Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 34
Filtrar
Más filtros

Banco de datos
Tipo del documento
Intervalo de año de publicación
1.
J Pharmacol Exp Ther ; 386(1): 80-92, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37142443

RESUMEN

Blocking chemokine receptor C-C chemoattractant cytokine (chemokine) receptor (CCR) 6-dependent T cell migration has therapeutic promise in inflammatory diseases. PF-07054894 is a novel CCR6 antagonist that blocked only CCR6, CCR7, and C-X-C chemoattractant cytokine (chemokine) receptor (CXCR) 2 in a ß-arrestin assay panel of 168 G protein-coupled receptors. Inhibition of CCR6-mediated human T cell chemotaxis by (R)-4-((2-(((1,4-Dimethyl-1H-pyrazol-3-yl)(1-methylcyclopentyl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxy-N,N-dimethylpicolinamide (PF-07054894) was insurmountable by CCR6 ligand, C-C motif ligand (CCL) 20. In contrast, blockade of CCR7-dependent chemotaxis in human T cells and CXCR2-dependent chemotaxis in human neutrophils by PF-07054894 were surmountable by CCL19 and C-X-C motif ligand 1, respectively. [3H]-PF-07054894 showed a slower dissociation rate for CCR6 than for CCR7 and CXCR2 suggesting that differences in chemotaxis patterns of inhibition could be attributable to offset kinetics. Consistent with this notion, an analog of PF-07054894 with fast dissociation rate showed surmountable inhibition of CCL20/CCR6 chemotaxis. Furthermore, pre-equilibration of T cells with PF-07054894 increased its inhibitory potency in CCL20/CCR6 chemotaxis by 10-fold. The functional selectivity of PF-07054894 for inhibition of CCR6 relative to CCR7 and CXCR2 is estimated to be at least 50- and 150-fold, respectively. When administered orally to naïve cynomolgus monkeys, PF-07054894 increased the frequency of CCR6+ peripheral blood T cells, suggesting that blockade of CCR6 inhibited homeostatic migration of T cells from blood to tissues. PF-07054894 inhibited interleukin-23-induced mouse skin ear swelling to a similar extent as genetic ablation of CCR6. PF-07054894 caused an increase in cell surface CCR6 in mouse and monkey B cells, which was recapitulated in mouse splenocytes in vitro. In conclusion, PF-07054894 is a potent and functionally selective CCR6 antagonist that blocks CCR6-mediated chemotaxis in vitro and in vivo. SIGNIFICANCE STATEMENT: The chemokine receptor, C-C chemoattractant cytokine (chemokine) receptor 6 (CCR6) plays a key role in the migration of pathogenic lymphocytes and dendritic cells into sites of inflammation. (R)-4-((2-(((1,4-Dimethyl-1H-pyrazol-3-yl)(1-methylcyclopentyl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxy-N,N-dimethylpicolinamide (PF-07054894) is a novel CCR6 small molecule antagonist that illustrates the importance of binding kinetics in achieving pharmacological potency and selectivity. Orally administered PF-07054894 blocks homeostatic and pathogenic functions of CCR6, suggesting that it is a promising therapeutic agent for the treatment of a variety of autoimmune and inflammatory diseases.


Asunto(s)
Quimiocinas CC , Interleucina-23 , Humanos , Animales , Ratones , Quimiocinas CC/genética , Receptores CCR7 , Ligandos , Linfocitos T , Inflamación , Receptores CCR6
2.
Toxicol Appl Pharmacol ; 473: 116582, 2023 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-37295732

RESUMEN

A high incidence of thymic lymphoma has been noted in mice deficient of retinoid-related orphan receptor γ2 (RORγ2), which is required for differentiation of naïve CD4+ T cells into TH17 cells. Using a RORγ homozygous knockout (KO) mouse model of thymic lymphoma, we characterized this tumor progression and investigated the utility of 5-hydroxymethylcytosine (5hmC) signatures as a non-invasive circulating biomarker for early prediction of malignancy. No evidence for malignancy was noted in the wild-type mice, while primary thymic lymphoma with multi-organ metastasis was observed microscopically in 97% of the homozygous RORγ KO mice. The severity of thymic lymphoma was not age-dependent in the KO mice of 2 to 4 months old. Differential enrichment of 5hmC in thymic DNA and plasma cell-free DNA (cfDNA) was compared across different stages of tumor progression. Random forest modeling of plasma cfDNA achieved good predictivity (AUC = 0.74) in distinguishing early non-metastatic thymic lymphoma compared to cancer-free controls, while perfect predictivity was achieved with advanced multi-organ metastatic disease (AUC = 1.00). Lymphoid-specific genes involved in thymocyte selection during T cell development (Themis, Tox) were differentially enriched in both plasma and thymic tissue. This could help in differentiating thymic lymphoma from other tumors commonly detected in rodent carcinogenicity studies used in pharmaceutical drug development to inform human malignancy risk. Overall, these results provide a proof-of-concept for using circulating cfDNA profiles in rodent carcinogenicity studies for early risk assessment of novel pharmaceutical targets.


Asunto(s)
Ácidos Nucleicos Libres de Células , Neoplasias , Animales , Humanos , Lactante , Ratones , Ácidos Nucleicos Libres de Células/genética , Ratones Noqueados , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares
3.
Toxicol Pathol ; 50(3): 280-293, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35128980

RESUMEN

Coronavirus disease 2019 (COVID-19) in humans has a wide range of presentations, ranging from asymptomatic or mild symptoms to severe illness. Suitable animal models mimicking varying degrees of clinical disease manifestations could expedite development of therapeutics and vaccines for COVID-19. Here we demonstrate that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection resulted in subclinical disease in rhesus macaques with mild pneumonia and clinical disease in Syrian hamsters with severe pneumonia. SARS-CoV-2 infection was confirmed by formalin-fixed, paraffin-embedded (FFPE) polymerase chain reaction (PCR), immunohistochemistry, or in situ hybridization. Replicating virus in the lungs was identified using in situ hybridization or virus plaque forming assays. Viral encephalitis, reported in some COVID-19 patients, was identified in one macaque and was confirmed with immunohistochemistry. There was no evidence of encephalitis in hamsters. Severity and distribution of lung inflammation were substantially more in hamsters compared with macaques and exhibited vascular changes and virus-induced cytopathic changes as seen in COVID-19 patients. Neither the hamster nor macaque models demonstrated evidence for multisystemic inflammatory syndrome (MIS). Data presented here demonstrate that macaques may be appropriate for mechanistic studies of mild asymptomatic COVID-19 pneumonia and COVID-19-associated encephalitis, whereas Syrian hamsters may be more suited to study severe COVID-19 pneumonia.


Asunto(s)
COVID-19 , Encefalitis , Animales , Vacunas contra la COVID-19 , Cricetinae , Modelos Animales de Enfermedad , Encefalitis/patología , Humanos , Pulmón/patología , Macaca mulatta , Mesocricetus , SARS-CoV-2
4.
BMC Genomics ; 21(1): 64, 2020 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-31959126

RESUMEN

BACKGROUND: The advent of Next Generation Sequencing has allowed transcriptomes to be profiled with unprecedented accuracy, but the high costs of full-length mRNA sequencing have posed a limit on the accessibility and scalability of the technology. To address this, we developed 3'Pool-seq: a simple, cost-effective, and scalable RNA-seq method that focuses sequencing to the 3'-end of mRNA. We drew from aspects of SMART-seq, Drop-seq, and TruSeq to implement an easy workflow, and optimized parameters such as input RNA concentrations, tagmentation conditions, and read depth specifically for bulk-RNA. RESULTS: Thorough optimization resulted in a protocol that takes less than 12 h to perform, does not require custom sequencing primers or instrumentation, and cuts over 90% of the costs associated with TruSeq, while still achieving accurate gene expression quantification (Pearson's correlation coefficient with ERCC theoretical concentration r = 0.96) and differential gene detection (ROC analysis of 3'Pool-seq compared to TruSeq AUC = 0.921). The 3'Pool-seq dual indexing scheme was further adapted for a 96-well plate format, and ERCC spike-ins were used to correct for potential row or column pooling effects. Transcriptional profiling of troglitazone and pioglitazone treatments at multiple doses and time points in HepG2 cells was then used to show how 3'Pool-seq could distinguish the two molecules based on their molecular signatures. CONCLUSIONS: 3'Pool-seq can accurately detect gene expression at a level that is on par with TruSeq, at one tenth of the total cost. Furthermore, its unprecedented TruSeq/Nextera hybrid indexing scheme and streamlined workflow can be applied in several different formats, including 96-well plates, which allows users to thoroughly evaluate biological systems under several conditions and timepoints. Care must be taken regarding experimental design and plate layout such that potential pooling effects can be accounted for and corrected. Lastly, further studies using multiple sets of ERCC spike-ins may be used to simulate differential gene expression in a system with known ground-state values.


Asunto(s)
RNA-Seq/métodos , Animales , Análisis Costo-Beneficio , Células Hep G2 , Humanos , Ratones , Pioglitazona/farmacología , RNA-Seq/economía , Transcriptoma/efectos de los fármacos , Troglitazona/farmacología
5.
J Neuroinflammation ; 15(1): 142, 2018 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-29759062

RESUMEN

BACKGROUND: Acute neurological insults caused by infection, systemic inflammation, ischemia, or traumatic injury are often associated with breakdown of the blood-brain barrier (BBB) followed by infiltration of peripheral immune cells, cytotoxic proteins, and water. BBB breakdown and extravasation of these peripheral components into the brain parenchyma result in inflammation, oxidative stress, edema, excitotoxicity, and neurodegeneration. These downstream consequences of BBB dysfunction can drive pathophysiological processes and play a substantial role in the morbidity and mortality of acute and chronic neurological insults, and contribute to long-term sequelae. Preserving or rescuing BBB integrity and homeostasis therefore represents a translational research area of high therapeutic potential. METHODS: Induction of general and localized BBB disruption in mice was carried out using systemic administration of LPS and focal photothrombotic ischemic insult, respectively, in the presence and absence of the monoacylglycerol lipase (MAGL) inhibitor, CPD-4645. The effects of CPD-4645 treatment were assessed by gene expression analysis performed on neurovascular-enriched brain fractions, cytokine and inflammatory mediator measurement, and functional assessment of BBB permeability. The mechanism of action of CPD-4645 was studied pharmacologically using inverse agonists/antagonists of the cannabinoid receptors CB1 and CB2. RESULTS: Here, we demonstrate that the neurovasculature exhibits a unique transcriptional signature following inflammatory insults, and pharmacological inhibition of MAGL using a newly characterized inhibitor rescues the transcriptional profile of brain vasculature and restores its functional homeostasis. This pronounced effect of MAGL inhibition on blood-brain barrier permeability is evident following both systemic inflammatory and localized ischemic insults. Mechanistically, the protective effects of the MAGL inhibitor are partially mediated by cannabinoid receptor signaling in the ischemic brain insult. CONCLUSIONS: Our results support considering MAGL inhibitors as potential therapeutics for BBB dysfunction and cerebral edema associated with inflammatory brain insults.


Asunto(s)
Ácidos Araquidónicos/antagonistas & inhibidores , Ácidos Araquidónicos/metabolismo , Barrera Hematoencefálica/metabolismo , Lesiones Encefálicas/tratamiento farmacológico , Lesiones Encefálicas/metabolismo , Permeabilidad Capilar/fisiología , Endocannabinoides/antagonistas & inhibidores , Endocannabinoides/metabolismo , Glicéridos/antagonistas & inhibidores , Glicéridos/metabolismo , Animales , Barrera Hematoencefálica/efectos de los fármacos , Lesiones Encefálicas/inducido químicamente , Permeabilidad Capilar/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Hidrólisis/efectos de los fármacos , Lipopolisacáridos , Masculino , Ratones , Ratones Endogámicos C57BL , Monoacilglicerol Lipasas/antagonistas & inhibidores , Monoacilglicerol Lipasas/metabolismo
6.
Neurobiol Dis ; 77: 220-7, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25796564

RESUMEN

Brain-derived neurotrophic factor (BDNF) signaling is integral to a range of neural functions, including synaptic plasticity and exhibits activity-dependent regulation of expression. As altered BDNF signaling has been implicated in multiple psychiatric diseases, here we report a quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNAs encoding TrkB, total BDNF, and the four most abundant BDNF transcripts (I, IIc, IV, and VI) in postmortem tissue from matched tetrads of subjects with schizophrenia, bipolar disorder, or major depressive disorder (MDD) and healthy comparison subjects. In all three regions examined, dorsolateral prefrontal cortex (DLPFC), associative striatum and hippocampus, total BDNF mRNA levels did not differ in any disease state. In DLPFC, BDNF IIc was significantly lower in schizophrenia relative to healthy comparison subjects. In hippocampus, BDNF I, IIc, and VI were lower in subjects with both schizophrenia and bipolar disorder relative to comparison subjects. In striatum, TrkB mRNA was lower in bipolar disorder and MDD, while BDNF IIc was elevated in MDD, relative to comparison subjects. These data highlight potential alterations in BDNF signaling in the corticohippocampal circuit in schizophrenia, and within the striatum in mood disorders. Novel therapies aimed at improving BDNF-TrkB signaling may therefore have potential to impact on a range of psychiatric disorders.


Asunto(s)
Trastorno Bipolar/patología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Encéfalo/metabolismo , Trastorno Depresivo Mayor/patología , Glicoproteínas de Membrana/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Esquizofrenia/patología , Adulto , Factor Neurotrófico Derivado del Encéfalo/genética , Cuerpo Estriado/metabolismo , Femenino , Hipocampo/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Corteza Prefrontal/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor trkB
7.
Proc Natl Acad Sci U S A ; 109(8): 3125-30, 2012 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-22315408

RESUMEN

Schizophrenia is characterized by affective, cognitive, neuromorphological, and molecular abnormalities that may have a neurodevelopmental origin. MicroRNAs (miRNAs) are small noncoding RNA sequences critical to neurodevelopment and adult neuronal processes by coordinating the activity of multiple genes within biological networks. We examined the expression of 854 miRNAs in prefrontal cortical tissue from 100 control, schizophrenic, and bipolar subjects. The cyclic AMP-responsive element binding- and NMDA-regulated microRNA miR-132 was significantly down-regulated in both the schizophrenic discovery cohort and a second, independent set of schizophrenic subjects. Analysis of miR-132 target gene expression in schizophrenia gene-expression microarrays identified 26 genes up-regulated in schizophrenia subjects. Consistent with NMDA-mediated hypofunction observed in schizophrenic subjects, administration of an NMDA antagonist to adult mice results in miR-132 down-regulation in the prefrontal cortex. Furthermore, miR-132 expression in the murine prefrontal cortex exhibits significant developmental regulation and overlaps with critical neurodevelopmental processes during adolescence. Adult prefrontal expression of miR-132 can be down-regulated by pharmacologic inhibition of NMDA receptor signaling during a brief postnatal period. Several key genes, including DNMT3A, GATA2, and DPYSL3, are regulated by miR-132 and exhibited altered expression either during normal neurodevelopment or in tissue from adult schizophrenic subjects. Our data suggest miR-132 dysregulation and subsequent abnormal expression of miR-132 target genes contribute to the neurodevelopmental and neuromorphological pathologies present in schizophrenia.


Asunto(s)
Encéfalo/crecimiento & desarrollo , Encéfalo/fisiopatología , Regulación de la Expresión Génica , MicroARNs/genética , Esquizofrenia/genética , Esquizofrenia/fisiopatología , Adulto , Animales , Antipsicóticos/farmacología , Antipsicóticos/uso terapéutico , Trastorno Bipolar/tratamiento farmacológico , Trastorno Bipolar/genética , Trastorno Bipolar/fisiopatología , Encéfalo/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Corteza Cerebral/fisiopatología , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , Bases de Datos Genéticas , Demografía , Modelos Animales de Enfermedad , Factor de Transcripción GATA2/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Ratones , MicroARNs/metabolismo , Proteínas Musculares/metabolismo , N-Metilaspartato/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Sistemas de Lectura Abierta/genética , Reacción en Cadena de la Polimerasa , Ratas , Receptores de N-Metil-D-Aspartato/metabolismo , Reproducibilidad de los Resultados , Esquizofrenia/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos
8.
Hum Gene Ther ; 35(15-16): 527-542, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-39049734

RESUMEN

Regulators and industry are actively seeking improvements and alternatives to current models and approaches to evaluate potential carcinogenicity of gene therapies (GTs). A meeting of invited experts was organized by NC3Rs/UKEMS (London, March 2023) to discuss this topic. This article describes the consensus reached among delegates on the definition of vector genotoxicity, sources of uncertainty, suitable toxicological endpoints for genotoxic assessment of GTs, and future research needs. The collected recommendations should inform the further development of regulatory guidelines for the nonclinical toxicological assessment of GT products.


Asunto(s)
Terapia Genética , Terapia Genética/efectos adversos , Terapia Genética/métodos , Humanos , Factores de Riesgo , Animales , Vectores Genéticos/efectos adversos , Consenso , Neoplasias/terapia , Neoplasias/genética , Medición de Riesgo
9.
Mol Ther Methods Clin Dev ; 29: 395-405, 2023 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-37251978

RESUMEN

Many current gene therapy targets use recombinant adeno-associated virus (AAV). The majority of delivered AAV therapeutics persist as episomes, separate from host DNA, yet some viral DNA can integrate into host DNA in different proportions and at genomic locations. The potential for viral integration leading to oncogenic transformation has led regulatory agencies to require investigation into AAV integration events following gene therapy in preclinical species. In the present study, tissues were collected from cynomolgus monkeys and mice 6 and 8 weeks, respectively, following administration of an AAV vector delivering transgene cargo. We compared three different next-generation sequencing approaches (shearing extension primer tag selection ligation-mediated PCR, targeted enrichment sequencing [TES], and whole-genome sequencing) to contrast the specificity, scope, and frequency of integration detected by each method. All three methods detected dose-dependent insertions with a limited number of hotspots and expanded clones. While the functional outcome was similar for all three methods, TES was the most cost-effective and comprehensive method of detecting viral integration. Our findings aim to inform the direction of molecular efforts to ensure a thorough hazard assessment of AAV viral integration in our preclinical gene therapy studies.

10.
Biomarkers ; 17(6): 524-31, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22672085

RESUMEN

Adult rats were treated acutely with peripheral kainic acid (KA), and changes in brain-derived neurotrophic factor (BDNF) mRNA and protein were tracked over time across multiple brain regions. Despite robust elevation in both mRNA and protein in multiple brain regions, plasma BDNF was unchanged and cerebrospinal fluid (CSF) BDNF levels remained undetectable. Primary neurons were then treated with KA. BDNF was similarly elevated within neurons, but was undetectable in neuronal media. Thus, while deficits in BDNF signaling have been implicated in a number of diseases, these data suggest that extracellular concentrations of BDNF may not be a facile biomarker for changes in neurons.


Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/sangre , Factor Neurotrófico Derivado del Encéfalo/genética , Expresión Génica , Hipocampo/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Factor Neurotrófico Derivado del Encéfalo/líquido cefalorraquídeo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Células Cultivadas , Plexo Coroideo/patología , Hipocampo/patología , Humanos , Ácido Kaínico , L-Lactato Deshidrogenasa/metabolismo , Masculino , Neuronas/enzimología , Neuronas/metabolismo , Cultivo Primario de Células , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Convulsiones/sangre , Convulsiones/líquido cefalorraquídeo , Convulsiones/inducido químicamente
11.
Hum Gene Ther ; 33(3-4): 175-187, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34931542

RESUMEN

Recombinant adeno-associated viruses (AAVs) have emerged as promising vectors for human gene therapy, but some variants have induced severe toxicity in Rhesus monkeys and piglets following high-dose intravenous (IV) administration. To characterize biodistribution, transduction, and toxicity among common preclinical species, an AAV9 neurotropic variant expressing the survival motor neuron 1 (SMN1) transgene (AAV-PHP.B-CBh-SMN1) was administered by IV bolus injection to Wistar Han rats and cynomolgus monkeys at doses of 2 × 1013, 5 × 1013, or 1 × 1014 vg/kg. A dose-dependent degeneration/necrosis of neurons without clinical manifestations occurred in dorsal root ganglia (DRGs) and sympathetic thoracic ganglia in rats, while liver injury was not observed in rats. In monkeys, one male at 5 × 1013 vg/kg was found dead on day 4. Clinical pathology data on days 3 and/or 4 at all doses suggested liver dysfunction and coagulation disorders, which led to study termination. Histologic evaluation of the liver in monkeys showed hepatocyte degeneration and necrosis without inflammatory cell infiltrates or intravascular thrombi, suggesting that hepatocyte injury is a direct effect of the vector following hepatocyte transduction. In situ hybridization demonstrated a dose-dependent expression of SMN1 transgene mRNA in the cytoplasm and DNA in the nucleus of periportal to panlobular hepatocytes, while quantitative polymerase chain reaction confirmed the dose-dependent presence of SMN1 transgene mRNA and DNA in monkeys. Monkeys produced a much greater amount of transgene mRNA compared with rats. In DRGs, neuronal degeneration/necrosis and accompanying findings were observed in monkeys as early as 4 days after test article administration. The present results show sensory neuron toxicity following IV delivery of AAV vectors at high doses with an early onset in Macaca fascicularis and after 1 month in rats, and suggest adding the autonomic system in the watch list for preclinical and clinical studies. Our data also suggest that the rat may be useful for evaluating the potential DRG toxicity of AAV vectors, while acute hepatic toxicity associated with coagulation disorders appears to be highly species-dependent.


Asunto(s)
Dependovirus , Vectores Genéticos , Animales , Dependovirus/genética , Vectores Genéticos/genética , Macaca fascicularis , Masculino , Neuronas Motoras , Necrosis , ARN Mensajero , Ratas , Ratas Wistar , Porcinos , Distribución Tisular , Transducción Genética
12.
J Pharmacol Exp Ther ; 336(1): 64-76, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20923867

RESUMEN

Inhibition of phosphodiesterase 10A (PDE10A) promotes cyclic nucleotide signaling, increases striatal activation, and decreases behavioral activity. Enhanced cyclic nucleotide signaling is a well established route to producing changes in gene expression. We hypothesized that chronic suppression of PDE10A activity would have significant effects on gene expression in the striatum. A comparison of the expression profile of PDE10A knockout (KO) mice and wild-type mice after chronic PDE10A inhibition revealed altered expression of 19 overlapping genes with few significant changes outside the striatum or after administration of a PDE10A inhibitor to KO animals. Chronic inhibition of PDE10A produced up-regulation of mRNAs encoding genes that included prodynorphin, synaptotagmin10, phosphodiesterase 1C, glutamate decarboxylase 1, and diacylglycerol O-acyltransferase and a down-regulation of mRNAs encoding choline acetyltransferase and Kv1.6, suggesting long-term suppression of the PDE10A enzyme is consistent with altered striatal excitability and potential utility as a antipsychotic therapy. In addition, up-regulation of mRNAs encoding histone 3 (H3) and down-regulation of histone deacetylase 4, follistatin, and claspin mRNAs suggests activation of molecular cascades capable of neuroprotection. We used lentiviral delivery of cAMP response element (CRE)-luciferase reporter constructs into the striatum and live animal imaging of 2-{4-[-pyridin-4-yl-1-(2,2,2-trifluoro-ethyl)-1H-pyrazol-3-yl]-phenoxymethyl}-quinoline succinic acid (TP-10)-induced luciferase activity to further demonstrate PDE10 inhibition results in CRE-mediated transcription. Consistent with potential neuroprotective cascades, we also demonstrate phosphorylation of mitogen- and stress-activated kinase 1 and H3 in vivo after TP-10 treatment. The observed changes in signaling and gene expression are predicted to provide neuroprotective effects in models of Huntington's disease.


Asunto(s)
Cuerpo Estriado/enzimología , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/enzimología , Neurotransmisores/antagonistas & inhibidores , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Enfermedad de Huntington/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Neurotransmisores/biosíntesis , Neurotransmisores/genética , Inhibidores de Fosfodiesterasa/uso terapéutico , Hidrolasas Diéster Fosfóricas/deficiencia , Hidrolasas Diéster Fosfóricas/genética , Pirazoles/farmacología , Pirazoles/uso terapéutico , Quinolinas/farmacología , Quinolinas/uso terapéutico , Transducción de Señal/genética
13.
Bioorg Med Chem Lett ; 21(9): 2631-6, 2011 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-21269825

RESUMEN

The synthesis and structure-activity relationship (SAR) of a novel series of di-substituted imidazoles, derived from modification of DAPT, are described. Subsequent optimization led to identification of a highly potent series of inhibitors that contain a ß-amine in the imidazole side-chain resulting in a robust in vivo reduction of plasma and brain Aß in guinea pigs. The therapeutic index between Aß reductions and changes in B-cell populations were studied for compound 10 h.


Asunto(s)
Enfermedad de Alzheimer , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Imidazoles/síntesis química , Imidazoles/farmacología , Aminación/efectos de los fármacos , Péptidos beta-Amiloides/sangre , Péptidos beta-Amiloides/metabolismo , Animales , Bioensayo , Diamida/síntesis química , Diamida/química , Diamida/farmacología , Inhibidores Enzimáticos/química , Cobayas , Células HeLa , Humanos , Imidazoles/química , Concentración 50 Inhibidora , Estructura Molecular , Relación Estructura-Actividad
14.
Bioorg Med Chem Lett ; 21(9): 2637-40, 2011 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-21269827

RESUMEN

A novel series of tetralin containing amino imidazoles, derived from modification of the corresponding phenyl acetic acid derivatives is described. Replacement of the amide led to identification of a potent series of tetralin-amino imidazoles with robust central efficacy. The reduction of brain Aß in guinea pigs in the absence of changes in B-cells suggested a potential therapeutic index with respect to APP processing compared with biomarkers of notch related toxicity. Optimization of the FTOC to plasma concentrations at the brain Aß EC(50) lead to the identification of compound 14f (PF-3084014) which was selected for clinical development.


Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Tetrahidronaftalenos/síntesis química , Tetrahidronaftalenos/farmacología , Valina/análogos & derivados , Animales , Bioensayo , Diseño de Fármacos , Inhibidores Enzimáticos/química , Cobayas , Imidazoles/síntesis química , Imidazoles/química , Imidazoles/farmacología , Concentración 50 Inhibidora , Estructura Molecular , Relación Estructura-Actividad , Tetrahidronaftalenos/química , Valina/síntesis química , Valina/química , Valina/farmacología
15.
Toxicol In Vitro ; 70: 105012, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33049313

RESUMEN

Alveolar type II (ATII) epithelial cells contain lamellar bodies (LBs) which synthesize and store lung surfactants. In animals, the inhibition or knockout of leucine-rich repeat kinase 2 (LRRK2) causes abnormal enlargement of LBs in ATII cells. This effect of LRRK2 inhibition in lung is largely accepted as being mediated directly through blocking of the kinase function; however, downstream consequences in the lung remain unknown. In this work we established an in vitro alveolar epithelial cell (AEC) model that recapitulates the in vivo phenotype of ATII cells and developed an assay to quantify changes in LB size in response to LRRK2 inhibitors. Culture of primary human AECs at the air-liquid interface on matrigel and collagen-coated transwell inserts in the presence of growth factors promoted the LB formation and apical microvilli and induced expression of LRRK2 and ATII cell markers. Treatment with a selective LRRK2 inhibitor resulted in pharmacological reduction of phospho-LRRK2 and a significant increase in LB size; effects previously reported in lungs of non-human primates treated with LRRK2 inhibitor. In summary, our human in vitro AEC model recapitulates the abnormal lung findings observed in LRRK2-perturbed animals and holds the potential for expanding current understanding of LRRK2 function in the lung.


Asunto(s)
Células Epiteliales Alveolares/patología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/antagonistas & inhibidores , Modelos Biológicos , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenocarcinoma del Pulmón/metabolismo , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/ultraestructura , Células Cultivadas , Evaluación Preclínica de Medicamentos , Expresión Génica , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Neoplasias Pulmonares/metabolismo , Proteína C Asociada a Surfactante Pulmonar/metabolismo
16.
Transl Psychiatry ; 11(1): 136, 2021 02 19.
Artículo en Inglés | MEDLINE | ID: mdl-33608496

RESUMEN

Schizophrenia is a complex and heterogenous disease that presents with abnormalities in glutamate signaling and altered immune and inflammatory signals. Genome-wide association studies have indicated specific genes and pathways that may contribute to schizophrenia. We assessed the impact of the functional missense variant SLC39A8 (ZIP8)-A391T (ZIP8A391T) on zinc transport, glutamate signaling, and the neuroinflammatory response. The ZIP8A391T mutation resulted in reduced zinc transport into the cell, suggesting a loss in the tight control of zinc in the synaptic cleft. Electrophysiological recordings from perturbed neurons revealed a significant reduction in NMDA- and AMPA-mediated spontaneous EPSCs (sEPSCs) and a reduction in GluN2A and GluA1/2/3 receptor surface expression. All phenotypes were rescued by re-expression of wild-type ZIP8 (ZIP8WT) or application of the membrane-impermeable zinc chelator ZX1. ZIP8 reduction also resulted in decreased BBB integrity, increased IL-6/IL-1ß protein expression, and increased NFκB following TNFα stimulation, indicating that ZIP8 loss-of-function may exacerbate immune and inflammatory signals. Together, our findings demonstrate that the A391T missense mutation results in alterations in glutamate and immune function and provide novel therapeutic targets relevant to schizophrenia.


Asunto(s)
Proteínas de Transporte de Catión , Esquizofrenia , Alelos , Proteínas de Transporte de Catión/genética , Estudio de Asociación del Genoma Completo , Ácido Glutámico , Humanos , Inmunidad Innata , Receptores de Glutamato , Esquizofrenia/genética
17.
J Pharmacol Exp Ther ; 334(1): 269-77, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-20363853

RESUMEN

PF-3084014 [(S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-3-ylamino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide] is a novel gamma-secretase inhibitor that reduces amyloid-beta (Abeta) production with an in vitro IC(50) of 1.2 nM (whole-cell assay) to 6.2 nM (cell-free assay). This compound inhibits Notch-related T- and B-cell maturation in an in vitro thymocyte assay with an EC(50) of 2.1 microM. A single acute dose showed dose-dependent reduction in brain, cerebrospinal fluid (CSF), and plasma Abeta in Tg2576 mice as measured by enzyme-linked immunosorbent assay and immunoprecipitation (IP)/mass spectrometry (MS). Guinea pigs were dosed with PF-3084014 for 5 days via osmotic minipump at 0.03 to 3 mg/kg/day and exhibited dose-dependent reduction in brain, CSF, and plasma Abeta. To further characterize Abeta dynamics in brain, CSF, and plasma in relation to drug exposure and Notch-related toxicities, guinea pigs were dosed with 0.03 to 10 mg/kg PF-3084014, and tissues were collected at regular intervals from 0.75 to 30 h after dose. Brain, CSF, and plasma all exhibited dose-dependent reductions in Abeta, and the magnitude and duration of Abeta lowering exceeded those of the reductions in B-cell endpoints. Other gamma-secretase inhibitors have shown high potency at elevating Abeta in the conditioned media of whole cells and the plasma of multiple animal models and humans. Such potentiation was not observed with PF-3084014. IP/MS analysis, however, revealed dose-dependent increases in Abeta11-40 and Abeta1-43 at doses that potently inhibited Abeta1-40 and Abeta1-42. PF-3084014, like previously described gamma-secretase inhibitors, preferentially reduced Abeta1-40 relative to Abeta1-42. Potency at Abeta relative to Notch-related endpoints in vitro and in vivo suggests that a therapeutic index can be achieved with this compound.


Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/farmacocinética , Tetrahidronaftalenos/farmacología , Tetrahidronaftalenos/farmacocinética , Valina/análogos & derivados , Animales , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Línea Celular , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/efectos adversos , Inhibidores Enzimáticos/química , Escherichia coli/genética , Femenino , Cobayas , Humanos , Recuento de Linfocitos , Masculino , Ratones , Ratones Endogámicos , Estructura Molecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Bazo/citología , Bazo/efectos de los fármacos , Tetrahidronaftalenos/efectos adversos , Tetrahidronaftalenos/química , Distribución Tisular , Transfección , Valina/efectos adversos , Valina/química , Valina/farmacocinética , Valina/farmacología
18.
PLoS One ; 15(6): e0233895, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32497062

RESUMEN

Deficits in fast-spiking inhibitory interneurons (FSINs) within the dorsolateral prefrontal cortex (dlPFC) are hypothesized to underlie cognitive impairment associated with schizophrenia. Though representing a minority of interneurons, this key cell type coordinates broad neural network gamma-frequency oscillations, associated with cognition and cognitive flexibility. Here we report expression of GluN2D mRNA selectively in parvalbumin positive cells of human postmortem dlPFC tissue, but not pyramidal neurons, with little to no GluN2C expression in either cell type. In acute murine mPFC slices the GluN2C/D selective positive allosteric modulator (PAM), CIQ(+), increased the intrinsic excitability as well as enhanced NMDAR-mediated EPSCs onto FSINs. This increase in intrinsic excitability with GluN2C/D PAM was also observed in the Dlx 5/6+/- FSIN developmental deficit model with reported FSIN hypoexcitability. Together these data speak to selective modulation of FSINs by a GluN2D PAM, providing a potential mechanism to counter the FSIN-deficit seen in schizophrenia.


Asunto(s)
Interneuronas/metabolismo , Parvalbúminas/metabolismo , Corteza Prefrontal/fisiología , Receptores de N-Metil-D-Aspartato/metabolismo , Potenciales de Acción , Adulto , Animales , Femenino , Expresión Génica , Humanos , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Inhibición Neural , Células Piramidales/metabolismo , ARN Mensajero/genética , Receptores de N-Metil-D-Aspartato/genética
19.
J Parkinsons Dis ; 10(2): 623-629, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32007961

RESUMEN

The phosphorylated form of LRRK2, pS935 LRRK2, has been proposed as a target modulation biomarker for LRRK2 inhibitors. The primary aim of the study was to characterize and qualify this biomarker for therapeutic trials of LRRK2 inhibitors in Parkinson's disease (PD). To this end, analytically validated assays were used to monitor levels of pS935 LRRK2 and total LRRK2 in peripheral blood mononuclear cells (PBMCs) from the following donor groups: healthy controls, idiopathic PD, and G2019S carriers with and without PD. Neither analyte correlated with age, gender, or disease severity. While total LRRK2 levels were similar across the four groups, there was a significant reduction in pS935 LRRK2 levels in disease-manifesting G2019S carriers compared to idiopathic PD. In aggregate, these data indicate that phosphorylation of LRRK2 at S935 may reflect a state marker for G2019S LRRK2-driven PD, the underlying biology for which requires investigation in future studies. This study also provides critical foundational data to inform the integration of pS935 and total LRRK2 levels as biomarkers in therapeutic trials of LRRK2 kinase inhibitors.


Asunto(s)
Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Leucocitos Mononucleares/metabolismo , Enfermedad de Parkinson/sangre , Enfermedad de Parkinson/genética , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Femenino , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Masculino , Persona de Mediana Edad , Fosforilación/fisiología
20.
Nat Neurosci ; 23(11): 1352-1364, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-33097921

RESUMEN

The mechanisms by which prenatal immune activation increase the risk for neuropsychiatric disorders are unclear. Here, we generated developmental cortical interneurons (cINs)-which are known to be affected in schizophrenia (SCZ) when matured-from induced pluripotent stem cells (iPSCs) derived from healthy controls (HCs) and individuals with SCZ and co-cultured them with or without activated microglia. Co-culture with activated microglia disturbed metabolic pathways, as indicated by unbiased transcriptome analyses, and impaired mitochondrial function, arborization, synapse formation and synaptic GABA release. Deficits in mitochondrial function and arborization were reversed by alpha lipoic acid and acetyl-L-carnitine treatments, which boost mitochondrial function. Notably, activated-microglia-conditioned medium altered metabolism in cINs and iPSCs from HCs but not in iPSCs from individuals with SCZ or in glutamatergic neurons. After removal of activated-microglia-conditioned medium, SCZ cINs but not HC cINs showed prolonged metabolic deficits, which suggests that there is an interaction between SCZ genetic backgrounds and environmental risk factors.


Asunto(s)
Corteza Cerebral/metabolismo , Interneuronas/metabolismo , Microglía/metabolismo , Esquizofrenia/metabolismo , Adulto , Técnicas de Cocultivo , Encefalitis/metabolismo , Expresión Génica , Ácido Glutámico/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Masculino , Persona de Mediana Edad , Mitocondrias/metabolismo , Adulto Joven , Ácido gamma-Aminobutírico/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA