scholarly journals Signatures of Nucleotide Analog Incorporation by an RNA-Dependent RNA Polymerase Revealed Using High-Throughput Magnetic Tweezers

Cell Reports ◽  
2017 ◽  
Vol 21 (4) ◽  
pp. 1063-1076 ◽  
Author(s):  
David Dulin ◽  
Jamie J. Arnold ◽  
Theo van Laar ◽  
Hyung-Suk Oh ◽  
Cheri Lee ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
High Throughput ◽  
Magnetic Tweezers ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotide Analog

scholarly journals The nucleotide addition cycle of the SARS-CoV-2 polymerase

2021 ◽  
Author(s):  
Subhas Chandra Bera ◽  
Mona Seifert ◽  
Robert N. Kirchdoerfer ◽  
Pauline van Nies ◽  
Yibulayin Wubulikasimu ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Rna Polymerase ◽  
Human Health ◽  
High Throughput ◽  
Magnetic Tweezers ◽  
Power Stroke ◽  
Rna Dependent Rna Polymerase ◽  
The Core ◽  
Nucleotide Addition ◽  
Important Structure

AbstractCoronaviruses have evolved elaborate multisubunit machines to replicate and transcribe their genomes. Central to these machines are the RNA-dependent RNA polymerase subunit (nsp12) and its intimately associated cofactors (nsp7 and nsp8). We have used a high-throughput magnetic-tweezers approach to develop a mechanochemical description of this core polymerase. The core polymerase exists in at least three catalytically distinct conformations, one being kinetically consistent with incorporation of incorrect nucleotides. We provide the first evidence that an RdRp uses a thermal ratchet instead of a power stroke to transition from the pre- to post-translocated state. Ultra-stable magnetic tweezers enables the direct observation of coronavirus polymerase deep and long-lived backtrack that are strongly stimulated by secondary structure in the template. The framework presented here elucidates one of the most important structure-dynamics-function relationships in human health today, and will form the grounds for understanding the regulation of this complex.

scholarly journals High-throughput identification of compounds targeting influenza RNA-dependent RNA polymerase activity

2010 ◽  
Vol 107 (45) ◽  
pp. 19151-19156 ◽  
Author(s):  
C.-Y. Su ◽  
T.-J. R. Cheng ◽  
M.-I. Lin ◽  
S.-Y. Wang ◽  
W.-I. Huang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
High Throughput ◽  
Polymerase Activity ◽  
Rna Dependent Rna Polymerase ◽  
Rna Polymerase Activity

scholarly journals Development of remdesivir repositioning as a nucleotide analog against COVID-19 RNA dependent RNA polymerase

2020 ◽  
pp. 1-9 ◽  
Author(s):  
Mohammad Mahdi Nejadi Babadaei ◽  
Anwarul Hasan ◽  
Yasaman Vahdani ◽  
Samir Haj Bloukh ◽  
Majid Sharifi ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotide Analog

scholarly journals Working Mechanism of Remdesivir and Discovering New Potential Inhibitors to SARS-CoV-2 RNA-Dependent RNA Polymerase (RdRp) using High-Throughput Virtual Screening and Molecular Dynamics Simulations .

2021 ◽  
Author(s):  
Dylan Brunt ◽  
Phillip Lakernick ◽  
CHUN WU
Keyword(s):  
Molecular Dynamics ◽  
Free Energy ◽  
Virtual Screening ◽  
Rna Polymerase ◽  
High Throughput ◽  
Binding Free Energy ◽  
Free Energy Calculations ◽  
Rna Dependent Rna Polymerase ◽  
Zinc Database ◽  
High Throughput Virtual Screening

Abstract RNA-dependent RNA polymerase (RdRp), is an enzyme essential component in the RNA replication within the life cycle of the severely acute respiratory coronavirus-2 (SARS-CoV-2), causing the deadly respiratory induced sickness COVID-19. Remdesivir is a prodrug that has seen some success in inhibiting this enzyme, however there is still the pressing need for effective alternatives. In this study, we present the discovery of four non-nucleoside small molecules that bind favorably to RdRp over adenosine-triphosphate (ATP) and active-form remdesivir-triphosphate (RTP) using high-throughput virtual screening (HTVS) coupled with extensive (total 4800 ns) molecular dynamics (MD) simulations with using the ZINC compounds database against SARS-CoV-2 RdRp (PDB: 7BV2). We found that the simulations with both ATP and RTP remained stable for the duration of their trajectories, and it was revealed that the phosphate tail of RTP was stabilized by a positive amino acid pocket near the entry channel of RTP and magnesium ions containing residues K551, R553, R555 and K621. It was also found that residues D623, D760, and N691 further stabilized the ribose portion of RTP with U10 on the template RNA strand forming hydrogen pairs with the adenosine motif. Using these models of RdRp, we employed them to screen the ZINC database of ~17 million molecules. Using docking and drug properties scoring, we narrowed down our selection to fourteen candidates. These were subjected to 200 ns simulations each underwent free energy calculations. We identified four hit compounds from the ZINC database that have similar binding poses to RTP while possessing lower overall binding free energies, with ZINC097971592 having a binding free energy two times lower than RTP.

scholarly journals Potential Novel Thioether-Amide or Guanidine-Linker Class of SARS-CoV-2 Virus RNA-Dependent RNA Polymerase Inhibitors Identified by High-Throughput Virtual Screening Coupled to Free-Energy Calculations

2021 ◽  
Vol 22 (20) ◽  
pp. 11143
Author(s):  
Marko Jukič ◽  
Dušanka Janežič ◽  
Urban Bren
Keyword(s):  
Virtual Screening ◽  
Rna Polymerase ◽  
High Throughput ◽  
Biological Evaluation ◽  
Free Energy Calculations ◽  
Rna Dependent Rna Polymerase ◽  
Linear Interaction ◽  
Energy Calculations ◽  
High Throughput Virtual Screening

SARS-CoV-2, or severe acute respiratory syndrome coronavirus 2, represents a new pathogen from the family of Coronaviridae that caused a global pandemic of COVID-19 disease. In the absence of effective antiviral drugs, research of novel therapeutic targets such as SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) becomes essential. This viral protein is without a human counterpart and thus represents a unique prospective drug target. However, in vitro biological evaluation testing on RdRp remains difficult and is not widely available. Therefore, we prepared a database of commercial small-molecule compounds and performed an in silico high-throughput virtual screening on the active site of the SARS-CoV-2 RdRp using ensemble docking. We identified a novel thioether-amide or guanidine-linker class of potential RdRp inhibitors and calculated favorable binding free energies of representative hits by molecular dynamics simulations coupled with Linear Interaction Energy calculations. This innovative procedure maximized the respective phase-space sampling and yielded non-covalent inhibitors representing small optimizable molecules that are synthetically readily accessible, commercially available as well as suitable for further biological evaluation and mode of action studies.

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 Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Yushen Du ◽  
Nicholas C. Wu ◽  
Lin Jiang ◽  
Tianhao Zhang ◽  
Danyang Gong ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Influenza A Virus ◽  
Structure Analysis ◽  
High Throughput ◽  
Influenza A ◽  
Sequence Conservation ◽  
Rna Dependent Rna Polymerase ◽  
Multiple Functions ◽  
Structure Information ◽  
Homologous Structure

ABSTRACT Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available. IMPORTANCE To fully comprehend the diverse functions of a protein, it is essential to understand the functionality of individual residues. Current methods are highly dependent on evolutionary sequence conservation, which is usually limited by sampling size. Sequence conservation-based methods are further confounded by structural constraints and multifunctionality of proteins. Here we present a method that can systematically identify and annotate functional residues of a given protein. We used a high-throughput functional profiling platform to identify essential residues. Coupling it with homologous-structure comparison, we were able to annotate multiple functions of proteins. We demonstrated the method with the PB1 protein of influenza A virus and identified novel functional residues in addition to its canonical function as an RNA-dependent RNA polymerase. Not limited to virology, this method is generally applicable to other proteins that can be functionally selected and about which homologous-structure information is available.

Sign in / Sign up

Export Citation Format

Share Document