displaying 1 - 10 records in total 249
    records per page




    Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp12/7/8 RNA-dependent RNA Polymerase PROTEIN

    Authors: Rupert Beale; Agustina P Bertolin; Berta Canal; John FX Diffley; Lucy S Drury; Michael Howell; Jennifer Milligan; Viktor Posse; Rachel Ulferts; Florian Weissmann; Mary Wu; Jingkun Zeng

    doi:10.1101/2021.04.07.438807 Date: 2021-04-08 Source: bioRxiv

    The coronavirus disease 2019 MESHD ( COVID-19 MESHD) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 MESHD etiologic agent is the severe acute respiratory syndrome coronavirus 2 MESHD (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication-transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase PROTEIN ( RdRp PROTEIN) holoenzyme. The RdRp PROTEIN is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologs in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes PROTEIN. We developed a novel fluorescence resonance energy transfer (FRET)-based strand displacement assay for monitoring SARS-CoV-2 RdRp PROTEIN activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp PROTEIN inhibitors. We identified 3 novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp PROTEIN activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.

    TMPRSS2 HGNC and RNA-dependent RNA polymerase PROTEIN are effective targets of therapeutic intervention for treatment of COVID-19 MESHD caused by SARS-CoV-2 variants (B.1.1.7 and B.1.351)

    Authors: Jihye Lee; JinAh Lee; Hyeon Ju Kim; Meehyun Ko; Youngmee Jee; Seungtaek Kim

    doi:10.1101/2021.04.06.438540 Date: 2021-04-08 Source: bioRxiv

    SARS-CoV-2 is a causative agent of COVID-19 pandemic MESHD and the development of therapeutic interventions is urgently needed. So far, monoclonal antibodies and drug repositioning are the main methods for drug development and this effort was partially successful. Since the beginning of COVID-19 pandemic MESHD, the emergence of SARS-CoV-2 variants has been reported in many parts of the world and the main concern is whether the current vaccines and therapeutics are still effective against these variant viruses. The viral entry and viral RNA-dependent RNA polymerase PROTEIN ( RdRp PROTEIN) are the main targets of current drug development, thus the inhibitory effects of TMPRSS2 HGNC and RdRp PROTEIN inhibitors were compared among the early SARS-CoV-2 isolate (lineage A) and the two recent variants (lineage B.1.1.7 and lineage B.1.351) identified in the UK and South Africa, respectively. Our in vitro analysis of viral replication showed that the drugs targeting TMPRSS2 HGNC and RdRp PROTEIN are equally effective against the two variants of concern.

    Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease PROTEIN ExoN

    Authors: Nicholas H Moeller; Ke Shi; Özlem Demir; Surajit Banerjee; Lulu Yin; Christopher Belica; Cameron Durfee; Rommie E Amaro; Hideki Aihara

    doi:10.1101/2021.04.02.438274 Date: 2021-04-04 Source: bioRxiv

    High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease PROTEIN (ExoN) in non-structural protein 14 PROTEIN (nsp14), which excises nucleotides including antiviral drugs mis-incorporated by the low-fidelity viral RNA-dependent RNA polymerase PROTEIN ( RdRp PROTEIN) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here we determined a 1.6-[A] resolution crystal structure of SARS-CoV-2 ExoN in complex with its essential co-factor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. Molecular dynamics simulations further show remarkable flexibility of multi-domain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA-binding to support its exoribonuclease PROTEIN activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anti-coronaviral drugs or strategies to attenuate the viral virulence.

    Screening of HLA-A HGNC restricted T cell epitopes of SARS-CoV-2 and induction of CD8+ T cell responses in HLA-A HGNC transgenic mice

    Authors: Xiaoxiao Jin; Yan Ding; Shihui Sun; Xinyi Wang; Zining Zhou; Xiaotao Liu; Miaomiao Li; Xian Chen; Anran Shen; Yandan Wu; Bicheng Liu; Jianqiong Zhang; Jian Li; Yi Yang; Haibo Qiu; Chuanlai Shen; Yuxian He; Guangyu Zhao

    doi:10.1101/2021.04.01.438020 Date: 2021-04-01 Source: bioRxiv

    While SARS-CoV-2-specific T cells have been characterized to play essential roles in host immune protection in COVID-19 MESHD patients, few researches focus on the functional validation of T cell epitopes and development of vaccines inducing specific T cell responses. In this study, 120 CD8 T cell epitopes from E, M, N, S and RdRp PROTEIN proteins of SARS-CoV-2 were validated by on-silicon prediction, DC-peptide-PBL costimulation with PBMCs of healthy donors and HLA-A HGNC molecule competitive binding experiments. Among them, 110, 15, 6, 14 and 12 epitopes were highly homologous with SARS-CoV MESHD, OC43, NL63, HKU1, and 229E, respectively. Thirty-one epitopes restricted by HLA-A2 molecule were used to generate peptide cocktail vaccines in combination with Poly(I:C), R848 or polylactic-co-glycolic acid nanoparticles, which elicited robust specific CD8 T cell responses in wild-type and HLA-A2/DR1 transgenic mice. Seven of the 31 epitopes were found to be cross-presented by HLA-A2 and H-2K/Db molecules. These data have provided a library of SARS-CoV-2 CD8 T cell epitopes which restricted by a series of high-frequency HLA-A HGNC allotypes and covered broad population in Asia, and initially confirmed the feasibility of human MHC class I molecule-restricted SARS-CoV2 epitope peptide cocktail vaccines, thus will facilitate the development of T cell epitope vaccines and specific cellular function detection kits.

    The nucleotide addition cycle of the SARS-CoV-2 polymerase

    Authors: Subhas Chandra Bera; Mona Seifert; Robert Kirchdoerfer; Pauline van Nies; Yibulayin Wubulikasimu; Salina Quack; Flavia S. Papini; Jamie J. Arnold; Bruno Canard; Craig E. Cameron; Martin Depken; David Dulin

    doi:10.1101/2021.03.27.437309 Date: 2021-03-27 Source: bioRxiv

    Coronaviruses have evolved elaborate multisubunit machines to replicate and transcribe their genomes. Central to these machines are the RNA-dependent RNA polymerase PROTEIN subunit (nsp12) and its intimately associated cofactors (nsp7 and nsp8). We have used a high-throughput magnetic-tweezers approach to develop a mechanochemical description of this core polymerase. The core polymerase exists in at least three catalytically distinct conformations, one being kinetically consistent with incorporation of incorrect nucleotides. We provide the first evidence that an RdRp PROTEIN uses a thermal ratchet instead of a power stroke MESHD to transition from the pre- to post-translocated state. Ultra-stable magnetic tweezers enables the direct observation of coronavirus polymerase deep and long-lived backtrack that are strongly stimulated by secondary structure in the template. The framework presented here elucidates one of the most important structure-dynamics-function relationships in human health today, and will form the grounds for understanding the regulation of this complex.

    SARS-CoV-2 N gene PROTEIN dropout and N gene PROTEIN Ct value shift as indicator for the presence of B.1.1.7 lineage in a widely used commercial multiplex PCR assay

    Authors: Paul Wollschlaeger; Nadja Gerlitz; Daniel Todt; Stephanie Pfaender; Thomas Bollinger; Andreas Sing; Alexandra Dangel; Nikolaus Ackermann; Klaus Korn; Armin Ensser; Eike Steinmann; Michael Buhl; Joerg Steinmann

    doi:10.1101/2021.03.23.21254171 Date: 2021-03-26 Source: medRxiv

    Objectives. Increased importance in detection and surveillance of SARS-CoV-2 has been demonstrated due to the emergence of variants of concern (VOCs). In this study we evaluated if a commercially available real-time SARS-CoV-2 PCR assay can identify B.1.1.7 lineage samples by a specific N gene PROTEIN dropout or Ct value shift compared to the S or RdRP PROTEIN gene. Methods. Patients samples with confirmed B.1.1.7 variant by whole-genome sequencing and variant-specific PCR (n=48) and non-B.1.1.7 samples (n=53) were tested by the Allplex SARS-CoV-2/FluA/FluB/RSV PCR assay for presence of S, RdRP PROTEIN and N gene PROTEIN of SARS CoV-2. The N gene PROTEIN coding sequence of SARS-CoV-2 with and without D3L mutation (specific for B.1.1.7) were cloned into pCR-TOPO vectors and Allplex SARS-CoV-2/FluA/FluB/RSV PCR assay was performed. Results. All studied B.1.1.7 patient samples showed significantly higher Ct values (delta 6-10, N-gene PROTEIN dropout on Ct values >29) in the N gene PROTEIN compared to the respective values of S and RdRP PROTEIN gene. Receiver operating characteristic (ROC) curve analysis resulted in 100% sensitivity and specificity for delta Ct N/ RdRP PROTEIN and delta Ct N/S. As a result of the reversed genetic experiments we found also the shift in Ct values for the 3L variant N-gene PROTEIN. Conclusions. N gene PROTEIN dropout or Ct value shift is specific for B.1.1.7 positive samples using the Allplex SARS-CoV-2/FluA/FluB/RSV PCR assay. This approach can be used as a rapid tool for B.1.1.7 detection in single assay high throughput diagnostics.

    Dimeric form of SARS-CoV-2 polymerase

    Authors:

    doi:10.1101/2021.03.23.436644 Date: 2021-03-24 Source: bioRxiv

    The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase PROTEIN ( RdRp PROTEIN) to replicate and transcribe its genome. Structures of the RdRp PROTEIN revealed a monomeric enzyme composed of the catalytic subunit nsp12, two copies of subunit nsp8, and one copy of subunit nsp7. Here we report an alternative, dimeric form of the coronavirus polymerase and resolve its structure at 4.8 [A] resolution. In this structure, the two RdRps PROTEIN contain only one copy of nsp8 and dimerize via their nsp7 subunits to adopt an antiparallel arrangement. We speculate that the RdRp PROTEIN dimer facilitates template switching during production of sub-genomic RNAs for transcription.

    Protein-primed RNA synthesis in SARS-CoVs MESHD and structural basis for inhibition by AT-527

    Authors: Ashleigh Shannon; Veronique Fattorini; Bhawna Sama; Barbara Selisko; Mikael Feracci; Camille Falcou; Pierre Gauffre; Priscila El Kazzi; Etienne Decroly; Nadia Rabah; Karine Toulon; Cecilia Eydoux; Jean-Claude Guillemot; Mathieu Noel; Francoise Debart; Jean-Jacques Vasseur; Adel Moussa; Steven Good; Kai Lin; Jean-Pierre Sommadossi; Yingxiao Zhu; Xiaodong Yan; Hui Shi; Francois Ferron; Bruno Canard

    doi:10.1101/2021.03.23.436564 Date: 2021-03-23 Source: bioRxiv

    How viruses from the Coronaviridae family initiate viral RNA synthesis is unknown. Here we show that the SARS-CoV-1 and -2 Nidovirus RdRp PROTEIN-Associated Nucleotidyltransferase (NiRAN) domain on nsp12 uridylates the viral cofactor nsp8, forming a UMP- Nsp HGNC8 covalent intermediate that subsequently primes RNA synthesis from a poly(A) template; a protein-priming mechanism reminiscent of Picornaviridae enzymes. In parallel, the RdRp PROTEIN active site of nsp12 synthesizes a pppGpU primer, which primes (-)ssRNA synthesis at the precise genome-poly(A) junction. The guanosine analogue 5'-triphosphate AT-9010 (prodrug: AT-527) tightly binds to the NiRAN and inhibits both nsp8-labeling and the initiation of RNA synthesis. A 2.98 A resolution Cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-(nsp8)2 /RNA/NTP quaternary complex shows AT-9010 simultaneously binds to both NiRAN and RdRp PROTEIN active site of nsp12, blocking their respective activities. AT-527 is currently in phase II clinical trials, and is a potent inhibitor of SARS-CoV-1 and -2, representing a promising drug for COVID-19 MESHD treatment.

    The SARS-CoV-2 replication-transcription complex is a priority target for broad-spectrum pan-coronavirus drugs

    Authors: Setayesh Yazdani; Nicola De Maio; Yining Ding; Vijay Shahani; Nick Goldman; Matthieu Schapira

    doi:10.1101/2021.03.23.436637 Date: 2021-03-23 Source: bioRxiv

    In the absence of effective treatment, COVID-19 MESHD is likely to remain a global disease burden. Compounding this threat is the near certainty that novel coronaviruses with pandemic potential will emerge in years to come. Pan-coronavirus drugs - agents active against both SARS-CoV-2 and other coronaviruses - would address both threats. A strategy to develop such broad-spectrum inhibitors is to pharmacologically target binding sites on SARS-CoV-2 proteins that are highly conserved in other known coronaviruses, the assumption being that any selective pressure to keep a site conserved across past viruses will apply to future ones. Here, we systematically mapped druggable binding pockets on the experimental structure of fifteen SARS-CoV-2 proteins and analyzed their variation across twenty-seven - and {beta}-coronaviruses and across thousands of SARS-CoV-2 samples from COVID-19 MESHD patients. We find that the two most conserved druggable sites are a pocket overlapping the RNA binding site of the helicase nsp13, and the catalytic site of the RNA-dependent RNA polymerase PROTEIN nsp12, both components of the viral replication-transcription complex. We present the data on a public web portal (https://www.thesgc.org/SARSCoV2_pocketome/) where users can interactively navigate individual protein structures and view the genetic variability of drug binding pockets in 3D.

    Identification of guanylyltransferase activity in the SARS-CoV-2 RNA polymerase

    Authors: Alexander P Walker; Haitian Fan; Jeremy R Keown; Jonathan Grimes; Ervin Fodor

    doi:10.1101/2021.03.17.435913 Date: 2021-03-18 Source: bioRxiv

    SARS-CoV-2 is a positive-sense RNA virus that is responsible for the ongoing Coronavirus Disease MESHD Coronavirus Disease 2019 MESHD ( COVID-19 MESHD) pandemic, which continues to cause significant morbidity, mortality and economic strain. SARS-CoV-2 can cause severe respiratory disease MESHD and death MESHD in humans, highlighting the need for effective antiviral therapies. The RNA synthesis machinery of SARS-CoV-2 is an ideal drug target and consists of non-structural protein 12 PROTEIN (nsp12), which is directly responsible for RNA synthesis, and numerous co-factors that are involved in RNA proofreading and 5' capping of viral mRNAs. The formation of the 5' cap-1 HGNC structure is known to require a guanylyltransferase (GTase) as well as 5' triphosphatase and methyltransferase activities. However, the mechanism of SARS-CoV-2 mRNA capping remains poorly understood. Here we show that the SARS-CoV-2 RNA polymerase nsp12 functions as a GTase. We characterise this GTase activity and find that the nsp12 NiRAN (nidovirus RdRP PROTEIN-associated nucleotidyltransferase) domain is responsible for carrying out the addition of a GTP nucleotide to the 5' end of viral RNA via a 5' to 5' triphosphate linkage. We also show that remdesivir triphosphate, the active form of the antiviral drug remdesivir, inhibits the SARS-CoV-2 GTase reaction as efficiently as RNA polymerase activity. These data improve understanding of coronavirus mRNA cap synthesis and highlight a new target for novel or repurposed antiviral drugs against SARS-CoV-2.

The ZB MED preprint Viewer preVIEW includes all COVID-19 related preprints from medRxiv and bioRxiv, from ChemRxiv, from ResearchSquare, from arXiv and from Preprints.org and is updated on a daily basis (7am CET/CEST).
The web page can also be accessed via API.

Sources


Annotations

All
None
MeSH Disease
HGNC Genes
SARS-CoV-2 Proteins


Export subcorpus as...

This service is developed in the project nfdi4health task force covid-19 which is a part of nfdi4health.

nfdi4health is one of the funded consortia of the National Research Data Infrastructure programme of the DFG.