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

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 (Mpro) 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.

Discovery of GS-5245 (Obeldesivir), an Oral Prodrug of Nucleoside GS-441524 That Exhibits Antiviral Efficacy in SARS-CoV-2-Infected African Green Monkeys.

Remdesivir 1 is an phosphoramidate prodrug that releases the monophosphate of nucleoside GS-441524 (2) into lung cells, thereby forming the bioactive triphosphate 2-NTP. 2-NTP, an analog of ATP, inhibits the SARS-CoV-2 RNA-dependent RNA polymerase replication and transcription of viral RNA. Strong clinical results for 1 have prompted interest in oral approaches to generate 2-NTP. Here, we describe the discovery of a 5'-isobutyryl ester prodrug of 2 (GS-5245, Obeldesivir, 3) that has low cellular cytotoxicity and 3-7-fold improved oral delivery of 2 in monkeys. Prodrug 3 is cleaved presystemically to provide high systemic exposures of 2 that overcome its less efficient metabolism to 2-NTP, leading to strong SARS-CoV-2 antiviral efficacy in an African green monkey infection model. Exposure-based SARS-CoV-2 efficacy relationships resulted in an estimated clinical dose of 350-400 mg twice daily. Importantly, all SARS-CoV-2 variants remain susceptible to 2, which supports development of 3 as a promising COVID-19 treatment.

Efficacy of the oral nucleoside prodrug GS-5245 (Obeldesivir) against SARS-CoV-2 and coronaviruses with pandemic potential.

Despite the wide availability of several safe and effective vaccines that can prevent severe COVID-19 disease, the emergence of SARS-CoV-2 variants of concern (VOC) that can partially evade vaccine immunity remains a global health concern. In addition, the emergence of highly mutated and neutralization-resistant SARS-CoV-2 VOCs such as BA.1 and BA.5 that can partially or fully evade (1) many therapeutic monoclonal antibodies in clinical use underlines the need for additional effective 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 RNA-dependent viral RNA polymerase (RdRp). Importantly, we show that GS-5245 is broadly potent in vitro against alphacoronavirus HCoV-NL63, severe acute respiratory syndrome coronavirus (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 in vitro and highly effective as antiviral therapy in mouse models of SARS-CoV, SARS-CoV-2 (WA/1), MERS-CoV and Bat-CoV RsSHC014 pathogenesis. In all these models of divergent coronaviruses, we observed protection and/or significant reduction of disease metrics such as weight loss, lung viral replication, acute lung injury, and degradation in pulmonary function in GS-5245-treated mice compared to vehicle controls. Finally, we demonstrate that GS-5245 in combination with the main protease (Mpro) inhibitor nirmatrelvir had increased efficacy in vivo against SARS-CoV-2 compared to each single agent. Altogether, our data supports the continuing clinical evaluation of GS-5245 in humans infected with COVID-19, including as part of a combination antiviral therapy, especially in populations with the most urgent need for more efficacious and durable interventions.

Intravenous delivery of GS-441524 is efficacious in the African green monkey model of SARS-CoV-2 infection.

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.

Journey of remdesivir from the inhibition of hepatitis C virus to the treatment of COVID-19.

If a planned path reaches a dead-end, one can simply stop. Or one can turn around, walk back to the last intersection and take another path, or one can consider taking few paths in parallel. The last scenario is reflective of the journey of remdesivir, the first antiviral for the treatment of COVID-19, that was approved by FDA less than 10 months after the isolation of SARS-CoV-2, the virus responsible for the COVID-19 pandemic. As of January 2022, 10 million COVID-19 patients have been treated with remdesivir worldwide, but the journey of this molecule started more than a decade earlier with the search for a cure of hepatitis C virus. The development path of remdesivir before the emergence of COVID-19 represents a valuable example of a preemptive pandemic preparedness, but the pursuit of this path would not have been possible without sustaining support of John C. Martin, whom we will sorely miss for his piercing vision, uncompromising leadership, and genuine compassion for patients suffering around the world.

Therapeutic treatment with an oral prodrug of the remdesivir parental nucleoside is protective against SARS-CoV-2 pathogenesis in mice.

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.

Phosphoramidate Prodrugs Continue to Deliver, The Journey of Remdesivir (GS-5734) from RSV to SARS-CoV-2.

Remdesivir (GS-5734) is a monophenol, 2-ethylbutylalanine phosphoramidate prodrug of a 1'-cyano-4-aza-7,9-dideazaadenosine C-nucleoside (GS-441524) that is FDA approved for the treatment of hospitalized patients with COVID-19. The prodrug, initially invented for respiratory syncytial virus, was later found to have activity toward emerging RNA viruses, including Ebola and coronaviruses. Remdesivir is among the first examples of a phosphoramidate prodrug aimed at delivering a nucleoside monophosphate into lung cells to efficiently generate the nucleoside triphosphate inhibitor of viral RNA polymerases. With remdesivir as the central case study, the present work describes the antiviral potency and in vitro metabolism evidence for lung cell activation of phosphoramidates, together with their in vivo pharmacokinetics, lung distribution, and antiviral efficacy toward respiratory viruses. The lung delivery of nucleoside monophosphate analogs using prodrugs warrants further investigation toward the development of novel respiratory antivirals.

Subcutaneous remdesivir administration prevents interstitial pneumonia in rhesus macaques inoculated with SARS-CoV-2.

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.

Inhaled remdesivir reduces viral burden in a nonhuman primate model of SARS-CoV-2 infection.

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.

Oral prodrug of remdesivir parent GS-441524 is efficacious against SARS-CoV-2 in ferrets.

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.

Therapeutic efficacy of an oral nucleoside analog of remdesivir against SARS-CoV-2 pathogenesis in mice.

The COVID-19 pandemic remains uncontrolled despite the rapid rollout of safe and effective 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. Additionally, the emergence of SARS-CoV-2 variants of concern with their potential to escape 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 parental nucleoside of remdesivir, which targets the highly conserved RNA-dependent RNA polymerase. GS-621763 exhibited significant antiviral activity in lung cell lines and two different human primary lung cell culture systems. 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 significantly reduced viral load, lung pathology, and improved pulmonary function in COVID-19 mouse model. A direct comparison of GS-621763 with molnupiravir, an oral nucleoside analog antiviral currently in human clinical trial, proved both drugs to be similarly efficacious. These data demonstrate that therapy with oral prodrugs of remdesivir can significantly improve outcomes in SARS-CoV-2 infected mice. Thus, GS-621763 supports the exploration of GS-441524 oral prodrugs for the treatment of COVID-19 in humans.

Prodrugs of a 1'-CN-4-Aza-7,9-dideazaadenosine C-Nucleoside Leading to the Discovery of Remdesivir (GS-5734) as a Potent Inhibitor of Respiratory Syncytial Virus with Efficacy in the African Green Monkey Model of RSV.

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.

Toll-Like Receptor 8 Agonist GS-9688 Induces Sustained Efficacy in the Woodchuck Model of Chronic Hepatitis B.

BACKGROUND AND AIMS: GS-9688 (selgantolimod) is an oral selective small molecule agonist of toll-like receptor 8 in clinical development for the treatment of chronic hepatitis B. In this study, we evaluated the antiviral efficacy of GS-9688 in woodchucks chronically infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to hepatitis B virus. APPROACH AND RESULTS: WHV-infected woodchucks received eight weekly oral doses of vehicle, 1 mg/kg GS-9688, or 3 mg/kg GS-9688. Vehicle and 1 mg/kg GS-9688 had no antiviral effect, whereas 3 mg/kg GS-9688 induced a >5 log10 reduction in serum viral load and reduced WHV surface antigen (WHsAg) levels to below the limit of detection in half of the treated woodchucks. In these animals, the antiviral response was maintained until the end of the study (>5 months after the end of treatment). GS-9688 treatment reduced intrahepatic WHV RNA and DNA levels by >95% in animals in which the antiviral response was sustained after treatment cessation, and these woodchucks also developed detectable anti-WHsAg antibodies. The antiviral efficacy of weekly oral dosing with 3 mg/kg GS-9688 was confirmed in a second woodchuck study. The antiviral response to GS-9688 did not correlate with systemic GS-9688 or cytokine levels but was associated with transient elevation of liver injury biomarkers and enhanced proliferative response of peripheral blood mononuclear cells to WHV peptides. Transcriptomic analysis of liver biopsies taken prior to treatment suggested that T follicular helper cells and various other immune cell subsets may play a role in the antiviral response to GS-9688. CONCLUSIONS: Finite, short-duration treatment with a clinically relevant dose of GS-9688 is well tolerated and can induce a sustained antiviral response in WHV-infected woodchucks; the identification of a baseline intrahepatic transcriptional signature associated with response to GS-9688 treatment provides insights into the immune mechanisms that mediate this antiviral effect.

Safety, pharmacokinetics and pharmacodynamics of selgantolimod, an oral Toll-like receptor 8 agonist: a Phase Ia study in healthy subjects.

BACKGROUND: Selgantolimod is a novel oral, selective Toll-like receptor 8 (TLR8) agonist in development for the treatment of chronic hepatitis B (CHB). TLR8 is an endosomal innate immune receptor and a target for treatment of viral infections. This first-in-human study investigated the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of selgantolimod in healthy volunteers. METHODS: Of 71 subjects enrolled, 59 received a single dose of selgantolimod (0.5, 1.5, 3 or 5 mg) or placebo, and 12 were evaluated for food effect. Safety, PK and PD activity by induction of cytokines, chemokines and acute phase proteins were assessed. PK/PD analyses were conducted. RESULTS: Single doses of 0.5-5 mg were generally safe. No serious adverse events (AEs) or AEs leading to discontinuation were reported, and most were Grade 1 in severity. Selgantolimod displayed rapid absorption and dose-proportional PK and PD activity. Food had minimal effect on PK but resulted in diminished PD activity. In PK/PD analyses, near-saturation of induction for most evaluated biomarkers occurred at the 5-mg dose. CONCLUSIONS: Single doses of up to 5 mg selgantolimod were safe and induced dose-dependent PD responses. These data support evaluation of selgantolimod in combination with other agents in future clinical studies of CHB. Australian New Zealand Clinical Trials Registration: ACTRN12616001646437.

Discovery of GS-9688 (Selgantolimod) as a Potent and Selective Oral Toll-Like Receptor 8 Agonist for the Treatment of Chronic Hepatitis B.

Toll-like receptor 8 (TLR8) recognizes pathogen-derived single-stranded RNA fragments to trigger innate and adaptive immune responses. Chronic hepatitis B (CHB) is associated with a dysfunctional immune response, and therefore a selective TLR8 agonist may be an effective treatment option. Structure-based optimization of a dual TLR7/8 agonist led to the identification of the selective TLR8 clinical candidate (R)-2-((2-amino-7-fluoropyrido[3,2-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (GS-9688, (R)-7). Potent TLR8 agonism (IL-12p40 EC50 = 220 nM) and >100-fold TLR7 selectivity (IFN-α EC50 > 50 µM) was observed in human peripheral blood mononuclear cells (PBMCs). The TLR8-ectodomain:(R)-7 complex confirmed TLR8 binding and a direct ligand interaction with TLR8 residue Asp545. Oral (R)-7 had good absorption and high first pass clearance in preclinical species. A reduction in viral markers was observed in HBV-infected primary human hepatocytes treated with media from PBMCs stimulated with (R)-7, supporting the clinical development of (R)-7 for the treatment of CHB.

Structure-guided discovery of a novel, potent, and orally bioavailable 3,5-dimethylisoxazole aryl-benzimidazole BET bromodomain inhibitor.

The bromodomain and extra-terminal (BET) family of proteins, consisting of the bromodomains containing protein 2 (BRD2), BRD3, BRD4, and the testis-specific BRDT, are key epigenetic regulators of gene transcription and has emerged as an attractive target for anticancer therapy. Herein, we describe the discovery of a novel potent BET bromodomain inhibitor, using a systematic structure-based approach focused on improving potency, metabolic stability, and permeability. The optimized dimethylisoxazole aryl-benzimidazole inhibitor exhibited high potency towards BRD4 and related BET proteins in biochemical and cell-based assays and inhibited tumor growth in two proof-of-concept preclinical animal models.

Discovery of a Potent and Orally Bioavailable Cyclophilin Inhibitor Derived from the Sanglifehrin Macrocycle.

Cyclophilins are a family of peptidyl-prolyl isomerases that are implicated in a wide range of diseases including hepatitis C. Our aim was to discover through total synthesis an orally bioavailable, non-immunosuppressive cyclophilin (Cyp) inhibitor with potent anti-hepatitis C virus (HCV) activity that could serve as part of an all oral antiviral combination therapy. An initial lead 2 derived from the sanglifehrin A macrocycle was optimized using structure based design to produce a potent and orally bioavailable inhibitor 3. The macrocycle ring size was reduced by one atom, and an internal hydrogen bond drove improved permeability and drug-like properties. 3 demonstrates potent Cyp inhibition ( Kd = 5 nM), potent anti-HCV 2a activity (EC50 = 98 nM), and high oral bioavailability in rat (100%) and dog (55%). The synthetic accessibility and properties of 3 support its potential as an anti-HCV agent and for interrogating the role of Cyp inhibition in a variety of diseases.

Phase 1 First-in-Human, Single- and Multiple-Ascending Dose, and Food Effect Studies to Assess the Safety, Tolerability, and Pharmacokinetics of Presatovir for the Treatment of Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV)-associated respiratory tract infection is a leading cause of hospitalizations in infants for which no effective treatment exists. RSV infection is also an important cause of respiratory disease in adults and immunocompromised patients. Presatovir (GS-5806) is an orally bioavailable antiviral agent that inhibits fusion of RSV with host cell membranes. Here, results from 2 phase 1 studies that evaluated safety, tolerability, and pharmacokinetics of presatovir in healthy adults following administration of single and multiple (7 days) once- or twice-daily ascending doses (first-in-human study) and in the presence or absence of food (food effect study) are described. Presatovir exhibited favorable safety and pharmacokinetic profiles that supported once-daily dosing. Presatovir exposure increased in an approximately dose-proportional manner across the evaluated dose range (single doses 25-300 mg; multiple doses 10-75 mg once daily for 7 days). Administration of presatovir with a high-fat meal did not alter exposure, supporting administration without regard to a meal in further clinical studies. These data were subsequently used to inform presatovir dosing regimens in a phase 2a challenge study of adults experimentally infected with RSV. Collectively, results from phase 1 evaluations and a phase 2a challenge study support further clinical investigation of presatovir for the treatment of RSV infection.

Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease.

Emerging coronaviruses (CoVs) cause severe disease in humans, but no approved therapeutics are available. The CoV nsp14 exoribonuclease (ExoN) has complicated development of antiviral nucleosides due to its proofreading activity. We recently reported that the nucleoside analogue GS-5734 (remdesivir) potently inhibits human and zoonotic CoVs in vitro and in a severe acute respiratory syndrome coronavirus (SARS-CoV) mouse model. However, studies with GS-5734 have not reported resistance associated with GS-5734, nor do we understand the action of GS-5734 in wild-type (WT) proofreading CoVs. Here, we show that GS-5734 inhibits murine hepatitis virus (MHV) with similar 50% effective concentration values (EC50) as SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Passage of WT MHV in the presence of the GS-5734 parent nucleoside selected two mutations in the nsp12 polymerase at residues conserved across all CoVs that conferred up to 5.6-fold resistance to GS-5734, as determined by EC50 The resistant viruses were unable to compete with WT in direct coinfection passage in the absence of GS-5734. Introduction of the MHV resistance mutations into SARS-CoV resulted in the same in vitro resistance phenotype and attenuated SARS-CoV pathogenesis in a mouse model. Finally, we demonstrate that an MHV mutant lacking ExoN proofreading was significantly more sensitive to GS-5734. Combined, the results indicate that GS-5734 interferes with the nsp12 polymerase even in the setting of intact ExoN proofreading activity and that resistance can be overcome with increased, nontoxic concentrations of GS-5734, further supporting the development of GS-5734 as a broad-spectrum therapeutic to protect against contemporary and emerging CoVs.IMPORTANCE Coronaviruses (CoVs) cause severe human infections, but there are no approved antivirals to treat these infections. Development of nucleoside-based therapeutics for CoV infections has been hampered by the presence of a proofreading exoribonuclease. Here, we expand the known efficacy of the nucleotide prodrug remdesivir (GS-5734) to include a group ß-2a CoV. Further, GS-5734 potently inhibits CoVs with intact proofreading. Following selection with the GS-5734 parent nucleoside, 2 amino acid substitutions in the nsp12 polymerase at residues that are identical across CoVs provide low-level resistance to GS-5734. The resistance mutations decrease viral fitness of MHV in vitro and attenuate pathogenesis in a SARS-CoV animal model of infection. Together, these studies define the target of GS-5734 activity and demonstrate that resistance is difficult to select, only partial, and impairs fitness and virulence of MHV and SARS-CoV, supporting further development of GS-5734 as a potential effective pan-CoV antiviral.