RESUMEN
Muscarinic receptors are G protein-coupled receptors (GPCRs) that play a role in various physiological functions. Previous studies have shown that these receptors, along with other GPCRs, are voltage-sensitive; both their affinity toward agonists and their activation are regulated by membrane potential. To our knowledge, whether the effect of antagonists on these receptors is voltage-dependent has not yet been studied. In this study, we used Xenopus oocytes expressing the M2 muscarinic receptor (M2R) to investigate this question. Our results indicate that the potencies of two M2R antagonists, atropine and scopolamine, are voltage-dependent; they are more effective at resting potential than under depolarization. In contrast, the M2R antagonist AF-DX 386 did not exhibit voltage-dependent potency.Furthermore, we discovered that the voltage dependence of M2R activation by acetylcholine remains unchanged in the presence of two allosteric modulators, the negative modulator gallamine and the positive modulator LY2119620. These findings enhance our understanding of GPCRs' voltage dependence and may have pharmacological implications.
Asunto(s)
Antagonistas Muscarínicos , Oocitos , Receptor Muscarínico M2 , Xenopus laevis , Animales , Receptor Muscarínico M2/antagonistas & inhibidores , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M2/agonistas , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/fisiología , Antagonistas Muscarínicos/farmacología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Oocitos/fisiología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Atropina/farmacología , Escopolamina/farmacología , Acetilcolina/metabolismo , Acetilcolina/farmacología , Femenino , Sulfonamidas , TiadiazolesRESUMEN
G protein-coupled receptors (GPCRs) transduce the effects of many neuromodulators including dopamine, serotonin, epinephrine, acetylcholine, and opioids. The localization of synthetic or endogenous GPCR agonists impacts their action on specific neuronal pathways. In this paper, we show a series of single-protein chain integrator sensors that are highly modular and could potentially be used to determine GPCR agonist localization across the brain. We previously engineered integrator sensors for the mu- and kappa-opioid receptor agonists called M- and K-Single-chain Protein-based Opioid Transmission Indicator Tool (SPOTIT), respectively. Here, we engineered red versions of the SPOTIT sensors for multiplexed imaging of GPCR agonists. We also modified SPOTIT to create an integrator sensor design platform called SPOTIT for all GPCRs (SPOTall). We used the SPOTall platform to engineer sensors for the beta 2-adrenergic receptor (B2AR), the dopamine receptor D1, and the cholinergic receptor muscarinic 2 agonists. Finally, we demonstrated the application of M-SPOTIT and B2AR-SPOTall in detecting exogenously administered morphine, isoproterenol, and epinephrine in the mouse brain via locally injected viruses. The SPOTIT and SPOTall sensor design platform has the potential for unbiased agonist detection of many synthetic and endogenous neuromodulators across the brain.
Asunto(s)
Receptores Acoplados a Proteínas G , Animales , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Humanos , Ratones , Células HEK293 , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/metabolismo , Isoproterenol/farmacología , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Morfina/farmacología , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/diagnóstico por imagen , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/metabolismo , Técnicas Biosensibles/métodosRESUMEN
The M2 muscarinic receptor (M2R) is a prototypical G-protein-coupled receptor (GPCR) that serves as a model system for understanding GPCR regulation by both orthosteric and allosteric ligands. Here, we investigate the mechanisms governing M2R signaling versatility using cryo-electron microscopy (cryo-EM) and NMR spectroscopy, focusing on the physiological agonist acetylcholine and a supra-physiological agonist iperoxo, as well as a positive allosteric modulator LY2119620. These studies reveal that acetylcholine stabilizes a more heterogeneous M2R-G-protein complex than iperoxo, where two conformers with distinctive G-protein orientations were determined. We find that LY2119620 increases the affinity for both agonists, but differentially modulates agonists efficacy in G-protein and ß-arrestin pathways. Structural and spectroscopic analysis suggest that LY211620 stabilizes distinct intracellular conformational ensembles from agonist-bound M2R, which may enhance ß-arrestin recruitment while impairing G-protein activation. These results highlight the role of conformational dynamics in the complex signaling behavior of GPCRs, and could facilitate design of better drugs.
Asunto(s)
Acetilcolina , Receptores Muscarínicos , Microscopía por Crioelectrón , Regulación Alostérica/fisiología , Receptores Muscarínicos/metabolismo , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Unión al GTP/metabolismo , Ligandos , beta-Arrestinas/metabolismoRESUMEN
Inhibitory regulation of the heart is determined by both cholinergic M2 receptors (M2R) and adenosine A1 receptors (A1R) that activate the same signaling pathway, the ACh-gated inward rectifier K+ (KACh) channels via Gi/o proteins. Previously, we have shown that the agonist-specific voltage sensitivity of M2R underlies several voltage-dependent features of IKACh, including the 'relaxation' property, which is characterized by a gradual increase or decrease of the current when cardiomyocytes are stepped to hyperpolarized or depolarized voltages, respectively. However, it is unknown whether membrane potential also affects A1R and how this could impact IKACh. Upon recording whole-cell currents of guinea-pig cardiomyocytes, we found that stimulation of the A1R-Gi/o-IKACh pathway with adenosine only caused a very slight voltage dependence in concentration-response relationships (~1.2-fold EC50 increase with depolarization) that was not manifested in the relative affinity, as estimated by the current deactivation kinetics (τ = 4074 ± 214 ms at -100 mV and τ = 4331 ± 341 ms at +30 mV; P = 0.31). Moreover, IKACh did not exhibit relaxation. Contrarily, activation of the M2R-Gi/o-IKACh pathway with acetylcholine induced the typical relaxation of the current, which correlated with the clear voltage-dependent effect observed in the concentration-response curves (~2.8-fold EC50 increase with depolarization) and in the IKACh deactivation kinetics (τ = 1762 ± 119 ms at -100 mV and τ = 1503 ± 160 ms at +30 mV; P = 0.01). Our findings further substantiate the hypothesis of the agonist-specific voltage dependence of GPCRs and that the IKACh relaxation is consequence of this property.
Asunto(s)
Acetilcolina/farmacología , Agonistas del Receptor de Adenosina A1/farmacología , Adenosina/farmacología , Activación del Canal Iónico/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Canales de Potasio/metabolismo , Receptor de Adenosina A1/metabolismo , Animales , Femenino , Cobayas , Masculino , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/metabolismoRESUMEN
BACKGROUND: Glioblastoma multiforme (GBM) is characterized by heterogeneous cell populations. Among these, the Glioblastoma Stem Cells (GSCs) fraction shares some similarities with Neural Stem Cells. GSCs exhibit enhanced resistance to conventional chemotherapy drugs. Our previous studies demonstrated that the activation of M2 muscarinic acetylcholine receptors (mAChRs) negatively modulates GSCs proliferation and survival. The aim of the present study was to analyze the ability of the M2 dualsteric agonist Iper-8-naphthalimide (N-8-Iper) to counteract GSCs drug resistance. METHODS: Chemosensitivity to M2 dualsteric agonist N-8-Iper and chemotherapy drugs such as temozolomide, doxorubicin, or cisplatin was evaluated in vitro by MTT assay in two different GSC lines. Drug efflux pumps expression was evaluated by RT-PCR and qRT-PCR. RESULTS: By using sub-toxic concentrations of N-8-Iper combined with the individual chemotherapeutic agents, we found that only low doses of the M2 agonist combined with doxorubicin or cisplatin or temozolomide were significantly able to counteract cell growth in both GSC lines. Moreover, we evaluated as the exposure to high and low doses of N-8-Iper downregulated the ATP-binding cassette (ABC) drug efflux pumps expression levels. CONCLUSIONS: Our results revealed the ability of the investigated M2 agonist to counteract drug resistance in two GSC lines, at least partially by downregulating the ABC drug efflux pumps expression. The combined effects of low doses of conventional chemotherapy and M2 agonists may thus represent a novel promising pharmacological approach to impair the GSC-drug resistance in the GBM therapy.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Resistencia a Antineoplásicos , Glioblastoma/tratamiento farmacológico , Agonistas Muscarínicos/farmacología , Naftalimidas/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Doxorrubicina/farmacología , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Transducción de Señal , Temozolomida/farmacologíaRESUMEN
One of the major limits of chemotherapy is depending on the ability of the cancer cells to elude and adapt to different drugs. Recently, we demonstrated how the activation of the M2 muscarinic receptor could impair neuroblastoma cell proliferation. In the present paper, we investigate the possible effects mediated by the preferential M2 receptor agonist arecaidine propargyl ester (APE) on drug resistance in two neuroblastoma cell lines, SK-N-BE and SK-N-BE(2C), a sub-clone presenting drug resistance. In both cell lines, we compare the expression of the M2 receptor and the effects mediated by the M2 agonist APE on cell cycle, demonstrating a decreased percentage of cells in S phase and an accumulation of SK-N-BE cells in G1 phase, while the APE treatment of SK-N-BE(2C) cells induced a block in G2/M phase. The withdrawal of the M2 agonist from the medium shows that only the SK-N-BE(2C) cells are able to rescue cell proliferation. Further, we demonstrate that the co-treatment of low doses of APE with doxorubicin or cisplatin significantly counteracts cell proliferation when compared with the single treatment. Analysis of the expression of ATP-binding cassette (ABC) efflux pumps demonstrates the ability of the M2 agonist to downregulate their expression and that this negative modulation may be dependent on N-MYC decreased expression induced by the M2 agonist. Our data demonstrate that the combined effect of low doses of conventional drugs and the M2 agonist may represent a new promising therapeutic approach in neuroblastoma treatment, in light of its significant impact on drug resistance and the possible reduction in the side effects caused by high doses of chemotherapy drugs.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Arecolina/análogos & derivados , Neuroblastoma/tratamiento farmacológico , Receptor Muscarínico M2/agonistas , Transportadoras de Casetes de Unión a ATP/genética , Arecolina/administración & dosificación , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/administración & dosificación , Doxorrubicina/administración & dosificación , Resistencia a Antineoplásicos , Expresión Génica/efectos de los fármacos , Humanos , Neuroblastoma/metabolismo , Neuroblastoma/patología , Receptor Muscarínico M2/genéticaRESUMEN
Triple negative tumors are more aggressive than other breast cancer subtypes and there is a lack of specific therapeutic targets on them. Since muscarinic receptors have been linked to tumor progression, we investigated the effect of metronomic therapy employing a traditional anti-cancer drug, paclitaxel plus muscarinic agonists at low doses on this type of tumor. We observed that MDA-MB231 tumor cells express muscarinic receptors, while they are absent in the non-tumorigenic MCF-10A cell line, which was used as control. The addition of carbachol or arecaidine propargyl ester, a non-selective or a selective subtype 2 muscarinic receptor agonist respectively, plus paclitaxel reduces cell viability involving a down-regulation in the expression of ATP "binding cassette" G2 drug transporter and epidermal growth factor receptor. We also detected an inhibition of tumor cell migration and anti-angiogenic effects produced by those drug combinations in vitro and in vivo (in NUDE mice) respectively. Our findings provide substantial evidence about subtype 2 muscarinic receptors as therapeutic targets for the treatment of triple negative tumors.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Agonistas Colinérgicos/administración & dosificación , Paclitaxel/administración & dosificación , Receptor Muscarínico M2/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Administración Metronómica , Animales , Arecolina/administración & dosificación , Arecolina/análogos & derivados , Carbacol/administración & dosificación , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Receptores ErbB/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Proteínas de Neoplasias/metabolismo , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/patología , ARN Interferente Pequeño/metabolismo , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/genética , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Neoplasias de la Mama Triple Negativas/patología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The septo-hippocampal cholinergic system is critical for hippocampal learning and memory. However, a quantitative description of the in vivo firing patterns and physiological function of medial septal (MS) cholinergic neurons is still missing. In this study, we combined optogenetics with multichannel in vivo recording and recorded MS cholinergic neuron firings in freely behaving male mice for 5.5-72 h. We found that their firing activities were highly correlated with hippocampal theta states. MS cholinergic neurons were highly active during theta-dominant epochs, such as active exploration and rapid eye movement sleep, but almost silent during non-theta epochs, such as slow-wave sleep (SWS). Interestingly, optogenetic activation of these MS cholinergic neurons during SWS suppressed CA1 ripple oscillations. This suppression could be rescued by muscarinic M2 or M4 receptor antagonists. These results suggest the following important physiological function of MS cholinergic neurons: maintaining high hippocampal acetylcholine level by persistent firing during theta epochs, consequently suppressing ripples and allowing theta oscillations to dominate.SIGNIFICANCE STATEMENT The major source of acetylcholine in the hippocampus comes from the medial septum. Early experiments found that lesions to the MS result in the disappearance of hippocampal theta oscillation, which leads to speculation that the septo-hippocampal cholinergic projection contributing to theta oscillation. In this article, by long-term recording of MS cholinergic neurons, we found that they show a theta state-related firing pattern. However, optogenetically activating these neurons shows little effect on theta rhythm in the hippocampus. Instead, we found that activating MS cholinergic neurons during slow-wave sleep could suppress hippocampal ripple oscillations. This suppression is mediated by muscarinic M2 and M4 receptors.
Asunto(s)
Potenciales de Acción/fisiología , Neuronas Colinérgicas/fisiología , Hipocampo/fisiología , Receptor Muscarínico M2/fisiología , Receptor Muscarínico M4/fisiología , Ritmo Teta/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Agonistas Colinérgicos/farmacología , Neuronas Colinérgicas/química , Neuronas Colinérgicas/efectos de los fármacos , Hipocampo/química , Hipocampo/efectos de los fármacos , Masculino , Ratones , Ratones Transgénicos , Antagonistas Muscarínicos/farmacología , Optogenética/métodos , Técnicas de Cultivo de Órganos , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/antagonistas & inhibidores , Receptor Muscarínico M4/agonistas , Receptor Muscarínico M4/antagonistas & inhibidores , Receptores Muscarínicos/fisiología , Ritmo Teta/efectos de los fármacosRESUMEN
Several drugs have been prepared to treat of heart failure using some protocols which require dangerous reagents and specific conditions. The aim of this study was to synthesize a series of steroid derivatives (compounds 2 to 18) using some chemical strategies. The biological activity of steroid derivatives against heart failure was evaluated using an ischemia/reperfusion model. In addition, the effect exerted by compounds 4 or 5 on left ventricular pressure was evaluated in the absence or presence of yohimbine, butaxamine and methoctramine. The results showed that 1) both compounds 4 or 5 significantly decrease the heart failure (translated as infarct area) compared with the compounds 2, 3 and 6-18. In addition, the compound 4 and 5 decreased the left ventricular pressure in a dose-dependent manner and this effect was significantly inhibited in the presence of methoctramine (p = 005). In conclusion, the compounds 4 or 5 decrease both the infarct area and left ventricular pressure via M2-muscarinic receptor activation.
Asunto(s)
Diseño de Fármacos , Insuficiencia Cardíaca/tratamiento farmacológico , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/metabolismo , Esteroides/farmacología , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Insuficiencia Cardíaca/metabolismo , Masculino , Conformación Molecular , Ratas , Ratas Wistar , Esteroides/síntesis química , Esteroides/química , Relación Estructura-ActividadRESUMEN
Despite the high incidence of acute and chronic pain in the general population, the efficacy of currently available medications is unsatisfactory. Insufficient management of pain has a profound impact on the quality of life and can have serious physical, psychological, social, and economic consequences. This unmet need reflects a failure to develop novel classes of analgesic drugs with superior clinical properties and lower risk of abuse. Nevertheless, recent advances in our understanding of the neurobiology of pain are offering new opportunities for developing different therapeutic approaches. Among those, the activation of M2 muscarinic acetylcholine receptors, which play a key role in the cholinergic regulation of the nociceptive transmission, constitutes one of the most promising strategies. We have recently developed a small library of novel pharmacological agents by merging the structures of known orthosteric and allosteric muscarinic ligands through their molecular hybridization, an emerging approach in medicinal chemistry based on the combination of pharmacophoric moieties of different bioactive substances to produce a new compound with improved pharmacological properties. Herein we report the functional characterization of the new ligands in vitro and the assessment of their efficacy as analgesic agents and tolerability in mice. This work provides new insights for the development and optimization of novel muscarinic hybrid compounds for the management of pain.
Asunto(s)
Analgésicos/farmacología , Descubrimiento de Drogas/métodos , Atrios Cardíacos/efectos de los fármacos , Receptor Muscarínico M2/agonistas , Regulación Alostérica , Sitio Alostérico , Analgésicos/efectos adversos , Analgésicos/química , Animales , Función del Atrio Izquierdo , Conducta Animal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Cobayas , Atrios Cardíacos/metabolismo , Técnicas In Vitro , Ligandos , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Dolor/tratamiento farmacológico , Receptor Muscarínico M2/antagonistas & inhibidoresRESUMEN
In mouse ileal myocytes, muscarinic receptor-mediated cationic current (mIcat) occurs mainly through synergism of M2 and M3 subtypes involving Gi/o-type GTP-binding proteins and phospholipase C (PLC). We have further studied the M2/M3 synergistic pathway. Carbachol-induced mIcat was markedly depressed by YM-254890, a Gq/11 protein inhibitor. However, the mIcat was unaffected by heparin, calphostin C, or chelerythrine, suggesting that mIcat activation does not involve signaling molecules downstream of phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown. M2-knockout (KO) mice displayed a reduced mIcat (~10% of wild-type mIcat) because of the lack of M2-Gi/o signaling. The impaired mIcat was insensitive to neuropeptide Y possessing a Gi/o-stimulating activity. M3-KO mice also displayed a reduced mIcat (~6% of wild-type mIcat) because of the lack of M3-Gq/11 signaling, and the mIcat was insensitive to prostaglandin F2α possessing a Gq/11-stimulating activity. These results suggest the importance of Gq/11/PLC-hydrolyzed PIP2 breakdown itself in mIcat activation and also support the idea that the M2/M3 synergistic pathway represents a signaling complex consisting of M2-Gi/o and M3-Gq/11-PLC systems in which both G proteins are special for this pathway but not general in receptor coupling.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Mucosa Intestinal/metabolismo , Miocitos del Músculo Liso/metabolismo , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M3/metabolismo , Animales , Agonistas Colinérgicos/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/agonistas , Cobayas , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Péptidos Cíclicos/farmacología , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M3/agonistasRESUMEN
The M2 muscarinic acetylcholine receptor (M2R) is a prototypical GPCR that plays important roles in regulating heart rate and CNS functions. Crystal structures provide snapshots of the M2R in inactive and active states, but the allosteric link between the ligand binding pocket and cytoplasmic surface remains poorly understood. Here we used solution NMR to examine the structure and dynamics of the M2R labeled with 13CH3-ε-methionine upon binding to various orthosteric and allosteric ligands having a range of efficacy for both G protein activation and arrestin recruitment. We observed ligand-specific changes in the NMR spectra of 13CH3-ε-methionine probes in the M2R extracellular domain, transmembrane core, and cytoplasmic surface, allowing us to correlate ligand structure with changes in receptor structure and dynamics. We show that the M2R has a complex energy landscape in which ligands with different efficacy profiles stabilize distinct receptor conformations.
Asunto(s)
Acetilcolina/química , Carbacol/química , Isoxazoles/química , Pilocarpina/química , Piridinas/química , Compuestos de Amonio Cuaternario/química , Receptor Muscarínico M2/química , Tiadiazoles/química , Acetilcolina/metabolismo , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Sitios de Unión , Carbacol/metabolismo , Clonación Molecular , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Isoxazoles/metabolismo , Cinética , Ligandos , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Pilocarpina/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Piridinas/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Termodinámica , Tiadiazoles/metabolismoRESUMEN
Light-triggered reversible modulation of physiological functions offers the promise of enabling on-demand spatiotemporally controlled therapeutic interventions. Optogenetics has been successfully implemented in the heart, but significant barriers to its use in the clinic remain, such as the need for genetic transfection. Herein, we present a method to modulate cardiac function with light through a photoswitchable compound and without genetic manipulation. The molecule, named PAI, was designed by introduction of a photoswitch into the molecular structure of an M2 mAChR agonist. In vitro assays revealed that PAI enables light-dependent activation of M2 mAChRs. To validate the method, we show that PAI photoisomers display different cardiac effects in a mammalian animal model, and demonstrate reversible, real-time photocontrol of cardiac function in translucent wildtype tadpoles. PAI can also effectively activate M2 receptors using two-photon excitation with near-infrared light, which overcomes the scattering and low penetration of short-wavelength illumination, and offers new opportunities for intravital imaging and control of cardiac function.
Asunto(s)
Agonistas Muscarínicos/farmacología , Sistema Nervioso Parasimpático/efectos de los fármacos , Receptor Muscarínico M2/agonistas , Sistema Nervioso Simpático/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Frecuencia Cardíaca/efectos de los fármacos , Rayos Infrarrojos , Simulación del Acoplamiento Molecular , Estructura Molecular , Agonistas Muscarínicos/síntesis química , Agonistas Muscarínicos/química , Procesos Fotoquímicos , Ratas , Ratas Wistar , Estereoisomerismo , Relación Estructura-Actividad , XenopusRESUMEN
Predicting the complete free energy landscape associated with protein-ligand unbinding may greatly help designing drugs with highly optimized pharmacokinetics. Here we investigate the unbinding of the iperoxo agonist to its target human neuroreceptor M2, embedded in a neuronal membrane. By feeding out-of-equilibrium molecular simulations data in a classification analysis, we identify the few essential reaction coordinates of the process. The full landscape is then reconstructed using an exact enhanced sampling method, well-tempered metadynamics in its funnel variant. The calculations reproduce well the measured affinity, provide a rationale for mutagenesis data, and show that the ligand can escape via two different routes. The allosteric modulator LY2119620 turns out to hamper both escapes routes, thus slowing down the unbinding process, as experimentally observed. This computationally affordable protocol is totally general, and it can be easily applied to determine the full free energy landscape of membrane receptors/drug interactions.
Asunto(s)
Isoxazoles/farmacología , Simulación de Dinámica Molecular , Compuestos de Amonio Cuaternario/farmacología , Receptor Muscarínico M2/agonistas , Termodinámica , Humanos , Isoxazoles/química , Ligandos , Compuestos de Amonio Cuaternario/química , Receptor Muscarínico M2/químicaRESUMEN
Muscarinic M2 and M4 receptors resemble each other in brain distribution, function, and Gi/o protein signaling. However, there is evidence from human recombinant receptors that the M4 receptor also couples to Gs protein whereas such an alternative signaling is of minor importance for its M2 counterpart. The question arises whether this property is shared by native receptors, e.g., the murine hippocampal M2- and the striatal M4-autoreceptor. To this end, the electrically evoked tritium overflow was studied in mouse hippocampal and striatal slices pre-incubated with 3H-choline. 3H-Acetylcholine release in either region was inhibited by the potent muscarinic receptor agonist iperoxo (pIC50 8.6-8.8) in an atropine-sensitive manner (apparent pA2 8.6-8.8); iperoxo was much more potent than oxotremorine (pIC50 6.5-6.6). In hippocampal slices, N-ethylmaleimide (NEM) 32 µM, which inactivates Gi/o proteins, tended to shift the concentration-response curve of iperoxo (pIC50 8.8) to the right (pIC50 8.5) and depressed its maximum from 85 to 69%. In striatal slices, the inhibitory effect of iperoxo declined at concentrations higher than 0.1 µM, yielding a biphasic curve with a pIC50 of 8.6 for the falling part and a pEC50 of 6.4 for the rising part of the curve. The inhibitory effect of iperoxo 10 µM (47%) after NEM pre-treatment was lower by about 35% compared to the maximum (74%) obtained without NEM. In conclusion, our data, which need to be confirmed by pertussis toxin, might suggest that in the striatum, unlike the hippocampus, stimulatory Gs protein comes into play at high concentrations of a muscarinic receptor agonist.
Asunto(s)
Acetilcolina/metabolismo , Autorreceptores/metabolismo , Cuerpo Estriado/efectos de los fármacos , Etilmaleimida/farmacología , Hipocampo/efectos de los fármacos , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M4/metabolismo , Animales , Cuerpo Estriado/metabolismo , Hipocampo/metabolismo , Isoxazoles/farmacología , Masculino , Ratones , Agonistas Muscarínicos/farmacología , Compuestos de Amonio Cuaternario/farmacología , Receptor Muscarínico M2/agonistas , TritioRESUMEN
Glioblastoma (GBM) is the most aggressive human brain tumor. The high growth potential and decreased susceptibility to apoptosis of the glioma cells is mainly dependent on genetic amplifications or mutations of oncogenic or pro-apoptotic genes, respectively. We have previously shown that the activation of the M2 acetylcholine muscarinic receptors inhibited cell proliferation and induced apoptosis in two GBM cell lines and cancer stem cells. The aim of this study was to delve into the molecular mechanisms underlying the M2-mediated cell proliferation arrest. Exploiting U87MG and U251MG cell lines as model systems, we evaluated the ability of M2 receptors to interfere with Notch-1 and EGFR pathways, whose activation promotes GBM proliferation. We demonstrated that the activation of M2 receptors, by agonist treatment, counteracted Notch and EGFR signaling, through different regulatory cascades depending, at least in part, on p53 status. Only in U87MG cells, which mimic p53-wild type GBMs, did M2 activation trigger a molecular circuitry involving p53, Notch-1, and the tumor suppressor mir-34a-5p. This regulatory module negatively controls Notch-1, which affects cell proliferation mainly through the Notch-1/EGFR axis. Our data highlighted, for the first time, a molecular circuitry that is deregulated in the p53 wild type GBM, based on the cross-talk between M2 receptor and the Notch-1/EGFR pathways, mediated by mir-34a-5p.
Asunto(s)
Receptores ErbB/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/metabolismo , MicroARNs/genética , Receptor Muscarínico M2/metabolismo , Receptor Notch1/metabolismo , Transducción de Señal , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Modelos Moleculares , Unión Proteica , Interferencia de ARN , Receptor Muscarínico M2/agonistas , Transducción de Señal/efectos de los fármacosRESUMEN
The muscarinic M2 acetylcholine receptor, one of the few G-protein coupled receptors that has not only been crystallized in both active and inactive conformations but also in the presence of a positive allosteric modulator, is an interesting system to study the molecular mechanisms of GPCR activation and ligand allosterism. Here, we have employed molecular dynamics (MD) simulations (adding to 14 µs in total) to study conformational changes triggered by the inverse agonist R-(-)-3-quinuclidinyl-benzilate (QNB) in the structure of the active M2 receptor (PBD ID 4MQS ) after replacement of the agonist iperoxo by the inverse agonist QNB. This permitted us to identify the sequence of events in the deactivation mechanism of the M2 acetylcholine receptor, which results first in the rearrangement of the transmission switch, the subsequent opening of the extracellular portion of the receptor and finally, the closure of the intracellular part. We also evaluate the effect of the positive allosteric modulator LY2119620 when bound simultaneously with the orthosteric agonist iperoxo and find that it restricts the conformation of Trp4227.35 in a position that modulates the orientation of the Tyr4267.39 at the orthosteric-binding pocket.
Asunto(s)
Simulación de Dinámica Molecular , Receptor Muscarínico M2/metabolismo , Regulación Alostérica/efectos de los fármacos , Agonismo Inverso de Drogas , Ligandos , Conformación Proteica/efectos de los fármacos , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/antagonistas & inhibidoresRESUMEN
In previous studies, we found that the orthosteric muscarinic agonist arecaidine propargyl ester (APE) (100⯵M) induced a decreased cell proliferation and severe apoptosis in glioblastoma cancer stem cells (GSCs). In this report, we have investigated the effects mediated by hybrid (orthosteric/allosteric) muscarinic agonists P-6-Iper and N-8-Iper on GSCs survival. At variance with APE, the agonist N-8-Iper inhibited cell growth in a dose dependent manner and also impaired cell survival at low doses. The inhibitory effects of the N-8-Iper action appear to be mediated by M2 receptor activation, since they were strongly reduced by co-administration of the selective M2 receptor antagonist methoctramine as well as upon M2 receptor silencing. Moreover, analysis of the expression of phosphorylated histone H2AX (γ-H2AX) indicated that the treatment with N-8-Iper produced a decreased cell survival by induction of DNA damage. The ability of N-8-Iper to produce a cytotoxic effect and apoptosis at low doses indicates that this muscarinic agonist is a suitable probe in a putative therapeutic intervention on glioblastoma through M2 receptor activation.
Asunto(s)
Glioblastoma/patología , Agonistas Muscarínicos/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Receptor Muscarínico M2/agonistas , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Daño del ADN/efectos de los fármacos , Daño del ADN/fisiología , Relación Dosis-Respuesta a Droga , Humanos , Receptor Muscarínico M2/metabolismoRESUMEN
The muscarinic M$_{2}$ receptor is a prominent member of the GPCR family and strongly involved in heart diseases. Recently published experimental work explored the cellular response to iperoxo-induced M$_{2}$ receptor stimulation in Chinese hamster ovary (CHO) cells. To better understand these responses, we modelled and analysed the muscarinic M$_{2}$ receptor-dependent signalling pathway combined with relevant secondary messenger molecules using mass action. In our literature-based joint signalling and secondary messenger model, all binding and phosphorylation events are explicitly taken into account in order to enable subsequent stoichiometric matrix analysis. We propose constraint flux sampling (CFS) as a method to characterize the expected shift of the steady state reaction flux distribution due to the known amount of cAMP production and PDE4 activation. CFS correctly predicts an experimentally observable influence on the cytoskeleton structure (marked by actin and tubulin) and in consequence a change of the optical density of cells. In a second step, we use CFS to simulate the effect of knock-out experiments within our biological system, and thus to rank the influence of individual molecules on the observed change of the optical cell density. In particular, we confirm the relevance of the protein RGS14, which is supported by current literature. A combination of CFS with Elementary Flux Mode analysis enabled us to determine the possible underlying mechanism. Our analysis suggests that mathematical tools developed for metabolic network analysis can also be applied to mixed secondary messenger and signalling models. This could be very helpful to perform model checking with little effort and to generate hypotheses for further research if parameters are not known.
Asunto(s)
Receptor Muscarínico M2/metabolismo , Animales , Células CHO , Cricetulus , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Isoxazoles/farmacología , Conceptos Matemáticos , Modelos Biológicos , Agonistas Muscarínicos/farmacología , Compuestos de Amonio Cuaternario/farmacología , Receptor Muscarínico M2/agonistas , Sistemas de Mensajero Secundario , Transducción de SeñalRESUMEN
Mesenchymal stem cells (MSCs), also known as stromal mesenchymal stem cells, are multipotent cells, which can be found in many tissues and organs as bone marrow, adipose tissue and other tissues. In particular MSCs derived from Adipose tissue (ADSCs) are the most frequently used in regenerative medicine because they are easy to source, rapidly expandable in culture and excellent differentiation potential into adipocytes, chondrocytes, and other cell types. Acetylcholine (ACh), the most important neurotransmitter in Central nervous system (CNS) and peripheral nervous system (PNS), plays important roles also in non-neural tissue, but its functions in MSCs are still not investigated. Although MSCs express muscarinic receptor subtypes, their role is completely unknown. In the present work muscarinic cholinergic effects were characterized in rat ADSCs. Analysis by RT-PCR demonstrates that ADSCs express M1-M4 muscarinic receptor subtypes, whereas M2 is one of the most expressed subtype. For this reason, our attention was focused on M2 subtype. By using the selective M2 against Arecaidine Propargyl Ester (APE) we performed cell proliferation and migration assays demonstrating that APE causes cell growth and migration inhibition without affecting cell survival. Our results indicate that ACh via M2 receptors, may contribute to the maintaining of the ADSCs quiescent status. These data are the first evidence that ACh, via muscarinic receptors, might contribute to control ADSCs physiology.