Conformational and biological studies of neuropeptide Y analogs containing structural alterations

We evaluated the alpha-helix content, the biological activities and the affinities of a series of neuropeptide Y (NPY) analogs containing structural alterations, mainly in the central portion of the molecule for which a putative alpha-helix arrangement has been proposed. First, we investigated the conformational and pharmacological characteristics of derivatives containing the N-terminal tetrapeptide linked to C-terminal peptide-amide segments of various lengths. In some of these, the missing portion was replaced with epsilon-aminocaproic acid, a flexible arm-linker. Data revealed that (1-4)-Aca-(18-36)NPY is a discontinuous analog almost as potent as the native peptide in a pharmacological preparation enriched in Y2 receptors (rat vas deferens), whereas it is about 5 times less potent in a Y1 bioassay (rabbit saphenous vein). This analog showed a similar profile in [125I]PYY binding assays performed in rat frontoparietal cortex (Y1) and hippocampus (Y2) membrane preparations. In a series of truncated derivatives obtained with the successive removal of the 5-13 to 5-17 segments of the NPY molecule, no apparent correlation was observed between the affinity or potency in bioassays and the alpha-helix content, as measured by circular dichroism spectroscopy. Other truncated analogs, obtained by linking the C-terminal 31-36 fragment to various N-terminal tetrapeptides were also investigated. None showed any affinity in brain membrane preparations (frontoparietal cortex and hippocampus) or activity in the rat vas deferens bioassay. However, a weak short-lasting contraction was measured with some of these analogs in the rabbit saphenous vein, thus suggesting that the 1-4 and 31-36 segments of the molecule contains pharmacophores recognized by the Y1 receptor subtype. The contribution of the arginine residues also was evaluated in relation with the alpha-helix. Their successive substitution with lysine, an excellent helix-promoter, showed that the replacement of Arg-19 or Arg-25, two residues found in the putative alpha-helix, gave active analogs. Furthermore, the substitution of Arg-19 with lysine increased the activity in the rat vas deferens as well as the affinity in the brain membrane binding assays. On the other hand, the substitution of Arg-33 produced a weak agonist, whereas the replacement of Arg-35 generated an inactive analog in the Y2-pharmacological preparation and a very weak competitor in the CNS binding assays. Interestingly, this latter analog was still active in the rabbit saphenous vein, thus identifying the position 35 as an additional potential target for the development of Y1 versus Y2 specific molecules.(ABSTRACT TRUNCATED AT 400 WORDS)