Synthesis and biological activity of analogs of dynorphin-A(1–13) substituted in positions 2 and 4: Design of [Ala2,Trp4]-Dyn-A(1–13) as a putative selective opioid antagonist

Abstract

Mono- and di-substituted analogs of dynorphin-A(1-13) (Dyn-A(1-13)) were synthesized by the solid-phase procedure. The products were purified and analyzed for their ability to inhibit the electrically evoked contractions of the guinea pig ileum (GPI) and mouse vas deferens (MVD) and to compete with the binding of [3H]etorphine ([3H]ET) and [3H]ethylketocyclazocine ([3H]EKC) to homogenates of ratbrain (.mu.-, .delta.-, .KAPPA.2-receptors) and guinea pig cerebellum (.KAPPA.-receptor), respectively. Introduction of Ala in position 2 caused a drastic decrease in the activity of the peptide on the smooth muscle preparations (IC50 of 104 and 2.250 nM in the GPI and the MVD as compared with 0.7 and 21 nM for the parent peptide, respectively). Conversely, this analog retained much of the opioid binding activity of Dyn-A(1-13) (relative binding potencies of 15 and 72% for the displacement of [3H]ET and [3H]EKC, respectively.) The replacement of Phe4 by Trp also caused drastic decreases in the activity of the peptide in the smooth muscle preparations relative potencies of 0.8 and 8.8% on the GPI and MVD while much of the binding potency to the opioid receptors was retained (31 and 67% for the displacement of [3H]ET and [3H]EKC, respectively). [Ala2,Trp4]-Dyn-A(1-13) was the least potent peptide tested in the smooth muscle assays (relative potencies: 0.1 and 0.6%). However, this latter analog still retained some opioid binding activity in the displacement of [3H]ET to rat brain homogenates (3%). Finally, the disubstituted peptide selectively anatgonized the activity of Dyn-A(1-13), .beta.-endorphin, and the .sigma.- and (or) .KAPPA.2-selective ligands SKF-10047 and cyclazocine in the GPI when applied prior to the addition of the opioid agonist. However, this compound did not display any antagonist activity against phencyclidine, a .sigma.-ligand, and any other selective ligands for .mu.-, .delta.-, or .KAPPA.-receptors. These data indicate that specific modifications in positions 2 and 4 of Dyn-A(1-13) can abolish most of its agonist potency while retaining its binding capacity to specific receptor types, presumably .KAPPA.2 for [Ala2,Trp4]-Dyn-A(1-13), thus providing a putative selective antagonist for the endogenous peptide.