Selective activation of the M1 muscarinic acetylcholine receptor achieved by allosteric potentiation

The forebrain cholinergic system promotes higher brain function in part by signaling through the M₁ muscarinic acetylcholine receptor (mAChR). During Alzheimer''s disease (AD), these cholinergic neurons degenerate, therefore selectively activating M₁ receptors could improve cognitive function in these patients while avoiding unwanted peripheral responses associated with non-selective muscarinic agonists. We describe here benzyl quinolone carboxylic acid (BQCA), a highly selective allosteric potentiator of the M₁ mAChR. BQCA reduces the concentration of ACh required to activate M₁ up to 129-fold with an inflection point value of 845 nM. No potentiation, agonism, or antagonism activity on other mAChRs is observed up to 100 μM. Furthermore studies in M₁^−/− mice demonstrates that BQCA requires M₁ to promote inositol phosphate turnover in primary neurons and to increase c-fos and arc RNA expression and ERK phosphorylation in the brain. Radioligand-binding assays, molecular modeling, and site-directed mutagenesis experiments indicate that BQCA acts at an allosteric site involving residues Y179 and W400. BQCA reverses scopolamine-induced memory deficits in contextual fear conditioning, increases blood flow to the cerebral cortex, and increases wakefulness while reducing delta sleep. In contrast to M₁ allosteric agonists, which do not improve memory in scopolamine-challenged mice in contextual fear conditioning, BQCA induces β-arrestin recruitment to M₁, suggesting a role for this signal transduction mechanism in the cholinergic modulation of memory. In summary, BQCA exploits an allosteric potentiation mechanism to provide selectivity for the M₁ receptor and represents a promising therapeutic strategy for cognitive disorders.Basal forebrain cholinergic neurons innervate information processing centers in the hippocampus and cortex to promote attention and memory. During AD, these neurons profoundly degenerate, contributing to cognitive impairment (1). While cholinesterase inhibitors demonstrate the therapeutic potential for boosting cholinergic function in AD, they are limited by tolerability and provide modest benefit, thus there remains a tremendous need for improved therapies (2). Selectively targeting the ACh receptors involved in memory, while sparing receptors involved in other physiological processes, could provide additional efficacy, a widely pursued approach that has yet to lead to new medicines.ACh signals by activating ligand-gated ion channels (nicotinic receptors) and metabotropic (muscarinic) G protein-coupled receptors (GPCRs) designated M₁–M₅. Among the mAChRs, M₁ is most abundantly expressed in the hippocampus, cortex, and striatum, and localizes to postsynaptic membranes (3), where it signals via G_q/G₁₁ G-proteins to phospholipase C and through other G-proteins to additional signaling systems (4, 5). M₁ regulates several ion channels including KCNQ inwardly rectifying K⁺ currents, voltage-gated calcium channels, and NMDA receptors (4–9). Thus M₁ could mediate much of the cognitive effects of ACh. Supporting this hypothesis xanomeline, an M₁/M₄ preferring agonist, improved cognition and behavior in AD patients but was not tolerated due to unwanted cholinergic effects (10). Additional studies suggest that M₁ activation could slow AD progression by reducing Aβ42 peptides (11). Thus a drug that activates M₁ could potentially improve cognition while over time slowing the progression of the disease. Unfortunately, conservation of the ACh binding site has precluded the discovery of selective agonists.Many GPCRs, including mAChRs (12), have allosteric binding sites bound by small molecules that activate the receptor in the absence of ligand (allosteric agonist) or enhance the response to native ligand (positive allosteric modulator) (13). As allosteric sites are theoretically under less evolutionary constraint, targeting them affords opportunities for selectivity. This concept was demonstrated by the M₁ allosteric agonist TBPB (14) and a collection of relatively selective positive allosteric modulators (15). Here we describe BQCA [1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid], an orally available drug-like molecule that regulates memory and brain function by potentiating M₁.