Functional Selectivity Revealed by N-Methylation Scanning of Human Urotensin II and Related Peptides

In accordance with their common but also divergent physiological actions, human urotensin II ( hU-II, 1) and urotensin II-related peptide (URP, 2) could stabilize specific urotensin II receptor (UTR) conformations, thereby activating different signalling pathways, a feature referred to as biased agonism or functional selectivity. Sequential N-methylation of the amides in the conserved core sequence of 1, 2 and fragment U-II4-11 (3) shed light on structural requirements involved in their functional selectivity. Thus, eighteen N-methylated UTR ligands were synthesized and their biological profiles evaluated using both in vitro competition binding assays, ex vivo rat aortic ring bioassays and BRET-based biosensor experiments. Biological activity diverged from that of the parent structures contingent on the location of amide methylation indicating relevant hydrogen-bond interactions for function of the endogenous peptides. Conformational analysis of selected N-methyl analogs indicated the importance of specific amide residues of 2 for the distinct pharmacology relative to 1 and 3.