1-, 2- and 3-AG as substrates of the endocannabinoid enzymes and endogenous ligands of the cannabinoid receptor 1.

2-Arachidonoylglycerol (2-AG) is the most potent and abundant endocannabinoid that acts as a full agonist at the cannabinoid 1 (CB1) and 2 (CB2) receptors. It serves as a substrate for several serine hydrolases, including monoacylglycerol lipase (MGL), α/ß hydrolase domain 6 (ABHD6) and fatty acid amide hydrolase (FAAH). However, 2-AG's rapid conversion to 1-AG (the S stereoisomer) and 3-AG (the R stereoisomer) complicates in vivo signaling. Here, we present the interaction profiles of 2-AG and its isomerization products, 1- and 3-AG, with the endocannabinoid MGL, ABHD6 and FAAH enzymes as well as the CB1 receptor. The 1- and 3-AG enantiomers are less prone to isomerization, and their affinities to endocannabinoid enzymes and potencies at CB1 receptor are quite different compared to 2-AG. Although MGL is the principal hydrolytic enzyme of 2-AG, 3-AG (the R isomer) appears to be the best substrate for hMGL. Contrarily, 1-AG (the S isomer) demonstrates the worst substrate profile, indicating that the stereochemistry of 1(3)-monoacylglycerols is very important for MGL enzyme. On the other hand, both 1- and 3-AG (the sn1 monoacylglycerols) are efficiently hydrolyzed by hABHD6 without preference, while 2-AG (the sn2 monoacylglycerol) has the lowest rate of hydrolysis. FAAH, the principal hydrolytic enzyme for arachidonoylethanolamide (anandamide, AEA), catalyzes the hydrolysis of all three isomers with similar efficiencies. In a functional cAMP assay at CB1 receptor, all three isomers behaved as agonists, with 2-AG being the most potent, followed by 3-AG then 1-AG. The presented data provides stereochemical insights to design chemically stable AG analogs with preferential stability against enzymes of interest.

Primary saturation of α, ß-unsaturated carbonyl containing fatty acids does not abolish electrophilicity.

Metabolism of polyunsaturated fatty acids results in the formation of hydroxylated fatty acids that can be further oxidized by dehydrogenases, often resulting in the formation of electrophilic, α,ß-unsaturated ketone containing fatty acids. As electrophiles are associated with redox signaling, we sought to investigate the metabolism of the oxo-fatty acid products in relation to their double bond architecture. Using an untargeted liquid chromatography mass spectrometry approach, we identified mono- and di-saturated products of the arachidonic acid-derived 11-oxoeicosatetraenoic acid (11-oxoETE) and mono-saturated metabolites of 15-oxoETE and docosahexaenoic acid-derived 17-oxodocosahexaenoinc acid (17-oxoDHA) in both human A549 lung carcinoma and umbilical vein endothelial cells. Notably, mono-saturated oxo-fatty acids maintained their electrophilicity as determined by nucleophilic conjugation to glutathione while a second saturation of 11-oxoETE resulted in a loss of electrophilicity. These results would suggest that prostaglandin reductase 1 (PTGR1), known only for its reduction of the α,ß-unsaturated double bond, was not responsible for the saturation of oxo-fatty acids at alternative double bonds. Surprisingly, knockdown of PTGR1 expression by shRNA confirmed its participation in the formation of 15-oxoETE and 17-oxoDHA mono-saturated metabolites. Furthermore, overexpression of PTGR1 in A549 cells increased the rate and total amount of oxo-fatty acid saturation. These findings will further facilitate the study of electrophilic fatty acid metabolism and signaling in the context of inflammatory diseases and cancer where they have been shown to have anti-inflammatory and anti-proliferative signaling properties.

New Pyridone-Based Derivatives as Cannabinoid Receptor Type 2 Agonists.

The activation of the human cannabinoid receptor type II (CB2R) is known to mediate analgesic and anti-inflammatory processes without the central adverse effects related to cannabinoid receptor type I (CB1R). In this work we describe the synthesis and evaluation of a novel series of N-aryl-2-pyridone-3-carboxamide derivatives tested as human cannabinoid receptor type II (CB2R) agonists. Different cycloalkanes linked to the N-aryl pyridone by an amide group displayed CB2R agonist activity as determined by intracellular [cAMP] levels. The most promising compound 8d exhibited a non-toxic profile and similar potency (EC50 = 112 nM) to endogenous agonists Anandamide (AEA) and 2-Arachidonoylglycerol (2-AG) providing new information for the development of small molecules activating CB2R. Molecular docking studies showed a binding pose consistent with two structurally different agonists WIN-55212-2 and AM12033 and suggested structural requirements on the pyridone substituents that can satisfy the orthosteric pocket and induce an agonist response. Our results provide additional evidence to support the 2-pyridone ring as a suitable scaffold for the design of CB2R agonists and represent a starting point for further optimization and development of novel compounds for the treatment of pain and inflammation.

New Disulfiram Derivatives as MAGL-Selective Inhibitors.

Monoacylglycerol lipase (MAGL) is a key enzyme in the human endocannabinoid system. It is also the main enzyme responsible for the conversion of 2-arachidonoyl glycerol (2-AG) to arachidonic acid (AA), a precursor of prostaglandin synthesis. The inhibition of MAGL activity would be beneficial for the treatment of a wide range of diseases, such as inflammation, neurodegeneration, metabolic disorders and cancer. Here, the author reports the pharmacological evaluation of new disulfiram derivatives as potent inhibitors of MAGL. These analogues displayed high inhibition selectivity over fatty acid amide hydrolase (FAAH), another endocannabinoid-hydrolyzing enzyme. In particular, compound 2i inhibited MAGL in the low micromolar range. However, it did not show any inhibitory activity against FAAH.

Central nervous system, peripheral and hemodynamic effects of nanoformulated anandamide in hypertension.

PURPOSE: Hypertensive lesions induce alterations at hemodynamic, peripheral, and central levels. Anandamide (N-arachidonoylethanolamine; AEA) protects neurons from inflammatory damage, but its free administration may cause central adverse effects. AEA controlled release by nanoformulations could reduce/eliminate its side effects. The present study aimed to evaluate the effects of nanoformulated AEA (nf-AEA) on systolic blood pressure (SBP), behavior, and central/peripheral inflammatory, oxidative, and apoptotic state in spontaneously hypertensive rats (SHR). MATERIALS/METHODS: Male rats were used, both Wistar Kyoto (WKY) and SHR (n â€‹= â€‹10 per group), with/without treatment with nf-AEA (obtained by electrospraying) at a weekly dose of 5 â€‹mg/kg IP for 4 weeks. SBP was measured and behavioral tests were performed. Inflammatory/oxidative markers were quantified at the central (brain cortex) and peripheral (serum) level. RESULTS: SHR showed hyperactivity, low anxiety, and high concentrations of central/peripheral inflammatory/oxidative markers, also higher apoptosis of brain cortical cells compared to WKY. As opposed to this group, treatment with nf-AEA in SHR significantly reduced SBP, peripheral/central inflammatory/oxidative makers, and central apoptosis. Nf-AEA also increased neuroprotective mechanisms mediated by intracellular heat shock protein 70 (Hsp70), which were attenuated in untreated SHR. Additionally, nf-AEA reversed the abnormal behaviors observed in SHR without producing central adverse effects. CONCLUSIONS: Our results suggest protective properties of nf-AEA, both peripherally and centrally, through a signaling pathway that would involve the type I angiotensin II receptor, Wilms tumor transcription factor 1, Hsp70, and iNOS. Considering non-nf-AEA limitations, this nanoformulation could contribute to the development of new antihypertensive and behavioral disorder treatments associated with neuroinflammation.

N­linoleyltyrosine protects PC12 cells against oxidative damage via autophagy: Possible involvement of CB1 receptor regulation.

Oxidative stress is one of the main pathogenic factors of neurodegenerative diseases. As the ligand of cannabinoid type 1 (CB1) and 2 (CB2) receptors, anandamide (AEA) exerts benign antioxidant activities. However, the instability of AEA results in low levels in vivo, which limit its further application. Based on the structure of AEA, N­linoleyltyrosine (NITyr) was synthesized in our laboratory and was hypothesized to possess a similar function to that of AEA. To the best of our knowledge, the present study demonstrates for the first time, the activities and mechanisms of NITyr. NITyr treatment attenuated hydrogen peroxide (H2O2)­induced cytotoxicity, with the most promiment effect observed at 1 µmol/l. Treatment with NITyr also suppressed the H2O2­induced elevation of reactive oxygen species (ROS) and enhanced the expression of the autophagy­related proteins, LC3­II, beclin­1, ATG 5 and ATG13. The autophagic inhibitor, 3­methyladenine, reversed the effects of NITyr on ROS levels and cellular viability. Furthermore, AM251, a CB1 receptor antagonist, but not AM630 (a CB2 receptor antagonist), diminished the effects of NITyr on cell viability, ROS generation and autophagy­related protein expression. However, NITyr increased the protein expression of both the CB1 and CB2 receptors. Therefore, NITyr was concluded to protect PC12 cells against H2O2­induced oxidative injury by inducing autophagy, a process which may involve the CB1 receptor.

Neuroprotective and Antioxidant Activity of Arachidonoyl Choline, Its Bis-Quaternized Analogues and Other Acylcholines.

The antioxidant activity and protective effect in the toxicity model of H2O2 were studied for arachidonic (AA-CHOL), docosahexaenoic (DHA-CHOL), linoleic (Ln-CHOL), and oleic (Ol-CHOL) fatty acids, as well as arachidonoyl dicholine (AA-diCHOL) and O-arachidonoyl bistetramethylaminoisopropanol (ABTAP). AA-CHOL, DHA-CHOL and Ln-CHOL provided a 20% increase in cell survival. AA-CHOL, AA-diCHOL, Ol-CHOL, and ABTAP had a radical-scavenging effect in the ABTS test, approximately equal to the activity of a standard radical scavenger Trolox.

Altered productions of prostaglandins and prostamides by human amnion in response to infectious and inflammatory stimuli identified by mutliplex mass spectrometry.

INTRODUCTION: Prostaglandins are critical for the onset and progression of labor in mammals, and are formed by the metabolism of arachidonic acid. The products of arachidonic acid, 2-arachidonoylglycerol (2-AG), and anandamide (AEA) have a similar lipid back bone but differing polar head groups, meaning that identification of these products by immunoassay can be difficult. MATERIALS AND METHODS: In the current study, we present the use of mass spectrometry as multiplex method of identifying the specific end products of arachidonic and anandamide metabolism by human derived amnion explants treated with either an infectious agent (LPS) or inflammatory mediator (IL-1ß or TNF-α). RESULTS: Human amnion tissue explants treated with LPS, IL-1ß, or TNF-α increased production of prostaglandin E2 (PGE2; p < 0.05) but decreased PGFM. Overall, PGE2 production was greater compared to the other prostaglandins and prostamides irrespective of treatment. CONCLUSIONS: The findings of the current study are in keeping with the literature which describes amnion tissues as predominantly producing PGE2. The use of mass spectrometry for the differential identification of prostaglandins, prostamides, and other eicosanoids may help better elucidate mechanisms of preterm labor, and lead to new targets for the prediction of risk for preterm labor and/or birth.

Synthesis, physicochemical characterisation and biological activity of anandamide/ɛ-polycaprolactone nanoparticles obtained by electrospraying.

Drug encapsulation in nanocarriers such as polymeric nanoparticles (Nps) may help to overcome the limitations associated with cannabinoids. In this study, the authors' work aimed to highlight the use of electrospraying techniques for the development of carrier Nps of anandamide (AEA), an endocannabinoid with attractive pharmacological effects but underestimated due to its unfavourable physicochemical and pharmacokinetic properties added to its undesirable effects at the level of the central nervous system. The authors characterised physicochemically and evaluated in vitro biological activity of anandamide/ɛ-polycaprolactone nanoparticles (Nps-AEA/PCL) obtained by electrospraying in epithelial cells of the human proximal tubule (HK2), to prove the utility of this method and to validate the biological effect of Nps-AEA/PCL. They obtained particles from 100 to 900 nm of diameter with a predominance of 200-400 nm. Their zeta potential was -20 ± 1.86 mV. They demonstrated the stable encapsulation of AEA in Nps-AEA/PCL, as well as its dose-dependent capacity to induce the expression of iNOS and NO levels and to decrease the Na+/K+ ATPase activity in HK2 cells. Obtaining Nps-AEA/PCL by electrospraying would represent a promising methodology for a novel AEA pharmaceutical formulation development with optimal physicochemical properties, physical stability and biological activity on HK2 cells.

Structure and Dynamics of the Acyl Chains in the Membrane Trafficking and Enzymatic Processing of Lipids.

The regulatory chemical mechanisms of lipid trafficking and degradation are involved in many pathophysiological processes, being implicated in severe pain, inflammation, and cancer. In addition, the processing of lipids is also relevant for industrial and environmental applications. However, there is poor understanding of the chemical features that control lipid membrane trafficking and allow lipid-degrading enzymes to efficiently select and hydrolyze specific fatty acids from a complex cellular milieu of bioactive lipids. This is particularly true for lipid acyl chains, which have diverse structures that can critically affect the many complex reactions needed to elongate, desaturate, or transport fatty acids. Building upon our own contributions in this field, we will discuss how molecular simulations, integrated with experimental evidence, have revealed that the structure and dynamics of the lipid tail are actively involved in modulating membrane trafficking at cellular organelles, and enzymatic reactions at cell membranes. Further evidence comes from recent crystal structures of lipid receptors and remodeling enzymes. Taken together, these recent works have identified those structural features of the lipid acyl chain that are crucial for the regioselectivity and stereospecificity of essential desaturation reactions. In this context, we will first illustrate how atomistic and coarse-grained simulations have elucidated the structure-function relationships between the chemical composition of the lipid's acyl chains and the molecular properties of lipid bilayers. Particular emphasis will be given to the prominent chemical role of the number of double carbon-carbon bonds along the lipid acyl chain, that is, discriminating between saturated, monounsaturated, and polyunsaturated lipids. Different levels of saturation in fatty acid molecules dramatically influence the biophysical properties of lipid assemblies and their interaction with proteins. We will then discuss the processing of lipids by membrane-bound enzymes. Our focus will be on lipids such as anandamide and 2-arachidonoylglycerol. These are the main molecules that act as neurotransmitters in the endocannabinoid system. Specifically, recent findings indicate a crucial interplay between the level of saturation of the lipid tail, its energetically and sterically favored conformations, and the hydrophobic accessory cavities in lipid-degrading enzymes, which help form catalytically active conformations of the selected substrate. This Account will emphasize how the specific chemical structure of acyl chains affects the molecular mechanisms for modulating membrane trafficking and selective hydrolysis. The results examined here show that, by using molecular simulations to investigate lipid plasticity and substrate flexibility, researchers can enrich their interpretation of experimental results about the structure-function relationships of lipids. This could positively impact chemical and biological studies in the field and ultimately support protein engineering studies and structure-based drug discovery to target lipid-processing enzymes.

Determination of endocannabinoids and endocannabinoid-like substances in human K3EDTA plasma - LC-MS/MS method validation and pre-analytical characteristics.

The determination of endocannabinoids and endocannabinoid-like substances in biological human samples is a vibrant field of research with great significance due to postulated relevance of these substances in diseases such as Alzheimer's disease, multiple sclerosis, cancer and cardiovascular diseases. For a possible use as biomarker in early prediction or diagnosis of a disease as well as examination of a successful treatment, the valid determination of the analytes in common accessible human samples, such as plasma or serum, is of great importance. A method for the determination of arachidonoyl ethanolamide, oleoyl ethanolamide, palmitoyl ethanolamide, 1-arachidonoyl glycerol and 2-arachidonoyl glycerol in human K3EDTA plasma using liquid-liquid-extraction in combination with liquid chromatography-tandem-mass spectrometry has been developed and validated for the quantification of the aforementioned analytes. Particular emphasis was placed on the chromatographic separation of the isomers 1-arachidonoyl glycerol and 2-arachidonoyl glycerol, arachidonoyl ethanolamide and O-arachidonoyl ethanolamine (virodhamine) as well as oleoyl ethanolamide and vaccenic acid ethanolamide. During the validation process, increasing concentrations of 1-arachidonoyl glycerol and 2-arachidonoyl glycerol while storing plasma samples were observed. In-depth investigation of pre-analytical sample handling revealed rising concentrations for both analytes in plasma and for arachidonoyl ethanolamide, oleoyl ethanolamide and palmitoyl ethanolamide in whole blood, dependent on the period and temperature of storage. Prevention of the increase in concentration was not possible, raising the question whether human K3EDTA plasma is suitable for the determination of endocannabinoids and endocannabinoid-like substances. Especially the common practice to calculate the concentration of 2-arachidonoyl glycerol as sum of 1-arachidonoyl glycerol and 2-arachidonoyl glycerol is highly questionable because the concentrations of both analytes increase unequally while storing the plasma samples in the fridge.

Cannabimimetic plants: are they new cannabinoidergic modulators?

MAIN CONCLUSION: Phytochemicals and secondary metabolites able to interact with the endocannabinoid system (Cannabimimetics) have been recently described in a broad range of plants and fruits. These findings can open new alternative avenues to explore for the development of novel therapeutic compounds. The cannabinoids regulate many physiological and pathological functions in both animals and plants. Cannabis sativa is the main plant that produces phytocannabinoids inside resins capable to defend the plant from the aggression of parasites and herbivores. Animals produce anandamide and 2-arachidonoyl glycerol, which thanks to binding with main receptors such as type-1 cannabinoid receptor (CB1R) and the type-2 cannabinoid receptor (CB2R) are involved in inflammation processes and several brain functions. Endogenous cannabinoids, enzymes for synthesis and degradation of cannabinoids, and CB1R and CB2R constitute the endocannabinoid system (ECS). Other plants can produce cannabinoid-like molecules such as perrottetinene extracted from Radula perrottetii, or anandamide and 2-arachidonoyl glycerol extracted from some bryophytes. Moreover, several other secondary metabolites can also interact with the ECS of animals and take the name of cannabimimetics. These phytoextracts not derived from Cannabis sativa can act as receptor agonists or antagonist, or enzyme inhibitors of ECS and can be involved in the inflammation, oxidative stress, cancer, and neuroprotection. Finally, given the evolutionary heterogeneity of the cannabimimetic plants, some authors speculated on the fascinating thesis of the evolutionary convergence between plants and animals regarding biological functions of ECS. The review aims to provide a critical and complete assessment of the botanical, chemical and therapeutic aspects of cannabimimetic plants to evaluate their spread in the world and medicinal potentiality.

Synthesis, molecular modelling studies and biological evaluation of new oxoeicosanoid receptor 1 agonists.

The oxoeicosanoid receptor 1 (OXER1) is a member of the G-protein coupled receptors (GPCR) family, and is involved in inflammatory processes and oncogenesis. As such it is an attractive target for pharmacological intervention. The present study aimed to shed light on the molecular fundaments of OXER1 modulation using chemical probes structurally related to the natural agonist 5-oxo-ETE. In a first step, 5-oxo-ETE and its closely related derivatives (5-oxo-EPE and 4-oxo-DHA) were obtained by conducting concise and high-yielding syntheses. The biological activity of obtained compounds was assessed in terms of potency (EC50) and efficacy (Emax) for arrestin recruitment. Finally, molecular modelling and simulation were used to explore binding characteristics of 5-oxo-ETE and derivatives with the aim to rationalize biological activity. Our data suggest that the tested 5-oxo-ETE derivatives (i) insert quickly into the membrane, (ii) access the receptor via transmembrane helices (TMs) 5 and 6 from the membrane side and (iii) drive potency and efficacy by differential interaction with TM5 and 7. Most importantly, we found that the methyl ester of 5-oxo-ETE (1a) showed even a higher maximum response than the natural agonist (1). In contrast, shifting the 5-oxo group into position 4 results in inactive compounds (4-oxo DHA compounds (3) and (3a)). All in all, our study provides relevant structural data that help understanding better OXER1 functionality and its modulation. The structural information presented herein will be useful for designing new lead compounds with desired signalling profiles.

Ceramide binding to anandamide increases its half-life and potentiates its cytotoxicity in human neuroblastoma cells.

Anandamide (AEA) is a ubiquitous lipid that exerts neurotransmitter functions but also controls important biological functions such as proliferation, survival, or programmed cell death. The latter effects are also regulated by ceramide, a lipid enzymatically generated from sphingomyelin hydrolysis by sphingomyelinase. Ceramide has been shown to increase the cellular toxicity of AEA, but the mechanisms controlling this potentiating effect remained unclear. Here we have used a panel of in silico, physicochemical, biochemical and cellular approaches to study the crosstalk between AEA and ceramide apoptotic pathways. Molecular dynamics simulations indicated that AEA and ceramide could form a stable complex in phosphatidylcholine membranes. Consistent with these data, we showed that AEA can specifically insert into ceramide monolayers whereas it did not penetrate into sphingomyelin membranes. Then we have studied the effects of ceramide on AEA-induced toxicity of human neuroblastoma cells. In these experiments, the cells have been either naturally enriched in ceramide by neutral sphingomyelinase pre-incubation or treated with C2-ceramide, a biologically active ceramide analog. Both treatments significantly increased the cytotoxicity of AEA as assessed by the MTS mitochondrial toxicity assay. This effect was correlated with the concomitant accumulation of natural ceramide (or its synthetic analog) and AEA in the cells. A kinetic study of AEA hydrolysis showed that ceramide inhibited the fatty acid amino hydrolase (FAAH) activity in cell extracts. Taken together, these data suggested that ceramide binds to AEA, increases its half-life and potentiates its cytotoxicity. Overall, these mechanisms account for a functional cross-talk between AEA and ceramide apoptotic pathways.

2-Arachidonoylglycerol ameliorates inflammatory stress-induced insulin resistance in cardiomyocytes.

Several studies have linked impaired glucose uptake and insulin resistance (IR) to functional impairment of the heart. Recently, endocannabinoids have been implicated in cardiovascular disease. However, the mechanisms involving endocannabinoid signaling, glucose uptake, and IR in cardiomyocytes are understudied. Here we report that the endocannabinoid 2-arachidonoylglycerol (2-AG), via stimulation of cannabinoid type 1 (CB1) receptor and Ca2+/calmodulin-dependent protein kinase ß, activates AMP-activated kinase (AMPK), leading to increased glucose uptake. Interestingly, we have observed that the mRNA expression of CB1 and CB2 receptors was decreased in diabetic mice, indicating reduced endocannabinoid signaling in the diabetic heart. We further establish that TNFα induces IR in cardiomyocytes. Treatment with 2-AG suppresses TNFα-induced proinflammatory markers and improves IR and glucose uptake. Conversely, pharmacological inhibition or knockdown of AMPK attenuates the anti-inflammatory effect and reversal of IR elicited by 2-AG. Additionally, in human embryonic stem cell-derived cardiomyocytes challenged with TNFα or FFA, we demonstrate that 2-AG improves insulin sensitivity and glucose uptake. In conclusion, 2-AG abates inflammatory responses, increases glucose uptake, and overcomes IR in an AMPK-dependent manner in cardiomyocytes.

Probing the orientation of inhibitor and epoxy-eicosatrienoic acid binding in the active site of soluble epoxide hydrolase.

Soluble epoxide hydrolase (sEH) is an important therapeutic target of many diseases, such as chronic obstructive pulmonary disease (COPD) and diabetic neuropathic pain. It acts by hydrolyzing and thus regulating specific bioactive long chain polyunsaturated fatty acid epoxides (lcPUFA), like epoxyeicosatrienoic acids (EETs). To better predict which epoxides could be hydrolyzed by sEH, one needs to dissect the important factors and structural requirements that govern the binding of the substrates to sEH. This knowledge allows further exploration of the physiological role played by sEH. Unfortunately, a crystal structure of sEH with a substrate bound has not yet been reported. In this report, new photoaffinity mimics of a sEH inhibitor and EET regioisomers were prepared and used in combination with peptide sequencing and computational modeling, to identify the binding orientation of different regioisomers and enantiomers of EETs into the catalytic cavity of sEH. Results indicate that the stereochemistry of the epoxide plays a crucial role in dictating the binding orientation of the substrate.

Keys to Lipid Selection in Fatty Acid Amide Hydrolase Catalysis: Structural Flexibility, Gating Residues and Multiple Binding Pockets.

The fatty acid amide hydrolase (FAAH) regulates the endocannabinoid system cleaving primarily the lipid messenger anandamide. FAAH has been well characterized over the years and, importantly, it represents a promising drug target to treat several diseases, including inflammatory-related diseases and cancer. But its enzymatic mechanism for lipid selection to specifically hydrolyze anandamide, rather than similar bioactive lipids, remains elusive. Here, we clarify this mechanism in FAAH, examining the role of the dynamic paddle, which is formed by the gating residues Phe432 and Trp531 at the boundary between two cavities that form the FAAH catalytic site (the "membrane-access" and the "acyl chain-binding" pockets). We integrate microsecond-long MD simulations of wild type and double mutant model systems (Phe432Ala and Trp531Ala) of FAAH, embedded in a realistic membrane/water environment, with mutagenesis and kinetic experiments. We comparatively analyze three fatty acid substrates with different hydrolysis rates (anandamide > oleamide > palmitoylethanolamide). Our findings identify FAAH's mechanism to selectively accommodate anandamide into a multi-pocket binding site, and to properly orient the substrate in pre-reactive conformations for efficient hydrolysis that is interceded by the dynamic paddle. Our findings therefore endorse a structural framework for a lipid selection mechanism mediated by structural flexibility and gating residues between multiple binding cavities, as found in FAAH. Based on the available structural data, this exquisite catalytic strategy for substrate specificity seems to be shared by other lipid-degrading enzymes with similar enzymatic architecture. The mechanistic insights for lipid selection might assist de-novo enzyme design or drug discovery efforts.

Molecular-interaction and signaling profiles of AM3677, a novel covalent agonist selective for the cannabinoid 1 receptor.

The cannabinoid 1 receptor (CB1R) is one of the most abundant G protein-coupled receptors (GPCRs) in the central nervous system. CB1R involvement in multiple physiological processes, especially neurotransmitter release and synaptic function, has made this GPCR a prime drug discovery target, and pharmacological CB1R activation has been demonstrated to be a tenable therapeutic modality. Accordingly, the design and profiling of novel, drug-like CB1R modulators to inform the receptor's ligand-interaction landscape and molecular pharmacology constitute a prime contemporary research focus. For this purpose, we report utilization of AM3677, a designer endocannabinoid (anandamide) analogue derivatized with a reactive electrophilic isothiocyanate functionality, as a covalent, CB1R-selective chemical probe. The data demonstrate that reaction of AM3677 with a cysteine residue in transmembrane helix 6 of human CB1R (hCB1R), C6.47(355), is a key feature of AM3677's ligand-binding motif. Pharmacologically, AM3677 acts as a high-affinity, low-efficacy CB1R agonist that inhibits forskolin-stimulated cellular cAMP formation and stimulates CB1R coupling to G protein. AM3677 also induces CB1R endocytosis and irreversible receptor internalization. Computational docking suggests the importance of discrete hydrogen bonding and aromatic interactions as determinants of AM3677's topology within the ligand-binding pocket of active-state hCB1R. These results constitute the initial identification and characterization of a potent, high-affinity, hCB1R-selective covalent agonist with utility as a pharmacologically active, orthosteric-site probe for providing insight into structure-function correlates of ligand-induced CB1R activation and the molecular features of that activation by the native ligand, anandamide.

Computational insights into function and inhibition of fatty acid amide hydrolase.

The Fatty Acid Amide Hydrolase (FAAH) enzyme is a membrane-bound serine hydrolase responsible for the deactivating hydrolysis of a family of naturally occurring fatty acid amides. FAAH is a critical enzyme of the endocannabinoid system, being mainly responsible for regulating the level of its main cannabinoid substrate anandamide. For this reason, pharmacological inhibition of FAAH, which increases the level of endogenous anandamide, is a promising strategy to cure a variety of diseases including pain, inflammation, and cancer. Much structural, mutagenesis, and kinetic data on FAAH has been generated over the last couple of decades. This has prompted several informative computational investigations to elucidate, at the atomic-level, mechanistic details on catalysis and inhibition of this pharmaceutically relevant enzyme. Here, we review how these computational studies - based on classical molecular dynamics, full quantum mechanics, and hybrid QM/MM methods - have clarified the binding and reactivity of some relevant substrates and inhibitors of FAAH. We also discuss the experimental implications of these computational insights, which have provided a thoughtful elucidation of the complex physical and chemical steps of the enzymatic mechanism of FAAH. Finally, we discuss how computations have been helpful for building structure-activity relationships of potent FAAH inhibitors.

Relationship between seminal plasma levels of anandamide congeners palmitoylethanolamide and oleoylethanolamide and semen quality.

OBJECTIVE: To determine whether changes in seminal plasma concentrations of the endogenous lipid signaling molecules palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) have significant effects on sperm quality. DESIGN: Biochemical and physiological studies of human seminal plasma and spermatozoa. SETTING: Academic tertiary care medical center. PATIENT(S): Ninety men attending an infertility clinic for semen analysis. INTERVENTION(S): Palmitoylethanolamide and OEA extracted from seminal plasma were quantified by ultra high-performance liquid chromatography (HPLC)-tandem mass spectrometry. Patient sperm from semen with normal parameters were exposed in vitro to PEA or OEA to determine effects on sperm motility, viability, and mitochondrial activity. MAIN OUTCOME MEASURE(S): The relationship between seminal plasma concentrations of PEA and OEA and sperm quality and the effect of these compounds on sperm motility, viability, and mitochondria activity in vitro. RESULT(S): Palmitoylethanolamide and OEA concentrations in seminal plasma were lower in men with asthenozoospermia and oligoasthenoteratozospermia compared with men with normal semen parameters. Palmitoylethanolamide and OEA rapidly and significantly improved sperm motility and maintained viability without affecting mitochondria activity in vitro. CONCLUSION(S): Maintenance of normal PEA and OEA tone in human seminal plasma may be necessary for the preservation of normal sperm function and male fertility. Exocannabinoids found in Cannabis, such as delta-9-tetrahydrocannabinol and cannabidiol, could compete with these endocannabinoids upsetting their finely balanced, normal functioning and resulting in male reproductive failure.