RESUMO
Deregulation of the Wnt/ß-catenin signaling pathway is associated with many serious disorders, including cancer and Alzheimer's disease. The pivotal player is ß-catenin, which avoids degradation after activation of the pathway and is translocated to the nucleus, where it interacts with TCF/LEF transcription factors and induces expression of genes involved in cell cycle and apoptosis regulation. The identification of small molecules that may affect Wnt/ß-catenin signaling remains an important target during the development of novel therapies. We used the TCF/LEF lentiviral vector and the Wnt-independent H1703 cell line to develop a luciferase reporter-based cell assay for screening of the Wnt/ß-catenin pathway modulators. Following the optimization of cell density, concentration of activator, and stimulation time, the reporter system was validated by demonstrating its specific and dose-dependent response to several established modulators of Wnt/ß-catenin signaling such as Wnt3a, small interfering RNA (siRNA) against ß-catenin, glycogen synthase kinase 3 (GSK-3), and ß-catenin/TCF transcription complex inhibitors. Two pilot screens of inhibitors and activators of Wnt/ß-catenin signaling identified potential novel modulators of this pathway. Our findings suggest that the H1703-7TFP assay constitutes a suitable model of low background and high sensitivity for the low- and high-scale screening of the Wnt/ß-catenin pathway modulators.
Assuntos
Bioensaio/métodos , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt3A/metabolismo , beta Catenina/metabolismo , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos/métodos , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Fatores de Transcrição TCF/genética , Fatores de Transcrição TCF/metabolismo , Proteína Wnt3A/genética , beta Catenina/genéticaRESUMO
Pur-alpha (Purα) plays an important role in a variety of cellular processes including transcriptional regulation, cell proliferation and oncogenic transformation. To better understand the role of Purα in the developing and mature brain, we generated Purα-deficient mice, which we were able to raise to the age of six months. Purα(-/-) mice were born with no obvious pathological condition. We obtained convincing evidence that lack of Purα prolongs the postnatal proliferation of neuronal precursor cells both in the hippocampus and in the cerebellum, however, without affecting the overall number of postmitotic neurons. Independent of these findings, we observed alterations in the expression and distribution of the dendritic protein MAP2, the translation of which has been proposed previously to be Purα-dependent. At the age of 2 weeks, Purα(-/-) mice generated a continuous tremor which persisted throughout lifetime. Finally, adult Purα(-/-) mice displayed a megalencephaly and histopathological findings including axonal swellings and hyperphosphorylation of neurofilaments. Our studies underline the importance of Purα in the proliferation of neuronal precursor cells during postnatal brain development and suggest a role for Purα in the regulation of the expression and cellular distribution of dendritic and axonal proteins. Since recent studies implicate a link between Purα and the fragile X tremor/ataxia syndrome, our Purα(-/-) mouse model will provide new opportunities for understanding the mechanisms of neurodegeneration.
Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/patologia , Proteínas de Ligação a DNA/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Animais , Axônios/metabolismo , Química Encefálica , Proliferação de Células , Cerebelo/citologia , Cerebelo/crescimento & desenvolvimento , Cerebelo/patologia , Cérebro/crescimento & desenvolvimento , Cérebro/patologia , Proteínas de Ligação a DNA/genética , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Hipertrofia , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/análise , Proteínas do Tecido Nervoso/genética , Proteínas de Neurofilamentos/metabolismo , FosforilaçãoRESUMO
Since the release of ICH E6(R2), multiple efforts have been made to interpret the requirements and suggest ways of implementing quality tolerance limits (QTLs) alongside existing risk-based quality management methodologies. While these efforts have contributed positively to developing a common understanding of QTLs, some uncertainty remains regarding implementable approaches. In this article, we review the approaches taken by some leading biopharmaceutical companies, offering recommendations for how to make QTLs most effective, what makes them ineffective, and several case studies to illustrate these concepts. This includes how best to choose QTL parameters and thresholds for a given study, how to differentiate QTLs from key risk indicators, and how QTLs relate to critical-to-quality factors and the statistical design of the trials.
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Produtos Biológicos , Locos de Características Quantitativas , Gestão de RiscosRESUMO
Background: Phosphodiesterase 10A (PDE10A) is expressed almost exclusively in the striatum and its inhibition is suggested to offer potential treatment in disorders associated with basal ganglia. We evaluated the selectivity, cytotoxicity, genotoxicity, pharmacokinetics and potential adverse effects of a novel PDE10A inhibitor, CPL500036, in vivo. Methods: The potency of CPL500036 was demonstrated by microfluidic technology, and selectivity was investigated in a radioligand binding assay against 44 targets. Cardiotoxicity in vitro was evaluated in human ether-a-go-go related gene (hERG)-potassium channel-overexpressing cells by the patch-clamp method and by assessing key parameters in 3D cardiac spheroids. Cytotoxicity was determined in H1299, HepG2 and SH-SY5Y cell lines. The Ames test was used for genotoxicity analyses. During in vivo studies, CPL500036 was administered by oral gavage. CPL500036 exposure were determined by liquid chromatography-tandem mass spectrometry and plasma protein binding was assessed. The bar test was employed to assess catalepsy. Prolactin and glucose levels in rat blood were measured by ELISAs and glucometers, respectively. Cardiovascular safety in vivo was investigated in dogs using a telemetry method. Results: CPL500036 inhibited PDE10A at an IC50 of 1 nM, and interacted only with the muscarinic M2 receptor as a negative allosteric modulator with an IC50 of 9.2 µM. Despite inhibiting hERG tail current at an IC25 of 3.2 µM, cardiovascular adverse effects were not observed in human cardiac 3D spheroids or in vivo. Cytotoxicity in vitro was observed only at > 60 µM and genotoxicity was not recorded during the Ames test. CPL500036 presented good bioavailability and penetration into the brain. CPL500036 elicited catalepsy at 0.6 mg/kg, but hyperprolactinemia or hyperglycemic effects were not observed in doses up to 3 mg/kg. Conclusion: CPL500036 is a potent, selective and orally bioavailable PDE10A inhibitor with a good safety profile distinct from marketed antipsychotics. CPL500036 may be a compelling drug candidate.
RESUMO
The International Council for Harmonisation (ICH) E6(R2) (International Council for Harmonisation (ICH). ICH harmonised guideline: integrated addendum to ICH E6(R1): guideline for good clinical practice E6(R2). 2016. https://database.ich.org/sites/default/files/E6_R2_Addendum.pdf . Accessed 5 Dec 2019) introduced Quality Tolerance Limits (QTLs) to the industry, and in doing so, modernized quality control for clinical trials. QTLs provide measured feedback on clinical trial parameters previously only used by statistical and clinical functions to track trial progress toward endpoints. Elevating these measures as part of the Quality Management System (QMS) provides greater visibility across clinical trial functions and the enterprise as well as to measures that are important indicators of the state of participant protection and reliability of trial results. In support of this new requirement, TransCelerate developed a framework to guide industry sponsors and their agents in implementing QTLs. This QTL Framework is intended to aid industry's ability to improve the quality of clinical research through the implementation of QTLs in a way that helps protect trial participants and reliability of trial results while meeting Health Authority (HA) expectations. The framework is intended to maximize efficiency and minimize confusion in the implementation of QTLs. The framework includes proposed approaches for implementation of QTLs for a clinical trial as defined in Section 5.0.4 and 5.0.7 of ICH E6(R2) (International Council for Harmonisation (ICH). ICH harmonised guideline: integrated addendum to ICH E6(R1): guideline for good clinical practice E6(R2). 2016. https://database.ich.org/sites/default/files/E6_R2_Addendum.pdf . Accessed 5 Dec 2019) and considerations for setting thresholds.
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Ensaios Clínicos como Assunto , Humanos , Reprodutibilidade dos TestesRESUMO
Mutations in presenilin 1 (PS1), which are the major cause of familial Alzheimer's disease (FAD), are involved in perturbations of cellular Ca2+ homeostasis. Attenuation of capacitative Ca2+ entry (CCE) is the most often observed alteration of Ca2+ homeostasis in cells bearing FAD PS1 mutations. However, molecular mechanisms underlying this CCE impairment remains elusive. We demonstrate that cellular levels of STIM1 and STIM2 proteins, which are key players in CCE, depend on presenilins. We found increased level of STIM1 and decreased level of STIM2 proteins in mouse embryonic fibroblasts lacking presenilins. Fura-2 ratiometric assays revealed that CCE is enhanced in these cells after Ca2+ stores depletion by thapsigargin treatment. In turn, overexpression of PS1 with FAD mutations in HEK293 cells led to an attenuation of CCE. Although, no changes in STIM protein levels were observed in these HEK293 cells, FAD mutations in endogenous PS1 in human B lymphocytes resulted in a decreased expression of STIM2 in parallel to an attenuation of CCE. Our experiments showing that knock-out of presenilins in MEF cells and FAD mutations in endogenous PS1 in lymphocytes affect both CCE and the cellular level of STIM proteins open new perspectives for studies on CCE in FAD.
Assuntos
Doença de Alzheimer/metabolismo , Moléculas de Adesão Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Presenilina-1/metabolismo , Presenilina-2/metabolismo , Idoso , Doença de Alzheimer/genética , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Canais de Cálcio , Moléculas de Adesão Celular/genética , Células Cultivadas , Humanos , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mutagênese Sítio-Dirigida , Proteínas de Neoplasias/genética , Presenilina-1/genética , Presenilina-2/genética , Molécula 1 de Interação Estromal , Molécula 2 de Interação EstromalRESUMO
Recent findings indicate that Store Operated Ca(2+) Entry (SOCE) in non-excitable cells is based on the interaction of ER calcium sensor STIM1 with the plasma membrane Ca(2+) channel protein ORAI1. However, despite physiological evidence for functional SOCE in neurons, its mechanism is not known. Using PCR, immunoblotting and immunohistochemical methods we show that STIM1 protein is present in the mouse brain. The protein and mRNA levels of STIM1 are similar in the thalamus, the hippocampus, the cortex and the amygdala and the higher level is observed in the cerebellum. Immunohistochemistry of the cortex and the hippocampus of brain sections shows that STIM1 is present in cell bodies and dendrites of pyramidal neurons. In the cerebellum STIM1 is present in Purkinje and granule cells. The same immunostaining pattern is observed in cultured hippocampal and cortical neurons. Localization of YFP-STIM1 and ORAI1 changes from a dispersed pattern in untreated cortical neurons to puncta-like pattern in cells with a Ca(2+) store depleted by thapsigargin treatment. The YFP-STIM1(D76A) dominant positive mutant, which is active regardless of the Ca(2+) level in ER, concentrates as puncta even without depletion of the neuronal Ca(2+) store. Also, this mutant forces ORAI1 redistribution to form puncta-like staining. We suggest that in neurons, just as in non-excitable cells, the STIM1 and ORAI1 proteins are involved in SOCE.
Assuntos
Encéfalo/metabolismo , Canais de Cálcio/metabolismo , Glicoproteínas de Membrana/genética , Neurônios/metabolismo , Animais , Cálcio/deficiência , Cálcio/metabolismo , Cerebelo/metabolismo , Córtex Cerebral/metabolismo , Primers do DNA , Expressão Gênica , Hipocampo/metabolismo , Imuno-Histoquímica , Glicoproteínas de Membrana/metabolismo , Camundongos , Proteína ORAI1 , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Molécula 1 de Interação Estromal , Tálamo/metabolismoRESUMO
BACKGROUND: Rituximab is used in the treatment of CD20+ B cell lymphomas and other B cell lymphoproliferative disorders. Its clinical efficacy might be further improved by combinations with other drugs such as statins that inhibit cholesterol synthesis and show promising antilymphoma effects. The objective of this study was to evaluate the influence of statins on rituximab-induced killing of B cell lymphomas. METHODS AND FINDINGS: Complement-dependent cytotoxicity (CDC) was assessed by MTT and Alamar blue assays as well as trypan blue staining, and antibody-dependent cellular cytotoxicity (ADCC) was assessed by a 51Cr release assay. Statins were found to significantly decrease rituximab-mediated CDC and ADCC of B cell lymphoma cells. Incubation of B cell lymphoma cells with statins decreased CD20 immunostaining in flow cytometry studies but did not affect total cellular levels of CD20 as measured with RT-PCR and Western blotting. Similar effects are exerted by other cholesterol-depleting agents (methyl-beta-cyclodextrin and berberine), but not filipin III, indicating that the presence of plasma membrane cholesterol and not lipid rafts is required for rituximab-mediated CDC. Immunofluorescence microscopy using double staining with monoclonal antibodies (mAbs) directed against a conformational epitope and a linear cytoplasmic epitope revealed that CD20 is present in the plasma membrane in comparable amounts in control and statin-treated cells. Atomic force microscopy and limited proteolysis indicated that statins, through cholesterol depletion, induce conformational changes in CD20 that result in impaired binding of anti-CD20 mAb. An in vivo reduction of cholesterol induced by short-term treatment of five patients with hypercholesterolemia with atorvastatin resulted in reduced anti-CD20 binding to freshly isolated B cells. CONCLUSIONS: Statins were shown to interfere with both detection of CD20 and antilymphoma activity of rituximab. These studies have significant clinical implications, as impaired binding of mAbs to conformational epitopes of CD20 elicited by statins could delay diagnosis, postpone effective treatment, or impair anti-lymphoma activity of rituximab.
Assuntos
Anticorpos Monoclonais/efeitos dos fármacos , Anticorpos Monoclonais/uso terapêutico , Antígenos CD20/efeitos dos fármacos , Antineoplásicos/antagonistas & inibidores , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Linfoma de Células B/tratamento farmacológico , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Murinos , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antígenos CD20/química , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Colesterol/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Humanos , Hipercolesterolemia/sangue , Hipercolesterolemia/tratamento farmacológico , Lovastatina/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , RituximabRESUMO
Alzheimer disease (AD) is the most common form of adult dementia. Its pathological hallmarks are synaptic degeneration, deposition of amyloid plaques and neurofibrillary tangles, leading to neuronal loss. A few hypotheses have been proposed to explain AD pathogenesis. The beta-amyloid (Abeta) and hyperphosphorylated tau hypotheses suggest that these proteins are the main players in AD development. Another hypothesis proposes that the dysregulation of calcium homeostasis may be a key factor in accelerating other pathological changes. Although Abeta and tau have been extensively studied, recently published data provide a growing body of evidence supporting the critical role of calcium signalling in AD. For example, presenilins, which are mutated in familial cases of AD, were demonstrated to form low conductance calcium channels in the ER and elevated cytosolic calcium concentration increases amyloid generation. Moreover, memantine, an antagonist of the NMDA-calcium channel receptor, has been found to have a beneficial effect for AD patients offering novel possibilities for a calcium signalling targeted therapy of AD. This review underscores the growing importance of calcium ions in AD development and focuses on the relevant aspects of calcium homeostasis.
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Doença de Alzheimer/metabolismo , Cálcio/metabolismo , Cálcio/fisiologia , Precursor de Proteína beta-Amiloide/metabolismo , Homeostase/fisiologia , Humanos , Fosforilação , Proteínas tau/metabolismoRESUMO
New compounds containing [1,2,4]triazolo [1,5-a]pyridine (I), pyrazolo [1,5-a]pyridine (II), 1H-1,3-benzodiazole (III) and imidazo [1,2-a]pyrimidine (IV) backbones were designed and synthesized for PDE10A interaction. Among these compounds, 1H-1,3-benzodiazoles and imidazo [1,2-a]pyrimidines showed the highest affinity for PDE10A enzyme as well as good metabolic stability. Both classes of compounds were identified as selective and potent PDE10A enzyme inhibitors.
Assuntos
Inibidores Enzimáticos/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Pirimidinas/farmacologia , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Pirimidinas/síntese química , Pirimidinas/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
In this paper we reviewed the latest literature on molecular techniques used in diagnosis and epidemiology of infections caused by pathogenic fungi. Traditional methods used for the identification and typing of medically relevant fungi include morphological and biochemical analysis. These methods are time-consuming and base on phenotypic features what makes them unreliable. We described the usefulness in mycological studies of fast and very sensitive molecular methods which rely on PCR and hybridization techniques.
Assuntos
Fungos/classificação , Técnicas de Tipagem Micológica , Micoses/diagnóstico , Micoses/epidemiologia , Reação em Cadeia da Polimerase/métodos , Impressões Digitais de DNA/métodos , DNA Fúngico/análise , Eletroforese em Gel de Campo Pulsado/métodos , Fungos/genética , Fungos/isolamento & purificação , Fungos/patogenicidade , Humanos , Epidemiologia Molecular , Micoses/microbiologia , Hibridização de Ácido Nucleico , Técnica de Amplificação ao Acaso de DNA PolimórficoRESUMO
The pathogenesis of schizophrenia involves several complex cellular mechanisms and is not well understood. Recent research has demonstrated an association between primary disturbances characteristic of the disease, including altered dopaminergic and glutamatergic neurotransmission, and impairments in neuronal calcium (Ca(2+)) homeostasis and signaling. Emerging Ca(2+) hypothesis links and unifies various cellular processes involved in the pathogenesis of schizophrenia and suggests a central role of dysregulation of Ca(2+) homeostasis in the etiology of the disease. This review explores the in vitro data on Ca(2+) homeostasis and signaling in schizophrenia. Major limitation in this research is the lack of schizophrenia markers and validated disease models. As indicated in this review, one way to overcome these limitations may be analyses of Ca(2+) signalosomes in peripheral cells from schizophrenia patients. Validation of animal models of schizophrenia may permit the application of advanced Ca(2+) imaging techniques in living animals.
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Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Homeostase/fisiologia , Esquizofrenia/metabolismo , Animais , Cálcio/fisiologia , Humanos , Neurônios/metabolismo , Neurônios/fisiologiaRESUMO
Gamma-secretase is an integral membrane protease, which is a complex of four membrane proteins. Improper functioning of gamma-secretase was found to be critical in the pathogenesis of Alzheimer's disease. Despite numerous efforts, the structure of the protease as well as its proteolytic mechanism remains poorly understood. In this work we constructed a model of interactions between two proteins forming gamma-secretase: APH-1 and presenilin. This interface is based on a highly conserved GxxxGxxxG motif in the APH-1 protein. It can form a tight contact with a small-residue AxxxAxxxG motif in presenilin. Here, four binding modes based on similar structures involving GxxxG motifs in glycophorin and aquaporin were proposed and verified. The resulting best model employs antiparallel orientations of interacting helices and is in agreement with the currently accepted topology of both proteins. This model can be used for further structural characterization of gamma-secretase and its components.
Assuntos
Proteínas de Membrana/química , Modelos Moleculares , Presenilina-1/química , Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/química , Endopeptidases , Genótipo , Humanos , Proteínas de Membrana/genética , Peptídeo HidrolasesRESUMO
BACKGROUND: Presenilin 1 (PS1) and presenilin 2 (PS2) are membranous proteins involved in the pathology of Alzheimer's disease. The development of specific therapies targeted at PS1 or PS2 requires the determination of biochemical properties of presenilins. Hence, in this study we analyzed the hydrophobic and ionic properties of endogenous presenilins. METHODS: Lysates of immortalized human B-lymphocytes were used as a source of endogenous presenilins. The presence of 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) detergent in lysates favored preservation of PS1 and PS2 native protein complexes. We compared Kyte-Doolittle hydropathicity profiles and hydrophobic interactions of PS1 and PS2 with phenyl-agarose. We also compared the ionic properties of presenilins using anion-exchange chromatography. RESULTS: The hydropathicity profiles of PS1 and PS2 revealed similarly located hydrophobic regions and more hydrophobic region in the C-terminal fragment of PS2. However, both PS1 and PS2 under physiological conditions showed no interactions with phenyl-agarose. Despite similar predicted isoelectric points, PS1 and PS2 exhibited different ionic behavior during anion-exchange chromatography. CONCLUSIONS: The different than expected hydrophobic and ionic behavior of PS1 and PS2 may be caused by interactions with other proteins present in complexes formed by endogenous presenilins. The observed difference in ionic properties of PS1 and PS2 can be further explained assuming that PS1 and PS2 form complexes with different sets of proteins. The composition of such variegated PS1 and PS2 complexes can be explored using a proteomic approach. The difference in PS1 and PS2 ionic behavior can be used for purification of endogenous PS1 from PS2, which has not yet been achieved by any other means.