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

Abstract

In order to stabilize the C-terminal dodecapeptide of neuropeptide Y (NPY) we replaced Leu 2s and Thr 32 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.2+ Signal transduction was investigated" by measuring Ca current inhibition in human SH-SY5Y cells and cyclic [Lys2S-Glu3Z] 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 Yz receptor is suggested.

Receptor Subtype-specific Docking of Asp6.59 with C-terminal Arginine Residues in Y Receptor Ligands

“…Then because of the inflexibility of Arg 33 within the ␣-helix, this important position can interact with Asp 6.59 (proximity model). This is further supported by the importance of the ␣-helix of the ligand for Y 2 R binding (26). A second reasonable model suggests that the first interaction site is identical in all YRs but also different from the interaction of Asp 6.59 .…”

“…However, the C-terminal pentapeptide of all natural ligands was identified as an essential region for binding to all YRs (23,24), and these residues of the ligand C terminus seem to represent a core contact domain. Interestingly, studies of analogs containing conformational constraints, when bound at the various YR subtypes, support the concept of structural differences between subtypes in this domain (25)(26)(27)(28). However, the two conserved Arg residues at positions 33 and 35 are important contact sites for all YRs.…”

Identification of neuropeptide Y cleavage products in human blood to improve metabolic stability

“…Cleavage of the peptides from the resin and identification of the CF-labeled peptides was performed as already described above. [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] ]NPY, and [Lys(CF) 4 , Ahx [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] ]NPY. The analytical data of these peptides are summarized in Table I.…”

“…21 Several proteolytic enzymes like neutral endopeptidase or metalloendopeptidase have been reported to affect the metabolic stability of peptides in plasma. 22 In the present study, we have studied the metabolic stability of NPY, the Y 2 -receptor selective analog [Ahx [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] For modification we chose labelling of the peptides with 5(6)-carboxyfluorescein (CF), in order to discriminate the peptide signal from plasma proteins by HPLC at its excitation/emission wavelengths as 450/517 nm, respectively. In addition to NPY itself the two selective compounds were modified at the N-terminus as well as at the side chain of Lys 4 in order to investigate the best position for modification with respect to metabolic stability.…”

“…Accordingly, we decided not to use any smaller reported analogs, e. g. NPY (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36), although this analog has been shown to exhibit some agonistic properties at Y 2 -receptors as well. 36,37 In case of CF-[Ahx [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] ]NPY, which is a centrally truncated Y 2 receptor selective analog, [17][18][19] an enzymatic degradation product CF-[Ahx [5][6][7][8][9][10][11][12][13][14][15][16][17]…”