Identification and Pharmacological Characterization of a Low-Liability Antinociceptive Bifunctional MOR/DOR Cyclic Peptide

Abstract

Peptide-based opioid ligands are important candidates for the development of novel, safer, and more effective analgesics to treat pain. To develop peptide-based safer analgesics, we synthesized a mixture-based cyclic pentapeptide library containing a total of 24,624 pentapeptides and screened the mixture-based library samples using a 55 °C warm water tail-withdrawal assay. Using this phenotypic screening approach, we deconvoluted the mixture-based samples to identify a novel cyclic peptide Tyr-[D-Lys-Dap(Ant)-Thr-Gly] (CycloAnt), which produced dose- and time-dependent antinociception with an ED₅₀ (and 95% confidence interval) of 0.70 (0.52-0.97) mg/kg i.p. mediated by the mu-opioid receptor (MOR). Additionally, higher doses (≥3 mg/kg, i.p.) of CycloAnt antagonized delta-opioid receptors (DOR) for at least 3 h. Pharmacological characterization of CycloAnt showed the cyclic peptide did not reduce breathing rate in mice at doses up to 15 times the analgesic ED₅₀ value, and produced dramatically less hyperlocomotion than the MOR agonist, morphine. While chronic administration of CycloAnt resulted in antinociceptive tolerance, it was without opioid-induced hyperalgesia and with significantly reduced signs of naloxone-precipitated withdrawal, which suggested reduced physical dependence compared to morphine. Collectively, the results suggest this dual MOR/DOR multifunctional ligand is an excellent lead for the development of peptide-based safer analgesics.

Keywords: antinociception; bifunctional ligand; cyclic peptide; delta-opioid receptor; mixed-based combinatorial library; mu-opioid receptor; opioid antagonist; opioid liabilities; structure–activity relationships.

Figure 1

The mixture-based cyclic peptide library and library composition. (Top). General peptide sequence in the library. (Bottom). The defined single amino acid (O) of each sample in the library.

Figure 2

Positional scan screening of the cyclic peptide library in vivo using the 55 °C warm water tail-withdrawal assay in young adult male C57BL/6J mice across an 8 h period. (A): library defined at position 2, (B): library defined at position 4, (C): library defined at position 5. Each sample was given to the mice (n = 8) i.p. at a dose of 5 mg/kg. The tail-withdrawal latencies were measured at six time points post-administration, with combined time to withdraw tails (s; y-axis) calculated by taking the sum of the average tail-withdrawal latencies from each time point. Dotted line = vehicle alone (i.p.); cyan line = morphine control (10 mg/kg, i.p.). (D): Summary of key functionality residues identified in the screening. Data represented the combined time to tail withdrawal ± SEM for each sample. Dark blue bars in (A–C) represent significantly different from vehicle (p < 0.05), but not morphine (p > 0.05); One-way ANOVA with Dunnett’s post hoc tests.

Figure 3

Antinociception produced by the top five mixture-based samples in the mouse 55 °C warm water tail-withdrawal assay is dose-dependent. (A) Dose–response of mixture-based samples 5, 6 and 16 defined at the R² position, 60 min after administration; (B) Dose–response of mixture-based samples 81 and 91 defined at the R⁵ position, 120 min after administration. All samples were administered by the i.p. route. Morphine is included as a positive control, with antinociception measured at the 30 min peak effect. All points = 8 mice (7–11 mice for morphine).

Scheme 1

Solution phase synthesis of the cyclic peptides using POSS-SH as a soluble support.

Figure 4

Acute antinociceptive activity in the 55 °C WWTW assay following i.p. administration in C57BL/6J mice. Shown as (A) dose–response and (B) time-course of antinociception following a maximally efficacious dose of CycloAnt or CycloAnt-Leu. Morphine and vehicle are included as controls where appropriate. Points represent average % Antinociception ± SEM of 6–11 mice for each compound presented.

Figure 5

Evaluation of opioid receptor involvement in the antinociceptive activity of CycloAnt (3 mg/kg, i.p.) 20 min after administration to wild-type (C57BL/6 J) or mice where individual opioid receptors have been knocked out. * Significantly different from vehicle-control response (p < 0.05); † Significantly different from wild-type mice response (p < 0.0001); one-way ANOVA with Dunnett’s post hoc test. Bars each represent average % Antinociception ± SEM of 8 mice.

Figure 6

Evaluation of opioid antagonist activity of CycloAnt in the 55 °C warm water tail-withdrawal assay. MOR KO mice were tested to avoid confounding antinociception mediated by this receptor. (A) A 60 min pretreatment with CycloAnt (3 or 10 mg/kg, i.p.) dose-dependently prevented SNC-80-induced antinociception (100 nmol, i.c.v.), but not that of U50,488 (10 mg/kg, i.p.). (B) Time-dependent DOR antagonism by CycloAnt. The antinociceptive effect of SNC-80 (100 nmol, i.c.v.) was determined in mice pretreated for 1, 3, 6 or 8 h with CycloAnt (10 mg/kg, i.p.). Points represent average % antinociception ± SEM from 6 to 12 mice for each bar. *significantly different from response of agonist alone (p < 0.05); one-way ANOVA with Dunnett’s post hoc test.

Figure 7

Effects of the hit on (A) respiration and (B) ambulation in C57BL/6J mice tested in the CLAMS/Oxymax system. Respiratory and ambulation were monitored after i.p. administration of vehicle (saline, i.p.), morphine (10 or 30 mg/kg, i.p.), or CycloAnt (3 or 10.6 mg/kg, i.p.). Data from 12-20 mice presented as % vehicle ± SEM; breaths per minute, BPM (A) or ambulation, XAMB (B). Dashed lines represent normalized vehicle response. * Significantly different from vehicle control response (p < 0.05); † Significantly different from response of morphine at either dose (p < 0.05); two-way RM ANOVA with Tukey’s multiple comparison post hoc test.

Figure 8

Assessment of chronic treatment on (A) induced hyperalgesia in the 48 °C warm water tail-withdrawal test, or (B) antinociceptive tolerance in the 55 °C warm water tail-withdrawal test. Mice were first tested on day 1 to establish baseline responses prior to any treatment. Mice were then subjected to a dosing regimen with morphine (10 mg/kg, i.p., twice a day; blue bars) or CycloAnt (3 mg/kg, i.p., twice a day; red bars) for 4 days. On day 5, tail-withdrawal latency from 48 °C water was assessed prior to final dosing (A), whereas tail-withdrawal latency from 55 °C water was tested 20 min (for CycloAnt, 3 mg/kg, i.p.) or 30 min (for morphine, 10 mg/kg, i.p.) after drug administration (B). Data are shown as mean ± SEM of (A) pre- and post-dosing testing or (B) responses to listed drug in naïve and chronically treated mice. * p < 0.01 versus baseline (A) or response in naïve mice (B), Two-way RM ANOVA with Sidak’s multiple comparisons post hoc test. n = 9–11 mice/treatment.