Aliphatic Azides as Selective Cysteine Labeling Reagents for Integral Membrane Proteins.

Upon ultraviolet activation, cannabinergic aliphatic azido (N3) 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.

The role of human monoacylglycerol lipase (hMAGL) binding pocket in breakup of unsaturated phospholipid membranes.

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.

Human Cannabinoid Receptor 2 Ligand-Interaction Motif: Transmembrane Helix 2 Cysteine, C2.59(89), as Determinant of Classical Cannabinoid Agonist Activity and Binding Pose.

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 [3H]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 [3H]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).

Mapping Cannabinoid 1 Receptor Allosteric Site(s): Critical Molecular Determinant and Signaling Profile of GAT100, a Novel, Potent, and Irreversibly Binding Probe.

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 ß-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 ß-arrestin1 recruitment, PLCß3 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.

Therapeutic Efficacy of an ω-3-Fatty Acid-Containing 17-ß Estradiol Nano-Delivery System against Experimental Atherosclerosis.

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-ß-estradiol (17-ßE)-loaded, CREKA-peptide-modified nanoemulsion system on experimental atherosclerosis. In vitro treatment of cultured human aortic endothelial cells (ECs) with the 17-ßE-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-ßE and/or ω-3 polyunsaturated fatty acid components of the nano-delivery system. At therapeutic doses, the 17-ßE-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-ßE-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.

Novel Electrophilic and Photoaffinity Covalent Probes for Mapping the Cannabinoid 1 Receptor Allosteric Site(s).

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.

17ß-estradiol (E2) in membranes: Orientation and dynamic properties.

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 17ß-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 17ß-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.

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.

Synthetic agents in the context of metabolic/bariatric surgery: expanding the scope and impact of diabetes drug discovery.

Type 2 diabetes (T2D) - particularly with concurrent obesity ('diabesity') - is an intensifying global public-health problem. Medical needs and market opportunities in the T2D space have propelled discovery efforts aimed at inventing new synthetic T2D drugs differentiable by improved safety and efficacy and/or the ability to modulate emerging T2D targets. Particularly for moderately and severely obese individuals, weight-loss (bariatric) surgery offers an effective means of reducing obesity-driven T2D that is superior in many respects to medical T2D management. Yet, not all overweight or obese individuals with T2D qualify for bariatric surgery, and current healthcare resources are inadequate for applying surgical T2D control to more than a very small segment of qualified patients. Bariatric surgery is no guarantee of 'curative' T2D abrogation, significant rates of T2D non-remission or re-emergence having been observed in diabesity patients following bariatric procedures. Preoperative glucose control by oral hypoglycemic drugs reduces the chance of T2D recurrence post-surgery, and diabesity patients in whom glycemic indices have been improved by bariatric surgery may still require some level of T2D pharmacotherapy. Laboratory and clinical data indicate that synthetic T2D drugs can improve T2D-related outcomes following bariatric procedures, and current T2D drug-discovery efforts are being informed by the metabolic advantages associated with bariatric surgery. These circumstances intensify the need for and extend the impact of T2D drug discovery by demonstrating multiple levels of interplay between medical and surgical approaches to improve the health of individuals with diabesity and, perhaps, approach the overarching goal of decreasing long-term cardiovascular mortality.

Engineering of an ω-3 polyunsaturated fatty acid-containing nanoemulsion system for combination C6-ceramide and 17ß-estradiol delivery and bioactivity in human vascular endothelial and smooth muscle cells.

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), 17ß-estradiol (17-ßE) 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-ßE 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-ßE loaded nanoemulsion inhibited VSMC proliferation and supported EC proliferation, responses associated with the mitogen-activated-protein-kinase (MAPK) signaling. Co-administration of 17-ßE 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-ßE 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-ßE 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.

Endocannabinoid enzyme engineering: soluble human thio-monoacylglycerol lipase (sol-S-hMGL).

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.

Therapeutic strategies for endothelial dysfunction.

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.

Mass spectrometry-based proteomics of human cannabinoid receptor 2: covalent cysteine 6.47(257)-ligand interaction affording megagonist receptor activation.

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.

hCB2 ligand-interaction landscape: cysteine residues critical to biarylpyrazole antagonist binding motif and receptor modulation.

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.

Identification by nuclear magnetic resonance spectroscopy of an active-site hydrogen-bond network in human monoacylglycerol lipase (hMGL): implications for hMGL dynamics, pharmacological inhibition, and catalytic mechanism.

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.

NMR solution structure of human cannabinoid receptor-1 helix 7/8 peptide: candidate electrostatic interactions and microdomain formation.

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.

Structural biology of human cannabinoid receptor-2 helix 6 in membrane-mimetic environments.

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.

Covalent inhibitors of human monoacylglycerol lipase: ligand-assisted characterization of the catalytic site by mass spectrometry and mutational analysis.

The active site of recombinant hexa-histidine-tagged human monoacylglycerol lipase (hMGL) is characterized by mass spectrometry using the inhibitors 5-((biphenyl-4-yl)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (AM6701), and N-arachidonylmaleimide (NAM) as probes. Carbamylation of Ser(129) by AM6701 in the putative hMGL catalytic triad demonstrates this residue's essential role in catalysis. Partial NAM alkylation of hMGL cysteine residues 215 and/or 249 was sufficient to achieve approximately 80% enzyme inhibition. Although Cys(215) and/or Cys(249) mutations to alanine(s) did not affect hMGL hydrolytic activity as compared with nonmutated hMGL, the C215A displayed heightened NAM sensitivity, whereas the C249A evidenced reduced NAM sensitivity. These data conclusively demonstrate a sulfhydryl-based mechanism for NAM inhibition of hMGL in which Cys(249) is of paramount importance. Identification of amino acids critical to the catalytic activity and pharmacological modulation of hMGL informs the design of selective MGL inhibitors as potential drugs.

Combination of paclitaxel and nitric oxide as a novel treatment for the reduction of restenosis.

The combination of a nitric oxide (NO) donor and a paclitaxel-NO donor conjugate coated on a vascular stent was tested in a rabbit iliac artery model of stenosis as a potential therapy for restenosis. Paclitaxel was conjugated with a NO donor at the 7-position to give compound 7. An adamantane-based NO donor 14 was synthesized and combined with 7 to provide a burst of NO in the first few critical hours following injury to the vessel wall. Both 7 and 14 demonstrated antiproliferative activity (IC(50) = 20 nM and 15 microM, respectively) and antiplatelet activity (IC(50) = 10 and 1 microM, respectively). Stents were coated with a layer of a polymer containing test compounds. The total amount of NO eluted from the stents after a 6 h implantation in the rabbit iliac artery was 35%, 95%, and 69% of the original content for the stents coated with 7, 14, and the combination of 7 and 14, respectively. The antistenotic activity of 7 and 14 was determined in a 28-day rabbit model with two control groups (uncoated stents and polymer-coated stents) and two study groups (paclitaxel-coated stents and stents coated with the combination of 7 and 14). Polymer-coated stents caused inflammation and increased stenosis by 39% when compared to the uncoated stents. The stents coated with 7 plus 14 were as good as the uncoated stents, 41% better than the polymer-coated stents and 34% better than the paclitaxel-coated stents. These data indicate a beneficial effect of adding NO to an antiproliferative agent (paclitaxel) and suggest a potential therapeutic combination for the treatment of stenotic vessel disease.