Activation of the cannabinoid CB1 receptor may involve a W6 48/F3 36 rotamer toggle switch.

The cannabinoid CB1 receptor, a member of the Rhodopsin (Rho) family of G protein coupled receptors (GPCRs), exhibits high levels of constitutive activity. In contrast, Rho exhibits an exquisite lack of constitutive activity. In Rho, W6.48(265) on transmembrane helix 6 (TMH6) is flanked by aromatic residues at positions i-4 (F6.44) and i + 3 (Y6.51), while in CB1 the residues i-4 and i + 3 to W6.48 are leucines (L6.44 and L6.51). Based upon spectroscopic evidence, W6.48 has been proposed to undergo a rotamer switch (chi1 g+ -->trans) upon activation of Rho. In the work reported here, the biased Monte Carlo method, Conformational Memories (CM) was used to test the hypothesis that the high constitutive activity exhibited by CB1 may be due, in part, to the lack of aromatic residues i-4 and i + 3 from W6.48. In this work, the W6.48 rotamer shift (chi1 g+ -->trans) was used as the criterion for activation. Conformational Memories (CM) calculations on WT CB1 TMH6 and L6.44F and L6.51Y mutant TMH6s revealed that an aromatic residue at 6.44 tends to disfavor the W6.48 chi1 g+ -->trans transition and an aromatic residue at 6.51 would require a concomitant movement of the Y6.51 chi1 from trans-->g+ when the W6.48 chi1 undergoes a g+ -->trans shift. In contrast, CM calculations on WT CB1 TMH6 revealed that the presence of leucines at 6.44 and 6.51 provide W6.48 with greater conformational mobility, with a W6.48 transchi1 preferred. Conformational Memories calculations also revealed that the W6.48 chi1 g+ -->trans transition in WT CB1 TMH6 is correlated with the degree of kinking in TMH6. The average proline kink angles for TMH6 were higher for helices with a W6.48 g+ chi1 than for those with a W6.48 transchi1. These results are consistent with experimental evidence that TMH6 straightens during activation. Transmembrane helix (TMH) bundle models of the inactive (R) and active (R*) states of CB1 were then probed for interactions that may constrain W6.48 in the inactive state of CB1. These studies revealed that F3.36 (transchi1) helps to constrain W6.48 in a g+ chi1 in the inactive (R) state of CB1. In the R* state, these studies suggest that F3.36 must assume a g+ chi1 in order to allow W6.48 to shift to a transchi1. These results suggest that the W6.48/F3.36 interaction may act as the 'toggle switch' for CB1 activation, with W6.48 chi1 g+/F3.36 chi1 trans representing the inactive (R) and W6.48 chi1 trans/F3.36 chi1 g+ representing the active (R*) state of CB1.

The difference between the CB(1) and CB(2) cannabinoid receptors at position 5.46 is crucial for the selectivity of WIN55212-2 for CB(2).

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).

Role of a conserved lysine residue in the peripheral cannabinoid receptor (CB2): evidence for subtype specificity.

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.

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'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.

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.

Cardiovascular manifestations of mixed connective tissue disease in adults.

To assess the nature and distribution of cardiovascular abnormalities associated with mixed connective tissue disease, we studied 38 patients with overlapping clinical manifestations of systemic lupus erythematosus, progressive systemic sclerosis and polymyositis, and circulating antibodies to nuclear ribonucleoprotein. The protocol included taking a medical history and a physical echocardiogram, and pulmonary function tests. Cardiac catheterization was performed on 17 patients. Postmortem examination was performed on four of the five patients who died during follow-up. Acute pericarditis and/or pericardial effusion was detected in 11 patients (29%) and mitral valve prolapse was identified in 10 patients (26%). Marked intimal hyperplasia of coronary arteries was observed in all four hearts that were autopsied and perivascular and myocardial leukocytic aggregates were present in two hearts. Pulmonary vascular resistance was elevated in 11 of the 17 patients who underwent cardiac catheterization. In summary, cardiovascular abnormalities associated with mixed connective tissue disease include acute pericarditis and/or effusion, mitral valve prolapse, intimal hyperplasia of coronary arteries, perivascular and myocardial leukocytic infiltrates, and pulmonary hypertension.