key: cord-0688993-un9g40aa authors: Li, Yingjun; Cao, Liu; Li, Ge; Cong, Feng; Li, Yunfeng; Sun, Jing; Luo, Yinzhu; Chen, Guijiang; Li, Guanguan; Wang, Ping; Xing, Fan; Ji, Yanxi; Zhao, Jincun; Zhang, Yu; Guo, Deyin; Zhang, Xumu title: Remdesivir Metabolite GS-441524 Effectively Inhibits SARS-CoV-2 Infection in Mice Models date: 2020-10-27 journal: bioRxiv DOI: 10.1101/2020.10.26.353300 sha: c5b11ae506606b57bb256747e20604b89c1b14c6 doc_id: 688993 cord_uid: un9g40aa The outbreak of coronavirus disease 2019 (COVID-19) rapidly spreads across worldwide and becomes a global pandemic. Remdesivir is the only COVID-19 treatment approved by U.S. Food and Drug Administration (FDA); however, its effectiveness is still under questioning as raised by the results of a large WHO Solidarity Trial. Herein, we report that the parent nucleotide of remdesivir, GS-441524, potently inhibits the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Vero E6 and other cells. It exhibits good plasma distribution and longer half-life (t1/2=4.8h) in rat PK study. GS-441524 is highly efficacious against SARS-CoV-2 in AAV-hACE2 transduced mice and murine hepatitis virus (MHV) in mice, reducing the viral titers in CoV-attacked organs, without noticeable toxicity. Given that GS-441524 was the predominant metabolite of remdesivir in the plasma, the anti-COVID-19 effect of remdesivir may partly come from the effect of GS-441524. Our results also supported that GS-441524 as a promising and inexpensive drug candidate in the treatment of COVID-19 and future emerging CoVs diseases. Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in December 2019. 1, 2 SARS-CoV-2 is a novel single-stranded RNA virus belonging to beta coronavirus. 3 Although most coronavirus infections cause only mild respiratory symptoms, infection with SARS-CoV-2 could progress to acute severe respiratory syndrome (ARDS) and be lethal. 4 Since the declaration of COVID-19 as a global pandemic by the World Health Organization (WHO), infection and mortality rapidly increased worldwide. As of 24 October 2020, more than 42,000,000 confirmed COVID-19 cases and >1,100,000 associated deaths were reported worldwide, according to the Johns Hopkins University COVID-19 global case dashboard. Effective therapies and drug candidates to treat COVID-19 are urgently needed. Although several virus-based and host-based therapeutics agents have been evaluated for the treatment of COVID-19, such as lopinavir/ritonavir, 5 immunoglobulin, hydroxychloroquine, 6 EIDD-2801, 7 baricitinib, 8 and AT-527. 9 Currently, only remdesivir, an RNA-dependent RNA polymerase (RdRP) inhibitor, recently received U.S. Food and Drug Administration (FDA) approval for COVID-19 treatment. 10, 11, 12, 13, 14 However, its effectiveness to reduce hospital stay and risk of death of COVID-19 patients is still under questioning, as revealed by WHO solidarity trial. 15 In addition, the broad applicability of remdesivir, especially accessibility in the developing country will be limited by its difficulty to synthesize and expensiveness. Structurally, remdesivir ( Figure 1 ) is a nucleotide McGuigan prodrug presuming to be intracellularly metabolized into an active analog of triphosphate (GS-443902). 16 The anionic phosphate moiety on remdesivir is masked by phenol and L-alaninate ethylbutyl ester, which is supposed to be enzymatically cleavedoff inside cells. However, pharmacokinetics (PK) study in nonhuman primates and healthy human subjects showed that remdesivir is rapidly metabolized to be GS-704277, GS-441524 (parent nucleoside), and GS-443902 in plasma following intravenous administration (Figure 1) . 17 19, 20 The contribution of its metabolites especially GS-441524, to clinical outcomes is not fully understood. In vitro studies revealed that GS-441524 was significantly less potent against Ebola virus (EBOV), hepatitis C virus (HCV) and respiratory syncytial virus (RSV), as compared to remdesivir. 12, 21 The activation of nucleoside analogs needs two enzymatic phosphorylation steps to convert to the pharmacologically active triphosphate (GS-443902). The conversion of GS-441524 to monophosphate is considered a rate-limiting step in the activation of GS-441524. The prodrug remdesivir is considered to be superior to GS-441524 by bypassing the perceived ratelimiting first phosphorylation step and also by improving cell permeability. 22 However, GS-441524 exhibited good in vitro efficacy against human coronaviruses (CoV), such as SARS-CoV, middle east respiratory syndrome coronavirus (MERS-CoV), and zoonotic coronaviruses. Feline infectious peritonitis (FIP) was caused by feline coronaviruses (FCoV) infection and has long been considered a fatal feline disease. A 96% cure rate was observed in GS-441524 treated cats with feline coronavirus infection. 16, 23, 24 These early studies indicate the potential of GS-441524 for the treatment of coronavirus diseases as well as COVID-19. 25, 26 Herein, we investigated and compared GS-441524 with remdesivir for their in vitro anti-SARS-CoV-2 activities, PK profile and in vivo efficacy against SARS-CoV-2 and MHV. Our results provided further experimental insights to emphasize GS-441524 as a potential antiviral for COVID-19 and future emerging CoVs diseases. Initially, we compared the anti-SARS-CoV-2 activity of GS-441524 and remdesivir in Vero The PK profile of remdesivir and its metabolites was studied with intravenous (iv) administration of remdesivir in cynomolgus monkey and human subjects. 20, 27 However, there is no reported PK about GS-441524 itself. Next, we determined the pharmacokinetics of GS-441524 in Sprague-Dawley (SD) rats via iv and intragastric (ig) injection with a dose of 30 mg/kg. GS-441524 exhibited encouraging iv PK parameters with a long half-life (t1/2) of 4.8 h and a high Cmax of 163616.6 μg/L. Although the bioavailability (~5 %) of GS-441524 is not ideal, the Cmax in ig administrated mice was 2708 μg/L (9.3μM), higher than the concentration required for > 50% SARS-CoV-2 inhibition, indicating that GS-441524 might be able to achieve in vivo efficacy relevant doses following an oral dosing regimen. Our results were consistent with the clinical PK study of remdesivir, which showed GS-441524, as a metabolite, has improved plasma stability as compared to that of remdesivir. We next proceeded to study the in vivo anti-SARS-CoV-2 efficacy of GS-441524. Although mice are the convenient animal for in vivo assessing the anti-COVID-19 activity of drugs, they are resistant to SARS-CoV-2. SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) to enter cells, but mouse ACE2 does not sensitize cells for infection. 28 We established the transduction mice with adenovirus associated virus (AAV) vector expressing hACE2 in Biosafety Level 3 (BSL-3) laboratory. AAV-hACE2 mice support SARS-CoV-2 replication and exhibit pulmonary inflammation and lung injury. 29, 30 In this study, 1×10 5 plaque forming unit (PFU) SARS-CoV-2 were intranasally inoculated to AAV-hACE2 mice and mice body weights were monitored over a The COVID-19 pandemic associated with high contagiousness, morbidity and mortality, emphasizing the imperative need for anti-viral agents. RdRP inhibitor remdesivir was the only anti-COVID-19 drug that accelerated the recovery in adults who were hospitalized with COVID-19 in a large randomized clinical trial, but controversy still remains. 15, 33 Due to the synthesis complexity, expensiveness of Active Pharmaceutical Ingredient (API) and obligatory intravenous administration, the application and accessibility of remdesivir will be limited. The parent nucleotide GS-441524 is superior to remdesivir in several aspects. First of all, GS-441524 is a structurally simple molecule and is easier to synthesize than remdesivir. Secondly, pre-clinical and clinical PK study of remdesivir revealed that GS-441524 was the predominant and persistent metabolite in circulation. Although other metabolites, such as alanine metabolite GS-704277, triphosphorylated GS-443902 was also detected in the plasma, these compounds are highly negative charged and have poor cell membrane permeability, which will then inevitably convert to GS-441524 or excrete from the body (Figure 1) . The relative mRNA expression level of each gene was normalized to GAPDH housekeeping gene expression in the untreated condition, and fold induction was calculated by the ΔΔCT method relative to those in untreated samples. To Adeno-associated virus 9 encoding hACE2 were purchased from Packgene (AAV-hACE2). The modified intratracheal aerosolization was used to intratracheally delivery AAV vector (5x10 11 GC) to lung tissue of 4-week-old BALB/c mice. 35 The viral titration in lung tissue was determined using focus FFA assay as previously described. 30 Mice lung dissections were fixed in zinc formalin and embedded with paraffin. Tissue sections (~4 μm) were stained with hematoxylin and eosin. Clinical features of patients infected with 2019 novel coronavirus in Wuhan Pathogenesis of COVID-19 from a cell biology perspective. The European respiratory journal A pneumonia outbreak associated with a new coronavirus of probable bat origin Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus-Infected Pneumonia A Trial of Lopinavir-Ritonavir in Adults Hospitalized with Severe Covid-19 Effect of High vs Low Doses of Chloroquine Diphosphate as Adjunctive Therapy for Patients Hospitalized With Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection A Randomized Clinical Trial An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice Immunomodulatory Drugs in the Management of SARS-CoV-2 COVID-19 treatment: close to a cure? A rapid review of pharmacotherapies for the novel coronavirus (SARS-CoV-2) Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro Remdesivir for 5 or 10 Days in Patients with Severe Covid-19 Adenine C-Nucleoside (GS-5734) for the Treatment of Ebola and Emerging Viruses Broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent RNA dependent RNA polymerase Structural Basis for RNA Replication by the SARS-CoV-2 Polymerase Repurposed antiviral drugs for COVID-19 -interim WHO SOLIDARITY trial results. medRxiv Synthesis and antiviral activity of a series of 1'-substituted 4-aza-7,9-dideazaadenosine C-nucleosides Clinical benefit of remdesivir in rhesus macaques infected with SARS-CoV-2 Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys Pharmacokinetics of remdesivir and GS-441524 in two critically ill patients who recovered from COVID-19 Safety, Tolerability, and Pharmacokinetics of Remdesivir, An Antiviral for Treatment of COVID-19, in Healthy Subjects GS-5734 and its parent nucleoside analog inhibit Filo The mechanism of action of beta-D-2'-deoxy-2'-fluoro-2'-Cmethylcytidine involves a second metabolic pathway leading to beta-D-2'-deoxy-2'-fluoro-2'-C-methyluridine 5'-triphosphate, a potent inhibitor of the hepatitis C virus RNAdependent RNA polymerase The nucleoside analog GS-441524 strongly inhibits feline infectious peritonitis (FIP) virus in tissue culture and experimental cat infection studies Antiviral treatment using the adenosine nucleoside analogue GS-441524 in cats with clinically diagnosed neurological feline infectious peritonitis Remdesivir Inhibits SARS-CoV-2 in Human Lung Cells and Chimeric SARS-CoV Expressing the SARS-CoV-2 RNA Polymerase in Mice Advantages of the Parent Nucleoside GS-441524 over Remdesivir for Covid-19 Treatment Development and validation of a UHPLC-MS/MS method for quantification of the prodrug remdesivir and its metabolite GS-441524: a tool for clinical pharmacokinetics of SARS-CoV-2/COVID-19 and Ebola virus disease A pneumonia outbreak associated with a new coronavirus of probable bat origin Mouse model of SARS-CoV-2 reveals inflammatory role of type I interferon signaling Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment Of Mice and Men: The Coronavirus MHV and Mouse Models as a Translational Approach to Understand SARS-CoV-2 Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease Remdesivir for the Treatment of Covid-19 -Final Report The ORF8 Protein of SARS-CoV-2 Mediates Immune Evasion through Potently Downregulating MHC-I Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment Intratracheal Administration of siRNA Triggers mRNA Silencing in the Lung to Modulate T Cell Immune Response and Lung Inflammation The project was supported by Shenzhen Science and Technology Innovation Committee (ZDSYS20190902093215877), Shenzhen Bay Laboratory (SZBL2019062801006) andTechnology and National Natural Science Foundation of China (grant #32041002). We thank Chuwen Lin from School of Medicine, Sun Yat-Sen University for the help in lung pathology analysis. X.Z. and D.G. initiated the project; X.Z., D.G., Y.Z. and Y.L. designed the project. Y.L. and L.C. wrote the manuscript. Y.L., G.L., and P.W. prepared the compounds; L.C., F.X. and Y.J. performed the antiviral activity experiments; G.L., F.C., J.S., Y-Z.L. and J.Z. carried out the MHV mice experiments; Y-F.L. performed the PK study. Y.L and L.C. analyzed the data. Y.Z., D.G and X.Z.supervised and supported the project. All authors reviewed and approved the manuscript. Supplementary information and chemical compound information are available in the online version of the paper. Reprints and permissions information is available online at www.nature.com/reprints. Correspondence and requests for materials should be addressed to Y.Z., D.G. or to X. Z. The authors declare no competing financial interests.