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1.
Nat Commun ; 12(1): 4878, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385447

RESUMO

A postprandial increase of translation mediated by eukaryotic Initiation Factor 6 (eIF6) occurs in the liver. Its contribution to steatosis and disease is unknown. In this study we address whether eIF6-driven translation contributes to disease progression. eIF6 levels increase throughout the progression from Non-Alcoholic Fatty Liver Disease (NAFLD) to hepatocellular carcinoma. Reduction of eIF6 levels protects the liver from disease progression. eIF6 depletion blunts lipid accumulation, increases fatty acid oxidation (FAO) and reduces oncogenic transformation in vitro. In addition, eIF6 depletion delays the progression from NAFLD to hepatocellular carcinoma, in vivo. Mechanistically, eIF6 depletion reduces the translation of transcription factor C/EBPß, leading to a drop in biomarkers associated with NAFLD progression to hepatocellular carcinoma and preserves mitochondrial respiration due to the maintenance of an alternative mTORC1-eIF4F translational branch that increases the expression of transcription factor YY1. We provide proof-of-concept that in vitro pharmacological inhibition of eIF6 activity recapitulates the protective effects of eIF6 depletion. We hypothesize the existence of a targetable, evolutionarily conserved translation circuit optimized for lipid accumulation and tumor progression.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Hepatopatia Gordurosa não Alcoólica/genética , Fatores de Iniciação de Peptídeos/genética , Biossíntese de Proteínas/genética , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Carcinoma Hepatocelular/metabolismo , Linhagem Celular , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Clofazimina/farmacologia , Dieta Hiperlipídica/efeitos adversos , Progressão da Doença , Inativação Gênica , Humanos , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/etiologia , Obesidade/genética , Obesidade/metabolismo , Fatores de Iniciação de Peptídeos/antagonistas & inibidores , Fatores de Iniciação de Peptídeos/metabolismo
2.
Sci Transl Med ; 13(588)2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33827977

RESUMO

Huntingtin (HTT)-lowering therapies hold promise to slow down neurodegeneration in Huntington's disease (HD). Here, we assessed the translatability and long-term durability of recombinant adeno-associated viral vector serotype 5 expressing a microRNA targeting human HTT (rAAV5-miHTT) administered by magnetic resonance imaging-guided convention-enhanced delivery in transgenic HD minipigs. rAAV5-miHTT (1.2 × 1013 vector genome (VG) copies per brain) was successfully administered into the striatum (bilaterally in caudate and putamen), using age-matched untreated animals as controls. Widespread brain biodistribution of vector DNA was observed, with the highest concentration in target (striatal) regions, thalamus, and cortical regions. Vector DNA presence and transgene expression were similar at 6 and 12 months after administration. Expression of miHTT strongly correlated with vector DNA, with a corresponding reduction of mutant HTT (mHTT) protein of more than 75% in injected areas, and 30 to 50% lowering in distal regions. Translational pharmacokinetic and pharmacodynamic measures in cerebrospinal fluid (CSF) were largely in line with the effects observed in the brain. CSF miHTT expression was detected up to 12 months, with CSF mHTT protein lowering of 25 to 30% at 6 and 12 months after dosing. This study demonstrates widespread biodistribution, strong and durable efficiency of rAAV5-miHTT in disease-relevant regions in a large brain, and the potential of using CSF analysis to determine vector expression and efficacy in the clinic.


Assuntos
Doença de Huntington , MicroRNAs , Animais , Modelos Animais de Doenças , Terapia Genética , Vetores Genéticos/genética , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/terapia , MicroRNAs/metabolismo , Suínos , Porco Miniatura/metabolismo , Distribuição Tecidual
3.
J Med Chem ; 64(4): 2139-2150, 2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33555858

RESUMO

The insulin-like peptide human relaxin-2 was identified as a hormone that, among other biological functions, mediates the hemodynamic changes occurring during pregnancy. Recombinant relaxin-2 (serelaxin) has shown beneficial effects in acute heart failure, but its full therapeutic potential has been hampered by its short half-life and the need for intravenous administration limiting its use to intensive care units. In this study, we report the development of long-acting potent single-chain relaxin peptide mimetics. Modifications in the B-chain of relaxin, such as the introduction of specific mutations and the trimming of the sequence to an optimal size, resulted in potent, structurally simplified peptide agonists of the relaxin receptor Relaxin Family Peptide Receptor 1 (RXFP1) (e.g., 54). Introduction of suitable spacers and fatty acids led to the identification of single-chain lipidated peptide agonists of RXFP1, with sub-nanomolar activity, high subcutaneous bioavailability, extended half-lives, and in vivo efficacy (e.g., 64).


Assuntos
Lipopeptídeos/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores de Peptídeos/agonistas , Relaxina/análogos & derivados , Relaxina/farmacologia , Sequência de Aminoácidos , Animais , Doenças Cardiovasculares , Linhagem Celular Tumoral , Células HEK293 , Meia-Vida , Humanos , Lipopeptídeos/genética , Lipopeptídeos/farmacocinética , Masculino , Simulação de Dinâmica Molecular , Estrutura Molecular , Mutação , Subunidades Proteicas , Ratos Sprague-Dawley , Relaxina/genética , Relação Estrutura-Atividade
4.
Free Radic Biol Med ; 162: 243-254, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33096251

RESUMO

Oxidative stress has been associated with pathogenesis in several diseases including Huntington's disease (HD), a neurodegenerative disorder caused by a mutation in the huntingtin gene. Oxidative stress induced reactive oxygen species (ROS) are normally controlled at the cellular level by the nuclear factor (erythroid-derived 2)-like 2 (NRF2) a transcription factor that regulates the expression of various antioxidants and detoxifying proteins. Normally NRF2 is largely inactivated in the cytoplasm by the Kelch-like ECH-associated protein 1 (KEAP1)/Cullin-3 (CUL3) mediated ubiquitination and subsequent proteosomal degradation. In the presence of ROS, KEAP1 sensor cysteines are directly or indirectly engaged resulting in NRF2 release, nuclear translocation, and activation of its target genes. Consequently the activation of NRF2 by a small-molecule drug may have the therapeutic potential to control oxidative stress by upregulation of the endogenous antioxidant responses. Here we attempted to validate the use of a reversible non-acidic KEAP1 binder (Compound 2) to activate NRF2 with better cellular activity than similar acidic compounds. When tested head to head with sulforaphane, a covalent KEAP1 binder, Compound 2 had a similar ability to induce the expression of genes known to be modulated by NRF2 in neurons and astrocytes isolated from wild-type rat, wild type mouse and zQ175 (an HD mouse model) embryos. However, while sulforaphane also negatively affected genes involved in neurotoxicity in these cells, Compound 2 showed a clean profile suggesting its mode of action has lower off-target activity. We show that Compound 2 was able to protect cells from an oxidative insult by preserving the ATP content and the mitochondrial potential of primary astrocytes, consistent with the hypothesis that neurotoxicity induced by oxidative stress can be limited by upregulation of innate antioxidant response.


Assuntos
Antioxidantes , Doença de Huntington , Animais , Astrócitos/metabolismo , Doença de Huntington/tratamento farmacológico , Doença de Huntington/genética , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Neurônios/metabolismo , Estresse Oxidativo , Ratos
5.
Sci Rep ; 10(1): 22137, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33335120

RESUMO

Huntington's disease (HD) is a monogenetic neurodegenerative disorder that is caused by the expansion of a polyglutamine region within the huntingtin (HTT) protein, but there is still an incomplete understanding of the molecular mechanisms that drive pathology. Expression of the mutant form of HTT is a key aspect of diseased tissues, and the most promising therapeutic approaches aim to lower expanded HTT levels. Consequently, the investigation of HTT expression in time and in multiple tissues, with assays that accurately quantify expanded and non-expanded HTT, are required to delineate HTT homeostasis and to best design and interpret pharmacodynamic readouts for HTT lowering therapeutics. Here we evaluate mutant polyglutamine-expanded (mHTT) and polyglutamine-independent HTT specific immunoassays for validation in human HD and control fibroblasts and use to elucidate the CSF/brain and peripheral tissue expression of HTT in preclinical HD models.


Assuntos
Expressão Gênica , Proteína Huntingtina/genética , Doença de Huntington/genética , Mutação , Animais , Biomarcadores , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Fibroblastos/metabolismo , Técnicas de Introdução de Genes , Humanos , Proteína Huntingtina/líquido cefalorraquidiano , Proteína Huntingtina/metabolismo , Doença de Huntington/tratamento farmacológico , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Imunoensaio , Imuno-Histoquímica , Camundongos , Ratos , Reprodutibilidade dos Testes
6.
PLoS Negl Trop Dis ; 14(10): e0008767, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33044962

RESUMO

Schistosomiasis is one of the most devastating neglected tropical parasitic diseases caused by trematodes of the genus Schistosoma. Praziquantel (PZQ) is today the only drug used in humans and animals for the treatment of schistosomiasis but unfortunately it is poorly effective on larval and juvenile stages of the parasite. Therefore, it is urgent the discovery of new drug targets and compounds. We have recently showed that the anti-anginal drug perhexiline maleate (PHX) is very active on multiple developmental stages of Schistosoma mansoni in vitro. It is well known that PHX impacts the lipid metabolism in mammals, but the final target on schistosomes still remains unknown. The aim of this study was to evaluate the ability of 1H nuclear magnetic resonance (NMR) spectroscopy in revealing metabolic perturbations due to PHX treatment of S. mansoni adult male worms. The effects of PHX were compared with the ones induced by vehicle and gambogic acid, in order to detect different metabolic profiles and specificity of the PHX action. Remarkably a list of metabolites associated to PHX-treatment was identified with enrichment in several connected metabolic pathways including also the Kennedy pathway mediating the glycerophospholipid metabolism. Our study represents the first 1H-NMR metabolomic approach to characterize the response of S. mansoni to drug treatment. The obtained "metabolic fingerprint" associated to PHX treatment could represent a strategy for displaying cellular metabolic changes for any given drug and to compare compounds targeting similar or distinct biochemical pathways.


Assuntos
Anti-Helmínticos/administração & dosagem , Monitoramento de Medicamentos/métodos , Espectroscopia de Prótons por Ressonância Magnética/métodos , Schistosoma mansoni/efeitos dos fármacos , Schistosoma mansoni/metabolismo , Esquistossomose mansoni/tratamento farmacológico , Adulto , Animais , Feminino , Humanos , Masculino , Metaboloma/efeitos dos fármacos , Camundongos Endogâmicos ICR , Perexilina/administração & dosagem , Perexilina/análogos & derivados , Praziquantel/administração & dosagem , Schistosoma mansoni/crescimento & desenvolvimento , Esquistossomose mansoni/parasitologia
7.
Bioorg Med Chem ; 28(21): 115738, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-33065433

RESUMO

Inhibition of KEAP1-NRF2 protein-protein interaction is considered a promising strategy to selectively and effectively activate NRF2, a transcription factor which is involved in several pathologies such as Huntington's disease (HD). A library of linear peptides based on the NRF2-binding motifs was generated on the nonapeptide lead Ac-LDEETGEFL-NH2 spanning residues 76-84 of the Neh2 domain of NRF2 with the aim to replace E78, E79 and E82 with non-acidic amino acids. A deeper understanding of the features and accessibility of the T80 subpocket was also targeted by structure-based design. Approaches to improve cell permeability were investigated using both different classes of cyclic peptides and conjugation to cell-penetrating peptides. This insight will guide future design of macrocycles, peptido-mimetics and, most importantly, small neutral brain-penetrating molecules to evaluate whether NRF2 activators have utility in HD.


Assuntos
Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Peptídeos Cíclicos/química , Peptídeos/química , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Desenho de Fármacos , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/antagonistas & inibidores , Simulação de Dinâmica Molecular , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Peptídeos/metabolismo , Peptídeos/farmacologia , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacologia , Ligação Proteica , Relação Estrutura-Atividade
8.
ACS Med Chem Lett ; 11(5): 740-746, 2020 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-32435379

RESUMO

The NRF2-ARE pathway is an intrinsic mechanism of defense against oxidative stress. Inhibition of the interaction between NRF2 and its main negative regulator KEAP1 is an attractive strategy toward neuroprotective agents. We report here the identification of nonacidic tetrahydroisoquinolines (THIQs) that inhibit the KEAP1/NRF2 protein-protein interaction. Peptide SAR at one residue is utilized as a tool to probe structural changes within a specific pocket of the KEAP1 binding site. We used structural information from peptide screening at the P2 pocket, noncovalent small-molecules inhibitors, and the outcome from an explorative SAR at position 5 of THIQs to identify a series of neutral THIQ analogs that bind to KEAP1 in the low micromolar range. These analogs establish new H-bond interactions at the P3 and P2 pockets allowing the replacement of the carboxylic acid functionality by a neutral primary carboxamide. X-ray crystallographic studies reveal the novel binding mode of these molecules to KEAP1.

9.
PLoS Negl Trop Dis ; 14(5): e0008339, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32437349

RESUMO

Trypanothione reductase (TR) is a key enzyme that catalyzes the reduction of trypanothione, an antioxidant dithiol that protects Trypanosomatid parasites from oxidative stress induced by mammalian host defense systems. TR is considered an attractive target for the development of novel anti-parasitic agents as it is essential for parasite survival but has no close homologue in humans. We report here the identification of spiro-containing derivatives as inhibitors of TR from Trypanosoma brucei (TbTR), the parasite responsible for Human African Trypanosomiasis. The hit series, identified by high throughput screening, was shown to bind TbTR reversibly and to compete with the trypanothione (TS2) substrate. The prototype compound 1 from this series was also found to impede the growth of Trypanosoma brucei parasites in vitro. The X-ray crystal structure of TbTR in complex with compound 1 solved at 1.98 Å allowed the identification of the hydrophobic pocket where the inhibitor binds, placed close to the catalytic histidine (His 461') and lined by Trp21, Val53, Ile106, Tyr110 and Met113. This new inhibitor is specific for TbTR and no activity was detected against the structurally similar human glutathione reductase (hGR). The central spiro scaffold is known to be suitable for brain active compounds in humans thus representing an attractive starting point for the future treatment of the central nervous system stage of T. brucei infections.


Assuntos
Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , NADH NADPH Oxirredutases/antagonistas & inibidores , Tolueno/análogos & derivados , Trypanosoma brucei brucei/efeitos dos fármacos , Antiprotozoários/isolamento & purificação , Sítios de Ligação , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/isolamento & purificação , Ensaios de Triagem em Larga Escala , NADH NADPH Oxirredutases/química , Ligação Proteica , Conformação Proteica , Tolueno/isolamento & purificação , Tolueno/farmacologia , Trypanosoma brucei brucei/enzimologia
10.
Cell Death Differ ; 27(10): 2921-2941, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32382110

RESUMO

Fibro/Adipogenic Progenitors (FAPs) are muscle-interstitial progenitors mediating pro-myogenic signals that are critical for muscle homeostasis and regeneration. In myopathies, the autocrine/paracrine constraints controlling FAP adipogenesis are released causing fat infiltrates. Here, by combining pharmacological screening, high-dimensional mass cytometry and in silico network modeling with the integration of single-cell/bulk RNA sequencing data, we highlighted the canonical WNT/GSK/ß-catenin signaling as a crucial pathway modulating FAP adipogenesis triggered by insulin signaling. Consistently, pharmacological blockade of GSK3, by the LY2090314 inhibitor, stabilizes ß-catenin and represses PPARγ expression abrogating FAP adipogenesis ex vivo while limiting fatty degeneration in vivo. Furthermore, GSK3 inhibition improves the FAP pro-myogenic role by efficiently stimulating, via follistatin secretion, muscle satellite cell (MuSC) differentiation into mature myotubes. Combining, publicly available single-cell RNAseq datasets, we characterize FAPs as the main source of WNT ligands inferring their potential in mediating autocrine/paracrine responses in the muscle niche. Lastly, we identify WNT5a, whose expression is impaired in dystrophic FAPs, as a crucial WNT ligand able to restrain the detrimental adipogenic differentiation drift of these cells through the positive modulation of the ß-catenin signaling.

11.
Methods Mol Biol ; 2151: 219-227, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32452008

RESUMO

Schistosomiasis is one of the major parasitic diseases with more than  200 million people infected worldwide every year. Praziquantel is the drug of choice against the schistosomiasis although the use of a single drug to treat such a large amount of infected people appears particularly worrisome. For this reason, the search of new schistosomicidal compounds is viewed as an urgent goal and a number of screening campaigns have been carried out in the past years. The larval stage of Schistosoma (schistosomula) has been widely used in order to identify new compounds against the parasite. Here we describe detailed practical procedures for a luminescence-based assay proven to be highly effective for the selection of schistosomicidal compounds on small and medium-high scale. The assay is based on the quantitation of the parasite ATP, a good indicator of metabolically active cells, as measure of schistosomula viability. This assay is fast and reproducible, and it is suitable either for manual or for semiautomated screenings.


Assuntos
Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala/métodos , Luminescência , Schistosoma mansoni/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sobrevivência Celular , Cercárias/genética , Larva/metabolismo , Transformação Genética
12.
Bioorg Med Chem Lett ; 30(8): 127052, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32113841

RESUMO

The identification of a new series of growth inhibitors of Trypanosoma cruzi, the causative agent of Chagas' disease, is described. In vitro screening of a subset of compounds from our in-house compound collection against the parasite led to the identification of hit compound 1 with low micromolar inhibition of T. cruzi growth. SAR exploration on the hit compound led to the identification of compounds that show nanomolar parasite growth inhibition (T. cruzi EC50 ≤ 100 nM) and no cytotoxicity in human cells (HeLa CC50 > 50 µM). Further investigation identified CYP51 inhibition (compound 11 CYP51 IC50 52 nM) as a possible mechanism of action of this new class of anti-parasitic agents.


Assuntos
Descoberta de Drogas , Inibidores do Crescimento/farmacologia , Piridinas/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores do Crescimento/síntese química , Inibidores do Crescimento/química , Humanos , Estrutura Molecular , Testes de Sensibilidade Parasitária , Piridinas/síntese química , Piridinas/química , Relação Estrutura-Atividade , Tripanossomicidas/síntese química , Tripanossomicidas/química
13.
Sci Rep ; 10(1): 5363, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32210313

RESUMO

Muscle resident fibro-adipogenic progenitors (FAPs), support muscle regeneration by releasing cytokines that stimulate the differentiation of myogenic stem cells. However, in non-physiological contexts (myopathies, atrophy, aging) FAPs cause fibrotic and fat infiltrations that impair muscle function. We set out to perform a fluorescence microscopy-based screening to identify compounds that perturb the differentiation trajectories of these multipotent stem cells. From a primary screen of 1,120 FDA/EMA approved drugs, we identified 34 compounds as potential inhibitors of adipogenic differentiation of FAPs isolated from the murine model (mdx) of Duchenne muscular dystrophy (DMD). The hit list from this screen was surprisingly enriched with compounds from the glucocorticoid (GCs) chemical class, drugs that are known to promote adipogenesis in vitro and in vivo. To shed light on these data, three GCs identified in our screening efforts were characterized by different approaches. We found that like dexamethasone, budesonide inhibits adipogenesis induced by insulin in sub-confluent FAPs. However, both drugs have a pro-adipogenic impact when the adipogenic mix contains factors that increase the concentration of cAMP. Gene expression analysis demonstrated that treatment with glucocorticoids induces the transcription of Gilz/Tsc22d3, an inhibitor of the adipogenic master regulator PPARγ, only in anti-adipogenic conditions. Additionally, alongside their anti-adipogenic effect, GCs are shown to promote terminal differentiation of satellite cells. Both the anti-adipogenic and pro-myogenic effects are mediated by the glucocorticoid receptor and are not observed in the presence of receptor inhibitors. Steroid administration currently represents the standard treatment for DMD patients, the rationale being based on their anti-inflammatory effects. The findings presented here offer new insights on additional glucocorticoid effects on muscle stem cells that may affect muscle homeostasis and physiology.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Glucocorticoides/farmacologia , Desenvolvimento Muscular/efeitos dos fármacos , Músculo Esquelético/citologia , Adipogenia/efeitos dos fármacos , Animais , Budesonida/administração & dosagem , Budesonida/farmacologia , Diferenciação Celular/fisiologia , Células Cultivadas , AMP Cíclico/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Microscopia de Fluorescência , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/patologia , PPAR gama/metabolismo , Receptores de Glucocorticoides/metabolismo , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Células Satélites de Músculo Esquelético/patologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/patologia , Fatores de Transcrição/metabolismo
14.
Cells ; 9(1)2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31936702

RESUMO

Eukaryotic initiation factor 6 (eIF6) is necessary for the nucleolar biogenesis of 60S ribosomes. However, most of eIF6 resides in the cytoplasm, where it acts as an initiation factor. eIF6 is necessary for maximal protein synthesis downstream of growth factor stimulation. eIF6 is an antiassociation factor that binds 60S subunits, in turn preventing premature 40S joining and thus the formation of inactive 80S subunits. It is widely thought that eIF6 antiassociation activity is critical for its function. Here, we exploited and improved our assay for eIF6 binding to ribosomes (iRIA) in order to screen for modulators of eIF6 binding to the 60S. Three compounds, eIFsixty-1 (clofazimine), eIFsixty-4, and eIFsixty-6 were identified and characterized. All three inhibit the binding of eIF6 to the 60S in the micromolar range. eIFsixty-4 robustly inhibits cell growth, whereas eIFsixty-1 and eIFsixty-6 might have dose- and cell-specific effects. Puromycin labeling shows that eIF6ixty-4 is a strong global translational inhibitor, whereas the other two are mild modulators. Polysome profiling and RT-qPCR show that all three inhibitors reduce the specific translation of well-known eIF6 targets. In contrast, none of them affect the nucleolar localization of eIF6. These data provide proof of principle that the generation of eIF6 translational modulators is feasible.


Assuntos
Fatores de Iniciação de Peptídeos/metabolismo , Biossíntese de Proteínas , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Linhagem Celular , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Sobrevivência Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Polirribossomos/efeitos dos fármacos , Polirribossomos/metabolismo , Ligação Proteica/efeitos dos fármacos , Puromicina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes
15.
Anal Bioanal Chem ; 411(29): 7669-7680, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31273412

RESUMO

There is a general agreement that pharmacologically mediated stimulation of human γ-globin gene expression and increase of production of fetal hemoglobin (HbF) is a potential therapeutic approach in the experimental therapy of ß-thalassemia and sickle cell anemia. Here, we report the development and characterization of cellular biosensors carrying enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP) genes under the control of the human γ-globin and ß-globin gene promoters, respectively; these dual-reporter cell lines are suitable to identify the induction ability of screened compounds on the transcription in erythroid cells of γ-globin and ß-globin genes by FACS with efficiency and reproducibility. Our experimental system allows to identify (a) HbF inducers stimulating to different extent the activity of the γ-globin gene promoter and (b) molecules that stimulate also the activity of the ß-globin gene promoter. A good correlation does exist between the results obtained by using the EGFP/RFP clones and experiments performed on erythroid precursor cells from ß-thalassemic patients, confirming that this experimental system can be employed for high-throughput screening (HTS) analysis. Finally, we have demonstrated that this dual-reporter cell line can be used for HTS in 384-well plate, in order to identify novel HbF inducers for the therapy of ß-thalassemia and sickle cell anemia. Graphical abstract.


Assuntos
Técnicas Biossensoriais , Diferenciação Celular/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Regiões Promotoras Genéticas , Transcrição Genética , Globinas beta/genética , gama-Globinas/genética , Eritrócitos/citologia , Hemoglobina Fetal/genética , Proteínas de Fluorescência Verde/genética , Humanos , Células K562 , Reprodutibilidade dos Testes
16.
Int J Syst Evol Microbiol ; 69(9): 2899-2906, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31274407

RESUMO

A facultative halo-tolerant Aspergillus strain was isolated from olive brine waste, the effluent from the debittering process of table olives. Phenotypic and molecular characteristics showed clearly that the isolate represents a novel species. Based on the source of isolation, the new species has been named Aspergillus olivimuriae. It was found tolerant to high concentrations of NaCl (15 %) or sucrose (60 %) and it exhibits substantial growth under these conditions. Although the new species grew profusely at 37 °C, no growth was observed at 40 °C, conidia en masse were avellaneous on all media. The description of the new species Aspergillus olivimuriae brings the total species of Aspergillus sect. Flavipedes to 15. The type strain of A. olivimuriae sp. nov. is NRRL 66783 (CCF 6208), its whole genome has been deposited as PRJNA498048.


Assuntos
Aspergillus/classificação , Microbiologia de Alimentos , Olea/microbiologia , Filogenia , Sais , Aspergillus/isolamento & purificação , DNA Fúngico/genética , Técnicas de Tipagem Micológica , Pigmentação , Análise de Sequência de DNA , Esporos Fúngicos
17.
J Clin Invest ; 129(6): 2390-2403, 2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-31063986

RESUMO

A disintegrine and metalloproteinase 10 (ADAM10) is implicated in synaptic function through its interaction with postsynaptic receptors and adhesion molecules. Here, we report that levels of active ADAM10 are increased in Huntington's disease (HD) mouse cortices and striata and in human postmortem caudate. We show that, in the presence of polyglutamine-expanded (polyQ-expanded) huntingtin (HTT), ADAM10 accumulates at the postsynaptic densities (PSDs) and causes excessive cleavage of the synaptic protein N-cadherin (N-CAD). This aberrant phenotype is also detected in neurons from HD patients where it can be reverted by selective silencing of mutant HTT. Consistently, ex vivo delivery of an ADAM10 synthetic inhibitor reduces N-CAD proteolysis and corrects electrophysiological alterations in striatal medium-sized spiny neurons (MSNs) of 2 HD mouse models. Moreover, we show that heterozygous conditional deletion of ADAM10 or delivery of a competitive TAT-Pro-ADAM10709-729 peptide in R6/2 mice prevents N-CAD proteolysis and ameliorates cognitive deficits in the mice. Reduction in synapse loss was also found in R6/2 mice conditionally deleted for ADAM10. Taken together, these results point to a detrimental role of hyperactive ADAM10 at the HD synapse and provide preclinical evidence of the therapeutic potential of ADAM10 inhibition in HD.


Assuntos
Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Disfunção Cognitiva/enzimologia , Doença de Huntington/enzimologia , Proteínas de Membrana/metabolismo , Densidade Pós-Sináptica/enzimologia , Proteína ADAM10/genética , Adulto , Idoso , Secretases da Proteína Precursora do Amiloide/genética , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Disfunção Cognitiva/genética , Disfunção Cognitiva/patologia , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Doença de Huntington/genética , Doença de Huntington/patologia , Masculino , Proteínas de Membrana/genética , Camundongos Transgênicos , Pessoa de Meia-Idade , Densidade Pós-Sináptica/genética , Densidade Pós-Sináptica/patologia
18.
ACS Med Chem Lett ; 10(4): 481-486, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30996783

RESUMO

The application of class I HDAC inhibitors as cancer therapies is well established, but more recently their development for nononcological indications has increased. We report here on the generation of improved class I selective human HDAC inhibitors based on an ethylketone zinc binding group (ZBG) in place of the hydroxamic acid that features the majority of HDAC inhibitors. We also describe a novel set of HDAC3 isoform selective inhibitors that show stronger potency and selectivity than the most commonly used HDAC3 selective tool compound RGFP966. These compounds are again based on an alternative ZBG with respect to the ortho-anilide that is featured in HDAC3 selective compounds reported to date.

19.
Dev Comp Immunol ; 96: 9-17, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30790604

RESUMO

The increasing resistance to conventional antibiotics is an urgent problem that can be addressed by the discovery of new antimicrobial drugs such as antimicrobial peptides (AMPs). AMPs are components of innate immune system of eukaryotes and are not prone to the conventional mechanisms that are responsible of drug resistance. Fish are an important source of AMPs and, recently, we have isolated and characterized a new 22 amino acid residues peptide, the chionodracine (Cnd), from the Antarctic icefish Chionodraco hamatus. In this paper we focused on a new Cnd-derived mutant peptide, namely Cnd-m3a, designed to improve the selectivity against prokaryotic cells and the antimicrobial activity against human pathogens of the initial Cnd template. Cnd-m3a was used for immunization of rabbits, which gave rise to a polyclonal antibody able to detect the peptide. The interaction kinetic of Cnd-m3a with the Antarctic bacterium Psychrobacter sp. (TAD1) was imaged using a transmission electron microscopy (TEM) immunogold method. Initially the peptide was associated with the plasma membrane, but after 180 min of incubation, it was found in the cytoplasm interacting with a DNA target inside the bacterial cells. Using fluorescent probes we showed that the newly designed mutant can create pores in the outer membrane of the bacteria E. coli and Psychrobacter sp. (TAD1), confirming the results of TEM analysis. Moreover, in vitro assays demonstrated that Cnd-m3a is able to bind lipid vesicles of different compositions with a preference toward negatively charged ones, which mimics the prokaryotic cell. The Cnd-m3a peptide showed quite low hemolytic activity and weak cytotoxic effect against human primary and tumor cell lines, but high antimicrobial activity against selected Gram - human pathogens. These results highlighted the high potential of the Cnd-m3a peptide as a starting point for developing a new human therapeutic agent.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Escherichia coli/efeitos dos fármacos , Proteínas de Peixes/farmacologia , Psychrobacter/efeitos dos fármacos , Animais , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Linhagem Celular Tumoral , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Citoplasma/efeitos dos fármacos , Citoplasma/ultraestrutura , Desenho de Fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/fisiologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Transmissão , Mutação , Psychrobacter/fisiologia , Coelhos , Testes de Toxicidade
20.
Parasit Vectors ; 11(1): 668, 2018 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-30587243

RESUMO

BACKGROUND: Novel anti-schistosomal multi-stage drugs are needed because only a single drug, praziquantel, is available for the treatment of schistosomiasis and is poorly effective on larval and juvenile stages of the parasite. Schistosomes have a complex life-cycle and multiple developmental stages in the intermediate and definitive hosts. Acetylation and deacetylation of histones play pivotal roles in chromatin structure and in the regulation of transcription in eukaryotic cells. Histone deacetylase (HDAC) inhibitors modulate acetylation of several other proteins localized both in the nucleus and in the cytoplasm and therefore impact on many signaling networks and biological processes. Histone post-translational modifications may provide parasites with the ability to readily adapt to changes in gene expression required for their development and adaptation to the host environment. The aim of the present study was to screen a HDAC class I inhibitor library in order to identify and characterize novel multi-stage hit compounds. METHODS: We used a high-throughput assay based on the quantitation of ATP in the Schistosoma mansoni larval stage (schistosomula) and screened a library of 1500 class I HDAC inhibitors. Subsequently, a few hits were selected and further characterized by viability assays and phenotypic analyses on adult parasites by carmine red and confocal microscopy. RESULTS: Three compounds (SmI-124, SmI-148 and SmI-558) that had an effect on the viability of both the schistosomula larval stage and the adult worm were identified. Treatment with sub-lethal doses of SmI-148 and SmI-558 also decreased egg production. Moreover, treatment of adult parasites with SmI-148, and to a lesser extent Sm-124, was associated with histone hyperacetylation. Finally, SmI-148 and SmI-558 treatments of worm pairs caused a phenotype characterized by defects in the parasite reproductive system, with peculiar features in the ovary. In addition, SmI-558 induced oocyte- and vitelline cell-engulfment and signs of degeneration in the uterus and/or oviduct. CONCLUSIONS: We report the screening of a small HDAC inhibitor library and the identification of three novel compounds which impair viability of the S. mansoni larval stage and adult pairs. These compounds are useful tools for studying deacetylase activity during parasite development and for interfering with egg production. Characterization of their specificity for selected S. mansoni versus human HDAC could provide insights that can be used in optimization and compound design.


Assuntos
Anti-Helmínticos/administração & dosagem , Inibidores de Histona Desacetilases/administração & dosagem , Óvulo/efeitos dos fármacos , Schistosoma mansoni/efeitos dos fármacos , Schistosoma mansoni/crescimento & desenvolvimento , Esquistossomose/tratamento farmacológico , Acetilação , Animais , Anti-Helmínticos/química , Feminino , Proteínas de Helminto/antagonistas & inibidores , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Inibidores de Histona Desacetilases/química , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Estágios do Ciclo de Vida/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Óvulo/crescimento & desenvolvimento , Óvulo/metabolismo , Schistosoma mansoni/enzimologia , Schistosoma mansoni/genética , Esquistossomose/parasitologia
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