Characterization of the M₄ muscarinic acetylcholine receptor (mAChR) positive allosteric modulator, VU0467154, at chemo-genetically modified M₄ receptors

P. Valuskova¹, S. J. Bradley¹, J. P. Conn², A. B. Tobin¹. ¹Institute of Molecular, Cell and Systems Biology, University of Glasgow, Glasgow, United Kingdom, ²Center for Neuroscience Drug Discovery, Vanderbilt University, Nashville.

Introduction: M₄ mAChRs play a crucial role in the control of basal locomotor activity (1), modulation of dopamine-dependent behaviours, neurotransmission and cognitive processes. Recently, the M₄ mAChR has been highlighted as a potential therapeutic target for neuropsychiatric disorders, such as schizophrenia, and pharmacological activation of M₄ mAChRs has been shown to alleviate some of the positive and cognitive symptoms associated with this disease (2).

Method: We have generated a transgenic mouse model whereby the wild-type M₄ receptor is replaced by a chemo-genetically modified M₄ mutant receptor (M₄ DREADD), where two mutations in the orthosteric binding pocket cause a loss of ACh activity but a gain in responsiveness to an otherwise inert ligand, clozapine-N-oxide (CNO). Here we conduct an in vitro pharmacological characterization of the M₄ positive allosteric modulator (M₄ PAM), VU0467154 (3), at M₄ DREADDs, to explore the utility of this ligand as a pharmacological tool in the M₄ DREADD mice.

Results: VU0467154 potentiated acetylcholine (ACh)-mediated displacement of [³H]-NMS binding at the M₄ wild-type receptor but had no effect on affinity of ACh at the M₄ DREADD. To further examine the functional modulation mediated by VU0467154, we performed allosteric interaction studies between orthosteric ligands and VU0467154 at both M₄ wild-type and M₄ DREADDs. VU0467154 dose-dependently potentiated the ability of ACh and CNO to stimulate ERK1/2 phosphorylation at the M₄ wild-type receptor. At M₄ DREADD, VU0467154 enhanced the potency of ACh to stimulate ERK1/2 phosphorylation, however, had minimal effect on the action of CNO at the M₄ DREADD in the same pathway. Our initial behavioural experiments with M₄ DREADD mice revealed reduced cued and contextual fear conditioning responses that suggested the impairment of amygdala-based and hippocampal-based learning and memory, respectively. Our future experiments will be focused on restoration of the behavioural deficits associated with a lack of M₄ mAChRs after activation of M₄ DREADDs with CNO and/or M₄ PAM.

Conclusions: We have found that the M₄ PAM, VU0467154, can potentiate ACh-stimulation of ERK1/2 phosphorylation at the M₄ DREADD, highlighting the potential utility of this compound to restore ACh signaling in M₄ DREADD mice.

References:

1. J. Gomeza et al.(1999). Proc. Natl. Acad. Sci. U.S.A. 96, 10483-10488.

2. M. Bubser et al.(2014). ACS Chem Neurosci. 5, 920-942.

3. M. R. Wood et al.(2017). Bioorg. Med. Chem. Lett. 27, 171-175 (2017).