Hydrogen Bonding (Base Pairing) in Antiviral Activity.

Base pairing based on hydrogen bonding has, since its inception, been crucial in the antiviral activity of arabinosyladenine, 2'-deoxyuridines (i.e., IDU, TFT, BVDU), acyclic nucleoside analogues (i.e., acyclovir) and nucleoside reverse transcriptase inhibitors (NRTIs). Base pairing based on hydrogen bonding also plays a key role in the mechanism of action of various acyclic nucleoside phosphonates (ANPs) such as adefovir, tenofovir, cidofovir and O-DAPYs, thus explaining their activity against a wide array of DNA viruses (human hepatitis B virus (HBV), human immunodeficiency (HIV) and human herpes viruses (i.e., human cytomegalovirus)). Hydrogen bonding (base pairing) also seems to be involved in the inhibitory activity of Cf1743 (and its prodrug FV-100) against varicella-zoster virus (VZV) and in the activity of sofosbuvir against hepatitis C virus and that of remdesivir against SARS-CoV-2 (COVID-19). Hydrogen bonding (base pairing) may also explain the broad-spectrum antiviral effects of ribavirin and favipiravir. This may lead to lethal mutagenesis (error catastrophe), as has been demonstrated with molnutegravir in its activity against SARS-CoV-2.

Dual computational and biological assessment of some promising nucleoside analogs against the COVID-19-Omicron variant.

Nucleoside analogs/derivatives (NAs/NDs) with potent antiviral activities are now deemed very convenient choices for the treatment of coronavirus disease 2019 (COVID-19) arisen by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. At the same time, the appearance of a new strain of SARS-CoV-2, the Omicron variant, necessitates multiplied efforts in fighting COVID-19. Counteracting the crucial SARS-CoV-2 enzymes RNA-dependent RNA polymerase (RdRp) and 3'-to-5' exoribonuclease (ExoN) jointly altogether using the same inhibitor is a quite successful new plan to demultiplicate SARS-CoV-2 particles and eliminate COVID-19 whatever the SARS-CoV-2 subtype is (due to the significant conservation nature of RdRps and ExoNs in the different SARS-CoV-2 strains). Successive in silico screening of known NAs finally disclosed six different promising NAs, which are riboprine/forodesine/tecadenoson/nelarabine/vidarabine/maribavir, respectively, that predictably can act through the planned dual-action mode. Further in vitro evaluations affirmed the anti-SARS-CoV-2/anti-COVID-19 potentials of these NAs, with riboprine and forodesine being at the top. The two NAs are able to effectively antagonize the replication of the new virulent SARS-CoV-2 strains with considerably minute in vitro anti-RdRp and anti-SARS-CoV-2 EC50 values of 189 and 408 nM for riboprine and 207 and 657 nM for forodesine, respectively, surpassing both remdesivir and the new anti-COVID-19 drug molnupiravir. Furthermore, the favorable structural characteristics of the two molecules qualify them for varied types of isosteric and analogistic chemical derivatization. In one word, the present important outcomes of this comprehensive dual study revealed the anticipating repurposing potentials of some known nucleosides, led by the two NAs riboprine and forodesine, to successfully discontinue the coronaviral-2 polymerase/exoribonuclease interactions with RNA nucleotides in the SARS-CoV-2 Omicron variant (BA.5 sublineage) and accordingly alleviate COVID-19 infections, motivating us to initiate the two drugs' diverse anti-COVID-19 pharmacological evaluations to add both of them betimes in the COVID-19 therapeutic protocols.

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.

Structural basis for substrate selection by the SARS-CoV-2 replicase.

The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication-transcription complex (RTC)1. Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogues, including remdesivir2. Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA. To be effective inhibitors, antiviral nucleoside analogues must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogues compete, has not been discerned in detail. Here, we use cryogenic-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation. Furthermore, we investigate the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart adenosine triphosphate3,4. Our results explain the suite of interactions required for NTP recognition, informing the rational design of antivirals. Our analysis also yields insights into nucleotide recognition by the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase), an enigmatic catalytic domain essential for viral propagation5. The NiRAN selectively binds guanosine triphosphate, strengthening proposals for the role of this domain in the formation of the 5' RNA cap6.

Nucleoside Analogs and Perylene Derivatives Modulate Phase Separation of SARS-CoV-2 N Protein and Genomic RNA In Vitro.

The life cycle of severe acute respiratory syndrome coronavirus 2 includes several steps that are supposedly mediated by liquid-liquid phase separation (LLPS) of the viral nucleocapsid protein (N) and genomic RNA. To facilitate the rational design of LLPS-targeting therapeutics, we modeled N-RNA biomolecular condensates in vitro and analyzed their sensitivity to several small-molecule antivirals. The model condensates were obtained and visualized under physiological conditions using an optimized RNA sequence enriched with N-binding motifs. The antivirals were selected based on their presumed ability to compete with RNA for specific N sites or interfere with non-specific pi-pi/cation-pi interactions. The set of antivirals included fleximers, 5'-norcarbocyclic nucleoside analogs, and perylene-harboring nucleoside analogs as well as non-nucleoside amphiphilic and hydrophobic perylene derivatives. Most of these antivirals enhanced the formation of N-RNA condensates. Hydrophobic perylene derivatives and 5'-norcarbocyclic derivatives caused up to 50-fold and 15-fold enhancement, respectively. Molecular modeling data argue that hydrophobic compounds do not hamper specific N-RNA interactions and may promote non-specific ones. These findings shed light on the determinants of potent small-molecule modulators of viral LLPS.

Discovery of SARS-CoV-2 antiviral synergy between remdesivir and approved drugs in human lung cells.

SARS coronavirus 2 (SARS-CoV-2) has caused an ongoing global pandemic with significant mortality and morbidity. At this time, the only FDA-approved therapeutic for COVID-19 is remdesivir, a broad-spectrum antiviral nucleoside analog. Efficacy is only moderate, and improved treatment strategies are urgently needed. To accomplish this goal, we devised a strategy to identify compounds that act synergistically with remdesivir in preventing SARS-CoV-2 replication. We conducted combinatorial high-throughput screening in the presence of submaximal remdesivir concentrations, using a human lung epithelial cell line infected with a clinical isolate of SARS-CoV-2. This identified 20 approved drugs that act synergistically with remdesivir, many with favorable pharmacokinetic and safety profiles. Strongest effects were observed with established antivirals, Hepatitis C virus nonstructural protein 5A (HCV NS5A) inhibitors velpatasvir and elbasvir. Combination with their partner drugs sofosbuvir and grazoprevir further increased efficacy, increasing remdesivir's apparent potency > 25-fold. We report that HCV NS5A inhibitors act on the SARS-CoV-2 exonuclease proofreader, providing a possible explanation for the synergy observed with nucleoside analog remdesivir. FDA-approved Hepatitis C therapeutics Epclusa® (velpatasvir/sofosbuvir) and Zepatier® (elbasvir/grazoprevir) could be further optimized to achieve potency and pharmacokinetic properties that support clinical evaluation in combination with remdesivir.

Synthesis and Anticancer and Antiviral Activities of C-2'-Branched Arabinonucleosides.

d-Arabinofuranosyl-pyrimidine and -purine nucleoside analogues containing alkylthio-, acetylthio- or 1-thiosugar substituents at the C2' position were prepared from the corresponding 3',5'-O-silylene acetal-protected nucleoside 2'-exomethylenes by photoinitiated, radical-mediated hydrothiolation reactions. Although the stereochemical outcome of the hydrothiolation depended on the structure of both the thiol and the furanoside aglycone, in general, high d-arabino selectivity was obtained. The cytotoxic effect of the arabinonucleosides was studied on tumorous SCC (mouse squamous cell) and immortalized control HaCaT (human keratinocyte) cell lines by MTT assay. Three pyrimidine nucleosides containing C2'-butylsulfanylmethyl or -acetylthiomethyl groups showed promising cytotoxicity at low micromolar concentrations with good selectivity towards tumor cells. SAR analysis using a methyl ß-d-arabinofuranoside reference compound showed that the silyl-protecting group, the nucleobase and the corresponding C2' substituent are crucial for the cell growth inhibitory activity. The effects of the three most active nucleoside analogues on parameters indicative of cytotoxicity, such as cell size, division time and cell generation time, were investigated by near-infrared live cell imaging, which showed that the 2'-acetylthiomethyluridine derivative induced the most significant functional and morphological changes. Some nucleoside analogues also exerted anti-SARS-CoV-2 and/or anti-HCoV-229E activity with low micromolar EC50 values; however, the antiviral activity was always accompanied by significant cytotoxicity.

Facile access to 4'-(N-acylsulfonamide) modified nucleosides and evaluation of their inhibitory activity against SARS-CoV-2 RNA cap N7-guanine-methyltransferase nsp14.

N-Acylsulfonamides possess an additional carbonyl function compared to their sulfonamide analogues. Due to their unique physico-chemical properties, interest in molecules containing the N-acylsulfonamide moiety and especially nucleoside derivatives is growing in the field of medicinal chemistry. The recent renewal of interest in antiviral drugs derived from nucleosides containing a sulfonamide function has led us to evaluate the therapeutic potential of N-acylsulfonamide analogues. While these compounds are usually obtained by a difficult acylation of sulfonamides, we report here the easy and efficient synthesis of 20 4'-(N-acylsulfonamide) adenosine derivatives via the sulfo-click reaction. The target compounds were obtained from thioacid and sulfonyl azide synthons in excellent yields and were evaluated as potential inhibitors of the SARS-CoV-2 RNA cap N7-guanine-methyltransferase nsp14.

The Nucleoside/Nucleotide Analogs Tenofovir and Emtricitabine Are Inactive against SARS-CoV-2.

The urgent response to the COVID-19 pandemic required accelerated evaluation of many approved drugs as potential antiviral agents against the causative pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using cell-based, biochemical, and modeling approaches, we studied the approved HIV-1 nucleoside/tide reverse transcriptase inhibitors (NRTIs) tenofovir (TFV) and emtricitabine (FTC), as well as prodrugs tenofovir alafenamide (TAF) and tenofovir disoproxilfumarate (TDF) for their antiviral effect against SARS-CoV-2. A comprehensive set of in vitro data indicates that TFV, TAF, TDF, and FTC are inactive against SARS-CoV-2. None of the NRTIs showed antiviral activity in SARS-CoV-2 infected A549-hACE2 cells or in primary normal human lung bronchial epithelial (NHBE) cells at concentrations up to 50 µM TAF, TDF, FTC, or 500 µM TFV. These results are corroborated by the low incorporation efficiency of respective NTP analogs by the SARS-CoV-2 RNA-dependent-RNA polymerase (RdRp), and lack of the RdRp inhibition. Structural modeling further demonstrated poor fitting of these NRTI active metabolites at the SARS-CoV-2 RdRp active site. Our data indicate that the HIV-1 NRTIs are unlikely direct-antivirals against SARS-CoV-2, and clinicians and researchers should exercise caution when exploring ideas of using these and other NRTIs to treat or prevent COVID-19.

Assessment of nucleotide/nucleoside analog intervention in primer-dependent viral RNA-dependent RNA polymerases.

Nucleotide/nucleoside analogs (NAs) are important compounds used in antiviral drug development. To understand the action mode of NA drugs, we present an enzymology protocol to initially evaluate the intervention mechanism of the NTP forms of NAs on a coronaviral RNA-dependent RNA polymerase (RdRP). We describe the preparation of SARS-CoV-2 RdRP proteins and RNA constructs, followed by a primer-dependent RdRP assay to assess NTP forms of NAs. Two representative NA drugs, sofosbuvir and remdesivir, are used for demonstration of this protocol. For complete details on the use and execution of this protocol, please refer to Wu et al. (2021).

Synthesis of Sugar and Nucleoside Analogs and Evaluation of Their Anticancer and Analgesic Potentials.

Chemical modification of sugars and nucleosides has a long history of producing compounds with improved selectivity and efficacy. In this study, several modified sugars (2-3) and ribonucleoside analogs (4-8) have been synthesized from α-d-glucose in a total of 21 steps. The compounds were tested for peripheral anti-nociceptive characteristics in the acetic acid-induced writhing assay in mice, where compounds 2, 7, and 8 showed a significant reduction in the number of writhes by 56%, 62%, and 63%, respectively. The compounds were also tested for their cytotoxic potential against human HeLa cell line via trypan blue dye exclusion test followed by cell counting kit-8 (CCK-8) assay. Compound 6 demonstrated significant cytotoxic activity with an IC50 value of 54 µg/mL. Molecular docking simulations revealed that compounds 2, 7, and 8 had a comparable binding affinity to cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzymes. Additionally, the bridged nucleoside analogs 7 and 8 potently inhibited adenosine kinase enzyme as well, which indicates an alternate mechanistic pathway behind their anti-nociceptive action. Cytotoxic compound 6 demonstrated strong docking with cancer drug targets human cytidine deaminase, proto-oncogene tyrosine-protein kinase Src, human thymidine kinase 1, human thymidylate synthase, and human adenosine deaminase 2. This is the first ever reporting of the synthesis and analgesic property of compound 8 and the cytotoxic potential of compound 6.

Lethal Mutagenesis of RNA Viruses and Approved Drugs with Antiviral Mutagenic Activity.

In RNA viruses, a small increase in their mutation rates can be sufficient to exceed their threshold of viability. Lethal mutagenesis is a therapeutic strategy based on the use of mutagens, driving viral populations to extinction. Extinction catastrophe can be experimentally induced by promutagenic nucleosides in cell culture models. The loss of HIV infectivity has been observed after passage in 5-hydroxydeoxycytidine or 5,6-dihydro-5-aza-2'-deoxycytidine while producing a two-fold increase in the viral mutation frequency. Among approved nucleoside analogs, experiments with polioviruses and other RNA viruses suggested that ribavirin can be mutagenic, although its mechanism of action is not clear. Favipiravir and molnupiravir exert an antiviral effect through lethal mutagenesis. Both drugs are broad-spectrum antiviral agents active against RNA viruses. Favipiravir incorporates into viral RNA, affecting the G→A and C→U transition rates. Molnupiravir (a prodrug of ß-d-N4-hydroxycytidine) has been recently approved for the treatment of SARS-CoV-2 infection. Its triphosphate derivative can be incorporated into viral RNA and extended by the coronavirus RNA polymerase. Incorrect base pairing and inefficient extension by the polymerase promote mutagenesis by increasing the G→A and C→U transition frequencies. Despite having remarkable antiviral action and resilience to drug resistance, carcinogenic risks and genotoxicity are important concerns limiting their extended use in antiviral therapy.

Non-nucleoside structured compounds with antiviral activity-past 10 years (2010-2020).

Nucleosides and their derivatives are a well-known and well-described class of compounds with antiviral activity. Currently, in the era of the COVID-19 pandemic, scientists are also looking for compounds not related to nucleosides with antiviral properties. This review aims to provide an overview of selected synthetic antiviral agents not associated to nucleosides developed against human viruses and introduced to preclinical and clinical trials as well as drugs approved for antiviral therapy over the last 10 years. The article describes for the first time the wide classification of such antiviral drugs and drug candidates and briefly summarizes the biological target and clinical applications of the compounds. The described compounds are arranged according to the antiviral mechanism of action. Knowledge of the drug's activity toward specific molecular targets may be the key to researching new antiviral compounds and repositioning drugs already approved for clinical use. The paper also briefly discusses the future directions of antiviral therapy. The described examples of antiviral compounds can be helpful for further drug development.

A Review on the New Indication of Nucleoside Reverse Transcriptase Inhibitors (NRTIs) in the Treatment of Coronavirus Disease 2019.

BACKGROUND: In December 2019, a new coronavirus (nCoV) emerged as a public health concern spreading all around the world. Several attempts have been made to discover effective drugs and vaccines. Up to now, multiple COVID-19 vaccines have been developed against this mysterious virus, and a lot of individuals have already got vaccinated. OBJECTIVE: Anti-viral drugs are effective in treating and managing COVID-19. Nucleoside reverse transcriptase inhibitors (NRTIs) are a collection of antiviral drugs for treating HIV and HBV infections. These drugs prevent virus replication by blocking reverse transcriptase (RT). In this review, we discuss the interaction of this class of anti- HIV drugs with specific functional proteins and enzymes of SARSCoV- 2. METHODS: A search of the databases, including Web of Science, Embase, PubMed, Scopus, and Google Scholar, was conducted from commencement to September 2020. The relevant articles on the potential effects of NRTIs on COVID-19 were collected. Finally, twenty-three articles were selected, including all in vitro, in vivo, and clinical studies. RESULTS: It was observed that RdRp, spike, ACE2, PNP, inflammatory cytokines, and nucleocapsid protein participate in the pathogenesis of SARS-CoV-2. NRTIs target these proteins by binding to them. CONCLUSION: This review is focused on the mechanisms of NRTIs to introduce them as potential therapies for COVID-19. However, further in vitro and in vivo investigations will provide helpful information for the identification of drug candidates as a part of COVID-19 management.

Nucleosides and emerging viruses: A new story.

With several US Food and Drug Administration (FDA)-approved drugs and high barriers to resistance, nucleoside and nucleotide analogs remain the cornerstone of antiviral therapies for not only herpesviruses, but also HIV and hepatitis viruses (B and C); however, with the exception of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for which vaccines have been developed at unprecedented speed, there are no vaccines or small antivirals yet available for (re)emerging viruses, which are primarily RNA viruses. Thus, herein, we present an overview of ribonucleoside analogs recently developed and acting as inhibitors of the viral RNA-dependent RNA polymerase (RdRp). They are new lead structures that will be exploited for the discovery of new antiviral nucleosides.

Pyrimidine inhibitors synergize with nucleoside analogues to block SARS-CoV-2.

The SARS-CoV-2 virus has infected more than 261 million people and has led to more than 5 million deaths in the past year and a half1 ( https://www.who.org/ ). Individuals with SARS-CoV-2 infection typically develop mild-to-severe flu-like symptoms, whereas infection of a subset of individuals leads to severe-to-fatal clinical outcomes2. Although vaccines have been rapidly developed to combat SARS-CoV-2, there has been a dearth of antiviral therapeutics. There is an urgent need for therapeutics, which has been amplified by the emerging threats of variants that may evade vaccines. Large-scale efforts are underway to identify antiviral drugs. Here we screened approximately 18,000 drugs for antiviral activity using live virus infection in human respiratory cells and validated 122 drugs with antiviral activity and selectivity against SARS-CoV-2. Among these candidates are 16 nucleoside analogues, the largest category of clinically used antivirals. This included the antivirals remdesivir and molnupiravir, which have been approved for use in COVID-19. RNA viruses rely on a high supply of nucleoside triphosphates from the host to efficiently replicate, and we identified a panel of host nucleoside biosynthesis inhibitors as antiviral. Moreover, we found that combining pyrimidine biosynthesis inhibitors with antiviral nucleoside analogues synergistically inhibits SARS-CoV-2 infection in vitro and in vivo against emerging strains of SARS-CoV-2, suggesting a clinical path forward.

Remdesivir induces persistent mitochondrial and structural damage in human induced pluripotent stem cell-derived cardiomyocytes.

AIMS: Remdesivir is a prodrug of an adenosine triphosphate analogue and is currently the only drug formally approved for the treatment of hospitalized coronavirus disease of 2019 (COVID-19) patients. Nucleoside/nucleotide analogues have been shown to induce mitochondrial damage and cardiotoxicity, and this may be exacerbated by hypoxia, which frequently occurs in severe COVID-19 patients. Although there have been few reports of adverse cardiovascular events associated with remdesivir, clinical data are limited. Here, we investigated whether remdesivir induced cardiotoxicity using an in vitro human cardiac model. METHODS AND RESULTS: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were exposed to remdesivir under normoxic and hypoxic conditions to simulate mild and severe COVID-19, respectively. Remdesivir induced mitochondrial fragmentation, reduced redox potential, and suppressed mitochondrial respiration at levels below the estimated plasma concentration under both normoxic and hypoxic conditions. Non-mitochondrial damage such as electrophysiological alterations and sarcomere disarray were also observed. Importantly, some of these changes persisted after the cessation of treatment, culminating in increased cell death. Mechanistically, we found that inhibition of DRP1, a regulator of mitochondrial fission, ameliorated the cardiotoxic effects of remdesivir, showing that remdesivir-induced cardiotoxicity was preventable and excessive mitochondrial fission might contribute to this phenotype. CONCLUSIONS: Using an in vitro model, we demonstrated that remdesivir can induce cardiotoxicity in hiPSC-CMs at clinically relevant concentrations. These results reveal previously unknown potential side-effects of remdesivir and highlight the importance of further investigations with in vivo animal models and active clinical monitoring to prevent lasting cardiac damage to patients.

Can natural products stop the SARS-CoV-2 virus? A docking and molecular dynamics study of a natural product database.

Background: The SARS-CoV-2 3CLpro is one of the primary targets for designing new and repurposing known drugs. Methodology: A virtual screening of molecules from the Natural Product Atlas was performed, followed by molecular dynamics simulations of the most potent inhibitor bound to two conformations of the protease and into two binding sites. Conclusion: Eight molecules with appropriate ADMET properties are suggested as potential inhibitors. The greatest benefit of this study is the demonstration that these ligands can bind in the catalytic site but also to the groove between domains II and III, where they interact with a series of residues which have an important role in the dimerization and the maturation process of the enzyme.

Advancement of Prodrug Approaches for Nucleotide Antiviral Agents.

Synthetic nucleoside or nucleotide analogues played a key role to the development of antiviral agents in past decades. However, low membrane permeability and insufficient cellular phosphorylation impaired the biological activity of polar nucleoside drugs because they have to penetrate the cell membrane and be phosphorylated to active metabolite stepwise by intracellular enzymes. To overcome these limitations, diverse lipophilic prodrug modifications based on nucleoside mono-, di-, and triphosphate were designed and put into practice to efficiently deliver nucleoside into the target site, and bypass the rate-limited phosphorylation step. As the most successful prodrug strategy, ProTide technology has led to the discovery of three FDA-approved antiviral agents, including sofosbuvir, tenofovir alafenadmide, and remdesivir, which has been authorized for emergency use in patients of COVID-19 in the US. In recent years, nucleoside di- and triphosphate prodrugs have also made the significant progress. This review will focus on the summary of design approach and metabolic activation path of different nucleotide prodrug strategies. The potential application of nucleotide prodrugs for the treatment of COVID-19 was also described due to the pandemic of SARS-CoV-2.