Discovery and Characterization of Novel Subtype-Selective Allosteric Agonists for the Investigation of M₁ Receptor Function in the Central Nervous System

Abstract

Cholinergic transmission in the forebrain is mediated primarily by five subtypes of muscarinic acetylcholine receptors (mAChRs), termed M1-M5. Of the mAChR subtypes, M1 is among the most heavily expressed in regions that are critical for learning and memory, and has been viewed as the most critical mAChR subtype for memory and attention mechanisms. Unfortunately, it has been difficult to develop selective activators of M1 and other individual mAChR subtypes, which has prevented detailed studies of the functional roles of selective activation of M1. Using a functional HTS screen and subsequent diversity-oriented synthesis approach we have discovered a novel series of highly selective M1 allosteric agonists. These compounds activate M1 with EC50 values in the 150 nM to 500 nM range and have unprecedented, clean ancillary pharmacology (no substantial activity at 10μM across a large panel of targets). Targeted mutagenesis revealed a potentially novel allosteric binding site in the third extracellular loop of the M1 receptor for these allosteric agonists. Optimized compounds, such as VU0357017, provide excellent brain exposure after systemic dosing and have robust in vivo efficacy in reversing scopolamine-induced deficits in a rodent model of contextual fear conditioning. This series of selective M1 allosteric agonists provides critical research tools to allow dissection of M1-mediated effects in the CNS and potential leads for novel treatments for Alzheimer’s disease and schizophrenia.

Antipsychotic Drug-Like Effects of the Selective M4 Muscarinic Acetylcholine Receptor Positive Allosteric Modulator VU0152100

“…However, as the higher doses of amphetamine used in the cognitive studies can induce increases in both dopamine and norepinephrine (McKittrick and Abercrombie 2007), it will be important in future studies to confirm that these effects of VU0152100 are mediated predominately through reversal of dopaminergic signaling. Finally, VU0152100 provides an important M 4 -selective tool compound that used along with recently reported M 1 -selective PAMs (Ma et al, 2009;Shirey et al, 2009) or allosteric agonists (Lebois et al, 2010;Digby et al, 2012) will allow a more complete understanding of the relative roles of these two mAChR subtypes in regulating multiple aspects of cognitive function.…”

“…Studies were conducted using conditioning chambers within sound attenuating cubicles as previously described (see Lebois et al, 2010). All rats were handled and injected with saline for 2 days before conditioning.…”

Molecular Mechanisms of Bitopic Ligand Engagement with the M1 Muscarinic Acetylcholine Receptor

“…) and ECL3 (Lys-392 E3 and Asp-393 E3 ) were also included based on the previous suggestion of involvement with allosteric agonist binding (27). Tables 1 and 2).…”

Chemical Modification of the M1 Agonist VU0364572 Reveals Molecular Switches in Pharmacology and a Bitopic Binding Mode

“…13−15 More recently, we reported discovery of VU0364572 and VU0357017 as highly selective M 1 agonists that appear to act at an allosteric site to activate the receptor. 7,16,17 Extensive profiling of VU0364572 and VU0357017 across a large panel of GPCRs revealed that these compounds have little or no activity at other GPCRs and are the most selective M 1 agonists reported to date. 7,16,18 It is thought that the allosteric mechanism of action of these novel M 1 agonists is critical for achieving high selectivity for M 1 relative to other mAChR subtypes.…”