Biological and conformational evaluation of bifunctional compounds for opioid receptor agonists and neurokinin 1 receptor antagonists possessing two penicillamines

Abstract

Neuropathic pain states and tolerance to opioids can result from system changes in the CNS, such as up-regulation of the NK1 receptor and substance P, lead to antiopioid effects in ascending or descending pain-signaling pathways. Bifunctional compounds, possessing both the NK1 antagonist pharmacophore and the opioid agonist pharmacophore with delta-selectivity, could counteract these system changes to have significant analgesic efficacy without undesirable side effects. As a result of the introduction of cyclic and topological constraints with penicillamines, 2 (Tyr-cyclo[d-Pen-Gly-Phe-Pen]-Pro-Leu-Trp-NH-[3',5'-(CF(3))(2)-Bzl]) was found as the best bifunctional compound with effective NK1 antagonist and potent opioid agonist activities, and 1400-fold delta-selectivity over the mu-receptor. The NMR structural analysis of 2 revealed that the relative positioning of the two connected pharmacophores as well as its cyclic and topological constraints might be responsible for its excellent bifunctional activities as well as its significant delta-opioid selectivity. Together with the observed high metabolic stability, 2 could be considered as a valuable research tool and possibly a promising candidate for a novel analgesic drug.

Figure 1

Sequences of opioid and NK1 receptor peptides.

Figure 2

Comparison of the in vitro stability of peptide derivatives for 1 and 2 incubated in rat plasma at 37°C (*, p < 0.05).

Figure 3

Ensembles of the best 10 calculated structures of 2 in 40-fold DPC micelle/pH 4.5 buffer with the lowest restraint energy, aligned on backbone atoms of residues 1–8. (A) Only backbone atoms in the aligned structures are illustrated with C-terminal benzyl moiety and disulfide bond. (B) All non-hydrogen atoms were displayed.

Figure 4

(A) Superimposed image of 2 (red) with the X-ray crystal structure of highly selective δ opioid agonist DPDPE (green). Superimposition was performed on all the backbone atoms of residues 1–5 (rmsd = 1.81 Å). Only backbone atoms are illustrated with C-terminal benzyl moiety and disulfide bond (yellow). Superimposed image of obtained NMR structure of 2 with the NMR structure of 1 (blue) at the lowest restraint energies. Superimposition was performed on all the backbone atoms of: (B) residues 1–8, rmsd = 2.34 Å; (C) residues 1–5, rmsd = 1.40 Å; (D) residues 5–8, rmsd = 0.65 Å.

Figure 5

The Ramachandran ϕ, ψ plots for 2 for residues 2–8 of 10 final structures.

Figure 6

Typical effect of Mn²⁺ ions on TOCSY Spectra. 2 with DPC micelles (top row) and with 200 μM Mn²⁺ (bottom), for H^N-H^α region (left column), aliphatic side-chain region (middle) and aromatic region (right). Preserved resonances (labeled) are shown by the addition of Mn²⁺. Spectra were compared from the same noise level.