Display options
Cite
Chan LJ, Smith CM, Chua BE, et al. Synthesis of fluorescent analogs of relaxin family peptides and their preliminary in vitro and in vivo characterization. Front Chem. 2013;1:30doi: 10.3389/fchem.2013.00030.
Chan, L. J., Smith, C. M., Chua, B. E., Lin, F., Bathgate, R. A., Separovic, F., Gundlach, A. L., Hossain, M. A., & Wade, J. D. (2013). Synthesis of fluorescent analogs of relaxin family peptides and their preliminary in vitro and in vivo characterization. Frontiers in chemistry, 130. https://doi.org/10.3389/fchem.2013.00030
Chan, Linda J, et al. "Synthesis of fluorescent analogs of relaxin family peptides and their preliminary in vitro and in vivo characterization." Frontiers in chemistry vol. 1 (2013): 30. doi: https://doi.org/10.3389/fchem.2013.00030
Chan LJ, Smith CM, Chua BE, Lin F, Bathgate RA, Separovic F, Gundlach AL, Hossain MA, Wade JD. Synthesis of fluorescent analogs of relaxin family peptides and their preliminary in vitro and in vivo characterization. Front Chem. 2013 Dec 06;1:30. doi: 10.3389/fchem.2013.00030. eCollection 2013. PMID: 24790958; PMCID: PMC3982560.
Copy Download .nbib Format: NLM
Share it on

Front Chem. 2013 Dec 06;1:30. doi: 10.3389/fchem.2013.00030. eCollection 2013.

Synthesis of fluorescent analogs of relaxin family peptides and their preliminary in vitro and in vivo characterization.

Frontiers in chemistry

Linda J Chan, Craig M Smith, Berenice E Chua, Feng Lin, Ross A D Bathgate, Frances Separovic, Andrew L Gundlach, Mohammed Akhter Hossain, John D Wade

Affiliations

  1. The Florey Institute of Neuroscience and Mental Health, The University of Melbourne VIC, Australia ; School of Chemistry, The University of Melbourne VIC, Australia.
  2. The Florey Institute of Neuroscience and Mental Health, The University of Melbourne VIC, Australia ; Florey Department of Neuroscience and Mental Health, The University of Melbourne VIC, Australia.
  3. The Florey Institute of Neuroscience and Mental Health, The University of Melbourne VIC, Australia.
  4. The Florey Institute of Neuroscience and Mental Health, The University of Melbourne VIC, Australia ; Florey Department of Neuroscience and Mental Health, The University of Melbourne VIC, Australia ; Department of Biochemistry and Molecular Biology, The University of Melbourne VIC, Australia.
  5. School of Chemistry, The University of Melbourne VIC, Australia.
  6. The Florey Institute of Neuroscience and Mental Health, The University of Melbourne VIC, Australia ; School of Chemistry, The University of Melbourne VIC, Australia ; Florey Department of Neuroscience and Mental Health, The University of Melbourne VIC, Australia.

PMID: 24790958 PMCID: PMC3982560 DOI: 10.3389/fchem.2013.00030

Abstract

Relaxin, a heterodimeric polypeptide hormone, is a key regulator of collagen metabolism and multiple vascular control pathways in humans and rodents. Its actions are mediated via its cognate G-protein-coupled receptor, RXFP1 although it also "pharmacologically" activates RXFP2, the receptor for the related, insulin-like peptide 3 (INSL3), which has specific actions on reproduction and bone metabolism. Therefore, experimental tools to facilitate insights into the distinct biological actions of relaxin and INSL3 are required, particularly for studies of tissues containing both RXFP1 and RXFP2. Here, we chemically functionalized human (H2) relaxin, the RXFP1-selective relaxin analog H2:A(4-24)(F23A), and INSL3 to accommodate a fluorophore without marked reduction in binding or activation propensity. Chemical synthesis of the two chains for each peptide was followed by sequential regioselective formation of their three disulfide bonds. Click chemistry conjugation of Cy5.5 at the B-chain N-terminus, with conservation of the disulfide bonds, yielded analogs displaying appropriate selective binding affinity and ability to activate RXFP1 and/or RXFP2 in vitro. The in vivo biological activity of Cy5.5-H2 relaxin and Cy5.5-H2:A(4-24)(F23A) was confirmed in mice, as acute intracerebroventricular (icv) infusion of these peptides (but not Cy5.5-INSL3) stimulated water drinking, an established behavioral response elicited by central RXFP1 activation. The central distribution of Cy5.5-conjugated peptides was examined in mice killed 30 min after infusion, revealing higher fluorescence within brain tissue near-adjacent to the cerebral ventricle walls relative to deeper brain areas. Production of fluorophore-conjugated relaxin family peptides will facilitate future pharmacological studies to probe the function of H2 relaxin/RXFP1 and INSL3/RXFP2 signaling in vivo while tracking their distribution following central or peripheral administration.

Keywords: Cy5.5 fluorophore; RXFP1; RXFP2; brain; click chemistry; relaxin

References

  1. Behav Brain Res. 2013 May 1;244:142-51 - PubMed
  2. Am J Physiol Cell Physiol. 2009 May;296(5):C1058-66 - PubMed
  3. Biochemistry. 2006 Jan 24;45(3):1043-53 - PubMed
  4. Endocrinology. 2004 Oct;145(10):4712-20 - PubMed
  5. J Biol Chem. 2006 Nov 17;281(46):34942-54 - PubMed
  6. J Neuroendocrinol. 1997 Jun;9(6):431-7 - PubMed
  7. J Biol Chem. 2008 Jun 20;283(25):17287-97 - PubMed
  8. Bioconjug Chem. 2009 Jul;20(7):1390-6 - PubMed
  9. Neuroscience. 2006 Aug 11;141(1):329-44 - PubMed
  10. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6413-7 - PubMed
  11. Eur J Neurosci. 2005 Oct;22(8):2117-22 - PubMed
  12. Mol Endocrinol. 2003 Dec;17(12):2639-46 - PubMed
  13. Anal Biochem. 1995 Apr 10;226(2):349-54 - PubMed
  14. Diabetes Obes Metab. 2010 Dec;12(12):1090-6 - PubMed
  15. Am J Physiol Endocrinol Metab. 2007 Mar;292(3):E913-9 - PubMed
  16. Bioconjug Chem. 2008 Jul;19(7):1456-63 - PubMed
  17. FEBS J. 2010 Dec;277(24):4990-7 - PubMed
  18. Brain Res. 1988 Jul 5;455(1):58-64 - PubMed
  19. Angew Chem Int Ed Engl. 2001 Jun 1;40(11):2004-2021 - PubMed
  20. Physiol Rev. 2013 Jan;93(1):405-80 - PubMed
  21. Protein Pept Lett. 2011 Mar;18(3):220-9 - PubMed
  22. J Endocrinol Invest. 2006 Jul-Aug;29(7):657-64 - PubMed
  23. Nat Rev Cardiol. 2010 Jan;7(1):48-58 - PubMed
  24. Biochemistry. 2009 Jul 21;48(28):6571-84 - PubMed
  25. Biochem Biophys Res Commun. 2012 Apr 6;420(2):253-6 - PubMed
  26. J Biol Chem. 2002 Aug 30;277(35):31283-6 - PubMed
  27. J Pept Res. 2001 May;57(5):374-82 - PubMed
  28. Neuroscience. 2008 Oct 2;156(2):319-33 - PubMed
  29. Ann N Y Acad Sci. 2009 Apr;1160:226-35 - PubMed
  30. Lancet. 2013 Jan 5;381(9860):29-39 - PubMed
  31. Vitam Horm. 2009;80:1-31 - PubMed
  32. Adv Exp Med Biol. 2007;612:119-37 - PubMed
  33. J Biol Chem. 2012 Nov 30;287(49):41152-64 - PubMed
  34. Proc Natl Acad Sci U S A. 2002 Feb 5;99(3):1701-6 - PubMed
  35. Ann N Y Acad Sci. 2005 May;1041:197-204 - PubMed
  36. Science. 2002 Jan 25;295(5555):671-4 - PubMed
  37. Endocrinology. 1998 May;139(5):2322-8 - PubMed
  38. Learn Mem. 2009 Oct 30;16(11):730-42 - PubMed
  39. Behav Pharmacol. 2012 Sep;23(5-6):516-25 - PubMed
  40. Neuropsychopharmacology. 2012 May;37(6):1323-37 - PubMed
  41. Chembiochem. 2008 Jul 21;9(11):1816-22 - PubMed

Publication Types