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X-ray structures of endothelin ETB receptor bound to clinical antagonist bosentan and its analog

Abstract

Endothelin receptors (ETRs) have crucial roles in vascular control and are targets for drugs designed to treat circulatory-system diseases and cancer progression. The nonpeptide dual-ETR antagonist bosentan is the first oral drug approved to treat pulmonary arterial hypertension. Here we report crystal structures of human endothelin ETB receptor bound to bosentan and to the ETB-selective analog K-8794, at 3.6-Å and 2.2-Å resolution, respectively. The K-8794-bound structure reveals the detailed water-mediated hydrogen-bonding network at the transmembrane core, which could account for the weak negative allosteric modulation of ETB by Na+ ions. The bosentan-bound structure reveals detailed interactions with ETB, which are probably conserved in the ETA receptor. A comparison of the two structures shows unexpected similarity between antagonist and agonist binding. Despite this similarity, bosentan sterically prevents the inward movement of transmembrane helix 6 (TM6), and thus exerts its antagonistic activity. These structural insights will facilitate the rational design of new ETR-targeting drugs.

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Figure 1: Overall structures of antagonist-bound ETB receptors.
Figure 2: Hydrogen-bonding network in the transmembrane core.
Figure 3: Drug-binding site.
Figure 4: Comparison of the binding modes of ET-1 and bosentan.

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References

  1. Yanagisawa, M. et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332, 411–415 (1988).

    Article  CAS  Google Scholar 

  2. Arai, H., Hori, S., Aramori, I., Ohkubo, H. & Nakanishi, S. Cloning and expression of a cDNA encoding an endothelin receptor. Nature 348, 730–732 (1990).

    Article  CAS  Google Scholar 

  3. Sakurai, T. et al. Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature 348, 732–735 (1990).

    Article  CAS  Google Scholar 

  4. Kedzierski, R.M. & Yanagisawa, M. Endothelin system: the double-edged sword in health and disease. Annu. Rev. Pharmacol. Toxicol. 41, 851–876 (2001).

    Article  CAS  Google Scholar 

  5. Kohan, D.E., Rossi, N.F., Inscho, E.W. & Pollock, D.M. Regulation of blood pressure and salt homeostasis by endothelin. Physiol. Rev. 91, 1–77 (2011).

    Article  CAS  Google Scholar 

  6. Rubanyi, G.M. & Polokoff, M.A. Endothelins: molecular biology, biochemistry, pharmacology, physiology, and pathophysiology. Pharmacol. Rev. 46, 325–415 (1994).

    CAS  PubMed  Google Scholar 

  7. Shin, M.K., Levorse, J.M., Ingram, R.S. & Tilghman, S.M. The temporal requirement for endothelin receptor-B signalling during neural crest development. Nature 402, 496–501 (1999).

    Article  CAS  Google Scholar 

  8. Remuzzi, G., Perico, N. & Benigni, A. New therapeutics that antagonize endothelin: promises and frustrations. Nat. Rev. Drug Discov. 1, 986–1001 (2002).

    Article  CAS  Google Scholar 

  9. Channick, R.N. et al. Effects of the dual endothelin-receptor antagonist bosentan in patients with pulmonary hypertension: a randomised placebo-controlled study. Lancet 358, 1119–1123 (2001).

    Article  CAS  Google Scholar 

  10. Rubin, L.J. et al. Bosentan therapy for pulmonary arterial hypertension. N. Engl. J. Med. 346, 896–903 (2002).

    Article  CAS  Google Scholar 

  11. Dhaun, N., Goddard, J. & Webb, D.J. The endothelin system and its antagonism in chronic kidney disease. J. Am. Soc. Nephrol. 17, 943–955 (2006).

    Article  CAS  Google Scholar 

  12. Rosanò, L., Spinella, F. & Bagnato, A. Endothelin 1 in cancer: biological implications and therapeutic opportunities. Nat. Rev. Cancer 13, 637–651 (2013).

    Article  Google Scholar 

  13. Clozel, M. et al. Pharmacological characterization of bosentan, a new potent orally active nonpeptide endothelin receptor antagonist. J. Pharmacol. Exp. Ther. 270, 228–235 (1994).

    CAS  PubMed  Google Scholar 

  14. Clozel, M. et al. Pathophysiological role of endothelin revealed by the first orally active endothelin receptor antagonist. Nature 365, 759–761 (1993).

    Article  CAS  Google Scholar 

  15. Neidhart, W. et al. The discovery of nonpeptide endothelin receptor antagonists. Progression towards bosentan. Chimia 50, 519–524 (1996).

    CAS  Google Scholar 

  16. Mucke, H.A. Pulmonary arterial hypertension: on the way to a manageable disease. Curr. Opin. Investig. Drugs 9, 957–962 (2008).

    CAS  PubMed  Google Scholar 

  17. Norman, P. Pulmonary arterial hypertension: a rare disease that encourages the development of multiple treatments. Expert Opin. Orphan Drugs 2, 1137–1145 (2014).

    Article  CAS  Google Scholar 

  18. Korn, J.H. et al. Digital ulcers in systemic sclerosis: prevention by treatment with bosentan, an oral endothelin receptor antagonist. Arthritis Rheum. 50, 3985–3993 (2004).

    Article  CAS  Google Scholar 

  19. Gatfield, J., Mueller Grandjean, C., Bur, D., Bolli, M.H. & Nayler, O. Distinct ETA receptor binding mode of macitentan as determined by site directed mutagenesis. PLoS One 9, e107809 (2014).

    Article  Google Scholar 

  20. Gatfield, J., Mueller Grandjean, C., Sasse, T., Clozel, M. & Nayler, O. Slow receptor dissociation kinetics differentiate macitentan from other endothelin receptor antagonists in pulmonary arterial smooth muscle cells. PLoS One 7, e47662 (2012).

    Article  CAS  Google Scholar 

  21. Krum, H., Viskoper, R.J., Lacourciere, Y., Budde, M. & Charlon, V. The effect of an endothelin-receptor antagonist, bosentan, on blood pressure in patients with essential hypertension. N. Engl. J. Med. 338, 784–790 (1998).

    Article  CAS  Google Scholar 

  22. Bolli, M.H. et al. The discovery of N-[5-(4-bromophenyl)-6-[2-[(5-bromo-2-pyrimidinyl)oxy]ethoxy]-4-pyrimidinyl]-N′-propylsulfamide (macitentan), an orally active, potent dual endothelin receptor antagonist. J. Med. Chem. 55, 7849–7861 (2012).

    Article  CAS  Google Scholar 

  23. Vatter, H. & Seifert, V. Ambrisentan, a non-peptide endothelin receptor antagonist. Cardiovasc. Drug Rev. 24, 63–76 (2006).

    Article  CAS  Google Scholar 

  24. Barst, R.J. et al. Sitaxsentan therapy for pulmonary arterial hypertension. Am. J. Respir. Crit. Care Med. 169, 441–447 (2004).

    Article  Google Scholar 

  25. Boss, C., Bolli, M.H. & Gatfield, J. From bosentan (Tracleer®) to macitentan (Opsumit®): the medicinal chemistry perspective. Bioorg. Med. Chem. Lett. 26, 3381–3394 (2016).

    Article  CAS  Google Scholar 

  26. Kholdani, C.A., Fares, W.H. & Trow, T.K. Macitentan for the treatment of pulmonary arterial hypertension. Vasc. Health Risk Manag. 10, 665–673 (2014).

    PubMed  PubMed Central  Google Scholar 

  27. Shihoya, W. et al. Activation mechanism of endothelin ETB receptor by endothelin-1. Nature 537, 363–368 (2016).

    Article  CAS  Google Scholar 

  28. Sawaki, M. et al. Chronic effects of an orally active selective endothelin-B-receptor antagonist in experimental congestive heart failure. J. Cardiovasc. Pharmacol. 36, S323–S326 (2000).

    Article  CAS  Google Scholar 

  29. Okuta, A., Tani, K., Nishimura, S., Fujiyoshi, Y. & Doi, T. Thermostabilization of the human endothelin type B receptor. J. Mol. Biol. 428, 2265–2274 (2016).

    Article  CAS  Google Scholar 

  30. Hattori, M., Hibbs, R.E. & Gouaux, E. A fluorescence-detection size-exclusion chromatography-based thermostability assay for membrane protein precrystallization screening. Structure 20, 1293–1299 (2012).

    Article  CAS  Google Scholar 

  31. Parker, M.S., Wong, Y.Y. & Parker, S.L. An ion-responsive motif in the second transmembrane segment of rhodopsin-like receptors. Amino Acids 35, 1–15 (2008).

    Article  CAS  Google Scholar 

  32. Katritch, V. et al. Allosteric sodium in class A GPCR signaling. Trends Biochem. Sci. 39, 233–244 (2014).

    Article  CAS  Google Scholar 

  33. Liu, W. et al. Structural basis for allosteric regulation of GPCRs by sodium ions. Science 337, 232–236 (2012).

    Article  CAS  Google Scholar 

  34. Miller-Gallacher, J.L. et al. The 2.1 Å resolution structure of cyanopindolol-bound β1-adrenoceptor identifies an intramembrane Na+ ion that stabilises the ligand-free receptor. PLoS One 9, e92727 (2014).

    Article  Google Scholar 

  35. Fenalti, G. et al. Molecular control of δ-opioid receptor signalling. Nature 506, 191–196 (2014).

    Article  CAS  Google Scholar 

  36. Zhang, C. et al. High-resolution crystal structure of human protease-activated receptor 1. Nature 492, 387–392 (2012).

    Article  CAS  Google Scholar 

  37. Harding, M.M. Metal-ligand geometry relevant to proteins and in proteins: sodium and potassium. Acta Crystallogr. D Biol. Crystallogr. 58, 872–874 (2002).

    Article  Google Scholar 

  38. White, J.F. et al. Structure of the agonist-bound neurotensin receptor. Nature 490, 508–513 (2012).

    Article  CAS  Google Scholar 

  39. Yin, J., Mobarec, J.C., Kolb, P. & Rosenbaum, D.M. Crystal structure of the human OX2 orexin receptor bound to the insomnia drug suvorexant. Nature 519, 247–250 (2015).

    Article  CAS  Google Scholar 

  40. Thompson, A.A. et al. Structure of the nociceptin/orphanin FQ receptor in complex with a peptide mimetic. Nature 485, 395–399 (2012).

    Article  CAS  Google Scholar 

  41. Takasuka, T., Sakurai, T., Goto, K., Furuichi, Y. & Watanabe, T. Human endothelin receptor ETB. Amino acid sequence requirements for super stable complex formation with its ligand. J. Biol. Chem. 269, 7509–7513 (1994).

    CAS  PubMed  Google Scholar 

  42. Rose, P.M. et al. Aspartate mutation distinguishes ETA but not ETB receptor subtype-selective ligand binding while abolishing phospholipase C activation in both receptors. FEBS Lett. 361, 243–249 (1995).

    Article  CAS  Google Scholar 

  43. Opgenorth, T.J. et al. Pharmacological characterization of A-127722: an orally active and highly potent ETA-selective receptor antagonist. J. Pharmacol. Exp. Ther. 276, 473–481 (1996).

    CAS  PubMed  Google Scholar 

  44. Winn, M. et al. 2,4-Diarylpyrrolidine-3-carboxylic acids—potent ETA selective endothelin receptor antagonists. 1. Discovery of A-127722. J. Med. Chem. 39, 1039–1048 (1996).

    Article  CAS  Google Scholar 

  45. Kikuchi, T. et al. Endothelin-1 analogues substituted at both position 18 and 19: highly potent endothelin antagonists with no selectivity for either receptor subtype ETA or ETB . J. Med. Chem. 36, 4087–4093 (1993).

    Article  CAS  Google Scholar 

  46. Tam, J.P. et al. Alanine scan of endothelin: importance of aromatic residues. Peptides 15, 703–708 (1994).

    Article  CAS  Google Scholar 

  47. Rovero, P., Patacchini, R. & Maggi, C.A. Structure-activity studies on endothelin (16-21), the C-terminal hexapeptide of the endothelins, in the guinea-pig bronchus. Br. J. Pharmacol. 101, 232–234 (1990).

    Article  CAS  Google Scholar 

  48. Henry, J.A., Horwell, D.C., Meecham, K.G. & Rees, D.C. A structure-affinity study of the amino-acid side-chains in neurotensin—N and C-terminal deletions and Ala-scan. Bioorg. Med. Chem. Lett. 3, 949–952 (1993).

    Article  CAS  Google Scholar 

  49. Guerrini, R. et al. Address and message sequences for the nociceptin receptor: a structure-activity study of nociceptin-(1-13)-peptide amide. J. Med. Chem. 40, 1789–1793 (1997).

    Article  CAS  Google Scholar 

  50. Nakajima, K. et al. Structure-activity relationship of endothelin: importance of charged groups. Biochem. Biophys. Res. Commun. 163, 424–429 (1989).

    Article  CAS  Google Scholar 

  51. Vagner, J., Qu, H. & Hruby, V.J. Peptidomimetics, a synthetic tool of drug discovery. Curr. Opin. Chem. Biol. 12, 292–296 (2008).

    Article  CAS  Google Scholar 

  52. Hruby, V.J. Designing peptide receptor agonists and antagonists. Nat. Rev. Drug Discov. 1, 847–858 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  54. Kabsch, W. Xds. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  Google Scholar 

  55. Ueno, G. et al. Remote access and automation of SPring-8 MX beamlines. AIP Conf. Proc. 1741, 050021 (2016).

    Article  Google Scholar 

  56. Kabsch, W. Processing of X-ray snapshots from crystals in random orientations. Acta Crystallogr. D Biol. Crystallogr. 70, 2204–2216 (2014).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  61. Inoue, A. et al. TGFα shedding assay: an accurate and versatile method for detecting GPCR activation. Nat. Methods 9, 1021–1029 (2012).

    Article  CAS  Google Scholar 

  62. Doi, T., Sugimoto, H., Arimoto, I., Hiroaki, Y. & Fujiyoshi, Y. Interactions of endothelin receptor subtypes A and B with Gi, Go, and Gq in reconstituted phospholipid vesicles. Biochemistry 38, 3090–3099 (1999).

    Article  CAS  Google Scholar 

  63. Wada, K. et al. Purification of an endothelin receptor from human placenta. Biochem. Biophys. Res. Commun. 167, 251–257 (1990).

    Article  CAS  Google Scholar 

  64. Elshourbagy, N.A. et al. Molecular cloning and characterization of the major endothelin receptor subtype in porcine cerebellum. Mol. Pharmacol. 41, 465–473 (1992).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank the members of the Nureki lab and the beamline staff at BL32XU of SPring-8 (Sayo, Japan) for technical assistance during data collection. We also thank Kowa Co., Ltd., for providing K-8794. pCAGGS expression plasmid vector was a kind gift from J. Miyazaki (Osaka University, Osaka, Japan). The diffraction experiments were performed at SPring-8 BL32XU (proposals 2015A1024, 2015A1057, 2015B2024, and 2015B2057). This work was supported by JSPS KAKENHI grants 16K07172 (T.D.), 26640102 (T.D.), 16H06294 (O.N.), 15H05775 (F.Y.), 15J09780 (S.W.), 17J30010 (S.W.), 17H05000 (T.N.) and 15H06862 (K.Y.), the Core Research for Evolutional Science, PRESTO from the Japan Science and Technology (JST) Technology Program; the Platform for Drug Discovery, Information, and Structural Life Science from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; the Japan Agency for Medical Research and Development (AMED); and the National Institute of Biomedical Innovation. A.I. was funded by JST, PRESTO (grant JPMJPR1331), and the PRIME from AMED. J.A. received funding from AMED-CREST and AMED, and a MEXT Grant-in-Aid for Scientific Research on Innovative Areas (grant 15H05897).

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Contributions

W.S. designed experiments; expressed, purified, and crystallized the antagonist-bound ETB receptor; collected data; and refined the structures. T.N. initially crystallized the K-8794-bound ETB receptor, assisted with the structural determination, and designed the construct ETB-Y4-mT4L. K.Y. and K.H. developed a pipeline for data collection and processing, and assisted with the structural determination. A.I., F.M.N.K., and J.A. performed and oversaw the cell-based assays. A.O. introduced K-8794 in the experimental design and characterized its pharmacology. K.T. initially designed the T4L-fused construct. T.D. performed the radiobinding assays. The manuscript was prepared by W.S., T.N., K.Y., A.I., K.H., K.T., Y.F., T.D., and O.N. Y.F., T.D., and O.N. supervised the research.

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Correspondence to Tomoko Doi or Osamu Nureki.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Crystallization.

a, Crystallization constructs of the ETB receptor are shown, with all of the modifications to the human wild-type the ETB receptor indicated. Residues interacting with both bosentan and K-8794 are colored green, the residue only interacting with bosentan is blue, and those only interacting with K-8794 are red, as shown in the figure. b, c, Effects of ET-1 on the release of AP-TGFα and antagonists on the ET-1-induced release of AP-TGFα in HEK293 cells expressing the endothelin receptors. In the competitive assays, the concentration of the agonist ET-1 was 0.2 nM, and the AP-TGFα release response in the ET-1 treatment alone was normalized to 100%. Symbols and error bars are means and s.e.m. (standard error of the mean), respectively. For most data points, the error bars are smaller than the symbols. d, e, Crystallographic data of the ETB-Y5-mT4L protein bound to K-8794 (d) and the ETB-Y4-mT4L protein bound to bosentan (e). The left panels show the crystals of the antagonist-bound ETB receptors. The middle and right panels show their crystal packings. T4L is shown as a grey cartoon, and the K-8794- and bosentan-bound ETB receptors are shown as orange and turquoise cartoons, respectively. Crystal lattices are indicated by black lines.

Supplementary Figure 2 Electron density.

a, b, Fo − Fc omit maps for K-8794 (a) and bosentan (b), contoured at 3.0 σ and 4.0 σ, respectively. TM6 and TM7 are omitted. c, The bosentan binding site, in which the colors represent the temperature factors ranging from 20 Å2 (blue) to 120 Å2 (red). d, Stereo view of the 2Fo−Fc map, contoured at 1.0 σ, for the residues within 4 Å contact distances of the ligand in the K-8794-bound ETB structure. e, Stereo views of the 2Fo−Fc maps, contoured at 1.0 σ, for the residues within 4 Å contact distances of the ligand in the bosentan-bound ETB structure. f, Stereo view of the composite omit map, contoured at 1.0 σ, for the residues within 4 Å contact distances of the ligand in the bosentan-bound ETB structure.

Supplementary Figure 3 Comparison with other peptide-activated GPCRs.

ac, Comparison of the antagonist binding sites of the peptide-activated GPCRs. Ribbon representations of the ETB receptor in complex with bosentan (a), Orexin receptor OX2 in complex with suvorexant (PDB accession number 4RNB) (b), and NOP receptor in complex with the peptidomimetic antagonist C-24 (PDB accession number 4EA3) (c) are aligned according to the position of Trp6.48, which is indicated by the stick model in each figure. The black dashed line indicates the position of the Cα atoms of Trp6.48. The small-molecule antagonists are represented by stick models. Like the ETB receptor, OX2 belongs to the β subfamily of the class A GPCRs, while NOP belongs to the γ subfamily. d, e, Electrostatic surfaces of the ETB structures bound to bosentan (d) and ET-1 (e), viewed from the extracellular side (left) and within the membrane plane (right). Bosentan and ET-1 are shown as sticks and transparent surfaces, colored blue and pink, respectively.

Supplementary Figure 4 Ligand-interaction diagrams.

Interaction diagrams of K-8794 (a) and bosentan (b) with the ETB receptor. Interactions within 4 Å are shown. Polar and hydrophobic contacts are represented as red dashed and green lines, respectively.

Supplementary Figure 5 Homology between ETB and ETA.

Amino acid sequence alignment of the human ETB (UniProt ID: P24530) and ETA (P25101) receptors. Secondary structure elements for α-helices and β-strands are indicated by cylinders and arrows, respectively. Conservation of the residues between ETA and ETB is indicated as follows: red panels for completely conserved, red letters for partially conserved, and black letters for not conserved. The residues involved in bosentan and K-8794 binding are shown as blue squares and orange diamonds, respectively.

Supplementary Figure 6 Small-molecule endothelin-receptor antagonists.

Chemical structures of major small-molecule endothelin receptor antagonists. Endothelin receptor antagonists commonly have negatively-charged moieties (sulfonamide or carboxylate).

Supplementary Figure 7 Comparison of structural changes on ligand binding.

a, b, Comparison of the ET-1 and bosentan binding modes, coloured as in Fig. 6. Receptors and ET-1 are represented by ribbons, and the side chains of ET-118-21 and bosentan are shown as sticks with transparent surfaces. c, The residues that interact with both ET-1 and bosentan are superimposed. df, Comparison of the structural changes upon ET-1 (d), bosentan (e), and K-8794 (f) binding, coloured as in Figs. 1 and 4.

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Shihoya, W., Nishizawa, T., Yamashita, K. et al. X-ray structures of endothelin ETB receptor bound to clinical antagonist bosentan and its analog. Nat Struct Mol Biol 24, 758–764 (2017). https://doi.org/10.1038/nsmb.3450

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