SARS-CoV-2 ANTIVIRAL ACTIVITY OF ZOTATIFIN, A HOST-TARGETING eIF4A INHIBITOR

Background: Zotatifin (eFT226) is a potent and selective inhibitor of eukaryotic initiation factor 4A (eIF4A), a host RNA helicase required for SARS-CoV-2 replication. Zotatifin selectively inhibits translation of ribonucleic acids (RNAs) containing specific short polypurine motifs in their 5-prime (5') regions. Two such highly conserved motifs are found in the SARS-CoV-2 genome. Zotatifin is currently being evaluated in a Phase 1b dose escalation study in 36 patients with mild to moderate COVID disease. In this in vitro study, we evaluated the selectivity of zotatifin's inhibition of SARS-CoV-2 translation, the antiviral activity of zotatifin alone against different human coronaviruses and the antiviral activity of zotatifin in combination with other antivirals against SARSCoV-2. Method(s): The selectivity of zotatifin for viral translation was evaluated in a cell-based reporter assay wherein luciferase translation was driven by 5'-sequences from SARS-CoV-2 or tubulin, a housekeeping gene. The antiviral activity of zotatifin was evaluated against SARS-CoV-1, SARS-CoV-2 variants (Wash/1/2020 (ancestral), delta, omicron BA.2), MERS-CoV and HCoV-299E in primary or established cell lines using cytopathic effect or infectious virus as endpoints. The antiviral activity of zotatifin in combination with remdesivir, N-hydroxycytidine (NHC;active nucleoside analogue metabolite of molnupiravir), nirmatrelvir, baricitinib or sotrovimab was evaluated against SARS-CoV-2 and analyzed by the method of Pritchard and Shipman. Result(s): Zotatifin inhibited the translation of the SARS-CoV-2 luciferase reporter construct with a mean IC50 of 3 nM and was ~14-fold less potent in inhibiting the tubulin reporter construct. Zotatifin potently inhibited the replication of all human coronaviruses tested with 50% effective concentrations (EC50s) ranging from 0.016 to 37.3 nM. The 50% cytotoxic concentration (CC50) value for zotatifin was 250 to >100,000 nM, yielding selectivity indices of 7 to >6250. Zotatifin was ~20 to >100-fold more potent than remdesivir, nirmatrelvir or NHC (figure) and demonstrated additive interactions when combined with remdesivir, NHC, nirmatrelvir, baricitinib or sotrovimab in vitro. Conclusion(s): The potent broad-spectrum activity of zotatifin against a variety of human coronaviruses and additive activity when combined with different anti-SARS-CoV-2 antivirals highlight the advantages of eIF4A as a target and warrant further evaluation in human clinical trials.

SARS-CoV-2 REPLICON SYSTEM FOR THE PHENOTYPIC EVALUATION OF NSP GENE SUBSTITUTIONS

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the current global pandemic of the COVID-19, which has persisted partly through the emergence of new variants. A non-infectious, convenient, and reproducible in vitro system is needed to assess drug susceptibility of new variants of concern and potential drug resistance mutations. Method(s): The SARS-CoV-2 replicon protocol was adapted and optimized based on {Zhang 2021}. The replicon RNA was produced by in vitro transcription of full-length replicon DNA assembled by ligation of plasmid fragments encoding for the SARS-CoV-2 non-structural proteins (Nsps), nucleoprotein and gaussia luciferase reporter protein. Wild-type and mutant replicon RNAs were transfected into Huh7-1CN cells by electroporation and treated with remdesivir (RDV). To determine EC50 values, luciferase activity was determined at 48 hours post transfection. A recombinant SARS-CoV-2 virus rescue system {Xie 2020} was used to generate matching Nsp mutants for comparison with the replicon system. Result(s): The selected substitutions reflective of Omicron BA.5 sub-lineage BF.7 variant: the triple mutants (Nsp12 (P323L) +Nsp13 (R392C) + Nsp14 (I42V), and a single Nsp12 L247F mutant as well as several specific Nsp12 mutations identified by in vitro resistance selection with RDV or RDV parent nucleoside analog GS-441524 were cloned into the replicon and tested for susceptibility to RDV. RDV inhibited the SARS-CoV-2 wild-type replicon with a mean EC50 value of 14.7 +/- 3.5 nM (N=9). The Nsp12 P323L substitution, a common polymorphism in all major variants of concern including Omicron, was fully susceptible to RDV with a 0.6-fold change in EC50 from the wild-type. The Omicron BF.7 triple mutants and L247F were also fully susceptible to RDV with 0.5- and 0.4-fold changes, respectively. Nsp12 substitutions F480L, V557L, V792I, S759A+V792I, and C799F resulting from in vitro resistance selections with RDV showed minimal to moderate levels of reduced susceptibility to RDV (1.8 to 18.3-fold change) (Table 1). The RDV EC50 fold changes correlated between the noninfectious replicon and recombinant infection virus system (Table 1). Conclusion(s): The replicon system is a convenient and reproducible model to test the susceptibility of SARS-CoV-2 mutant variants to RDV and potentially other antivirals. The common Nsp12 polymorphisms in all variants including the highly transmissible Omicron variant were fully susceptible to RDV.

Liver and COVID-19 - A Review and Clinical Approach

Liver enzyme abnormalities occur frequently in patients diagnosed with Coronavirus disease 2019 (COVID-19). It has been suggested that patients with severe acute liver injury are more likely to be admitted to intensive care, require intubation or renal replacement therapy and their mortality rate is higher than patients without severe acute liver injury. This review article explores the possible aetiologies of liver dysfunction seen in patients with COVID-19 and also the effect of COVID-19 on patients with pre-existing liver disease. Finally, we suggest clinical approaches to treating a patient with liver enzyme disturbance and COVID-19 and also caring for patients who require liver transplantation in the COVID-19 era.Copyright © 2022 Bentham Science Publishers.

Plant-Derived Natural Non-Nucleoside Analog Inhibitors (NNAIs) against RNA-Dependent RNA Polymerase Complex (nsp7/nsp8/nsp12) of SARS-CoV-2.

The emergence of fast-spreading SARS-CoV-2 mutants has sparked a new phase of COVID-19 pandemic. There is a dire necessity for antivirals targeting highly conserved genomic domains on SARS-CoV-2 that are less prone to mutation. The nsp12, also known as the RNA-dependent RNA-polymerase (RdRp), the core component of 'SARS-CoV-2 replication-transcription complex', is a potential well-conserved druggable antiviral target. Several FDA-approved RdRp 'nucleotide analog inhibitors (NAIs)' such as remdesivir, have been repurposed to treat COVID-19 infections. The NAIs target RdRp protein translation and competitively block the nucleotide insertion into the RNA chain, resulting in the inhibition of viral replication. However, the replication proofreading function of nsp14-ExoN could provide resistance to SARS-CoV-2 against many NAIs. Conversely, the 'non-nucleoside analog inhibitors (NNAIs)' bind to allosteric sites on viral polymerase surface, change the redox state; thereby, exert antiviral activity by altering interactions between the enzyme substrate and active core catalytic site of the RdRp. NNAIs neither require metabolic activation (unlike NAIs) nor compete with intracellular pool of nucleotide triphosphates (NTPs) for anti-RdRp activity. The NNAIs from phytonutrient origin are potential antiviral candidates compared to their synthetic counterparts. Several in-silico studies reported the antiviral spectrum of natural phytonutrient-NNAIs such as Suramin, Silibinin (flavonolignan), Theaflavin (tea polyphenol), Baicalein (5,6,7-trihydroxyflavone), Corilagin (gallotannin), Hesperidin (citrus bioflavonoid), Lycorine (pyrrolidine alkaloid), with superior redox characteristics (free binding energy, hydrogen-bonds, etc.) than antiviral drugs (i.e. remdesivir, favipiravir). These phytonutrient-NNAIs also exert anti-inflammatory, antioxidant, immunomodulatory and cardioprotective functions, with multifunctional therapeutic benefits in the clinical management of COVID-19.

Can plant-derived anti-HIV compounds be used in COVID-19 cases?

People living with HIV are more exposed to the adverse health effects of the worldwide COVID-19 pandemic. The pandemic's health and social repercussions may promote drug abuse and inadequate HIV management among this demographic. The coronavirus pandemic of 2019 (COVID-19) has caused unprecedented disruption worldwide in people's lives and health care. When the COVID-19 epidemic was identified, people with HIV faced significant obstacles and hurdles to achieving optimal care results. The viral spike protein (S-Protein) and the cognate host cell receptor angiotensin-converting enzyme 2 (ACE2) are both realistic and appropriate intervention targets. Calanolides A, Holy Basil, Kuwanon-L, and Patentiflorin have anti-HIV effects. Our computational biology study investigated that these compounds all had interaction binding scores related to S protein of coronavirus of -9.0 kcal /mol, -7.1 kcal /mol, -9.1 kcal /mol, and -10.3 kcal/mol/mol, respectively. A combination of plant-derived anti-HIV compounds like protease inhibitors and nucleoside analogs, which are commonly used to treat HIV infection, might be explored in clinical trials for the treatment of COVID-19.

Inhibition of Viral RNA-Dependent RNA Polymerases by Nucleoside Inhibitors: An Illustration of the Unity and Diversity of Mechanisms.

RNA-dependent RNA polymerase (RdRP) is essential for the replication and expression of RNA viral genomes. This class of viruses comprise a large number of highly pathogenic agents that infect essentially all species of plants and animals including humans. Infections often lead to epidemics and pandemics that have remained largely out of control due to the lack of specific and reliable preventive and therapeutic regimens. This unmet medical need has led to the exploration of new antiviral targets, of which RdRP is a major one, due to the fact of its obligatory need in virus growth. Recent studies have demonstrated the ability of several synthetic nucleoside analogs to serve as mimics of the corresponding natural nucleosides. These mimics cause stalling/termination of RdRP, or misincorporation, preventing virus replication or promoting large-scale lethal mutations. Several such analogs have received clinical approval and are being routinely used in therapy. In parallel, the molecular structural basis of their inhibitory interactions with RdRP is being elucidated, revealing both traditional and novel mechanisms including a delayed chain termination effect. This review offers a molecular commentary on these mechanisms along with their clinical implications based on analyses of recent results, which should facilitate the rational design of structure-based antiviral drugs.

The First Domestic Original Drug for the Treatment of COVID-19: Azvudine.

OBJECTIVE To summarize the basic information, mechanism of action, pharmacokinetics, efficacy, safety, interactions, and precautions of azvudine, to provide references for its clinical use. METHODS Literatures related to azvudine from the official website of Chinese clinical trial regi stry, clinicaltrials.gov, Pubmed, CNKI and Wanfang were systematically searched and summarized. RESULTS Azvudine is an oral small-molecule corona virus disease(COVID-19) treatment drug independently developed by China. As a nucleoside analogue targeting to viral RNA-dependent RNA polymerases (RdRp), it can inhibit RNA virus reverse transcription process and replication process. The results of phase III clinical trials showed that azvudine could significantly shorten the time of nucleic acid conversion in patients with mild to moderate corona virus disease (COVID-19). Compared with the control group, the azvudine group can significantly shorten the improvement time of pneumonia. For moderate and severe patients, azvudine treatment also showed significant therapeutic effects in the time of nucleic acid conversion, discharge, and rehabilitation. CONCLUSION The drug possesses good safety and tolerability in patients, which provide a choice for the clinical treatment of COVID-19. Copyright © 2022 Chinese Pharmaceutical Association. All rights reserved.

Improving properties of the nucleobase analogs T-705/T-1105 as potential antiviral.

In this minireview we describe our work on the improvement of the nucleobase analogs Favipiravir (T-705) und its non-fluorinated derivative T-1105 as influenza and SARS-CoV-2 active compounds. Both nucleobases were converted into nucleotides and then included in our nucleotide prodrugs technologies cycloSal-monophosphates, DiPPro-nucleoside diphosphates and TriPPPro-nucleoside triphosphates. Particularly the DiPPro-derivatives of T-1105-RDP proved to be very active against influenza viruses. T-1105-derivatives in general were found to be more antivirally active as compared to their T-705 counterpart. This may be due to the low chemical stability of all ribosylated derivatives of T-705. The ribosyltriphosphate derivative of T-1105 was studied for the potential to act as a inhibitor of the SARS-CoV-2 RdRp and was found to be an extremely potent compound causing lethal mutagenesis. The pronucleotide technologies, the chemical synthesis, the biophysical properties and the biological effects of the compounds will be addressed as well.

A Series of Adenosine Analogs as the First Efficacious Anti-SARS-CoV-2 Drugs against the B.1.1.529.4 Lineage: A Preclinical Repurposing Research Study.

Given the rapid progression of the coronavirus disease 2019 (COVID-19) pandemic, an ultrafast response was urgently required to handle this major public crisis. To contain the pandemic, investments are required to develop diagnostic tests, prophylactic vaccines, and novel therapies. Lately, nucleoside analog (NA) antivirals topped the scene as top options for the treatment of COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Meanwhile, the continuous generation of new lineages of the SARS-CoV-2 Omicron variant caused a new challenge in the persistent COVID-19 battle. Hitting the two crucial SARS-CoV-2 enzymes RNA-dependent RNA polymerase (RdRp) and 3'-to-5' exoribonuclease (ExoN) collectively together using only one single ligand is a very successful new approach to stop SARS-CoV-2 multiplication and combat COVID-19 irrespective of the SARS-CoV-2 variant type because RdRps and ExoNs are broadly conserved among all SARS-CoV-2 strains. Herein, the current comprehensive study investigated most NAs libraries, searching for the most ideal drug candidates expectedly able to perfectly act through this double tactic. Gradual computational filtration gave rise to six different promising NAs, which are riboprine, forodesine, tecadenoson, nelarabine, vidarabine, and maribavir, respectively. Further biological assessment proved for the first time, using the in vitro anti-RdRp/ExoN and anti-SARS-CoV-2 bioassays, that riboprine and forodesine, among all the six tested NAs, are able to powerfully inhibit the replication of the new virulent strains of SARS-CoV-2 with extremely minute in vitro anti-RdRp and anti-SARS-CoV-2 EC50 values of about 0.22 and 0.49 µM for riboprine and about 0.25 and 0.73 µM for forodesine, respectively, surpassing both remdesivir and the new anti-COVID-19 drug molnupiravir. The prior in silico data supported these biochemical findings, suggesting that riboprine and forodesine molecules strongly hit the key catalytic pockets of the SARS-CoV-2 (Omicron variant) RdRp's and ExoN's main active sites. Additionally, the ideal pharmacophoric features of riboprine and forodesine molecules render them typical dual-action inhibitors of SARS-CoV-2 replication and proofreading, with their relatively flexible structures open for diverse types of chemical derivatization. In Brief, the current important results of this comprehensive study revealed the interesting repurposing potentials of, mainly, the two nucleosides riboprine and forodesine to effectively shut down the polymerase/exoribonuclease-RNA nucleotides interactions of the SARS-CoV-2 Omicron variant and consequently treat COVID-19 infections, motivating us to rapidly begin the two drugs' broad preclinical/clinical anti-COVID-19 bioevaluations, hoping to combine both drugs soon in the COVID-19 treatment protocols.

The potential of remdesivir to affect function, metabolism and proliferation of cardiac and kidney cells in vitro.

Remdesivir is a prodrug of a nucleoside analog and the first antiviral therapeutic approved for coronavirus disease. Recent cardiac safety concerns and reports on remdesivir-related acute kidney injury call for a better characterization of remdesivir toxicity and understanding of the underlying mechanisms. Here, we performed an in vitro toxicity assessment of remdesivir around clinically relevant concentrations (Cmax 9 µM) using H9c2 rat cardiomyoblasts, neonatal mouse cardiomyocytes (NMCM), rat NRK-52E and human RPTEC/TERT1 cells as cell models for the assessment of cardiotoxicity or nephrotoxicity, respectively. Due to the known potential of nucleoside analogs for the induction of mitochondrial toxicity, we assessed mitochondrial function in response to remdesivir treatment, early proteomic changes in NMCM and RPTEC/TERT1 cells and the contractile function of NMCM. Short-term treatments (24 h) of H9c2 and NRK-52E cells with remdesivir adversely affected cell viability by inhibition of proliferation as determined by significantly decreased 3H-thymidine uptake. Mitochondrial toxicity of remdesivir (1.6-3.1 µM) in cardiac cells was evident by a significant decrease in oxygen consumption, a collapse of mitochondrial membrane potential and an increase in lactate secretion after a 24-48-h treatment. This was supported by early proteomic changes of respiratory chain proteins and intermediate filaments that are typically involved in mitochondrial reorganization. Functionally, an impedance-based analysis showed that remdesivir (6.25 µM) affected the beat rate and contractility of NMCM. In conclusion, we identified adverse effects of remdesivir in cardiac and kidney cells at clinically relevant concentrations, suggesting a careful evaluation of therapeutic use in patients at risk for cardiovascular or kidney disease.

Identifying Small-Molecule Inhibitors of SARS-CoV-2 RNA-Dependent RNA Polymerase by Establishing a Fluorometric Assay.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2), a member of the coronavirus family, appeared in 2019 and has caused the largest global public health and economic emergency in recent history, affecting almost all sectors of society. SARS-CoV-2 is a single-stranded positive-sense RNA virus that relies on RNA-dependent RNA polymerase (RdRp) activity in viral transcription and replication. Due to its high sequence and structural conservation in coronavirus and new SARS-CoV-2 variants, RdRp has been recognized as the key therapeutic target to design novel antiviral strategies. Nucleotide analogs (NAs), such as remdesivir, is the most promising class of RdRp inhibitors to be used in the treatment of COVID-19. However, the presence of exonucleases in SARS-CoV-2 caused a great challenge to NAs; the excision of incorporated NAs will lead to viral resistance to this group of inhibitors. Here, we expressed active RdRp protein in both a eukaryotic expression system of baculovirus-infected insect cells and a prokaryotic expression system of Escherichia coli cells. Nsp7 and nsp8 of the functional RdRp holoenzyme were generated in E. coli. An in vitro RdRp activity assay has been established with a reconstituted nsp12/nsp7/nsp8 complex and biotin-labeled self-priming RNAs, and the activity of the RdRp complex was determined by detecting binding and extension of RNAs. Moreover, to meet the needs of high-throughput drug screening, we developed a fluorometric approach based on dsRNA quantification to assess the catalytic activity of the RdRp complex, which is also suitable for testing in 96-well plates. We demonstrated that the active triphosphate form of remdesivir (RTP) and several reported non-nucleotide analog viral polymerase inhibitors blocked the RdRp in the in vitro RdRp activity assay and high-throughput screening model. This high-throughput screening model has been applied to a custom synthetic chemical and natural product library of thousands of compounds for screening SARS-CoV-2 RdRp inhibitors. Our efficient RdRp inhibitor discovery system provides a powerful platform for the screening, validation, and evaluation of novel antiviral molecules targeting SARS-CoV-2 RdRp, particularly for non-nucleotide antivirals drugs (NNAs).

Nucleoside analog GS-441524: pharmacokinetics in different species, safety, and potential effectiveness against Covid-19.

GS-441524, the parent nucleoside of remdesivir, has been proposed to be effective against Covid-19 based on in vitro studies and studies in animals. However, randomized clinical trials of the agent to treat Covid-19 have not been conducted. Here, we evaluated GS-441524 for Covid-19 treatment based on studies reporting pharmacokinetic parameters of the agent in mice, rats, cats, dogs, monkeys, and the single individual in the first-in-human trial supplemented with information about its activity against severe acute respiratory syndrome coronavirus 2 and safety. A dosing interval of 8 h was considered clinically relevant and used to calculate steady-state plasma concentrations of GS-441524. These ranged from 0.27 to 234.41 µM, reflecting differences in species, doses, and administration routes. Fifty percent maximal inhibitory concentrations of GS-441524 against severe acute respiratory syndrome coronavirus 2 ranged from 0.08 µM to above 10 µM with a median of 0.87 µM whereas concentrations required to produce 90% of the maximal inhibition of the virus varied from 0.18 µM to more than 20 µM with a median of 1.42 µM in the collected data. Most of these concentrations were substantially lower than the calculated steady-state plasma concentrations of the agent. Plasma exposures to orally administered GS-441524, calculated after normalization of doses, were larger for dogs, mice, and rats than cynomolgus monkeys and humans, probably reflecting interspecies differences in oral uptake with reported oral bioavailabilities below 8.0% in cynomolgus monkeys and values as high as 92% in dogs. Reported oral bioavailabilities in rodents ranged from 12% to 57%. Using different presumptions, we estimated human oral bioavailability of GS-441524 at 13% and 20%. Importantly, doses of GS-441524 lower than the 13 mg/kg dose used in the first-in-human trial may be effective against Covid-19. Also, GS-441524 appears to be well-tolerated. In conclusion, GS-441524 has potential for oral treatment of Covid-19.

EARLY TREATMENT WITH PREDNISOLONE AND ACYCLOVIR IN SARS-COV-2 COMPLICATED BELL'S PALSY: A CASE REPORT

Bell's palsy is an idiopathic, unilateral, acute weakness of the face in a pattern consistent with peripheral facial nerve dysfunction and may be partial or complete, occurring with equal frequency on either side of the face. The incidence is about 20 in 100,000 people a year, with about 1 in 60-lifetime risks. Bell's palsy has a peak incidence between the ages of 15 and 40 years. Viral infections are commonly associated with facial nerve pathology, which leads to peripheral facial paralysis. A potential cause of peripheral facial paralysis might be COVID-19 and neurological symptoms could be the first and only manifestation of the disease. Possible mechanisms related to nerve damage in idiopathic facial nerve paralysis include ischemia of vasa nervorum and demyelination induced by an inflammatory process. Direct viral damage or an autoimmune reaction toward the nerve-producing inflammation would be alternative or contributing mechanisms to dysfunction. Acyclovir (aciclovir) is a nucleoside analogue antiviral drug active against some of the herpes virus groups of DNA viruses and RNA viruses. The mechanism of prednisolone may involve modulation of the immune response to the causative agent or direct reduction of edema around the facial nerve within the facial canal. Prednisolone and acyclovir are commonly prescribed separately and in combination, although evidence of their effectiveness is weak. We report a case diagnosed with COVID-19 after presenting with isolated peripheral facial palsy.

Safety, tolerability, and pharmacokinetics of VV116, an oral nucleoside analog against SARS-CoV-2, in Chinese healthy subjects.

VV116 (JT001) is an oral drug candidate of nucleoside analog against SARS-CoV-2. The purpose of the three phase I studies was to evaluate the safety, tolerability, and pharmacokinetics of single and multiple ascending oral doses of VV116 in healthy subjects, as well as the effect of food on the pharmacokinetics and safety of VV116. Three studies were launched sequentially: Study 1 (single ascending-dose study, SAD), Study 2 (multiple ascending-dose study, MAD), and Study 3 (food-effect study, FE). A total of 86 healthy subjects were enrolled in the studies. VV116 tablets or placebo were administered per protocol requirements. Blood samples were collected at the scheduled time points for pharmacokinetic analysis. 116-N1, the metabolite of VV116, was detected in plasma and calculated for the PK parameters. In SAD, AUC and Cmax increased in an approximately dose-proportional manner in the dose range of 25-800 mg. T1/2 was within 4.80-6.95 h. In MAD, the accumulation ratio for Cmax and AUC indicated a slight accumulation upon repeated dosing of VV116. In FE, the standard meal had no effect on Cmax and AUC of VV116. No serious adverse event occurred in the studies, and no subject withdrew from the studies due to adverse events. Thus, VV116 exhibited satisfactory safety and tolerability in healthy subjects, which supports the continued investigation of VV116 in patients with COVID-19.

Anti-influenza Virus Activity of Methylthio-Formycin Distinct From That of T-705.

Seasonal influenza virus epidemics result in severe illness, and occasionally influenza pandemics cause significant morbidity and mortality, although vaccines and anti-influenza virus drugs are available. By screening an in-house library, we identified methylthio-formycin (SMeFM), an adenosine analog, as a potent inhibitor of influenza virus propagation. SMeFM inhibited the propagation of influenza A and B viruses (IC50: 34.1 and 37.9 nM, respectively) and viruses showing reduced susceptibility to baloxavir and neuraminidase inhibitors but not T-705 (Favipiravir). However, the combination of T-705 and SMeFM inhibited the propagation of the influenza virus not in an antagonistic but in a slightly synergistic manner, suggesting that SMeFM has targets distinct from that of T-705. SMeFM induced A-to-C transversion mutations in virus genome RNA, and SMeFM triphosphate did not inhibit in vitro viral RNA synthesis. Our results show that SMeFM inhibits the propagation of the influenza virus by a mechanism different from that of T-705 and is a potential drug candidate to develop for anti-influenza drug.

Recapitulating Trafficking of Nucleosides Into the Active Site of Polymerases of RNA Viruses: The Challenge and the Prize.

Nucleoside analogs are very effective antiviral agents with currently over 25 compounds approved for the therapy of viral infections. Still, their successful use against RNA viruses is very recent, despite RNA viruses comprising some of the most damaging human pathogens (e.g., Coronaviruses, Influenza viruses, or Flaviviridae such as dengue, Zika and hepatitis C viruses). The breakthrough came in 2013-2014, when the nucleoside analog Sofosbuvir became one of the cornerstones of current curative treatments for hepatitis C virus (HCV). An analog designed on the same principles, Remdesivir, has been the first approved compound against SARS-CoV-2, the coronavirus that causes the current COVID-19 pandemic. Both of these nucleoside analogs target the RNA-dependent RNA polymerase (RdRp) (NS5B for HCV, nsp12 for SARS-CoV-2). RdRps of RNA viruses display a peculiar elaboration of the classical polymerase architecture that leads to their active site being caged. Thus, triphosphate nucleosides and their analogs must access this active site in several steps along a narrow and dynamic tunnel. This makes straightforward computational approaches such as docking unsuitable for getting atomic-level details of this process. Here we give an account of ribose-modified nucleoside analogs as inhibitors of viral RdRps and of why taking into account the dynamics of these polymerases is necessary to understand nucleotide selection by RdRps. As a case study we use a computational protocol we recently described to examine the approach of the NTP tunnel of HCV NS5B by cellular metabolites of Sofosbuvir. We find major differences with natural nucleotides even at this early stage of nucleotide entry.

Activity of Galidesivir in a Hamster Model of SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) has claimed the lives of millions of people worldwide since it first emerged. The impact of the COVID-19 pandemic on public health and the global economy has highlighted the medical need for the development of broadly acting interventions against emerging viral threats. Galidesivir is a broad-spectrum antiviral compound with demonstrated in vitro and in vivo efficacy against several RNA viruses of public health concern, including those causing yellow fever, Ebola, Marburg, and Rift Valley fever. In vitro studies have shown that the antiviral activity of galidesivir also extends to coronaviruses. Herein, we describe the efficacy of galidesivir in the Syrian golden hamster model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Treatment with galidesivir reduced lung pathology in infected animals compared with untreated controls when treatment was initiated 24 h prior to infection. These results add to the evidence of the applicability of galidesivir as a potential medical intervention for a range of acute viral illnesses, including coronaviruses.

An update on the progress of galidesivir (BCX4430), a broad-spectrum antiviral.

Galidesivir (BCX4430) is an adenosine nucleoside analog that is broadly active in cell culture against several RNA viruses of various families. This activity has also been shown in animal models of viral disease associated with Ebola, Marburg, yellow fever, Zika, and Rift Valley fever viruses. In many cases, the compound is more efficacious in animal models than cell culture activity would predict. Based on favorable data from in vivo animal studies, galidesivir has recently undergone evaluation in several phase I clinical trials, including against severe acute respiratory syndrome coronavirus 2, and as a medical countermeasure for the treatment of Marburg virus disease.

Combining SARS-CoV-2 Proofreading Exonuclease and RNA-Dependent RNA Polymerase Inhibitors as a Strategy to Combat COVID-19: A High-Throughput in silico Screening.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions of people worldwide. Currently, many clinical trials in search of effective COVID-19 drugs are underway. Viral RNA-dependent RNA polymerase (RdRp) remains the target of choice for prophylactic or curative treatment of COVID-19. Nucleoside analogs are the most promising RdRp inhibitors and have shown effectiveness in vitro, as well as in clinical settings. One limitation of such RdRp inhibitors is the removal of incorporated nucleoside analogs by SARS-CoV-2 exonuclease (ExoN). Thus, ExoN proofreading activity accomplishes resistance to many of the RdRp inhibitors. We hypothesize that in the absence of highly efficient antivirals to treat COVID-19, combinatorial drug therapy with RdRp and ExoN inhibitors will be a promising strategy to combat the disease. To repurpose drugs for COVID-19 treatment, 10,397 conformers of 2,240 approved drugs were screened against the ExoN domain of nsp14 using AutoDock VINA. The molecular docking approach and detailed study of interactions helped us to identify dexamethasone metasulfobenzoate, conivaptan, hesperidin, and glycyrrhizic acid as potential inhibitors of ExoN activity. The results were further confirmed using molecular dynamics (MD) simulations and molecular mechanics combined with generalized Born model and solvent accessibility method (MM-GBSA) calculations. Furthermore, the binding free energy of conivaptan and hesperidin, estimated using MM-GBSA, was -85.86 ± 0.68 and 119.07 ± 0.69 kcal/mol, respectively. Based on docking, MD simulations and known antiviral activities, and conivaptan and hesperidin were identified as potential SARS-CoV-2 ExoN inhibitors. We recommend further investigation of this combinational therapy using RdRp inhibitors with a repurposed ExoN inhibitor as a potential COVID-19 treatment.

Structural insight into the recognition of S-adenosyl-L-homocysteine and sinefungin in SARS-CoV-2 Nsp16/Nsp10 RNA cap 2'-O-Methyltransferase.

The recent ongoing coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to rapidly spread across the world. To date, neither a specific antiviral drug nor a clinically effective vaccine is available. Among the 15 viral non-structural proteins (nsps), nsp16 methyltransferase has been considered as a potential target due to its crucial role in RNA cap 2'-O-methylation process, preventing the virus detection by cell innate immunity mechanisms. In the present study, molecular recognition between the two natural nucleoside analogs (S-adenosyl-l-homocysteine (SAH) and sinefungin (SFG)) and the SARS-CoV-2 nsp16/nsp10/m7GpppAC5 was studied using all-atom molecular dynamics simulations and free energy calculations based on MM/GBSA and WaterSwap approaches. The binding affinity and the number of hot-spot residues, atomic contacts, and H-bond formations of SFG/nsp16 complex were distinctly higher than those of SAH/nsp16 system, consistent with the lower water accessibility at the enzyme active site. Notably, only SFG could electrostatically interact with the 2'-OH and N3 of RNA's adenosine moiety, mimicking the methyl transfer reaction of S-adenosyl-l-methionine substrate. The atomistic binding mechanism obtained from this work paves the way for further optimizations and designs of more specific SARS-CoV-2 nsp16 inhibitors in the fight against COVID-19.