Bivalent argininamide-type neuropeptide Y Y1 antagonists do not support the hypothesis of receptor dimerisation

Abstract

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 Y1 receptor (Y1R) antagonist, we prepared a series of bivalent Y1R ligands using a wide range of linker lengths (8 to 36 atoms). Exploiting the high eudismic ratio (>1000) of the parent compound we synthesized sets of (R,R)-, (R,S)- and (S,S)-configured bivalent ligands to get a hint to the “bridging” of two Y1Rs by simultaneous interaction with both binding sites of a putative receptor dimer. Except for the (S,S)-isomers the bivalent ligands were high affinity Y1R antagonists (Ca2+ assay on HEL cells; radioligand competition assay on human Y1R expressing SK-N-MC and MCF-7 cells). Whereas the (R,R)-enantiomers were most potent, there were no marked differences compared to the corresponding meso forms. The difference between (R,R)- and (R,S)-diastereomers was most pronounced (about 6-fold) in case of the Y1R antagonist containing a spacer of 20 atoms in length. Among the (R,R)-enantiomers length and structural diversity of the linker had little effect on Y1R affinity. Although the bivalent ligands preferentially bind to the Y1R, the selectivity relative to human Y2, Y4 and Y5 receptors was markedly reduced compared to monovalent argininamides. The results of this study do neither support the existence of Y1R dimers nor the simultaneous occupation of both binding pockets by the twin compounds. However, as the interaction with Y1R dimers can not 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 synthesized, containing an N-propionylated amino-functionalized branched linker (Ki ≥ 18 nM), which can be easily prepared in a tritiated form.