Abstract
Positive allosteric modulators (PAMs) that target the M1 muscarinic acetylcholine (ACh) receptor (M1 mAChR) are potential treatments for cognitive deficits in conditions such as Alzheimer disease and schizophrenia. We recently reported novel 4-phenylpyridine-2-one and 6-phenylpyrimidin-4-one M1 mAChR PAMs with the potential to display different modes of positive allosteric modulation and/or agonism but whose molecular mechanisms of action remain undetermined. The current study compared the pharmacology of three such novel PAMs with the prototypical first-generation PAM, benzyl quinolone carboxylic acid (BQCA), in a recombinant Chinese hamster ovary (CHO) cell line stably expressing the human M1 mAChR. Interactions between the orthosteric agonists and the novel PAMs or BQCA suggested their allosteric effects were solely governed by modulation of agonist affinity. The greatest degree of positive co-operativity was observed with higher efficacy agonists, whereas minimal potentiation was observed when the modulators were tested against the lower efficacy agonist, xanomeline. Each PAM was investigated for its effects on the endogenous agonist ACh on three different signaling pathways [extracellular signal-regulated kinases 1/2 phosphorylation, inositol monophosphate (IP1) accumulation, and β-arrestin-2 recruitment], revealing that the allosteric potentiation generally tracked with the efficiency of stimulus-response coupling, and that there was little pathway bias in the allosteric effects. Thus, despite the identification of novel allosteric scaffolds targeting the M1 mAChR, the molecular mechanism of action of these compounds is largely consistent with a model of allostery previously described for BQCA, suggesting that this may be a more generalized mechanism for M1 mAChR PAM effects than previously appreciated.
Highlights
Each positive allosteric modulator (PAM) was investigated for its effects on the endogenous agonist ACh on three different signaling pathways [extracellular signalregulated kinases 1/2 phosphorylation, inositol monophosphate (IP1) accumulation, and b-arrestin-2 recruitment], revealing that the allosteric potentiation generally tracked with the efficiency of stimulus-response coupling, and that there was little pathway bias in the allosteric effects
When assessed for effects on the IP1 signaling pathway, our preliminary pharmacological characterization indicated that MIPS1674 was an allosteric agonist with minimal PAM activity against ACh, MIPS1745 was a “pure” PAM of ACh with no direct allosteric agonism, whereas MIPS1780 behaved as a mixed PAM-agonist in modulating ACh function
The fact that these three PAMs potentially exhibited three different “allosteric phenotypes” (Mistry et al, 2016) formed the basis for selecting them for further pharmacological evaluation. These compounds were initially tested to ensure they were selective for the M1 muscarinic acetylcholine receptor (mAChR) over other mAChR subtypes using an extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation assay
Summary
The muscarinic acetylcholine (ACh) receptors (mAChRs) belong to the rhodopsin-like (class A) family of G protein-. A.C. is a senior principal and P.M.S. a principal research fellow of the National Health and Medical Research Council of Australia. Distinct mAChR subtypes (denoted M1–M5) exhibit a widespread distribution throughout the central nervous system and peripheral organs (Caulfield, 1993; Nathanson, 2008; Kruse et al, 2014). An ever-growing array of additional signaling pathways, including those not necessarily mediated by G proteins, has been linked to mAChR activation (Lanzafame et al, 2003)
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