4’-fluorouridine as a potential COVID-19 oral drug?: a review [version 2; peer review: 1 approved with reservations, 1 not approved]

The available antiviral drugs against coronavirus disease 2019 (COVID-19) are limited. Oral drugs that can be prescribed to non-hospitalized patients are required. The 4′-fluoruridine, a nucleoside analog similar to remdesivir, is one of the promising candidates for COVID-19 oral therapy due to its ability to stall viral RdRp. Available data suggested that 4'-fluorouridine has antiviral activity against the respiratory syncytial virus, hepatitis C virus, lymphocytic choriomeningitis virus,

sity
Al Baha4781Saudi Arabia

Steven De Jonghe 0000-0002-3872-6558
Leuven, BelgiumK U Leuven 
M Slobodan 
School of Nursing
Wollongong University
2522WollongongNSWAustralia

Department of Microbiology, Immunology and Transplantation
Laboratory of Virology and Chemotherapy
Rega Institute for Medical Research
KU Leuven
LeuvenBelgium

Janković 

Department of Biology
Faculty of Mathematics and Natural Sciences
Sam Ratulangi University
Manado


North Sulawesi
95115Indonesia


Division of Pathology
ICAR-Indian Veterinary Research Institute
Izatnagar, Bareilly


Uttar Pradesh
243122India


Department of Community Medicine and Health Care for Pilgrims
Faculty of Medicine
Umm Al-Qura University
21955MakkahSaudi Arabia


University of Kragujevac
Kragujeva

erbia

4'-flu
rouridine as a potential COVID-19 oral drug?: a review [version 2; peer review: 1 approved with reservations, 1 not approved] Previously titled: 4'-fluorouridine and its derivatives as potential COVID-19 oral drugs: a review
22 MAY 2023549E67BA1FDE8FD387CD151D4EFC67DD10.12688/f1000research.109701.14'-fluorouridine, SARS-CoV-2, antiviral drug, COVID-19, RNAdependent RNA polymerase Investigation, Writing -Original Draft PreparationTallei TE: Conceptualization, Formal Analysis, Validation, Visualization, Writing -Original Draft Preparation, Writing -Review & EditingFatimawali F: Data Curation, Writing -Original Draft Preparation, Writing -Review & EditingCelik I: Data Curation, Writing -Review & EditingAlhumaydhi FA: Formal Analysis, Validation, Visualization, Writing -Review & EditingEmran TB: Formal Analysis, Validation, Visualization, Writing -Review & EditingDhama K: Validation, Writing -Review & Edi ingRabaan AA: Data Curation, Funding Acquisition, Investigation, Methodology, Writing -Review & EditingGarout MA: Funding Acquisition, Investigation, Methodology, Software, Writing -Review & EditingHalwani MA: Data Curation, Funding Acquisition, Writing -Review & EditingAl Mutair A: Data Curation, Funding Acquisition, Software, Writing -Review & EditingAlhumaid S: Funding Acquisition, Investigation, Methodology, Software, Writing -Review & EditingHarapan H: Supervision, Writing -Review & Editing
The available antiviral drugs against coronavirus disease 2019 (COVID-19) are limited.Oral drugs that can be prescribed to non-hospitalized patients are required.The 4′-fluoruridine, a nucleoside analog similar to remdesivir, is one of the promising candidates for COVID-19 oral therapy due to its ability to stall viral RdRp.Available data suggested that 4'-fluorouridine has antiviral activity against the respiratory syncytial virus, hepatitis C virus, lymphocytic choriomeningitis virus,Open Peer ReviewApproval Status1 2 version 2(revision)

Introduction

Coronavirus disease 2019 (COVID- 19) is a respiratory disease that emerged in December 2019.The World Health Organization (WHO) declared COVID-19 as a pandemic, that is still ongoing, caused by the novel, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 1SARS-CoV-2 is enclosed, positive-single-stranded RNA virus that could infect humans, as well as a variety of other animals. 2 SARS-CoV-2 enters into host cells via the spike (S) glycoprotein, which has a receptor-binding domain (RBD) that mediates direct contact with a cellular receptor, angiotensin-converting enzyme 2 (ACE2). 2 The S glycoprotein, which consists of two subunits, S1 and S2, is an inactive precursor that must be cleaved to mediate membrane fusion. 3Accordingly, the S1/S2 polybasic cleavage site is proteolytically cleaved by cellular cathepsin L and transmembrane protease serine 2 (TMPRSS2) 4,5 to facilitate viral entry.

COVID-19 was initially detected as a zoonotically transmitted disease in Wuhan, China and has since spread throughout the world and caused high burdens in society. 6,7The disease has a broad spectrum of symptoms, ranging from mild and common symptoms,

ch as fever and cough,
to the more severe presentations of pneumonia and even organ failure. 8ased on WHO COVID-19 Dashboard, more than 757 million cases of COVID-19 have been reported as of February 20, 2023, with over 6.8 million deaths. 9The causative agent, SARS-CoV-2, is easily transmitted through the air via respiratory droplets, direct contact with contaminated surfaces, and fecal-oral transmission, 10 making it spread rapidly with a reproduction number (R0) of 2.69 and a case fatality rate (CFR) of 2.67. 11Along with its rapid spread, it has a high mutation rate, 12,13 making the fight against this virus a race between drugs and vaccines and the virus itself. 14e repurposing of existing drugs is currently being extensively explored, as these drugs have the potential to significantly accelerate the prevention of viral spread and transmission, as well as the development of new COVID-19 therapies. 15,16Some of antiviral have been tested against COVID-19 with some strength and limitation. 17For example, molnupiravir, a potent ribonucleoside analog that inhibits viral replication, has been shown to reduce the risk of hospitalization or death in COVID-19 patients 18 by inserting itself into the viral genome during its synthesis by RNAdependent RNA polymerase (RdRp). 19However, there are some concerns regarding its mechanism of action and its possible detrimental effects on human cells. 20Amid these concerns, there is rising hope in the emergence of 4 0 -fluorouridine (4 0 -FIU), whose mechanism of action involves the inhibition of R Rp activity.4 0 -FIU was reported to inhibit the replication of the virus in cells without impairing cell metabolism. 21Additionally, it inhibited SARS-CoV-2 and the agents of several other pandemic-potential viral infections, such as avian influenza. 21In this review, the antiviral activity of 4 0 -FlU and its The promising benefits of 4 0 -FlU against COVID-19 are highlighted in this review.


The SARS-CoV-2 genome

The genome of SARS-CoV-2 comprises approximately 30,000 nucleotide-long single-stranded positive-sense RNA, similar to the genomes of other human coronaviruses. 22,235][26] The viral genome encodes a 7096-residue polyprotein with a variety of structural and non-structural proteins (NSPs).The large portion of the genomic content is composed of sequences encoding two non-structural proteins, followed by ORF1a, and ORF1 b, and subsequently the sequences encoding the structural proteins.ORFs1a and 1b encode the polyproteins pp1a and pp1ab, respectively, with the polyprotein pp1ab being encoded by the ribosomal frameshift mechanism of gene 1b.These polyproteins are then digested by virally encoded proteinases, which results in the production of 16 proteins that are highly conserved across all coronaviruses of the same family. 27The viral genome has a guanine-cytosine (GC) content of 38% 28 and 11 protein-coding genes, with 12 expressed proteins.The hemagglutinin-esterase gene, which is found in several beta-coronaviruses, is absent in SARS-CoV-2. 230][31][32] The ORFs are organized into replicase and protease genes, as well as genes encoding the REVISED Amendments from Version 1 In the revised version, we have corrected and revised our article to ensure that all data are now correct based on the updated information.We have updated our manuscript using the current available data related to the COVID-19, and the drugs.We have revised our article title into: 4'-fluorouridine as a potential COVID-19 oral drug?: a review.We also have revised our abstract to provide better fact with the latest information.We have updated the information of molnupiravir stating that it has been approved by FDA under emergency use authorization (EUA).The stereochemistry has been added under the Figure 2. We have provided the pharmacokinetics and pharmacokinetic parameters in to a table from animal study.We also have provided the toxicity of 4 0 -fluorouridine from animal study under the evidence from in vivo study heading.

Any further responses from the reviewers can be found at the end of the article important S, E, M, and N proteins, which are present in a regular 5 0 -3 0 order and are important drug/vaccine targets.The products of these genes facilitate viral entry, fusion, and survival in host cells. 32,33The SARS-CoV-2 genome, similar to the genomes of other human coronaviruses such as SARS-CoV and MERS-CoV, has a m7G-cap structure, with m7GpppA1, at the 5 0 end, and an approximately 30-60 nucleotide long poly A tail at the 3 0 end. 34RS-CoV-2 is a member of genus Betacoronavirus (β-CoV) in the family Coronaviridae of the order Nidovirales. 35,36ARS-CoV-2 is relatively more infectious than SARS-CoV and MERS-CoV.Other mammalian species may have served as intermediate or amplifying hosts, with eventual ecological isolation resulting in the acquisition of some or all of the mutations required for efficient human transmission.37 Since its discovery, the SARS-CoV-2 genome has shown genomic variability.38 In a recent study, 48,635 complete genomes of SARS-CoV-2 were compared with the reference Wuhan genome NC_045512.2and were evaluated to exhibit an average of 7.23 mutations per sample.39 The study demonstrated single nucleotide transitions to be the most common type of mutations prevalent in SARS-CoV-2 isolates globally.39 Another study also found that 5,775 distinct genomic variants of SARS-CoV-2 emerged compared to Wuhan genome NC_045512.2with multiple mutations including in-frame deletions, non-coding deletions and insertions, and frameshift deletions and insertions. 40e host RNA editing machinery, in which adenosine deaminase acts on RNA (ADAR deaminase (APOBEC) targets dsRNA for adenosine deamination to inosines (A-to-I) and apolipoprotein B mRNA editing enzyme catalytic subunit) deaminates cytosines to uracils (C-to-U) on ssRNA or ssDNA, and may play a role in the induction of the SARS-CoV-2 genome mutations and modifications found during viral infection.22 While mutations in the viral genome are natural, during this pandemic, mutations in the SARS-CoV-2 genome have been a scourge. Ths is because of the numerous mutations that have enabled this virus to repeatedly evade antibody neutralization.41,42 This is especially true when there are multiple mutations in the gene that encodes the S glycoprotein.43 Special attention should be given to mutations in genes encoding non-structural proteins, as they may impair the effectiveness of antiviral drugs.Even though some variations are rare in specific regions, the C14408T and A23403G mutations on the Nsp12 and S proteins, respectively, have been found to be the most common worldwide and both induce missense mutations.44 Accordingly, emerging variants of SARS-CoV-2 are very influential and pose a significant threat.Even if individuals have been exposed to SARS-CoV-2 previously, they can still become infected with other variants of SARS-CoV-2 and contract COVID-19, indicating that prior exposure to the virus does not guarantee complete immunity against the disease.45


RdRp of SARS-CoV-2 as a potential drug target

The structural and non-structural proteins of SARS-CoV-2 are all potential drug/vaccine targets. 26The main protease (M pro ), a key enzyme for the viral polyprotein proteolytic process, and RdRp, a key viral enzyme that is critical in mediating viral replication and transcription, are two attractive drug targets for SARS-CoV-2. 27,46,47Rp (Figure 1) catalyzes the replication of RNA from an RNA template.It catalyzes the production of complementary RNA strands for a given RNA template.It differs from the traditional DNA-dependent RNA polymerases, which are found in all species and catalyze the transcription of RNA from a DNA template. 48RdRp is encoded in the genomes of the majority of RNA viruses that do not have a "DNA stage," including SARS-CoV-2. 49,50Structural modeling of RdRps from positive-sense RNA viruses (HCV and SARS-CoV-2) and negative-sense RNA viruses (i.e., influenza) have confirmed the differences between the two types of RdRps. 51Rps are not required for the survival of eukaryotic cells.As a result, they have the potential to be used as therapeutic targets against viral infections.The replication of RNA from a template strand is prevented when RdRp is inhibited the RdRp of SARS-CoV-2 differs from the RdRp of SARS-CoV and MERS-COV 27 the RdRps of all three coronaviruses are highly conserved, suggesting that RdRp is a promising broad-spectrum antiviral target for coronaviruses. 52,53me antivirals against SARS-CoV-2 such as remdesivir and molnupiravir targeted RdRp. 54,55Remdesivir has been demonstrated to shorten recovery time in COVID-19 patients 56,57 and is currently licensed for clinical usage. 58RdRp can use GS-441524 triphosphate (an active metabolite of remdesivir) as a substrate, thus causing premature chain termination and inhibition of viral replication.Remdesivir is a nucleotide analog that prevents further elongation of the RNA polynucleotide by covalently binding to and interfering with the termination of the nascent RNA either early or late, or by inhibiting further elongation of the RNA polynucleotide.As a result of the premature termination, nonfunctional RNA is produced, which is then degraded via the normal cellular functions of the body. 57,59Molnupiravir, another RdRp, has been reported to reduce the risk of hospitalization or mortality in at-risk patients by 50%. 18Molnupiravir, another RdRp inhibitor, is a highly effective ribonucleoside analog that prevents viral replication. 554 0 -FIU is another nucleotide analog that has been tested to have antiviral activity against SARS-CoV-2 by causing the stalling of RdRp. 21This compound has been shown to have antiviral activity against several viruses. 21,60,614 0 -fluorouridine (4 0 -FIU) in potential drug against SARS-CoV-2 Uridine, a ribonucleoside that forms nucleic acids, is a glycosylated pyrimidine analog of uracil that is attached by a -N1glycosidic bond to a ribofuranose moiety. 62Uridine replaces thymidine in the RNA.Uridine analogs are often developed as antiviral for RNA viruses.The general hypothesis is that these uridine analogs interfere with or inhibit the replication of RNA viruses in host cells.One such example is the use of molnupiravir, a nucleoside analog, that mimics the cytidine or uridine. 63The possibility of 4 0 -fluorouridine being a new oral drug for COVID-19 with a different mechanism of action than molnupiravir is being inve tigated.It is a uridine nucleoside analog that contains uridine and fluorine.The 4 0 -FIU structure and its similarity to uridine described in Figure 2 were created using ChemDraw. 64The antiviral activity of 4 0 -FlU has led to the suggestion that this is due to the small atomic radius and strong stereo-electronic effect of fluorine.This configuration may have an effect on the conformational flexibility of the backbone, thereby increasing metabolic stability. 21,65wever, several studies have stated that 4 0 -FIU is quite unstable 66,67 compared to its derivatives. 674 0 -FIU derivatives have been proven to have high enough stability for biological studies. 67Examples of 4 0 -FIU derivatives are 4 0 -fluorouridine-5-monophosphate and 4 0 -fluorouridine 5 0 -O-triphospate. 61,66Two patents, WO2019173602A1 and WO2021137913A2, showed that 4 0 -FIU, 4 0 -fluorouridine 5 0 -triphosphate (EIDD-02991) and 4 0 -fluorouridine-5 0isobutyl ester (EIDD-02947) (Figure 2) have antiviral activity against positive-and negative-sense RNA viruses by blocking their RdRp.


Evidence from in vitro studies

A study revealed that 4 0 -FIU was active at submicromolar concentrations against paramyxovirus, rhabdovirus, measles virus (MeV), human parainfluenza virus type 3 (HPIV3), Sendai virus (SeV), vesicular stomatitis virus (VSV), and rabies virus (RabV). 21The compound rapidly accumulates intracellularly in human airway epithelial (HAE) cells, resulting in 3.42 nmol/million cells after 1 h of exposure.Additionally, the metabolic activities of tested cell lines such as Hep-2, Madin-Darby canine kidney (MDCK) cells, hamster kidney fibroblasts (BHK-T7), and human bronchial epithelium


cells (BEAS-2B

) when exposed with 500 μM 4 0 -FIU remains unaffected, implying that the antiviral effect is due to cytotoxicity. 21cording to patent WO2019173602A1, 4 0 -FIU has a half maximal effective concentration (EC 50 ) of 1.86 μM in the Vero E6 cell line, cytotoxicity with a CC 50 of 380 μM, and stability in human plasma against Junin virus.CC 50 is the concentration of a drug required to achieve a 50% reduction in cell viability.A study found revealed that 4 0 -FIU has effective antiviral activity against strains of Arenaviridae and inhibits lymphocytic choriomeningitis virus (LCMV) with an EC 50 of 7.22 μM in the Vero E6 cell line. 60This compound had a significant action against recombinant respiratory syncytial viruses (RSV) A2-line19F and clinical RSV isolates, with EC 50 values ranging from 0.61 to 1.2 μM. 68In cellbased minireplicon systems, 4 0 -FIU inhibited RSV RdRp complex activity during the initial mechanistic characterization.In a human tissue model, HAE cells, including ciliate and mucus-producing cells, with 4 0 -FIU added to the basolateral chamber after RSV infection, showed that this compound can reduce apical viral shedding with an EC 50 of 55 nM. 68onfocal microscopy confirmed the efficacy of 4 0 -FIU at the basolateral position after the pseudostratified organization of the epithelium with tight junctions and the observation of a rare positive strain of RSV antigen. 68In addition to RSV, 4 0 -FIU can also reduce paramyxovirus RdRp complex activity in cell-based mini eplicon systems, and in a Nipah virus (NIV) minireplicon reporter test, 4 0 -FIU effectively suppressed the RdRp activity of NIV. 21veral studies on 4 0 -FIU derivatives have also revealed that they possess antiviral properties.The 4 0 -FIU 5 0 -O-triphosphate was shown to effectively inhibit the key hepatitis C virus (HCV) enzymes, nucleoside triphosphate (NTP)-dependent RNA polymerase NS5B, and NTP-dependent NTPase/helicase NS3.This compound successfully inhibited NTP-dependent RNA polymerase with an IC 50 value of 2 μM.For the NTP-dependent NTPase/helicase NS3, 4 0 -FIU 5 0 -O-triphosphate was found to be a substrate for the NTPase reaction and did not inhibit helicase activity, but this reaction was found to be slightly weaker than adenosine triphosphate (ATP). 61 in vitro study on 4 0 -FIU-5 0 -triphosphate showed that RSV RdRp recognized and incorporated 4 0 -FIU-5 0 -triphosphate in place of uridine triphosphate (UTP) during the synthesis of the genome.Full addition of cytidine triphosphate (CTP) and 4 0 -FIU caused limited elongation and not the full-length expected product, indicating that 4 0 -FIU delayed polymerase stalling. 21Another study showed that 4 0 -FIU derivatives potently inhibited HCV infection.The IC 50 of the 4 0 -FIU derivative in the polymerase NS5B assay was as low as 27 nM.Human DNA and RNA polymerase also showed little inhibition by the 4 0 -FIU derivative. 69wever, while several studies have shown that 4 0 -FIU derivatives have antiviral activity, they are different from 4 0 -FIU analogs.A study conducted to determine the antiviral activity of 4 0 -FIU analogs for the varicella-zoster virus (VZV) and human cytomegalovirus (HMCV) showed that there was no antiviral activity with an EC 50 value >100 μM. 70Another study showed that SARS-CoV-2 was particularly sensitive to 4 0 -FIU, with EC 50 values ranging from 0.2 to 0.6 μM. 21dditionally, this study demonstrated that when 4 0 -FIU 5 0 -triphosphate was incorporated in place of UTP in SARS-CoV-2 RdRp, the enzyme did not immediately stall, but it stalled after multiple incorporations and was prominent when 4 0 -FIU 5 0 -triphosphate was incorporated.This is a distinctive mode of action in comparison to molnupiravir, which induces lethal viral mutagenesis once it is incorporated into the viral genomic RNA. 21idence from in vivo study Fluorouridine has been shown to be effective at inhibiting biological activity, particularly cell division, in Tetrahymena pyriformis. 71After ingestion, compound 4 0 -FIU is further phosphorylated in the cell to form the active metabolite 4 0 -FIU (Figure 3A).A previous study 21 described the pharmacokinetics of 4 0 -FlU in ferrets, which were used as a model to test the efficacy of the compound against SARS-CoV-2.The study determined the pharmacokinetic profiles of 4 0 -FlU after single oral dose of 15 or 50 mg/kg in ferrets.The results showed that the peak plasma concentrations (C max ) of 4 0 -FlU were 34.8 and 63.3 μM, respectively, and the overall exposure was 154 AE 27.6 and 413.1 AE 78.1 hoursÂnmol/ml, respectively.The pharmacokinetics of 4 0 -FlU showed good oral dose-proportionality. Based on these pharmacokinetic parameters, the study selected a once-daily oral dose of 20 mg/kg for efficacy tests against SARS-CoV-2 in ferrets.The study found that therapeutic treatment with 4 0 -FlU initiated 12 hours after infection reduced virus burden in nasal lavages by approximately three orders of magnitude for the SARS-CoV-2 isolates tested.All three variants of SARS-CoV-2 (alpha, gamma, and delta) were highly sensitive to 4 0 -FlU, remaining below the level of detection 36 to 48 hours after the onset of oral treatment.The shedding of infectious particles ceased completely in all animals after 2.5 days of treatment (3 days postinfection).Summary of pharmacokinetic parameters of 4 0 -FIU in animal study is presented in Table 1.

While the 4 0 -FlU demonstrates a potent dose-dependent activity against several viruses including SARS-CoV-2, the toxicity of the compound cannot be ignored.A study reported that global metabolic activity of established human and animal cell lines remained unaltered after exposure to 4 0 -FlU, indicating that the antiviral effect is not due to cytotoxicity. 21However, when tested on disease-relevant primary human airway epithelial cells, 4 0 -FlU showed low cytotoxicity levels with a CC 50 of 169 μM. 21Therefore, further research is needed to determine its safety and efficacy before considering it for clinical use.Patent WO2019173602A1 demonstrated that 4 0 -FIU was extremely well tolerated in an in vivo study in mice.The compound was shown to reduce RSV load in the lungs, with the most effective dose being 5 mg/kg body weight.After day 5 post-infection, animals treated with 4 0 -FIU demonstrated a significant decrease in bioluminescence intensity in the lungs. 21Currently, there are no clinical trials of 4 0 -FIU available in international repositories including Clinicaltrials.gov,EU Clinical Trials Registry, WHO-International Clinical Trials Registry Platform and others.4 0 fluorouridine (4 0 -FIU) antiviral activity compared to other antiviral drugs against SARS-CoV-2 Drug repositioning is the only feasible option for addressing the COVID-19 global challenge in the short term.2][73][74][75] Bemnifosbuvir is a is an orally viral replication inhibitor and some of the clinical trials have been conducted or are ongoing with no available results.Favipiravir (T-705 or 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) is a guanine analog that was synthesized by modifying a pyrazine analog.It is an antiviral agent that selectively and potently inhibits the RdRp of RNA viruses. 76The compound is phosphorylated intracellularly to produce the active form, favipiravir-RTP (favipiravir ribo-furanosyl-5-tr phosphate) (Figure 3B), which is recognized as a substrate by RdRp and inhibits the activity of RNA polymerase. 77The mechanism of action of favipiravir involves lethal mutagenesis. 78Favipiravir demonstrated protection against RNA virus influenza in a mouse model and Crimean-Congo hemorrhagic fever virus (CCHFV) in macaques. 79However, several cell lines showed reduced or missing selectivity against SARS-CoV-2. 80It differs from 4 0

FIU, which shows p
omising results both in vitro and in vivo.

cular by employing cells that have different metab
lic profiles.Additionally, favipiravir has been linked to an increased risk of teratogenicity an embryotoxicity 81 and a poor pharmacokinetic (PK) profile. 82mdesivir is a promising antiviral agent for tackling SARS-CoV-2. 56Remdesivir is an adenosine analog and a monophos horamidate prodrug.Its activity involves the incorporation of its active form (remdesivir triphosphate, Figure 3C) into nascent viral RNA, halting RNA synthesis. 59In vitro, a study revealed that remdesivir successfully suppressed SARS-CoV-2 83 and has now been granted approval. 58Similar to remdesivir, 4 0 -FIU is also a nucleoside analog and the mechanism of action of 4 0 -FIU as potential oral drug for COVID-19 is by inhibiting RdRp to prevent viral RNA replication 21 (Figure 4).The advantage is that 4 0 -F