A ‘deep dive’ into the SARS-Cov-2 polymerase assembly: identifying novel allosteric sites and analyzing the hydrogen bond networks and correlated dynamics

Barakat, Khaled; Ahmed, Marawan; Tabana, Yasser; Ha, Minwoo

doi:10.1080/07391102.2021.1930162

Cited by 8 publications

(4 citation statements)

References 55 publications

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“…This lower dynamic movement in several domains and in the catalytic site is due to the allosteric effect, which resulted from the binding of ligand to the allosteric site. The catalytic site of the RdRp of SARS-CoV-2 is present in the palm subdomain part and made of motif A-G [ 41 ]. This active site comprises D618, D760 and D761, and other residues from various motifs that include T680, T710, F753, N767, L775, E796, H810, V820, K912, E921, K500 and S518 [ 41 ].…”

Section: Resultsmentioning

confidence: 99%

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Computational Study of SARS-CoV-2 RNA Dependent RNA Polymerase Allosteric Site Inhibition

et al. 2021

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The COVID-19 pandemic has caused millions of fatalities since 2019. Despite the availability of vaccines for this disease, new strains are causing rapid ailment and are a continuous threat to vaccine efficacy. Here, molecular docking and simulations identify strong inhibitors of the allosteric site of the SARS-CoV-2 virus RNA dependent RNA polymerase (RdRp). More than one hundred different flavonoids were docked with the SARS-CoV-2 RdRp allosteric site through computational screening. The three top hits were Naringoside, Myricetin and Aureusidin 4,6-diglucoside. Simulation analyses confirmed that they are in constant contact during the simulation time course and have strong association with the enzyme’s allosteric site. Absorption, distribution, metabolism, excretion and toxicity (ADMET) data provided medicinal information of these top three hits. They had good human intestinal absorption (HIA) concentrations and were non-toxic. Due to high mutation rates in the active sites of the viral enzyme, these new allosteric site inhibitors offer opportunities to drug SARS-CoV-2 RdRp. These results provide new information for the design of novel allosteric inhibitors against SARS-CoV-2 RdRp.

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Section: Resultsmentioning

confidence: 99%

“…The catalytic site of the RdRp of SARS-CoV-2 is present in the palm subdomain part and made of motif A-G [ 41 ]. This active site comprises D618, D760 and D761, and other residues from various motifs that include T680, T710, F753, N767, L775, E796, H810, V820, K912, E921, K500 and S518 [ 41 ].…”

Section: Resultsmentioning

confidence: 99%

Computational Study of SARS-CoV-2 RNA Dependent RNA Polymerase Allosteric Site Inhibition

et al. 2021

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“…Moreover, these mutations may also represent an obstacle in the designing of non-nucleoside or allosteric RdRp inhibitors. Particularly, the nsp8-nsp12 interaction interface and the interface domain of nsp12 have been identified as potential hot spots for small molecule inhibitors, with the binding site in nsp12 directly involving the P323 or surrounding amino acid residues [58][59][60] .…”

Section: Discussionmentioning

confidence: 99%

Biochemical characterization of naturally occurring mutations in SARS-CoV-2 RNA-dependent RNA polymerase

Danda,

Klimešová,

Kušková

et al. 2024

Preprint

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Since the emergence of SARS-CoV-2, mutations in all subunits of the RNA-dependent RNA polymerase (RdRp) of the virus have been repeatedly reported. Although RdRp represents a primary target for antiviral drugs, experimental studies exploring the phenotypic effect of these mutations have been limited. This study focuses on the phenotypic effects of substitutions in the three RdRp subunits: nsp7, nsp8, and nsp12, selected based on their occurrence rate and potential impact. We employed nano-differential scanning fluorimetry and microscale thermophoresis to examine the impact of these mutations on protein stability and RdRp complex assembly. We observed diverse impacts; notably, a single mutation in nsp8 significantly increased its stability as evidenced by a 13 [deg]C increase in melting temperature, whereas certain mutations in nsp7 and nsp8 reduced their binding affinity to nsp12 during RdRp complex formation. Using a fluorometric enzymatic assay, we assessed the overall effect on RNA polymerase activity. We found that most of the examined mutations altered the polymerase activity, often as a direct result of changes in stability or affinity to the other components of the RdRp complex. Intriguingly, a combination of nsp8 A21V and nsp12 P323L mutations resulted in a 50% increase in polymerase activity. Additionally, some of the examined substitutions in the RdRp subunits notably influenced the sensitivity of RdRp to Remdesivir, highlighting their potential implications for therapeutic strategies. To our knowledge, this is the first biochemical study to demonstrate the impact of amino acid mutations across all components constituting the RdRp complex in emerging SARS-CoV-2 subvariants.

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“…Both nsp12 active sites are essential for viral growth [19] and targeting either site is expected to inhibit SARS-CoV-2. Fourth, computational and functional studies suggest that, like multi-subunit bacterial RNAPs, SARS-CoV-2 RdRp may be controlled allosterically [17,23] and several hypothetical allosteric pockets have been identified in nsp12 [24][25][26]. Finally, the transcribing RdRp associates with other nsps to form a large replication-transcription complex [16], which presents multiple hypothetical targets for small-molecule inhibitors.…”

Section: Introductionmentioning

confidence: 99%

Going Retro, Going Viral: Experiences and Lessons in Drug Discovery from COVID-19

Wang

Svetlov²,

Bartikofsky

et al. 2022

Molecules

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The severity of the COVID-19 pandemic and the pace of its global spread have motivated researchers to opt for repurposing existing drugs against SARS-CoV-2 rather than discover or develop novel ones. For reasons of speed, throughput, and cost-effectiveness, virtual screening campaigns, relying heavily on in silico docking, have dominated published reports. A particular focus as a drug target has been the principal active site (i.e., RNA synthesis) of RNA-dependent RNA polymerase (RdRp), despite the existence of a second, and also indispensable, active site in the same enzyme. Here we report the results of our experimental interrogation of several small-molecule inhibitors, including natural products proposed to be effective by in silico studies. Notably, we find that two antibiotics in clinical use, fidaxomicin and rifabutin, inhibit RNA synthesis by SARS-CoV-2 RdRp in vitro and inhibit viral replication in cell culture. However, our mutagenesis studies contradict the binding sites predicted computationally. We discuss the implications of these and other findings for computational studies predicting the binding of ligands to large and flexible protein complexes and therefore for drug discovery or repurposing efforts utilizing such studies. Finally, we suggest several improvements on such efforts ongoing against SARS-CoV-2 and future pathogens as they arise.

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A ‘deep dive’ into the SARS-Cov-2 polymerase assembly: identifying novel allosteric sites and analyzing the hydrogen bond networks and correlated dynamics

Abstract: 2021): A 'deep dive' into the SARS-Cov-2 polymerase assembly: identifying novel allosteric sites and analyzing the hydrogen bond networks and correlated dynamics, Journal of Biomolecular Structure and Dynamics,

Cited by 8 publications

References 55 publications

Computational Study of SARS-CoV-2 RNA Dependent RNA Polymerase Allosteric Site Inhibition

Computational Study of SARS-CoV-2 RNA Dependent RNA Polymerase Allosteric Site Inhibition

Biochemical characterization of naturally occurring mutations in SARS-CoV-2 RNA-dependent RNA polymerase

Going Retro, Going Viral: Experiences and Lessons in Drug Discovery from COVID-19

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