In the December 2 Nature Chemical Biology, researchers led by Jeffrey Conn at Vanderbilt University, Nashville, Tennessee, report that they have developed a small molecule that not only boosts the response to acetylcholine by almost 50-fold, but more importantly, that acts specifically on the M4-type muscarinic acetylcholine receptors (mAChRs) predominantly found in the central nervous system. Called VU10010, the chemical potentiates the effect of acetylcholine in tissue from the rodent hippocampus. This brain region is intimately associated with learning and memory and deteriorates progressively in Alzheimer disease (AD). It is not clear if such a molecule could prove useful in the treatment of AD, but it could help researchers develop a detailed understanding of the functional roles of M4 receptors in brain circuits, suggest the authors.

Three acetylcholinesterase inhibitors are currently approved for the treatment of mild to moderate Alzheimer disease (AD) in the U.S. While they extend the half-life of acetylcholine in the central nervous system, they also heighten acetylcholinergic transmission in peripheral tissues, which leads to side effects including a slow heart rate, gastrointestinal irritation, and sweating. These can limit the amount of drug a patient can tolerate. Such limits could be overcome by drugs that specifically target mAChRs in the central nervous system.

The difficulty in developing drugs for specific subtypes of any neurotransmitter receptor is that the transmitter binding site is normally highly conserved, so any chemical that blocks or potentiates one receptor usually has the same effect on the others. One way around this is to design molecules that modulate auxiliary components, such as the G proteins that couple to the receptors. Such allosteric modulators with modest effects have been reported for the M1 and M4 mAChRs (see Lazareno et al., 1998 and Lazareno et al., 2004, respectively). It was this approach that Conn and colleagues took. As a starting point, joint first authors Jana Shirey, Zixiu Xiang, and colleagues focused on a chemical (LY2033298) previously reported to be a specific allosteric modulator of M4 muscarinic receptors. They searched a chemical library for chemicals with a similar core structure and then narrowed down 232 potential candidates to three molecules that robustly potentiate M4 receptor activation but have no effect on the other four muscarinic receptor subtypes: M1, M2, M3 and M5. The best compound decreased by about eightfold the amount of acetylcholine needed to increase intracellular calcium in Chinese hamster ovary cells stably expressing both rat M4 receptor and a chimeric G protein that mobilizes calcium stores. This chimera is a widely used tool to study the activation of inhibitory G proteins (see Coward et al., 1999).

By comparing the activity and structure of these three compounds, the researchers identified various modifications that might make the chemicals more potent. These modifications led to a fresh series of compounds, VU10000 to VU10010. Putting this small library through its paces, the researchers found that the best compound, VU10010, decreased the amount of acetylcholine needed to get a half-maximal response by 47-fold. VU10010 had no effect on the other muscarinic receptors or on two unrelated G-protein-coupled receptors (GPCR), the family I GPCR, P2Y1, and mGluR5, a family III GPCR.

Next the researchers turned to rat hippocampal slices. There, muscarinic agonists depress transmission at excitatory glutamatergic synapses and reduce transmission at inhibitory GABAergic synapses. Shirey and colleagues found that the compound had no effect on these pathways alone, but greatly potentiated the effect of the cholinergic agonist cabachol on excitatory pathways. In the presence of VU10010, cabachol-induced depression of excitatory post-synaptic currents increased by about 37 percent. In contrast, the compound had no effect on cabachol-induced reduction of inhibitory post-synaptic currents. It also had no effect on excitatory currents in hippocampal slices from M4 knockout mice, supporting the contention that the compound is a specific M4 modulator.

How does VU10010 work? The authors found that it cannot displace the acetylcholine antagonist scopolamine from the active site of the receptor, which is in keeping with the compound acting on some allosteric site. The researchers found that it does increase the affinity of acetylcholine for the receptor by about 14-fold, but that alone would not explain the nearly 50-fold increase in calcium mobilization the compound achieves. That may be due to VU10010’s actions on G proteins. The researchers found that the compound increases maximal GTP binding to membranes in CHO cells that express the rat M4 receptor. Thus, the compound seems to increase the M4 receptor’s affinity for acetylcholine and its coupling of the receptor to G proteins, though how it does that is not clear.

Though compounds that specifically target the M1 receptor are currently being evaluated in AD clinical trials (see ARF related news story), it is not clear if an M4 potentiator might be useful for this disease. “The bulk of the evidence has pointed towards a more relevant role of the M1 receptor in AD, because it is the muscarinic receptor subtype most closely linked to cognition and behavior, as well as to synaptic plasticity, excitability, and amyloidogenesis,” Allan Levey, an AD clinician at Emory University in Atlanta, Georgia, told ARF via e-mail. “Unfortunately, we still do not have selective drugs for M1 available, and many of the drugs touted as being M1-selective appear to bind to other receptors, including M3 and M4,” he said. Levey added that the M4 receptor is predominantly found in the striatum and that studies in animals, including the M4 knockout mouse, point to a key role for this receptor in motor function. “However, there is also some reason to believe that it could be important in behavior as well,” he added.

In the hippocampus, a major site of neurodegeneration in AD patients, the VU10010 compound seems to selectively depress glutamatergic transmission. “This selective regulation of excitatory synaptic transmission is a first critical step in developing a detailed understanding of the roles of M4 in modulating hippocampal function,” write the authors. Levey agreed with that sentiment. “The development of truly selective and potent drugs, as described in this study by Jeffrey Conn’s group, is an exciting first step in having pharmacological tools to really sort out the functions of the M4 receptor and its potential utility as a target for drugs for neurodegenerative diseases, including Alzheimer and Parkinson diseases,” he wrote.—Tom Fagan


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News Citations

  1. Cholinergic Transmission and Aβ: Boosting M1 Receptors Treats Model

Paper Citations

  1. . Subtype-selective positive cooperative interactions between brucine analogues and acetylcholine at muscarinic receptors: radioligand binding studies. Mol Pharmacol. 1998 Mar;53(3):573-89. PubMed.
  2. . Thiochrome enhances acetylcholine affinity at muscarinic M4 receptors: receptor subtype selectivity via cooperativity rather than affinity. Mol Pharmacol. 2004 Jan;65(1):257-66. PubMed.
  3. . Chimeric G proteins allow a high-throughput signaling assay of Gi-coupled receptors. Anal Biochem. 1999 Jun 1;270(2):242-8. PubMed.

Further Reading

Primary Papers

  1. . An allosteric potentiator of M4 mAChR modulates hippocampal synaptic transmission. Nat Chem Biol. 2008 Jan;4(1):42-50. PubMed.