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 (1) and urotensin II-related
peptide (2) could stabilize specific urotensin II receptor
(UTR) conformations, thereby activating different signaling 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, 18 N-methylated UTR ligands were synthesized and their biological profiles
evaluated using 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 the 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.