NEUROPEPTIDE-Y - Y-1 AND Y-2 AFFINITIES OF THE COMPLETE SERIES OF ANALOGS WITH SINGLE D-RESIDUE SUBSTITUTIONS

In an effort to gain insight into the bioactive conformation of neurop eptide Y upon interaction with its receptors, all single-point D-amino acid substituted NPY analogues were prepared, and their Y-1 and Y-2 r eceptor binding affinities were evaluated using the human neuroblastom a cell lines, SK-N-MC and SK-N-BE2, respectively. Solid-phase synthesi s (Boc strategy) followed by preparative HPLC purification produced an alogues of high purity that were characterized by RP-HPLC, AAA, LSIMS, CZE, and optical rotation. Of the 37 isomers (a naturally occurring g lycine at position 9 was replaced by Ala and D-Ala), Y-1 receptor bind ing was most perturbed by chiral inversion of residues at the C-termin us (residues 20, 27, 29-35, K-i greater than or equal to 300 nM). Subs titutions at residues 2-5, 28, and 36 had K-i values ranging from 40 t o 260 nM. Substitutions at all other positions yielded analogues with affinities ranging from 1.5 to 20 nM. Binding affinities to the Y-2 cl ass of receptors all measured in the low or sub-nanomolar concentratio ns, with the exception of C-terminally modified isomers (residues 30-3 5). Only [D-Arg(33)]-,and [D-Gln(34)]NPY displayed no measurable bindi ng affinity to Y-2 receptors at the highest concentration tested (1000 nM). Representative analogues were selected on the basis of their bin ding affinities and position in the sequence for structural analysis u sing circular dichroism (CD) spectroscopy. Of the nine peptide evaluat ed ([D-Pro(5)]-, [Ala(9)]-, [D-Ala(9)]-, [D-Glu(10)]-, [D-Asp(11)]-, [ D-Ala(18)]-, [D-Tyr(20)]-, [D,Tyr(27)]-, and [D-Arg(33)] NPY), only [D -Tyr(27)] NPY expressed a definitive correlation between loss of bindi ng affinity and disruption of secondary structure by having the propen sity to form beta-sheets at the expense of alpha-helical content. It w as concluded that although the incorporation of a single D-amino acid within the sequence of NPY may confer a conformational perturbation, t he receptor interaction was only affected when certain critical residu es were modified, findings that provide a basis for the identification of the binding pharmacophore of NPY.