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

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.

Synthetic Covalently Linked Dimeric Form of H2 Relaxin Retains Native RXFP1 Activity and Has ImprovedIn VitroSerum Stability

“…This linker was chosen because it is sufficiently longer to space the two peptide units apart and also because it is monodisperse thus simplifying characterization. Our previous studies have shown that the N-terminus of the B-chain can be truncated by six residues [ 34 ] or modified to accommodate a functional moiety such as a biotin [ 35 ] or large fluorophore [ 18 ] without significant loss of activity. This is because this site is far from its primary active site which consists of the B-chain C-terminal α -helical region [ 2 ].…”

“…Following simultaneous cleavage and side chain deprotection and purification of the crude A- and B-chains, stepwise formation of the three disulfide bonds was carried out via successive oxidation, thiolysis, and iodolysis as previously described [ 18 ].…”

Novel Methods for the Chemical Synthesis of Insulin Superfamily Peptides and of Analogues Containing Disulfide Isosteres

“…We subsequently found that this strategy worked well for the first acquisition of human relaxin-3 (H3 relaxin), which was previously shown to be refractive to the random chain refolding approach, and enabled its tertiary structure determination by NMR spectroscopy . The synthetic strategy was used to prepare other members of the relaxin subfamily of peptides including INSL3, INSL5, and R3/I5 and to determine their tertiary structures, ,− as well as relaxin-2 from other species including horse and mouse. − Numerous analogues of relaxin subfamily peptides, including versions that are size-reduced and site-specifically labeled with europium or fluorescent tags for receptor interaction studies have also been obtained. − A successful assembly of Drosophila insulin-like peptide 2 was also achieved . A modified strategy was also developed in which the C-terminus of the A-chain of insulin glargine was assembled on the solid support containing penta-Lys attached by a base-labile 4-hydroxymethylbenzoic acid (HMBA) linker .…”

Relaxin-3/RXFP3 networks: an emerging target for the treatment of depression and other neuropsychiatric diseases?

“…Studies which have centrally administered RXFP3 agonists have mainly employed the icv route, and although it is often assumed that peptides are able to access receptors throughout the whole brain ( Bittencourt and Sawchenko, 2000 ), recent studies in our laboratory using fluorophore-conjugated relaxin family peptides suggest that periventricular regions such as the PVN may be exposed to higher concentrations of peptide ( Chan et al, 2013 ). Although RXFP3 agonists or antagonists have been locally infused into the bed nucleus of the stria terminalis ( Ryan et al, 2013b ), central amygdala ( Ma et al, 2010 ), medial septum ( Ma et al, 2009a ), and hypothalamic nuclei ( McGowan et al, 2007 ) of rodents, in connection with actions on reward, fear, spatial memory, and feeding, respectively; many other RXFP3-rich brain regions including those distal to the ventricular system remain to be targeted, including the median raphé, superior and inferior colliculus, intergeniculate leaflet, IPN, supramammillary nucleus, diagonal band of Broca, fields within the dorsal and ventral hippocampus, and the retrosplenial and cingulate cortices (see Figure 1 ).…”