The oral nucleoside prodrug GS-5245 is efficacious against SARS-CoV-2 and other endemic, epidemic, and enzootic coronaviruses

Abstract

Despite the wide availability of several safe and effective vaccines that prevent severe COVID-19, the persistent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that can evade vaccine-elicited immunity remains a global health concern. In addition, the emergence of SARS-CoV-2 VOCs that can evade therapeutic monoclonal antibodies underscores the need for additional, variant-resistant treatment strategies. Here, we characterize the antiviral activity of GS-5245, obeldesivir (ODV), an oral prodrug of the parent nucleoside GS-441524, which targets the highly conserved viral RNA-dependent RNA polymerase (RdRp). We show that GS-5245 is broadly potent in vitro against alphacoronavirus HCoV-NL63, SARS-CoV, SARS-CoV-related bat-CoV RsSHC014, Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2 WA/1, and the highly transmissible SARS-CoV-2 BA.1 Omicron variant. Moreover, in mouse models of SARS-CoV, SARS-CoV-2 (WA/1 and Omicron B1.1.529), MERS-CoV, and bat-CoV RsSHC014 pathogenesis, we observed a dose-dependent reduction in viral replication, body weight loss, acute lung injury, and pulmonary function with GS-5245 therapy. Last, we demonstrate that a combination of GS-5245 and main protease (M^pro) inhibitor nirmatrelvir improved outcomes in vivo against SARS-CoV-2 compared with the single agents. Together, our data support the clinical evaluation of GS-5245 against coronaviruses that cause or have the potential to cause human disease.

Fig. 1.. GS-5245 is broadly active against a panel of enzootic, endemic and pandemic coronaviruses in primary human airway cells.

(A) The mean percent inhibition and cytotoxicity of SARS-CoV-2 WA/1 replication by GS-5245 and control compounds GS-441524, remdesivir, and nirmatrelvir in A459-hACE2 cells is shown (triplicate samples were analyzed). Cells were infected at an MOI of 0.5. Data are representative of two independent experiments. (B) Antiviral activity of GS-5245 is shown for primary human airway epithelial cells from donor 1 against SARS-CoV and related bat-CoV SHC014, SARS-CoV-2 WA1 and SARS-CoV-2 Omicron BA.1 and MERS-CoV. HAEs were treated with indicated doses of drug or DMSO and then infected at an MOI of 0.5. At 2 hpi, input virus was removed, cultures were washed once, and infectious virus titers in plaque forming units (PFU) was measured in apical washes at 72 hpi. Cultures remained in the presence of drug for the duration of the study. (C) Antiviral activity of GS-5245 in primary human airway epithelial cell from donor 2 against SARS-CoV and related bat-CoV SHC014, SARS-CoV-2 WA1, and SARS-CoV-2 Omicron BA.1 and MERS-CoV. This was performed similarly but independent of the study in (B). For (B and C), each symbol represents the virus titer per triplicate culture and the line is at the mean and asterisks indicate statistical significance as determined by One-Way ANOVA with Tukey’s multiple comparison test where * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. The dashed lines in (A) denote the IC₅₀ values. The dashed lines in (B and C) denote the lower limit of detection.

Fig. 2.. GS-5245 exhibits dose-dependent therapeutic efficacy against SARS-CoV-2 MA10 in BALB/c mice.

(A) Shown is the percent change compared with starting weight in 10-week-old female BALB/c mice infected with SARS-CoV-2 MA10 at 1 x 10⁴ PFU. Mice were treated BID with vehicle, 3 mg/kg GS-5245, 10 mg/kg GS-5245, 30 mg/kg GS-5245, or 100 mg/kg molnupiravir starting at 12 hpi. n = 10 mice per group. Rx = drug. (B) SARS-CoV-2 MA10 lung infectious viral titers were measured at 4 dpi in mice treated with vehicle (veh), GS-5245 at increasing concentrations, or 100 mg/kg molnupiravir (MOV). LOD, limit of detection. (C) Macroscopic lung discoloration was evaluated at 4 dpi in therapeutically treated mice compared to vehicle. (D) Pulmonary function (PenH) was monitored by whole-body plethysmography from day zero through 4 dpi in SARS-CoV-2-infected treated mice. The boxes encompass the 25th to 75th percentile, whereas the whiskers represent the range. (E) Microscopic ATS acute lung injury pathology scoring at 4 dpi is shown for vehicle, GS-5245, and molnupiravir-treated mice. (F) Microscopic DAD acute lung injury pathology scoring at 4 dpi is shown for vehicle, GS-5245, and molnupiravir-treated mice. For (A), (B), (C), (E) and (F), each symbol represents data for one mouse and the line is at the mean. For (A and D), asterisks denote significant p values from a two-way ANOVA after a Tukey’s multiple comparisons test where * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. For (B), (C), (E) and (F), asterisks denote significant p values from a Kruskal-Wallis test after a Dunn’s multiple comparisons test where * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001. This study was performed once but some conditions were repeated in fig. S8 and S9.

Fig. 3.. Therapeutic efficacy of GS-5245 against pre-emergent bat SARS-related RsSHC014-CoV in K18-hACE2 mice.

(A) Shown is the percent change compared with starting weight in 10-week-old female K18-hACE2 mice infected with RsSHC014-CoV at 1 x 10⁴ PFU. Mice were treated BID with vehicle, 10 mg/kg GS-5245, 30 mg/kg GS-5245 at 12 hpi or 24 hpi. n = 10 mice per group. Asterisks denote significant p values from a two-way ANOVA after a Tukey’s multiple comparisons test where * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001. (B) RsSHC014-CoV lung infectious viral titers were measured 4 dpi in mice treated with vehicle (veh) or GS-5245 at 10 and 30 mg/kg at either 12 or 24 hpi. LOD = limit of detection. (C) Macroscopic lung discoloration was evaluated at 4 dpi in therapeutically treated K18-hACE2 mice compared to vehicle. (D) Microscopic ATS acute lung injury pathology scoring at 4 dpi is shown for vehicle vs. GS-5245-treated mice. (E) Microscopic DAD acute lung injury pathology scoring at 4 dpi is shown for vehicle vs. GS-5245-treated mice. For all panels, each symbol represents data for one mouse and the line is at the mean. For (B), (C), (D) and (E), Asterisks denote significant p values from a Kruskal-Wallis test after a Dunn’s multiple comparisons test where * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001. This study was performed once.

Fig. 4.. GS-5245 protects against SARS-CoV MA15 mortality in BALB/c mice.

(A) Shown is the percent change compared with starting weight in 10-week-old female BALB/c mice infected with SARS-CoV MA15 at 1 x 10⁴ PFU . Mice were treated BID with vehicle, 10 mg/kg GS-5245, 30 mg/kg GS-5245 at 12 hpi or at 24 hpi. n = 10 mice per group. (B) SARS-CoV MA15 lung infectious viral titers were measured 4 dpi in mice treated with vehicle or GS-5245 at 10 and 30 mg/kg at either 12 or 24 hpi. (C) Macroscopic lung discoloration at 4 dpi is shown for therapeutically treated BALB/c mice compared to vehicle. (D) Shown is the percent survival in vehicle vs GS-5245-treated mice. Survival curves are different (p = 0.0013) using a Log-rank Mantel-Cox test. (E) Microscopic ATS acute lung injury pathology scoring at 4 dpi is shown for vehicle vs. GS-5245-treated mice. (F) Microscopic DAD acute lung injury pathology scoring at 4 dpi is shown for vehicle vs. GS-5245-treated mice. (G) Shown is pulmonary function monitored by whole-body plethysmography from day zero through 4 dpi in SARS-CoV-infected treated mice. The boxes encompass the 25th to 75th percentile, whereas the whiskers represent the range. For (A), (B), (C), (E) and (F), each symbol represents data for one mouse and the line is at the mean. For (A) and (G), asterisks denote significant p values from a mixed-effects analysis (REML) with a Tukey’s multiple comparisons test where * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. For (B), (C), (E), and (F), asterisks denote significant p values from a Kruskal-Wallis test after a Dunn’s multiple comparisons test where * p ≤ 0.05, ** p ≤ 0.01. This study was performed once.

Fig. 5.. GS-5245 therapy protects against MERS-CoV pathogenesis in mice.

(A) Shown is the percent change compared with starting weight in 20-week-old male and female 288/330⁺⁺ DPP4-modified mice infected with a mouse-adapted MERS-CoV at 5 x 10⁴ PFU. Mice were treated BID starting at 12 hpi with vehicle, 10 mg/kg GS-5245, or 30 mg/kg GS-5245. n = 8 total mice per group. Male and female mice were distributed as equally as possible in each group. (B) MERS-CoV lung infectious viral titers were measured at 5 dpi in mice treated with vehicle or GS-5245 at 10 and 30 mg/kg at 12 hpi. (C) Macroscopic lung discoloration at 5 dpi is shown for therapeutically treated mice compared to vehicle. (D) Shown is pulmonary function monitored by whole-body plethysmography from day 0 through 5 dpi in MERS-CoV-infected treated 288/330⁺⁺ DPP4 mice. The boxes encompass the 25th to 75th percentile, whereas the whiskers represent the range. (E) Microscopic ATS acute lung injury pathology scoring at 5 dpi is shown for vehicle vs. GS-5245-treated mice. (F) Microscopic DAD acute lung injury pathology scoring at 5 dpi is shown for vehicle vs. GS-5245-treated mice. For (A), (B), (C), (E) and (F), each symbol represents data for one mouse and the line is at the mean. For (A) and (D), asterisks denote significant p values from a two-way ANOVA after a Tukey’s multiple comparisons test where * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. For (B), (C), (E) and (F), asterisks denote significant p values from a Kruskal-Wallis test after a Dunn’s multiple comparisons test where * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001. This study was performed once.

Fig. 6.. GS-5245 therapy potently reduces SARS-CoV-2 Omicron replication in K18-hACE2 mice.

(A) Shown is the percent change compared with starting weight in 10-week-old female K18-hACE2 mice infected with a SARS-CoV-2 Omicron BA.1 clinical isolate at 1 x 10⁴ PFU. Mice were treated BID starting at 12 hpi with vehicle and 30 mg/kg GS-5245. n = 10 mice per group. (B) BA.1 lung infectious viral titers were measured at 2, 3, and 4 dpi in mice treated with vehicle or GS-5245 and 30 mg/kg at 12 hpi. Asterisks denote significant p values from a Kruskal-Wallis test after a Dunn’s multiple comparisons test where * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. (C) Macroscopic lung discoloration at 4 dpi is shown for therapeutically treated mice compared to vehicle (D) Microscopic ATS acute lung injury pathology scoring at 4 dpi is shown for vehicle vs. GS-5245-treated mice. (E) Microscopic DAD acute lung injury pathology scoring at 4 dpi is shown for vehicle vs. GS-5245-treated mice. For all panels, each symbol represents data for one mouse and the line is at the mean. For (C to E), Asterisks denote significant p values from a Mann-Whitney test where * p ≤ 0.05, **** p ≤ 0.0001. This study was performed once.

Fig. 7.. Combination GS-5245 and nirmatrelvir therapy diminishes SARS-CoV-2 pathogenesis in mice.

(A) Shown is the percent change compared with starting weight in 10-week-old female BALB/c mice infected with SARS-CoV-2 MA10 at 1 x 10⁴ PFU. Mice were treated BID starting at 12 hpi with vehicle (n = 28), 4 mg/kg GS-5245 (n = 30), 40 mg/kg nirmatrelvir (n = 29), or a combination of 4mg/kg GS-5245 and 40 mg/kg nirmatrelvir (n = 29). Asterisks indicate statistical significance by mixed-effects model followed by a Tukey’s multiple comparisons test where * p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001. (B) Pulmonary function as measured by whole-body plethysmography in SARS-CoV-2-infected mice is shown starting at day 0 through 4 dpi. n = 4 to 5 mice per group were evaluated each time. The boxes encompass the 25th to 75th percentile, whereas the whiskers represent the range. (C) SARS-CoV-2 lung titers measured at 2dpi. n = 10 per group. (D) SARS-CoV-2 lung titers measured at 4dpi. Vehicle n = 18, GS-5245 n = 20, nirmatrelvir n = 19, combination n = 19. (E) Macroscopic lung discoloration at measured at 4 dpi in mice. Vehicle n = 8, GS-5245 n = 10, nirmatrelvir n = 9, combination n = 9. (F) Microscopic ATS acute lung injury score and (G) Diffuse alveolar damage (DAD) score measured at 4dpi. Vehicle n = 8, GS-5245 n = 10, nirmatrelvir n = 9, combination n = 9. For (A), (C), (D), (E), (F) and (G), each symbol represents data for one mouse and the line is at the mean. For (B), (C), (D), (E), (F) and (G), asterisks indicate statistical significance by Mann-Whitney test where * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. For (A) and (D), data shown are combined from two independent experiments. All other panels are from a single study.