Characterization of a putative orexin receptor in Ciona intestinalis sheds light on the evolution of the orexin/hypocretin system in chordates.

Tunicates are evolutionary model organisms bridging the gap between vertebrates and invertebrates. A genomic sequence in Ciona intestinalis (CiOX) shows high similarity to vertebrate orexin receptors and protostome allatotropin receptors (ATR). Here, molecular phylogeny suggested that CiOX is divergent from ATRs and human orexin receptors (hOX1/2). However, CiOX appears closer to hOX1/2 than to ATR both in terms of sequence percent identity and in its modelled binding cavity, as suggested by molecular modelling. CiOX was heterologously expressed in a recombinant HEK293 cell system. Human orexins weakly but concentration-dependently activated its Gq signalling (Ca2+ elevation), and the responses were inhibited by the non-selective orexin receptor antagonists TCS 1102 and almorexant, but only weakly by the OX1-selective antagonist SB-334867. Furthermore, the 5-/6-carboxytetramethylrhodamine (TAMRA)-labelled human orexin-A was able to bind to CiOX. Database mining was used to predict a potential endogenous C. intestinalis orexin peptide (Ci-orexin-A). Ci-orexin-A was able to displace TAMRA-orexin-A, but not to induce any calcium response at the CiOX. Consequently, we suggested that the orexin signalling system is conserved in Ciona intestinalis, although the relevant peptide-receptor interaction was not fully elucidated.

Recent advances in generative biology for biotherapeutic discovery.

Generative biology combines artificial intelligence (AI), advanced life sciences technologies, and automation to revolutionize the process of designing novel biomolecules with prescribed properties, giving drug discoverers the ability to escape the limitations of biology during the design of next-generation protein therapeutics. Significant hurdles remain, namely: (i) the inherently complex nature of drug discovery, (ii) the bewildering number of promising computational and experimental techniques that have emerged in the past several years, and (iii) the limited availability of relevant protein sequence-function data for drug-like molecules. There is a need to focus on computational methods that will be most practically effective for protein drug discovery and on building experimental platforms to generate the data most appropriate for these methods. Here, we discuss recent advances in computational and experimental life sciences that are most crucial for impacting the pace and success of protein drug discovery.

Sex-Dependent Attentional Impairments in a Subchronic Ketamine Mouse Model for Schizophrenia.

Background: The development of more effective treatments for schizophrenia targeting cognitive and negative symptoms has been limited, partly due to a disconnect between rodent models and human illness. Ketamine administration is widely used to model symptoms of schizophrenia in both humans and rodents. In mice, subchronic ketamine treatment reproduces key dopamine and glutamate dysfunction; however, it is unclear how this translates into behavioral changes reflecting positive, negative, and cognitive symptoms. Methods: In male and female mice treated with either subchronic ketamine or saline, we assessed spontaneous and amphetamine-induced locomotor activity to measure behaviors relevant to positive symptoms, and used a touchscreen-based progressive ratio task of motivation and the rodent continuous performance test of attention to capture specific negative and cognitive symptoms, respectively. To explore neuronal changes underlying the behavioral effects of subchronic ketamine treatment, we quantified expression of the immediate early gene product, c-Fos, in key corticostriatal regions using immunofluorescence. Results: We showed that spontaneous locomotor activity was unchanged in male and female subchronic ketamine-treated animals, and amphetamine-induced locomotor response was reduced. Subchronic ketamine treatment did not alter motivation in either male or female mice. In contrast, we identified a sex-specific effect of subchronic ketamine on attentional processing wherein female mice performed worse than control mice due to increased nonselective responding. Finally, we showed that subchronic ketamine treatment increased c-Fos expression in prefrontal cortical and striatal regions, consistent with a mechanism of widespread disinhibition of neuronal activity. Conclusions: Our results highlight that the subchronic ketamine mouse model reproduces a subset of behavioral symptoms that are relevant for schizophrenia.

Ligand-directed biased agonism at human histamine H3 receptor isoforms across Gαi/o- and ß-arrestin2-mediated pathways.

The histamine H3 receptor (H3R) is a neurotransmitter receptor that is primarily found in the brain, where it controls the release and synthesis of histamine, as well as the release of other neurotransmitters (e.g. dopamine, serotonin). Notably, 20 H3R isoforms are differentially expressed in the human brain as a consequence of alternative gene splicing. The hH3R-445, -415, -365 and -329 isoforms contain the prototypical GPCR (7TM) structure, yet exhibit deletions in the third intracellular loop, a structural domain that is pivotal for G protein-coupling, signaling and regulation. To date, the physiological relevance underlying the individual and combinatorial function of hH3R isoforms remains poorly understood. Nevertheless, given their significant implication in physiological processes (e.g. cognition, homeostasis) and neurological disorders (e.g. Alzheimer's and Parkinson's disease, schizophrenia), widespread targeting of hH3R isoforms by drugs may lead to on-target side effects in brain regions that are unaffected by disease. To this end, isoform- and/or pathway-selective targeting of hH3R isoforms by biased agonists could be of therapeutic relevance for the development of region- and disease-specific drugs. Hence, we have evaluated ligand biased signaling at the hH3R-445, -415, -365 and -329 isoforms across various Gαi/o-mediated (i.e. [35S]GTPγS accumulation, cAMP inhibition, pERK1/2 activation, pAKT T308/S473 activation) and non Gαi/o-mediated (i.e. ß-arrestin2 recruitment) endpoints that are relevant to neurological diseases. Our findings indicate that H3R agonists display significantly altered patterns in their degree of ligand bias, in a pathway- and isoform-dependent manner, underlining the significance to investigate GPCRs with multiple isoforms to improve development of selective drugs. SUBJECT CATEGORY: Neuropharmacology.

Gpr88 Deletion Impacts Motivational Control Without Overt Disruptions to Striatal Dopamine.

Background: Disrupted motivational control is a common-but poorly treated-feature of psychiatric disorders, arising via aberrant mesolimbic dopaminergic signaling. GPR88 is an orphan G protein-coupled receptor that is highly expressed in the striatum and therefore well placed to modulate disrupted signaling. While the phenotype of Gpr88 knockout mice suggests a role in motivational pathways, it is unclear whether GPR88 is involved in reward valuation and/or effort-based decision making in a sex-dependent manner and whether this involves altered dopamine function. Methods: In male and female Gpr88 knockout mice, we used touchscreen-based progressive ratio, with and without reward devaluation, and effort-related choice tasks to assess motivation and cost/benefit decision making, respectively. To explore whether these motivational behaviors were related to alterations in the striatal dopamine system, we quantified expression of dopamine-related genes and/or proteins and used [18F]DOPA positron emission tomography and GTPγ[35S] binding to assess presynaptic and postsynaptic dopamine function, respectively. Results: We showed that male and female Gpr88 knockout mice displayed greater motivational drive than wild-type mice, which was maintained following reward devaluation. Furthermore, we showed that cost/benefit decision making was impaired in male, but not female, Gpr88 knockout mice. Surprisingly, we found that Gpr88 deletion had no effect on striatal dopamine by any of the measures assessed. Conclusions: Our results highlight that GPR88 regulates motivational control but that disruption of such behaviors following Gpr88 deletion occurs independently of gross perturbations to striatal dopamine at a gene, protein, or functional level. This work provides further insights into GPR88 as a drug target for motivational disorders.

Development of in silico models to predict viscosity and mouse clearance using a comprehensive analytical data set collected on 83 scaffold-consistent monoclonal antibodies.

Biologic drug discovery pipelines are designed to deliver protein therapeutics that have exquisite functional potency and selectivity while also manifesting biophysical characteristics suitable for manufacturing, storage, and convenient administration to patients. The ability to use computational methods to predict biophysical properties from protein sequence, potentially in combination with high throughput assays, could decrease timelines and increase the success rates for therapeutic developability engineering by eliminating lengthy and expensive cycles of recombinant protein production and testing. To support development of high-quality predictive models for antibody developability, we designed a sequence-diverse panel of 83 effector functionless IgG1 antibodies displaying a range of biophysical properties, produced and formulated each protein under standard platform conditions, and collected a comprehensive package of analytical data, including in vitro assays and in vivo mouse pharmacokinetics. We used this robust training data set to build machine learning classifier models that can predict complex protein behavior from these data and features derived from predicted and/or experimental structures. Our models predict with 87% accuracy whether viscosity at 150 mg/mL is above or below a threshold of 15 centipoise (cP) and with 75% accuracy whether the area under the plasma drug concentration-time curve (AUC0-672 h) in normal mouse is above or below a threshold of 3.9 × 106 h x ng/mL.

"We don't want to run before we walk": the attitudes of Australian stakeholders towards using psychedelics for mental health conditions.

OBJECTIVES: This study was aimed at understanding the attitudes and positions of key Australian organisational and political stakeholders towards using psychedelic agents in medically supervised environments to treat mental health conditions. Specifically, this research was designed to identify some of the issues that might impede the clinical implementation of psychedelics. METHODS: Semi-structured interviews were conducted with four Australian politicians and nine representatives of key stakeholder organisations between September 2022 and January 2023. Data analyses were completed using pattern-based inductive thematic analysis. RESULTS: Participants were cautiously optimistic about using psychedelics to treat mental health conditions, with hesitancy emerging due to the perceived inadequacy of research into the efficacy and feasibility of these treatments. Politicians consistently mentioned that negative stigma prevented them and their peers from supporting the use of psychedelics in Australia. Effective, evidence-based, clear messaging that refutes misconceptions, uses persuasive messaging and provides clear information to inform implementation is needed to improve knowledge and challenge attitudes, biases and emotions that can influence the debate around psychedelics. CONCLUSIONS: Stakeholder representatives and politicians agree that insufficient evidence exists to support the widespread clinical implementation of psychedelics in Australia. Politicians also perceive the stigma associated with psychedelics might negatively influence progressive legislation. Additional research and a clear presentation of this research are needed before the clinical use of psychedelics can be supported.

Molecular and structural insights into the 5-HT2C receptor as a therapeutic target for substance use disorders.

Substance use disorder (SUD) is a chronic condition, with maintained abuse of a substance leading to physiological and psychological alterations and often changes in cognitive and social behaviours. Current therapies include psychotherapy coupled with medication; however, high relapse rates reveal the shortcomings of these therapies. The signalling, expression profile, and neurological function of the serotonin 2C receptor (5-HT2C receptor) make it a candidate of interest for the treatment of SUD. Recently, psychedelics, which broadly act at 5-HT2 receptors, have indicated potential for the treatment of SUD, implicating the 5-HT2C receptor. The modern psychedelic movement has rekindled interest in the 5-HT2C receptor, resulting in many new studies, especially structural analyses. This review explores the structural, molecular and cellular mechanisms governing 5-HT2C receptor function in the context of SUD. This provides the basis of the preclinical and clinical evidence for their role in SUD and highlights the potential for future exploration.

Molecular insights into orphan G protein-coupled receptors relevant to schizophrenia.

Schizophrenia remains a sizable socio-economic burden that continues to be treated with therapeutics based on 70-year old science. All currently approved therapeutics primarily target the dopamine D2 receptor to achieve their efficacy. Whilst dopaminergic dysregulation is a key feature in this disorder, the targeting of dopaminergic machinery has yielded limited efficacy and an appreciable side effect burden. Over the recent decades, numerous drugs that engage non-dopaminergic G protein-coupled receptors (GPCRs) have yielded a promise of efficacy without the deleterious side effect profile, yet none have successfully completed clinical studies and progressed to the market. More recently, there has been increased attention around non-dopaminergic GPCR-targeting drugs, which demonstrated efficacy in some schizophrenia symptom domains. This provides renewed hope that effective schizophrenia treatment may lie outside of the dopaminergic space. Despite the potential for muscarinic receptor- (and other well-characterised GPCR families) targeting drugs to treat schizophrenia, they are often plagued with complications such as lack of receptor subtype selectivity and peripheral on-target side effects. Orphan GPCR studies have opened a new avenue of exploration with many demonstrating schizophrenia-relevant mechanisms and a favourable expression profile, thus offering potential for novel drug development. This review discusses centrally expressed orphan GPCRs: GPR3, GPR6, GPR12, GPR52, GPR85, GPR88 and GPR139 and their relationship to schizophrenia. We review their expression, signalling mechanisms and cellular function, in conjunction with small molecule development and structural insights. We seek to provide a snapshot of the growing evidence and development potential of new classes of schizophrenia therapeutics.

Identifying promising sequences for protein engineering using a deep transformer protein language model.

Protein engineers aim to discover and design novel sequences with targeted, desirable properties. Given the near limitless size of the protein sequence landscape, it is no surprise that these desirable sequences are often a relative rarity. This makes identifying such sequences a costly and time-consuming endeavor. In this work, we show how to use a deep transformer protein language model to identify sequences that have the most promise. Specifically, we use the model's self-attention map to calculate a Promise Score that weights the relative importance of a given sequence according to predicted interactions with a specified binding partner. This Promise Score can then be used to identify strong binders worthy of further study and experimentation. We use the Promise Score within two protein engineering contexts-Nanobody (Nb) discovery and protein optimization. With Nb discovery, we show how the Promise Score provides an effective way to select lead sequences from Nb repertoires. With protein optimization, we show how to use the Promise Score to select site-specific mutagenesis experiments that identify a high percentage of improved sequences. In both cases, we also show how the self-attention map used to calculate the Promise Score can indicate which regions of a protein are involved in intermolecular interactions that drive the targeted property. Finally, we describe how to fine-tune the transformer protein language model to learn a predictive model for the targeted property, and discuss the capabilities and limitations of fine-tuning with and without knowledge transfer within the context of protein engineering.

Targeting muscarinic receptors for the treatment of alcohol use disorders: Opportunities and hurdles for clinical development.

Emerging evidence suggests muscarinic acetylcholine receptors represent novel targets to treat alcohol use disorder. In this review, we draw from literature across medicinal chemistry, molecular biology, addiction and learning/cognition fields to interrogate the proposition for muscarinic receptor ligands in treating various aspects of alcohol use disorder, including cognitive dysfunction, motivation to consume alcohol and relapse. In support of this proposition, we describe cholinergic dysfunction in the pathophysiology of alcohol use disorder at a network level, including alcohol-induced adaptations present in both human post-mortem brains and reverse-translated rodent models. Preclinical behavioural pharmacology implicates specific muscarinic receptors, in particular, M4 and M5 receptors, as potential therapeutic targets worthy of further interrogation. We detail how these receptors can be selectively targeted in vivo by the use of subtype-selective allosteric modulators, a strategy that overcomes the issue of targeting a highly conserved orthosteric site bound by acetylcholine. Finally, we highlight the intense pharma interest in allosteric modulators of muscarinic receptors for other indications that provide an opportunity for repurposing into the alcohol use disorder space and provide some currently unanswered questions as a roadmap for future investigation.

Building the foundation for a community-generated national research blueprint for inherited bleeding disorders: facilitating research through infrastructure, workforce, resources and funding.

BACKGROUND: The National Hemophilia Foundation (NHF) conducted extensive, inclusive community consultations to guide prioritization of research in coming decades in alignment with its mission to find cures and address and prevent complications enabling people and families with blood disorders to thrive. RESEARCH DESIGN AND METHODS: With the American Thrombosis and Hemostasis Network, NHF recruited multidisciplinary expert working groups (WG) to distill the community-identified priorities into concrete research questions and score their feasibility, impact, and risk. WG6 was charged with identifying the infrastructure, workforce development, and funding and resources to facilitate the prioritized research. Community input on conclusions was gathered at the NHF State of the Science Research Summit. RESULTS: WG6 detailed a minimal research capacity infrastructure threshold, and opportunities to enable its attainment, for bleeding disorders centers to participate in prospective, multicenter national registries. They identified challenges and opportunities to recruit, retain, and train the diverse multidisciplinary care and research workforce required into the future. Innovative collaborative approaches to trial design, resource networking, and funding to surmount obstacles facing research in rare disorders were elucidated. CONCLUSIONS: The innovations in infrastructure, workforce development, and resources and funding proposed herein may contribute to facilitating a National Research Blueprint for Inherited Bleeding Disorders.

Research is critical to advancing the diagnosis and care of people with inherited bleeding disorders (PWIBD). This research requires significant infrastructure, including people and resources. Hemophilia treatment centers (HTC) need many different skilled care professionals including doctors, nurses, and other providers; also statisticians, data managers, and other experts to process patients' clinical information into research. Attracting diverse qualified professionals to the clinical and research work requires long-term planning, recruiting individuals in training programs and retaining them as they become experts. Research infrastructure includes physical servers running database software, networks that link them, and the environment in which these components function. US Centers for Disease Control and Prevention (CDC) and American Thrombosis and Hemostasis Network (ATHN) coordinate and fund data collection at HTCs on the health and well-being of thousands of PWIBD into a registry used in research studies.National Hemophilia Foundation (NHF) and ATHN asked our group of health care professionals, technology experts, and lived experience experts (LEE) to identify the infrastructure, workforce, and resources needed to do the research most important to PWIBD. We identified the types of CDC/ATHN studies all HTCs should be able to perform, and the physical and human infrastructure this requires. We prioritized finding the best clinical trial designs to study inherited bleeding disorders, identifying ways to share personnel and tools between HTCs, and innovating how research is governed and funded. Involving LEEs in designing, managing, and carrying out research will be key in conducting research to improve the lives of PWIBD.

Structural and Molecular Determinants for Isoform Bias at Human Histamine H3 Receptor Isoforms.

The human histamine H3 receptor (hH3R) is predominantly expressed in the CNS, where it regulates the synthesis and release of histamine and other neurotransmitters. Due to its neuromodulatory role, the hH3R has been associated with various CNS disorders, including Alzheimer's and Parkinson's disease. Markedly, the hH3R gene undergoes extensive splicing, resulting in 20 isoforms, of which 7TM isoforms exhibit variations in the intracellular loop 3 (IL3) and/or C-terminal tail. Particularly, hH3R isoforms that display variations in IL3 (e.g., hH3R-365) are shown to differentially signal via Gαi-dependent pathways upon binding of biased agonists (e.g., immepip, proxifan, imetit). Nevertheless, the mechanisms underlying biased agonism at hH3R isoforms remain unknown. Using a structure-function relationship study with a broad range of H3R agonists, we thereby explored determinants underlying isoform bias at hH3R isoforms that exhibit variations in IL3 (i.e., hH3R-445, -415, -365, and -329) in a Gαi-dependent pathway (cAMP inhibition). Hence, we systematically characterized hH3R isoforms on isoform bias by comparing various ligand properties (i.e., structural and molecular) to the degree of isoform bias. Importantly, our study provides novel insights into the structural and molecular basis of receptor isoform bias, highlighting the importance to study GPCRs with multiple isoforms to better tailor drugs.

Opportunities and challenges for the development of M1 muscarinic receptor positive allosteric modulators in the treatment for neurocognitive deficits.

Targeting allosteric sites of M1 muscarinic acetylcholine receptors (M1 receptors) is a promising strategy to treat neurocognitive disorders, such as Alzheimer's disease and schizophrenia. Indeed, the last two decades have seen an impressive body of work focussing on the design and development of positive allosteric modulators (PAMs) for the M1 receptor. This has led to the identification of a structurally diverse range of highly selective M1 PAMs. In preclinical models, M1 PAMs have shown rescue of cognitive deficits and improvement of endpoints predictive of symptom domains of schizophrenia. Yet, to date only a few M1 PAMs have reached early-stage clinical trials, with many of them failing to progress further due to on-target mediated cholinergic adverse effects that have plagued the development of this class of ligand. This review covers the recent preclinical and clinical studies in the field of M1 receptor drug discovery for the treatment of Alzheimer's disease and schizophrenia, with a specific focus on M1 PAM, highlighting both the undoubted potential but also key challenges for the successful translation of M1 PAMs from bench-side to bedside.

Beyond antipsychotics: a twenty-first century update for preclinical development of schizophrenia therapeutics.

Despite 50+ years of drug discovery, current antipsychotics have limited efficacy against negative and cognitive symptoms of schizophrenia, and are ineffective-with the exception of clozapine-against any symptom domain for patients who are treatment resistant. Novel therapeutics with diverse non-dopamine D2 receptor targets have been explored extensively in clinical trials, yet often fail due to a lack of efficacy despite showing promise in preclinical development. This lack of translation between preclinical and clinical efficacy suggests a systematic failure in current methods that determine efficacy in preclinical rodent models. In this review, we critically evaluate rodent models and behavioural tests used to determine preclinical efficacy, and look to clinical research to provide a roadmap for developing improved translational measures. We highlight the dependence of preclinical models and tests on dopamine-centric theories of dysfunction and how this has contributed towards a self-reinforcing loop away from clinically meaningful predictions of efficacy. We review recent clinical findings of distinct dopamine-mediated dysfunction of corticostriatal circuits in patients with treatment-resistant vs. non-treatment-resistant schizophrenia and suggest criteria for establishing rodent models to reflect such differences, with a focus on objective, translational measures. Finally, we review current schizophrenia drug discovery and propose a framework where preclinical models are validated against objective, clinically informed measures and preclinical tests of efficacy map onto those used clinically.

Trace Amine-Associated Receptor 1 (TAAR1): Molecular and Clinical Insights for the Treatment of Schizophrenia and Related Comorbidities.

Schizophrenia is a complex and severe mental illness. Current treatments for schizophrenia typically modulate dopaminergic neurotransmission by D2-receptor blockade. While reducing positive symptoms of schizophrenia, current antipsychotic drugs have little clinical effect on negative symptoms and cognitive impairments. For the last few decades, discovery efforts have sought nondopaminergic compounds with the aim to effectively treat the broad symptoms of schizophrenia. In this viewpoint, we provide an overview on trace-amine associated receptor-1 (TAAR1), which presents a clinically validated nondopaminergic target for treating schizophrenia and related disorders, with significantly less overall side-effect burden. TAAR1 agonists may also be specifically beneficial for the substance abuse comorbidity and metabolic syndrome that is often present in patients with schizophrenia.

Multipathway In Vitro Pharmacological Characterization of Specialized Proresolving G Protein-Coupled Receptors.

Specialized proresolving mediators (SPMs) and their cognate G protein-coupled receptors are implicated in autoimmune disorders, including chronic inflammation, rheumatoid arthritis, systemic scleroderma, and lupus erythematosus. To date, six G protein-coupled receptors (GPCRs) have been paired with numerous endogenous and synthetic ligands. However, the function and downstream signaling of these receptors remains unclear. To address this knowledge gap, we systematically expressed each receptor in a human embryonic kindney 293 (HEK293)-Flp-In-CD8a-FLAG cell system. Each receptor was pharmacologically characterized with both synthetic and putative endogenous ligands across different signaling assays, covering both G protein-dependent (Gs, Gi, and Gq) and independent mechanisms (ß-arrestin2 recruitment). Three orphan GPCRs previously identified as SPM receptors (GPR 18, GPR32 and GPR37) failed to express in HEK 293 cells. Although we were unsuccessful in identifying an endogenous ligand for formyl peptide receptor 2 (FPR2)/lipoxin A4 receptor (ALX), with only a modest response to N-formylmethionine-leucyl-phenylalanine (fMLP), we did reveal clear signaling bias away from extracelluar signal-related kinase (ERK) 1/2 phosphorylation for the clinically tested agonist N-(2-{[4-(1,1-difluoroethyl)-1,3-oxazol-2-yl]methyl}-2H-1,2,3-triazol-4-yl)-2-methyl-5-(3-methylphenyl)-1,3-oxazole-4-carboxamide (ACT-389949), adding further evidence for its poor efficacy in two phase I studies. We also identified neuroprotectin D1 as a new leukotriene B4 receptor 1 (BLT1) agonist, implying an alternative target for the neuroprotective effects of the ligand. We confirmed activity for resolvin E1 (RvE1) at BLT1 but failed to observe any response at the chemerin1 receptor. This study provides some much-needed clarity around published receptor-ligand pairings but indicates that the expression and function of these SPM GPCRs remains very much context-dependent. In addition, the identification of signaling bias at FPR2/ALX may assist in guiding design of new FPR2/ALX agonists for the treatment of autoimmune disorders. SIGNIFICANCE STATEMENT: To our knowledge, this is the first study to comprehensibly show how several natural mediators and synthetic ligands signal through three specialized proresolving mediator GPCRs using multiple ligands from different classes across four-six endpoint signaling assays. This study discovers new ligand pairings, refutes others, reveals poly-pharmacology, and identifies biased agonism in formyl peptide receptor 2/lipoxin A4 receptor pharmacology. This study highlights the potential of these receptors in treating specific autoimmune diseases, including rheumatoid arthritis, systemic scleroderma, and systemic lupus erythematosus.

M1 muscarinic receptor activation decreases alcohol consumption via a reduction in consummatory behavior.

Muscarinic acetylcholine receptors (mAChRs) have been shown to mediate alcohol consumption and seeking. Both M4 and M5 mAChRs have been highlighted as potential novel treatment targets for alcohol use disorders (AUD). Similarly, M1 mAChRs are expressed throughout reward circuitry, and their signaling has been implicated in cocaine consumption. However, whether the same effects are seen for alcohol consumption, or whether natural reward intake is inadvertently impacted is still unknown. To determine the role of M1 mAChRs in alcohol consumption, we tested operant self-administration of alcohol under both fixed ratio (FR3) and progressive ratio (PR3-4) schedules. Enhancing M1 mAChR signaling (via the M1 PAM-Agonist PF-06767832, 1 mg/kg, i.p.) reduced operant alcohol consumption on a fixed schedule but had no effect on motivation to acquire alcohol. To determine whether these actions were specific to alcohol, we examined the effects of M1 enhancement on natural reward (sucrose) self-administration. Systemic administration of PF-06767832 (1 mg/kg, i.p.) also reduced operant sucrose self-administration, suggesting the actions of the M1 receptor may be non-selective across drug and natural rewards. Finally, to understand whether this reduction extended to natural consummatory behaviors, we assessed home cage standard chow and water consumption. M1 enhancement via systemic PF-06767832 administration reduced food and water consumption. Together our results suggest the M1 PAM-agonist, PF-06767832, non-specifically reduces consummatory behaviors that are not associated with motivational strength for the reward. These data highlight the need to further characterize M1 agonists, PAMs, and PAM-agonists, which may have varying degrees of utility in the treatment of neuropsychiatric disorders including AUD.