Synthesis, conformational analysis and biological activity of cyclic analogs of the octadecaneuropeptide ODN -: Design of a potent endozepine antagonist

The octadecaneuropeptide (ODN; QATVGDVNTDRPG LLDLK) and its C-terminal octapeptide (OP; RPGLLDLK), which exert anxiogenic activity, have been previously shown to increase intracellular calcium concentration ([Ca2+](i)) in cultured rat astrocytes through activation of a metabotropic receptor positively coupled to phospholipase C. It has also been found that the [D-Leu5]OP analog possesses a weak antagonistic activity. The aim of the present study was to synthesize and characterize cyclic analogs of OP and [D-Leu5]OP. On-resin homodetic backbone cyclization of OP yielded an analog, cyclo(1-8) OP, which was three times more potent and 1.4-times more efficacious than OP to increase [Ca2+](i) in cultured rat astrocytes. Cyclo(1-8) OP also mimicked the effect of both OP and ODN on polyphosphoinositide turnover. Conversely, the cyclo(1-8) [D-Leu5]OP analog was totally devoid of agonistic activity but suppressed the effect of OP and ODN on [Ca2+](i) and phosphoinositide metabolism in astrocytes. The structure of these cyclic analogs has been determined by two-dimensional H-1-NMR and molecular dynamics. Cycio(1-8) OP exhibited a single conformation characterized by a gamma turn comprising residues Pro2-Leu4 and a type III beta turn encompassing residues Leu5-Lys8. Cyclo(1-8) [D-Leu(5)]OP was present as two equimolar conformers resulting from cis/trans isomerization of the Arg-Pro peptide bond. These pharmacological and structural data should prove useful for the rational design of non peptidic ODN analogs.