Two conserved oligomer interfaces of NSP7 and NSP8 underpin the dynamic assembly of SARS-CoV-2 RdRP

Biswal, Mahamaya; Diggs, Stephen; Xu, Duo; Khudaverdyan, Nelli; Lu, Jiuwei; Fang, Jian; Blaha, Gregor; Hai, Rong; Song, Jikui

doi:10.1093/nar/gkab370

Cited by 50 publications

(82 citation statements)

References 33 publications

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“…Interestingly, while the top poses for αTOS were spread to a few locations across the surface of NSP12, our docking studies on the interaction of αTOS with NSP7 and NSP8 each converged to a single region (Figure S8A-C). In each case, the most energetically favorable binding poses localized with residues that participate in one of the two recently described hydrophobic interfaces that is required for the assembly of the functional RdRp (Figure 3G; Figure S8D) 6 . The residues within this interface are highly conserved across coronaviruses, and site directed mutagenesis of several residues within this interface reduced or ablated the transcriptional activity of the SARS-CoV-2 RdRp 6 .…”

Section: Tocopherols Inhibit Sars-cov-2 Rdrpmentioning

confidence: 91%

“…In each case, the most energetically favorable binding poses localized with residues that participate in one of the two recently described hydrophobic interfaces that is required for the assembly of the functional RdRp (Figure 3G; Figure S8D) 6 . The residues within this interface are highly conserved across coronaviruses, and site directed mutagenesis of several residues within this interface reduced or ablated the transcriptional activity of the SARS-CoV-2 RdRp 6 . Taken together, these findings strongly support a mechanism by which TPGS prevents or destabilizes the assembly of the SARS-CoV-2 RdRp.…”

Section: Tocopherols Inhibit Sars-cov-2 Rdrpmentioning

confidence: 91%

“…Docking experiments were performed using Autodock Vina 61 . Individual NSP7 and NSP8 structures were isolated from the structure of the SARS-CoV-2 NSP7-NSP8 complex (PDB ID: 7JLT) 6 while SARS-CoV-2 NSP12 was isolated from the SARS-CoV-2 RNA-dependent RNA polymerase structure (PDB ID: 6M71) 62 . These structures and α-tocopherol succinate were modeled in Autodock Tools with α-tocopherol succinate possessing 16 rotatable bonds.…”

Section: Docking Studiesmentioning

confidence: 99%

“…CoV-2 RdRp 6 . Taken together, these findings strongly support a mechanism by which TPGS prevents or destabilizes the assembly of the SARS-CoV-2 RdRp.…”

Section: Tocopherols Inhibit Sars-cov-2 Rdrpmentioning

confidence: 99%

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Water-soluble tocopherol derivatives inhibit SARS-CoV-2 RNA-dependent RNA polymerase

Pacl

Tipper

Sevalkar

et al. 2021

Preprint

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The recent emergence of a novel coronavirus, SARS-CoV-2, has led to the global pandemic of the severe disease COVID-19 in humans. While efforts to quickly identify effective antiviral therapies have focused largely on repurposing existing drugs, the current standard of care, remdesivir, remains the only authorized antiviral intervention of COVID-19 and provides only modest clinical benefits. Here we show that water-soluble derivatives of α-tocopherol have potent antiviral activity and synergize with remdesivir as inhibitors of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Through an artificial-intelligence-driven in silico screen and in vitro viral inhibition assay, we identified D-α-tocopherol polyethylene glycol succinate (TPGS) as an effective antiviral against SARS-CoV-2 and β-coronaviruses more broadly that also displays strong synergy with remdesivir. We subsequently determined that TPGS and other water-soluble derivatives of α-tocopherol inhibit the transcriptional activity of purified SARS-CoV-2 RdRp and identified affinity binding sites for these compounds within a conserved, hydrophobic interface between SARS-CoV-2 nonstructural protein 7 and nonstructural protein 8 that is functionally implicated in the assembly of the SARS-CoV-2 RdRp. In summary, we conclude that solubilizing modifications to α-tocopherol allow it to interact with the SARS-CoV-2 RdRp, making it an effective antiviral molecule alone and even more so in combination with remdesivir. These findings are significant given that many tocopherol derivatives, including TPGS, are considered safe for humans, orally bioavailable, and dramatically enhance the activity of the only approved antiviral for SARS-CoV-2 infection.

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Section: Tocopherols Inhibit Sars-cov-2 Rdrpmentioning

confidence: 91%

Section: Tocopherols Inhibit Sars-cov-2 Rdrpmentioning

confidence: 91%

Section: Docking Studiesmentioning

confidence: 99%

“…CoV-2 RdRp 6 . Taken together, these findings strongly support a mechanism by which TPGS prevents or destabilizes the assembly of the SARS-CoV-2 RdRp.…”

Section: Tocopherols Inhibit Sars-cov-2 Rdrpmentioning

confidence: 99%

See 2 more Smart Citations

Water-soluble tocopherol derivatives inhibit SARS-CoV-2 RNA-dependent RNA polymerase

Pacl

Tipper

Sevalkar

et al. 2021

Preprint

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“…The replicase-transcriptase complex(RTC) consists of 16 nsps in which multiple enzymes, including nsp3 (papain like protease) ,3CL protease nsp5(chymotripsin like main protease), primase complex, RdRp(nsp12),nsp13(helicase)and nsp14(exoribonuclease) [6]. The nsp12 is the main content of the replication machinery which involves the transcriptional activity of RNA-dependent RNA polymerase (RdRp) [7]. It is a popular target for a selective antiviral strategy against SARS COV 2 because RNA synthesis by RNA-dependent RNA polymerase does not occur in mammalian cells [8].…”

Section: Introductionmentioning

confidence: 99%

Comparative Analysis of Small Moleclues and Natural Plants Compounds as Therapeutic Inhibitors Targeting RdRp and Nucleocapsid Proteins of SARS CoV 2: An in Silico Approach

Poleboyina

2021

Preprint

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Nucleocapsid protein and RNA-dependent RNA polymerase (RdRp) activity in viral structural membrane, transcription and replication has been recognized as an attractive target to design novel antiviral strategies. The essential feature of the N protein of SARS COV 2 is to bind to the viral genome to promote the exact folding of the hammerhead ribozyme averting unproductive RNA confirmations and lead them to right into a helical capsid shape or RNP complex, whose packaging is crucial to viability. RdRp is an essential enzyme that helps in RNA synthesis by catalyzing the RNA template-dependent development of phosphodiester bonds. RdRp makes a complex with two cofactors nsp7 and nsp8 to play a key role in RNA synthesis, transcription and replication of the SARS-CoV-2. In our study we used small molecules and natural plants compounds as therapeutic inhibitors targeting RdRp and N proteins of SARS COV 2. Their structures were geometrically optimized and energetically minimized using Hyperchem software. Molecular docking was performed using Molegro virtual docker and top ligands were selected based on MolDock score,Rerank score and H-bonding energy. Our results showed that 9 compounds against N protein and 7 compounds against RdRp protein forming better inhibitory effect with most lowest MolDock score − 285.68kcal/mol and − 201.5kcal/mol respectively. we hope that these small molecules and natural plants compounds can inhibit the viral enzymes and helps the patients in reducing specific symptoms of SARS-CoV-2 infection. However in vivo experimental studies and clinical trials need to get that more favorable result.

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Optimized Recombinant Expression and Purification of the SARS‐CoV‐2 Polymerase Complex

Podadera,

Campo,

Rehman

et al. 2024

Current Protocols

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An optimized protocol has been developed to express and purify the core RNA‐dependent RNA polymerase (RdRP) complex from the severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2). The expression and purification of active core SARS‐CoV‐2 RdRp complex is challenging due to the complex multidomain fold of nsp12, and the assembly of the multimeric complex involving nsp7, nsp8, and nsp12. Our approach adapts a previously published method to express the core SARS‐CoV‐2 RdRP complex in Escherichia coli and combines it with a procedure to express the nsp12 fusion with maltose‐binding protein in insect cells to promote the efficient assembly and purification of an enzymatically active core polymerase complex. The resulting method provides a reliable platform to produce milligram amounts of the polymerase complex with the expected 1:2:1 stoichiometry for nsp7, nsp8, and nsp12, respectively, following the removal of all affinity tags. This approach addresses some of the limitations of previously reported methods to provide a reliable source of the active polymerase complex for structure, function, and inhibition studies of the SARS‐CoV‐2 RdRP complex using recombinant plasmid constructs that have been deposited in the widely accessible Addgene repository. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Expression and production of SARS‐CoV‐2 nsp7, nsp8, and nsp12 in E. coli cellsSupport Protocol: Establishment and maintenance of insect cell culturesBasic Protocol 2: Generation of recombinant baculovirus in Sf9 cells and production of nsp12 fusion protein in T. ni cellsBasic Protocol 3: Purification of the SARS‐CoV‐2 core polymerase complex

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Two conserved oligomer interfaces of NSP7 and NSP8 underpin the dynamic assembly of SARS-CoV-2 RdRP

Cited by 50 publications

References 33 publications

Water-soluble tocopherol derivatives inhibit SARS-CoV-2 RNA-dependent RNA polymerase

Water-soluble tocopherol derivatives inhibit SARS-CoV-2 RNA-dependent RNA polymerase

Comparative Analysis of Small Moleclues and Natural Plants Compounds as Therapeutic Inhibitors Targeting RdRp and Nucleocapsid Proteins of SARS CoV 2: An in Silico Approach

Optimized Recombinant Expression and Purification of the SARS‐CoV‐2 Polymerase Complex

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