Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 16 de 16
Filter
1.
Molecules ; 27(13)2022 Jun 30.
Article in English | MEDLINE | ID: covidwho-1917636

ABSTRACT

The urgent response to the COVID-19 pandemic required accelerated evaluation of many approved drugs as potential antiviral agents against the causative pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using cell-based, biochemical, and modeling approaches, we studied the approved HIV-1 nucleoside/tide reverse transcriptase inhibitors (NRTIs) tenofovir (TFV) and emtricitabine (FTC), as well as prodrugs tenofovir alafenamide (TAF) and tenofovir disoproxilfumarate (TDF) for their antiviral effect against SARS-CoV-2. A comprehensive set of in vitro data indicates that TFV, TAF, TDF, and FTC are inactive against SARS-CoV-2. None of the NRTIs showed antiviral activity in SARS-CoV-2 infected A549-hACE2 cells or in primary normal human lung bronchial epithelial (NHBE) cells at concentrations up to 50 µM TAF, TDF, FTC, or 500 µM TFV. These results are corroborated by the low incorporation efficiency of respective NTP analogs by the SARS-CoV-2 RNA-dependent-RNA polymerase (RdRp), and lack of the RdRp inhibition. Structural modeling further demonstrated poor fitting of these NRTI active metabolites at the SARS-CoV-2 RdRp active site. Our data indicate that the HIV-1 NRTIs are unlikely direct-antivirals against SARS-CoV-2, and clinicians and researchers should exercise caution when exploring ideas of using these and other NRTIs to treat or prevent COVID-19.


Subject(s)
Anti-HIV Agents , COVID-19 , HIV Infections , HIV-1 , Anti-HIV Agents/pharmacology , Anti-HIV Agents/therapeutic use , COVID-19/drug therapy , Emtricitabine/pharmacology , Emtricitabine/therapeutic use , HIV Infections/drug therapy , Humans , Nucleosides/pharmacology , Nucleosides/therapeutic use , Nucleotides/pharmacology , Pandemics , RNA, Viral , RNA-Dependent RNA Polymerase , SARS-CoV-2 , Tenofovir/pharmacology , Tenofovir/therapeutic use
2.
Antimicrob Agents Chemother ; 66(6): e0022222, 2022 06 21.
Article in English | MEDLINE | ID: covidwho-1832332

ABSTRACT

Genetic variation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence and rapid spread of multiple variants throughout the pandemic, of which Omicron is currently the predominant variant circulating worldwide. SARS-CoV-2 variants of concern/variants of interest (VOC/VOI) have evidence of increased viral transmission, disease severity, or decreased effectiveness of vaccines and neutralizing antibodies. Remdesivir (RDV [VEKLURY]) is a nucleoside analog prodrug and the first FDA-approved antiviral treatment of COVID-19. Here, we present a comprehensive antiviral activity assessment of RDV and its parent nucleoside, GS-441524, against 10 current and former SARS-CoV-2 VOC/VOI clinical isolates by nucleoprotein enzyme-linked immunosorbent assay (ELISA) and plaque reduction assay. Delta and Omicron variants remained susceptible to RDV and GS-441524, with 50% effective concentration (EC50) values 0.30- to 0.62-fold of those observed against the ancestral WA1 isolate. All other tested variants exhibited EC50 values ranging from 0.13- to 2.3-fold of the observed EC50 values against WA1. Analysis of nearly 6 million publicly available variant isolate sequences confirmed that Nsp12, the RNA-dependent RNA polymerase (RdRp) target of RDV and GS-441524, is highly conserved across variants, with only 2 prevalent changes (P323L and G671S). Using recombinant viruses, both RDV and GS-441524 retained potency against all viruses containing frequent variant substitutions or their combination. Taken together, these results highlight the conserved nature of SARS-CoV-2 Nsp12 and provide evidence of sustained SARS-CoV-2 antiviral activity of RDV and GS-441524 across the tested variants. The observed pan-variant activity of RDV supports its continued use for the treatment of COVID-19 regardless of the SARS-CoV-2 variant.


Subject(s)
COVID-19 , SARS-CoV-2 , Adenosine/analogs & derivatives , Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacology , COVID-19/drug therapy , Humans , SARS-CoV-2/genetics
3.
Antiviral Res ; 203: 105329, 2022 07.
Article in English | MEDLINE | ID: covidwho-1819427

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, has infected over 260 million people over the past 2 years. Remdesivir (RDV, VEKLURY®) is currently the only antiviral therapy fully approved by the FDA for the treatment of COVID-19. The parent nucleoside of RDV, GS-441524, exhibits antiviral activity against numerous respiratory viruses including SARS-CoV-2, although at reduced in vitro potency compared to RDV in most assays. Here we find in both human alveolar and bronchial primary cells, GS-441524 is metabolized to the pharmacologically active GS-441524 triphosphate (TP) less efficiently than RDV, which correlates with a lower in vitro SARS-CoV-2 antiviral activity. In vivo, African green monkeys (AGM) orally dosed with GS-441524 yielded low plasma levels due to limited oral bioavailability of <10%. When GS-441524 was delivered via intravenous (IV) administration, although plasma concentrations of GS-441524 were significantly higher, lung TP levels were lower than observed from IV RDV. To determine the required systemic exposure of GS-441524 associated with in vivo antiviral efficacy, SARS-CoV-2 infected AGMs were treated with a once-daily IV dose of either 7.5 or 20 mg/kg GS-441524 or IV RDV for 5 days and compared to vehicle control. Despite the reduced lung TP formation compared to IV dosing of RDV, daily treatment with IV GS-441524 resulted in dose-dependent efficacy, with the 20 mg/kg GS-441524 treatment resulting in significant reductions of SARS-CoV-2 replication in the lower respiratory tract of infected animals. These findings demonstrate the in vivo SARS-CoV-2 antiviral efficacy of GS-441524 and support evaluation of its orally bioavailable prodrugs as potential therapies for COVID-19.


Subject(s)
COVID-19 , Adenosine/analogs & derivatives , Animals , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Chlorocebus aethiops , Humans , Pandemics , SARS-CoV-2
4.
Sci Transl Med ; 14(643): eabm3410, 2022 05 04.
Article in English | MEDLINE | ID: covidwho-1752762

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic remains uncontrolled despite the rapid rollout of safe and effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, underscoring the need to develop highly effective antivirals. In the setting of waning immunity from infection and vaccination, breakthrough infections are becoming increasingly common and treatment options remain limited. In addition, the emergence of SARS-CoV-2 variants of concern, with their potential to escape neutralization by therapeutic monoclonal antibodies, emphasizes the need to develop second-generation oral antivirals targeting highly conserved viral proteins that can be rapidly deployed to outpatients. Here, we demonstrate the in vitro antiviral activity and in vivo therapeutic efficacy of GS-621763, an orally bioavailable prodrug of GS-441524, the parent nucleoside of remdesivir, which targets the highly conserved virus RNA-dependent RNA polymerase. GS-621763 exhibited antiviral activity against SARS-CoV-2 in lung cell lines and two different human primary lung cell culture systems. GS-621763 was also potently antiviral against a genetically unrelated emerging coronavirus, Middle East respiratory syndrome CoV (MERS-CoV). The dose-proportional pharmacokinetic profile observed after oral administration of GS-621763 translated to dose-dependent antiviral activity in mice infected with SARS-CoV-2. Therapeutic GS-621763 administration reduced viral load and lung pathology; treatment also improved pulmonary function in COVID-19 mouse model. A direct comparison of GS-621763 with molnupiravir, an oral nucleoside analog antiviral that has recently received EUA approval, proved both drugs to be similarly efficacious in mice. These data support the exploration of GS-441524 oral prodrugs for the treatment of COVID-19.


Subject(s)
COVID-19 , Coronavirus Infections , Prodrugs , Adenosine/analogs & derivatives , Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Coronavirus Infections/drug therapy , Humans , Mice , Nucleosides , Parents , Prodrugs/pharmacology , Prodrugs/therapeutic use , SARS-CoV-2
5.
Sci Transl Med ; 14(633): eabl8282, 2022 Feb 23.
Article in English | MEDLINE | ID: covidwho-1598975

ABSTRACT

Remdesivir (RDV) is a nucleotide analog prodrug with demonstrated clinical benefit in patients with coronavirus disease 2019 (COVID-19). In October 2020, the US FDA approved intravenous (IV) RDV as the first treatment for hospitalized COVID-19 patients. Furthermore, RDV has been approved or authorized for emergency use in more than 50 countries. To make RDV more convenient for non-hospitalized patients earlier in disease, alternative routes of administration are being evaluated. Here, we investigated the pharmacokinetics and efficacy of RDV administered by head dome inhalation in African green monkeys (AGM). Relative to an IV administration of RDV at 10 mg/kg, an approximately 20-fold lower dose administered by inhalation produced comparable concentrations of the pharmacologically active triphosphate in lower respiratory tract tissues. Distribution of the active triphosphate into the upper respiratory tract was also observed following inhaled RDV exposure. Inhalation RDV dosing resulted in lower systemic exposures to RDV and its metabolites as compared with IV RDV dosing. An efficacy study with repeated dosing of inhaled RDV in an AGM model of SARS-CoV-2 infection demonstrated reductions in viral replication in bronchoalveolar lavage fluid and respiratory tract tissues compared with placebo. Efficacy was observed with inhaled RDV administered once daily at a pulmonary deposited dose of 0.35 mg/kg beginning approximately 8 hours post-infection. Moreover, the efficacy of inhaled RDV was similar to that of IV RDV administered once at 10 mg/kg followed by 5 mg/kg daily in the same study. Together, these findings support further clinical development of inhalation RDV.


Subject(s)
COVID-19 , Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Animals , Antiviral Agents/pharmacokinetics , COVID-19/drug therapy , Chlorocebus aethiops , Humans , Primates , SARS-CoV-2 , Viral Load
6.
J Biol Chem ; 298(2): 101529, 2022 02.
Article in English | MEDLINE | ID: covidwho-1587355

ABSTRACT

Remdesivir (RDV) is a direct-acting antiviral agent that is approved in several countries for the treatment of coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2. RDV exhibits broad-spectrum antiviral activity against positive-sense RNA viruses, for example, severe acute respiratory syndrome coronavirus and hepatitis C virus, and nonsegmented negative-sense RNA viruses, for example, Nipah virus, whereas segmented negative-sense RNA viruses such as influenza virus or Crimean-Congo hemorrhagic fever virus are not sensitive to the drug. The reasons for this apparent efficacy pattern are unknown. Here, we expressed and purified representative RNA-dependent RNA polymerases and studied three biochemical parameters that have been associated with the inhibitory effects of RDV-triphosphate (TP): (i) selective incorporation of the nucleotide substrate RDV-TP, (ii) the effect of the incorporated RDV-monophosphate (MP) on primer extension, and (iii) the effect of RDV-MP in the template during incorporation of the complementary UTP. We found a strong correlation between antiviral effects and efficient incorporation of RDV-TP. Inhibition in primer extension reactions was heterogeneous and usually inefficient at higher NTP concentrations. In contrast, template-dependent inhibition of UTP incorporation opposite the embedded RDV-MP was seen with all polymerases. Molecular modeling suggests a steric conflict between the 1'-cyano group of the inhibitor and residues of the structurally conserved RNA-dependent RNA polymerase motif F. We conclude that future efforts in the development of nucleotide analogs with a broader spectrum of antiviral activities should focus on improving rates of incorporation while capitalizing on the inhibitory effects of a bulky 1'-modification.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Models, Molecular , RNA Viruses/enzymology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/pharmacology , Alanine/chemistry , Alanine/pharmacology , Antiviral Agents/pharmacology , Hepacivirus/drug effects , Hepacivirus/enzymology , Negative-Sense RNA Viruses/drug effects , Negative-Sense RNA Viruses/enzymology , Nipah Virus/drug effects , Nipah Virus/enzymology , Positive-Strand RNA Viruses/drug effects , Positive-Strand RNA Viruses/enzymology , RNA Viruses/drug effects , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Virus Replication/drug effects
7.
Nat Commun ; 12(1): 6415, 2021 11 05.
Article in English | MEDLINE | ID: covidwho-1503781

ABSTRACT

Remdesivir is an antiviral approved for COVID-19 treatment, but its wider use is limited by intravenous delivery. An orally bioavailable remdesivir analog may boost therapeutic benefit by facilitating early administration to non-hospitalized patients. This study characterizes the anti-SARS-CoV-2 efficacy of GS-621763, an oral prodrug of remdesivir parent nucleoside GS-441524. Both GS-621763 and GS-441524 inhibit SARS-CoV-2, including variants of concern (VOC) in cell culture and human airway epithelium organoids. Oral GS-621763 is efficiently converted to plasma metabolite GS-441524, and in lungs to the triphosphate metabolite identical to that generated by remdesivir, demonstrating a consistent mechanism of activity. Twice-daily oral administration of 10 mg/kg GS-621763 reduces SARS-CoV-2 burden to near-undetectable levels in ferrets. When dosed therapeutically against VOC P.1 gamma γ, oral GS-621763 blocks virus replication and prevents transmission to untreated contact animals. These results demonstrate therapeutic efficacy of a much-needed orally bioavailable analog of remdesivir in a relevant animal model of SARS-CoV-2 infection.


Subject(s)
Adenosine/analogs & derivatives , Antiviral Agents/pharmacology , COVID-19/drug therapy , Prodrugs/pharmacology , SARS-CoV-2/drug effects , Adenosine/pharmacology , Animals , COVID-19/metabolism , COVID-19/virology , Cell Line , Chlorocebus aethiops , Cricetinae , Disease Models, Animal , Female , Ferrets , Humans , SARS-CoV-2/isolation & purification
8.
J Biol Chem ; 297(4): 101218, 2021 10.
Article in English | MEDLINE | ID: covidwho-1433454

ABSTRACT

The SARS-CoV-2 replication-transcription complex is an assembly of nonstructural viral proteins that collectively act to reproduce the viral genome and generate mRNA transcripts. While the structures of the individual proteins involved are known, how they assemble into a functioning superstructure is not. Applying molecular modeling tools, including protein-protein docking, to the available structures of nsp7-nsp16 and the nucleocapsid, we have constructed an atomistic model of how these proteins associate. Our principal finding is that the complex is hexameric, centered on nsp15. The nsp15 hexamer is capped on two faces by trimers of nsp14/nsp16/(nsp10)2, which then recruit six nsp12/nsp7/(nsp8)2 polymerase subunits to the complex. To this, six subunits of nsp13 are arranged around the superstructure, but not evenly distributed. Polymerase subunits that coordinate dimers of nsp13 are capable of binding the nucleocapsid, which positions the 5'-UTR TRS-L RNA over the polymerase active site, a state distinguishing transcription from replication. Analysis of the viral RNA path through the complex indicates the dsRNA that exits the polymerase passes over the nsp14 exonuclease and nsp15 endonuclease sites before being unwound by a convergence of zinc fingers from nsp10 and nsp14. The template strand is then directed away from the complex, while the nascent strand is directed to the sites responsible for mRNA capping. The model presents a cohesive picture of the multiple functions of the coronavirus replication-transcription complex and addresses fundamental questions related to proofreading, template switching, mRNA capping, and the role of the endonuclease.


Subject(s)
Endoribonucleases/metabolism , Models, Molecular , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Binding Sites , COVID-19/pathology , COVID-19/virology , Dimerization , Endoribonucleases/chemistry , Endoribonucleases/genetics , Humans , Molecular Docking Simulation , Protein Structure, Quaternary , RNA, Double-Stranded/chemistry , RNA, Double-Stranded/metabolism , SARS-CoV-2/isolation & purification , Transcription, Genetic , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Virus Replication
9.
Antimicrob Agents Chemother ; 65(9): e0060221, 2021 08 17.
Article in English | MEDLINE | ID: covidwho-1434879

ABSTRACT

Remdesivir (RDV; GS-5734, Veklury), the first FDA-approved antiviral to treat COVID-19, is a single-diastereomer monophosphoramidate prodrug of an adenosine analogue. RDV is taken up in the target cells and metabolized in multiple steps to form the active nucleoside triphosphate (TP) (GS-443902), which, in turn, acts as a potent and selective inhibitor of multiple viral RNA polymerases. In this report, we profiled the key enzymes involved in the RDV metabolic pathway with multiple parallel approaches: (i) bioinformatic analysis of nucleoside/nucleotide metabolic enzyme mRNA expression using public human tissue and lung single-cell bulk mRNA sequence (RNA-seq) data sets, (ii) protein and mRNA quantification of enzymes in human lung tissue and primary lung cells, (iii) biochemical studies on the catalytic rate of key enzymes, (iv) effects of specific enzyme inhibitors on the GS-443902 formation, and (v) the effects of these inhibitors on RDV antiviral activity against SARS-CoV-2 in cell culture. Our data collectively demonstrated that carboxylesterase 1 (CES1) and cathepsin A (CatA) are enzymes involved in hydrolyzing RDV to its alanine intermediate MetX, which is further hydrolyzed to the monophosphate form by histidine triad nucleotide-binding protein 1 (HINT1). The monophosphate is then consecutively phosphorylated to diphosphate and triphosphate by cellular phosphotransferases. Our data support the hypothesis that the unique properties of RDV prodrug not only allow lung-specific accumulation critical for the treatment of respiratory viral infection such as COVID-19 but also enable efficient intracellular metabolism of RDV and its MetX to monophosphate and successive phosphorylation to form the active TP in disease-relevant cells.


Subject(s)
COVID-19 , SARS-CoV-2 , Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacology , COVID-19/drug therapy , Humans , Lung , Nerve Tissue Proteins
12.
Antiviral Res ; 192: 105122, 2021 08.
Article in English | MEDLINE | ID: covidwho-1283915

ABSTRACT

There are, besides remdesivir, no approved antivirals for the treatment of SARS-CoV-2 infections. To aid in the search for antivirals against this virus, we explored the use of human tracheal airway epithelial cells (HtAEC) and human small airway epithelial cells (HsAEC) grown at the air-liquid interface (ALI). These cultures were infected at the apical side with one of two different SARS-CoV-2 isolates. Each virus was shown to replicate to high titers for extended periods of time (at least 8 days) and, in particular an isolate with the D614G in the spike (S) protein did so more efficiently at 35 °C than 37 °C. The effect of a selected panel of reference drugs that were added to the culture medium at the basolateral side of the system was explored. Remdesivir, GS-441524 (the parent nucleoside of remdesivir), EIDD-1931 (the parent nucleoside of molnupiravir) and IFN (ß1 and λ1) all resulted in dose-dependent inhibition of viral RNA and infectious virus titers collected at the apical side. However, AT-511 (the free base form of AT-527 currently in clinical testing) failed to inhibit viral replication in these in vitro primary cell models. Together, these results provide a reference for further studies aimed at selecting SARS-CoV-2 inhibitors for further preclinical and clinical development.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , SARS-CoV-2/drug effects , Virus Replication/drug effects , Animals , Cell Line , Chlorocebus aethiops , Drug Evaluation, Preclinical/methods , Epithelial Cells/virology , Humans , RNA, Viral , SARS-CoV-2/isolation & purification , Vero Cells
13.
J Med Chem ; 64(8): 5001-5017, 2021 04 22.
Article in English | MEDLINE | ID: covidwho-1174625

ABSTRACT

A discovery program targeting respiratory syncytial virus (RSV) identified C-nucleoside 4 (RSV A2 EC50 = 530 nM) as a phenotypic screening lead targeting the RSV RNA-dependent RNA polymerase (RdRp). Prodrug exploration resulted in the discovery of remdesivir (1, GS-5734) that is >30-fold more potent than 4 against RSV in HEp-2 and NHBE cells. Metabolism studies in vitro confirmed the rapid formation of the active triphosphate metabolite, 1-NTP, and in vivo studies in cynomolgus and African Green monkeys demonstrated a >10-fold higher lung tissue concentration of 1-NTP following molar normalized IV dosing of 1 compared to that of 4. A once daily 10 mg/kg IV administration of 1 in an African Green monkey RSV model demonstrated a >2-log10 reduction in the peak lung viral load. These early data following the discovery of 1 supported its potential as a novel treatment for RSV prior to its development for Ebola and approval for COVID-19 treatment.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacology , Prodrugs/pharmacology , Respiratory Syncytial Virus Infections/drug therapy , Respiratory Syncytial Virus, Human/drug effects , Adenosine Monophosphate/pharmacology , Alanine/pharmacology , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Caco-2 Cells , Cells, Cultured , Chlorocebus aethiops , Disease Models, Animal , Dogs , Drug Evaluation, Preclinical/methods , Epithelial Cells/virology , Humans , Macaca fascicularis , Male , Prodrugs/chemistry , Prodrugs/pharmacokinetics , Rats, Sprague-Dawley , Respiratory Syncytial Virus Infections/virology , Structure-Activity Relationship , Tissue Distribution , Tubercidin/analogs & derivatives , Tubercidin/chemistry , Viral Load
14.
Nature ; 592(7852): 116-121, 2021 04.
Article in English | MEDLINE | ID: covidwho-892040

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein substitution D614G became dominant during the coronavirus disease 2019 (COVID-19) pandemic1,2. However, the effect of this variant on viral spread and vaccine efficacy remains to be defined. Here we engineered the spike D614G substitution in the USA-WA1/2020 SARS-CoV-2 strain, and found that it enhances viral replication in human lung epithelial cells and primary human airway tissues by increasing the infectivity and stability of virions. Hamsters infected with SARS-CoV-2 expressing spike(D614G) (G614 virus) produced higher infectious titres in nasal washes and the trachea, but not in the lungs, supporting clinical evidence showing that the mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increase transmission. Sera from hamsters infected with D614 virus exhibit modestly higher neutralization titres against G614 virus than against D614 virus, suggesting that the mutation is unlikely to reduce the ability of vaccines in clinical trials to protect against COVID-19, and that therapeutic antibodies should be tested against the circulating G614 virus. Together with clinical findings, our work underscores the importance of this variant in viral spread and its implications for vaccine efficacy and antibody therapy.


Subject(s)
COVID-19/transmission , COVID-19/virology , Genetic Fitness , Mutation , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/genetics , Animals , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/therapeutic use , COVID-19/immunology , COVID-19 Vaccines/immunology , Cricetinae , Disease Models, Animal , Humans , Lung/virology , Male , Mesocricetus/virology , Models, Biological , Nasal Mucosa/virology , Neutralization Tests , Protein Stability , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Tissue Culture Techniques , Trachea/virology , Viral Load , Virion/chemistry , Virion/pathogenicity , Virion/physiology , Virus Replication/genetics
15.
Nat Commun ; 11(1): 5214, 2020 10 15.
Article in English | MEDLINE | ID: covidwho-872699

ABSTRACT

A high-throughput platform would greatly facilitate coronavirus disease 2019 (COVID-19) serological testing and antiviral screening. Here we present a high-throughput nanoluciferase severe respiratory syndrome coronavirus 2 (SARS-CoV-2-Nluc) that is genetically stable and replicates similarly to the wild-type virus in cell culture. SARS-CoV-2-Nluc can be used to measure neutralizing antibody activity in patient sera within 5 hours, and it produces results in concordance with a plaque reduction neutralization test (PRNT). Additionally, using SARS-CoV-2-Nluc infection of A549 cells expressing human ACE2 receptor (A549-hACE2), we show that the assay can be used for antiviral screening. Using the optimized SARS-CoV-2-Nluc assay, we evaluate a panel of antivirals and other anti-infective drugs, and we identify nelfinavir, rupintrivir, and cobicistat as the most selective inhibitors of SARS-CoV-2-Nluc (EC50 0.77 to 2.74 µM). In contrast, most of the clinically approved antivirals, including tenofovir alafenamide, emtricitabine, sofosbuvir, ledipasvir, and velpatasvir were inactive at concentrations up to 10 µM. Collectively, this high-throughput platform represents a reliable tool for rapid neutralization testing and antiviral screening for SARS-CoV-2.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , High-Throughput Screening Assays/methods , Neutralization Tests/methods , Pneumonia, Viral/diagnosis , A549 Cells , Angiotensin-Converting Enzyme 2 , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Betacoronavirus/genetics , Betacoronavirus/immunology , COVID-19 , Chlorocebus aethiops , Coronavirus Infections/virology , Humans , Luciferases/genetics , Pandemics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/virology , SARS-CoV-2 , Vero Cells , Virus Internalization/drug effects , Virus Replication/drug effects
16.
Cell Rep ; 32(3): 107940, 2020 07 21.
Article in English | MEDLINE | ID: covidwho-635658

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the novel viral disease COVID-19. With no approved therapies, this pandemic illustrates the urgent need for broad-spectrum antiviral countermeasures against SARS-CoV-2 and future emerging CoVs. We report that remdesivir (RDV) potently inhibits SARS-CoV-2 replication in human lung cells and primary human airway epithelial cultures (EC50 = 0.01 µM). Weaker activity is observed in Vero E6 cells (EC50 = 1.65 µM) because of their low capacity to metabolize RDV. To rapidly evaluate in vivo efficacy, we engineered a chimeric SARS-CoV encoding the viral target of RDV, the RNA-dependent RNA polymerase of SARS-CoV-2. In mice infected with the chimeric virus, therapeutic RDV administration diminishes lung viral load and improves pulmonary function compared with vehicle-treated animals. These data demonstrate that RDV is potently active against SARS-CoV-2 in vitro and in vivo, supporting its further clinical testing for treatment of COVID-19.

SELECTION OF CITATIONS
SEARCH DETAIL