Catherine A. Gehrig

Topographically designed analogs of [cyclic] [D-Pen2,D-Pen5]enkephalin

The conformationally restricted, cyclic disulfide-containing delta opioid receptor selective enkephalin analogue [D-Pen2,D-Pen5]enkephalin (1, DPDPE) was systematically modified topographically by addition of a methyl group at either the pro-S or pro-R position of the beta carbon of an L-Phe4 or D-Phe4 residue to give [(2S,3S)-beta-MePhe4]DPDPE (2), [(2R,3R)-beta-MePhe4]DPDPE (3), [(2S,3R)-beta-MePhe4]DPDPE (4), and [(2R,3S)-beta-MePhe4]DPDPE (5). The four corresponding isomers were prepared in which the beta-methylphenylalanine residue was p-nitro substituted, that is with a beta-methyl-p-nitrophenylalanine (beta-Me-p-NO2Phe) residue, to give [(2S,3S)-beta-Me-p-NO2Phe4]DPDPE (6), [(2R,3R)-beta-Me-p-NO2Phe4]DPDPE (7), [(2S,3R)-beta-Me-p-NO2Phe4] DPDPE (8), and [(2R,3S)-beta-Me-p-NO2Phe4]DPDPE (9), respectively. The potency and selectivity (delta vs mu opioid receptor) were evaluated by radioreceptor binding assays in the rat brain using [3H]CTOP (mu ligand) and [3H]DPDPE (delta ligand) and by bioassay with mouse vas deferens (MVD, delta receptor assay) and guinea pig ileum (GPI, mu receptor assay). The eight analogues of DPDPE showed highly variable binding and bioassay activities particularly at the delta opioid receptor (4 orders of magnitude), but also at the mu opioid receptor, which led to large differences (3 orders of magnitude) in receptor selectivity. For example, [(2S,3S)-beta-MePhe4]DPDPE (2) is 1800-fold selective in binding to the delta vs mu receptor, making it one of the most selective delta opioid receptor ligands in the enkephalin series as assessed by the rat brain binding assay, whereas the corresponding (2R,3R)-beta-Me-p-NO2Phe-containing analogue 9 is only 4.5-fold selective (nonselective) in this same assay. On the other hand, in the bioassay systems, [(2S,3S)-beta-Me-p-NO2Phe4]DPDPE (5) is more potent than DPDPE and 8800-fold selective for the MVD (delta receptor) vs the GPI (mu receptor), making it the most highly selective ligand in this series for the delta opioid receptor on the basis of these bioassays. In these assay systems, the (2R,3S)-beta-MePhe4-containing analogue 5 had very weak potency and virtually no receptor selectivity (4.4-fold). These results demonstrate that topographical modification alone in a conformationally restricted peptide ligand can significantly modulate both potency and receptor selectivity of peptide ligands that have multiple sites of biological activity and suggest that this approach may have general application to peptide ligand design.

DPen2-[DPen5]enkephalin, a delta opioid receptor-selective analog of [Leu]enkephalin, impairs avoidance learning in an automated shelf-jump task in rats.

Both [Leu]enkephalin and DPen2-[DPen5]enkephalin, a delta opioid receptor selective analog of [Leu]enkephalin, impaired acquisition of an automated shelf-jump response in rats. A similar level of impairment was produced by equimolar doses of the two enkephalins. As is seen for [Leu]enkephalin when tested in a one-way active avoidance task, the dose-response function for the impairment produced by DPen2-[DPen5]enkephalin in the automated shelf-jump task is U-shaped. These results, together with our previous findings that DPen2-[DPen5]enkephalin and [Leu]enkephalin both impair acquisition of a one-way active avoidance response in mice, and that [Leu]enkephalin impairs acquisition of that same response in rats, support our suggestion that delta opioid receptors are implicated in the effects of [Leu]enkephalin on conditioning. In addition, these results indicate that the involvement of delta opioid receptors in acquisition impairment extends to two species of rodents and to two different avoidance conditioning tasks.