Quercetin and Luteolin Are Single-digit Micromolar Inhibitors of the SARS-CoV-2 RNA-dependent RNA Polymerase

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly become a global health pandemic. Among the viral proteins, RNA-dependent RNA polymerase (RdRp) is responsible for viral genome replication and has emerged as one of the most promising targets for pharmacological intervention against SARS-CoV-2. To this end, we experimentally tested luteolin and quercetin for their ability to inhibit the RdRp enzyme. These two compounds are ancestors of flavonoid natural compounds known for a variety of basal pharmacological activities. Luteolin and quercetin returned a single-digit IC50 of 4.6 µM and 6.9 µM, respectively. Then, through dynamic docking simulations, we identified possible binding modes of these compounds to a recently published cryo-EM structure of RdRp. Collectively, these data indicate that these two compounds are a valid starting point for further optimization and development of a new class of RdRp inhibitors to treat SARS-CoV-2 and potentially other viral infections.

Download Full-text

The RNA-Dependent RNA Polymerase NIb of Potyviruses Plays Multifunctional, Contrasting Roles during Viral Infection

Viruses ◽

10.3390/v12010077 ◽

2020 ◽

Vol 12 (1) ◽

pp. 77 ◽

Cited By ~ 7

Author(s):

Wentao Shen ◽

Yan Shi ◽

Zhaoji Dai ◽

Aiming Wang

Keyword(s):

Rna Polymerase ◽

Mosaic Virus ◽

Current Knowledge ◽

Soybean Mosaic Virus ◽

Virus Multiplication ◽

Future Research ◽

Plum Pox Virus ◽

Genome Replication ◽

Rna Dependent Rna Polymerase ◽

Viral Genome Replication

Potyviruses represent the largest group of known plant RNA viruses and include many agriculturally important viruses, such as Plum pox virus, Soybean mosaic virus, Turnip mosaic virus, and Potato virus Y. Potyviruses adopt polyprotein processing as their genome expression strategy. Among the 11 known viral proteins, the nuclear inclusion protein b (NIb) is the RNA-dependent RNA polymerase responsible for viral genome replication. Beyond its principal role as an RNA replicase, NIb has been shown to play key roles in diverse virus–host interactions. NIb recruits several host proteins into the viral replication complexes (VRCs), which are essential for the formation of functional VRCs for virus multiplication, and interacts with the sumoylation pathway proteins to suppress NPR1-mediated immunity response. On the other hand, NIb serves as a target of selective autophagy as well as an elicitor of effector-triggered immunity, resulting in attenuated virus infection. These contrasting roles of NIb provide an excellent example of the complex co-evolutionary arms race between plant hosts and potyviruses. This review highlights the current knowledge about the multifunctional roles of NIb in potyvirus infection, and discusses future research directions.

Download Full-text

A dynamic regulatory interface on SARS-CoV-2 RNA polymerase

10.1101/2020.07.30.229187 ◽

2020 ◽

Cited By ~ 1

Author(s):

Wei Shi ◽

Ming Chen ◽

Yang Yang ◽

Wei Zhou ◽

Shiyun Chen ◽

...

Keyword(s):

Rna Polymerase ◽

Active Center ◽

Antiviral Drug ◽

Polymerase Activity ◽

Genome Replication ◽

Specific Interactions ◽

Global Public Health ◽

Rna Dependent Rna Polymerase ◽

Viral Genome Replication

AbstractThe RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is the core machinery responsible for the viral genome replication and transcription and also a major antiviral target. Here we report the cryo-electron microscopy structure of a post-translocated SARS-CoV-2 RdRp core complex, comprising one nsp12, one separate nsp8(I) monomer, one nsp7-nsp8(II) subcomplex and a replicating RNA substrate. Compared with the recently reported SARS-CoV-2 RdRp complexes, the nsp8(I)/nsp7 interface in this RdRp complex shifts away from the nsp12 polymerase. Further functional characterizations suggest that specific interactions between the nsp8(I) and nsp7, together with the rearrangement of nsp8(I)/nsp7 interface, ensure the efficient and processive RNA synthesis by the RdRp complex. Our findings provide a mechanistic insight into how nsp7 and nsp8 cofactors regulate the polymerase activity of nsp12 and suggest a potential new intervention interface, in addition to the canonical polymerase active center, in RdRp for antiviral design.Author summarySince it was first discovered and reported in late 2019, the coronavirus disease 2019 (COVID-19) pandemic caused by highly contagious SARS-CoV-2 virus is wreaking havoc around the world. Currently, no highly effective and specific antiviral drug is available for clinical treatment. Therefore, the threat of COVID-19 transmission necessitates the discovery of more effective antiviral strategies. Viral RNA-dependent RNA polymerase (RdRp) is an important antiviral drug target. Here, our cryo-EM structure of a SARS-CoV-2 RdRp/RNA replicating complex reveals a previously uncharacterized overall shift of the cofactor nsp8(I)/nsp7 interface, leading to its rearrangement. Through in vitro functional test, we found that the specific interactions on the interface are important to the efficient RNA polymerase activity of SARS-CoV-2 RdRp. These observations let us to suggest this interface as a potential new drug intervention site, outside of the canonical polymerase active center, in RdRp for antiviral design. Our findings would provide new insights into regulatory mechanism of this novel SARS-CoV-2 RdRp, contribute to the design of antiviral drugs against SARS-CoV-2, and benefit the global public health.

Download Full-text

Dimerisation of the influenza virus RNA polymerase during viral genome replication

Access Microbiology ◽

10.1099/acmi.ac2019.po0145 ◽

2019 ◽

Vol 1 (1A) ◽

Author(s):

Alexander Walker ◽

Haitian Fan ◽

Loic Carrique ◽

Jeremy Keown ◽

David Bauer ◽

...

Keyword(s):

Influenza Virus ◽

Rna Polymerase ◽

Viral Genome ◽

Genome Replication ◽

Virus Rna ◽

Viral Genome Replication

Download Full-text

Hitchhiking of Viral Genomes on Cellular Chromosomes

Annual Review of Virology ◽

10.1146/annurev-virology-092818-015716 ◽

2019 ◽

Vol 6 (1) ◽

pp. 275-296 ◽

Cited By ~ 3

Author(s):

Tami L. Coursey ◽

Alison A. McBride

Keyword(s):

Viral Genome ◽

Viral Infections ◽

Genome Replication ◽

Mitotic Chromosomes ◽

Dna Viruses ◽

Viral Genomes ◽

Barr Virus ◽

Dividing Cells ◽

Viral Genome Replication ◽

Epstein Barr

Persistent viral infections require a host cell reservoir that maintains functional copies of the viral genome. To this end, several DNA viruses maintain their genomes as extrachromosomal DNA minichromosomes in actively dividing cells. These viruses typically encode a viral protein that binds specifically to viral DNA genomes and tethers them to host mitotic chromosomes, thus enabling the viral genomes to hitchhike or piggyback into daughter cells. Viruses that use this tethering mechanism include papillomaviruses and the gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. This review describes the advantages and consequences of persistent extrachromosomal viral genome replication.

Download Full-text

Hydrophobic and Charged Residues in the C-Terminal Arm of Hepatitis C Virus RNA-Dependent RNA Polymerase Regulate Initiation and Elongation

Journal of Virology ◽

10.1128/jvi.01106-14 ◽

2014 ◽

Vol 89 (4) ◽

pp. 2052-2063 ◽

Cited By ~ 5

Author(s):

Amy L. Cherry ◽

Caitriona A. Dennis ◽

Andrew Baron ◽

Leslie E. Eisele ◽

Pia A. Thommes ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

De Novo ◽

Structural Features ◽

Primer Extension ◽

Hcv Rna ◽

Genome Replication ◽

Rna Dependent Rna Polymerase ◽

Subgenomic Replicon

ABSTRACTThe RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV) is essential for viral genome replication. Crystal structures of the HCV RdRp reveal two C-terminal features, a β-loop and a C-terminal arm, suitably located for involvement in positioning components of the initiation complex. Here we show that these two elements intimately regulate template and nucleotide binding, initiation, and elongation. We constructed a series of β-loop and C-terminal arm mutants, which were used forin vitroanalysis of RdRpde novoinitiation and primer extension activities. All mutants showed a substantial decrease in initiation activities but a marked increase in primer extension activities, indicating an ability to form more stable elongation complexes with long primer-template RNAs. Structural studies of the mutants indicated that these enzyme properties might be attributed to an increased flexibility in the C-terminal features resulting in a more open polymerase cleft, which likely favors the elongation process but hampers the initiation steps. A UTP cocrystal structure of one mutant shows, in contrast to the wild-type protein, several alternate conformations of the substrate, confirming that even subtle changes in the C-terminal arm result in a more loosely organized active site and flexible binding modes of the nucleotide. We used a subgenomic replicon system to assess the effects of the same mutations on viral replication in cells. Even the subtlest mutations either severely impaired or completely abolished the ability of the replicon to replicate, further supporting the concept that the correct positioning of both the β-loop and C-terminal arm plays an essential role during initiation and in HCV replication in general.IMPORTANCEHCV RNA polymerase is a key target for the development of directly acting agents to cure HCV infections, which necessitates a thorough understanding of the functional roles of the various structural features of the RdRp. Here we show that even highly conservative changes, e.g., Tyr→Phe or Asp→Glu, in these seemingly peripheral structural features have profound effects on the initiation and elongation properties of the HCV polymerase.

Download Full-text

Computationally repurposed drugs and natural products against RNA dependent RNA polymerase as potential COVID-19 therapies

Molecular Biomedicine ◽

10.1186/s43556-021-00050-3 ◽

2021 ◽

Vol 2 (1) ◽

Author(s):

Sakshi Piplani ◽

Puneet Kumar Singh ◽

David A. Winkler ◽

Nikolai Petrovsky

Keyword(s):

Natural Products ◽

Rna Polymerase ◽

Kinase Inhibitor ◽

Binding Energies ◽

Dynamics Simulation ◽

Rna Dependent Rna Polymerase ◽

Starting Point ◽

Potential Activity ◽

Repurposed Drugs ◽

Multiple Drugs

AbstractRepurposing of existing drugs and drug candidates is an ideal approach to identify new potential therapies for SARS-CoV-2 that can be tested without delay in human trials of infected patients. Here we applied a virtual screening approach using Autodock Vina and molecular dynamics simulation in tandem to calculate binding energies for repurposed drugs against the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). We thereby identified 80 promising compounds with potential activity against SARS-Cov2, consisting of a mixture of antiviral drugs, natural products and drugs with diverse modes of action. A substantial proportion of the top 80 compounds identified in this study had been shown by others to have SARS-CoV-2 antiviral effects in vitro or in vivo, thereby validating our approach. Amongst our top hits not previously reported to have SARS-CoV-2 activity, were eribulin, a macrocyclic ketone analogue of the marine compound halichondrin B and an anticancer drug, the AXL receptor tyrosine kinase inhibitor bemcentinib. Our top hits from our RdRp drug screen may not only have utility in treating COVID-19 but may provide a useful starting point for therapeutics against other coronaviruses. Hence, our modelling approach successfully identified multiple drugs with potential activity against SARS-CoV-2 RdRp.

Download Full-text

Docking studies of tetra substituted pyrazolone derivatives as potential antiviral agents

JOURNAL OF PHARMACEUTICAL CHEMISTRY ◽

10.14805/jphchem.2018.art103 ◽

2018 ◽

Vol 5 (2) ◽

pp. 5-8

Author(s):

Jyothi Achuthanandhan ◽

Baskar Lakshmanan

Keyword(s):

Rna Polymerase ◽

Inhibitory Activity ◽

In Silico ◽

Antiviral Agents ◽

Docking Studies ◽

Binding Modes ◽

Binding Interaction ◽

Rna Dependent Rna Polymerase ◽

Adme Prediction ◽

Pyrazolone Derivatives

In an attempt to find potential antiviral agents, a series of pyrazolones (PA1-PA6& PC1-PC6) were designed and evaluated for their DENVNS5 (RNA-dependent RNA polymerase) inhibitory activity. Molecular docking studies of all the designed compounds into the binding site of DENVNS5 (PDB Code: 4C11) were performed to gain a comprehensive understanding into rational binding modes. These compounds were also screened for in silico drug-likeliness properties on the basis of the absorption, distribution, metabolism and excretion (ADME) prediction. Among all the synthesized compounds, analogue PA6showed superior inhibitory activity against RNA dependent RNA polymerase. SAR study indicated that the presence of an electron withdrawing substitution on pyrazolone derivatives significantly improves its binding interaction with the protein.Results of ADME prediction revealed that most of these compounds showed in silico drug-likeliness.

Download Full-text

Protein Nucleotidylylation in +ssRNA Viruses

Viruses ◽

10.3390/v13081549 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1549

Author(s):

Alice-Roza Eruera ◽

Alice M. McSweeney ◽

Geena M. McKenzie-Goldsmith ◽

Vernon K. Ward

Keyword(s):

Life Cycle ◽

Rna Polymerase ◽

Viral Replication ◽

Viral Protein ◽

Rna Viruses ◽

Replication Initiation ◽

Genome Replication ◽

Rna Dependent Rna Polymerase ◽

Nucleotidyl Transferase ◽

Initiation Of Translation

Nucleotidylylation is a post-transcriptional modification important for replication in the picornavirus supergroup of RNA viruses, including members of the Caliciviridae, Coronaviridae, Picornaviridae and Potyviridae virus families. This modification occurs when the RNA-dependent RNA polymerase (RdRp) attaches one or more nucleotides to a target protein through a nucleotidyl-transferase reaction. The most characterized nucleotidylylation target is VPg (viral protein genome-linked), a protein linked to the 5′ end of the genome in Caliciviridae, Picornaviridae and Potyviridae. The nucleotidylylation of VPg by RdRp is a critical step for the VPg protein to act as a primer for genome replication and, in Caliciviridae and Potyviridae, for the initiation of translation. In contrast, Coronaviridae do not express a VPg protein, but the nucleotidylylation of proteins involved in replication initiation is critical for genome replication. Furthermore, the RdRp proteins of the viruses that perform nucleotidylylation are themselves nucleotidylylated, and in the case of coronavirus, this has been shown to be essential for viral replication. This review focuses on nucleotidylylation within the picornavirus supergroup of viruses, including the proteins that are modified, what is known about the nucleotidylylation process and the roles that these modifications have in the viral life cycle.

Download Full-text

Dual Effect of Organogermanium Compound THGP on RIG-I-Mediated Viral Sensing and Viral Replication during Influenza a Virus Infection

Viruses ◽

10.3390/v13091674 ◽

2021 ◽

Vol 13 (9) ◽

pp. 1674

Author(s):

Sunanda Baidya ◽

Yoko Nishimoto ◽

Seiichi Sato ◽

Yasuhiro Shimada ◽

Nozomi Sakurai ◽

...

Keyword(s):

Immune Responses ◽

Influenza A ◽

Viral Infections ◽

Genome Replication ◽

Type I ◽

Innate Immune Responses ◽

Viral Polymerase ◽

Microbial Infections ◽

Viral Genome Replication ◽

Vesicular Stomatitis

The interaction of viral nucleic acid with protein factors is a crucial process for initiating viral polymerase-mediated viral genome replication while activating pattern recognition receptor (PRR)-mediated innate immune responses. It has previously been reported that a hydrolysate of Ge-132, 3-(trihydroxygermyl) propanoic acid (THGP), shows a modulatory effect on microbial infections, inflammation, and immune responses. However, the detailed mechanism by which THGP can modify these processes during viral infections remained unknown. Here, we show that THGP can specifically downregulate type I interferon (IFN) production in response to stimulation with a cytosolic RNA sensor RIG-I ligand 5′-triphosphate RNA (3pRNA) but not double-stranded RNA, DNA, or lipopolysaccharide. Consistently, treatment with THGP resulted in the dose-dependent suppression of type I IFN induction upon infections with influenza virus (IAV) and vesicular stomatitis virus, which are known to be mainly sensed by RIG-I. Mechanistically, THGP directly binds to the 5′-triphosphate moiety of viral RNA and competes with RIG-I-mediated recognition. Furthermore, we found that THGP can directly counteract the replication of IAV but not EMCV (encephalitismyocarditis virus), by inhibiting the interaction of viral polymerase with RNA genome. Finally, IAV RNA levels were significantly reduced in the lung tissues of THGP-treated mice when compared with untreated mice. These results suggest a possible therapeutic implication of THGP and show direct antiviral action, together with the suppressive activity of innate inflammation.

Download Full-text

A Library of Nucleotide Analogues Terminate RNA Synthesis Catalyzed by Polymerases of Coronaviruses Causing SARS and COVID-19

10.1101/2020.04.23.058776 ◽

2020 ◽

Cited By ~ 2

Author(s):

Steffen Jockusch ◽

Chuanjuan Tao ◽

Xiaoxu Li ◽

Thomas K. Anderson ◽

Minchen Chien ◽

...

Keyword(s):

Rna Polymerase ◽

Safety Profile ◽

Rna Synthesis ◽

Viral Infections ◽

Nucleoside Triphosphate ◽

Exonuclease Activity ◽

Base Pairs ◽

Rna Dependent Rna Polymerase ◽

Nucleotide Analogues ◽

Tenofovir Alafenamide

AbstractSARS-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 additional 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 with regard to their ability to be incorporated by the RdRps in the polymerase reaction and then prevent further incorporation. While all 11 molecules tested displayed incorporation, 6 exhibited immediate termination of the polymerase reaction (Carbovir triphosphate, Ganciclovir triphosphate, Stavudine triphosphate, Entecavir triphosphate, 3’-O-methyl UTP and Biotin-16-dUTP), 2 showed delayed termination (Cidofovir diphosphate and 2’-O-methyl UTP), and 3 did not terminate the polymerase reaction (2’-fluoro-dUTP, 2’-amino-dUTP and Desthiobiotin-16-UTP). The coronavirus genomes encode an exonuclease that apparently requires a 2’ -OH group to excise mismatched bases at the 3’-terminus. In this study, all of the nucleoside triphosphate analogues we evaluated form Watson-Cricklike base pairs. All the nucleotide analogues which demonstrated termination either lack a 2’-OH, have a blocked 2’-OH, or show delayed termination. These nucleotides may thus have the potential to resist exonuclease activity, a property that we will investigate in the future. Furthermore, prodrugs of five of these nucleotide analogues (Brincidofovir/Cidofovir, Abacavir, Valganciclovir/Ganciclovir, Stavudine and Entecavir) are FDA approved for other viral infections, and their safety profile is well known. Thus, they can be evaluated rapidly as potential therapies for COVID-19.

Download Full-text