THE BIOACTIVE CONFORMATION OF NEUROPEPTIDE-Y ANALOGS AT THE HUMAN Y-2-RECEPTOR

Several attempts to investigate the bioactive conformation of neuropep tide Y have been made so far. As cyclic peptides are much more rigid t han linear ones, we decided to synthesise cyclic analogues of the C-te rminal dodekapeptide amide neuropeptide Y Ac-25-36. Cyclisation was pe rformed by side chain lactamisation of ornithine or lysine and glutami c or aspartic acid. The affinity of the 19 peptides ranged from K-i 0. 6 nM to greater than 10000 nM. We found that the size, position, orien tation, configuration, and the location of the cycle plays an importan t role for receptor recognition. Circular dichroic studies have been p erformed to characterise the secondary structure of each peptide. Rece ptor binding studies were carried out on human neuroblastoma cell line s SK-N-MC (Y-1) and SMS-KAN (Y-2), and on rabbit kidney membranes (Y-2 ). The pharmacological and spectral data showed that the alpha-helix c ontent was not the predominant factor for high Y-2-receptor affinity. instead, the location and the size of the hydrophobic lactam bridge, a nd the conserved C-terminal tetrapeptide (Arg-Glu-Arg-Tyr) seemed to b e the main parameters. Using molecular dynamics, the structures of fou r cyclic peptides (i,i+4) have been investigated and compared with the previously published NMR structure of one of the cyclic peptide analo gues. Significant differences have been found in the overall three-dim ensional fold of the peptides. The distances between the N- and the C- terminus allow discrimination between peptides with high binding affin ity and those with low binding affinity, because of the correlation th at was found with the measured affinity. Thus, this study suggests tha t a turn-like structure and the orientation of the C-terminus towards the N-terminus play major roles for high affinity binding of cyclic do decapeptides to the Y-2-receptor. None of the cyclic segments exhibits significant affinity to the Y-1-receptor. Thus, these results support the hypothesis of a discontinuous binding site of neuropeptide Y at t he Y-1-receptor.