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

Chan, Linda J., Smith, Craig M., Chua, Berenice E., Lin, Feng, Bathgate, Ross A.D., Separovic, Frances, Gundlach, Andrew L., Hossain, Mohammed Akhter and Wade, John D. 2013, Synthesis of fluorescent analogs of relaxin family peptides and their preliminary in vitro and in vivo characterization, Frontiers in chemistry, vol. 1, pp. 1-9, doi: 10.3389/fchem.2013.00030.

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Title Synthesis of fluorescent analogs of relaxin family peptides and their preliminary in vitro and in vivo characterization
Formatted title Synthesis of fluorescent analogs of relaxin family peptides and their preliminary in vitro and in vivo characterization
Author(s) Chan, Linda J.
Smith, Craig M.
Chua, Berenice E.
Lin, Feng
Bathgate, Ross A.D.
Separovic, Frances
Gundlach, Andrew L.
Hossain, Mohammed Akhter
Wade, John D.
Journal name Frontiers in chemistry
Volume number 1
Article ID 30
Start page 1
End page 9
Total pages 9
Publisher Frontiers Research Foundation
Place of publication Lausanne, Switzerland
Publication date 2013-12
ISSN 2296-2646
Keyword(s) Cy5.5 fluorophore
RXFP1
RXFP2
brain
click chemistry
relaxin
Science & Technology
Physical Sciences
Chemistry, Multidisciplinary
Chemistry
Summary 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.
Language eng
DOI 10.3389/fchem.2013.00030
Field of Research 030199 Analytical Chemistry not elsewhere classified
Socio Economic Objective 0 Not Applicable
HERDC Research category C1.1 Refereed article in a scholarly journal
ERA Research output type C Journal article
Copyright notice ©2013, The Authors
Free to Read? Yes
Use Rights Creative Commons Attribution licence
Persistent URL http://hdl.handle.net/10536/DRO/DU:30093785

Document type: Journal Article
Collections: School of Medicine
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Every reasonable effort has been made to ensure that permission has been obtained for items included in DRO. If you believe that your rights have been infringed by this repository, please contact drosupport@deakin.edu.au.