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1.
Medicine (Baltimore) ; 98(26): e15858, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31261494

RESUMO

This analysis aims to describe the outcomes of two nonambulatory patients with Duchenne muscular dystrophy (DMD) who participated in two clinical studies. The two consecutive trials of eteplirsen (studies 201 and 202) were conducted in patients with DMD (N = 12) and confirmed genetic mutations amenable to exon 51 skipping.In study 201, 12 patients were randomized to receive once-weekly, double-blind intravenous infusions of eteplirsen 30 or 50 mg/kg or placebo for 24 weeks; patients then received open-label eteplirsen during weeks 25 through 28. All 12 patients continued onto open-label extension study 202 and received long-term treatment with eteplirsen. We compared cardiac, pulmonary, and upper limb function and dystrophin production in the nonambulatory twin patients versus the 10 ambulatory patients through 240 combined treatment weeks.Ten study patients remained ambulatory through both studies, while the identical twin patients both experienced early, rapid loss of ambulation. The twin patients had greater disease severity at baseline (6-minute walk test [6MWT], 330 and 256 m) versus the other patients (n = 10; 6MWT range, 341-418 m). They maintained cardiac and upper limb function through combined week 240, with outcomes similar to those of the patients who remained ambulatory. Dystrophin production was confirmed following eteplirsen treatment.Despite the loss of ambulation, other markers of disease progression remained relatively stable in the eteplirsen-treated twin patients and were similar to those of the ambulatory patients.


Assuntos
Morfolinos/uso terapêutico , Distrofia Muscular de Duchenne/tratamento farmacológico , Criança , Progressão da Doença , Doenças em Gêmeos , Método Duplo-Cego , Distrofina/genética , Distrofina/metabolismo , Humanos , Masculino , Morfolinos/efeitos adversos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/fisiopatologia , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Índice de Gravidade de Doença , Resultado do Tratamento , Teste de Caminhada , Caminhada
2.
J Antibiot (Tokyo) ; 72(4): 225-236, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30737453

RESUMO

In bacteria, RNase III cleaves the initial long primary ribosomal RNA transcripts/precursors (pre-rRNAs), thereby releasing the pre-16S and pre-23S rRNAs for maturation. This cleavage is specified by the double-stranded secondary structures flanking the mature rRNAs, and not necessarily by the nucleotide sequences. Inhibition of this cleavage would lead to a build-up of pre-rRNA molecules. Doxycycline has earlier been shown to bind synthetic double-stranded RNAs and inhibit their cleavage by RNase III. Since bacterial rRNA processing is primarily dependent on RNase III cleavage (which is inhibited by doxycycline), doxycycline could therefore inhibit the normal processing of bacterial rRNA. In this study, the effect of doxycycline on bacterial rRNA processing was investigated by analyzing the amounts of various rRNAs in growing Escherichia coli cells treated with doxycycline. The results showed a doxycycline dose-dependent decrease in mature 16S and 23S rRNAs, concurrent with an accumulation of the initial rRNA transcripts and long precursors. Morphologically, treated cells were elongated at low drug concentrations, while nucleoid degeneration indicative of cell death occurred at higher drug concentrations. These observations suggest that doxycycline inhibits the cleavage and processing of bacterial rRNA transcripts/precursors, leading to impaired formation of mature rRNAs, and the consequent inhibition of protein synthesis for which the tetracycline group of antibiotics are renowned. Since rRNA structure and processing pathway is conserved among bacterial species, this mechanism may account for the broad spectrum of antibiotic activity and selective microbial protein synthesis inhibition of doxycycline and the tetracyclines.


Assuntos
Antibacterianos/farmacologia , Doxiciclina/farmacologia , Escherichia coli/efeitos dos fármacos , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Ribossômico 16S/metabolismo , RNA Ribossômico 23S/metabolismo
3.
Curr Med Chem ; 26(33): 6020-6032, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30501594

RESUMO

BACKGROUND: Inherent or acquired chemo resistance in cancer patients has been a perpetual limitation in cancer treatment. Expanding knowledge on essential cellular processes opens a new window for therapeutic targeting. Ribosome biogenesis is a process that shows potential due to its fundamental role in cell development and contribution to tumorigenesis as a result of its upregulation. Inhibiting components of ribosome biogenesis has been explored and has shown interesting results. Yet, an important key component, methyltransferase Fibrillarin (FBL), which influences both the abundance and composition of ribosomes, has not been exploited thus far. METHODS: In this literature review, we describe relevant aspects of ribosome biogenesis in cancer to emphasize the potential of FBL as a therapeutic target, in order to lower the genotoxic effects of anti-cancer treatment. RESULTS: Remarkably, the amplification of the 19q13 cytogenetic band, including the gene coding for FBL, correlated to cell viability and resistance in pancreatic cells as well as to a trend toward a shorter survival in pancreatic cancer patients. Targeting ribosome biogenesis, more specifically compared to the secondary effects of chemotherapeutics such as 5-fluorouracil or oxaliplatin, has been achieved by compound CX-5461. The cell dependent activity of this Pol I inhibitor has been reported in ovarian cancer, melanoma and leukemia models with active or mutated p53 status, presenting a promising mechanism to evade p53 resistance. CONCLUSION: Targeting critical ribosome biogenesis components in order to decrease the genotoxic activity in cancer cell looks promising. Hence, we believe that targeting key protein rRNA methyltransferase FBL shows great potential, due to its pivotal role in ribosome biogenesis, its correlation to an improved survival rate at low expression in breast cancer patients and its association with p53.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Resistência a Medicamentos , Ribossomos/metabolismo , Antimetabólitos Antineoplásicos/química , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , Proteínas Cromossômicas não Histona/química , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Ribossomos/genética , Transcrição Genética/efeitos dos fármacos , Viroses/tratamento farmacológico , Viroses/metabolismo
4.
Methods Mol Biol ; 1870: 249-262, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30539561

RESUMO

Expression of genetic information is a multistep process which needs to be tightly regulated. One of the regulatory mechanisms is posttranscriptional modification of RNA, which can alter the stability, expression, or protein composition. Therefore, misregulation of this important cellular process can lead to pathological consequences, such as cancer development. It has been shown that alteration in the expression of certain RNA-modifying genes can promote tumorigenesis. Here, we present a mRNA expression analysis-based approach to comprehensively determine the expression of RNA readers/writers/erasers using DNA damage as an example, and then to validate the effect of altered RNA reader/writer/erasers in regulating the DNA damage response.


Assuntos
Dano ao DNA , Regulação da Expressão Gênica , RNA/genética , Antineoplásicos/farmacologia , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Raios gama , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/efeitos da radiação , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
5.
Cell Rep ; 25(12): 3519-3529.e2, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30566874

RESUMO

Cells control their size by coordinating cell cycle progression with volume growth. Size control is typically studied at specific cell cycle transitions that are delayed or accelerated depending on size. This focus is well suited for revealing mechanisms acting at these transitions, but neglects the dynamics in other cell cycle phases, and is therefore inherently limited for studying how the characteristic cell size is determined. We address this limitation through a formalism that intuitively visualizes the characteristic size emerging from integrated cell cycle dynamics of individual cells. Applying this formalism to budding yeast, we describe the contributions of the un-budded (G1) and budded (S-G2-M) phase to size adjustments following environmental or genetic perturbations. We show that although the budded phase can be perturbed with little consequences for G1 dynamics, perturbations in G1 propagate to the budded phase. Our study provides an integrated view on cell size determinants in budding yeast.


Assuntos
Tamanho Celular , Saccharomyces cerevisiae/citologia , Carbono/farmacologia , Ciclo Celular/efeitos dos fármacos , Mutação/genética , Biossíntese de Proteínas/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento
6.
Development ; 145(20)2018 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-30337486

RESUMO

A growing number of tissue-specific inherited disorders are associated with impaired ribosome production, despite the universal requirement for ribosome function. Recently, mutations in RPSA, a protein component of the small ribosomal subunit, were discovered to underlie approximately half of all isolated congenital asplenia cases. However, the mechanisms by which mutations in this ribosome biogenesis factor lead specifically to spleen agenesis remain unknown, in part due to the lack of a suitable animal model for study. Here we reveal that RPSA is required for normal spleen development in the frog, Xenopus tropicalis Depletion of Rpsa in early embryonic development disrupts pre-rRNA processing and ribosome biogenesis, and impairs expression of the key spleen patterning genes nkx2-5, bapx1 and pod1 in the spleen anlage. Importantly, we also show that whereas injection of human RPSA mRNA can rescue both pre-rRNA processing and spleen patterning, injection of human mRNA bearing a common disease-associated mutation cannot. Together, we present the first animal model of RPSA-mediated asplenia and reveal a crucial requirement for RPSA in pre-rRNA processing and molecular patterning during early Xenopus development.


Assuntos
Estudos de Associação Genética , Síndromes de Imunodeficiência/genética , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA/genética , Proteínas Ribossômicas/genética , Baço/anormalidades , Baço/embriologia , Proteínas de Xenopus/genética , Xenopus/embriologia , Xenopus/genética , Animais , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Síndromes de Imunodeficiência/embriologia , Morfolinos/farmacologia , Mutação/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Proteínas Ribossômicas/metabolismo , Baço/efeitos dos fármacos , Baço/metabolismo , Proteínas de Xenopus/metabolismo
7.
Sci Rep ; 8(1): 13780, 2018 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-30214075

RESUMO

Post-transcriptional processes have been recognised as pivotal in the control of gene expression, and impairments in RNA processing are reported in several pathologies (i.e., cancer and neurodegeneration). Focusing on RNA-binding proteins (RBPs), the involvement of Embryonic Lethal Abnormal Vision (ELAV) or Hu proteins and their complexes with target mRNAs in the aetiology of various dysfunctions, has suggested the great potential of compounds able to interfere with the complex stability as an innovative pharmacological strategy for the treatment of numerous diseases. Here, we present a rational follow-up investigation of the interaction between ELAV isoform HuR and structurally-related compounds (i.e., flavonoids and coumarins), naturally decorated with different functional groups, by means of STD-NMR and Molecular Modelling. Our results represent the foundation for the development of potent and selective ligands able to interfere with ELAV-RNA complexes.


Assuntos
Cumarínicos/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Flavonoides/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Proteína Semelhante a ELAV 1/genética , Humanos , Ligantes , Imagem por Ressonância Magnética , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , RNA Mensageiro/genética
8.
PLoS Pathog ; 14(9): e1007315, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30252911

RESUMO

Kinetoplastid parasites-trypanosomes and leishmanias-infect millions of humans and cause economically devastating diseases of livestock, and the few existing drugs have serious deficiencies. Benzoxaborole-based compounds are very promising potential novel anti-trypanosomal therapies, with candidates already in human and animal clinical trials. We investigated the mechanism of action of several benzoxaboroles, including AN7973, an early candidate for veterinary trypanosomosis. In all kinetoplastids, transcription is polycistronic. Individual mRNA 5'-ends are created by trans splicing of a short leader sequence, with coupled polyadenylation of the preceding mRNA. Treatment of Trypanosoma brucei with AN7973 inhibited trans splicing within 1h, as judged by loss of the Y-structure splicing intermediate, reduced levels of mRNA, and accumulation of peri-nuclear granules. Methylation of the spliced leader precursor RNA was not affected, but more prolonged AN7973 treatment caused an increase in S-adenosyl methionine and methylated lysine. Together, the results indicate that mRNA processing is a primary target of AN7973. Polyadenylation is required for kinetoplastid trans splicing, and the EC50 for AN7973 in T. brucei was increased three-fold by over-expression of the T. brucei cleavage and polyadenylation factor CPSF3, identifying CPSF3 as a potential molecular target. Molecular modeling results suggested that inhibition of CPSF3 by AN7973 is feasible. Our results thus chemically validate mRNA processing as a viable drug target in trypanosomes. Several other benzoxaboroles showed metabolomic and splicing effects that were similar to those of AN7973, identifying splicing inhibition as a common mode of action and suggesting that it might be linked to subsequent changes in methylated metabolites. Granule formation, splicing inhibition and resistance after CPSF3 expression did not, however, always correlate and prolonged selection of trypanosomes in AN7973 resulted in only 1.5-fold resistance. It is therefore possible that the modes of action of oxaboroles that target trypanosome mRNA processing might extend beyond CPSF3 inhibition.


Assuntos
Benzoxazóis/farmacologia , RNA de Protozoário/metabolismo , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/metabolismo , Animais , Benzoxazóis/química , Bovinos , Resistência a Medicamentos/genética , Cabras , Humanos , Camundongos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , Trans-Splicing/efeitos dos fármacos , Tripanossomicidas/química , Trypanosoma brucei brucei/genética , Trypanosoma congolense/efeitos dos fármacos , Trypanosoma congolense/genética , Trypanosoma congolense/metabolismo , Trypanosoma vivax/efeitos dos fármacos , Trypanosoma vivax/genética , Trypanosoma vivax/metabolismo , Tripanossomíase/tratamento farmacológico , Tripanossomíase/parasitologia
9.
Proc Natl Acad Sci U S A ; 115(38): 9616-9621, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30185555

RESUMO

African trypanosomes cause lethal and neglected tropical diseases, known as sleeping sickness in humans and nagana in animals. Current therapies are limited, but fortunately, promising therapies are in advanced clinical and veterinary development, including acoziborole (AN5568 or SCYX-7158) and AN11736, respectively. These benzoxaboroles will likely be key to the World Health Organization's target of disease control by 2030. Their mode of action was previously unknown. We have developed a high-coverage overexpression library and use it here to explore drug mode of action in Trypanosoma brucei Initially, an inhibitor with a known target was used to select for drug resistance and to test massive parallel library screening and genome-wide mapping; this effectively identified the known target and validated the approach. Subsequently, the overexpression screening approach was used to identify the target of the benzoxaboroles, Cleavage and Polyadenylation Specificity Factor 3 (CPSF3, Tb927.4.1340). We validated the CPSF3 endonuclease as the target, using independent overexpression strains. Knockdown provided genetic validation of CPSF3 as essential, and GFP tagging confirmed the expected nuclear localization. Molecular docking and CRISPR-Cas9-based editing demonstrated how acoziborole can specifically block the active site and mRNA processing by parasite, but not host CPSF3. Thus, our findings provide both genetic and chemical validation for CPSF3 as an important drug target in trypanosomes and reveal inhibition of mRNA maturation as the mode of action of the trypanocidal benzoxaboroles. Understanding the mechanism of action of benzoxaborole-based therapies can assist development of improved therapies, as well as the prediction and monitoring of resistance, if or when it arises.


Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/fisiologia , Tripanossomíase Africana/prevenção & controle , Animais , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Compostos de Boro/farmacologia , Compostos de Boro/uso terapêutico , Sistemas CRISPR-Cas , Núcleo Celular/genética , Núcleo Celular/metabolismo , Fator de Especificidade de Clivagem e Poliadenilação/genética , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Resistência a Medicamentos/efeitos dos fármacos , Resistência a Medicamentos/genética , Técnicas de Silenciamento de Genes , Biblioteca Gênica , Ensaios de Triagem em Larga Escala/métodos , Humanos , Simulação de Acoplamento Molecular , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Mensageiro/metabolismo , RNA de Protozoário/metabolismo , Tripanossomicidas/uso terapêutico , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/transmissão , Tripanossomíase Africana/veterinária , Valina/análogos & derivados , Valina/farmacologia , Valina/uso terapêutico
10.
JCI Insight ; 3(15)2018 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-30089721

RESUMO

MicroRNAs (miRs) posttranscriptionally regulate mRNA and its translation into protein, and are considered master controllers of genes modulating normal physiology and disease. There is growing interest in how miRs change with drug treatment, and leveraging this for precision guided therapy. Here we contrast 2 closely related therapies, inhibitors of phosphodiesterase type 5 or type 9 (PDE5-I, PDE9-I), given to mice subjected to sustained cardiac pressure overload (PO). Both inhibitors augment cyclic guanosine monophosphate (cGMP) to activate protein kinase G, with PDE5-I regulating nitric oxide (NO) and PDE9-I natriuretic peptide-dependent signaling. While both produced strong phenotypic improvement of PO pathobiology, they surprisingly showed binary differences in miR profiles; PDE5-I broadly reduces more than 120 miRs, including nearly half those increased by PO, whereas PDE9-I has minimal impact on any miR (P < 0.0001). The disparity evolves after pre-miR processing and is organ specific. Lastly, even enhancing NO-coupled cGMP by different methods leads to altered miR regulation. Thus, seemingly similar therapeutic interventions can be barcoded by profound differences in miR signatures, and reversing disease-associated miR changes is not required for therapy success.


Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Cardiopatias/tratamento farmacológico , MicroRNAs/metabolismo , Inibidores da Fosfodiesterase 5/farmacologia , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Animais , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Modelos Animais de Doenças , Cardiopatias/etiologia , Humanos , Masculino , Camundongos , Peptídeos Natriuréticos/metabolismo , Óxido Nítrico/metabolismo , Inibidores da Fosfodiesterase 5/uso terapêutico , Transdução de Sinais
11.
Bioorg Chem ; 80: 492-497, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29990897

RESUMO

miRNAs are key cellular regulators and their dysregulation is associated with many human diseases. They are usually produced locally in a spatiotemporally controlled manner to target mRNAs and regulate gene expression. Thus, developing chemical tools for manipulating miRNA with spatiotemporal precise is critical for studying miRNA. Herein, we designed a strategy to control miRNA biogenesis with light controllable inhibitor targeting the pre-miRNA processing by Dicer. By conjugating two non-inhibiting units, a low affinity Dicer inhibitor and a pre-miRNA binder, through a photocleavable linker, the bifunctional molecule obtained could inhibit miRNA production. Taking advantage of the photocleavable property of the linker, the bifunctional inhibitor can be fragmented into separate non-inhibiting units and therefore be deactivated by light. We expect that this strategy could be applied to generate chemical biological tools that allow light-mediated spatiotemporal control of miRNA maturation and contribute to the study of miRNA function.


Assuntos
RNA Helicases DEAD-box/metabolismo , MicroRNAs/metabolismo , Ribonuclease III/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Desenho de Fármacos , Humanos , Luz , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/efeitos da radiação , Proteínas Recombinantes/metabolismo , Transcrição Genética/efeitos dos fármacos , Transcrição Genética/efeitos da radiação
12.
Nat Commun ; 9(1): 1875, 2018 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-29760464

RESUMO

It has been generally thought that tRNA modifications are stable and static, and their frequencies are rarely regulated. N6-threonylcarbamoyladenosine (t6A) occurs at position 37 of five mitochondrial (mt-)tRNA species. We show that YRDC and OSGEPL1 are responsible for t6A37 formation, utilizing L-threonine, ATP, and CO2/bicarbonate as substrates. OSGEPL1-knockout cells exhibit respiratory defects and reduced mitochondrial translation. We find low level of t6A37 in mutant mt-tRNA isolated from the MERRF-like patient's cells, indicating that lack of t6A37 results in pathological consequences. Kinetic measurements of t6A37 formation reveal that the Km value of CO2/bicarbonate is extremely high (31 mM), suggesting that CO2/bicarbonate is a rate-limiting factor for t6A37 formation. Consistent with this, we observe a low frequency of t6A37 in mt-tRNAs isolated from human cells cultured without bicarbonate. These findings indicate that t6A37 is regulated by sensing intracellular CO2/bicarbonate concentration, implying that mitochondrial translation is modulated in a codon-specific manner under physiological conditions.


Assuntos
Bicarbonatos/farmacologia , Dióxido de Carbono/farmacologia , Síndrome MERRF/metabolismo , Mitocôndrias/metabolismo , Proteínas/metabolismo , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA de Transferência/química , Adenosina/análogos & derivados , Adenosina/química , Adenosina/metabolismo , Proteínas Reguladoras de Apoptose , Pareamento de Bases , Bicarbonatos/metabolismo , Sistemas CRISPR-Cas , Dióxido de Carbono/metabolismo , Linhagem Celular , Respiração Celular , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Deleção de Genes , Células HEK293 , Células HT29 , Células HeLa , Humanos , Síndrome MERRF/genética , Síndrome MERRF/patologia , Mitocôndrias/patologia , Modelos Biológicos , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Mioblastos/patologia , Conformação de Ácido Nucleico , Proteínas/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
13.
J Pharmacokinet Pharmacodyn ; 45(4): 557-575, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29704219

RESUMO

A multiscale pharmacodynamic model was developed to characterize the receptor-mediated, transcriptomic, and proteomic determinants of corticosteroid (CS) effects on clinically relevant hepatic processes following a single dose of methylprednisolone (MPL) given to adrenalectomized (ADX) rats. The enhancement of tyrosine aminotransferase (TAT) mRNA, protein, and enzyme activity were simultaneously described. Mechanisms related to the effects of MPL on glucose homeostasis, including the regulation of CCAAT-enhancer binding protein-beta (C/EBPß) and phosphoenolpyruvate carboxykinase (PEPCK) as well as insulin dynamics were evaluated. The MPL-induced suppression of circulating lymphocytes was modeled by coupling its effect on cell trafficking with pharmacogenomic effects on cell apoptosis via the hepatic (STAT3-regulated) acute phase response. Transcriptomic and proteomic time-course profiles measured in steroid-treated rat liver were utilized to model the dynamics of mechanistically relevant gene products, which were linked to associated systemic end-points. While time-courses of TAT mRNA, protein, and activity were well described by transcription-mediated changes, additional post-transcriptional processes were included to explain the lack of correlation between PEPCK mRNA and protein. The immune response model quantitatively discerned the relative roles of cell trafficking versus gene-mediated lymphocyte apoptosis by MPL. This systems pharmacodynamic model provides insights into the contributions of selected molecular events occurring in liver and explores mechanistic hypotheses for the multi-factorial control of clinically relevant pharmacodynamic outcomes.


Assuntos
Fígado/efeitos dos fármacos , Fígado/metabolismo , Metilprednisolona/farmacologia , Transdução de Sinais/efeitos dos fármacos , Corticosteroides/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Glucocorticoides/genética , Glucocorticoides/metabolismo , Insulina/genética , Masculino , Modelos Biológicos , Proteômica/métodos , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , Ratos , Ratos Wistar , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Transdução de Sinais/genética , Transcrição Genética/efeitos dos fármacos , Transcrição Genética/genética , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Tirosina Transaminase/genética
14.
Sci Rep ; 8(1): 1667, 2018 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-29374231

RESUMO

MicroRNAs are key factors in the regulation of gene expression and their deregulation has been directly linked to various pathologies such as cancer. The use of small molecules to tackle the overexpression of oncogenic miRNAs has proved its efficacy and holds the promise for therapeutic applications. Here we describe the screening of a 640-compound library and the identification of polyamine derivatives interfering with in vitro Dicer-mediated processing of the oncogenic miR-372 precursor (pre-miR-372). The most active inhibitor is a spermine-amidine conjugate that binds to the pre-miR-372 with a KD of 0.15 µM, and inhibits its in vitro processing with a IC50 of 1.06 µM. The inhibition of miR-372 biogenesis was confirmed in gastric cancer cells overexpressing miR-372 and a specific inhibition of proliferation through de-repression of the tumor suppressor LATS2 protein, a miR-372 target, was observed. This compound modifies the expression of a small set of miRNAs and its selective biological activity has been confirmed in patient-derived ex vivo cultures of gastric carcinoma. Polyamine derivatives are promising starting materials for future studies about the inhibition of oncogenic miRNAs and, to the best of our knowledge, this is the first report about the application of functionalized polyamines as miRNAs interfering agents.


Assuntos
Antineoplásicos/farmacologia , MicroRNAs/metabolismo , Poliaminas/farmacologia , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Antineoplásicos/isolamento & purificação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Concentração Inibidora 50 , Poliaminas/isolamento & purificação , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Supressoras de Tumor/biossíntese
15.
ACS Chem Biol ; 13(12): 3243-3250, 2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-29313662

RESUMO

The recent discovery of reversible chemical modifications on mRNA has opened a new era of post-transcriptional gene regulation in eukaryotes. Among the 15 types of modifications identified in mRNA of eukaryotes, N7-methylguanosine (m7G) is unique owing to its presence in the 5' cap structure. It remains unknown whether m7G is also present internally in mRNA, and this is largely attributed to the lack of an appropriate analytical method to differentiate internal m7G in mRNA from that in the 5' cap. To address this analytical challenge, we developed a novel strategy of combining differential enzymatic digestion with liquid chromatography-tandem mass spectrometry analysis to quantify the levels of these two types of m7G modifications in mRNA. In particular, we found that S1 nuclease and phosphodiesterase I exhibit differential activities toward internal and 5'-terminal m7G. By using this method, we found that internal m7G was present in mRNA of cultured human cells as well as plants and rat tissue. In addition, our results showed that plants contain higher levels of internal m7G in mRNA than mammals. We also observed that exposure of rice to cadmium (Cd) stimulated marked diminution in the levels of m7G at both the 5' cap and internal positions of mRNA, which was correlated with the Cd-induced elevated expression of m7G-decapping enzymes. Taken together, we reported here a strategy to distinguish internal and 5'-terminal m7G in mRNA, and by using this method, we demonstrated the prevalence of internal m7G modification in mRNA, which we believe will stimulate future functional studies of m7G on post-transcriptional gene regulation in eukaryotes.


Assuntos
Endorribonucleases/química , Guanina/análogos & derivados , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Animais , Cádmio/farmacologia , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Endorribonucleases/genética , Endorribonucleases/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Guanina/química , Humanos , Masculino , Espectrometria de Massas/métodos , Oryza/enzimologia , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Mensageiro/síntese química , RNA Mensageiro/genética , Ratos Sprague-Dawley
16.
Antiviral Res ; 149: 191-201, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29133129

RESUMO

In pursuit of novel therapeutics targeting the hepatitis B virus (HBV) infection, we evaluated a dihydroquinolizinone compound (DHQ-1) that in the nanomolar range reduced the production of virion and surface protein (HBsAg) in tissue culture. This compound also showed broad HBV genotype coverage, but was inactive against a panel of DNA and RNA viruses of other species. Oral administration of DHQ-1 in the AAV-HBV mouse model resulted in a significant reduction of serum HBsAg as soon as 4 days following the commencement of treatment. Reduction of HBV markers in both in vitro and in vivo experiments was related to the reduced amount of viral RNA including pre-genomic RNA (pgRNA) and 2.4/2.1 kb HBsAg mRNA. Nuclear run-on and subcellular fractionation experiments indicated that DHQ-1 mediated HBV RNA reduction was the result of accelerated viral RNA degradation in the nucleus, rather than the consequence of inhibition of transcription initiation. Through mutagenesis of HBsAg gene sequences, we found induction of HBsAg mRNA decay by DHQ-1 required the presence of the HBV posttranscriptional regulatory element (HPRE), with a 109 nucleotides sequence within the central region of the HPRE alpha sub-element being the most critical. Taken together, the current study shows that a small molecule can reduce the overall levels of HBV RNA, especially the HBsAg mRNA, and viral surface proteins. This may shed light on the development of a new class of HBV therapeutics.


Assuntos
Antivirais/farmacologia , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/genética , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Viral/genética , Elementos de Resposta , Sítios de Ligação , Genótipo , Humanos , Ligação Proteica , Estabilidade de RNA/efeitos dos fármacos , Transfecção , Replicação Viral
17.
Sci Rep ; 7(1): 17900, 2017 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-29263339

RESUMO

Portal hypertension (PH) is a major cause of morbidity and mortality in chronic liver disease. Infection and inflammation play a role in potentiating PH and pro-inflammatory cytokines, including TNF, are associated with severity of PH. In this study, cirrhotic bile duct ligated (BDL) rats with PH were treated with Infliximab (IFX, a monoclonal antibody against TNF) and its impact on modulation of vascular tone was assessed. BDL rats had increased TNF and NFkB compared to sham operated rats, and their reduction by IFX was associated with a reduction in portal pressure. IFX treatment also reduced hepatic oxidative stress, and biochemical markers of hepatic inflammation and injury. IFX treatment was associated with an improvement in eNOS activity and increased L-arginine/ADMA ratio and DDAH1 expression. In vitro analysis of HepG2 hepatocytes showed that DDAH1 protein expression is reduced by oxidative stress, and this is in part mediated by post-transcriptional regulation by the 3'UTR. This study supports a role for the DDAH1/ADMA axis on the effect of inflammation and oxidative stress in PH and provides insight for new therapies.


Assuntos
Amidoidrolases/genética , Hipertensão Portal/genética , Cirrose Hepática/genética , Óxido Nítrico Sintase Tipo III/genética , Processamento Pós-Transcricional do RNA/genética , Fatores de Necrose Tumoral/genética , Animais , Arginina/genética , Arginina/metabolismo , Ductos Biliares/efeitos dos fármacos , Ductos Biliares/metabolismo , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Células Hep G2 , Humanos , Hipertensão Portal/tratamento farmacológico , Hipertensão Portal/metabolismo , Inflamação/genética , Inflamação/metabolismo , Infliximab/farmacologia , Ligadura/métodos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Masculino , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Pressão na Veia Porta/efeitos dos fármacos , Pressão na Veia Porta/genética , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
18.
J Nat Prod ; 80(12): 3186-3193, 2017 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-29148754

RESUMO

The C-19 quassinoid eurycomalactone (1) has recently been shown to be a potent (IC50 = 0.5 µM) NF-κB inhibitor in a luciferase reporter model. In this study, we show that 1 with similar potency inhibited the expression of the NF-κB-dependent target genes ICAM-1, VCAM-1, and E-selectin in TNFα-activated human endothelial cells (HUVECtert) by flow cytometry experiments. Surprisingly, 1 (2 µM) did not inhibit TNFα-induced IKKα/ß or IκBα phosphorylation significantly. Also, the TNFα-induced degradation of IκBα remained unchanged in response to 1 (2 µM). In addition, pretreatment of HUVECtert with 1 (2 µM) had no statistically significant effect on TNFα-mediated nuclear translocation of the NF-κB subunit p65 (RelA). Quantitative RT-PCR revealed that 1 (0.5-5 µM) exhibited diverse effects on the TNFα-induced transcription of ICAM-1, VCAM-1, and SELE genes since the mRNA level either remained unchanged (ICAM-1, E-selectin, and VCAM-1 at 0.5 µM 1), was reduced (VCAM-1 at 5 µM 1), or even increased (E-selectin at 5 µM 1). Finally, the time-dependent depletion of a short-lived protein (cyclin D1) as well as the measurement of de novo protein synthesis in the presence of 1 (2-5 µM) suggested that 1 might act as a protein synthesis inhibitor rather than an inhibitor of early NF-κB signaling.


Assuntos
Moléculas de Adesão Celular/antagonistas & inibidores , Células Endoteliais/efeitos dos fármacos , Quassinas/farmacologia , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/biossíntese , Linhagem Celular , Ciclina D1/metabolismo , Selectina E/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Eurycoma/química , Células Endoteliais da Veia Umbilical Humana , Humanos , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Quassinas/química , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
20.
J Biol Chem ; 292(44): 18129-18144, 2017 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-28893905

RESUMO

Lysine acetylation is a widespread posttranslational modification affecting many biological pathways. Recent studies indicate that acetylated lysine residues mainly exhibit low acetylation occupancy, but challenges in sample preparation and analysis make it difficult to confidently assign these numbers, limiting understanding of their biological significance. Here, we tested three common sample preparation methods to determine their suitability for assessing acetylation stoichiometry in three human cell lines, identifying the acetylation occupancy in more than 1,300 proteins from each cell line. The stoichiometric analysis in combination with quantitative proteomics also enabled us to explore their functional roles. We found that higher abundance of the deacetylase sirtuin 1 (SIRT1) correlated with lower acetylation occupancy and lower levels of ribosomal proteins, including those involved in ribosome biogenesis and rRNA processing. Treatment with the SIRT1 inhibitor EX-527 confirmed SIRT1's role in the regulation of pre-rRNA synthesis and processing. Specifically, proteins involved in pre-rRNA transcription, including subunits of the polymerase I and SL1 complexes and the RNA polymerase I-specific transcription initiation factor RRN3, were up-regulated after SIRT1 inhibition. Moreover, many protein effectors and regulators of pre-rRNA processing needed for rRNA maturation were also up-regulated after EX-527 treatment with the outcome that pre-rRNA and 28S rRNA levels also increased. More generally, we found that SIRT1 inhibition down-regulates metabolic pathways, including glycolysis and pyruvate metabolism. Together, these results provide the largest data set thus far of lysine acetylation stoichiometry (available via ProteomeXchange with identifier PXD005903) and set the stage for further biological investigations of this central posttranslational modification.


Assuntos
Regulação da Expressão Gênica , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , Sirtuína 1/metabolismo , Acetilação/efeitos dos fármacos , Métodos Analíticos de Preparação de Amostras , Carbazóis/farmacologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Cinética , Mapeamento de Peptídeos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteômica/métodos , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Ribossômico 28S/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/genética , Espectrometria de Massas em Tandem
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