Pentapeptides displaying mu opioid receptor agonist and delta opioid receptor partial agonist/antagonist properties

Abstract

Chronic use of mu-opioid agonists has been shown to cause neurochemical adaptations resulting in tolerance and dependence. While the analgesic effects of these drugs are mediated by mu-opioid receptors (MOR), several studies have shown that antagonism or knockdown of delta-opioid receptors (DOR) can lessen or prevent development of tolerance and dependence. On the basis of computational modeling of putative active and inactive conformations of MOR and DOR, we have synthesized a series of pentapeptides with the goal of developing a MOR agonist/DOR antagonist peptide with similar affinity at both receptors as a tool to probe functional opioid receptor interaction(s). The eight resulting naphthylalanine-substituted cyclic pentapeptides displayed variable mixed-efficacy profiles. The most promising peptide (9; Tyr-c(S-CH(2)-S)[D-Cys-Phe-2-Nal-Cys]NH(2)) displayed a MOR agonist and DOR partial agonist/antagonist profile and bound with equipotent affinity (K(i) approximately 0.5 nM) to both receptors, but also showed kappa opioid receptor (KOR) agonist activity.

Figure 1

Modeling of peptide 1 in the binding pocket of putative active and inactive conformations of mouse MOR and human DOR. Peptide 1 docked in the putative active (A) and inactive (B) conformation of MOR shows no noticeable unfavorable interactions between ligand side chains and residues from the receptor binding pocket. Peptide 1 docked in the active conformation of DOR (C) shows a steric overlap of the peptide Phe⁴ side chain with the side chain of receptor Trp²⁸⁴ from TM6 (arrow), while peptide 1 in the inactive conformation of DOR (D) does not show a similar steric hindrance.

Figure 2

Naphthylalanine-containing analogs of peptide 1.

Figure 3

Pentapeptide 9 docked in the binding pocket of putative active and inactive conformations of mouse MOR and human DOR. The 2-naphthylalanine⁴ side chain of peptide 9 shows minimal hindrance with receptor residue Lys³⁰³ in the MOR active conformation (A), but an increased steric overlap with Trp²⁸⁴ side chain (arrow) in the DOR active conformation (C). These hindrances are absent in the inactive conformations of both MOR (B) and DOR (D).

Figure 4

Pharmacological analysis of peptide 9. (A) Activity of peptide 9 in the [³⁵S]GTPγS binding assay at MOR, DOR and KOR. Results are plotted as percent stimulation compared to a 10 µM concentration of standard compounds (MOR standard agonist DAMGO, DOR standard agonist SNC80, and KOR standard agonist U69,593). Peptide 9 has 10-fold higher potency at MOR (EC₅₀: 1.2 ± 0.05 nM) than KOR (EC₅₀: 12 ± 0.1 nM) and 10 µM 9 produces only 6.9 ± 2.3% of SNC80-induced stimulation at DOR. (B) DOR antagonism of peptide 9 in the [³⁵S]GTPγS binding assay. Peptide 9 (100 nM) produces a 3.1-fold rightward shift in the SNC80 dose-response curve, indicating DOR antagonism. Calculated Ke = 48 ± 9.5 nM. Results are plotted as percentage of the maximum level of SNC80-stimulated [³⁵S]GTPγS binding. (C) Partial agonism of peptide 9 at DOR as illustrated by inhibition of adenylyl cyclase. Results are shown as percent inhibition of 5 µM forskolin-stimulated adenylyl cyclase production in C₆-DOR cells. Standard DOR agonist SNC80 (1 µM) gave 67 ± 4.4% inhibition of adenylyl cyclase production with a calculated maximal effect of 76 ± 8.8% while 1 µM peptide 9 produced 37 ± 4.2% inhibition with a calculated maximum of 43 ± 9.0% inhibition. Peptide 9 is more potent (EC₅₀ = 36 ± 4.8 nM) than SNC80 (EC₅₀ = 166 ± 43 nM) in this assay. The DOR antagonist naltrindole (NTI) did not inhibit cAMP accumulation.