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1.
Int J Mol Sci ; 22(4)2021 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-33557278

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. The virus still spreads globally through human-to-human transmission. Nevertheless, there are no specific treatments clinically approved. This study aimed to compare antiviral activity of gemcitabine and its analogue 2'-fluoro-2'-deoxycytidine (2FdC) against SARS-CoV-2 as well as cytotoxicity in vitro. Fluorescent image-based antiviral assays revealed that gemcitabine was highly potent, with a 50% effective concentration (EC50) of 1.2 µM, more active than the well-known nucleoside monophosphate remdesivir (EC50 = 35.4 µM). In contrast, 2FdC was marginally active (EC50 = 175.2 µM). For all three compounds, the 50% cytotoxic concentration (CC50) values were over 300 µM toward Vero CCL-81 cells. Western blot and quantitative reverse-transcription polymerase chain reaction analyses verified that gemcitabine blocked viral protein expression in virus-infected cells, not only Vero CCL-81 cells but also Calu-3 human lung epithelial cells in a dose-dependent manner. It was found that gemcitabine has a synergistic effect when combined with remdesivir. This report suggests that the difluoro group of gemcitabine is critical for the antiviral activity and that its combination with other evaluated antiviral drugs, such as remdesivir, could be a desirable option to treat SARS-CoV-2 infection.


Asunto(s)
Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Desoxicitidina/análogos & derivados , /efectos de los fármacos , Adenosina Monofosfato/farmacología , Alanina/farmacología , Animales , Antivirales/farmacología , /virología , Línea Celular , Chlorocebus aethiops , Desoxicitidina/farmacología , Quimioterapia Combinada , Humanos , Concentración 50 Inhibidora , Células Vero , Replicación Viral/efectos de los fármacos
2.
Nat Commun ; 12(1): 279, 2021 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-33436624

RESUMEN

Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analog and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3'-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3'-nucleotide of the RNA product is matched and located with the template base in the active center, and this may impair proofreading by the viral 3'-exonuclease. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.


Asunto(s)
Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/farmacología , /efectos de los fármacos , Antivirales/farmacología , Aptámeros de Nucleótidos , /efectos de los fármacos , Nucleótidos , ARN Viral , /enzimología , Replicación Viral/efectos de los fármacos
3.
Nat Commun ; 12(1): 668, 2021 01 28.
Artículo en Inglés | MEDLINE | ID: mdl-33510133

RESUMEN

Except remdesivir, no specific antivirals for SARS-CoV-2 infection are currently available. Here, we characterize two small-molecule-compounds, named GRL-1720 and 5h, containing an indoline and indole moiety, respectively, which target the SARS-CoV-2 main protease (Mpro). We use VeroE6 cell-based assays with RNA-qPCR, cytopathic assays, and immunocytochemistry and show both compounds to block the infectivity of SARS-CoV-2 with EC50 values of 15 ± 4 and 4.2 ± 0.7 µM for GRL-1720 and 5h, respectively. Remdesivir permitted viral breakthrough at high concentrations; however, compound 5h completely blocks SARS-CoV-2 infection in vitro without viral breakthrough or detectable cytotoxicity. Combination of 5h and remdesivir exhibits synergism against SARS-CoV-2. Additional X-ray structural analysis show that 5h forms a covalent bond with Mpro and makes polar interactions with multiple active site amino acid residues. The present data suggest that 5h might serve as a lead Mpro inhibitor for the development of therapeutics for SARS-CoV-2 infection.


Asunto(s)
/tratamiento farmacológico , /efectos de los fármacos , /efectos de los fármacos , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Animales , Antivirales/farmacología , Línea Celular , Chlorocebus aethiops , Humanos , Indoles/farmacología , Células Vero , /metabolismo
4.
Ann Palliat Med ; 10(1): 707-720, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33440983

RESUMEN

The whole world is battling through coronavirus disease 2019 (COVID-19) which is a fatal pandemic. In the early 2020, the World Health Organization (WHO) declared it as a global health emergency without definitive treatments and preventive approaches. In the absence of definitive therapeutic agents, this thorough review summarizes and outlines the potency and safety of all molecules and therapeutics which may have potential antiviral effects. A number of molecules and therapeutics licensed or being tested for some other conditions were found effective in different in vitro studies as well as in many small sample-sized clinical trials and independent case studies. However, in those clinical trials, there were some limitations which need to be overcome to find the most promising antiviral against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In conclusion, many of above-mentioned antivirals seems to have some therapeutic effects but none of them have been shown to have a strong evidence for their proper recommendation and approval in the treatment of COVID-19. Constantly evolving new evidences, exclusive adult data, language barrier, and type of study (observational, retrospective, small-sized clinical trials, or independent case series) resulted to the several limitations of this review. The need for multicentered, large sample-sized, randomized, placebo-controlled trials on COVID-19 patients to reach a proper conclusion on the most promising antiviral agent is warranted.


Asunto(s)
Antivirales/uso terapéutico , /terapia , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Adenosina Monofosfato/uso terapéutico , Alanina/análogos & derivados , Alanina/farmacología , Alanina/uso terapéutico , Amidas/farmacología , Amidas/uso terapéutico , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Azetidinas/farmacología , Azetidinas/uso terapéutico , Cloroquina/farmacología , Cloroquina/uso terapéutico , Combinación de Medicamentos , Humanos , Hidroxicloroquina/farmacología , Hidroxicloroquina/uso terapéutico , Inmunización Pasiva , Indoles/farmacología , Indoles/uso terapéutico , Interferones/farmacología , Interferones/uso terapéutico , Ivermectina/farmacología , Ivermectina/uso terapéutico , Lopinavir/farmacología , Lopinavir/uso terapéutico , Oseltamivir/farmacología , Oseltamivir/uso terapéutico , Purinas/farmacología , Purinas/uso terapéutico , Pirazinas/farmacología , Pirazinas/uso terapéutico , Pirazoles/farmacología , Pirazoles/uso terapéutico , Ribavirina/farmacología , Ribavirina/uso terapéutico , Ritonavir/farmacología , Ritonavir/uso terapéutico , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Tiazoles/farmacología , Tiazoles/uso terapéutico
5.
Cell Stem Cell ; 28(2): 331-342.e5, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33450186

RESUMEN

ApoE4, a strong genetic risk factor for Alzheimer disease, has been associated with increased risk for severe COVID-19. However, it is unclear whether ApoE4 alters COVID-19 susceptibility or severity, and the role of direct viral infection in brain cells remains obscure. We tested the neurotropism of SARS-CoV2 in human-induced pluripotent stem cell (hiPSC) models and observed low-grade infection of neurons and astrocytes that is boosted in neuron-astrocyte co-cultures and organoids. We then generated isogenic ApoE3/3 and ApoE4/4 hiPSCs and found an increased rate of SARS-CoV-2 infection in ApoE4/4 neurons and astrocytes. ApoE4 astrocytes exhibited enlarged size and elevated nuclear fragmentation upon SARS-CoV-2 infection. Finally, we show that remdesivir treatment inhibits SARS-CoV2 infection of hiPSC neurons and astrocytes. These findings suggest that ApoE4 may play a causal role in COVID-19 severity. Understanding how risk factors impact COVID-19 susceptibility and severity will help us understand the potential long-term effects in different patient populations.


Asunto(s)
Apolipoproteínas E/metabolismo , Encéfalo/patología , Encéfalo/virología , Células Madre Pluripotentes Inducidas/virología , Tropismo/fisiología , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Animales , Antivirales/farmacología , Astrocitos/efectos de los fármacos , Astrocitos/patología , Astrocitos/virología , Diferenciación Celular , Chlorocebus aethiops , Humanos , Degeneración Nerviosa/patología , Neuritas/patología , Neuronas/efectos de los fármacos , Neuronas/patología , Neuronas/virología , Organoides/efectos de los fármacos , Organoides/patología , Organoides/virología , Isoformas de Proteínas/metabolismo , Sinapsis/patología , Células Vero
6.
Sci Rep ; 11(1): 2229, 2021 01 26.
Artículo en Inglés | MEDLINE | ID: mdl-33500537

RESUMEN

The development of specific antiviral compounds to SARS-CoV-2 is an urgent task. One of the obstacles for the antiviral development is the requirement of biocontainment because infectious SARS-CoV-2 must be handled in a biosafety level-3 laboratory. Replicon, a non-infectious self-replicative viral RNA, could be a safe and effective tool for antiviral evaluation. Herein, we generated a PCR-based SARS-CoV-2 replicon. Eight fragments covering the entire SARS-CoV-2 genome except S, E, and M genes were amplified with HiBiT-tag sequence by PCR. The amplicons were ligated and in vitro transcribed to RNA. The cells electroporated with the replicon RNA showed more than 3000 times higher luminescence than MOCK control cells at 24 h post-electroporation, indicating robust translation and RNA replication of the replicon. The replication was drastically inhibited by remdesivir, an RNA polymerase inhibitor for SARS-CoV-2. The IC50 of remdesivir in this study was 0.29 µM, generally consistent to the IC50 obtained using infectious SARS-CoV-2 in a previous study (0.77 µM). Taken together, this system could be applied to the safe and effective antiviral evaluation without using infectious SARS-CoV-2. Because this is a PCR-based and transient replicon system, further improvement including the establishment of stable cell line must be achieved.


Asunto(s)
Antivirales/farmacología , Diseño de Fármacos , /efectos de los fármacos , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Animales , Células CHO , Chlorocebus aethiops , Cricetulus , Evaluación Preclínica de Medicamentos , Electroporación , Genoma Viral , Células HEK293 , Humanos , Concentración 50 Inhibidora , Cinética , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , ARN Viral , Regiones no Traducidas , Células Vero , Virión , Replicación Viral/efectos de los fármacos
7.
PLoS Pathog ; 17(1): e1009292, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33507952

RESUMEN

The human airway epithelium is the initial site of SARS-CoV-2 infection. We used flow cytometry and single cell RNA-sequencing to understand how the heterogeneity of this diverse cell population contributes to elements of viral tropism and pathogenesis, antiviral immunity, and treatment response to remdesivir. We found that, while a variety of epithelial cell types are susceptible to infection, ciliated cells are the predominant cell target of SARS-CoV-2. The host protease TMPRSS2 was required for infection of these cells. Importantly, remdesivir treatment effectively inhibited viral replication across cell types, and blunted hyperinflammatory responses. Induction of interferon responses within infected cells was rare and there was significant heterogeneity in the antiviral gene signatures, varying with the burden of infection in each cell. We also found that heavily infected secretory cells expressed abundant IL-6, a potential mediator of COVID-19 pathogenesis.


Asunto(s)
Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Antivirales/farmacología , /virología , Tropismo Viral , Adenosina Monofosfato/farmacología , Alanina/farmacología , /genética , Epitelio/inmunología , Epitelio/virología , Humanos , Interferones/genética , Interferones/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Pulmón/inmunología , Pulmón/virología , Tropismo Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacos
8.
Gene ; 764: 145081, 2021 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-32860897

RESUMEN

Metalaxyl is one of the main fungicides used to control pepper blight caused by Phytophthora capsici. Metalaxyl resistance of P. capsici, caused by the long-term intense use of this fungicide, has become one of the most serious challenges facing pest management. In this study, a conserved domain RPOLA-N of the RPA190 gene of P. capsici (RPA190-pc) was identified from the P. capsici SD1-9 strain. The role of the RPA190-pc underlying the metalaxyl resistance of P. capsici was investigated. Three P. capsici mutants, two with downregulated RPA190-pc (SD1-9C-3 and C-4) expression and one showing upregulation (OESD1-9-1), were obtained by Polyethylene Glycol (PEG) mediated protoplast transformations of P. capsici SD1-9. Quantitative real-time reverse transcription PCR results showed that RPA190-pc was downregulated by more than 60% in SD1-9C-3/C-4 and upregulated 3-fold in OESD1-9-1 compared with that of the control strain SD1-9. Evaluation of the metalaxyl resistance of these three transformants showed that the EC50 values of metalaxyl against SD1-9C-3, SD1-9C-4, and OESD1-9-1 were 120.0 µg·mL-1, 24.4 µg·mL-1, and 15573.0 µg·mL-1, respectively, corresponding to 63.3% decrease, 92.5% decrease, and 47.7-fold increase relative to the EC50 value in SD1-9. Compared with SD1-9, the mycelia of transformants SD1-9C-3, SD1-9C-4, and OESD1-9-1 showed more branches and shorter branches; and the transformants had different pathogenicity to different hosts plants. The expression of the candidate gene RPA190-pc during 10 life-history stages was further studied, the results showed that expression level reached a maximum at the zoospores stage, and it gradually increased with the increase of SD1 and SD1-9 infection time of pepper leaves, indicated that RPA190-pc may be related to the growth and pathogenicity of P. capsici. These results indicate that the expression of RPA190-pc is involved in the regulation of P. capsici resistance to metalaxyl.


Asunto(s)
Farmacorresistencia Fúngica/genética , Proteínas Fúngicas/genética , Fungicidas Industriales/farmacología , Phytophthora/genética , ARN Polimerasa I/genética , Alanina/análogos & derivados , Alanina/farmacología , Capsicum/microbiología , Regulación del Desarrollo de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Estadios del Ciclo de Vida/genética , Mutación , Micelio/genética , Micelio/crecimiento & desarrollo , Phytophthora/efectos de los fármacos , Phytophthora/crecimiento & desarrollo , Phytophthora/patogenicidad , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/prevención & control , Hojas de la Planta/microbiología , Dominios Proteicos/genética , Esporangios/genética , Esporangios/crecimiento & desarrollo , Virulencia/genética
9.
Acta Pharm ; 71(2): 163-174, 2021 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-33151166

RESUMEN

The current outbreak of novel coronavirus (COVID-19) infections urges the need to identify potential therapeutic agents. Therefore, the repurposing of FDA-approved drugs against today's diseases involves the use of de-risked compounds with potentially lower costs and shorter development timelines. In this study, the recently resolved X-ray crystallographic structure of COVID-19 main protease (Mpro) was used to generate a pharmacophore model and to conduct a docking study to capture antiviral drugs as new promising COVID-19 main protease inhibitors. The developed pharmacophore successfully captured five FDA-approved antiviral drugs (lopinavir, remdesivir, ritonavir, saquinavir and raltegravir). The five drugs were successfully docked into the binding site of COVID-19 Mpro and showed several specific binding interactions that were comparable to those tying the co-crystallized inhibitor X77 inside the binding site of COVID-19 Mpro. Three of the captured drugs namely, remdesivir, lopinavir and ritonavir, were reported to have promising results in COVID-19 treatment and therefore increases the confidence in our results. Our findings suggest an additional possible mechanism of action for remdesivir as an antiviral drug inhibiting COVID-19 Mpro. Additionally, a combination of structure-based pharmacophore modeling with a docking study is expected to facilitate the discovery of novel COVID-19 Mpro inhibitors.


Asunto(s)
Infecciones por Coronavirus/enzimología , Neumonía Viral/enzimología , Inhibidores de Proteasas/farmacología , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/química , Adenosina Monofosfato/farmacología , Adenosina Monofosfato/uso terapéutico , Alanina/análogos & derivados , Alanina/química , Alanina/farmacología , Alanina/uso terapéutico , Antivirales/química , Antivirales/farmacología , Infecciones por Coronavirus/tratamiento farmacológico , Cristalografía por Rayos X , Descubrimiento de Drogas/métodos , Reposicionamiento de Medicamentos , Humanos , Modelos Químicos , Simulación del Acoplamiento Molecular , Estructura Molecular , Pandemias , Neumonía Viral/tratamiento farmacológico , Inhibidores de Proteasas/química , Relación Estructura-Actividad
10.
Ned Tijdschr Geneeskd ; 1642020 11 12.
Artículo en Holandés | MEDLINE | ID: mdl-33331727

RESUMEN

Much has changed in the medical treatment of COVID-19 after the first patient with an infection with SARS-CoV-2 in the Netherlands was diagnosed in February 2020. On the basis of limited data, at first only off-label use of (hydroxy)chloroquine seemed to be a treatment option. However, now based on the findings of several randomized studies, other medicines have been included in the Dutch guidelines about the treatment of COVID-19. In this article, we will briefly discuss the current state of affairs with regard to the drugs (hydroxy) chloroquine, remdesivir and corticosteroids. Again, it appears that only well-executed randomized clinical trials can determine the status of various supposedly effective drugs.


Asunto(s)
Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Reposicionamiento de Medicamentos , Glucocorticoides , Hidroxicloroquina , /efectos de los fármacos , Adenosina Monofosfato/farmacología , Adenosina Monofosfato/uso terapéutico , Alanina/farmacología , Alanina/uso terapéutico , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Antivirales/farmacología , Antivirales/uso terapéutico , /epidemiología , Reposicionamiento de Medicamentos/métodos , Reposicionamiento de Medicamentos/normas , Glucocorticoides/farmacología , Glucocorticoides/uso terapéutico , Humanos , Hidroxicloroquina/farmacología , Hidroxicloroquina/uso terapéutico , Países Bajos/epidemiología , Ensayos Clínicos Controlados Aleatorios como Asunto , Resultado del Tratamiento
11.
Clin Microbiol Rev ; 34(1)2020 12 16.
Artículo en Inglés | MEDLINE | ID: mdl-33055231

RESUMEN

Patients and physicians worldwide are facing tremendous health care hazards that are caused by the ongoing severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) pandemic. Remdesivir (GS-5734) is the first approved treatment for severe coronavirus disease 2019 (COVID-19). It is a novel nucleoside analog with a broad antiviral activity spectrum among RNA viruses, including ebolavirus (EBOV) and the respiratory pathogens Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV, and SARS-CoV-2. First described in 2016, the drug was derived from an antiviral library of small molecules intended to target emerging pathogenic RNA viruses. In vivo, remdesivir showed therapeutic and prophylactic effects in animal models of EBOV, MERS-CoV, SARS-CoV, and SARS-CoV-2 infection. However, the substance failed in a clinical trial on ebolavirus disease (EVD), where it was inferior to investigational monoclonal antibodies in an interim analysis. As there was no placebo control in this study, no conclusions on its efficacy in EVD can be made. In contrast, data from a placebo-controlled trial show beneficial effects for patients with COVID-19. Remdesivir reduces the time to recovery of hospitalized patients who require supplemental oxygen and may have a positive impact on mortality outcomes while having a favorable safety profile. Although this is an important milestone in the fight against COVID-19, approval of this drug will not be sufficient to solve the public health issues caused by the ongoing pandemic. Further scientific efforts are needed to evaluate the full potential of nucleoside analogs as treatment or prophylaxis of viral respiratory infections and to develop effective antivirals that are orally bioavailable.


Asunto(s)
Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Antivirales/farmacología , Infecciones por Coronavirus/tratamiento farmacológico , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Neumonía Viral/tratamiento farmacológico , Síndrome Respiratorio Agudo Grave/tratamiento farmacológico , Adenosina Monofosfato/farmacocinética , Adenosina Monofosfato/farmacología , Alanina/farmacocinética , Alanina/farmacología , Antivirales/farmacocinética , Betacoronavirus/efectos de los fármacos , Betacoronavirus/crecimiento & desarrollo , Betacoronavirus/patogenicidad , Ensayos Clínicos como Asunto , Ensayos de Uso Compasivo/métodos , Infecciones por Coronavirus/mortalidad , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Esquema de Medicación , Ebolavirus/efectos de los fármacos , Ebolavirus/crecimiento & desarrollo , Ebolavirus/patogenicidad , Fiebre Hemorrágica Ebola/mortalidad , Fiebre Hemorrágica Ebola/patología , Fiebre Hemorrágica Ebola/virología , Humanos , Coronavirus del Síndrome Respiratorio de Oriente Medio/efectos de los fármacos , Coronavirus del Síndrome Respiratorio de Oriente Medio/crecimiento & desarrollo , Coronavirus del Síndrome Respiratorio de Oriente Medio/patogenicidad , Pandemias , Seguridad del Paciente , Neumonía Viral/mortalidad , Neumonía Viral/patología , Neumonía Viral/virología , Virus del SRAS/efectos de los fármacos , Virus del SRAS/crecimiento & desarrollo , Virus del SRAS/patogenicidad , Síndrome Respiratorio Agudo Grave/mortalidad , Síndrome Respiratorio Agudo Grave/patología , Síndrome Respiratorio Agudo Grave/virología , Análisis de Supervivencia , Resultado del Tratamiento
12.
SAR QSAR Environ Res ; 31(11): 857-867, 2020 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-33100032

RESUMEN

A novel coronavirus recently identified in Wuhan, China (2019-nCoV) has resulted in an increasing number of patients globally, and has become a highly lethal pathogenic member of the coronavirus family affecting humans. 2019-nCoV has established itself as one of the most threatening pandemics that human beings have faced, and therefore analysis and evaluation of all possible responses against infection is required. One such strategy includes utilizing the knowledge gained from the SARS and MERS outbreaks regarding existing antivirals. Indicating a potential for success, one of the drugs, remdesivir, under repurposing studies, has shown positive results in initial clinical studies. Therefore, in the current work, the authors have attempted to utilize the remdesivir-RdRp complex - RdRp (RNA-dependent RNA polymerase) being the putative target for remdesivir - to screen a library of the already reported RdRp inhibitor database. Further clustering on the basis of structural features and scoring refinement was performed to filter out false positive hits. Finally, molecular dynamics simulation was carried out to validate the identification of hits as RdRp inhibitors against novel coronavirus 2019-nCoV. The results yielded two putative hits which can inhibit RdRp with better potency than remdesivir, subject to further biological evaluation.


Asunto(s)
Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Antivirales/farmacología , Simulación del Acoplamiento Molecular , /antagonistas & inhibidores , Adenosina Monofosfato/química , Adenosina Monofosfato/farmacología , Alanina/química , Alanina/farmacología , Antivirales/química , Betacoronavirus/efectos de los fármacos , Betacoronavirus/enzimología , Infecciones por Coronavirus/tratamiento farmacológico , Pandemias , Neumonía Viral , Relación Estructura-Actividad Cuantitativa , Proteínas Virales/efectos de los fármacos
13.
Sci Rep ; 10(1): 16577, 2020 10 06.
Artículo en Inglés | MEDLINE | ID: mdl-33024223

RESUMEN

SARS-CoV-2 is responsible for COVID-19, resulting in the largest pandemic in over a hundred years. After examining the molecular structures and activities of hepatitis C viral inhibitors and comparing hepatitis C virus and coronavirus replication, we previously postulated that the FDA-approved hepatitis C drug EPCLUSA (Sofosbuvir/Velpatasvir) might inhibit SARS-CoV-2. We subsequently demonstrated that Sofosbuvir triphosphate is incorporated by the relatively low fidelity SARS-CoV and SARS-CoV-2 RNA-dependent RNA polymerases (RdRps), serving as an immediate polymerase reaction terminator, but not by a host-like high fidelity DNA polymerase. Other investigators have since demonstrated the ability of Sofosbuvir to inhibit SARS-CoV-2 replication in lung and brain cells; additionally, COVID-19 clinical trials with EPCLUSA and with Sofosbuvir plus Daclatasvir have been initiated in several countries. SARS-CoV-2 has an exonuclease-based proofreader to maintain the viral genome integrity. Any effective antiviral targeting the SARS-CoV-2 RdRp must display a certain level of resistance to this proofreading activity. We report here that Sofosbuvir terminated RNA resists removal by the exonuclease to a substantially higher extent than RNA terminated by Remdesivir, another drug being used as a COVID-19 therapeutic. These results offer a molecular basis supporting the current use of Sofosbuvir in combination with other drugs in COVID-19 clinical trials.


Asunto(s)
Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/tratamiento farmacológico , Exonucleasas/metabolismo , Neumonía Viral/tratamiento farmacológico , Profármacos/farmacología , ARN Viral/efectos de los fármacos , Sofosbuvir/farmacología , Adenosina Monofosfato/química , Adenosina Monofosfato/farmacología , Adenosina Monofosfato/uso terapéutico , Alanina/química , Alanina/farmacología , Alanina/uso terapéutico , Antivirales/química , Antivirales/uso terapéutico , Betacoronavirus/enzimología , Infecciones por Coronavirus/virología , Descubrimiento de Drogas/métodos , Reposicionamiento de Medicamentos/métodos , Hepacivirus/efectos de los fármacos , Hepacivirus/enzimología , Hepatitis C/tratamiento farmacológico , Hepatitis C/virología , Humanos , Pandemias , Neumonía Viral/virología , Profármacos/uso terapéutico , ARN Viral/química , ARN Viral/metabolismo , /metabolismo , Sofosbuvir/química , Sofosbuvir/uso terapéutico , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacos
14.
Proc Natl Acad Sci U S A ; 117(43): 26946-26954, 2020 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-33028676

RESUMEN

Remdesivir is a broad-spectrum antiviral nucleotide prodrug that has been clinically evaluated in Ebola virus patients and recently received emergency use authorization (EUA) for treatment of COVID-19. With approvals from the Federal Select Agent Program and the Centers for Disease Control and Prevention's Institutional Biosecurity Board, we characterized the resistance profile of remdesivir by serially passaging Ebola virus under remdesivir selection; we generated lineages with low-level reduced susceptibility to remdesivir after 35 passages. We found that a single amino acid substitution, F548S, in the Ebola virus polymerase conferred low-level reduced susceptibility to remdesivir. The F548 residue is highly conserved in filoviruses but should be subject to specific surveillance among novel filoviruses, in newly emerging variants in ongoing outbreaks, and also in Ebola virus patients undergoing remdesivir therapy. Homology modeling suggests that the Ebola virus polymerase F548 residue lies in the F-motif of the polymerase active site, a region that was previously identified as susceptible to resistance mutations in coronaviruses. Our data suggest that molecular surveillance of this region of the polymerase in remdesivir-treated COVID-19 patients is also warranted.


Asunto(s)
Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Antivirales/farmacología , Betacoronavirus/enzimología , Ebolavirus/enzimología , Proteínas no Estructurales Virales/química , Adenosina Monofosfato/farmacología , Alanina/farmacología , Betacoronavirus/química , Línea Celular , Tolerancia a Medicamentos/genética , Ebolavirus/efectos de los fármacos , Ebolavirus/genética , Humanos , Modelos Moleculares , Mutación , Proteínas no Estructurales Virales/genética , Replicación Viral/efectos de los fármacos
15.
Proc Natl Acad Sci U S A ; 117(43): 26915-26925, 2020 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-33046644

RESUMEN

Zoonotic coronaviruses represent an ongoing threat, yet the myriads of circulating animal viruses complicate the identification of higher-risk isolates that threaten human health. Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered, highly pathogenic virus that likely evolved from closely related HKU2 bat coronaviruses, circulating in Rhinolophus spp. bats in China and elsewhere. As coronaviruses cause severe economic losses in the pork industry and swine are key intermediate hosts of human disease outbreaks, we synthetically resurrected a recombinant virus (rSADS-CoV) as well as a derivative encoding tomato red fluorescent protein (tRFP) in place of ORF3. rSADS-CoV replicated efficiently in a variety of continuous animal and primate cell lines, including human liver and rectal carcinoma cell lines. Of concern, rSADS-CoV also replicated efficiently in several different primary human lung cell types, as well as primary human intestinal cells. rSADS-CoV did not use human coronavirus ACE-2, DPP4, or CD13 receptors for docking and entry. Contemporary human donor sera neutralized the group I human coronavirus NL63, but not rSADS-CoV, suggesting limited human group I coronavirus cross protective herd immunity. Importantly, remdesivir, a broad-spectrum nucleoside analog that is effective against other group 1 and 2 coronaviruses, efficiently blocked rSADS-CoV replication in vitro. rSADS-CoV demonstrated little, if any, replicative capacity in either immune-competent or immunodeficient mice, indicating a critical need for improved animal models. Efficient growth in primary human lung and intestinal cells implicate SADS-CoV as a potential higher-risk emerging coronavirus pathogen that could negatively impact the global economy and human health.


Asunto(s)
Alphacoronavirus/fisiología , Infecciones por Coronavirus/virología , Susceptibilidad a Enfermedades/virología , Replicación Viral , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Alphacoronavirus/genética , Alphacoronavirus/crecimiento & desarrollo , Animales , Células Cultivadas , Chlorocebus aethiops , Infecciones por Coronavirus/transmisión , Expresión Génica , Especificidad del Huésped , Humanos , Proteínas Luminiscentes/genética , Ratones , Células Vero , Replicación Viral/efectos de los fármacos
16.
Sci Rep ; 10(1): 16200, 2020 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-33004837

RESUMEN

The current coronavirus (COVID-19) pandemic is exacerbated by the absence of effective therapeutic agents. Notably, patients with COVID-19 and comorbidities such as hypertension and cardiac diseases have a higher mortality rate. An efficient strategy in response to this issue is repurposing drugs with antiviral activity for therapeutic effect. Digoxin (DIG) and ouabain (OUA) are FDA drugs for heart diseases that have antiviral activity against several coronaviruses. Thus, we aimed to assess antiviral activity of DIG and OUA against SARS-CoV-2 infection. The half-maximal inhibitory concentrations (IC50) of DIG and OUA were determined at a nanomolar concentration. Progeny virus titers of single-dose treatment of DIG, OUA and remdesivir were approximately 103-, 104- and 103-fold lower (> 99% inhibition), respectively, than that of non-treated control or chloroquine at 48 h post-infection (hpi). Furthermore, therapeutic treatment with DIG and OUA inhibited over 99% of SARS-CoV-2 replication, leading to viral inhibition at the post entry stage of the viral life cycle. Collectively, these results suggest that DIG and OUA may be an alternative treatment for COVID-19, with potential additional therapeutic effects for patients with cardiovascular disease.


Asunto(s)
Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Digoxina/farmacología , Ouabaína/farmacología , Replicación Viral , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Animales , Betacoronavirus/fisiología , Chlorocebus aethiops , Cloroquina/farmacología , Concentración 50 Inhibidora , Células Vero
17.
Drugs ; 80(13): 1355-1363, 2020 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-32870481

RESUMEN

The antiviral agent remdesivir (Veklury®; Gilead Sciences), nucleotide analogue prodrug, has broad-spectrum activity against viruses from several families. Having demonstrated potent antiviral activity against coronaviruses in preclinical studies, remdesivir emerged as a candidate drug for the treatment of the novel coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, during the current global pandemic. Phase III evaluation of remdesivir in the treatment of COVID-19 commenced in early 2020 and has thus far yielded promising results. In late May 2020, Taiwan conditionally approved the use of remdesivir in patients with severe COVID-19. This was followed by a rapid succession of conditional approvals in various countries/regions including the EU and Canada. Preceding these conditional approvals, an emergency use authorization for remdesivir had been granted in the USA (on 1 May 2020) and a special approval for emergency use was granted in Japan (on 7 May 2020). This article summarizes the milestones in the development of remdesivir leading to its first conditional approval for the treatment of COVID-19.


Asunto(s)
Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Infecciones por Coronavirus , Desarrollo de Medicamentos , Pandemias , Neumonía Viral , Adenosina Monofosfato/farmacología , Alanina/farmacología , Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/epidemiología , Humanos , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/epidemiología
19.
Biomolecules ; 10(9)2020 09 21.
Artículo en Inglés | MEDLINE | ID: mdl-32967116

RESUMEN

We report the results of our in silico study of approved drugs as potential treatments for COVID-19. The study is based on the analysis of normal modes of proteins. The drugs studied include chloroquine, ivermectin, remdesivir, sofosbuvir, boceprevir, and α-difluoromethylornithine (DMFO). We applied the tools we developed and standard tools used in the structural biology community. Our results indicate that small molecules selectively bind to stable, kinetically active residues and residues adjoining them on the surface of proteins and inside protein pockets, and that some prefer hydrophobic sites over other active sites. Our approach is not restricted to viruses and can facilitate rational drug design, as well as improve our understanding of molecular interactions, in general.


Asunto(s)
Antivirales/farmacología , Infecciones por Coronavirus/tratamiento farmacológico , Pandemias , Neumonía Viral/tratamiento farmacológico , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/química , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/química , Alanina/farmacología , Anticuerpos Antivirales/inmunología , Reacciones Antígeno-Anticuerpo , Antivirales/química , Antivirales/uso terapéutico , Betacoronavirus , Sitios de Unión , Cloroquina/química , Cloroquina/farmacología , Infecciones por Coronavirus/prevención & control , Reposicionamiento de Medicamentos , Eflornitina/química , Eflornitina/farmacología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ivermectina/química , Ivermectina/farmacología , L-Lactato Deshidrogenasa/química , L-Lactato Deshidrogenasa/efectos de los fármacos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Pandemias/prevención & control , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/efectos de los fármacos , Neumonía Viral/prevención & control , Prolina/análogos & derivados , Prolina/química , Prolina/farmacología , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Receptores de Glicina/química , Receptores de Glicina/efectos de los fármacos , Saposinas/química , Saposinas/efectos de los fármacos , Sofosbuvir/química , Sofosbuvir/farmacología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/efectos de los fármacos , Relación Estructura-Actividad
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