RESUMEN
Genome-wide association studies have identified risk loci associated with the development of inflammatory bowel disease, while epidemiological studies have emphasized that pathogenesis likely involves host interactions with environmental elements whose source and structure need to be defined. Here, we identify a class of compounds derived from dietary, microbial, and industrial sources that are characterized by the presence of a five-membered oxazole ring and induce CD1d-dependent intestinal inflammation. We observe that minimal oxazole structures modulate natural killer T cell-dependent inflammation by regulating lipid antigen presentation by CD1d on intestinal epithelial cells (IECs). CD1d-restricted production of interleukin 10 by IECs is limited through activity of the aryl hydrocarbon receptor (AhR) pathway in response to oxazole induction of tryptophan metabolites. As such, the depletion of the AhR in the intestinal epithelium abrogates oxazole-induced inflammation. In summary, we identify environmentally derived oxazoles as triggers of CD1d-dependent intestinal inflammatory responses that occur via activation of the AhR in the intestinal epithelium.
Asunto(s)
Colitis/patología , Dieta , Intestinos/patología , Oxazoles/farmacología , Receptores de Hidrocarburo de Aril/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Antígenos CD1d/genética , Antígenos CD1d/metabolismo , Colitis/inducido químicamente , Colitis/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interleucina-10/metabolismo , Intestinos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células T Asesinas Naturales/inmunología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Receptores de Hidrocarburo de Aril/genética , Triptófano/metabolismoRESUMEN
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
Artificial control of neuronal activity enables the study of neural circuits and restoration of neural functions. Direct, rapid, and sustained photocontrol of intact neurons could overcome the limitations of established electrical stimulation such as poor selectivity. We have developed fast photoswitchable ligands of glutamate receptors (GluRs) to enable neuronal control in the auditory system. The new photoswitchable ligands induced photocurrents in untransfected neurons upon covalently tethering to endogenous GluRs and activating them reversibly with visible light pulses of a few milliseconds. As a proof of concept of these molecular prostheses, we applied them to the ultrafast synapses of auditory neurons of the cochlea that encode sound and provide auditory input to the brain. This drug-based method afforded the optical stimulation of auditory neurons of adult gerbils at hundreds of hertz without genetic manipulation that would be required for their optogenetic control. This indicates that the new photoswitchable ligands are also applicable to the spatiotemporal control of fast spiking interneurons in the brain.
Asunto(s)
Cóclea , Optogenética , Cóclea/fisiología , Ligandos , Neuronas , Optogenética/métodos , Prótesis e ImplantesRESUMEN
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.
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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/fisiologíaRESUMEN
Breakthrough cancer pain (BTcP) refers to a sudden and transient exacerbation of pain, which develops in patients treated with opioid analgesics. Fast-onset analgesia is required for the treatment of BTcP. Light-activated drugs offer a novel potential strategy for the rapid control of pain without the typical adverse effects of systemic analgesic drugs. mGlu5 metabotropic glutamate receptor antagonists display potent analgesic activity, and light-induced activation of one of these compounds (JF-NP-26) in the thalamus was found to induce analgesia in models of inflammatory and neuropathic pain. We used an established mouse model of BTcP based on the injection of cancer cells into the femur, followed, 16 days later, by systemic administration of morphine. BTcP was induced by injection of endothelin-1 (ET-1) into the tumor, 20 min after morphine administration. Mice were implanted with optic fibers delivering light in the visible spectrum (405 nm) in the thalamus or prelimbic cortex to locally activate systemically injected JF-NP-26. Light delivery in the thalamus caused rapid and substantial analgesia, and this effect was specific because light delivery in the prelimbic cortex did not relieve BTcP. This finding lays the groundwork for the use of optopharmacology in the treatment of BTcP.
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Analgesia , Dolor Irruptivo , Dolor en Cáncer , Neoplasias , Receptores de Glutamato Metabotrópico , Analgesia/efectos adversos , Analgésicos/farmacología , Analgésicos/uso terapéutico , Analgésicos Opioides/efectos adversos , Animales , Dolor Irruptivo/tratamiento farmacológico , Dolor Irruptivo/etiología , Dolor en Cáncer/tratamiento farmacológico , Dolor en Cáncer/etiología , Modelos Animales de Enfermedad , Ratones , Morfina/farmacología , Morfina/uso terapéutico , Neoplasias/tratamiento farmacológico , Dimensión del Dolor , TálamoRESUMEN
Catecholamine-triggered ß-adrenoceptor (ß-AR) signaling is essential for the correct functioning of the heart. Although both ß1 - and ß2 -AR subtypes are expressed in cardiomyocytes, drugs selectively targeting ß1 -AR have proven this receptor as the main target for the therapeutic effects of beta blockers in the heart. Here, we report a new strategy for the light-control of ß1 -AR activation by means of photoswitchable drugs with a high level of ß1 -/ß2 -AR selectivity. All reported molecules allow for an efficient real-time optical control of receptor function in vitro. Moreover, using confocal microscopy we demonstrate that the binding of our best hit, pAzo-2, can be reversibly photocontrolled. Strikingly, pAzo-2 also enables a dynamic cardiac rhythm management on living zebrafish larvae using light, thus highlighting the therapeutic and research potential of the developed photoswitches. Overall, this work provides the first proof of precise control of the therapeutic target ß1 -AR in native environments using light.
Asunto(s)
Receptores Adrenérgicos beta 2 , Pez Cebra , Antagonistas Adrenérgicos beta/farmacología , Animales , Ligandos , Miocitos Cardíacos/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Pez Cebra/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.
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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
Four series of C7N aminocyclitol analogues of glucose were synthesized by stereocontrolled epoxide opening of hydroxyl protected forms of the cyclohexane epoxides cyclophellitol and 1,6-epi-cyclophellitol. The resulting hydroxymethyl substituted aminocyclitols were tested as glycosidase inhibitors. Cyclitols having an amino group in an α configuration at a position equivalent to the anomeric in the sugar were found to be low micromolar inhibitors of the α-glucosidase from baker's yeast with Ki's near to 2 µM. On the other hand, N-octyl aminocyclitols having the nitrogen substituents in an α or ß configuration were found to be good inhibitors of recombinant ß-glucocerebrosidase with Ki values between 8.3 and 17 µM, and also inhibited lysosomal ß-glucosidase activity in live cells at low-micromolar concentrations. A computational docking study suggests a differential binding among the different series of ß-glucocerebrosidase inhibitors. In agreement with the experimental results, the binding poses obtained indicate that the presence of an alkyl lipid substituent in the inhibitor mimicking one of the lipid chains in the substrate is critical for potency. In contrast, the matching of hydroxymethyl substituents in the aminocyclitols and the parent glucosylceramide does not seem to be strictly necessary for potent inhibition, indicating the risk of simplifying structural analogies in sugar mimetic design.
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Ciclitoles/síntesis química , Ciclohexanoles/síntesis química , Inhibidores Enzimáticos/química , Glucosilceramidasa/antagonistas & inhibidores , Glucosilceramidasa/química , beta-Glucosidasa/antagonistas & inhibidores , beta-Glucosidasa/química , Ciclitoles/química , Ciclohexanoles/química , Cinética , Relación Estructura-Actividad , alfa-GlucosidasasRESUMEN
A new type of galactose mimetics has been synthesized following a straightforward synthetic approach based on cyclohexene olefin aziridination reactions directed by hydroxyl substituents. These enantiomerically pure galacto-configured N-aminoaziridines are potent irreversible inhibitors of Aspergillus oryzae and Escherichia coliß-galactosidases.
Asunto(s)
Aspergillus oryzae/enzimología , Aziridinas/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Escherichia coli/enzimología , Galactosa/química , beta-Galactosidasa/antagonistas & inhibidores , Aspergillus oryzae/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Concentración de Iones de Hidrógeno , Cinética , beta-Galactosidasa/metabolismoRESUMEN
A library of sphingolipid analogues is designed and tested as inhibitors against mammalian and fungal sphingolipid enzymes. The synthesis of sphingolipid analogues is based on the nucleophilic ring-opening reactions of N-activated aziridine derivatives with thiols, ß-thioglycosyl thiols, phosphorothioates, phosphates, and amines to afford compounds having different lipid backbones and substituents representative of the naturally occurring sphingolipid families. The screening on mammalian sphingomyelin synthase (SMS) and glucosylceramide synthase (GCS) and yeast inositol phosphorylceramide synthase (IPCS) enzymes identified several inhibitors of GCS and IPCS, but no inhibition of SMS was observed among the tested compounds.
Asunto(s)
Aziridinas/química , Glucosiltransferasas/antagonistas & inhibidores , Glucosiltransferasas/química , Esfingolípidos/síntesis química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/antagonistas & inhibidores , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Animales , Saccharomyces cerevisiae , Esfingolípidos/química , Esfingolípidos/farmacologíaRESUMEN
Metabotropic glutamate receptors (mGluRs) are high-profile G-protein coupled receptors drug targets because of their involvement in several neurological disease states, and mGluR5 in particular is a subtype whose controlled allosteric modulation, both positive and negative, can potentially be useful for the treatment of schizophrenia and relief of chronic pain, respectively. Here we model mGluR5 with a collection of positive and negative allosteric modulators (PAMs and NAMs) in both active and inactive receptor states, in a manner that is consistent with experimental information, using a specialized protocol that includes homology to increase docking accuracy, and receptor relaxation to generate an individual induced fit with each allosteric modulator. Results implicate two residues in particular for NAM and PAM function: NAM interaction with W785 for receptor inactivation, and NAM/PAM H-bonding with S809 for receptor (in)activation. Models suggest the orientation of the H-bond between allosteric modulator and S809, controlled by PAM/NAM chemistry, influences the position of TM7, which in turn influences the shape of the allosteric site, and potentially the receptor state. NAM-bound and PAM-bound mGluR5 models also reveal that although PAMs and NAMs bind in the same pocket and share similar binding modes, they have distinct effects on the conformation of the receptor. Our models, together with the identification of a possible activation mechanism, may be useful in the rational design of new allosteric modulators for mGluR5.
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Descubrimiento de Drogas , Simulación del Acoplamiento Molecular , Receptor del Glutamato Metabotropico 5/química , Receptor del Glutamato Metabotropico 5/metabolismo , Regulación Alostérica/efectos de los fármacos , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Activation of type I NKT (iNKT) cells by CD1d-presented agonists is a potent immunotherapeutic tool. α-Galactosylceramide (α-GalCer) is the prototypic agonist, but its excessive potency with simultaneous production of both pro- and anti-inflammatory cytokines hampers its potential therapeutic use. In search for novel agonists, we have analyzed the structure and function of HS44, a synthetic aminocyclitolic ceramide analog designed to avoid unrestrained iNKT cell activation. HS44 is a weaker agonist compared with α-GalCer in vitro, although in vivo it induces robust IFN-γ production, and highly reduced but still functional Th2 response. The characteristic cytokine storm produced upon α-GalCer activation was not induced. Consequently, HS44 induced a very efficient iNKT cell-dependent antitumoral response in B16 animal model. In addition, intranasal administration showed the capacity to induce lung inflammation and airway hyperreactivity, a cardinal asthma feature. Thus, HS44 is able to elicit functional Th1 or Th2 responses. Structural studies show that HS44 binds to CD1d with the same conformation as α-GalCer. The TCR binds to HS44 similarly as α-GalCer, but forms less contacts, thus explaining its weaker TCR affinity and, consequently, its weaker recognition by iNKT cells. The ability of this compound to activate an efficient, but not massive, tailored functional immune response makes it an attractive reagent for immune manipulation.
Asunto(s)
Ciclitoles/química , Galactosilceramidas/química , Galactosilceramidas/farmacología , Factores Inmunológicos/química , Factores Inmunológicos/farmacología , Células T Asesinas Naturales/efectos de los fármacos , Células T Asesinas Naturales/inmunología , Relación Estructura-Actividad Cuantitativa , Animales , Hiperreactividad Bronquial/tratamiento farmacológico , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/patología , Células Cultivadas , Cristalografía por Rayos X , Ciclitoles/agonistas , Ciclitoles/farmacología , Modelos Animales de Enfermedad , Femenino , Galactosilceramidas/agonistas , Factores Inmunológicos/clasificación , Activación de Linfocitos/efectos de los fármacos , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células T Asesinas Naturales/patologíaRESUMEN
Purpose: Severe side effects prevent the utilization of otherwise promising drugs in treatments. These side effects arise when drugs affect untargeted tissues due to poor target specificity. In photopharmacology, light controls the timing and the location of drug delivery, improving treatment specificity and pharmacokinetic control. Photopharmaceuticals have not seen widespread adoption in part because researchers do not always have access to reliable and reproducible light delivery devices at prices which fit within the larger research budget. Method: In this work, we present a customizable photomodulator for use in both wearable and implantable devices. For experimental validation of the photomodulator, we photolyse JF-NP-26 in rats. Results: We successfully drive in vivo photopharmacology with a tethered photomodulator and demonstrate modifications which enable the photomodulator to operate wirelessly. Conclusion: By documenting our photomodulator development, we hope to introduce researchers to a simple solution which significantly lowers the engineering barriers to photopharmacology research. Graphical abstract: Researchers present a photomodulator, a device designed to facilitate in vivo photopharmacology. They demonstrate the in vivo capabilities of the photomodulator by photoreleasing raseglurant, an mGluR5 inhibitor, to treat pain in an acute rat model and follow this study by showing how to reconfigure the photomodulator to work wirelessly and interface with other biomedical devices. Supplementary Information: The online version contains supplementary material available at 10.1007/s13534-023-00334-3.
RESUMEN
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
The (poly)pharmacology of drug metabolites is seldom comprehensively characterized in drug discovery. However, some drug metabolites can reach high plasma concentrations and display in vivo activity. Here, we use computational and experimental methods to comprehensively characterize the kinase polypharmacology of M324, the major metabolite of the PARP1 inhibitor rucaparib. We demonstrate that M324 displays unique PLK2 inhibition at clinical concentrations. This kinase activity could have implications for the efficacy and safety of rucaparib and therefore warrants further clinical investigation. Importantly, we identify synergy between the drug and the metabolite in prostate cancer models and a complete reduction of α-synuclein accumulation in Parkinson's disease models. These activities could be harnessed in the clinic or open new drug discovery opportunities. The study reported here highlights the importance of characterizing the activity of drug metabolites to comprehensively understand drug response in the clinic and exploit our current drug arsenal in precision medicine.
Asunto(s)
Indoles , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Animales , Humanos , Masculino , Ratones , Línea Celular Tumoral , Sinergismo Farmacológico , Indoles/farmacología , Indoles/química , Indoles/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patologíaRESUMEN
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
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
NKT cells respond to a variety of CD1d-restricted glycolipid Ags that are structurally related to the prototypic Ag α-galactosylceramide (α-GalCer). A modified analog of α-GalCer with a carbon-based glycosidic linkage (α-C-GalCer) has generated great interest because of its apparent ability to promote prolonged, Th1-biased immune responses. In this study, we report the activation of spleen NKT cells to α-C-GalCer, and related C-glycoside ligands, is weaker than that of α-GalCer. Furthermore, the Vß8.2 and Vß7 NKT TCR affinity for CD1d-α-C-GalCer, and some related analogs, is â¼10-fold lower than that for the NKT TCR-CD1d-α-GalCer interaction. Nevertheless, the crystal structure of the Vß8.2 NKT TCR-CD1d-α-C-GalCer complex is similar to that of the corresponding NKT TCR-CD1d-α-GalCer complex, although subtle differences at the interface provide a basis for understanding the lower affinity of the NKT TCR-CD1d-α-C-GalCer interaction. Our findings support the concept that for CD1d-restricted NKT cells, altered glycolipid ligands can promote markedly different responses while adopting similar TCR-docking topologies.