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

Abstract

Emerging viral infections are difficult to control because heterogeneous members periodically cycle in and out of humans and zoonotic hosts, complicating the development of specific antiviral therapies and vaccines. Coronaviruses (CoVs) have a proclivity to spread rapidly into new host species causing severe disease. Severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) successively emerged, causing severe epidemic respiratory disease in immunologically naïve human populations throughout the globe. Broad-spectrum therapies capable of inhibiting CoV infections would address an immediate unmet medical need and could be invaluable in the treatment of emerging and endemic CoV infections. We show that a nucleotide prodrug, GS-5734, currently in clinical development for treatment of Ebola virus disease, can inhibit SARS-CoV and MERS-CoV replication in multiple in vitro systems, including primary human airway epithelial cell cultures with submicromolar IC50 values. GS-5734 was also effective against bat CoVs, prepandemic bat CoVs, and circulating contemporary human CoV in primary human lung cells, thus demonstrating broad-spectrum anti-CoV activity. In a mouse model of SARS-CoV pathogenesis, prophylactic and early therapeutic administration of GS-5734 significantly reduced lung viral load and improved clinical signs of disease as well as respiratory function. These data provide substantive evidence that GS-5734 may prove effective against endemic MERS-CoV in the Middle East, circulating human CoV, and, possibly most importantly, emerging CoV of the future.

Conflict of interest statement

Competing interests: The authors affiliated with Gilead Sciences are employees of the company and may own company stock. M.O.C., J.Y.F, R.J., R.L.M., A.S.R., and D.S. are listed as inventors on International Application No. PCT/US2016/052092 filed by Gilead Sciences, Inc., directed to methods of treating coronaviridae virus infections. Travel of M.R.D. to Gilead Sciences, Inc. to discuss this project was paid for by Gilead Sciences.

Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Figures

Figure 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 nLuc at an MOI of 0.08 in the presence of varying concentrations of GS-5734 for 48hr after which replication was measured through quantitation of MERS-CoV expressed nano luciferase (nLUC). (B) Cytoxicity in 2B4 cells treated similarly to those in A. Viability was measured via CellTiter-Glo. Data for A and B is representative of three independent experiments. (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 LC/MS.

Figure 2. GS-5734 prevents SARS- and MERS-CoV replication in human airway epithelial cells

(A) Antiviral efficacy of GS-5734 against MERS-CoV in primary human airway epithelial (HAE) cell cultures. HAE cells were infected with MERS-CoV RFP at an MOI of 0.5 in duplicate in the presence of GS-5734 for 48hr after which apical washes were collected for virus titration. Representative data from two separate experiments with three different cell donors is displayed. (B) Antiviral efficacy of GS-5734 against SARS-CoV in HAE cells. Cultures were infected with SARS-CoV GFP and treated and analyzed as described in A. (C) Quantitative RT-PCR for MERS-CoV ORF1 and ORFN mRNA. Total RNA was isolated from the cultures in panel A for RT-PCR analysis. (D) Quantitative RT-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 prior to fluorescent imaging.

Figure 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 (RNA dependent RNA polymerase) and CoV Spike glycoprotein, which mediates host tropism and entry into cells. Text color of virus strain label corresponds to virus host species on left. (B) Top panel, antiviral efficacy of GS-5734 in HAE cells against group 1 human circulating CoV (hCoV-NL63) and bat CoV from group 2b (HKU3, SHC014, WIV1) and 2c (HKU5). HAE cells were infected at an MOI of 0.5 in the presence of GS-5734 in duplicate. After 48hr, virus produced was titrated via plaque assay. Each data point represents the titer per culture. Bottom panel, quantitative RT-PCR for CoV ORF1 and ORFN mRNA in total RNA from the cultures in top panel.

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

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

Figure 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 -1dpi with either vehicle (n = 42), 25mg/kg twice daily (BID, n = 25) or 50mg/kg once daily (QD, n = 28) GS-5734. (B) SARS-CoV lung titer in panel A mice at 2dpi (left) (Vehicle N = 11, 50mg/kg N = 11, 25mg/kg N = 5) or 5dpi (right) (Vehicle N = 13, 50mg/kg N = 13, 25mg/kg N = 4). (C) Quantitation of SARS-CoV antigen in lung sections in panel A mice at 2dpi (left) (Vehicle N = 15, 50mg/kg N = 12, 25mg/kg N = 7) or 5dpi (right) (Vehicle N = 10, 50mg/kg N = 12, 25mg/kg N = 4). (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 2dpi highlighting the conducting airway lumen (aw). (F) Whole-body plethysmography (WBP) to measure pulmonary function in panel A mice. Penh is a surrogate measure of bronchoconstriction. Expiration time is the time taken to release one breath. End of expiratory pause is the time between breaths. Symbols and error bars for panels A, B, D, and I represent the mean and standard deviation. The boxes encompass the 25–75th percentile while the whiskers represent the range in C, E and F. Asterisk indicates statistical significance (p<0.05) by two-way ANOVA with Tukey’s multiple comparison test for D, F, and I and Kruskal-Wallis in E.

Figure 6. Therapeutic post-exposure administration of GS-5734 mitigates disease

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