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Pepducins and Other Lipidated Peptides as Mechanistic Probes and Therapeutics

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Cell-Penetrating Peptides

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1324))

Abstract

Lipopeptides based on the intracellular loops of cell-surface receptors, known as “Pepducins,” represent a promising new class of compounds used for the study of membrane proteins and as potential therapeutics in a variety of diseases. Detailed knowledge of the three-dimensional structure of G-protein-coupled receptors (GPCRs) and delineation of the mechanisms of pepducin activation and biased G-protein signaling has facilitated the development of even more potent pepducin allosteric modulators.

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References

  1. Audet M, Bouvier M (2012) Restructuring G-protein- coupled receptor activation. Cell 151:14–23

    Article  CAS  PubMed  Google Scholar 

  2. O’Callaghan K, Kuliopulos A, Covic L (2012) Turning receptors on and off with intracellular pepducins: new insights into G-protein-coupled receptor drug development. J Biol Chem 287:12787–12796

    Article  PubMed Central  PubMed  Google Scholar 

  3. Tressel SL, Koukos G, Tchernychev B, Jacques SL, Covic L, Kuliopulos A (2011) Pharmacology, biodistribution, and efficacy of GPCR-based pepducins in disease models. Methods Mol Biol 683:259–275

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Covic L, Gresser AL, Talavera J, Swift S, Kuliopulos A (2002) Activation and inhibition of G protein-coupled receptors by cell-penetrating membrane-tethered peptides. Proc Natl Acad Sci U S A 99:643–648

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Covic L, Misra M, Badar J, Singh C, Kuliopulos A (2002) Pepducin-based intervention of thrombin-receptor signaling and systemic platelet activation. Nat Med 8:1161–1165

    Article  CAS  PubMed  Google Scholar 

  6. Wielders SJ, Bennaghmouch A, Reutelingsperger CP, Bevers EM, Lindhout T (2007) Anticoagulant and antithrombotic properties of intracellular protease-activated receptor antagonists. J Thromb Haemost 5:571–576

    Article  CAS  PubMed  Google Scholar 

  7. Fontanini KB, Janz J, Looby R, Hamilton JA (2010) Rapid binding and transmembrane diffusion of pepducins in phospholipid bilayers. Biophys J 98:278a

    Article  Google Scholar 

  8. Janz JM, Ren Y, Looby R, Kazmi MA, Sachdev P, Grunbeck A, Haggis L, Chinnapen D, Lin AY, Seibert C, McMurry T, Carlson KE, Muir TW, Hunt S 3rd, Sakmar TP (2011) Direct interaction between an allosteric agonist pepducin and the chemokine receptor CXCR4. J Am Chem Soc 133:15878–15881

    Article  CAS  PubMed  Google Scholar 

  9. Tsuji M, Ueda S, Hirayama T, Okuda K, Sakaguchi Y, Isono A, Nagasawa H (2013) FRET-based imaging of transbilayer movement of pepducin in living cells by novel intracellular bioreductively activatable fluorescent probes. Org Biomol Chem 11:3030–3037

    Article  CAS  PubMed  Google Scholar 

  10. Overington JP, Al-Lazikani B, Hopkins AL (2006) How many drug targets are there? Nat Rev Drug Discov 5:993–996

    Article  CAS  PubMed  Google Scholar 

  11. Rask-Andersen M, Almen MS, Schioth HB (2011) Trends in the exploitation of novel drug targets. Nat Rev Drug Discov 10:579–590

    Article  CAS  PubMed  Google Scholar 

  12. Rasmussen SG, DeVree BT, Zou Y, Kruse AC, Chung KY, Kobilka TS, Thian FS, Chae PS, Pardon E, Calinski D, Mathiesen JM, Shah ST, Lyons JA, Caffrey M, Gellman SH, Steyaert J, Skiniotis G, Weis WI, Sunahara RK, Kobilka BK (2011) Crystal structure of the beta2 adrenergic receptor-Gs protein complex. Nature 477:549–555

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Standfuss J, Edwards PC, D’Antona A, Fransen M, Xie G, Oprian DD, Schertler GF (2011) The structural basis of agonist-induced activation in constitutively active rhodopsin. Nature 471:656–660

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. Chung KY, Rasmussen SG, Liu T, Li S, DeVree BT, Chae PS, Calinski D, Kobilka BK, Woods VL Jr, Sunahara RK (2011) Conformational changes in the G protein Gs induced by the beta2 adrenergic receptor. Nature 477:611–615

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. McKeown SC, Zecri FJ, Fortier E, Taggart A, Sviridenko L, Adams CM, McAllister KH, Pin SS (2014) The design and implementation of a generic lipopeptide scanning platform to enable the identification of ‘locally acting’ agonists for the apelin receptor. Bioorg Med Chem Lett 24:4871–4875

    Article  CAS  PubMed  Google Scholar 

  16. Leger AJ, Jacques SL, Badar J, Kaneider NC, Derian CK, Andrade-Gordon P, Covic L, Kuliopulos A (2006) Blocking the protease-activated receptor 1-4 heterodimer in platelet-mediated thrombosis. Circulation 113:1244–1254

    Article  CAS  PubMed  Google Scholar 

  17. Zhang P, Gruber A, Kasuda S, Kimmelstiel C, O’Callaghan K, Cox DH, Bohm A, Baleja JD, Covic L, Kuliopulos A (2012) Suppression of arterial thrombosis without affecting hemostatic parameters with a cell-penetrating PAR1 pepducin. Circulation 126:83–91

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Trivedi V, Boire A, Tchernychev B, Kaneider NC, Leger AJ, O’Callaghan K, Covic L, Kuliopulos A (2009) Platelet matrix metalloprotease-1 mediates thrombogenesis by activating PAR1 at a cryptic ligand site. Cell 137:332–343

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. Kimmelstiel C, Zhang P, Kapur NK, Weintraub A, Krishnamurthy B, Castaneda V, Covic L, Kuliopulos A (2011) Bivalirudin is a dual inhibitor of thrombin and collagen-dependent platelet activation in patients undergoing percutaneous coronary intervention. Circ Cardiovasc Interv 4:171–179

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Foley CJ, Fanjul-Fernandez M, Bohm A, Nguyen N, Agarwal A, Austin K, Koukos G, Covic L, Lopez-Otin C, Kuliopulos A (2014) Matrix metalloprotease 1a deficiency suppresses tumor growth and angiogenesis. Oncogene 33:2264

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. Foley CJ, Luo C, O’Callaghan K, Hinds PW, Covic L, Kuliopulos A (2012) Matrix metalloprotease-1a promotes tumorigenesis and metastasis. J Biol Chem 287:24330–24338

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Kamath L, Meydani A, Foss F, Kuliopulos A (2001) Signaling from protease-activated receptor-1 inhibits migration and invasion of breast cancer cells. Cancer Res 61:5933–5940

    CAS  PubMed  Google Scholar 

  23. Yang E, Boire A, Agarwal A, Nguyen N, O’Callaghan K, Tu P, Kuliopulos A, Covic L (2009) Blockade of PAR1 signaling with cell-penetrating pepducins inhibits Akt survival pathways in breast cancer cells and suppresses tumor survival and metastasis. Cancer Res 69:6223–6231

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Agarwal A, Tressel SL, Kaimal R, Balla M, Lam FH, Covic L, Kuliopulos A (2010) Identification of a metalloprotease-chemokine signaling system in the ovarian cancer microenvironment: implications for antiangiogenic therapy. Cancer Res 70:5880–5890

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. Tressel SL, Kaneider NC, Kasuda S, Foley C, Koukos G, Austin K, Agarwal A, Covic L, Opal SM, Kuliopulos A (2011) A matrix metalloprotease-PAR1 system regulates vascular integrity, systemic inflammation and death in sepsis. EMBO Mol Med 3:370–384

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Kaneider NC, Agarwal A, Leger AJ, Kuliopulos A (2005) Reversing systemic inflammatory response syndrome with chemokine receptor pepducins. Nat Med 11:661–665

    Article  CAS  PubMed  Google Scholar 

  27. Sevigny LM, Zhang P, Bohm A, Lazarides K, Perides G, Covic L, Kuliopulos A (2011) Interdicting protease-activated receptor-2-driven inflammation with cell-penetrating pepducins. Proc Natl Acad Sci U S A 108:8491–8496

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Carr R 3rd, Du Y, Quoyer J, Panettieri RA Jr, Janz JM, Bouvier M, Kobilka BK, Benovic JL (2014) Development and characterization of pepducins as Gs-biased allosteric agonists. J Biol Chem 289:35668

    Article  CAS  PubMed  Google Scholar 

  29. Tchernychev B, Ren Y, Sachdev P, Janz JM, Haggis L, O’Shea A, McBride E, Looby R, Deng Q, McMurry T, Kazmi MA, Sakmar TP, Hunt S 3rd, Carlson KE (2010) Discovery of a CXCR4 agonist pepducin that mobilizes bone marrow hematopoietic cells. Proc Natl Acad Sci U S A 107:22255–22259

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Scheerer P, Park JH, Hildebrand PW, Kim YJ, Krauss N, Choe HW, Hofmann KP, Ernst OP (2008) Crystal structure of opsin in its G-protein-interacting conformation. Nature 455:497–502

    Article  CAS  PubMed  Google Scholar 

  31. Rasmussen SG, Choi HJ, Fung JJ, Pardon E, Casarosa P, Chae PS, Devree BT, Rosenbaum DM, Thian FS, Kobilka TS, Schnapp A, Konetzki I, Sunahara RK, Gellman SH, Pautsch A, Steyaert J, Weis WI, Kobilka BK (2011) Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor. Nature 469:175–180

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Forsman H, Bylund J, Oprea TI, Karlsson A, Boulay F, Rabiet MJ, Dahlgren C (2013) The leukocyte chemotactic receptor FPR2, but not the closely related FPR1, is sensitive to cell-penetrating pepducins with amino acid sequences descending from the third intracellular receptor loop. Biochim Biophys Acta 1833:1914–1923

    Article  CAS  PubMed  Google Scholar 

  33. Kaneider NC, Leger AJ, Agarwal A, Nguyen N, Perides G, Derian C, Covic L, Kuliopulos A (2007) ‘Role reversal’ for the receptor PAR1 in sepsis-induced vascular damage. Nat Immunol 8:1303–1312

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Quoyer J, Janz JM, Luo J, Ren Y, Armando S, Lukashova V, Benovic JL, Carlson KE, Hunt SW 3rd, Bouvier M (2013) Pepducin targeting the C-X-C chemokine receptor type 4 acts as a biased agonist favoring activation of the inhibitory G protein. Proc Natl Acad Sci U S A 110:E5088–E5097

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  35. Hollenberg MD, Mihara K, Polley D, Suen JY, Han A, Fairlie DP, Ramachandran R (2014) Biased signalling and proteinase-activated receptors (PARs): targeting inflammatory disease. Br J Pharmacol 171:1180–1194

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Tautermann CS (2014) GPCR structures in drug design, emerging opportunities with new structures. Bioorg Med Chem Lett 24:4073–4079

    Article  CAS  PubMed  Google Scholar 

  37. Edwards RJ, Moran N, Devocelle M, Kiernan A, Meade G, Signac W, Foy M, Park SD, Dunne E, Kenny D, Shields DC (2007) Bioinformatic discovery of novel bioactive peptides. Nat Chem Biol 3:108–112

    Article  CAS  PubMed  Google Scholar 

  38. Valente P, Fernandez-Carvajal A, Camprubi-Robles M, Gomis A, Quirce S, Viana F, Fernandez-Ballester G, Gonzalez-Ros JM, Belmonte C, Planells-Cases R, Ferrer-Montiel A (2011) Membrane-tethered peptides patterned after the TRP domain (TRPducins) selectively inhibit TRPV1 channel activity. FASEB J 25:1628–1640

    Article  CAS  PubMed  Google Scholar 

  39. Robbins J, Marsh SJ, Brown DA (2006) Probing the regulation of M (Kv7) potassium channels in intact neurons with membrane-targeted peptides. J Neurosci 26:7950–7961

    Article  CAS  PubMed  Google Scholar 

  40. Gong H, Shen B, Flevaris P, Chow C, Lam SC, Voyno-Yasenetskaya TA, Kozasa T, Du X (2010) G protein subunit Galpha13 binds to integrin alphaIIbbeta3 and mediates integrin “outside-in” signaling. Science 327:340–343

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  41. Huang JS, Dong L, Kozasa T, Le Breton GC (2007) Signaling through G(alpha)13 switch region I is essential for protease-activated receptor 1-mediated human platelet shape change, aggregation, and secretion. J Biol Chem 282:10210–10222

    Article  CAS  PubMed  Google Scholar 

  42. Koukos G, Sevigny L, Zhang P, Covic L, Kuliopulos A (2011) Serine and metalloprotease signaling through PAR1 in arterial thrombosis and vascular injury. IUBMB Life 63:412–418

    Article  CAS  PubMed  Google Scholar 

  43. Zhang P, Covic L, Kuliopulos A (2012) Protease-activated receptors. Platelets 31:249

    CAS  Google Scholar 

  44. Boire A, Covic L, Agarwal A, Jacques S, Sherifi S, Kuliopulos A (2005) PAR1 is a matrix metalloprotease-1 receptor that promotes invasion and tumorigenesis of breast cancer cells. Cell 120:303–313

    Article  CAS  PubMed  Google Scholar 

  45. Cisowski J, O’Callaghan K, Kuliopulos A, Yang J, Nguyen N, Deng Q, Yang E, Fogel M, Tressel S, Foley C, Agarwal A, Hunt SW 3rd, McMurry T, Brinckerhoff L, Covic L (2011) Targeting protease-activated receptor-1 with cell-penetrating pepducins in lung cancer. Am J Pathol 179:513–523

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  46. Agarwal A, Covic L, Sevigny LM, Kaneider NC, Lazarides K, Azabdaftari G, Sharifi S, Kuliopulos A (2008) Targeting a metalloprotease-PAR1 signaling system with cell-penetrating pepducins inhibits angiogenesis, ascites, and progression of ovarian cancer. Mol Cancer Ther 7:2746–2757

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  47. Covic L, Gresser AL, Kuliopulos A (2000) Biphasic kinetics of activation and signaling for PAR1 and PAR4 thrombin receptors in platelets. Biochemistry 39:5458–5467

    Article  CAS  PubMed  Google Scholar 

  48. Jacques SL, Kuliopulos A (2003) Protease-activated receptor-4 uses dual prolines and an anionic retention motif for thrombin recognition and cleavage. Biochem J 376:733–740

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  49. Kuliopulos A, Covic L (2003) Blocking receptors on the inside: pepducin-based intervention of PAR signaling and thrombosis. Life Sci 74:255–262

    Article  CAS  PubMed  Google Scholar 

  50. Leger AJ, Covic L, Kuliopulos A (2006) Protease-activated receptors in cardiovascular diseases. Circulation 114:1070–1077

    Article  CAS  PubMed  Google Scholar 

  51. Covic L, Tchernychev B, Jacques S, Kuliopulos A (2007) Pharmacology and in vivo efficacy of pepducins in hemostasis and arterial thrombosis. In: Langel U (ed) Handbook of cell-penetrating peptides. Taylor & Francis, New York, NY, pp 245–257

    Google Scholar 

  52. Covic L, Singh C, Smith H, Kuliopulos A (2002) Role of the PAR4 thrombin receptor in stabilizing platelet-platelet aggregates as revealed by a patient with Hermansky-Pudlak syndrome. Thromb Haemost 87:722–727

    CAS  PubMed  Google Scholar 

  53. Trumel C, Payrastre B, Plantavid M, Hechler B, Viala C, Presek P, Martinson EA, Cazenave J-P, Chap H, Gachet C (1999) A key role of adenosine diphosphate in the irreversible platelet aggregation induced by the PAR1-activating peptide through the late activation of phosphoinositide 3-kinase. Blood 94:4156–4165

    CAS  PubMed  Google Scholar 

  54. Michael ES, Kuliopulos A, Covic L, Steer ML, Perides G (2013) Pharmacological inhibition of PAR2 with the pepducin P2pal-18S protects mice against acute experimental biliary pancreatitis. Am J Physiol Gastrointest Liver Physiol 304:G516–G526

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  55. Allen CD, Ansel KM, Low C, Lesley R, Tamamura H, Fujii N, Cyster JG (2004) Germinal center dark and light zone organization is mediated by CXCR4 and CXCR5. Nat Immunol 5:943–952

    Article  CAS  PubMed  Google Scholar 

  56. Mantovani A, Allavena P, Sica A, Balkwill F (2008) Cancer-related inflammation. Nature 454:436–444

    Article  CAS  PubMed  Google Scholar 

  57. O’Callaghan K, Lee L, Nguyen N, Hsieh MY, Kaneider NC, Klein AK, Sprague K, Van Etten RA, Kuliopulos A, Covic L (2012) Targeting CXCR4 with cell-penetrating pepducins in lymphoma and lymphocytic leukemia. Blood 119:1717–1725

    Article  PubMed Central  PubMed  Google Scholar 

  58. Severino B, Incisivo GM, Fiorino F, Bertolino A, Frecentese F, Barbato F, Manganelli S, Maggioni G, Capasso D, Caliendo G, Santagada V, Sorrentino R, Roviezzo F, Perissutti E (2013) Identification of a pepducin acting as S1P3 receptor antagonist. J Pept Sci 19:717–724

    Article  CAS  PubMed  Google Scholar 

  59. Fu H, Karlsson J, Bylund J, Movitz C, Karlsson A, Dahlgren C (2006) Ligand recognition and activation of formyl peptide receptors in neutrophils. J Leukoc Biol 79:247–256

    Article  CAS  PubMed  Google Scholar 

  60. Ye RD, Boulay F, Wang JM, Dahlgren C, Gerard C, Parmentier M, Serhan CN, Murphy PM (2009) International union of basic and clinical pharmacology. LXXIII. Nomenclature for the formyl peptide receptor (FPR) family. Pharmacol Rev 61:119–161

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  61. Winther M, Gabl M, Oprea TI, Jonsson B, Boulay F, Bylund J, Dahlgren C, Forsman H (2014) Antibacterial activity of pepducins, allosterical modulators of formyl peptide receptor signaling. Antimicrob Agents Chemother 58:2985–2988

    Article  PubMed Central  PubMed  Google Scholar 

  62. Lee HY, Kim SD, Shim JW, Kim HJ, Kwon JY, Kim JM, Baek SH, Park JS, Bae YS (2010) Activation of human monocytes by a formyl peptide receptor 2-derived pepducin. FEBS Lett 584:4102–4108

    Article  CAS  PubMed  Google Scholar 

  63. Kubo S, Ishiki T, Doe I, Sekiguchi F, Nishikawa H, Kawai K, Matsui H, Kawabata A (2006) Distinct activity of peptide mimetic intracellular ligands (pepducins) for proteinase-activated receptor-1 in multiple cells/tissues. Ann N Y Acad Sci 1091:445–459

    Article  CAS  PubMed  Google Scholar 

  64. Lin C, Duitman J, Daalhuisen J, Ten Brink M, von der Thusen J, van der Poll T, Borensztajn K, Spek CA (2013) Targeting protease activated receptor-1 with P1pal-12 limits bleomycin-induced pulmonary fibrosis. Thorax 69:152–160

    Article  PubMed  Google Scholar 

  65. Gabl M, Winther M, Skovbakke SL, Bylund J, Dahlgren C, Forsman H (2014) A pepducin derived from the third intracellular loop of FPR2 is a partial agonist for direct activation of this receptor in neutrophils but a full agonist for cross-talk triggered reactivation of FPR2. PLoS One 9:e109516

    Article  PubMed Central  PubMed  Google Scholar 

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Authors and Affiliations

  1. Center for Hemostasis and Thrombosis Research, Tufts Medical Center, Tufts University School of Medicine, Box 7510, 800 Washington Street, Boston, MA, 02111, USA

    Ping Zhang, Lidija Covic & Athan Kuliopulos

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  1. Ping Zhang
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  2. Lidija Covic
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  3. Athan Kuliopulos
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Correspondence to Athan Kuliopulos .

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  1. Department of Neurochemistry, Stockholm University, Stockholm, Sweden

    Ülo Langel

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Zhang, P., Covic, L., Kuliopulos, A. (2015). Pepducins and Other Lipidated Peptides as Mechanistic Probes and Therapeutics. In: Langel, Ü. (eds) Cell-Penetrating Peptides. Methods in Molecular Biology, vol 1324. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2806-4_13

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  • DOI: https://doi.org/10.1007/978-1-4939-2806-4_13

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2805-7

  • Online ISBN: 978-1-4939-2806-4

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