Effects of natural polymorphisms in SARS-CoV-2 RNA-dependent RNA polymerase on its activity and sensitivity to inhibitors in vitro

Miropolskaya, Nataliya; Kozlov, M. V.; Petushkov, Ivan; Prostova, Maria A; Pupov, Danil; Esyunina, Daria; Kochetkov, Sergey N.; Kulbachinskiy, Andrey

doi:10.1016/j.biochi.2022.10.007

Cited by 5 publications

(10 citation statements)

References 52 publications

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“…RdRp was incubated with each of the compounds and the reaction was initiated by adding a pre-annealed primer–template RNA substrate ( Figure 3 , top) and nucleotides. In accordance with our previous analysis of RdRp activity with this RNA substrate [ 32 ], in the absence of inhibitors, RdRp extended the RNA primer until the end of the template strand ( Figure 3 , control reactions with DMSO). It was found that several of the tested compounds could inhibit RNA extension ( Figure 3 A,D).…”

Section: Resultssupporting

confidence: 90%

“…High-resolution mass spectra were recorded on a Bruker Daltonics MicrOTOF-Q II device using electrospray ionization mass spectrometry (ESI-MS). The measurements were carried out in the mode of positive ions in accordance with the previously applied conditions [ 32 ].…”

Section: Methodsmentioning

confidence: 99%

“…SARS-CoV-2 RdRp holoenzyme containing the catalytic subunit nsp12 and fused nsp7-nsp8 subunits (separated by a His6-tag) was expressed in E. coli BL-21(DE3) and purified using Ni-affinity and anion-exchange chromatography steps as described previously [ 32 , 43 ]. RNA oligonucleotides for in vitro reactions were prepared by DNA synthesis (Moscow, Russia).…”

Section: Methodsmentioning

confidence: 99%

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Nucleoside Analogs That Inhibit SARS-CoV-2 Replication by Blocking Interaction of Virus Polymerase with RNA

Matyugina

Petushkov

Суржиков

et al. 2023

IJMS

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The SARS-CoV-2 betacoronavirus pandemic has claimed more than 6.5 million lives and, despite the development and use of COVID-19 vaccines, remains a major global public health problem. The development of specific drugs for the treatment of this disease remains a very urgent task. In the context of a repurposing strategy, we previously screened a library of nucleoside analogs showing different types of biological activity against the SARS-CoV-2 virus. The screening revealed compounds capable of inhibiting the reproduction of SARS-CoV-2 with EC50 values in the range of 20–50 µM. Here we present the design and synthesis of various analogs of the leader compounds, the evaluation of their cytotoxicity and antiviral activity against SARS-CoV-2 in cell cultures, as well as experimental data on RNA-dependent RNA polymerase inhibition. Several compounds have been shown to prevent the interaction between the SARS-CoV-2 RNA-dependent RNA polymerase and the RNA substrate, likely inhibiting virus replication. Three of the synthesized compounds have also been shown to inhibit influenza virus. The structures of these compounds can be used for further optimization in order to develop an antiviral drug.

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Section: Resultssupporting

confidence: 90%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Nucleoside Analogs That Inhibit SARS-CoV-2 Replication by Blocking Interaction of Virus Polymerase with RNA

Matyugina

Petushkov

Суржиков

et al. 2023

IJMS

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“…Our results showed that even a single amino acid substitution in any of the protein subunits of SARS-CoV-2 RdRp could alter the enzymatic activity of the complex (Fig 4 , C). Noteworthy, substitutions within nsp7 and nsp8 did not change the RdRp activity as substantially as P323L in nsp12, and even in comparison with other studied mutations in nsp12 19 , the impact was less significant. As anticipated, the differences in enzymatic activity align with alterations in the biophysical properties of the proteins.…”

Section: Discussionmentioning

confidence: 74%

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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“…Their activity against SARS-CoV-2 has not been verified so far. We tested Flex-nt10 (an ATP analog) for RdRp inhibition using a previously developed assay [ 34 ] ( Supplementary Text Box S1 and Figure S6 ) and observed relatively weak activity. The apparent EC50 (half-maximal effective concentration) value was ≥300 µM in the presence of 10 µM ATP.…”

Section: Resultsmentioning

confidence: 99%

Nucleoside Analogs and Perylene Derivatives Modulate Phase Separation of SARS-CoV-2 N Protein and Genomic RNA In Vitro

Svetlova

Knizhnik

Manuvera

et al. 2022

IJMS

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The life cycle of severe acute respiratory syndrome coronavirus 2 includes several steps that are supposedly mediated by liquid–liquid phase separation (LLPS) of the viral nucleocapsid protein (N) and genomic RNA. To facilitate the rational design of LLPS-targeting therapeutics, we modeled N-RNA biomolecular condensates in vitro and analyzed their sensitivity to several small-molecule antivirals. The model condensates were obtained and visualized under physiological conditions using an optimized RNA sequence enriched with N-binding motifs. The antivirals were selected based on their presumed ability to compete with RNA for specific N sites or interfere with non-specific pi–pi/cation–pi interactions. The set of antivirals included fleximers, 5′-norcarbocyclic nucleoside analogs, and perylene-harboring nucleoside analogs as well as non-nucleoside amphiphilic and hydrophobic perylene derivatives. Most of these antivirals enhanced the formation of N-RNA condensates. Hydrophobic perylene derivatives and 5′-norcarbocyclic derivatives caused up to 50-fold and 15-fold enhancement, respectively. Molecular modeling data argue that hydrophobic compounds do not hamper specific N-RNA interactions and may promote non-specific ones. These findings shed light on the determinants of potent small-molecule modulators of viral LLPS.

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Effects of natural polymorphisms in SARS-CoV-2 RNA-dependent RNA polymerase on its activity and sensitivity to inhibitors in vitro

Cited by 5 publications

References 52 publications

Nucleoside Analogs That Inhibit SARS-CoV-2 Replication by Blocking Interaction of Virus Polymerase with RNA

Nucleoside Analogs That Inhibit SARS-CoV-2 Replication by Blocking Interaction of Virus Polymerase with RNA

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

Nucleoside Analogs and Perylene Derivatives Modulate Phase Separation of SARS-CoV-2 N Protein and Genomic RNA In Vitro

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