The Florey Institute of Neuroscience and Mental Health, The University of Melbourne VIC, Australia ; School of Chemistry, The University of Melbourne VIC, Australia.
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.
Front Chem. 2013 Dec 6;1:30. doi: 10.3389/fchem.2013.00030. eCollection 2013.
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.
松弛素是一种二聚体多肽激素，是人类和啮齿动物胶原代谢和多种血管控制途径的关键调节剂。其作用是通过其同源 G 蛋白偶联受体 RXFP1 介导的，尽管它也“药理学上”激活了 RXFP2，即相关胰岛素样肽 3 (INSL3) 的受体，后者对生殖和骨代谢有特定作用。因此，需要实验工具来促进对松弛素和 INSL3 的不同生物学作用的深入了解，特别是对于含有 RXFP1 和 RXFP2 的组织的研究。在这里，我们对人（H2）松弛素、RXFP1 选择性松弛素类似物 H2:A(4-24)(F23A) 和 INSL3 进行了化学功能化，以适应荧光团的存在，而不会明显降低结合或激活倾向。每个肽的两条链的化学合成后，通过顺序区域选择性形成它们的三个二硫键。B 链 N 端的 Cy5.5 通过点击化学偶联，同时保持二硫键的完整性，得到的类似物显示出适当的选择性结合亲和力和体外激活 RXFP1 和/或 RXFP2 的能力。Cy5.5-H2 松弛素和 Cy5.5-H2:A(4-24)(F23A) 的体内生物学活性在小鼠中得到了证实，因为这些肽（但不是 Cy5.5-INSL3）的急性脑室内（icv）输注刺激了饮水，这是中央 RXFP1 激活引起的一种已建立的行为反应。在输注后 30 分钟杀死的小鼠中检查了 Cy5.5 缀合肽的中枢分布，发现与大脑深部区域相比，靠近脑室壁的脑组织中荧光强度更高。荧光团缀合的松弛素家族肽的生产将促进未来的药理学研究，以在体内探测 H2 松弛素/RXFP1 和 INSL3/RXFP2 信号的功能，同时跟踪它们在中枢或外周给药后的分布。
