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Deciphering biased inverse agonism of cangrelor and ticagrelor at P2Y12 receptor

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Abstract

P2Y12 receptor (P2Y12-R) is one of the major targets for drug inhibiting platelet aggregation in the treatment/prevention of arterial thrombosis. However, the clinical use of P2Y12-R antagonists faces some limitations, such as a delayed onset of action (clopidogrel) or adverse effect profile (ticagrelor, cangrelor), justifying the development of a new generation of P2Y12-R antagonists with a better clinical benefit–risk balance. Although the recent concept of biased agonism offers the possibility to alleviate undesirable adverse effects while preserving therapeutic outcomes, it has never been explored at P2Y12-R. For the first time, using highly sensitive BRET2-based probes, we accurately delineated biased ligand efficacy at P2Y12-R in living HEK293T cells on G protein activation and downstream effectors. We demonstrated that P2Y12-R displayed constitutive Gi/o-dependent signaling that is impaired by the R122C mutation, previously associated with a bleeding disorder. More importantly, we reported the biased inverse agonist efficacy of cangrelor and ticagrelor that could underlie their clinical efficacy. Our study points out that constitutive P2Y12-R signaling is a normal feature of the receptor that might be essential for platelets to respond faster to a vessel injury. From a therapeutic standpoint, our data suggest that the beneficial advantages of antiplatelet drugs might be more related to inverse agonism at P2Y12-R than to antagonism of ADP-mediated signaling. In the future, deciphering P2Y12-R constitutive activity should allow the discovery of more selective biased P2Y12-R blockers demonstrating therapeutic advantages over classical antiplatelet drugs by improving therapeutic outcomes and concomitantly relieving undesirable adverse effects.

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References

  1. Sabouret P, Rushton-Smith SK, Kerneis M, Silvain J, Collet JP, Montalescot G (2015) Dual antiplatelet therapy: optimal timing, management, and duration. Eur Heart J Cardiovasc Pharmacother 1:198–204

    Article  PubMed  Google Scholar 

  2. Hollopeter G, Jantzen HM, Vincent D, Li G, England L, Ramakrishnan V et al (2001) Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature 409:202–207

    Article  CAS  PubMed  Google Scholar 

  3. Bonello L, Tantry US, Marcucci R, Blindt R, Angiolillo DJ, Becker R et al (2010) Consensus and future directions on the definition of high on-treatment platelet reactivity to adenosine diphosphate. J Am Coll Cardiol 56:919–933

    Article  CAS  PubMed  Google Scholar 

  4. Gurbel PA, Kereiakes DJ, Tantry US (2010) Ticagrelor for the treatment of arterial thrombosis. Expert Opin Pharmacother 11:2251–2259

    Article  CAS  PubMed  Google Scholar 

  5. Husted S, van Giezen JJ (2009) Ticagrelor: the first reversibly binding oral P2Y12 receptor antagonist. Cardiovasc Ther 27:259–274

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Qamar A, Bhatt DL (2016) Current status of data on cangrelor. Pharmacol Ther 159:102–109

    Article  CAS  PubMed  Google Scholar 

  7. Testa L, Biondi Zoccai GG, Valgimigli M, Latini RA, Pizzocri S, Lanotte S et al (2010) Current concepts on antiplatelet therapy: focus on the novel thienopyridine and non-thienopyridine agents. Adv Hematol 2010:595934

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Wallentin L, Becker RC, Budaj A, Cannon CP, Emanuelsson H, Held C et al (2009) Ticagrelor versus clopidogrel in patients with acute coronary syndromes. N Engl J Med 361:1045–1057

    Article  CAS  PubMed  Google Scholar 

  9. Serebruany VL, Sibbing D, DiNicolantonio JJ (2014) Dyspnea and reversibility of antiplatelet agents: ticagrelor, elinogrel, cangrelor, and beyond. Cardiology 127:20–24

    Article  CAS  PubMed  Google Scholar 

  10. Unverdorben M, Parodi G, Pistolesi M, Storey RF (2016) Dyspnea related to reversibly-binding P2Y12 inhibitors: a review of the pathophysiology, clinical presentation and diagnostics. Int J Cardiol 202:167–173

    Article  PubMed  Google Scholar 

  11. Scirica BM, Cannon CP, Emanuelsson H, Michelson EL, Harrington RA, Husted S et al (2011) The incidence of bradyarrhythmias and clinical bradyarrhythmic events in patients with acute coronary syndromes treated with ticagrelor or clopidogrel in the PLATO (Platelet Inhibition and Patient Outcomes) trial: results of the continuous electrocardiographic assessment substudy. J Am Coll Cardiol 57:1908–1916

    Article  CAS  PubMed  Google Scholar 

  12. Zhang N, Zhang Z, Yang Y, Xu Y, Li G, Liu T (2015) Ticagrelor-related gout: an underestimated side effect. Int J Cardiol 192:11–13

    Article  PubMed  Google Scholar 

  13. Zhang J, Zhang K, Gao ZG, Paoletta S, Zhang D, Han GW et al (2014) Agonist-bound structure of the human P2Y12 receptor. Nature 509:119–122

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Zhang K, Zhang J, Gao ZG, Zhang D, Zhu L, Han GW et al (2014) Structure of the human P2Y12 receptor in complex with an antithrombotic drug. Nature 509:115–118

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Kenakin T, Christopoulos A (2013) Signalling bias in new drug discovery: detection, quantification and therapeutic impact. Nat Rev Drug Discov 12:205–216

    Article  CAS  PubMed  Google Scholar 

  16. Galandrin S, Denis C, Boularan C, Marie J, M’Kadmi C, Pilette C et al (2016) Cardioprotective angiotensin-(1-7) peptide acts as a natural-biased ligand at the angiotensin II type 1 receptor. Hypertension 68:1365–1374

    Article  CAS  PubMed  Google Scholar 

  17. Kingwell K (2015) Pioneering biased ligand offers efficacy with reduced on-target toxicity. Nat Rev Drug Discov 14:809–810

    Article  CAS  PubMed  Google Scholar 

  18. Viscusi ER, Webster L, Kuss M, Daniels S, Bolognese JA, Zuckerman S et al (2016) A randomized, phase 2 study investigating TRV130, a biased ligand of the mu-opioid receptor, for the intravenous treatment of acute pain. Pain 157:264–272

    Article  CAS  PubMed  Google Scholar 

  19. Pons V, Serhan N, Gayral S, Malaval C, Nauze M, Malet N et al (2014) Role of the ubiquitin-proteasome system in the regulation of P2Y13 receptor expression: impact on hepatic HDL uptake. Cell Mol Life Sci 71:1775–1788

    Article  CAS  PubMed  Google Scholar 

  20. Sauliere A, Bellot M, Paris H, Denis C, Finana F, Hansen JT et al (2012) Deciphering biased-agonism complexity reveals a new active AT1 receptor entity. Nat Chem Biol 8:622–630

    Article  CAS  PubMed  Google Scholar 

  21. Onfroy L, Galandrin S, Pontier SM, Seguelas MH, N’Guyen D, Senard JM et al (2017) G protein stoichiometry dictates biased agonism through distinct receptor-G protein partitioning. Sci Rep 7:7885

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Jiang LI, Collins J, Davis R, Lin KM, DeCamp D, Roach T et al (2007) Use of a cAMP BRET sensor to characterize a novel regulation of cAMP by the sphingosine 1-phosphate/G13 pathway. J Biol Chem 282:10576–10584

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Sanchez-Soto M, Bonifazi A, Cai NS, Ellenberger MP, Newman AH, Ferre S et al (2016) Evidence for noncanonical neurotransmitter activation: norepinephrine as a dopamine D2-like receptor agonist. Mol Pharmacol 89:457–466

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Bodor ET, Waldo GL, Hooks SB, Corbitt J, Boyer JL, Harden TK (2003) Purification and functional reconstitution of the human P2Y12 receptor. Mol Pharmacol 64:1210–1216

    Article  CAS  PubMed  Google Scholar 

  25. M’Kadmi C, Leyris JP, Onfroy L, Gales C, Sauliere A, Gagne D et al (2015) Agonism, antagonism, and inverse agonism bias at the Ghrelin receptor signaling. J Biol Chem 290:27021–27039

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Nisar S, Daly ME, Federici AB, Artoni A, Mumford AD, Watson SP et al (2011) An intact PDZ motif is essential for correct P2Y12 purinoceptor traffic in human platelets. Blood 118:5641–5651

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Patel YM, Lordkipanidze M, Lowe GC, Nisar SP, Garner K, Stockley J et al (2014) A novel mutation in the P2Y12 receptor and a function-reducing polymorphism in protease-activated receptor 1 in a patient with chronic bleeding. J Thromb Haemost 12:716–725

    Article  CAS  PubMed  Google Scholar 

  28. Denis C, Sauliere A, Galandrin S, Senard JM, Gales C (2012) Probing heterotrimeric G protein activation: applications to biased ligands. Curr Pharm Des 18:128–144

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. He Q, Zhu Y, Corbin BA, Plagge A, Bastepe M (2015) The G protein alpha subunit variant XLalphas promotes inositol 1,4,5-trisphosphate signaling and mediates the renal actions of parathyroid hormone in vivo. Sci Signal. 8:ra84

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Jeon JP, Roh SE, Wie J, Kim J, Kim H, Lee KP et al (2013) Activation of TRPC4beta by Galphai subunit increases Ca2 + selectivity and controls neurite morphogenesis in cultured hippocampal neuron. Cell Calcium 54:307–319

    Article  CAS  PubMed  Google Scholar 

  31. Kenakin T (2004) Efficacy as a vector: the relative prevalence and paucity of inverse agonism. Mol Pharmacol 65:2–11

    Article  CAS  PubMed  Google Scholar 

  32. Aungraheeta R, Conibear A, Butler M, Kelly E, Nylander S, Mumford A et al (2016) Inverse agonism at the P2Y12 receptor and ENT1 transporter blockade contribute to platelet inhibition by ticagrelor. Blood 128:2717–2728

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Van Giesen JJJ, Nilsson L, Berntsson P, Wissing BM, Giordanetto F, Tomlinson W et al (2009) Ticagrelor binds to human P2Y(12) independently from ADP but antagonizes ADP-induced receptor signaling and platelet aggregation. J Thromb Haemost 7:1556–1565

    Article  CAS  Google Scholar 

  34. Gales C, Van Durm JJ, Schaak S, Pontier S, Percherancier Y, Audet M et al (2006) Probing the activation-promoted structural rearrangements in preassembled receptor-G protein complexes. Nat Struct Mol Biol 13:778–786

    Article  CAS  PubMed  Google Scholar 

  35. Kim S, Jin J, Kunapuli SP (2004) Akt activation in platelets depends on Gi signaling pathways. J Biol Chem 279:4186–4195

    Article  CAS  PubMed  Google Scholar 

  36. Schwarz UR, Geiger J, Walter U, Eigenthaler M (1999) Flow cytometry analysis of intracellular VASP phosphorylation for the assessment of activating and inhibitory signal transduction pathways in human platelets–definition and detection of ticlopidine/clopidogrel effects. Thromb Haemost 82:1145–1152

    Article  CAS  PubMed  Google Scholar 

  37. Bonello L, Laine M, Kipson N, Mancini J, Helal O, Fromonot J et al (2014) Ticagrelor increases adenosine plasma concentration in patients with an acute coronary syndrome. J Am Coll Cardiol 63:872–877

    Article  CAS  PubMed  Google Scholar 

  38. Cattaneo M, Schulz R, Nylander S (2014) Adenosine-mediated effects of ticagrelor: evidence and potential clinical relevance. J Am Coll Cardiol 63:2503–2509

    Article  CAS  PubMed  Google Scholar 

  39. Nylander S, Femia EA, Scavone M, Berntsson P, Asztely AK, Nelander K et al (2013) Ticagrelor inhibits human platelet aggregation via adenosine in addition to P2Y12 antagonism. J Thromb Haemost 11:1867–1876

    CAS  PubMed  Google Scholar 

  40. Hu L, Chang L, Zhang Y, Zhai L, Zhang S, Qi Z et al (2017) Platelets express activated P2Y12 receptor in patients with diabetes mellitus. Circulation 136:817–833

    Article  CAS  PubMed  Google Scholar 

  41. Fontana P, Dupont A, Gandrille S, Bachelot-Loza C, Reny JL, Aiach M et al (2003) Adenosine diphosphate-induced platelet aggregation is associated with P2Y12 gene sequence variations in healthy subjects. Circulation 108:989–995

    Article  CAS  PubMed  Google Scholar 

  42. Li MP, Tang J, Wen ZP, Zhang YJ, Zhang W, Zhou HH et al (2015) Influence of P2Y12 polymorphisms on platelet activity but not ex vivo antiplatelet effect of ticagrelor in healthy Chinese male subjects. Blood Coagul Fibrinolysis 26:874–881

    Article  CAS  PubMed  Google Scholar 

  43. Zoheir N, Abd Elhamid S, Abulata N, El Sobky M, Khafagy D, Mostafa A (2013) P2Y12 receptor gene polymorphism and antiplatelet effect of clopidogrel in patients with coronary artery disease after coronary stenting. Blood Coagul Fibrinolysis 24:525–531

    Article  CAS  PubMed  Google Scholar 

  44. Kim KA, Song WG, Lee HM, Joo HJ, Park JY (2013) Effect of P2Y1 and P2Y12 genetic polymorphisms on the ADP-induced platelet aggregation in a Korean population. Thromb Res 132:221–226

    Article  CAS  PubMed  Google Scholar 

  45. Lee SJ, Jung IS, Jung EJ, Choi JY, Yeo CW, Cho DY et al (2011) Identification of P2Y12 single-nucleotide polymorphisms and their influences on the variation in ADP-induced platelet aggregation. Thromb Res 127:220–227

    Article  CAS  PubMed  Google Scholar 

  46. Cui G, Zhang S, Zou J, Chen Y, Chen H (2017) P2Y12 receptor gene polymorphism and the risk of resistance to clopidogrel: a meta-analysis and review of the literature. Adv Clin Exp Med 26:343–349

    Article  Google Scholar 

  47. Yang HH, Chen Y, Gao CY (2016) Associations of P2Y12R gene polymorphisms with susceptibility to coronary heart disease and clinical efficacy of antiplatelet treatment with clopidogrel. Cardiovasc Ther 34:460–467

    Article  CAS  PubMed  Google Scholar 

  48. Ango F, Prezeau L, Muller T, Tu JC, Xiao B, Worley PF et al (2001) Agonist-independent activation of metabotropic glutamate receptors by the intracellular protein Homer. Nature 411:962–965

    Article  CAS  PubMed  Google Scholar 

  49. Corder G, Doolen S, Donahue RR, Winter MK, Jutras BL, He Y et al (2013) Constitutive mu-opioid receptor activity leads to long-term endogenous analgesia and dependence. Science 341:1394–1399

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Morisset S, Rouleau A, Ligneau X, Gbahou F, Tardivel-Lacombe J, Stark H et al (2000) High constitutive activity of native H3 receptors regulates histamine neurons in brain. Nature 408:860–864

    Article  CAS  PubMed  Google Scholar 

  51. Lane JR, May LT, Parton RG, Sexton PM, Christopoulos A (2017) A kinetic view of GPCR allostery and biased agonism. Nat Chem Biol 13:929–937

    Article  CAS  PubMed  Google Scholar 

  52. Irannejad R, Pessino V, Mika D, Huang B, Wedegaertner PB, Conti M et al (2017) Functional selectivity of GPCR-directed drug action through location bias. Nat Chem Biol 13:799–806

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Galandrin S, Onfroy L, Poirot MC, Senard JM, Gales C (2016) Delineating biased ligand efficacy at 7TM receptors from an experimental perspective. Int J Biochem Cell Biol 77:251–263

    Article  CAS  PubMed  Google Scholar 

  54. Shukla AK, Violin JD, Whalen EJ, Gesty-Palmer D, Shenoy SK, Lefkowitz RJ (2008) Distinct conformational changes in beta-arrestin report biased agonism at seven-transmembrane receptors. Proc Natl Acad Sci USA 105:9988–9993

    Article  PubMed  Google Scholar 

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Acknowledgements

We thank Lauriane Onfroy for critical reading of the manuscript. We thank the imaging facility (INSERM UMR1048, Toulouse) and the DNA sequencing platform (Plateau de génomique GeT-Purpan de la plateforme Génome et Transcriptome de la Génopole de Toulouse Midi-Pyrénées). BP, JMS, C Galés and VP are supported by the Institut National de la santé et de la Recherche Médicale (INSERM). C Galés is also supported by the Fondation Bettencourt-Schueller (C Galés) and BP by the Fondation pour la Recherche Médicale (FRM) and the Institut Universitaire de France (IUF).

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  1. Cédric Garcia and Agnès Maurel-Ribes equally contribute to this work.

Authors and Affiliations

  1. Laboratoire d’Hématologie, Centre Hospitalier Universitaire de Toulouse, 31000, Toulouse, France

    Cédric Garcia, Agnès Maurel-Ribes & Bernard Payrastre

  2. INSERM U1048, Institut des Maladies Métaboliques et Cardiovasculaires, Université de Toulouse, 1 Avenue Jean Poulhès, BP 84225, 31432, Toulouse Cedex 04, France

    Michel Nauze, Du N’Guyen, Laurent O. Martinez, Bernard Payrastre, Jean-Michel Sénard, Céline Galés & Véronique Pons

  3. Service de Pharmacologie Clinique, Centre Hospitalier Universitaire de Toulouse, Université de Toulouse, 31000, Toulouse, France

    Jean-Michel Sénard

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  1. Cédric Garcia
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18_2018_2960_MOESM1_ESM.tif

Supplementary Figure S1:Relative expression of Gα protein and CAMYEL probes. a-b. Relative expression of RLuc8-Gα protein probes (a) or CAMYEL sensor (b) was assessed by luminescence measurement in each experiment. Data represent the mean ± SEM of at least three independent experiments. (TIFF 4930 kb)

18_2018_2960_MOESM2_ESM.tif

Supplementary Figure S2:Constitutive activation of Gai2 protein depends on P2Y12-R expression. a. Basal BRET signal was measured in HEK293T/17 cells co-expressing Gαi2-RLuc8, GFP2-Gγ2, Gβ1 in the absence (0µg) or in the presence of increasing amounts of WT P2Y12-R (ranging from 0,01 to 4 µg/dish). b. Relative expression of RLuc8-Gα protein probes was assessed by luminescence measurement in each condition. a-b. Data represent the mean ± SEM of at least six independent experiments and statistical significance between cells expressing increasing amounts of WT P2Y12-R or not (0 µg) was assessed using one-way ANOVA followed by a Dunnett’s multiple comparison test (** P < 0.01; *** P < 0.001; ns, not statistically significant). (TIFF 4176 kb)

18_2018_2960_MOESM3_ESM.tif

Supplementary Figure S3:WT and mutant P2Y12-R are expressed at the cell surface of HEK293T/17 cells. a. HEK293T/17 cells expressing N-terminally Myc-tagged WT or mutant P2Y12-R were processed for immunofluorescence using an anti-Myc antibody. Nuclei were stained with 5 μg/mL DAPI. Scale bar indicates 10 μm. b. HEK293T/17 cells were transfected with increasing amounts of vectors encoding N-terminally Myc-tagged WT or mutant P2Y12-R (ranging from 15 to 500 ng/well). Then, cell surface expression of the different receptors was quantified using an anti-Myc antibody. Data represent the mean ± SEM of five independent experiments. Statistical significance between cell surface expression of WT and mutant P2Y12-R was assessed using two-way ANOVA followed by a Bonferroni post-test (ns, not statistically significant). (TIFF 23037 kb)

18_2018_2960_MOESM4_ESM.tif

Supplementary Figure S4:Inverse agonist efficacy of cangrelor depends on P2Y12-R expression. Gαi2 protein activation was measured in HEK293T/17 cells co-expressing Gαi2-RLuc8, GFP2-Gγ2 and Gβ1 alone (0µg) or with increasing amounts of vectors encoding WT P2Y12-R (ranging from 0,01 to 4 µg/dish), after stimulation or not with cangrelor (10 μM). Results are expressed as the difference in the BRET signal measured in the presence and in the absence of cangrelor. Data represent the mean ± SEM of six independent experiments. Statistical significance between cells expressing the WT P2Y12-R or not was assessed using one-way ANOVA followed by Kruskal-Wallis and Dunn’s multiple comparison post-tests (* P < 0.05; ** P < 0.01; ns indicates not statistically significant). (TIFF 4048 kb)

18_2018_2960_MOESM5_ESM.tif

Supplementary Figure S5:Dose–response curves of Gαi/o protein activation mediated by cangrelor or ticagrelor at WT P2Y12-R. Dose–response curves of each Gαi/o protein activation were assessed in HEK293T/17 cells co-expressing Gαi/o-RLuc8, GFP2-Gγ2, Gβ1 and WT P2Y12-R after stimulation or not with increasing concentrations of cangrelor or ticagrelor (values from Fig. 5). Results are expressed as the difference in the BRET signal measured in the presence and the absence of ligand. Data represent the mean ± SEM of at least three independent experiments. Statistical significance between unstimulated cells and cells stimulated with the different concentrations of cangrelor (*) or ticagrelor (#) was assessed using one-way ANOVA followed by a Dunnett’s multiple comparison test (* or # P < 0.05; ** or ## P < 0.01; *** or ### P < 0.001). (TIFF 4529 kb)

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Garcia, C., Maurel-Ribes, A., Nauze, M. et al. Deciphering biased inverse agonism of cangrelor and ticagrelor at P2Y12 receptor. Cell. Mol. Life Sci. 76, 561–576 (2019). https://doi.org/10.1007/s00018-018-2960-3

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