Design of a True Bivalent Ligand with Picomolar Binding Affinity for a G Protein-Coupled Receptor Homodimer

Bivalent ligands
have emerged as chemical tools to study G protein-coupled
receptor dimers. Using a combination of computational, chemical, and
biochemical tools, here we describe the design of bivalent ligand 13 with high affinity (KDB1 =
21 pM) for the dopamine D2 receptor (D2R) homodimer.
Bivalent ligand 13 enhances the binding affinity relative
to monovalent compound 15 by 37-fold, indicating simultaneous
binding at both protomers. Using synthetic peptides with amino acid
sequences of transmembrane (TM) domains of D2R, we provide
evidence that TM6 forms the interface of the homodimer. Notably, the
disturber peptide TAT-TM6 decreased the binding of bivalent ligand 13 by 52-fold and had no effect on monovalent compound 15, confirming the D2R homodimer through TM6 ex
vivo. In conclusion, by using a versatile multivalent chemical platform,
we have developed a precise strategy to generate a true bivalent ligand
that simultaneously targets both orthosteric sites of the D2R homodimer.