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.

Pregnenolone is considered the inactive precursor of all steroid hormones and its potential functional effects have been largely neglected. The administration of the main active principle of Cannabis sativa (marijuana) Δ9-tetrahydrocannabinol (THC) substantially increases the synthesis of pregnenolone in the brain via the activation of type-1 cannabinoid (CB1) receptor. Pregnenolone then, acting as a signaling specific inhibitor of the CB1 receptor, reduces several effects of THC. This negative feedback mediated by pregnenolone reveals an unknown paracrine/autocrine loop protecting the brain from CB1 receptor over-activation that could open an unforeseen novel approach for the treatment of cannabis intoxication and addiction.

The cannabinoid receptors belong to the class A (rhodopsin family) of G protein-coupled receptors (GPCRs).2 The second cannabinoid receptor subtype, CB2 (2), is highly expressed throughout the immune system (3, 4) and has been described in the central nervous system under both pathological (5) and physiological conditions (6). All known CB2 ligands are highly lipophilic. In fact, the CB2 endogenous cannabinoid, sn-2-arachidonoylglycerol (2-AG) (7,8), is synthesized on demand from the lipid bilayer itself in a two-step process in which phospholipase C-␤ hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol, which is then hydrolyzed by diacylglycerol lipase to yield 2-AG (9, 10). After 2-AG interaction with the membraneembedded CB receptor, it is hydrolyzed to arachidonic acid and glycerol by a membrane-associated enzyme, monoacylglycerol lipase (11). As revealed by the crystal structures of rhodopsin (12-15), the ␤ 2 -adrenergic receptor (AR) (16 -18), ␤ 1 -AR (19), and adenosine A2A receptor (20), the general topology of a GPCR includes the following: 1) an extracellular (EC) N terminus; 2) seven transmembrane ␣-helices (TMHs) arranged to form a closed bundle; 3) loops connecting TMHs that extend intra-and extracellularly; and 4) an intracellular (IC) C terminus that begins with a short helical segment (helix 8) oriented parallel to the membrane surface. Agonists bind inside the crevice formed by the TMH bundle and produce conformational changes on the IC face of the receptor that uncover previously masked G protein-binding sites (21), which then lead to G protein coupling. Biophysical studies using a variety of techniques indicate that ligand-induced receptor activation produces the following changes: 1) a conformational change in the W6.48 "toggle switch" within the ligand binding pocket (22); 2) a change in the relative orientations of TMH3 and -6 that breaks an IC "ionic lock" (23-28), with the intracellular end of TMH6 moving away from TMH3 by hinging and moving up toward lipid (27); 3) the uptake of two protons (29); and 4) an influx of water (30).Recent isothiocyanate covalent labeling studies have suggested that a classical cannabinoid, AM841, enters the CB2 receptor via the lipid bilayer (1). However, the sequence of steps involved in such a lipid pathway entry has not yet been elucidated. We report here microsecond time scale unbiased

In superior cervical ganglion neurons, N-(piperidiny-1-yl)-5-(4-

The biarylpyrazole, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716; 1) has been shown to act as an inverse agonist/antagonist at the cannabinoid CB1 receptor. Our previous mutant cycle study suggested that K3.28(192) is involved in a direct interaction with the C-3 substituent of 1 in wild-type (WT) CB1.(1) However, these results did not establish what part of the C-3 substituent of 1 is involved in the K3.28(192) hydrogen bond, the carboxamide oxygen or the piperidine nitrogen. Furthermore, our previous calcium channel assay results for 5-(4- chlorophenyl)-3-[(E)-2-cyclohexylethenyl]-1-(2,4-dichlorophenyl)-4- methyl-1H-pyrazole (VCHSR; 2) (an analogue of 1 that lacks hydrogen-bonding capability in its C-3 substituent) showed that this compound acts as a neutral antagonist, a result that is in contrast to 1, which acts as an inverse agonist in this same assay.(1) These results suggested a relationship between biarylpyrazole interaction with K3.28(192) at CB1 and inverse agonism, but these results were for a single pair of compounds (1 and 2). The work presented here was designed to test two hypotheses derived from our modeling and mutant cycle results. The hypotheses are as follows: (1) it is the carboxamide oxygen of the C-3 substituent of 1 that interacts directly with K3.28(192) and (2) the interaction with K3.28(192) is crucial for the production of inverse agonism for biarylpyrazoles such as 1. To determine whether the carboxamide oxygen or the piperidine nitrogen of the C-3 substituent may be the interaction site for K3.28(192), we designed, synthesized, and evaluated a new set of analogues of 1 (3-6, Chart 1) in which modifications only to the C-3 substituent of 1 have been made. In each case, the modifications that were made preserved the geometry of this substituent in 1. The absence of the piperidine nitrogen was not found to affect affinity, whereas the absence of the carboxamide oxygen resulted in a reduction in affinity. CB1 docking studies in an inactive state model of CB1 resulted in the trend, 3,1<5,4<2<6 for ligand/CB1 interaction energies. This trend was consistent with the trend in WT CB1 Ki values versus [3H]CP55,940 reported here. In calcium channel assays, all analogues with carboxamide oxygens (1, 3, and 4) were found to be inverse agonists, whereas those that lacked this group (2, 5, and 6) were found to be neutral antagonists. Taken together, these results support the hypothesis that it is the carboxamide oxygen of the C-3 substituent of 1 that engages in a hydrogen bond with K3.28(192) in WT CB1. Furthermore, functional results for 1-6 support the hypothesis that the interaction of 1 with K3.28(192) may be key to its inverse agonism.

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.

Endocannabinoid stucture-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 saturation in at least the last five carbons of the acyl chain. To probe the molecular 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 (K(i) = 34.4 +/- 3.2 nM); 20:4, n-6 (K(i) = 39.2 +/- 5.7 nM); 20:3, n-6 (K(i) = 53.4 +/- 5.5 nM); and 20:2, n-6 (K(i) > 1500 nM). CM studies indicated that each analogue 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 analogues in the series, the U/J-shaped family had the higher population. In addition, the 20:2, n-6 ethanolamide U-shaped family was not as tightly curved as were those of the other analogues 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 average radii of curvature (with their 95% confidence intervals) were found to be 5.8 A (5.3-6.2) for 20:2, n-6; 4.4 A (4.1-4.7) for 20:3, n-6; 4.0 A (3.7-4.2) for 20:4, n-6; and 4.0 A (3.6-4.5) for 22:4, n-6. These results suggest that higher CB1 affinity is associated 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 dimethyl anandamide analogues (R)-N-(1-methyl-2-hydroxyethyl)-2-(R)-methyl-arachidonamide (K(i) = 7.42 +/- 0.86 nM), (R)-N-(1-methyl-2-hydroxyethyl)-2-(S)-methyl-arachidonamide (K(i) = 185 +/- 12 nM), (S)-N-(1-methyl-2-hydroxyethyl)-2-(S)-methyl-arachidonamide (K(i) = 389 +/- 72 nM), and (S)-N-(1-methyl-2-hydroxyethyl)-2-(R)-methyl-arachidonamide (K(i) = 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 intramolecular hydrogen bond between the carboxamide oxygen and the headgroup hydroxyl that orients the C2 and C1' methyl 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.pi 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 bind...

The time step of atomistic molecular dynamics (MD) simulations is determined by the fastest motions in the system and is typically limited to 2 fs. An increasingly popular approach is to increase the mass of the hydrogen atoms to ∼3 amu and decrease the mass of the parent atom by an equivalent amount. This approach, known as hydrogen-mass repartitioning (HMR), permits time steps up to 4 fs with reasonable simulation stability. While HMR has been applied in many published studies to date, it has not been extensively tested for membrane-containing systems. Here, we compare the results of simulations of a variety of membranes and membrane–protein systems run using a 2 fs time step and a 4 fs time step with HMR. For pure membrane systems, we find almost no difference in structural properties, such as area-per-lipid, electron density profiles, and order parameters, although there are differences in kinetic properties such as the diffusion constant. Conductance through a porin in an applied field, partitioning of a small peptide, hydrogen-bond dynamics, and membrane mixing show very little dependence on HMR and the time step. We also tested a 9 Å cutoff as compared to the standard CHARMM cutoff of 12 Å, finding significant deviations in many properties tested. We conclude that HMR is a valid approach for membrane systems, but a 9 Å cutoff is not.