dynorphin-(1-17) and dynorphin-(1-8)

In order to study the structure-activity relationships of dynorphin A-(1-8) amide [Dyn(1-8)-NH2], 20 analogues were synthesized by the solution method. Their biological activities were determined in the three bioassays [guinea pig ileum (GPI), mouse vas deferens (MVD), and rabbit vas deferens (RVD)] and in the mouse tail-pinch test after intravenous administration. Some analogues that showed interesting activity in the bioassays and/or in the analgesic tests were further characterized in mu-, delta-, and kappa-representative binding assays. The obtained data indicate that modification of the enkephalin segment to give metabolically stable analogues with high affinity and selectivity for the kappa receptor is strictly limited and that introduction of MeArg in position 7 protects the Arg6-Arg-7 bond from enzymatic degradation without potency drop and change of opioid receptor selectivity. [MeTyr1,MeArg7,D-Leu8]Dyn(1-8)-NHEt (18) [IC50 (nM) = 0.3 (GPI), 7.4 (MVD), and 2.6 (RVD); tail pinch ED50 (mg/kg) = 0.75] showed opioid activity similar to that of dynorphin A in the three bioassays and relatively high kappa-receptor selectivity in the binding assays and produced a 2.5-fold more potent analgesic effect than morphine. [D-Cys2-Cys5,MeArg7,D-Leu8]Dyn(1-8)-NHEt (20) showed a 40-60-fold more potent opioid activity than 18 in the three bioassays and produced a 3.4-fold more potent analgesic effect than 18. In the binding assays, however, 20 showed higher affinity for mu and delta receptors than for the kappa receptor.

Topics: Amino Acid Sequence; Analgesia; Animals; Biological Assay; Dynorphins; Guinea Pigs; Male; Mice; Molecular Sequence Data; Muscle, Smooth; Peptide Fragments; Rabbits; Receptors, Opioid; Receptors, Opioid, kappa; Structure-Activity Relationship

Following incubation of [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9) with suspensions of guinea pig brain membranes, analysis of the supernatants by HPLC has shown that both peptides are degraded at 25 degrees C and at 0 degrees C. Bestatin and captopril reduce degradation at 0 degrees C but for a similar degree of protection at 25 degrees C arginine-containing dipeptides are also required. The effects of these peptidase inhibitors on the degradation profiles indicate that [3H]dynorphin A (1-8) has three main sites of cleavage: the Tyr1-Gly2, Arg6-Arg7, and Leu5-Arg6 bonds. With [3H]dynorphin A (1-9) as substrate the Arg7-Ile8 and Ile8-Arg9 bonds are also liable to cleavage. In binding assays, in contrast to the effects of peptidase inhibitors on the degradation of unbound [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9), bestatin and captopril have little effect on the binding characteristics of the tritiated dynorphin A fragments at the kappa-site at 0 degrees C. However, at 25 degrees C binding is low in the absence of peptidase inhibitors. When binding at mu- and delta-sites is prevented, the maximal binding capacities of [3H]dynorphin A (1-8), [3H]dynorphin A (1-9), and [3H](-)-bremazocine at the kappa-site are similar; [3H]dynorphin A (1-9) has 5-10 times higher affinity for the kappa-site than [3H]dynorphin A (1-8). Comparison of the effects of peptidase inhibitors on unbound dynorphin A fragments with their effects in binding assays suggests that the bound peptides are protected from the action of peptidases.

Topics: Animals; Benzomorphans; Binding Sites; Brain; Dynorphins; Guinea Pigs; Kinetics; Membranes; Peptide Fragments; Protease Inhibitors; Receptors, Opioid; Receptors, Opioid, kappa; Substrate Specificity; Temperature