Original Article

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

Authors: Jian Shen, Lei Zhang, Wan-ling Song, Tao Meng, Xin Wang, Lin Chen, Lin-yin Feng, Ye-chun Xu, Jing-kang Shen
DOI: 10.1038/aps.2012.151


Jian SHEN, Lei ZHANG, Wan-ling SONG, Tao MENG, Xin WANG, Lin CHEN, Lin-yin FENG*, Ye-chun XU*, Jing-kang SHEN*
The State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

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.

Keywords: G protein-coupled receptors; adenosine; dopamine; A1 receptor; D1 receptor; heterodimers; bivalent ligands; radiolabeled binding assay; FRET; molecular modeling

This study was supported by the ‘100 Talents Project’ of CAS to Ye-chun XU and National S&T Major Project (No 2012ZX09301-001-005).
* To whom correspondence should be addressed.
E-mail jkshen@mail.shcnc.ac.cn (Jing-kang SHEN); lyfeng@mail.shcnc.ac.cn (Lin-ying FENG); ycxu@mail.shcnc.ac.cn (Ye-chun XU)
Received 2012-05-31 Accepted 2012-10-08

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