Bivalent argininamide-type neuropeptide y y(1) antagonists do not support the hypothesis of receptor dimerisation.

Bivalent ligands are potential tools to investigate the dimerisation of G-protein-coupled receptors. Based on the (R)-argininamide BIBP 3226, a potent and selective neuropeptide Y Y(1) receptor (Y(1)R) antagonist, we prepared a series of bivalent Y(1)R ligands with a wide range of linker lengths (8-36 atoms). Exploiting the high eudismic ratio (>1000) of the parent compound, we synthesised sets of R,R-, R,S- and S,S-configured bivalent ligands to gain insight into the "bridging" of two Y(1)Rs by simultaneous interaction with both binding sites of a putative receptor dimer. Except for the S,S isomers, the bivalent ligands are high-affinity Y(1)R antagonists, as determined by Ca(2+) assays on HEL cells and radioligand competition assays on human Y(1)R-expressing SK-N-MC and MCF-7 cells. Whereas the R,R enantiomers are most potent, no marked differences were observed relative to the corresponding meso forms. The difference between R,R and R,S diastereomers was most pronounced (about sixfold) in the case of the Y(1)R antagonist containing a spacer of 20 atoms in length. Among the R,R enantiomers, linker length and structural diversity had little effect on Y(1)R affinity. Although the bivalent ligands preferentially bind to the Y(1)R, the selectivity toward human Y(2), Y(4), and Y(5) receptors was markedly lower than that of the monovalent argininamides. The results of this study neither support the presence of Y(1)R dimers nor the simultaneous occupation of both binding pockets by the twin compounds. However, as the interaction with Y(1)R dimers cannot be unequivocally ruled out, the preparation of a bivalent radioligand is suggested to determine the ligand-receptor stoichiometry. Aiming at such radiolabelled pharmacological tools, prototype twin compounds were synthesised, containing an N-propionylated amino-functionalised branched linker (K(i)> or =18 nM), a tritiated form of which can be easily prepared.