Heterobivalent Ligand for the Adenosine A_2A-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_2AR) and dopamine D₂ (D₂R) receptors. Here, we have designed and synthesized heterobivalent ligands for the A_2AR-D₂R 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_2AR) = 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_2AR-D₂R heteromer.