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1.
Proc Natl Acad Sci U S A ; 116(27): 13680-13689, 2019 07 02.
Artículo en Inglés | MEDLINE | ID: mdl-31196955

RESUMEN

The physiological activity of proteins is often studied with loss-of-function genetic approaches, but the corresponding phenotypes develop slowly and can be confounding. Photopharmacology allows direct, fast, and reversible control of endogenous protein activity, with spatiotemporal resolution set by the illumination method. Here, we combine a photoswitchable allosteric modulator (alloswitch) and 2-photon excitation using pulsed near-infrared lasers to reversibly silence metabotropic glutamate 5 (mGlu5) receptor activity in intact brain tissue. Endogenous receptors can be photoactivated in neurons and astrocytes with pharmacological selectivity and with an axial resolution between 5 and 10 µm. Thus, 2-photon pharmacology using alloswitch allows investigating mGlu5-dependent processes in wild-type animals, including synaptic formation and plasticity, and signaling pathways from intracellular organelles.


Asunto(s)
Encéfalo/fisiología , Optogenética/métodos , Fotones , Receptores de Superficie Celular/metabolismo , Animales , Astrocitos/metabolismo , Astrocitos/fisiología , Encéfalo/metabolismo , Calcio/metabolismo , Neuronas/metabolismo , Neuronas/fisiología , Ratas , Ratas Sprague-Dawley , Receptor del Glutamato Metabotropico 5/metabolismo , Receptor del Glutamato Metabotropico 5/fisiología , Receptores de Superficie Celular/fisiología
2.
Nat Chem Biol ; 10(10): 813-5, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25173999

RESUMEN

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/fisiología
3.
J Org Chem ; 80(20): 9915-25, 2015 Oct 16.
Artículo en Inglés | MEDLINE | ID: mdl-26414427

RESUMEN

A new azobenzene-based photoswitch, 2, has been designed to enable optical control of ionotropic glutamate receptors in neurons via sensitized two-photon excitation with NIR light. In order to develop an efficient and versatile synthetic route for this molecule, a modular strategy is described which relies on the use of a new linear fully protected glutamate derivative stable in basic media. The resulting compound undergoes one-photon trans-cis photoisomerization via two different mechanisms: direct excitation of its azoaromatic unit and irradiation of the pyrene sensitizer, a well-known two-photon sensitive chromophore. Moreover, 2 presents large thermal stability of its cis isomer, in contrast to other two-photon responsive switches relying on the intrinsic nonlinear optical properties of push-pull substituted azobenzenes. As a result, the molecular system developed herein is a very promising candidate for evoking large photoinduced biological responses during the multiphoton operation of neuronal glutamate receptors with NIR light, which require accumulation of the protein-bound cis state of the switch upon repeated illumination.


Asunto(s)
Compuestos Azo/química , Receptores Ionotrópicos de Glutamato/química , Compuestos Azo/síntesis química , Ligandos , Estructura Molecular , Neuronas/química , Procesos Fotoquímicos , Estereoisomerismo
4.
J Am Chem Soc ; 136(24): 8693-701, 2014 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-24857186

RESUMEN

Synthetic photochromic compounds can be designed to control a variety of proteins and their biochemical functions in living cells, but the high spatiotemporal precision and tissue penetration of two-photon stimulation have never been investigated in these molecules. Here we demonstrate two-photon excitation of azobenzene-based protein switches and versatile strategies to enhance their photochemical responses. This enables new applications to control the activation of neurons and astrocytes with cellular and subcellular resolution.


Asunto(s)
Compuestos Azo/química , Proteínas/química , Protones , Compuestos Azo/síntesis química , Células Cultivadas , Células HEK293 , Humanos , Estructura Molecular , Procesos Fotoquímicos
5.
Cell Rep ; 40(13): 111426, 2022 09 27.
Artículo en Inglés | MEDLINE | ID: mdl-36170823

RESUMEN

The prefrontal cortex (PFC) is a hub for cognitive control, and dopamine profoundly influences its functions. In other brain regions, astrocytes sense diverse neurotransmitters and neuromodulators and, in turn, orchestrate regulation of neuroactive substances. However, basic physiology of PFC astrocytes, including which neuromodulatory signals they respond to and how they contribute to PFC function, is unclear. Here, we characterize divergent signaling signatures in mouse astrocytes of the PFC and primary sensory cortex, which show differential responsiveness to locomotion. We find that PFC astrocytes express receptors for dopamine but are unresponsive through the Gs/Gi-cAMP pathway. Instead, fast calcium signals in PFC astrocytes are time locked to dopamine release and are mediated by α1-adrenergic receptors both ex vivo and in vivo. Further, we describe dopamine-triggered regulation of extracellular ATP at PFC astrocyte territories. Thus, we identify astrocytes as active players in dopaminergic signaling in the PFC, contributing to PFC function though neuromodulator receptor crosstalk.


Asunto(s)
Dopamina , Receptores Adrenérgicos alfa 1 , Adenosina Trifosfato/metabolismo , Animales , Astrocitos/metabolismo , Calcio/metabolismo , Dopamina/metabolismo , Ratones , Corteza Prefrontal/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo
6.
Nat Neurosci ; 22(11): 1936-1944, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31570865

RESUMEN

Recent work examining astrocytic physiology centers on fluorescence imaging, due to development of sensitive fluorescent indicators and observation of spatiotemporally complex calcium activity. However, the field remains hindered in characterizing these dynamics, both within single cells and at the population level, because of the insufficiency of current region-of-interest-based approaches to describe activity that is often spatially unfixed, size-varying and propagative. Here we present an analytical framework that releases astrocyte biologists from region-of-interest-based tools. The Astrocyte Quantitative Analysis (AQuA) software takes an event-based perspective to model and accurately quantify complex calcium and neurotransmitter activity in fluorescence imaging datasets. We apply AQuA to a range of ex vivo and in vivo imaging data and use physiologically relevant parameters to comprehensively describe the data. Since AQuA is data-driven and based on machine learning principles, it can be applied across model organisms, fluorescent indicators, experimental modes, and imaging resolutions and speeds, enabling researchers to elucidate fundamental neural physiology.


Asunto(s)
Astrocitos/metabolismo , Calcio/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Neurotransmisores/metabolismo , Imagen Óptica/métodos , Adenoviridae , Algoritmos , Animales , Animales Recién Nacidos , Vectores Genéticos , Ratones , Programas Informáticos , Transfección , Corteza Visual/metabolismo
7.
ACS Cent Sci ; 3(1): 81-91, 2017 Jan 25.
Artículo en Inglés | MEDLINE | ID: mdl-28149957

RESUMEN

Phenylazopyridines are photoisomerizable compounds with high potential to control biological functions with light. We have obtained a series of phenylazopyridines with light dependent activity as negative allosteric modulators (NAM) of metabotropic glutamate receptor subtype 5 (mGlu5). Here we describe the factors needed to achieve an operational molecular photoisomerization and its effective translation into in vitro and in vivo receptor photoswitching, which includes zebrafish larva motility and the regulation of the antinociceptive effects in mice. The combination of light and some specific phenylazopyridine ligands displays atypical pharmacological profiles, including light-dependent receptor overactivation, which can be observed both in vitro and in vivo. Remarkably, the localized administration of light and a photoswitchable compound in the peripheral tissues of rodents or in the brain amygdalae results in an illumination-dependent analgesic effect. The results reveal a robust translation of the phenylazopyridine photoisomerization to a precise photoregulation of biological activity.

8.
Cell Chem Biol ; 23(8): 929-34, 2016 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-27478159

RESUMEN

OptoGluNAM4.1, a negative allosteric modulator (NAM) of metabotropic glutamate receptor 4 (mGlu4) contains a reactive group that covalently binds to the receptor and a blue-light-activated, fast-relaxing azobenzene group that allows reversible receptor activity photocontrol in vitro and in vivo. OptoGluNAM4.1 induces light-dependent behavior in zebrafish and reverses the activity of the mGlu4 agonist LSP4-2022 in a mice model of chronic pain, defining a photopharmacological tool to better elucidate the physiological roles of the mGlu4 receptor in the nervous system.


Asunto(s)
Compuestos Azo/farmacología , Luz , Ácido Pirrolidona Carboxílico/análogos & derivados , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/efectos de la radiación , Animales , Compuestos Azo/química , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Umbral del Dolor/efectos de los fármacos , Ácido Pirrolidona Carboxílico/química , Ácido Pirrolidona Carboxílico/farmacología , Receptores de Glutamato Metabotrópico/metabolismo , Relación Estructura-Actividad , Factores de Tiempo , Pez Cebra/metabolismo
9.
Curr Neuropharmacol ; 14(5): 441-54, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26391742

RESUMEN

Metabotropic glutamate receptors (mGluRs) are important drug targets because of their involvement in several neurological diseases. Among mGluRs, mGlu5 is a particularly high-profile target because its positive or negative allosteric modulation can potentially treat schizophrenia or anxiety and chronic pain, respectively. Here, we computationally and experimentally probe the functional binding of a novel photoswitchable mGlu5 NAM, termed alloswitch-1, which loses its NAM functionality under violet light. We show alloswitch-1 binds deep in the allosteric pocket in a similar fashion to mavoglurant, the co-crystallized NAM in the mGlu5 transmembrane domain crystal structure. Alloswitch-1, like NAM 2-Methyl-6-(phenylethynyl)pyridine (MPEP), is significantly affected by P655M mutation deep in the allosteric pocket, eradicating its functionality. In MD simulations, we show alloswitch-1 and MPEP stabilize the co-crystallized water molecule located at the bottom of the allosteric site that is seemingly characteristic of the inactive receptor state. Furthermore, both NAMs form H-bonds with S809 on helix 7, which may constitute an important stabilizing interaction for NAM-induced mGlu5 inactivation. Alloswitch-1, through isomerization of its amide group from trans to cis is able to form an additional interaction with N747 on helix 5. This may be an important interaction for amide-containing mGlu5 NAMs, helping to stabilize their binding in a potentially unusual cis-amide state. Simulated conformational switching of alloswitch-1 in silico suggests photoisomerization of its azo group from trans to cis may be possible within the allosteric pocket. However, photoexcited alloswitch-1 binds in an unstable fashion, breaking H-bonds with the protein and destabilizing the co-crystallized water molecule. This suggests photoswitching may have destabilizing effects on mGlu5 binding and functionality.


Asunto(s)
Regulación Alostérica , Luz , Procesos Fotoquímicos , Receptor del Glutamato Metabotropico 5/metabolismo , Receptor del Glutamato Metabotropico 5/efectos de la radiación , Sitio Alostérico , Antagonistas de Aminoácidos Excitadores/farmacología , Células HEK293 , Humanos , Enlace de Hidrógeno , Isomerismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mutación , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Piridinas/farmacología , Receptor del Glutamato Metabotropico 5/antagonistas & inhibidores , Receptor del Glutamato Metabotropico 5/genética , Agua/química
10.
Nat Commun ; 7: 12221, 2016 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-27436051

RESUMEN

Light-regulated drugs allow remotely photoswitching biological activity and enable plausible therapies based on small molecules. However, only freely diffusible photochromic ligands have been shown to work directly in endogenous receptors and methods for covalent attachment depend on genetic manipulation. Here we introduce a chemical strategy to covalently conjugate and photoswitch the activity of endogenous proteins and demonstrate its application to the kainate receptor channel GluK1. The approach is based on photoswitchable ligands containing a short-lived, highly reactive anchoring group that is targeted at the protein of interest by ligand affinity. These targeted covalent photoswitches (TCPs) constitute a new class of light-regulated drugs and act as prosthetic molecules that photocontrol the activity of GluK1-expressing neurons, and restore photoresponses in degenerated retina. The modularity of TCPs enables the application to different ligands and opens the way to new therapeutic opportunities.


Asunto(s)
Luz , Óptica y Fotónica/métodos , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Animales , Química Clic , Femenino , Ganglios Espinales/metabolismo , Células HEK293 , Humanos , Isomerismo , Ligandos , Ratones , Modelos Moleculares , Neuronas/metabolismo , Receptores de Superficie Celular/química , Retina/metabolismo
11.
J Neurosci Methods ; 201(1): 46-54, 2011 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-21782847

RESUMEN

Chronic orofacial pain encompasses a range of debilitating conditions, however in contrast to other body regions, few animal models are available to investigate mechanisms and treatments in the trigeminal area. Particularly, there is a lack of reliable models and testing methods in mice. We have behaviourally tested C57BL/6 mice subjected to unilateral chronic constriction injury (CCI) of the infraorbital nerve (IoN) or unilateral injections of Complete Freunds Adjuvant (CFA) into the vibrissal pad region with the aid of von Frey filaments and air-puffs and the use of a newly designed restraining device. These models were validated by suppressing the pain responses with appropriate drugs. The IoN-CCI group showed significant hyperalgesia on the ipsilateral side in comparison to baseline values for up to 20 days post-CCI following von Frey and air-puff stimulation. Gabapentin (60mg/kg), but not saline, temporarily reversed the hyperalgesia. Animals that received a CFA injection showed hyperresponsivity to both von Frey and air-puff stimulation for up to 4 days post injection. These effects were transiently reversed with 3mg/kg i.p. morphine but not saline. Our study proposes a new restraining device for mice, and validates a behavioural testing procedure of several facial pain models in mice, allowing for reproducible and robust assessment of the effects of pain-related agents and treatments, or phenotyping of genetically modified animals.


Asunto(s)
Modelos Animales de Enfermedad , Dimensión del Dolor/métodos , Neuralgia del Trigémino/fisiopatología , Animales , Inflamación/diagnóstico , Inflamación/tratamiento farmacológico , Inflamación/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Morfina/farmacología , Morfina/uso terapéutico , Dimensión del Dolor/efectos de los fármacos , Dimensión del Dolor/instrumentación , Umbral del Dolor/efectos de los fármacos , Umbral del Dolor/fisiología , Distribución Aleatoria , Neuralgia del Trigémino/diagnóstico , Neuralgia del Trigémino/tratamiento farmacológico
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