Active Triphosphate Form Research Articles

Mechanism and spectrum of inhibition of a 4′-cyano modified nucleotide analog against diverse RNA polymerases of prototypic respiratory RNA viruses

The development of safe and effective broad-spectrum antivirals that target the replication machinery of respiratory viruses is of high priority in pandemic preparedness programs. Here, we studied the mechanism of action of a newly discovered nucleotide analog against diverse RNA-dependent RNA polymerases (RdRp) of prototypic respiratory viruses. GS-646939 is the active 5′-triphosphate (TP) metabolite of a 4ʹ-cyano modified C-adenosine analog phosphoramidate prodrug GS-7682. Enzyme kinetics show that the RdRps of human rhinovirus type 16 (HRV-16) and enterovirus 71 (EV-71) incorporate GS-646939 with unprecedented selectivity; GS-646939 is incorporated 20-50-fold more efficiently than its natural ATP counterpart. The RdRp complex of respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) incorporate GS-646939 and ATP with similar efficiency. In contrast, influenza B RdRp shows a clear preference for ATP and human mitochondrial RNA polymerase (h-mtRNAP) does not show significant incorporation of GS-646939. Once incorporated into the nascent RNA strand, GS-646939 acts as a chain-terminator although higher NTP concentrations can partially overcome inhibition for some polymerases. Modeling and biochemical data suggest that the 4ʹ-modification inhibits RdRp translocation. Comparative studies with GS-443902, the active triphosphate form of the 1′-cyano modified prodrugs remdesivir and obeldesivir, reveal not only different mechanisms of inhibition, but also differences in the spectrum of inhibition of viral polymerases. In conclusion, 1ʹ-cyano and 4ʹ-cyano modifications of nucleotide analogs provide complementary strategies to target the polymerase of several families of respiratory RNA viruses.

SAMHD1 single nucleotide polymorphisms impact outcome in children with newly diagnosed acute myeloid leukemia.

Cytarabine arabinoside (Ara-C) has been the cornerstone of acute myeloid leukemia (AML) chemotherapy for decades. After cellular uptake, it is phosphorylated into its active triphosphate form (Ara-CTP), which primarily exerts its cytotoxic effects by inhibiting DNA synthesis in proliferating cells. Interpatient variation in the enzymes involved in the Ara-C metabolic pathway has been shown to affect intracellular abundance of Ara-CTP and, thus, its therapeutic benefit. Recently, SAMHD1 (SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase 1) has emerged to play a role in Ara-CTP inactivation, development of drug resistance, and, consequently, clinical response in AML. Despite this, the impact of genetic variations in SAMHD1 on outcome in AML has not been investigated in depth. In this study, we evaluated 25 single nucleotide polymorphisms (SNPs) within the SAMHD1 gene for association with clinical outcome in 400 pediatric patients with newly diagnosed AML from 2 clinical trials, AML02 and AML08. Three SNPs, rs1291128, rs1291141, and rs7265241 located in the 3' region of SAMHD1 were significantly associated with at least 1 clinical outcome: minimal residual disease after induction I, event-free survival (EFS), or overall survival (OS) in the 2 cohorts. In an independent cohort of patients from the COG-AAML1031 trial (n= 854), rs7265241 A>G remained significantly associated with EFS and OS. In multivariable analysis, all the SNPs remained independent predictors of clinical outcome. These results highlight the relevance of the SAMHD1 pharmacogenomics in context of response to Ara-C in AML and warrants the need for further validation in expanded patient cohorts.

Discovery of a 2'-Fluoro,2'-Bromouridine Phosphoramidate Prodrug Exhibiting Anti-Yellow Fever Virus Activity in Culture and in Mice.

Yellow fever virus (YFV) is a potentially lethal, zoonotic, blood-borne flavivirus transmitted to humans and non-human primates by mosquitoes. Owing to multiple deadly epidemics, the WHO classifies YFV as a "high impact, high threat disease" with resurgent epidemic potential. At present, there are no approved antiviral therapies to combat YFV infection. Herein we report on 2'-halogen-modified nucleoside analogs as potential anti-YFV agents. Of 11 compounds evaluated, three showed great promise with low toxicity, high intracellular metabolism into the active nucleoside triphosphate form, and sub-micromolar anti-YFV activity. Notably, we investigated a 2'-fluoro,2'-bromouridine phosphate prodrug (C9), a known anti-HCV agent with good stability in human blood and favorable metabolism. Predictive modeling revealed that C9 could readily bind the active site of the YFV RdRp, conferring its anti-YFV activity. C9 displayed potent anti-YFV activity in primary human macrophages, 3D hepatocyte spheroids, and in mice. In an A129 murine model, shortly after infection, C9 significantly reduced YFV replication and protected against YFV-induced liver inflammation and pathology with no adverse effects. Collectively, this work identifies a potent new anti-YFV agent with strong therapeutic promise.

Treatment of chronic hepatitis B: virological and pharmacological aspects

Tenofovir is a nucleotidic analog inhibitor used in monotherapy as first line treatment of chronic Hepatitis B virus (HBV) infection. This drug requires a two-step phosphorylation by cellular kinases. The active triphosphate form inhibits viral DNA polymerase. Tenofovir has a very low oral bioavailability following oral administration. Hence, its oral administration requires the use of prodrugs: tenofovir disoproxil fumararate (TDF) or tenofovir alafénamide (TAF). TAF demonstrated a lower kidney and bone toxicity than TDF. TAF and TDF treatments allow prevention of chronic hepatitis B complications, which are cirrhosis and hepatocellular carcinoma. Prevention of these complications requires a virological response to the treatment, defined as undetectable HBV DNA. TDF and TAF are associated with virological response rates from 64 to 94 % after one year of treatment. This rate depends on HBV viral load at diagnostic, HBe antigen status, mutations in the HBV polymerase gene (Pol/RT) and patient compliance to the treatment. Tenofovir has a high genetic barrier and resistance mutations to this drug have not yet been described. However, mutations rt181T/V and/or rtN236T have been associated with reduced susceptibility to TDF/TAF in vitro and delayed response in vivo. Recently described mutations CYEI and rtA194T have been associated with reduced susceptibility to TDF/TAF in vitro without any change in viral response in vivo. Patient compliance can be improved using cognitive behavioral therapy, supporter interventions and use of short message service. Finally, some genetic polymorphisms in MRP(multidrug resistance-associated protein)-2 and MRP4 efflux transporters could be associated with TDF toxicity and virological response to TDF or TAF. In the perspective of functional HBV cure, TDF and TAF are likely to be still used, in association with new class of antivirals. For this reason, it is important to further characterize the pharmacological and virological factors associated with partial virological response to tenofovir.

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).

Cladribine as a Potential Object of Nucleoside Transporter-Based Drug Interactions.

Cladribine is a nucleoside analog that is phosphorylated in its target cells (B and T-lymphocytes) to its active triphosphate form (2-chlorodeoxyadenosine triphosphate). Cladribine tablets 10 mg (Mavenclad®), administered for up to 10 days per year in 2 consecutive years (3.5-mg/kg cumulative dose over 2 years), are used to treat patients with relapsing multiple sclerosis. Cladribine has been shown to be a substrate of various nucleoside transporters (NTs). Intestinal absorption and distribution of cladribine throughout the body appear to be essentially mediated by equilibrative NTs (ENTs) and concentrative NTs (CNTs), specifically by ENT1, ENT2, ENT4, CNT2 (low affinity), and CNT3. Other efficient transporters of cladribine are the ABC efflux transporters, specifically breast cancer resistance protein, which likely modulates the oral absorption and renal excretion of cladribine. A key transporter for the intracellular uptake of cladribine into B and T-lymphocytes is ENT1 with ancillary contributions of ENT2 and CNT2. Transporter-based drug interactions affecting absorption and target cellular uptake of a prodrug such as cladribine are likely to reduce systemic bioavailability and target cell exposure, thereby possibly hampering clinical efficacy. In order to manage optimized therapy, i.e., to ensure uncompromised target cell uptake to preserve the full therapeutic potential of cladribine, it is important that clinicians are aware of the existence of NT-inhibiting medicinal products, various lifestyle drugs, and food components. This article reviews the existing knowledge on inhibitors of NT, which may alter cladribine absorption, distribution, and uptake into target cells, thereby summarizing the existing knowledge on optimized methods of administration and concomitant drugs that should be avoided during cladribine treatment.

Programmable nano-reactors for stochastic sensing

Chemical reactions of single molecules, caused by rapid formation or breaking of chemical bonds, are difficult to observe even with state-of-the-art instruments. A biological nanopore can be engineered into a single molecule reactor, capable of detecting the binding of a monatomic ion or the transient appearance of chemical intermediates. Pore engineering of this type is however technically challenging, which has significantly restricted further development of this technique. We propose a versatile strategy, “programmable nano-reactors for stochastic sensing” (PNRSS), by which a variety of single molecule reactions of hydrogen peroxide, metal ions, ethylene glycol, glycerol, lactic acid, vitamins, catecholamines or nucleoside analogues can be observed directly. PNRSS presents a refined sensing resolution which can be further enhanced by an artificial intelligence algorithm. Remdesivir, a nucleoside analogue and an investigational anti-viral drug used to treat COVID-19, can be distinguished from its active triphosphate form by PNRSS, suggesting applications in pharmacokinetics or drug screening.

Quantitative analysis of binary mixtures of entecavir using solid-state analytical techniques with chemometric methods

Entecavir (ETV), a guanosine analogue, can be rapidly phosphorylated to the active intracellular triphosphate form that inhibits replication of the hepatitis B virus (HBV). This work aims to develop useful methods to quantify the content of ETV-A in ETV-H and ETV-A binary mixtures. The integral enthalpy value of the phase transformation in the DSC thermogram was selected to quantify the content of ETV-A in binary mixture. Different ranges (full spectrum and partial spectrum) of the PXRD and ATR-FTIR data versus ETV-A content were used to develop partial least squares (PLS) calibration curves with different pre-processing algorithms including multiplicative scatter correction (MSC), standard normal variate (SNV), wavelet transform (WT) or their combinations. The standard curve obtained for the DSC technique is Y = 100.455 + 7.255X, R2 = 0.997, in the ETV-A contents range of 0–100% w/w% in binary mixtures. Limit of detection (LOD) of ETV-A was 0.909% and limit of quantitation (LOQ) is 2.755%. The best correction models for PXRD and ATR-FTIR techniques, in the ETV-A contents range of 0–100% w/w% in binary mixtures, were Y = 0.346 + 0.999X, R2 = 0.999 in the 2θ range of 4-6° with 14-34° and Y = 0.767 + 0.991X, R2 = 0.991 in the range of 3500–2500 cm−1 with 2000–400 cm−1 pretreated by MSC + WT, respectively. Limit of detection (LOD) of ETV-A was 1.394% and limit of quantitation (LOQ) was 4.224% for PXRD technique. LOD and LOQ for ATR-FTIR technique were 9.124% and 27.648%. The results show that DSC, PXRD and ATR-FTIR techniques are precise and accurate, and could be used for the measurement of ETV-A content in the binary mixtures.

Review of Transporter Substrate, Inhibitor, and Inducer Characteristics of Cladribine.

Cladribine is a nucleoside analog that is phosphorylated in its target cells (B- and T-lymphocytes) to its active adenosine triphosphate form (2-chlorodeoxyadenosine triphosphate). Cladribine tablets 10 mg (Mavenclad®) administered for up to 10 days per year in 2 consecutive years (3.5-mg/kg cumulative dose over 2 years) are used to treat patients with relapsing multiple sclerosis. The ATP-binding cassette, solute carrier, and nucleoside transporter substrate, inhibitor, and inducer characteristics of cladribine are reviewed in this article. Available evidence suggests that the distribution of cladribine across biological membranes is facilitated by a number of uptake and efflux transporters. Among the key ATP-binding cassette efflux transporters, only breast cancer resistance protein has been shown to be an efficient transporter of cladribine, while P-glycoprotein does not transport cladribine well. Intestinal absorption, distribution throughout the body, and intracellular uptake of cladribine appear to be exclusively mediated by equilibrative and concentrative nucleoside transporters, specifically by ENT1, ENT2, ENT4, CNT2 (low affinity), and CNT3. Renal excretion of cladribine appears to be most likely driven by breast cancer resistance protein, ENT1, and P-glycoprotein. The latter may play a role despite its poor cladribine transport efficiency in view of the renal abundance of P-glycoprotein. There is no evidence that solute carrier uptake transporters such as organic anion transporting polypeptides, organic anion transporters, and organic cation transporters are involved in the transport of cladribine. Available in vitro studies examining the inhibitor characteristics of cladribine for a total of 13 major ATP-binding cassette, solute carrier, and CNT transporters indicate that in vivo inhibition of any of these transporters by cladribine is unlikely.

Abstract 1059: Combination of gemcitabine and cytidine deaminase inhibitor: A breakthrough cocktail for the treatment of pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma is a very common type of pancreatic cancer and is one of the deadliest cancers. Gemcitabine (Gem), is still the mainstay drug for the treatment of PDAC. Gem is a hydrophilic drug which requires nucleoside transporters for its efficient uptake. Once inside the cell, Gem is converted to an active triphosphate form which inhibits DNA synthesis and causes apoptosis. However, Gem gets inactivated by cytidine deaminase (CDA). Long term treatment with Gem leads to increased CDA production thereby developing resistance. We hypothesize that combination of Gem with CDA inhibitor like Zebularine (Zeb), could prevent the intracellular degradation of Gem. Combination can potentially result in enhancing the anti-cancer activity of Gem, reduced dose and dosing frequency and minimizing side effects. The objectives of our study were; (a) to investigate the cytotoxic interaction of Gem and Zeb alone and in combination (b) Evaluating effect of various apoptosis and proliferation markers using flow cytometry and western blot assay. Initially we explored the anti-proliferative effects of Gem and Zeb as a single drug and in combination on MiaPaca-2 cells using MTT assay. Combination index was calculated using chou talalay method and Combenefit software. Effect of Gem, Zeb, and combination treatment on the cell viability of 3D tumor spheroids and its area was carried out using fluorescence microscopy and ImageJ software respectively. Apoptosis study was carried out using Annexin V apoptosis kit.Gem and Zeb exhibited dose-dependent inhibition in viability of MiaPaca-2 cells with an IC50 of 0.48 and 67.82 µM, respectively. The combination index of 0.09 suggest “very strong synergism” (Table 1). 3D spheroid treated with combination exhibited a highly distorted surface with significantly higher number of dead cells. Our research confirms that this synergistic combination can be a breakthrough approach for the treatment of PDAC. Table 1.IC50 of Gem, Zeb and combination of Gem+Zeb in Miapaca2 cellsDrugMolar RatioIC50 of GemIC50 of ZebCombination IndexSingle-0.4867.82Gem1:100.0440.40.09 Citation Format: Manali Patki, Aishwarya Saraswat, Shraddha Bhutkar, Vikas Dukhande, Ketankumar Patel. Combination of gemcitabine and cytidine deaminase inhibitor: A breakthrough cocktail for the treatment of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1059.

β-d-N4-hydroxycytidine Inhibits SARS-CoV-2 Through Lethal Mutagenesis But Is Also Mutagenic To Mammalian Cells.

Mutagenic ribonucleosides can act as broad-based antiviral agents. They are metabolized to the active ribonucleoside triphosphate form and concentrate in genomes of RNA viruses during viral replication. β-d-N4-hydroxycytidine (NHC, initial metabolite of molnupiravir) is >100-fold more active than ribavirin or favipiravir against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with antiviral activity correlated to the level of mutagenesis in virion RNA. However, NHC also displays host mutational activity in an animal cell culture assay, consistent with RNA and DNA precursors sharing a common intermediate of a ribonucleoside diphosphate. These results indicate highly active mutagenic ribonucleosides may hold risk for the host.

A cell-based assay to discover inhibitors of SARS-CoV-2 RNA dependent RNA polymerase

Antiviral therapeutics is one effective avenue to control and end this devastating COVID-19 pandemic. The viral RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 has been recognized as a valuable target of antivirals. However, the cell-free SARS-CoV-2 RdRp biochemical assay requires the conversion of nucleotide prodrugs into the active triphosphate forms, which regularly occurs in cells yet is a complicated multiple-step chemical process in vitro, and thus hinders the utility of this cell-free assay in the rapid discovery of RdRp inhibitors. In addition, SARS-CoV-2 exoribonuclease provides the proof-reading capacity to viral RdRp, thus creates relatively high resistance threshold of viral RdRp to nucleotide analog inhibitors, which must be examined and evaluated in the development of this class of antivirals. Here, we report a cell-based assay to evaluate the efficacy of nucleotide analog compounds against SARS-CoV-2 RdRp and assess their tolerance to viral exoribonuclease-mediated proof-reading. By testing seven commonly used nucleotide analog viral polymerase inhibitors, Remdesivir, Molnupiravir, Ribavirin, Favipiravir, Penciclovir, Entecavir and Tenofovir, we found that both Molnupiravir and Remdesivir showed the strong inhibition of SARS-CoV-2 RdRp, with EC50 value of 0.22 μM and 0.67 μM, respectively. Moreover, our results suggested that exoribonuclease nsp14 increases resistance of SARS-CoV-2 RdRp to nucleotide analog inhibitors. We also determined that Remdesivir presented the highest resistance to viral exoribonuclease activity in cells. Therefore, we have developed a cell-based SARS-CoV-2 RdRp assay which can be deployed to discover SARS-CoV-2 RdRp inhibitors that are urgently needed to treat COVID-19 patients.

Off-Target In Vitro Profiling Demonstrates that Remdesivir Is a Highly Selective Antiviral Agent.

Remdesivir (RDV, GS-5734), the first FDA-approved antiviral for the treatment of COVID-19, is a single diastereomer monophosphoramidate prodrug of an adenosine analogue. It is intracellularly metabolized into the active triphosphate form, which in turn acts as a potent and selective inhibitor of multiple viral RNA polymerases. RDV has broad-spectrum activity against members of the coronavirus family, such as SARS-CoV-2, SARS-CoV, and MERS-CoV, as well as filoviruses and paramyxoviruses. To assess the potential for off-target toxicity, RDV was evaluated in a set of cellular and biochemical assays. Cytotoxicity was evaluated in a set of relevant human cell lines and primary cells. In addition, RDV was evaluated for mitochondrial toxicity under aerobic and anaerobic metabolic conditions, and for the effects on mitochondrial DNA content, mitochondrial protein synthesis, cellular respiration, and induction of reactive oxygen species. Last, the active 5'-triphosphate metabolite of RDV, GS-443902, was evaluated for potential interaction with human DNA and RNA polymerases. Among all of the human cells tested under 5 to 14 days of continuous exposure, the 50% cytotoxic concentration (CC50) values of RDV ranged from 1.7 to >20 μM, resulting in selectivity indices (SI, CC50/EC50) from >170 to 20,000, with respect to RDV anti-SARS-CoV-2 activity (50% effective concentration [EC50] of 9.9 nM in human airway epithelial cells). Overall, the cellular and biochemical assays demonstrated a low potential for RDV to elicit off-target toxicity, including mitochondria-specific toxicity, consistent with the reported clinical safety profile.

Template-dependent inhibition of coronavirus RNA-dependent RNA polymerase by remdesivir reveals a second mechanism of action

Remdesivir (RDV) is a direct-acting antiviral agent that is used to treat patients with severe coronavirus disease 2019 (COVID-19). RDV targets the viral RNA-dependent RNA polymerase (RdRp) of severe acute respiratory syndrome coronavirus 2 (SARS–CoV-2). We have previously shown that incorporation of the active triphosphate form of RDV (RDV-TP) at position i causes delayed chain termination at position i + 3. Here we demonstrate that the S861G mutation in RdRp eliminates chain termination, which confirms the existence of a steric clash between Ser-861 and the incorporated RDV-TP. With WT RdRp, increasing concentrations of NTP pools cause a gradual decrease in termination and the resulting read-through increases full-length product formation. Hence, RDV residues could be embedded in copies of the first RNA strand that is later used as a template. We show that the efficiency of incorporation of the complementary UTP opposite template RDV is compromised, providing a second opportunity to inhibit replication. A structural model suggests that RDV, when serving as the template for the incoming UTP, is not properly positioned because of a significant clash with Ala-558. The adjacent Val-557 is in direct contact with the template base, and the V557L mutation is implicated in low-level resistance to RDV. We further show that the V557L mutation in RdRp lowers the nucleotide concentration required to bypass this template-dependent inhibition. The collective data provide strong evidence to show that template-dependent inhibition of SARS–CoV-2 RdRp by RDV is biologically relevant.

Nucleotide analogues as inhibitors of SARS-CoV Polymerase.

SARS‐CoV‐2, a member of the coronavirus family, has caused a global public health emergency. Based on our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously reasoned that the FDA‐approved hepatitis C drug EPCLUSA (Sofosbuvir/Velpatasvir) should inhibit coronaviruses, including SARS‐CoV‐2. Here, using model polymerase extension experiments, we demonstrate that the active triphosphate form of Sofosbuvir is incorporated by low‐fidelity polymerases and SARS‐CoV RNA‐dependent RNA polymerase (RdRp), and blocks further incorporation by these polymerases; the active triphosphate form of Sofosbuvir is not incorporated by a host‐like high‐fidelity DNA polymerase. Using the same molecular insight, we selected 3’‐fluoro‐3’‐deoxythymidine triphosphate and 3’‐azido‐3’‐deoxythymidine triphosphate, which are the active forms of two other anti‐viral agents, Alovudine and AZT (an FDA‐approved HIV/AIDS drug) for evaluation as inhibitors of SARS‐CoV RdRp. We demonstrate the ability of two of these HIV reverse transcriptase inhibitors to be incorporated by SARS‐CoV RdRp where they also terminate further polymerase extension. Given the 98% amino acid similarity of the SARS‐CoV and SARS‐CoV‐2 RdRps, we expect these nucleotide analogues would also inhibit the SARS‐CoV‐2 polymerase. These results offer guidance to further modify these nucleotide analogues to generate more potent broad‐spectrum anti‐coronavirus agents.

Nucleotide Analogues as Inhibitors of SARS-CoV-2 Polymerase, a Key Drug Target for COVID-19.

SARS-CoV-2 is responsible for the current COVID-19 pandemic. On the basis of our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously demonstrated that three nucleotide analogues (the triphosphates of Sofosbuvir, Alovudine, and AZT) inhibit the SARS-CoV RNA-dependent RNA polymerase (RdRp). We also demonstrated that a library of additional nucleotide analogues terminate RNA synthesis catalyzed by the SARS-CoV-2 RdRp, a well-established drug target for COVID-19. Here, we used polymerase extension experiments to demonstrate that the active triphosphate form of Sofosbuvir (an FDA-approved hepatitis C drug) is incorporated by SARS-CoV-2 RdRp and blocks further incorporation. Using the molecular insight gained from the previous studies, we selected the active triphosphate forms of six other antiviral agents, Alovudine, Tenofovir alafenamide, AZT, Abacavir, Lamivudine, and Emtricitabine, for evaluation as inhibitors of the SARS-CoV-2 RdRp and demonstrated the ability of these viral polymerase inhibitors to be incorporated by SARS-CoV-2 RdRp, where they terminate further polymerase extension with varying efficiency. These results provide a molecular basis for inhibition of the SARS-CoV-2 RdRp by these nucleotide analogues. If sufficient efficacy of some of these FDA-approved drugs in inhibiting viral replication in cell culture is established, they may be explored as potential COVID-19 therapeutics.

A library of nucleotide analogues terminate RNA synthesis catalyzed by polymerases of coronaviruses that cause SARS and COVID-19

SARS-CoV-2, a member of the coronavirus family, is responsible for the current COVID-19 worldwide pandemic. We previously demonstrated that five nucleotide analogues inhibit the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), including the active triphosphate forms of Sofosbuvir, Alovudine, Zidovudine, Tenofovir alafenamide and Emtricitabine. We report here the evaluation of a library of nucleoside triphosphate analogues with a variety of structural and chemical features as inhibitors of the RdRps of SARS-CoV and SARS-CoV-2. These features include modifications on the sugar (2′ or 3′ modifications, carbocyclic, acyclic, or dideoxynucleotides) or on the base. The goal is to identify nucleotide analogues that not only terminate RNA synthesis catalyzed by these coronavirus RdRps, but also have the potential to resist the viruses' exonuclease activity. We examined these nucleotide analogues for their ability to be incorporated by the RdRps in the polymerase reaction and to prevent further incorporation. While all 11 molecules tested displayed incorporation, 6 exhibited immediate termination of the polymerase reaction (triphosphates of Carbovir, Ganciclovir, Stavudine and Entecavir; 3′-OMe-UTP and Biotin-16-dUTP), 2 showed delayed termination (Cidofovir diphosphate and 2′-OMe-UTP), and 3 did not terminate the polymerase reaction (2′-F-dUTP, 2′–NH2–dUTP and Desthiobiotin-16-UTP). The coronaviruses possess an exonuclease that apparently requires a 2′-OH at the 3′-terminus of the growing RNA strand for proofreading. In this study, all nucleoside triphosphate analogues evaluated form Watson-Crick-like base pairs. The nucleotide analogues demonstrating termination either lack a 2′-OH, have a blocked 2′-OH, or show delayed termination. Thus, these nucleotide analogues are of interest for further investigation to evaluate whether they can evade the viral exonuclease activity. Prodrugs of five of these nucleotide analogues (Cidofovir, Abacavir, Valganciclovir/Ganciclovir, Stavudine and Entecavir) are FDA-approved medications for treatment of other viral infections, and their safety profiles are well established. After demonstrating potency in inhibiting viral replication in cell culture, candidate molecules can be rapidly evaluated as potential therapies for COVID-19.

Comparative Antiviral Activity of Remdesivir and Anti-HIV Nucleoside Analogs Against Human Coronavirus 229E (HCoV-229E).

Remdesivir is a nucleotide prodrug that is currently undergoing extensive clinical trials for the treatment of COVID-19. The prodrug is metabolized to its active triphosphate form and interferes with the action of RNA-dependent RNA polymerase of SARS-COV-2. Herein, we report the antiviral activity of remdesivir against human coronavirus 229E (HCoV-229E) compared to known anti-HIV agents. These agents included tenofovir (TFV), 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA), alovudine (FLT), lamivudine (3TC), and emtricitabine (FTC), known as nucleoside reverse-transcriptase inhibitors (NRTIs), and a number of 5′-O-fatty acylated anti-HIV nucleoside conjugates. The anti-HIV nucleosides interfere with HIV RNA-dependent DNA polymerase and/or act as chain terminators. Normal human fibroblast lung cells (MRC-5) were used to determine the cytotoxicity of the compounds. The study revealed that remdesivir exhibited an EC50 value of 0.07 µM against HCoV-229E with TC50 of > 2.00 µM against MRC-5 cells. Parent NRTIs were found to be inactive against (HCoV-229E) at tested concentrations. Among all the NRTIs and 5′-O-fatty acyl conjugates of NRTIs, 5′-O-tetradecanoyl ester conjugate of FTC showed modest activity with EC50 and TC50 values of 72.8 µM and 87.5 µM, respectively. These data can be used for the design of potential compounds against other coronaviruses.

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.

BackgroundRemdesivir has received significant attention for its potential application in the treatment of COVID-19, caused by SARS-CoV-2. Remdesivir has already been tested for Ebola virus disease treatment and found to have activity against SARS and MERS coronaviruses. The remdesivir core contains GS-441524, which interferes with RNA-dependent RNA polymerases alone. In non-human primates, following IV administration, remdesivir is rapidly distributed into PBMCs and converted within 2 h to the active nucleoside triphosphate form, while GS-441524 is detectable in plasma for up to 24 h. Nevertheless, remdesivir pharmacokinetics and pharmacodynamics in humans are still unexplored, highlighting the need for a precise analytical method for remdesivir and GS-441524 quantification.ObjectivesThe validation of a reliable UHPLC-MS/MS method for remdesivir and GS-441524 quantification in human plasma.MethodsRemdesivir and GS-441524 standards and quality controls were prepared in plasma from healthy donors. Sample preparation consisted of protein precipitation, followed by dilution and injection into the QSight 220 UHPLC-MS/MS system. Chromatographic separation was obtained through an Acquity HSS T3 1.8 μm, 2.1 × 50 mm column, with a gradient of water and acetonitrile with 0.05% formic acid. The method was validated using EMA and FDA guidelines.ResultsAnalyte stability has been evaluated and described in detail. The method successfully fulfilled the validation process and it was demonstrated that, when possible, sample thermal inactivation could be a good choice in order to improve biosafety.ConclusionsThis method represents a useful tool for studying remdesivir and GS-441524 clinical pharmacokinetics, particularly during the current COVID-19 outbreak.

Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency

Effective treatments for coronavirus disease 2019 (COVID-19) are urgently needed to control this current pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Replication of SARS-CoV-2 depends on the viral RNA-dependent RNA polymerase (RdRp), which is the likely target of the investigational nucleotide analogue remdesivir (RDV). RDV shows broad-spectrum antiviral activity against RNA viruses, and previous studies with RdRps from Ebola virus and Middle East respiratory syndrome coronavirus (MERS-CoV) have revealed that delayed chain termination is RDV's plausible mechanism of action. Here, we expressed and purified active SARS-CoV-2 RdRp composed of the nonstructural proteins nsp8 and nsp12. Enzyme kinetics indicated that this RdRp efficiently incorporates the active triphosphate form of RDV (RDV-TP) into RNA. Incorporation of RDV-TP at position i caused termination of RNA synthesis at position i+3. We obtained almost identical results with SARS-CoV, MERS-CoV, and SARS-CoV-2 RdRps. A unique property of RDV-TP is its high selectivity over incorporation of its natural nucleotide counterpart ATP. In this regard, the triphosphate forms of 2′-C-methylated compounds, including sofosbuvir, approved for the management of hepatitis C virus infection, and the broad-acting antivirals favipiravir and ribavirin, exhibited significant deficits. Furthermore, we provide evidence for the target specificity of RDV, as RDV-TP was less efficiently incorporated by the distantly related Lassa virus RdRp, and termination of RNA synthesis was not observed. These results collectively provide a unifying, refined mechanism of RDV-mediated RNA synthesis inhibition in coronaviruses and define this nucleotide analogue as a direct-acting antiviral.