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Muscarinic Receptor Agonists: Not Created Equal

11 July 2012. Agonists of the M1 muscarinic acetylcholine receptor show promise as schizophrenia drug targets. However, in a new study published June 20 in the Journal of Neuroscience, P. Jeffrey Conn of Vanderbilt University in Nashville, Tennessee, and colleagues support the idea that substantial variability is present among different agonists, and that these differences can alter their therapeutic potential.

Current pharmacological treatments for schizophrenia leave a lot to be desired. Antipsychotic drugs ameliorate or eliminate positive symptoms in most patients, but leave negative and cognitive symptoms largely unaffected. Given that the latter are the best predictor of functional outcome, developing new treatments for schizophrenia is of paramount importance. Recently, muscarinic acetylcholine receptors have been gaining momentum as viable drug targets for cognitive disorders including schizophrenia and Alzheimer’s disease (Bolbecker and Shekhar, 2012; see SRF related news story; SRF news story). For example, M1 receptors are known to be important in various domains of cognition, and a pilot study of the M1- and M4-selective agonist xanomeline found a reduction in positive and negative symptoms, as well as cognitive improvements, in schizophrenia (Shekhar et al., 2008).

Lack of muscarinic agonists with selectivity for individual receptor subtypes has hampered past attempts at drug development. However, compounds that exhibit very high selectivity for single subtypes, such as M1, and particularly for allosteric sites away from the main ligand binding site, have recently been developed. M1 agonists are known to activate a variety of signaling pathways involving calcium, β-arrestin, and extracellular regulated signal kinase (ERK), and recent data suggest that different allosteric M1 agonists may activate different signaling pathways (Thomas et al., 2008). Thus, Conn and colleagues reasoned that if specific drugs activate only some of the M1-mediated responses, the effects of individual drugs, and their therapeutic potential, may vary widely. In the present study, the researchers examined this idea by comparing the signaling, electrophysiological, and behavioral properties of their two recently developed, highly selective M1 allosteric agonists—VU0357017 and VU0364572 (Lebois et al., 2010; Lebois et al., 2011).

Using non-neuronal cell lines, first author Gregory Digby and colleagues found that both agonists increased calcium mobilization and activated ERK1/2 phosphorylation, but neither had a significant effect on β-arrestin recruitment. Since both agonists, especially VU0357017, exhibited relatively weak effects on calcium and phosphorylated ERK1/2, the researchers hypothesized that the drugs may be partial agonists, and thus would behave differently in systems with low receptor expression. In fact, both drugs did behave as partial agonists, though VU0364572 had a stronger effect on both calcium mobilization and ERK1/2 phosphorylation.

Digby and colleagues next probed the physiological and behavioral function of their agonists. Using rat hippocampal slices, they examined the effect on synaptic plasticity, thought to underlie the memory-enhancing effects of M1 activation. Both drugs induced long-term potentiation in hippocampal slices (similar to what has been reported for other M1 agonists), though only VU0364572 induced long-term depression. To examine the behavioral correlates of these electrophysiological studies, the researchers assessed hippocampal-dependent cognitive functioning using the Morris water maze, an assay of spatial learning and memory, and contextual fear conditioning, another measure of learning. Although intraperitoneal injection of VU0357017 had no effect on water maze performance, injection of VU0364572 produced enhanced performance. Both drugs improved the acquisition of contextual fear.

Next, the researchers turned to the medial prefrontal cortex, a brain region implicated in the working memory and learning improvements seen with M1 agonists. A prior study of an M1 agonist from Conn’s group had found increased excitatory postsynaptic current (EPSC) activity in the medial prefrontal cortex and improved reversal learning (Shirey et al. 2009). In the current study, however, neither agonist affected the inter-event interval of firing, indicating no change in single EPSC frequency. Reversal learning was not measured. Thus, the lack of strong effects in medial prefrontal cortex with the new agents buttresses the idea of variability among different M1 agonists.

Conn’s group also assessed the ability of the agonists to reverse amphetamine-induced hyperlocomotion, thought to be an effect of M1 receptors on dopamine neurotransmission in the striatum and hypothesized to be the mechanism underlying the antipsychotic effect of xanomeline. Unfortunately, unlike a previous report from his group, neither agonist demonstrated an effect on hyperlocomotion, and exhibited only weak effects on the excitability of medium spiny neurons in the striatum, suggesting that these drugs will have limited antipsychotic properties.

Taken together, the results of the current study suggest that different allosteric agonists can have distinct effects on M1-mediated responses that produce varying therapeutic effects. As noted by the authors in the discussion, these data have implications for future drug development. “The present findings suggest that reliance on a streamlined strategy of optimizing with a single readout of M1 function could yield compounds that may not have the desired effects…. Also, measuring physiological effects of advanced compounds in multiple CNS systems is important to reduce the risk of inadvertently advancing drug candidates that have more restricted CNS actions.”—Allison A. Curley.

Digby GJ, Noetzel MJ, Bubser M, Utley TJ, Walker AG, Byun NE, Lebois EP, Xiang Z, Sheffler DJ, Cho HP, Davis AA, Nemirovsky NE, Mennenga SE, Camp BW, Bimonte-Nelson HA, Bode J, Italiano K, Morrison R, Daniels JS, Niswender CM, Olive MF, Lindsley CW, Jones CK, Conn PJ. Novel Allosteric Agonists of M1 Muscarinic Acetylcholine Receptors Induce Brain Region-Specific Responses That Correspond with Behavioral Effects in Animal Models. J Neurosci . 2012 Jun 20 ; 32(25):8532-8544. Abstract

Comments on News and Primary Papers
Comment by:  Bryan Roth, SRF Advisor
Submitted 15 July 2012
Posted 16 July 2012
  I recommend the Primary Papers

This is an interesting and important paper which serves as a cautionary reminder regarding the potential activity of allosteric modulators to have "functionally selective" effects on GPCR signaling (Urban et al., 2007; Allen and Roth, 2011). Thus, it has been known for decades (see Urban et al., 2007, for review) that GPCR "agonists" and "antagonists" can activate distinct signaling pathways and that a drug can appear to be an "agonist" for one pathway and have "antagonist" activity at another. As we have recently suggested, such activity could have potentially therapeutic implications for schizophrenia and related disorders (Allen et al., 2011).

Although it has been recognized for some time that allosteric modulators of GPCR activity may also have functionally selective actions (Sheffler and Conn, 2008) in vitro, it was unknown if these effects of signaling bias were therapeutically relevant.

What is important here is that Digby et al. rigorously demonstrate signaling bias in vitro and in situ, and then follow these findings up by showing that the signaling "fingerprint" is relevant to the apparent electrophysiological effects of the drugs in vivo.

Although the drugs lacked apparent antipsychotic drug-like actions, they clearly had different effects on synaptic physiology.

Taken together with many other papers of this sort which have appeared over the past several years, the findings reinforce the principle that relying on a single cellular readout for demonstrating the "agonist," "antagonist," or "allosteric" actions of a small molecule could ultimately be problematic for therapeutics.


Allen JA, Roth BL. Strategies to discover unexpected targets for drugs active at G protein-coupled receptors. Annu Rev Pharmacol Toxicol. 2011 Feb 10;51:117-44. Abstract

Allen JA, Yost JM, Setola V, Chen X, Sassano MF, Chen M, Peterson S, Yadav PN, Huang XP, Feng B, Jensen NH, Che X, Bai X, Frye SV, Wetsel WC, Caron MG, Javitch JA, Roth BL, Jin J. Discovery of β-arrestin-biased dopamine D2 ligands for probing signal transduction pathways essential for antipsychotic efficacy. Proc Natl Acad Sci U S A. 2011 Nov 8;108(45):18488-93. Abstract

Sheffler DJ, Conn PJ. Allosteric potentiators of metabotropic glutamate receptor subtype 1a differentially modulate independent signaling pathways in baby hamster kidney cells. Neuropharmacology. 2008 Sep;55(4):419-27. Abstract

Urban JD, Clarke WP, von Zastrow M, Nichols DE, Kobilka B, Weinstein H, Javitch JA, Roth BL, Christopoulos A, Sexton PM, Miller KJ, Spedding M, Mailman RB. Functional selectivity and classical concepts of quantitative pharmacology. J Pharmacol Exp Ther. 2007 Jan;320(1):1-13. Abstract

View all comments by Bryan RothComment by:  Rick Neubig
Submitted 19 July 2012
Posted 20 July 2012

This is a very useful paper, but it is worth pointing out that there is really no evidence for "ligand bias" or "functional selectivity" of these compounds. The VU analogs are partial agonists but show no bias toward one signal pathway or another. The low ERK and α-arrestin signaling are due to the lower degree of receptor reserve for those two pathways and the requirement for more efficacious agonists to signal. The correlation of the in-vitro studies with the different in-vivo readouts does provide very important guidance for pharmacologists about what properties are needed for M1 agonists to produce different physiological effects. It doesn't, however, give any real information about "biased agonists."

View all comments by Rick NeubigComment by:  Anantha ShekharAmanda Bolbecker
Submitted 27 July 2012
Posted 27 July 2012

The discovery of M1 allosteric agonists caused a great deal of excitement because they act on the M1 receptor fairly exclusively. The new research by Digby et al. suggests that this newer class of compounds can influence very specific molecular signaling pathways initiated by M1 activation, and that these pathways mediate specific aspects of cognition. These findings could have profound effects on the development of novel therapeutic agents for cognitive impairments seen in schizophrenia and other severe neuropsychiatric disorders.

View all comments by Anantha Shekhar
View all comments by Amanda BolbeckerComment by:  Bryan Roth, SRF Advisor
Submitted 1 August 2012
Posted 1 August 2012

Regarding the comment by Rick Neubig: while it could be argued that the paper does not provide "gold standard" pharmacological data for bona fide functional selectivity/stimulus bias, in fairness to the authors I provide their conclusions directly from the paper:

"Thus, the differential effects of these M1 agonists on CNS responses may reflect a combination of partial agonist activity that is impacted by differences in receptor reserve and by an inherent stimulus bias at M1 so that these compounds are not capable of fully activating some responses, even in systems in which the receptor is highly expressed…. Thus, different levels of M1 expression are likely to contribute to the differential responses to VU0357017 and VU0364572 observed in these studies. However, it was interesting to find that VU0357017 never achieved full efficacy in activation of ERK1/2 phosphorylation, even in cell lines with strong induction of M1 expression to levels that induced high receptor reserve in the calcium mobilization assay. Also, VU0357017 did not induce robust β-arrestin responses in the original cell line or in the TREx hM1 cells. Thus, the differential effects of these M1 agonists on CNS responses may reflect a combination of partial agonist activity that is impacted by differences in receptor reserve and by an inherent stimulus bias at M1 so that these compounds are not capable of fully activating some responses, even in systems in which the receptor is highly expressed."

View all comments by Bryan Roth

Comments on Related News

Related News: TBPB or Not to Be—The Latest on Muscarinic Receptor Agonists

Comment by:  Brian DeanElizabeth Scarr
Submitted 19 November 2008
Posted 19 November 2008

The data implicating acetylcholine in the treatment and neurobiology of schizophrenia continues to grow and has generated the hypothesis that activating specific cholinergic receptors will 1) lead to improvements in the cognitive deficits associated with the disorder and 2) prove to be an alternative mechanism for reducing psychotic symptoms. However, translating these concepts into clinically effective moieties has proven difficult. One problem in designing agonists for the muscarinic receptors is the highly conserved acetylcholine (orthosteric) binding site (Langmead et al., 2008). This has made it difficult to produce centrally active drugs that do not also activate peripheral M2 and M3 receptors, causing cholinergic side effects such as bradycardia, gastrointestinal problems and salivation. Thus, the discovery of allosteric binding sites on both muscarinic (Spalding et al., 2002; Langmead et al., 2006) and nicotinic (Bertrand and Gopalakrishnan, 2007), for review including principles of allosteric modulation) receptors was significant as these sites seem to have a lower degree of homology within receptor families and may offer alternative targets for drug development. The recent characterization of the allosteric M1 agonist, TBPB (1-(1’-2-methylbenzyl)-1,4’-bipiperidin-4-yl) (Jones et al., 2008), is the latest addition to a growing pharmacopeia of novel compounds that may prove to be effective in treating the symptoms of schizophrenia.

This news story contains commentary on other potential allosteric agonists that have been reported to target muscarinic M4 receptors (LY2033298, VU0152099, and VU0152100) and their effectiveness in animal models predictive of antipsychotic action. It also mentions AC-260584, a M1 receptor allosteric agonist, which has been shown to reduce amphetamine and MK-801-induced hyperactivity as well as improving spatial memory performance (Vanover et al., 2008). Another selective M1 allosteric agonist (77-LH-28-1) has been shown to mobilize calcium and enhance NMDA-mediated excitation in rat hippocampus (Langmead et al., 2008), as well as demonstrating procognitive effects in preclinical trials (Watson et al., 2008). Together, these data sustain the promise that muscarinic allosteric agonists for the M4 receptor may be beneficial for psychotic symptoms whereas those that target the M1 receptor may improve both cognitive processing and psychotic symptoms.

With regard to the nicotinic receptors it has been reported that galantamine, a combined acetylcholinesterase inhibitor and allosteric potentiator of nicotinic α7 and α4β2 receptors, may perform better than acetylcholinesterase inhibitors (Deutsch et al., 2008). Although only modestly brain-penetrant, the α7 nicotinic allosteric potentiator NS1738 (1-(5-chloro-2-hydroxy-phenyl)-3-(2-chloro-5-trifluoromethylphenyl)-urea), reduces the scopolamine-induced deficits seen in acquisition of water-maze learning tasks and improved social recognition in rats (Timmermann et al., 2007). Other α7 nicotinic allosteric potentiators have been reported to improve performance in a variety of learning and/or memory tasks and to normalize sensory gating deficits (Ng et al., 2007; Hurst et al., 2007; Kohlhaas et al., 2006). Interestingly, 17β-estradiol appears to act as a positive allosteric modulator at human but not rat α4β2 nicotinic receptors (Paradiso et al., 2001) but to date few clinical studies have used 17β-estradiol as an adjunct to antipsychotics; those studies that have used this approach have not yielded compelling results (see, e.g., (Bergemann et al., 2008a; Bergemann et al., 2007; Bergemann et al., 2008b). Again, current data lend credence to the theory that activating nicotinic receptors may improve cognitive dysfunction and improve sensory gating, as indeed nicotine does for the P50 sensory gating deficit in some patients with schizophrenia. It should be noted however, that improved P50 gating did not correspond to any improvement in the clinical ratings of patients (Adler et al., 2005) and thus may not constitute a clinical improvement.

As well as showing improved selectivity for the receptor of interest, allosteric modulation offers a number of other benefits. It allows the system to continue integrating the temporal and spatial signaling patterns of the native transmitter, both of which are essential for normal neurotransmission. Furthermore, current data suggests that allosteric agonists do not cause appreciable receptor internalization (Watson et al., 2008), which should reduce the rate of desensitization compared to orthosteric agonists. Despite the promising preclinical results obtained with the allosteric agents, they are still a long way from clinical use and a number of problems may arise. Firstly, as pointed out in the article, there is the possibility that these agents interact with allosteric sites on other receptors. Secondly, it has been shown that allosteric agonists can cause differential activation of second messenger systems (Thomas et al., 2008), which may limit their therapeutic usefulness. Finally, it is highly likely that schizophrenia is a syndrome, consisting of a number of diseases. Thus, it is improbable that any single treatment is going to be effective across the entire syndrome; rather, as is the case for existing antipsychotic medications, cholinergic potentiators may prove to be efficacious in a subgroup of people with the disorder. A group that might benefit from treatment with drugs which selectively activate components of the cholinergic system is muscarinic-receptor-deficit schizophrenia, a subgroup within schizophrenia that has lost, on average, 75 percent of their cortical M1 receptors (Scarr et al., 2008). Despite these potential issues, the allosteric agonists/modulators are the result of novel approaches to the development of drugs to treat schizophrenia. It is hoped they also mark the start of a very stimulating era in schizophrenia research.


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