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

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

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.

Highlights

Relaxin is a peptide hormone that has long been recognized for its pleiotropic roles in peripheral tissues, especially during pregnancy (Hisaw, 1926; Bathgate et al, 2013)
In experimental systems H2 relaxin interacts with RXFP2, which is structurally similar to RXFP1 (Hsu et al, 2002), and is the native receptor for the related peptide, insulin-like peptide 3 (INSL3) (Kumagai et al, 2002) there is no strong evidence that this interaction is physiologically significant (Bogatcheva et al, 2003; Kamat et al, 2004)
Several investigators have reported that intracerebroventricular infusion of H2 relaxin in rodents induces a drinking response, which is thought to be mediated via activation of RXFP1 expressed by neurons in the circumventricular organs, such as the subfornical organ

Summary

Introduction

Relaxin is a peptide hormone that has long been recognized for its pleiotropic roles in peripheral tissues, especially during pregnancy (Hisaw, 1926; Bathgate et al, 2013). Relaxin is mainly produced in the corpus luteum during pregnancy, but is present in the placenta and prostate gland, in addition to a host of non-reproductive tissues including the brain (Shabanpoor et al, 2009; Bathgate et al, 2013). It interacts with relaxin family peptide receptor 1 (RXFP1), a G-protein-coupled receptor, to exert its biological effects. Several investigators have reported that intracerebroventricular (icv) infusion of H2 relaxin in rodents induces a drinking (dipsogenic) response, which is thought to be mediated via activation of RXFP1 expressed by neurons in the circumventricular organs, such as the subfornical organ

Methods

Results

Conclusion