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1.
Viruses ; 14(6)2022 May 29.
Article in English | MEDLINE | ID: covidwho-1869826

ABSTRACT

Measles virus (MV) is a highly contagious respiratory virus responsible for outbreaks associated with significant morbidity and mortality among children and young adults. Although safe and effective measles vaccines are available, the COVID-19 pandemic has resulted in vaccination coverage gaps that may lead to the resurgence of measles when restrictions are lifted. This puts individuals who cannot be vaccinated, such as young infants and immunocompromised individuals, at risk. Therapeutic interventions are complicated by the long incubation time of measles, resulting in a narrow treatment window. At present, the only available WHO-advised option is treatment with intravenous immunoglobulins, although this is not approved as standard of care. Antivirals against measles may contribute to intervention strategies to limit the impact of future outbreaks. Here, we review previously described antivirals and antiviral assays, evaluate the antiviral efficacy of a number of compounds to inhibit MV dissemination in vitro, and discuss potential application in specific target populations. We conclude that broadly reactive antivirals could strengthen existing intervention strategies to limit the impact of measles outbreaks.

2.
Antimicrob Agents Chemother ; 66(6): e0022222, 2022 Jun 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 Jul.
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 ; : eabo0718, 2022 Apr 28.
Article in English | MEDLINE | ID: covidwho-1816673

ABSTRACT

The nucleoside analog remdesivir (RDV) is a Food and Drug Administration (FDA)-approved antiviral for treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Thus, it is critical to understand factors that promote or prevent RDV resistance. We passaged SARS-CoV-2 in the presence of increasing concentrations of GS-441524, the parent nucleoside of RDV. After 13 passages, we isolated three viral lineages with phenotypic resistance as defined by increases in half-maximal effective concentration (EC50) from 2.7-to 10.4-fold. Sequence analysis identified non-synonymous mutations in nonstructural protein 12 RNA-dependent RNA polymerase (nsp12-RdRp): V166A, N198S, S759A, V792I and C799F/R. Two lineages encoded the S759A substitution at the RdRp Ser759-Asp-Asp active motif. In one lineage, the V792I substitution emerged first, then combined with S759A. Introduction of S759A and V792I substitutions at homologous nsp12 positions in murine hepatitis virus (MHV) demonstrated transferability across betacoronaviruses; introduction of these substitutions resulted in up to 38-fold RDV resistance and a replication defect. Biochemical analysis of SARS-CoV-2 RdRp encoding S759A demonstrated a roughly 10-fold decreased preference for RDV-triphosphate (RDV-TP) as a substrate, whereas nsp12-V792I diminished the uridine-triphosphate (UTP) concentration needed to overcome template-dependent inhibition associated with RDV. The in vitro-selected substitutions identified in this study were rare or not detected in the greater than 6 million publicly available nsp12-RdRp consensus sequences in the absence of RDV selection. The results define genetic and biochemical pathways to RDV resistance and emphasize the need for additional studies to define the potential for emergence of these or other RDV resistance mutations in clinical settings.

5.
Antiviral Res ; 198: 105246, 2022 02.
Article in English | MEDLINE | ID: covidwho-1639070

ABSTRACT

The utility of remdesivir treatment in COVID-19 patients is currently limited by the necessity to administer this antiviral intravenously, which has generally limited its use to hospitalized patients. Here, we tested a novel, subcutaneous formulation of remdesivir in the rhesus macaque model of SARS-CoV-2 infection that was previously used to establish the efficacy of remdesivir against this virus in vivo. Compared to vehicle-treated animals, macaques treated with subcutaneous remdesivir from 12 h through 6 days post inoculation showed reduced signs of respiratory disease, a reduction of virus replication in the lower respiratory tract, and an absence of interstitial pneumonia. Thus, early subcutaneous administration of remdesivir can protect from lower respiratory tract disease caused by SARS-CoV-2.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Lung Diseases, Interstitial/prevention & control , SARS-CoV-2/drug effects , Adenosine Monophosphate/administration & dosage , Adenosine Monophosphate/pharmacokinetics , Adenosine Monophosphate/therapeutic use , Administration, Cutaneous , Alanine/administration & dosage , Alanine/pharmacokinetics , Alanine/therapeutic use , Animals , Antiviral Agents/administration & dosage , Antiviral Agents/pharmacokinetics , Disease Models, Animal , Female , Lung/pathology , Lung/virology , Macaca mulatta , Male , Viral Load/drug effects , Virus Replication/drug effects
6.
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
7.
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
9.
Open forum infectious diseases ; 8(Suppl 1):S806-S807, 2021.
Article in English | EuropePMC | ID: covidwho-1564252

ABSTRACT

Background Remdesivir (RDV) is a potent nucleotide prodrug inhibitor of the SARS-CoV-2 RNA-dependent RNA polymerase that has demonstrated efficacy in the treatment of patients hospitalized with moderate to severe COVID-19. This Phase 3 (GS-US-540–9012) double-blind, placebo-controlled study compared the efficacy and safety of 3 days of RDV to standard of care in non-hospitalized, high-risk participants with confirmed COVID-19. Table 1. COVID-19 related hospitalization or death, COVID-19 related medically attended visits or death, and Treatment Emergent Adverse Events Methods Participants were randomly assigned 1:1 to receive intravenous (IV) RDV (200 mg on day 1, 100 mg on days 2 to 3) or placebo. The primary efficacy endpoint was composite COVID-19 hospitalization or all-cause death by day 28 and compared using Cox proportional hazards model with baseline stratification factors as covariates. The primary safety endpoint was proportion of participants with treatment-emergent adverse events. Study enrollment was terminated early for administrative reasons in light of the evolving pandemic. Results 562 patients underwent randomization and started their assigned treatment (279, RDV;283, placebo). Baseline demographics and characteristics were balanced across arms. Overall, 52% were male, 44% were Hispanic/Latino ethnicity and 30% were ≥ 60 years old. The most common comorbidities were diabetes mellitus (62%), obesity (56%;median BMI, 30.7), and hypertension (48%). Median baseline SARS-CoV-2 RNA nasopharyngeal viral load was 6.2 log10 copies/mL. Treatment with RDV significantly reduced COVID-19 hospitalization or all-cause death by day 28 (HR, 0.13;95% CI, 0.03 – 0.59;p = 0.008;Table 1) compared to placebo. Participants receiving RDV also had significantly lower risk for COVID-19-related medically attended visits or all-cause death by day 28 compared to placebo (HR, 0.19;95% CI, 0.07 – 0.56;p = 0.002;Table 1). No deaths occurred in either arm by day 28. There was no difference between arms in time-weighted average change in nasopharyngeal viral loads from baseline up to day 7. The proportion of patients with AEs was similar between arms (Table 1);the most common AEs in the RDV arm were nausea (11%), headache (6%), and diarrhea (4%). Conclusion A 3-day course of IV RDV was safe, well tolerated and highly effective at preventing COVID-19 related hospitalization or death in high-risk non-hospitalized COVID-19 patients. Disclosures Joshua A. Hill, MD, Allogene (Individual(s) Involved: Self): Consultant;Allovir (Individual(s) Involved: Self): Consultant, Grant/Research Support;Amplyx (Individual(s) Involved: Self): Consultant;Covance/CSL (Individual(s) Involved: Self): Consultant;CRISPR (Individual(s) Involved: Self): Consultant;Gilead (Individual(s) Involved: Self): Consultant, Grant/Research Support;Karius: Grant/Research Support, Scientific Research Study Investigator;Medscape (Individual(s) Involved: Self): Consultant;Octapharma (Individual(s) Involved: Self): Consultant;OptumHealth (Individual(s) Involved: Self): Consultant;Takeda (Individual(s) Involved: Self): Consultant, Grant/Research Support, Scientific Research Study Investigator Roger Paredes, MD, PhD, Gilead Sciences, Inc (Grant/Research Support, Scientific Research Study Investigator, Advisor or Review Panel member) Carlos Vaca, MD, Gilead Sciences, Inc (Scientific Research Study Investigator) Jorge Mera, MD, Gilead Sciences, Inc (Consultant, Study Investigator (payment to employer not self)) Gilberto Perez, MD, Gilead Sciences, Inc (Scientific Research Study Investigator) Godson Oguchi, MD, Gilead Sciences, Inc (Scientific Research Study Investigator) Pablo Ryan, MD PhD, Gilead Sciences, Inc (Grant/Research Support, Scientific Research Study Investigator, Advisor or Review Panel member) Jan Gerstoft, MD, Gilead Sciences, Inc (Other Financial or Material Support, Study Investigator (payment to employer)) Michael Brown, FRCP PhD, Gilead Sciences, Inc (Scientific Research Study Inve tigator, Investigator for numerous remdesivir trials (employer received compensation)) Morgan Katz, MD, MHS, Roche (Individual(s) Involved: Self): Advisor or Review Panel member;Skinclique (Individual(s) Involved: Self): Consultant Gregory Camus, PhD, Gilead Sciences (Employee, Shareholder) Danielle P. Porter, PhD, Gilead Sciences (Employee, Shareholder) Robert H. Hyland, DPhil, Gilead Sciences, Inc (Shareholder, Other Financial or Material Support, Employee during the conduct of this trial) Shuguang Chen, PhD, Gilead Sciences, Inc (Employee, Shareholder) Kavita Juneja, MD, Gilead Sciences, Inc (Employee) Anu Osinusi, MD, Gilead Sciences, Inc (Employee, Shareholder) Frank Duff, MD, Gilead Sciences, Inc (Employee, Shareholder) Robert L. Gottlieb, MD, Eli Lilly (Scientific Research Study Investigator, Advisor or Review Panel member)Gilead Sciences (Scientific Research Study Investigator, Advisor or Review Panel member, Other Financial or Material Support, Gift in kind to Baylor Scott and White Research Institute for NCT03383419)GSK (Advisor or Review Panel member)Johnson and Johnson (Scientific Research Study Investigator)Kinevant (Scientific Research Study Investigator)Roche/Genentech (Scientific Research Study Investigator)

10.
Sci Rep ; 11(1): 19458, 2021 09 30.
Article in English | MEDLINE | ID: covidwho-1447326

ABSTRACT

Efficacious therapeutics for Ebola virus disease are in great demand. Ebola virus infections mediated by mucosal exposure, and aerosolization in particular, present a novel challenge due to nontypical massive early infection of respiratory lymphoid tissues. We performed a randomized and blinded study to compare outcomes from vehicle-treated and remdesivir-treated rhesus monkeys in a lethal model of infection resulting from aerosolized Ebola virus exposure. Remdesivir treatment initiated 4 days after exposure was associated with a significant survival benefit, significant reduction in serum viral titer, and improvements in clinical pathology biomarker levels and lung histology compared to vehicle treatment. These observations indicate that remdesivir may have value in countering aerosol-induced Ebola virus disease.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacology , Ebolavirus/drug effects , Hemorrhagic Fever, Ebola/drug therapy , Adenosine Monophosphate/administration & dosage , Adenosine Monophosphate/pharmacology , Administration, Intravenous , Aerosols , Alanine/administration & dosage , Alanine/pharmacology , Animals , Antiviral Agents/administration & dosage , Disease Models, Animal , Female , Hemorrhagic Fever, Ebola/blood , Kaplan-Meier Estimate , Liver/drug effects , Liver/virology , Lung/pathology , Lung/virology , Lymph Nodes/drug effects , Lymph Nodes/pathology , Lymph Nodes/virology , Macaca mulatta , Male , Random Allocation , Systemic Inflammatory Response Syndrome/drug therapy , Systemic Inflammatory Response Syndrome/virology , Viral Load/drug effects , Viremia/drug therapy
11.
Nat Commun ; 12(1): 1891, 2021 03 25.
Article in English | MEDLINE | ID: covidwho-1387333

ABSTRACT

Monoclonal antibodies (mAbs) and remdesivir, a small-molecule antiviral, are promising monotherapies for many viruses, including members of the genera Marburgvirus and Ebolavirus (family Filoviridae), and more recently, SARS-CoV-2. One of the major challenges of acute viral infections is the treatment of advanced disease. Thus, extending the window of therapeutic intervention is critical. Here, we explore the benefit of combination therapy with a mAb and remdesivir in a non-human primate model of Marburg virus (MARV) disease. While rhesus monkeys are protected against lethal infection when treatment with either a human mAb (MR186-YTE; 100%), or remdesivir (80%), is initiated 5 days post-inoculation (dpi) with MARV, no animals survive when either treatment is initiated alone beginning 6 dpi. However, by combining MR186-YTE with remdesivir beginning 6 dpi, significant protection (80%) is achieved, thereby extending the therapeutic window. These results suggest value in exploring combination therapy in patients presenting with advanced filovirus disease.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antibodies, Monoclonal/therapeutic use , Antibodies, Viral/therapeutic use , Marburg Virus Disease/drug therapy , Marburgvirus/drug effects , Adenosine Monophosphate/therapeutic use , Alanine/therapeutic use , Animals , Disease Models, Animal , Drug Therapy, Combination , Macaca mulatta , Marburg Virus Disease/prevention & control , Viral Load/drug effects
13.
Cell Rep ; 36(4): 109450, 2021 07 27.
Article in English | MEDLINE | ID: covidwho-1306890

ABSTRACT

Improving clinical care for individuals infected with SARS-CoV-2 variants is a global health priority. Small-molecule antivirals like remdesivir (RDV) and biologics such as human monoclonal antibodies (mAbs) have demonstrated therapeutic efficacy against SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19). It is not known whether combination RDV/mAb will improve outcomes over single-agent therapies or whether antibody therapies will remain efficacious against variants. Here, we show that a combination of two mAbs in clinical trials, C144 and C135, have potent antiviral effects against even when initiated 48 h after infection and have therapeutic efficacy in vivo against the B.1.351 variant of concern (VOC). Combining RDV and antibodies provided a modest improvement in outcomes compared with single agents. These data support the continued use of RDV to treat SARS-CoV-2 infections and the continued clinical development of the C144 and C135 antibody combination to treat patients infected with SARS-CoV-2 variants.


Subject(s)
Antibodies, Monoclonal/pharmacology , COVID-19/drug therapy , SARS-CoV-2/drug effects , Animals , Antibodies, Monoclonal/immunology , Antiviral Agents/pharmacology , Humans , Mice , SARS-CoV-2/pathogenicity
14.
J Biol Chem ; 295(47): 16156-16165, 2020 11 20.
Article in English | MEDLINE | ID: covidwho-793655

ABSTRACT

Remdesivir (RDV) is a direct-acting antiviral agent that is used to treat patients with severe coronavirus disease 2019 (COVID-19). RDV targets the viral RNA-dependent RNA polymerase (RdRp) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We have previously shown that incorporation of the active triphosphate form of RDV (RDV-TP) at position i causes delayed chain termination at position i + 3. Here we demonstrate that the S861G mutation in RdRp eliminates chain termination, which confirms the existence of a steric clash between Ser-861 and the incorporated RDV-TP. With WT RdRp, increasing concentrations of NTP pools cause a gradual decrease in termination and the resulting read-through increases full-length product formation. Hence, RDV residues could be embedded in copies of the first RNA strand that is later used as a template. We show that the efficiency of incorporation of the complementary UTP opposite template RDV is compromised, providing a second opportunity to inhibit replication. A structural model suggests that RDV, when serving as the template for the incoming UTP, is not properly positioned because of a significant clash with Ala-558. The adjacent Val-557 is in direct contact with the template base, and the V557L mutation is implicated in low-level resistance to RDV. We further show that the V557L mutation in RdRp lowers the nucleotide concentration required to bypass this template-dependent inhibition. The collective data provide strong evidence to show that template-dependent inhibition of SARS-CoV-2 RdRp by RDV is biologically relevant.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacology , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Transcription Termination, Genetic/drug effects , Adenosine Monophosphate/pharmacology , Alanine/pharmacology , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Models, Chemical , Mutation , Nucleotides/metabolism , SARS-CoV-2/genetics , Templates, Genetic , Virus Replication/drug effects
15.
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.

16.
Nature ; 585(7824): 273-276, 2020 09.
Article in English | MEDLINE | ID: covidwho-592386

ABSTRACT

Effective therapies to treat coronavirus disease 2019 (COVID-19) are urgently needed. While many investigational, approved, and repurposed drugs have been suggested as potential treatments, preclinical data from animal models can guide the search for effective treatments by ruling out those that lack efficacy in vivo. Remdesivir (GS-5734) is a nucleotide analogue prodrug with broad antiviral activity1,2 that is currently being investigated in COVID-19 clinical trials and recently received Emergency Use Authorization from the US Food and Drug Administration3,4. In animal models, remdesivir was effective against infection with Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV)2,5,6. In vitro, remdesivir inhibited replication of SARS-CoV-27,8. Here we investigate the efficacy of remdesivir in a rhesus macaque model of SARS-CoV-2 infection9. Unlike vehicle-treated animals, macaques treated with remdesivir did not show signs of respiratory disease; they also showed reduced pulmonary infiltrates on radiographs and reduced virus titres in bronchoalveolar lavages twelve hours after the first dose. Virus shedding from the upper respiratory tract was not reduced by remdesivir treatment. At necropsy, remdesivir-treated animals had lower lung viral loads and reduced lung damage. Thus, treatment with remdesivir initiated early during infection had a clinical benefit in rhesus macaques infected with SARS-CoV-2. Although the rhesus macaque model does not represent the severe disease observed in some patients with COVID-19, our data support the early initiation of remdesivir treatment in patients with COVID-19 to prevent progression to pneumonia.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Disease Models, Animal , Macaca mulatta/virology , Pneumonia, Viral/prevention & control , Adenosine Monophosphate/pharmacokinetics , Adenosine Monophosphate/pharmacology , Adenosine Monophosphate/therapeutic use , Alanine/pharmacokinetics , Alanine/pharmacology , Alanine/therapeutic use , Animals , Betacoronavirus/genetics , Betacoronavirus/pathogenicity , Bronchoalveolar Lavage Fluid/virology , COVID-19 , Coronavirus Infections/pathology , Coronavirus Infections/physiopathology , DNA Mutational Analysis , Disease Progression , Drug Resistance, Viral , Female , Lung/drug effects , Lung/pathology , Lung/physiopathology , Lung/virology , Male , Pandemics , Pneumonia, Viral/drug therapy , Pneumonia, Viral/pathology , Pneumonia, Viral/physiopathology , Pneumonia, Viral/virology , SARS-CoV-2 , Secondary Prevention , Time Factors , Viral Load/drug effects , Virus Replication/drug effects , Virus Shedding/drug effects
17.
J Biol Chem ; 295(20): 6785-6797, 2020 05 15.
Article in English | MEDLINE | ID: covidwho-52576

ABSTRACT

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.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacology , Betacoronavirus/enzymology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Virus Replication/drug effects , Adenosine Monophosphate/pharmacology , Alanine/pharmacology , Animals , Betacoronavirus/physiology , Models, Molecular , SARS-CoV-2 , Sf9 Cells , Spodoptera
18.
J Biol Chem ; 295(15): 4773-4779, 2020 04 10.
Article in English | MEDLINE | ID: covidwho-1988

ABSTRACT

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.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacology , Middle East Respiratory Syndrome Coronavirus/enzymology , Nucleic Acid Synthesis Inhibitors/pharmacology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Virus Replication/drug effects , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/pharmacology , Alanine/chemistry , Alanine/pharmacology , Animals , Antiviral Agents/chemistry , Coronavirus/enzymology , Ebolavirus/enzymology , Gene Expression , Nucleic Acid Synthesis Inhibitors/chemistry , RNA , RNA-Dependent RNA Polymerase/genetics , Sf9 Cells , Viral Nonstructural Proteins/genetics
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