Functional and Binding Characterizations of Urotensin II-Related Peptides in Human and Rat Urotensin II-Receptor Assay

摘要

Urotensin II (U-II; cyclo~5-10[H-Glu-Thr-Pro-Asp-Cys-Phe-Trp-Lys-Tyt-Cys-Val-OH]) is a potent vasoconstrictor in mammals, and it is postulated that it plays a central role in cardiovascular homeostasis. Thus,we initiated a structure-to-function analysis of this peptide characterized by a N-terminal tail and a cyclic core formed through a disulfide briding. A total of 41 analogs focusing on these characteristics were developed and evaluated using a binding assay on membranes from a stable HEK-293 cell line containing the human or rat U-II receptor, a functional assay for Ca~2+ mobilization on transiently transfected CHO-K1 cells with the human or rat U-II receptor, and a rat thoracic aorta bioassay. At first, the focus was applied on peptide compounds containing exocyclic modification. From this series, it appeared that only valine-11 played a significant role although it is not an essential amino acid. Similarly, endocyclic and ring transformations of Hu-II were also studied. In most cases, a detrimental effect on affinity and biological activity was observed. However, two compounds, [Tyr~6]Hu-II and [Phe~9]hU-II, retained affinity and activity. So far, our binding, functional, and pharmacological data clearly demonstrated the minor contribution of the N-terminal segment and the essential role of the cyclic structure. More particularly, three residues within the loop, i.e., Trp-7, Lys-8, and Tyr-9, are required for receptor recognition and activation. This three-pole feature, kept by the disulfide bond in a correct spatial arrangement, appears as the key pharmacophore for the U-II receptor.