RESUMEN
Metabotropic glutamate (mGlu) receptors respond to glutamate, the major excitatory neurotransmitter in the mammalian brain, mediating a modulatory role that is critical for higher-order brain functions such as learning and memory. Since the first mGlu receptor was cloned in 1992, eight subtypes have been identified along with many isoforms and splice variants. The mGlu receptors are transmembrane-spanning proteins belonging to the class C G protein-coupled receptor family and represent attractive targets for a multitude of central nervous system disorders. Concerted drug discovery efforts over the past three decades have yielded a wealth of pharmacological tools including subtype-selective agents that competitively block or mimic the actions of glutamate or act allosterically via distinct sites to enhance or inhibit receptor activity. Herein, we review the physiologic and pathophysiological roles for individual mGlu receptor subtypes including the pleiotropic nature of intracellular signal transduction arising from each. We provide a comprehensive analysis of the in vitro and in vivo pharmacological properties of prototypical and commercially available orthosteric agonists and antagonists as well as allosteric modulators, including ligands that have entered clinical trials. Finally, we highlight emerging areas of research that hold promise to facilitate rational design of highly selective mGlu receptor-targeting therapeutics in the future. SIGNIFICANCE STATEMENT: The metabotropic glutamate receptors are attractive therapeutic targets for a range of psychiatric and neurological disorders. Over the past three decades, intense discovery efforts have yielded diverse pharmacological tools acting either competitively or allosterically, which have enabled dissection of fundamental biological process modulated by metabotropic glutamate receptors and established proof of concept for many therapeutic indications. We review metabotropic glutamate receptor molecular pharmacology and highlight emerging areas that are offering new avenues to selectively modulate neurotransmission.
Asunto(s)
Fenómenos Biológicos , Enfermedades del Sistema Nervioso Central , Farmacología Clínica , Receptores de Glutamato Metabotrópico , Animales , Humanos , Transducción de SeñalRESUMEN
Neuropathic pain is a common health problem resulting in exacerbated response to noxious and non noxious stimuli, as well as impaired emotional and cognitive responses. Unfortunately, neuropathic pain is also one of the most difficult pain syndromes to manage, highlighting the importance of better understanding the brain regions and neuromodulatory mechanisms involved in its regulation. Among the many interconnected brain areas which process pain, the amygdala is known to play an important role in the integration of sensory and emotional pain signals. Here we questioned the ability of a recently identified neuromodulatory mechanism associated with the metabotropic glutamate receptors mGlu4 in the amygdala to modulate neuropathic pain. In a murine model of peripheral mononeuropathy, we demonstrate that pharmacological activation of amygdala mGlu4 efficiently alleviates sensory and depressive-like symptoms in both male and female mice. Moreover, we reveal a differential modulation of these symptoms. Activating mGlu4 in the contralateral amygdala relative to the side of the mononeuropathy, is necessary and sufficient to relieve both sensory and depressive-like symptoms, while ipsilateral activation solely reduces depressive-like symptoms. Furthermore, using photopharmacology, a recent strategy allowing precise photocontrol of endogenous proteins, we further demonstrate the dynamic alleviation of neuropathic pain through light-dependent facilitation of mGlu4 by a photoswitchable positive allosteric modulator. Finally, coupling photopharmacology and analgesic conditioned place preference, we show a significant pain-reducing effect of mGlu4 activation. Taken together, these data highlight the analgesic potential of enhancing amygdala mGlu4 activity to counteract neuropathy reinforcing its therapeutic interest for the treatment of pathological pain.
Asunto(s)
Neuralgia , Receptores de Glutamato Metabotrópico , Femenino , Ratones , Masculino , Animales , Neuralgia/tratamiento farmacológico , Amígdala del Cerebelo/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Encéfalo/metabolismoRESUMEN
Mass spectrometry (MS) binding assays are a label-free alternative to radioligand or fluorescence binding assays, so the readout is based on direct mass spectrometric detection of the test ligand. The study presented here describes the development and validation of a highly sensitive, rapid, and robust MS binding assay for the quantification of the binding of the metabotropic glutamate 5 (mGlu5) negative allosteric modulator (NAM), MPEP (2-methyl-6-phenylethynylpyridine) at the mGlu5 allosteric binding site. The LC-ESI-MS/MS (liquid chromatography-electrospray ionization-tandem mass spectrometric) analytical method was established and validated with a deuterated analogue of MPEP as an internal standard. The developed MS binding assay described here allowed for the determination of MS binding affinity estimates that were in agreement with affinity estimates obtained from a tritiated MPEP radioligand saturation binding assay, indicating the suitability of this methodology for determining affinity estimates for compounds that target mGlu5 allosteric binding sites. Graphical abstract.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Receptor del Glutamato Metabotropico 5/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Sitio Alostérico , Células HEK293 , Humanos , Ligandos , Límite de Detección , Unión Proteica , Ensayo de Unión Radioligante , Reproducibilidad de los ResultadosRESUMEN
Controlling drug activity with light offers the possibility of enhancing pharmacological selectivity with spatial and temporal regulation, thus enabling highly localized therapeutic effects and precise dosing patterns. Here we report on the development and characterization of what is to our knowledge the first photoswitchable allosteric modulator of a G protein-coupled receptor. Alloswitch-1 is selective for the metabotropic glutamate receptor mGlu5 and enables the optical control of endogenous mGlu5 receptors.
Asunto(s)
Compuestos Azo/química , Piridinas/farmacología , Receptor del Glutamato Metabotropico 5/metabolismo , Regulación Alostérica/efectos de la radiación , Sitio Alostérico , Animales , Animales Recién Nacidos , Astrocitos/citología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/efectos de la radiación , Expresión Génica , Células HEK293 , Humanos , Larva/efectos de los fármacos , Larva/fisiología , Larva/efectos de la radiación , Luz , Procesos Fotoquímicos , Cultivo Primario de Células , Piridinas/síntesis química , Ratas , Receptor del Glutamato Metabotropico 5/agonistas , Receptor del Glutamato Metabotropico 5/antagonistas & inhibidores , Receptor del Glutamato Metabotropico 5/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Transfección , Xenopus/fisiologíaRESUMEN
Type 4 metabotropic glutamate (mGlu4) receptors are emerging targets for the treatment of various disorders. Accordingly, numerous mGlu4-positive allosteric modulators (PAMs) have been identified, some of which also display agonist activity. To identify the structural bases for their allosteric action, we explored the relationship between the binding pockets of mGlu4 PAMs with different chemical scaffolds and their functional properties. By use of innovative mGlu4 biosensors and second-messenger assays, we show that all PAMs enhance agonist action on the receptor through different degrees of allosteric agonism and positive cooperativity. For example, whereas VU0155041 and VU0415374 display equivalent efficacies [log(τ(B)) = 1.15 ± 0.38 and 1.25 ± 0.44, respectively], they increase the ability of L-AP4 to stabilize the active conformation of the receptor by 4 and 39 times, respectively. Modeling and docking studies identify 2 overlapping binding pockets as follows: a first site homologous to the pocket of natural agonists of class A GPCRs linked to allosteric agonism and a second one pointing toward a site topographically homologous to the Na(+) binding pocket of class A GPCRs, occupied by PAMs exhibiting the strongest cooperativity. These results reveal that intrinsic efficacy and cooperativity of mGlu4 PAMs are correlated with their binding mode, and vice versa, integrating structural and functional knowledge from different GPCR classes.
Asunto(s)
Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/metabolismo , Regulación Alostérica , Sitio Alostérico , Anilidas/farmacología , Técnicas Biosensibles , Ácidos Ciclohexanocarboxílicos/farmacología , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Ligandos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación Proteica , Receptores de Glutamato Metabotrópico/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sistemas de Mensajero SecundarioRESUMEN
Metabotropic glutamate receptors (mGluRs) play key roles in the modulation of many synapses. Chloride (Cl(-)) is known to directly bind and regulate the function of different actors of neuronal activity, and several studies have pointed to the possible modulation of mGluRs by Cl(-). Herein, we demonstrate that Cl(-) behaves as a positive allosteric modulator of mGluRs. For example, whereas glutamate potency was 3.08 ± 0.33 µM on metabotropic glutamate (mGlu) 4 receptors in high-Cl(-) buffer, signaling activity was almost abolished in low Cl(-) in cell-based assays. Cl(-) potency was 78.6 ± 3.5 mM. Cl(-) possesses a high positive cooperativity with glutamate (Hill slope ≈6 on mGlu4), meaning that small variations in [Cl(-)] lead to large variations in glutamate action. Using molecular modeling and mutagenesis, we have identified 2 well-conserved Cl(-) binding pockets in the extracellular domain of mGluRs. Moreover, modeling of activity-dependent Cl(-) variations at GABAergic synapses suggests that these variations may be compatible with a dynamic modulation of the most sensitive mGluRs present in these synapses. Taken together, these data reveal a necessary role of Cl(-) for the glutamate activation of many mGluRs. Exploiting Cl(-) binding pockets may yield to the development of innovative regulators of mGluR activity.
Asunto(s)
Cloruros/metabolismo , Espacio Extracelular/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Regulación Alostérica , Sitio Alostérico , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Cloruros/farmacología , Células HEK293 , Humanos , Modelos Moleculares , Mutación , Unión Proteica , Estructura Terciaria de Proteína/efectos de los fármacos , Ratas , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/genética , Homología de Secuencia de AminoácidoRESUMEN
The four stereoisomers of 1-amino-2-fluoro-2-(phosphonomethyl)cyclopropane-1-carboxylic acid (FAP4) were synthesized via diastereoselective Rh(II)-catalysed cyclopropanation of a phosphonylated fluoroalkene. Different isomers of FAP4 and the corresponding non-fluorinated analogs showed a similar pharmacological profile against the isoforms of metabotropic glutamate receptor (mGluR). Within the fluorinated series, (-)-(Z)-FAP4 and (-)-(E)-FAP4 demonstrated the highest agonist activity against mGlu4 (EC50 0.10 µM). Our results suggest that fluorocyclopropanes bearing an amino-acid function can be suitable for the development of potent conformationally restricted mGluR agonists.
Asunto(s)
Ácidos Carboxílicos/química , Receptores de Glutamato Metabotrópico/agonistas , Ácidos Carboxílicos/síntesis química , Ácidos Carboxílicos/metabolismo , Ciclopropanos/química , Ácido Glutámico/química , Ácido Glutámico/metabolismo , Unión Proteica , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , EstereoisomerismoRESUMEN
A series of phosphinic glutamate derivatives (e.g.LSP1-2111) have been proven to be potent agonists of metabotropic glutamate (mGlu) receptors and shown promising in vivo activity. However, so far all were synthesized and tested as a mixture of two diastereomers whose absolute and relative configurations are not known. In this study, the stereomers were separated on a Crownpack CR(+) column and their absolute configuration was assessed by means of a diastereoselective synthesis. Both separated L-stereomers activated the mGlu4 receptor with EC50's of 0.72 and 4.4 µM for (1S,1'S)-and (1S,1'R)-LSP1-2111, respectively.
Asunto(s)
Ácido Glutámico/química , Compuestos Organofosforados/química , Ácido Glutámico/farmacología , Células HEK293 , Humanos , Modelos Moleculares , Conformación Molecular , Receptores de Glutamato Metabotrópico/agonistasRESUMEN
Hyperactivity of the glutamatergic system is involved in the development of central sensitization in the pain neuraxis, associated with allodynia and hyperalgesia observed in patients with chronic pain. Herein we study the ability of type 4 metabotropic glutamate receptors (mGlu4) to regulate spinal glutamate signaling and alleviate chronic pain. We show that mGlu4 are located both on unmyelinated C-fibers and spinal neurons terminals in the inner lamina II of the spinal cord where they inhibit glutamatergic transmission through coupling to Cav2.2 channels. Genetic deletion of mGlu4 in mice alters sensitivity to strong noxious mechanical compression and accelerates the onset of the nociceptive behavior in the inflammatory phase of the formalin test. However, responses to punctate mechanical stimulation and nocifensive responses to thermal noxious stimuli are not modified. Accordingly, pharmacological activation of mGlu4 inhibits mechanical hypersensitivity in animal models of inflammatory or neuropathic pain while leaving acute mechanical perception unchanged in naive animals. Together, these results reveal that mGlu4 is a promising new target for the treatment of chronic pain.
Asunto(s)
Agonistas de Aminoácidos Excitadores/uso terapéutico , Hiperalgesia/tratamiento farmacológico , Receptores de Glutamato Metabotrópico/agonistas , Animales , Western Blotting , Carragenina , Enfermedad Crónica , Constricción Patológica/patología , Fenómenos Electrofisiológicos/fisiología , Técnica del Anticuerpo Fluorescente , Inmersión/fisiopatología , Interneuronas/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Vaina de Mielina/fisiología , Dimensión del Dolor/efectos de los fármacos , Técnicas de Placa-Clamp , Ácidos Fosfínicos/administración & dosificación , Ácidos Fosfínicos/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato Metabotrópico/biosíntesis , Receptores de Glutamato Metabotrópico/genética , Rizotomía , Células Receptoras Sensoriales/fisiología , Médula Espinal/citología , Médula Espinal/fisiología , Transmisión Sináptica/fisiologíaRESUMEN
In recent years, there has been growing interest in the potential therapeutic use of inhibitors of adenosine A2A receptors (A2AR) for the treatment of neurodegenerative diseases and cancer. Nevertheless, the widespread expression of A2AR throughout the body emphasizes the importance of temporally and spatially selective ligands. Photopharmacology is an emerging strategy that utilizes photosensitive ligands to attain high spatiotemporal precision and regulate the function of biomolecules using light. In this study, we combined photochemistry and cellular and in vivo photopharmacology to investigate the light sensitivity of the FDA-approved antagonist istradefylline and its potential use as an A2AR photopharmacological tool. Our findings reveal that istradefylline exhibits rapid trans-to-cis isomerization under near-UV light, and prolonged exposure results in the formation of photocycloaddition products. We demonstrate that exposure to UV light triggers a time-dependent decrease in the antagonistic activity of istradefylline in A2AR-expressing cells and enables real-time optical control of A2AR signaling in living cells and zebrafish. Together, these data demonstrate that istradefylline is a photoinactivatable A2AR antagonist and that this property can be utilized to perform photopharmacological experiments in living cells and animals.
Asunto(s)
Receptor de Adenosina A2A , Pez Cebra , Animales , Receptor de Adenosina A2A/metabolismo , Pez Cebra/metabolismo , Purinas/farmacología , Transducción de Señal , Antagonistas del Receptor de Adenosina A2/uso terapéuticoRESUMEN
Metabotropic glutamate (mGlu) receptors play a key role in modulating most synapses in the brain. The mGlu7 receptors inhibit presynaptic neurotransmitter release and offer therapeutic possibilities for post-traumatic stress disorders or epilepsy. Screening campaigns provided mGlu7-specific allosteric modulators as the inhibitor XAP044 (Gee et al. J. Biol. Chem. 2014). In contrast to other mGlu receptor allosteric modulators, XAP044 does not bind in the transmembrane domain but to the extracellular domain of the mGlu7 receptor and not at the orthosteric site. Here, we identified the mode of action of XAP044, combining synthesis of derivatives, modeling and docking experiments, and mutagenesis. We propose a unique mode of action of these inhibitors, preventing the closure of the Venus flytrap agonist binding domain. While acting as a noncompetitive antagonist of L-AP4, XAP044 and derivatives act as apparent competitive antagonists of LSP4-2022. These data revealed more potent XAP044 analogues and new possibilities to target mGluRs.
Asunto(s)
Receptores de Glutamato Metabotrópico , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de Glutamato Metabotrópico/química , Humanos , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad , Animales , Dominios Proteicos , Regulación Alostérica/efectos de los fármacos , Células HEK293 , Sitios de UniónRESUMEN
We used light-sensitive drugs to identify the brain region-specific role of mGlu5 metabotropic glutamate receptors in the control of pain. Optical activation of systemic JF-NP-26, a caged, normally inactive, negative allosteric modulator (NAM) of mGlu5 receptors, in cingulate, prelimbic, and infralimbic cortices and thalamus inhibited neuropathic pain hypersensitivity. Systemic treatment of alloswitch-1, an intrinsically active mGlu5 receptor NAM, caused analgesia, and the effect was reversed by light-induced drug inactivation in the prelimbic and infralimbic cortices, and thalamus. This demonstrates that mGlu5 receptor blockade in the medial prefrontal cortex and thalamus is both sufficient and necessary for the analgesic activity of mGlu5 receptor antagonists. Surprisingly, when the light was delivered in the basolateral amygdala, local activation of systemic JF-NP-26 reduced pain thresholds, whereas inactivation of alloswitch-1 enhanced analgesia. Electrophysiological analysis showed that alloswitch-1 increased excitatory synaptic responses in prelimbic pyramidal neurons evoked by stimulation of presumed BLA input, and decreased BLA-driven feedforward inhibition of amygdala output neurons. Both effects were reversed by optical silencing and reinstated by optical reactivation of alloswitch-1. These findings demonstrate for the first time that the action of mGlu5 receptors in the pain neuraxis is not homogenous, and suggest that blockade of mGlu5 receptors in the BLA may limit the overall analgesic activity of mGlu5 receptor antagonists. This could explain the suboptimal effect of mGlu5 NAMs on pain in human studies and validate photopharmacology as an important tool to determine ideal target sites for systemic drugs.
Asunto(s)
Luz , Receptor del Glutamato Metabotropico 5 , Receptor del Glutamato Metabotropico 5/metabolismo , Receptor del Glutamato Metabotropico 5/antagonistas & inhibidores , Animales , Masculino , Ratones , Neuralgia/metabolismo , Tálamo/efectos de los fármacos , Tálamo/metabolismo , Complejo Nuclear Basolateral/metabolismo , Complejo Nuclear Basolateral/efectos de los fármacos , Analgésicos/farmacología , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Ratones Endogámicos C57BLRESUMEN
Knowing the site of drug action is important to optimize effectiveness and address any side effects. We used light-sensitive drugs to identify the brain region-specific role of mGlu5 metabotropic glutamate receptors in the control of pain. Optical activation of systemic JF-NP-26, a caged, normally inactive, negative allosteric modulator (NAM) of mGlu5 receptors, in cingulate, prelimbic and infralimbic cortices and thalamus inhibited neuropathic pain hypersensitivity. Systemic treatment of alloswitch-1, an intrinsically active mGlu5 receptor NAM, caused analgesia, and the effect was reversed by light-induced drug inactivation in in the prelimbic and infralimbic cortices, and thalamus. This demonstrates that mGlu5 receptor blockade in the medial prefrontal cortex and thalamus is both sufficient and necessary for the analgesic activity of mGlu5 receptor antagonists. Surprisingly, when light was delivered in the basolateral amygdala, local activation of systemic JF-NP-26 reduced pain thresholds, whereas inactivation of alloswitch-1 enhanced analgesia. Electrophysiological analysis showed that alloswitch-1 increased excitatory synaptic responses in prelimbic pyramidal neurons evoked by stimulation of BLA input, and decreased feedforward inhibition of amygdala output neurons by BLA. Both effects were reversed by optical silencing and reinstated by optical reactivation of alloswitch-1. These findings demonstrate for the first time that the action of mGlu5 receptors in the pain neuraxis is not homogenous, and suggest that blockade of mGlu5 receptors in the BLA may limit the overall analgesic activity of mGlu5 receptor antagonists. This could explain the suboptimal effect of mGlu5 NAMs on pain in human studies and validate photopharmacology as an important tool to determine ideal target sites for systemic drugs.
RESUMEN
Metabotropic glutamate receptors (mGlu) regulate multiple functions in the nervous systems and are involved in several neurological disorders. However, selectively targeting individual mGlu subtypes with spatiotemporal precision is still an unmet need. Photopharmacology can address this concern through the utilization of photoswitchable compounds such as optogluram, which is a positive allosteric modulator (PAM) of mGlu4 that enables the precise control of physiological responses using light but does not have an optimal selectivity profile. Optogluram analogs were developed to obtain photoswitchable PAMs of mGlu4 receptor with an improved selectivity. Among them, optogluram-2 emerged as a photoswitchable ligand for mGlu4 receptor with activity as both PAM and allosteric agonists. It presents a higher selectivity and offers improved photoswitching of mGlu4 activity. These improved properties make optogluram-2 an excellent candidate to study the role of mGlu4 with a high spatiotemporal precision in systems where mGlu4 can be co-expressed with other mGlu receptors.
RESUMEN
Metabotropic glutamate (mGlu) receptors are promising targets to treat numerous brain disorders. So far, allosteric modulators are the only subtype selective ligands, but pure agonists still have strong therapeutic potential. Here, we aimed at investigating the possibility of developing subtype-selective agonists by extending the glutamate-like structure to hit a nonconsensus binding area. We report the properties of the first mGlu4-selective orthosteric agonist, derived from a virtual screening hit, LSP4-2022 using cell-based assays with recombinant mGlu receptors [EC(50): 0.11 ± 0.02, 11.6 ± 1.9, 29.2 ± 4.2 µM (n>19) in calcium assays on mGlu4, mGlu7, and mGlu8 receptors, respectively, with no activity at the group I and -II mGlu receptors at 100 µM]. LSP4-2022 inhibits neurotransmission in cerebellar slices from wild-type but not mGlu4 receptor-knockout mice. In vivo, it possesses antiparkinsonian properties after central or systemic administration in a haloperidol-induced catalepsy test, revealing its ability to cross the blood-brain barrier. Site-directed mutagenesis and molecular modeling was used to identify the LSP4-2022 binding site, revealing interaction with both the glutamate binding site and a variable pocket responsible for selectivity. These data reveal new approaches for developing selective, hydrophilic, and brain-penetrant mGlu receptor agonists, offering new possibilities to design original bioactive compounds with therapeutic potential.
Asunto(s)
Agonistas de Aminoácidos Excitadores/química , Agonistas de Aminoácidos Excitadores/farmacología , Ligandos , Ácidos Fosfínicos/química , Ácidos Fosfínicos/farmacología , Receptores de Glutamato Metabotrópico/agonistas , Animales , Antiparkinsonianos/química , Antiparkinsonianos/metabolismo , Antiparkinsonianos/farmacología , Sitios de Unión , Relación Dosis-Respuesta a Droga , Agonistas de Aminoácidos Excitadores/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Ratones Noqueados , Estructura Molecular , Mutagénesis Sitio-Dirigida , Técnicas de Placa-Clamp , Ácidos Fosfínicos/metabolismo , Ratas , Ratas Wistar , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Transmisión Sináptica/efectos de los fármacosRESUMEN
Herein we describe the diastereoselective synthesis of glutamic acid analogs and the evaluation of their agonist activity towards metabotropic glutamate receptor subtype 4 (mGluR4). These analogs are based on a monofluorinated cyclopropane core substituted with an α-aminoacid function. The potential of this new building block as a tool for the development of a novel class of drugs is demonstrated with racemic analog 11a that displayed the best agonist activity with an EC50 of 340 nM.
Asunto(s)
Ácidos Carboxílicos/farmacología , Ciclopropanos/farmacología , Diseño de Fármacos , Ácido Glutámico/farmacología , Receptores de Glutamato Metabotrópico/agonistas , Ácidos Carboxílicos/síntesis química , Ácidos Carboxílicos/química , Células Cultivadas , Ciclopropanos/síntesis química , Ciclopropanos/química , Relación Dosis-Respuesta a Droga , Ácido Glutámico/síntesis química , Ácido Glutámico/química , Células HEK293 , Humanos , Estructura Molecular , Proteínas Recombinantes/agonistas , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Class C GPCRs, that include metabotropic glutamate receptors (mGlu), taste receptors, GABAB receptor and Calcium-sensing receptor, are unusual in terms of their molecular architecture and allosteric regulation. They all form obligatory dimers, dimerization being fundamental for their function. More specifically, the mGlu are activated by the main excitatory neurotransmitter, L-glutamate. mGlu activation by glutamate binding in the venus flytrap domain (VFT) triggers conformational changes that are transmitted, through the Cystein-Rich Domain (CRD), to the conserved fold of 7 transmembrane helices (7TM), that couples to intracellular G protein. mGlu activity can also be allosterically modulated by positive (PAM) or negative (NAM) allosteric modulators binding to the 7TM. Recent progress in cryo-electron microscopy (cryoEM) has allowed unprecedented advances in deciphering the structural and molecular basis of their activation mechanism. The agonist induces a large movement between the subunits, bringing the 7TMs together and stabilizing a 7TM conformation structurally similar to the inactive state. The diversity of inactive conformations for the class C was unexpected but allows PAM stabilising a 7TM active conformation independent of the conformational changes induced by agonists, representing an alternative mode of mGlu activation. Here we present and discuss recent structural characterisation of mGlu receptors, highlighting findings that make the class C of GPCR unique. Understanding the structural basis of mGlu dimer signaling represents a landmark achievement and paves the way for structural investigation of GPCR dimer signaling in general. Structural information will open new avenues for structure-based drug design.
Title: Les avancées récentes dans le domaine de la biologie structurale des récepteurs couplés aux protéines G de la classe C : Le récepteur métabotropique du glutamate 5. Abstract: La classe C des Récepteurs Couplés aux Protéines G (RCPG) comprend plusieurs membres aux fonctions physiologiques importantes comme par exemple les récepteurs des principaux neurotransmetteurs excitateurs (glutamate) et inhibiteurs (GABA) du système nerveux, les récepteurs des goûts umami et sucré et les récepteurs sensibles au calcium. Ces récepteurs possèdent une architecture moléculaire particulière, caractérisée par la présence d'un large domaine extracellulaire (ECD) relié à un domaine membranaire composé de 7 hélices transmembranaires (7TM). De plus, ils forment tous des dimères obligatoires, la dimérisation étant fondamentale pour leur fonction. La fixation d'agoniste dans l'ECD induit l'activation du récepteur. L'activité des agonistes peut être modulée de manière allostérique par des modulateurs positifs (PAM) ou négatifs (NAM), se liant au domaine 7TM. Il est important de comprendre comment les changements de conformation induits par la liaison des agonistes au sein du domaine extracellulaire sont transmis au domaine transmembranaire mais aussi de comprendre les bases structurales et moléculaires de la régulation allostérique des récepteurs de la classe C. Les progrès récents de la microscopie électronique en conditions cryogéniques (cryoEM) ont permis des avancées sans précédent dans le décryptage des bases structurelles et moléculaires des mécanismes d'activation des RCPG de classe C, et notamment du récepteur métabotropique du glutamate de type 5 (mGlu5). Le glutamate entraîne une fermeture et un changement d'orientation des domaines extracellulaires qui induit un mouvement important entre les sous-unités, rapprochant les 7TM et stabilisant la conformation active du récepteur. La diversité de conformations inactives pour les récepteurs de la classe C était inattendue mais propice à une activation possible par des PAM. Ces derniers stabilisent une conformation active des 7TM, indépendante des changements conformationnels induits par les agonistes, représentant un mode alternatif d'activation des récepteurs mGlu. Nous présentons et discutons ici les caractérisations structurales récentes des récepteurs de classe C, en soulignant les résultats qui rendent cette famille de récepteurs unique. La compréhension de la base structurelle de la signalisation des dimères de mGlu représente une réalisation historique et ouvre la voie à l'analyse de la signalisation des dimères de RCPG en général. Ces analyses structurales devraient également ouvrir de nouvelles voies pour la conception de médicaments ciblant cette famille de récepteurs qui sont aussi des cibles thérapeutiques.
Asunto(s)
Receptor del Glutamato Metabotropico 5 , Receptores Acoplados a Proteínas G , Regulación Alostérica , Microscopía por Crioelectrón , Humanos , Receptor del Glutamato Metabotropico 5/química , Receptor del Glutamato Metabotropico 5/metabolismo , Receptor del Glutamato Metabotropico 5/ultraestructura , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/clasificación , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/ultraestructuraRESUMEN
Metabotropic glutamate receptors (mGluRs) are dimeric G-protein-coupled receptors activated by the main excitatory neurotransmitter, L-glutamate. mGluR activation by agonists binding in the venus flytrap domain is regulated by positive (PAM) or negative (NAM) allosteric modulators binding to the 7-transmembrane domain (7TM). We report the cryo-electron microscopy structures of fully inactive and intermediate-active conformations of mGlu5 receptor bound to an antagonist and a NAM or an agonist and a PAM, respectively, as well as the crystal structure of the 7TM bound to a photoswitchable NAM. The agonist induces a large movement between the subunits, bringing the 7TMs together and stabilizing a 7TM conformation structurally similar to the inactive state. Using functional approaches, we demonstrate that the PAM stabilizes a 7TM active conformation independent of the conformational changes induced by agonists, representing an alternative mode of mGlu activation. These findings provide a structural basis for different mGluR activation modes.
Asunto(s)
Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Receptor del Glutamato Metabotropico 5/agonistas , Receptor del Glutamato Metabotropico 5/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Microscopía por Crioelectrón , Cristalografía por Rayos X , Agonistas de Aminoácidos Excitadores/metabolismo , Antagonistas de Aminoácidos Excitadores/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Subunidades de Proteína , Receptor del Glutamato Metabotropico 5/metabolismo , Receptor del Glutamato Metabotropico 5/ultraestructura , Relación Estructura-ActividadRESUMEN
Developing nondopaminergic palliative treatments for Parkinson's disease represents a major challenge to avoid the debilitating side effects produced by L-DOPA therapy. Increasing interest is addressed to the selective targeting of group III metabotropic glutamate (mGlu) receptors that inhibit transmitter release at presumably overactive synapses in the basal ganglia. Here we characterize the functional action of a new orthosteric group III mGlu agonist, LSP1-2111, with a preferential affinity for mGlu4 receptor. In mouse brain slices, LSP1-2111 inhibits striatopallidal GABAergic transmission by selectively activating the mGlu4 receptor but has no effect at a synapse modulated solely by the mGlu7 and mGlu8 receptors. Intrapallidal LSP1-2111 infusion reverses the akinesia produced by nigrostriatal dopamine depletion in a reaction time task, whereas an mGlu8-receptor agonist has no effect. Finally, systemic administration of LSP1-2111 counteracts haloperidol-induced catalepsy, opening promising perspectives for the development of antiparkinsonian therapeutic strategies focused on orthosteric mGlu4-receptor agonists.
Asunto(s)
Aminobutiratos/uso terapéutico , Antiparkinsonianos/uso terapéutico , Trastornos Parkinsonianos/tratamiento farmacológico , Trastornos Parkinsonianos/fisiopatología , Ácidos Fosfínicos/uso terapéutico , Receptores de Glutamato Metabotrópico/agonistas , Animales , Conducta/efectos de los fármacos , Catalepsia/inducido químicamente , Catalepsia/tratamiento farmacológico , Línea Celular , Modelos Animales de Enfermedad , Fenómenos Electrofisiológicos , Globo Pálido/efectos de los fármacos , Globo Pálido/fisiopatología , Haloperidol/farmacología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratas , Transmisión Sináptica/efectos de los fármacosRESUMEN
Glutamate and gamma-amino butyric acid (GABA) are respectively two major excitatory and inhibitory neurotransmitters of the adult mammalian central nervous system. These neurotransmitters exert their action through two types of receptors: ionotropic and metabotropic receptors. While ionotropic receptors are ligand gated ion channels involved in fast synaptic transmission, metabotropic receptors belong to the superfamily of G-protein coupled receptors (GPCRs) and are responsible for the neuromodulatory effect of glutamate and GABA. Metabotropic glutamate receptors (mGluRs) and metabotropic GABA receptors (GABA-B) are present at different levels of the pain neuraxis where they regulate nociceptive transmission and pain. The present review will focus on the role of these receptors in the modulation of pain perception.