Research ArticleEMERGING INFECTIONS

Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses

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Science Translational Medicine  28 Jun 2017:
Vol. 9, Issue 396, eaal3653
DOI: 10.1126/scitranslmed.aal3653
  • Fig. 1. MERS-CoV antiviral efficacy, toxicity, and metabolism of GS-5734 in 2B4 cells.

    (A) Mean percent inhibition of MERS-CoV replication by GS-5734. 2B4 cells were infected in triplicate with MERS-CoV nanoluciferase (nLUC) at a multiplicity of infection (MOI) of 0.08 in the presence of varying concentrations of GS-5734 for 48 hours, after which replication was measured through quantitation of MERS-CoV–expressed nLUC. (B) Cytotoxicity in 2B4 cells treated similarly to that in (A). Viability was measured via CellTiter-Glo. Data for (A) and (B) are representative of three independent experiments. DMSO, dimethyl sulfoxide. (C) Measurement of intracellular nucleotide triphosphate (NTP) in 2B4 cells. In three independent experiments, triplicate wells of cells were treated with 1 μM GS-5734 and harvested over time to measure NTP via liquid chromatography–mass spectrometry (LC-MS).

  • Fig. 2. GS-5734 prevents SARS-CoV and MERS-CoV replication in HAE cells.

    (A) Antiviral efficacy of GS-5734 against MERS-CoV in primary HAE cell cultures. HAE cells were infected with MERS-CoV red fluorescent protein (RFP) at an MOI of 0.5 in duplicate in the presence of GS-5734 for 48 hours, after which apical washes were collected for virus titration. Representative data from two separate experiments with three different cell donors are displayed. PFU, plaque-forming units. (B) Antiviral efficacy of GS-5734 against SARS-CoV in HAE cells. Cultures were infected with SARS-CoV green fluorescent protein (GFP), treated, and analyzed as described in (A). (C) qRT-PCR for MERS-CoV ORF1 and ORFN mRNA. Total RNA was isolated from cultures in (A) for qRT-PCR analysis. (D) qRT-PCR for SARS-CoV ORF1 and ORFN in cells from (C), as described in (B). (E) HAE cells were infected with MERS-CoV RFP and treated with GS-5734 as in (A). Nuclei were stained with Hoechst 33258 before fluorescent imaging.

  • Fig. 3. GS-5734 is effective against a diverse array of human and zoonotic CoV in HAE.

    (A) Neighbor-joining trees created with representatives from all four CoV genogroups showing the genetic similarity of CoV nsp12 (RdRp) and CoV spike glycoprotein, which mediates host tropism and entry into cells. Text color of the virus strain label corresponds to virus host species on the left. The heatmap adjacent to each neighbor-joining tree depicts percent amino acid identity (% A.A. identity) against SARS-CoV or MERS-CoV. (B) Top: Antiviral efficacy of GS-5734 in HAE cells against circulating group 1 human CoV (HCoV-NL63) and bat CoV from group 2b (HKU3, SHC014, and WIV1) and 2c (HKU5). HAE cells were infected at an MOI of 0.5 in the presence of GS-5734 in duplicate. After 48 hours, virus produced was titrated via plaque assay. Each data point represents the titer per culture. Bottom: qRT-PCR for CoV ORF1 and ORFN mRNA in total RNA from cultures in the top panel.

  • Fig. 4. Pharmacokinetics of GS-5734 in Ces1c−/− mice.

    (A) Pharmacokinetics in Ces1c−/− mouse plasma after subcutaneous administration of GS-5734 (25 mg/kg). Longitudinal plasma samples were taken to measure prodrug GS-5734, intermediate metabolites Ala-Met, and nucleoside by LC-MS. (B) Lung TP in Ces1c−/− mouse lung 4 hours after subcutaneous administration of GS-5734 (50 mg/kg). Nucleotide monophosphate (Nuc-MP), diphosphate (Nuc-DP), triphosphate (Nuc-TP), and total nucleotide (sum of Nuc-MP, Nuc-DP, and Nuc-TP) are displayed. (C) Pharmacokinetics of total nucleotide in Ces1c−/− mouse lung after subcutaneous administration of GS-5734 at 25 mg/kg BID or 50 mg/kg QD.

  • Fig. 5. Prophylactic treatment with GS-5734 reduces SARS-CoV disease.

    (A) Percent starting weight of Ces1c−/− mice infected with 104 PFU SARS-CoV MA15 treated beginning at −1 dpi with either vehicle (n = 42) or GS-5734 [25 mg/kg BID (n = 25) or 50 mg/kg QD (n = 28)]. (B) SARS-CoV lung titer of mice in (A) at 2 dpi (vehicle, n = 11; 50 mg/kg, n = 11; 25 mg/kg, n = 5) (left) or 5 dpi (vehicle, n = 13; 50 mg/kg, n = 13; 25 mg/kg, n = 4) (right). n.s., not significant. (C) Quantitation of SARS-CoV antigen in lung sections of mice in (A) at 2 dpi (left) (vehicle, n = 15; 50 mg/kg, n = 12; 25 mg/kg, n = 7) (left) or 5 dpi (vehicle, n = 10; 50 mg/kg, n = 12; 25 mg/kg, n = 4) (right). (D) Photomicrographs of SARS-CoV antigen staining (brown) and nuclei (blue) in lung sections from 2 and 5 dpi. (E) Photomicrographs of hematoxylin and eosin–stained mouse lung sections from 2 dpi highlighting the conducting airway lumen (aw). (F) WBP was used to measure the pulmonary function of mice in (A). Penh is a surrogate measure of bronchoconstriction. Expiration time (te) is the time taken to release one breath. End of expiratory pause (EEP) is the time between breaths. Symbols and error bars for (A), (B), (D), and (I) represent the mean and SD. The boxes encompass the 25th to 75th percentile, whereas the whiskers represent the range in (C), (E), and (F). Asterisk indicates statistical significance (P < 0.05) by two-way analysis of variance (ANOVA) with Tukey’s multiple comparison test for (D), (F), and (I) and with Kruskal-Wallis test for (E).

  • Fig. 6. Therapeutic postexposure administration of GS-5734 mitigates disease.

    (A) Percent starting weight of 27- to 28-week-old female Ces1c−/− mice infected with 103 PFU SARS-CoV MA15 and treated BID with vehicle or GS-5734 (25 mg/kg) beginning on either −1 dpi (vehicle, n = 5; GS-5734, n = 10) or +1 dpi (vehicle, n = 4; GS-5734, n = 11). Weights of GS-5734–treated animals were statistically different (P < 0.05) from those of vehicle-treated animals at 3 and 4 dpi for prophylactic groups and at 4 dpi for therapeutic groups by two-way ANOVA with Tukey’s multiple comparison test. (B) Percent starting weights of mice in (A) at 4 dpi. (C) SARS-CoV lung titer in mice infected and treated as described in (A). Asterisks indicate statistical significance (P < 0.05) by Mann-Whitney test for (B) and (C). (D) WBP was used to measure the pulmonary function in mice infected and treated as described in (A). Penh is a surrogate measure of bronchoconstriction or airway obstruction. Asterisks indicate statistical significance by two-way ANOVA with Šidák’s multiple comparison test.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/396/eaal3653/DC1

    Materials and Methods

    Fig. S1. In vitro toxicity and efficacy of GS-5734 in 2B4 cells.

    Fig. S2. In vitro toxicity of GS-5734 in primary HAE cell cultures.

    Fig. S3. In vitro toxicity of GS-5734 in primary NHBE cell cultures.

    Fig. S4. SARS-CoV in vivo pathogenesis is similar in WT and Ces1c−/− mice.

    Fig. S5. Metabolism in NHBE cells and pharmacokinetic analysis in nonhuman primates.

    Fig. S6. GS-5734 diminishes SARS-CoV–induced lung pathology.

    Fig. S7. Therapeutic administration of GS-5734 beginning at 2 dpi does not provide a therapeutic benefit.

    Fig. S8. A comprehensive platform approach to evaluate therapeutics against emerging viral infections.

    Table S1. CoV genomic and subgenomic real-time primer sets.

    Table S2. Primer/probe sets for indicators of cellular apoptosis/toxicity qRT-PCR.

    Table S3. Primary data.

  • Supplementary Material for:

    Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses

    Timothy P. Sheahan, Amy C. Sims, Rachel L. Graham, Vineet D. Menachery, Lisa E. Gralinski, James B. Case, Sarah R. Leist, Krzysztof Pyrc, Joy Y. Feng, Iva Trantcheva, Roy Bannister, Yeojin Park, Darius Babusis, Michael O. Clarke, Richard L. Mackman, Jamie E. Spahn, Christopher A. Palmiotti, Dustin Siegel, Adrian S. Ray, Tomas Cihlar, Robert Jordan, Mark R. Denison,* Ralph S. Baric*

    *Corresponding author. Email: mark.denison{at}vanderbilt.edu (M.R.D.); rbaric{at}email.unc.edu (R.S.B.)

    Published 28 June 2017, Sci. Transl. Med. 9, eaal3653 (2017)
    DOI: 10.1126/scitranslmed.aal3653

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. In vitro toxicity and efficacy of GS-5734 in 2B4 cells.
    • Fig. S2. In vitro toxicity of GS-5734 in primary HAE cell cultures.
    • Fig. S3. In vitro toxicity of GS-5734 in primary NHBE cell cultures.
    • Fig. S4. SARS-CoV in vivo pathogenesis is similar in WT and Ces1c−/− mice.
    • Fig. S5. Metabolism in NHBE cells and pharmacokinetic analysis in nonhuman primates.
    • Fig. S6. GS-5734 diminishes SARS-CoV–induced lung pathology.
    • Fig. S7. Therapeutic administration of GS-5734 beginning at 2 dpi does not provide a therapeutic benefit.
    • Fig. S8. A comprehensive platform approach to evaluate therapeutics against emerging viral infections.
    • Table S1. CoV genomic and subgenomic real-time primer sets.
    • Table S2. Primer/probe sets for indicators of cellular apoptosis/toxicity qRT-PCR.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S3 (Microsoft Excel format). Primary data.

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