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A correction to the article "Nucleoside Diphosphate Kinase and the Activation of Antiviral Phosphonate Analogs of Nucleotides: Binding Mode and Phosphorylation of Tenofovir Derivatives," that was published in volume 28 issue is presented.

Remdesivir (RDV) is a direct-acting antiviral agent that is approved in several countries for the treatment of Coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2. RDV exhibits broad-spectrum antiviral activity against positive-sense RNA viruses, for example, severe acute respiratory syndrome coronavirus and hepatitis C virus, and nonsegmented negative-sense RNA viruses, for example, Nipah virus, whereas segmented negative-sense RNA viruses such as influenza virus or Crimean-Congo hemorrhagic fever virus are not sensitive to the drug. The reasons for this apparent efficacy pattern are unknown. Here, we expressed and purified representative RNA-dependent RNA polymerases and studied three biochemical parameters that have been associated with the inhibitory effects of RDV-triphosphate (TP): (i) selective incorporation of the nucleotide substrate RDV-TP, (ii) the effect of the incorporated RDV-monophosphate (MP) on primer extension, and (iii) the effect of RDV-MP in the template during incorporation of the complementary UTP. We found a strong correlation between antiviral effects and efficient incorporation of RDV-TP. Inhibition in primer extension reactions was heterogeneous and usually inefficient at higher NTP concentrations. In contrast, template-dependent inhibition of UTP incorporation opposite the embedded RDV-MP was seen with all polymerases. Molecular modeling suggests a steric conflict between the 1'-cyano group of the inhibitor and residues of the structurally conserved RNA-dependent RNA polymerase motif F. We conclude that future efforts in the development of nucleotide analogs with a broader spectrum of antiviral activities should focus on improving rates of incorporation while capitalizing on the inhibitory effects of a bulky 1'-modification. [ABSTRACT FROM AUTHOR]

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 (MERSCoV) 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'-Cmethylated compounds, including sofosbuvir, approved for the management of hepatitisCvirus 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 ofRNAsynthesis 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. [ABSTRACT FROM AUTHOR]

Acute respiratory viral infections, such as pneumovirus and respiratory picornavirus infections, exacerbate disease in COPD and asthma patients. A research program targeting respiratory syncytial virus (RSV) led to the discovery of GS-7682 ( 1 ), a novel phosphoramidate prodrug of a 4'-CN-4-aza-7,9-dideazaadenosine C -nucleoside GS-646089 ( 2 ) with broad antiviral activity against RSV (EC 50 = 3-46 nM), human metapneumovirus (EC 50 = 210 nM), human rhinovirus (EC 50 = 54-61 nM), and enterovirus (EC 50 = 83-90 nM). Prodrug optimization for cellular potency and lung cell metabolism identified 5'-methyl [( S )-hydroxy(phenoxy)phosphoryl]-l-alaninate in combination with 2',3'-diisobutyrate promoieties as being optimal for high levels of intracellular triphosphate formation in vitro and in vivo . 1 demonstrated significant reductions of viral loads in the lower respiratory tract of RSV-infected African green monkeys when administered once daily via intratracheal nebulized aerosol. Together, these findings support additional evaluation of 1 and its analogues as potential therapeutics for pneumo- and picornaviruses.

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 (RdRps) of prototypic respiratory viruses. GS-646939 is the active 5'-triphosphate metabolite of a 4'-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 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 and human metapneumovirus incorporate GS-646939 and ATP with similar efficiency. In contrast, influenza B RdRp shows a clear preference for ATP and human mitochondrial RNA polymerase 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 4'-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, 1'-cyano and 4'-cyano modifications of nucleotide analogs provide complementary strategies to target the polymerase of several families of respiratory RNA viruses., Competing Interests: Conflict of interest M. G. received funding from Gilead Sciences in support for studies on the mechanism of action of nucleotide analog polymerase inhibitors. J. P., D. S. S., J. K. P., J. Y. F., and J. P. B. are current or former Gilead employees., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Despite the wide availability of several safe and effective vaccines that prevent severe COVID-19, the persistent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that can evade vaccine-elicited immunity remains a global health concern. In addition, the emergence of SARS-CoV-2 VOCs that can evade therapeutic monoclonal antibodies underscores the need for additional, variant-resistant treatment strategies. Here, we characterize the antiviral activity of GS-5245, obeldesivir (ODV), an oral prodrug of the parent nucleoside GS-441524, which targets the highly conserved viral RNA-dependent RNA polymerase (RdRp). We show that GS-5245 is broadly potent in vitro against alphacoronavirus HCoV-NL63, SARS-CoV, SARS-CoV-related bat-CoV RsSHC014, Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2 WA/1, and the highly transmissible SARS-CoV-2 BA.1 Omicron variant. Moreover, in mouse models of SARS-CoV, SARS-CoV-2 (WA/1 and Omicron B1.1.529), MERS-CoV, and bat-CoV RsSHC014 pathogenesis, we observed a dose-dependent reduction in viral replication, body weight loss, acute lung injury, and pulmonary function with GS-5245 therapy. Last, we demonstrate that a combination of GS-5245 and main protease (M pro ) inhibitor nirmatrelvir improved outcomes in vivo against SARS-CoV-2 compared with the single agents. Together, our data support the clinical evaluation of GS-5245 against coronaviruses that cause or have the potential to cause human disease.

Remdesivir 1 is an phosphoramidate prodrug that releases the monophosphate of nucleoside GS-441524 ( 2 ) into lung cells, thereby forming the bioactive triphosphate 2-NTP . 2-NTP , an analog of ATP, inhibits the SARS-CoV-2 RNA-dependent RNA polymerase replication and transcription of viral RNA. Strong clinical results for 1 have prompted interest in oral approaches to generate 2-NTP . Here, we describe the discovery of a 5'-isobutyryl ester prodrug of 2 (GS-5245, Obeldesivir, 3 ) that has low cellular cytotoxicity and 3-7-fold improved oral delivery of 2 in monkeys. Prodrug 3 is cleaved presystemically to provide high systemic exposures of 2 that overcome its less efficient metabolism to 2-NTP , leading to strong SARS-CoV-2 antiviral efficacy in an African green monkey infection model. Exposure-based SARS-CoV-2 efficacy relationships resulted in an estimated clinical dose of 350-400 mg twice daily. Importantly, all SARS-CoV-2 variants remain susceptible to 2 , which supports development of 3 as a promising COVID-19 treatment.

Remdesivir (GS-5734; VEKLURY) is a single diastereomer monophosphoramidate prodrug of an adenosine analog (GS-441524). Remdesivir is taken up by target cells and metabolized in multiple steps to form the active nucleoside triphosphate (GS-443902), which acts as a potent inhibitor of viral RNA-dependent RNA polymerases. Remdesivir and GS-441524 have antiviral activity against multiple RNA viruses. Here, we expand the evaluation of remdesivir's antiviral activity to members of the families Flaviviridae, Picornaviridae, Filoviridae, Orthomyxoviridae, and Hepadnaviridae. Using cell-based assays, we show that remdesivir can inhibit infection of flaviviruses (such as dengue 1-4, West Nile, yellow fever, Zika viruses), picornaviruses (such as enterovirus and rhinovirus), and filoviruses (such as various Ebola, Marburg, and Sudan virus isolates, including novel geographic isolates), but is ineffective or is significantly less effective against orthomyxoviruses (influenza A and B viruses), or hepadnaviruses B, D, and E. In addition, remdesivir shows no antagonistic effect when combined with favipiravir, another broadly acting antiviral nucleoside analog, and has minimal interaction with a panel of concomitant medications. Our data further support remdesivir as a broad-spectrum antiviral agent that has the potential to address multiple unmet medical needs, including those related to antiviral pandemic preparedness., (© 2023. The Author(s).)

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 remdesivir 2 . 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 triphosphate 3,4 . 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 propagation 5 . The NiRAN selectively binds guanosine triphosphate, strengthening proposals for the role of this domain in the formation of the 5' RNA cap 6 ., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Some of the most efficacious antiviral therapeutics are ribonucleos(t)ide analogs. The presence of a 3'-to-5' proofreading exoribonuclease (ExoN) in coronaviruses diminishes the potency of many ribonucleotide analogs. The ability to interfere with ExoN activity will create new possibilities for control of SARS-CoV-2 infection. ExoN is formed by a 1:1 complex of nsp14 and nsp10 proteins. We have purified and characterized ExoN using a robust, quantitative system that reveals determinants of specificity and efficiency of hydrolysis. Double-stranded RNA is preferred over single-stranded RNA. Nucleotide excision is distributive, with only one or two nucleotides hydrolyzed in a single binding event. The composition of the terminal basepair modulates excision. A stalled SARS-CoV-2 replicase in complex with either correctly or incorrectly terminated products prevents excision, suggesting that a mispaired end is insufficient to displace the replicase. Finally, we have discovered several modifications to the 3'-RNA terminus that interfere with or block ExoN-catalyzed excision. While a 3'-OH facilitates hydrolysis of a nucleotide with a normal ribose configuration, this substituent is not required for a nucleotide with a planar ribose configuration such as that present in the antiviral nucleotide produced by viperin. Design of ExoN-resistant, antiviral ribonucleotides should be feasible., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Chronic hepatitis B (CHB) is a global health care challenge and a major cause of liver disease. To find new therapeutic avenues with a potential to functionally cure chronic Hepatitis B virus (HBV) infection, we performed a focused screen of epigenetic modifiers to identify potential inhibitors of replication or gene expression. From this work we identified isonicotinic acid inhibitors of the histone lysine demethylase 5 (KDM5) with potent anti-HBV activity. To enhance the cellular permeability and liver accumulation of the most potent KDM5 inhibitor identified (GS-080) an ester prodrug was developed (GS-5801) that resulted in improved bioavailability and liver exposure as well as an increased H3K4me3:H3 ratio on chromatin. GS-5801 treatment of HBV-infected primary human hepatocytes reduced the levels of HBV RNA, DNA and antigen. Evaluation of GS-5801 antiviral activity in a humanized mouse model of HBV infection, however, did not result in antiviral efficacy, despite achieving pharmacodynamic levels of H3K4me3:H3 predicted to be efficacious from the in vitro model. Here we discuss potential reasons for the disconnect between in vitro and in vivo efficacy, which highlight the translational difficulties of epigenetic targets for viral diseases., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Gilmore et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The therapeutic targeting of the nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase (RdRp) of the Hepatitis C Virus (HCV) with nucleotide analogs led to a deep understanding of this enzymes structure, function and substrate specificity. Unlike previously studied DNA polymerases including the reverse transcriptase of Human Immunodeficiency Virus, development of biochemical assays for HCV RdRp proved challenging due to low solubility of the full-length protein and inefficient acceptance of exogenous primer/templates. Despite the poor apparent specific activity, HCV RdRp was found to support rapid and processive transcription once elongation is initiated in vitro consistent with its high level of viral replication in the livers of patients. Understanding of the substrate specificity of HCV RdRp led to the discovery of the active triphosphate of sofosbuvir as a nonobligate chain-terminator of viral RNA transcripts. The ternary crystal structure of HCV RdRp, primer/template, and incoming nucleotide showed the interaction between the nucleotide analog and the 2'-hydroxyl binding pocket and how an unfit mutation of serine 282 to threonine results in resistance by interacting with the uracil base and modified 2'-position of the analog. Host polymerases mediate off-target toxicity of nucleotide analogs and the active metabolite of sofosbuvir was found to not be efficiently incorporated by host polymerases including the mitochondrial RNA polymerase (POLRMT). Knowledge from studying inhibitors of HCV RdRp serves to advance antiviral drug discovery for other emerging RNA viruses including the discovery of remdesivir as an inhibitor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), the virus that causes COVID-19., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Middle East respiratory syndrome coronavirus (MERS-CoV) is the causative agent of a severe respiratory disease associated with more than 2468 human infections and over 851 deaths in 27 countries since 2012. There are no approved treatments for MERS-CoV infection although a combination of lopinavir, ritonavir and interferon beta (LPV/RTV-IFNb) is currently being evaluated in humans in the Kingdom of Saudi Arabia. Here, we show that remdesivir (RDV) and IFNb have superior antiviral activity to LPV and RTV in vitro. In mice, both prophylactic and therapeutic RDV improve pulmonary function and reduce lung viral loads and severe lung pathology. In contrast, prophylactic LPV/RTV-IFNb slightly reduces viral loads without impacting other disease parameters. Therapeutic LPV/RTV-IFNb improves pulmonary function but does not reduce virus replication or severe lung pathology. Thus, we provide in vivo evidence of the potential for RDV to treat MERS-CoV infections.

Bruton's tyrosine kinase (BTK) is a clinically validated target for B-cell leukemias and lymphomas with FDA-approved small-molecule inhibitors ibrutinib and acalabrutinib. Tirabrutinib (GS-4059/ONO-4059, Gilead Sciences, Inc., Foster City, CA) is a second-generation, potent, selective, irreversible BTK inhibitor in clinical development for lymphoid malignancies, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL). An accurate pharmacodynamic assay to assess tirabrutinib target coverage in phase 1/2 clinical studies will inform dose and schedule selection for advanced clinical evaluation. We developed a novel duplex homogeneous BTK occupancy assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) to measure free and total BTK levels in a multiplexed format. The dual-wavelength emission property of terbium-conjugated anti-BTK antibody served as the energy donor for two fluorescent energy acceptors with distinct excitation and emission spectra. The assay was characterized and qualified using full-length purified recombinant human BTK protein and peripheral blood mononuclear cells derived from healthy volunteers and patients with CLL. We demonstrated assay utility using cells derived from lymph node and bone marrow samples from patients with CLL and DLBCL. Our TR-FRET-based BTK occupancy assay provides accurate, quantitative assessment of BTK occupancy in the clinical trial program for tirabrutinib and is in use in ongoing clinical studies.

Background: Sofosbuvir is a nucleoside analogue inhibitor of the HCV NS5B polymerase approved for treatment of HCV-infected patients in combination with ribavirin or with other antivirals. It has activity against all genotypes of HCV. Resistance to sofosbuvir in genotype-1 and -2 HCV is conferred by the S282T substitution in NS5B., Methods: To begin to define the correlates of resistance to sofosbuvir in other genotypes, we performed selection experiments in cell culture using cell lines containing subgenomic replicons derived from genotypes-1b, -2a, -3a and -4a, or chimeric replicons in a genotype-1b background but encoding genotype-2b, -5a and -6a NS5B polymerase., Results: In every case, S282T was selected following passage in the presence of increasing concentrations of sofosbuvir for 10 to 15 weeks. When introduced as a site-directed mutant, S282T conferred reductions in sofosbuvir susceptibility of between 2.4 and 19.4-fold. Other substitutions observed during the selections had relatively less impact on susceptibility, such as N237S in genotype-6a (2.5-fold). Replication capacity was affected by the introduction of S282T in all genotypes to variable extents (3.2% to 22% of wild type)., Conclusions: These results confirm that S282T is the primary sofosbuvir resistance-associated substitution and that replication capacity is reduced when it is present in all genotypes of HCV.

Hepatitis C virus (HCV) infection presents an unmet medical need requiring more effective treatment options. Nucleoside inhibitors (NI) of HCV polymerase (NS5B) have demonstrated pan-genotypic activity and durable antiviral response in the clinic, and they are likely to become a key component of future treatment regimens. NI candidates that have entered clinical development thus far have all been N-nucleoside derivatives. Herein, we report the discovery of a C-nucleoside class of NS5B inhibitors. Exploration of adenosine analogs in this class identified 1'-cyano-2'-C-methyl 4-aza-7,9-dideaza adenosine as a potent and selective inhibitor of NS5B. A monophosphate prodrug approach afforded a series of compounds showing submicromolar activity in HCV replicon assays. Further pharmacokinetic optimization for sufficient oral absorption and liver triphosphate loading led to identification of a clinical development candidate GS-6620. In a phase I clinical study, the potential for potent activity was demonstrated but with high intra- and interpatient pharmacokinetic and pharmacodynamic variability.

Background: The K65R mutation in human immunodeficiency virus type 1 reverse transcriptase can be selected by abacavir, didanosine, tenofovir, and stavudine in vivo resulting in reduced susceptibility to these drugs and decreased viral replication capacity. In clinical isolates, K65R is frequently accompanied by the A62V and S68G reverse transcriptase mutations., Methods: The role of A62V and S68G in combination with K65R was investigated using phenotypic, viral growth competition, pre-steady-state kinetic, and excision analyses., Results: Addition of A62V and S68G to K65R caused no significant change in human immunodeficiency virus type 1 resistance to abacavir, didanosine, tenofovir, or stavudine but partially restored the replication defect of virus containing K65R. The triple mutant K65R+A62V+S68G still showed some replication defect compared with wild-type virus. Pre-steady-state kinetic analysis demonstrated that K65R resulted in a decreased rate of incorporation (kpol) for all natural dNTPs, which were partially restored to wild-type levels by addition of A62V and S68G. When added to K65R and S68G, the A62V mutation seemed to restore adenosine triphosphate-mediated excision of tenofovir to wild-type levels., Conclusions: A62V and S68G serve as partial compensatory mutations for the K65R mutation in reverse transcriptase by improving the viral replication capacity, which is likely due to increased incorporation efficiency of the natural substrates.

Mitochondrial toxicity is a limiting factor in the use of some nucleoside reverse transcriptase inhibitors of HIV. To further understand the impact of structural features on the incorporation and exonuclease removal of nucleoside monophosphate (MP) analogues by human mitochondrial DNA polymerase (pol gamma), transient kinetic studies were done with analogues of 2'-deoxyguanosine triphosphate. The kinetic parameters for the incorporation and removal of carbovir (CBV)-MP, dioxolane guanosine (DXG)-MP and 2',3'-dideoxy-2',3'-didehydroguanosine (d4G)-MP were studied with pol gamma holoenzyme. The importance of the ribose oxygen in incorporation by pol gamma was illustrated by an approximate 3,000-fold decrease in the incorporation efficiency of an analogue lacking the ribose oxygen (CBV-TP) relative to those containing a ribose oxygen (DXG-TP and d4G-TP). As a result, a comparison with previous data for the incorporation by HIV reverse transcriptase showed CBV-TP to be approximately 800-8,000-fold more selective for its antiviral target over pol gamma relative to the other guanosine analogues. However, DXG-TP and d4G-TP were found to be much more selective than previously reported values for mitochondrial toxic nucleoside analogues. Structural modelling based on sequence homology with other polymerase A family members suggests that an interaction between the ribose oxygen and arginine 853 in pol gamma may play a critical role in causing this differential incorporation. Exonuclease removal of a chain-terminating CBV-MP was also found to be more efficient by pol gamma. These results help to further elucidate the structure activity relationships for pol gamma and should aid in the design of more selective antiviral agents.

During mitochondrial DNA (mtDNA) replication, DNA/RNA heteroduplex intermediates are formed. To understand how and why ribonucleotides are involved in mtDNA replication, we have studied the novel RNA-associated activities of human mitochondrial DNA polymerase (Pol gamma), including reverse transcription, RNA-directed 3' --> 5' DNA excision, RNA-primed DNA synthesis, and ribonucleotide incorporation. Remarkably, Pol gamma catalyzes reverse transcription with a slightly higher efficiency than HIV-1 reverse transcriptase, suggesting that the activity may be physiologically significant, and furthermore, proofreading activity with an RNA template was also observed. RNA-primed DNA synthesis activity is required for initiation of mtDNA replication, and we have found that Pol gamma holoenzyme is capable of performing this reaction at a physiologically relevant rate and that the accessory subunit plays an essential role in the initiation steps. Single ribonucleotides have been found scattered in the mtDNA genome, although their role and significance are not yet defined. Our finding that Pol gamma also incorporates ribonucleotide triphosphates into a DNA primer offers a plausible enzymatic pathway for the origin of the RNA-containing mtDNA genome.