CONFORMATIONAL AND BIOLOGICAL STUDIES OF NEUROPEPTIDE-Y ANALOGS CONTAINING STRUCTURAL ALTERATIONS

We evaluated the alpha-helix content, the biological activities and th e affinities of a series of neuropeptide Y (NPY) analogs containing st ructural alterations, mainly in the central portion of the molecule fo r which a putative alpha-helix arrangement has been proposed. First, w e investigated the conformational and pharmacological characteristics of derivatives containing the N-terminal tetrapeptide linked to C-term inal peptide-amide segments of various lengths. In some of these, the missing portion was replaced with epsilon-aminocaproic acid, a flexibl e arm-linker. Data revealed that (1-4)-Aca-(18-36)NPY is a discontinuo us analog almost as potent as the native peptide in a pharmacological preparation enriched in Y-2 receptors (rat vas deferens), whereas it i s about 5 times less potent in a Y-1 bioassay(rabbit saphenous vein). This analog showed a similar profile in [I-125]PYY binding assays perf ormed in rat frontoparietal cortex (Y-1) and hippocampus (Y-2) membran e 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 i n bioassays and the alpha-helix content, as measured by circular dichr oism 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 def erens bioassay. However, a weak short-lasting contraction was measured with some of these analogs in the rabbit saphenous vein, thus suggest ing that the 1-4 and 31-36 segments of the molecule contains pharmacop hores recognized by the Y-1 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-promote r, showed that the replacement of Arg-19 or Arg-25, two residues found in the putative alpha-helix, gave active analogs. Furthermore, the su bstitution 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 agonis t, whereas the replacement of Arg-35 generated an inactive analog in t he Y-2-pharmacological preparation and a very weak competitor in the C NS binding assays. Interestingly, this latter analog was still active in the rabbit saphenous vein, thus identifying the position 35 as an a dditional potential target for the development of Y-1 versus Y-2 speci fic molecules. Overall, these pharmacological and spectral data showed that the alpha-helix content is not a predominant factor for the main tenance of affinity and activity of the NPY analogs. Instead, our resu lts suggest that the key parameter is the folding of the NPY molecule, which ensures an adequate orientation of the N- and C-terminal residu es.