Heterobivalent Ligand for the Adenosine A2A-Dopamine D2 Receptor Heteromer

A G protein-coupled receptor heteromer that fulfills the established criteria for its existence in vivo is the complex between adenosine A(2A) (A(2A)R) and dopamine D-2 (D2R) receptors. Here, we have designed and synthesized heterobivalent ligands for the A(2A)R-D-2R heteromer with various spacer lengths. The indispensable simultaneous binding of these ligands to the two different orthosteric sites of the heteromer has been evaluated by radioligand competition-binding assays in the absence and presence of specific peptides that disrupt the formation of the heteromer, label-free dynamic mass redistribution assays in living cells, and molecular dynamic simulations. This combination of techniques has permitted us to identify compound 26 [K-DB1 (A(2A)R) = 2.1 nM, K-DB1 (D2R) = 0.13 nM], with a spacer length of 43-atoms, as a true bivalent ligand that simultaneously binds to the two different orthosteric sites. Moreover, bioluminescence resonance energy transfer experiments indicate that 26 favors the stabilization of the A(2A)R-D2R heteromer.