Improving metabolic stability by glycosylation: bifunctional peptide derivatives that are opioid receptor agonists and neurokinin 1 receptor antagonists

Abstract

In order to obtain a metabolically more stable analgesic peptide derivative, O-beta-glycosylated serine (Ser(Glc)) was introduced into TY027 (Tyr-d-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-3',5'-Bzl(CF(3))(2)) which was a previously reported bifunctional compound with delta/micro opioid agonist and neurokinin-1 receptor antagonist activities and with a half-life of 4.8 h in rat plasma. Incorporation of Ser(Glc) into various positions of TY027 gave analogues with variable bioactivities. Analogue 6 (Tyr-d-Ala-Gly-Phe-Nle-Pro-Leu-Ser(Glc)-Trp-NH-3',5'-Bzl(CF(3))(2)) was found to have effective bifunctional activities with a well-defined conformation with two beta-turns based on the NMR conformational analysis in the presence of DPC micelles. In addition, 6 showed significant improvement in its metabolic stability (70 + or - 9% of 6 was intact after 24 h incubation in rat plasma). This improved metabolic stability, along with its effective and delta selective bifunctional activities, suggests that 6 could be an interesting research tool and possibly a promising candidate as a novel analgesic drug.

Figure 1

Sequences of opioid and NK1 receptor peptides.

Figure 2

Comparison of the in vitro metabolic stability for 1 (open circle), 2 (open triangle), 5 (crossing) and 6 (filled circle) incubated in rat plasma at 37°C. Calculated half lives of peptide derivatives (T_1/2) were 4.8 h for 1 and > 6 h for 2, 5 and 6. 70 ± 9 % of 6 was found intact after 24 h incubation. The samples were tested in three independent experiments (n = 3) and the mean values were used for the analysis with the SD. Statistical significance was determined by Kruskal-Wallis test followed by Tukey’s test. Asterisks denote significant differences (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 3

Diagram of H^N-H^α coupling constants, NOE connectivities, and H^α chemical shift index (CSI) for the (A) 3, (B) 4 and (C) 5 and (D) 6. The H^α CSI was calculated using the random-coil values reported by Andersen et al.^,

Figure 4

Ensembles of the best 20 calculated structures in 40-fold DPC micelle / pH 4.5 buffer for 1, 3, 4, 5 and 6, respectively, with the lowest restraint energy, (a) aligned on backbone atoms of residues 5-8. The aligned structures are illustrated with the C-terminal benzyl moiety (purple) and glucose (orange). (b) The most stable conformers are shown with all heavy atoms (C, N and O).

Figure 5

The Glycosylated Ser (crosses) and Gly (open circle) were indicated in the Ramachandran φ,ψ plots for (A) 3, (B) 4 and (C) 5 for residues 2-7 and (D) 6 for residues 2-8 of 20 final structures. Ser(OGlc) (crossing) and Gly³ (open diamond) were specified. The Nle⁵ with negative φ angle in 4 (circled) were illustrated in (B). Angular order parameters for φ (E) and ψ (F) angles calculated from the 20 final structures for 1 (open circle), 3 (filled triangle), 4 (open diamond), 5 (crossing) and 6 (filled circle), respectively. For calculating the ψ angles of Trp⁸, the nitrogen atoms of C-terminal benzyl amide were used instead of N (i + 3), respectively.

Figure 6

The paramagnetic effects on TOCSY Spectra of 6. 6 with DPC micelles (top row) and with 200 μM Mn²⁺ (bottom). Preserved resonances (labeled) are in a phase not missed by the phase-specific radical probe (Mn²⁺). Spectra were compared from the same noise level. X10 represents the cross-peaks derived from the corresponding aromatic protons of benzyl moiety.