What are the therapeutic candidates targeting M4?

Introduction to M4 Receptor
The muscarinic receptor M4 is one of the five subtypes belonging to the G protein‐coupled receptor (GPCR) family, which are activated by the neurotransmitter acetylcholine. M4 receptors are particularly interesting in the field of neuropharmacology due to their role in modulating dopaminergic neurotransmission and thus impacting a wide range of neurophysiological processes, including cognition, motor control, and emotional regulation. Their localization in brain regions such as the basal ganglia and the cortex underlies a prominent role in controlling the excitability of neuronal circuits related to psychosis and movement disorders. In targeted drug discovery, the high conservation of the orthosteric binding sites across muscarinic subtypes has hampered the development of selective agonists. This challenge has led researchers to explore the allosteric modulation of M4 receptors, which provides unique opportunities for achieving selectivity while also minimizing adverse peripheral side effects associated with non‐selective cholinergic stimulation.

Structure and Function of M4 Receptor
Structurally, M4 receptors share the characteristic seven-transmembrane domain architecture of GPCRs. Like its muscarinic siblings, the receptor comprises extracellular domains that are critical for ligand binding and intracellular loops that interact with G proteins. A key functional aspect of M4 is its preferred coupling to the Gi/o family of G proteins. Activation of M4 results in the inhibition of adenylyl cyclase, leading to decreased cyclic adenosine monophosphate (cAMP) production and subsequent modulation of downstream signaling pathways. Although structural studies have highlighted the similarity in the orthosteric binding regions between subtypes such as M1 and M4, subtle differences—often revealed by advanced techniques such as cryo-electron microscopy—are being exploited in drug design. These differences enable the development of therapeutics that bind selectively to the receptor’s allosteric sites, thereby fine-tuning its activity without triggering the widespread activation that can lead to deleterious side effects.

Role of M4 in Human Physiology
The physiological role of the M4 receptor is diverse and intricately linked with the regulation of central dopaminergic tone. M4 receptors are primarily expressed in subcortical regions like the striatum, where they act as critical modulators of dopamine release. By inhibiting adenylyl cyclase activity through Gi/o proteins, M4 receptor activation creates a feedback mechanism that helps control the firing rate of dopaminergic neurons, thus maintaining the balance necessary for proper motor function and cognitive performance. Clinical and preclinical evidence suggests that a reduction in M4 receptor function or a decrease in receptor density can lead to disinhibition of dopamine release, a hallmark characteristic in the pathophysiology of disorders such as schizophrenia. This observation has sparked intense research into candidate compounds that can augment or mimic the effects of acetylcholine at the M4 receptor, thereby offering a novel approach to treat psychosis and related conditions.

Current Therapeutic Candidates Targeting M4
Recent advancements in drug discovery have led to the development of multiple therapeutic candidates that modulate M4 receptor activity. These candidates vary in their mode of action, ranging from full agonists to positive allosteric modulators (PAMs). They are being extensively evaluated in preclinical models and clinical trials, especially for the treatment of schizophrenia and neurocognitive disorders.

Overview of Existing Candidates
Among the therapeutic candidates targeting M4 receptors, several compounds have drawn significant attention by virtue of their ability to modulate receptor activity with high specificity:

• Xanomeline-trospium (formulated as KarXT) is a combination compound where xanomeline, an M1/M4 receptor-preferring agonist, is paired with trospium to mitigate undesired peripheral cholinergic effects. KarXT has been at the forefront of clinical advances, emerging as a candidate in phase 2 and phase 3 clinical trials for treating schizophrenia. Its unique mechanism capitalizes on the agonistic activity at M4 to modulate dopaminergic tone without the dopamine D2 receptor blockade that is characteristic of traditional antipsychotics.

• Emraclidine, a highly selective positive allosteric modulator (PAM) of the M4 receptor, represents another promising candidate. Emraclidine has recently been advanced into phase 1b clinical studies in patients with schizophrenia. The compound is designed to enhance the receptor’s response to endogenous acetylcholine, thereby selectively reducing excessive dopamine release in key brain regions implicated in psychosis.

• NMRA-266 is another investigational candidate emerging from recent developmental programs. Indications suggest that NMRA-266, which selectively targets the M4 muscarinic receptor, has successfully achieved IND clearance. This progress underscores the clinical validation of the M4 receptor as a therapeutic target for schizophrenia and possibly other neuropsychiatric disorders.

• There are also dual M1/M4 receptor candidates developed by companies such as Neurocrine Biosciences and Sosei Heptares. These compounds are engineered to provide synergistic modulation of both receptor subtypes, potentially offering broader efficacy in improving cognitive and psychotic symptoms while optimizing the side-effect profile through receptor selectivity.

• In addition to these agonists and modulators, early-stage research and preclinical studies have also explored a range of structurally distinct compounds, including newer generations of positive allosteric modulators such as VU154 and LY298. These compounds, while not yet in advanced clinical trials, have shown promising pharmacological activity in preclinical models by selectively enhancing M4 receptor signaling and subsequently reducing hyperdopaminergic activity.

Developmental Stages and Clinical Trials
The pathway from candidate identification to clinical application is characterized by rigorous preclinical studies followed by progressive phases of clinical trials. KarXT, for instance, has not only demonstrated compelling efficacy in animal models but has also progressed into later-phase clinical trials (phase 3) with initial success in schizophrenia populations. Emraclidine, though slightly less advanced in its developmental timeline, has completed phase 1b studies with early safety and efficacy signals that indicate a favorable tolerability profile and robust antipsychotic activity.
NMRA-266 has recently been granted IND clearance, positioning it to enter early human trials, while dual M1/M4 receptor candidates are currently being evaluated in early clinical studies aimed at refining dosing strategies and establishing clinical endpoints. Preclinical candidates such as the M4 PAMs like VU154 and LY298 have already provided the groundwork for understanding the molecular determinants of M4 allosteric modulation, and their optimization may pave the way for next-generation therapeutics. Overall, the clinical landscape is moving rapidly, with a trend toward identifying compounds that combine potent efficacy with an improved side-effect profile by leveraging the intrinsic benefits of allosteric modulation.

Mechanisms of Action
Understanding how therapeutic candidates interact with the M4 receptor is critical to both optimizing their selective activity and minimizing adverse effects. Mechanistic studies have laid a strong foundation for the rational design of M4 modulators.

How Candidates Interact with M4
Candidate compounds targeting the M4 receptor can be broadly grouped into orthosteric agonists and allosteric modulators. Orthosteric agonists bind directly to the acetylcholine-binding site of the receptor; however, due to the high degree of sequence conservation across muscarinic subtypes, achieving subtype selectivity at this site is challenging. Xanomeline functions as an orthosteric agonist and, when paired with trospium in KarXT, offers targeted central cholinergic stimulation while restricting peripheral activation.

In contrast, allosteric modulators interact with sites that are distinct from the traditional acetylcholine-binding domain. These sites allow for greater “druggability” and subtype selectivity. Emraclidine, for example, is a positive allosteric modulator (PAM) of M4 that binds at an allosteric pocket to enhance the binding affinity and efficacy of endogenous acetylcholine. This cooperative binding can result in a 400-fold increase in acetylcholine affinity in some cases, although the exact magnitude depends on the compound and cellular context. Modulators such as VU154 and LY298 also operate via allosteric mechanisms, biasing receptor signaling toward desired downstream pathways while mitigating the risk of receptor desensitization commonly associated with continuous orthosteric stimulation.

The allosteric ligands achieve their selectivity by engaging amino acid residues that are less conserved among muscarinic receptors. Structural studies, including cryo-electron microscopy, have provided detailed insights into these binding modes and interactions. For instance, one study revealed that allosteric modulators can occupy a vertical binding pose within the receptor’s allosteric pocket, which not only facilitates their selectivity but also determines the precise modulation of receptor conformation critical for downstream signaling. This architectural insight explains why compounds such as emraclidine have been able to demonstrate strong receptor-specific efficacy with reduced peripheral side effects.

Biological Pathways Involved
Activation of the M4 receptor, whether by orthosteric agonism or allosteric modulation, sets in motion several intracellular signaling cascades. The predominant downstream pathway involves coupling to Gi/o proteins, culminating in the inhibition of adenylyl cyclase and subsequent reduction in cAMP levels. This decrease in cAMP initiates a cascade that ultimately restrains excessive dopamine release in the striatum—a key mechanism underlying the antipsychotic effects observed with M4 activation.

In addition to cAMP modulation, evidence suggests that M4 receptor activation can indirectly influence other neurotransmitter systems. For example, M4-mediated effects on dopamine have been shown to also require the co-activation of CB2 receptors. Studies have demonstrated that selective blockade of CB2 receptors attenuates the effects of M4 modulators on extracellular dopamine levels, suggesting a synergistic interplay between M4 and cannabinoid receptor signaling in regulating dopaminergic neurotransmission. Furthermore, the engagement of M4 receptors on specific populations of GABAergic neurons in the striatum facilitates a nuanced modulation of neuronal excitability, influencing both motor activity and cognitive processes.

Thus, the biological pathways impacted by M4-targeting treatments encompass both direct receptor signaling as well as the modulation of interconnected neurotransmitter systems, including dopamine and endocannabinoids. This makes M4 an attractive target for a range of therapeutic applications where downregulation of pathologically excessive dopaminergic activity is desired.

Potential Applications and Efficacy
The therapeutic implications of selectively targeting the M4 receptor are broad and promising, with particular emphasis on psychiatric disorders.

Therapeutic Areas
The majority of research focus has been on schizophrenia, where hyperdopaminergic states—especially in subcortical regions—are linked to psychotic symptoms. M4 agonists and PAMs have shown potential in reducing these symptoms by modulating dopamine release in key brain areas such as the striatum.
• Schizophrenia and Psychosis: Xanomeline-trospium (KarXT) and emraclidine have been developed specifically to address both cognitive deficits and positive symptoms of schizophrenia. Their ability to reduce excessive dopamine release offers an alternative to traditional antipsychotics that primarily block dopamine receptors, thereby potentially reducing treatment-limiting side effects such as extrapyramidal symptoms.
• Alzheimer’s Disease with Psychotic Features: Given the shared pathways involving cholinergic deficits and dysregulated dopaminergic tone in Alzheimer’s disease, M4 receptor activation is being explored as a strategy to mitigate psychotic symptoms and even cognitive deterioration in this population.
• Addiction and Substance Abuse: Some preliminary research indicates that by modulating dopaminergic neurotransmission, M4-targeted therapies may also have potential applications in the treatment of addictions, where dysregulated dopamine pathways play a critical role.
• Movement Disorders: Although less central to current development programs, the role of M4 receptors in moderating motor activity positions them as potential therapeutic targets in certain movement disorders where dopaminergic imbalance is implicated.

Efficacy in Preclinical and Clinical Studies
Preclinical studies have consistently demonstrated that M4 receptor modulators produce robust antipsychotic-like effects in animal models. For example, administration of M4 PAMs in rodent models results in a significant reduction of amphetamine-induced hyperlocomotion and improvement in sensorimotor gating deficits—key behavioral correlates of schizophrenia. Additionally, studies have shown that M4 modulators reduce extracellular dopamine levels in the striatum, a hallmark of their therapeutic mechanism.

In clinical settings, early-phase trials have generated encouraging data. KarXT, which combines xanomeline and trospium, has progressed through phase 2 studies and is now in phase 3 clinical trials with promising efficacy and tolerability profiles. These trials have reported meaningful improvements in both positive and negative symptoms of schizophrenia, alongside a reduction in side effects typically associated with dopamine receptor blockade. Similarly, emraclidine’s phase 1b studies have rounded out a preliminary clinical proof-of-concept that highlights its potential antipsychotic efficacy combined with a favorable cardiovascular and extrapyramidal side effect profile.

Moreover, dual M1/M4 candidates and other experimental compounds are being evaluated for their synergistic effects on modulating not only psychosis but also cognitive deficits observed in neurodegenerative conditions. The translation of robust preclinical effects into early clinical efficacy is fueling optimism that M4-targeting therapeutics will soon become part of the standard treatment armamentarium for psychiatric disorders.

Challenges and Future Research
Despite the declared promise of M4-targeted therapeutics, numerous challenges remain that may affect the clinical translation and long-term success of these candidates.

Current Challenges in Targeting M4
One of the principal challenges in targeting M4 receptors arises from the high structural conservation of the orthosteric binding sites among the muscarinic receptor family. This similarity increases the risk of cross-reactivity and off-target effects when using orthosteric agonists, as seen with earlier efforts that led to adverse peripheral cholinergic side effects. Xanomeline’s initial development encountered such issues until the combination strategy with trospium was implemented to restrict peripheral activation.

Another challenge relates to receptor desensitization. Continuous stimulation of M4 receptors by direct agonists can lead to receptor internalization and reduced sensitivity over time. Allosteric modulators have been proposed as a solution to this problem by enhancing the efficacy of endogenous acetylcholine only when and where it is naturally present, thus mitigating rapid desensitization. However, designing allosteric modulators that maintain their selectivity and do not inadvertently affect other signaling pathways remains a nuanced balancing act.

In addition, the complexity of the biological network involving M4 receptors—as evidenced by the interplay with CB2 receptors and other neurotransmitter systems—adds an additional layer of complexity to understanding the full spectrum of therapeutic effects. This necessitates comprehensive pharmacodynamic studies and the development of robust biomarkers to predict and monitor therapeutic outcomes. Moreover, long-term safety, particularly in chronic indications such as schizophrenia or Alzheimer’s disease, is a critical regulatory concern.

Future Directions and Research Opportunities
Future research in M4-targeted therapeutics should focus on leveraging the advances in structural biology and computational drug design. Improved resolution in receptor structure determination, for instance through cryo-electron microscopy, can provide deeper insights into the distinctive allosteric pockets of M4 receptors. Such insights will allow the rational design of molecules that achieve superior selectivity and minimize unintended activation of other muscarinic receptors.

Another promising direction involves the use of biased signaling. By developing ligands that preferentially activate beneficial signaling pathways (e.g., Gi/o pathways) over deleterious ones (e.g., those leading to desensitization or adverse side effects), researchers may enhance therapeutic efficacy. Novel compounds that exploit this mechanism could offer prolonged clinical benefits with reduced tolerance and adverse effects.

There is also a growing interest in dual-target strategies. As seen with the dual M1/M4 receptor candidates, addressing multiple aspects of cholinergic dysfunction could yield more comprehensive improvements in cognitive and psychotic symptoms. Further research into the synergistic interactions between M1 and M4 signaling pathways may uncover additional therapeutic benefits and provide a basis for combination therapies.

Clinical research is also moving toward personalized medicine approaches. Given that schizophrenia and related disorders are heterogeneous syndromes, stratifying patients based on receptor expression patterns or genetic biomarkers could help identify those who are most likely to benefit from M4 modulation. Future studies could incorporate blood transcriptome analysis and neuroimaging modalities to monitor receptor occupancy and downstream signaling changes, thus refining patient selection and dosing algorithms.

Moreover, expanding the potential therapeutic applications of M4 modulators beyond psychiatric disorders is another area ripe for exploration. While the current focus is understandably on schizophrenia and Alzheimer’s disease, the modulation of dopaminergic tone by M4 receptors may also have implications in the treatment of substance abuse disorders and specific movement disorders. Preclinical studies examining these possibilities could open up new avenues for drug development and eventual clinical translation.

In addition to optimizing the molecules per se, future research must address the challenges of drug delivery. For instance, ensuring central nervous system (CNS) penetration while avoiding peripheral cholinergic side effects is critical. Innovative formulation strategies, such as lipid-based nanoparticles or prodrug approaches, could facilitate targeted delivery of M4 modulators to the brain, thereby enhancing clinical efficacy and patient compliance.

Finally, another key research opportunity is the investigation of long-term effects and safety profiles for M4-targeted compounds. Chronic studies in animal models, followed by long-duration human clinical trials, will be essential to understand the full spectrum of effects—both therapeutic and adverse—of sustained M4 receptor modulation. Such studies should ideally be multidisciplinary, integrating pharmacokinetic, pharmacodynamic, and safety data to support reliable dose optimization and minimize risks over extended treatment periods.

Conclusion
In summary, multiple therapeutic candidates targeting M4 receptors are being developed with the aim of providing a new strategy for treating neuropsychiatric disorders, predominantly schizophrenia. Starting with the pioneering xanomeline-trospium combination (KarXT), which leverages both orthosteric agonism and peripheral restriction, to highly selective M4 positive allosteric modulators like emraclidine and NMRA-266, the field has evolved significantly in recent years. These compounds work by enhancing M4 receptor activity either directly or through allosteric pathways, leading to a reduction in excess extracellular dopamine release—a central mechanism implicated in psychosis.

Beyond schizophrenia, these modulators hold promise in addressing cognitive deficits seen in Alzheimer’s disease and potentially other disorders characterized by dopaminergic dysregulation. Preclinical studies have consistently shown significant antipsychotic-like effects, while early clinical trials indicate promising tolerability and efficacy profiles. Underlying these clinical advances is a deepening understanding of the structural nuances of the M4 receptor, which has enabled the development of compounds capable of selective binding at allosteric sites; this specificity is integral in mitigating off-target effects and improving the overall therapeutic index.

However, considerable challenges remain. The need for increased receptor selectivity, prevention of receptor desensitization, careful modulation of complex neurotransmitter networks, and the delivery of these agents across the blood–brain barrier are issues that must be addressed through further research. Future directions include the exploration of biased signaling mechanisms, dual-target approaches that combine M1 and M4 modulation, and the application of advanced computational and structural biology techniques to optimize drug design. Furthermore, a focus on long-term safety and personalized medicine approaches will help facilitate the translation of these promising candidates into effective treatments for patients.

Overall, while the journey from bench to bedside is complex and fraught with challenges, the advancements in M4 receptor research underscore the therapeutic potential of these candidates. By combining robust preclinical findings with innovative clinical strategies, the next generation of M4-targeted treatments is poised to offer significant improvements over traditional therapies, ultimately providing patients with more effective and better-tolerated options for managing schizophrenia and related disorders.