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Discovery of allosteric modulators for GABAA receptors by ligand-directed chemistry

Abstract

The fast inhibitory actions of γ-aminobutyric acid (GABA) are mainly mediated by GABAA receptors (GABAARs) in the brain. The existence of multiple ligand-binding sites and a lack of structural information have hampered the efficient screening of drugs capable of acting on GABAARs. We have developed semisynthetic fluorescent biosensors for orthosteric and allosteric GABAAR ligands on live cells via coupling of affinity-based chemical labeling reagents to a bimolecular fluorescence quenching and recovery system. These biosensors were amenable to the high-throughput screening of a chemical library, leading to the discovery of new small molecules capable of interacting with GABAARs. Electrophysiological measurements revealed that one hit, 4,4′,4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), was a novel negative allosteric modulator capable of strongly suppressing GABA-induced chloride currents. Thus, these semisynthetic biosensors represent versatile platforms for screening drugs to treat GABAAR-related neurological disorders, and this strategy can be extended to structurally complicated membrane proteins.

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Figure 1: Construction of GABAAR-based semisynthetic fluorescent biosensors by coupling affinity-based chemical labeling with BFQR system.
Figure 2: Chemical labeling of GABAARs in HEK293T cells using CGAM reagents.
Figure 3: Construction of GABAAR-based semisynthetic fluorescent biosensors for orthosteric ligands.
Figure 4: Construction of GABAAR-based fluorescent biosensors for allosteric benzodiazepine site ligands.
Figure 5: High-throughput screening of small molecules bound to benzodiazepine site on GABAAR using the semisynthetic biosensors, HEK(Bzp).
Figure 6: Functional characterization of hit chemicals.

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Acknowledgements

We thank S. Moss (Tufts University) for GABAAR constructs. The pCAGGS vector was provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. We thank Y. Yasueda (Kyoto University) and E. Kusaka (Kyoto University) for excellent technical assistance. This work was funded by the Japan Science and Technology Agency (JST) Core Research for Evolutional Science and Technology (CREST) of Molecular Technologies and JSPS KAKENHI (JP15H01637) to I.H. and by a SUNBOR Grant from Suntory Foundation for Life Sciences to S.K.

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Contributions

S.K. and I.H. initiated and designed the project. K.Y. performed synthesis and chemical labeling in cultured cells. K.Y. and S.K. performed molecular biology experiments. S.K. performed radioisotope experiments. T.N. and R.I. performed electrophysiological experiments. K.Y., S.K., and I.H. wrote the manuscript. All authors discussed and commented on the manuscript.

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Correspondence to Shigeki Kiyonaka or Itaru Hamachi.

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Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1 and 2 and Supplementary Figures 1–21. (PDF 4813 kb)

Supplementary Data Set 1

The entire list of pharmacologically active compounds (LOPAC1280). (XLSX 195 kb)

Supplementary Note

Synthetic Procedures (PDF 944 kb)

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Yamaura, K., Kiyonaka, S., Numata, T. et al. Discovery of allosteric modulators for GABAA receptors by ligand-directed chemistry. Nat Chem Biol 12, 822–830 (2016). https://doi.org/10.1038/nchembio.2150

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