Targeting endocannabinoid signaling: FAAH and MAG lipase inhibitors

Inspired by the medicinal properties of the plant Cannabis sativa and its principal component (−)-trans-Δ9-tetrahydrocannabinol (THC), researchers have developed a variety of compounds to modulate the endocannabinoid system in the human brain. Inhibitors of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), which are the enzymes responsible for the inactivation of the endogenous cannabinoids anandamide and 2-arachidonoylglycerol, respectively, may exert therapeutic effects without inducing the adverse side effects associated with direct cannabinoid CB1 receptor stimulation by THC. Here we review the FAAH and MAGL inhibitors that have reached clinical trials, discuss potential caveats, and provide an outlook on where the field is headed.NWOMolecular Physiolog

MODELING OF A METHANE FUELLED DIRECT CARBON FUEL CELL SYSTEM

ABSTRACT Energy conversion today is subject to high thermodynamic losses. About 50 to 90 % of the exergy of primary fuels is lost during conversion into power or heat. The fast increasing world energy demand makes a further increase of conversion efficiencies inevitable. The substantial thermodynamic losses (exergy losses of 20 to 30 %) of thermal fuel conversion will limit future improvements of power plant efficiencies. Electrochemical conversion of fuel enables fuel conversion with minimum losses. Various fuel cell systems have been investigated at the Delft University of Technology during the past twenty years. It appeared that exergy analyses can be very helpful in understanding the extent and causes of thermodynamic losses in fuel cell systems. More than 50 % of the losses in high temperature fuel cell (MCFC and SOFC) systems can be caused by heat transfer. Therefore system optimisation must focus on reducing the need for heat transfer as well as improving the conditions for the unavoidable heat transfer. Various options for reducing the need for heat transfer are discussed in this paper. High temperature fuel cells, eventually integrated into gas turbine processes, can replace the combustion process in future power plants. High temperature fuel cells will be necessary to obtain conversion efficiencies up to 80 % in case of large scale electricity production in the future. The introduction of fuel cells is considered to be a first step in the integration of electrochemical conversion in future energy conversion systems. Keywords: Fuel cell systems; Exergy analysis; Thermodynamic analysis; System modelling; Cycle Tempo; PEMFC; MCFC; SOFC; Applications INTRODUCTION Today our world strongly depends on the availability of energy for almost all of their activities. Total energy demand is growing fast, in particular due to the development of the large Asian countries. To ensure our future energy supply the losses of energy conversion have to be reduced and the utilisation of available sources, in particular renewable sources, should be stimulated. Conversion of primary fuels into electricity, power, heat or secondary fuels is necessary to fulfil our energy demands. These conversions are involved with substantial thermodynamic losses. These losses should be presented as exergy losses to get a true representation of the thermodynamic performance of conversion systems. Exergy is the potential to obtain work from an amount of energy or from an energy flow; exergy values do represent a true yardstick for all relevant thermodynamic characteristics Exergy efficiencies are some percentage points lower than thermal efficiencies (based on the lower heating value of the fuel) in case of the conversion of primary energy into power. As thermal efficiencies of less than 50 % are normal practice exergy losses will on average be higher than 50 %. Thermal efficiencies are in general high (around 90 % or more) in case of heat generating systems, but exergy efficiencies are much lower depending on the temperature level at which i

Direct and two-step bioorthogonal probes for Bruton's tyrosine kinase based on ibrutinib: a comparative study

Molecular Physiolog

Inhibition of diacylglycerol lipase beta modulates lipid and endocannabinoid levels in the ex vivo human placenta

IntroductionLipids and fatty acids are key components in metabolic processes of the human placenta, thereby contributing to the development of the fetus. Placental dyslipidemia and aberrant activity of lipases have been linked to diverse pregnancy associated complications, such as preeclampsia and preterm birth. The serine hydrolases, diacylglycerol lipase alpha and beta (DAGL alpha, DAGL beta) catalyze the degradation of diacylglycerols, leading to the formation of monoacylglycerols (MAG), including one main endocannabinoid 2-arachidonoylglycerol (2-AG). The major role of DAGL in the biosynthesis of 2-AG is evident from various studies in mice but has not been investigated in the human placenta. Here, we report the use of the small molecule inhibitor DH376, in combination with the ex vivo placental perfusion system, activity-based protein profiling (ABPP) and lipidomics, to determine the impact of acute DAGL inhibition on placental lipid networks. MethodsDAGL alpha and DAGL beta mRNA expression was detected by RT-qPCR and in situ hybridization in term placentas. Immunohistochemistry staining for CK7, CD163 and VWF was applied to localize DAGL beta transcripts to different cell types of the placenta. DAGL beta activity was determined by in- gel and MS-based activity-based protein profiling (ABPP) and validated by addition of the enzyme inhibitors LEI-105 and DH376. Enzyme kinetics were measured by EnzChek (TM) lipase substrate assay. Ex vivo placental perfusion experiments were performed +/- DH376 [1 mu M] and changes in tissue lipid and fatty acid profiles were measured by LC-MS. Additionally, free fatty acid levels of the maternal and fetal circulations were determined. ResultsWe demonstrate that mRNA expression of DAGL beta prevails in placental tissue, compared to DAGL alpha (p <= 0.0001) and that DAGL beta is mainly located to CK7 positive trophoblasts (p <= 0.0001). Although few DAGL alpha transcripts were identified, no active enzyme was detected applying in-gel or MS-based ABPP, which underlined that DAGL beta is the principal DAGL in the placenta. DAGL beta dependent substrate hydrolysis in placental membrane lysates was determined by the application of LEI-105 and DH376. Ex vivo pharmacological inhibition of DAGL beta by DH376 led to reduced MAG tissue levels (p <= 0.01), including 2-AG (p <= 0.0001). We further provide an activity landscape of serine hydrolases, showing a broad spectrum of metabolically active enzymes in the human placenta. DiscussionOur results emphasize the role of DAGL beta activity in the human placenta by determining the biosynthesis of 2-AG. Thus, this study highlights the special importance of intra-cellular lipases in lipid network regulation. Together, the activity of these specific enzymes may contribute to the lipid signaling at the maternal-fetal interface, with implications for function of the placenta in normal and compromised pregnancies.Molecular Physiolog

Clickable Vitamins as a New Tool to Track Vitamin A and Retinoic Acid in Immune Cells.

The vitamin A derivative, retinoid acid (RA) is key player in guiding adaptive mucosal immune responses. However, data on the uptake and metabolism of vitamin A within human immune cells has remained largely elusive because retinoids are small, lipophilic molecules which are difficult to detect. To overcome this problem and to be able to study the effect of vitamin A metabolism in human immune cell subsets, we have synthesized novel bio-orthogonal retinoid-based probes (clickable probes), which are structurally and functionally indistinguishable from vitamin A. The probes contain a functional group (an alkyne) to conjugate to a fluorogenic dye to monitor retinoid molecules in real-time in immune cells. We demonstrate, by using flow cytometry and microscopy, that multiple immune cells have the capacity to internalize retinoids to varying degrees, including human monocyte-derived dendritic cells (DCs) and naïve B lymphocytes. We observed that naïve B cells lack the enzymatic machinery to produce RA, but use exogenous retinoic acid to enhance CD38 expression. Furthermore, we showed that human DCs metabolize retinal into retinoic acid, which in co-culture with naïve B cells led to of the induction of CD38 expression. These data demonstrate that in humans, DCs can serve as an exogenous source of RA for naïve B cells. Taken together, through the use of clickable vitamins our data provide valuable insight in the mechanism of vitamin A metabolism and its importance for human adaptive immunity.Molecular Physiolog

Discovery of Novel Small Molecule Activators of β-Catenin Signaling

Wnt/β-catenin signaling plays a major role in embryonic development and adult stem cell maintenance. Reduced activation of the Wnt/β-catenin pathway underlies neurodegenerative disorders and aberrations in bone formation. Screening of a small molecule compound library with a β-galactosidase fragment complementation assay measuring β-catenin nuclear entry revealed bona fide activators of β-catenin signaling. The compounds stabilized cytoplasmic β-catenin and activated β–catenin-dependent reporter gene activity. Although the mechanism through which the compounds activate β-catenin signaling has yet to be determined, several key regulators of Wnt/β-catenin signaling, including glycogen synthase kinase 3 and Frizzled receptors, were excluded as the molecular target. The compounds displayed remarkable selectivity, as they only induced β-catenin signaling in a human osteosarcoma U2OS cell line and not in a variety of other cell lines examined. Our data indicate that differences in cellular Wnt/β-catenin signaling machinery can be exploited to identify cell type-specific activators of Wnt/β-catenin signaling

Structure–Activity Relationship Studies of α-Ketoamides as Inhibitors of the Phospholipase A and Acyltransferase Enzyme Family

The phospholipase A and acyltransferase (PLAAT) family of cysteine hydrolases consists of five members, which are involved in the Ca2+-independent production of N-acylphosphatidylethanolamines (NAPEs). NAPEs are lipid precursors for bioactive N-acylethanolamines (NAEs) that are involved in various physiological processes such as food intake, pain, inflammation, stress, and anxiety. Recently, we identified alpha-ketoamides as the first pan-active PLAAT inhibitor scaffold that reduced arachidonic acid levels in PLAAT3-overexpressing U2OS cells and in HepG2 cells. Here, we report the structure-activity relationships of the alpha-ketoamide series using activity-based protein profiling. This led to the identification of LEI-301, a nanomolar potent inhibitor for the PLAAT family members. LEI-301 reduced the NAE levels, including anandamide, in cells overexpressing PLAAT2 or PLAAT5. Collectively, LEI-301 may help to dissect the physiological role of the PLAATs

Identification of α,β-Hydrolase Domain Containing Protein 6 as a Diacylglycerol Lipase in Neuro-2a Cells

The endocannabinoid 2-arachidonoylglycerol (2-AG) is involved in neuronal differentiation. This study aimed to identify the biosynthetic enzymes responsible for 2-AG production during retinoic acid (RA)-induced neurite outgrowth of Neuro-2a cells. First, we confirmed that RA stimulation of Neuro-2a cells increases 2-AG production and neurite outgrowth. The diacylglycerol lipase (DAGL) inhibitor DH376 blocked 2-AG production and reduced neuronal differentiation. Surprisingly, CRISPR/Cas9-mediated knockdown of DAGLα and DAGLβ in Neuro-2a cells did not reduce 2-AG levels, suggesting another enzyme capable of producing 2-AG in this cell line. Chemical proteomics revealed DAGLβ and α,β-hydrolase domain containing protein (ABHD6) as the only targets of DH376 in Neuro-2a cells. Biochemical, genetic and lipidomic studies demonstrated that ABHD6 possesses DAGL activity in conjunction with its previously reported monoacylglycerol lipase activity. RA treatment of Neuro-2a cells increased by three-fold the amount of active ABHD6. Our study shows that ABHD6 exhibits significant DAG lipase activity in Neuro-2a cells in addition to its known MAG lipase activity and suggest it is involved in neuronal differentiation.Analytical BioScience

Comparative photoaffinity profiling of omega-3 signaling lipid probes reveals prostaglandin reductase 1 as a metabolic hub in human macrophages

The fish oil constituent docosahexaenoic acid (DHA, 22:6 n-3) is a signaling lipid with anti-inflammatory properties. The molecular mechanisms underlying the biological effect of DHA are poorly understood. Here, we report the design, synthesis, and application of a complementary pair of bio-orthogonal, photoreactive probes based on the polyunsaturated scaffold DHA and its oxidative metabolite 17-hydroxydocosahexaenoic acid (17-HDHA). In these probes, an alkyne serves as a handle to introduce a fluorescent reporter group or a biotin-affinity tag via copper(I)-catalyzed azide-alkyne cycloaddition. This pair of chemical probes was used to map specific targets of the omega-3 signaling lipids in primary human macrophages. Prostaglandin reductase 1 (PTGR1) was identified as an interaction partner that metabolizes 17-oxo-DHA, an oxidative metabolite of 17-HDHA. 17-oxo-DHA reduced the formation of pro-inflammatory lipids 5-HETE and LTB4 in human macrophages and neutrophils. Our results demonstrate the potential of comparative photoaffinity protein profiling for the discovery of metabolic enzymes of bioactive lipids and highlight the power of chemical proteomics to uncover new biological insights.Bio-organic SynthesisMolecular Physiolog

Cannabinoid type 1 receptor inverse agonism attenuates dyslipidemia and atherosclerosis in APOE∗3-Leiden.CETP mice

Pharmacological blockade of the cannabinoid type 1 receptor, a G protein-coupled receptor expressed in the central nervous system and various peripheral tissues, reverses diet-induced obesity and dyslipidemia through the reduction of food intake and altered nutrient partitioning. This strategy is being explored for a number of therapeutic applications; however, its potency for the treatment of atherosclerotic cardiovascular disease via improvements in lipid metabolism remains unclear. Therefore, here, we aimed to investigate whether inhibition of the endocannabinoid system can attenuate atherosclerosis development through improvement of dyslipidemia. Lean, dyslipidemic female APOE∗3-Leiden.CETP transgenic mice were fed a Western-type diet supplemented with or without the cannabinoid type 1 receptor inverse agonist rimonabant (20 mg·kg body weight-1 day-1) for up to 20 weeks. Plasma lipids and bile acids were determined, and atherosclerotic lesions were scored in the aortic valve region. Rimonabant lowered plasma levels of triglyceride (TG) (-56%) and non-HDL-C (-19%) and increased HDL-C (+57%). These effects were explained by decreased VLDL-TG production (-52%) and accelerated VLDL-TG turnover accompanied by pronounced browning of white adipose tissue. In addition, rimonabant attenuated reverse cholesterol transport (-30%), increased plasma bile acid levels (+160%), and increased hepatic cholesterol accumulation (+88%). Importantly, rimonabant markedly lowered atherosclerotic lesion size (-64%), which coincided with decreased lesion severity (28% vs. 56% severe lesions) and which strongly correlated with non-HDL-C exposure (R2 = 0.60). Taken together, inhibition of the endocannabinoid system potently reverses dyslipidemia and prevents atherogenesis, even in the absence of obesity.Molecular Physiolog