http://pubs.acs.org/journal/acsodf Article Evaluating Allosteric Perturbations in Cannabinoid Receptor 1 by In Silico Single-Point Mutation Oscar Díaz, Pedro Renault,* and Jesús Giraldo* Cite This: ACS Omega 2022, 7, 37873−37884 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Cannabinoid receptor 1 (CB1) is a promising drug target involved in many physiological processes. Using atomistic molecular dynamics (MD) simulations, we examined the structural effect of F237L mutation on CB1, a mutation that has qualitatively similar effects to allosteric ligand ORG27569 binding. This mutation showed a global effect on CB1 conformations. Among the observed effects, TM6 outward movement and the conformational change of the NPxxY motif upon receptor activation by CB1 agonist CP55940 were hindered compared to wt CB1. Within the orthosteric binding site, CP55940 interactions with CB1 were altered. Our results revealed that allosteric perturbations introduced by the mutation had a global impact on receptor conformations, suggesting that the mutation site is a key region for allosteric modulation in CB1. ■ INTRODUCTION Cannabinoid receptor 1 (CB1) is one of the key components to avoid overdose) and greater GPCR subtype-selectivity.8,9 Allosteric modulators can increase (positive allosteric modu- of the endocannabinoid system. CB1 belongs to the class A G lators, PAMs) and decrease (negative allosteric modulators, protein-coupled receptor (GPCR) family, is one of the most NAMs) the functional response exerted by orthosteric ligands abundantly expressed GPCRs in the brain, and is involved in or the receptor itself10 or provide bias toward a particular the modulation of pain, behavior, memory, and cognition.1,2 signaling pathway (biased allosteric modulators, BAMs).11 The CB1 is one of the targets of Δ9-tetrahydrocannabinol (THC), first allosteric modulator targeting CB1 (ORG27569) was the major psychoactive component of the cannabis plant, discovered in 2005,12 but many others have since been which has been consumed over millennia for recreational and discovered.13−16 medical uses.3 Thus, the pharmacological properties of drugs Currently, a crystal structure of CB1 with bound ligands that bind to cannabinoid receptors (cannabinoids) have been CP55940 (orthosteric agonist) and ORG27569 (allosteric extensively studied over the years, including their endogenous modulator) is available.17 In this structure, the orthosteric ligands (endocannabinoids), cannabinoids present in the binding site is adapted for CP55940 binding but the cannabis plant such as THC, and synthetic cannabinoids.2,4,5 intracellular region is in an inactive-like conformation. This Although synthetic THC and a synthetic analogue (nabilone) structure confirmed the known pharmacologic behavior of are approved for medical use in the United States and in ORG27569: positive allosterism for binding and negative several countries in the EU (see ref 6 for a review on approved allosterism for function,12 known as a PAM-antagonist.18 medications in the EU countries), the development of Moreover, this crystal structure shows ORG27569 bound to synthetic cannabinoid drugs with clinical use has been difficult. the intracellular region of transmembrane helix (TM) 2 and Specifically, inverse agonist SR141716A (rimonabant) was TM4, in an extrahelical cavity in contact with the membrane.17 withdrawn from the market due to its apparent association to This binding site overlaps with a cholesterol binding site in depression and suicidal ideation.5,7 Given the difficulty of CB1 as shown by agonist-bound crystal structures of CB1.19 developing drugs that target the CB1 orthosteric site, allosteric Consequently, ORG27569 competes with cholesterol binding, modulation has emerged as an interesting approach to regulate as described previously in rat brain membranes.20 Interestingly, the effects exhibited by orthosteric ligands. Importantly, allosteric compounds have known advantages over orthosteric ligands, such as a ceiling effect (in contrast to the effect of Received: August 4, 2022 competition with a different orthosteric ligand, which can Accepted: September 28, 2022 indefinitely shift the response of the endogenous ligand in a Published: October 14, 2022 dose-dependent manner, the functional effects of allosteric modulators are “saturable”, with a ceiling driven by the cooperativity between ligands and thus may offer an advantage © 2022 The Authors. Published by American Chemical Society https://doi.org/10.1021/acsomega.2c04980 37873 ACS Omega 2022, 7, 37873−37884 ACS Omega http://pubs.acs.org/journal/acsodf Article cholesterol enrichment reduces CP55940 binding,21 while of CB1,24 we performed unbiased, microsecond-length cholesterol depletion decreases inverse agonist binding,20 molecular dynamics (MD) simulations in the apo state and suggesting cholesterol behaves as a NAM. This indicates this also bound to the agonist CP55940, and observed spontaneous cavity contains key residues that affect orthosteric ligand transitions to active-like states. This model included the binding and receptor activation, despite not being part of the protonation of some selected residues shown to favor active- orthosteric binding site or the G protein binding site. like conformations (see Methods section). Thus, it provides a Recently, parallel tempering metadynamics simulations reference for comparison with subsequent studies on CB1. indicated that a shift in F1552.42 (Ballesteros-Weinstein Here, we perform additional MD simulations to explore the numbering scheme included as a superscript22), a residue perturbations on CB1 introduced by F237L mutation, as a located in this cavity, is critical for receptor activation, and this model for allosteric perturbation at the allosteric site observed rearrangement displayed a higher energy barrier than TM6 in crystal structures,17,19 and evaluate their effects on outward movement.23 This shift of F1552.42 can be observed by CP55940-mediated CB1 activation. To do this, we employ comparing inactive and active G protein-bound crystal the same CB1 model as presented in Diaz ́ et al.27 so as to structures of CB1, where F1552.42 points toward the over- compare the F237L mutant to wt CB1. lapping binding site of ORG27569 and cholesterol in the active receptor.24−26 Another residue located at the overlapping binding pocket is F2374.46. Mutation of this residue to leucine ■ MATERIALS AND METHODS The wt CB1 model employed was previously used in Diaz ́ et has been shown to increase CP55940 affinity and reduce 27 al. Briefly, the model was built from starting coordinates of inverse agonist SR141716A affinity.17 As for its functional the inactive crystal structure of CB124 obtained from the effect, it has been proposed that F2374.46 has a role in the Protein Data Bank (PDB 5U09). After removal of the activation process.17 In active crystal structures of CB1 with cocrystallized fusion protein, T210A mutation was restored cocrystallized G protein,25,26 F2374.46 is displaced toward the to wt and the missing regions of TM5, TM6, and ICL3 were extrahelical site and away from TM2 and TM3 (overlapping modeled using MODELER v9.14.28 Then, the receptor was with the binding pose of ORG27569) and its space is replaced energy minimized, and CP55940 was docked into the by F1552.42, which shifts toward TM4 (Figure 1). This shift of orthosteric binding site using AUTODOCK4.2.29 After our simulations with this model in Diaz ́ et al.,27 an inactive structure of CB1 with cocrystallized CP55940 was published.17 Nevertheless, to allow direct comparisons with these earlier simulations, we kept the same protocol here and used the model built with docking. In the present study, the F237L mutant CB1 was generated from this previous model by replacing F2374.46 with the leucine rotamer with highest probability in CHIMERA 1.1430 according to the Dunbrack library.31 Then, models of F237L CB1 and wt CB1 were minimized in CHIMERA 1.14 performing 1000 steps of steepest descent and 100 conjugate gradient steps in the AMBER14SB force field. Molecular dynamics (MD) simulations of wt CB1 analyzed in this study at apo and CP55940-bound conditions were previously published in Diaź et al.27 In two replicas of these simulations, starting from the inactive state, wt CB1 achieved Figure 1. (A) View of the inactive wt CB1 model with bound active-like conformations when bound to CP55940. Therefore, CP55940 and with conserved sequence motifs in class A GPCRs they provide a good point of comparison to evaluate CB1 highlighted. (B) Close up view of the extrahelical cavity in the inactive activation. The same MD simulation approach was employed state (green, from PDB ID 5U09) and active state (orange, from PDB for F237L CB1. The C257ECL2-C264ECL2 disulfide bridge was ID 6KPG) containing ORG27569 and cholesterol contact residues. maintained, and charges for D1632.50, D2133.49, E323ICL3, D324ICL3, D3336.25, and D3386.30 were neutralized, while F1552.42 and F2374.46 is not tied to intracellular conformational H3025.66, H3045.68, and H320ICL3 were protonated.27 Com- changes in TM6 and TM7, which can be observed in active- pared to the expected residue protonation state at pH 7, this like agonist-bound crystal structures of CB1 without bound G alternative protonation state was shown to promote activation protein,19 since F1552.42 and F2374.46 are in an “inactive-like” of CB127 and other class A GPCRs.32−34 Systems were state in these structures. Interestingly, a cholesterol molecule is embedded in 80 × 80 Å 1-palmitoyl-2-oleoyl-sn-glycero-3- bound to the extrahelical cavity in these active-like crystal phosphocoline (POPC) homogeneous membranes, and the structures. Overall, this indicates CB1 activation is sensitive to system charge was neutralized at 0.15 M concentration of KCl. perturbations in this region, which may be introduced by Simulations were performed using ACEMD software35 in the allosteric ligand binding, residue mutation or membrane lipid CHARMM36 force field36 at 300 K and 1 atm, while CP55940 composition. Furthermore, F2374.46 is not part of well-known was parametrized in the CHARMM GenFF37 using Para- sequence motifs or activation microswitches in class A GPCRs mChem.38 Equilibration consisted in 8 ns of simulation time despite its capability to modulate ligand binding. progressively releasing restraints on protein and ligand heavy In this study, we investigate structural and dynamical effects atoms followed by 20 ns of simulation without restraints, and of the F237L mutation. This work follows a previous study on then each production run consisted of 2 μs of simulation time. the molecular mechanism of agonist-mediated CB1 activa- MD simulation input files are provided in Supporting tion.27 In that study, starting from an inactive crystal structure Information. Three replicas were performed for each 37874 https://doi.org/10.1021/acsomega.2c04980 ACS Omega 2022, 7, 37873−37884 ACS Omega http://pubs.acs.org/journal/acsodf Article simulation condition (apo and agonist-bound wt CB1; apo and In order to characterize differences in the interaction agonist-bound mutant), adding up to a total of 24 μs of frequencies between agonist and contacting residues for both simulation time analyzed in this study. wt and F237L CB1, first the distance between the closest heavy Trajectories were analyzed by examining MD-generated atoms of CP55940 and the receptor for each trajectory frame snapshots at an interval of 4 ns of simulation time. These MD- was measured using gmx pairdist in GROMACS.40 Then, generated conformations were visually inspected and distances contact frequencies for each residue were calculated consid- between Cα atoms of R2143.50-D3386.30 (residues implicated in ering that a contact is present if the distance is <4 Å. Finally, the formation of the ionic lock), side chain hydroxyl atoms of the frequencies for CP55940-F237L CB1 contacts were Y2945.58-Y3977.53 and the χ1 dihedral angles of W3566.48, subtracted from the frequencies for wt CB1 for each respective F2003.36, F1552.42, and F2374.46 were measured using VMD residue to generate a frequency difference. As a second 1.9.3.39 Distances between aromatic moieties of F1552.42, approach, ΔGbind was estimated using gmx_MMPBSA,42 F2374.46, and W2414.50 were measured from the center of mass including an implicit membrane with thickness determined of the heavy atoms of aromatic rings in VMD, and the presence by CHARMM-GUI initial setup. Solvent, membrane, and of aromatic interactions was considered when this distance was internal dielectric constants used were 80, 7, and 2, within a 7.0 Å distance cutoff. respectively. Residues within 4 Å of the starting CP55940 Protein root-mean square deviation (RMSD) was also binding position and residues that showed average difference measured in VMD based on Cα atoms of transmembrane in contact frequency over 0.1 were considered for decom- helices and taking the initial structure as reference. Ligand position analysis. Average differences in free energy decom- RMSD was based on its heavy atoms and measured against the position were evaluated by a two-sample, two-tailed Student’s t initial docking pose. The initial CP55940 docking pose was test, with equal or unequal variances as assessed by an F-test. compared to crystal structures with cocrystallized CP55940 Water occupancy was defined as the percentage of frames in (PDB 6KQI17 and 7WV923), and contact residues were which a given protein residue established a contact (distance defined as those within 4 Å of CP55940 heavy atoms. within 4 Å) with a water molecule. Distances between protein Principal component analysis (PCA) was performed with and water were measured with the tool gmx pairdist in GROMACS.40 To do this, all replicas of agonist-bound wt and GROMACS.40 agonist-bound mutant CB1 were concatenated and aligned to the Cα atoms of the initial model. The N-terminal and the highly flexible ICL3 were excluded and not considered in the ■ RESULTS Overall, MD trajectories were stable, as indicated by the analysis. Therefore, only residues from A1181.34 to R3075.71 and RMSD curves in Figure S1. Higher values were observed for from A3356.27 to F412H8 were included in PC calculations. two of the replicas of agonist-bound wt receptor (Figure S1A), Then, we built and diagonalized the covariance matrix and this is consistent with the conformational changes seen in corresponding to this concatenated and aligned trajectory. these trajectories, where CB1 achieved active-like conforma- The elements of this matrix were Cij = ⟨(ri − ⟨ri ⟩) (rj − ⟨rj⟩)⟩ tions,27 characterized by a movement of TM6 (Figure S1E). In where i and j denote the Cartesian coordinates of the Cα comparison to wt systems, the RMSD was higher in trajectories atoms of the receptor. The vector ri indicates the instantaneous of the mutant, and this can be explained by the relaxation that value of coordinate i and ⟨ri⟩ is the average value of this followed the perturbation introduced by the mutation (Figure coordinate in the ensemble of conformations. Each individual S1B, D, F). Plots of the root mean square fluctuation (RMSF) trajectory was then projected on the space of the first two also point to a consistent pattern of CB1 fluctuations in all principal components. trajectories (Figure S2). The statistical analysis of pairwise residue distances was The RMSD of the agonist CP55940 was relatively high performed using the Ensemble Difference Distance Matrix, (Figure S3B, C), and probably reflected inaccuracies implemented in the Bio3D package in R.41 Residue distances introduced by docking. As shown in Figure S3A, the docking were compared between two groups, based on the results of pose differs from the crystal structure (PDB 6KQI) mainly in PCA: (i) replicas 2 and 3 of agonist-bound wt CB1 and (ii) all the position of the C ring and the orientation of its OH groups replicas of agonist-bound F237L CB1. The distance between (Figure S4A). As a result, native contacts between the ligand two residues was defined as the closest distance between any and TM2 are missing from the docking pose. Overall, from 19 heavy atoms in the residues (side chains included). Residue protein−ligand contacts present in the crystal structure, 12 pairs whose distances were greater than 10 Å across all frames were found in docking (63%). In addition, contacts with of all trajectories were excluded from the analysis. In the same I267ECL2 and L2765.40 were frequently formed during the manner as PCA, the N-terminal and the ICL3 were also simulations, raising the percentage of native contacts retained excluded (only residues from A1181.34 to R3075.71 and from from the crystal structure to 74%. More details about the A3356.27 to F412H8 were considered). Matrices of the average frequency of CB1-CP55940 contacts in the simulations can be distances between residues were built for the two groups and a found in Table S1. Nevertheless, CP55940 remained in its difference distance matrix was obtained by subtracting the binding site throughout the MD trajectories (Figure S3B, C), matrix of the mutant from that of the wt receptor; however, despite fluctuations observed mostly due to movement of its only statistically significant differences greater than 1 Å were alkyl chain and, to a lesser extent, its C-ring (Figure S4). retained. Statistical significance was determined by a two- Moreover, as active-like conformations were generated in sample Wilcoxon test, with p-value lower than 0.005. To CP55940-bound CB1 but not in apo,27 this indicates that our identify close contacts that were present in one group but not model captures native contacts that are likely important for in other, we selected a subset of the significant differences in receptor activation. Taken together, these observations suggest which the average residue distance was lower than a 4 Å cutoff that, despite inaccuracies introduced by docking, and in one group and greater than this cutoff in the other. considering that MD simulations of F237L CB1 started from 37875 https://doi.org/10.1021/acsomega.2c04980 ACS Omega 2022, 7, 37873−37884 ACS Omega http://pubs.acs.org/journal/acsodf Article Figure 2. Distribution of the R2143.50-D3386.30 distance in MD simulations of (A) apo CB1 and (B) CP55940-bound CB1. Data from all replicas; MD snapshots were collected at a 4 ns time interval in each one of them. Histograms for wt CB1 were calculated from MD simulations published in ́ et al. 2019. Diaz Figure 3. Distribution of the distance between side chain oxygen atoms of Y2945.58 and Y3977.53 in MD simulations of (A) apo CB1 and (B) CP55940-bound CB1. Data from all replicas; MD snapshots were collected at a 4 ns time interval in each one of them. Histograms for wt CB1 were calculated from MD simulations published in Diaź et al.27 the same initial model, it was still possible to discern the effect This indicates the outward movement of TM6 upon receptor of the mutation on collective motions of the receptor. activation by CP55940 was hindered by F237L mutation, and F237L Mutation Hindered Outward Movement of this is consistent with a role of F2374.46 on receptor TM6. The hallmark of class A GPCR activation is the breakage activation.17,25 of the salt-bridge formed by residues 3.50 and 6.30 (ionic lock) F237L Mutation Blocked Y3977.53 Conformational and consequent outward movement of TM6 away from Change. At the receptor level, the binding of a signaling TM3,43 which can be observed by comparing inactive and protein is the final consequence of receptor activation. In this active CB1 crystal structures.19,23−26,44 In our MD simulations regard, the NPxxY motif has been linked to G protein binding, of apo CB1, TM6 remained in inactive-like conformations as Y397 7.53 shifts toward Y294 5.58 in active receptor both in wt and F237L CB1, as shown by the distance between conformations to facilitate G protein binding and blocking residues R2143.50-D3386.30 (Figure 2A), which is expected TM6 inactivation.25,43 In MD simulations of apo CB1, Y3977.53 because CB1 was in the inactive conformation at the start of remained in an inactive-like conformation overall, as shown by MD simulations. In CP55940-bound wt CB1 MD simulations, the distance between Y3977.53 and Y2945.58 (Figure 3A), TM6 moved outward, reaching active-like states in two of the regardless of F237L mutation. In contrast, a new population three replicas performed.27 Thus, two subpopulations of TM6 was observed in wt CB1 bound to CP55940 centered at ∼3 Å, conformation can be observed: an inactive-like conformation indicating the presence of active-like conformations27 (blue that is represented by a closer R2143.50-D3386.30, and an active- curve in Figure 3B). However, this subpopulation was not like conformation represented by a larger TM3-TM6 distance, clearly represented in CP55940-bound F237L CB1 (red curve indicating a broken ionic lock (blue curve in Figure 2B). On in Figure 3B), where Y3977.53 remained in inactive-like the contrary, this did not occur in F237L mutant CB1 (red conformations such as in apo CB1. This suggests that F237L curve in Figure 2B) even when CP55940 was bound to the mutation blocks the conformational change in Y3977.53 receptor. Here, TM6 remained mostly in an inactive-like observed in active crystal structures of CB1.25,26 Together conformation, similar to the observations in apo conditions. with the hindrance of TM6 movement, our results indicate that 37876 https://doi.org/10.1021/acsomega.2c04980 ACS Omega 2022, 7, 37873−37884 ACS Omega http://pubs.acs.org/journal/acsodf Article Figure 4. χ1 dihedral angles of (A, B) W3566.48 and (C, D) F2003.36 along MD simulation time for three trajectories of (A, C) wt CB1* and (B, D) F237L CB1. *Data from MD simulations of wt CB1 taken from Diaz ́ et al.27 F237L mutation interfered with receptor activation during the between CP55940 and CB1 were calculated for MD simulations. simulations of wt and mutant receptors. The difference in W3566.48/F2003.36 Rotameric Switch Was Unaffected average frequencies between wt and mutant suggests there is a by F237L Mutation. The residue pair W3566.48/F2003.36 difference on how CP55940 interacts with CB1, with some forms an additional microswitch for receptor activation in interactions favored for wt and others for F237L (Figure 5). CB1, consisting of a double rotameric switch where W3566.48 Moreover, interaction differences were consistent across and F2003.36 are rearranged upon receptor activation. This is simulation time (Figures S5 and S6). accompanied by an upward movement of TM3.27 This Additionally, the affinity of CP55940 for wt and F237L CB1 rearrangement has been proposed to be mediated by changes was estimated using Molecular Mechanics Poisson−Boltzmann in the χ1 dihedral angles of W3566.48 and F2003.36 that result in Surface Area (MMPBSA), decomposed for each residue within the breakage of aromatic stacking interactions between these 4 Å of CP55940 starting conformation, in addition to residues two residues upon receptor activation.25 McAllister et al., 2004 identified by contact frequency difference analysis. Overall, the proposed W3566.48 shifts its χ1 dihedral angle from gauche(+) difference between global ΔGbind for wt and F237L was not to trans, followed by a transition in the χ1 dihedral angle of statistically significant as calculated by a two-sample Student’s t F2003.36 from trans to gauche(+).45 This was observed in MD test (ΔGbindwt = −12.22 ± 0.52 kcal/mol, ΔGbindF237L = −12.48 simulations of wt CB1 as described in Diaź et al., 2019 (Figure ± 0.71 kcal/mol (mean ± S.E.M.), p = 0.583, for n = 3 4A, C), although W3566.48 conformation may return to trajectories). From both contact frequency difference and gauche(+) after TM3 upward movement while the receptor MMPBSA decomposition, residues could be grouped accord- is in an active-like conformation, as shown by active CB1 ing to whether their interaction with CP55940 was favored in crystal structures.25,26 In MD simulations of CP55940-bound wt or in F237L. According to frequency differences, residues F237L CB1 (Figure 4B, D), the χ1 dihedral angle from favored by wt CB1 were most notably S1231.39, F1702.57, and W3566.48 transitioned from gauche(+) to trans and χ1 dihedral M3847.40, while residues favored by F237L CB1 were angle from F2003.36 transitioned from trans to gauche(+), P269ECL2, A3807.36, C3827.38, and C3867.42. MMPBSA decom- suggesting that the double rotameric switch was unaffected by position (Figure 5C) showed residues S1231.39, F1702.57, F237L mutation. F2003.36, L2765.40, M3847.40, and L3877.43 were favored toward Contacts between CP55940 and CB1 Were Affected wt CB1, while residues L3596.51, F3797.35, A3807.36, and by F237L Mutation. To evaluate the effect of F237L C3867.42 were favored toward F237L CB1. To a lesser extent, mutation on agonist binding, the frequencies of contact this analysis also indicated that interaction with residues 37877 https://doi.org/10.1021/acsomega.2c04980 ACS Omega 2022, 7, 37873−37884 ACS Omega http://pubs.acs.org/journal/acsodf Article Figure 5. Contact analysis between CP55940 and CB1 in MD simulations. (A) Structural representation of contact frequencies favored by wt CB1 (blue) and by F237L CB1 (orange). (B) Difference between average contact frequencies per residue (residues are considered in contact if any of their heavy atoms is at <4 Å from the ligand). (C) Average difference in ΔGbind decomposed for each residue interacting with CP55940, in wt and F237L CB1 (in kcal/mol). Positive values represent CP55940-CB1 interactions favored in wt CB1 (higher contact frequency or more negative ΔG), while negative values represent interactions favored in F237L CB1. MD trajectories of wt CB1 taken from Diaz ́ et al.27 I267ECL2, F268ECL2, and P269ECL2 was favored in F237L CB1. F237L Mutation Had a Global Effect on CB1. Distance MMPBSA analysis is in qualitative agreement with the contact distributions of residues R2143.50-D3386.30 and of residues frequency analysis except for residue T1973.33 (Figure 5B). Y2945.58-Y3977.53 (Figures 2 and 3, respectively) suggest that However, none of the detected differences between wt and the effect of the mutation is more evident when CB1 is bound F237L CB1 was statistically significant (p-values summarized to the agonist. In order to obtain a general assessment of the in Table S2). impact of the mutation on the structure and dynamics of CB1 Interestingly, many of the residues favored by F237L are during the simulations of this condition, we performed a close to the extracellular region of the receptor, including principal component analysis (PCA). We projected the I267ECL2, F268ECL2, and P269ECL2 which are in extracellular trajectories of wt and mutant CB1 bound to CP55940 on a loop 2 (ECL2), and F3797.35 in TM7, identified by both common vector space (obtained after concatenation of the contact frequency and MMPBSA decomposition analyses. trajectories; see details in the Methods section). These ECL2 is a critical region for CP55940 binding, as alanine projections are shown in Figure 6A, where conformations explored in wt CB1 simulations are represented in blue and substitutions at every position between P269ECL2 and I271ECL2 those in mutant CB1 simulations are in red. It is possible to see abolish CP55940 binding.46 On the other hand, a mutation to a substantial overlap between the region of the conformational A or W in F1702.57, whose interaction with CP55940 was space which was visited by replica 1 of the wt receptor (light favored in wt CB1, does not alter the potency of CP55940.44 blue) and the space collectively explored by the three replicas Critical residues whose contacts with CP55940 were favored in of the mutant receptor (in red). We note that replica 1 was wt CB1 include M3636.55 and S3837.39,47,48 which were precisely the only one in which wt CB1 did not achieve active- identified by MMPBSA decomposition analysis and not by like conformations, and no consistent outward movement of contact frequency analysis, indicating that interaction of TM6 was observed (see Figure S1). In contrast, the two other CP55940 with M3636.55 and S3837.39, while present in F237L replicas of the wt system explored regions of the conforma- CB1, were slightly stronger in wt CB1. Overall, our results tional space which were inaccessible to the mutant receptor, suggest the contact pattern between CP55940 and F237L CB1 indicating that conformational changes were restricted by the is altered in comparison with wt CB1 and interactions with mutation. residues located at extracellular regions of the receptor are The features of such changes can be clarified by an favored in F237L CB1. inspection of the directions of collective motion that 37878 https://doi.org/10.1021/acsomega.2c04980 ACS Omega 2022, 7, 37873−37884 ACS Omega http://pubs.acs.org/journal/acsodf Article conditions: (i) replicas 2 and 3 of wt CB1 (where active-like conformations were observed); (ii) the three replicas of the mutant. We then determined statistically significant differences in average residue pairwise distances between these two groups. Residue pairs with distances always longer than 10 Å in all trajectories were excluded from the analysis. A list of the 1378 residue pairs whose variations in distance were significantly higher than 1 Å is available in the Supporting Information. Considering the magnitude of distance variations, the top 35 (variations in the range 4.6−6.7 Å) corresponded to distances that were larger in the wt receptor; interestingly, they involved residue pairs in TM6-TM2 and TM6-ICL2. It is necessary to descend to the 36th position in the ranking to find a residue pair with increased distance in the mutant, and it involved residues in TM7 and TM3. The 10 largest differences are listed in Table 1 and schematically depicted in Figure 7A, Table 1. Top 10 Highest Differences in Average Residue Pairwise Distance between MD Simulations of wt (Replicas 2 and 3) and F237L CB1 (All Replicas) with Bound CP55940 avg. avg. distance distance in in F237L delta residue 1 residue 2 wt (Å) (Å) (Å) p value P1512.38 D3386.30 20.22 13.52 6.70 1.9 × 10−291 F1552.42 L3456.37 11.98 6.00 5.98 1.9 × 10−254 S1522.45 A3426.34 17.16 11.43 5.73 4.9 × 10−284 Figure 6. (A) Projection on the principal components PC1 and PC2 P1512.38 L3416.33 15.88 10.19 5.69 2.9 × 10−275 of the wt and F237L CB1 MD-generated conformations with bound F1552.42 A3426.34 17.25 11.57 5.69 8.7 × 10−248 CP55940. (B) Extreme conformations along the direction of PC1 F1552.42 V3466.38 16.15 10.45 5.69 9.6 × 10−244 (minimum value in blue, maximum value in red). A transition from P1512.38 M3376.29 19.72 14.04 5.68 1.1 × 10−257 the red to the blue conformation corresponds to a movement from S1522.39 L3416.33 12.68 7.04 5.64 7.5 × 10−277 positive to negative projections along the direction of PC1. (C) Extreme conformations along the direction of PC2 (minimum value S1522.39 D3386.30 17.34 11.76 5.58 3.4 × 10−284 in blue, maximum value in cyan). A transition from the blue to the S1522.39 I3396.31 19.59 14.06 5.53 3.7 × 10−276 cyan conformation corresponds to a movement from negative to positive projections along the direction of PC2. correspond to PCs 1 and 2. Figure 6B shows that going from positive to negative values of the projection along PC1 (from right to left in the PC map in Figure 6A) corresponds to an outward motion of TM6. In the case of PC2, Figure 6C shows the motion from negative to positive values of the projection (from bottom to top in the PC map) corresponds to a bending of TM7 that contributes to open the intracellular cavity of the receptor (besides a collective rearrangement around the orthosteric site). Therefore, open and active-like conforma- tions of CB1 are scarcely explored by the mutant. Taken together, these results indicate that the mutation had an impact on the dynamical behavior of CB1 and shifted the receptor Figure 7. (A) Ten largest distance differences in MD simulations between wt CB1 (replicas 2 and 3) and F237L CB1 (all replicas) with population toward the inactive state. bound CP55940. (B) Differences in contacts between wt CB1 As detailed in the Methods section, PCA was based on the (replicas 2 and 3) and F237L CB1 (all replicas) with bound Cα atoms of CB1, thus providing information on motions of CP55940. Contacts only present in wt CB1 are represented in blue the main chain. However, we also wanted to investigate how and contacts only present in F237L are represented in red. the mutation affected the side chains of the receptor. The residues analyzed in the previous sections are either micro- switches of CB1 activation well established in the literature25,26 which shows that in wt CB1 there was a larger distance or form the binding cavity of the orthosteric ligand, and thus between TM6 and TM2 in comparison to the mutant. This were selected as a first indicator of the effect of F237L increased distance was consistent across simulation time mutation. To characterize other residues that may be affected (P1512.38-D3386.30 distance shown in Figure S7). This is in by F237L mutation, we calculated distance matrices of wt and agreement with our other results and indicates that an outward mutant CB1, both in the presence of the agonist. PCA results movement of TM6 was hindered by the mutation. It is also allowed us to separate two groups for comparison in order to worth noting that the largest distance observed between TM7 reduce noise and focus on differences between the two and TM3 in the mutant is also consistent with the hypothesis 37879 https://doi.org/10.1021/acsomega.2c04980 ACS Omega 2022, 7, 37873−37884 ACS Omega http://pubs.acs.org/journal/acsodf Article that the mutation suppresses collective motions that lead to substitution of phenylalanine by leucine would disrupt such activation, since the approach between these two helices is well aromatic interactions between F2374.46 and W2414.50 character- described in the activation of class A GPCRs.49 istic of states coupled to the G protein. Presumably, this Moreover, the hindrance of TM6 movement by the modulation might extend to ligand binding to this region mutation was reflected by the higher frequency of water because of the fact that ORG27569 and cholesterol bind to molecules in contact with residues A3426.34 and I3967.52 (close CB1 with an inactive-like conformation of F1552.42 and to the G protein binding cavity) in wt CB1 compared to F237L F2374.46.17,19,23 CB1 (Figure S8). The presence of these additional contacts with water molecules in this region suggests an entrance of water molecules as a consequence of the outward movement of TM6, and can be seen as an indicator of conformational ■ DISCUSSION AND CONCLUSIONS In this study, we have interrogated the effect of the F237L change, which occurred in wt receptor and was hindered by the mutation upon CB1 activation through molecular dynamics mutation. simulations. Here, we performed MD simulations of apo and In a complementary analysis, we also identified residues CP55940-bound F237L CB1, starting from the inactive state of which formed close contacts (distance < 4 Å for at least 50% of the receptor. We compared our observations with results from the simulation time) in one group but not in the other (Table our previous work, in which we performed simulations of wt 2 and Figure 7B). In this category, we found residue pairs in CB1.27 In that work, we observed that the agonist-bound wt receptor achieved active-like conformations during the Table 2. Differences in Close Residue Contacts (Distance < trajectories; in contrast, in the simulations of the mutant 4 Å) between MD Simulations of wt (replicas 2 and 3) and performed here CB1 remained inactive, even in the presence of F237L CB1 (All Replicas) with Bound CP55940 the agonist. Notably, the mutation was associated to a suppression of a large amplitude outward movement of TM6 residue 1 residue 2 contact in wt contact in F237L in the simulations. S2033.39 S3907.46 yes no In both of our studies�our previous work with wt CB1 and Y2945.58 I3486.40 yes no the present one with the mutant�systems were setup in the M2955.59 L3496.49 yes no same way, and we followed the same simulation protocol. This D1632.50 T3917.47 yes no included the insertion of the receptor in a very simple model T1301.46 S3907.46 no yes membrane, composed of POPC. Though it has been shown N1341.50 S3907.46 no yes that cholesterol can play an important role as an allosteric T2103.46 Y2945.58 no yes modulator of GPCRs,51 and particularly of CB1,20,21,52 it was Y2945.58 V3466.38 no yes not included in our simulations. This allowed us to make direct I2905.54 L3496.41 no yes comparisons with our previous work and also to decouple the D1632.50 N3937.49 no yes effect of the mutation from allosteric effects exerted by cholesterol. Moreover, F2374.46 is located in a cholesterol TM6-TM3 and TM6-TM5; and also pairs between TM7 and binding site of CB1,19 and an investigation of the impact of the TM1, and between and TM2 and TM3. Residues D1632.50, mutation on cholesterol binding or the combined effects of Y2945.58, S3907.46, and L3496.41 were each one of them involved cholesterol and the mutation were beyond the scope of this in more than one of these contacts which were switched on or paper. However, it should be important to consider the off, suggesting an important role for these residues in the behavior of the mutant receptor in the presence of cholesterol, collective dynamics of CB1. Notably, many of these changing and we believe this topic deserves attention in the future. contacts, as well as the large amplitude distance variations, Crystal structures show that the conformation of F2374.46 occurred far from the site of the mutation, involving several changes upon receptor activation, and this conformational other helices and distinct regions of the receptor, pointing to change is accompanied by a rearrangement of F1552.42 (see the global structural and dynamical effect of the mutation. Figure 1B). In the inactive state,24 F1552.42 points toward the F237L Substitution Disrupted Aromatic Interactions G protein binding cavity, interacting with residues L2073.43, in the Mutation Site. F237L mutation consists of a T2103.46, L3456.37, I3486.40, and Y3977.53 in other TMs. Upon substitution of an aromatic residue with an aliphatic one. In activation,25,26 F1552.42 shifts and points toward the extra- crystal structures of CB1, F2374.46 is in the vicinity of two helical cavity and interacts with residues L2093.45, D2133.49, aromatic residues, F1552.42 and W2414.50; the latter is a highly A2334.42, and L2374.46. This shift therefore leads to a loss of conserved residue in class A GPCRs50 which participates in contacts between TM2 and TM6 which possibly facilitates the allosteric modulator ORG27569 binding.17 In one of the MD uncoupling between these two helices. The important role of simulation replicas of wt CB1 bound to CP55940,27 a F1552.42 in CB1 activation has been recently highlighted by conformational change of F1552.42 and F2374.46 was observed. metadynamics simulations, 23 which indicated that the In this MD trajectory, active-like conformations were rearrangement of this residue (and, more generally, a observed27 and F1552.42 shifted toward TM4 generating an rearrangement of TM2) is a critical step in CB1 activation. intermediate state in which F1552.42 and F2374.46 briefly Importantly, these simulations revealed that the free energy formed aromatic tilted T-shape interactions (Figure S9), after barrier to the conformational change of F1552.42 is much which F2374.46 shifted away from TM3. This configuration of higher than the one associated to the outward movement of F1552.42 and F2374.46 (Figure S9C) can be seen in active crystal TM6. Our results are consistent with this picture, since the structures of CB1 with cocrystallized G protein,25,26 but not in outward movement of TM6 was more frequently observed in active-like crystal structures without the G protein.19 Aromatic our simulations than the reorientation of F1552.42. Importantly, interactions between F2374.46 and W2414.50 are favored in this we should note that in our trajectories the F237L mutation “displaced” configuration of F2374.46 (Figure S10). Thus, hindered this relatively easier movement of TM6, suggesting 37880 https://doi.org/10.1021/acsomega.2c04980 ACS Omega 2022, 7, 37873−37884 ACS Omega http://pubs.acs.org/journal/acsodf Article that the mutation contributes to heighten the energy barrier to with F2374.46 in its “displaced” conformation (Figures S9 and the displacement of this helix. S10), and such interactions would be lost in the F237L mutant The active-like states observed in our simulations were then CB1. Though contacts between F2374.46 and W2414.50 are frequently characterized by TM6 and F1552.42 in active and observed in crystal structures of the receptor coupled to the G inactive conformations, respectively (this also characterizes protein,25,26 suggesting aromatic interactions between F2374.46 conformational states explored in the metadynamics simu- and W2414.50 are characteristic of the active state, the lations referred to above, in which TM6 could switch between disruption of such interactions is likely not the only factor inward and outward states while F1552.42 remained in its involved in the effect of F237L mutation upon activation. inactive conformation). The existence of such distinct Actually, one of the important results in this study is the global intermediate states is consistent with models of GPCR effect of the mutation. While the perturbation of local aromatic dynamics that propose the existence of multiple conforma- interactions is probably relevant, we observed that the effects tional states in dynamical equilibrium.53 Other molecules�for are nonlocal and widespread, affecting pairwise residue instance, orthosteric or allosteric ligands, membrane lipids or distances and contacts across CB1 structure. The dynamical intracellular proteins such as G proteins�would be able to coupling between the local network of aromatic residues and shift this equilibrium and stabilize certain conformations, thus the global state of the receptor deserves further investigation. modulating the receptor function. For example, recent NMR F1552.42, F2374.46, and W2414.50 may work as an “allosteric experiments with CB1 in the presence of different ligands micro-switch” that could be modulated by mutations, allosteric (including the agonist CP55940 and the allosteric modulator ligands, or membrane lipids. We believe this hypothesis should ORG27569) detected a variety of conformational states, be further explored through new simulations with mutations of leading the authors to propose a model for activation and these residues, and also through metadynamics simulations, to allosteric modulation including not only inactive and active probe free energy barriers associated to their conformational states but also preactive and active-like conformations; each changes. one of them would be favored by the interaction with distinct Experimental results showed that F237L increases CP55940 partners.54 In the particular case of the conformations of TM6 binding and reduces inverse agonist SR141716A binding.17 and F1552.42, it is possible that the stabilization of both in the Our simulations indicated that the F237L mutation had an fully active conformation requires coupling to the G protein. impact on residues on the CP55940 binding site, and therefore Our results suggest that the F237L mutation renders G protein they are consistent with the experimental verification that coupling less probable, because it shifts the dynamical perturbations in F2374.46 can affect orthosteric ligand binding. equilibrium toward the inactive state of the receptor. However, we have not been able to capture significant Structural differences between wt and mutant CB1 in the differences between wt and mutant affinities for CP55940. simulations were accompanied by modifications in the This can be explained by the relatively low difference in the hydration of the receptor. In particular, wt CB1 exhibited a experimentally determined CP55940 affinity between wt and higher occupancy of water molecules in specific residues close F237L CB1 (Kd in wt CB1:8.7 ± 0.7 nM; Kd F237L CB1:5.0 ± to the G-protein binding site. The entrance of these water 0.2 nM; data from Shao et al.17) and also by limitations in molecules was possibly facilitated by the outward movement of TM6, that opened the intracellular cavity. It constituted MMPBSA methodology, such as considering an implicit another indicator of conformational changes observed in wt solvent and membrane. Also, the entropic component was CB1 and suppressed by the mutation. However, the observed not considered, which might be an important factor differences in the pattern of hydration between wt and mutant contributing to the free energy of binding, or perhaps altered CB1 are complex (Figure S8), and it remains to be verified if by F237L mutation. Despite these limitations and inaccuracies water molecules play an active part in triggering CB1 introduced by docking, our results suggest that multiple conformational changes, as suggested for the activation of residues in the CP55940 binding site are affected by F237L other GPCRs.55,56 mutation, as shown by contact analysis and MMPBSA free It seems logical to attribute the behavior of F237L mutation energy decomposition (Figure 5). These results indicate a to the loss of aromatic interactions in the mutated receptor due complex effect, with some protein−ligand interactions being to the substitution of an aromatic residue for an aliphatic one. more probable in wt CB1 and others favored in the mutant. It should be noted that, although our simulations indicated Among the residues that display different contact frequencies that stacking or T-shape aromatic interactions between with the agonist, I267ECL2, P269ECL2, and D272ECL2 are located F1552.42 and F2374.46 may form transiently in a dynamic in the ECL2; notably, contacts between the agonist and context (Figure S9), aromatic interactions between these I267ECL2 and P269ECL2 are favored in the mutant. This is an residues are not present in CB1 crystal structures. Moreover, in interesting result, since it has been shown that ECL2 is a mutagenesis experiments the replacement of F1552.42 by V, an critical region for CP55940 binding.46 One hypothesis is that aliphatic but smaller side chain, was shown to increase Gi the F237L mutation, by favoring these contacts, leads to a signaling; in contrast, mutation to the aromatic but larger W stronger interaction with ECL2 and facilitate agonist binding had the opposite effect.23 These results suggest the aromaticity into a higher affinity state. The small difference in experimental of F1552.42 might not be the determinant factor for its role in affinities for CP55940 between wt and F237L CB1 poses a the activation process, and also the possibility that the size of challenge to the corroboration of this hypothesis through the side chain at this location might be important. simulations. Nevertheless, it could be interesting to use more On the other hand, W2414.50 is a highly conserved aromatic sophisticated free energy estimation methods (e.g., funnel residue in GPCRs (conserved at 97% in class A GPCRs50) metadynamics57) to interrogate more closely the role of these located one helix turn from F2374.46 and is also a contact ECL2 residues in the process of agonist binding. Mutations at residue for ORG2756917 and cholesterol.19 In our trajectories, these ECL2 sites could be tested both theoretically and W2414.50 could form stacking or T-shape aromatic interactions experimentally to test the validity of our observations. 37881 https://doi.org/10.1021/acsomega.2c04980 ACS Omega 2022, 7, 37873−37884 ACS Omega http://pubs.acs.org/journal/acsodf Article The F237L mutation exhibits a qualitatively similar effect to Statistically significant differences in average pairwise allosteric ligand binding. Mutations whose effect is similar to residue distance between MD simulations of wt and allosteric ligand binding have been reported before for other F237L CB1 bound to CP55940 (ODS), protein and GPCRs. For example, in the metabotropic glutamate receptor CP55940 RMSD over time for each replica, RMSF of all 2 (mGluR2), it has been observed that Q679V and C770A residues for each replica, structural comparison of mutations converted a partial agonist into a full agonist, docked CP55940 to cocrystallized CP55940, distance therefore acting in a similar way as a positive allosteric between CP55940 and P269 ECL2 , CP55940 and modulator (PAM).58 ORG27569 is one of the most studied M3847.40, and P1512.38 and D3386.30 in MD simulations CB1 allosteric modulators and has intriguing pharmacological of wt and F237L CB1 bound to CP55940, differences in properties in that it displays positive binding cooperativity and occupancy of water molecules for each residue in MD negative functional cooperativity with CB1 agonists such as simulations of wt and F237L CB1 bound to CP55940, CP55940. Allosteric modulators with this behavior have been distances between residues F1552.42, F2374.46, and previously denominated PAM-antagonists.18 However, the W2414.50 and their configurations during MD simu- mechanism by which ORG27569 exerts this effect is still lations, contact frequencies of CP55940 with the unknown. In the CP55940 and ORG27569-bound crystal receptor in MD simulations of wt and F237L CB1, p- structure of CB1,17 ORG27569 can be observed bound to the values of performed statistical tests for the difference in intracellular side of the receptor, in an extrahelical binding site CP55940 binding residue energy decomposition be- formed by TM2 and TM4 that partially overlaps with a tween wt and F237L CB1 (PDF) cholesterol binding site.17,19 F2374.46 is a contact residue for Atomic coordinates for all models and input files for MD both ORG27569 and cholesterol binding as shown in these simulations and MMPBSA calculations (ZIP) crystal structures. Moreover, F237L mutation causes an Differences in average residue pairwise distances increase in the affinity of CP55940 for CB117 and, as examined between agonist-bound wt and agonist-bound mutant in this study, impairs receptor activation. Furthermore, it has receptor (TXT) been shown that ORG27569 promotes receptor internal- ization,20,59 an effect that is also observed in homologous F238L mutation in rat CB1.60 When considered together, all these data and the shared effects between ORG27569 binding ■ AUTHOR INFORMATION Corresponding Authors and F237L mutation, combined with the fact that F2374.46 is a Jesús Giraldo − Laboratory of Molecular Neuropharmacology contact residue for ORG27569, suggest that F237 4.46 and Bioinformatics, Unitat de Bioestadística and Institut de contributes to mediate its allosteric effects. Neurociències and Unitat de Neurociència Traslacional, Parc This study was limited to the analysis of F237L mutation. Taulí Hospital Universitari, Institut d’Investigació i Innovació Since this mutation has a global effect on the receptor Parc Taulí (I3PT), Institut de Neurociències, Universitat conformational properties, this indicates that this region is a Autònoma de Barcelona, Bellaterra 08193, Spain; Instituto potential hotspot for allosteric modulation in CB1. It would be de Salud Carlos III, Centro de Investigación Biomédica en expected that other perturbations in this region such as those Red de Salud Mental (CIBERSAM), Madrid 28029, Spain; resulting from ligand binding would affect distant regions of orcid.org/0000-0001-7082-4695; Email: Jesus.Giraldo@ the receptor, thus suggesting a chemical space where a variety uab.es of allosteric ligands may be developed. Finally, we conclude Pedro Renault − Laboratory of Molecular that MD simulations on mutant receptor models, despite their Neuropharmacology and Bioinformatics, Unitat de inherent limitations and taken with the necessary caution,61 Bioestadística and Institut de Neurociències and Unitat de can be used as an exploratory tool to provide clues for Neurociència Traslacional, Parc Taulí Hospital Universitari, subsequent structure-based drug design of allosteric com- Institut d’Investigació i Innovació Parc Taulí (I3PT), Institut pounds. de Neurociències, Universitat Autònoma de Barcelona, ■ ASSOCIATED CONTENT Data Availability Statement Bellaterra 08193, Spain; Instituto de Salud Carlos III, Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Madrid 28029, Spain; orcid.org/0000- Atomic coordinates for the initial MD systems for wt and 0002-5649-3057; Email: Pedro.Renault@uab.cat F237L CB1, both apo and bound to CP55940, as well as input files for MD simulations with ACEMD and MMPBSA Author calculations are provided in the Supporting Information. The Oscar Díaz − Laboratory of Molecular Neuropharmacology following software was used: CHARMM-GUI (https://www. and Bioinformatics, Unitat de Bioestadística and Institut de charmm-gui.org/), ACEMD2 (https://www.acellera.com/ Neurociències and Unitat de Neurociència Traslacional, Parc acemd/), MODELER 9.14 (https://salilab.org/modeller/), Taulí Hospital Universitari, Institut d’Investigació i Innovació AUTODOCK 4.2 (https://autodock.scripps.edu/), CHI- Parc Taulí (I3PT), Institut de Neurociències, Universitat MERA 1.14 (https://www.cgl.ucsf.edu/chimera/), VMD Autònoma de Barcelona, Bellaterra 08193, Spain; Instituto 1.9.4 (https://www.ks.uiuc.edu/Research/vmd/), Bio3D de Salud Carlos III, Centro de Investigación Biomédica en 2.4.1 (http://thegrantlab.org/bio3d/), gmx_MMPBSA 1.4.3 Red de Salud Mental (CIBERSAM), Madrid 28029, Spain (https://pypi.org/project/gmx-MMPBSA/) and GRO- Complete contact information is available at: MACS2020.4 (https://www.gromacs.org/). Graphs were https://pubs.acs.org/10.1021/acsomega.2c04980 generated with Matplotlib 2.2.5 (https://matplotlib.org/). * sı Supporting Information Author Contributions The Supporting Information is available free of charge at MD simulations were performed and analyzed by Ó .D. P.R. https://pubs.acs.org/doi/10.1021/acsomega.2c04980. and J.G. designed and supervised the study. The manuscript 37882 https://doi.org/10.1021/acsomega.2c04980 ACS Omega 2022, 7, 37873−37884 ACS Omega http://pubs.acs.org/journal/acsodf Article was written through contributions of all authors. All authors (13) Horswill, J. G.; Bali, U.; Shaaban, S.; Keily, J. F.; Jeevaratnam, have given approval to the final version of the manuscript. P.; Babbs, A. J.; Reynet, C.; Wong Kai In, P. PSNCBAM-1, a Novel Allosteric Antagonist at Cannabinoid CB 1 Receptors with Notes Hypophagic Effects in Rats. Br. J. Pharmacol. 2007, 152 (5), 805−814. The authors declare no competing financial interest. (14) Immadi, S. S.; Dopart, R.; Wu, Z.; Fu, B.; Kendall, D. A.; Lu, D. ■ ACKNOWLEDGMENTS This project has received funding from the European Union’s Exploring 6-Azaindole and 7-Azaindole Rings for Developing Cannabinoid Receptor 1 Allosteric Modulators. Cannabis Cannabinoid Res. 2018, 3 (1), 252−258. (15) Laprairie, R. B.; Kulkarni, P. M.; Deschamps, J. R.; Kelly, M. E. Horizon2020 research and innovation programme under grant M.; Janero, D. R.; Cascio, M. G.; Stevenson, L. A.; Pertwee, R. G.; agreement 848068 and from Ministerio de Ciencia e Kenakin, T. P.; Denovan-Wright, E. M.; Thakur, G. A. Enantiospecific Innovación (Spain) under grant agreement PID2020- Allosteric Modulation of Cannabinoid 1 Receptor. ACS Chem. 119136RB-I00. This publication reflects only the authors’ Neurosci. 2017, 8 (6), 1188−1203. view and the European Commission is not responsible for any (16) Nguyen, T.; Li, J.-X.; Thomas, B. F.; Wiley, J. L.; Kenakin, T. use that may be made of the information it contains. We thank P.; Zhang, Y. Allosteric Modulation: An Alternate Approach Xin Yang for providing the MD-generated receptor struc- Targeting the Cannabinoid CB1 Receptor. Med. Res. Rev. 2017, 37 tures23 at the end of this project. (3), 441−474. (17) Shao, Z.; Yan, W.; Chapman, K.; Ramesh, K.; Ferrell, A. J.; Yin, ■ ABBREVIATIONS CB1, cannabinoid receptor 1; GPCR, G protein-coupled J.; Wang, X.; Xu, Q.; Rosenbaum, D. M. Structure of an Allosteric Modulator Bound to the CB1 Cannabinoid Receptor. Nat. Chem. Biol. 2019, 15, 1199−1205. receptor; THC, Δ9-tetrahydrocannabinol; TM, transmem- (18) Kenakin, T.; Strachan, R. T. PAM-Antagonists: A Better Way to brane helix; ICL, intracellular loop; ECL, extracellular loop; Block Pathological Receptor Signaling? Trends Pharmacol. Sci. 2018, PDB, Protein Data Bank; NAM, negative allosteric modulator; 39 (8), 748−765. PAM, positive allosteric modulator; POPC, 1-palmitoyl-2- (19) Hua, T.; Vemuri, K.; Nikas, S. P.; Laprairie, R. B.; Wu, Y.; Qu, oleoyl-sn-glycero-3-phosphocoline; MMPBSA, molecular me- L.; Pu, M.; Korde, A.; Jiang, S.; Ho, J.-H.; Han, G. W.; Ding, K.; Li, chanics Poisson−Boltzmann surface area X.; Liu, H.; Hanson, M. A.; Zhao, S.; Bohn, L. M.; Makriyannis, A.; Stevens, R. C.; Liu, Z.-J. Crystal Structures of Agonist-Bound Human ■ REFERENCES (1) Marsicano, G.; Lutz, B. Expression of the Cannabinoid Receptor Cannabinoid Receptor CB1. Nature 2017, 547 (7664), 468−471. (20) Stornaiuolo, M.; Bruno, A.; Botta, L.; Regina, G. La; Cosconati, S.; Silvestri, R.; Marinelli, L.; Novellino, E. Endogenous vs Exogenous CB1 in Distinct Neuronal Subpopulations in the Adult Mouse Allosteric Modulators in GPCRs: A Dispute for Shuttling CB1 among Forebrain. Eur. J. Neurosci. 1999, 11 (12), 4213−4225. Different Membrane Microenvironments. Sci. Rep. 2015, 5 (1), 1−13. (2) Reggio, P. H. Endocannabinoid Binding to the Cannabinoid (21) Bari, M.; Paradisi, A.; Pasquariello, N.; Maccarrone, M. 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