Synthesis, Biological Activity and Molecular Docking of Chimeric Peptides Targeting Opioid and NOP Receptors

Abstract

Recently, mixed opioid/NOP agonists came to the spotlight for their favorable functional profiles and promising outcomes in clinical trials as novel analgesics. This study reports on two novel chimeric peptides incorporating the fragment Tyr-c[D-Lys-Phe-Phe]Asp-NH₂ (RP-170), a cyclic peptide with high affinity for µ and κ opioid receptors (or MOP and KOP, respectively), conjugated with the peptide Ac-RYYRIK-NH₂, a known ligand of the nociceptin/orphanin FQ receptor (NOP), yielding RP-170-RYYRIK-NH₂ (KW-495) and RP-170-Gly₃-RYYRIK-NH₂ (KW-496). In vitro, the chimeric KW-496 gained affinity for KOP, hence becoming a dual KOP/MOP agonist, while KW-495 behaved as a mixed MOP/NOP agonist with low nM affinity. Hence, KW-495 was selected for further in vivo experiments. Intrathecal administration of this peptide in mice elicited antinociceptive effects in the hot-plate test; this action was sensitive to both the universal opioid receptor antagonist naloxone and the selective NOP antagonist SB-612111. The rotarod test revealed that KW-495 administration did not alter the mice motor coordination performance. Computational studies have been conducted on the two chimeras to investigate the structural determinants at the basis of the experimental activities, including any role of the Gly₃ spacer.

Keywords: antitociceptive test; calcium mobilization assay; chimeric peptides; docking studies; nociceptin receptor; opioid receptors.

Figure 5

Side views of the predicted complexes of KW-495/KOP and KW-496/KOP obtained with the receptor models extracted from PDB ID 6B73. The ligands are rendered in thick lines; C is rendered in gray, N in blue and O in red. Figure obtained with PacDOCK web server [56,57].

Figure 1

Structures of the parent peptides RP-170 and Ac-RYYRIK-NH₂ and of the hybrid derivatives KW-495 and KW-496, and the synthetic strategy.

Figure 2

Effect of different doses of KW-495 chimera and EM-2 in the mouse hot-plate test. Results are expressed as percentage (mean ± SEM) of the maximal possible effect (%MPE) for the inhibition of jumping induced by i.t. injection of KW-495 or EM-2. n = 6–10 mice for each experimental group.

Figure 3

Antagonist effect of naloxone hydrochloride (NAL) or SB-612111 hydrochloride (SB), both at 1 mg/kg i.p., on the inhibition of jumping by administration of KW-495 (6.2 nmol/animal, i.t.) in the mouse hot-plate test. Results are expressed as percentage (mean ± SEM) of the maximal possible effect (%MPE). n = 10–12 mice for each experimental group. Statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test. * is for p < 0.05 and ** is for p < 0.01 as compared with KW-495.

Figure 4

Effects of KW-495 (6.2 nmol/animal, i.t.) on falling latencies of mice in the rotarod test, n = 7–8 mice for each experimental group.

Figure 6

Side views of the predicted complexes of KW-495/NOP and KW-496/NOP obtained with the receptor models extracted from PDB ID 5DHG. The ligands are rendered in thick lines; C is rendered in gray, N in blue and O in red. Figure obtained with PacDOCK web server [56,57].