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Introduction: Escalating costs and inherent uncertainties associated with drug discovery invite initiatives to improve its efficiency and de-risk campaigns for inventing better therapeutics. One such initiative involves recognizing and exploiting current approaches in therapeutics invention with molecular mechanisms of action that hold promise for designing and targeting new chemical entities as drugs., Areas Covered: This perspective considers the current contextual framework around three drug-discovery approaches and evaluates their potential to help identify new targets/modalities in small-molecule molecular pharmacology: diversifying ligand-directed phenotypes for G protein-coupled receptor (GPCR) pharmacotherapeutic signaling; developing therapeutic-protein degraders and stabilizers for proximity-inducing pharmacology; and mining organelle biology for druggable therapeutic targets., Expert Opinion: The contemporary drug-discovery approaches examined appear generalizable and versatile to have applications in therapeutics invention beyond those case studies discussed herein. Accordingly, they may be considered strategic trends worthy of note in advancing the field toward novel ways of addressing pharmacotherapeutically unmet medical needs.

Allosteric modulators of cannabinoid 1 receptor (CB1R) show translational promise over orthosteric ligands due to their potential to elicit therapeutic benefit without cannabimimetic side effects. The prototypic 2-phenylindole CB1R allosteric modulator, GAT211 (1), demonstrates preclinical efficacy in various disease models. The limited systematic structure-activity relationship (SAR) data at the C2 position of the indole ring within GAT211 invites the opportunity for further modifications to improve GAT211's pharmacological profile while serving to amplify and variegate this library of therapeutically attractive agents. These considerations prompted this focused SAR study in which we substituted the GAT211 C2-phenyl ring with heteroaromatic substituents. The synthesized GAT211 analogs were then evaluated in vitro as CB1R allosteric modulators in cAMP and β-arrestin2 assays with CP55,940 as the orthosteric ligand. Furan and thiophene rings (15c-f and 15m) were the best-tolerated substituents at the C2 position of GAT211 for engagement with human CB1R (hCB1R). The SAR around the novel ligands reported allowed direct experimental characterization of the interaction profile of that pharmacophore with its binding domain in functional, human CB1R, thus offering guidance for accessing subsequent-generation hCB1R allosteric modulators as potential therapeutics. The most potent analog, 15d, markedly promoted orthosteric ligand binding to hCB1R. Pharmacological profiling in the GTPγS and mouse vas deferens assays demonstrated that 15d behaves as a CB1R agonist-positive allosteric modulator (ago-PAM), as confirmed electrophysiologically in autoptic neurons. In vivo, 15d was efficacious as a topical agent that significantly reduced intraocular pressure (IOP) in the ocular normotensive murine model of glaucoma. Since elevated IOP is a decisive risk factor for glaucoma and attendant vision loss, our data support the proposition that the 2-phenylindole class of CB1R ago-PAMs has therapeutic potential for glaucoma and other diseases where potentiation of CB1R signaling may be therapeutic., (Copyright © 2021. Published by Elsevier Ltd.)

We apply the magic methyl effect to improve the potency/efficacy of GAT211, the prototypic 2-phenylindole-based cannabinoid type-1 receptor (CB1R) agonist-positive allosteric modulator (ago-PAM). Introducing a methyl group at the α-position of nitro group generated two diastereomers, the greater potency and efficacy of erythro , (±)- 9 vs threo , (±)- 10 constitutes the first demonstration of diastereoselective CB1R-allosteric modulator interaction. Of the (±)- 9 enantiomers, (-)-( S , R )- 13 evidenced improved potency over GAT211 as a CB1R ago-PAM, whereas (+)-( R , S )- 14 was a CB1R allosteric agonist biased toward G protein- vs β-arrestin1/2-dependent signaling. (-)-( S , R )- 13 and (+)-( R , S )- 14 were devoid of undesirable side effects (triad test), and (+)-( R , S )- 14 reduced intraocular pressure with an unprecedentedly long duration of action in a murine glaucoma model. (-)-( S , R )- 13 docked into both a CB1R extracellular PAM and intracellular allosteric-agonist site(s), whereas (+)-( R , S )- 14 preferentially engaged only the latter. Exploiting G-protein biased CB1R-allosteric modulation can offer safer therapeutic candidates for glaucoma and, potentially, other diseases.

Specific tuning of cannabinoid 1 receptor (CB1R) activity by small-molecule allosteric modulators is a therapeutic modality with multiple properties inherently advantageous to therapeutic applications. We previously generated a library of unique CB1R positive allosteric modulators (PAMs) derived from GAT211, which has three pharmacophoric sites critical to its ago-PAM activity. To elaborate our CB1R PAM library, we report the rational design and molecular-pharmacology profiling of several 2-phenylindole analogs modified at the "site-III" aromatic ring. The comprehensive structure-activity relationship (SAR) investigation demonstrates that attaching small lipophilic functional groups on the ortho-position of the GAT211 site-III phenyl ring could markedly enhance CB1R ago-PAM activity. Select site-III modifications also improved GAT211's water solubility. The SAR reported both extends the structural diversity of this compound class and demonstrates the utility of GAT211's site-III for improving the parent compound's drug-like properties of potency and/or aqueous solubility., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cannabinoid 1 receptor (CB1R) allosteric ligands hold a far-reaching therapeutic promise. We report the application of fluoro- and nitrogen-walk approaches to enhance the drug-like properties of GAT211, a prototype CB1R allosteric agonist-positive allosteric modulator (ago-PAM). Several analogs exhibited improved functional potency (cAMP, β-arrestin 2), metabolic stability, and aqueous solubility. Two key analogs, GAT591 ( 6r ) and GAT593 ( 6s ), exhibited augmented allosteric-agonist and PAM activities in neuronal cultures, improved metabolic stability, and enhanced orthosteric agonist binding (CP55,940). Both analogs also exhibited good analgesic potency in the CFA inflammatory-pain model with longer duration of action over GAT211 while being devoid of adverse cannabimimetic effects. Another analog, GAT592 ( 9j ), exhibited moderate ago-PAM potency and improved aqueous solubility with therapeutic reduction of intraocular pressure in murine glaucoma models. The SAR findings and the enhanced allosteric activity in this class of allosteric modulators were accounted for in our recently developed computational model for CB1R allosteric activation and positive allosteric modulation.

Upon ultraviolet activation, cannabinergic aliphatic azido (N 3 ) ligands covalently label cannabinoid receptors, prominent G-protein-coupled receptor (GPCR) drug targets. We report here the mechanism of covalent attachment to selected substrates of the high-affinity CBR inverse agonist AM1335 and its deuterated analog AM1335(d10), arylpyrazole compounds with an azide moiety at their n-pentyl side chain. To model the receptor interaction, we utilized the human cannabinoid 2 receptor (hCB2R) transmembrane helix 6 (TMH6) peptide and an N-acyl-protected cysteine (NAC). The photochemical reaction products of model substrates with AM1335 and AM1335(d10) were analyzed with tandem electrospray ionization mass spectrometry fragmentation and deuterium exchange mass spectrometry. The nitrene initially formed after photoreaction undergoes rearrangement to an imine which then interacts with the cysteine sulfhydryl group, resulting in ligand attachment. Our results demonstrate that covalent probes carrying aliphatic azides behave more selectively than originally thought and can be used to label protein cysteine residues preferentially.

Human monoacylglycerol lipase (hMAGL) plays a key role in homeostatic tuning of the endocannabinoid signaling system and supports aggressive tumorogenesis, making this enzyme a promising therapeutic target. hMAGL features a membrane-associated lid domain that regulates entry of endocannabinoid lipid substrates into the hydrophobic channel accessing the active site, likely from the membrane bilayer. The present work applied simultaneous surface plasmon resonance and electrochemical impedance spectroscopy measurements to show that, in absence of the substrate, hMAGL can remove phospholipid molecules from the membrane and, thereby, disintegrate pre-formed, intact, tethered phospholipid bilayer membrane mimetics (tBLMs) composed of unsaturated phosphatidylcholines. To probe the mechanism of hMAGL-induced on tBLMs compromise, we investigated the effect of wild type and mutant hMAGLs and hMAGL rendered catalytically inactive, as a function of concentration and in the presence of chemically distinct active-site inhibitors. Our data show that hMAGL's lid domain and hydrophobic substrate-binding pocket play important roles in hMAGL-induced bilayer lipid mobilization, whereas hydrolytic activity of the enzyme does not appear to be a factor., (Published by Elsevier Inc.)

The cannabinoid 1 receptor (CB1R) is one of the most widely expressed metabotropic G protein-coupled receptors in brain, and its participation in various (patho)physiological processes has made CB1R activation a viable therapeutic modality. Adverse psychotropic effects limit the clinical utility of CB1R orthosteric agonists and have promoted the search for CB1R positive allosteric modulators (PAMs) with the promise of improved drug-like pharmacology and enhanced safety over typical CB1R agonists. In this study, we describe the synthesis and in vitro and ex vivo pharmacology of the novel allosteric CB1R modulator GAT211 (racemic) and its resolved enantiomers, GAT228 (R) and GAT229 (S). GAT211 engages CB1R allosteric site(s), enhances the binding of the orthosteric full agonist [ 3 H]CP55,490, and reduces the binding of the orthosteric antagonist/inverse agonist [ 3 H]SR141716A. GAT211 displayed both PAM and agonist activity in HEK293A and Neuro2a cells expressing human recombinant CB1R (hCB1R) and in mouse-brain membranes rich in native CB1R. GAT211 also exhibited a strong PAM effect in isolated vas deferens endogenously expressing CB1R. Each resolved and crystallized GAT211 enantiomer showed a markedly distinctive pharmacology as a CB1R allosteric modulator. In all biological systems examined, GAT211's allosteric agonist activity resided with the R-(+)-enantiomer (GAT228), whereas its PAM activity resided with the S-(-)-enantiomer (GAT229), which lacked intrinsic activity. These results constitute the first demonstration of enantiomer-selective CB1R positive allosteric modulation and set a precedent whereby enantiomeric resolution can decisively define the molecular pharmacology of a CB1R allosteric ligand.

Cannabinoid receptor 2 (CB2R)-dependent signaling is implicated in neuronal physiology and immune surveillance by brain microglia. Selective CB2R agonists hold therapeutic promise for inflammatory and other neurological disorders. Information on human CB2R (hCB2R) ligand-binding and functional domains is needed to inform the rational design and optimization of candidate druglike hCB2R agonists. Prior demonstration that hCB2R transmembrane helix 2 (TMH2) cysteine C2.59(89) reacts with small-molecule methanethiosulfonates showed that this cysteine residue is accessible to sulfhydryl derivatization reagents. We now report the design and application of two novel, pharmacologically active, high-affinity molecular probes, AM4073 and AM4099, as chemical reporters to interrogate directly the interaction of classical cannabinoid agonists with hCB2R cysteine residues. AM4073 has one electrophilic isothiocyanate (NCS) functionality at the C9 position of its cyclohexenyl C-ring, whereas AM4099 has NCS groups at that position and at the terminus of its aromatic A-ring C3 side chain. Pretreatment of wild-type hCB2R with either probe reduced subsequent [ 3 H]CP55,940 specific binding by ∼60%. Conservative serine substitution of any hCB2R TMH cysteine residue except C2.59(89) did not affect the reduction of [ 3 H]CP55,940 specific binding by either probe, suggesting that AM4073 and AM4099 interact irreversibly with this TMH2 cysteine. In contrast, AM841, an exceptionally potent hCB2R megagonist and direct AM4073/4099 congener bearing a single electrophilic NCS group at the terminus of its C3 side chain, had been demonstrated to bind covalently to TMH6 cysteine C6.47(257) and not C2.59(89). Molecular modeling indicates that the AM4073-hCB2R* interaction at C2.59(89) orients this classical cannabinoid away from TMH6 and toward the TMH2-TMH3 interface in the receptor's hydrophobic binding pocket, whereas the AM841-hCB2R* interaction at C6.47(257) favors agonist orientation toward TMH6/7. These data constitute initial evidence that TMH2 cysteine C2.59(89) is a component of the hCB2R binding pocket for classical cannabinoids. The results further demonstrate how interactions between classical cannabinoids and specific amino acids within the hCB2R* ligand-binding domain act as determinants of agonist pharmacological properties and the architecture of the agonist-hCB2R* conformational ensemble, allowing the receptor to adopt distinct activity states, such that interaction of classical cannabinoids with TMH6 cysteine C6.47(257) favors a binding pose more advantageous for agonist potency than does their interaction with TMH2 cysteine C2.59(89).

The phenomenon of functional selectivity, whereby a ligand preferentially directs the information output of a G-protein coupled receptor (GPCR) along (a) particular effector pathway(s) and away from others, has redefined traditional GPCR signaling paradigms to provide a new approach to structure-based drug design. The two principal cannabinoid receptors (CBRs) 1 and 2 belong to the class-A GPCR subfamily and are considered tenable therapeutic targets for several indications. Yet conventional orthosteric ligands (agonists, antagonists/inverse agonists) for these receptors have had very limited clinical utility due to their propensity to incite on-target adverse events. Chemically distinct classes of cannabinergic ligands exhibit signaling bias at CBRs towards individual subsets of signal transduction pathways. In this review, we discuss the known signaling pathways regulated by CBRs and examine the current evidence for functional selectivity at CBRs in response to endogenous and exogenous cannabinergic ligands as biased agonists. We further discuss the receptor and ligand structural features allowing for selective activation of CBR-dependent functional responses. The design and development of biased ligands may offer a pathway to therapeutic success for novel CBR-targeted drugs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Also expressed in various peripheral tissues, the type-1 cannabinoid receptor (CB1R) is the predominant G protein-coupled receptor (GPCR) in brain, where it is responsible for retrograde control of neurotransmitter release. Cellular signaling mediated by CB1R is involved in numerous physiological processes, and pharmacological CB1R modulation is considered a tenable therapeutic approach for diseases ranging from substance-use disorders and glaucoma to metabolic syndrome. Despite the design and synthesis of a variety of bioactive small molecules targeted to the CB1R orthosteric ligand-binding site, the potential of CB1R as a therapeutic GPCR has been largely unrealized due to adverse events associated with typical orthosteric CB1R agonists and antagonists/inverse agonists. Modulation of CB1R-mediated signal transmission by targeting alternative allosteric ligand-binding site(s) on the receptor has garnered interest as a potentially safer and more effective therapeutic modality. This chapter highlights the design and synthesis of novel, pharmacologically active CB1R allosteric modulators and emphasizes how their molecular properties and the positive and negative allosteric control they exert can lead to improved CB1R-targeted pharmacotherapeutics, as well as designer covalent probes that can be used to map CB1R allosteric binding domains and inform structure-based drug design., (© 2017 Elsevier Inc. All rights reserved.)

Detailed characterization of the ligand-binding motifs and structure-function correlates of the principal GPCRs of the endocannabinoid-signaling system, the cannabinoid 1 (CB1R) and cannabinoid 2 (CB2R) receptors, is essential to inform the rational design of drugs that modulate CB1R- and CB2R-dependent biosignaling for therapeutic gain. We discuss herein an experimental paradigm termed "ligand-assisted protein structure" (LAPS) that affords a means of characterizing, at the amino acid level, CB1R and CB2R structural features key to ligand engagement and receptor-dependent information transmission. For this purpose, LAPS integrates three key disciplines and methodologies: (a) medicinal chemistry: design and synthesis of high-affinity, pharmacologically active probes as reporters capable of reacting irreversibly with particular amino acids at (or in the immediate vicinity of) the ligand-binding domain of the functionally active receptor; (b) molecular and cellular biology: introduction of discrete, conservative point mutations into the target GPCR and determination of their effect on probe binding and pharmacological activity; (c) analytical chemistry: identification of the site(s) of probe-GPCR interaction through focused, bottom-up, amino acid-level proteomic identification of the probe-receptor complex using liquid chromatography tandem mass spectrometry. Subsequent in silico methods including ligand docking and computational modeling provide supplementary data on the probe-receptor interaction as defined by LAPS. Examples of LAPS as applied to human CB2R orthosteric binding site characterization for a biarylpyrazole antagonist/inverse agonist and a classical cannabinoid agonist belonging to distinct chemical classes of cannabinergic compounds are given as paradigms for further application of this methodology to other therapeutic protein targets. LAPS is well positioned to complement other experimental and in silico methods in contemporary structural biology such as X-ray crystallography., (© 2017 Elsevier Inc. All rights reserved.)

Introduction: Allosteric modulators of G-protein coupled receptors (GPCRs) hold the promise of improved pharmacology and safety over typical orthosteric GPCR ligands. These features are particularly relevant to the cannabinoid receptor 1 (CB1R) GPCR, since typical orthosteric CB1R ligands are associated with adverse events that limit their translational potential. Areas covered: The contextual basis for applying allostery to CB1R is considered from pharmacological, drug-discovery, and medicinal standpoints. Rational design of small-molecule CB1R allosteric modulators as potential pharmacotherapeutics would be greatly facilitated by direct experimental characterization of structure-function correlates underlying the biological activity of chemically-diverse CB1R allosteric modulators, CB1R allosteric ligand-binding binding pockets, and amino acid contact residues critical to allosteric ligand engagement and activity. In these regards, designer covalent probes exhibiting well-characterized molecular pharmacology as CB1R allosteric modulators are emerging as valuable molecular reporters enabling experimental interrogation of CB1R allosteric site(s) and informing the design of new CB1R agents as drugs. Expert opinion: Synthesis and pharmacological profiling of CB1R allosteric ligands will continue to provide valuable insights into CB1R structure-function correlates. The resulting data should expand the repertoire of novel agents capable of exerting therapeutic benefit by modulating CB1R-dependent signaling.

Introduction: Drug discovery depends critically upon published results from the academy. The reproducibility of preclinical research findings reported by academia in the peer-reviewed literature has been called into question, seriously jeopardizing the value of academic science for inventing therapeutics., Areas Covered: The corrosive effects of the reproducibility issue on drug discovery are considered. Purported correctives imposed upon academia from the outside deal mainly with expunging fraudulent literature and imposing punitive sanctions on the responsible authors. The salutary influence of such post facto actions on the reproducibility of discovery-relevant preclinical research data from academia appears limited. Rather, intentional doctoral-scientist education focused on data replicability and translationally-meaningful science and active participation of university entities charged with research innovation and asset commercialization toward ensuring data quality are advocated as key academic initiatives for addressing the reproducibility issue., Expert Opinion: A mindset shift on the part of both senior university faculty and the academy to take responsibility for the data reproducibility crisis and commit proactively to positive educational, incentivization, and risk- and reward-sharing practices will be fundamental for improving the value of published preclinical academic research to drug discovery.

Endothelial dysfunction has been implicated in the pathophysiology of multiple cardiovascular diseases and involves components of both innate and acquired immune mechanisms. Identifying signature patterns and targets associated with endothelial dysfunction can help in the development of novel nanotherapeutic platforms for treatment of vascular diseases. This review discusses nucleic acid-based regulation of endothelial function and the different nucleic acid-based nanotherapeutic approaches designed to target endothelial dysfunction in cardiovascular disorders.

One of the most abundant G-protein coupled receptors (GPCRs) in brain, the cannabinoid 1 receptor (CB1R), is a tractable therapeutic target for treating diverse psychobehavioral and somatic disorders. Adverse on-target effects associated with small-molecule CB1R orthosteric agonists and inverse agonists/antagonists have plagued their translational potential. Allosteric CB1R modulators offer a potentially safer modality through which CB1R signaling may be directed for therapeutic benefit. Rational design of candidate, druglike CB1R allosteric modulators requires greater understanding of the architecture of the CB1R allosteric endodomain(s) and the capacity of CB1R allosteric ligands to tune the receptor's information output. We have recently reported the synthesis of a focused library of rationally designed, covalent analogues of Org27569 and PSNCBAM-1, two prototypic CB1R negative allosteric modulators (NAMs). Among the novel, pharmacologically active CB1R NAMs reported, the isothiocyanate GAT100 emerged as the lead by virtue of its exceptional potency in the [(35)S]GTPγS and β-arrestin signaling assays and its ability to label CB1R as a covalent allosteric probe with significantly reduced inverse agonism in the [(35)S]GTPγS assay as compared to Org27569. We report here a comprehensive functional profiling of GAT100 across an array of important downstream cell-signaling pathways and analysis of its potential orthosteric probe-dependence and signaling bias. The results demonstrate that GAT100 is a NAM of the orthosteric CB1R agonist CP55,940 and the endocannabinoids 2-arachidonoylglycerol and anandamide for β-arrestin1 recruitment, PLCβ3 and ERK1/2 phosphorylation, cAMP accumulation, and CB1R internalization in HEK293A cells overexpressing CB1R and in Neuro2a and STHdh(Q7/Q7) cells endogenously expressing CB1R. Distinctively, GAT100 was a more potent and efficacious CB1R NAM than Org27569 and PSNCBAM-1 in all signaling assays and did not exhibit the inverse agonism associated with Org27569 and PSNCBAM-1. Computational docking studies implicate C7.38(382) as a key feature of GAT100 ligand-binding motif. These data help inform the engineering of newer-generation, druggable CB1R allosteric modulators and demonstrate the utility of GAT100 as a covalent probe for mapping structure-function correlates characteristic of the druggable CB1R allosteric space.

The serine hydrolase monoacylglycerol lipase (MGL) functions as the main metabolizing enzyme of 2-arachidonoyl glycerol, an endocannabinoid signaling lipid whose elevation through genetic or pharmacological MGL ablation exerts therapeutic effects in various preclinical disease models. To inform structure-based MGL inhibitor design, we report the direct NMR detection of a reversible equilibrium between active and inactive states of human MGL (hMGL) that is slow on the NMR time scale and can be modulated in a controlled manner by pH, temperature, and select point mutations. Kinetic measurements revealed that hMGL substrate turnover is rate-limited across this equilibrium. We identify a network of aromatic interactions and hydrogen bonds that regulates hMGL active-inactive state interconversion. The data highlight specific inter-residue interactions within hMGL modulating the enzymes function and implicate transitions between active (open) and inactive (closed) states of the hMGL lid domain in controlling substrate access to the enzymes active site., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Atherosclerosis and its consequences remain prevalent clinical challenges throughout the world. Initiation and progression of atherosclerosis involves a complex, dynamic interplay among inflammation, hyperlipidemia, and endothelial dysfunction. A multicomponent treatment approach targeted for delivery within diseased vessels could prove beneficial in treating atherosclerosis. This study was undertaken to evaluate the multimodal effects of a novel ω-3-fatty acid-rich, 17-β-estradiol (17-βE)-loaded, CREKA-peptide-modified nanoemulsion system on experimental atherosclerosis. In vitro treatment of cultured human aortic endothelial cells (ECs) with the 17-βE-loaded, CREKA-peptide-modified nanoemulsion system increased cellular nitrate/nitrite, indicating improved nitric oxide formation. In vivo, systemic administration of this nanoemulsion system to apolipoprotein-E knock out (ApoE-/-) mice fed a high-fat diet significantly improved multiple parameters related to the etiology and development of occlusive atherosclerotic vasculopathy: lesion area, circulating plasma lipid levels, and expression of aortic-wall inflammatory markers. These salutary effects were attributed selectively to the 17-βE and/or ω-3 polyunsaturated fatty acid components of the nano-delivery system. At therapeutic doses, the 17-βE-loaded, CREKA-peptide modified nanoemulsion system appeared to be biocompatible in that it elicited no apparent adverse/toxic effects, as indexed by body weight, plasma alanine aminotransferase/aspartate aminotransferase levels, and liver and kidney histopathology. The study demonstrates the therapeutic potential of a novel, 17-βE-loaded, CREKA-peptide-modified nanoemulsion system against atherosclerosis in a multimodal fashion by reducing lesion size, lowering the levels of circulating plasma lipids and decreasing the gene expression of inflammatory markers associated with the disease.

Non-genomic membrane effects of estrogens are of great interest because of the diverse biological activities they may elicit. To further our understanding of the molecular features of the interaction between estrogenic hormones and membrane bilayers, we have determined the preferred orientation, location, and dynamic properties of 17β-estradiol (E2) in two different phospholipid membrane environments using (2)H-NMR and 2D (1)H-(13)C HSQC in conjunction with molecular dynamics simulations. Unequivocal spectral assignments to specific (2)H labels were made possible by synthesizing six selectively deuterated E2 molecules. The data allow us to conclude that the E2 molecule adopts a nearly "horizontal" orientation in the membrane bilayer with its long axis essentially perpendicular to the lipid acyl-chains. All four rings of the E2 molecule are located near the membrane interface, allowing both the E2 3-OH and the 17β-OH groups to engage in hydrogen bonding and electrostatic interactions with polar phospholipid groups. The findings augment our knowledge of the molecular interactions between E2 and membrane bilayer and highlight the asymmetric nature of the dynamic motions of the rigid E2 molecule in a membrane environment., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Undesirable side effects associated with orthosteric agonists/antagonists of cannabinoid 1 receptor (CB1R), a tractable target for treating several pathologies affecting humans, have greatly limited their translational potential. Recent discovery of CB1R negative allosteric modulators (NAMs) has renewed interest in CB1R by offering a potentially safer therapeutic avenue. To elucidate the CB1R allosteric binding motif and thereby facilitate rational drug discovery, we report the synthesis and biochemical characterization of first covalent ligands designed to bind irreversibly to the CB1R allosteric site. Either an electrophilic or a photoactivatable group was introduced at key positions of two classical CB1R NAMs: Org27569 (1) and PSNCBAM-1 (2). Among these, 20 (GAT100) emerged as the most potent NAM in functional assays, did not exhibit inverse agonism, and behaved as a robust positive allosteric modulator of binding of orthosteric agonist CP55,940. This novel covalent probe can serve as a useful tool for characterizing CB1R allosteric ligand-binding motifs.

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.

Endocananbnoid-system G-protein coupled receptors (GPCRs) and transient receptor potential (TRP) cation channels are critical components of cellular biosignaling networks. These plasma-membrane proteins are pleiotropic in their ability to interact with and engage structurally diverse ligands. The endocannabinoid and TRP signaling systems overlap in their recognition properties with respect to select naturally occurring plant-derived ligands that belong to the terpene and lipid chemical classes, the overlap establishing a physiological connectivity between these two ubiquitous cell-signaling systems. Identification and pharmacological profiling of phytochemicals engaged by cannabinoid GPCRs and/or TRP channels has inspired the synthesis of novel designer ligands that interact with cannabinoid receptors and/or TRP channels as xenobiotics. Functional interplay between the endocannabinoid and TRP-channel signaling systems is responsible for the antinocifensive action of some synthetic cananbinoids (WIN55,212-2 and AM1241), vasorelaxation by the endocannabinoid N-arachidonylethanolamide (anandamide), and the pain-relief afforded by the synthetic anandamide analogue N-arachidonoylaminophenol (AM404), the active metabolite of the widely used nonprescription analgesic and antipyretic acetaminophen (paracetamol). The biological actions of some plant-derived cannabinoid-receptor (e.g., Δ(9)-tetrahydrocannabinol) or TRP-channel (e.g,, menthol) ligands either carry abuse potential themselves or promote the use of other addictive substances, suggesting the therapeutic potential for modulating these signaling systems for abuse-related disorders. The pleiotropic nature of and therapeutically relevant interactions between cananbinergic and TRP-channel signaling suggest the possibility of dual-acting ligands as drugs.

Introduction: The significant contribution of substance-use disorders (SUDs) to the global-disease burden and associated unmet medical needs has not engendered a commensurate level of pharma-industry research and development (R&D) for novel SUD therapeutics invention. Analysis of contextual factors shaping this position suggests potential routes toward incentivizing R&D commitment for that purpose., Areas Covered: This article considers multiple primary factors that have consorted to disincentivize pharma industry's operating in the SUD space: ill-understood pathology; variegated treatments and patient profiles; involved clinical trials; and - with particular reference to SUDs-negative cultural/business stigmas and shallow commercial precedent. Industry incentivization for SUD drug innovation requires progress on several fronts, including: translational experimental data and systems; personalized, holistic SUD treatment approaches; interactions among pharma, nonindustry constituencies, and the medical profession with vested interests in countering negative stereotypes and expanding SUD treatment options; and public-private alliances focused on improving SUD pharmacotherapy., Expert Opinion: Given the well-entrenched business stance whereby the prospect of future profits in major markets largely determines drug-company R&D investment trajectory, strategic initiatives offering substantial reductions in the risks and opportunity (i.e., time and money) costs associated with SUD drug discovery are likely to be the most potent drivers for encouraging mainstream industry positioning in this therapeutic area. Such initiatives could originate from front-loaded R&D operational and back-loaded patent, regulatory, marketing and health-care policy reforms. These may be too involved and protracted for the turbulent pharmaceutical industry to entertain amid its recent retrenchment from psychiatric/CNS diseases and intense pressures to increase productivity and shareholder value.

Delayed endothelial cell (EC) regeneration and the medial vascular smooth muscle cells (VSMCs) proliferation contribute to arterial restenosis. Although ω-3-polyunsaturated fatty acids (PUFAs), 17β-estradiol (17-βE) and C6-ceramide (CER) have shown therapeutic promise in addressing restenosis, extensive protein binding and lipophilicity complicate their (co-)delivery to cellular targets. We report engineering of an ω-3-PUFA-rich oil-in-water nanoemulsion formulation that effectively delivers 17-βE and CER cargo to cultured vascular cells. The cargo-free, ω-3-PUFA-rich nanoemulsion itself typically reduced growth factor-stimulated cellular proliferation, as did nanoemulsion-delivered CER alone, through enhanced pro-apoptotic caspase 3/7 activity. 17-βE loaded nanoemulsion inhibited VSMC proliferation and supported EC proliferation, responses associated with the mitogen-activated-protein-kinase (MAPK) signaling. Co-administration of 17-βE and CER loaded nanoemulsions exerted an anti-proliferative effect more pronounced on VSMCs than ECs. These therapeutically beneficial responses to ω-3-PUFA, CER, and/or 17-βE in our nanoemulsion formulation invite evaluation of this novel approach in animal models of restenosis and other occlusive vasculopathies., From the Clinical Editor: This team of investigators report the engineering of an ω-3-PUFA-rich oil-in-water nanoemulsion formulation that effectively delivers 17-βE and C6-ceramide cargo to cultured vascular cells in an effort to address vascular restenosis. Further preclinical studies will be needed in animal models before this approach could be considered for clinical trials., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Human monoacylglycerol lipase (hMGL) regulates endocannabinoid signaling primarily by deactivating the lipid messenger 2-arachidonoylglycerol. Agents that carbamylate hMGLs catalytic Ser(122) constitute a leading class of therapeutically promising hMGL inhibitors. We have applied peptide-level hydrogen/deuterium exchange mass spectrometry to characterize hMGL's conformational responses to two potent carbamylating inhibitors, AM6580 (irreversible) and AM6701 (slowly reversible). A dynamic, solvent-exposed lid domain is characteristic of hMGL's solution conformation. Both hMGL inhibitors restricted backbone enzyme motility in the active-site region and increased substrate binding-pocket solvent exposure. Covalent reaction of AM6580 with hMGL generates a bulkier carbamylated Ser(122) residue as compared to the more discrete Ser(122) modification by AM6701, a difference reflected in AM6580's more pronounced effect upon hMGL conformation. We demonstrate that structurally distinct carbamylating hMGL inhibitors generate particular conformational ensembles characterized by region-specific hMGL dynamics. By demonstrating the distinctive influences of two hMGL inhibitors on enzyme conformation, this study furthers our understanding at the molecular level of the dynamic features of hMGL interaction with small-molecule ligands.

The membrane-associated serine hydrolase, monoacylglycerol lipase (MGL), is a well-recognized therapeutic target that regulates endocannabinoid signaling. Crystallographic studies, while providing structural information about static MGL states, offer no direct experimental insight into the impact of MGL's membrane association upon its structure-function landscape. We report application of phospholipid bilayer nanodiscs as biomembrane models with which to evaluate the effect of a membrane system on the catalytic properties and conformational dynamics of human MGL (hMGL). Anionic and charge-neutral phospholipid bilayer nanodiscs enhanced hMGL's kinetic properties [apparent maximum velocity (Vmax) and substrate affinity (Km)]. Hydrogen exchange mass spectrometry (HX MS) was used as a conformational analysis method to profile experimentally the extent of hMGL-nanodisc interaction and its impact upon hMGL structure. We provide evidence that significant regions of hMGL lid-domain helix α4 and neighboring helix α6 interact with the nanodisc phospholipid bilayer, anchoring hMGL in a more open conformation to facilitate ligand access to the enzyme's substrate-binding channel. Covalent modification of membrane-associated hMGL by the irreversible carbamate inhibitor, AM6580, shielded the active site region, but did not increase solvent exposure of the lid domain, suggesting that the inactive, carbamylated enzyme remains intact and membrane associated. Molecular dynamics simulations generated conformational models congruent with the open, membrane-associated topology of active and inhibited, covalently-modified hMGL. Our data indicate that hMGL interaction with a phospholipid membrane bilayer induces regional changes in the enzyme's conformation that favor its recruiting lipophilic substrate/inhibitor from membrane stores to the active site via the lid, resulting in enhanced hMGL catalytic activity and substrate affinity., (© 2013 The Protein Society.)

Aims: AM-1241, a novel, racemic cannabinoid-2 receptor (CB2) ligand, is the primary experimental agonist used to characterize the role of CB2-mediated lipid signaling in health and disease, including substance abuse disorders. In vivo pharmacological effects have been used as indirect proxies for AM-1241 biotransformation processes that could modulate CB2 activity. We report the initial pre-clinical characterization of AM-1241 biotransformation and in vivo distribution., Main Methods: AM-1241 metabolism was characterized in a variety of predictive in vitro systems (Caco-2 cells; mouse, rat and human microsomes) and in the mouse in vivo. Liquid chromatography and mass spectrometry techniques were used to quantify AM-1241 tissue distribution and metabolic conversion., Key Findings: AM-1241 bound extensively to plasma protein/albumin. A pharmacological AM-1241 dose (25mg/kg, i.v.) was administered to mice for direct determination of its plasma half-life (37 min), following which AM-1241 was quantified in brain, spleen, liver, and kidney. After p.o. administration, AM-1241 was detected in plasma, spleen, and kidney; its oral bioavailability was ~21%. From Caco-2 permeability studies and microsomal-based hepatic clearance estimates, in vivo AM-1241 absorption was moderate. Hepatic microsomal metabolism of AM-1241 in vitro generated hydroxylation and demethylation metabolites. Species-dependent differences were discovered in AM-1241's predicted hepatic clearance. Our data demonstrate that AM-1241 has the following characteristics: a) short plasma half-life; b) limited oral bioavailability; c) extensive plasma/albumin binding; d) metabolic substrate for hepatic hydroxylation and demethylation; e) moderate hepatic clearance., Significance: These results should help inform the design, optimization, and pre-clinical profiling of CB2 ligands as pharmacological tools and medicines., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In the mammalian central nervous system, monoacylglycerol lipase (MGL) is principally responsible for inactivating the endocannabinoid signaling lipid 2-arachidonoylglycerol (2-AG) and modulates cannabinoid-1 receptor (CB1R) desensitization and signal intensity. MGL is also a drug target for diseases in which CB1R stimulation may be therapeutic. To inform the design of human MGL (hMGL) inhibitors, we have engineered a Leu(Leu(169);Leu(176))-to-Ser(Ser(169);Ser(176)) double hMGL mutant (sol-hMGL) which exhibited enhanced solubility properties, and we further mutated this variant by substituting its catalytic-triad Ser(122) with Cys (sol-S-hMGL). The hMGL variants hydrolyzed both 2-AG and a fluorogenic reporter substrate with comparable affinities. Our results suggest that the hMGL cysteine mutant maintains the same overall architecture as wild-type hMGL. The results also underscore the superior nucleophilic nature of the reactive catalytic Ser(122) residue as compared to that of Cys(122) in the sol-S-hMGL mutant and suggest that the nucleophilic character of the Cys(122) residue is not commensurately enhanced within the three dimensional architecture of hMGL. The interaction of the sol-hMGL variants with the irreversible inhibitors AM6580 and N-arachidonylmaleimide (NAM) and the reversible inhibitor AM10212 was profiled. LC/MS analysis of tryptic digests from sol-S-hMGL directly demonstrate covalent modification of this variant by NAM and AM6580, consistent with enzyme thiol alkylation and carbamoylation, respectively. These data provide insight into hMGL catalysis, the key role of the nucleophilic character of Ser(122), and the mechanisms underlying hMGL inhibition by different classes of small molecules.

Introduction: Addiction to chemical substances with abuse potential presents medical needs largely unsolved by extant therapeutic strategies. Signal transmission through the cannabinoid-1 receptor (CB1R) in the central nervous system (CNS) modulates neurotransmitters/neuronal pathways contributing to the rewarding properties and hedonic effects of certain nondrug stimuli (e.g., food) and many prototypical addictive drugs, promoting excessive intake and its pathological consequences. Typical CB1R antagonists/inverse agonists reduce the rewarding effects and normalize behavioral phenotypes associated with food and abused drugs, but carry an unacceptable adverse-event profile that may reflect, at least partly, their intrinsic ability to alter basal homeostatic CB1R signaling in the CNS and elicit a negative efficacy response. Alternatively, peripherally biased CB1R inverse agonists with limited CNS permeability and putative CB1R neutral antagonists expressing modest (if any) inverse-agonist efficacy are garnering attention for treating obesity and related cardiometabolic complications with a potentially enhanced benefit-to-risk profile., Areas Covered: This mini-review calls attention to the proposition that CB1R neutral antagonists offer attractive opportunities for pharmacotherapeutic exploitation in the substance abuse/drug addiction space, whereas the restricted CNS accessibility of peripherally biased CB1R inverse agonists circumscribes their therapeutic utility for this indication., Expert Opinion: The unique preclinical pharmacology, efficacy profiles, and reduced adverse-event risk of CB1R neutral antagonists make them worthy of translational study for their potential therapeutic application beyond obesity/cardiometabolic disease to include substance-abuse/drug-addiction disorders.

Introduction: A functionally compromised vascular endothelium is associated with tissue-damaging responses including inflammation, immune stimulation, oxidative stress and platelet activation/aggregation and can lead to severe end-organ damage, as implicated in the pathology of several cardiac, cerebral and renal disorders. Multiple noninvasive techniques are available for assessing endothelial dysfunction in clinical settings. Diverse interventions have been identified as having therapeutic potential for treating endothelial dysfunction and preventing its pathophysiological sequellae., Areas Covered: Evaluation techniques and interventional treatment approaches for endothelial dysfunction, with particular reference to prevalent cardiovascular and metabolic disorders such as coronary artery disease and diabetes. Limitations of the current treatments and avenues for improved endothelium-targeted therapies., Expert Opinion: Beneficial pleiotropic effects of various agents (cardiovascular medicines, antioxidants, nutritional supplements) on vascular endothelial function in humans notwithstanding, a growing body of preclinical data suggests that protein-, cell- and gene-based approaches hold promise for selective therapeutic targeting of the dysfunctional vascular endothelium. Additional efficacy data in appropriate animal models of vascular injury and cardiometabolic disease, further refinement of delivery modalities and continued investigation of the mechanisms underlying endothelial repair and regeneration should help identify the most promising therapeutic approaches for improving endothelial function that merit evaluation in human trials.

The lack of experimental characterization of the structures and ligand-binding motifs of therapeutic G-protein coupled receptors (GPCRs) hampers rational drug discovery. The human cannabinoid receptor 2 (hCB2R) is a class-A GPCR and promising therapeutic target for small-molecule cannabinergic agonists as medicines. Prior mutational and modeling data constitute provisional evidence that AM-841, a high-affinity classical cannabinoid, interacts with cysteine C6.47(257) in hCB2R transmembrane helix 6 (TMH6) to afford improved hCB2R selectivity and unprecedented agonist potency. We now apply bottom-up mass spectrometry (MS)-based proteomics to define directly the hCB2R-AM-841 interaction at the amino-acid level. Recombinant hCB2R, overexpressed as an N-terminal FLAG-tagged/C-terminal 6His-tagged protein (FLAG-hCB2R-6His) with a baculovirus system, was solubilized and purified by immunochromatography as functional receptor. A multiplex multiple reaction monitoring (MRM)-MS method was developed that allowed us to observe unambiguously all seven discrete TMH peptides in the tryptic digest of purified FLAG-hCB2R-6His and demonstrate that AM-841 modifies hCB2R TMH6 exclusively. High-resolution mass spectra of the TMH6 tryptic peptide obtained by Q-TOF MS/MS analysis demonstrated that AM-841 covalently and selectively modifies hCB2R at TMH6 cysteine C6.47(257). These data demonstrate how integration of MS-based proteomics into a ligand-assisted protein structure (LAPS) experimental paradigm can offer guidance to structure-enabled GPCR agonist design.

Introduction: Obesity and related cardiometabolic derangements are spiraling global health problems urgently in need of safe, effective and durable pharmacotherapy., Areas Covered: As an orexigenic and anabolic biosignaling network, the endocannabinoid system interacts with other information-transducing pathways to help ensure metabolic homeostasis. Hyperphagia stimulates reinforcing neuronal circuits favoring energy intake and conservation, inviting overweight/obesity and cardiometabolic risk factors ('metabolic syndrome'). Associated increases in cannabinoid 1 G protein-coupled receptor (CB1R) activity/expression further exacerbate food consumption and the metabolic shift toward fat production and accumulation. The role of CB1R activity in hyperphagia and weight gain spurred the development of rimonabant (SR141716; Acomplia), the first-in-class CB1R antagonist/inverse agonist weight-loss drug. Rimonabant and similar CB1R inverse agonists also exert pleiotropic actions in addition to weight-loss effects that help correct obesity-related metabolic derangements and reduce cardiovascular risk in humans. The medicinal utility of these agents was crippled by clinically significant central and peripheral adverse effects that appear to reflect CB1R inverse agonists as a class. Consequently, increased attention is being given to CB1R neutral antagonists, CB1R blockers with intrinsically weak, if any, functional potency to elicit the negative-efficacy responses associated with inverse agonists. Laboratory studies demonstrate that CB1R neutral antagonists - whether readily accessible to the central nervous system or not (i.e., 'periphero-neutral' antagonists) - retain the salient therapeutic effects of CB1R inverse agonists on hyperphagia, weight-gain, and obesity-driven metabolic abnormalities with the distinct advantage of being associated with significantly less preclinical adverse events than are conventional CB1R inverse agonists such as rimonabant., Expert Opinion: CB1R (periphero-)neutral antagonists merit continued analysis of their molecular pharmacology and evaluation of their therapeutic significance and translational potential as new-generation medicines for obesity-related derangements, including nonalcoholic fatty liver disease and type 2 diabetes, if not obesity itself.

G protein-coupled receptors (GPCRs) play critical physiological and therapeutic roles. The human cannabinoid 1 GPCR (hCB1) is a prime pharmacotherapeutic target for addiction and cardiometabolic disease. Our prior biophysical studies on the structural biology of a synthetic peptide representing the functionally significant hCB1 transmembrane helix 7 (TMH7) and its cytoplasmic extension, helix 8 (H8), [hCB1(TMH7/H8)] demonstrated that the helices are oriented virtually perpendicular to each other in membrane-mimetic environments. We identified several hCB1(TMH7/H8) structure-function determinants, including multiple electrostatic amino-acid interactions and a proline kink involving the highly conserved NPXXY motif. In phospholipid bicelles, TMH7 structure, orientation, and topology relative to H8 are dynamically modulated by the surrounding membrane phospholipid bilayer. These data provide a contextual basis for the present solid-state NMR study to investigate whether intermolecular interactions between hCB1(TMH7/H8) and its phospholipid environment may affect membrane-bilayer structure. For this purpose, we measured (1)H-(13)C heteronuclear dipolar couplings for the choline, glycerol, and acyl-chain regions of dimyristoylphosphocholine in a magnetically aligned hCB1(TMH7/H8) bicelle sample. The results identify discrete regional interactions between hCB1(TMH7/H8) and membrane lipid molecules that increase phospholipid motion and decrease phospholipid order, indicating that the peptide's partial traversal of the bilayer alters membrane structure. These data offer new insight into hCB1(TMH7/H8) properties and support the concept that the membrane bilayer itself may serve as a mechanochemical mediator of hCB1/GPCR signal transduction. Since interaction with its membrane environment has been implicated in hCB1 function and its modulation by small-molecule therapeutics, our work should help inform hCB1 pharmacology and the design of hCB1-targeted drugs.

The human cannabinoid 2 GPCR (hCB2) is a prime therapeutic target. To define potential cysteine-related binding motifs critical to hCB2-ligand interaction, a library of hCB2 cysteine-substitution mutants and a novel, high-affinity biarylpyrazole hCB2 antagonist/inverse agonist (AM1336) functionalized to serve as a covalent affinity probe to target cysteine residues within (or in the microenvironment of) its hCB2 binding pocket were generated. The data provide direct experimental demonstration that both hCB2 TMH7 cysteines [i.e., C7.38(284) and C7.42(288)] are critical to optimal hCB2-AM1336 binding interaction and AM1336 pharmacological activity in a cell-based functional assay (cAMP formation). Elongating the AM1336 aliphatic side chain generated another novel hCB2 inverse agonist that binds covalently and selectively to C7.42(288) only. Identification of specific cysteine residues critical to hCB2 ligand interaction and function informs the structure-based design of hCB2-targeted medicines., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Intramolecular hydrogen bonding is an important determinant of enzyme structure, catalysis, and inhibitor action. Monoacylglycerol lipase (MGL) modulates cannabinergic signaling as the main enzyme responsible for deactivating 2-arachidonoylglycerol (2-AG), a primary endocannabinoid lipid messenger. By enhancing tissue-protective 2-AG tone, targeted MGL inhibitors hold therapeutic promise for managing pain and treating inflammatory and neurodegenerative diseases. We report study of purified, solubilized human MGL (hMGL) to explore the details of hMGL catalysis by using two known covalent hMGL inhibitors, the carbamoyl tetrazole AM6701 and N-arachidonoylmaleimide (NAM), that act through distinct mechanisms. Using proton nuclear magnetic resonance spectroscopy (NMR) with purified wild-type and mutant hMGLs, we have directly observed a strong hydrogen-bond network involving Asp239 and His269 of the catalytic triad and neighboring Leu241 and Cys242 residues. hMGL inhibition by AM6701 alters this hydrogen-bonding pattern through subtle active-site structural rearrangements without influencing hydrogen-bond occupancies. Rapid carbamoylation of hMGL Ser122 by AM6701 and elimination of the leaving group is followed by a slow hydrolysis of the carbamate group, ultimately regenerating catalytically competent hMGL. In contrast, hMGL titration with NAM, which leads to cysteine alkylation, stoichiometrically decreases the population of the active-site hydrogen bonds. NAM prevents reformation of this network, and in this manner inhibits hMGL irreversibly. These data provide detailed molecular insight into the distinctive mechanisms of two covalent hMGL inhibitors and implicate a hydrogen-bond network as a structural feature of hMGL catalytic function.

The endocannabinoid metabolome consists of a growing, (patho)physiologically important family of fatty-acid derived signaling lipids. Diet is a major source of fatty acid substrate for mammalian endocannabinoid biosynthesis. The principal long-chain PUFA found in mammalian brain, docosahexaenoic acid (DHA), supports neurological function, retinal development, and overall health. The extent to which dietary DHA supplementation influences endocannabinoid-related metabolites in brain, within the context of the circulating endocannabinoid profile, is currently unknown. We report the first lipidomic analysis of acute 2-week DHA dietary supplementation effects on the physiological state of 15 fatty-acid, N-acylethanolamine, and glycerol-ester endocannabinoid metabolome constituents in murine plasma and brain. The DHA-rich diet markedly elevated DHA, eicosapentaenoic acid, 2-eicosapentanoylglycerol (EPG), and docosahexanoylethanolamine in both compartments. Dietary DHA enhancement generally affected the synthesis of the N-acyl-ethanolamine and glycerol-ester metabolites to favor the docosahexaenoic and eicosapentaenoic vs. arachidonoyl and oleoyl homologs in both brain and plasma. The greater overall responsiveness of the endocannabinoid metabolome in plasma versus brain may reflect a more circumscribed homeostatic response range of brain lipids to dietary DHA supplementation. The ability of short-term DHA enhancement to modulate select constituents of the physiological brain and plasma endocannabinoid metabolomes carries metabolic and therapeutic implications.

Aims: This review posits that fatty acid amide hydrolase (FAAH) inhibition has therapeutic potential against neuropathological states including traumatic brain injury; Alzheimer's, Huntington's, and Parkinson's diseases; and stroke., Main Methods: This proposition is supported by data from numerous in vitro and in vivo experiments establishing metabolic and pharmacological contexts for the neuroprotective role of the endogenous cannabinoid ("endocannabinoid") system and selective FAAH inhibitors., Key Findings: The systems biology of endocannabinoid signaling involves two main cannabinoid receptors, the principal endocannabinoid lipid mediators N-arachidonoylethanolamine ("anandamide") (AEA) and 2-arachidonoyl glycerol (2-AG), related metabolites, and the proteins involved in endocannabinoid biosynthesis, biotransformation, and transit. The endocannabinoid system is capable of activating distinct signaling pathways on-demand in response to pathogenic events or stimuli, thereby enhancing cell survival and promoting tissue repair. Accumulating data suggest that endocannabinoid system modulation at discrete targets is a promising pharmacotherapeutic strategy for treating various medical conditions. In particular, neuronal injury activates cannabinoid signaling in the central nervous system as an intrinsic neuroprotective response. Indirect potentiation of this salutary response through pharmacological inhibition of FAAH, an endocannabinoid-deactivating enzyme, and consequent activation of signaling pathways downstream from cannabinoid receptors have been shown to promote neuronal maintenance and function., Significance: This therapeutic modality has the potential to offer site- and event-specific neuroprotection under conditions where endocannabinoids are being produced as part of a physiological protective mechanism. In contrast, direct application of cannabinoid receptor agonists to the central nervous system may activate CB receptors indiscriminately and invite unwanted psychotrophic effects., (Copyright 2009 Elsevier Inc. All rights reserved.)

The human cannabinoid 1 receptor (hCB1), a ubiquitous G protein-coupled receptor (GPCR), transmits cannabinergic signals that participate in diverse (patho)physiological processes. Pharmacotherapeutic hCB1 targeting is considered a tractable approach for treating such prevalent diseases as obesity, mood disorders, and drug addiction. The hydrophobic nature of the transmembrane helices of hCB1 presents a formidable difficulty to its direct structural analysis. Comprehensive experimental characterization of functional hCB1 by mass spectrometry (MS) is essential to the targeting of affinity probes that can be used to define directly hCB1 binding domains using a ligand-assisted experimental approach. Such information would greatly facilitate the rational design of hCB1-selective agonists/antagonists with therapeutic potential. We report the first high-coverage MS analysis of the primary sequence of the functional hCB1 receptor, one of the few such comprehensive MS-based analyses of any GPCR. Recombinant C-terminal hexa-histidine-tagged hCB1 (His6-hCB1) was expressed in cultured insect (Spodoptera frugiperda) cells, solubilized by a procedure devised to enhance receptor purity following metal-affinity chromatography, desalted by buffer exchange, and digested in solution with (chymo)trypsin. "Bottom-up" nanoLC-MS/MS of the (chymo)tryptic digests afforded a degree of overall hCB1 coverage (>94%) thus far reported for only two other GPCRs. This MS-compatible procedure devised for His6-hCB1 sample preparation, incorporating in-solution (chymo)trypsin digestion in the presence of a low concentration of CYMAL-5 detergent, may be applicable to the MS-based proteomic characterization of other GPCRs. This work should help enable future ligand-assisted structural characterization of hCB1 binding motifs at the amino-acid level using rationally designed and targeted covalent cannabinergic probes.

We report the NMR solution structure of a synthetic 40-mer (T(377)-E(416)) that encompasses human cannabinoid receptor-1 (hCB1) transmembrane helix 7 (TMH7) and helix 8 (H8) [hCB1(TMH7/H8)] in 30% trifluoroethanol/H(2)O. Structural features include, from the peptide's amino terminus, a hydrophobic alpha-helix (TMH7); a loop-like, 11 residue segment featuring a pronounced Pro-kink within the conserved NPxxY motif; a short amphipathic alpha-helix (H8) orthogonal to TMH7 with cationic and hydrophobic amino-acid clusters; and an unstructured C-terminal end. The hCB1(TMH7/H8) NMR solution structure suggests multiple electrostatic amino-acid interactions, including an intrahelical H8 salt bridge and a hydrogen-bond network involving the peptide's loop-like region. Potential cation-pi and cation-phenolic OH interactions between Y(397) in the TMH7 NPxxY motif and R(405) in H8 are identified as candidate structural forces promoting interhelical microdomain formation. This microdomain may function as a flexible molecular hinge during ligand-induced hCB1 conformer transitions.

We detail the structure and dynamics of a synthetic peptide corresponding to transmembrane helix 6 (TMH6) of human cannabinoid receptor-2 (hCB2) in biomembrane-mimetic environments. The peptide's NMR structural biology is characterized by two alpha-helical domains bridged by a flexible, nonhelical hinge region containing a highly-conserved CWFP motif with an environmentally sensitive, Pro-based conformational switch. Buried within the peptide's flexible region, W(258) may hydrogen-bond with L(255) to help stabilize the Pro-kinked hCB2 TMH6 structure and position C(257) advantageously for interaction with agonist ligands. These characteristics of hCB2 TMH6 are potential structural features of ligand-induced hCB2 activation in vivo.

The influence of membrane environment on human cannabinoid 1 (hCB(1)) receptor transmembrane helix (TMH) conformational dynamics was investigated by solid-state NMR and site-directed spin labeling/EPR with a synthetic peptide, hCB(1)(T377-E416), corresponding to the receptor's C-terminal component, i.e., TMH7 and its intracellular alpha-helical extension (H8) (TMH7/H8). Solid-state NMR experiments with mechanically aligned hCB(1)(T377-E416) specifically (2)H- or (15)N-labeled at Ala380 and reconstituted in membrane-mimetic dimyristoylphosphocholine (DMPC) or 1-palmitoyl-2-oleoyl-sn-glycerophosphocholine (POPC) bilayers demonstrate that the conformation of the TMH7/H8 peptide is more heterogeneous in the thinner DMPC bilayer than in the thicker POPC bilayer. As revealed by EPR studies on hCB(1)(T377-E416) spin-labeled at Cys382 and reconstituted into the phospholipid bilayers, the spin label partitions actively between hydrophobic and hydrophilic environments. In the DMPC bilayer, the hydrophobic component dominates, regardless of temperature. Mobility parameters (DeltaH(0)(-1)) are 0.3 and 0.73 G for the peptide in the DMPC or POPC bilayer environment, respectively. Interspin distances of doubly labeled hCB(1)(T377-E416) peptide reconstituted into a TFE/H(2)O mixture or a POPC or DMPC bilayer were estimated to be 10.6 +/- 0.5, 16.8 +/- 1, and 11.6 +/- 0.8 A, respectively. The extent of coupling (>or=50%) between spin labels located at i and i + 4 in a TFE/H(2)O mixture or a POPC bilayer is indicative of an alpha-helical TMH conformation, whereas the much lower coupling (14%) when the peptide is in a DMPC bilayer suggests a high degree of peptide conformational heterogeneity. These data demonstrate that hCB(1)(T377-E416) backbone dynamics as well as spin-label rotameric freedom are sensitive to and altered by the peptide's phospholipid bilayer environment, which exerts a dynamic influence on the conformation of a TMH critical to signal transmission by the hCB(1) receptor.

Little direct information is available regarding the influence of membrane environment on transmembrane (TM) G-protein-coupled receptor (GPCR) conformation and dynamics. The human CB1 cannabinoid receptor (hCB1) is a prominent GPCR pharmacotherapeutic target in which helix 7 appears critical to ligand recognition. We have chemically synthesized a hCB1 peptide corresponding to a segment of TM helix 7 and the entire contiguous helix 8 domain (fourth cytoplasmic loop) and reconstituted it in defined phospholipid-bilayer model membranes. Using an NMR-based strategy combined with molecular dynamics simulations, we provide the first direct experimental description of the orientation of hCB1 helix 7 in phospholipid membranes of varying thickness and the mechanism by which helix-7 conformation adjusts to avoid hydrophobic mismatch. Solid-state (15)N NMR data show that hCB1 helices 7 and 8 reconstituted into phospholipid bilayers are oriented in a TM and in-plane (i.e., parallel to the phospholipid membrane surface) fashion, respectively. TM helix orientation is influenced by the thickness of the hydrophobic membrane bilayer as well as the interaction of helix 8 with phospholipid polar headgroups. Molecular dynamics simulations show that a decrease in phospholipid chain-length induces a kink at P394 in TM helix 7 to avoid hydrophobic mismatch. Thus, the NP(X)nY motif found in hCB1 and highly conserved throughout the GPCR superfamily is important for flexing helix 7 to accommodate bilayer thickness. Dynamic modulation of hCB1-receptor TM helix conformation by its membrane environment may have general relevance to GPCR structure and function.