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    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.

    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.

    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.

    Dual targeting of cytokine storm and viral replication in COVID-19 MESHD by plant-derived steroidal pregnanes in silico

    Authors: Gideon A. Gyebi; Oludare M. Ogunyemi; Ibrahim M. Ibrahim; Saheed O. Afolabi; Joseph O. Adebayo

    doi:10.21203/rs.3.rs-329239/v1 Date: 2021-03-14 Source: ResearchSquare

    The high morbidity and mortality rate of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) infection MESHD arises majorly from the Acute Respiratory Distress Syndrome MESHD and “cytokine storm” syndrome, which is sustained by an aberrant systemic inflammatory response and elevated pro-inflammatory cytokines. Thus, phytocompounds with broad-spectrum anti-inflammatory activity that target multiple SARS-CoV-2 proteins will enhance the development of effective drugs against the disease. In this study, an in-house library of 106 steriodal plant-derived pregnanes (PDPs) was docked in the active regions of human glucocorticoid receptors (hGRs) in a comparative molecular docking analysis. Based on the minimal binding energy and a comparative dexamethason binding mode analysis, a list of top twenty ranked PDPs docked in the agonist conformation of hGR, with binding energies ranging between -9.8 and -11.2 Kcal/mol, was obtained and analyzed for interactions with the human Janus kinases 1 and Interleukins-6 and SARS-CoV-2 3-chymotrypsin- like protease, Papain PROTEIN-like protease and RNA-dependent RNA polymerase PROTEIN. For each target protein, the top three ranked PDPs were selected. Eight PDPs (bregenin, hirundigenin, anhydroholantogenin, atratogenin A, atratogenin B, glaucogenin A, glaucogenin C and glaucogenin MESHD D) with high binding tendencies to the catalytic residues of multiple targets were identified. A high degree of structural stability was observed from the 100 ns molecular dynamics simulation analyses of glaucogenin C MESHD and hirundigenin complexes of hGR. The selected top-eight ranked PDPs demonstrated favourable druggable and in silico ADMET properties. Thus, the therapeutic potentials of glaucogenin C MESHD and hirundigenin can be explored for further in vitro and in vivo studies.

    Multiplex real-time RT-PCR method for the diagnosis of SARS-CoV-2 by targeting viral N2, RdRP PROTEIN and human RP genes

    Authors: Huseyin Tombuloglu; Hussein Sabit; Ebtesam Al-Suhaimi; Hamoud Al-Khallaf; Juma Kabanja; Najat Al-Saleh

    doi:10.21203/rs.3.rs-308136/v1 Date: 2021-03-07 Source: ResearchSquare

    Corona Virus Disease MESHD 2019 ( COVID-19 MESHD) is a disease caused by severe acute respiratory syndrome coronavirus 2 MESHD (SARS-CoV-2). This pandemic has brought the world to a standstill and threatened human lives. Many methods are known to date to detect this virus. Due to their relative accuracy, polymerase chain reaction (PCR)-based assays are the most frequently applied and considered the gold standard. However, some of these assays have the disadvantages of taking time to show the result and might produce false-negative and false-positive ones. Therefore, designing rapid and accurate PCR-based testing assay is of paramount importance for early detection of this virus and for more efficient control of the spread of this disease. We, here, describe a fast, reliable, easy-to- use, and high-throughput multiplex SARS-CoV-2 RT-PCR detection method. The assay was designed to detect two viral genes (N2 and RdRP PROTEIN) and a human gene (RP) simultaneously. The performance and the accuracy of the assay was tested in 28 SARS-CoV-2 positive samples and compared with commercial kits, which showed 100% positive percent agreement with a limit of detection (LOD) value of 1.25 copies/µL or 5 copies/reaction. The current assay is found accurate, reliable, simple, sensitive, and specific. It can be used as an optimized SARS-CoV-2 diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.

    Analysis of SARS-CoV-2 Mutations Over Time Reveals Increasing Prevalence of Variants in the Spike Protein PROTEIN and RNA-Dependent RNA Polymerase PROTEIN

    Authors: William M Showers; Sonia M Leach; Katerina Kechris; Michael Strong

    doi:10.1101/2021.03.05.433666 Date: 2021-03-05 Source: bioRxiv

    Amid the ongoing COVID-19 pandemic MESHD, it has become increasingly important to monitor the mutations that arise in the SARS-CoV-2 virus, to prepare public health strategies and guide the further development of vaccines and therapeutics. The spike (S) protein PROTEIN and the proteins comprising the RNA-Dependent RNA Polymerase PROTEIN ( RdRP PROTEIN) are key vaccine and drug targets, respectively, making mutation surveillance of these proteins of great importance. Full protein sequences for the spike proteins PROTEIN and RNA-dependent RNA polymerase PROTEIN proteins were downloaded from the GISAID database, aligned, and the variants identified. Polymorphisms in the protein sequence were investigated at the protein structural level and examined longitudinally in order to identify sequence and strain variants that are emerging over time. Our analysis revealed a group of variants in the spike protein PROTEIN and the polymerase complex that appeared in August, and account for around five percent of the genomes analyzed up to the last week of October. A structural analysis also facilitated investigation of several unique variants in the receptor binding domain and the N-terminal domain of the spike protein PROTEIN, with high-frequency mutations occurring more commonly in these regions. The identification of new variants emphasizes the need for further study on the effects of these mutations and the implications of their increased prevalence, particularly as these mutations may impact vaccine or therapeutic efficacy.

    Atorvastatin effectively inhibits late replicative cycle steps of SARS-CoV-2 in vitro

    Authors: Maria Isabel Zapata-Cardona; Lizdany Flórez-Álvarez; Wildeman Zapata-Builes; Ariadna Guerra-Sandoval; Carlos Guerra-Almonacid; Jaime Hincapié-García; Maria Teresa Rugeles; Juan Carlos Hernández

    doi:10.1101/2021.03.01.433498 Date: 2021-03-03 Source: bioRxiv

    Introduction: SARS-CoV-2 has caused a pandemic of historic proportions and continues to spread worldwide. Currently, there is no effective therapy against this virus. This article evaluated the in vitro antiviral effect of Atorvastatin against SARS-CoV-2 and also identified the interaction affinity between Atorvastatin and three SARS-CoV-2 proteins MESHD, using in silico structure-based molecular docking approach. Materials and methods: The antiviral activity of Atorvastatin against SARS-CoV-2 was evaluated by three different treatment strategies using a clinical isolate of SARS-CoV-2. The interaction of Atorvastatin with Spike, RNA-dependent RNA polymerase PROTEIN ( RdRp PROTEIN) and 3C-like protease ( 3CLpro PROTEIN) was evaluated by molecular docking. Results: Atorvastatin showed anti-SARS-CoV-2 activity of 79%, 54.8%, 22.6% and 25% at 31.2, 15.6, 7.9, and 3.9 {micro}M, respectively, by pre-post-treatment strategy. In addition, atorvastatin demonstrated an antiviral effect of 26.9% at 31.2 {micro}M by pre-infection treatment. This compound also inhibited SARS-CoV-2 in 66.9%, 75%, 27.9% and 29.2% at concentrations of 31.2, 15.6, 7.9, and 3.9 {micro}M, respectively, by post-infection treatment. The interaction of atorvastatin with SARS-CoV-2 Spike PROTEIN SARS-CoV-2 Spike MESHD, RdRp PROTEIN and 3CL protease PROTEIN yielded a binding affinity of -8.5 Kcal/mol, -6.2 Kcal/mol, and -7.5 Kcal/mol, respectively. Conclusion: Our study demonstrated the in vitro anti-SARS-CoV-2 activity of Atorvastatin, mainly against the late steps of the viral replicative cycle. A favorable binding affinity with viral proteins by bioinformatics methods was also shown. Due to its low cost, availability, well-established safety and tolerability, and the extensive clinical experience of atorvastatin, it could prove valuable in reducing morbidity and mortality from COVID-19 MESHD.

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MeSH Disease
HGNC Genes
SARS-CoV-2 Proteins

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