RESUMO
The M4 muscarinic acetylcholine receptor (M4 mAChR) has emerged as a drug target of high therapeutic interest due to its expression in regions of the brain involved in the regulation of psychosis, cognition, and addiction. The mAChR agonist, xanomeline, has provided significant improvement in the Positive and Negative Symptom Scale (PANSS) scores in a Phase II clinical trial for the treatment of patients suffering from schizophrenia. Here we report the active state cryo-EM structure of xanomeline bound to the human M4 mAChR in complex with the heterotrimeric Gi1 transducer protein. Unexpectedly, two molecules of xanomeline were found to concomitantly bind to the monomeric M4 mAChR, with one molecule bound in the orthosteric (acetylcholine-binding) site and a second molecule in an extracellular vestibular allosteric site. Molecular dynamic simulations supports the structural findings, and pharmacological validation confirmed that xanomeline acts as a dual orthosteric and allosteric ligand at the human M4 mAChR. These findings provide a basis for further understanding xanomeline's complex pharmacology and highlight the myriad of ways through which clinically relevant ligands can bind to and regulate GPCRs.
Assuntos
Comportamento Aditivo , Humanos , Sítio Alostérico , Encéfalo , CogniçãoRESUMO
A drug's selectivity for target receptors is essential to its therapeutic utility, but achieving selectivity between similar receptors is challenging. The serendipitous discovery of ligands that stimulate target receptors more strongly than closely related receptors, despite binding with similar affinities, suggests a solution. The molecular mechanism of such 'efficacy-driven selectivity' has remained unclear, however, hindering design of such ligands. Here, using atomic-level simulations, we reveal the structural basis for the efficacy-driven selectivity of a long-studied clinical drug candidate, xanomeline, between closely related muscarinic acetylcholine receptors (mAChRs). Xanomeline's binding mode is similar across mAChRs in their inactive states but differs between mAChRs in their active states, with divergent effects on active-state stability. We validate this mechanism experimentally and use it to design ligands with altered efficacy-driven selectivity. Our results suggest strategies for the rational design of ligands that achieve efficacy-driven selectivity for many pharmaceutically important G-protein-coupled receptors.
Assuntos
Receptores Muscarínicos , Tiadiazóis , Ligantes , Receptores Muscarínicos/química , Receptores Muscarínicos/metabolismo , Piridinas , Tiadiazóis/química , Receptores Acoplados a Proteínas G/químicaRESUMO
Modern interest in muscarinic acetylcholine receptor (mAChR) activators for schizophrenia began in the 1990s when xanomeline, an M1/M4-preferring mAChR agonist developed for cognitive symptoms of Alzheimer's disease (AD), had unexpected antipsychotic activity. However, strategies to address tolerability concerns associated with activation of peripheral mAChRs were not available at that time. The discovery of specific targeted ligands and combination treatments to reduce peripheral mAChR engagement have advanced the potential of mAChR activators as effective treatments for psychotic disorders. This review provides perspectives on the background of the identification of mAChRs as potential antipsychotics, advances in the preclinical understanding of mAChRs as targets, and the current state of mAChR activators under active clinical development for schizophrenia.
Assuntos
Transtornos Psicóticos , Esquizofrenia , Humanos , Agonistas Muscarínicos/farmacologia , Agonistas Muscarínicos/uso terapêutico , Receptores Muscarínicos , Esquizofrenia/tratamento farmacológico , Transtornos Psicóticos/tratamento farmacológico , Acetilcolina , Receptor Muscarínico M1/agonistasRESUMO
Schizophrenia remains a challenging disease to treat effectively with current antipsychotic medications due to their limited efficacy across the entire spectrum of core symptoms as well as their often burdensome side-effect profiles and poor tolerability. An unmet need remains for novel, mechanistically unique, and better tolerated therapeutic agents for treating schizophrenia, especially those that treat not only positive symptoms but also the negative and cognitive symptoms of the disease. Almost 25 years ago, the muscarinic acetylcholine receptor (mAChR) agonist xanomeline was reported to reduce psychotic symptoms and improve cognition in patients with Alzheimer's disease. The antipsychotic and procognitive properties of xanomeline were subsequently confirmed in a small study of acutely psychotic patients with chronic schizophrenia. These unexpected clinical findings have prompted considerable efforts across academia and industry to target mAChRs as a new approach to potentially treat schizophrenia and other psychotic disorders. The authors discuss recent advances in mAChR biology and pharmacology and the current understanding of the relative roles of the various mAChR subtypes, their downstream cellular effectors, and key neural circuits mediating the reduction in the core symptoms of schizophrenia in patients treated with xanomeline. They also provide an update on the status of novel mAChR agonists currently in development for potential treatment of schizophrenia and other neuropsychiatric disorders.
Assuntos
Antipsicóticos , Agonistas Muscarínicos , Transtornos Psicóticos , Esquizofrenia , Antipsicóticos/farmacologia , Antipsicóticos/uso terapêutico , Humanos , Agonistas Muscarínicos/farmacologia , Agonistas Muscarínicos/uso terapêutico , Transtornos Psicóticos/tratamento farmacológico , Receptores Muscarínicos , Esquizofrenia/tratamento farmacológicoRESUMO
Many Food and Drug Administration (FDA)-approved drugs are structural analogues of the endogenous (natural) ligands of G protein-coupled receptors (GPCRs). However, it is becoming appreciated that chemically distinct ligands can bind to GPCRs in conformations that lead to different cellular signaling events, a phenomenon termed biased agonism. Despite this, the rigorous experimentation and analysis required to identify biased agonism are often not undertaken in most clinical candidates and go unrealized. Recently, xanomeline, a muscarinic acetylcholine receptor (mAChR) agonist, has entered phase III clinical trials for the treatment of schizophrenia. If successful, xanomeline will be the first novel FDA-approved antipsychotic drug in almost 50 years. Intriguingly, xanomeline's potential for biased agonism at the mAChRs and, in particular, the M4 mAChR, the most promising receptor target for schizophrenia, has not been assessed. Here, we quantify the biased agonism profile of xanomeline and three other mAChR agonists in Chinese hamster ovary cells recombinantly expressing the M4 mAChR. Agonist activity was examined across nine distinct signaling readouts, including the activation of five different G protein subtypes, ERK1/2 phosphorylation, ß-arrestin recruitment, calcium mobilization, and cAMP regulation. Relative to acetylcholine (ACh), xanomeline was biased away from ERK1/2 phosphorylation and calcium mobilization compared to Gαi2 protein activation. These findings likely have important implications for our understanding of the therapeutic action of xanomeline and call for further investigation into the in vivo consequences of biased agonism in drugs targeting the M4 mAChR for the treatment of schizophrenia.
Assuntos
Cálcio , Tiadiazóis , Acetilcolina/metabolismo , Acetilcolina/farmacologia , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Ligantes , Agonistas Muscarínicos/farmacologia , Agonistas Muscarínicos/uso terapêutico , Piridinas , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M4/agonistas , Receptores Acoplados a Proteínas G , Receptores Muscarínicos , Tiadiazóis/químicaRESUMO
Opioid misuse and addiction are a public health crisis resulting in debilitation, deaths, and significant social and economic impact. Curbing this crisis requires collaboration among academic, government, and industrial partners toward the development of effective nonaddictive pain medications, interventions for opioid overdose, and addiction treatments. A 2-day meeting, The Opioid Crisis and the Future of Addiction and Pain Therapeutics: Opportunities, Tools, and Technologies Symposium, was held at the National Institutes of Health (NIH) to address these concerns and to chart a collaborative path forward. The meeting was supported by the NIH Helping to End Addiction Long-TermSM (HEAL) Initiative, an aggressive, trans-agency effort to speed scientific solutions to stem the national opioid crisis. The event was unique in bringing together two research disciplines, addiction and pain, in order to create a forum for crosscommunication and collaboration. The output from the symposium will be considered by the HEAL Initiative; this article summarizes the scientific presentations and key takeaways. Improved understanding of the etiology of acute and chronic pain will enable the discovery of novel targets and regulatable pain circuits for safe and effective therapeutics, as well as relevant biomarkers to ensure adequate testing in clinical trials. Applications of improved technologies including reagents, assays, model systems, and validated probe compounds will likely increase the delivery of testable hypotheses and therapeutics to enable better health outcomes for patients. The symposium goals were achieved by increasing interdisciplinary collaboration to accelerate solutions for this pressing public health challenge and provide a framework for focused efforts within the research community. SIGNIFICANCE STATEMENT: This article summarizes key messages and discussions resulting from a 2-day symposium focused on challenges and opportunities in developing addiction- and pain-related medications. Speakers and attendees came from 40 states in the United States and 15 countries, bringing perspectives from academia, industry, government, and healthcare by researchers, clinicians, regulatory experts, and patient advocates.
Assuntos
Analgésicos Opioides/uso terapêutico , Comportamento Aditivo/terapia , Dor Crônica/tratamento farmacológico , Congressos como Assunto/tendências , National Institutes of Health (U.S.)/tendências , Epidemia de Opioides/tendências , Analgésicos Opioides/efeitos adversos , Comportamento Aditivo/epidemiologia , Dor Crônica/epidemiologia , Previsões , Humanos , Epidemia de Opioides/prevenção & controle , Transtornos Relacionados ao Uso de Opioides/epidemiologia , Transtornos Relacionados ao Uso de Opioides/prevenção & controle , Estados Unidos/epidemiologiaRESUMO
Allosteric modulation of G protein coupled receptors (GPCRs) is rapidly becoming a standard option for development of therapeutics headed to the clinic. Although GPCRs represent about 35% of marketed drugs, to date only two allosteric modulators have been approved for human use. However, many are now in early clinical development are can provide unique regulation of GPCRs including high selectivity along with physiologic temporal and spatial signaling. These molecules bind to a site that is distinct from the site where the endogenous agonist binds yet can provide robust modulation effects that span from the positive to the negative. Along with classical chemogenomic techniques, newer technology is being directly applied to their development including three dimensional biophysical structure-function analysis and in silico molecular dynamic simulations. The goal is to provide rationally designed molecules from well informed physical and in silico data to speed the discovery and development of the next generation therapeutics. In this chapter an example of the evolution of allosteric drug discovery targeting the muscarinic receptor family should serve to inform of progress in this exciting area of research and early drug development.
Assuntos
Descoberta de Drogas , Receptores Acoplados a Proteínas G/metabolismo , Regulação Alostérica , Sítio Alostérico , Animais , Avaliação Pré-Clínica de Medicamentos , Humanos , Ligantes , Receptores Acoplados a Proteínas G/químicaRESUMO
Allosteric modulators are highly desirable as drugs, particularly for G-protein-coupled receptor (GPCR) targets, because allosteric drugs can achieve selectivity between closely related receptors. The mechanisms by which allosteric modulators achieve selectivity remain elusive, however, particularly given recent structures that reveal similar allosteric binding sites across receptors. Here we show that positive allosteric modulators (PAMs) of the M1 muscarinic acetylcholine receptor (mAChR) achieve exquisite selectivity by occupying a dynamic pocket absent in existing crystal structures. This cryptic pocket forms far more frequently in molecular dynamics simulations of the M1 mAChR than in those of other mAChRs. These observations reconcile mutagenesis data that previously appeared contradictory. Further mutagenesis experiments validate our prediction that preventing cryptic pocket opening decreases the affinity of M1-selective PAMs. Our findings suggest opportunities for the design of subtype-specific drugs exploiting cryptic pockets that open in certain receptors but not in other receptors with nearly identical static structures.
Assuntos
Receptor Muscarínico M1/química , Receptores Acoplados a Proteínas G/química , Regulação Alostérica , Sítio Alostérico , Cristalografia por Raios X , Desenho de Fármacos , Ligantes , Simulação de Dinâmica Molecular , Mutagênese Sítio-DirigidaRESUMO
Nonselective glutamate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists are efficacious in chronic pain but have significant tolerability issues, likely arising from the ubiquitous expression of AMPA receptors in the central nervous system (CNS). Recently, LY3130481 has been shown to selectively block AMPA receptors coassembled with the auxiliary protein, transmembrane AMPA receptor regulatory protein (TARP) γ8, which is highly expressed in the hippocampus but also in pain pathways, including anterior cingulate (ACC) and somatosensory cortices and the spinal cord, suggesting that selective blockade of γ8/AMPA receptors may suppress nociceptive signaling with fewer CNS side effects. The potency of LY3130481 on recombinant γ8-containing AMPA receptors was modulated by coexpression with other TARPs; γ2 subunits affected activity more than γ3 subunits. Consistent with these findings, LY3130481 had decreasing potency on receptors from rat hippocampal, cortical, spinal cord, and cerebellar neurons that was replicated in tissue from human brain. LY3130481 partially suppressed, whereas the nonselective AMPA antagonist GYKI53784 completely blocked, AMPA receptor-dependent excitatory postsynaptic potentials in ACC and spinal neurons in vitro. Similarly, LY3130481 attenuated short-term synaptic plasticity in spinal sensory neurons in vivo in response to stimulation of peripheral afferents. LY3130481 also significantly reduced nocifensive behaviors after intraplantar formalin that was correlated with occupancy of CNS γ8-containing AMPA receptors. In addition, LY3130481 dose-dependently attenuated established gait impairment after joint damage and tactile allodynia after spinal nerve ligation, all in the absence of motor side effects. Collectively, these data demonstrate that LY3130481 can suppress excitatory synaptic transmission and plasticity in pain pathways containing γ8/AMPA receptors and significantly reduce nocifensive behaviors, suggesting a novel, effective, and safer therapy for chronic pain conditions.
Assuntos
Canais de Cálcio/metabolismo , Dor Crônica/tratamento farmacológico , Dor Crônica/metabolismo , Terapia de Alvo Molecular , Receptores de AMPA/metabolismo , Animais , Benzotiazóis/farmacologia , Benzotiazóis/uso terapêutico , Dor Crônica/fisiopatologia , Masculino , Plasticidade Neuronal/efeitos dos fármacos , Nociceptividade/efeitos dos fármacos , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Ratos , Ratos Sprague-Dawley , Transmissão Sináptica/efeitos dos fármacos , Distribuição TecidualRESUMO
Pharmaceutical companies are relying more often on external sources of innovation to boost their discovery research productivity. However, more in-depth knowledge about how external innovation may translate to successful product launches is still required in order to better understand how to best leverage the innovation ecosystem. We analyzed the pre-approval publication histories for FDA-approved new molecular entities (NMEs) and new biologic entities (NBEs) launched by 13 top research pharma companies during the last decade (2006-2016). We found that academic institutions contributed the majority of pre-approval publications and that publication subject matter is closely aligned with the strengths of the respective innovator. We found this to also be true for candidate drugs terminated in Phase 3, but the volume of literature on these molecules is substantially less than for approved drugs. This may suggest that approved drugs are often associated with a more robust dataset provided by a large number of institutes. Collectively, the results of our analysis support the hypothesis that a collaborative research innovation environment spanning across academia, industry and government is highly conducive to successful drug approvals.
Assuntos
Aprovação de Drogas/estatística & dados numéricos , Indústria Farmacêutica/estatística & dados numéricos , Parcerias Público-Privadas , Produtos Biológicos , Estados Unidos , United States Food and Drug Administration , Universidades/estatística & dados numéricosRESUMO
BACKGROUND AND PURPOSE: We aimed to identify and develop novel, selective muscarinic M1 receptor agonists as potential therapeutic agents for the symptomatic treatment of Alzheimer's disease. EXPERIMENTAL APPROACH: We developed and utilized a novel M1 receptor occupancy assay to drive a structure activity relationship in a relevant brain region while simultaneously tracking drug levels in plasma and brain to optimize for central penetration. Functional activity was tracked in relevant native in vitro assays allowing translational (rat-human) benchmarking of structure-activity relationship molecules to clinical comparators. KEY RESULTS: Using this paradigm, we identified a series of M1 receptor selective molecules displaying desirable in vitro and in vivo properties and optimized key features, such as central penetration while maintaining selectivity and a partial agonist profile. From these compounds, we selected spiropiperidine 1 (SPP1). In vitro, SPP1 is a potent, partial agonist of cortical and hippocampal M1 receptors with activity conserved across species. SPP1 displays high functional selectivity for M1 receptors over native M2 and M3 receptor anti-targets and over a panel of other targets. Assessment of central target engagement by receptor occupancy reveals SPP1 significantly and dose-dependently occupies rodent cortical M1 receptors. CONCLUSIONS AND IMPLICATIONS: We report the discovery of SPP1, a novel, functionally selective, brain penetrant partial orthosteric agonist at M1 receptors, identified by a novel receptor occupancy assay. SPP1 is amenable to in vitro and in vivo study and provides a valuable research tool to further probe the role of M1 receptors in physiology and disease.
Assuntos
Osteopontina/agonistas , Piperidinas/farmacologia , Receptor Muscarínico M1/agonistas , Compostos de Espiro/farmacologia , Animais , Células CHO , Células Cultivadas , Cricetulus , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Piperidinas/química , Ratos , Ratos Sprague-Dawley , Compostos de Espiro/química , Relação Estrutura-Atividade , XenopusRESUMO
In the search for improved symptomatic treatment options for neurodegenerative and neuropsychiatric diseases, muscarinic acetylcholine M1 receptors (M1 mAChRs) have received significant attention. Drug development efforts have identified a number of novel ligands, some of which have advanced to the clinic. However, a significant issue for progressing these therapeutics is the lack of robust, translatable, and validated biomarkers. One valuable approach to assessing target engagement is to use positron emission tomography (PET) tracers. In this study we describe the pharmacological characterization of a selective M1 agonist amenable for in vivo tracer studies. We used a novel direct binding assay to identify nonradiolabeled ligands, including LSN3172176, with the favorable characteristics required for a PET tracer. In vitro functional and radioligand binding experiments revealed that LSN3172176 was a potent partial agonist (EC50 2.4-7.0 nM, Emax 43%-73%), displaying binding selectivity for M1 mAChRs (Kd = 1.5 nM) that was conserved across species (native tissue Kd = 1.02, 2.66, 8, and 1.03 at mouse, rat, monkey, and human, respectively). Overall selectivity of LSN3172176 appeared to be a product of potency and stabilization of the high-affinity state of the M1 receptor, relative to other mAChR subtypes (M1 > M2, M4, M5 > M3). In vivo, use of wild-type and mAChR knockout mice further supported the M1-preferring selectivity profile of LSN3172176 for the M1 receptor (78% reduction in cortical occupancy in M1 KO mice). These findings support the development of LSN3172176 as a potential PET tracer for assessment of M1 mAChR target engagement in the clinic and to further elucidate the function of M1 mAChRs in health and disease.
Assuntos
Tomografia por Emissão de Pósitrons/métodos , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/metabolismo , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Humanos , Cinética , Camundongos , Traçadores Radioativos , Ratos , Reprodutibilidade dos TestesRESUMO
The realization of the therapeutic potential of targeting the M1 muscarinic acetylcholine receptor (mAChR) for the treatment of cognitive decline in Alzheimer's disease has prompted the discovery of M1 mAChR ligands showing efficacy in alleviating cognitive dysfunction in both rodents and humans. Among these is GSK1034702 (7-fluoro-5-methyl-3-[1-(oxan-4-yl)piperidin-4-yl]-1H-benzimidazol-2-one), described previously as a potent M1 receptor allosteric agonist, which showed procognitive effects in rodents and improved immediate memory in a clinical nicotine withdrawal test but induced significant side effects. Here we provide evidence using ligand binding, chemical biology and functional assays to establish that rather than the allosteric mechanism claimed, GSK1034702 interacts in a bitopic manner at the M1 mAChR such that it can concomitantly span both the orthosteric and an allosteric binding site. The bitopic nature of GSK1034702, together with the intrinsic agonist activity and a lack of muscarinic receptor subtype selectivity reported here, all likely contribute to the adverse effects of this molecule in clinical trials. Although they impart beneficial effects on learning and memory, we conclude that these properties are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data support the notion that "pure" positive allosteric modulators showing selectivity for the M1 mAChR with low levels of intrinsic activity would be preferable to provide clinical efficacy with low adverse responses.
Assuntos
Acetilcolina/metabolismo , Agonistas Muscarínicos/farmacologia , Receptor Muscarínico M1/metabolismo , Receptores Muscarínicos/metabolismo , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico/efeitos dos fármacos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Animais , Benzimidazóis/farmacologia , Sítios de Ligação/efeitos dos fármacos , Células CHO , Linhagem Celular , Ensaios Clínicos como Assunto , Cricetinae , Cricetulus , Humanos , Aprendizagem/efeitos dos fármacos , Masculino , Memória/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
The cholinergic signalling system has been an attractive pathway to seek targets for modulation of arousal, cognition, and attention which are compromised in neurodegenerative and neuropsychiatric diseases. The acetylcholine muscarinic receptor M1 and M4 subtypes which are highly expressed in the central nervous system, in cortex, hippocampus and striatum, key areas of cognitive and neuropsychiatric control, have received particular attention. Historical muscarinic drug development yielded first generation agonists with modest selectivity for these two receptor targets over M2 and M3 receptors, the major peripheral sub-types hypothesised to underlie the dose-limiting clinical side effects. More recent compound screening and medicinal chemistry optimization of orthosteric and allosteric agonists, and positive allosteric modulators binding to sites distinct from the highly homologous acetylcholine binding pocket have yielded a collection of highly selective tool compounds for preclinical validation studies. Several M1 selective ligands have progressed to early clinical development and in time will hopefully lead to useful therapeutics for treating symptoms of Alzheimer's disease and related disorders. This article is part of the Special Issue entitled 'Neuropharmacology on Muscarinic Receptors'.
Assuntos
Agonistas Muscarínicos/farmacologia , Doenças Neurodegenerativas/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M4/agonistas , Animais , Humanos , Agonistas Muscarínicos/uso terapêutico , Doenças Neurodegenerativas/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M4/metabolismoRESUMO
Rodent genomic alignment sequences support a 2-exon model for muscarinic M4 receptor. Using this model a novel N-terminal extension was discovered in the human muscarinic acetylcholine M4 receptor. An open reading frame was discovered in the human, mouse and rat with a common ATG (methionine start codon) that extended the N-terminus of the muscarinic acetylcholine M4 receptor subtype by 155 amino acids resulting in a longer variant. Transcriptional evidence for this splice variant was confirmed by RNA-Seq and RT-PCR experiments performed from human donor brain prefrontal cortices. We detected a human upstream exon indicating the translation of the mature longer M4 receptor transcript. The predicted size for the longer two-exon M4 receptor splice variant with the additional 155 amino acid N-terminal extension, designated M4L is 69.7 kDa compared to the 53 kDa canonical single exon M4 receptor (M4S). Western blot analysis from a mammalian overexpression system, and saturation radioligand binding with [3H]-NMS (N-methyl-scopolamine) demonstrated the expression of this new splice variant. Comparative pharmacological characterization between the M4L and M4S receptors revealed that both the orthosteric and allosteric binding sites for both receptors were very similar despite the addition of an N-terminal extension.
Assuntos
Splicing de RNA , Receptor Muscarínico M4/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Éxons , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Camundongos , Reação em Cadeia da Polimerase , Córtex Pré-Frontal/metabolismo , Ensaio Radioligante , Ratos , Receptor Muscarínico M4/genética , Análise de Sequência de RNA , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: Conventional antidepressants lack efficacy for many patients (treatmentresistant depression or TRD) and generally take weeks to produce full therapeutic response in others. Emerging data has identified certain drugs such as ketamine as rapidly-acting antidepressants for major depressive disorder and TRD. Scopolamine, a drug used to treat motion sickness and nausea, has also been demonstrated to function as a rapidly-acting antidepressant. The mechanisms associated with efficacy in TRD patients and rapid onset of action have been suggested to involve a-Amino-3-hydroxy- 5-methyl-4-isoxazolepropionic acid (AMPA) receptor and mammalian target of rapamycin (mTOR) signaling. Since the work on these mechanisms with scopolamine has been limited, the present set of experiments was designed to further explore these mechanisms of action. METHOD: Male, NIH Swiss mice demonstrated a robust and immediate antidepressant signature with ketamine or scopolamine when studied under the forced-swim test. RESULTS: The AMPA receptor antagonist NBQX prevented this antidepressant-like effect of scopolamine and ketamine. An orally-bioavilable mTOR inhibitor (AZD8055) also attenuated the antidepressant- like effects of scopolamine and ketamine. Scopolamine was also shown to augment the antidepressant- like effect of the selective serotonin reuptake inhibitor citalopram. When given in combination, scopolamine and ketamine acted synergistically to produce antidepressant-like effects. Although drug interaction data suggested that additional mechanisms might be at play, metabolomic analysis of frontal cortex and plasma from muscarinic M1+/+ and M1 -/- mice given scopolamine or vehicle did not reveal any hints as to the nature of these additional mechanisms of action. CONCLUSION: Overall, the data substantiate and extend the idea that AMPA and mTOR signaling pathways are necessary for the antidepressant-like effects of scopolamine and ketamine, mechanisms that appear to be of general significance for TRD therapeutic agents.
Assuntos
Antidepressivos/farmacologia , Transtorno Depressivo/tratamento farmacológico , Escopolamina/farmacologia , Animais , Citalopram/farmacologia , Transtorno Depressivo/metabolismo , Interações Medicamentosas , Quimioterapia Combinada , Antagonistas de Aminoácidos Excitatórios/farmacologia , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/metabolismo , Ketamina/farmacologia , Masculino , Metaboloma/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfolinas/farmacologia , Quinoxalinas/farmacologia , Receptor Muscarínico M1/genética , Receptor Muscarínico M1/metabolismo , Receptores de AMPA/antagonistas & inibidores , Receptores de AMPA/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismoRESUMO
LY2812223 [(1R,2S,4R,5R,6R)-2-amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid] was identified via structure-activity studies arising from the potent metabotropic glutamate mGlu2/3 receptor agonist LY354740 [(+)-2-aminobicyclo[3.1.0] hexane-2,6-dicarboxylic acid] as an mGlu2-preferring agonist. This pharmacology was determined using stably transfected cells containing either the human mGlu2 or mGlu3 receptor. We extended the pharmacological evaluation of LY2812223 to native brain tissues derived from relevant species used for preclinical drug development as well as human postmortem brain tissue. This analysis was conducted to ensure pharmacological translation from animals to human subjects in subsequent clinical studies. A guanosine 5'-O-(3-[35S]thio)triphosphate (GTPγS) functional binding assay, a method for measuring Gi-coupled signaling that is inherent to the group 2 mGlu receptors, was used to evaluate LY2812223 pharmacology of native mGlu receptors in mouse, rat, nonhuman primate, and human cortical brain tissue samples. In native tissue membranes, LY2812223 unexpectedly acted as a partial agonist across all species tested. Activity of LY2812223 was lost in cortical membranes collected from mGlu2 knockout mice, but not those from mGlu3 knockout mice, providing additional support for mGlu2-preferring activity. Other signal transduction assays were used for comparison with the GTP binding assay (cAMP, calcium mobilization, and dynamic mass redistribution). In ectopic cell line-based assays, LY2812223 displayed near maximal agonist responses at the mGlu2 receptor across all assay formats, while it showed no functional agonist activity at the mGlu3 receptor except in the cAMP assay. In native brain slices or membranes that express both mGlu2 and mGlu3 receptors, LY2812223 displayed unexpected partial agonist activity, which may suggest a functional interplay between these receptor subtypes in the brain.
Assuntos
Encéfalo/efeitos dos fármacos , Compostos Bicíclicos com Pontes/farmacologia , Agonismo Parcial de Drogas , Agonistas de Aminoácidos Excitatórios/farmacologia , Receptores de Glutamato Metabotrópico/agonistas , Triazóis/farmacologia , Animais , Encéfalo/metabolismo , Compostos Bicíclicos com Pontes/metabolismo , Relação Dose-Resposta a Droga , Agonistas de Aminoácidos Excitatórios/metabolismo , Humanos , Camundongos , Camundongos Knockout , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato Metabotrópico/metabolismo , Pesquisa Translacional Biomédica , Triazóis/metabolismoRESUMO
The current frontline symptomatic treatment for Alzheimer's disease (AD) is whole-body upregulation of cholinergic transmission via inhibition of acetylcholinesterase. This approach leads to profound dose-related adverse effects. An alternative strategy is to selectively target muscarinic acetylcholine receptors, particularly the M1 muscarinic acetylcholine receptor (M1 mAChR), which was previously shown to have procognitive activity. However, developing M1 mAChR-selective orthosteric ligands has proven challenging. Here, we have shown that mouse prion disease shows many of the hallmarks of human AD, including progressive terminal neurodegeneration and memory deficits due to a disruption of hippocampal cholinergic innervation. The fact that we also show that muscarinic signaling is maintained in both AD and mouse prion disease points to the latter as an excellent model for testing the efficacy of muscarinic pharmacological entities. The memory deficits we observed in mouse prion disease were completely restored by treatment with benzyl quinolone carboxylic acid (BQCA) and benzoquinazoline-12 (BQZ-12), two highly selective positive allosteric modulators (PAMs) of M1 mAChRs. Furthermore, prolonged exposure to BQCA markedly extended the lifespan of diseased mice. Thus, enhancing hippocampal muscarinic signaling using M1 mAChR PAMs restored memory loss and slowed the progression of mouse prion disease, indicating that this ligand type may have clinical benefit in diseases showing defective cholinergic transmission, such as AD.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Hipocampo/metabolismo , Transtornos da Memória/tratamento farmacológico , Doenças Priônicas/tratamento farmacológico , Quinolinas/farmacologia , Receptor Muscarínico M1/metabolismo , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Animais , Hipocampo/fisiopatologia , Humanos , Transtornos da Memória/genética , Transtornos da Memória/metabolismo , Transtornos da Memória/fisiopatologia , Camundongos , Camundongos Knockout , Doenças Priônicas/genética , Doenças Priônicas/metabolismo , Doenças Priônicas/fisiopatologia , Receptor Muscarínico M1/genéticaRESUMO
Identification of synthetic ligands selective for muscarinic receptor subtypes has been challenging due to the high sequence identity and structural homology among the five muscarinic acetylcholine receptors. Here, we report the pharmacological characterization of PCS1055, a novel muscarinic M4 receptor antagonist. PCS1055 inhibited radioligand [(3)H]-NMS binding to the M4 receptor with a Ki=6.5nM. Though the potency of PCS1055 is lower than that of pan-muscarinic antagonist atropine, it has better subtype selectivity over previously reported M4-selective reagents such as the muscarinic-peptide toxins (Karlsson et al., 1994; Santiago and Potter, 2001a) at the M1 subtype, and benzoxazine ligand PD102807 at the M3-subtype (Bohme et al., 2002). A detailed head-to-head comparison study using [(3)H]-NMS competitive binding assays characterizes the selectivity profiles of PCS1055 to that of other potent muscarinic-antagonist compounds PD102807, tropicamide, AF-DX-384, pirenzapine, and atropine. In addition to binding studies, the subtype specificity of PCS1055 is also demonstrated by functional receptor activation as readout by GTP-γ-[(35)S] binding. These GTP-γ-[(35)S] binding studies showed that PCS1055 exhibited 255-, 69.1-, 342- and >1000-fold greater inhibition of Oxo-M activity at the M4 versus the M1-, M2(-), M3-or M5 receptor subtypes, respectively. Schild analyses indicates that PCS1055 acts as a competitive antagonist to muscarinic M4 receptor, and confirms the affinity of the ligand to be low nanomolar, Kb=5.72nM. Therefore, PCS1055 represents a new M4-preferring antagonist that may be useful in elucidating the roles of M4 receptor signaling.