Design, synthesis and biological evaluation of bivalent ligands against A1-D1 receptor heteromers.

Aim:To design and synthesize bivalent ligands for adenosine A₁-dopamine D₁ receptor heteromers (A₁-D₁R), and evaluate their pharmacological activities.Methods:Bivalent ligands and their corresponding A₁R monovalent ligands were designed and synthesized. The affinities of the bivalent ligands for A₁R and D₁R in rat brain membrane preparation were examined using radiolabeled binding assays. To demonstrate the formation of A₁-D₁R, fluorescence resonance energy transfer (FRET) was conducted in HEK293 cells transfected with D₁-CFP and A₁-YFP. Molecular modeling was used to analyze the possible mode of protein-protein and protein-ligand interactions.Results:Two bivalent ligands for A₁R and D₁R (20a, 20b), as well as the corresponding A₁R monovalent ligands (21a, 21b) were synthesized. In radiolabeled binding assays, the bivalent ligands showed affinities for A₁R 10-100 times higher than those of the corresponding monovalent ligands. In FRET experiments, the bivalent ligands significantly increased the heterodimerization of A1R and D₁R compared with the corresponding monovalent ligands. A heterodimer model with the interface of helixes 3, 4, 5 of A1R and helixes 1, 6, 7 from D₁R was established with molecular modeling. The distance between the two ligand binding sites in the heterodimer model was approximately 48.4 Å, which was shorter than the length of the bivalent ligands.Conclusion:This study demonstrates the existence of A₁-D₁R in situ and a simultaneous interaction of bivalent ligands with both the receptors. [ABSTRACT FROM AUTHOR]