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An allosteric potentiator of M4 mAChR modulates hippocampal synaptic transmission

Abstract

Muscarinic acetylcholine receptors (mAChRs) provide viable targets for the treatment of multiple central nervous system disorders. We have used cheminformatics and medicinal chemistry to develop new, highly selective M4 allosteric potentiators. VU10010, the lead compound, potentiates the M4 response to acetylcholine 47-fold while having no activity at other mAChR subtypes. This compound binds to an allosteric site on the receptor and increases affinity for acetylcholine and coupling to G proteins. Whole-cell patch clamp recordings revealed that selective potentiation of M4 with VU10010 increases carbachol-induced depression of transmission at excitatory but not inhibitory synapses in the hippocampus. The effect was not mimicked by an inactive analog of VU10010 and was absent in M4 knockout mice. Selective regulation of excitatory transmission by M4 suggests that targeting of individual mAChR subtypes could be used to differentially regulate specific aspects of mAChR modulation of function in this important forebrain structure.

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Figure 1: Database mining yields a series of compounds with allosteric potentiator activity at the rat M4 receptor.
Figure 2: Lead compounds robustly potentiate M4-mediated calcium mobilization.
Figure 3: Synthesis of compounds in the VU1000 series.
Figure 4: Chemical optimization generates compounds that potentiate M4-mediated calcium mobilization with greater efficacy than lead compound 16.
Figure 5: Compound 20j is selective for M4 receptor relative to the other mAChRs.
Figure 6: Compound 20j binds to an allosteric site on M4 mAChR and causes an increase in affinity for ACh and M4-mediated [35S]GTPγS binding.
Figure 7: Compound 20j potentiates CCh-induced reduction of EPSCs but not IPSCs at rat hippocampal Shaffer collateral–CA1 synapses.

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Acknowledgements

The authors thank K. Hemstapat, D.J. Sheffler, E.L. Days and N.T. Nalywajko for technical support and discussion of data. The authors also thank A. Levey (Emory University) for stable mAChR cell lines and T.I. Bonner (US National Institute of Mental Health) for the rM4 DNA construct. This work was supported by grants from the US National Institute of Mental Health and National Institute of Neurological Disorders and Stroke. Vanderbilt is a site in the US National Institutes of Health–supported Molecular Libraries Screening Center Network.

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J.K.S., cell-based and binding assays and handling of the manuscript; Z.X., electrophysiology; D.O. and R.W., chemical synthesis and chemical characterization; A.E.B., M4 cell line establishment; K.A.J. and C.M.N., human M4 data; J.E.A., electrophysiology; A.L.R., substructure search and chemical library selection; J.W., generated M4 knockout mice; D.W., analysis template design; P.J.C., principal investigator.

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Correspondence to P Jeffrey Conn.

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Shirey, J., Xiang, Z., Orton, D. et al. An allosteric potentiator of M4 mAChR modulates hippocampal synaptic transmission. Nat Chem Biol 4, 42–50 (2008). https://doi.org/10.1038/nchembio.2007.55

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