition further suggest a self-reinforcing effect of cPLA2α inhibition in response to TNF. Collectively, these results support that cPLA2α is an attractive therapeutic target candidate as its inhibition reduces the production of multiple key pro-inflammatory factors involved in RA pathogenesis.

As a regulator of cellular inflammation and proliferation, cytosolic phospholipase A2 α (cPLA2α) is a promising therapeutic target for psoriasis; indeed, the cPLA2α inhibitor AVX001 has shown efficacy against plaque psoriasis in a phase I/IIa clinical trial. To improve our understanding of the anti-psoriatic properties of AVX001, we sought to determine how the compound modulates inflammation and keratinocyte hyperproliferation, key characteristics of the psoriatic epidermis. We measured eicosanoid release from human peripheral blood mononuclear cells (PBMC) and immortalized keratinocytes (HaCaT) and studied proliferation in HaCaT grown as monolayers and stratified cultures. We demonstrated that inhibition of cPLA2α using AVX001 produced a balanced reduction of prostaglandins and leukotrienes; significantly limited prostaglandin E2 (PGE2) release from both PBMC and HaCaT in response to pro-inflammatory stimuli; attenuated growth factor-induced arachidonic acid and PGE2 release from HaCaT; and inhibited keratinocyte proliferation in the absence and presence of exogenous growth factors, as well as in stratified cultures. These data suggest that the anti-psoriatic properties of AVX001 could result from a combination of anti-inflammatory and anti-proliferative effects, probably due to reduced local eicosanoid availability.