Monoamine Oxidase Inhibition by Major Tanshinones from Salvia miltiorrhiza and Selective Muscarinic Acetylcholine M4 Receptor Antagonism by Tanshinone I.

Monoamine oxidases (MAOs) and muscarinic acetylcholine receptors (mAChRs) are considered important therapeutic targets for Parkinson's disease (PD). Lipophilic tanshinones are major phytoconstituents in the dried roots of Salvia miltiorrhiza that have demonstrated neuroprotective effects against dopaminergic neurotoxins and the inhibition of MAO-A. Since MAO-B inhibition is considered an effective therapeutic strategy for PD, we tested the inhibitory activities of three abundant tanshinone congeners against recombinant human MAO (hMAO) isoenzymes through in vitro experiments. In our study, tanshinone I (1) exhibited the highest potency against hMAO-A, followed by tanshinone IIA and cryptotanshinone, with an IC50 less than 10 µM. They also suppressed hMAO-B activity, with an IC50 below 25 µM. Although tanshinones are known to inhibit hMAO-A, their enzyme inhibition mechanism and binding sites have yet to be investigated. Enzyme kinetics and molecular docking studies have revealed the mode of inhibition and interactions of tanshinones during enzyme inhibition. Proteochemometric modeling predicted mAChRs as possible pharmacological targets of 1, and in vitro functional assays confirmed the selective M4 antagonist nature of 1 (56.1% ± 2.40% inhibition of control agonist response at 100 µM). These findings indicate that 1 is a potential therapeutic molecule for managing the motor dysfunction and depression associated with PD.

Discovery of a novel 2,3-dimethylimidazo[1,2-a]pyrazine-6-carboxamide M4 positive allosteric modulator (PAM) chemotype.

This Letter details our efforts to discover structurally unique M4 PAMs containing 5,6-heteroaryl ring systems. In an attempt to improve the DMPK profiles of the 2,3-dimethyl-2H-indazole-5-carboxamide and 1-methyl-1H-benzo[d][1,2,3]triazole-6-carboxamide cores, we investigated a plethora of core replacements. This exercise identified a novel 2,3-dimethylimidazo[1,2-a]pyrazine-6-carboxamide core that provided improved M4 PAM activity and CNS penetration.

Discovery of structurally distinct tricyclic M4 positive allosteric modulator (PAM) chemotypes.

This Letter details our efforts to develop new M4 PAM scaffolds with improved pharmacological properties. This endeavor involved replacing the 3,4-dimethylpyridazine core with two novel cores: a 2,3-dimethyl-2H-indazole-5-carboxamide core or a 1-methyl-1H-benzo[d][1,2,3]triazole-6-carboxamide core. Due to shallow SAR, these cores were further evolved into two unique tricyclic cores: an 8,9-dimethyl-8H-pyrazolo[3,4-h]quinazoline core and an 1-methyl-1H-[1,2,3]triazolo[4,5-h]quinazoline core. Both tricyclic cores displayed low nanomolar potency against both human and rat M4.

Evaluation of Operational Models of Agonism and Allosterism at Receptors with Multiple Orthosteric Binding Sites.

Current operational models of agonism and allosterism quantify ligand actions at receptors where agonist concentration-response relationships are nonhyperbolic by introduction of a transducer slope that relates receptor occupancy to response. However, for some receptors nonhyperbolic concentration-response relationships arise from multiple endogenous agonist molecules binding to a receptor in a cooperative manner. Thus, we developed operational models of agonism in systems with cooperative agonist binding and evaluated the models by simulating data describing agonist effects. The models were validated by analyzing experimental data demonstrating the effects of agonists and allosteric modulators at receptors where agonist binding follows hyperbolic (M4 muscarinic acetylcholine receptors) or nonhyperbolic relationships (metabotropic glutamate receptor 5 and calcium-sensing receptor). For hyperbolic agonist concentration-response relationships, no differences in estimates of ligand affinity, efficacy, or cooperativity were observed when the slope was assigned to either a transducer slope or agonist binding slope. In contrast, for receptors with nonhyperbolic agonist concentration-response relationships, estimates of ligand affinity, efficacy, or cooperativity varied depending on the assignment of the slope. The extent of this variation depended on the magnitude of the slope value and agonist efficacy, and for allosteric modulators on the magnitude of cooperativity. The modified operational models described herein are well suited to analyzing agonist and modulator interactions at receptors that bind multiple orthosteric agonists in a cooperative manner. Accounting for cooperative agonist binding is essential to accurately quantify agonist and drug actions. SIGNIFICANCE STATEMENT: Some orthosteric agonists bind to multiple sites on a receptor, but current analytical methods to characterize such interactions are limited. Herein, we develop and validate operational models of agonism and allosterism for receptors with multiple orthosteric binding sites, and demonstrate that such models are essential to accurately quantify agonist and drug actions. These findings have important implications for the discovery and development of drugs targeting receptors such as the calcium-sensing receptor, which binds at least five calcium ions.

Discovery of a novel 3,4-dimethylcinnoline carboxamide M4 positive allosteric modulator (PAM) chemotype via scaffold hopping.

This Letter details our efforts to replace the 2,4-dimethylquinoline carboxamide core of our previous M4 PAM series, which suffered from high predicted hepatic clearance and protein binding. A scaffold hopping exercise identified a novel 3,4-dimethylcinnoline carboxamide core that provided good M4 PAM activity and improved clearance and protein binding profiles.

Discovery of a novel 2,4-dimethylquinoline-6-carboxamide M4 positive allosteric modulator (PAM) chemotype via scaffold hopping.

This Letter details our efforts to replace the 3-amino moiety, an essential pharmacophore for M4 PAM activity in most M4 PAMs to date, within the thieno[2,3-b]pyridine core, as the ß-amino carboxamide motif has been shown to engender poor solubility, varying degrees of P-gp efflux and represents a structural alert. A scaffold hopping exercise identified a novel 2,4-dimethylquinoline carboxamide core that provided M4 PAM activity and good CNS penetration without an amino moiety. In addition, MacMillan photoredox catalysis chemistry was essential for construction of the 2,4-dimethylquinoline core.

Crystal structures of the M1 and M4 muscarinic acetylcholine receptors.

Muscarinic M1-M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer's disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. Here we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 and M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. We also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains.

In Silico Analysis of Binding Interaction of Mamba Toxins with M4 and M2 Muscarinic Acetylcholine Receptors for Therapeutic Use in Alzheimer's Disease.

Muscarinic acetylcholine receptors are stimulated by the neurotransmitter acetylcholine and are involved in various functions across the human body. These receptors have surfaced for their potential use as targets in treatment of Alzheimer's disease. Muscarinic receptors have been reported to show binding interaction with various mamba toxins, such as dendrotoxins and muscarinic toxins that act as antagonists of these receptors. Therefore, in our study we have focused on the binding analysis of these mamba toxins with the M4 and M2 muscarinic acetylcholine autoreceptors for their potential use as targets in treating cognitive symptoms associated with Alzheimer's disease. A ligand dataset was developed that consisted of dendrotoxins and muscarinic toxins originating from various mamba species. Receptor dataset consisted of M4 and M2 muscarinic acetylcholine autoreceptors. Docking studies were performed using AutoDock 4.2 between these ligands with each receptor and further analysis was done using various computational tools. Docking experiments were performed and analyzed to check the binding compatibilities between mamba toxins and muscarinic acetylcholine autoreceptors. Detail analysis revealed that these ligands bind to active site residues of both receptors. Therefore by these in silico results, we suggest that the mamba toxins can be potential antagonists of the M4 and M2 muscarinic acetylcholine autoreceptors.

Biochemical characterization of a heterotrimeric G(i)-protein activator peptide designed from the junction between the intracellular third loop and sixth transmembrane helix in the m4 muscarinic acetylcholine receptor.

Muscarinic acetylcholine receptors (mAChRs) are G-protein coupled receptors (GPCRs) that are activated by acetylcholine released from parasympathetic nerves. The mAChR family comprises 5 subtypes, m1-m5, each of which has a different coupling selectivity for heterotrimeric GTP-binding proteins (G-proteins). m4 mAChR specifically activates the Gi/o family by enhancing the guanine nucleotide exchange factor (GEF) reaction with the Gα subunit through an interaction that occurs via intracellular segments. Here, we report that the m4 mAChR mimetic peptide m4i3c(14)Gly, comprising 14 residues in the junction between the intracellular third loop (i3c) and transmembrane helix VI (TM-VI) extended with a C-terminal glycine residue, presents GEF activity toward the Gi1 α subunit (Gαi1). The m4i3c(14)Gly forms a stable complex with guanine nucleotide-free Gαi1 via three residues in the VTI(L/F) motif, which is conserved within the m2/4 mAChRs. These results suggest that this m4 mAChR mimetic peptide, which comprises the amino acid of the mAChR intracellular segments, is a useful tool for understanding the interaction between GPCRs and G-proteins.

The role of the M4 lipid-sensor in the folding, trafficking, and allosteric modulation of nicotinic acetylcholine receptors.

With the availability of high resolution structural data, increasing attention has focused on the mechanisms by which drugs and endogenous compounds allosterically modulate nicotinic acetylcholine receptor (nAChR) function. Lipids are potent modulators of the nAChR from Torpedo. Membrane lipids influence nAChR function by both conformational selection and kinetic mechanisms, stabilizing varying proportions of pre-existing resting, open, desensitized, and uncoupled conformations, as well as influencing the transitions between these conformational states. Structural and functional data highlight a role for the lipid-exposed M4 transmembrane α-helix of each subunit in lipid sensing, and suggest that lipids influence gating by altering the binding of M4 to the adjacent transmembrane α-helices, M1 and M3. M4 has also been implicated in both the folding and trafficking of nAChRs to the cell surface, as well as in the potentiation of nAChR gating by neurosteroids. Here, we discuss the roles of M4 in the folding, trafficking, and allosteric modulation of nAChRs. We also consider the hypothesis that variable chemistry at the M4-M1/M3 transmembrane α-helical interface in different nAChR subunits governs the capacity for potentiation by activating lipids. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'.

Detection and quantification of allosteric modulation of endogenous m4 muscarinic acetylcholine receptor using impedance-based label-free technology in a neuronal cell line.

Allosteric modulators of G protein-coupled receptors have the potential to achieve greater receptor subtype selectivity compared with ligands targeting the orthosteric site of this receptor family. However, the high attrition rate in GPCR drug discovery programs has highlighted the need to better characterize lead compounds in terms of their allosteric action, as well as the signals they elicit. Recently, the use of label-free technologies has been proposed as an approach to overcome some limitations of endpoint-based assays and detect global changes in the ligand-stimulated cell. In this study, we assessed the ability of an impedance-based label-free technology, xCELLigence, to detect allosteric modulation in a neuronal cell line natively expressing rodent M4 muscarinic acetylcholine receptors. We were able to demonstrate that positive allosteric modulation of the endogenous M4 muscarinic acetylcholine receptor can be detected using this technology. Importantly, the allosteric parameters estimated from the label-free approach are comparable to those estimated from endpoint-based assays.

Role of the third intracellular loop in the subtype-specific internalization and recycling of muscarinic M2 and M4 receptors.

Muscarinic M2, M4, and M2-M4 chimera receptors were transiently expressed in HEK-293 tsA201 cells, and agonist-dependent internalization of these receptors and recycling of internalized receptors were examined by measuring the amount of cell-surface receptors as [3H]N-methylscopolamine (NMS) binding activity. Coexpression of a dominant negative form of dynamin (DN-dynamin,dynamin K44A) greatly reduced the agonist-dependent internalization of M4 receptors but not of M2 receptors, as was reported by Vögler et al. (J Biol Chem 273, 12155-12160, 1998).The agonist-dependent internalization of M2/M4-i3/M2 chimera receptors (M2 receptors with the i3 loop replaced by that of M4 receptors) was greatly reduced by co-expression of DN-dynamin as was the case for M4 receptors, whereas the agonist-dependent internalization of M4/M2-i3/M4 chimera receptors was hardly affected by co-expression of DN-dynamin as was the case for M2 receptors.Internalized M2/M4-i3/M2 receptors as well as internalized M4 receptors were shown to be recycled back to the cell surface after removal of agonists, whereas no recycling was observed for M4/M2-i3/M4 receptors as well as M2 receptors. These results indicate that the i3 loops of M2 and M4 receptors take a major role in their agonist-dependent internalization and recycling.

Development of a radioligand, [(3)H]LY2119620, to probe the human M(2) and M(4) muscarinic receptor allosteric binding sites.

In this study, we characterized a muscarinic acetylcholine receptor (mAChR) potentiator, LY2119620 (3-amino-5-chloro-N-cyclopropyl-4-methyl-6-[2-(4-methylpiperazin-1-yl)-2-oxoethoxy]thieno[2,3-b]pyridine-2-carboxamide) as a novel probe of the human M2 and M4 allosteric binding sites. Since the discovery of allosteric binding sites on G protein-coupled receptors, compounds targeting these novel sites have been starting to emerge. For example, LY2033298 (3-amino-5-chloro-6-methoxy-4-methyl-thieno(2,3-b)pyridine-2-carboxylic acid cyclopropylamid) and a derivative of this chemical scaffold, VU152100 (3-amino-N-(4-methoxybenzyl)-4,6-dim​ethylthieno[2,3-b]pyridine carboxamide), bind to the human M4 mAChR allosteric pocket. In the current study, we characterized LY2119620, a compound similar in structure to LY2033298 and binds to the same allosteric site on the human M4 mAChRs. However, LY2119620 also binds to an allosteric site on the human M2 subtype. [(3)H]NMS ([(3)H]N-methylscopolamine) binding experiments confirm that LY2119620 does not compete for the orthosteric binding pocket at any of the five muscarinic receptor subtypes. Dissociation kinetic studies using [(3)H]NMS further support that LY2119620 binds allosterically to the M2 and M4 mAChRs and was positively cooperative with muscarinic orthosteric agonists. To probe directly the allosteric sites on M2 and M4, we radiolabeled LY2119620. Cooperativity binding of [(3)H]LY2119620 with mAChR orthosteric agonists detects significant changes in Bmax values with little change in Kd, suggesting a G protein-dependent process. Furthermore, [(3)H]LY2119620 was displaced by compounds of similar chemical structure but not by previously described mAChR allosteric compounds such as gallamine or WIN 62,577 (17-ß-hydroxy-17-α-ethynyl-δ-4-androstano[3,2-b]pyrimido[1,2-a]benzimidazole). Our results therefore demonstrate the development of a radioligand, [(3)H]LY2119620 to probe specifically the human M2 and M4 muscarinic receptor allosteric binding sites.

Antipsychotic drug-like effects of the selective M4 muscarinic acetylcholine receptor positive allosteric modulator VU0152100.

Accumulating evidence suggests that selective M4 muscarinic acetylcholine receptor (mAChR) activators may offer a novel strategy for the treatment of psychosis. However, previous efforts to develop selective M4 activators were unsuccessful because of the lack of M4 mAChR subtype specificity and off-target muscarinic adverse effects. We recently developed VU0152100, a highly selective M4 positive allosteric modulator (PAM) that exerts central effects after systemic administration. We now report that VU0152100 dose-dependently reverses amphetamine-induced hyperlocomotion in rats and wild-type mice, but not in M4 KO mice. VU0152100 also blocks amphetamine-induced disruption of the acquisition of contextual fear conditioning and prepulse inhibition of the acoustic startle reflex. These effects were observed at doses that do not produce catalepsy or peripheral adverse effects associated with non-selective mAChR agonists. To further understand the effects of selective potentiation of M4 on region-specific brain activation, VU0152100 alone and in combination with amphetamine were evaluated using pharmacologic magnetic resonance imaging (phMRI). Key neural substrates of M4-mediated modulation of the amphetamine response included the nucleus accumbens (NAS), caudate-putamen (CP), hippocampus, and medial thalamus. Functional connectivity analysis of phMRI data, specifically assessing correlations in activation between regions, revealed several brain networks involved in the M4 modulation of amphetamine-induced brain activation, including the NAS and retrosplenial cortex with motor cortex, hippocampus, and medial thalamus. Using in vivo microdialysis, we found that VU0152100 reversed amphetamine-induced increases in extracellular dopamine levels in NAS and CP. The present data are consistent with an antipsychotic drug-like profile of activity for VU0152100. Taken together, these data support the development of selective M4 PAMs as a new approach to the treatment of psychosis and cognitive impairments associated with psychiatric disorders such as schizophrenia.

Discovery of a selective M4 positive allosteric modulator based on the 3-amino-thieno[2,3-b]pyridine-2-carboxamide scaffold: development of ML253, a potent and brain penetrant compound that is active in a preclinical model of schizophrenia.

Herein we report a next generation muscarinic receptor 4 (M(4)) positive allosteric modulator (PAM), ML253 which exhibits nanomolar activity at both the human (EC(50)=56 nM) and rat (EC(50)=176 nM) receptors and excellent efficacy by the left-ward shift of the ACh concentration response curve (fold shift, human=106; rat=50). In addition, ML253 is selective against the four other muscarinic subtypes, displays excellent CNS exposure and is active in an amphetamine-induced hyperlocomotion assay.

Discovery of N-(4-methoxy-7-methylbenzo[d]thiazol-2-yl)isonicatinamide, ML293, as a novel, selective and brain penetrant positive allosteric modulator of the muscarinic 4 (M4) receptor.

Herein we describe the discovery and development of a novel class of M(4) positive allosteric modulators, culminating in the discovery of ML293. ML293 exhibited modest potency at the human M4 receptor (EC(50)=1.3 µM) and excellent efficacy as noted by the 14.6-fold leftward shift of the agonist concentration-response curve. ML293 was also selective versus the other muscarinic subtypes and displayed excellent in vivo PK properties in rat with low IV clearance (11.6 mL/min/kg) and excellent brain exposure (PO PBL, 10 mg/kg at 1h, [Brain]=10.3 µM, B:P=0.85).

The role of transmembrane domain 3 in the actions of orthosteric, allosteric, and atypical agonists of the M4 muscarinic acetylcholine receptor.

Despite the discovery of a diverse range of novel agonists and allosteric modulators of the M(4) muscarinic acetylcholine (ACh) receptor (mAChR), little is known about how such ligands activate the receptor. We used site-directed mutagenesis of conserved residues in transmembrane 3 (TMIII), a key region involved in G protein-coupled receptor activation, to probe the binding and function of prototypical orthosteric mAChR agonists, allosteric modulators, and "atypical" agonists. We found that most mutations did not affect the binding of the allosteric modulators, with the exception of W108(3.28)A and L109(3.29)A (which may contribute directly to the interface between allosteric and orthosteric sites) and mutation D112(3.32)N (which may cause a global disruption of a hydrogen bond network). Although numerous mutations affected signaling, we did not identify amino acids that were important for the functional activity of any one class of agonist (orthosteric, allosteric, or atypical) to the exclusion of any others, suggesting that TMIII is key for the transmission of stimulus irrespective of the agonist. We also identified two key residues, Trp108(3.28) and Asp112(3.32), that are essential for the transmission of binding cooperativity between 3-amino-5-chloro-6-methoxy-4-methyl-thieno[2,3-b]pyridine- 2-carboxylic acid cyclopropylamide (LY2033298) and ACh. Finally, we found that LY2033298 was able to rescue functionally impaired signaling of ACh at the majority of mutants tested in a manner that was inversely correlated with the ACh signaling efficacy, indicating that a key part of the mechanism of the positive cooperativity mediated by LY2033298 on the endogenous agonist involves a global drive of the receptor toward an active conformation.

Impact of species variability and 'probe-dependence' on the detection and in vivo validation of allosteric modulation at the M4 muscarinic acetylcholine receptor.

BACKGROUND AND PURPOSE: We recently characterized LY2033298 as a novel allosteric modulator and agonist at M(4) muscarinic acetylcholine receptors (mAChRs). Evidence also suggested a difference in the potency of LY2033298 at rodent relative to human M(4) mAChRs. The current study investigated the basis for the species difference of this modulator and used this knowledge to rationalize its in vivo actions. EXPERIMENTAL APPROACH: LY2033298 was investigated in vitro in CHO cells stably expressing human or mouse M(4) mAChRs, using assays of agonist-induced ERK1/2 or GSK-3α phosphorylation, [(35) S]-GTPγS binding, or effects on equilibrium binding of [(3) H]-NMS and ACh. The in vivo actions of LY2033298 were investigated in a mouse model of amphetamine-induced locomotor activity. The function of LY2033298 was examined in combination with ACh, oxotremorine or xanomeline. KEY RESULTS: LY2033298 had similar affinities for the human and mouse M(4) mAChRs. However, LY2033298 had a lower positive co-operativity with ACh at the mouse relative to the human M(4) mAChR. At the mouse M(4) mAChR, LY2033298 showed higher co-operativity with oxotremorine than with ACh or xanomeline. The different degrees of co-operativity between LY2033298 and each agonist at the mouse relative to the human M(4) mAChR necessitated the co-administration of LY2033298 with oxotremorine in order to show in vivo efficacy of LY2033298. CONCLUSIONS AND IMPLICATIONS: These results provide evidence for species variability when comparing the allosteric interaction between LY2033298 and ACh at the M(4) mAChR, and also highlight how the interaction between LY2033298 and different orthosteric ligands is subject to 'probe dependence'. This has implications for the validation of allosteric modulator actions in vivo.

Structural determinants of allosteric agonism and modulation at the M4 muscarinic acetylcholine receptor: identification of ligand-specific and global activation mechanisms.

The recently identified small molecule, 3-amino-5-chloro-6-methoxy-4-methylthieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298), is the first selective allosteric modulator of the muscarinic acetylcholine receptors (mAChRs) that mediates both receptor activation and positive modulation of the endogenous agonist, acetylcholine (ACh), via the same allosteric site on the M(4) mAChR. We thus utilized this novel chemical tool, as well as ACh, the bitopic (orthosteric/allosteric) agonist, McN-A-343, and the clinically efficacious M(1)/M(4) mAChR-preferring agonist, xanomeline, in conjunction with site-directed mutagenesis of four different regions of the M(4) mAChR (extracellular loops 1, 2, and 3, and transmembrane domain 7), to identify regions that govern ligand-specific modes of binding, signaling, and allosteric modulation. In the first extracellular loop (E1), we identified Ile(93) and Lys(95) as key residues that specifically govern the signaling efficacy of LY2033298 and its binding cooperativity with ACh, whereas Phe(186) in the E2 loop was identified as a key contributor to the binding affinity of the modulator for the allosteric site, and Asp(432) in the E3 loop appears to be involved in the functional (activation) cooperativity between the modulator and the endogenous agonist. In contrast, the highly conserved transmembrane domain 7 residues, Tyr(439) and Tyr(443), were identified as contributing to a key activation switch utilized by all classes of agonists. These results provide new insights into the existence of multiple activation switches in G protein-coupled receptors (GPCRs), some of which can be selectively exploited by allosteric agonists, whereas others represent global activation mechanisms for all classes of ligand.

Molecular mechanisms of action and in vivo validation of an M4 muscarinic acetylcholine receptor allosteric modulator with potential antipsychotic properties.

We recently identified LY2033298 as a novel allosteric potentiator of acetylcholine (ACh) at the M(4) muscarinic acetylcholine receptor (mAChR). This study characterized the molecular mode of action of this modulator in both recombinant and native systems. Radioligand-binding studies revealed that LY2033298 displayed a preference for the active state of the M(4) mAChR, manifested as a potentiation in the binding affinity of ACh (but not antagonists) and an increase in the proportion of high-affinity agonist-receptor complexes. This property accounted for the robust allosteric agonism displayed by the modulator in recombinant cells in assays of [(35)S]GTPgammaS binding, extracellular regulated kinase 1/2 phosphorylation, glycogen synthase kinase 3beta phosphorylation, and receptor internalization. We also found that the extent of modulation by LY2033298 differed depending on the signaling pathway, indicating that LY2033298 engenders functional selectivity in the actions of ACh. This property was retained in NG108-15 cells, which natively express rodent M(4) mAChRs. Functional interaction studies between LY2033298 and various orthosteric and allosteric ligands revealed that its site of action overlaps with the allosteric site used by prototypical mAChR modulators. Importantly, LY2033298 reduced [(3)H]ACh release from rat striatal slices, indicating retention of its ability to allosterically potentiate endogenous ACh in situ. Moreover, its ability to potentiate oxotremorine-mediated inhibition of condition avoidance responding in rodents was significantly attenuated in M(4) mAChR knockout mice, validating the M(4) mAChR as a key target of action of this novel allosteric ligand.