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1.
Biol Pharm Bull ; 2020 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-32581190

RESUMO

Early diagnosis of Niemann-Pick diseases (NPDs) is important for better prognosis of such diseases. N-Palmitoyl-O-phosphocholine-serine (PPCS) is a new NPD biomarker possessing high sensitivity, and with its combination with sphingosylphosphocholine (SPC) it may be possible to distinguish NPD-C from NPD-A/B. In this study, a rapid liquid chromatography/tandem mass spectrometry (LC/MS/MS) method (method 1) and a validated LC/MS/MS analysis (method 2) of PPCS and SPC were developed, and we have proposed a diagnostic screening strategy for NPDs using a combination of serum PPCS and SPC concentrations.Nexera and API 5000 were used as LC/MS/MS systems. C18 columns with lengths of 10 mm and 50 mm were used for method 1 and 2, respectively. 2H3-labeled PPCS (PPCS-2H3_ and nor-SPC were used as internal standards. Selective reaction monitoring in positive-ion mode was used for MS/MS. Run times of 1.2 min and 8 min were set for methods 1 and 2, respectively.In both methods 1 and 2, two analytes showed high linearity in the range of 1-4000 ng/mL. Method 2 provided high accuracy and precision in method validation. Serum concentrations of both analytes were significantly higher in NPD-C patients than those of healthy subjects in both methods. Serum PPCS correlated between methods 1 and 2; however, it was different in the case of SPC. The serum PPCS/SPC ratio was different in healthy subjects, NPD-C, and NPD-A/B. These results suggest that using a combination of the two LC/MS/MS analytical methods for PPCS and SPC is useful for diagnostic screening of NPDs.

2.
J Lipid Res ; 61(7): 972-982, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32457038

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus (SARS-CoV)-2 has resulted in the death of more than 328,000 persons worldwide in the first 5 months of 2020. Herculean efforts to rapidly design and produce vaccines and other antiviral interventions are ongoing. However, newly evolving viral mutations, the prospect of only temporary immunity, and a long path to regulatory approval pose significant challenges and call for a common, readily available, and inexpensive treatment. Strategic drug repurposing combined with rapid testing of established molecular targets could provide a pause in disease progression. SARS-CoV-2 shares extensive structural and functional conservation with SARS-CoV-1, including engagement of the same host cell receptor (angiotensin-converting enzyme 2) localized in cholesterol-rich microdomains. These lipid-enveloped viruses encounter the endosomal/lysosomal host compartment in a critical step of infection and maturation. Niemann-Pick type C (NP-C) disease is a rare monogenic neurodegenerative disease caused by deficient efflux of lipids from the late endosome/lysosome (LE/L). The NP-C disease-causing gene (NPC1) has been strongly associated with viral infection, both as a filovirus receptor (e.g., Ebola) and through LE/L lipid trafficking. This suggests that NPC1 inhibitors or NP-C disease mimetics could serve as anti-SARS-CoV-2 agents. Fortunately, there are such clinically approved molecules that elicit antiviral activity in preclinical studies, without causing NP-C disease. Inhibition of NPC1 may impair viral SARS-CoV-2 infectivity via several lipid-dependent mechanisms, which disturb the microenvironment optimum for viral infectivity. We suggest that known mechanistic information on NPC1 could be utilized to identify existing and future drugs to treat COVID-19.

3.
Int J Mol Sci ; 21(3)2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32019132

RESUMO

Niemann-Pick disease type C (NPC) is an autosomal recessive disorder characterized by abnormal accumulation of free cholesterol and sphingolipids in lysosomes. The iminosugar miglustat, which inhibits hexosylceramide synthesis, is used for NPC treatment, and 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), a cyclic oligosaccharide derivative, is being developed to treat NPC. Moreover, therapeutic potential of 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD) was shown in NPC models, although its mechanism of action remains unclear. Here, we investigated the effects of HP-ß-CD, HP-γ-CD, and their homolog 2-hydroxypropyl-α-cyclodextrin (HP-α-CD) on lipid accumulation in Npc1-null Chinese hamster ovary (CHO) cells compared with those of miglustat. HP-ß-CD and HP-γ-CD, unlike HP-α-CD, reduced intracellular free cholesterol levels and normalized the lysosome changes in Npc1-null cells but not in wild-type CHO cells. In contrast, miglustat did not normalize intracellular free cholesterol accumulation or lysosome changes in Npc1-null cells. However, miglustat decreased the levels of hexosylceramide and tended to increase those of sphingomyelins in line with its action as a glucosylceramide synthase inhibitor in both Npc1-null and wild-type CHO cells. Interestingly, HP-ß-CD and HP-γ-CD, unlike HP-α-CD, reduced sphingomyelins in Npc1-null, but not wild-type, cells. In conclusion, HP-ß-CD and HP-γ-CD reduce the accumulation of sphingolipids, mainly sphingomyelins, and free cholesterol as well as lysosome changes in Npc1-null, but not in wild-type, CHO cells.

4.
Chem Commun (Camb) ; 55(85): 12845-12848, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31596280

RESUMO

Multivalent mannosides with inherent macrophage recognition abilities, built on ß-cyclodextrin, RAFT cyclopeptide or peptide dendrimer cores, trigger selective inhibition of lysosomal ß-glucocerebrosidase or α-mannosidase depending on valency and topology, offering new opportunities in multitargeted drug design.


Assuntos
Desenho de Fármacos , Manosídeos/química , Glucosilceramidase/antagonistas & inibidores , Lectinas/química , Macrófagos/metabolismo , Manosídeos/metabolismo , Peptídeos Cíclicos/química , alfa-Manosidase/antagonistas & inibidores , beta-Ciclodextrinas/química
5.
Int J Mol Sci ; 20(20)2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31658747

RESUMO

Niemann-Pick disease type C (NPC) is an autosomal recessive disorder caused by the mutation of cholesterol-transporting proteins. In addition, early treatment is important for good prognosis of this disease because of the progressive neurodegeneration. However, the diagnosis of this disease is difficult due to a variety of clinical spectrum. Lysosphingomyelin-509, which is one of the most useful biomarkers for NPC, was applied for the rapid and easy detection of NPC. The fact that its chemical structure was unknown until recently implicates the unrevealed pathophysiology and molecular mechanisms of NPC. In this study, we aimed to elucidate the structure of lysosphingomyelin-509 by various mass spectrometric techniques. As our identification strategy, we adopted analytical and organic chemistry approaches to the serum of patients with NPC. Chemical derivatization and hydrogen abstraction dissociation-tandem mass spectrometry were used for the determination of function groups and partial structure, respectively. As a result, we revealed the exact structure of lysosphingomyelin-509 as N-acylated and O-phosphocholine adducted serine. Additionally, we found that a group of metabolites with N-acyl groups were increased considerably in the serum/plasma of patients with NPC as compared to that of other groups using targeted lipidomics analysis. Our techniques were useful for the identification of lysosphingomyelin-509.


Assuntos
Lipídeos/química , Lipídeos/isolamento & purificação , Doença de Niemann-Pick Tipo C/diagnóstico , Fosforilcolina/química , Fosforilcolina/isolamento & purificação , Serina/química , Biomarcadores/sangue , Feminino , Humanos , Masculino , Doença de Niemann-Pick Tipo C/metabolismo , Fosforilcolina/metabolismo , Serina/metabolismo , Espectrometria de Massas em Tandem/métodos
6.
Yonago Acta Med ; 62(2): 240-243, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31341401

RESUMO

Graves' disease occurs predominantly in women. Epstein-Barr virus (EBV) mainly persists in human B lymphocytes, and its reactivation stimulates antibody production. We previously suggested that the EBV reactivation-induced production of TRAb and IgM at 100 nM estradiol (pregnant level) was lower than that at 0 nM estradiol and that class switch recombination may be increased by estradiol. In this study, we examined the effect of estradiol on EBV reactivation. We identified the expression of EBV-glycoprotein 350/220 (gp350/220) in the late phase of reactivation and plasma cell differentiation of EBV-infected cells using 72A1 antibody and CD138 antibody, respectively. We found the mean ratio of gp 350/220(+) CD138(+) cells at 100 nM estradiol was higher than that at 0 nM estradiol. These results suggested that EBV-infected cells could survive with keeping the ability of antibody production in 100 nM estradiol, which is consistent with the improvement of Graves' disease during maternity and exacerbation postpartum.

7.
J Med Chem ; 62(12): 5832-5843, 2019 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-31017416

RESUMO

α-Mannosidosis (AM) results from deficient lysosomal α-mannosidase (LAMAN) activity and subsequent substrate accumulation in the lysosome, leading to severe pathology. Many of the AM-causative mutations compromise enzyme folding and could be rescued with purpose-designed pharmacological chaperones (PCs). We found that PCs combining a LAMAN glycone-binding motif based on the 5 N,6 O-oxomethylidenemannojirimycin (OMJ) glycomimetic core and different aglycones, in either mono- or multivalent displays, elicit binding modes involving glycone and nonglycone enzyme regions that reinforce the protein folding and stabilization potential. Multivalent derivatives exhibited potent enzyme inhibition that generally prevailed over the chaperone effect. On the contrary, monovalent OMJ derivatives with LAMAN aglycone binding area-fitting substituents proved effective as activity enhancers for several mutant LAMAN forms in AM patient fibroblasts and/or transfected MAN2 B1-KO cells. This translated into a significant improvement in endosomal/lysosomal function, reverting not only the primary LAMAN substrate accumulation but also the additional downstream consequences such as cholesterol accumulation.

8.
Int J Mol Sci ; 20(5)2019 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-30845767

RESUMO

Niemann-Pick disease Type C (NPC) is a rare lysosomal storage disease characterized by the dysfunction of intracellular cholesterol trafficking with progressive neurodegeneration and hepatomegaly. We evaluated the potential of 6-O-α-maltosyl-ß-cyclodextrin (G2-ß-CD) as a drug candidate against NPC. The physicochemical properties of G2-ß-CD as an injectable agent were assessed, and molecular interactions between G2-ß-CD and free cholesterol were studied by solubility analysis and two-dimensional proton nuclear magnetic resonance spectroscopy. The efficacy of G2-ß-CD against NPC was evaluated using Npc1 deficient Chinese hamster ovary (CHO) cells and Npc1 deficient mice. G2-ß-CD in aqueous solution showed relatively low viscosity and surface activity; characteristics suitable for developing injectable formulations. G2-ß-CD formed higher-order inclusion complexes with free cholesterol. G2-ß-CD attenuated dysfunction of intercellular cholesterol trafficking and lysosome volume in Npc1 deficient CHO cells in a concentration dependent manner. Weekly subcutaneous injections of G2-ß-CD (2.9 mmol/kg) ameliorated abnormal cholesterol metabolism, hepatocytomegaly, and elevated serum transaminases in Npc1 deficient mice. In addition, a single cerebroventricular injection of G2-ß-CD (21.4 µmol/kg) prevented Purkinje cell loss in the cerebellum, body weight loss, and motor dysfunction in Npc1 deficient mice. In summary, G2-ß-CD possesses characteristics favorable for injectable formulations and has therapeutic potential against in vitro and in vivo NPC models.


Assuntos
Colesterol/metabolismo , Proteína C1 de Niemann-Pick/deficiência , Doença de Niemann-Pick Tipo C/tratamento farmacológico , beta-Ciclodextrinas/administração & dosagem , Animais , Células CHO , Cricetinae , Cricetulus , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Injeções Subcutâneas , Camundongos , Doença de Niemann-Pick Tipo C/metabolismo , Ressonância Magnética Nuclear Biomolecular , Resultado do Tratamento , beta-Ciclodextrinas/farmacologia
9.
Hum Mol Genet ; 28(11): 1894-1904, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30689867

RESUMO

Parkinson's disease (PD) is characterized by dopaminergic (DA) cell loss and the accumulation of pathological alpha synuclein (asyn), but its precise pathomechanism remains unclear, and no appropriate animal model has yet been established. Recent studies have shown that a heterozygous mutation of glucocerebrosidase (gba) is one of the most important genetic risk factors in PD. To create mouse model for PD, we crossed asyn Bacterial Artificial Chromosome transgenic mice with gba heterozygous knockout mice. These double-mutant (dm) mice express human asyn in a physiological manner through its native promoter and showed an increase in phosphorylated asyn in the regions vulnerable to PD, such as the olfactory bulb and dorsal motor nucleus of the vagus nerve. Only dm mice showed a significant reduction in DA cells in the substantia nigra pars compacta, suggesting these animals were suitable for a prodromal model of PD. Next, we investigated the in vivo mechanism by which GBA insufficiency accelerates PD pathology, focusing on lipid metabolism. Dm mice showed an increased level of glucosylsphingosine without any noticeable accumulation of glucosylceramide, a direct substrate of GBA. In addition, the overexpression of asyn resulted in decreased GBA activity in mice, while dm mice tended to show an even further decreased level of GBA activity. In conclusion, we created a novel prodromal mouse model to study the disease pathogenesis and develop novel therapeutics for PD and also revealed the mechanism by which heterozygous gba deficiency contributes to PD through abnormal lipid metabolism under conditions of an altered asyn expression in vivo.


Assuntos
Glucosilceramidase/genética , Doença de Parkinson/genética , alfa-Sinucleína/genética , Animais , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Humanos , Metabolismo dos Lipídeos/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Parte Compacta da Substância Negra/metabolismo , Parte Compacta da Substância Negra/patologia , Sintomas Prodrômicos
10.
Viral Immunol ; 31(8): 540-547, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30222515

RESUMO

Immunoglobulin (Ig) G4-related disease (IgG4-RD) is a newly recognized systemic fibroinflammatory disease with characteristic histological findings and high serum IgG4 levels. Epstein-Barr virus (EBV) is a persistent herpesvirus in B lymphocytes, and we previously reported EBV reactivation-induced Ig production. We showed that EBV reactivation induced the production of thyrotropin receptor antibodies, the causative antibodies of Graves' disease. In the present study, we investigated whether EBV reactivation induced IgG4 production and if EBV-positive B cells or IgG4-positive plasma cells are present in the thyroid tissues of Graves' disease patients with lymphoplasmacytic infiltration. EBV-encoded small RNA1 (EBER1) in situ hybridization and immunohistochemistry for IgG and IgG4 were performed on seven resected thyroid tissues with lymphoplasmacytic infiltration collected from the thyroids of 11 Graves' disease patients. We then cultured the lymphocytes of 13 Graves' disease patients and 14 controls and induced EBV reactivation to measure IgG4 levels in culture fluids. We detected EBER1-positive cells and IgG4-positive plasma cells in the same area of thyroid tissues. EBV-reactivated cells with IgG4 on their surface were observed in culture cells, and IgG4 production was detected in culture fluids. The IgG4/IgG percentage was higher than that in normal serum level. A subset of Graves' disease is an IgG4-RD-like condition, not an IgG4-RD. EBV reactivation stimulates IgG4 production, which may result in high serum IgG4 levels and promote IgG4-positive plasma cell infiltration. EBER1 needs to be examined when an increase in IgG4-positive plasma cell numbers is noted.


Assuntos
Linfócitos B/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Doença de Graves/imunologia , Herpesvirus Humano 4/imunologia , Imunoglobulina G/biossíntese , Ativação Viral/imunologia , Adulto , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Linfócitos B/virologia , Estudos de Casos e Controles , Infecções por Vírus Epstein-Barr/complicações , Feminino , Doença de Graves/patologia , Humanos , Imunoglobulina G/análise , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , RNA Viral/análise , RNA Viral/imunologia , Adulto Jovem
11.
Molecules ; 23(4)2018 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-29673163

RESUMO

A series of sp²-iminosugar glycomimetics differing in the reducing or nonreducing character, the configurational pattern (d-gluco or l-ido), the architecture of the glycone skeleton, and the nature of the nonglycone substituent has been synthesized and assayed for their inhibition properties towards commercial glycosidases. On the basis of their affinity and selectivity towards GH1 ß-glucosidases, reducing and nonreducing bicyclic derivatives having a hydroxylation profile of structural complementarity with d-glucose and incorporating an N'-octyl-isourea or -isothiourea segment were selected for further evaluation of their inhibitory/chaperoning potential against human glucocerebrosidase (GCase). The 1-deoxynojirimycin (DNJ)-related nonreducing conjugates behaved as stronger GCase inhibitors than the reducing counterparts and exhibited potent chaperoning capabilities in Gaucher fibroblasts hosting the neuronopathic G188S/G183W mutation, the isothiourea derivative being indeed one of the most efficient chaperone candidates reported up to date (70% activity enhancement at 20 pM). At their optimal concentration, the four selected compounds promoted mutant GCase activity enhancements over 3-fold; yet, the inhibitor/chaperoning balance became unfavorable at much lower concentration for nonreducing as compared to reducing derivatives.


Assuntos
Inibidores Enzimáticos/uso terapêutico , Doença de Gaucher/tratamento farmacológico , Doença de Gaucher/enzimologia , Glucosilceramidase/antagonistas & inibidores , Glucosilceramidase/genética , Imino Açúcares/uso terapêutico , Chaperonas Moleculares/uso terapêutico , 1-Desoxinojirimicina/uso terapêutico , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Doença de Gaucher/genética , Glucosamina/análogos & derivados , Glucosamina/uso terapêutico , Humanos , Mutação
12.
J Mol Cell Cardiol ; 115: 158-169, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29355491

RESUMO

The human ether-a-go-go-related gene (hERG) encodes the α subunit of a rapidly activating delayed-rectifier potassium (IKr) channel. Mutations of the hERG cause long QT syndrome type 2 (LQT2). Acetylation of lysine residues occurs in a subset of non-histone proteins and this modification is controlled by both histone acetyltransferases and deacetylases (HDACs). The aim of this study was to clarify effects of HDAC(s) on wild-type (WT) and mutant hERG proteins. WThERG and two trafficking-defective mutants (G601S and R752W) were transiently expressed in HEK293 cells, which were treated with a pan-HDAC inhibitor Trichostatin A (TSA) or an isoform-selective HDAC6 inhibitor Tubastatin A (TBA). Both TSA and TBA increased protein levels of WThERG and induced expression of mature forms of the two mutants. Immunoprecipitation showed an interaction between HDAC6 and immature forms of hERG. Coexpression of HDAC6 decreased acetylation and, reciprocally, increased ubiquitination of hERG, resulting in its decreased expression. siRNA against HDAC6, as well as TBA, exerted opposite effects. Immunochemistry revealed that HDAC6 knockdown increased expression of the WThERG and two mutants both in the endoplasmic reticulum and on the cell surface. Electrophysiology showed that HDAC6 knockdown or TBA treatment increased the hERG channel current corresponding to the rapidly activating delayed-rectifier potassium current (IKr) in HEK293 cells stably expressing the WT or mutants. Three lysine residues (K116, K495 and K757) of hERG were predicted to be acetylated. Substitution of these lysine residues with arginine eliminated HDAC6 effects. In HL-1 mouse cardiomyocytes, TBA enhanced endogenous ERG expression, increased IKr, and shortened action potential duration. These results indicate that hERG is a substrate of HDAC6. HDAC6 inhibition induced acetylation of hERG which counteracted ubiquitination leading its stabilization. HDAC6 inhibition may be a novel therapeutic option for LQT2.


Assuntos
Canal de Potássio ERG1/metabolismo , Desacetilase 6 de Histona/metabolismo , Proteínas Mutantes/metabolismo , Acetilação/efeitos dos fármacos , Animais , Canal de Potássio ERG1/química , Células HEK293 , Inibidores de Histona Desacetilases/farmacologia , Humanos , Lisina/metabolismo , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos
13.
Carbohydr Res ; 455: 54-61, 2018 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-29172126

RESUMO

We aimed to investigate whether 6-O-α-maltosyl-ß-cyclodextrin (Mal-ßCD) is incorporated into cells and lysosomes during the release of unesterified cholesterol in Npc1-deficient Chinese hamster ovary (CHO) cells (Npc1 KO cells) and CHO-JP17 cells (JP17 cells). Internalization of Mal-ßCD in cells and lysosomes and extracellular release of lysosomal unesterified cholesterol were demonstrated by LC/MS/MS and LC/MS, respectively. Internalization of Mal-ßCD was greater in Npc1 KO cells than in JP17 cells. The majority of internalized Mal-ßCD in both cell types was metabolized by lysosomal α-glucosidase to 6-O-α-D-glucosyl-ß-cyclodextrin (Glc-ßCD). However, Mal-ßCD did not directly enter the lysosomes prepared from cell homogenates. Mal-ßCD-treated Npc1 KO cells and JP17 cells both released Mal-ßCD and Glc-ßCD, together with unesterified cholesterol, out of cells. The release of unesterified cholesterol by Mal-ßCD in Npc1 KO cells was much greater than that in JP17 cells. This study is the first to report the influx of Mal-ßCD into the lysosomes of Npc1 KO cells and JP17 cells, followed by generation of Glc-ßCD, and the efflux of Mal-ßCD/Glc-ßCD and unesterified cholesterol to the extracellular fluid, based on the quantitative LC/MS analysis.


Assuntos
Colesterol/química , Lisossomos/química , beta-Ciclodextrinas/química , Animais , Células CHO , Cromatografia Líquida , Cricetinae , Cricetulus , Endocitose/fisiologia , Espectrometria de Massas
14.
PLoS One ; 12(8): e0183225, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28817660

RESUMO

Cardiac progenitor cells have a limited proliferative capacity. The CREB-binding protein/p300-interacting transactivator, with the Glu/Asp-rich carboxy-terminal domain (Cited) gene family, regulates gene transcription. Increased expression of the Cited4 gene in an adult mouse is associated with exercise-induced cardiomyocyte hypertrophy and proliferation. However, the expression patterns and functional roles of the Cited4 gene during cardiogenesis are largely unknown. Therefore, in the present study, we investigated the expression patterns and functional roles of the Cited4 gene during in vitro cardiogenesis. Using embryoid bodies formed from mouse embryonic stem cells, we evaluated the expression patterns of the Cited4 gene by quantitative reverse transcriptase-polymerase chain reaction. Cited4 gene expression levels increased and decreased during the early and late phases of cardiogenesis, respectively. Moreover, Cited4 gene levels were significantly high in the cardiac progenitor cell population. A functional assay of the Cited4 gene in cardiac progenitor cells using flow cytometry indicated that overexpression of the Cited4 gene significantly increased the cardiac progenitor cell population compared with the control and knockdown groups. A cell proliferation assay, with 5-ethynyl-2'-deoxyuridine incorporation and Ki67 expression during the late phase of cardiogenesis, indicated that the number of troponin T-positive embryonic stem cell-direived cardiomyocytes with proliferative capacity was significantly greater in the overexpression group than in the control and knockdown groups. Our study results suggest that the Cited4 gene is related to cardiac differentiation and maintenance of proliferation capacity of embryonic stem cell-derived cardiomyocytes during in vitro cardiogenesis. Therefore, manipulation of Cited4 gene expression may be of great interest for cardiac regeneration.


Assuntos
Proliferação de Células , Células-Tronco Embrionárias/citologia , Coração/embriologia , Miócitos Cardíacos/citologia , Fatores de Transcrição/metabolismo , Animais , Separação Celular , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/genética
15.
J Arrhythm ; 33(3): 226-233, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28607619

RESUMO

BACKGROUND: Pilsicainide, classified as a relatively selective Na+ channel blocker, also has an inhibitory action on the rapidly-activating delayed-rectifier K+ current (IKr ) through human ether-a-go-go-related gene (hERG) channels. We studied the effects of chronic exposure to pilsicainide on the expression of wild-type (WT) hERG proteins and WT-hERG channel currents, as well as on the expression of mutant hERG proteins, in a heterologous expression system. METHODS: HEK293 cells stably expressing WT or mutant hERG proteins were subjected to Western blotting, immunofluorescence microscopy and patch-clamp experiments. RESULTS: Acute exposure to pilsicainide at 0.03-10 µM influenced neither the expression of WT-hERG proteins nor WT-hERG channel currents. Chronic treatment with 0.03-10 µM pilsicainide for 48 h, however, increased the expression of WT-hERG proteins and channel currents in a concentration-dependent manner. Chronic treatment with 3 µM pilsicainide for 48 h delayed degradation of WT-hERG proteins and increased the channels expressed on the plasma membrane. A cell membrane-impermeant pilsicainide derivative did not influence the expression of WT-hERG, indicating that pilsicainide stabilized the protein inside the cell. Pilsicainide did not influence phosphorylation of Akt (protein kinase B) or expression of heat shock protein families such as HSF-1, hsp70 and hsp90. E4031, a chemical chaperone for hERG, abolished the pilsicainide effect on hERG. Chronic treatment with pilsicainide could also increase the protein expression of trafficking-defective mutant hERG, G601S and R752W. CONCLUSIONS: Pilsicainide penetrates the plasma membrane, stabilizes WT-hERG proteins by acting as a chemical chaperone, and enhances WT-hERG channel currents. This mechanism could also be applicable to modulations of certain mutant-hERG proteins.

16.
J Med Chem ; 60(5): 1829-1842, 2017 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-28171725

RESUMO

Amphiphilic glycomimetics encompassing a rigid, undistortable nortropane skeleton based on 1,6-anhydro-l-idonojirimycin and a polyfluorinated antenna, when formulated as the corresponding inclusion complexes with ß-cyclodextrin (ßCD), have been shown to behave as pharmacological chaperones (PCs) that efficiently rescue lysosomal ß-glucocerebrosidase mutants associated with the neuronopathic variants of Gaucher disease (GD), including the highly refractory L444P/L444P and L444P/P415R single nucleotide polymorphs, in patient fibroblasts. The body of work here presented includes the design criteria for the PC prototype, the synthesis of a series of candidates, the characterization of the PC:ßCD complexes, the determination of the selectivity profiles toward a panel of commercial and human lysosomal glycosidases, the evaluation of the chaperoning activity in type 1 (non-neuronopathic), type 2 (acute neuronopathic), and type 3 (adult neuronopathic) GD fibroblasts, the confirmation of the rescuing mechanism by immunolabeling, and the analysis of the PC:GCase binding mode by docking experiments.


Assuntos
Flúor/química , Doença de Gaucher/enzimologia , Glucosilceramidase/metabolismo , Chaperonas Moleculares/metabolismo , beta-Ciclodextrinas/química , Células Cultivadas , Humanos , Simulação de Acoplamento Molecular
17.
J Biol Chem ; 292(11): 4395-4410, 2017 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-28031458

RESUMO

Niemann-Pick type C (NP-C) disease is a fatal genetic lipidosis for which there is no Food and Drug Administration (FDA)-approved therapy. Vorinostat, an FDA-approved inhibitor of histone deacetylases, ameliorates lysosomal lipid accumulation in cultured NP-C patient fibroblasts. To assess the therapeutic potential of histone deacetylase inhibition, we pursued these in vitro observations in two murine models of NP-C disease. Npc1nmf164 mice, which express a missense mutation in the Npc1 gene, were treated intraperitoneally, from weaning, with the maximum tolerated dose of vorinostat (150 mg/kg, 5 days/week). Disease progression was measured via gene expression, liver function and pathology, serum and tissue lipid levels, body weight, and life span. Transcriptome analyses of treated livers indicated multiple changes consistent with reversal of liver dysfunction that typifies NP-C disease. Significant improvements in liver pathology and function were achieved by this treatment regimen; however, NPC1 protein maturation and levels, disease progression, weight loss, and animal morbidity were not detectably altered. Vorinostat concentrations were >200 µm in the plasma compartment of treated animals but were almost 100-fold lower in brain tissue. Apolipoprotein B metabolism and the expression of key components of lipid homeostasis in primary hepatocytes from null (Npc1-/-) and missense (Npc1nmf164 ) mutant mice were altered by vorinostat treatment, consistent with a response by these cells independent of the status of the Npc1 locus. These results suggest that HDAC inhibitors have utility to treat visceral NP-C disease. However, it is clear that improved blood-brain barrier penetration will be required to alleviate the neurological symptoms of human NP-C disease.


Assuntos
Inibidores de Histona Desacetilases/uso terapêutico , Ácidos Hidroxâmicos/uso terapêutico , Fígado/efeitos dos fármacos , Fígado/fisiopatologia , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Doença de Niemann-Pick Tipo C/genética , Proteínas/genética , Animais , Apolipoproteínas B/metabolismo , Células Cultivadas , Colesterol/genética , Colesterol/metabolismo , Modelos Animais de Doenças , Inibidores de Histona Desacetilases/farmacocinética , Homeostase/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/farmacocinética , Peptídeos e Proteínas de Sinalização Intracelular , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto , Doença de Niemann-Pick Tipo C/patologia , Doença de Niemann-Pick Tipo C/fisiopatologia , Proteínas/metabolismo , Transcriptoma/efeitos dos fármacos , Vorinostat
19.
Eur J Med Chem ; 126: 160-170, 2017 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-27750150

RESUMO

This report is about the identification, synthesis and initial biological characterization of derivatives of 4-epi-isofagomine as pharmacological chaperones (PC) for human lysosomal ß-galactosidase. The two epimers of 4-epi-isofagomine carrying a pentyl group at C-5a, namely (5aR)- and (5aS)-5a-C-pentyl-4-epi-isofagomine, were prepared by an innovative procedure involving in the key step the addition of nitrohexane to a keto-pentopyranoside. Both epimers were evaluated as inhibitors of the human ß-galactosidase: the (5aR)-stereoisomer (compound 1) was found to be a very potent inhibitor of the enzyme (IC50 = 8 nM, 30× more potent than 4-epi-isofagomine at pH 7.3) with a high selectivity for this glycosidase whereas the (5aS) epimer was a much weaker inhibitor. In addition, compound 1 showed a remarkable activity as a PC. It significantly enhanced the residual activity of mutant ß-galactosidase in 15 patient cell lines out of 23, with enhancement factors greater than 3.5 in 10 cell lines and activity restoration up to 91% of normal. Altogether, these results indicated that (5aR)-5a-C-pentyl-4-epi-isofagomine constitutes a promising PC-based drug candidate for the treatment of GM1-gangliosidosis and Morquio disease type B.


Assuntos
Inibidores Enzimáticos/farmacologia , Gangliosidose GM1/genética , Imino Piranoses/farmacologia , Lisossomos/enzimologia , Mucopolissacaridose IV/genética , Mutação , beta-Galactosidase/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Fibroblastos/efeitos dos fármacos , Gangliosidose GM1/enzimologia , Gangliosidose GM1/patologia , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Imino Piranoses/síntese química , Imino Piranoses/química , Mucopolissacaridose IV/enzimologia , Mucopolissacaridose IV/patologia , Desnaturação Proteica , beta-Galactosidase/química , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
20.
Circ J ; 80(12): 2443-2452, 2016 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-27803431

RESUMO

BACKGROUND: Long QT syndrome 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). Most of its mutations give rise to unstable hERG proteins degraded by the proteasome. Recently, carbachol was reported to stabilize the wild-type hERG-FLAG via activation of the muscarinic type 3 receptor (M3-mAChR). Its action on mutant hERG-FLAG, however, remains uninvestigated.Methods and Results:A novel mutant hERG-FLAG carried 2 mutations: an amino acid substitution G572S and an in-frame insertion D1037_V1038insGD. When expressed in HEK293 cells, this mutant hERG-FLAG was degraded by the proteasome and failed to be transported to the cell surface. Carbachol restored stability of the mutant hERG-FLAG and facilitated cell-surface expression. Carbachol activated PKC, augmented phosphorylation of heat shock factor 1 (HSF1) and enhanced expression of heat shock proteins (hsps), hsp70 and hsp90. Both a M3-mAChR antagonist, 4-DAMP, and a PKC inhibitor, bisindolylmaleimide, abolished carbachol-induced stabilization of the mutant hERG-FLAG. CONCLUSIONS: M3-mAChR activation leads to enhancement of hsp expression via PKC-dependent phosphorylation of HSF1, thereby stabilizing the mutant hERG-FLAG protein. Thus, M3-mAChR activators may have a therapeutic value for patients with LQT2. (Circ J 2016; 80: 2443-2452).


Assuntos
Proteínas de Ligação a DNA/metabolismo , Canal de Potássio ERG1 , Síndrome do QT Longo , Mutação , Receptor Muscarínico M3/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Adolescente , Proteínas de Ligação a DNA/genética , Canal de Potássio ERG1/genética , Canal de Potássio ERG1/metabolismo , Células HEK293 , Fatores de Transcrição de Choque Térmico , Humanos , Síndrome do QT Longo/genética , Síndrome do QT Longo/metabolismo , Masculino , Fosforilação/genética , Estabilidade Proteica , Receptor Muscarínico M3/genética , Fatores de Transcrição/genética , Transfecção
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