Urolinin: The First Linear Peptidic Urotensin-II Receptor Agonist

Abstract

This study investigated the role of individual U-II amino acid positions and side chain characteristics important for U-IIR activation. A complete permutation library of 209 U-II variants was studied in an activity screen that contained single substitution variants of each position with one of the other 19 proteinogenic amino acids. Receptor activation was measured using a cell-based high-throughput fluorescence calcium mobilization assay. We generated the first complete U-II substitution map for U-II receptor activation, resulting in a detailed view into the structural features required for receptor activation, accompanied by complementary information from receptor modeling and ligand docking studies. On the basis of the systematic SAR study of U-II, we created 33 further short and linear U-II variants from eight to three amino acids in length, including d- and other non-natural amino acids. We identified the first high-potency linear U-II analogues. Urolinin, a linear U-II agonist (nWWK-Tyr(3-NO)-Abu), shows low nanomolar potency as well as improved metabolic stability.

Octreotide Does Not Inhibit Proliferation in Five Neuroendocrine Tumor Cell Lines

“…Peptide iodination and radioligand-binding assays were performed as previously described ( 21 ). Briefly, 10 nmol Tyr 11 -somatostatin-14 (Tyr 11 –SST14, Bachem, Bubendorf, Switzerland) were iodinated by the chloramin T method ( 22 ) with 1 mCi carrier-free Na 125 I (NEZ033L010MC, Perkin Elmer, Waltham, MA, USA) and purified by HPLC (Analytic HPLC 1200 Series, Agilent, Santa Clara, CA, USA) (see Figure S1 in Supplementary Material).…”

AGTR1 is overexpressed in neuroendocrine neoplasms, regulates secretion and may serve as a target for molecular imaging and therapy

“…Peptide iodination was performed as previously described (18). Briefly, 10 nmol angiotensin II (Sigma, Deisenhofen, Germany) were iodinated with 1 mCi carrier-free Na 125 I (NEZ033L010MC, Perkin Elmer, Waltham, MA, USA) and purified by HPLC (Analytic HPLC 1200 Series, Agilent, Santa Clara, CA, USA; see Figure S1 in supplementary material).…”

“…Experiments in 96-well format were performed as previously described (18). For 384-well experiments, 15,000 to 20,000 cells per well were seeded after plate coating and for any incubation and washing steps 20 µl total volume were used.…”

New directions for urotensin II receptor ligands

“…Those two studies underline the crucial role played by the disulfide bridge in the binding and activation processes and suggest that bridge replacement might open up a new avenue for the conception of modulators specifically targeting URP‐associated functions. Interestingly, a linear peptidic UT analog was recently discovered through permutation library . This derivative, termed Urolinin (nWWK‐Tyr(3‐NO 2 )‐Abu‐NH 2 , Figure ), displays a very low binding affinity on competition assay, is unable to trigger aortic contraction but can induce calcium mobilization with a potency similar to what is observed for UII.…”

“…This derivative, termed Urolinin (nWWK‐Tyr(3‐NO 2 )‐Abu‐NH 2 , Figure ), displays a very low binding affinity on competition assay, is unable to trigger aortic contraction but can induce calcium mobilization with a potency similar to what is observed for UII. The absence of contraction despite a potent calcium mobilization has arisen the possibility that such ligands could indeed function as biased UT agonist . Hence, linear peptides, derived from urolinin, could represent new molecular template for the generation of UT‐biased ligands.…”