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Allosteric modulation of a cannabinoid G protein-coupled receptor: binding site elucidation and relationship to G protein signaling.

ABSTRACT: The cannabinoid 1 (CB1) allosteric modulator, 5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)-ethyl]-amide) (ORG27569), has the paradoxical effect of increasing the equilibrium binding of [(3)H](-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl]cyclohexan-1-ol (CP55,940, an orthosteric agonist) while at the same time decreasing its efficacy (in G protein-mediated signaling). ORG27569 also decreases basal signaling, acting as an inverse agonist for the G protein-mediated signaling pathway. In ligand displacement assays, ORG27569 can displace the CB1 antagonist/inverse agonist, N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide(SR141716A). The goal of this work was to identify the binding site of ORG27569 at CB1. To this end, we used computation, synthesis, mutation, and functional studies to identify the ORG27569-binding site in the CB1 TMH3-6-7 region. This site is consistent with the results of K3.28(192)A, F3.36(200)A, W5.43(279)A, W6.48(356)A, and F3.25(189)A mutation studies, which revealed the ORG27569-binding site overlaps with our previously determined binding site of SR141716A but extends extracellularly. Additionally, we identified a key electrostatic interaction between the ORG27569 piperidine ring nitrogen and K3.28(192) that is important for ORG27569 to act as an inverse agonist. At this allosteric site, ORG27569 promotes an intermediate conformation of the CB1 receptor, explaining ORG27569's ability to increase equilibrium binding of CP55,940. This site also explains ORG27569's ability to antagonize the efficacy of CP55,940 in three complementary ways. 1) ORG27569 sterically blocks movements of the second extracellular loop that have been linked to receptor activation. 2) ORG27569 sterically blocks a key electrostatic interaction between the third extracellular loop residue Lys-373 and D2.63(176). 3) ORG27569 packs against TMH6, sterically hindering movements of this helix that have been shown to be important for receptor activation.

SUBMITTER: Shore DM

PROVIDER: S-EPMC3937654 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Allosteric modulation of a cannabinoid G protein-coupled receptor: binding site elucidation and relationship to G protein signaling.

Shore Derek M DM Baillie Gemma L GL Hurst Dow H DH Navas Frank F Seltzman Herbert H HH Marcu Jahan P JP Abood Mary E ME Ross Ruth A RA Reggio Patricia H PH

The Journal of biological chemistry 20131223 9

The cannabinoid 1 (CB1) allosteric modulator, 5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)-ethyl]-amide) (ORG27569), has the paradoxical effect of increasing the equilibrium binding of [(3)H](-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl]cyclohexan-1-ol (CP55,940, an orthosteric agonist) while at the same time decreasing its efficacy (in G protein-mediated signaling). ORG27569 also decreases basal signaling, acting as an inverse agonist for the G ...[more]

PMID: 24366865

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Project description:Metabotropic glutamate receptors (mGluR) are mainly expressed in the central nervous system (CNS) and contain eight receptor subtypes, named mGluR1 to mGluR8. The crystal structures of mGluR1 and mGluR5 that are bound with the negative allosteric modulator (NAM) were reported recently. These structures provide a basic model for all class C of G-protein coupled receptors (GPCRs) and may aid in the design of new allosteric modulators for the treatment of CNS disorders. However, these structures are only combined with NAMs in the previous reports. The conformations that are bound with positive allosteric modulator (PAM) or agonist of mGluR1/5 remain unknown. Moreover, the structural information of the other six mGluRs and the comparisons of the mGluRs family have not been explored in terms of their binding pockets, the binding modes of different compounds, and important binding residues. With these crystal structures as the starting point, we built 3D structural models for six mGluRs by using homology modeling and molecular dynamics (MD) simulations. We systematically compared their allosteric binding sites/pockets, the important residues, and the selective residues by using a series of comparable dockings with both the NAM and the PAM. Our results show that several residues played important roles for the receptors' selectivity. The observations of detailed interactions between compounds and their correspondent receptors are congruent with the specificity and potency of derivatives or compounds bioassayed in vitro. We then carried out 100 ns MD simulations of mGluR5 (residue 26-832, formed by Venus Flytrap domain, a so-called cysteine-rich domain, and 7 trans-membrane domains) bound with antagonist/NAM and with agonist/PAM. Our results show that both the NAM and the PAM seemed stable in class C GPCRs during the MD. However, the movements of "ionic lock," of trans-membrane domains, and of some activation-related residues in 7 trans-membrane domains of mGluR5 were congruent with the findings in class A GPCRs. Finally, we selected nine representative bound structures to perform 30 ns MD simulations for validating the stabilities of interactions, respectively. All these bound structures kept stable during the MD simulations, indicating that the binding poses in this present work are reasonable. We provided new insight into better understanding of the structural and functional roles of the mGluRs family and facilitated the future structure-based design of novel ligands of mGluRs family with therapeutic potential.

Project description:One of the most abundant G-protein coupled receptors (GPCRs) in brain, the cannabinoid 1 receptor (CB1R), is a tractable therapeutic target for treating diverse psychobehavioral and somatic disorders. Adverse on-target effects associated with small-molecule CB1R orthosteric agonists and inverse agonists/antagonists have plagued their translational potential. Allosteric CB1R modulators offer a potentially safer modality through which CB1R signaling may be directed for therapeutic benefit. Rational design of candidate, druglike CB1R allosteric modulators requires greater understanding of the architecture of the CB1R allosteric endodomain(s) and the capacity of CB1R allosteric ligands to tune the receptor's information output. We have recently reported the synthesis of a focused library of rationally designed, covalent analogues of Org27569 and PSNCBAM-1, two prototypic CB1R negative allosteric modulators (NAMs). Among the novel, pharmacologically active CB1R NAMs reported, the isothiocyanate GAT100 emerged as the lead by virtue of its exceptional potency in the [(35)S]GTP?S and ?-arrestin signaling assays and its ability to label CB1R as a covalent allosteric probe with significantly reduced inverse agonism in the [(35)S]GTP?S assay as compared to Org27569. We report here a comprehensive functional profiling of GAT100 across an array of important downstream cell-signaling pathways and analysis of its potential orthosteric probe-dependence and signaling bias. The results demonstrate that GAT100 is a NAM of the orthosteric CB1R agonist CP55,940 and the endocannabinoids 2-arachidonoylglycerol and anandamide for ?-arrestin1 recruitment, PLC?3 and ERK1/2 phosphorylation, cAMP accumulation, and CB1R internalization in HEK293A cells overexpressing CB1R and in Neuro2a and STHdh(Q7/Q7) cells endogenously expressing CB1R. Distinctively, GAT100 was a more potent and efficacious CB1R NAM than Org27569 and PSNCBAM-1 in all signaling assays and did not exhibit the inverse agonism associated with Org27569 and PSNCBAM-1. Computational docking studies implicate C7.38(382) as a key feature of GAT100 ligand-binding motif. These data help inform the engineering of newer-generation, druggable CB1R allosteric modulators and demonstrate the utility of GAT100 as a covalent probe for mapping structure-function correlates characteristic of the druggable CB1R allosteric space.

Dataset Information

Allosteric modulation of a cannabinoid G protein-coupled receptor: binding site elucidation and relationship to G protein signaling.

Publications

Allosteric modulation of a cannabinoid G protein-coupled receptor: binding site elucidation and relationship to G protein signaling.

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