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An Aromatic Microdomain at the Cannabinoid CB1 Receptor Constitutes an Agonist/Inverse Agonist Binding Region
Abstract
The cannabinoid CB(1) receptor transmembrane helix (TMH) 3-4-5-6 region includes an aromatic microdomain comprised of residues F3.25, F3.36, W4.64, Y5.39, W5.43, and W6.48. In previous work, we have demonstrated that aromaticity at position 5.39 in CB(1) is crucial for proper function of CB(1). Modeling studies reported here suggest that in the inactive state of CB(1), the binding site of the CB(1) inverse agonist/antagonist SR141716A is within the TMH3-4-5-6 aromatic microdomain and involves direct aromatic stacking interactions with F3.36, Y5.39, and W5.43, as well as hydrogen bonding with K3.28. Further, modeling studies suggest that in the active state of CB(1), the CB agonist WIN55,212-2 binds in this same aromatic microdomain, with direct aromatic stacking interactions with F3.36, W5.43, and W6.48. In contrast, in the binding pocket model, the CB agonist anandamide binds in the TMH2-3-6-7 region in which hydrogen bonding and C-H.pi interactions appear to be important. Only one TMH3 aromatic residue, F3.25, was found to be part of the anandamide binding pocket. To probe the importance of the TMH3-4-5-6 aromatic microdomain to ligand binding, stable transfected cell lines were created for single-point mutations of each aromatic microdomain residue to alanine. Improper cellular expression of the W4.64A was observed and precluded further characterization of this mutation. The affinity of the cannabinoid agonist CP55,940 was unaffected by the F3.25A, F3.36A, W5.43A, or W6.48A mutations, making CP55,940 an appropriate choice as the radioligand for binding studies. The binding of SR141716A and WIN55,212-2 were found to be affected by the F3.36A, W5.43A, and W6.48A mutations, suggesting that these residues are part of the binding site for these two ligands. Only the F3.25A mutation was found to affect the binding of anandamide, suggesting a divergence in binding site regions for anandamide from WIN55,212-2, as well as SR141716A. Taken together, these results support modeling studies that identify the TMH3-4-5-6 aromatic microdomain as the binding region of SR141716A and WIN55,212-2, but not of anandamide.
... In addition, the S1P receptors are like CB1/CB2 in the presence of E1.49 at TMH1. E1.49 has been reported to be a key interaction site for pregnenolone (an endogenous negative allosteric modulator that protects the brain from cannabis intoxication) with CB1 [15], while the LPA 1-3 receptors share a W5.43 with CB1/CB2 that has been shown to affect antagonist binding to the cannabinoid receptors [16]. In addition, S1P 1 , and the cannabinoid receptors recognize lipid-derived ligands that have been shown to bind to the receptor by diffusing from bulk lipid towards the binding site via a transmembrane portal [6,7,14,17,18]. ...
... Results suggested that the loss of potencies of anandamide, and the classical and non-classical cannabinoids, but not WIN55212 at the K3.28A mutant is due to their low affinities to the receptor, and a basic residue at 3.28 is required for CP55940 binding. Based on mutation data, modeling studies suggested a hydrogen bond interaction between K3.28 and the amide oxygen of anandamide [16,24], and with classical and non-classical cannabinoids [25][26][27]. While Shim argued later that K3.28 is important for stabilizing the binding site for the endocannabinoids and the classical and non-classical cannabinoids and not directly involved in their binding [28]. ...
... Different studies determined binding affinity of CP55940 to F3.25A mutant; in one study, the binding affinity of CP55940 determined by saturation binding against [ 3 H]SR141716A was 60-fold lower in F3.25A hCB1 stably transfected in CHO-K1 cells compared to WT [38]. In other studies, CP55940 affinity was not affected in F3.25A mCB1 mutant receptor stably transfected into HEK293 cells, affinity was determined using [ 3 H]CP55940 [16,46]. The discrepancy in binding affinities here could be due to species differences. ...
... Aromatic stacking within CB1 has also been shown to exist. Some of the aromatic stacking interactions that have been proposed include Tyr215, Phe208, Phe289, Tyr292, Ty2296 ,Trp356, Phe170 and Phe200 (McAllister et al., 2003;Shim, 2009;Shim and Howlett, 2006). The interaction between Trp356 of the CWXXP motif and Phe200 in the inactive state of the receptor is highly conserved throughout family A GPCRs and is believed to also exist within CB1 (McAllister et al., 2004;Singh et al., 2002). ...
... binding experiments, suggesting some commonality between their binding sites. Thus, Glide was used to generate a grid centered on the center-of-mass of our previously reported binding site for SR141716A at the CB 1 receptor McAllister et al., 2004;McAllister et al., 2003). The grid dimensions were 26 Å x 26 Å x 26 Å; this grid size allowed Glide to thoroughly explore the receptor for possible binding site(s). ...
... for the mutant receptors were not significantly different from the K d at the wild-type CB 1 receptor (K d for mutants = 6 nM, 4 nM, and 3 nM respectively) suggesting these receptors were properly folded. Therefore, competition binding assays on these mutants were performed using suggest that this mutation participates in direct aromatic stacking interactions with one of the fluorophenyl rings of LDK1229 consistent with prior studies (McAllister et al., 2003). As shown in Table 4, we found here that enlarging the residue at position 7.42 via the C7.42 386 M mutation results in a 5-fold loss in affinity for LDK1229 consistent with previous studies by Farrens and colleagues (Fay et al., 2005). ...
The regulation of G protein coupled receptors (GPCRs) plays a fundamental role in physiologic homeostasis. There are two cannabinoid receptors: CB1, found on neurons of the central and peripheral nervous system and regulate neuromodulatory processes and CB2, found in peripheral tissues, particularly in immune tissues. In this research, three different modulation mechanisms of the cannabinoid receptors are studied. Chapter 2 focuses on the structure-activity relationships of novel allosteric modulators from the indole-2-carboxamide class of compounds. These novel allosteric modulators were optimized for their KB (binding affinity to the allosteric site) and their cooperativity factor α (magnitude by which affinity of orthosteric ligand is changed). Although these positive allosteric modulators enhanced orthosteric CP55, 950 agonist affinity and decreased SR141716A inverse agonist affinity to CB1 consistent with an active receptor conformation, they antagonized basal and agonist-induced G protein coupling. Chapter 3 focuses on the characterization of a group of new inverse agonists from the class of benzhydryl piperazine analogs. These compounds exhibit high nanomolar binding affinities to CB1, antagonize basal as well as agonist-induced G protein coupling and increase receptor cell surface localization, consistent with inverse agonist behavior. Docking and mutational analyses revealed SR141716A-like interactions in the CB1 binding pocket. However, this benzhydryl piperazine scaffold is structurally distinct from first generation CB1 inverse agonists and holds promise for developing peripherally active CB1 inverse agonists with fewer psychiatric side effects. Chapter 4 focuses on the development and optimization of a novel technique using the photo-cross-linking unnatural amino acid p-benzoyl-L-phenylalanine (pBpa) in live cells. This will elucidate the binding partners of the CB2 receptor at defined locations and time points in real-time using mass spectrometry. To this end, I determined the best detergent to solubilize CB2, the ratios of DNA to tRNA and pBpa-synthetase needed, the optimal concentration of pBpa in the media and the ideal UV exposure times needed for the efficient incorporation of pBpa into the full-length CB2 receptor. Additionally, pilot experiments using different CB2 ligands and varying treatment times were performed and possible CB2-binding partner complexes were observed. These results will aid in the discovery of more effective and selective GPCR ligands.
... 54 This leads to a unique set of optimized residue side chains for each of the 100 ligand poses. In this process we did not replace the W5.43 residue with alanine because we consider this tryptophan to be critical for ligand interaction based on site-directed mutagenesis data, 26 leaving its side chain in the form predicted by SCREAM for each of the ten protein conformations. ...
... We predict that rimonabant is anchored by hydrogen bonds to W5. 43 63 ], and indeed previous sitedirected mutagenesis data indicate that mutations of W5.43 and K3.28 to alanine have the largest effect in decreasing experimental binding affinity upon mutation to alanine with >1000-fold and 17.2-fold, respectively. 26,28 The components of the predicted binding energies for this complex are shown in Table 1, showing contributions from each residue in the binding site. The most important residue is W5.43 with a predicted binding contribution to rimonabant of −9.36 kcal/ mol, consistent with the decrease by a factor of >1000 upon mutation to alanine. ...
... The most important residue is W5.43 with a predicted binding contribution to rimonabant of −9.36 kcal/ mol, consistent with the decrease by a factor of >1000 upon mutation to alanine. 26 Indeed our predicted pharmacophore for rimonabant ( Figure 1B) has the significant polar and hydrophobic contacts with the CB1 receptor expected for a strongly bonding ligand. ...
Human cannabinoid type 1 (CB1) G-protein coupled receptor is a potential therapeutic target for obesity. The previously predicted and experimentally validated ensemble of ligand-free conformations of CB1 [Scott, C.E. et al. Protein Sci. 2013, 22, 101-113; Ahn, K.H. et al. Proteins 2013, 81, 1304-1317] are used here to predict the binding sites for known CB1-selective inverse agonists including rimonabant and its seven known derivatives. This binding pocket, which differs significantly from previously published models, is used to identify 16 novel compounds expected to be CB1 inverse agonists by exploiting potential new interactions. We show experimentally that two of these compounds exhibit inverse agonist properties including inhibition of basal and agonist-induced G-protein coupling activity, as well as an enhanced level of CB1 cell surface localization. This demonstrates the utility of using the predicted binding sites for an ensemble of CB1 receptor structures for designing new CB1 inverse agonists.
... 48 AM3677 was found to bind within the TMH3-4-5-6 region, which is rich in aromatic residues ( Figure 6). 49,50 Based upon CB1R mutation studies that identified K3. 28(192) as an essential point of interaction for AEA at CB1R, 51 K3. 28(192) was considered the primary interaction site for AM3677 at CB1R*. In the resulting hydrogen bond, the AM3677 amide oxygen/ CB1R K3. 28(192) interaction is consistent with studies demonstrating that F3.25(189) mutation affects AEA binding 49,50 and that F3.25(189) is part of the CB1R binding pocket. ...
... 49,50 Based upon CB1R mutation studies that identified K3. 28(192) as an essential point of interaction for AEA at CB1R, 51 K3. 28(192) was considered the primary interaction site for AM3677 at CB1R*. In the resulting hydrogen bond, the AM3677 amide oxygen/ CB1R K3. 28(192) interaction is consistent with studies demonstrating that F3.25(189) mutation affects AEA binding 49,50 and that F3.25(189) is part of the CB1R binding pocket. 52 The modeled complex is also consistent with a previously published AEA-hCB1R* complex 49 in which interactions between AEA and hCB1R* also occur at residues F3.25(189) and K3. ...
... 28(192). However, the AEA fatty-acyl chain adopted a curved, U- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 shaped conformation in that complex, 49 whereas AM3677 adopts another low free-energy shape, a Jshaped conformation, in the complex reported here. 53,54 This difference likely reflects prevention of AM3677 from adopting the more compact U-shape by its covalent attachment at C6. 47 In class-A GPCRs, C6.47 is part of the highly-conserved CWXP hinge motif that participates in receptor conformational changes accompanying ligand-induced activation. ...
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) analog 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.
... Previous CB1 modeling studies used homology modeling to bRho or other crystallized GPCRs as templates, followed in some cases by molecular dynamics. [47][48][49][50][51][52][53] These studies are summarized in-depth in Supporting Information Section S5.2. Software programs such as Modeller 54 have also been used previously to predict CB1's structure. ...
... Furthermore, ligandbinding residues are not accessible to the binding site of these homology built structures. 47,49 For example W5.43A leads to a $1000-fold drop in binding affinity for the antagonist rimonabant, 49 but the ModBase prediction, has this residue facing into the lipid membrane region. In our predicted structures, this residue points into the binding site. ...
... Furthermore, ligandbinding residues are not accessible to the binding site of these homology built structures. 47,49 For example W5.43A leads to a $1000-fold drop in binding affinity for the antagonist rimonabant, 49 but the ModBase prediction, has this residue facing into the lipid membrane region. In our predicted structures, this residue points into the binding site. ...
Transmembrane signal transduction is achieved by activation of G protein-coupled
receptors (GPCRs) like the human cannabinoid CB1 receptor. These receptors exist
in an ensemble of conformations, each of which might bind to different signaling
molecules. Mutating a single residue, Thr 210, to Ile in the third transmembrane
(TM3) domain, makes it more active then WT, whereas mutating it to Ala makes it
fully inactive. We used the Gensemble method to predict 3D structures of these
receptors. We find conformational differences that explain the CB1 receptor’s
activation mechanism. These predictions were validated by designing double
mutants that were expected to switch the inactive T210A back to WT levels of
activation. The 2nd mutation for T210A is predicted to cause an important saltbridge
between TMs 2 and 6 to break. GTPδ binding assays show a large
increase in G protein-coupling for the double mutants indicating increased
activation. We docked known agonists to these receptors and then performed 50
ns of molecular dynamics. The inactive T210A receptor with the docked agonist
WIN55212 -2 maintains two stable interhelical salt-bridges. Similarly, the WT
receptor maintains a salt-bridge between TMs 3 and 6, which suggests that a G
protein is necessary to stabilize the active conformation.
... However, an isoleucine (Y5.39I) mutation results in a loss of ligand binding along with pronounced topological changes in TMHs 3-4-5. Such systematic analysis of aromatic microdomain demonstrates that the cannabinoids SR141716A and WIN 55,212 require aromatic stacking interactions of TMHs 3-4-5-6 to activate the CB1 receptor (McAllister et al., 2003). This study also revealed a selective binding region for aminoalkylindoles and diaryl pyrazole cannabinoids, but not for endogenous (anandamide) and bicyclic (CP 55,940) cannabinoids. ...
... Other residues may have more dramatic effects on protein folding or conformation. For example, tryptophan 4.64 (W4.64) is conserved between CB1 and CB2 receptors and mutational analysis suggests this aromatic residue is important for ligand binding and signaling (McAllister et al., 2003;Rhee, 2002). Substituting W4.64 with the nonaromatic residues leucine (W4.64L) and alanine (W4.64A) resulted in drastic reductions-W4.64L and W4.64A mutants displayed a significant loss of ligand binding and signal transduction. ...
Mutational analysis of the cannabinoid receptor will surely contribute to our base of knowledge, building increasingly accurate molecular models of cannabinoid receptors. The ability to tailor future cannabinoid ligands to the receptor will hinge on how well this receptor system is understood. This understanding will potentially yield useful synthetic/modified cannabinergic therapeutic drugs and will very probably serve as future treatment modalities for symptoms of drug withdrawal and addiction. One possibility, on the forefront of pharmacogenomics research, is to create compounds that may activate cannabinoid receptors when there is a loss of endogenous ligand activity or ligand recognition owing to a mutation of those genes encoding the cannabinoid receptors.
... Further to this, the position of residue TRP287 6.48 was shown to be distinct for β-arrestin-biased agonist WMS-X600. Mutation of TRP287 6.48 to ALA had a greater effect on the potency of β-arrestin2 recruitment for WMS-X600 compared to U50,488, the same effect was observed for nalfurafine, though this residue has been tied to general receptor efficacy in the past in MOR and in other class A GPCRS [35], [85][86][87][88][89]. ...
Drugs acting at the opioid receptor family are clinically used to treat chronic and acute pain, though they represent the second line of treatment behind GABA analogs, antidepressants and SSRI’s. Within the opioid family mu and kappa opioid receptor are commonly targeted. However, activation of the mu opioid receptor has side effects of constipation, tolerance, dependence, euphoria, and respiratory depression; activation of the kappa opioid receptor leads to dysphoria and sedation. The side effects of mu opioid receptor activation have led to mu receptor drugs being widely abused with great overdose risk. For these reasons, newer safer opioid analgesics are in high demand. For many years a focus within the opioid field was finding drugs that activated the G protein pathway at mu opioid receptor, without activating the β-arrestin pathway, known as biased agonism. Recent advances have shown that this may not be the way forward to develop safer analgesics at mu opioid receptor, though there is still some promise at the kappa opioid receptor. Here we discuss recent novel approaches to develop safer opioid drugs including efficacy vs bias and fine-tuning receptor activation by targeting sub-pockets in the orthosteric site, we explore recent works on the structural basis of bias, and we put forward the suggestion that Gα subtype selectivity may be an exciting new area of interest.
... Regarding the interaction of anandamide with the cannabinoid receptors, a mutagenesis study demonstrated the importance of the amino acid F3.25 (following B&W nomenclature) for binding anandamide in the binding site of the CB1 receptor while a computational docking study showed interactions between anandamide, in a hairpin conformation, with the same receptor (McAllister et al., 2003). In a crystallographic study where the structure of the CB1 receptor was solved, the authors used the X-ray structure to dock various agonist molecules, including anandamide and found that the molecule prefers a hairpin conformation in the binding site of the receptor (Hua et al., 2016). ...
The anandamide is a relevant ligand due to its capacity of interacting with several proteins, including the T-type calcium channels, which play an important role in neuropathic pain and depression disorders. Hence, a detailed characterization of the chemical properties and conformational stability of anandamide may provide valuable information to understand its behavior in a biological context. Herein, conceptual DFT and QTAIM analyses were performed to theoretically characterize the chemical reactivity properties and the structural stability of conformations of anandamide, using the BP86/cc-pVTZ level of theory. Global reactivity description, based on conceptual DFT, indicates that the hardness increases and the electrophilicity index decreases for both, the hairpin and U-shape conformers relative to the extended conformers. Also, an increase in the chemical potential value and a decrease in the electronegativity and the electrophilicity index is observed in the ethanolamide open ring conformers in comparison with the corresponding closed ring structures. In addition, regarding the characterization of local reactivity descriptors, the maximum values of the Fukui and Parr functions indicate that the most probable location for a nucleophilic attack is either the hydroxyl oxygen located in the ethanolamide closed ring conformers or the carbonyl oxygen present in the open ring conformers. The most probable location for an electrophilic attack is in the alkyl double bond region in all anandamide conformers. According to the QTAIM results, the intramolecular hydrogen bond formation stabilizing the structure of anandamide has interaction energy values for the closed ring conformations of 12.33–12.46 kcal mol⁻¹, indicating a strong interaction. Lastly, molecular docking calculations determined that a region in the pore, denominate as pore-blocking, is a probable site for the interaction of anandamide with the human Cav3.2 isoform of the T-type calcium channel family. The pore-blocking site contains hydrophobic residues where the non-polar part in the final alkyl region of anandamide established mainly alkyl-alkyl interactions, while the polar part (the ethanolamide group) interacts with the polar residue S900. The information based on conceptual DFT presented may aid in the design of drugs with similar chemical characteristics as those identified in anandamide so as to bind anandamide-interacting proteins, including the T-type calcium channels.
... 92 Multiple aromatic stacking interactions have also been observed in silico between highaffinity CB1R ligands and the transmembrane domains 3−6 of CB1R, which is a region rich with tyrosine, phenylalanine, and tryptophan residues. 101 Furthermore, several compounds used in these docking studies were JWH-series analogues which specifically lacked the carbonyl oxygen yet still retained CB1R activity, impugning a central tenet of the three-point theory, and supporting the π-stacking interpretation. 100 However, this π-stacking CB1R agonist binding theory, which was sufficient to explain the affinity of naphthoylindole SCBs, was unable to explain the generations of SCBs that came to follow. ...
This review article covers the background, pharmacology, adverse effects, synthesis, pharmacokinetics, metabolism, and history of synthetic cannabinoid compounds. Synthetic cannabinoids are a class of novel psychoactive substances that act as agonists at cannabinoid receptors. This class of compounds is structurally diverse and rapidly changing, with multiple generations of molecules having been developed in the last decade. The structural diversity of synthetic cannabinoids is supported by the breadth of chemical space available for exploitation by clandestine chemists, and incentivized by attempts to remain ahead of legal pressures. As a class, synthetic cannabinoid products have a more serious adverse effect profile than traditional phytocannabinoids, including notable risks of lethality, as well as a history of dangerous adulteration. Most synthetic cannabinoids are rapidly metabolized to active species with prolonged residence times and peripheral tissue distribution, and analytical confirmation of use of these compounds remains challenging. Overall, the emergence of synthetic cannabinoids serves as a noteworthy example of the pressing public health challenges associated with the increasing development of easily synthesized, structurally-flexible, highly-potent, psychoactive drugs.
... 9-HODE was approximately 10-fold more efficacious than acidic pH. 13 to CB 1 . 54 Together, our data raise the possibility of a comparable pocket on two lipid receptors, CB 1 and hGPR132a, able to accommodate CP-55,940. A limitation of our functional assay data is that it does not distinguish mutational effects on efficacy and affinity. ...
The G-protein-coupled receptor GPR132, also known as G2A, is activated by 9-hydroxy-octadecadienoic acid (9-HODE) and other oxidized fatty acids. Other suggested GPR132 agonists including lysophosphatidylcholine (LPC) have not been readily reproduced. Here, we identify N-acylamides in particular N-acylglycines, as lipid activators of GPR132 with comparable activity to 9-HODE. The order-of-potency is N-palmitoylglycine > 9-HODE ≈ N-linoleoylglycine > linoleamide > N-oleoylglycine ≈ N-stereoylglycine > N-arachidonoyl-glycine > N-docosehexanoylglycine. Physiological concentrations of N-acylglycines in tissue are sufficient to activate GPR132. N-linoleoylglycine and 9-HODE also activate rat and mouse GPR132, despite limited sequence conservation to human. We describe pharmacological tools for GPR132, identified through drug screening. SKF-95667 is a novel GPR132 agonist. SB-583831 and SB-583355 are peptidomimetic molecules containing core amino acids (glycine and phenylalanine, respectively), and structurally related to previously described ligands. A telmisartan analog, GSK1820795A, antagonizes the actions of N-acylamides at GPR132. The synthetic cannabinoid CP-55 940 also activates GPR132. Molecular docking to a homology model suggested a site for lipid binding, predicting the acyl side-chain to extend into the membrane bilayer between TM4 and TM5 of GPR132. Small-molecule ligands are envisaged to occupy a "classical" site encapsulated in the 7TM bundle. Structure-directed mutagenesis indicates a critical role for arginine at position 203 in transmembrane domain 5 to mediate GPR132 activation by N-acylamides. Our data suggest distinct modes of binding for small-molecule and lipid agonists to the GPR132 receptor. Antagonists, such as those described here, will be vital to understand the physiological role of this long-studied target.
... 8 As such, researchers frequently utilized CB1 receptor homology models constructed based on the limited GPCR crystal structures available for studying structure-function relationships, activation mechanisms, predicting ligand binding, and for drug discovery applications such as virtual screening. [9][10][11][12][13][14][15] However, the performance of homology models has been shown to be inferior to crystal structures in drug discovery applications such as predicting the binding poses of ligands and in virtual screening. 16,17 Following several recent breakthroughs in GPCR crystallography, the crystal structure of the inactive-state CB1 receptor bound to the inverse agonist taranabant and antagonist AM6538 was nally solved in late 2016. ...
The therapeutic potential of the CB1 cannabinoid receptor remains underexploited with only a few synthetic ligands on the market. The crystal structures of both the inactive and active-state CB1 receptor have recently been solved, allowing for unprecedented opportunities in structure-based drug discovery applications such as virtual screening. In this study, we have investigated the virtual screening performance of the active and inactive-state CB1 crystal structures and their ability to discriminate between agonist and inverse agonist/antagonist ligands. The ligands of inactive and active-state CB1 receptor crystal structures were then swapped via cross-docking and the resulting structures were subjected to microsecond molecular dynamics (MD) simulations, followed by virtual screening of the MD-extracted structures. The original crystal structures were found to be biased towards ligands matching their activation state during virtual screening. MD simulations of the cross-docked CB1 structures resulted in a minor shift of receptor conformation towards the inactive state for the active-state CB1 structure complexed with the inverse agonist taranabant. Effects on virtual screening were more pronounced, as MD simulations of the cross-docked receptor-ligand complexes reversed the ligand bias in virtual screening observed with the original crystal structures. The simulations also produced receptor conformations that outperformed the crystal structures in virtual screening and in predicting the binding pose of the cognate ligand. The findings of this study highlight the potential of cross-docking and MD simulations to reverse the ligand bias of crystal structures, which may be useful when the crystal structure of only one activation state is available.
... Flexing in the CWxP motif is produced by ligand binding, which alters the χ1 dihedral angles of the "toggle switch" residues. For CB1, these are F3.36 in TMH3 and a tryptophan residue (W6.48) which is part of the CWxP motif [69][70][71][72]. The F3.36A CB1 mutation resulted in increased basal [ 35 S]GTPγS binding of the receptor. ...
The endocannabinoid system has emerged as a promising target for the treatment of numerous diseases, including cancer, neurodegenerative disorders, and metabolic syndromes. Thus far, two cannabinoid receptors, CB1 and CB2, have been discovered, which are found predominantly in the central nervous system (CB1) or the immune system (CB2), among other organs and tissues. CB1 receptor ligands have been shown to induce a complex pattern of intracellular effects. The binding of a ligand induces distinct conformational changes in the receptor, which will eventually translate into distinct intracellular signaling pathways through coupling to specific intracellular effector proteins. These proteins can mediate receptor desensitization, trafficking, or signaling. Ligand specificity and selectivity, complex cellular components, and the concomitant expression of other proteins (which either regulate the CB1 receptor or are regulated by the CB1 receptor) will affect the therapeutic outcome of its targeting. With an increased interest in G protein-coupled receptors (GPCR) research, in-depth studies using mutations, biological assays, and spectroscopic techniques (such as NMR, EPR, MS, FRET, and X-ray crystallography), as well as computational modelling, have begun to reveal a set of concerted structural features in Class A GPCRs which relate to signaling pathways and the mechanisms of ligand-induced activation, deactivation, or activity modulation. This review will focus on the structural features of the CB1 receptor, mutations known to bias its signaling, and reported studies of CB1 receptor ligands to control its specific signaling.
... This movement (of ~2 Å) of CP55,940 towards the two toggle switch residues, indicates the activation of the CB2 receptor because the distance between toggle switch residue Trp258 6.48 and CP55,940 (to the terminal carbon of dimethyl heptyl side chain of CP55,940) remained short, ~3.8 Å, throughout the remainder of the MD simulation after t = 40 ns (Fig 13). A modeling study which compared the relative position of the toggle switch residues reported for the CB1 receptor (McAllister et al., 2004;McAllister et al., 2003;Singh et al., 2002), proposed that a similar toggle switch mechanism could exist between Phe117 3.36 and Trp258 6.48 within the CB2 receptor (Hurst, et al., 2010;Latek et al., 2011). In addition, the reports on the CB1 active-state crystal structure also described the synergistic movement of two residues, Phe200 3.36 and Trp356 6.48 known as a toggle switch (Hua et al., 2017). ...
Selective activation of the cannabinoid receptor subtype 2 (CB2) shows promise for treating pain, inflammation, multiple sclerosis, cancer, ischemic/reperfusion injury, and osteoporosis. Target selectivity and off-target side effects are two major limiting factors for orthosteric ligands, and, therefore, the search for allosteric modulators (AMs) is a widely used drug discovery approach. To date, only a limited number of CB2 AMs have been identified, possessing only micromolar activity at best, and the CB2 receptor’s allosteric site(s) are not well characterized. Herein, we used computational approaches including receptor modeling, site mapping, docking, MD simulations and binding free-energy calculations to predict, characterize and validate sites within the complex of the CB2 receptor with bound orthosteric agonist CP55,940. After docking of known negative CB2 allosteric modulators (NAMs), dihydro-gambogic acid (DHGA) and trans-β-caryophyllene (TBC) (note that TBC also shows agonist activity) at the predicted allosteric sites, the best total complex with CB2, CP55,940 and NAM was embedded into a hydrated lipid-bilayer and subjected to a 200 ns molecular dynamics simulation. The presence of an AM affected the CB2–CP55,940 complex, altering the relative positioning of the toggle switch residues and promoting a strong π-π interaction between Phe1173.36 and Trp2586.48. Binding of either TBC or DHGA to a putative allosteric pocket directly adjacent to the orthosteric ligand reduced the binding free energy of CP55,940, which is consistent with the expected effect of a negative AM. The identified allosteric sites present immense scope for the discovery of novel classes of CB2 allosteric modulators.
... An important approach for rational drug design is to identify residues and domains that are crucial for receptor function. Within the CB1 receptor there are defined domains involved in ligand binding (McAllister et al., 2003;Kapur et al., 2008;Shim et al., 2011), switching between active and inactive form (Nie and Lewis, 2001;Ahn et al., 2013;Marcu et al., 2013;Scott et al., 2013), and G-protein binding (Shim et al., 2013). Specific phosphorylation sites of the C-terminal tail of the CB1 receptor have been implicated in the regulation of CB1 receptor endocytosis and post-endocytic trafficking (Garcia et al., 1998;Hsieh et al., 1999;Jin et al., 1999;Daigle et al., 2008). ...
Defining functional domains and amino acid residues in G protein coupled receptors (GPCRs) represent an important way to improve rational drug design for this major class of drug targets. The cannabinoid type 1 (CB1) receptor is one of the most abundant GPCRs in the central nervous system and is involved in many physiological and pathophysiological processes. Interestingly, cannabinoid type 1 receptor with a phenylalanine 238 to leucine mutation (CB1F238L) has been already linked to a number of both in vitro and in vivo alterations. While CB1F238L causes significantly reduced presynaptic neurotransmitter release at the cellular level, behaviorally this mutation induces increased risk taking, social play behavior and reward sensitivity in rats. However, the molecular mechanisms underlying these changes are not fully understood. In this study, we tested whether the F238L mutation affects trafficking and axonal/presynaptic polarization of the CB1 receptor in vitro. Steady state or ligand modulated surface expression and lipid raft association was analyzed in human embryonic kidney 293 (HEK293) cells stably expressing either wild-type cannabinoid type 1 receptor (CB1wt) or CB1F238L receptor. Axonal/presynaptic polarization of the CB1F238L receptor was assessed in transfected primary hippocampal neurons. We show that in vitro the CB1F238L receptor displays increased association with lipid rafts, which coincides with increased lipid raft mediated constitutive endocytosis, leading to a reduction in steady state surface expression of the CB1F238L receptor. Furthermore, the CB1F238L receptor showed increased axonal polarization in primary hippocampal neurons. These data demonstrate that endocytosis of the CB1 receptor is an important mediator of axonal/presynaptic polarization and that phenylalanine 238 plays a key role in CB1 receptor trafficking and axonal polarization.
... Rimonabant, the inverse agonist, binds to the CB 1 and interaction is thought to exist through hydrogen bonding between the carbonyl group of Rimonabant and the Lys192 residue of the CB 1 receptor, shown in Fig. 3. This bond stabilizes the Lys192-Asp366 salt bridge in CB 1 helices 3 and 6, believed to be specific to the inactive CB 1 state ( Lange & Kruse, 2005;McAllister et al., 2003). Rimonabant, through direct stacking of its 2,4-dichlorophenyl ring to the Trp279/Phe200/Trp356 residues (on CB 1 ) on one end and the para-chlorophenyl ring (on Rimonabant) to the Tyr275/Trp255/ Phe278 (on CB 1 ) on the other end, binds within the transmembrane3-4-5-6 aromatic microdomain of the CB 1 ( Fan et al., 2009;Lange & Kruse, 2005). ...
Opioid receptors (ORs), μOR, δOR, κOR and ORL1 mediate numerous signaling cascades, most importantly, through the modulation of ion channels. Research demonstrates the role of OR mediated signal transduction in treating pain, cancer, neurodegenerative disorders and cardiac insults. Yet, the primary application of drugs that modulate ORs is analgesia. Current opioids like morphine that are mainly μOR orthosteric agonists attract many undesirable side-effects (constipation, urinary retention, respiratory depression and hypotension) and the existing modus operandi against these is the inclusion of a μOR antagonist (for example. naloxone) which itself produces side-effects. As such, there is a current thrust to delineate the anti-nociceptive pathways mediated by ORs from the pathways involved in their induction of debilitating side-effects, in order to develop enhanced lead molecules. This review discusses the effects of natural products on the OR-induced signaling cascades and compares these to current synthetic leads and drugs. Important to these discussions is the complexity of OR signaling which involves OR trafficking, de- and re-sensitization, homo- and hetero-dimerization, the type of ligand binding (agonist, antagonist, reverse antagonist, orthosteric and allosteric agonist and antagonist in the context of biased agonism) and reasons for dysregulation that primarily occur because of inter-individual variations. Our current understanding of the different forms of ORs has expanded, thus introducing the concept of allosterism, which is also discussed. The authors present possible combination therapies to be explored towards developing the 'Holy Grail' of analgesics, for example, ignavine, the natural μOR positive allosteric modulator (PAM) with codeine and the natural fascaplysin, a balanced agonist with fentanyl. There remain many gaps in natural products research on ORs, more so on ORL1 and δ- and ҡ receptors. Furthermore, additional exploration of ORs' modulation is needed for ameliorating other associated disease conditions of global concern.
... The residue W293 6.48 has previously been identified as a rotamer toggle switch for GPCR activation in the conserved polar network and participating in coordination of a sodium ion [6,[41][42][43][44][45]. The MD simulations described here showed that the full agonist norbuprenorphine came into close contact with this residue, affecting the preferred rotamer of the W293 6.48 side chain, whereas the low efficacy agonist buprenorphine and antagonist diprenorphine did not. ...
The μ-opioid receptor (MOPr) is a clinically important G protein-coupled receptor (GPCR) which couples to Gi/o proteins and arrestins. At present the receptor conformational changes that occur following agonist binding and activation are poorly understood. This study has employed molecular dynamics simulations to investigate the binding mode and receptor conformational changes induced by structurally similar opioid ligands of widely differing intrinsic agonist efficacy, norbuprenorphine, buprenorphine and diprenorphine. Bioluminescence resonance energy transfer (BRET) assays for Gi activation and arrestin-3 recruitment in HEK 293 cells confirmed that norbuprenorphine is a high efficacy agonist, buprenorphine a low efficacy agonist, and diprenorphine an antagonist at the MOPr. Molecular dynamics simulations revealed that these ligands adopt distinct binding poses and engage different subsets of residues, despite sharing a common morphinan scaffold. Notably, norbuprenorphine interacted with sodium ion - coordinating residues W2936.48 and N1503.35, whilst buprenorphine and diprenorphine did not. Principal component analysis of the movements of the receptor transmembrane domains showed that the buprenorphine-bound receptor occupied a distinct set of conformations to the norbuprenorphine-bound receptor. Addition of an allosteric sodium ion caused the receptor and ligand to adopt an inactive conformation. The differences in ligand-residue interactions and receptor conformations observed here may underlie the differing efficacies for cellular signalling outputs for these ligands.
... Intriguingly, plasmon-waveguide resonance (PWR) spectroscopy has demonstrated that CP55,940 and WIN55,212-2 produced distinct spectral changes (PWR shifts in opposite directions) on binding to the hCB 1 indicating that the two agonists produce qualitatively distinct active conformations of the receptor, which have differing affinity for Ga i . 114 Differential signaling by WIN55,212-2 and CP55,940 is consistent with the suggestion that these ligands have overlapping but distinct binding sites, 115,116 a finding supported by molecular docking in the recently described crystal structure of CB 1 . 117,118 Laprairie et al. 20 investigated the biased signaling of WIN55,212-2, CP55,940, 2-AG, anandamide, THC, cannabidiol, and the combination THC + cannabidiol on several signaling pathways. ...
By contrast to traditional single-point assays of cAMP accumulation or ERK phosphorylation, GPCR modulation of ion channels provides a continuous and relatively direct readout of receptor activity, particularly when Gβγ inhibition of ICa or activation of GIRK is measured.⁷⁵ Unfortunately, there are very few cell lines where native CB1 receptors coexist with voltage-gated ICa or GIRK. NG-108-15 and N18 cells, with endogenous CB1, were used for the very earliest descriptions of CB1 receptor inhibition of ICa,76–78 however, receptor regulation was not examined. Recombinant CB1 receptors expressed in murine AtT-20 cells couple to both inhibition of ICa and activation of GIRK,⁷⁹ and desensitization of GIRK activation has been reported.⁸⁰ AtT-20 cells and Xenopus oocytes have been used to provide some insight into CB1 regulation of ion channels.80,81 In oocytes, desensitization of rCB1-mediated activation of GIRK was shown to be stimulated by coexpression of both GRK3 and β-arrestin-2, but not affected by either protein alone.⁸⁰ The efficiency of other members of the GRK family, or of β-arrestin-1, was not addressed in the oocyte studies, nor were the effects of coexpression of these molecules on basal coupling of CB1 to GIRK.
... F 189 interacts with the AEA amide oxygen and an F 189A mutation in CB 1 decreases AEA binding sixfold (McAllister et al., 2004). The AEA amide oxygen also interacts with a charged residue at position 192 (K in CB 1 , D in NPR-19) and the AEA hydroxyl forms a hydrogen bond with S 383 (McAllister et al., 2003). These data highlight the effective coupling of a human G-proteincoupled receptor to endogenous C. elegans G-proteins and strongly support the hypothesis that NPR-19 is a mammalian cannabinoid receptor ortholog. ...
Cannabis sativa, or marijuana, a popular recreational drug, alters sensory perception and exerts a range of potential medicinal benefits. The present study demonstrates that theendogenouscannabinoid receptor agonists 2-arachidonoylglycerol (2-AG)andanandamide(AEA)activate a canonical cannabinoid receptor in Caenorhabditis elegans and also modulate monoaminergic signaling at multiple levels. 2-AG orAEAinhibit nociception and feeding through a pathway requiring the cannabinoid-like receptor NPR-19. 2-AG or AEA activate NPR-19 directly and cannabinoid-dependent inhibition can be rescued in npr-19-null animals by the expression of ahumancannabinoid receptor,CB1, highlighting the orthology of the receptors. Cannabinoids also modulate nociception and locomotion through an NPR-19-independent pathway requiring an α2A-adrenergic-like octopamine (OA) receptor, OCTR-1, and a 5-HT1A-like serotonin (5-HT) receptor, SER-4, that involves a complex interaction among cannabinoid, octopaminergic, and serotonergic signaling. 2-AG activates OCTR-1 directly. In contrast, 2-AG does not activate SER-4 directly, but appears to enhance SER-4-dependent serotonergic signaling by increasing endogenous 5-HT. This study defines a conserved cannabinoid signaling system in C. elegans, demonstrates the cannabinoid-dependent activation of monoaminergic signaling, and highlights the advantages of studying cannabinoid signaling in a genetically tractable whole-animal model.
... region. The binding site is located deep in the receptor core and residues in direct contact with SR141716A include F200 3.36 , W279 5.43 , W356 6.48 (McAllister et al., 2003) and C386 7.42 (Fay et al., 2005). The two residues, W279 5.43 and W356 6.48 are part of the aromatic microdomain of CB 1 . ...
... The N-substituents reach the end of the long channel and interact with helix V. The binding mode of JWH-018 and WIN 55,212-2 is supported by mutations on helix V (McAllister et al., 2003;Song et al., 1999) and SAR study of N-alkyl chain length (Aung et al., 2000). Notably, all of the agonists interact with Phe268 ECL2 and Phe379 7.35 in our docking poses, which is consistent with mutagenesis studies on ECL2 (Ahn et al., 2009) and Phe379 7.35 ( Figure S4I). ...
... The N-substituents reach the end of the long channel and interact with helix V. The binding mode of JWH-018 and WIN 55,212-2 is supported by mutations on helix V (McAllister et al., 2003;Song et al., 1999) and SAR study of N-alkyl chain length (Aung et al., 2000). Notably, all of the agonists interact with Phe268 ECL2 and Phe379 7.35 in our docking poses, which is consistent with mutagenesis studies on ECL2 (Ahn et al., 2009) and Phe379 7.35 ( Figure S4I). ...
Cannabinoid receptor 1 (CB1) is the principal target of Δ⁹-tetrahydrocannabinol (THC), a psychoactive chemical from Cannabis sativa with a wide range of therapeutic applications and a long history of recreational use. CB1 is activated by endocannabinoids and is a promising therapeutic target for pain management, inflammation, obesity, and substance abuse disorders. Here, we present the 2.8 Å crystal structure of human CB1 in complex with AM6538, a stabilizing antagonist, synthesized and characterized for this structural study. The structure of the CB1-AM6538 complex reveals key features of the receptor and critical interactions for antagonist binding. In combination with functional studies and molecular modeling, the structure provides insight into the binding mode of naturally occurring CB1 ligands, such as THC, and synthetic cannabinoids. This enhances our understanding of the molecular basis for the physiological functions of CB1 and provides new opportunities for the design of next-generation CB1-targeting pharmaceuticals.
... The observation that mutating hCB1R tryptophan W5.43 abrogated Org27569's ability to inhibit CP55,940 signaling implicated this residue in the Org27569 binding site [32]. W5.43 mutation also adversely affected binding of the orthosteric aminoalkylindole agonist WIN55,212 [119], suggesting the possibility that overlap between the binding pockets for WIN55,212 and Org27569 facilitates their allosteric cooperativity. A subsequent study identified a putative Org27569 allosteric binding site comprised of regions from CB1R's TMH3, TMH6, and TMH7 [33]. ...
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.
... Screening results of peripheral selectivity and hCB1R affinity on our early analogues redirected ligand design. The peripheral selectivity of the high affinity compounds was tested in the Madin−Darby canine kidney (MDCK) epithelial cell line assay as a model of the BBB 29 and showed an association with the npentyl indoles and morpholinoethyl indenes, respectively. Thus, the N-pentyl 4-carboxy methyl ester indole 1-4a was compared to the N-morpholinoethyl 4-carboxy methyl ester indole analogue 1-4o. ...
Alleviation of neuropathic pain by cannabinoids is limited by their central nervous system (CNS) side effects. Indole and indene compounds were engineered for high hCB1R affinity, peripheral selectivity, metabolic stability, and in vivo efficacy. An epithelial cell line assay identified candidates with <1% blood-brain barrier penetration for testing in a rat neuropathy induced by unilateral sciatic nerve entrapment (SNE). The SNE-induced mechanical allodynia was reversibly suppressed, partially or completely, after intraperitoneal or oral administration of several indenes. At doses that relieve neuropathy symptoms, the indenes completely lacked, while the brain-permeant CB1R agonist HU-210 (1) exhibited strong CNS side effects, in catalepsy, hypothermia, and motor incoordination assays. Pharmacokinetic findings of ~0.001 cerebrospinal fluid:plasma ratio further supported limited CNS penetration. Pretreatment with selective CB1R or CB2R blockers suggested mainly CB1R contribution to an indene’s anti-allodynic effects. Therefore, this class of CB1R agonists holds promise as a viable treatment for neuropathic pain.
... Some active GPCR models have been constructed through homology modeling using the X-ray inactive bovine rhodopsin as a template. The so-built models have been modified to take into account the above-mentioned activation studies, as in the case of human cannabinoid CB 1 and CB 2 [133,134], chemokine CCR 5 [135] by chemokines involves an aromatic cluster and -adrenergic [69] receptors. In this last case, a previously obtained -adrenergic model was also used as a template. ...
G protein-coupled receptors (GPCRs) represent the largest family of signal transduction membrane proteins and play a critical role in many key physiological processes such as neurotransmission, cellular metabolism, secretion, cell growth, immune defence, and differentiation. Therefore, it is not surprising that these receptors represent a realized and ongoing opportunity for drug development. In this scenario, structure-based drug design techniques turned out to be a really attractive approach, leading to a breakthrough in the discovery of novel therapeutic agents. Indeed, much of this success has to be attributed to the pioneering elucidation of the bovine rhodopsin crystal structure, which represents a milestone in the understanding of GPCRs structures. Starting from the experimentally found rhodopsin 3D coordinates, the tandem application of homology building techniques and molecular docking has become one the most important approaches for structure and ligand binding analysis. Nevertheless, the construction of realistic models of certain GPCRs still remains time consuming and requires many refinements of the models in close association with experiments. This review is aimed at providing a deep view into the current status of GPCR modeling, highlighting the recent progresses made in the rhodopsin-based homology building together with alternative computational approaches. The application of these techniques in the detection of GPCR ligands and the elucidation on how they impact the world of drug discovery is also discussed.
... CB1R intramembrane loops were proposed to shape a "binding pocket" that 2-AG could reach through a gap allowing lipidic ligands to enter from membrane bilayer, without need of extracellular access (Hurst et al., 2013). Aminoalkylindole cannabinoids such as WIN bind at a different site (McAllister et al., 2003;Hurst et al., 2013), so WIN could act as a positive allosteric modulator for 2-AG, by increasing 2-AG-induced constitutive CB1R activation, leading to enhanced inhibition of cAMP/PKA signaling in the somatodendritic compartment. Interestingly, the agonist CP55,940 binds at a different site than WIN (Kapur et al., 2007) and dissimilarly to WIN, CP55,940-mediated inhibition of somatodendritic PKA activity is significantly stronger after DAGL inhibition. ...
Neurons display important differences in plasma membrane composition between somatodendritic and axonal compartments, potentially leading to currently unexplored consequences in G-protein-coupled-receptor signaling. Here, by using highly-resolved biosensor imaging to measure local changes in basal levels of key signaling components, we explored features of type-1 cannabinoid receptor (CB1R) signaling in individual axons and dendrites of cultured rat hippocampal neurons. Activation of endogenous CB1Rs led to rapid, Gi/o-protein- and cAMP-mediated decrease of cyclic-AMP-dependent protein kinase (PKA) activity in the somatodendritic compartment. In axons, PKA inhibition was significantly stronger, in line with axonally-polarized distribution of CB1Rs. Conversely, inverse agonist AM281 produced marked rapid increase of basal PKA activation in somata and dendrites, but not in axons, removing constitutive activation of CB1Rs generated by local production of the endocannabinoid 2-arachidonoylglycerol (2-AG). Interestingly, somatodendritic 2-AG levels differently modified signaling responses to CB1R activation by Δ(9)-THC, the psychoactive compound of marijuana, and by the synthetic cannabinoids WIN55,212-2 and CP55,940. These highly contrasted differences in sub-neuronal signaling responses warrant caution in extrapolating pharmacological profiles, which are typically obtained in non-polarized cells, to predict in vivo responses of axonal (i.e., presynaptic) GPCRs. Therefore, our results suggest that enhanced comprehension of GPCR signaling constraints imposed by neuronal cell biology may improve the understanding of neuropharmacological action.
Anandamide, an endogenous fatty acid, displays a wide conformational space due to the nature of its chemical structure, particularly its polyunsaturated aliphatic chain component (omega‐6 fatty acid). Six main minima are considered after a conformational search based on the MM+ method, namely, extended shape, U‐shape, and hairpin shape with either an open or a closed conformation of the ethanolamide (EA) ring. For these six conformers, DFT calculations were performed to theoretically characterize their structural stability, NMR and IR spectroscopic, and electronic properties using the BP86/cc‐pVTZ level of theory with the solute‐implicit solvent model PCM. DLPNO‐CCSD(T) level of theory was used for comparison with DFT results. Our results indicate that the conformers with closed EA ring are more stable than their corresponding open ring counterparts. With the NMR and IR spectroscopies was characterized the formation of the intramolecular hydrogen bond in the closed conformers of the EA ring. The electronic properties investigated include the calculation of the frontier molecular orbitals (FMO), the molecular electrostatic potential (MEP), and the natural bond orbitals (NBO). Additionally, the multiscale ONIOM QM1/QM2 model was used to simulate a solute‐explicit solvent system and molecular dynamics simulations were used to simulate the anandamide systems embedded in a hydrated symmetric POPC membrane and in aqueous solution. The results suggest that alkyl‐middle and EA groups in anandamide may play an important role in the ligand‐receptor interaction. Anandamide displays a wide conformational space due to its chemical structure, particularly its polyunsaturated aliphatic chain. Three main minima are characterized as the preferred conformations after a DFT conformational search, ONIOM‐DFT calculations, and molecular dynamics simulations. NMR and IR spectra of the conformers of anandamide are analyzed to characterize the intramolecular hydrogen bond. Ethanolamide and middle alkyl groups are important in the ligand‐receptor interaction.
The endocannabinoid signalling (ECS) system is a complex lipid signalling pathway that modulates diverse physiological processes in both vertebrate and invertebrate systems. In nematodes, knowledge of endocannabinoid (EC) biology is derived primarily from the free-living model species Caenorhabditis elegans, where ECS has been linked to key aspects of nematode biology. The conservation and complexity of nematode ECS beyond C. elegans is largely uncharacterised, undermining the understanding of ECS biology in nematodes including species with key importance to human, veterinary and plant health. In this study we exploited publicly available omics datasets, in silico bioinformatics and phylogenetic analyses to examine the presence, conservation and life stage expression profiles of EC-effectors across phylum Nematoda. Our data demonstrate that: (i) ECS is broadly conserved across phylum Nematoda, including in therapeutically and agriculturally relevant species; (ii) EC-effectors appear to display clade and lifestyle-specific conservation patterns; (iii) filarial species possess a reduced EC-effector complement; (iv) there are key differences between nematode and vertebrate EC-effectors; (v) life stage-, tissue- and sex-specific EC-effector expression profiles suggest a role for ECS in therapeutically relevant parasitic nematodes. To our knowledge, this study represents the most comprehensive characterisation of ECS pathways in phylum Nematoda and inform our understanding of nematode ECS complexity. Fundamental knowledge of nematode ECS systems will seed follow-on functional studies in key nematode parasites to underpin novel drug target discovery efforts.
X-ray crystallography and cryogenic electronic microscopy have provided significant advancement in the knowledge of GPCR structure and have allowed the rational design of GPCR ligands. The class A GPCRs cannabinoid receptor type 1 and type 2 are implicated in many pathophysiological processes and thus rational design of drug and tool compounds is of great interest. Recent structural insight into cannabinoid receptors has already led to a greater understanding of ligand binding sites and receptor residues that likely contribute to ligand selectivity. Herein, classes of heterocyclic covalent cannabinoid receptor ligands are reviewed in light of the recent advances in structural knowledge of cannabinoid receptors, with particular discussion regarding covalent ligand selectivity and rationale design.
This study reports on the isothiouronium salt catalyzed transfer hydrogenation of various 3‐substituted‐2H‐1,4‐benzoxazines, with Hantzsch esters as a hydrogen source. Transfer hydrogenation of 1,4‐benzoxazines has been successfully carried out with small amount of 1 mol% S‐benzyl‐N,N′‐diphenyl isothiouronium iodide as a catalyst. Various 3,4‐dihydro‐2H‐1,4‐benzoxazines were obtained in quantitative yield at room temperature.
Herein, a transition-metal-free multicomponent cascade reaction of readily available α-halogenated ketones, ortho-aminophenols, and aldehydes using a novel dipeptide-based phosphonium salt catalyst was developed for the efficient construction of various 2H-1,4-benzoxazine derivatives with excellent functional-group tolerance. The method represents an unprecedented approach for trapping active 1,5-bifunctional intermediates with α-halogenated ketones to access biologically important benzoxazine scaffolds bearing two stereogenic centers with excellent asymmetric induction.
The cannabinoid (CB) receptors (CB1R and CB2R) represent a promising therapeutic target for several indications such as nociception and obesity. The ligands with nonselectivity can be traced to the high similarity in the binding sites of both cannabinoid receptors. Therefore, the need for selectivity, potency, and G-protein coupling bias has further complicated the design of desired compounds. The bias of currently studied cannabinoid agonists is seldom investigated, and agonists that do exhibit bias are typically nonselective. However, certain long-chain endocannabinoids represent a class of selective and potent CB1R agonists. The binding mode for this class of compounds has remained elusive, limiting the implementation of its binding features to currently studied agonists. Hence, in the present study, the binding poses for these long-chain cannabinoids, along with other interesting ligands, with the receptors have been determined, by using a combination of molecular docking and molecular dynamics (MD) simulations along with molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) binding free energy calculations. The binding poses for the long-chain cannabinoids implicate that a site surrounded by the transmembrane (TM)2, TM7, and extracellular loop (ECL)2 is vital for providing the long-chain ligands with the selectivity for CB1R, especially I267 of CB1R (corresponding to L182 of CB2R). Based on the obtained binding modes, the calculated relative binding free energies and selectivity are all in good agreement with the corresponding experimental data, suggesting that the determined binding poses are reasonable. The computational strategy used in this study may also prove fruitful in applications with other GPCRs or membrane-bound proteins.
Due to the different reactivity of hydrogenation reaction by metal-free FLPs catalyst for 2,3-disubstituted 2H-1,4-benzoxazine, we explored the reaction mechanism by density functional theory calculations. We have chosen three kinds of substrates with different hydrogenation reactivity as the prototype substrates and toluene as the solvent to calculate the potential energy profile for the FLPs-catalysed hydrogenation reaction at M06-2X/6-311++G(d,p) level with polarized continuum model (PCM) to simulate the solvent effect. From the potential energy profile, we found that when B(C 6 F 5 ) 3 encounters with 2,3-diphenyl 2H-1,4-benzoxazine (1o) or 2-methyl-3-phenyl 2H-1,4-benzoxazine (1p) in toluene, it mainly generates the mixture of Lewis acid-base adducts and Frustrated Lewis Pairs, which has almost similar stability suggesting the transformation of each other by intermolecular rearrangement. However, it reveals big difference when the B(C 6 F 5 ) 3 encounters with 2,3-dimethyl 2H-1,4-benzoxazine (1q), where the Lewis acid-base adducts is the preference rather than the mixture of Lewis acid-base adducts and Frustrated Lewis Pairs or Frustrated Lewis Pairs since the lower stability energy. Due to the big energy gap (10.9 kcal/mol) between Lewis acid-base adducts and Frustrated Lewis Pairs, the generated Lewis acid-base adducts could not transform into Frustrated Lewis Pairs in the FLPs-catalysed hydrogenation of 1q at 298 K. That is the main reason why 1q is an inert substrate for the hydrogenation catalysed by FLPs. Natural Bond Orbital, Mulliken charge analysis and the proton affinity energy of N4 site was carried out to assess the electric effect of substituent at C3 on N4 site. It reveals negligible effect of substituent at C3 on N4 charge (basicity) and thus proposes that steric hindrance effect is the major factor affecting the stability energy of Lewis acid-base adducts and Frustrated Lewis Pairs. This is confirmed further by calculative investigation about the substituent effect (-CH 2 CH 3 , -CH(CH 3 ) 2 and C(CH 3 ) 3 ) on the stability of Lewis acid-base adducts and Frustrated Lewis Pairs in 2-methyl-3-substituted 2H-1,4-benzoxazine, where with the increased steric hindrance effect, Lewis acid-base adducts tend to have similar stability with Frustrated Lewis Pairs even though less stability. These results clearly illustrate the elusive phenomenon in our previous experiment and may provide new insight for the design of another novel FLPs-catalysed hydrogenation reaction. © 2019 Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences.
A biocatalytic reduction of 2H-1,4-benzoxazines using imine reductases is reported. This process enables a smooth and enantioselective synthesis of the resulting cyclic amines under mild conditions in aqueous media by means of a catalytic amount of the cofactor NADPH as hydride source as well as glucose as reducing agent used in stoichiometric amount for in situ-cofactor recycling. Several substrates were studied, and the 3,4-dihydro-2H-1,4-benzoxazines were obtained with up to 99% ee. In addition, the efficiency of this reduction process based on imine reductases as catalysts has been demonstrated for one 2H-1,4-benzoxazine on elevated lab scale running at a substrate loading of 10 g L⁻¹ in the presence of a tailor-made whole cell-catalyst.
Synthesis and isolation of highly unstable azirinobenzoxazole and benzoxazines in a chemodivergent fashion from aryl azido vinylogous carbonates by simple change in transition metal acetate is described. Thermal or rhodium(II) acetate‐mediated decomposition of these azides gave dihydroazirino benzoxazole. Their nickel(II) acetate‐promoted reaction gave 4‐dihydro‐2H‐benzoxazines, whereas copper(II) acetate led to the corresponding oxidized imine derivatives. Benzaoxazine derivative could be kinetically resolved using a proline‐catalyzed Mannich reaction. The benzoxazines were rapidly elaborated to angularly fused tetracyclic systems and coumarin‐fused derivatives in a “one pot” fashion.
In this study, we have developed solvent‐controlled regioselective Mannich reaction of cyclicimines (benzoxazinones) with various ketones by using ʟ‐proline as catalyst to afford Mannich products in high yields with excellent enantio‐ and diastereoselectivity. The unsymmetrical ketones produced the linear isomer as the major product in chloroform solvent. On contrary, using the DMSO solvent favoured the formation of branch isomer product with excellent enantio‐ and diastereoselectivity. The aromatic auxiliary was successfully removed and afforded N‐protected chiral amino acid derivative.
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The organized tightly regulated signaling relays engaged by the cannabinoid receptors (CBs) and their ligands, G proteins and other effectors, together constitute the endocannabinoid system (ECS). This system governs many biological functions including cell proliferation, regulation of ion transport and neuronal messaging. This review will firstly examine the physiology of the ECS, briefly discussing some anomalies in the relay of the ECS signaling as these are consequently linked to maladies of global concern including neurological disorders, cardiovascular disease and cancer. While endogenous ligands are crucial for dispatching messages through the ECS, there are also commonalities in binding affinities with copious exogenous ligands, both natural and synthetic. Therefore, this review provides a comparative analysis of both types of exogenous ligands with emphasis on natural products given their putative safer efficacy and the role of Δ9-tetrahydrocannabinol (Δ9-THC) in uncovering the ECS. Efficacy is congruent to both types of compounds but noteworthy is the effect of a combination therapy to achieve efficacy without the unideal side-effects. An example is Sativex that displayed promise in treating Huntington's disease (HD) in preclinical models allowing for its transition to current clinical investigation. Despite the in vitro and preclinical efficacy of Δ9-THC to treat neurodegenerative ailments, its psychotropic effects limit its clinical applicability to treating feeding disorders. We therefore propose further investigation of other compounds and their combinations such as the triterpene, α,β-amyrin that exhibited greater binding affinity to CB1 than CB2 and was more potent than Δ9-THC and the N-alkylamides that exhibited CB2 selective affinity, the latter can be explored towards peripherally exclusive ECS modulation. The synthetic CB1 antagonist, Rimonabant was pulled from market for the treatment of diabetes, however its analogue SR144528 maybe an ideal lead molecule towards this end and HU-210 and Org27569 are also promising synthetic small molecules.
In the last few years, cannabinoid type-2 receptor (CB2R) selective ligands have shown a great potential as novel therapeutic drugs in several diseases. With the aim of discovering new selective cannabinoid ligands, a series of pyridazinone-4-carboxamides was designed and synthesized, and the new derivatives tested for their affinity toward the hCB1R and hCB2R. The 6-(4-chloro-3-methylphenyl)-2-(4-fluorobenzyl)-N-(cis-4-methylcyclohexyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide (9) displayed high CB2-affinity (KiCB2 = 2.0 ± 0.81 nM) and a notable selectivity (KiCB1/KiCB2 > 2000). In addition, 9 and other active new synthesized entities have demonstrated to behave as CB2R inverse agonists in [³⁵S]-GTPγS binding assay. ADME predictions of the newly synthesized CB2R ligands suggest a favourable pharmacokinetic profile. Docking studies disclosed the specific pattern of interactions of these derivatives. Our results support that pyridazinone-4-carboxamides represent a new promising scaffold for the development of potent and selective CB2R ligands.
A metal-free hydrogenation of 3-substituted 2H-1,4-benzoxazines has been successfully realized with 2.5 mol% of B(C6F5)3 as a catalyst to furnish a variety of 3,4-dihydro-2H-1,4-benzoxazines in 93-99% yields. Up to 42% ee was also achieved for the asymmetric hydrogenation with the use of a chiral diene and HB(C6F5)2.
The present invention overcomes the problems associated with existing drug delivery systems by delivering cannabinoids transdermally. Preferably, the cannabinoids are delivered via an occlusive body (i.e., a patch) to alleviate harmful side effects and avoid gastrointestinal (first-pass) metabolism of the drug by the patient. A first aspect of the invention provides a method for relieving symptoms associated with illness or associated with the treatment of illness in a mammalian subject, comprising the steps of selecting at least one cannabinoid from the group consisting of cannabinol, cannabidiol, nabilone, levonantradol, (−)-HU-210, (+)-HU-210, 11-hydroxy-Δ9-THC, Δ8-THC-11-oic acid, CP 55,940, and R(+)-WIN 55,212-2, selecting at least one permeation enhancer from the group consisting of propylene glycol monolaurate, diethylene glycol monoethyl ether, an oleoyl macrogolglyceride, a caprylocaproyl macrogolglyceride, and an oleyl alcohol, and delivering the selected cannabinoid and permeation enhancer transdermally to treat an illness.
In a previous study, we have identified 3-alkyl-1,5-diaryl-1
H
-1,2,4-triazoles to be a novel class of cannabinoid type 1 receptor (CB
1
R) antagonists. In order to expand the number of cannabinoid ligands with a central 1,2,4-triazole scaffold, we have synthesized a novel series of 1-benzyl-1
H
-1,2,4-triazoles, and some of them were evaluated by CB
1
R radioligand binding assays. Compound
12a
showed the most interesting pharmacological properties, possessing a CB
1
R affinity in the nanomolar range.
The first copper-catalyzed asymmetric formal [4 + 2] cycloaddition of o-aminophenol derivatives with propargylic esters as the bis-electrophilic C2 synthons for the stereoselective construction of chiral 2,3,4-trisubstituted 2H-1,4-benzoxazines bearing an exocyclic double bond has been developed. By using a structurally modified chiral ketimine P,N,N-ligand, a wide range of optically active 2H-1,4-benzoxazines were prepared in high yields and with excellent enantioselectivities (up to 97% ee).
Peptides may act through G protein-coupled receptors to influence cardiovascular performance; thus, delineating mechanisms involved in signaling is a molecular-based strategy to influence health. Molecular switches, often represented by conserved motifs, maintain a receptor in an inactive state. However, once the switch is broken, the transmembrane regions move and activation occurs. The molecular switches of Drosophila melanogaster myosuppressin (MS) receptors were previously identified to include a unique ionic lock and novel 3-6 lock, as well as transmission and tyrosine toggle switches. In addition to MS, cardioactive ligands structurally related by a C-terminal RF-NH2 include sulfakinin, neuropeptide F (NPF), short NPF, and FMRF-NH2-containing peptide subfamilies. We hypothesized receptor molecular switch motifs were conserved within a RF-NH2 subfamily and across species. Thus, we investigated RF-NH2 receptor (RFa-R) molecular switches in D. melanogaster, Tribolium castaneum, Anopheles gambiae, Rhodnius prolixus, and Bombyx mori. Adipokinetic hormone (AKH), which does not contain a RF-NH2, was also examined. The tyrosine toggle switch and ionic lock showed a higher degree of conservation within a RF-NH2 subfamily than the transmission switch and 3-7 lock. AKH receptor motifs were not representative of a RF-NH2 subfamily. The motifs and interactions of switches in the RFa-Rs were consistent with receptor activation and ligand-specific binding.
Copyright © 2015. Published by Elsevier Inc.
6-Alkoxy-5-aryl-3-pyridincarboxamides, including the brain penetrant compound 14g and its peripherally restricted analog 14h, have been recently introduced as selective, high affinity antagonists of the human cannabinoid receptor-1 (hCB1R, J. Med. Chem. 56, 9874-96, 2013). Binding analyses revealed two orders of magnitude lower affinity of these compounds for mouse and rat versus human CB1R, whereas the affinity of rimonabant is comparable for all three CB1Rs. Modeling of ligand binding to CB1R and binding assays with native and mutant (Ile105Met) hCB1Rs indicate that the Ile105 to Met mutation in rodent CB1Rs accounts for the species-dependent affinity of 14g and 14h. Our work identifies Ile105 as a new pharmacophore component for developing better hCB1R antagonists, and invalidates rodent models for assessing the antiobesity efficacy of 14g and 14h.
The American Society for Pharmacology and Experimental Therapeutics.
The highly enantioselective hydrogenation of 3-aryl-2H-1,4-benzoxazines was achieved using the (cyclooctadiene)iridium chloride dimer/(S)-SegPhos/iodine {[Ir(COD)Cl]2/(S)-SegPhos/I2} system as catalyst with up to 92% ee. The 3-styryl-2H-1,4-benzoxazine derivatives were also hydrogenated by the iridium catalyst and Pd/C in two consecutive steps whereby 93–95% ee values were obtained.
The association of the adipocyte lipid-binding protein (ALBP) with arachidonic acid (all cis, 20:4 delta 5,8,11,14) and oleic acid (cis, 18:1 delta 9) has been examined by titration calorimentry. In addition, the crystal structure of ALBP with bound arachidonic acid has also been obtained. Crystallographic analysis of the arachidonic acid.ALBP complex along with the previously reported oleic acid-ALBP structure (Xu, Z., Bernlohr, D. A., and Banaszak, L. J. (1993) J. Biol. Chem. 268, 7874-7884) provides a framework for the molecular examination of protein-lipid association. Isothermal titration calorimetry revealed high affinity association of both unsaturated fatty acids with the protein. The calorimetric data yielded the following thermodynamic parameters for arachidonic acid: Kd = 4.4 microM, n = 0.8, delta G = -7370 cal/mol, delta H = -6770 cal/mol, and T delta S = +600 cal/mol. For oleic acid, the thermodynamic parameters were Kd = 2.4 microM, n = 0.9, delta G = -7770 cal/mol, delta H = -6050 cal/mol, and T delta S = +1720 cal/mol. The identification of thermodynamically dominating enthalpic factors for both fatty acids are consistent with the crystallographic studies demonstrating the interaction of the fatty acid carboxylate with a combination of Arg106, Arg126, and Tyr128. The crystallographic refinement of the protein-arachidonate complex was carried out to 1.6 A with the resultant R factor of 0.19. Within the cavity of the crystalline binding protein, the arachidonate was found in a hairpin conformation. The conformation of the bound ligand is consistent with acceptable torsional angles and the four cis double bonds in arachidonate. These results demonstrate that arachidonate is a ligand for ALBP. They provide thermodynamic and structural data concerning the physical basis for protein-lipid interaction and suggest that intracellular lipid-binding proteins may mediate the biological effects of polyunsaturated fatty acids in vivo.
Agonist binding to G protein-coupled receptors is believed to promote a conformational change that leads to the formation of the active receptor state. However, the character of this conformational change which provides the important link between agonist binding and G protein coupling is not known. Here we report evidence that agonist binding to the 2 adrenoceptor induces a conformational change around 125Cys in transmembrane domain (TM) III and around 285Cys in TM VI. A series of mutant 2 adrenoceptors with a limited number of cysteines available for chemical derivatization were purified, site-selectively labeled with the conformationally sensitive, cysteine-reactive fluorophore IANBD and analyzed by fluorescence spectroscopy. Like the wild-type receptor, mutant receptors containing 125Cys and/or 285Cys showed an agonist-induced decrease in fluorescence, while no agonist-induced response was observed in a receptor where these two cysteines were mutated. These data suggest that IANBD bound to 125Cys and 285Cys are exposed to a more polar environment upon agonist binding, and indicate that movements of transmembrane segments III and VI are involved in activation of G protein-coupled receptors.
This chapter discusses the integrated methods for the construction of three-dimensional models and computational probing of structure–function relations in G protein-coupled receptors (GPCR). The rapid pace of cloning and expression of G protein-coupled receptors offers attractive opportunities to probe the structural basis of signal transduction mechanisms at the level of these cell-surface receptors. Major insights have emerged from comparisons and classifications of the amino acid sequences of GPCRs into families defined by evolutionary developments and adapted to perform selective functions. Structural data on GPCRs, based on biochemical, immunological, and biophysical approaches have validated consensus architecture of GPCRs with an extracellular N-terminus, a cytoplasmic C-terminus, and a transmembrane portion comprised of seven-transmembrane helical domains connected by loops. Developments in the molecular modeling and computational exploration of GPCR proteins indicate a tantalizing potential to alleviate some of these difficulties. These expectations are based on the increased rate of success achieved by molecular modeling and computational simulation methods in providing structural insights relevant to the functions of biological molecules.
To further characterize neuronal cannabinoid receptors, we compared the ability of known and novel cannabinoid analogs to compete for receptor sites labeled with either [3H]SR141716A or [3H]CP-55,940. These efforts were also directed toward extending the structure-activity relationships for cannabinoid agonists and antagonists. A series of alternatively halogenated analogs of SR141716A were synthesized and tested in rat brain membrane binding assays along with the classical cannabinoids, Delta9-tetrahydrocannabinol, cannabinol, cannabidiol, the nonclassical cannabinoid CP-55,940, the aminoalkylindole WIN55212-2 and the endogenous fatty acid ethanolamide, anandamide. Saturation binding isotherms were performed with both radioligands, as were displacement studies, allowing an accurate comparison to be made between the binding of these various compounds. Competition studies demonstrated that all of the compounds were able to displace the binding of [3H]CP-55,940 with rank order potencies that agreed with previous studies. However, the rank order potencies of these compounds in competition studies with [3H]SR141716A differed significantly from those determined with [3H]CP-55,940. These results suggest that CP-55,940, WIN55212-2 and other agonists interact with cannabinoid binding sites within the brain which are distinguishable from the population of binding sites for SR141716A, its analogs and cannabidiol. Structural modification of SR141716A significantly altered the affinity of the compound and its relative ability to displace either [3H]CP-55,940 or [3H]SR141716A preferentially within the rat brain receptor membrane preparation.
A cDNA clone encoding a receptor protein which presents all the characteristics of a guanine-nucleotide-binding protein (G-protein)-coupled receptor was isolated from a human brain stem cDNA library. The probe used (HGMP08) was a 600 bp DNA fragment amplified by a low-stringency PCR, using human genomic DNA as template and degenerate oligonucleotide primers corresponding to conserved sequences amongst the known G-protein-coupled receptors. The deduced amino acid sequence encodes a protein of 472 residues which shares 97.3% identity with the rat cannabinoid receptor cloned recently [Matsuda, Lolait, Brownstein, Young & Bronner (1990) Nature (London) 346, 561-564]. Abundant transcripts were detected in the brain, as expected, but lower amounts were also found in the testis. The same probe was used to screen a human testis cDNA library. The cDNA clones obtained were partially sequenced, demonstrating the identity of the cannabinoid receptors expressed in both tissues. Specific binding of the synthetic cannabinoid ligand [3H]CP55940 was observed on membranes from Cos-7 cells transfected with the recombinant receptor clone. In stably transfected CHO-K1 cell lines, cannabinoid agonists mediated a dose-dependent and stereoselective inhibition of forskolin-induced cyclic AMP accumulation. The ability to express the human cannabinoid receptor in mammalian cells should help in developing more selective drugs, and should facilitate the search for the endogenous cannabinoid ligand(s).
The association of the adipocyte lipid-binding protein (ALBP) with arachidonic acid (all cis, 20:4 delta 5,8,11,14) and oleic acid (cis, 18:1 delta 9) has been examined by titration calorimentry. In addition, the crystal structure of ALBP with bound arachidonic acid has also been obtained. Crystallographic analysis of the arachidonic acid.ALBP complex along with the previously reported oleic acid-ALBP structure (Xu, Z., Bernlohr, D. A., and Banaszak, L. J. (1993) J. Biol. Chem. 268, 7874-7884) provides a framework for the molecular examination of protein-lipid association. Isothermal titration calorimetry revealed high affinity association of both unsaturated fatty acids with the protein. The calorimetric data yielded the following thermodynamic parameters for arachidonic acid: Kd = 4.4 microM, n = 0.8, delta G = -7370 cal/mol, delta H = -6770 cal/mol, and T delta S = +600 cal/mol. For oleic acid, the thermodynamic parameters were Kd = 2.4 microM, n = 0.9, delta G = -7770 cal/mol, delta H = -6050 cal/mol, and T delta S = +1720 cal/mol. The identification of thermodynamically dominating enthalpic factors for both fatty acids are consistent with the crystallographic studies demonstrating the interaction of the fatty acid carboxylate with a combination of Arg106, Arg126, and Tyr128. The crystallographic refinement of the protein-arachidonate complex was carried out to 1.6 A with the resultant R factor of 0.19. Within the cavity of the crystalline binding protein, the arachidonate was found in a hairpin conformation. The conformation of the bound ligand is consistent with acceptable torsional angles and the four cis double bonds in arachidonate. These results demonstrate that arachidonate is a ligand for ALBP. They provide thermodynamic and structural data concerning the physical basis for protein-lipid interaction and suggest that intracellular lipid-binding proteins may mediate the biological effects of polyunsaturated fatty acids in vivo.
The binding site of the beta2 adrenergic receptor, like that of other homologous G-protein-coupled receptors, is contained within a water-accessible crevice formed among its seven membrane-spanning segments. Methanethiosulfonate ethylammonium (MTSEA), a charged, hydrophilic, lipophobic, sulfhydryl-specific reagent, had no effect on the binding of agonist or antagonist to wild-type beta2 receptor expressed in HEK 293 cells. This suggested that no endogenous cysteines are accessible in the binding site crevice. In contrast, in a constitutively active beta2 receptor, MTSEA significantly inhibited antagonist binding, and isoproterenol slowed the rate of reaction of MTSEA. This implies that at least one endogenous cysteine becomes accessible in the binding site crevice of the constitutively active beta2 receptor. Cys-285, in the sixth membrane-spanning segment, is responsible for the inhibitory effect of MTSEA on ligand binding to the constitutively active mutant. The acquired accessibility of Cys-285 in the constitutively active mutant may result from a rotation and/or tilting of the sixth membrane-spanning segment associated with activation of the receptor. This rearrangement could bring Cys-285 to the margin of the binding site crevice where it becomes accessible to MTSEA.
In the present study, we showed that Chinese hamster ovary (CHO) cells transfected with human central cannabinoid receptor (CB1) exhibit high constitutive activity at both levels of mitogen-activated protein kinase (MAPK) and adenylyl cyclase. These activities could be blocked by the CB1-selective ligand, SR 141716A, that functions as an inverse agonist. Moreover, binding studies showed that guanine nucleotides decreased the binding of the agonist CP-55,940, an effect usually observed with agonists, whereas it enhanced the binding of SR 141716A, a property of inverse agonists. Unexpectedly, we found that CB1-mediated effects of SR 141716A included inhibition of MAPK activation by pertussis toxin-sensitive receptor-tyrosine kinase such as insulin or insulin-like growth factor 1 receptors but not by pertussis toxin-insensitive receptor-tyrosine kinase such as the fibroblast growth factor receptor. We also observed similar results when cells were stimulated with Mas-7, a mastoparan analog, that directly activates the Gi protein. Furthermore, SR 141716A inhibited guanosine 5'-0-(thiotriphosphate) uptake induced by CP-55,940 or Mas-7 in CHO-CB1 cell membranes. This indicates that, in addition to the inhibition of autoactivated CB1, SR 141716A can deliver a biological signal that blocks the Gi protein and consequently abrogates most of the Gi-mediated responses. By contrast, SR 141716A had no effect on MAPK activation by insulin or IGF1 in CHO cells lacking CB1 receptors, ruling out the possibility of a direct interaction of SR 141716A with the Gi protein. This supports the notion that the Gi protein may act as a negative intracellular signaling cross-talk molecule. From these original results, which considerably enlarge the biological properties of the inverse agonist, we propose a novel model for receptor/ligand interactions.
The rhodopsin-like superfamily of 7-transmembrane receptors is the largest class of signalling molecules in the mammalian genome. Recently, a combination of mutagenesis, biophysical and modelling studies have suggested a credible model for the alpha-carbon backbone in the transmembrane region of the 7-transmembrane receptors, and have started to reveal the structural basis of the conformational switch from the inactive to the active state. A key feature may be the replacement of a network of radial constraints, centred on transmembrane helix three, which stabilise the inactive ground state of the receptor by a new set of axial interactions which help to stabilise the activated state. Transmembrane helix three may act as a rotary switch in the activation mechanism.
Heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) respond to a variety of different
external stimuli and activate G proteins. GPCRs share many structural features, including a bundle of seven transmembrane
α helices connected by six loops of varying lengths. We determined the structure of rhodopsin from diffraction data extending
to 2.8 angstroms resolution. The highly organized structure in the extracellular region, including a conserved disulfide bridge,
forms a basis for the arrangement of the seven-helix transmembrane motif. The ground-state chromophore, 11-cis-retinal, holds the transmembrane region of the protein in the inactive conformation. Interactions of the chromophore with
a cluster of key residues determine the wavelength of the maximum absorption. Changes in these interactions among rhodopsins
facilitate color discrimination. Identification of a set of residues that mediate interactions between the transmembrane helices
and the cytoplasmic surface, where G-protein activation occurs, also suggests a possible structural change upon photoactivation.
The environmentally sensitive, sulfhydryl-reactive, fluorescent probe N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene-diamine (IANBD) was used as a molecular reporter of agonist-induced conformational changes in the beta(2) adrenergic receptor, a prototype hormone-activated G protein-coupled receptor. In the background of a mutant beta(2) adrenergic receptor, with a minimal number of endogenous cysteine residues, new cysteines were introduced in positions 269(6.31), 270(6.32), 271(6.33), and 272(6.34) at the cytoplasmic side of transmembrane segment (TM) 6. The resulting mutant receptors were fully functional and bound both agonists and antagonist with high affinities also upon IANBD labeling. Fluorescence spectroscopy analysis of the purified and site-selectively IANBD-labeled mutants suggested that the covalently attached fluorophore was exposed to a less polar environment at all four positions upon agonist binding. Whereas evidence for only a minor change in the molecular environment was obtained for positions 269(6.31) and 270(6.32), the full agonist isoproterenol caused clear dose-dependent and reversible increases in fluorescence emission at positions 271(6.33) and 272(6.34). The data suggest that activation of G protein-coupled receptors, which are activated by "diffusible" ligands, involves a structural rearrangement corresponding to the cytoplasmic part of TM 6. The preferred conformations of the IANBD moiety attached to the inserted cysteines were predicted by employing a computational method that incorporated the complex hydrophobic/hydrophilic environment in which the cysteines reside. Based on these preferred conformations, it is suggested that the spectral changes reflect an agonist-promoted movement of the cytoplasmic part of TM 6 away from the receptor core and upwards toward the membrane bilayer.
Extracellular signals are transduced across membranes via conformational changes in the transmembrane domains (TMs) of ion channels and G-protein-coupled receptors (GPCRs). Experimental and simulation studies indicate that such conformational switches in transmembrane (alpha-helices can be generated by proline-containing motifs that form molecular hinges. Computational approaches tested on model channel-forming peptides (e.g. alamethicin) reveal functional mechanisms in gap-junction proteins (such as connexin) and voltage-gated K+ channels. Similarly, functionally important roles for proline-based switches in TM6 and TM7 were identified in GPCRs. However, hinges in transmembrane helices are not confined to proline-containing sequence motifs, as evidenced by a non-proline hinge in the M2 helix of the nicotinic acetylcholine receptor. This helix lines the pore and plays a key role in the gating of this channel.
Synthesis of an antagonist, SR141716A, that selectively binds to brain cannabinoid (CB(1)) receptors without producing cannabimimetic activity in vivo, suggests that recognition and activation of cannabinoid receptors are separable events. In the present study, a series of SR141716A analogs were synthesized and were tested for CB(1) binding affinity and in a battery of in vivo tests, including hypomobility, antinociception, and hypothermia in mice. These analogs retained the central pyrazole structure of SR141716A with replacement of the 1-, 3-, 4-, and/or 5-substituents by alkyl side chains or other substituents known to impart potent agonist activity in traditional tricyclic cannabinoid compounds. Although none of the analogs alone produced the profile of cannabimimetic effects seen with full agonists, several of the 3-substituent analogs with higher binding affinities showed partial agonism for one or more measures. Cannabimimetic activity was most noted when the 3-substituent of SR141716A was replaced with an alkyl amide or ketone group. None of the 3-substituted analogs produced antagonist effects when tested in combination with 3 mg/kg Delta(9)-tetrahydrocannabinol (Delta(9)-THC). In contrast, antagonism of Delta(9)-THC's effects without accompanying agonist or partial agonist effects was observed with substitutions at positions 1, 4, and 5. These results suggest that the structural properties of 1- and 5-substituents are primarily responsible for the antagonist activity of SR141716A.
The movements of transmembrane segments (TMs) 3 and 6 at the cytoplasmic side of the membrane play an important role in the activation of G-protein-coupled receptors. Here we provide evidence for the existence of an ionic lock that constrains the relative mobility of the cytoplasmic ends of TM3 and TM6 in the inactive state of the beta(2)-adrenergic receptor. We propose that the highly conserved Arg-131(3.50) at the cytoplasmic end of TM3 interacts both with the adjacent Asp-130(3.49) and with Glu-268(6.30) at the cytoplasmic end of TM6. Such a network of ionic interactions has now been directly supported by the high-resolution structure of the inactive state of rhodopsin. We hypothesized that the network of interactions would serve to constrain the receptor in the inactive state, and the release of this ionic lock could be a key step in receptor activation. To test this hypothesis, we made charge-neutralizing mutations of Glu-268(6.30) and of Asp-130(3.49) in the beta(2)-adrenergic receptor. Alone and in combination, we observed a significant increase in basal and pindolol-stimulated cAMP accumulation in COS-7 cells transiently transfected with the mutant receptors. Moreover, based on the increased accessibility of Cys-285(6.47) in TM6, we provide evidence for a conformational rearrangement of TM6 that is highly correlated with the extent of constitutive activity of the different mutants. The present experimental data together with the recent high-resolution structure of rhodopsin suggest that ionic interactions between Asp/Glu(3.49), Arg(3.50), and Glu(6.30) may constitute a common switch governing the activation of many rhodopsin-like G-protein-coupled receptors.
The G protein-coupled cannabinoid receptor subtypes CB1 and CB2 have been cloned from several species. The CB1 receptor is highly conserved across species, whereas the CB2 receptor shows considerable cross-species variations. The two human receptors share only 44% overall identity, ranging from 35% to 82% in the transmembrane regions. Despite this structural disparity, the most potent cannabinoid agonists currently available are largely undiscriminating and are therefore unsatisfactory tools for investigating the architecture of ligand binding sites. However, the availability of two highly specific antagonists, SR 141716A for the CB1 receptor and SR 144528 for the CB2 receptor, has allowed us to adopt a systematic approach to defining their respective binding sites through the use of chimeric CB1 receptor/CB2 receptor constructs, coupled with site-directed mutagenesis. We identified the region encompassed by the fourth and fifth transmembrane helices as being critical for antagonist specificity. Both the wild type human receptors overexpressed in heterologous systems are autoactivated; SR 141716A and SR 144528 exhibit classical inverse agonist properties with their respective target receptors. In addition, through its interaction with the CB1 receptor SR 141716A blocks the Gi protein-mediated activation of mitogen-activated protein kinase stimulated by insulin or insulin-like growth factor I. An in-depth analysis of this discovery has led to a modified three-state model for the CB1 receptor, one of which implicates the SR 141716A-mediated sequestration of Gi proteins, with the result that the growth factor-stimulated intracellular pathways are effectively impeded.
Aminoalkylindoles (AAIs) are a novel series of cannabinoid receptor ligands. In this report we disclose the structural features of AAIs which are important for binding to this receptor as measured by inhibition of binding of [ 3 H]Win 55212-2 (5). Functional activity in the mouse vas deferens is also noted and used to distinguish agonists from potential antagonists. The key structural features for potent cannabinoid activity in this series are a bicyclic (naphthyl) substituent at the 3-position, a small (H) substituent at the 2-position, and an aminoethyl (morpholinoethyl) substituent at the 1-position. A 6-bromo analog, Win 54461 (31), has been identified as a potential cannabinoid receptor antagonist. Modeling experiments were done to develop a pharmacophore and also to compare AAI structures with those of classical cannabinoids. The fact that the cannabinoid AAIs arose out of work on a series of cyclooxygenase inhibitors makes sense now that an endogenous cannabinoid ligand has been identified which is a derivative of arachidonic acid. Because of their unique structures and physical properties, AAIs provide useful tools to study the structure and function of the cannabinoid receptor(s).
Molecular modeling has been employed to design a new group of cannabimimetic 1-alkyl-2-methyl-3-(1-naphthoyl)indoles. Cannabinoid activity was evaluated in vivo in the mouse and in vitro by determining the binding to the cannabinoid receptor. Maximum activity was found for the 1-butyl, pentyl and hexyl analogs. A rationalization for the alignment of these indoles with traditional cannabinoids is presented.
Our modeling studies have suggested that β branched amino acids Val, Ile, or Thr located (i,i + 3) or (i,i + 4) apart on an alpha helix can form a groove into which a ligand alkyl chain can fit. Exptl. support for this idea comes from the crystal structure of adipocyte lipid binding protein complexed with stearic acid. We hypothesized that the transmembrane helix 6 (TMH 6) βXXβ motif of the CB1/CB2 receptors (V6.43/I6.46), which immediately precedes the conserved TMH 6 CWXP motif, serves as an interaction site for the alkyl tail of cannabinoid (CB) ligands and that interaction with this motif may trigger receptor activation. Conformational memories (CM) calcns. on TMH 6 of CB1 and CB2 were used to explore the conformation of TMH 6 in unbound and complexed states. The conserved Pro 6.50 generated a kink in the α-helical structure that behaved as a flexible hinge. In the context of a three-dimensional model of the CB1 receptor, a helix from the more kinked family of CB1 TMH 6 conformers calcd. by CM brought the intracellular portion of TMH 6 in proximity to TMH 3, analogous to the inactive state TMH 3/6 conformation seen in the X-ray crystal structure of rhodopsin. A CM study of CB1 TMH 6 in which a pentane mol. (as a model system for the CB ligand side chain) interacts with the V6.43/I6.46 groove was also conducted. The results of this calcn. showed that alkyl chain interaction with the V6.43/I6.46 groove directly modulates the overall conformation of TMH 6, biasing the population of TMH 6 conformers toward the family of less-kinked CB1 TMH 6 conformations calcd. by CM. In the context of the CB1 bundle, this conformational change would cause the intracellular end of TMH 6 to move away from TMH 3. Such a movement is consistent with recent exptl. results for agonist induced conformational changes at the intracellular side of TMH 6 in the β2-adrenergic receptor. In contrast to results for CB1, CB2 TMH 6 showed a smaller range of kink angles possible for unbound TMH 6, with no significant shift in the populations of TMH 6 when the V6.43/I6.46 groove was occupied by pentane. We hypothesized that the profound flexibility differences between wild-type (WT) CB1 vs. WT CB2 TMH 6 revealed by CM calcns. may be due to the size of residue 6.49 which immediately precedes P6.50 of the CWXP motif (G6.49 in WT CB1 and F6.49 in WT CB2). To test this hypothesis, using CM, we compared the flexibilities of WT CB1 and CB2 TMH 6 with those of the switch mutants, CB1 G6.49F and CB2 F6.49G. Consistent with results reported above, the degree of kinking (av. of 100 conformers) was distinctly different between CB1 (40.9°; std. dev. ± 16.9°) and CB2 (24.6°; std. dev. ± 4.3°), with CB1 TMH 6 exhibiting a noticeably wider range of kink angles than CB2. These flexibilities were essentially switched in the mutants [CB1 G6.49F mutant (25.3°; std. dev. ± 5.7°) and a CB2 F6.49G mutant (44.3°; std. dev. ± 21.4°)]. Taken together, these results suggest that TMH 6 in CB1, but not in CB2, is sensitive to conformational modulation by an alkyl chain bound in the V6.43/I6.46 groove. Furthermore, results suggest that the small size of residue 6.49 in CB1 facilitates the P6.50 flexible hinge motion of CB1 TMH 6.
The predominant animal model in which the pharmacology of cannabinoids is studied is the mouse. Nonetheless, the structure and functional expression of the mouse cannabinoid receptor (CB1) gene have not been reported. We have cloned and expressed the gene for the mouse CB1 receptor and compared its properties with those of native mouse CB1 receptors in brain and N18TG2 neuroblastoma cells. The mouse CB1 gene was isolated from a mouse 129 strain genomic library. Sequence analysis of a 6-kb BamHI fragment of the mouse CB1 genomic clone indicates 95% nucleic acid identity between mouse and rat (99.5% amino acid identity) and 90% nucleic acid identity (97% amino acid identity) between mouse and human. Examination of the 5′ untranslated sequence of the mouse CB1 genomic clone revealed a splice junction site approximately 60 bp upstream from the translation start site, indicating the possibility of splice variants of the CB1 receptors. The coding region of the mouse CB1 receptor was stably expressed in 293 cells, and binding by [3H]SR 141716A and [3H]CP-55,940 was determined. The Bmax and Kd values obtained with [3H]SR 141716A (921 ± 58 fmol/mg and 0.73 ± 0.13 nM, respectively) were similar to those of native mouse CB1 receptors in brain (Bmax of 1.81 ± 0.44 pmol/mg, Kd of 0.16 ± 0.01 nM) and N18TG2 cells (Bmax of 197 ± 29 fmol/mg, Kd of 0.182 ± 0.08 nM). The mouse CB1 receptor genomic clone will be a useful tool for studying the function and regulation of the CB1 receptor in mice.
A series of 1-pentyl-1H-indol-3-yl-(1-naphthyl)methanes (9–11) and 2-methyl-1-pentyl-1H-indol-3-yl-(1-naphthyl)methanes (12–14) have been synthesized to investigate the hypothesis that cannabimimetic 3-(1-naphthoyl)indoles interact with the CB1 receptor by hydrogen bonding to the carbonyl group. Indoles 9–11 have significant (Ki=17–23nM) receptor affinity, somewhat less than that of the corresponding naphthoylindoles (5, 15, 16). 2-Methyl-1-indoles 12–14 have little affinity for the CB1 receptor, in contrast to 2-methyl-3-(1-naphthoyl)indoles 17–19, which have affinities comparable to those of 5, 15, 16. A cannabimimetic indene hydrocarbon (26) was synthesized and found to have Ki=26±4nM. Molecular modeling and receptor docking studies of naphthoylindole 16, its 2-methyl congener (19) and indolyl-1-naphthylmethanes 11 and 14, combined with the receptor affinities of these cannabimimetic indoles, strongly suggest that these cannabinoid receptor ligands bind primarily by aromatic stacking interactions in the transmembrane helix 3-4-5-6 region of the CB1 receptor.
The majority of extracellular physiologic signaling molecules act by stimulating GTP-binding protein (G-protein)-coupled receptors (GPCRs). To monitor directly the formation of the active state of a prototypical GPCR, we devised a method to site specifically attach fluorescein to an endogenous cysteine (Cys-265) at the cytoplasmic end of transmembrane 6 (TM6) of the adrenergic receptor (2AR), adjacent to the G-protein-coupling domain. We demonstrate that this tag reports agonist-induced conformational changes in the receptor, with agonists causing a decline in the fluorescence intensity of fluorescein-2AR that is proportional to the biological efficacy of the agonist. We also find that agonists alter the interaction between the fluorescein at Cys-265 and fluorescence-quenching reagents localized to different molecular environments of the receptor. These observations are consistent with a rotation and/or tilting of TM6 on agonist activation. Our studies, when compared with studies of activation in rhodopsin, indicate a general mechanism for GPCR activation; however, a notable difference is the relatively slow kinetics of the conformational changes in the 2AR, which may reflect the different energetics of activation by diffusible ligands.
As a potent, specific antagonist for the brain cannabinoid receptor (CB1), the biarylpyrazole N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A; 1) was the lead compound for initiating studies designed to examine the structure−activity relationships of related compounds and to search for more selective and potent cannabimimetic ligands. A series of pyrazole derivatives was designed and synthesized to aid in the characterization of the cannabinoid receptor binding sites and also to serve as potentially useful pharmacological probes. Therapeutically, such compounds may have the ability to antagonize harmful side effects of cannabinoids and cannabimimetic agents. Structural requirements for potent and selective brain cannabinoid CB1 receptor antagonistic activity included (a) a para-substituted phenyl ring at the 5-position, (b) a carboxamido group at the 3-position, and (c) a 2,4-dichlorophenyl substituent at the 1-position of the pyrazole ring. The most potent compound of this series contained a p-iodophenyl group at the 5-position, a piperidinyl carboxamide at the 3-position, and a 2,4-dichlorophenyl group at the 1-position of the pyrazole ring. The iodinated nature of this compound offers additional utility as a γ-enriching SPECT (single photon emission computed tomography) ligand that may be useful in characterizing brain CB1 receptor binding in vivo.
The conformational properties of cis-5,8,11,14-eicosatetraenoylethanolamide (anandamide) were analysed by the combined use of NMR experimental results plus molecular simulations. The structure of anandamide was found to be a predominantly linear with a seven-atom ring of the ethanolamine group having a hydrogen bond which stabilizes the molecule. The vinylic group present has a cis conformation in solution. The terminal chain has a linear conformation and undergoes isotropic fast motion typical of this structure. Copyright © 2001 John Wiley & Sons, Ltd.
A theoretical analysis has been made of the relationship between the inhibition constant (KI) of a substance and the (I50) value which expresses the concentration of inhibitor required to produce 50 per cent inhibition of an enzymic reaction at a specific substrate concentration. A comparison has been made of the relationships between KI and I50 for monosubstrate reactions when noncompetitive or uncompetitive inhibition kinetics apply, as well as for bisubstrate reactions under conditions of competitive, noncompetitive and uncompetitive inhibition kinetics. Precautions have been indicated against the indiscriminate use of I50 values in agreement with the admonitions previously described in the literature. The analysis described shows KI does not equal I50 when competitive inhibition kinetics apply; however, KI is equal to I50 under conditions of either noncompetitive or uncompetitive kinetics.
Pravadoline (1) is an (aminoalkyl)indole analgesic agent which is an inhibitor of cyclooxygenase and, in contrast to other NSAIDs, inhibits neuronally stimulated contractions in mouse vas deferens (MVD) preparations (IC50 = 0.45 microM). A number of conformationally restrained heterocyclic analogues of pravadoline were synthesized in which the morpholinoethyl side chain was tethered to the indole nucleus. Restraining the morpholine diminished the ability of these pravadoline analogues to inhibit prostaglandin synthesis in vitro. In contrast, mouse vas deferens inhibitory activity was enhanced in [2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl] pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-(4-methoxyphenyl)methano ne (20). Only the R enantiomer of 20 was active (IC50 = 0.044 microM). An optimal orientation of the morpholine nitrogen for MVD inhibitory activity within the analogues studied was in the lower right quadrant, below the plane defined by the indole ring. A subseries of analogues of 20 and a radioligand of the most potent analogue, (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo [1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (21) were prepared. Inhibition of radioligand binding in rat cerebellar membranes was observed to correlate with functional activity in mouse vas deferens preparations. Binding studies with this ligand (Win 55212-2) have helped demonstrate that the (aminoalkyl)indole binding site is functionally equivalent with the CP-55,940 cannabinoid binding site. These compounds represent a new class of cannabinoid receptor agonists.
The geometries of aromatic-aromatic interactions between phenylalanine residues in proteins are analysed in detail and correlated with energy calculations. A new definition of the interplanar angle is important for distinguishing favourable edge-to-face and unfavourable face-to-face orientations. The experimental observations are scattered over a wide range of conformational space, with no strongly preferred single orientation. However, Phe-Phe interactions occur almost exclusively in electrostatically attractive geometries: electrostatically unfavourable regions are only sparsely populated. Electrostatics dominate the geometry of interaction, while van der Waals' interactions are less significant, probably due to the hydrophobic environment of the protein core. The observations on proteins support the Hunter-Sanders rules for pi-pi interactions. In particular, offset stacked geometries, which theory predicts to be favourable, are observed experimentally. For monocyclic aromatics, use of a C-H dipole, the approach used in molecular mechanics calculations, accounts well for these aromatic-aromatic interactions. Comparison with the results obtained from the small molecules database indicates that the protein and small molecule crystal environments are very different.
Protein engineering is a powerful tool for studying relationships between receptor structure and function--providing that it is used and interpreted appropriately. Site-directed mutagenesis, deletion mutagenesis and construction of chimaeric proteins have all been used to characterize receptors. In this review, Walter Ward, David Timms and Alan Fersht describe the application of protein engineering, illustrating important concepts with experimental data. They explain that detailed study of function requires careful dissection of mechanistic steps. Care must also be taken when selecting replacement residues; mutation should not cause delocalized structural reorganization or else the true significance of functional change will remain unclear.
Analysis of neighboring aromatic groups in four biphenyl peptides or peptide analogs and 34 proteins reveals a specific aromatic-aromatic interaction. Aromatic pairs (less than 7 A between phenyl ring centroids) were analyzed for the frequency of pair type, their interaction geometry (separation and dihedral angle), their nonbonded interaction energy, the secondary structural locations of interacting residues, their environment, and their conservation in related molecules. The results indicate that on average about 60 percent of aromatic side chains in proteins are involved in aromatic pairs, 80 percent of which form networks of three or more interacting aromatic side chains. Phenyl ring centroids are separated by a preferential distance of between 4.5 and 7 A, and dihedral angles approaching 90 degrees are most common. Nonbonded potential energy calculations indicate that a typical aromatic-aromatic interaction has energy of between -1 and -2 kilocalories per mole. The free energy contribution of the interaction depends on the environment of the aromatic pair. Buried or partially buried pairs constitute 80 percent of the surveyed sample and contribute a free energy of between -0.6 and -1.3 kilocalories per mole to the stability of the protein's structure at physiologic temperature. Of the proteins surveyed, 80 percent of these energetically favorable interactions stabilize tertiary structure, and 20 percent stabilize quaternary structure. Conservation of the interaction in related molecules is particularly striking.
Over the past few years, the concept that the activation of G protein-coupled receptors and transmitter-gated ion channels depends on a conformational change has received increasingly widespread acceptance. As a result, these two structurally distinct families of receptors can now be considered to obey a similar two-state mechanism. However, traditional receptor theory has largely overlooked this concept. In this article, Paul Leff explains and illustrates the predictions of the two-state model of receptor activation and discusses its impact on the analysis and interpretation of agonist-receptor interactions.
The goal of this study was to determine the ends and orientations of the seven transmembrane helices of the cannabinoid (CB1) receptor, a G-protein coupled receptor (GPCR). After initial sequence alignment, Fourier transform methods were used with the nPRIFT hydrophobicity scale and with a variability profile to calculate the alpha-helical periodicity (AP) in the primary amino acid sequence of the human CB1 receptor and of its alignment. AP plots were used to identify the amino acids which comprise each of the seven CB1 transmembrane helices. An intracellular alpha helix extension of Helix 7 was characterized by analyzing the relative direction of variability and hydrophobic moment vectors. Variability moment vectors were then used to delineate the orientation of each helix in the membrane. Based upon these vector calculations, a tentative helix bundle arrangement was obtained. This arrangement is largely consistent with the proposed transmembrane helix bundle arrangement in rhodopsin, a GPCR.
The beta 2-adrenergic receptor undergoes isomerization between an inactive conformation (R) and an active conformation (R*). The formation of the active conformation of the receptor molecule can be promoted by adrenergic agonists or by mutations in the third cytoplasmic domain that constitutively activate the receptor. Here we show that, of several beta-adrenergic receptor-blocking drugs tested, only two, ICI 118551 and betaxolol, inhibit the basal signaling activity of the beta 2-adrenergic receptor, thus acting as negative antagonists. We document the molecular properties of the more efficacious ICI 118551; (i) it shows higher affinity for the inactive form of the receptor and (ii) it inhibits the spontaneous formation of a beta-adrenergic receptor kinase substrate by the receptor. These properties are opposite those of adrenergic agonists, indicating that, in a fashion reciprocal to that of agonists, negative antagonists promote the formation of an inactive conformation of the receptor.
The cannabinoid receptor in brain (CB1) specifically binds delta 9-tetrahydrocannabinol, the predominant central nervous system-active component of marijuana. An eicosanoid found in brain, N-(2-hydroxyethyl)arachidonylamide (anandamide), binds to CB1 with similar affinity. This report considers structure-activity requirements for a series of novel amides and rigid hairpin conformations typified by N-(2-hydroxyethyl)prostaglandin amides, assayed with phenylmethylsulfonyl fluoride inactivation of esterases/amidases. Arachidonyl esters were 30-fold less potent than N-(2-hydroxyethyl)arachidonylamide, showing a rank order of potency of methyl = ethyl > propyl = isopropyl. Within the N-(hydroxyalkyl)arachidonylamide series, a one-carbon increase in chain length increased the potency 2-fold, but continued extension decreased affinity. Substituting the amide for the N-(2-hydroxyethyl)amide function produced a 4-fold loss of affinity. The N-(propyl)-, N-(butyl)-, and N-(benzyl)arachidonylamide derivatives exhibited a 3-fold increase, no change, and a 5-fold decrease, respectively, in affinity, compared with N-(2-hydroxyethyl)arachidonylamide. Both the methoxy ether and the formamide derivatives suffered > 20-fold loss of potency, compared with N-(2-hydroxyethyl)arachidonylamide. N-(2-Aminoethyl)arachidonylamide interacted poorly with CB1. At 100 microM, N-(2-hydroxyethyl)amide analogs of prostaglandin E2, A2, B2, and B1 failed to alter [3H]CP55940 binding to CB1. N-(2-Hydroxyethyl)arachidonylamide inhibited adenylate cyclase with lesser potency but with similar efficacy, compared with desacetyllevonantradol. Extending the length of the hydroxyalkyl moiety by one carbon increased the apparent potency by 1 order of magnitude. The N-(propyl) derivative exhibited a 5-fold greater potency than did the N-(2-hydroxyethyl) analog. It appears that the bulk and length of the moiety appended to arachidonic acid are more important determinants of affinity for CB1 than is hydrogen-bonding capability.
Lys192 in the third transmembrane domain of the human CB1 cannabinoid receptor was converted to an alanine to study its role in receptor recognition and activation by agonists. HU-210, CP-55940, WIN55212-2, and anandamide, four cannabinoid agonists with distinct chemical structures, were used to characterize the wild-type and the mutant receptors. In human embryonal kidney 293 cells stably expressing the wild-type receptor, specific binding to [3H]WIN55212-2 and inhibition of cAMP accumulation by cannabinoid agonists were demonstrated, with different ligands exhibiting the expected rank orders of potency and stereoselectivity in competition binding and functional assays. In cells expressing the mutant receptor, the binding affinity of the receptor for [3H]WIN55212-2 was only slightly affected (the Kd for the mutant receptor was twice that of the wild-type), and the ability of WIN55212-2 to inhibit cAMP accumulation was unchanged. However, HU-210, CP-55940, and anandamide were unable to compete for [3H]WIN55212-2 binding to the mutant receptor. In addition, the potencies of HU-210, CP-55940, and anandamide in inhibiting cAMP accumulation were reduced by > 100-fold. These results demonstrate that Lys192 is critical for receptor binding by HU-210, CP-55940, and anandamide. Because Lys192 is not important for receptor binding and activation by WIN55212-2, WIN55212-2 must interact with the cannabinoid receptor through at least one point of interaction that is distinct from those of the three other agonists.
The antagonist SR 141716A has a high specificity for the central CB1 cannabinoid receptor and negligeable affinity for the
peripheral CB2 receptor, making it an excellent tool for probing receptor structure-activity relationships. From binding experiments
with mutated CB1 and with chimeric CB1/CB2 receptors we have begun to identify the domains of CB1 implicated in the recognition
of SR 141716A. Receptors were transiently expressed in COS-3 cells, and their binding characteristics were studied with SR
141716A and with CP 55,940, an agonist recognized equally well by the two receptors. The region delineated by the fourth and
fifth transmembrane helices of CB1 proved to be crucial for high affinity binding of SR 141716A. The CB1 and CB2 second extracellular
loops, e2, were exchanged, modifications that had no effect on SR 141716A binding in the CB1 variant but that eliminated CP
55,940 binding in both mutants. The replacement of the conserved cysteine residues in e2 of CB2 by serine also eliminated
CP 55,940 binding, but replacement of those in CB1 resulted in the sequestration of the mutated receptors in the cell cytoplasm.
The e2 domain thus plays some role in CP 55,940 binding but none in SR 141716A recognition, binding of the latter clearly
implicating residues in the adjoining transmembrane helices.
The difference of rhodopsin and metarhodopsin II (MII) absorption spectra exhibits a characteristic pattern in the UV wavelength range, consisting of peaks at 278, 286, 294, 302 nm. These difference bands are thought to result from the perturbation of the environments of tryptophan and/or tyrosine residues. We used site-directed mutagenesis to investigate the contribution of tryptophan absorption to these spectral features. Each of the five tryptophan residues in bovine rhodopsin was replaced by either a phenylalanine or a tyrosine. The mutant pigments (W35F, W126F, W161F, W175F, W265F/Y) were prepared and studied by UV-visible photobleaching difference spectroscopy. The difference spectra of the W35F and W175F mutants were identical to that of rhodopsin, whereas in the W161F mutant, the magnitudes of the 294- and 302-nm bands were slightly lowered. The differential absorbance at 294 nm was reduced by over 50% in the W126F and W265F/Y mutants. The difference peak at 302 nm was reduced in the W265F/Y mutants, but was almost completely absent in the W126F mutant. These data indicate that the difference bands at 294 and 302 nm originate from the perturbations of Trp126 and Trp265 environments resulting from a general conformational change concomitant with MII formation and receptor activation. Model studies on tryptophan absorption indicate that the difference peak at 294 nm is due to the differential shift of the Lb absorption of the indole side chain as a result of decreased hydrophobicity or polarizability of the Trp126 and Trp265 environments. The resolution of the 302-nm band, assigned to the differential shift of the indole La absorption, is consistent with hydrogen-bonding interactions of the indole N-H groups of Trp126 and Trp265 becoming weaker in MII. These results suggest that the photoactivation of rhodopsin involves a change in the relative disposition of transmembrane helices 3 and 6, which contain Trp126 and Trp265 respectively, within the alpha-helical bundle of the receptor.
Conformational changes are thought to underlie the activation of heterotrimeric GTP-binding protein (G protein)—coupled receptors.
Such changes in rhodopsin were explored by construction of double cysteine mutants, each containing one cysteine at the cytoplasmic
end of helix C and one cysteine at various positions in the cytoplasmic end of helix F. Magnetic dipolar interactions between
spin labels attached to these residues revealed their proximity, and changes in their interaction upon rhodopsin light activation
suggested a rigid body movement of helices relative to one another. Disulfide cross-linking of the helices prevented activation
of transducin, which suggests the importance of this movement for activation of rhodopsin.
The cannabinoid receptors, CB1 and CB2, are members of the G-protein coupled receptor family and share many of this family's structural features. A highly conserved aspartic acid residue in the second transmembrane domain of G-protein coupled receptors has been shown for many of these receptors to be functionally important for agonist binding and/or G-protein coupling. To determine whether this residue is involved in cannabinoid receptor function, we used site-directed mutagenesis of receptor cDNA followed by expression of the mutant receptor in HEK 293 cells. Aspartate 163 (in CB1) and aspartate 80 (in CB2) were substituted with either asparagine or glutamate. Stably transfected cell lines were tested for radioligand binding and inhibition of cAMP accumulation. Binding of the cannabinoid receptor agonist [3H]CP-55,940 was not affected by either mutation in either the CB1 or CB2 receptor, nor were the affinities of anandamide or (-)-delta 9-tetrahydrocannabinol. Binding of the CB1-selective receptor antagonist SR141716A also was unaltered. However, the affinity of WIN 55,212-2 was attenuated significantly in the CB1, but not the CB2, mutant receptors. Studies examining inhibition of cAMP accumulation showed reduced effects of cannabinoid agonists in the mutated receptors. Our data suggest that this aspartate residue is not generally important for ligand recognition in the cannabinoid receptors; however, it is required for communication with G proteins and signal transduction.
The endogenous cannabinoid anandamide (N-arachidonoylethanolamide) has been shown to possess higher affinity for the cannabinoid CB1 receptor than for the CB2 receptor. Carrier-mediated transport has been proposed to be essential for the termination of the biological effects of anandamide, while hydrolysis of anandamide is performed by a membrane-bound amidohydrolase, fatty acid amidohydrolase (FAAH). As interaction of anandamide with each of these targets occurs in different environments, the conformations of anandamide for interaction with each target may differ. To ascertain what conformations of anandamide, a highly flexible molecule, are favored in polar and nonpolar environments, the new method of Conformational Memories (CM) was used. CM has been shown to achieve complete conformational sampling of highly flexible ligands, to converge in a very practical number of steps, and to be capable of overcoming energy barriers very efficiently (Guarnieri et al. J. Am. Chem. Soc. 1996, 118, 5580). The generalized Born/surface area (GB/SA) continuum solvation models for chloroform and for water were used in the CM calculations. As a means of validation, CM was first applied to arachidonic acid because both experimental and theoretical conformational studies of arachidonic acid have been reported. CM was also applied to sn-2-arachidonylglycerol (2-AG), another endogenous CB ligand; to a 1,1-dimethylheptyl derivative of anandamide, an analogue with higher CB1 affinity than anandamide; and to N-(2-hydroxyethyl)prostaglandin-B2-ethanolamide (PGB2-EA), a prostanoid ligand which does not bind to CB1. Consistent with the literature, arachidonic acid was found to exist in an extended, angle-iron shape and in back-folded conformations which were U, J, or helical in shape. The angle-iron and U-shapes were both highly populated conformations with the angle-iron preferred in CHCl3 and the U-shape preferred in H2O. Results for anandamide and 2-AG paralleled those for arachidonic acid with the exception that anandamide in water does not adopt a pure extended conformation but, rather, favors a hybrid-extended/U-shape. For the dimethyl-heptyl derivative of anandamide, the U-shape was found to be predominant in both environments (42% in CHCl3, 38% in H2O), but the population of the angle-iron shape was still significant (25% in CHCl3, 29% in H2O). For all of these ligands, J-shaped conformers constituted from 7% to 17% of the conformer population, while the helical shape was less than 5%. In both CHCl3 and H2O, the presence of the five-membered ring attenuates the ability of PGB2-EA to adopt an extended conformation. PGB2-EA was found instead to exist predominantly in an L-shape (i.e., distorted U-shape). The low probability of PGB2-EA adopting an extended conformation may be why PGB2-EA shows such low affinity for the CB1 receptor. The conformational information obtained here for anandamide and 2-AG may be useful in the design of rigid analogues which mimic the preferred molecular conformations (shapes) of these ligands. Such rigid analogues may be useful in deducing the bioactive conformation of these endogenous cannabinoids, not only at the CB receptors but also at the FAAH enzyme active site and possibly at the binding site(s) on the newly proposed anandamide transporter.
The CB1 cannabinoid receptor antagonist SR 141716A abolished the inhibition of Ca2+ currents by the agonist WIN 55,212-2. However, SR 141716A alone increased Ca2+ currents, with an EC50 of 32 nM, in neurons that had been microinjected with CB1 cRNA. For an antagonist to elicit an effect, some receptors must be tonically active. Evidence for tonically active CB1 receptors was seen as enhanced tonic inhibition of Ca2+ currents. Preincubation with anandamide failed to enhance the effect of SR 141716A, indicating that anandamide did not cause receptor activity. Under Ca2+-free conditions designed to block the Ca2+-dependent formation of anandamide and sn-2-arachidonylglycerol, SR 141716A again increased the Ca2+ current. The Ca2+ current was tonically inhibited in neurons expressing the mutant K192A receptor, which has no affinity for anandamide, demonstrating that this receptor is also tonically active. SR 141716A had no effect on the Ca2+ current in these neurons, but SR 141716A could still antagonize the effect of WIN 55, 212-2. Thus, the K192 site is critical for the inverse agonist activity of SR 141716A. SR 141716A appeared to become a neutral antagonist at the K192A mutant receptor. Native cannabinoid receptors were studied in male rat major pelvic ganglion neurons, where it was found that WIN 55,212-2 inhibited and SR 141716A increased Ca2+ currents. Taken together, our results demonstrate that a population of native and cloned CB1 cannabinoid receptors can exist in a tonically active state that can be reversed by SR 141716A, which acts as an inverse agonist.
The aminoalkylindoles (AAIs) are agonists at both the cannabinoid CB1 and CB2 receptors. To determine whether the s-trans or s-cis form of AAIs is their receptor-appropriate conformation, two pairs of rigid AAI analogues were studied. These rigid analogues are naphthylidene-substituted aminoalkylindenes that lack the carbonyl oxygen of the AAIs. Two pairs of (E)- and (Z)-naphthylidene indenes (C-2 H and C-2 Me) were considered. In each pair, the E geometric isomer is intended to mimic the s-trans form of the AAIs, while the Z geometric isomer is intended to mimic the s-cis form. Complete conformational analyses of two AAIs, pravadoline (2) and WIN-55, 212-2 (1), and of each indene were performed using the semiempirical method AM1. S-trans and s-cis conformations of 1 and 2 were identified. AM1 single-point energy calculations revealed that when 1 and each indene were overlayed at their corresponding indole/indene rings, the (E)- and (Z)-indenes were able to overlay naphthyl rings with the corresponding s-trans or s-cis conformer of 1 with an energy expense of 1.13/0.69 kcal/mol for the C-2 H (E/Z)-indenes and 0.82/0.74 kcal/mol for the C-2 Me (E/Z)-indenes. On the basis of the hypothesis that aromatic stacking is the predominant interaction of AAIs such as 1 at the CB receptors and on the demonstration that the C-2 H (E/Z)- and C-2 Me (E/Z)-indene isomers can mimic the positions of the aromatic systems in the s-trans and s-cis conformers of 1, the modeling results support the previously established use of indenes as rigid analogues of the AAIs. A synthesis of the naphthylidene indenes was developed using Horner-Wittig chemistry that afforded the Z isomer in the C-2 H series, which was not produced in significant amounts from an earlier reported indene/aldehyde condensation reaction. This approach was extended to the C-2 Me series as well. Photochemical interconversions in both the C-2 H and C-2 Me series were also successful in obtaining the less favored isomer. Thus, the photochemical process can be used to provide quantities of the minor isomers C-2 H/Z and C-2 Me/E. The CB1 and CB2 affinities as well as the activity of each compound in the twitch response of the guinea pig ileum (GPI) assay were assessed. The E isomer in each series was found to have the higher affinity for both the CB1 and CB2 receptors. In the rat brain membrane assay versus [3H]CP-55,940, the Ki's for the C-2 H/C-2 Me series were 2.72/2.89 nM (E isomer) and 148/1945 nM (Z isomer). In membrane assays versus [3H]SR141716A, a two-site model was indicated for the C-2 H/C-2 Me (E isomers) with Ki's of 10. 8/9.44 nM for the higher-affinity site and 611/602 nM for the lower-affinity site. For the Z isomers, a one-site model was indicated with Ki's of 928/2178 nM obtained for the C2 H/C-2 Me analogues, respectively. For the C-2 H/C-2 Me series, the CB2 Ki's obtained using a cloned cell line were 2.72/2.05 nM (E isomer) and 132/658 nM (Z isomer). In the GPI assay, the relative order of potency was C-2 H E > C-2 Me E > C-2 H Z > C-2 Me Z. The C-2 H E isomer was found to be equipotent with 1, while the C-2 Me Z isomer was inactive at concentrations up to 3.16 microM. Thus, results indicate that the E geometric isomer in each pair of analogues is the isomer with the higher CB1 and CB2 affinities and the higher pharmacological potency. Taken together, results reported here support the hypothesis that the s-trans conformation of AAIs such as 1 is the preferred conformation for interaction at both the CB1 and CB2 receptors and that aromatic stacking may be an important interaction for AAIs at these receptors.
The human cannabinoid receptors, central cannabinoid receptor (CB1) and peripheral cannabinoid receptor (CB2), share only 44% amino acid identity overall, yet most ligands do not discriminate between receptor subtypes. Site-directed mutagenesis was employed as a means of mapping the ligand recognition site for the human CB2 cannabinoid receptor. A lysine residue in the third transmembrane domain of the CB2 receptor (K109), which is conserved between the CB1 and CB2 receptors, was mutated to alanine or arginine to determine the role of this charged amino acid in receptor function. The analogous mutation in the CB1 receptor (K192A) was found to be crucial for recognition of several cannabinoid compounds excluding (R)-(+)-[2, 3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1, 4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN 55,212-2). In contrast, in human embryonic kidney (HEK)-293 cells expressing the mutant or wild-type CB2 receptors, we found no significant differences in either the binding profile of several cannabinoid ligands nor in inhibition of cAMP accumulation. We identified a high-affinity site for (-)-3-[2-hydroxyl-4-(1, 1-dimethylheptyl)phenyl]-4-[3-hydroxyl propyl] cyclohexan-1-ol (CP-55,940) in the region of helices 3, 6, and 7, with S3.31(112), T3.35(116), and N7.49(295) in the K109A mutant using molecular modeling. The serine residue, unique to the CB2 receptor, was then mutated to glycine in the K109A mutant. This double mutant, K109AS112G, retains the ability to bind aminoalkylindoles but loses affinity for classical cannabinoids, as predicted by the molecular model. Distinct cellular localization of the mutant receptors observed with immunofluorescence also suggests differences in receptor function. In summary, we identified amino acid residues in the CB2 receptor that could lead to subtype specificity.
Two subtypes of the cannabinoid receptor (CB1 and CB2) are expressed in mammalian tissues. Although selective antagonists are available for each of the subtypes, most of the available cannabinoid agonists bind to both CB1 and CB2 with similar affinities. We have synthesized two analogs of N-arachidonylethanolamine (AEA), arachidonylcyclopropylamide (ACPA) and arachidonyl-2-chloroethylamide (ACEA), that bind to the CB1 receptor with very high affinity (KI values of 2.2 +/- 0.4 nM and 1.4 +/- 0.3 nM, respectively) and to the CB2 receptor with low affinity (KI values of 0.7 +/- 0.01 microM and 3.1 +/- 1.0 microM, respectively). Both ACPA and ACEA have the characteristics of agonists at the CB1 receptor; both inhibit forskolin-induced accumulation of cAMP in Chinese hamster ovary cells expressing the human CB1 receptor, and both analogs increase the binding of [35S]GTPgammaS to cerebellar membranes and inhibit electrically evoked contractions of the mouse vas deferens. ACPA and ACEA produce hypothermia in mice, and this effect is inhibited by coadministration of the CB1 receptor antagonist SR141716A. Therefore, ACPA and ACEA are high-affinity agonists of the CB1 receptor but do not bind the CB2 receptor, suggesting that structural analogs of AEA can be designed with considerable selectivity for the CB1 receptor over the CB2 receptor.
It has been reported that WIN55212-2, a prototypic aminoalkylindole, has higher affinity for CB(2) than for CB(1). To explain the selectivity of WIN55212-2 for CB(2), molecular modeling studies were performed to probe the interacting sites between WIN55212-2 and cannabinoid receptors. In TMH5 the position 5.46 is a Phe in CB(2) versus a Val in CB(1). Docking of WIN55212-2 into the models of CB(1) and CB(2) predicts that F5.46 will result in a greater aromatic stacking of CB(2) with WIN55212-2. Using site-directed mutagenesis, this hypothesis was tested by exchanging the amino acids at position 5.46 between CB(1) and CB(2). Two mutations, including a Phe to Val mutation at the position 5.46 in CB(2) (CB2F5. 46V), and a corresponding Val to Phe mutation at the position 5.46 in CB(1) (CB(1)V5.46F), were made. The mutant receptors were transfected into 293 cells, and stable cell lines expressing similar numbers of receptors as wild-type receptors were chosen for additional ligand binding and cAMP accumulation studies. In ligand- binding assays, the CB(2)F5.46V mutation decreased the affinity of WIN55212-2 for CB(2) by 14-fold. In contrast, the CB(1)V5.46F mutation increased the affinity of WIN55212-2 for CB(1) by 12-fold. However, these mutations did not change the affinity of HU-210, CP-55940, and anandamide for CB(1) and CB(2). In cAMP accumulation assays, the changes in EC(50) values of WIN55212-2 were consistent with the changes in its binding affinity caused by the mutations. These results strongly support the hypothesis that the selectivity of WIN55212-2 for CB(2) over CB(1) is attributable to the change from Val in CB(1) at position 5.46 to Phe in CB(2).
Two subtypes of the human cannabinoid receptor have been identified. The CB1 receptor is primarily distributed in the central nervous system, whereas the CB2 receptor is associated with peripheral tissue, including the spleen. These two subtypes are also distinguished by their ligand-binding profiles. The goal of this study was to identify critical residues in transmembrane region III (TM3) of the receptors that contribute to subtype specificity in ligand binding. For this purpose, a chimeric cannabinoid receptor [CB1/2(TM3)] was generated in which the TM3 of CB1 was replaced with the corresponding region of CB2. These receptors were stably expressed in Chinese hamster ovary cells for evaluation. The binding affinities of CB1/2(TM3) and the wild-type CB1 receptor to several prototype ligands were similar with one notable exception: the chimeric receptor exhibited a 4-fold enhancement in binding affinity to WIN 55,212-2 (K(d) = 4.8 nM) relative to that observed with CB1 (K(d) = 21.7 nM). Two additional aminoalkylindoles, JWH 015 and JWH 018, also bound the chimeric receptor (K(i) = 1.0 microM and 1.4 nM, respectively) with higher affinity compared with the wild-type CB1 (K(i) = 5.2 microM and 9.8 nM, respectively). Furthermore, the increase in binding affinities of the aminoalkylindoles were reflected in the EC(50) values for the ligand-induced inhibition of intracellular cAMP levels mediated by the chimeric receptor. This pattern mirrors the selectivity of WIN 55,212-2 binding to CB2 compared with CB1. Site-specific mutagenesis of the most notable amino acid changes in the chimeric receptor, Gly195 to Ser and Ala198 to Met, revealed that the enhancement in WIN 55,212-2 binding is contributed to by the Ser but not by the Met residue. The data indicate that the amino acid differences in TM3 between CB1 and CB2 play a critical role in subtype selectivity for this class of compounds.
Prostaglandin H synthase-1 and -2 (PGHS-1 and -2) catalyze the committed step in prostaglandin synthesis and are targets for nonsteroidal anti-inflammatory drugs (NSAIDs) like aspirin. We have determined the structure of PGHS-1 at 3 angstrom resolution with arachidonic acid (AA) bound in a chemically productive conformation. The fatty acid adopts an extended L-shaped conformation that positions the 13proS hydrogen of AA for abstraction by tyrosine-385, the likely radical donor. A space also exists for oxygen addition on the antarafacial surface of the carbon in the 11-position (C-11). While this conformation allows endoperoxide formation between C-11 and C-9, it also implies that a subsequent conformational rearrangement must occur to allow formation of the C-8/C-12 bond and to position C-15 for attack by a second molecule of oxygen.
Two subtypes of cannabinoid receptors are currently recognized, CB(1), found in brain and neuronal cells, and CB(2), found in spleen and immune cells. We have characterized 1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxyl ic acid phenylamide (CP-272871) as a novel aryl pyrazole antagonist for the CB(1) receptor. CP-272871 competed for binding of the cannabinoid agonist (3)H-labeled (-)-3-[2-hydroxy-4-(1, 1-dimethylheptyl)-phenyl]-4-[3-hydroxypropyl]cyclohexan-1-ol ([(3)H]CP-55940) at the CB(1) receptor in rat brain membranes with a K(d) value 20-fold greater than that of N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl (SR141716A). CP-272871 also competed for binding with the aminoalkylindole agonist (3)H-labeled (R)-(+)-[2, 3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]1, 4-benzoxazin-6-yl](1-naphthyl)methanone ([(3)H]WIN-55212-2), as well as the aryl pyrazole antagonist [(3)H]SR141716A. Inverse agonist as well as antagonist properties were observed for both SR141716A and CP-272871 in signal transduction assays in biological preparations in which the CB(1) receptor is endogenously expressed. SR141716A augmented secretin-stimulated cyclic AMP (cAMP) accumulation in intact N18TG2 neuroblastoma cells, and this response was reversed by the agonist desacetyllevonantradol. CP-272871 antagonized desacetyllevonantradol-mediated inhibition of adenylyl cyclase in N18TG2 membranes, and increased adenylyl cyclase activity in the absence of agonist. SR141716A and CP-272871 antagonized desacetyllevonantradol-stimulated (35)S-labeled guanosine-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) binding to brain membrane G-proteins, and decreased basal [(35)S]GTPgammaS binding to G-proteins. K(+) enhanced CP-272871 and SR141716A inverse agonist activity compared with Na(+) or NMDG(+) in the assay. These results demonstrated that the aryl pyrazoles SR141716A and CP-272871 behave as antagonists and as inverse agonists in G-protein-mediated signal transduction in preparations of endogenously expressed CB(1) receptors.
The relationship between the Ser, Thr, and Cys side-chain conformation (chi(1) = g(-), t, g(+)) and the main-chain conformation (phi and psi angles) has been studied in a selection of protein structures that contain alpha-helices. The statistical results show that the g(-) conformation of both Ser and Thr residues decreases their phi angles and increases their psi angles relative to Ala, used as a control. The additional hydrogen bond formed between the O(gamma) atom of Ser and Thr and the i-3 or i-4 peptide carbonyl oxygen induces or stabilizes a bending angle in the helix 3-4 degrees larger than for Ala. This is of particular significance for membrane proteins. Incorporation of this small bending angle in the transmembrane alpha-helix at one side of the cell membrane results in a significant displacement of the residues located at the other side of the membrane. We hypothesize that local alterations of the rotamer configurations of these Ser and Thr residues may result in significant conformational changes across transmembrane helices, and thus participate in the molecular mechanisms underlying transmembrane signaling. This finding has provided the structural basis to understand the experimentally observed influence of Ser residues on the conformational equilibrium between inactive and active states of the receptor, in the neurotransmitter subfamily of G protein-coupled receptors.
Several tryptophan (Trp) residues are conserved in G protein-coupled receptors (GPCRs). Relatively little is known about the contribution of these residues and especially of those in the fourth transmembrane domain in the function of the CB(2) cannabinoid receptor. Replacing W158 (very highly conserved in GPCRs) and W172 (conserved in CB(1) and CB(2) cannabinoid receptors but not in many other GPCRs) of the human CB(2) receptor with A or L or with F or Y produced different results. We found that the conservative change of W172 to F or Y retained cannabinoid binding and downstream signaling (inhibition of adenylyl cyclase), whereas removal of the aromatic side chain by mutating W172 to A or L eliminated agonist binding. W158 was even more sensitive to being mutated. We found that the conservative W158F mutation retained wild-type binding and signaling activities. However, W158Y and W158A mutants completely lost ligand binding capacity. Thus, the Trp side chains at positions 158 and 172 seem to have a critical, but different, role in cannabinoid binding to the human CB(2) receptor.
A non-redundant set of 1154 protein structures from the Protein Data Bank was examined with respect to close interactions between C-H-donor and pi-acceptor groups. A total of 31,087 interactions were found to satisfy our selection criteria. Their geometric parameters suggest that these interactions can be classified as weak hydrogen bonds.A set of 12 interaction classes were defined based on the division of the donors into three groups and the acceptors into four groups. These classes were examined separately, and the respective interactions described in detail in each class. Most prominent were interactions between aliphatic C-H donors and aromatic pi-acceptors and interactions between aromatic C-H donors and aromatic pi-acceptors. About three-quarters of the Trp-rings, half of all Phe and Tyr-rings and a quarter of all His-rings were found to be involved as acceptors in C-H...pi-interactions. On the donor side, a preference for aromatic C-H groups was observed, but also for the aliphatic side-chains of the long, extended amino acid residues Lys, Arg and Met, and the Pro ring. The average distance between the C-donor and the center-of-mass of the pi-acceptor was observed to be significantly longer in the 174 protein structures determined at >2.5 A resolution. Also, the distribution is significantly wider. This resolution dependence suggests that the force fields commonly used for the refinement of protein structures may not be adequate. C-H...pi-interactions involving aromatic groups either as donor or as acceptor groups are found mostly in the interior of the protein. The more hydrophilic the participating groups are, the closer to the surface are the interactions located. About 40 % of all C-H...pi-interactions occur between amino acid residue side-chains that are separated by nine or less residues in sequence. Dependent on the interaction class, different preferences for secondary structure, residue type and side-chain conformation were observed. It is likely that the C-H...pi-interactions contribute significantly to the overall stability of a protein.