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2.
Nat Commun ; 12(1): 2295, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33863887

RESUMO

The COVID-19 pandemic progresses unabated in many regions of the world. An effective antiviral against SARS-CoV-2 that could be administered orally for use following high-risk exposure would be of substantial benefit in controlling the COVID-19 pandemic. Herein, we show that MK-4482, an orally administered nucleoside analog, inhibits SARS-CoV-2 replication in the Syrian hamster model. The inhibitory effect of MK-4482 on SARS-CoV-2 replication is observed in animals when the drug is administered either beginning 12 h before or 12 h following infection in a high-risk exposure model. These data support the potential utility of MK-4482 to control SARS-CoV-2 infection in humans following high-risk exposure as well as for treatment of COVID-19 patients.


Assuntos
Antivirais/administração & dosagem , Citidina/análogos & derivados , Hidroxilaminas/administração & dosagem , Replicação Viral/efeitos dos fármacos , Administração Oral , Animais , Chlorocebus aethiops , Citidina/administração & dosagem , Modelos Animais de Doenças , Humanos , Mesocricetus , /fisiologia , Células Vero
3.
Nature ; 591(7850): 451-457, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33561864

RESUMO

All coronaviruses known to have recently emerged as human pathogens probably originated in bats1. Here we use a single experimental platform based on immunodeficient mice implanted with human lung tissue (hereafter, human lung-only mice (LoM)) to demonstrate the efficient in vivo replication of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as two endogenous SARS-like bat coronaviruses that show potential for emergence as human pathogens. Virus replication in this model occurs in bona fide human lung tissue and does not require any type of adaptation of the virus or the host. Our results indicate that bats contain endogenous coronaviruses that are capable of direct transmission to humans. Our detailed analysis of in vivo infection with SARS-CoV-2 in human lung tissue from LoM showed a predominant infection of human lung epithelial cells, including type-2 pneumocytes that are present in alveoli and ciliated airway cells. Acute infection with SARS-CoV-2 was highly cytopathic and induced a robust and sustained type-I interferon and inflammatory cytokine and chemokine response. Finally, we evaluated a therapeutic and pre-exposure prophylaxis strategy for SARS-CoV-2 infection. Our results show that therapeutic and prophylactic administration of EIDD-2801-an oral broad-spectrum antiviral agent that is currently in phase II/III clinical trials-markedly inhibited SARS-CoV-2 replication in vivo, and thus has considerable potential for the prevention and treatment of COVID-19.


Assuntos
/tratamento farmacológico , Citidina/análogos & derivados , Hidroxilaminas/administração & dosagem , Hidroxilaminas/uso terapêutico , Administração Oral , Células Epiteliais Alveolares/imunologia , Células Epiteliais Alveolares/patologia , Células Epiteliais Alveolares/virologia , Animais , Quimioprevenção , Quirópteros/virologia , Ensaios Clínicos Fase II como Assunto , Ensaios Clínicos Fase III como Assunto , Citidina/administração & dosagem , Citidina/uso terapêutico , Citocinas/imunologia , Células Epiteliais/virologia , Feminino , Xenoenxertos , Humanos , Imunidade Inata , Interferon Tipo I/imunologia , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Transplante de Pulmão , Masculino , Camundongos , Profilaxia Pós-Exposição , Profilaxia Pré-Exposição , /patogenicidade , Replicação Viral
4.
Expert Opin Ther Pat ; 31(4): 325-337, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33475441

RESUMO

Introduction: The current SARS-CoV-2 pandemic urgently demands for both prevention and treatment strategies. RNA-dependent RNA-polymerase (RdRp), which has no counterpart in human cells, is an excellent target for drug development. Given the time-consuming process of drug development, repurposing drugs approved for other indications or at least successfully tested in terms of safety and tolerability, is an attractive strategy to rapidly provide an effective medication for severe COVID-19 cases.Areas covered: The currently available data and upcominSg studies on RdRp which can be repurposed to halt SARS-CoV-2 replication, are reviewed.Expert opinion: Drug repurposing and design of novel compounds are proceeding in parallel to provide a quick response and new specific drugs, respectively. Notably, the proofreading SARS-CoV-2 exonuclease activity could limit the potential for drugs designed as immediate chain terminators and favor the development of compounds acting through delayed termination. While vaccination is awaited to curb the SARS-CoV-2 epidemic, even partially effective drugs from repurposing strategies can be of help to treat severe cases of disease. Considering the high conservation of RdRp among coronaviruses, an improved knowledge of its activity in vitro can provide useful information for drug development or drug repurposing to combat SARS-CoV-2 as well as future pandemics.


Assuntos
Antivirais/farmacologia , /antagonistas & inibidores , /efeitos dos fármacos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Amidas/farmacologia , Citidina/análogos & derivados , Citidina/farmacologia , Desenvolvimento de Medicamentos , Reposicionamento de Medicamentos , Humanos , Hidroxilaminas/farmacologia , Pirazinas/farmacologia , /enzimologia
5.
Int J Mol Sci ; 22(1)2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-33466545

RESUMO

Involvement of epigenetic mechanisms in the regulation of telomeres and transposable elements (TEs), genomic regions with the protective and potentially detrimental function, respectively, has been frequently studied. Here, we analyzed telomere lengths in Arabidopsis thaliana plants of Columbia, Landsberg erecta and Wassilevskija ecotypes exposed repeatedly to the hypomethylation drug zebularine during germination. Shorter telomeres were detected in plants growing from seedlings germinated in the presence of zebularine with a progression in telomeric phenotype across generations, relatively high inter-individual variability, and diverse responses among ecotypes. Interestingly, the extent of telomere shortening in zebularine Columbia and Wassilevskija plants corresponded to the transcriptional activation of TEs, suggesting a correlated response of these genomic elements to the zebularine treatment. Changes in lengths of telomeres and levels of TE transcripts in leaves were not always correlated with a hypomethylation of cytosines located in these regions, indicating a cytosine methylation-independent level of their regulation. These observations, including differences among ecotypes together with distinct dynamics of the reversal of the disruption of telomere homeostasis and TEs transcriptional activation, reflect a complex involvement of epigenetic processes in the regulation of crucial genomic regions. Our results further demonstrate the ability of plant cells to cope with these changes without a critical loss of the genome stability.


Assuntos
Arabidopsis/genética , Citidina/análogos & derivados , Elementos de DNA Transponíveis/genética , Telômero/genética , Arabidopsis/metabolismo , Citidina/genética , Citosina/metabolismo , Metilação de DNA/genética , Epigênese Genética/genética , Células Vegetais/metabolismo , Homeostase do Telômero/genética , Encurtamento do Telômero/genética , Ativação Transcricional/genética
6.
Nucleic Acids Res ; 49(2): 1006-1022, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33330931

RESUMO

The highly abundant N6-methyladenosine (m6A) RNA modification affects most aspects of mRNA function, yet the precise function of the rarer 5-methylcytidine (m5C) remains largely unknown. Here, we map m5C in the human transcriptome using methylation-dependent individual-nucleotide resolution cross-linking and immunoprecipitation (miCLIP) combined with RNA bisulfite sequencing. We identify NSUN6 as a methyltransferase with strong substrate specificity towards mRNA. NSUN6 primarily targeted three prime untranslated regions (3'UTR) at the consensus sequence motif CTCCA, located in loops of hairpin structures. Knockout and rescue experiments revealed enhanced mRNA and translation levels when NSUN6-targeted mRNAs were methylated. Ribosome profiling further demonstrated that NSUN6-specific methylation correlated with translation termination. While NSUN6 was dispensable for mouse embryonic development, it was down-regulated in human tumours and high expression of NSUN6 indicated better patient outcome of certain cancer types. In summary, our study identifies NSUN6 as a methyltransferase targeting mRNA, potentially as part of a quality control mechanism involved in translation termination fidelity.


Assuntos
Citidina/análogos & derivados , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , tRNA Metiltransferases/metabolismo , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Linhagem Celular Tumoral , Uso do Códon , Sequência Consenso , Citidina/metabolismo , Células-Tronco Embrionárias , Técnicas de Inativação de Genes , Genes Reporter , Células HEK293 , Humanos , Imunoprecipitação , Metilação , Camundongos , Camundongos Knockout , Mutagênese Sítio-Dirigida , RNA Mensageiro/genética , Transcriptoma , tRNA Metiltransferases/deficiência
7.
Methods Mol Biol ; 2198: 301-310, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32822040

RESUMO

Methylated DNA immunoprecipitation is a large scale purification technique. It enables the isolation of methylated DNA fragments for subsequent locus-specific or genome-wide analysis. Here we describe an immunoprecipitation protocol using a monoclonal mouse anti 5-methyl-cytidine antibody followed by next-generation sequencing (MeDIP-Seq).


Assuntos
Citidina/análogos & derivados , Imunoprecipitação/métodos , Análise de Sequência de DNA/métodos , Animais , Sequência de Bases , Mapeamento Cromossômico , Citidina/imunologia , DNA/genética , DNA/imunologia , Metilação de DNA/genética , Metilação de DNA/imunologia , Genoma , Estudo de Associação Genômica Ampla/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos
8.
Nucleic Acids Res ; 49(5): e27, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33313824

RESUMO

Cellular RNAs are subject to a myriad of different chemical modifications that play important roles in controlling RNA expression and function. Dysregulation of certain RNA modifications, the so-called 'epitranscriptome', contributes to human disease. One limitation in studying the functional, physiological, and pathological roles of the epitranscriptome is the availability of methods for the precise mapping of individual RNA modifications throughout the transcriptome. 3-Methylcytidine (m3C) modification of certain tRNAs is well established and was also recently detected in mRNA. However, methods for the specific mapping of m3C throughout the transcriptome are lacking. Here, we developed a m3C-specific technique, Hydrazine-Aniline Cleavage sequencing (HAC-seq), to profile the m3C methylome at single-nucleotide resolution. We applied HAC-seq to analyze ribosomal RNA (rRNA)-depleted total RNAs in human cells. We found that tRNAs are the predominant m3C-modified RNA species, with 17 m3C modification sites on 11 cytoplasmic and 2 mitochondrial tRNA isoacceptors in MCF7 cells. We found no evidence for m3C-modification of mRNA or other non-coding RNAs at comparable levels to tRNAs in these cells. HAC-seq provides a novel method for the unbiased, transcriptome-wide identification of m3C RNA modification at single-nucleotide resolution, and could be widely applied to reveal the m3C methylome in different cells and tissues.


Assuntos
Citidina/análogos & derivados , RNA de Transferência/química , Análise de Sequência de RNA/métodos , Compostos de Anilina/química , Citidina/análise , Citidina/metabolismo , Humanos , Hidrazinas/química , Células MCF-7 , RNA de Transferência/metabolismo , Transcriptoma
9.
Adv Chronic Kidney Dis ; 27(5): 434-441, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-33308510

RESUMO

Coronavirus disease 2019, the disease caused by the severe acute respiratory syndrome coronavirus 2 virus, was first identified in the Hubei Province of China in late 2019. Currently, the only role for therapy is treatment of the disease, as opposed to postexposure prophylaxis, however multiple clinical trials are currently ongoing for both treatment and prophylaxis. Treating coronavirus disease 2019 relies on two components; the first is inhibition of the viral entrance and replication within the body and the second is inhibition of an exacerbated immune response which can be seen in patients with severe disease. Many drugs have shown in vitro antiviral activity; however, clinical trials have not been as promising. This review summarizes the current data for the most commonly used drugs for coronavirus disease 2019 and will cover the unique factors that may affect the dosing of these medications in patients with CKD. While clinical trials are ongoing, most are in patients with normal kidney function. During a pandemic, when patients with CKD are at higher risk of both infection and death, it is imperative to include patients these patients in the clinical trials.


Assuntos
Anti-Inflamatórios/uso terapêutico , Antivirais/uso terapêutico , Insuficiência Renal Crônica/metabolismo , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/uso terapêutico , Alanina/análogos & derivados , Alanina/uso terapêutico , Amidas/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , /prevenção & controle , /uso terapêutico , Cloroquina/uso terapêutico , Creatinina/metabolismo , Citidina/análogos & derivados , Citidina/uso terapêutico , Dexametasona/uso terapêutico , Combinação de Medicamentos , Interações Medicamentosas , Humanos , Hidroxicloroquina/uso terapêutico , Hidroxilaminas/uso terapêutico , Imunização Passiva , Interferons/uso terapêutico , Inibidores de Janus Quinases/uso terapêutico , Lopinavir/uso terapêutico , Pirazinas/uso terapêutico , Eliminação Renal , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/terapia , Terapia de Substituição Renal , Ribavirina/uso terapêutico , Ritonavir/uso terapêutico
11.
Mol Cell ; 79(5): 710-727, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32853546

RESUMO

The coronavirus disease 2019 (COVID-19) that is wreaking havoc on worldwide public health and economies has heightened awareness about the lack of effective antiviral treatments for human coronaviruses (CoVs). Many current antivirals, notably nucleoside analogs (NAs), exert their effect by incorporation into viral genomes and subsequent disruption of viral replication and fidelity. The development of anti-CoV drugs has long been hindered by the capacity of CoVs to proofread and remove mismatched nucleotides during genome replication and transcription. Here, we review the molecular basis of the CoV proofreading complex and evaluate its potential as a drug target. We also consider existing nucleoside analogs and novel genomic techniques as potential anti-CoV therapeutics that could be used individually or in combination to target the proofreading mechanism.


Assuntos
Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/epidemiologia , Genoma Viral , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/epidemiologia , RNA Viral/genética , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/uso terapêutico , Alanina/análogos & derivados , Alanina/química , Alanina/uso terapêutico , Amidas/química , Amidas/uso terapêutico , Antivirais/química , Betacoronavirus/genética , Betacoronavirus/patogenicidade , Infecções por Coronavirus/virologia , Citidina/análogos & derivados , Humanos , Hidroxilaminas , Terapia de Alvo Molecular/métodos , Mutação , Pneumonia Viral/virologia , Pirazinas/química , Pirazinas/uso terapêutico , RNA Viral/antagonistas & inibidores , RNA Viral/metabolismo , Ribonucleosídeos/química , Ribonucleosídeos/uso terapêutico , Índice de Gravidade de Doença , Transcrição Genética , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
12.
Nature ; 583(7817): 638-643, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32555463

RESUMO

N4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA1-3. However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. Here we report ac4C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac4C at single-nucleotide resolution. In human and yeast mRNAs, ac4C sites are not detected but can be induced-at a conserved sequence motif-via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac4C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac4C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac4C and its potential thermoadaptive role. Our studies quantitatively define the ac4C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease4-6.


Assuntos
Acetilação , Citidina/análogos & derivados , Células Eucarióticas/metabolismo , Evolução Molecular , RNA/química , RNA/metabolismo , Archaea/química , Archaea/citologia , Archaea/genética , Archaea/crescimento & desenvolvimento , Sequência Conservada , Microscopia Crioeletrônica , Citidina/metabolismo , Células Eucarióticas/citologia , Células HeLa , Humanos , Modelos Moleculares , Acetiltransferases N-Terminal/metabolismo , RNA Arqueal/química , RNA Arqueal/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , Temperatura
13.
In Vivo ; 34(3 Suppl): 1567-1588, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32503814

RESUMO

BACKGROUND: On March 11, 2020, the World Health Organization (WHO) declared the outbreak of coronavirus disease (COVID-19) a pandemic. Since then, thousands of people have suffered and died, making the need for a treatment of severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) more crucial than ever. MATERIALS AND METHODS: The authors carried out a search in PubMed, ClinicalTrials.gov and New England Journal of Medicine (NEJM) for COVID-19 to provide information on the most promising treatments against SARS-CoV-2. RESULTS: Possible COVID-19 agents with promising efficacy and favorable safety profile were identified. The results support the combination of copper, N-acetylcysteine (NAC), colchicine and nitric oxide (NO) with candidate antiviral agents, remdesivir or EIDD-2801, as a treatment for patients positive for SARS-CoV-2. CONCLUSION: The authors propose to study the effects of the combination of copper, NAC, colchicine, NO and currently used experimental antiviral agents, remdesivir or EIDD-2801, as a potential treatment scheme for SARS-COV-2.


Assuntos
Acetilcisteína/uso terapêutico , Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/uso terapêutico , Colchicina/uso terapêutico , Cobre/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Óxido Nítrico/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Ribonucleosídeos/uso terapêutico , Acetilcisteína/administração & dosagem , Acetilcisteína/farmacologia , Monofosfato de Adenosina/administração & dosagem , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/uso terapêutico , Alanina/administração & dosagem , Alanina/farmacologia , Alanina/uso terapêutico , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/uso terapêutico , Antivirais/administração & dosagem , Antivirais/farmacologia , Autofagia/efeitos dos fármacos , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/fisiologia , Colchicina/administração & dosagem , Colchicina/farmacologia , Cobre/administração & dosagem , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/fisiopatologia , Citidina/análogos & derivados , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Hidroxilaminas , Inflamação , Óxido Nítrico/administração & dosagem , Óxido Nítrico/farmacologia , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/fisiopatologia , Pró-Fármacos/administração & dosagem , Pró-Fármacos/uso terapêutico , Ribonucleosídeos/administração & dosagem , Ribonucleosídeos/farmacologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
14.
Sci Transl Med ; 12(541)2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32253226

RESUMO

Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2, the causative agent of COVID-19. Here, we show that the ribonucleoside analog ß-d-N4-hydroxycytidine (NHC; EIDD-1931) has broad-spectrum antiviral activity against SARS-CoV-2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c bat-CoVs, as well as increased potency against a CoV bearing resistance mutations to the nucleoside analog inhibitor remdesivir. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC prodrug (ß-d-N4-hydroxycytidine-5'-isopropyl ester), improved pulmonary function and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral, but not host cell RNA, supporting a mechanism of lethal mutagenesis in CoV. The potency of NHC/EIDD-2801 against multiple CoVs and oral bioavailability highlights its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic CoVs.


Assuntos
Antivirais/administração & dosagem , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Ribonucleosídeos/administração & dosagem , Replicação Viral/efeitos dos fármacos , Monofosfato de Adenosina/administração & dosagem , Monofosfato de Adenosina/análogos & derivados , Alanina/administração & dosagem , Alanina/análogos & derivados , Animais , Antibioticoprofilaxia , Betacoronavirus/fisiologia , Linhagem Celular , Infecções por Coronavirus/patologia , Citidina/administração & dosagem , Citidina/análogos & derivados , Modelos Animais de Doenças , Farmacorresistência Viral , Humanos , Hidroxilaminas , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Modelos Moleculares , Mutação/efeitos dos fármacos , Pandemias , Pneumonia Viral/patologia , Cultura Primária de Células , RNA Viral , /genética , Distribuição Aleatória , Sistema Respiratório/citologia
15.
Transl Res ; 220: 33-42, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32088166

RESUMO

Influenza viruses are a major threat to human health globally. In addition to further improving vaccine prophylaxis, disease management through antiviral therapeutics constitutes an important component of the current intervention strategy to prevent advance to complicated disease and reduce case-fatality rates. Standard-of-care is treatment with neuraminidase inhibitors that prevent viral dissemination. In 2018, the first mechanistically new influenza drug class for the treatment of uncomplicated seasonal influenza in 2 decades was approved for human use. Targeting the PA endonuclease subunit of the viral polymerase complex, this class suppresses viral replication. However, the genetic barrier against viral resistance to both drug classes is low, pre-existing resistance is observed in circulating strains, and resistant viruses are pathogenic and transmit efficiently. Addressing the resistance problem has emerged as an important objective for the development of next-generation influenza virus therapeutics. This review will discuss the status of influenza therapeutics including the endonuclease inhibitor baloxavir marboxil after its first year of clinical use and evaluate a subset of direct-acting antiviral candidates in different stages of preclinical and clinical development.


Assuntos
Antivirais/uso terapêutico , Influenza Humana/tratamento farmacológico , Amidas/uso terapêutico , Anticorpos Neutralizantes/sangue , Citidina/análogos & derivados , Dibenzotiepinas , Farmacorresistência Viral , Humanos , Hidroxilaminas , Morfolinas , Neuraminidase/antagonistas & inibidores , Oxazinas/uso terapêutico , Pirazinas/uso terapêutico , Piridinas/uso terapêutico , Piridonas , Ribonucleosídeos/uso terapêutico , Tiepinas/uso terapêutico , Triazinas/uso terapêutico , Replicação Viral/efeitos dos fármacos
17.
Transl Res ; 218: 16-28, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31945316

RESUMO

Seasonal influenza viruses cause major morbidity and mortality worldwide, threatening in particular older adults and the immunocompromised. Two classes of influenza therapeutics dominate current disease management, but both are compromised by pre-existing or rapidly emerging viral resistance. We have recently reported a novel ribonucleoside analog clinical candidate, EIDD-2801, that combines potent antiviral efficacy in ferrets and human airway epithelium cultures with a high barrier against viral escape. In this study, we established fundamental EIDD-2801 efficacy paradigms against pandemic and seasonal influenza A virus (IAV) strains in ferrets that can be used to inform exposure targets and treatment regimens. Based on reduction of shed virus titers, alleviation of clinical signs, and lowered virus burden in upper and lower respiratory tract tissues, lowest efficacious oral dose concentrations of EIDD-2801, given twice daily, were 2.3 and 7 mg/kg of body weight against seasonal and pandemic IAVs, respectively. The latest opportunity for initiation of efficacious treatment was 36 hours after infection of ferrets. Administered in 12-hour intervals, three 7 mg/kg doses of EIDD-2801 were sufficient for maximal therapeutic benefit against a pandemic IAV and significantly shortened the time to resolution of clinical signs. Ferrets infected with pandemic IAV and treated following the minimally efficacious EIDD-2801 regimen demonstrated significantly less shed virus and inflammatory cellular infiltrates in nasal lavages, but mounted a robust humoral antiviral response after recovery that was indistinguishable from that of vehicle-treated animals. These results provide an experimental basis in a human disease-relevant influenza animal model for clinical testing of EIDD-2801.


Assuntos
Antivirais/uso terapêutico , Modelos Animais de Doenças , Furões , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/tratamento farmacológico , Ribonucleosídeos/uso terapêutico , Animais , Antivirais/administração & dosagem , Antivirais/farmacologia , Citidina/análogos & derivados , Cães , Relação Dose-Resposta a Droga , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Células HEK293 , Humanos , Hidroxilaminas , Vírus da Influenza A/isolamento & purificação , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Mutação , Ribonucleosídeos/administração & dosagem , Ribonucleosídeos/farmacologia
18.
J Mol Biol ; 432(4): 913-929, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31945376

RESUMO

Three of six arginine codons (CGU, CGC, and CGA) are decoded by two Escherichia coli tRNAArg isoacceptors. The anticodon stem and loop (ASL) domains of tRNAArg1 and tRNAArg2 both contain inosine and 2-methyladenosine modifications at positions 34 (I34) and 37 (m2A37). tRNAArg1 is also modified from cytidine to 2-thiocytidine at position 32 (s2C32). The s2C32 modification is known to negate wobble codon recognition of the rare CGA codon by an unknown mechanism, while still allowing decoding of CGU and CGC. Substitution of s2C32 for C32 in the Saccharomyces cerevisiae tRNAIleIAU anticodon stem and loop domain (ASL) negates wobble decoding of its synonymous A-ending codon, suggesting that this function of s2C at position 32 is a generalizable property. X-ray crystal structures of variously modified ASLArg1ICG and ASLArg2ICG constructs bound to cognate and wobble codons on the ribosome revealed the disruption of a C32-A38 cross-loop interaction but failed to fully explain the means by which s2C32 restricts I34 wobbling. Computational studies revealed that the adoption of a spatially broad inosine-adenosine base pair at the wobble position of the codon cannot be maintained simultaneously with the canonical ASL U-turn motif. C32-A38 cross-loop interactions are required for stability of the anticodon/codon interaction in the ribosomal A-site.


Assuntos
Códon/genética , Citidina/análogos & derivados , RNA de Transferência/metabolismo , Biologia Computacional , Cristalografia por Raios X , Citidina/metabolismo , Inosina/metabolismo , Nucleosídeos/metabolismo , RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Termodinâmica
19.
Nucleic Acids Res ; 48(3): 1225-1238, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31807777

RESUMO

Tet3 regulates the dynamic balance between 5-methylcyotsine (5mC) and 5-hydroxymethylcytosine (5hmC) in DNA during brain development and homeostasis. However, it remains unclear how its functions are modulated in a context-dependent manner during neuronal differentiation. Here, we show that cyclin-dependent kinase 5 (cdk5) phosphorylates Tet3 at the highly conserved serine 1310 and 1379 residues within its catalytic domain, changing its in vitro dioxygenase activity. Interestingly, when stably expressed in Tet1, 2, 3 triple-knockout mouse embryonic stem cells (ESCs), wild-type Tet3 induces higher level of 5hmC and concomitant expression of genes associated with neurogenesis whereas phosphor-mutant (S1310A/S1379A) Tet3 causes elevated 5hmC and expression of genes that are linked to metabolic processes. Consistent with this observation, Tet3-knockout mouse ESCs rescued with wild-type Tet3 have higher level of 5hmC at the promoter of neuron-specific gene BRN2 when compared to cells that expressed phosphor-mutant Tet3. Wild-type and phosphor-mutant Tet3 also exhibit differential binding affinity to histone variant H2A.Z. The differential 5hmC enrichment and H2A.Z occupancy at BRN2 promoter is correlated with higher gene expression and more efficient neuronal differentiation of ESCs that expressed wild-type Tet3. Taken together, our results suggest that cdk5-mediated phosphorylation of Tet3 is required for robust activation of neuronal differentiation program.


Assuntos
Quinase 5 Dependente de Ciclina/genética , Citidina/análogos & derivados , Dioxigenases/genética , Neurogênese/genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Diferenciação Celular/genética , Citidina/genética , Citidina/metabolismo , Metilação de DNA/genética , Proteínas de Ligação a DNA , Regulação da Expressão Gênica no Desenvolvimento/genética , Histonas/genética , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Fatores do Domínio POU/genética , Fosforilação , Regiões Promotoras Genéticas
20.
Biochem Biophys Res Commun ; 523(2): 368-374, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31866007

RESUMO

Trans-activator (Tat)-mediated human immunodeficiency virus type 1 (HIV-1) transcription is essential for the replication of HIV-1 and is considered a potent therapeutic target for HIV-1 inhibition. In this study, the Library of Pharmacologically Active Compounds (LOPAC1280) was screened using our dual-reporter screening system for repositioning as Tat-inhibitory compounds. Consequently, two compounds were found to be potent, with low cytotoxicity. Of these two compounds, Roscovitine (CYC202) is already known to be a Tat inhibitor, while gemcitabine has been newly identified as an inhibitor of Tat-mediated transcription linked to viral production and replication. In an additional screening using the ribonucleoside analogues of gemcitabine, two analogues (2'-C-methylcytidine and 3-deazauridine) showed a specific Tat-inhibitory effect linked to their anti-HIV-1 activity. Interestingly, these compounds did not affect Tat protein directly, while the mechanism underlying their inhibition of Tat-mediated transcription was linked to pyrimidine biosynthesis, rather than to alteration of the dNTP pool, influenced by the inhibition of ribonucleotide reductase. Taken together, the proposed functional screening system is a useful tool for the identification of inhibitors of Tat-mediated HIV-1 transcription from among a large number of compounds, and the inhibitory effect of HIV-1 transcription by gemcitabine and its analogues may suggest a strategy for developing a new class of therapeutic anti-HIV drugs.


Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/antagonistas & inibidores , 3-Desazauridina/farmacologia , Linhagem Celular , Citidina/análogos & derivados , Citidina/farmacologia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Reposicionamento de Medicamentos , HIV-1/genética , HIV-1/fisiologia , Ensaios de Triagem em Larga Escala , Humanos , Roscovitina/farmacologia , Bibliotecas de Moléculas Pequenas , Transcrição Genética/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo
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