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Structure of an allosteric modulator bound to the CB1 cannabinoid receptor

Nature Chemical Biology volume 15, pages 1199–1205 (2019)Cite this article

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Abstract

The CB1 receptor mediates the central nervous system response to cannabinoids, and is a drug target for pain, anxiety and seizures. CB1 also responds to allosteric modulators, which influence cannabinoid binding and efficacy. To understand the mechanism of these compounds, we solved the crystal structure of CB1 with the negative allosteric modulator (NAM) ORG27569 and the agonist CP55940. The structure reveals that the NAM binds to an extrahelical site within the inner leaflet of the membrane, which overlaps with a conserved site of cholesterol interaction in many G protein-coupled receptors (GPCRs). The ternary structure with ORG27569 and CP55940 captures an intermediate state of the receptor, in which aromatic residues at the base of the agonist-binding pocket adopt an inactive conformation despite the large contraction of the orthosteric pocket. The structure illustrates a potential strategy for drug modulation of CB1 and other class A GPCRs.

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Fig. 1: Two-dimensional structures of CB1 ligands.
Fig. 2: Overall structure of CB1 bound to CP55940 and ORG27569.
Fig. 3: Interaction of CP55940 with the CB1 receptor.
Fig. 4: Dual toggle switch of CB1 (F2003.36 and W3566.48) in different states.
Fig. 5: Binding of ORG27569 to the CB1 receptor.
Fig. 6: Effects of ORG27569 on ligand binding to wild-type CB1 and the F237 mutant.

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Data availability

Structural data have been deposited in the Protein Data Bank (PDB) with coordinate accession number 6KQI. All other data generated or analyzed during this study are included in this published article (and its supplementary information files) or are available from the corresponding author on reasonable request.

References

  1. Mechoulam, R. & Parker, L. A. The endocannabinoid system and the brain. Annu. Rev. Psychol. 64, 21–47 (2013).

    PubMed  Google Scholar 

  2. Whiting, P. F. et al. Cannabinoids for medical use: a systematic review and meta-analysis. JAMA 313, 2456–2473 (2015).

    CAS  PubMed  Google Scholar 

  3. Devinsky, O. et al. Trial of cannabidiol for drug-resistant seizures in the Dravet syndrome. N. Engl. J. Med. 376, 2011–2020 (2017).

    CAS  PubMed  Google Scholar 

  4. Kunos, G., Osei-Hyiaman, D., Bátkai, S., Sharkey, K. A. & Makriyannis, A. Should peripheral CB1 cannabinoid receptors be selectively targeted for therapeutic gain? Trends Pharmacol. Sci. 30, 1–7 (2009).

    CAS  PubMed  Google Scholar 

  5. Thal, D. M., Glukhova, A., Sexton, P. M. & Christopoulos, A. Structural insights into G-protein-coupled receptor allostery. Nature 559, 45–53 (2018).

    CAS  PubMed  Google Scholar 

  6. Price, M. R. et al. Allosteric modulation of the cannabinoid CB1 receptor. Mol. Pharmacol. 68, 1484–1495 (2005).

    CAS  PubMed  Google Scholar 

  7. Baillie, G. L. et al. CB1 receptor allosteric modulators display both agonist and signaling pathway specificity. Mol. Pharmacol. 83, 322–338 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Fay, J. F. & Farrens, D. L. A key agonist-induced conformational change in the cannabinoid receptor CB1 is blocked by the allosteric ligand Org 27569. J. Biol. Chem. 287, 33873–33882 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Fay, J. F. & Farrens, D. L. Structural dynamics and energetics underlying allosteric inactivation of the cannabinoid receptor CB1. Proc. Natl Acad. Sci. USA 112, 8469–8474 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Khurana, L., Mackie, K., Piomelli, D. & Kendall, D. A. Modulation of CB1 cannabinoid receptor by allosteric ligands: pharmacology and therapeutic opportunities. Neuropharmacology 124, 3–12 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Shao, Z. et al. High-resolution crystal structure of the human CB1 cannabinoid receptor. Nature 540, 602–608 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Suno, R. et al. Structural insights into the subtype-selective antagonist binding to the M2 muscarinic receptor. Nat. Chem. Biol. 14, 1150–1158 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Hua, T. et al. Crystal structures of agonist-bound human cannabinoid receptor CB1. Nature 547, 468–471 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Krishna Kumar, K. et al. Structure of a signaling cannabinoid receptor 1–G protein complex. Cell 176, 448–458.e12 (2019).

    CAS  PubMed  Google Scholar 

  15. Johnson, M. R. et al. Selective and potent analgetics derived from cannabinoids. J. Clin. Pharmacol. 21, 271S–282S (1981).

    CAS  PubMed  Google Scholar 

  16. Herkenham, M. et al. Cannabinoid receptor localization in brain. Proc. Natl Acad. Sci. USA 87, 1932–1936 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Shim, J.-Y., Bertalovitz, A. C. & Kendall, D. A. Identification of essential cannabinoid-binding domains: structural insights into early dynamic events in receptor activation. J. Biol. Chem. 286, 33422–33435 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Murphy, J. W. & Kendall, D. A. Integrity of extracellular loop 1 of the human cannabinoid receptor 1 is critical for high-affinity binding of the ligand CP 55,940 but not SR 141716A. Biochem. Pharmacol. 65, 1623–1631 (2003).

    CAS  PubMed  Google Scholar 

  19. Ahn, K. H., Bertalovitz, A. C., Mierke, D. F. & Kendall, D. A. Dual role of the second extracellular loop of the cannabinoid receptor 1: ligand binding and receptor localization. Mol. Pharmacol. 76, 833–842 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Singh, R. et al. Activation of the cannabinoid CB1 receptor may involve a W6 48/F3 36 rotamer toggle switch. J. Pept. Res. 60, 357–370 (2002).

    CAS  PubMed  Google Scholar 

  21. Rosenbaum, D. M. et al. GPCR engineering yields high-resolution structural insights into β2-adrenergic receptor function. Science 318, 1266–1273 (2007).

    CAS  PubMed  Google Scholar 

  22. Stornaiuolo, M. et al. Endogenous vs exogenous allosteric modulators in GPCRs: a dispute for shuttling CB1 among different membrane microenvironments. Sci. Rep. 5, 15453 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Shore, D. M. et al. Allosteric modulation of a cannabinoid G protein-coupled receptor: binding site elucidation and relationship to G protein signaling. J. Biol. Chem. 289, 5828–5845 (2014).

    CAS  PubMed  Google Scholar 

  24. Bari, M., Battista, N., Fezza, F., Finazzi-Agrò, A. & Maccarrone, M. Lipid rafts control signaling of type-1 cannabinoid receptors in neuronal cells. Implications for anandamide-induced apoptosis. J. Biol. Chem. 280, 12212–12220 (2005).

    CAS  PubMed  Google Scholar 

  25. Vallée, M. et al. Pregnenolone can protect the brain from cannabis intoxication. Science 343, 94–98 (2014).

    PubMed  PubMed Central  Google Scholar 

  26. Lu, J. et al. Structural basis for the cooperative allosteric activation of the free fatty acid receptor GPR40. Nat. Struct. Mol. Biol. 24, 570–577 (2017).

    CAS  PubMed  Google Scholar 

  27. Liu, H. et al. Orthosteric and allosteric action of the C5a receptor antagonists. Nat. Struct. Mol. Biol. 25, 472–481 (2018).

    CAS  PubMed  Google Scholar 

  28. Liu, X. et al. Mechanism of intracellular allosteric β2AR antagonist revealed by X-ray crystal structure. Nature 548, 480–484 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Zheng, Y. et al. Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists. Nature 540, 458–461 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Kruse, A. C. et al. Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 504, 101–106 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. De Lean, A., Stadel, J. M. & Lefkowitz, R. J. A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled β-adrenergic receptor. J. Biol. Chem. 255, 7108–7117 (1980).

    PubMed  Google Scholar 

  32. Hanson, M. A. et al. Crystal structure of a lipid G protein-coupled receptor. Science 335, 851–855 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Ahn, K. H., Mahmoud, M. M., Shim, J.-Y. & Kendall, D. A. Distinct roles of β-arrestin 1 and β-arrestin 2 in ORG27569-induced biased signaling and internalization of the cannabinoid receptor 1 (CB1). J. Biol. Chem. 288, 9790–9800 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Kang, Y. et al. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser. Nature 523, 561–567 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Gamage, T. F., Anderson, J. C. & Abood, M. E. CB1 allosteric modulator Org27569 is an antagonist/inverse agonist of ERK1/2 signaling. Cannabis Cannabinoid Res. 1, 272–280 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. D’Ambra, T. E. et al. Conformationally restrained analogues of pravadoline: nanomolar potent, enantioselective, (aminoalkyl)indole agonists of the cannabinoid receptor. J. Med. Chem. 35, 124–135 (1992).

    PubMed  Google Scholar 

  37. Kulkarni, P. M. et al. Novel electrophilic and photoaffinity covalent probes for mapping the cannabinoid 1 receptor allosteric site(s). J. Med. Chem. 59, 44–60 (2016).

    CAS  PubMed  Google Scholar 

  38. Horswill, J. G. et al. PSNCBAM-1, a novel allosteric antagonist at cannabinoid CB1 receptors with hypophagic effects in rats. Br. J. Pharmacol. 152, 805–814 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Di Marzo, V. New approaches and challenges to targeting the endocannabinoid system. Nat. Rev. Drug Discov. 17, 623–639 (2018).

    PubMed  Google Scholar 

  40. Osei-Hyiaman, D. et al. Hepatic CB1 receptor is required for development of diet-induced steatosis, dyslipidemia, and insulin and leptin resistance in mice. J. Clin. Invest. 118, 3160–3169 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Fernández-Ruiz, J. et al. Cannabidiol for neurodegenerative disorders: important new clinical applications for this phytocannabinoid? Br. J. Clin. Pharmacol. 75, 323–333 (2013).

    PubMed  Google Scholar 

  42. Thomas, A. et al. Cannabidiol displays unexpectedly high potency as an antagonist of CB1 and CB2 receptor agonists in vitro. Br. J. Pharmacol. 150, 613–623 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Hua, T. et al. Crystal structure of the human cannabinoid receptor CB1. Cell 167, 750–762.e14 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data. Methods Enzymol. 276, 307–326 (1997).

    CAS  PubMed  Google Scholar 

  45. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Horcajada, C., Guinovart, J. J., Fita, I. & Ferrer, J. C. Crystal structure of an archaeal glycogen synthase: insights into oligomerization and substrate binding of eukaryotic glycogen synthases. J. Biol. Chem. 281, 2923–2931 (2006).

    CAS  PubMed  Google Scholar 

  47. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Schüttelkopf, A. W. & van Aalten, D. M. F. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr. D 60, 1355–1363 (2004).

    PubMed  Google Scholar 

  50. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010).

    CAS  PubMed  Google Scholar 

  51. Morales-Perez, C. L., Noviello, C. M. & Hibbs, R. E. Manipulation of subunit stoichiometry in heteromeric membrane proteins. Structure 24, 797–805 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank the staff of the GM/CA-CAT beamline 23ID at the Advanced Photon Source for support during data collection. This project was supported by the Edward Mallinckrodt, Jr. Foundation (Scholar Award to D.M.R.), the Welch Foundation (grant no. I-1770 to D.M.R.) and the National Young Thousand Talents Program of China (to Z.S.). APS is a US Department of Energy Office of Science User Facility operated for the Department of Energy Office of Science by Argonne National Laboratory (no. DE-AC02-06CH11357).

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Authors and Affiliations

  1. Division of Nephrology and Kidney Research Institute, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, China

    Zhenhua Shao, Wei Yan & Xuehui Wang

  2. Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, TX, USA

    Karen Chapman, Karthik Ramesh, Aaron J. Ferrell, Jie Yin & Daniel M. Rosenbaum

  3. GM/CA, Advanced Photon Source, Argonne National Laboratory, Argonne, IL, USA

    Qingping Xu

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Contributions

Z.S. developed the CB1 construct and purification; expressed, purified and crystallized the receptor; collected diffraction data and solved and refined the structures. W.Y. and X.W. assisted in receptor expression, purification and structure refinement. K.C., K.R. and A.J.F. performed ligand-binding assays on CB1 constructs. J.Y. and Q.X. assisted in diffraction data collection and analysis. D.M.R. supervised the overall project, assisted with collection of diffraction data and wrote the manuscript.

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Correspondence to Daniel M. Rosenbaum.

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Shao, Z., Yan, W., Chapman, K. et al. Structure of an allosteric modulator bound to the CB1 cannabinoid receptor. Nat Chem Biol 15, 1199–1205 (2019). https://doi.org/10.1038/s41589-019-0387-2

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