Screening of SARS-CoV-2 antivirals through a cell-based RNA-dependent RNA polymerase (RdRp) reporter assay

Uppal, Timsy; Tuffo, Kai; Khaiboullina, Svetlana; Reganti, Sivani; Pandori, Mark; Verma, Subhash C.

doi:10.1016/j.cellin.2022.100046

Cited by 7 publications

(5 citation statements)

References 29 publications

(32 reference statements)

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“…Reduced RdRp activity has been shown to substantially slow down viral replication in RNA viruses such as in tick-borne flavivirus [ 54 ] and can alter the RNA synthesis process in tomato mosaic virus [ 55 ]. Also, inhibitors of RdRp of SARS-CoV-2 such as remdesivir slow down viral replication by reducing and inhibiting the viral RdRp efficiency [ 56 ]. This highlights the biological significance of the stabilizing and destabilizing effects of mutations on the viral replication protein complex in the context of viral replication fitness.…”

Section: Discussionmentioning

confidence: 99%

Bio-Chemoinformatics-Driven Analysis of nsp7 and nsp8 Mutations and Their Effects on Viral Replication Protein Complex Stability

Subong,

Ozawa

2024

CIMB

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The nonstructural proteins 7 and 8 (nsp7 and nsp8) of SARS-CoV-2 are highly important proteins involved in the RNA-dependent polymerase (RdRp) protein replication complex. In this study, we analyzed the global mutation of nsp7 and nsp8 in 2022 and 2023 and analyzed the effects of mutation on the viral replication protein complex using bio-chemoinformatics. Frequently occurring variants are found to be single amino acid mutations for both nsp7 and nsp8. The most frequently occurring mutations for nsp7 which include L56F, L71F, S25L, M3I, D77N, V33I and T83I are predicted to cause destabilizing effects, whereas those in nsp8 are predicted to cause stabilizing effects, with the threonine to isoleucine mutation (T89I, T145I, T123I, T148I, T187I) being a frequent mutation. A conserved domain database analysis generated critical interaction residues for nsp7 (Lys-7, His-36 and Asn-37) and nsp8 (Lys-58, Pro-183 and Arg-190), which, according to thermodynamic calculations, are prone to destabilization. Trp-29, Phe-49 of nsp7 and Trp-154, Tyr-135 and Phe-15 of nsp8 cause greater destabilizing effects to the protein complex based on a computational alanine scan suggesting them as possible new target sites. This study provides an intensive analysis of the mutations of nsp7 and nsp8 and their possible implications for viral complex stability.

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Section: Discussionmentioning

confidence: 99%

Bio-Chemoinformatics-Driven Analysis of nsp7 and nsp8 Mutations and Their Effects on Viral Replication Protein Complex Stability

Subong,

Ozawa

2024

CIMB

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“…Additionally, in the presence of RdRp activity, the synthesis of (+) Fluc RNA is amplified, while the levels of RLuc RNA (used as an internal control for transcription and translation) remain unaffected. Any compounds that inhibit RdRp activity will cause a decrease in FLuc levels, serving as an indicator of their inhibitory effects [ 82 ]. So, they established a cell-based fluorescence high-throughput screening method for anti-SARS-CoV-2 antiviral drugs.…”

Section: Fluorescence-based Drug Screening Methods For Novel Coronavi...mentioning

confidence: 99%

Development of Fluorescence-Based Assays for Key Viral Proteins in the SARS-CoV-2 Infection Process and Lifecycle

Deng,

Zhang,

Yan

et al. 2024

IJMS

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Since the appearance of SARS-CoV-2 in 2019, the ensuing COVID-19 (Corona Virus Disease 2019) pandemic has posed a significant threat to the global public health system, human health, life, and economic well-being. Researchers worldwide have devoted considerable efforts to curb its spread and development. The latest studies have identified five viral proteins, spike protein (Spike), viral main protease (3CLpro), papain-like protease (PLpro), RNA-dependent RNA polymerase (RdRp), and viral helicase (Helicase), which play crucial roles in the invasion of SARS-CoV-2 into the human body and its lifecycle. The development of novel anti-SARS-CoV-2 drugs targeting these five viral proteins holds immense promise. Therefore, the development of efficient, high-throughput screening methodologies specifically designed for these viral proteins is of utmost importance. Currently, a plethora of screening techniques exists, with fluorescence-based assays emerging as predominant contenders. In this review, we elucidate the foundational principles and methodologies underpinning fluorescence-based screening approaches directed at these pivotal viral targets, hoping to guide researchers in the judicious selection and refinement of screening strategies, thereby facilitating the discovery and development of lead compounds for anti-SARS-CoV-2 pharmaceuticals.

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“…A general assay was performed with SARS-CoV-2 WT and its variants Omicron (HIAE-W.A; GISAID: EPI_ISL_6901961) and Gamma (IMT-MAN87209; GISAID: EPI_ISL_1060981). To this, Vero E6 and A549 cells were seeded at a density of 1 × 10 5 cells per well into 24-well plates prior to the infection with virus at a multiplicity of infection (MOI) of 0.01 in the presence or absence of lectins at non-toxic concentrations for 24 h. The supernatant of all assays were collected and frozen for carrying out the following steps of extraction of viral RNA, cDNA synthesis, and real time PCR for viral titers quantification [ 52 ].…”

Section: Methodsmentioning

confidence: 99%

Mannose-Binding Lectins as Potent Antivirals against SARS-CoV-2

Grosche,

Souza,

Ferreira

et al. 2023

Viruses

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The SARS-CoV-2 entry into host cells is mainly mediated by the interactions between the viral spike protein (S) and the ACE-2 cell receptor, which are highly glycosylated. Therefore, carbohydrate binding agents may represent potential candidates to abrogate virus infection. Here, we evaluated the in vitro anti-SARS-CoV-2 activity of two mannose-binding lectins isolated from the Brazilian plants Canavalia brasiliensis and Dioclea violacea (ConBR and DVL). These lectins inhibited SARS-CoV-2 Wuhan-Hu-1 strain and variants Gamma and Omicron infections, with selectivity indexes (SI) of 7, 1.7, and 6.5, respectively for ConBR; and 25, 16.8, and 22.3, for DVL. ConBR and DVL inhibited over 95% of the early stages of the viral infection, with strong virucidal effect, and also protected cells from infection and presented post-entry inhibition. The presence of mannose resulted in the complete lack of anti-SARS-CoV-2 activity by ConBR and DVL, recovering virus titers. ATR-FTIR, molecular docking, and dynamic simulation between SARS-CoV-2 S and either lectins indicated molecular interactions with predicted binding energies of −85.4 and −72.0 Kcal/Mol, respectively. Our findings show that ConBR and DVL lectins possess strong activities against SARS-CoV-2, potentially by interacting with glycans and blocking virus entry into cells, representing potential candidates for the development of novel antiviral drugs.

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Screening of SARS-CoV-2 antivirals through a cell-based RNA-dependent RNA polymerase (RdRp) reporter assay

Cited by 7 publications

References 29 publications

Bio-Chemoinformatics-Driven Analysis of nsp7 and nsp8 Mutations and Their Effects on Viral Replication Protein Complex Stability

Bio-Chemoinformatics-Driven Analysis of nsp7 and nsp8 Mutations and Their Effects on Viral Replication Protein Complex Stability

Development of Fluorescence-Based Assays for Key Viral Proteins in the SARS-CoV-2 Infection Process and Lifecycle

Mannose-Binding Lectins as Potent Antivirals against SARS-CoV-2

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