Elucidating the molecular logic of a metabotropic glutamate receptor heterodimer.

Metabotropic glutamate (mGlu) receptor protomers can heterodimerize, leading to different pharmacology compared to their homodimeric counterparts. Here, we use complemented donor-acceptor resonance energy transfer (CODA-RET) technology that distinguishes signaling from defined mGlu heterodimers or homodimers, together with targeted mutagenesis of receptor protomers and computational docking, to elucidate the mechanism of activation and differential pharmacology in mGlu2/4 heteromers. We demonstrate that positive allosteric modulators (PAMs) that bind an upper allosteric pocket in the mGlu4 transmembrane domain are active at both mGlu4/4 homomers and mGlu2/4 heteromers, while those that bind a lower allosteric pocket within the same domain are efficacious in homomers but not heteromers. We further demonstrate that both protomers of mGlu2/4 heteromers are cis-activated by their orthosteric agonists, signaling independently with no trans-activation detected. Intriguingly, however, upper pocket mGlu4 PAMs enable trans-activation in mGlu2/4 heteromers from mGlu4 to the mGlu2 protomer and also enhance cis-activation of the mGlu2 protomer. While mGlu2 PAMs enhanced mGlu2 cis-activation in the heterodimer, we were unable to detect trans-activation in the opposite direction from mGlu2 to the mGlu4 protomer, suggesting an asymmetry of signaling. These insights into the molecular logic of this receptor heteromer are critical to building toward precision targeted therapies for multiple neuropsychiatric disorders.

Discovery of VU6016235: A Highly Selective, Orally Bioavailable, and Structurally Distinct Tricyclic M4 Muscarinic Acetylcholine Receptor Positive Allosteric Modulator (PAM).

Herein, we report structure-activity relationship (SAR) studies to develop novel tricyclic M4 PAM scaffolds with improved pharmacological properties. This endeavor involved a "tie-back" strategy to replace a 5-amino-2,4-dimethylthieno[2,3-d]pyrimidine-6-carboxamide core, which led to the discovery of two novel tricyclic cores. While both tricyclic cores displayed low nanomolar potency against both human and rat M4 and were highly brain-penetrant, the 2,4-dimethylpyrido[4',3':4,5]thieno[2,3-d]pyrimidine tricycle core provided lead compound, VU6016235, with an overall superior pharmacological and drug metabolism and pharmacokinetics (DMPK) profile, as well as efficacy in a preclinical antipsychotic animal model.

Development of VU6036864: A Triazolopyridine-Based High-Quality Antagonist Tool Compound of the M5 Muscarinic Acetylcholine Receptor.

While the muscarinic acetylcholine receptor mAChR subtype 5 (M5) has been studied over decades, recent findings suggest that more in-depth research is required to elucidate a thorough understanding of its physiological function related to neurological and psychiatric disorders. Our efforts to identify potent, selective, and pharmaceutically favorable next-generation M5 antagonist tool compounds have led to the discovery of a novel triazolopyridine-based series. In particular, VU6036864 (45) showed exquisite potency (human M5 IC50 = 20 nM), good subtype selectivity (>500 fold selectivity against human M1-4), desirable brain exposure (Kp = 0.68, Kp,uu = 0.65), and high oral bioavailability (%F > 100%). VU6036864 (45) and its close analogues will support further studies of M5 as advanced antagonist tool compounds and play an important role in the emerging biology of M5.

Discovery of VU6007496: Challenges in the Development of an M1 Positive Allosteric Modulator Backup Candidate.

Herein we report progress toward a backup clinical candidate to the M1 positive allosteric modulator (PAM) VU319/ACP-319. Scaffold-hopping from the pyrrolo[2,3-b]pyridine-based M1 PAM VU6007477 to isomeric pyrrolo[3,2-b]pyridine and thieno[3,2-b]pyridine congeners identified several backup contenders. Ultimately, VU6007496, a pyrrolo[3,2-b]pyridine, advanced into late stage profiling, only to be plagued with unanticipated, species-specific metabolism and active/toxic metabolites which were identified in our phenotypic seizure liability in vivo screen, preventing further development. However, VU6007496 proved to be a highly selective and CNS penetrant M1 PAM, with minimal agonism, that displayed excellent multispecies IV/PO pharmacokinetics (PK), CNS penetration, no induction of long-term depression (or cholinergic toxicity) and robust efficacy in novel object recognition (minimum effective dose = 3 mg/kg p.o.). Thus, VU6007496 can serve as another valuable in vivo tool compound in rats and nonhuman primates, but not mouse, to study selective M1 activation.

Discovery of VU6008677: A Structurally Distinct Tricyclic M4 Positive Allosteric Modulator with Improved CYP450 Profile.

This Letter details our efforts to develop novel tricyclic muscarinic acetylcholine receptor subtype 4 (M4) positive allosteric modulator (PAM) scaffolds with improved pharmacological properties. This endeavor involved a "tie-back" strategy to replace the 3-amino-5-chloro-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide core, which led to the discovery of two novel tricyclic cores: an 8-chloro-9-methylpyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4-amine core and 8-chloro-7,9-dimethylpyrido[3',2':4,5]furo[3,2-d]pyrimidin-4-amine core. Both tricyclic cores displayed low nanomolar potency against human M4 and greatly reduced cytochrome P450 inhibition when compared with parent compound ML253.

MeCP2 gene therapy ameliorates disease phenotype in mouse model for Pitt Hopkins syndrome.

The neurodevelopmental disorder Pitt Hopkins syndrome (PTHS) causes clinical symptoms similar to Rett syndrome (RTT) patients. However, RTT is caused by MECP2 mutations whereas mutations in the TCF4 gene lead to PTHS. The mechanistic commonalities underling these two disorders are unknown, but their shared symptomology suggest that convergent pathway-level disruption likely exists. We reprogrammed patient skin derived fibroblasts into induced neuronal progenitor cells. Interestingly, we discovered that MeCP2 levels were decreased in PTHS patient iNPCs relative to healthy controls and that both iNPCs and iAstrocytes displayed defects in function and differentiation in a mutation-specific manner. When Tcf4+/- mice were genetically crossed with mice overexpressing MeCP2, molecular and phenotypic defects were significantly ameliorated, underlining and important role of MeCP2 in PTHS pathology. Importantly, post-natal intracerebroventricular gene replacement therapy with adeno-associated viral vector serotype 9 (AAV9)-expressing MeCP2 (AAV9.P546.MeCP2) significantly improved iNPC and iAstrocyte function and effectively ameliorated histological and behavioral defects in Tcf4+/- mice. Combined, our data suggest a previously unknown role of MeCP2 in PTHS pathology and common pathways that might be affected in multiple neurodevelopmental disorders. Our work highlights potential novel therapeutic targets for PTHS, including upregulation of MeCP2 expression or its downstream targets or, potentially, MeCP2-based gene therapy.

Potentiation of the muscarinic acetylcholine receptor 1 modulates neurophysiological features in a mouse model of Rett syndrome.

Rett syndrome (RTT) is a neurodevelopmental disorder primarily caused by mutations in the X chromosome-linked gene Methyl-CpG Binding Protein 2 (MECP2). Restoring MeCP2 expression after disease onset in a mouse model of RTT reverses phenotypes, providing hope for development of treatments for RTT. Translatable biomarkers of improvement and treatment responses have the potential to accelerate both preclinical and clinical evaluation of targeted therapies in RTT. Studies in people with and mouse models of RTT have identified neurophysiological features, such as auditory event-related potentials, that correlate with disease severity, suggesting that they could be useful as biomarkers of disease improvement or early treatment response. We recently demonstrated that treatment of RTT mice with a positive allosteric modulator (PAM) of muscarinic acetylcholine subtype 1 receptor (M1) improved phenotypes, suggesting that modulation of M1 activity is a potential therapy in RTT. To evaluate whether neurophysiological features could be useful biomarkers to assess the effects of M1 PAM treatment, we acutely administered the M1 PAM VU0486846 (VU846) at doses of 1, 3, 10 and 30 â€‹mg/kg in wildtype and RTT mice. This resulted in an inverted U-shaped dose response with maximal improvement of AEP features at 3 â€‹mg/kg but with no marked effect on basal EEG power or epileptiform discharges in RTT mice and no significant changes in wildtype mice. These findings suggest that M1 potentiation can improve neural circuit synchrony to auditory stimuli in RTT mice and that neurophysiological features have potential as pharmacodynamic or treatment-responsive biomarkers for preclinical and clinical evaluation of putative therapies in RTT.

Novel pharmacological targets for GABAergic dysfunction in ADHD.

Attention deficit/hyperactivity disorder (ADHD) is a neurodevelopment disorder that affects approximately 5% of the population. The disorder is characterized by impulsivity, hyperactivity, and deficits in attention and cognition, although symptoms vary across patients due to the heterogenous and polygenic nature of the disorder. Stimulant medications are the standard of care treatment for ADHD patients, and their effectiveness has led to the dopaminergic hypothesis of ADHD in which deficits in dopaminergic signaling, especially in cortical brain regions, mechanistically underly ADHD pathophysiology. Despite their effectiveness in many individuals, almost one-third of patients do not respond to stimulant treatments and the long-term negative side effects of these medications remain unclear. Emerging clinical evidence is beginning to highlight an important role of dysregulated excitatory/inhibitory (E/I) balance in ADHD. These deficits in E/I balance are related to functional abnormalities in glutamate and Gamma-Aminobutyric Acid (GABA) signaling in the brain, with increasing emphasis placed on GABAergic interneurons driving specific aspects of ADHD pathophysiology. Recent genome-wide association studies (GWAS) have also highlighted how genes associated with GABA function are mutated in human populations with ADHD, resulting in the generation of several new genetic mouse models of ADHD. This review will discuss how GABAergic dysfunction underlies ADHD pathophysiology, and how specific receptors/proteins related to GABAergic interneuron dysfunction may be pharmacologically targeted to treat ADHD in subpopulations with specific comorbidities and symptom domains. This article is part of the Special Issue on "PFC circuit function in psychiatric disease and relevant models".

Therapeutic Potential for Metabotropic Glutamate Receptor 7 Modulators in Cognitive Disorders.

Metabotropic glutamate receptor 7 (mGlu7) is the most highly conserved and abundantly expressed mGlu receptor in the human brain. The presynaptic localization of mGlu7, coupled with its low affinity for its endogenous agonist, glutamate, are features that contribute to the receptor's role in modulating neuronal excitation and inhibition patterns, including long-term potentiation, in various brain regions. These characteristics suggest that mGlu7 modulation may serve as a novel therapeutic strategy in disorders of cognitive dysfunction, including neurodevelopmental disorders that cause impairments in learning, memory, and attention. Primary mutations in the GRM7 gene have recently been identified as novel causes of neurodevelopmental disorders, and these patients exhibit profound intellectual and cognitive disability. Pharmacological tools, such as agonists, antagonists, and allosteric modulators, have been the mainstay for targeting mGlu7 in its endogenous homodimeric form to probe effects of its function and modulation in disease models. However, recent research has identified diversity in dimerization, as well as trans-synaptic interacting proteins, that also play a role in mGlu7 signaling and pharmacological properties. These novel findings represent exciting opportunities in the field of mGlu receptor drug discovery and highlight the importance of further understanding the functions of mGlu7 in complex neurologic conditions at both the molecular and physiologic levels. SIGNIFICANCE STATEMENT: Proper expression and function of mGlu7 is essential for learning, attention, and memory formation at the molecular level within neural circuits. The pharmacological targeting of mGlu7 is undergoing a paradigm shift by incorporating an understanding of receptor interaction with other cis- and trans- acting synaptic proteins, as well as various intracellular signaling pathways. Based upon these new findings, mGlu7's potential as a drug target in the treatment of cognitive disorders and learning impairments is primed for exploration.

Activation of Metabotropic Glutamate Receptor 3 Modulates Thalamo-accumbal Transmission and Rescues Schizophrenia-like Physiological and Behavioral Deficits.

BACKGROUND: Polymorphisms in the gene encoding for metabotropic glutamate receptor 3 (mGlu3) are associated with an increased likelihood of schizophrenia diagnosis and can predict improvements in negative symptoms following treatment with antipsychotics. However, the mechanisms by which mGlu3 can regulate brain circuits involved in schizophrenia pathophysiology are not clear. METHODS: We employed selective pharmacological tools and a variety of approaches including whole-cell patch-clamp electrophysiology, slice optogenetics, and fiber photometry to investigate the effects of mGlu3 activation on phencyclidine (PCP)-induced impairments in thalamo-accumbal transmission and sociability deficits. A chemogenetic approach was used to evaluate the role of thalamo-accumbal transmission in PCP-induced sociability deficits. RESULTS: We first established that PCP treatment augmented excitatory transmission onto dopamine D1 receptor-expressing medium spiny neurons (D1-MSNs) in the nucleus accumbens (NAc) and induced sociability deficits. Our studies revealed a selective increase in glutamatergic synaptic transmission from thalamic afferents to D1-MSNs in the NAc shell. Chemogenetic silencing of thalamo-accumbal inputs rescued PCP-induced sociability deficits. Pharmacological activation of mGlu3 normalized PCP-induced impairments in thalamo-accumbal transmission and sociability deficits. Mechanistic studies revealed that mGlu3 activation induced robust long-term depression at synapses from the thalamic projections onto D1-MSNs in the NAc shell. CONCLUSIONS: These data demonstrate that activation of mGlu3 decreases thalamo-accumbal transmission and thereby rescues sociability deficits in mouse modeling schizophrenia-like symptoms. These findings provide novel insights into the NAc-specific mechanisms and suggest that agents modulating glutamatergic signaling in the NAc may provide a promising approach for treating negative symptoms in schizophrenia.

Development of a Selective and High Affinity Radioligand, [3H]VU6013720, for the M4 Muscarinic Receptor.

M4 muscarinic receptors are highly expressed in the striatum and cortex, brain regions that are involved in diseases such as Parkinson's disease, schizophrenia, and dystonia. Despite potential therapeutic advantages of specifically targeting the M4 receptor, it has been historically challenging to develop highly selective ligands, resulting in undesired off-target activity at other members of the muscarinic receptor family. Recently, we have reported first-in-class, potent, and selective M4 receptor antagonists. As an extension of that work, we now report the development and characterization of a radiolabeled M4 receptor antagonist, [3H]VU6013720, with high affinity (pKd of 9.5 ± 0.2 at rat M4, 9.7 at mouse M4, and 10 ± 0.1 at human M4 with atropine to define nonspecific binding) and no significant binding at the other muscarinic subtypes. Binding assays using this radioligand in rodent brain tissues demonstrate loss of specific binding in Chrm4 knockout animals. Dissociation kinetics experiments with various muscarinic ligands show differential effects on the dissociation of [3H]VU6013720 from M4 receptors, suggesting a binding site that is overlapping but may be distinct from the orthosteric site. Overall, these results demonstrate that [3H]VU6013720 is the first highly selective antagonist radioligand for the M4 receptor, representing a useful tool for studying the basic biology of M4 as well for the support of M4 receptor-based drug discovery. SIGNIFICANCE STATEMENT: This manuscript describes the development and characterization of a novel muscarinic (M) acetylcholine subtype 4 receptor antagonist radioligand, [3H]VU6013720. This ligand binds to or overlaps with the acetylcholine binding site, providing a highly selective radioligand for the M4 receptor that can be used to quantify M4 protein expression in vivo and probe the selective interactions of acetylcholine with M4 versus the other members of the muscarinic receptor family.

GRM7 gene mutations and consequences for neurodevelopment.

The metabotropic glutamate receptor 7 (mGlu7), encoded by the GRM7 gene in humans, is a presynaptic, G protein-coupled glutamate receptor that is essential for modulating neurotransmission. Mutations in or reduced expression of GRM7 have been identified in different genetic neurodevelopmental disorders (NDDs), and rare biallelic missense variants have been proposed to underlie a subset of NDDs. Clinical GRM7 variants have been associated with a range of symptoms consistent with neurodevelopmental molecular features, including hypomyelination, brain atrophy and defects in axon outgrowth. Here, we review the newest findings regarding the cellular and molecular defects caused by GRM7 variants in NDD patients.

Differential Activity of Orthosteric Agonists and Allosteric Modulators at Metabotropic Glutamate Receptor 7.

Metabotropic glutamate receptor 7 (mGlu7) is a G protein coupled receptor that has demonstrated promise as a therapeutic target across a number of neurologic and psychiatric diseases. Compounds that modulate the activity of mGlu7, such as positive and negative allosteric modulators, may represent new therapeutic strategies to modulate receptor activity. The endogenous neurotransmitter associated with the mGlu receptor family, glutamate, exhibits low efficacy and potency in activating mGlu7, and surrogate agonists, such as the compound L-(+)-2-Amino-4-phosphonobutyric acid (L-AP4), are often used for receptor activation and compound profiling. To understand the implications of the use of such agonists in the development of positive allosteric modulators (PAMs), we performed a systematic evaluation of receptor activation using a system in which mutations can be made in either protomer of the mGlu7 dimer; we employed mutations that prevent interaction with the orthosteric site as well as the G-protein coupling site of the receptor. We then measured increases in calcium levels downstream of a promiscuous G protein to assess the effects of mutations in one of the two protomers in the presence of two different agonists and three positive allosteric modulators. Our results reveal that distinct PAMs, for example N-[3-Chloro-4-[(5-chloro-2-pyridinyl)oxy]phenyl]-2-pyridinecarboxamide (VU0422288) and 3-(2,3-Difluoro-4-methoxyphenyl)-2,5-dimethyl-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine (VU6005649), do exhibit different maximal levels of potentiation with L-AP4 versus glutamate, but there appear to be common stable receptor conformations that are shared among all of the compounds examined here. SIGNIFICANCE STATEMENT: This manuscript describes the systematic evaluation of the mGlu7 agonists glutamate and L-(+)-2-Amino-4-phosphonobutyric acid (L-AP4) in the presence and absence of three distinct potentiators examining possible mechanistic differences. These findings demonstrate that mGlu7 potentiators display subtle variances in response to glutamate versus L-AP4.

Persistent challenges in the development of an mGlu7 PAM in vivo tool compound: The discovery of VU6046980.

Herein, we report on the further chemical optimization of the first reported mGlu7 positive allosteric modulator (PAM), VU6027459. Replacement of the quinoline core by a cinnoline scaffold increased mGlu7 PAM potency by âˆ¼ 10-fold, and concomitant introduction of a chiral tricyclic motif led to potent mGlu7 PAMs with enantioselective mGlu receptor selectivity profiles. Of these, VU6046980 emerged as a putative in vivo tool compound with excellent CNS penetration (Kp = 4.1; Kp,uu = 0.7) and efficacy in preclinical models. However, either off-target activity at the sigma-1 receptor or activity at a target not elucidated by large ancillary pharmacology panels led to sedation not driven by activation of mGlu7 (validated in Grm7 knockout mice). Thus, despite a significant advance, a viable mGlu7 PAM in vivo tool remains elusive.

Discovery of a potent M5 antagonist with improved clearance profile. Part 2: Pyrrolidine amide-based antagonists.

This Letter describes our ongoing effort to improve the clearance of selective M5 antagonists. Herein, we report the replacement of the previously disclosed piperidine amide (4, disclosed in Part 1) with a pyrrolidine amide core. Several compounds within this series provided good potency, subtype selectivity, and low to moderate clearance profiles. Interestingly, the left-hand side SAR for this series diverged from our earlier efforts.

Discovery of a potent M5 antagonist with improved clearance profile. Part 1: Piperidine amide-based antagonists.

The lack of potent and selective tool compounds with pharmaceutically favorable properties limits the in vivo understanding of muscarinic acetylcholine receptor subtype 5 (M5) biology. Previously, we presented a highly potent and selective M5 antagonist VU6019650 with a suboptimal clearance profile as our second-generation tool compound. Herein, we disclose our ongoing efforts to generate next-generation M5 antagonists with improved clearance profiles. A mix and match approach between VU6019650 (lead) and VU0500325 (HTS hit) generated a piperidine amide-based novel M5 antagonist series. Several analogs within this series, including 29f, provided good on-target potency with improved clearance profiles, though room for improvement remains.

Differential activity of mGlu7 allosteric modulators provides evidence for mGlu7/8 heterodimers at hippocampal Schaffer collateral-CA1 synapses.

Glutamate acts at eight metabotropic glutamate (mGlu) receptor subtypes expressed in a partially overlapping fashion in distinct brain circuits. Recent evidence indicates that specific mGlu receptor protomers can heterodimerize and that these heterodimers can exhibit different pharmacology when compared to their homodimeric counterparts. Group III mGlu agonist-induced suppression of evoked excitatory potentials and induction of long-term potentiation at Schaffer collateral-CA1 (SC-CA1) synapses in the rodent hippocampus can be blocked by the selective mGlu7 negative allosteric modulator (NAM), ADX71743. Curiously, a different mGlu7 NAM, 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazonolo[4,5-c]pyridin-4(5H)-one, failed to block these responses in brain slices despite its robust activity at mGlu7 homodimers in vitro. We hypothesized that this might result from heterodimerization of mGlu7 with another mGlu receptor protomer and focused on mGlu8 as a candidate given the reported effects of mGlu8-targeted compounds in the hippocampus. Here, we used complemented donor acceptor-resonance energy transfer to study mGlu7/8 heterodimer activation in vitro and observed that ADX71743 blocked responses of both mGlu7/7 homodimers and mGlu7/8 heterodimers, whereas 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazonolo[4,5-c]pyridin-4(5H)-one only antagonized responses of mGlu7/7 homodimers. Taken together with our electrophysiology observations, these results suggest that a receptor with pharmacology consistent with an mGlu7/8 heterodimer modulates the activity of SC-CA1 synapses. Building on this hypothesis, we identified two additional structurally related mGlu7 NAMs that also differ in their activity at mGlu7/8 heterodimers, in a manner consistent with their ability to inhibit synaptic transmission and plasticity at SC-CA1. Thus, we propose that mGlu7/8 heterodimers are a key molecular target for modulating the activity of hippocampal SC-CA1 synapses.

Development and profiling of mGlu7 NAMs with a range of saturable inhibition of agonist responses in vitro.

We describe here a series of metabotropic glutamate receptor 7 (mGlu7) negative allosteric modulators (NAMs) with a saturable range of activity in inhibiting responses to an orthosteric agonist in two distinct in vitro pharmacological assays. The range of inhibition among compounds in this scaffold provides highly structurally related ligands with differential degrees of receptor blockade that can be used to understand inhibitory efficacy profiles in native tissue or in vivo.

Clinical and Preclinical Evidence for M1 Muscarinic Acetylcholine Receptor Potentiation as a Therapeutic Approach for Rett Syndrome.

Rett syndrome (RTT) is a neurodevelopmental disorder that is characterized by developmental regression, loss of communicative ability, stereotyped hand wringing, cognitive impairment, and central apneas, among many other symptoms. RTT is caused by loss-of-function mutations in a methyl-reader known as methyl-CpG-binding protein 2 (MeCP2), a protein that links epigenetic changes on DNA to larger chromatin structure. Historically, target identification for RTT has relied heavily on Mecp2 knockout mice; however, we recently adopted the alternative approach of performing transcriptional profiling in autopsy samples from RTT patients. Through this mechanism, we identified muscarinic acetylcholine receptors (mAChRs) as potential therapeutic targets. Here, we characterized a cohort of 40 temporal cortex samples from individuals with RTT and quantified significantly decreased levels of the M1, M2, M3, and M5 mAChRs subtypes relative to neurotypical controls. Of these four subtypes, M1 expression demonstrated a linear relationship with MeCP2 expression, such that M1 levels were only diminished in contexts where MeCP2 was also significantly decreased. Further, we show that M1 potentiation with the positive allosteric modulator (PAM) VU0453595 (VU595) rescued social preference, spatial memory, and associative memory deficits, as well as decreased apneas in Mecp2+/- mice. VU595's efficacy on apneas in Mecp2+/- mice was mediated by the facilitation of the transition from inspiration to expiration. Molecular analysis correlated rescue with normalized global gene expression patterns in the brainstem and hippocampus, as well as increased Gsk3ß inhibition and NMDA receptor trafficking. Together, these data suggest that M1 PAMs could represent a new class of RTT therapeutics.

Optimized Administration of the M4 PAM VU0467154 Demonstrates Broad Efficacy, but Limited Effective Concentrations in Mecp2+/- Mice.

Hypofunction of cholinergic circuits and diminished cholinergic tone have been associated with the neurodevelopmental disorder Rett syndrome (RTT). Specifically, deletion of Mecp2 in cholinergic neurons evokes the same social and cognitive phenotypes in mice seen with global Mecp2 knockout, and decreased choline acetyltransferase activity and vesamicol binding have been reported in RTT autopsy samples. Further, we recently identified significant decreases in muscarinic acetylcholine receptor subtype 4 (M4) expression in both the motor cortex and cerebellum of RTT patient autopsies and established proof of concept that an acute dose of the positive allosteric modulator (PAM) VU0467154 (VU154) rescued phenotypes in Mecp2+/- mice. Here, we expand the assessment of M4 PAMs in RTT to address clinically relevant questions of tolerance, scope of benefit, dose response, chronic treatment, and mechanism. We show that VU154 has efficacy on anxiety, social preference, cognitive, and respiratory phenotypes in Mecp2+/- mice; however, the therapeutic range is narrow, with benefits seen at 3 mg/kg concentrations, but not 1 or 10 mg/kg. Further, sociability was diminished in VU154-treated Mecp2+/- mice, suggestive of a potential adverse effect. Compound efficacy on social, cognitive, and respiratory phenotypes was conserved with a 44-day treatment paradigm, with the caveat that breath rate was moderately decreased with chronic treatment in Mecp2+/+ and Mecp2+/- mice. VU154 effects on respiratory function correlated with an increase in Gsk3ß inhibition in the brainstem. These results identify the core symptom domains where efficacy and adverse effects may present with M4 administration in RTT model mice and advocate for the continued evaluation as potential RTT therapeutics.