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Heptapeptide SFLLRNP is a receptor−tethered ligand of protease-activated receptor 1 (PAR-1), and its Phe at position 2 is essential for the aggregation of human platelets. To validate the structural elements of the Phe-phenyl group in receptor activation, we have synthesized a complete set of S/Phe/LLRNP peptides comprising different series of fluorophenylalanine isomers (Fn)Phe, where n = 1, 2, 3, and 5. Phe-2-phenyl was strongly suggested to be involved in the edge-to-face CH/π interaction with the receptor aromatic group. In the present study, to prove this receptor interaction definitively, we synthesized another series of peptide analogs containing (F4)Phe-isomers, with the phenyl group of each isomer possessing only one hydrogen atom at the ortho, meta, or para position. When the peptides were assayed for their platelet aggregation activity, S/(2,3,4,6-F4)Phe/LLRNP and S/(2,3,4,5-F4)Phe/LLRNP exhibited noticeable activity (34% and 6% intensities of the native peptide, respectively), whereas S/(2,3,5,6-F4)Phe/LLRNP was completely inactive. The results indicated that, at the ortho and meta positions but not at the para position, benzene-hydrogen atoms are required for the CH/π interaction to activate the receptor. The results provided a decisive evidence of the molecular recognition property of Phe, the phenyl benzene-hydrogen atom of which participates directly in the interaction with the receptor aromatic π plane.

IsoVa-RYYRIK-NH2 is a highly specific antagonist ligand of the opioid receptor-like 1 (ORL1) receptor, an endogenous ligand of which is 17-mer peptide nociceptin. ORL1 antagonists have potential for clinical use as analgesic and antineuropathic drugs, and thus information on the receptor-binding characteristics of antagonists is very important for rational drug design. In the present study, we prepared tritium-labelled isova-RYYRIK-NH2 from its precursor with the 3-methylcrotonyl (CH3)2C CHCO group by a catalytic reduction using tritium gas. The resulting [3H]isoVa-RYYRIK-NH2 was evaluated in a saturation binding assay using the COS-7 cell membrane preparations of transiently expressed ORL1. It exhibited more than 90% specific binding with a dissociation constant of 1.21 ± 0.03 nM. From the mutual heterologous binding assays using [3H]isoVa-RYYRIK-NH2 and [3H]nociceptin, isoVa-RYYRIK-NH2 and nociceptin were found to share the receptor-binding site, but each also had a separate specific binding site of its own. They differentiated the two different binding states or conformations of ORL1, which might represent the agonist-active and antagonist-inactive conformations of ORL1. [3H]isoVa-RYYRIK-NH2 is thus a key tracer to uncover the amino acid residues important for receptor inactivation.