Design, synthesis and biological evaluation of bivalent ligands against A(1)-D(1) receptor heteromers

Abstract

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.

Figure 1

General structure of bivalent ligands.

Figure 2

Fluorescence resonance energy transfer (FRET) experiment confirmed bivalent ligands promoting the formation of heterodimers of A₁R and D₁R. ^bP<0.05, ^cP<0.01 vs negative control group.

Figure 3

Analysis of sequence and alignment of A₁ receptor and D₁ receptor in silico receptor.

Figure 4

Energy minimized heterodimer model with compound 20b binding. The left is D₁ receptor and the right A₁ receptor. (A) The side view of the compound 20b binding in the heterodimer. Ligand colored grey and the residues interacting with it are shown in lines. (B) The top view of the compound 20b binding in the heterodimer. Residues that form hydrogen bonds with PEG linker are shown in sticks and colored magentas. (C) Bivalent ligand is shown in sticks and colored white. Residues that form hydrogen bonds with PEG linker are shown in sticks and colored black. These residues include: Glu153, Lys173 from A₁ receptor and Asn311, Ser310 from D₁ receptor.

Scheme 1

Synthesis of the bivalent ligands linked by PEG.

Scheme 2

Synthesis of the monovalent ligand compounds. Reagents and conditions: a) EDCI, HOBt, DIPEA, DMF, rt, overnight (70%).