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2.
J Transl Med ; 18(1): 185, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32370758

RESUMO

A new human coronavirus named SARS-CoV-2 was identified in several cases of acute respiratory syndrome in Wuhan, China in December 2019. On March 11 2020, WHO declared the SARS-CoV-2 infection to be a pandemic, based on the involvement of 169 nations. Specific drugs for SARS-CoV-2 are obviously not available. Currently, drugs originally developed for other viruses or parasites are currently in clinical trials based on empiric data. In the quest of an effective antiviral drug, the most specific target for an RNA virus is the RNA-dependent RNA-polymerase (RdRp) which shows significant differences between positive-sense and negative-sense RNA viruses. An accurate evaluation of RdRps from different viruses may guide the development of new drugs or the repositioning of already approved antiviral drugs as treatment of SARS-CoV-2. This can accelerate the containment of the SARS-CoV-2 pandemic and, hopefully, of future pandemics due to other emerging zoonotic RNA viruses.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , RNA Replicase/antagonistas & inibidores , RNA Replicase/química , Sequência de Aminoácidos , Betacoronavirus/isolamento & purificação , Sequência Conservada , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/transmissão , Reposicionamento de Medicamentos , Humanos , Modelos Moleculares , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Pneumonia Viral/transmissão , RNA Replicase/metabolismo , Alinhamento de Sequência , Replicação Viral/efeitos dos fármacos , Eliminação de Partículas Virais/efeitos dos fármacos
3.
Nature ; 581(7808): 252-255, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32415276

Assuntos
Antivirais/farmacologia , Betacoronavirus/química , Betacoronavirus/imunologia , Desenho de Fármacos , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Vacinas Virais , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Alanina/análogos & derivados , Alanina/farmacologia , Alanina/uso terapêutico , Animais , Antivirais/química , Azóis/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , China , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Microscopia Crioeletrônica , Cristalização , Cristalografia por Raios X , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Avaliação Pré-Clínica de Medicamentos , Alemanha , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , National Institutes of Health (U.S.)/economia , National Institutes of Health (U.S.)/organização & administração , Compostos Organosselênicos/farmacologia , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Inibidores de Proteases/farmacologia , RNA Replicase/antagonistas & inibidores , RNA Replicase/química , RNA Replicase/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Síncrotrons , Fatores de Tempo , Reino Unido , Estados Unidos , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/imunologia , Vacinas Virais/química , Vacinas Virais/imunologia
4.
J Biol Chem ; 295(15): 4780-4781, 2020 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-32277065

RESUMO

The nucleotide analogue remdesivir is an investigational drug for the treatment of human coronavirus infection. Remdesivir is a phosphoramidate prodrug and is known to target viral RNA-dependent RNA polymerases. In this issue, Gordon et al. identify that remdesivir acts as a delayed RNA chain terminator for MERS-CoV polymerase complexes.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Coronavirus/efeitos dos fármacos , Coronavirus/enzimologia , RNA Replicase/antagonistas & inibidores , Monofosfato de Adenosina/farmacologia , Alanina/farmacologia , Animais , Coronavirus/fisiologia , Infecções por Coronavirus/virologia , Exonucleases , Humanos , Pandemias , Replicação Viral/efeitos dos fármacos
5.
J Transl Med ; 18(1): 179, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32321524

RESUMO

BACKGROUND: SARS-CoV-2 is a RNA coronavirus responsible for the pandemic of the Severe Acute Respiratory Syndrome (COVID-19). RNA viruses are characterized by a high mutation rate, up to a million times higher than that of their hosts. Virus mutagenic capability depends upon several factors, including the fidelity of viral enzymes that replicate nucleic acids, as SARS-CoV-2 RNA dependent RNA polymerase (RdRp). Mutation rate drives viral evolution and genome variability, thereby enabling viruses to escape host immunity and to develop drug resistance. METHODS: We analyzed 220 genomic sequences from the GISAID database derived from patients infected by SARS-CoV-2 worldwide from December 2019 to mid-March 2020. SARS-CoV-2 reference genome was obtained from the GenBank database. Genomes alignment was performed using Clustal Omega. Mann-Whitney and Fisher-Exact tests were used to assess statistical significance. RESULTS: We characterized 8 novel recurrent mutations of SARS-CoV-2, located at positions 1397, 2891, 14408, 17746, 17857, 18060, 23403 and 28881. Mutations in 2891, 3036, 14408, 23403 and 28881 positions are predominantly observed in Europe, whereas those located at positions 17746, 17857 and 18060 are exclusively present in North America. We noticed for the first time a silent mutation in RdRp gene in England (UK) on February 9th, 2020 while a different mutation in RdRp changing its amino acid composition emerged on February 20th, 2020 in Italy (Lombardy). Viruses with RdRp mutation have a median of 3 point mutations [range: 2-5], otherwise they have a median of 1 mutation [range: 0-3] (p value < 0.001). CONCLUSIONS: These findings suggest that the virus is evolving and European, North American and Asian strains might coexist, each of them characterized by a different mutation pattern. The contribution of the mutated RdRp to this phenomenon needs to be investigated. To date, several drugs targeting RdRp enzymes are being employed for SARS-CoV-2 infection treatment. Some of them have a predicted binding moiety in a SARS-CoV-2 RdRp hydrophobic cleft, which is adjacent to the 14408 mutation we identified. Consequently, it is important to study and characterize SARS-CoV-2 RdRp mutation in order to assess possible drug-resistance viral phenotypes. It is also important to recognize whether the presence of some mutations might correlate with different SARS-CoV-2 mortality rates.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Evolução Molecular , Genoma Viral/genética , Mutação , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , RNA Replicase/genética , Adulto , Ásia/epidemiologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/mortalidade , Farmacorresistência Viral/genética , Europa (Continente)/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Taxa de Mutação , América do Norte/epidemiologia , Oceania/epidemiologia , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/mortalidade , RNA Replicase/antagonistas & inibidores , RNA Replicase/metabolismo
6.
Science ; 368(6492): 779-782, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32277040

RESUMO

A novel coronavirus [severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2)] outbreak has caused a global coronavirus disease 2019 (COVID-19) pandemic, resulting in tens of thousands of infections and thousands of deaths worldwide. The RNA-dependent RNA polymerase [(RdRp), also named nsp12] is the central component of coronaviral replication and transcription machinery, and it appears to be a primary target for the antiviral drug remdesivir. We report the cryo-electron microscopy structure of COVID-19 virus full-length nsp12 in complex with cofactors nsp7 and nsp8 at 2.9-angstrom resolution. In addition to the conserved architecture of the polymerase core of the viral polymerase family, nsp12 possesses a newly identified ß-hairpin domain at its N terminus. A comparative analysis model shows how remdesivir binds to this polymerase. The structure provides a basis for the design of new antiviral therapeutics that target viral RdRp.


Assuntos
Betacoronavirus/enzimologia , RNA Replicase/química , RNA Replicase/ultraestrutura , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/ultraestrutura , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/metabolismo , Alanina/farmacologia , Antivirais/metabolismo , Antivirais/farmacologia , Domínio Catalítico , Microscopia Crioeletrônica , Desenho de Fármacos , Modelos Moleculares , Conformação Proteica em Folha beta , Domínios Proteicos , RNA Replicase/antagonistas & inibidores , RNA Replicase/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
7.
J Microbiol Immunol Infect ; 53(3): 436-443, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32307245

RESUMO

An outbreak related to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in Wuhan, China in December 2019. An extremely high potential for dissemination resulted in the global coronavirus disease 2019 (COVID-19) pandemic in 2020. Despite the worsening trends of COVID-19, no drugs are validated to have significant efficacy in clinical treatment of COVID-19 patients in large-scale studies. Remdesivir is considered the most promising antiviral agent; it works by inhibiting the activity of RNA-dependent RNA polymerase (RdRp). A large-scale study investigating the clinical efficacy of remdesivir (200 mg on day 1, followed by 100 mg once daily) is on-going. The other excellent anti-influenza RdRp inhibitor favipiravir is also being clinically evaluated for its efficacy in COVID-19 patients. The protease inhibitor lopinavir/ritonavir (LPV/RTV) alone is not shown to provide better antiviral efficacy than standard care. However, the regimen of LPV/RTV plus ribavirin was shown to be effective against SARS-CoV in vitro. Another promising alternative is hydroxychloroquine (200 mg thrice daily) plus azithromycin (500 mg on day 1, followed by 250 mg once daily on day 2-5), which showed excellent clinical efficacy on Chinese COVID-19 patients and anti-SARS-CoV-2 potency in vitro. The roles of teicoplanin (which inhibits the viral genome exposure in cytoplasm) and monoclonal and polyclonal antibodies in the treatment of SARS-CoV-2 are under investigation. Avoiding the prescription of non-steroidal anti-inflammatory drugs, angiotensin converting enzyme inhibitors, or angiotensin II type I receptor blockers is advised for COVID-19 patients.


Assuntos
Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/uso terapêutico , Alanina/análogos & derivados , Alanina/uso terapêutico , Amidas/uso terapêutico , Azitromicina/uso terapêutico , Infecções por Coronavirus/terapia , Combinação de Medicamentos , Humanos , Hidroxicloroquina/uso terapêutico , Imunização Passiva/métodos , Lopinavir/uso terapêutico , Pandemias , Pirazinas/uso terapêutico , RNA Replicase/antagonistas & inibidores , Ritonavir/uso terapêutico , Teicoplanina/uso terapêutico
8.
J Biol Chem ; 295(20): 6785-6797, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32284326

RESUMO

Effective treatments for coronavirus disease 2019 (COVID-19) are urgently needed to control this current pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Replication of SARS-CoV-2 depends on the viral RNA-dependent RNA polymerase (RdRp), which is the likely target of the investigational nucleotide analogue remdesivir (RDV). RDV shows broad-spectrum antiviral activity against RNA viruses, and previous studies with RdRps from Ebola virus and Middle East respiratory syndrome coronavirus (MERS-CoV) have revealed that delayed chain termination is RDV's plausible mechanism of action. Here, we expressed and purified active SARS-CoV-2 RdRp composed of the nonstructural proteins nsp8 and nsp12. Enzyme kinetics indicated that this RdRp efficiently incorporates the active triphosphate form of RDV (RDV-TP) into RNA. Incorporation of RDV-TP at position i caused termination of RNA synthesis at position i+3. We obtained almost identical results with SARS-CoV, MERS-CoV, and SARS-CoV-2 RdRps. A unique property of RDV-TP is its high selectivity over incorporation of its natural nucleotide counterpart ATP. In this regard, the triphosphate forms of 2'-C-methylated compounds, including sofosbuvir, approved for the management of hepatitis C virus infection, and the broad-acting antivirals favipiravir and ribavirin, exhibited significant deficits. Furthermore, we provide evidence for the target specificity of RDV, as RDV-TP was less efficiently incorporated by the distantly related Lassa virus RdRp, and termination of RNA synthesis was not observed. These results collectively provide a unifying, refined mechanism of RDV-mediated RNA synthesis inhibition in coronaviruses and define this nucleotide analogue as a direct-acting antiviral.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/farmacologia , Betacoronavirus/enzimologia , RNA Replicase/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Monofosfato de Adenosina/farmacologia , Alanina/farmacologia , Animais , Betacoronavirus/fisiologia , Modelos Moleculares , Células Sf9 , Spodoptera
9.
Life Sci ; 248: 117477, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32119961

RESUMO

AIMS: A newly emerged Human Coronavirus (HCoV) is reported two months ago in Wuhan, China (COVID-19). Until today >2700 deaths from the 80,000 confirmed cases reported mainly in China and 40 other countries. Human to human transmission is confirmed for COVID-19 by China a month ago. Based on the World Health Organization (WHO) reports, SARS HCoV is responsible for >8000 cases with confirmed 774 deaths. Additionally, MERS HCoV is responsible for 858 deaths out of about 2500 reported cases. The current study aims to test anti-HCV drugs against COVID-19 RNA dependent RNA polymerase (RdRp). MATERIALS AND METHODS: In this study, sequence analysis, modeling, and docking are used to build a model for Wuhan COVID-19 RdRp. Additionally, the newly emerged Wuhan HCoV RdRp model is targeted by anti-polymerase drugs, including the approved drugs Sofosbuvir and Ribavirin. KEY FINDINGS: The results suggest the effectiveness of Sofosbuvir, IDX-184, Ribavirin, and Remidisvir as potent drugs against the newly emerged HCoV disease. SIGNIFICANCE: The present study presents a perfect model for COVID-19 RdRp enabling its testing in silico against anti-polymerase drugs. Besides, the study presents some drugs that previously proved its efficiency against the newly emerged viral infection.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/química , Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Guanosina Monofosfato/análogos & derivados , Pneumonia Viral/tratamento farmacológico , RNA Replicase/antagonistas & inibidores , Ribavirina/química , Sofosbuvir/química , Proteínas Virais/antagonistas & inibidores , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Alanina/química , Alanina/metabolismo , Alphacoronavirus/enzimologia , Alphacoronavirus/genética , Sequência de Aminoácidos , Antivirais/metabolismo , Betacoronavirus/genética , Domínio Catalítico , Biologia Computacional/métodos , Infecções por Coronavirus/virologia , Reposicionamento de Medicamentos/métodos , Guanosina Monofosfato/química , Guanosina Monofosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Humanos , Simulação de Acoplamento Molecular , Pneumonia Viral/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Replicase/química , RNA Replicase/metabolismo , Ribavirina/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sofosbuvir/metabolismo , Termodinâmica , Uridina Trifosfato/química , Uridina Trifosfato/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo
10.
Life Sci ; 253: 117592, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32222463

RESUMO

AIMS: A new human coronavirus (HCoV), which has been designated SARS-CoV-2, began spreading in December 2019 in Wuhan City, China causing pneumonia called COVID-19. The spread of SARS-CoV-2 has been faster than any other coronaviruses that have succeeded in crossing the animal-human barrier. There is concern that this new virus will spread around the world as did the previous two HCoVs-Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS)-each of which caused approximately 800 deaths in the years 2002 and 2012, respectively. Thus far, 11,268 deaths have been reported from the 258,842 confirmed infections in 168 countries. MAIN METHODS: In this study, the RNA-dependent RNA polymerase (RdRp) of the newly emerged coronavirus is modeled, validated, and then targeted using different anti-polymerase drugs currently on the market that have been approved for use against various viruses. KEY FINDINGS: The results suggest the effectiveness of Ribavirin, Remdesivir, Sofosbuvir, Galidesivir, and Tenofovir as potent drugs against SARS-CoV-2 since they tightly bind to its RdRp. In addition, the results suggest guanosine derivative (IDX-184), Setrobuvir, and YAK as top seeds for antiviral treatments with high potential to fight the SARS-CoV-2 strain specifically. SIGNIFICANCE: The availability of FDA-approved anti-RdRp drugs can help treat patients and reduce the danger of the mysterious new viral infection COVID-19. The drugs mentioned above can tightly bind to the RdRp of the SARS-CoV-2 strain and thus may be used to treat the disease. No toxicity measurements are required for these drugs since they were previously tested prior to their approval by the FDA.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Simulação de Acoplamento Molecular , Inibidores da Síntese de Ácido Nucleico/química , RNA Replicase/antagonistas & inibidores , Antivirais/química , Infecções por Coronavirus/tratamento farmacológico , Humanos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Nucleosídeos/análogos & derivados
11.
J Biol Chem ; 295(15): 4773-4779, 2020 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-32094225

RESUMO

Antiviral drugs for managing infections with human coronaviruses are not yet approved, posing a serious challenge to current global efforts aimed at containing the outbreak of severe acute respiratory syndrome-coronavirus 2 (CoV-2). Remdesivir (RDV) is an investigational compound with a broad spectrum of antiviral activities against RNA viruses, including severe acute respiratory syndrome-CoV and Middle East respiratory syndrome (MERS-CoV). RDV is a nucleotide analog inhibitor of RNA-dependent RNA polymerases (RdRps). Here, we co-expressed the MERS-CoV nonstructural proteins nsp5, nsp7, nsp8, and nsp12 (RdRp) in insect cells as a part a polyprotein to study the mechanism of inhibition of MERS-CoV RdRp by RDV. We initially demonstrated that nsp8 and nsp12 form an active complex. The triphosphate form of the inhibitor (RDV-TP) competes with its natural counterpart ATP. Of note, the selectivity value for RDV-TP obtained here with a steady-state approach suggests that it is more efficiently incorporated than ATP and two other nucleotide analogs. Once incorporated at position i, the inhibitor caused RNA synthesis arrest at position i + 3. Hence, the likely mechanism of action is delayed RNA chain termination. The additional three nucleotides may protect the inhibitor from excision by the viral 3'-5' exonuclease activity. Together, these results help to explain the high potency of RDV against RNA viruses in cell-based assays.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/farmacologia , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , RNA Replicase/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Monofosfato de Adenosina/química , Monofosfato de Adenosina/farmacologia , Alanina/química , Alanina/farmacologia , Animais , Antivirais/química , Coronavirus/enzimologia , Ebolavirus/enzimologia , Expressão Gênica , Inibidores da Síntese de Ácido Nucleico/química , RNA , RNA Replicase/genética , Células Sf9 , Proteínas não Estruturais Virais/genética
12.
PLoS Negl Trop Dis ; 13(11): e0007894, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31738758

RESUMO

Dengue is a mosquito-borne viral infection that has spread globally in recent years. Around half of the world's population, especially in the tropics and subtropics, is at risk of infection. Every year, 50-100 million clinical cases are reported, and more than 500,000 patients develop the symptoms of severe dengue infection: dengue haemorrhagic fever and dengue shock syndrome, which threaten life in Asia and Latin America. No antiviral drug for dengue is available. The dengue virus (DENV) non-structural protein 5 (NS5), which possesses the RNA-dependent RNA polymerase (RdRp) activity and is responsible for viral replication and transcription, is an attractive target for anti-dengue drug development. In the present study, 16,240 small-molecule compounds in a fragment library were screened for their capabilities to inhibit the DENV type 2 (DENV2) RdRp activities in vitro. Based on in cellulo antiviral and cytotoxity assays, we selected the compound RK-0404678 with the EC50 value of 6.0 µM for DENV2. Crystallographic analyses revealed two unique binding sites for RK-0404678 within the RdRp, which are conserved in flavivirus NS5 proteins. No resistant viruses emerged after nine rounds of serial passage of DENV2 in the presence of RK-0404678, suggesting the high genetic barrier of this compound to the emergence of a resistant virus. Collectively, RK-0404678 and its binding sites provide a new framework for antiviral drug development.


Assuntos
Antivirais/isolamento & purificação , Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , RNA Replicase/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Sítios de Ligação , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Testes de Sensibilidade Microbiana , Ligação Proteica , RNA Replicase/química , RNA Replicase/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
13.
Curr Pharm Des ; 25(12): 1392-1401, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31258065

RESUMO

BACKGROUND: The recent Nipah virus (NiV) outbreak in India has caused a state of chaos, with potential to become the next international pandemic. There is still a great deal to learn about NiV for the development of a potent treatment against it. The NiV non-structural proteins play important roles in the lifecycle of the virus, with the RNA-dependent RNA-polymerase (RdRp) being a vital component in viral replication. In this study, we not only provide a comprehensive overview of all the literature concerning NiV, we also propose a model of the NiV RdRp and screen for potential inhibitors of the viral enzyme. METHODS: In this study, computational tools were utilized in the design of a NiV RdRp homology model. The active site of RdRp was then identified and potential inhibitors of the protein were discovered with the use of pharmacophore-based screening. RESULTS: Ramachandran plot analysis revealed a favourable model. Upon binding of nucleoside analog, 4'- Azidocytidine, active site residues Trp1714 and Ser1713 took part in stabilizing hydrogen bonds, while Thr1716, Ser1478, Ser1476 and Glu1465 contributed to hydrophobic interactions. Pharmacophore based screening yielded 18 hits, of which ZINC00085930 demonstrated the most optimal binding energy (-8.1 kcal/mol), validating its use for further analysis as an inhibitor of NiV. CONCLUSION: In this study we provide a critical guide, elucidating on the in silico requirements of the drug design and discovery process against NiV. This material lays a foundation for future research into the design and development of drugs that inhibit NiV.


Assuntos
Antivirais/farmacologia , Vírus Nipah/enzimologia , RNA Replicase/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Domínio Catalítico , Desenho de Fármacos , Vírus Nipah/patogenicidade , Vírus Nipah/fisiologia , Replicação Viral
14.
Int J Antimicrob Agents ; 54(4): 502-506, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31310806

RESUMO

The current outbreak of Zika virus (ZIKV) is the impetus for novel, safe and efficacious anti-ZIKV agents. ZIKV non-structural protein 5 RNA-dependent RNA polymerase (RdRp) is essential for viral replication and is logically regarded as an attractive drug target. This study used a fluorescence-based polymerase assay to find an anti-infective drug 10-undecenoic acid zinc salt (UA) which could inhibit RdRp activity with a half maximal inhibitory concentration (IC50) of 1.13-1.25 µM. Molecular docking and site-directed mutagenesis analyses identified D535 as the key amino acid in the interaction between RdRp and UA. Importantly, the surface plasmon resonance assay showed that UA had strong direct binding with ZIKV wild-type RdRp and a relatively weak interaction with D535A-RdRp. As a control, the nucleoside inhibitor sofosbuvir triphosphate (PSI-7409) conferred insensitivity to the fluorescence-based RdRp assay and cannot bind directly with RdRp. Moreover, UA showed anti-ZIKV activity comparable to sofosbuvir. All these results indicate that UA is likely to be a promising lead compound against ZIKV, exhibiting a different mechanism than sofosbuvir.


Assuntos
Antivirais/isolamento & purificação , Antivirais/farmacologia , RNA Replicase/antagonistas & inibidores , Ácidos Undecilênicos/isolamento & purificação , Ácidos Undecilênicos/farmacologia , Zika virus/enzimologia , Avaliação Pré-Clínica de Medicamentos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , RNA Replicase/genética , Ressonância de Plasmônio de Superfície
15.
Viruses ; 11(4)2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30987343

RESUMO

Remdesivir (GS-5734) is a 1'-cyano-substituted adenosine nucleotide analogue prodrug that shows broad-spectrum antiviral activity against several RNA viruses. This compound is currently under clinical development for the treatment of Ebola virus disease (EVD). While antiviral effects have been demonstrated in cell culture and in non-human primates, the mechanism of action of Ebola virus (EBOV) inhibition for remdesivir remains to be fully elucidated. The EBOV RNA-dependent RNA polymerase (RdRp) complex was recently expressed and purified, enabling biochemical studies with the relevant triphosphate (TP) form of remdesivir and its presumptive target. In this study, we confirmed that remdesivir-TP is able to compete for incorporation with adenosine triphosphate (ATP). Enzyme kinetics revealed that EBOV RdRp and respiratory syncytial virus (RSV) RdRp incorporate ATP and remdesivir-TP with similar efficiencies. The selectivity of ATP against remdesivir-TP is ~4 for EBOV RdRp and ~3 for RSV RdRp. In contrast, purified human mitochondrial RNA polymerase (h-mtRNAP) effectively discriminates against remdesivir-TP with a selectivity value of ~500-fold. For EBOV RdRp, the incorporated inhibitor at position i does not affect the ensuing nucleotide incorporation event at position i+1. For RSV RdRp, we measured a ~6-fold inhibition at position i+1 although RNA synthesis was not terminated. Chain termination was in both cases delayed and was seen predominantly at position i+5. This pattern is specific to remdesivir-TP and its 1'-cyano modification. Compounds with modifications at the 2'-position show different patterns of inhibition. While 2'-C-methyl-ATP is not incorporated, ara-ATP acts as a non-obligate chain terminator and prevents nucleotide incorporation at position i+1. Taken together, our biochemical data indicate that the major contribution to EBOV RNA synthesis inhibition by remdesivir can be ascribed to delayed chain termination. The long distance of five residues between the incorporated nucleotide analogue and its inhibitory effect warrant further investigation.


Assuntos
Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/farmacologia , Ebolavirus/enzimologia , Inibidores Enzimáticos/farmacologia , RNA Replicase/antagonistas & inibidores , Ribonucleotídeos/farmacologia , Proteínas Virais/antagonistas & inibidores , Adenosina/química , Adenosina/metabolismo , Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Alanina/química , Alanina/metabolismo , Alanina/farmacologia , Antivirais/química , Antivirais/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Cinética , Estrutura Molecular , Pró-Fármacos , RNA Replicase/metabolismo , RNA Viral/biossíntese , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/enzimologia , Vírus Sincicial Respiratório Humano/fisiologia , Ribonucleotídeos/química , Ribonucleotídeos/metabolismo , Especificidade por Substrato , Proteínas Virais/metabolismo
16.
Viruses ; 11(4)2019 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-31013994

RESUMO

To date, two plant genes encoding RNA-dependent RNA polymerases (RdRs) that play major roles in the defense against RNA viruses have been identified: (i) RdR1, which is responsible for the viral small RNAs (vsRNAs) found in virus-infected plants, and, (ii) RdR6, which acts as a surrogate in the absence of RdR1. In this study, the role of RdR6 in the defense against viroid infection was examined by knock-down of RdR6 followed by potato spindle tuber viroid (PSTVd) infection. The suppression of RdR6 expression increased the plant's growth, as was illustrated by the plant's increased height. PSTVd infection of RdR6 compromised plants resulted in an approximately three-fold increase in the accumulation of viroid RNA as compared to that seen in control plants. Additionally, RNA gel blot assay revealed an increase in the number of viroids derived small RNAs in RdR6 suppressed plants as compared to control plants. These data provide a direct correlation between RdR6 and viroid accumulation and indicate the role of RDR6 in the plant's susceptibility to viroid infection.


Assuntos
Vírus de Plantas/fisiologia , RNA Replicase/antagonistas & inibidores , Tabaco/virologia , Viroides/fisiologia , Técnicas de Silenciamento de Genes , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Interferência de RNA , RNA Replicase/genética , Vírus de RNA/patogenicidade , RNA Viral/análise , RNA Viral/genética , Tabaco/genética
17.
Curr Opin Infect Dis ; 32(2): 176-186, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30724789

RESUMO

PURPOSE OF REVIEW: We review antivirals inhibiting subunits of the influenza polymerase complex that are advancing in clinical development. RECENT FINDINGS: Favipiravir, pimodivir, and baloxavir are inhibitory in preclinical models for influenza A viruses, including pandemic threat viruses and those resistant to currently approved antivirals, and two (favipiravir and baloxavir) also inhibit influenza B viruses. All are orally administered, although the dosing regimens vary. The polymerase basic protein 1 transcriptase inhibitor favipiravir has shown inconsistent clinical effects in uncomplicated influenza, and is teratogenic effects in multiple species, contraindicating its use in pregnancy. The polymerase basic protein 2 cap-binding inhibitor pimodivir displays antiviral effects alone and in combination with oseltamivir in uncomplicated influenza, although variants with reduced susceptibility emerge frequently during monotherapy. Single doses of the polymerase acidic protein cap-dependent endonuclease inhibitor baloxavir are effective in alleviating symptoms and rapidly inhibiting viral replication in otherwise healthy and higher risk patients with acute influenza, although variants with reduced susceptibility emerge frequently during monotherapy. Combinations of newer polymerase inhibitors with neuraminidase inhibitors show synergy in preclinical models and are currently undergoing clinical testing in hospitalized patients. SUMMARY: These new polymerase inhibitors promise to add to the clinical management options and overall control strategies for influenza virus infections.


Assuntos
Antivirais/administração & dosagem , Desenvolvimento de Medicamentos/tendências , Endorribonucleases/antagonistas & inibidores , Influenza Humana/tratamento farmacológico , Orthomyxoviridae/enzimologia , RNA Replicase/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Antivirais/farmacologia , Ensaios Clínicos como Assunto , Humanos
18.
Antiviral Res ; 164: 91-96, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30771405

RESUMO

Baloxavir Marboxil (BXM) is an influenza polymerase inhibitor antiviral that binds to the endonuclease region in the PA subunit of influenza A and B viruses. To establish the baseline susceptibility of viruses circulating prior to licensure of BXM and to monitor for susceptibility post-BXM use, a cell culture-based focus reduction assay was developed to determine the susceptibility of 286 circulating seasonal influenza viruses, A(H1N1)pdm09, A(H3N2), B (Yamagata/Victoria) lineage viruses, including neuraminidase inhibitor (NAI) resistant viruses, to Baloxavir Acid (BXA), the active metabolic form of BXM. BXA was effective against all influenza subtypes tested with mean EC50 values (minimum-maximum) of 0.7 ±â€¯0.5 nM (0.1-2.1 nM), 1.2 ±â€¯0.6 nM (0.1-2.4), 7.2 ±â€¯3.5 nM (0.7-14.8), and 5.8 ±â€¯4.5 nM (1.8-15.5) obtained for A(H1N1)pdm09, A(H3N2), B(Victoria lineage), and B(Yamagata lineage) influenza viruses, respectively. Using reverse genetics, amino acid substitutions known to alter BXA susceptibility were introduced into the PA protein resulting in EC50 fold change increases that ranged from 2 to 65. Our study demonstrates that currently circulating viruses are susceptible to BXA and that the newly developed focus reduction assay is well suited to susceptibility monitoring in reference laboratories.


Assuntos
Antivirais/farmacologia , Orthomyxoviridae/efeitos dos fármacos , Oxazinas/farmacologia , Piridinas/farmacologia , RNA Replicase/antagonistas & inibidores , Tiepinas/farmacologia , Triazinas/farmacologia , Proteínas Virais/antagonistas & inibidores , Farmacorresistência Viral , Inibidores Enzimáticos/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/enzimologia , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/enzimologia , Vírus da Influenza B/efeitos dos fármacos , Vírus da Influenza B/enzimologia , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Orthomyxoviridae/enzimologia
19.
BMC Immunol ; 20(1): 6, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30669993

RESUMO

BACKGROUND: Enterovirus A 71 (EV71) is a neurotropic virus that may lead to acute flaccid paralysis, encephalitis, cardiopulmonary failure or even death. No vaccine and defensive drug controlling EV71 is currently available, novel and efficient antiviral drug or vaccine is therefore urgently needed. 3Dpol (RNA-dependent RNA polymerase (RdRp)) has been an important target for anti-EV71 drug development. METHODS: A panel of monoclonal IgG antibodies (mAbs) against EV71 3Dpol were generated by traditional cell fusion methods. And the antibody affinity and specificity to EV71 3Dpol were evaluated by Enzyme-linked Immunosorbent Assay (ELISA), Indirect Fluorescent Assay (IFA) and Western blotting. Antiviral activities of these antibodies were also determined in vitro and in vivo. RESULTS: Two mAbs towards EV71 3Dpol were able to effectively suppress EV71 replication in Vero-1008 cell when intracellarly delivered. And they also dampened the RNA polymerase activity of 3Dpol in vitro. More importantly, these mAbs provided partial protection in EV71-challenged neonatal murine challenge model. CONCLUSIONS: These results showed that two of mAbs against EV71 3Dpol inhibited EV71 replication and could be utilized as promising therapeutic drug candidate.


Assuntos
Anticorpos Monoclonais/farmacologia , Antivirais/farmacologia , Enterovirus Humano A/efeitos dos fármacos , RNA Replicase/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Sítios de Ligação , Modelos Animais de Doenças , Enterovirus Humano A/genética , Enterovirus Humano A/imunologia , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/mortalidade , Infecções por Enterovirus/virologia , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Camundongos , Ligação Proteica , RNA Replicase/metabolismo
20.
Artigo em Inglês | MEDLINE | ID: mdl-30642926

RESUMO

West Nile virus (WNV) is a medically important emerging arbovirus causing serious neuroinfections in humans and against which no approved antiviral therapy is currently available. In this study, we demonstrate that 2'-C-methyl- or 4'-azido-modified nucleosides are highly effective inhibitors of WNV replication, showing nanomolar or low micromolar anti-WNV activity and negligible cytotoxicity in cell culture. One representative of C2'-methylated nucleosides, 7-deaza-2'-C-methyladenosine, significantly protected WNV-infected mice from disease progression and mortality. Twice daily treatment at 25 mg/kg starting at the time of infection resulted in 100% survival of the mice. This compound was highly effective, even if the treatment was initiated 3 days postinfection, at the time of a peak of viremia, which resulted in a 90% survival rate. However, the antiviral effect of 7-deaza-2'-C-methyladenosine was absent or negligible when the treatment was started 8 days postinfection (i.e., at the time of extensive brain infection). The 4'-azido moiety appears to be another important determinant for highly efficient inhibition of WNV replication in vitro However, the strong anti-WNV effect of 4'-azidocytidine and 4'-azido-aracytidine was cell type dependent and observed predominantly in porcine kidney stable (PS) cells. The effect was much less pronounced in Vero cells. Our results indicate that 2'-C-methylated or 4'-azidated nucleosides merit further investigation as potential therapeutic agents for treating WNV infections as well as infections caused by other medically important flaviviruses.


Assuntos
Antivirais/uso terapêutico , Tubercidina/análogos & derivados , Febre do Nilo Ocidental/tratamento farmacológico , Vírus do Nilo Ocidental/efeitos dos fármacos , Animais , Linhagem Celular , Chlorocebus aethiops , Modelos Animais de Doenças , Progressão da Doença , Feminino , Camundongos , Camundongos Endogâmicos BALB C , RNA Replicase/antagonistas & inibidores , Suínos , Tubercidina/uso terapêutico , Células Vero , Viremia/tratamento farmacológico , Replicação Viral/efeitos dos fármacos , Febre do Nilo Ocidental/patologia , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/genética
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