Remdesivir-bound and ligand-free simulations reveal the probable mechanism of inhibiting the RNA dependent RNA polymerase of severe acute respiratory syndrome coronavirus 2

Drug repurposing studies targeting inhibition of RNA dependent RNA polymerase (RdRP) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have exhibited the potential effect of small molecules. In the present work a detailed interaction study between the phytochemicals from Indian medicinal plants and the RdRP of SARS-CoV-2 has been performed. The top four phytochemicals obtained through molecular docking were, swertiapuniside, cordifolide A, sitoindoside IX, and amarogentin belonging to Swertia chirayita, Tinospora cordifolia and Withania somnifera. These ligands bound to the RdRP were further studied using molecular dynamics simulations. The principal component analysis of these systems showed significant conformational changes in the finger and thumb subdomain of the RdRP. Hydrogen bonding, salt-bridge and water mediated interactions supported by MM-GBSA free energy of binding revealed strong binding of cordifolide A and sitoindoside IX to RdRP. The ligand-interacting residues belonged to either of the seven conserved motifs of the RdRP. These residues were polar and charged amino acids, namely, ARG 553, ARG 555, ASP 618, ASP 760, ASP 761, GLU 811, and SER 814. The glycosidic moieties of the phytochemicals were observed to form favourable interactions with these residues. Hence, these phytochemicals may hold the potential to act as RdRP inhibitors owing to their stability in binding to the druggable site.

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Conformational changes in human Norovirus polymerase

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314084046 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1595-C1595

Author(s):

Kenneth Ng ◽

Dmitry Zamyatkin ◽

Hayeong Rho ◽

Elesha Hoffarth ◽

Gabriela Jurca ◽

...

Keyword(s):

Rna Polymerase ◽

Conformational Changes ◽

Rna Binding ◽

Divalent Metal ◽

Rna Dependent Rna Polymerase ◽

Human Norovirus ◽

Conformational States ◽

Crystal Forms ◽

Divalent Metal Cations ◽

Positive Strand Rna

Human Noroviruses (NV) belong in the Caliciviridae family and are a major cause of gastroenteritis outbreaks throughout the world. Crystal structures of the RNA-dependent RNA polymerase from the human Norovirus have been determined in over ten different crystal forms in the presence and absence of divalent metal cations, nucleoside triphosphates, inhibitors and primer-template duplex RNA. These structures show how the polymerase enzyme can adopt a range of conformations in which the thumb, fingers and palm domains change orientations depending on the step of the enzymatic cycle trapped in different crystal forms. We discuss how the evidence from crystallographic and biochemical experiments combine to better understand how viral RNA polymerase enzymes from human Norovirus and related positive-strand RNA viruses can adopt a range of conformational states to facilitate RNA binding, NTP binding, catalysis, RNA translocation and pyrophosphate release. The detailed structural and mechanistic understanding of these conformational changes is important for providing a sound basis for understanding viral replication in general, as well as for the design of novel inhibitors capable of trapping the enzyme in specific conformational states.

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013679 ◽

2020 ◽

Vol 295 (20) ◽

pp. 6785-6797 ◽

Cited By ~ 164

Author(s):

Calvin J. Gordon ◽

Egor P. Tchesnokov ◽

Emma Woolner ◽

Jason K. Perry ◽

Joy Y. Feng ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Rna Polymerase ◽

Rna Synthesis ◽

Ebola Virus ◽

Lassa Virus ◽

Nonstructural Proteins ◽

Rna Dependent Rna Polymerase ◽

Direct Acting Antiviral ◽

Nucleotide Analogue ◽

Direct Acting

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.

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Complementary Mutations in the N and L Proteins for Restoration of Viral RNA Synthesis

Journal of Virology ◽

10.1128/jvi.01417-18 ◽

2018 ◽

Vol 92 (22) ◽

Cited By ~ 3

Author(s):

Weike Li ◽

Ryan H. Gumpper ◽

Yusuf Uddin ◽

Ingeborg Schmidt-Krey ◽

Ming Luo

Keyword(s):

Rna Polymerase ◽

Conformational Changes ◽

Rna Synthesis ◽

Large Subunit ◽

Viral Rna ◽

Structural Motif ◽

General Mechanism ◽

Rna Dependent Rna Polymerase ◽

N Protein ◽

Rna Genome

ABSTRACTDuring viral RNA synthesis by the viral RNA-dependent RNA polymerase (vRdRp) of vesicular stomatitis virus, the sequestered RNA genome must be released from the nucleocapsid in order to serve as the template. Unveiling the sequestered RNA by interactions of vRdRp proteins, the large subunit (L) and the phosphoprotein (P), with the nucleocapsid protein (N) must not disrupt the nucleocapsid assembly. We noticed that a flexible structural motif composed of an α-helix and a loop in the N protein may act as the access gate to the sequestered RNA. This suggests that local conformational changes in this structural motif may be induced by interactions with the polymerase to unveil the sequestered RNA, without disrupting the nucleocapsid assembly. Mutations of several residues in this structural motif—Glu169, Phe171, and Leu174—to Ala resulted in loss of viral RNA synthesis in a minigenome assay. After implementing these mutations in the viral genome, mutant viruses were recovered by reverse genetics and serial passages. Sequencing the genomes of the mutant viruses revealed that compensatory mutations in L, P, and N were required to restore the viral viability. Corresponding mutations were introduced in L, P, and N, and their complementarity to the N mutations was confirmed by the minigenome assay. Introduction of the corresponding mutations is also sufficient to rescue the mutant viruses. These results suggested that the interplay of the N structural motif with the L protein may play a role in accessing the nucleotide template without disrupting the overall structure of the nucleocapsid.IMPORTANCEDuring viral RNA synthesis of a negative-strand RNA virus, the viral RNA-dependent RNA polymerase (vRdRp) must gain access to the sequestered RNA in the nucleocapsid to use it as the template, but at the same time may not disrupt the nucleocapsid assembly. Our structural and mutagenesis studies showed that a flexible structural motif acts as a potential access gate to the sequestered RNA and plays an essential role in viral RNA synthesis. Interactions of this structural motif within the vRdRp may be required for unveiling the sequestered RNA. This mechanism of action allows the sequestered RNA to be released locally without disrupting the overall structure of the nucleocapsid. Since this flexible structural motif is present in the N proteins of many NSVs, release of the sequestered RNA genome by local conformational changes in the N protein may be a general mechanism in NSV viral RNA synthesis.

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

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.737745369.793573786 ◽

2020 ◽

Cited By ~ 1

Author(s):

Benoit Coulombe

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Rna Polymerase ◽

High Potency ◽

Rna Dependent Rna Polymerase ◽

Direct Acting Antiviral ◽

Direct Acting

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Prediction of Small Molecule Inhibitors Targeting the Severe Acute Respiratory Syndrome Coronavirus-2 RNA-dependent RNA Polymerase

ACS Omega ◽

10.1021/acsomega.0c02096 ◽

2020 ◽

Vol 5 (29) ◽

pp. 18356-18366 ◽

Cited By ~ 1

Author(s):

Mohammed Ahmad ◽

Abhisek Dwivedy ◽

Richard Mariadasse ◽

Satish Tiwari ◽

Deepsikha Kar ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Rna Polymerase ◽

Small Molecule ◽

Small Molecule Inhibitors ◽

Rna Dependent Rna Polymerase

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Early combination treatment with existing HIV antivirals: an effective treatment for COVID-19?

10.21203/rs.3.rs-35791/v1 ◽

2020 ◽

Author(s):

Roberto Nico Dallocchio ◽

Alessandro Dessì ◽

Andrea De Vito ◽

Giovanna Delogu ◽

Pier Andrea ◽

...

Keyword(s):

Clinical Trials ◽

Molecular Docking ◽

Severe Acute Respiratory Syndrome ◽

Rna Polymerase ◽

Protease Inhibitor ◽

Combination Treatment ◽

Binding Sites ◽

Nucleoside Analogues ◽

Rna Dependent Rna Polymerase ◽

Protein Pocket

Abstract Since no effective therapy exists, we aimed to test existing HIV antivirals for combination treatment of Coronavirus disease 19 (COVID-19). Our molecular docking findings suggest that lopinavir, ritonavir, darunavir, and atazanavir activated interactions with the key binding sites of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) protease with a better Ki for lopinavir, ritonavir, and darunavir. Furthermore, we evidenced the ability of remdesivir, tenofovir, emtricitabine, and lamivudine to be incorporated in SARS-CoV-2 RNA-dependent RNA polymerase in the same protein pocket where poses the corresponding natural nucleoside substrates with comparable Ki and activating similar interactions. In principle, the four antiviral nucleotides might be used effectively against SARS-CoV-2. The combination of a protease inhibitor and two nucleoside analogues should be evaluated in clinical trials for the treatment of COVID-19.

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TMPRSS2 and RNA-Dependent RNA Polymerase Are Effective Targets of Therapeutic Intervention for Treatment of COVID-19 Caused by SARS-CoV-2 Variants (B.1.1.7 and B.1.351)

Microbiology Spectrum ◽

10.1128/spectrum.00472-21 ◽

2021 ◽

Author(s):

Jihye Lee ◽

JinAh Lee ◽

Hyeon Ju Kim ◽

Meehyun Ko ◽

Youngmee Jee ◽

...

Keyword(s):

Public Health ◽

Monoclonal Antibodies ◽

Severe Acute Respiratory Syndrome ◽

Rna Polymerase ◽

Causative Agent ◽

Therapeutic Intervention ◽

Human Society ◽

Rna Dependent Rna Polymerase ◽

Global Problems

The coronavirus disease 2019 (COVID-19) pandemic is causing unprecedented global problems in both public health and human society. While some vaccines and monoclonal antibodies were successfully developed very quickly and are currently being used, numerous variants of the causative agent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are emerging and threatening the efficacy of vaccines and monoclonal antibodies.

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