RESUMEN
BACKGROUND: AZD2811 is a potent, selective Aurora kinase B inhibitor. We report the dose-escalation phase of a first-in-human study assessing nanoparticle-encapsulated AZD2811 in advanced solid tumours. METHODS: AZD2811 was administered in 12 dose-escalation cohorts (2-h intravenous infusion; 15â600 mg; 21-/28-day cycles) with granulocyte colony-stimulating factor (G-CSF) at higher doses. The primary objective was determining safety and maximum tolerated/recommended phase 2 dose (RP2D). RESULTS: Fifty-one patients received AZD2811. Drug exposure was sustained for several days post-dose. The most common AZD2811-related adverse events (AEs) were fatigue (27.3%) at ≤200 mg/cycle and neutropenia (37.9%) at ≥400 mg/cycle. Five patients had dose-limiting toxicities: grade (G)4 decreased neutrophil count (n = 1, 200 mg; Days 1, 4; 28-day cycle); G4 decreased neutrophil count and G3 stomatitis (n = 1 each, both 400 mg; Day 1; 21-day cycle); G3 febrile neutropenia and G3 fatigue (n = 1 each, both 600 mg; Day 1; 21-day cycle +G-CSF). RP2D was 500 mg; Day 1; 21-day cycle with G-CSF on Day 8. Neutropenia/neutrophil count decrease were on-target AEs. Best overall responses were partial response (n = 1, 2.0%) and stable disease (n = 23, 45.1%). CONCLUSIONS: At RP2D, AZD2811 was tolerable with G-CSF support. Neutropenia was a pharmacodynamic biomarker. CLINICAL TRIAL REGISTRATION: NCT02579226.
Asunto(s)
Antineoplásicos , Neoplasias , Neutropenia , Humanos , Aurora Quinasa B/uso terapéutico , Neoplasias/patología , Neutropenia/inducido químicamente , Fatiga/inducido químicamente , Factor Estimulante de Colonias de Granulocitos/efectos adversos , Dosis Máxima Tolerada , Relación Dosis-Respuesta a DrogaRESUMEN
Intracellular neurofibrillary tangles (NFTs) are the hallmark of Alzheimer's disease and other tauopathies in which tau, a microtubule-associated protein, loses its ability to stabilize microtubules. Several post-translational modifications including phosphorylation and truncation increase tau's propensity to aggregate thus forming NFTs; however, the mechanisms underlying tau conformational change and aggregation still remain to be defined. Caspase activation and subsequent proteolytic cleavage of tau is thought to be a potential trigger of this disease-related pathological conformation. The aim of this work was to investigate the link between caspase activation and a disease-related conformational change of tau in a neuroblastoma cell-based model of spontaneous tau aggregation. We demonstrated that caspase induction initiates proteolytic cleavage of tau and generation of conformationally altered and aggregated tau recognized by the MC1 conformational antibody. Most importantly, these events were shown to be attenuated with caspase inhibitors. This implies that therapeutics aimed at inhibiting caspase-mediated tau cleavage may prove beneficial in slowing cleavage and aggregation, thus potentially halting tau pathology and disease progression.
Asunto(s)
Inhibidores de Caspasas/farmacología , Caspasas/metabolismo , ATPasas Transportadoras de Cobre/metabolismo , Agregación Patológica de Proteínas/metabolismo , Proteínas tau/metabolismo , Animales , Línea Celular Tumoral , ATPasas Transportadoras de Cobre/química , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , Agregación Patológica de Proteínas/patología , Conformación Proteica/efectos de los fármacos , Estaurosporina/farmacología , Proteínas tau/químicaRESUMEN
The interneuronal propagation of aggregated tau is believed to play an important role in the pathogenesis of human tauopathies. It requires the uptake of seed-competent tau into cells, seeding of soluble tau in recipient neurons and release of seeded tau into the extracellular space to complete the cycle. At present, it is not known which tau species are seed-competent. Here, we have dissected the molecular characteristics of seed-competent tau species from the TgP301S tau mouse model using various biochemical techniques and assessed their seeding ability in cell and animal models. We found that sucrose gradient fractions from brain lysates seeded cellular tau aggregation only when large (>10 mer) aggregated, hyperphosphorylated (AT8- and AT100-positive) and nitrated tau was present. In contrast, there was no detectable seeding by fractions containing small, oligomeric (<6 mer) tau. Immunodepletion of the large aggregated AT8-positive tau strongly reduced seeding; moreover, fractions containing these species initiated the formation and spreading of filamentous tau pathology in vivo, whereas fractions containing tau monomers and small oligomeric assemblies did not. By electron microscopy, seed-competent sucrose gradient fractions contained aggregated tau species ranging from ring-like structures to small filaments. Together, these findings indicate that a range of filamentous tau aggregates are the major species that underlie the spreading of tau pathology in the P301S transgenic model. Significance statement: The spread of tau pathology from neuron to neuron is postulated to account for, or at least to contribute to, the overall propagation of tau pathology during the development of human tauopathies including Alzheimer's disease. It is therefore important to characterize the native tau species responsible for this process of seeding and pathology spreading. Here, we use several biochemical techniques to dissect the molecular characteristics of native tau protein conformers from TgP301S tau mice and show that seed-competent tau species comprise small fibrils capable of seeding tau pathology in cell and animal models. Characterization of seed-competent tau gives insight into disease mechanisms and therapeutic interventions.
Asunto(s)
Amiloide/genética , Encéfalo , Ovillos Neurofibrilares/genética , Tauopatías/genética , Proteínas tau/genética , Animales , Encéfalo/patología , Femenino , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ovillos Neurofibrilares/patología , Tauopatías/patologíaRESUMEN
Intracellular Tau inclusions are a pathological hallmark of several neurodegenerative diseases, collectively known as the tauopathies. They include Alzheimer disease, tangle-only dementia, Pick disease, argyrophilic grain disease, chronic traumatic encephalopathy, progressive supranuclear palsy, and corticobasal degeneration. Tau pathology appears to spread through intercellular propagation, requiring the formation of assembled "prion-like" species. Several cell and animal models have been described that recapitulate aspects of this phenomenon. However, the molecular characteristics of seed-competent Tau remain unclear. Here, we have used a cell model to understand the relationships between Tau structure/phosphorylation and seeding by aggregated Tau species from the brains of mice transgenic for human mutant P301S Tau and full-length aggregated recombinant P301S Tau. Deletion of motifs (275)VQIINK(280) and (306)VQIVYK(311) abolished the seeding activity of recombinant full-length Tau, suggesting that its aggregation was necessary for seeding. We describe conformational differences between native and synthetic Tau aggregates that may account for the higher seeding activity of native assembled Tau. When added to aggregated Tau seeds from the brains of mice transgenic for P301S Tau, soluble recombinant Tau aggregated and acquired the molecular properties of aggregated Tau from transgenic mouse brain. We show that seeding is conferred by aggregated Tau that enters cells through macropinocytosis and seeds the assembly of endogenous Tau into filaments.
Asunto(s)
Agregado de Proteínas , Agregación Patológica de Proteínas/metabolismo , Tauopatías/metabolismo , Proteínas tau/química , Animales , Encéfalo/metabolismo , Encéfalo/patología , Citoesqueleto/metabolismo , Citoesqueleto/patología , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Ratones , Ratones Transgénicos , Fosforilación , Conformación Proteica , Tauopatías/patología , Proteínas tau/biosíntesis , Proteínas tau/metabolismoRESUMEN
Neurofibrillary tangles, one of the hallmarks of Alzheimer disease (AD), are composed of paired helical filaments of abnormally hyperphosphorylated tau. The accumulation of these proteinaceous aggregates in AD correlates with synaptic loss and severity of dementia. Identifying the kinases involved in the pathological phosphorylation of tau may identify novel targets for AD. We used an unbiased approach to study the effect of 352 human kinases on their ability to phosphorylate tau at epitopes associated with AD. The kinases were overexpressed together with the longest form of human tau in human neuroblastoma cells. Levels of total and phosphorylated tau (epitopes Ser(P)-202, Thr(P)-231, Ser(P)-235, and Ser(P)-396/404) were measured in cell lysates using AlphaScreen assays. GSK3α, GSK3ß, and MAPK13 were found to be the most active tau kinases, phosphorylating tau at all four epitopes. We further dissected the effects of GSK3α and GSK3ß using pharmacological and genetic tools in hTau primary cortical neurons. Pathway analysis of the kinases identified in the screen suggested mechanisms for regulation of total tau levels and tau phosphorylation; for example, kinases that affect total tau levels do so by inhibition or activation of translation. A network fishing approach with the kinase hits identified other key molecules putatively involved in tau phosphorylation pathways, including the G-protein signaling through the Ras family of GTPases (MAPK family) pathway. The findings identify novel tau kinases and novel pathways that may be relevant for AD and other tauopathies.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Glucógeno Sintasa Quinasa 3/metabolismo , Proteína Quinasa 13 Activada por Mitógenos/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Animales , Línea Celular Tumoral , Corteza Cerebral/enzimología , Corteza Cerebral/patología , Epítopos/genética , Epítopos/metabolismo , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Humanos , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Transgénicos , Proteína Quinasa 13 Activada por Mitógenos/genética , Neuronas/enzimología , Neuronas/patología , Fosforilación , Proteínas tau/genéticaRESUMEN
The novel 7-transmembrane receptor MrgX1 is located predominantly in the dorsal root ganglion and has consequently been implicated in the perception of pain. Here we describe the discovery and optimization of a small molecule agonist and initial docking studies of this ligand into the receptor in order to provide a suitable lead and tool compound for the elucidation of the physiological function of the receptor.
Asunto(s)
Piperazinas/síntesis química , Piridazinas/síntesis química , Receptores Acoplados a Proteínas G/agonistas , Compuestos de Bifenilo/síntesis química , Compuestos de Bifenilo/farmacología , Calcio/metabolismo , Técnicas Químicas Combinatorias , Diseño de Fármacos , Humanos , Piperazinas/farmacología , Piridazinas/farmacología , Receptores Acoplados a Proteínas G/fisiología , Relación Estructura-ActividadRESUMEN
Amyloid beta (Abeta) peptides are the major constituent of amyloid plaques, one of the hallmark pathologies of Alzheimer's disease. Accurate and precise quantitation of these peptides in biological fluids is a critical component of Alzheimer's disease research. The current most established assay for analysis of Abeta peptides in preclinical research is enzyme-linked immunosorbent assay (ELISA), which, although sensitive and of proven utility, is a multistep, labor-intensive assay that is difficult to automate completely. To overcome these limitations, the authors have developed and optimized simple, sensitive, homogeneous 384-well assays for Abeta1-42 and Abeta1-40 using AlphaScreen technology. The assays are capable of detecting Abeta peptides at concentrations <2 pg/mL and, using a final assay volume of 20 microL, routinely generate Z' values >0.85. The AlphaScreen format has the following key advantages: substantially less hands-on time to run, easier to automate, higher throughput, and less expensive to run than the traditional ELISA. The results presented here show that AlphaScreen technology permits robust, efficient, and cost-effective quantitation of Abeta peptides.
Asunto(s)
Enfermedad de Alzheimer/diagnóstico , Péptidos beta-Amiloides/aislamiento & purificación , Tamizaje Masivo/métodos , Fragmentos de Péptidos/aislamiento & purificación , Secuencia de Aminoácidos , Péptidos beta-Amiloides/análisis , Péptidos beta-Amiloides/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Química Encefálica , Calibración , Células Cultivadas , Análisis Costo-Beneficio , Medios de Cultivo/química , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Tamizaje Masivo/economía , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Datos de Secuencia Molecular , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/inmunología , Sensibilidad y Especificidad , Extractos de Tejidos/análisis , Extractos de Tejidos/químicaAsunto(s)
Sistemas de Liberación de Medicamentos , Factores de Crecimiento Nervioso/uso terapéutico , Fármacos Neuroprotectores/administración & dosificación , Enfermedad de Parkinson/terapia , Trasplante de Células Madre , Animales , Humanos , Enfermedad de Parkinson/etiología , Enfermedad de Parkinson/fisiopatología , Receptores AMPA/agonistasRESUMEN
RATIONALE: Nicotine produces behavioural effects that are potentially related to its interaction with diverse nicotinic acetylcholine receptor populations. Evidence from gene deletion studies suggests that the interoceptive stimulus properties of nicotine are mediated by heteromeric high-affinity receptors containing alpha4beta2 subunits. Mice lacking beta2 subunits do not discriminate nicotine (Shoaib et al., Neuropharmacology, 42:530-539, 2002), and nicotine does not elicit dopamine release in these animals (Grady et al., J Neurochem, 76:258-268, 2001). The stimulus properties of nicotine can be detected in rats using a two-lever operant drug discrimination paradigm, allowing them to be classified pharmacologically using ligands with selectivity for receptors containing alpha4beta2, alpha3beta4 or alpha7 subunits. MATERIALS AND METHODS: Rats trained to discriminate 0.4 mg/kg nicotine from vehicle were given the nicotinic receptor agonists, cytisine, varenicline, TC2559, ABT-594, A-85380 (all having high affinity but varying selectivity for alpha4beta2-containing receptors), and WO 03/062224 and WO 01/60821A1 (selective for beta4- and alpha7-containing receptors, respectively). In separate studies, WO 03/062224 was used as the training stimulus. RESULTS: Nicotine, TC-2559, A-85380 and ABT-594 showed dose-dependent and complete stimulus substitution, whilst WO 03/062224 and WO 01/60821A1 were completely without effect. Cytisine and varenicline showed partial generalisation, consistent with their partial agonist activity at nicotinic receptors eliciting dopamine release in rat striatal slices. After almost 50 training sessions with WO 03/062224, there was no clear evidence that an alpha3beta4 receptor agonist could sustain a discriminable stimulus. CONCLUSION: Substitution to the nicotine discriminative stimulus required high-affinity and high intrinsic activity at beta2 but not at beta4- or at alpha7-containing nicotinic receptors.
Asunto(s)
Aprendizaje Discriminativo/fisiología , Motivación , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/fisiología , Tabaquismo/fisiopatología , Animales , Conducta Apetitiva/efectos de los fármacos , Conducta Apetitiva/fisiología , Azetidinas/farmacología , Benzazepinas/farmacología , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/fisiopatología , Cistina/farmacología , Aprendizaje Discriminativo/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Masculino , Piridinas/farmacología , Quinoxalinas/farmacología , Ratas , Ratas Endogámicas , Vareniclina , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
The neuropeptide neuromedin U (NmU) shows considerable structural conservation across species. Within the body, it is widely distributed and in mammals has been implicated in physiological roles, including the regulation of feeding, anxiety, pain, blood flow, and smooth muscle contraction. Human NmU-25 (hNmU-25) and other NmU analogs were recently identified as ligands for two human orphan G protein-coupled receptors, subsequently named hNmU-R1 and hNmU-R2. These receptors have approximately 50% amino acid homology, and, at least in mammalian species, NmU-R1 and NmU-R2 are expressed predominantly in the periphery and central nervous system, respectively. Here, we have characterized signaling mediated by hNmU-R1 and hNmU-R2 expressed as recombinant proteins in human embryonic kidney 293 cells, particularly to define their G protein coupling and the activation and regulation of signal transduction pathways. We show that these receptors couple to both Galpha(q/11) and Galpha(i). Activation of either receptor type causes a pertussis toxin-insensitive activation of both phospholipase C and mitogen activated-protein kinase and a pertussis toxin-sensitive inhibition of adenylyl cyclase with subnanomolar potency for each. Activation of phospholipase C is sustained, but despite this capacity for prolonged receptor activation, repetitive application of hNmU-25 does not cause repetitive intracellular Ca2+ signaling by either recombinant receptors or those expressed endogenously in isolated smooth muscle cells from rat fundus. Using several strategies, we show this to be a consequence of essentially irreversible binding of hNmU-25 to its receptors and that this is followed by ligand internalization. Despite structural differences between receptors, there were no apparent differences in their activation, coupling, or regulation.
Asunto(s)
Señalización del Calcio/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Proteínas de la Membrana/fisiología , Receptores de Neurotransmisores/fisiología , Línea Celular , Membrana Celular/metabolismo , AMP Cíclico/fisiología , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Ligandos , Proteínas de la Membrana/metabolismo , Subunidades de Proteína/metabolismo , Receptores de Neurotransmisores/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal , Transfección , Fosfolipasas de Tipo C/metabolismoRESUMEN
Neuromedin U (NmU) is a smooth muscle contracting peptide. Recently, two G-protein-coupled receptors for NmU (NmU1R and NmU2R) have been cloned having approximately 50% homology. They have distinct patterns of expression suggesting they may have different biological functions. This study provides a comprehensive characterization of both NmU receptors expressed in human embryonic kidney 293 cells. [125I]hNmU binding to the recombinant NmU receptors was rapid, saturable, of high affinity and to a single population of binding sites. Exposure of these cells to NmU isopeptides resulted in an increase in intracellular [Ca2+]i release (EC50 value of 0.50 +/- 0.10 nmol/l) and inositol phosphate formation (EC50 1.6 +/- 0.2 and 1.50 +/- 0.4 nmol/l for NmU1R and NmU2R respectively). Furthermore, hNmU inhibited forskolin (3 micromol/l)-stimulated accumulation of cAMP in intact HEK-293 cells expressing either NmU1R or NmU2R. The inhibitory effect was significant for the cells expressing NmU2R with IC50 value of 0.80 +/- 0.21 nmol/l. In summary, both NmU1R and NmU2R in HEK-293 cells have similar signaling capability.
Asunto(s)
Proteínas de la Membrana/metabolismo , Receptores de Neurotransmisores/metabolismo , Sitios de Unión , Células Cultivadas , Células Clonales , Humanos , Riñón/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Ensayo de Unión Radioligante , Receptores de Neurotransmisores/genética , Receptores de Neurotransmisores/fisiología , Transducción de SeñalRESUMEN
RATIONALE: Neuromedin-U (NmU) is an agonist at NMU1R and NMU2R. The brain distribution of NmU and its receptors, in particular NMU2R, suggests widespread central roles for NmU. In agreement, centrally administered NmU affects feeding behaviour, energy expenditure and pituitary output. Further central nervous system (CNS) roles for NmU warrant investigation. OBJECTIVES: To investigate the CNS role of NmU by mapping NMU1R and NMU2R mRNA and measuring the behavioural, endocrine, neurochemical and c-fos response to intracerebroventricular (i.c.v.) NmU. METHODS: Binding affinity and functional potency of rat NmU was determined at human NMU1R and NMU2R. Expression of NMU1R and NMU2R mRNA in rat and human tissue was determined using semi-quantitative reverse-transcription polymerase chain reaction. In in-vivo studies, NmU was administered i.c.v. to male Sprague-Dawley rats, and changes in grooming, motor activity and pre-pulse inhibition (PPI) were assessed. In further studies, plasma endocrine hormones, [DOPAC + HVA]/[dopamine] and [5-HIAA]/[5-HT] ratios and levels of Fos-like immunoreactivity (FLI) were measured 20 min post-NmU (i.c.v.). RESULTS: NmU bound to NMU1R ( K(I), 0.11+/-0.02 nM) and NMU2R ( K(I), 0.21+/-0.05 nM) with equal affinity and was equally active at NMU1R (EC(50), 1.25+/-0.05 nM) and NMU2R (EC(50), 1.10+/-0.20 nM) in a functional assay. NMU2R mRNA expression was found at the highest levels in the CNS regions of both rat and human tissues. NMU1R mRNA expression was restricted to the periphery of both species with the exception of the rat amygdala. NmU caused a marked increase in grooming and motor activity but did not affect PPI. Further, NmU decreased plasma prolactin but did not affect levels of corticosterone, luteinising hormone or thyroid stimulating hormone. NmU elevated levels of 5-HT in the frontal cortex and hypothalamus, with decreased levels of its metabolites in the hippocampus and hypothalamus, but did not affect dopamine function. NmU markedly increased FLI in the nucleus accumbens, frontal cortex and central amygdala. CONCLUSIONS: These data provide further evidence for widespread roles for NmU and its receptors in the brain.
Asunto(s)
Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/metabolismo , Proteínas de la Membrana/agonistas , Proteínas de la Membrana/metabolismo , Neuropéptidos/administración & dosificación , Receptores de Neurotransmisores/agonistas , Receptores de Neurotransmisores/metabolismo , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Inyecciones Intraventriculares , Ratas , Ratas Sprague-Dawley , PorcinosRESUMEN
Neuromedin U (NmU) is a structurally highly conserved neuropeptide. It is ubiquitously distributed, with highest levels found in the gastrointestinal tract and pituitary. Originally isolated from porcine spinal cord, it has since been isolated and sequenced from several species. Amino acid alignment of NmU from different species reveals a high level of conservation, and particular features within its structure are important for bioactivity. Specifically, the C terminus, including a terminal asparagine-linked amidation, is essential for activity. The conservation of NmU across a wide range of species indicates a strong evolutionary pressure to conserve this peptide and points to its physiological significance. Despite this, the precise physiological and indeed pathophysiological roles of NmU have remained elusive. NmU was first isolated based on its ability to contract rat uterine smooth-muscle (hence the suffix "U") and has since been implicated in the regulation of smooth-muscle contraction, blood pressure and local blood flow, ion transport in the gut, stress responses, cancer, gastric acid secretion, pronociception, and feeding behavior. Two G-protein-coupled receptors for NmU have recently been cloned. These receptors are widespread throughout the body but have differential distributions suggesting diverse but specific roles for the receptor subtypes. Here we detail the isolation and characterization of NmU, describe the discovery, cloning, distribution, and structure of its two receptors, and outline its possible roles in both physiology and pathophysiology. Ultimately the development of receptor-specific ligands and the generation of animals in which the receptors have been selectively knocked out will hopefully reveal the true extent of the biological roles of NmU and suggest novel therapeutic indications for selective activation or blockade of either of its receptors.
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Proteínas de la Membrana/metabolismo , Neuropéptidos/metabolismo , Receptores de Neurotransmisores/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Neuropéptidos/química , Receptores de Neurotransmisores/fisiologíaRESUMEN
We have recently shown that UDP-glucose, and some related UDP-sugars, are potent agonists of the novel G protein-coupled receptor GPR105 (recently re-named P2Y(14)). GPR105 is widely expressed throughout many brain regions and peripheral tissues of human and rodents, and couples to a pertussis toxin-sensitive G protein. To further characterise the role of GPR105, we demonstrate by immunohistochemistry with receptor-specific antiserum that GPR105 protein is widely distributed throughout the post mortem human brain where it is localised to glial cells, and specifically co-localises with astrocytes. Using quantitative RT-PCR we also show that GPR105 mRNA exhibits a restricted expression profile in an array of human cell lines and primary cells, with prominent expression detected in immune cells including neutrophils, lymphocytes, and megakaryocytic cells. To investigate the G protein selectivity of GPR105, we used chimeric Galpha subunits (Galpha(qi5), Galpha(qo5), and Galpha(qs5)) and an intracellular Ca(2+) mobilisation assay to demonstrate that GPR105 couples to Galpha subunits of the G(i/o) family but not to G(s) family proteins or to endogenous G(q/11) proteins in HEK-293 cells. Finally, we show that expression of GPR105 mRNA in the rat brain is up-regulated by immunologic challenge with lipopolysaccharide. Based on these observations, we propose that G(i/o)-coupled GPR105 might play an important role in peripheral and neuroimmune function in response to extracellular UDP-sugars.
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Astrocitos/metabolismo , Encéfalo/metabolismo , Leucocitos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Inmunológicos/metabolismo , Receptores Purinérgicos P2/metabolismo , Animales , Astrocitos/inmunología , Encéfalo/inmunología , Línea Celular , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Glucosa/inmunología , Humanos , Inmunohistoquímica , Leucocitos/inmunología , Lipopolisacáridos/inmunología , Masculino , Neuroinmunomodulación/inmunología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Mensajero/metabolismo , Ratas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/inmunología , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/inmunología , Receptores Purinérgicos P2Y , Regulación hacia Arriba/inmunología , Uridina Difosfato/inmunologíaRESUMEN
Nicotinic acid has been used clinically for over 40 years in the treatment of dyslipidemia producing a desirable normalization of a range of cardiovascular risk factors, including a marked elevation of high density lipoprotein and a reduction in mortality. The precise mechanism of action of nicotinic acid is unknown, although it is believed that activation of a G(i)-G protein-coupled receptor may contribute. Utilizing available information on the tissue distribution of nicotinic acid receptors, we identified candidate orphan receptors. The selected orphan receptors were screened for responses to nicotinic acid, in an assay for activation of G(i)-G proteins. Here we describe the identification of the G protein-coupled receptor HM74 as a low affinity receptor for nicotinic acid. We then describe the subsequent identification of HM74A in follow-up bioinformatics searches and demonstrate that it acts as a high affinity receptor for nicotinic acid and other compounds with related pharmacology. The discovery of HM74A as a molecular target for nicotinic acid may facilitate the discovery of superior drug molecules to treat dyslipidemia.
Asunto(s)
Niacina/farmacología , Receptores Nicotínicos/química , Secuencia de Aminoácidos , Animales , Células CHO , Membrana Celular/metabolismo , Cricetinae , ADN Complementario/metabolismo , Bases de Datos como Asunto , Relación Dosis-Respuesta a Droga , Femenino , Furanos/farmacología , Humanos , Hiperlipidemias/metabolismo , Hipolipemiantes/farmacología , Concentración 50 Inhibidora , Masculino , Datos de Secuencia Molecular , Niacina/química , Oocitos/metabolismo , Unión Proteica , Pirazinas/farmacología , ARN Mensajero/metabolismo , Ratas , Receptores Nicotínicos/metabolismo , Homología de Secuencia de Aminoácido , Distribución Tisular , XenopusRESUMEN
GPR41 and GPR43 are related members of a homologous family of orphan G protein-coupled receptors that are tandemly encoded at a single chromosomal locus in both humans and mice. We identified the acetate anion as an agonist of human GPR43 during routine ligand bank screening in yeast. This activity was confirmed after transient transfection of GPR43 into mammalian cells using Ca(2+) mobilization and [(35)S]guanosine 5'-O-(3-thiotriphosphate) binding assays and by coexpression with GIRK G protein-regulated potassium channels in Xenopus laevis oocytes. Other short chain carboxylic acid anions such as formate, propionate, butyrate, and pentanoate also had agonist activity. GPR41 is related to GPR43 (52% similarity; 43% identity) and was activated by similar ligands but with differing specificity for carbon chain length, with pentanoate being the most potent agonist. A third family member, GPR42, is most likely a recent gene duplication of GPR41 and may be a pseudogene. GPR41 was expressed primarily in adipose tissue, whereas the highest levels of GPR43 were found in immune cells. The identity of the cognate physiological ligands for these receptors is not clear, although propionate is known to occur in vivo at high concentrations under certain pathophysiological conditions.
Asunto(s)
Ácidos Carboxílicos/farmacología , Propionatos/farmacología , Receptores de Superficie Celular/agonistas , Receptores Acoplados a Proteínas G , Secuencia de Aminoácidos , Animales , Cartilla de ADN , Humanos , Inmunohistoquímica , Datos de Secuencia Molecular , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMEN
GPR40 is a member of a subfamily of homologous G protein-coupled receptors that include GPR41 and GPR43 and that have no current function or ligand ascribed. Ligand fishing experiments in HEK293 cells expressing human GPR40 revealed that a range of saturated and unsaturated carboxylic acids with carbon chain lengths greater than six were able to induce an elevation of [Ca(2+)](i), measured using a fluorometric imaging plate reader. 5,8,11-Eicosatriynoic acid was the most potent fatty acid tested, with a pEC(50) of 5.7. G protein coupling of GPR40 was examined in Chinese hamster ovary cells expressing the G alpha(q/i)-responsive Gal4-Elk1 reporter system. Expression of human GPR40 led to a constitutive induction of luciferase activity, which was further increased by exposure of the cells to eicosatriynoic acid. Neither the constitutive nor ligand-mediated luciferase induction was inhibited by pertussis toxin treatment, suggesting that GPR40 was coupled to G alpha(q/11.) Expression analysis by quantitative reverse transcription-PCR showed that GPR40 was specifically expressed in brain and pancreas, with expression in rodent pancreas being localized to insulin-producing beta-cells. These data suggest that some of the physiological effects of fatty acids in pancreatic islets and brain may be mediated through a cell-surface receptor.
Asunto(s)
Ácidos Grasos/farmacología , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G , Animales , Secuencia de Bases , Calcio/metabolismo , Línea Celular , Clonación Molecular , Cricetinae , Cartilla de ADN , Ácidos Grasos/genética , Humanos , Hibridación in Situ , Luciferasas/genética , Datos de Secuencia Molecular , Receptores de Superficie Celular/genéticaRESUMEN
The superfamily of G protein-coupled receptors (GPCRs; 7TMs) is one of the largest families of genes identified in humans, and has a proven history of being an excellent source of drug targets. The near completion of the human genome sequencing project has allowed the identification of a plethora of sequences encoding "orphan" GPCRs--putative receptors whose natural ligand(s) remain to be discovered. In many cases, the level of sequence homology with known receptors is insufficient to be able to predict the natural ligand for these orphan receptors, although it is usually possible to determine the likely nature of the cognate ligand e.g. peptide, lipid, nucleotide etc. Deorphanizing these novel GPCRs and evaluating their biological function has become a major target of many of the major pharmaceutical companies as well as several academic groups. Since 1995 more than 50 ligands for orphan GPCRs have been discovered by using the orphan receptor as a biosensor and screening candidate compounds looking for a biological response (the so-called "reverse pharmacology" approach). Identification of the natural ligands for these receptors marks the beginning of the process of understanding the biology of these newly discovered signalling systems and the development of novel therapies targeted at them. This article will focus on the functional assays which have been used to discover ligands for orphan GPCRs.