Synthesis, biological evaluation and structural characterization of novel glycopeptide analogues of nociceptin N/OFQ

Abstract

To examine if the biological activity of the N/OFQ peptide, which is the native ligand of the pain-related and viable drug target NOP receptor, could be modulated by glycosylation and if such effects could be conformationally related, we have synthesized three N/OFQ glycopeptide analogues, namely: [Thr(5)-O-α-D-GalNAc-N/OFQ] (glycopeptide 1), [Ser(10)-O-α-D-GalNAc]-N/OFQ (glycopeptide 2) and [Ser(10)-O-β-D-GlcNAc]-N/OFQ] (glycopeptide 3). They were tested for biological activity in competition binding assays using the zebrafish animal model in which glycopeptide 2 exhibited a slightly improved binding affinity, whereas glycopeptide 1 showed a remarkably reduced binding affinity compared to the parent compound and glycopeptide 3. The structural analysis of these glycopeptides and the parent N/OFQ peptide by NMR and circular dichroism indicated that their aqueous solutions are mainly populated by random coil conformers. However, in membrane mimic environments a certain proportion of the molecules of all these peptides exist as α-helix structures. Interestingly, under these experimental conditions, glycopeptide 1 (glycosylated at Thr-5) exhibited a population of folded hairpin-like geometries. From these facts it is tempting to speculate that nociceptin analogues showing linear helical structures are more complementary and thus interact more efficiently with the native NOP receptor than folded structures, since glycopeptide 1 showed a significantly reduced binding affinity for the NOP receptor.

Structure- and conformation-activity studies of nociceptin/orphanin FQ receptor dimeric ligands

“…5) in accordance with previous conformational studies on the full length N/OFQ5657. Consistent violation of a few NOE-derived distances and the comparison of the Hα chemical shift deviation of peptide 11 and N/OFQ (Figure S1) indicate that the helix is not completely stable.…”

“…The shielding effect of Phe4 on Phe1 is particularly evident considering the chemical shift of the aromatic protons of the last (especially H ζ , with Δδ ~ −0.40 ppm compared to the corresponding proton in an unshielded phenylalanine)59. Interestingly, similar shielding is observed for the NMR signals of N/OFQ (H ζ of Phe1: Δδ ~ −0.60 ppm)57, nonetheless, this interaction that regards key pharmacophore residues has never been described.…”

“…The increase of the local concentration and the reduction of the rotational and translational freedom of the neuropeptide are membrane-mediated events acting as determinant steps for the conformational transition of the peptide. Hence micelle solutions have been extensively used for peptide hormones conformational studies including opioids5455, and N/OFQ5657.…”

Modulation of the Interaction between a Peptide Ligand and a G Protein-Coupled Receptor by Halogen Atoms

“…28 Indeed, in their free state in water solution, and independently of the substitution, no medium-range or long-range NOEs were detected. These data indicate that these molecules behave similarly to other endogenous enkephalin peptides and analogues, with a remarkable flexibility and major extended conformations.…”

Potential of Nociceptin/Orphanin FQ Peptide Analogs for Drug Development

“…Attachment of sugar moieties to various peptides, including opioids can cause important changes in their native conformation, stability and activity [66] . To examine if the biological activity of the N/OFQ could be modulated by glycosylation and if such effect could be conformationally related, three N/OFQ glycopeptide analogs ( Figure 1) were synthesized [67] . Two obvious O‐glycosylation sites in the N/OFQ sequence are hydroxyl groups of Thr 5 and Ser 10 .…”