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

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