scholarly journals Remdesivir is a delayed translocation inhibitor of SARS CoV-2 replication in vitro

2020 ◽  
Author(s):  
Jack PK Bravo ◽  
Tyler L Dangerfield ◽  
David W Taylor ◽  
Kenneth A Johnson
Keyword(s):  
Rna Polymerase ◽  
Active Site ◽  
Rational Design ◽  
Antiviral Drugs ◽  
Chain Termination ◽  
Nucleoside Analog ◽  
Rna Dependent Rna Polymerase ◽  
Polymerase Complex

Remdesivir is a nucleoside analog approved by the FDA for treatment of COVID-19. Here, we present a 3.9-Å-resolution cryoEM reconstruction of a remdesivir-stalled RNA-dependent RNA polymerase complex, revealing full incorporation of three copies of remdesivir monophosphate (RMP) and a partially incorporated fourth RMP in the active site. The structure reveals that RMP blocks RNA translocation after incorporation of three bases following RMP, resulting in delayed chain termination, which can guide the rational design of improved antiviral drugs.

scholarly journals Development of a Simple In Vitro Assay To Identify and Evaluate Nucleotide Analogs against SARS-CoV-2 RNA-Dependent RNA Polymerase

2020 ◽  
Vol 65 (1) ◽  
pp. e01508-20
Author(s):  
Gaofei Lu ◽  
Xi Zhang ◽  
Weinan Zheng ◽  
Jialei Sun ◽  
Lan Hua ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Antiviral Treatment ◽  
Screening Method ◽  
Chain Termination ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotide Analogs ◽  
Vitro Assay ◽  
Assay Conditions ◽  
Incorporation Efficiency

ABSTRACTNucleotide analogs targeting viral RNA polymerase have been proved to be an effective strategy for antiviral treatment and are promising antiviral drugs to combat the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. In this study, we developed a robust in vitro nonradioactive primer extension assay to quantitatively evaluate the efficiency of incorporation of nucleotide analogs by SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Our results show that many nucleotide analogs can be incorporated into RNA by SARS-CoV-2 RdRp and that the incorporation of some of them leads to chain termination. The discrimination values of nucleotide analogs over those of natural nucleotides were measured to evaluate the incorporation efficiency of nucleotide analog by SARS-CoV-2 RdRp. In agreement with the data published in the literature, we found that the incorporation efficiency of remdesivir-TP is higher than that of ATP and incorporation of remdesivir-TP caused delayed chain termination, which can be overcome by higher concentrations of the next nucleotide to be incorporated. Our data also showed that the delayed chain termination pattern caused by remdesivir-TP incorporation is different for different template sequences. Multiple incorporations of remdesivir-TP caused chain termination under our assay conditions. Incorporation of sofosbuvir-TP is very low, suggesting that sofosbuvir may not be very effective in treating SARS-CoV-2 infection. As a comparison, 2′-C-methyl-GTP can be incorporated into RNA efficiently, and the derivative of 2′-C-methyl-GTP may have therapeutic application in treating SARS-CoV-2 infection. This report provides a simple screening method that should be useful for evaluating nucleotide-based drugs targeting SARS-CoV-2 RdRp and for studying the mechanism of action of selected nucleotide analogs.

Development of a simple in vitro assay to identify and evaluate nucleotide analogs against SARS-CoV-2 RNA-dependent RNA polymerase

2020 ◽  
Author(s):  
Gaofei Lu ◽  
Xi Zhang ◽  
Weinan Zheng ◽  
Jialei Sun ◽  
Lan Hua ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Mechanism Of Action ◽  
Antiviral Treatment ◽  
Chain Termination ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotide Analogs ◽  
Vitro Assay ◽  
Nucleotide Analog ◽  
Incorporation Efficiency

AbstractNucleotide analogs targeting viral RNA polymerase have been approved to be an effective strategy for antiviral treatment and are attracting antiviral drugs to combat the current SARS-CoV-2 pandemic. In this report, we develop a robust in vitro nonradioactive primer extension assay to evaluate the incorporation efficiency of nucleotide analog by SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) quantitively. Our results show that many nucleotide analogs can be incorporated into RNA by SARS-CoV-2 RdRp, and that the incorporation of some of them leads to chain termination. The discrimination values of nucleotide analog over those of natural nucleotide were measured to evaluate the incorporation efficiency of nucleotide analog by RdRp. We found that the incorporation efficiency of Remdesivir-TP is higher than ATP, and we did not observe chain termination or delayed chain termination caused by single Remdesivir-TP incorporation, while multiple incorporations of Remdesivir-TP caused chain termination in our assay condition. The incorporation efficiency of Ribavirin-TP and Favipiravir-TP is very low either as ATP or GTP analogs, which suggested that mutagenesis may not be the mechanism of action of those two drugs against SARS-CoV-2. Incorporation of Sofosbuvir-TP is also very low suggesting that sofosbuvir may not be very effective in treating SARS-CoV-2 infection. As a comparison, 2’-C-Methyl-GTP can be incorporated into RNA efficiently, and the derivative of 2’-C-Methyl-GTP may have therapeutic application in treating SARS-CoV-2 infection. This report provides a simple screening method that should be useful in evaluating nucleotide-based drugs targeting SARS-CoV-2 RdRp, and for studying the mechanism of action of selected nucleotide analog.

scholarly journals Bioactive Molecules of Tea as Potential Inhibitors for RNA-Dependent RNA Polymerase of SARS-CoV-2

2021 ◽  
Vol 8 ◽  
Author(s):  
Vijay Kumar Bhardwaj ◽  
Rahul Singh ◽  
Jatin Sharma ◽  
Vidya Rajendran ◽  
Rituraj Purohit ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Drug Repurposing ◽  
Docking Studies ◽  
Antiviral Drugs ◽  
Bioactive Molecules ◽  
Rna Dependent Rna Polymerase ◽  
Dynamics Simulations ◽  
Potential Inhibitors

The coronavirus disease (COVID-19), a worldwide pandemic, is caused by the severe acute respiratory syndrome-corona virus-2 (SARS-CoV-2). At this moment in time, there are no specific therapeutics available to combat COVID-19. Drug repurposing and identification of naturally available bioactive molecules to target SARS-CoV-2 are among the key strategies to tackle the notorious virus. The enzyme RNA-dependent RNA polymerase (RdRp) performs a pivotal role in replicating the virus. RdRp is a prime target for Remdesivir and other nucleotides analog-based antiviral drugs. In this study, we showed three bioactive molecules from tea (epicatechin-3,5-di-O-gallate, epigallocatechin-3,5-di-O-gallate, and epigallocatechin-3,4-di-O-gallate) that showed better interaction with critical residues present at the catalytic center and the NTP entry channel of RdRp than antiviral drugs Remdesivir and Favipiravir. Our computational approach to identify these molecules included molecular docking studies, followed by robust molecular dynamics simulations. All the three molecules are readily available in tea and could be made accessible along with other medications to treat COVID-19 patients. However, these results require validation by further in vitro and in vivo studies.

Recent Advances towards Drug Design Targeting the Protease of 2019 novel coronavirus (2019-nCoV)

2020 ◽  
Vol 27 ◽  
Author(s):  
Sehrish Bano ◽  
Abdul Hameed ◽  
Mariya Al-Rashida ◽  
Shafia Iftikhar ◽  
Jamshed Iqbal
Keyword(s):  
Drug Design ◽  
Rna Polymerase ◽  
Drug Targets ◽  
Antiviral Agents ◽  
Structural Features ◽  
Antiviral Drugs ◽  
Rna Dependent Rna Polymerase ◽  
Mortality And Morbidity ◽  
Functional Relationships ◽  
Novel Coronavirus

Background: The 2019 novel coronavirus (2019-nCoV), also known as coronavirus 2 (SARS-CoV-2) acute respiratory syndrome has recently emerged and continued to spread rapidly with high level of mortality and morbidity rates. Currently, no efficacious therapy is available to relieve coronavirus infections. As new drug design and development takes much time, there is a possibility to find an effective treatment from existing antiviral agents. Objective: In this case, there is a need to find out the relationship between possible drug targets and mechanism of action of antiviral drugs. This review discusses about the efforts to develop drug from known or new molecules. Methods: Viruses usually have two structural integrities, proteins and nucleic acids, both of which can be possible drug targets. Herein, we systemically discuss the structural-functional relationships of the spike, 3-chymotrypsin-like protease (3CLpro), papain like protease (PLpro) and RNA-dependent RNA polymerase (RdRp), as these are prominent structural features of corona virus. Certain antiviral drugs such as Remdesivir are RNA dependent RNA polymerase inhibitor. It has the ability to terminate RNA replication by inhibiting ATP. Results: It is reported that ATP is involved in synthesis of coronavirus non-structural proteins from 3CLpro and PLpro. Similarly, mechanisms of action of many other antiviral agents has been discussed in this review. It will provide new insights into the mechanism of inhibition, and let us develop new therapeutic antiviral approaches against novel SARS-CoV-2 coronavirus. Conclusion: In conclusion, this review summarizes recent progress in developing protease inhibitors for SARS-CoV-2.

scholarly journals Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

2016 ◽  
Vol 61 (3) ◽  
Author(s):  
Gaofei Lu ◽  
Gregory R. Bluemling ◽  
Paul Collop ◽  
Michael Hager ◽  
Damien Kuiper ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Zika Virus ◽  
Nonstructural Protein ◽  
Rna Dependent Rna Polymerase ◽  
Antiviral Compounds ◽  
Double Stranded Dna ◽  
Virus Rna ◽  
Potential Inhibitors

ABSTRACT Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an in vitro nonradioactive primer extension assay employing a fluorescently labeled primer-template pair. Both purified NS5 and NS5pol can carry out in vitro RNA-dependent RNA synthesis in this assay. Our results show that Mn2+ is required for enzymatic activity, while Mg2+ is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5′-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2′-C-methyl- and 2′-C-ethynyl-substituted analog 5′-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of in vitro polymerase assays in the identification of potential ZIKV inhibitors.

scholarly journals RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1738
Author(s):  
Alesia A. Levanova ◽  
Eeva J. Vainio ◽  
Jarkko Hantula ◽  
Minna M. Poranen
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Rna Virus ◽  
Polymerization Reaction ◽  
Rna Dependent Rna Polymerase ◽  
Independent Manner ◽  
Nucleotidyl Transferase ◽  
Rna Polymerization ◽  
The Family

Heterobasidion RNA virus 6 (HetRV6) is a double-stranded (ds)RNA mycovirus and a member of the recently established genus Orthocurvulavirus within the family Orthocurvulaviridae. The purpose of the study was to determine the biochemical requirements for RNA synthesis catalyzed by HetRV6 RNA-dependent RNA polymerase (RdRp). HetRV6 RdRp was expressed in Escherichia coli and isolated to near homogeneity using liquid chromatography. The enzyme activities were studied in vitro using radiolabeled UTP. The HetRV6 RdRp was able to initiate RNA synthesis in a primer-independent manner using both virus-related and heterologous single-stranded (ss)RNA templates, with a polymerization rate of about 46 nt/min under optimal NTP concentration and temperature. NTPs with 2′-fluoro modifications were also accepted as substrates in the HetRV6 RdRp-catalyzed RNA polymerization reaction. HetRV6 RdRp transcribed viral RNA genome via semi-conservative mechanism. Furthermore, the enzyme demonstrated terminal nucleotidyl transferase (TNTase) activity. Presence of Mn2+ was required for the HetRV6 RdRp catalyzed enzymatic activities. In summary, our study shows that HetRV6 RdRp is an active replicase in vitro that can be potentially used in biotechnological applications, molecular biology, and biomedicine.

scholarly journals The human H5N1 influenza A virus polymerase complex is active in vitro over a broad range of temperatures, in contrast to the WSN complex, and this property can be attributed to the PB2 subunit

2008 ◽  
Vol 89 (12) ◽  
pp. 2923-2932 ◽  
Author(s):  
Birgit G. Bradel-Tretheway ◽  
Z. Kelley ◽  
Shikha Chakraborty-Sett ◽  
Toru Takimoto ◽  
Baek Kim ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Influenza A Virus ◽  
Influenza A ◽  
Elevated Temperatures ◽  
Expression System ◽  
Lung Epithelial Cells ◽  
Upper Respiratory Tract ◽  
Polymerase Complex ◽  
H5n1 Influenza

Influenza A virus (IAV) replicates in the upper respiratory tract of humans at 33 °C and in the intestinal tract of birds at close to 41 °C. The viral RNA polymerase complex comprises three subunits (PA, PB1 and PB2) and plays an important role in host adaptation. We therefore developed an in vitro system to examine the temperature sensitivity of IAV RNA polymerase complexes from different origins. Complexes were prepared from human lung epithelial cells (A549) using a novel adenoviral expression system. Affinity-purified complexes were generated that contained either all three subunits (PA/PB1/PB2) from the A/Viet/1203/04 H5N1 virus (H/H/H) or the A/WSN/33 H1N1 strain (W/W/W). We also prepared chimeric complexes in which the PB2 subunit was exchanged (H/H/W, W/W/H) or substituted with an avian PB2 from the A/chicken/Nanchang/3-120/01 H3N2 strain (W/W/N). All complexes were functional in transcription, cap-binding and endonucleolytic activity. Complexes containing the H5N1 or Nanchang PB2 protein retained transcriptional activity over a broad temperature range (30–42 °C). In contrast, complexes containing the WSN PB2 protein lost activity at elevated temperatures (39 °C or higher). The E627K mutation in the avian PB2 was not required for this effect. Finally, the avian PB2 subunit was shown to confer enhanced stability to the WSN 3P complex. These results show that PB2 plays an important role in regulating the temperature optimum for IAV RNA polymerase activity, possibly due to effects on the functional stability of the 3P complex.

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