Structural Mimicry in Class A G Protein-coupled Receptor Rotamer Toggle Switches

In this study, we tested the hypothesis that a CB1 TMH3-4-5-6 aromatic microdomain, which includes F3.25(190), F3.36(201), W5.43(280), and W6.48(357), is centrally involved in CB1 receptor activation, with the F3.36(201)/W6.48(357) interaction key to the maintenance of the CB1-inactive state. We have shown previously that when F3.36(201), W5.43(280), and W6.48(357) are individually mutated to alanine, a significant reduction in ligand binding affinity is observed in the presence of WIN 55,212-2 and SR141716A but not CP55,940 and anandamide. In the work presented here, we report a detailed functional analysis of the F3.36(201)A, F3.25(190)A, W5.43(280)A, and W6.48(357)A mutant receptors in stable cell lines created in HEK cells for agonist-stimulated guanosine 5′-3-O-(thio)triphosphate (GTPγS) binding and GIRK1/4 channel current effects in Xenopus oocytes where the mutant proteins were expressed transiently. The F3.36(201)A mutation showed statistically significant increases in ligand-independent stimulation of GTPγS binding versus wild type CB1, although basal levels for the W6.48(357)A mutant were not statistically different from wild type CB1. F3.36(201)A demonstrated a limited activation profile in the presence of multiple agonists. In contrast, enhanced agonist activation was produced by W6.48(357)A. These results suggest that a F3.36(201)/W6.48(357)-specific contact is an important constraint for the CB1-inactive state that may need to break during activation. Modeling studies suggest that the F3.36(201)/W6.48(357) contact can exist in the inactive state of CB1 and be broken in the activated state via a χ1 rotamer switch (F3.36(201) trans, W6.48(357) g+) → (F3.36(201) g+, W6.48(357) trans). The F3.36(201)/W6.48(357) interaction therefore may represent a “toggle switch” for activation of CB1

Structural characterization of omega loop peptides from cytochrome c

M.S.Martha S. Brigg

Exploration of Biologically Relevant Conformations of Anandamide, 2-Arachidonylglycerol, and Their Analogues Using Conformational Memories

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

Conformational Memories and the Endocannabinoid Binding Site at the Cannabinoid CB1 Receptor

Endocannabinoid structure-activity relationships (SAR) indicate that the CB1 receptor recognizes ethanolamides whose fatty acid acyl chains have 20 or 22 carbons, with at least three homoallylic double bonds and satn. in at least the last five carbons of the acyl chain. To probe the mol. basis for these acyl chain requirements, the method of conformational memories (CM) was used to study the conformations available to an n-6 series of ethanolamide fatty acid acyl chain congeners: 22:4, n-6 (Ki = 34.4±3.2 nM); 20:4, n-6 (Ki = 39.2±5.7 nM); 20:3, n-6 (Ki = 53.4±5.5 nM); and 20:2, n-6 (Ki \u3e 1500 nM). CM studies indicated that each analog could form both extended and U/J-shaped families of conformers. However, for the low affinity 20:2, n-6 ethanolamide, the higher populated family was the extended conformer family, while for the other analogs in the series, the U/J-shaped family had the higher population. In addn., the 20:2, n-6 ethanolamide U-shaped family was not as tightly curved as were those of the other analogs studied. To quantitate this variation in curvature, the radius of curvature (in the C-3 to C-17 region) of each member of each U/J-shaped family was measured. The av. radii of curvature (with their 95% confidence intervals) were found to be 5.8 Å (5.3-6.2) for 20:2, n-6; 4.4 Å (4.1-4.7) for 20:3, n-6; 4.0 Å (3.7-4.2) for 20:4, n-6; and 4.0 Å (3.6-4.5) for 22:4, n-6. These results suggest that higher CB1 affinity is assocd. with endocannabinoids that can form tightly curved structures. Endocannabinoid SAR also indicate that the CB1 receptor does not tolerate large endocannabinoid headgroups; however, it does recognize both polar and nonpolar moieties in the headgroup region. To identify a headgroup orientation that results in high CB1 affinity, a series of di-Me anandamide analogs (R)-N-(1-methyl-2-hydroxyethyl)-2-(R)-methyl-arachidonamide (Ki = 7.42±0.86 nM), (R)-N-(1-methyl-2-hydroxyethyl)-2-(S)-methyl-arachidonamide (Ki = 185±12 nM), (S)-N-(1-methyl-2-hydroxyethyl)-2-(S)-methyl-arachidonamide (Ki = 389±72 nM), and (S)-N-(1-methyl-2-hydroxyethyl)-2-(R)-methyl-arachidonamide (Ki = 233±69 nM) were then studied using CM and computer receptor docking studies in an active state (R*) model of CB1. These studies suggested that the high CB1 affinity of the R,R stereoisomer is due to the ability of the headgroup to form an intramol. hydrogen bond between the carboxamide oxygen and the headgroup hydroxyl that orients the C2 and C1\u27 Me groups to have hydrophobic interactions with valine 3.32(196), while the carboxamide oxygen forms a hydrogen bond with lysine 3.28(192) at CB1. In this position in the CB1 binding pocket, the acyl chain has hydrophobic and C-H···π interactions with residues in the transmembrane helix (TMH) 2-3-7 region. Taken together, the studies reported here suggest that anandamide and its congeners adopt tightly curved U/J-shaped conformations at CB1 and suggest that the TMH 2-3-7 region is the endocannabinoid binding region at CB1

An Aromatic Microdomain at the Cannabinoid CB1 Receptor Constitutes an Agonist/Inverse Agonist Binding Region

The cannabinoid CB1 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 CB1 is crucial for proper function of CB1. Modeling studies reported here suggest that in the inactive state of CB1, the binding site of the CB1 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 CB1, 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···π 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

N-(Piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A) Interaction with LYS 3.28(192) Is Crucial for Its Inverse Agonism at the Cannabinoid CB1 Receptor

In superior cervical ganglion neurons, N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A) competitively antagonizes the Ca2+ current effect of the cannabinoid (CB) agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55212-2), and behaves as an inverse agonist by producing opposite current effects when applied alone. In contrast, in neurons expressing CB1 with a K→A mutation at residue 3.28(192) (i.e., K3.28A), SR141716A competitively antagonizes the effects of WIN55212-2, but behaves as a neutral antagonist by producing no current effects itself. Receptor modeling studies suggested that in the CB1 inactive (R) state, SR1417A16A stabilizes transmembrane helix 6 in its inactive conformation via arom. stacking with F3.36/W6.48. In this binding site, SR141716A would exhibit higher affinity for CB1 R due to a hydrogen bond between the SR141716A C3 substituent and K3.28(192), a residue available to SR141716A only in R. To test this hypothesis, a mutant thermodn. cycle was constructed that combined the evaluation of SR141716A affinity at WT CB1 and K3.28A with an evaluation of the wild-type CB1 and K3.28A affinities of an SR141716A analog, 5-(4-chlorophenyl)-3-[(E)-2-cyclohexylethenyl]-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole (VCHSR), that lacks hydrogen bonding potential at C3. Binding affinities suggested that K3.28 is involved in a strong interaction with SR141716A in WT CB1, but does not interact with VCHSR. Thermodn. cycle calcns. indicated that a direct interaction occurs between the C3 substituent of SR141716A and K3.28 in WT CB1. Consistent with these results, VCHSR acted as a neutral antagonist at WT CB1. These results support the hypothesis that hydrogen bonding of the SR141716A C3 substituent with K3.28 is responsible for its higher affinity for the inactive R state, leading to its inverse agonism

A Critical Role for a Tyrosine Residue in the Cannabinoid Receptors for Ligand Recognition

Previous mutation and modeling studies have identified an aromatic cluster in the transmembrane helix (TMH) 3-4-5 region as important for ligand binding at the CB1 and CB2 cannabinoid receptors. Through novel mixed mode Monte Carlo/Stochastic Dynamics (MC/SD) calculations, we tested the importance of aromaticity at position 5.39(275) in CB1. MC/SD calculations were performed on wild-type (WT) CB1 and two mutants, Y5.39(275)F and Y5.39(275)I. Results indicated that while the CB1 Y5.39(275)F mutant is very similar to WT, the Y5.39(275)I mutant shows pronounced topology changes in the TMH 3-4-5 region. Site-directed mutagenesis studies of tyrosine 5.39 to phenylalanine (Y→F) or isoleucine (Y→I) in both CB1 and CB2 were performed to determine the functional role of this amino acid in each receptor subtype. HEK 293 cells transfected with mutant receptor cDNAs were evaluated in radioligand binding and cyclic AMP assays. The CB1 mutant and WT receptors were also co-expressed with G-protein-coupled inwardly rectifying channels (GIRK1 and GIRK4) in Xenopus oocytes to assess functional coupling. The Y→F mutation resulted in cannnabinoid receptors with subtle differences in WT binding and signal transduction. In contrast, the Y→I mutations produced receptors that could not produce signal transduction or bind to multiple cannabinoid compounds. However, immunofluorescence data indicate that the Y→I mutation was compartmentalized and expressed at a level similar to that of the WT cannabinoid receptor. These results underscore the importance of aromaticity at position CB1 5.39(275) and CB2 5.39(191) for ligand recognition in the cannabinoid receptors

The Bioactive Conformation of Aminoalkylindoles at the Cannabinoid CB1 and CB2 Receptors: Insights Gained from (E)- and (Z)-Naphthylidene Indenes

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\u27s 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\u27s 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\u27s 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\u27s 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 \u3e C-2 Me E \u3e C-2 H Z \u3e 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 μM. 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