Receptor binding and G-protein activation by new Met5-enkephalin-Arg6-Phe7 derived peptides

Met(5)-enkephalin-Arg(6)-Phe(7) (Tyr-Gly-Gly-Phe-Met-Arg-Phe, MERF) is a naturally occurring heptapeptide that binds to opioid and non-opioid recognition sites in the central nervous system. Four synthetic analogs with single or double amino acid substitutions were prepared by solid phase peptide synthesis to achieve proteolytically more stable structures: Tyr-D-Ala-Gly-Phe-Met-Arg-Phe (I), Tyr-D-Ala-Gly-Phe-D-Nle-Arg-Phe (II), Tyr-D-Ala-Gly-Phe-L-Nle-Arg-Phe (III) and Tyr-Gry-Gly-Phe-L-Nle-Arg-Phe (IV). In this study receptor binding characteristics and G-protein activation of MERF and its derivatives were compared in crude membrane fractions of frog and rat brain. Synthetic MERF-derived peptides were potent competitors for [H-3]MERF and [H-3]naloxone binding sites with the exception of analog (II) which turned to be substantially less active. The presence of 100 mM NaCl or 100 mu M 5'-guanylylimidodiphosphate, Gpp(NH)p, decreased the affinity of the peptides in [H-3]naloxone binding assays, suggesting that these ligands might act as agonists at the opioid receptors. Some of the compounds were also used to stimulate guanosine-5'-O-(3-[gamma-[S-35]thio)triphosphate ([S-35]GTP gamma S) binding in rat and frog brain membranes at concentrations of 10(-9)-10(-5) M. The EC50 values Of analog (I) were the highest in both tissues. Analog (I) was as effective as MERF in rat brain membranes, but showed lower maximal stimulation in frog brain preparation. Again, analog (II) seemed to be the least efficacious peptide that stimulated [S-35]GTP gamma S binding only by 59 %. Specificity of the peptides was further investigated by the inhibition of agonist-stimulated [S-35]GTP gamma S binding in the presence of selective antagonists for the opioid receptor types. The mu-selective antagonist cyprodime displayed the lowest potency in inhibiting the effects of the peptides, whereas norbinaltorphimine (kappa-selective antagonist) and naltrindole (delta-selective antagonist) were quite potent in both tissues. We concluded that MERF and its derivatives are able to activate G-proteins mainly via kappa- and delta-opioid receptors.