Beate Rist

Modified, cyclic dodecapeptide analog of neuropeptide Y is the smallest full agonist at the human Y2 receptor

In order to stabilize the C‐terminal dodecapeptide of neuropeptide Y (NPY) we replaced Leu28 and Thr32 by Lys and Glu, respectively, and subsequently linked these residues by lactamization. This peptide analog of NPY shows a more than 100‐fold increase in affinity compared to the C‐terminal linear dodecapeptide in receptor binding studies performed at human neuroblastoma cells SMS‐KAN, which exclusively express the Y2 receptor subtype. Signal transduction was investigated by measuring Ca2+ current inhibition in human SH‐SY5Y cells and cyclic [Lys28‐Glu32] NPY Ac‐25–36 and NPY were shown to be equipotent in this assay. Thus, this molecule is the smallest Y2 receptor selective full agonist of NPY. Using 2D‐NMR experiments and molecular modelling techniques, the structures of the linear and cyclic peptides have been investigated and significant differences have been found, which may explain the improvement in biological activity. Thus, a model of the bioactive conformation of NPY at the human Y2 receptor is suggested.

CGRP 27-37 analogues with high affinity to the CGRP1 receptor show antagonistic properties in a rat blood flow assay

CGRP Y0-28-37 is known as a selective CGRP1 receptor antagonist. We succeeded in optimising the CGRP1 receptor affinity of this fragment by multiple amino acid replacement. The analogues [p34, F35]CGRP 27-37 and [D31, p34, F35]CGRP 27-37 exhibit a 100-fold increased affinity compared to the unmodified segment. Receptor binding studies were performed with human neuroblastoma cells SK-N-MC, which selectively express the hCGRP1 receptor. Blood flow, which is increased by exogenous CGRP, was measured in the right femoral artery. Preincubation of the rats with [p34, F35]CGRP 27-37 and [D31, p34, F35]CGRP 27-37 led to a significant decrease in CGRP induced increase in vascular conductance indicating the antagonistic properties of these compounds. Interestingly, an exchange of the amino acid Asn31 to Asp31 in [p34, F35]CGRP 27-37 shortened the period of the antagonistic effect significantly, suggestive of a different rate of metabolism for the two ligands. Secondary structure investigations obtained by circular dichroism measurements revealed that an increase in ordered structure correlates with high binding affinity.

Structure-affinity studies of C-terminally modified analogs of neuropeptide Y led to a novel class of peptidic Y1 receptor antagonist

A novel type of C-terminally modified analogs of the 36-mer peptide hormone neuropeptide Y has been synthesized, characterized and tested with respect to receptor affinity and biological activity in various systems. The compounds were obtained by synthesizing the fully protected peptide fragment NPY 1-35 or analogs of this, and coupling it in solution to various amines, alcohols, and modified tyrosine residues. It could be confirmed, that the C-terminal tyrosineamide of NPY is essential for its affinity to the Y1 receptor subtype. Obviously, the amino group of the amide part is more important than the oxygene atom of the carbonyl group, as NPY 1-35-tyrosinol has a lower affinity than NPY 1-35-tyrosinethioamide. NPY 1-35-tyramide could be shown to act as an antagonist in a Ca2+ release assay in human neuroblastoma cells. Analogs of NPY 1-35-tyramide showed the same structure-affinity relationships as NPY itself, suggesting, that there exists the same binding mode for the agonist and the antagonist.

Low potency inhibition of Ca2+ channel currents in human neuroblastoma (SH-SY5Y) cells by [Ala31]NPY, an l-alanine substituted analogue of neuropeptide Y ☆

Whole-cell Ca2+ channel currents were recorded in human neuroblastoma (SH-SY5Y) cells, using the perforated-patch technique with 10 mM Ba2+ as charge carrier. Neuropeptide Y (NPY; 10 nM to 1 microM) caused concentration-dependent inhibition of Ca2+ channel currents which were associated with a slowing in current activation kinetics. [Ala31]NPY, a residue 31 L-alanine substituted analogue of NPY, also inhibited Ca2+ channel currents and caused slowing of activation kinetics, but with approximately 6-fold lower potency. In the presence of 100 nM [Ala31]NPY (which itself had little or no effect on currents), the actions of NPY were similar in magnitude to its effects in the absence of the analogue. Our results suggest that substitution of isoleucine for alanine at residue 31 results in a NPY analogue which is a full agonist but with lower affinity for Y2 receptors.

Development of the first CGRP-antagonist with nanomolar affinity

Calcitonin gene-related peptide (CGRP) is a 37 amino acid peptide generated by alternative tissue-specific splicing of the primary transcript of the calcitonin. In contrast to calcitonin which is predominantly expressed in the C-cells of the thyroid, two forms ( and ) of CGRP are produced in a variety of human tissues which are mostly of neuronal origin. In fact, CGRP is co-localised with substance P in sensory nerves, with acetylcholine in motorneurones, and with various other transmitters in the brain [1]. CGRP consists of 37 amino acids, contains one disulfide bond and is C-terminally amidated. It binds to two receptors, which have been named and and which belong to the family of G-protein coupled receptors. The neuropeptide CGRP plays an important role in migraine because it acts as a central vasodilator. Potent antagonists are suggested for possible drugs in migraine therapy. CGRP 8-37, which has been used to characterize -receptors, has been the only shortened analog with high affinity.