Advertisement

Chimeric protein probes for C5a receptors through fusion of the anaphylatoxin C5a core region with a small-molecule antagonist

  • Chao Zuo
  • Wei-Wei Shi
  • Xiao-Xu Chen
  • Marie Glatz
  • Bernd Riedl
  • Ingo Flamme
  • Elisabeth Pook
  • Jiawei Wang
  • Ge-Min Fang
  • Donald Bierer
  • Lei LiuEmail author
Articles

Abstract

Blockade of the interaction of anaphylatoxin C5a with its receptor C5aR1 has been actively studied as a potential treatment for many inflammatory diseases; but current C5a antagonists exhibit inadequate potency and poor species cross-reactivity, and novel biochemical tools are needed to investigate whether the core region of C5a contains important interaction epitopes that can explain these limitations. Herein, we report the development of chimeric protein C5a probes containing both the complete core region of rat or human C5a, and the small-molecule antagonist PMX53-1. These probes were chemically synthesized through hydrazide-based native chemical ligation of a linear peptide hydrazide with the requisite cyclopeptidic antagonist, both of which were made by solid-phase synthesis. Quasi-racemic X-ray crystallography established that attachment of PMX53-1 did not affect the structure of the core region of C5a. Subsequent C5aR1 activity assays demonstrated the probes can provide valuable insights into the development of C5a antagonists; for example, they exhibited significantly better binding affinity and much improved species cross-reactivity than PMX53-1, supporting the notion that the effect of some epitopes outside the C-terminus of C5a should be taken into consideration when designing better C5a antagonists. Surprisingly, the core region of C5a was found to partially agonize C5aR1, suggesting the presence of more than one agonistic interaction in the binding of C5a to C5aR1. This study exemplifies the value of chemical protein synthesis in developing novel receptor probes for drug discovery research.

Keywords

anaphylatoxin C5a PMX53 quasi-racemic X-ray crystallography native chemical ligation 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Notes

Acknowledgements

This work was supported by the National Key R&D Program of China (2017YFA0505200), and the National Natural Science Foundation of China (21532004, 91753205, 81621002, 21621003).

Supplementary material

11426_2019_9513_MOESM1_ESM.pdf (1.1 mb)
Chimeric protein probes for C5a receptors through fusion of the anaphylatoxin C5a core region with a small-molecule antagonist

References

  1. 1(a).
    Shin HS, Snyderman R, Friedman E, Mellors A, Mayer MM. Science, 1968, 162: 361–363CrossRefGoogle Scholar
  2. 1(b).
    Manthey HD, Woodruff TM, Taylor SM, Monk PN. Int J Biochem Cell Biol, 2009, 41: 2114–2117CrossRefGoogle Scholar
  3. 1(c).
    Sarma JV, Ward PA. Cell Health Cytoskelet, 2012, 4: 73–82Google Scholar
  4. 2(a).
    Ward PA. Nat Rev Immunol, 2004, 4: 133–142CrossRefGoogle Scholar
  5. 2(b).
    Nozaki M, Raisler BJ, Sakurai E, Vidya Sarma J, Barnum SR, Lambris JD, Chen Y, Zhang K, Ambati BK, Baffi JZ, Ambati J. Proc Natl Acad Sci USA, 2006, 103: 2328–2333CrossRefGoogle Scholar
  6. 2(c).
    Markiewski MM, DeAngelis RA, Benencia F, Ricklin-Lichtsteiner SK, Koutoulaki A, Gerard C, Coukos G, Lambris JD. Nat Immunol, 2008, 9: 1225–1235CrossRefGoogle Scholar
  7. 2(d).
    Ricklin D, Hajishengallis G, Yang K, Lambris JD. Nat Immunol, 2010, 11: 785–797CrossRefGoogle Scholar
  8. 3(a).
    Chenoweth DE, Hugli TE. Proc Natl Acad Sci USA, 1978, 75: 3943–3947CrossRefGoogle Scholar
  9. 3(b).
    Mollison KW, Mandecki W, Zuiderweg ERP, Fayer L, Fey TA, Krause RA, Conway RG, Miller L, Edalji RP, Shallcross MA, Lane B, Fox JL, Greer J, Carter GW. Proc Natl Acad Sci USA, 1989, 86: 292–296CrossRefGoogle Scholar
  10. 3(c).
    Kawai M, Quincy DA, Lane B, Mollison KW, Luly JR, Carter GW. J Med Chem, 1991, 34: 2068–2071CrossRefGoogle Scholar
  11. 3(d).
    Siciliano SJ, Rollins TE, DeMartino J, Konteatis Z, Malkowitz L, Van Riper G, Bondy S, Rosen H, Springer MS. Proc Natl Acad Sci USA, 1994, 91: 1214–1218CrossRefGoogle Scholar
  12. 4(a).
    Ricklin D, Lambris JD. Nat Biotechnol, 2007, 25: 1265–1275CrossRefGoogle Scholar
  13. 4(b).
    Monk PN, Scola AM, Madala P, Fairlie DP. British JPharmacol, 2007, 152: 429–448CrossRefGoogle Scholar
  14. 5(a).
    Wong AK, Finch AM, Pierens GK, Craik DJ, Taylor SM, Fairlie DP. J Med Chem, 1998, 41: 3417–3425CrossRefGoogle Scholar
  15. 5(b).
    Finch AM, Wong AK, Paczkowski NJ, Wadi SK, Craik DJ, Fairlie DP, Taylor SM. J Med Chem, 1999, 42: 1965–1974CrossRefGoogle Scholar
  16. 5(c).
    March DR, Proctor LM, Stoermer MJ, Sbaglia R, Abbenante G, Reid RC, Woodruff TM, Wadi K, Paczkowski N, Tyndall JDA, Taylor SM, Fairlie DP. Mol Pharmacol, 2004, 65: 868–879CrossRefGoogle Scholar
  17. 6(a).
    Köhl J. Curr OpinMol Ther, 2006, 8: 529–538Google Scholar
  18. 6(b).
    Klos A, Wende E, Wareham KJ, Monk PN. Pharmacol Rev, 2013, 65: 500–543CrossRefGoogle Scholar
  19. 7.
    Vlattas I, Sytwu II, Dellureficio J, Stanton J, Braunwalder AF, Galakatos N, Kramer R, Seligmann B, Sills MA, Wasvary J. J Med Chem, 1994, 37: 2783–2790CrossRefGoogle Scholar
  20. 8(a).
    Bandlow V, Liese S, Lauster D, Ludwig K, Netz RR, Herrmann A, Seitz O. J Am Chem Soc, 2017, 139: 16389–16397CrossRefGoogle Scholar
  21. 8(b).
    Dubel N, Liese S, Scherz F, Seitz O. Angew Chem Int Ed, 2019, 58: 907–911CrossRefGoogle Scholar
  22. 9(a).
    Paes C, Ingalls J, Kampani K, Sulli C, Kakkar E, Murray M, Kotelnikov V, Greene TA, Rucker JB, Doranz BJ. J Am Chem Soc, 2009, 131: 6952–6954CrossRefGoogle Scholar
  23. 9(b).
    Coin I, Katritch V, Sun T, Xiang Z, Siu FY, Beyermann M, Stevens RC, Wang L. Cell, 2013, 155: 1258–1269CrossRefGoogle Scholar
  24. 10(a).
    Tharakaraman K, Robinson LN, Hatas A, Chen YL, Siyue L, Raguram S, Sasisekharan V, Wogan GN, Sasisekharan R. Proc Natl Acad Sci USA, 2013, 110: E1555–E1564CrossRefGoogle Scholar
  25. 10(b).
    Kadam RU, Juraszek J, Brandenburg B, Buyck C, Schepens WBG, Kesteleyn B, Stoops B, Vreeken RJ, Vermond J, Goutier W, Tang C, Vogels R, Friesen RHE, Goudsmit J, van Dongen MJP, Wilson IA. Science, 2017, 358: 496–502CrossRefGoogle Scholar
  26. 11(a).
    White R, Rusconi C, Scardino E, Wolberg A, Lawson J, Hoffman M, Sullenger B. Mol Ther, 2001, 4: 567–573CrossRefGoogle Scholar
  27. 11(b).
    Oney S, Lam RTS, Bompiani KM, Blake CM, Quick G, Heidel JD, Liu JYC, Mack BC, Davis ME, Leong KW, Sullenger BA. Nat Med, 2009, 15: 1224–1228CrossRefGoogle Scholar
  28. 12(a).
    Hartley O, Gaertner H, Wilken J, Thompson D, Fish R, Ramos A, Pastore C, Dufour B, Cerini F, Melotti A, Heveker N, Picard L, Alizon M, Mosier D, Kent S, Offord R. Proc Natl Acad Sci USA, 2004, 101: 16460–16465CrossRefGoogle Scholar
  29. 12(b).
    Clark RJ, Jensen J, Nevin ST, Callaghan BP, Adams DJ, Craik DJ. Angew Chem Int Ed, 2010, 49: 6545–6548CrossRefGoogle Scholar
  30. 12(c).
    Ghassemian A, Wang CIA, Yau MK, Reid RC, Lewis RJ, Fairlie DP, Alewood PF, Durek T. Chem Commun, 2013, 49: 2356–2358CrossRefGoogle Scholar
  31. 12(d).
    Lam HY, Zhang Y, Liu H, Xu J, Wong CTT, Xu C, Li X. J Am Chem Soc, 2013, 135: 6272–6279CrossRefGoogle Scholar
  32. 12(e).
    Harmand TJ, Pattabiraman VR, Bode JW. Angew Chem Int Ed, 2017, 56: 12639–12643CrossRefGoogle Scholar
  33. 12(f).
    Wang X, Sanchez J, Stone MJ, Payne RJ. Angew Chem Int Ed, 2017, 56: 8490–8494CrossRefGoogle Scholar
  34. 12(g).
    Thompson RE, Liu X, Ripoll-Rozada J, Alonso-García N, Parker BL, Pereira PJB, Payne RJ. Nat Chem, 2017, 9: 909–917CrossRefGoogle Scholar
  35. 12(h).
    Jin AH, Dekan Z, Smout MJ, Wilson D, Dutertre S, Vetter I, Lewis RJ, Loukas A, Daly NL, Alewood PF. Angew Chem Int Ed, 2017, 56: 14973–14976CrossRefGoogle Scholar
  36. 12(i).
    Wang D, Qin X, Zhao H, Li Z. Sci China Chem, 2017, 60: 689–700CrossRefGoogle Scholar
  37. 12(j).
    Yang J, Zhao J. Sci China Chem, 2018, 61: 97–112CrossRefGoogle Scholar
  38. 13(a).
    Dawson PE, Muir TW, Clark-Lewis I, Kent SBH. Science, 1994, 266: 776–779CrossRefGoogle Scholar
  39. 13(b).
    Fang GM, Li YM, Shen F, Huang YC, Li JB, Lin Y, Cui HK, Liu L. Angew Chem Int Ed, 2011, 50: 7645–7649CrossRefGoogle Scholar
  40. 13(c).
    Fang GM, Wang JX, Liu L. Angew Chem Int Ed, 2012, 51: 10347–10350CrossRefGoogle Scholar
  41. 13(d).
    Flood DT, Hintzen JCJ, Bird MJ, Cistrone PA, Chen JS, Dawson PE. Angew Chem Int Ed, 2018, 57: 11634–11639CrossRefGoogle Scholar
  42. 13(e).
    Guo XQ, Liang J, Li Y, Zhang Y, Huang D, Tian C. Chin Chem Lett, 2018, 29: 1139–1142CrossRefGoogle Scholar
  43. 13(f).
    Li H, Dong S. Sci China Chem, 2017, 60: 201–213CrossRefGoogle Scholar
  44. 13(g).
    Liu J, Dong S. Chin Chem Lett, 2018, 29: 1131–1134CrossRefGoogle Scholar
  45. 13(h).
    Pan M, Zheng Q, Ding S, Zhang L, Qu Q, Wang T, Hong D, Ren Y, Liang L, Chen C, Mei Z, Liu L. Angew Chem Int Ed, 2019, 58: 2627–2631CrossRefGoogle Scholar
  46. 14.
    Cui L, Carney DF, Hugli TE. Protein Sci, 1994, 3: 1169–1177CrossRefGoogle Scholar
  47. 15(a).
    Zheng JS, Tang S, Qi YK, Wang ZP, Liu L. Nat Protoc, 2013, 8: 2483–2495CrossRefGoogle Scholar
  48. 15(b).
    He Q, Li J, Qi Y, Wang Z, Huang Y, Liu L. Sci China Chem, 2017, 60: 621–627CrossRefGoogle Scholar
  49. 15(c).
    Si Y, Liang L, Tang S, Qi Y, Huang Y, Liu L. Sci China Chem, 2018, 61: 412–417CrossRefGoogle Scholar
  50. 15(d).
    Zheng JS, Yu M, Qi YK, Tang S, Shen F, Wang ZP, Xiao L, Zhang L, Tian CL, Liu L. J Am Chem Soc, 2014, 136: 3695–3704CrossRefGoogle Scholar
  51. 16.
    Morgan WT, Vallota EH, Müller-Eberhard HJ. Biochem BioPhys Res Commun, 1974, 57: 572–577CrossRefGoogle Scholar
  52. 17(a).
    Klco JM, Wiegand CB, Narzinski K, Baranski TJ. Nat Struct Mol Biol, 2005, 12: 320–326CrossRefGoogle Scholar
  53. 17(b).
    Reis ES, Chen H, Sfyroera G, Monk PN, Köhl J, Ricklin D, Lambris JD. J Immunol, 2012, 189: 4797–4805CrossRefGoogle Scholar
  54. 18.
    Schatz-Jakobsen JA, Yatime L, Larsen C, Petersen SV, Klos A, Andersen GR. Acta Crystlogr D Biol Crystlogr, 2014, 70: 1704–1717CrossRefGoogle Scholar
  55. 19(a).
    Zuiderweg ERP, Nettesheim DG, Mollison KW, Carter GW. Biochemistry, 1989, 28: 172–185CrossRefGoogle Scholar
  56. 19(b).
    Zuiderweg ERP, Fesik SW. Biochemistry, 1989, 28: 2387–2391CrossRefGoogle Scholar
  57. 19(c).
    Zhang X, Boyar W, Toth MJ, Wennogle L, Gonnella NC. Proteins, 1997, 28: 261–267CrossRefGoogle Scholar
  58. 19(d).
    Zhang X, Boyar W, Galakatos N, Gonnella NC. Protein Sci, 1997, 6: 65–72CrossRefGoogle Scholar
  59. 20(a).
    Mackay AL. Nature, 1989, 342: 133–133CrossRefGoogle Scholar
  60. 20(b).
    Mandal K, Uppalapati M, Ault-Riché D, Kenney J, Lowitz J, Sidhu SS, Kent SBH. Proc Natl Acad Sci USA, 2012, 109: 14779–14784CrossRefGoogle Scholar
  61. 20(c).
    Avital-Shmilovici M, Mandal K, Gates ZP, Phillips NB, Weiss MA, Kent SBH. J Am Chem Soc, 2013, 135: 3173–3185CrossRefGoogle Scholar
  62. 20(d).
    Bunker RD, Mandal K, Bashiri G, Chaston JJ, Pentelute BL, Lott JS, Kent SBH, Baker EN. Proc Natl Acad Sci USA, 2015, 112: 4310–4315CrossRefGoogle Scholar
  63. 20(e).
    Yeung H, Squire CJ, Yosaatmadja Y, Panjikar S, López G, Molina A, Baker EN, Harris PWR, Brimble MA. Angew Chem Int Ed, 2016, 55: 7930–7933CrossRefGoogle Scholar
  64. 21.
    McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ. J Appl Crystallogr, 1993, 26: 658–674Google Scholar
  65. 22.
    Liu H, Kim HR, Deepak RNVK, Wang L, Chung KY, Fan H, Wei Z, Zhang C. Nat Struct Mol Biol, 2018, 25: 472–481CrossRefGoogle Scholar
  66. 23.
    Stewart E, Federico SM, Chen X, Shelat AA, Bradley C, Gordon B, Karlstrom A, Twarog NR, Clay MR, Bahrami A, Freeman BB, Xu B, Zhou X, Wu J, Honnell V, Ocarz M, Blankenship K, Dapper J, Mardis ER, Wilson RK, Downing J, Zhang J, Easton J, Pappo A, Dyer MA. Nature, 2017, 549: 96–100CrossRefGoogle Scholar
  67. 24.
    Konteatis ZD, Siciliano SJ, Riper GV, Molineaux CJ, Pandya S, Fischer P, Rosen H, Mumford RA, Springer MS. J Immunol, 1994, 153: 4200–4205Google Scholar
  68. 25(a).
    Pentelute BL, Gates ZP, Tereshko V, Dashnau JL, Vanderkooi JM, Kossiakoff AA, Kent SBH. J Am Chem Soc, 2008, 130: 9695–9701CrossRefGoogle Scholar
  69. 25(b).
    Hayouka Z, Thomas NC, Mortenson DE, Satyshur KA, Weisblum B, Forest KT, Gellman SH. J Am Chem Soc, 2015, 137: 11884–11887CrossRefGoogle Scholar
  70. 25(c).
    Kreitler DF, Mortenson DE, Forest KT, Gellman SH. J Am Chem Soc, 2016, 138: 6498–6505CrossRefGoogle Scholar
  71. 25(d).
    Pan M, Gao S, Zheng Y, Tan X, Lan H, Tan X, Sun D, Lu L, Wang T, Zheng Q, Huang Y, Wang J, Liu L. J Am Chem Soc, 2016, 138: 7429–7435CrossRefGoogle Scholar
  72. 25(e).
    Gao S, Pan M, Zheng Y, Huang Y, Zheng Q, Sun D, Lu L, Tan X, Tan X, Lan H, Wang J, Wang T, Wang J, Liu L. J Am Chem Soc, 2016, 138: 14497–14502CrossRefGoogle Scholar
  73. 25(f).
    Chen CC, Gao S, Ai HS, Qu Q, Tian CL, Li YM. Sci China Chem, 2018, 61: 702–707CrossRefGoogle Scholar
  74. 25(g).
    Li Z, Zhang B, Zuo C, Liu L. Chin J Org Chem, 2018, 38: 2412–2419CrossRefGoogle Scholar
  75. 25(h).
    Chen C, Gao S, Qu Q, Mi P, Tao A, Li M. Chin Chem Lett, 2018, 29: 1135–1138CrossRefGoogle Scholar
  76. 26.
    Zhang L, Mallik B, Morikis D. Biopolymers, 2008, 90: 803–815CrossRefGoogle Scholar

Copyright information

© Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Chao Zuo
    • 1
  • Wei-Wei Shi
    • 1
  • Xiao-Xu Chen
    • 2
  • Marie Glatz
    • 3
  • Bernd Riedl
    • 3
  • Ingo Flamme
    • 4
  • Elisabeth Pook
    • 5
  • Jiawei Wang
    • 1
  • Ge-Min Fang
    • 2
  • Donald Bierer
    • 3
  • Lei Liu
    • 1
    Email author
  1. 1.Tsinghua-Peking Center for Life Sciences, Ministry of Education Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Center for Synthetic and Systems Biology, Department of ChemistryTsinghua UniversityBeijingChina
  2. 2.School of Life Science, Institutes of Physical Science and Information TechnologyAnhui UniversityHefeiChina
  3. 3.Department of Medicinal ChemistryBayer AGWuppertalGermany
  4. 4.Cardiovascular ResearchBayer AGWuppertalGermany
  5. 5.Lead DiscoveryBayer AGWuppertalGermany

Personalised recommendations