Unexpected opioid activity profiles of analogues of the novel peptide kappa opioid receptor ligand CJ-15,208

Abstract

An alanine scan was performed on the novel κ opioid receptor (KOR) peptide ligand CJ-15,208 to determine which residues contribute to the potent in vivo agonist activity observed for the parent peptide. These cyclic tetrapeptides were synthesized by a combination of solid-phase peptide synthesis of the linear precursors, followed by cyclization in solution. Like the parent peptide, each of the analogues exhibited agonist activity and KOR antagonist activity in an antinociceptive assay in vivo. Unlike the parent peptide, the agonist activity of the potent analogues was mediated predominantly, if not exclusively, by μ opioid receptors (MOR). Thus analogues 2 and 4, in which one of the phenylalanine residues was replaced by alanine, exhibited both potent MOR agonist activity and KOR antagonist activity in vivo. These peptides represent novel lead compounds for the development of peptide-based opioid analgesics.

Figure 1

Structures of a) the cyclic tetrapeptide CJ-15,208, 1, and b) alanine analogs 2-5. The residues are numbered 1-4, arbitrarily starting with the Phe C-terminal to the Trp residue.

Figure 1

Structures of a) the cyclic tetrapeptide CJ-15,208, 1, and b) alanine analogs 2-5. The residues are numbered 1-4, arbitrarily starting with the Phe C-terminal to the Trp residue.

Figure 2

The antinociceptive activity of the cyclic tetrapeptides was assessed in vivo following i.c.v. administration in the 55°C warm-water tail-withdrawal assay in C57Bl/6J mice. All points represent antinociception at peak response, which was 20 min (for 4), 30 min (for peptides 1, 2 and 5) or 40 min (peptide 3). All points represent average % antinociception ± SEM from 7-8 mice. Data for 1 from reference ^[12a].

Figure 3

Cyclic tetrapeptide induced antinociception is opioid-receptor mediated. Peak antinociceptive activity of peptides 2 (3 nmol), 3 (10 nmol), 4 (0.3 nmol) and 5 (30 nmol) was determined in the 55°C warm-water tail-withdrawal assay after i.c.v. administration to C57Bl/6J mice (open bars). Naloxone pretreatment (15 nmol i.c.v., striped bars) 25 min prior to peptide administration significantly antagonized the effect of each cyclic tetrapeptide. Tail-withdrawal latencies were measured 30 minutes after injection of the cyclic tetrapeptide. Data represents average % antinociception ± SEM from 7-8 mice. *=significantly different from response of matching administered compound alone, p<0.05, One-way ANOVA followed by Tukey’s HSD post hoc test.

Figure 4

Opioid-receptor-selective agonism by the cyclic tetrapeptides. The antinociceptive activity of peptides 2 (3 nmol), 3 (3 nmol), 4 (0.1 nmol) and 5 (30 nmol) was determined in the 55°C warm-water tail-withdrawal assay after i.c.v. administration to C57Bl/6J mice (solid bars). Antinociception was also assessed 24 h after administration in mice pretreated with β-FNA (5 mg/kg, s.c.; diagonally striped bars) or nor-BNI (10 mg/kg, i.p., wave-filled bars). Additional mice were pretreated with naltrindole (20 mg/kg, i.p., –15 min; hatched bars) before administration of one of the cyclic tetrapeptides. Tail-withdrawal latencies were measured in the mouse 55°C warm-water tail-withdrawal test 30 minutes after injection of the cyclic tetrapeptides 2, 4 and 5, or 40 min after peptide 3. Data represents average % antinociception ± S.E.M. from 8-16 mice. * = significantly different from response of matching administered compound alone, p<0.05, One-way ANOVA followed by Tukey’s HSD post hoc test.

Figure 5

Dose-dependent antagonism of U50,488-induced antinociception by tested cyclic tetrapeptides. The antinociceptive effects of U50,488 (10 mg/kg, i.p.; thatched bar) were determined 40 minutes after administration in mice pretreated 3 h with peptides 1 (diamonds), 2 (circles), 3 (inverted triangles), 4 (filled triangles) and 5 (squares) in the 55°C warm-water tail-withdrawal assay after i.c.v. administration. Data represents average % antinociception ± S.E.M. from 8 mice. * = significantly different from response of U50,488 p<0.05, One-way ANOVA followed by Tukey’s HSD post hoc test. Data for 1 from reference ^[12a].

Figure 6

Duration of cyclic tetrapeptide-mediated antagonism of U50,488-induced antinociception in the mouse 55°C warm-water tail-withdrawal test. Antinociception of U50,488 (10 mg/kg, i.p.; thatched bar) was determined in mice pretreated 3, 6, 18 or 24 h with peptides 2 (circles), 3 (inverted triangles), 4 (filled triangles) and 5 (squares), and for peptide 1 (diamonds) at 8, 18 and 24 h.^[12a] Tail-withdrawal latencies were determined 40 minutes after agonist administration. Data represents average % antinociception ± S.E.M. from 8 mice/point. * = significantly different from response of U50,488 p<0.05, One-way ANOVA followed by Tukey’s HSD post hoc test.

Figure 7

Receptor selectivity of the antagonism by the cyclic tetrapeptides in the mouse 55°C warm-water tail-withdrawal test. Antinociceptive activity of morphine (10 mg/kg, i.p., left set of bars) was not reduced by a 3 h pretreatment of the mice with the cyclic tetrapeptides 2 (10 nmol, striped black bar), 3 (1 nmol, striped white bar), 4 (1 nmol, striped gray bar) or 5 (10 nmol, striped dark gray bar). However, the antinociceptive effect of U50,488 (10 mg/kg, i.p., center set of bars) was significantly antagonized by pretreatment of the mice with any of the cyclic tetrapeptides. (For this set of tests peptide 2 was administered at 3 nmol, i.c.v.). In contrast, the antinociceptive effect of SNC-80 (100 nmol, i.c.v., right set of bars) was significantly prevented only by pretreatment with peptides 3 and 5. Tail-withdrawal latencies were measured in the mouse 55°C warm-water tail-withdrawal test 40 minutes after injection of the known selective agonists. Data represents average % antinociception ± S.E.M. from 8-16 mice. * = significantly different from response of matching administered agonist alone, p<0.05, One-way ANOVA followed by Tukey’s HSD post hoc test.

Scheme 1

Synthesis of peptide 2.