Neuropeptide Y N-Terminal Deletion Fragments: Correlation between Solution Structure and Receptor Binding Activity at Y1 Receptors in Rat Brain Cortex

Abstract

N alpha-Acetyl (Ac), N-terminal deletion fragments of porcine neuropeptide Y (NPY) have been synthesized and characterized for solution conformation properties by circular dichroism and for receptor binding activity at benextramine-sensitive Y1 binding sites in rat brain cortex. Sequential deletion of Tyr1, Pro2, and Ser3 had no effect on the structural (alpha-helical content of 32.5, 30.6, and 30.7%, respectively, at 1 x 10(-5) M) or aggregation (monomer to dimer transition for N alpha-Ac-NPY3-36 and N alpha-Ac-NPY4-36) properties of NPY. In contrast, deletion of Tyr1 decreased receptor binding activity in rat brain cortex by 4-fold (IC50 = 13.0 nM versus 3.75 nM for NPY), but further deletion of Pro2-Ser3 had no additional detrimental effect on receptor binding activity relative to the desTyr1 analog. Thus, Pro2 and Ser3 do not contribute either to the stability of the NPY tertiary structure nor directly to the receptor-ligand interactions. Additional removal of N-terminal amino acids Lys4-Pro5 decreased the helical content and abolished aggregation to a dimeric form of the resultant analog, results suggesting that the residues around Pro5 are important for formation of NPY's compact, pancreatic polypeptide (PP)-fold structure. This loss in structure also correlated with a further 2-3 fold drop in receptor binding activity. These structure-activity correlations provide evidence for the importance of the PP-fold structure in the activity of NPY at Y1 receptors in rat brain cortex.

A rational approach for the development of reduced‐size analogues of neuropeptide Y with high affinity to the Y1 receptor

“…These results are in agreement with our studies. We can confirm the great significance of the N-terminal segment NPY(l-6) and of the C-terminal part NPY (22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). Additionally, it seems that further amino acids around the spacer (Ahx) are not directly involved in receptor recognition.…”

“…For an N-terminus consisting of five or six amino acids, a C-terminus should consist of the residues 20-36, for one with seven amino acids the latter should contain 2 1-36 residues. If there are eight N-terminal amino acids, the most favourable Cterminal segment is NFV (18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36) (Figure 1).…”

“…This is, however, not surprising, since much smaller analogues such as [ A~X~~* I N P Y have been identitled as full Y2 receptor agonists [5,101. NPY,[AhxSYNPY and [Ahxsa)NPY, the four peptides consisting of the C-terminal part NPY (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36) showed the most reduced receptor affinity ( [ A~X~~~I N P Y , ICsO 940 n~; IAhx7-22]NPY, IC50 2 8 0 n~; [Ahx8-221NPY, ICsO 260nM; [Ahxs 22]NFY, ICsO 780 nM). In each case the extension of the C-terminal segment by one amino acid leads to considerably lower ICm values.…”

“…Exact concentrations were determined by quantitative amino acid analysis after gasphase hydrolysis ( -1 140/420, Applied Biosystems, Weiterstadt). 17.1 (I) 16.9 (I) 22.9 (11) 19.0 (I) 24.0 (11) 17.0 (I) 17.5 (I) 23.1 (II) 19.3 (I) 23.5 (11)…”

“…Hu et aL [23] investigated the Y1 binding sites in rat brain cortex using a series of N-terminal deletion fragments of NPY. 'QT' and the residues around Pro5 were identifled as important in maintaining the o v e d tertiary structure.…”