Allosteric activators of muscarinic receptors as novel approaches for treatment of CNS disorders.

Muscarinic acetylcholine receptors (mAChRs) represent exciting therapeutic targets for the treatment of multiple CNS disorders. The high degree of conservation of amino acids comprising the orthosteric acetylcholine (ACh) binding site between individual mAChR subtypes has hindered the development of subtype-selective compounds that bind to this site. As a result, many academic and industry researchers are now focusing on developing allosteric activators of mAChRs including both positive allosteric modulators (PAMs) and allosteric agonists. In the past 10 years major advances have been achieved in the discovery of allosteric ligands that possess much greater selectivity for individual mAChR subtypes when compared to previously developed orthosteric agents. These novel allosteric modulators of mAChRs may provide therapeutic potential for treatment of a number of CNS disorders such as Alzheimer's disease and schizophrenia.

A selective allosteric potentiator of the M1 muscarinic acetylcholine receptor increases activity of medial prefrontal cortical neurons and restores impairments in reversal learning.

M(1) muscarinic acetylcholine receptors (mAChRs) may represent a viable target for treatment of disorders involving impaired cognitive function. However, a major limitation to testing this hypothesis has been a lack of highly selective ligands for individual mAChR subtypes. We now report the rigorous molecular characterization of a novel compound, benzylquinolone carboxylic acid (BQCA), which acts as a potent, highly selective positive allosteric modulator (PAM) of the rat M(1) receptor. This compound does not directly activate the receptor, but acts at an allosteric site to increase functional responses to orthosteric agonists. Radioligand binding studies revealed that BQCA increases M(1) receptor affinity for acetylcholine. We found that activation of the M(1) receptor by BQCA induces a robust inward current and increases spontaneous EPSCs in medial prefrontal cortex (mPFC) pyramidal cells, effects which are absent in acute slices from M(1) receptor knock-out mice. Furthermore, to determine the effect of BQCA on intact and functioning brain circuits, multiple single-unit recordings were obtained from the mPFC of rats that showed BQCA increases firing of mPFC pyramidal cells in vivo. BQCA also restored discrimination reversal learning in a transgenic mouse model of Alzheimer's disease and was found to regulate non-amyloidogenic APP processing in vitro, suggesting that M(1) receptor PAMs have the potential to provide both symptomatic and disease modifying effects in Alzheimer's disease patients. Together, these studies provide compelling evidence that M(1) receptor activation induces a dramatic excitation of PFC neurons and suggest that selectively activating the M(1) mAChR subtype may ameliorate impairments in cognitive function.

Discovery and characterization of novel allosteric potentiators of M1 muscarinic receptors reveals multiple modes of activity.

Activators of M(1) muscarinic acetylcholine receptors (mAChRs) may provide novel treatments for schizophrenia and Alzheimer's disease. Unfortunately, the development of M(1)-active compounds has resulted in nonselective activation of the highly related M(2) to M(5) mAChR subtypes, which results in dose-limiting side effects. Using a functional screening approach, we identified several novel ligands that potentiated agonist activation of M(1) with low micromolar potencies and induced 5-fold or greater leftward shifts of the acetylcholine (ACh) concentration-response curve. These ligands did not compete for binding at the ACh binding site, indicating that they modulate receptor activity by binding to allosteric sites. The two most selective compounds, cyclopentyl 1,6-dimethyl-4-(6-nitrobenzo[d][1,3]-dioxol-5-yl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (VU0090157) and (E)-2-(4-ethoxyphenylamino)-N'-((2-hydroxynaphthalen-1-yl)methylene)acetohydrazide (VU0029767), induced progressive shifts in ACh affinity at M(1) that were consistent with their effects in a functional assay, suggesting that the mechanism for enhancement of M(1) activity by these compounds is by increasing agonist affinity. These compounds were strikingly different, however, in their ability to potentiate responses at a mutant M(1) receptor with decreased affinity for ACh and in their ability to affect responses of the allosteric M(1) agonist, 1-[1'-(2-tolyl)-1,4'-bipiperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one. Furthermore, these two compounds were distinct in their abilities to potentiate M(1)-mediated activation of phosphoinositide hydrolysis and phospholipase D. The discovery of multiple structurally distinct positive allosteric modulators of M(1) is an exciting advance in establishing the potential of allosteric modulators for selective activation of this receptor. These data also suggest that structurally diverse M(1) potentiators may act by distinct mechanisms and differentially regulate receptor coupling to downstream signaling pathways.

Centrally active allosteric potentiators of the M4 muscarinic acetylcholine receptor reverse amphetamine-induced hyperlocomotor activity in rats.

Previous clinical and animal studies suggest that selective activators of M(1) and/or M(4) muscarinic acetylcholine receptors (mAChRs) have potential as novel therapeutic agents for treatment of schizophrenia and Alzheimer's disease. However, highly selective centrally penetrant activators of either M(1) or M(4) have not been available, making it impossible to determine the in vivo effects of selective activation of these receptors. We previously identified VU10010 [3-amino-N-(4-chlorobenzyl)-4, 6-dimethylthieno[2,3-b]pyridine-2-carboxamide] as a potent and selective allosteric potentiator of M(4) mAChRs. However, unfavorable physiochemical properties prevented use of this compound for in vivo studies. We now report that chemical optimization of VU10010 has afforded two centrally penetrant analogs, VU0152099 [3-amino-N-(benzo[d][1,3]dioxol-5-ylmethyl)-4,6-dimethylthieno[2,3-b]pyridine carboxamide] and VU0152100 [3-amino-N-(4-methoxybenzyl)-4,6-dimethylthieno[2,3-b]pyridine carboxamide], that are potent and selective positive allosteric modulators of M(4). VU0152099 and VU0152100 had no agonist activity but potentiated responses of M(4) to acetylcholine. Both compounds were devoid of activity at other mAChR subtypes or at a panel of other GPCRs. The improved physiochemical properties of VU0152099 and VU0152100 allowed in vivo dosing and evaluation of behavioral effects in rats. Interestingly, these selective allosteric potentiators of M(4) reverse amphetamine-induced hyperlocomotion in rats, a model that is sensitive to known antipsychotic agents and to nonselective mAChR agonists. This is consistent with the hypothesis that M(4) plays an important role in regulating midbrain dopaminergic activity and raises the possibility that positive allosteric modulation of M(4) may mimic some of the antipsychotic-like effects of less selective mAChR agonists.

An allosteric potentiator of M4 mAChR modulates hippocampal synaptic transmission.

Muscarinic acetylcholine receptors (mAChRs) provide viable targets for the treatment of multiple central nervous system disorders. We have used cheminformatics and medicinal chemistry to develop new, highly selective M4 allosteric potentiators. VU10010, the lead compound, potentiates the M4 response to acetylcholine 47-fold while having no activity at other mAChR subtypes. This compound binds to an allosteric site on the receptor and increases affinity for acetylcholine and coupling to G proteins. Whole-cell patch clamp recordings revealed that selective potentiation of M4 with VU10010 increases carbachol-induced depression of transmission at excitatory but not inhibitory synapses in the hippocampus. The effect was not mimicked by an inactive analog of VU10010 and was absent in M4 knockout mice. Selective regulation of excitatory transmission by M4 suggests that targeting of individual mAChR subtypes could be used to differentially regulate specific aspects of mAChR modulation of function in this important forebrain structure.