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An allosteric modulator binds to a conformational hub in the β2 adrenergic receptor

Nature Chemical Biology volume 16, pages 749–755 (2020)Cite this article

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Abstract

Most drugs acting on G-protein-coupled receptors target the orthosteric binding pocket where the native hormone or neurotransmitter binds. There is much interest in finding allosteric ligands for these targets because they modulate physiologic signaling and promise to be more selective than orthosteric ligands. Here we describe a newly developed allosteric modulator of the β2-adrenergic receptor (β2AR), AS408, that binds to the membrane-facing surface of transmembrane segments 3 and 5, as revealed by X-ray crystallography. AS408 disrupts a water-mediated polar network involving E1223.41 and the backbone carbonyls of V2065.45 and S2075.46. The AS408 binding site is adjacent to a previously identified molecular switch for β2AR activation formed by I3.40, P5.50 and F6.44. The structure reveals how AS408 stabilizes the inactive conformation of this switch, thereby acting as a negative allosteric modulator for agonists and positive allosteric modulator for inverse agonists.

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Fig. 1: Hit-to-lead optimization, pharmacological characterization and structure of allosteric modulator AS408 bound to β2AR.
Fig. 2: Structural basis of the negative allosteric activity of AS408 on agonist binding to β2AR.
Fig. 3: Structure–activity relationships of AS408 analogs.
Fig. 4: AS408 helps to stabilize the inactive conformation.
Fig. 5: AS408 uses E1223.41 of β2AR, a residue that participates in an allosteric network.

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Data availability

Atomic coordinates and structure factors have been deposited in the PDB under accession code 6OBA.

References

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Digby, G. J., Shirey, J. K. & Conn, P. J. Allosteric activators of muscarinic receptors as novel approaches for treatment of CNS disorders. Mol. Biosyst. 6, 1345–1354 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Mohr, K., Trankle, C. & Holzgrabe, U. Structure/activity relationships of M2 muscarinic allosteric modulators. Recept. Channels 9, 229–240 (2003).

    CAS  PubMed  Google Scholar 

  4. Wootten, D., Christopoulos, A. & Sexton, P. M. Emerging paradigms in GPCR allostery: implications for drug discovery. Nat. Rev. Drug Discov. 12, 630–644 (2013).

    Article  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).

    Article  CAS  PubMed  Google Scholar 

  6. Ahn, S. et al. Allosteric ‘beta-blocker’ isolated from a DNA-encoded small molecule library. Proc. Natl Acad. Sci. USA 114, 1708–1713 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Ahn, S. et al. Small-molecule positive allosteric modulators of the beta2-adrenoceptor isolated from DNA-encoded libraries. Mol. Pharmacol. 94, 850–861 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Liu, X. et al. Mechanism of intracellular allosteric beta2AR antagonist revealed by X-ray crystal structure. Nature 548, 480–484 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Liu, X. et al. Mechanism of beta2AR regulation by an intracellular positive allosteric modulator. Science 364, 1283–1287 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Rasmussen, S. G. et al. Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor. Nature 469, 175–180 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Masureel, M. et al. Structural insights into binding specificity, efficacy and bias of a beta2AR partial agonist. Nat. Chem. Biol. 14, 1059–1066 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Ballesteros, J. A. & Weinstein, H. in Methods in Neurosciences Vol. 25 (ed. Sealfon, S. C.) 366–428 (Academic Press, 1995).

  13. Leach, K., Sexton, P. M. & Christopoulos, A. Allosteric GPCR modulators: taking advantage of permissive receptor pharmacology. Trends Pharmacol. Sci. 28, 382–389 (2007).

    Article  CAS  PubMed  Google Scholar 

  14. Aurelio, L. et al. Allosteric modulators of the adenosine A1 receptor: synthesis and pharmacological evaluation of 4-substituted 2-amino-3-benzoylthiophenes. J. Med. Chem. 52, 4543–4547 (2009).

    Article  CAS  PubMed  Google Scholar 

  15. Christopoulos, A. & Kenakin, T. G protein-coupled receptor allosterism and complexing. Pharmacol. Rev. 54, 323–374 (2002).

    Article  CAS  PubMed  Google Scholar 

  16. Srivastava, A. et al. High-resolution structure of the human GPR40 receptor bound to allosteric agonist TAK-875. Nature 513, 124–127 (2014).

    Article  CAS  PubMed  Google Scholar 

  17. Robertson, N. et al. Structure of the complement C5a receptor bound to the extra-helical antagonist NDT9513727. Nature 553, 111–114 (2018).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  19. Roth, C. B., Hanson, M. A. & Stevens, R. C. Stabilization of the human beta2-adrenergic receptor TM4-TM3-TM5 helix interface by mutagenesis of Glu122(3.41), a critical residue in GPCR structure. J. Mol. Biol. 376, 1305–1319 (2008).

    Article  CAS  PubMed  Google Scholar 

  20. Schonegge, A. M. et al. Evolutionary action and structural basis of the allosteric switch controlling beta2AR functional selectivity. Nat. Commun. 8, 2169(2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Du, Y. et al. Assembly of a GPCR-G protein complex. Cell 177, e1211 (2019).

    Article  CAS  Google Scholar 

  22. Szlenk, C. T., Gc, J. B. & Natesan, S. Does the lipid bilayer orchestrate access and binding of ligands to transmembrane orthosteric/allosteric sites of G protein-coupled receptors? Mol. Pharmacol. 96, 527–541 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Lu, S. & Zhang, J. Small molecule allosteric modulators of G-protein-coupled receptors: drug-target interactions. J. Med. Chem. 62, 24–45 (2019).

    Article  CAS  PubMed  Google Scholar 

  24. Pettersen, E. F. et al. UCSF Chimera – a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  25. Klicić, J. J., Friesner, R. A., Liu, S.-Y. & Guida, W. C. Accurate prediction of acidity constants in aqueous solution via density functional theory and self-consistent reaction field methods. J. Phys. Chem. A. 106, 1327–1335 (2002).

    Article  CAS  Google Scholar 

  26. Bochevarov, A. D., Watson, M. A., Greenwood, J. R. & Philipp, D. M. Multiconformation, density functional theory-based pKa prediction in application to large, flexible organic molecules with diverse functional groups. J. Chem. Theory Comput. 12, 6001–6019 (2016).

    Article  CAS  PubMed  Google Scholar 

  27. Yu, H. S., Watson, M. A. & Bochevarov, A. D. Weighted averaging scheme and local atomic descriptor for pKa prediction based on density functional theory. J. Chem. Inf. Model 58, 271–286 (2018).

    Article  CAS  PubMed  Google Scholar 

  28. Lomize, M. A., Lomize, A. L., Pogozheva, I. D. & Mosberg, H. I. OPM: orientations of proteins in membranes database. Bioinformatics 22, 623–625 (2006).

    Article  CAS  PubMed  Google Scholar 

  29. Wolf, M. G., Hoefling, M., Aponte-Santamaria, C., Grubmuller, H. & Groenhof, G. g_membed: efficient insertion of a membrane protein into an equilibrated lipid bilayer with minimal perturbation. J. Comput. Chem. 31, 2169–2174 (2010).

    Article  CAS  PubMed  Google Scholar 

  30. Case, D. A. et al. AMBER 2017 (University of California, San Francisco, 2017).

  31. Wang, J., Wolf, R. M., Caldwell, J. W., Kollman, P. A. & Case, D. A. Development and testing of a general amber force field. J. Comput. Chem. 25, 1157–1174 (2004).

    Article  CAS  PubMed  Google Scholar 

  32. Dickson, C. J. et al. Lipid14: the Amber lipid force field. J. Chem. Theory Comput. 10, 865–879 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Maier, J. A. et al. ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB. J. Chem. Theory Comput. 11, 3696–3713 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Berendsen, H. J. C., Grigera, J. R. & Straatsma, T. P. The missing term in effective pair potentials. J. Phys. Chem. 91, 6269–6271 (1987).

    Article  CAS  Google Scholar 

  35. Frisch, M. J. et al. Gaussian 09, revision B.01 (Gaussian, Inc., Wallingford, CT, 2009).

  36. Bayly, C. I., Cieplak, P., Cornell, W. D. & Kollman, P. A. A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges—the Resp model. J. Phys. Chem. 97, 10269–10280 (1993).

    Article  CAS  Google Scholar 

  37. Van Der Spoel, D. et al. GROMACS: fast, flexible, and free. J. Comput. Chem. 26, 1701–1718 (2005).

    Article  CAS  Google Scholar 

  38. Abraham, M. J. et al. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1-2, 19–25 (2015).

    Article  Google Scholar 

  39. Hess, B., Bekker, H., Berendsen, H. J. C. & Fraaije, J. G. E. M. LINCS: a linear constraint solver for molecular simulations. J. Comput. Chem. 18, 1463–1472 (1997).

    Article  CAS  Google Scholar 

  40. Darden, T., York, D. & Pedersen, L. Particle mesh Ewald—an n.log(n) method for Ewald sums in large systems. J. Chem. Phys. 98, 10089–10092 (1993).

    Article  CAS  Google Scholar 

  41. Hunter, J. D. Matplotlib: a 2D graphics environment. Comput. Sci. Eng. 9, 90–95 (2007).

    Article  Google Scholar 

  42. Schrödinger Release 2018-1: Maestro, Schrödinger, LLC, New York, NY, 2018.

  43. Rosenbaum, D. M. et al. GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function. Science 318, 1266–1273 (2007).

    Article  CAS  PubMed  Google Scholar 

  44. Kobilka, B. K. Amino and carboxyl terminal modifications to facilitate the production and purification of a G protein-coupled receptor. Anal. Biochem. 231, 269–271(1995).

    Article  CAS  PubMed  Google Scholar 

  45. Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic mesophases. Nat. Protocol. 4, 706–731 (2009).

    Article  CAS  Google Scholar 

  46. Kabsch, W. XDS. Acta Crystallogr. Sect. D 66, 125–132 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  49. Ten Eyck, L. F. Fast Fourier transform calculation of electron density maps. Methods Enzymol. 115, 324–337 (1985).

    Article  PubMed  Google Scholar 

  50. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. Sect. D 67, 235–242 (2011).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  52. Hubner, H. et al. Structure-guided development of heterodimer-selective GPCR ligands. Nat. Commun. 7, 12298 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. DeVree, B. T. et al. Allosteric coupling from G protein to the agonist-binding pocket in GPCRs. Nature 535, 182–186 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Whorton, M. R. et al. A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein. Proceed. Natl Acad. Sci. USA 104, 7682–7687 (2007).

    Article  CAS  Google Scholar 

  55. Vedel, L., Brauner-Osborne, H. & Mathiesen, J. M. A cAMP biosensor-based high-throughput screening assay for identification of Gs-coupled GPCR ligands and phosphodiesterase inhibitors. J. Biomol. Screen 20, 849–857 (2015).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We acknowledge support from the US NIH grant nos. GM106990 (B.K.S., P.G. and R.K.S.), and the DFG Grants Gm 13/10 and GRK 1910 (P.G.), and Beijing Advanced Innovation Center for Structural Biology, Tsinghua University (X.L.), as well as the compute resources provided by the RRZE. B.K.K. is a Chan Zuckerberg Biohub Investigator. We thank D. Steffen and M. Gilardi for help with confocal microscopy. We also thank A. Christopoulos for his critical insight with the analysis of cooperativity.

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  1. These authors contributed equally: Xiangyu Liu, Jonas Kaindl.

Authors and Affiliations

  1. School of Pharmaceutical Sciences, Tsinghua University, Beijing, China

    Xiangyu Liu

  2. Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, China

    Xiangyu Liu, Xinyu Xu & Brian K. Kobilka

  3. Department of Chemistry and Pharmacy, Medicinal Chemistry, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany

    Jonas Kaindl, Anne Stößel, Daniela Dengler, Markus Stanek, Harald Hübner & Peter Gmeiner

  4. Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, USA

    Magdalena Korczynska & Brian K. Shoichet

  5. Department of Pharmacology, University of California San Diego School of Medicine, La Jolla, CA, USA

    Mary J. Clark, Jake Mahoney & Roger K. Sunahara

  6. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA

    Rachel Ann Matt & Brian K. Kobilka

  7. School of Medicine, Tsinghua University, Beijing, China

    Xinyu Xu & Brian K. Kobilka

  8. Advanced Photon Technology Division, Research Infrastructure Group, SR Life Science Instrumentation Unit, RIKEN/SPring-8 Center Sayo-gun, Hyogo, Japan

    Kunio Hirata

  9. Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama, Japan

    Kunio Hirata

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Contributions

X.L. expressed and purified the receptor, crystallized the receptor–ligand complex and solved the crystal structure. K.H. performed the automatic data collection and processing. A.S., D.D. and M.S. synthesized and analytically characterized the chemical compounds. H.H., M.J.C., R.A.M., J.M., X.L., D.D. and X.X. performed ligand binding and signaling experiments. M.K. performed docking studies. J.K. performed MD simulations. The manuscript was written by B.K.K., X.L. and P.G. with editing and suggestions from R.K.S. and input from J.K. and H.H., P.G. supervised chemical synthesis of compounds, B.K.K., R.K.S. and P.G. supervised binding and signaling experiments. B.K.S. supervised docking. X.L. supervised structure determination. The project was conceived by B.K.K., P.G., R.K.S. and B.K.S.

Corresponding authors

Correspondence to Roger K. Sunahara, Brian K. Kobilka or Peter Gmeiner.

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Competing interests

B.K.K. is a cofounder of and consultant for ConfometRx, Inc.

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Liu, X., Kaindl, J., Korczynska, M. et al. An allosteric modulator binds to a conformational hub in the β2 adrenergic receptor. Nat Chem Biol 16, 749–755 (2020). https://doi.org/10.1038/s41589-020-0549-2

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