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    Bead-assisted SARS-CoV-2 multi-antigen serological test allows effective identification of patients

    Authors: Yaiza Caceres-Martell; Daniel Fernandez-Soto; Carmen Campos-Silva; Eva Maria Garcia-Cuesta; Jose M. Casasnovas; David Navas-Herrera; Alexandra Beneitez-Martinez; Pedro Martinez-Fleta; Arantzazu Alfranca; Francisco Sanchez-Madrid; Gabriela Escudero-Lopez; Carlos Vilches; Ricardo Jara-Acevedo; Hugh T. Reyburn; Jose M. Rodriguez-Frade; Mar Vales-Gomez

    doi:10.1101/2021.04.08.21254348 Date: 2021-04-10 Source: medRxiv

    Many new aspects of COVID-19 MESHD disease, including different clinical manifestations, have been identified during the pandemic. The wide array of symptoms and variation in disease severity after SARS-CoV-2 infection MESHD might be related to heterogeneity in the immune responses of different patients. Here we describe a new method for a simple multi-antigen serological test that generates a full picture of seroconversion in a single reaction. The assay is based on the detection by flow cytometry of multiple immunoglobulin classes (isotypes) specific for four SARS-CoV-2 antigens: the Spike glycoprotein PROTEIN (one of the highly immunogenic proteins), its RBD fragment (the major target for neutralising antibodies), the nucleocapsid protein PROTEIN and the main cysteine-like protease PROTEIN. Until now, most diagnostic serological tests measured antibodies to only one antigen and some patients seemed to not make any antibody response. Our data reveal that while most patients respond against all the viral antigens tested, others show a marked bias to make antibodies against either proteins exposed on the viral particle or those released after cellular infection. Combining all the four antigens and using machine learning techniques, it was possible to clearly discriminate between patients and healthy controls with 100% confidence. Further, combination of antigens and different immunoglobulin isotypes in this multi-antigen assay improved the classification of patients with mild and severe disease. Introduction of this method will facilitate massive screenings of patients to evaluate their immune response. It could also support vaccination campaigns both to select non-immune individuals and to distinguish infected MESHD patients from vaccine responders.

    Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of nsp5 Main Protease PROTEIN

    Authors: Clovis Basier; Rupert Beale; Ganka Bineva-Todd; Berta Canal; Joseph F Curran; Tom D Deegan; John FX Diffley; Ryo Fujisawa; Michael Howell; Dhira Joshi; Karim Labib; Chew Theng Lim; Jennifer Milligan; Hema Nagaraj; George Papageorgiou; Christelle Soudy; Kang Wei Tan; Rachel Ulferts; Florian Weissmann; Mary Wu; Theresa U Zeisner

    doi:10.1101/2021.04.07.438806 Date: 2021-04-08 Source: bioRxiv

    The coronavirus 2019 ( COVID-19 MESHD) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 MESHD (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5,000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.

    Combined computational and cellular screening identifies synergistic inhibition of SARS-CoV-2 by lenvatinib and remdesivir

    Authors: Marie O. Pohl; Idoia Busnadiego; Francesco Marrafino; Lars Wiedmer; Annika Hunziker; Sonja Fernbach; Elena Omori; Benjamin G Hale; Amedeo Caflisch; Stertz Silke

    doi:10.1101/2021.03.19.435806 Date: 2021-03-19 Source: bioRxiv

    Rapid repurposing of existing drugs as new therapeutics for COVID-19 MESHD has been an important strategy in the management of disease severity during the ongoing SARS-CoV-2 pandemic. Here, we screened by high-throughput docking 6,000 compounds within the DrugBank library for their potential to bind and inhibit the SARS-CoV-2 3CL main protease PROTEIN, a chymotrypsin-like enzyme that is essential for viral replication. For 19 candidate hits, parallel in vitro fluorescence-based protease-inhibition assays and Vero-CCL81 cell-based SARS-CoV-2 replication-inhibition assays were performed. One hit, diclazuril (an investigational anti-protozoal compound), was validated as a SARS-CoV-2 3CL main protease PROTEIN inhibitor in vitro (IC50 value of 29 M) and modestly inhibited SARS-CoV-2 replication in Vero-CCL81 cells. Another hit, lenvatinib (approved for use in humans as an anti-cancer treatment), could not be validated as a SARS-CoV-2 3CL main protease PROTEIN inhibitor in vitro, but serendipitously exhibited a striking functional synergy with the approved nucleoside analogue remdesivir to inhibit SARS-CoV-2 replication in Vero-CCL81 cells. Lenvatinib is a broadly-acting host receptor tyrosine kinase (RTK) inhibitor, but the synergistic effect with remdesivir was not observed with other approved RTK inhibitors (such as pazopanib or sunitinib), suggesting that the mechanism-of-action is independent of host RTKs. Furthermore, time-of-addition studies revealed that lenvatinib/remdesivir synergy probably targets SARS-CoV-2 replication subsequent to host-cell entry. Our study shows that combining computational and cellular screening is an efficient means to identify existing drugs with repurposing potential as antiviral compounds. Future studies should aim at understanding and optimizing the lenvatinib/remdesivir synergistic mechanism as a therapeutic option.

    SARS-CoV-2 Nsp5 HGNC Protein Causes Acute Lung Inflammation MESHD: A Dynamical Mathematical Model

    Authors: José Díaz; Elena R. Álvarez-Buylla; Antonio Bensussen

    id:10.20944/preprints202012.0749.v2 Date: 2021-03-15 Source: Preprints.org

    In the present work we propose a dynamical mathematical model of the lung cells inflammation process MESHD in response to SARS-CoV-2 infection MESHD. In this scenario the main protease PROTEIN Nsp5 HGNC enhances the inflammatory process, increasing the levels of NF kB, IL-6 HGNC, Cox2 HGNC, and PGE2 with respect to a reference state without the virus. In presence of the virus the translation rates of NF kB and IkB arise to a high constant value, and when the translation rate of IL-6 HGNC also increases above the threshold value of 7 pg mL-1 s-1 the model predicts a persistent over stimulated immune state with high levels of the cytokine IL-6 HGNC. Our model shows how such over stimulated immune state becomes autonomous of the signals from other immune cells such as macrophages and lymphocytes, and does not shut down by itself. We also show that in the context of the dynamical model presented here, Dexamethasone or Nimesulide have little effect on such inflammation MESHD state of the infected lung cell, and the only form to suppress it is with the inhibition of the activity of the viral protein Nsp5 HGNC.To that end, our model suggest that drugs like Saquinavir may be useful. In this form, our model suggests that Nsp5 HGNC is effectively a central node underlying the severe acute lung inflammation MESHD during SARS-CoV-2 infection MESHD. The persistent production of IL-6 HGNC by lung cells can be one of the causes of the cytokine storm observed in critical patients with COVID19 MESHD. Nsp5 HGNC seems to be the switch to start inflammation MESHD, the consequent overproduction of the ACE2 HGNC receptor, and an important underlying cause of the most severe cases of COVID19 MESHD.

    Disulfiram associated with lower risk of Covid-19 MESHD: a retrospective cohort study

    Authors: Nathanael Fillmore; Steven Bell; Ciyue Shen; Vinh Nguyen; Jennifer La; Maureen Dubreuil; Judith Strymish; Mary Brophy; Gautam Mehta; Hao Wu; Judy Lieberman; Nhan Do; Chris Sander

    doi:10.1101/2021.03.10.21253331 Date: 2021-03-12 Source: medRxiv

    In the global COVID-19 pandemic MESHD, there is a substantial need for effective, low-cost therapeutics. We investigated the potential effects of disulfiram on the incidence and outcomes of COVID-19 MESHD in an observational study in a large database of US Veterans Administration clinical records, the VA Corporate Data Warehouse (CDW). The study is motivated by the unique properties of disulfiram, which has been used as an anti-alcoholism drug since 1948, is non-toxic, easy to manufacture and inexpensive. Disulfiram reduces hyperinflammation in mammalian cells by inhibition of the gasdermin D HGNC pore. In a mouse model of sepsis MESHD, disulfiram reduced inflammatory cytokines and mortality. Disulfiram also is a low micromolar inhibitor of the Mpro PROTEIN and PLpro PROTEIN viral proteases of SARS-CoV-2. To investigate the potential effects of disulfiram on the incidence and severity of COVID-19 MESHD, we carried out an epidemiological study in the CDW. The VA dataset used has 944,127 patients tested for SARS-Cov-2, 167,327 with a positive test, and 2,233 on disulfiram, of which 188 had a positive SARS-Cov-2 test. A multivariable Cox regression adjusted for age, gender, race/ethnicity, region, a diagnosis of alcohol use disorders, and Charlson comorbidity score revealed a reduced incidence of COVID-19 MESHD with disulfiram use with a hazard ratio of 0.66 and 95% confidence interval of 0.57 to 0.76 (P < 0.001). There were no deaths among the 188 SARS-Cov-2 positive patients treated with disulfiram. The expected number of deaths would have been 5-6 according to the 3% death rate among the untreated (P-value 0.03). Our finding of a lower hazard ratio and less severe outcomes for COVID-19 MESHD in patients treated with disulfiram compared to those not treated is a statistical association and does not prove any causative effect of disulfiram. However, the results of this study suggest that there is a pharmacological contribution to the reduced incidence and severity of COVID-19 MESHD with the use of disulfiram. Given the known anti-inflammatory and viral anti-protease effects of disulfiram, it is reasonable and urgent to initiate accelerated clinical trials to assess whether disulfiram reduces SARS-CoV-2 infection MESHD, disease severity and death MESHD.

    Novel inhibition mechanism of SARS-CoV-2 main protease PROTEIN by ebselen and its derivatives

    Authors: Kangsa Amporndanai; Xiaoli Meng; Haitao Yang; Zhie-Jie Liu; Leike Zhang; Yao Zhao; Samar Hasnain; Zihe Rao; Zhenming Jin; Weijuan Shang

    doi:10.1101/2021.03.11.434764 Date: 2021-03-11 Source: bioRxiv

    The global emergence of SARS-CoV-2 has triggered numerous efforts to develop therapeutic options for COVID-19 pandemic MESHD. The main protease PROTEIN of SARS-CoV-2 (Mpro PROTEIN), which is a critical enzyme for transcription and replication of SARS-CoV-2, is a key target for therapeutic development against COVID-19 MESHD. An organoselenium drug called ebselen has recently been demonstrated to have strong inhibition against Mpro PROTEIN and antiviral activity but its molecular mode of action is unknown preventing further development. We have examined the binding modes of ebselen and its derivative in Mpro PROTEIN via high resolution co-crystallography and investigated their chemical reactivity via mass spectrometry. Stronger Mpro PROTEIN inhibition than ebselen and potent ability to rescue infected cells were observed for a number of ebselen derivatives. A free selenium atom bound with cysteine 145 of Mpro PROTEIN catalytic dyad has been revealed by crystallographic studies of Mpro PROTEIN with ebselen and MR6-31-2 suggesting hydrolysis of the enzyme bound organoselenium covalent adduct, formation of a phenolic by-product is confirmed by mass spectrometry. The target engagement of these compounds with an unprecedented mechanism of SARS-CoV-2 Mpro PROTEIN inhibition suggests wider therapeutic applications of organo-selenium compounds in SARS-CoV-2 and other zoonotic beta-corona viruses.

    A novel conformational state for SARS-CoV-2 main protease PROTEIN

    Authors: Emanuele Fornasier; Maria Ludovica Macchia; Gabriele Giachin; Alice Sosic; Matteo Pavan; Mattia Sturlese; Cristiano Salata; Stefano Moro; Barbara Gatto; Massimo Bellanda; Roberto Battistutta

    doi:10.1101/2021.03.04.433882 Date: 2021-03-04 Source: bioRxiv

    The SARS-CoV-2 main protease PROTEIN ( Mpro PROTEIN) has a pivotal role in mediating viral genome replication and transcription of coronavirus, making it a promising target for drugs against Covid-19 pandemic MESHD. Here we present a crystal structure of Mpro PROTEIN disclosing new structural features of key regions of the enzyme. We show that the oxyanion loop, involved in substrate recognition and enzymatic activity, can adopt a new conformation, which is stable and significantly different from the known ones. In this new state the S1 subsite of the substrate binding region is completely reshaped and a new cavity near the S2 subsite is created. This new structural information expands the knowledge of the conformational space available to Mpro PROTEIN, paving the way for the design of novel classes of inhibitors specifically designed to target this unprecedented binding site conformation, thus enlarging the chemical space for urgent antiviral drugs against Covid-19 pandemic MESHD.

    A pharmacophore model for SARS-CoV-2 3CLpro PROTEIN small molecule inhibitors and in vitro experimental validation of computationally screened inhibitors

    Authors: Enrico Glaab; Ganesh Babu Manoharan; Daniel Abankwa

    doi:10.1101/2021.03.02.433618 Date: 2021-03-03 Source: bioRxiv

    Among the biomedical efforts in response to the current coronavirus ( COVID-19 MESHD) pandemic, pharmacological strategies to reduce viral load in patients with severe forms of the disease are being studied intensively. One of the main drug target proteins proposed so far is the SARS-CoV-2 viral protease 3CLpro PROTEIN (also called Mpro PROTEIN), an essential component for viral replication. Ongoing ligand- and receptor-based computational screening efforts would be facilitated by an improved understanding of the electrostatic, hydrophobic and steric features that characterize small molecule inhibitors binding stably to 3CLpro PROTEIN, as well as by an extended collection of known binders. Here, we present combined virtual screening, molecular dynamics simulation, machine learning and in vitro experimental validation analyses which have led to the identification of small molecule inhibitors of 3CLpro PROTEIN with micromolar activity, and to a pharmacophore model that describes functional chemical groups associated with the molecular recognition of ligands by the 3CLpro PROTEIN binding pocket. Experimentally validated inhibitors using a ligand activity assay include natural compounds with available prior knowledge on safety and bioavailability properties, such as the natural compound rottlerin (IC50 = 37 mcM), and synthetic compounds previously not characterized (e.g. compound CID 46897844, IC50 = 31 mcM). In combination with the developed pharmacophore model, these and other confirmed 3CLpro PROTEIN inhibitors may provide a basis for further similarity-based screening in independent compound databases and structural design optimization efforts, to identify 3CLpro PROTEIN ligands with improved potency and selectivity. Overall, this study suggests that the integration of virtual screening, molecular dynamics simulations and machine learning can facilitate 3CLpro PROTEIN-targeted small molecule screening investigations. Different receptor-, ligand- and machine learning-based screening strategies provided complementary information, helping to increase the number and diversity of identified active compounds. Finally, the resulting pharmacophore model and experimentally validated small molecule inhibitors for 3CLpro PROTEIN provide resources to support follow-up computational screening efforts for this drug target.

    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.

    Nature Potential for COVID-19 MESHD: Targeting SARS-CoV-2 Mpro PROTEIN Inhibitor with Bioactive Compound

    Authors: Kaushik Kumar Bharadwaj; Tanmay Sarkar; Arabinda Ghosh; Debabrat Baishya; Bijuli Rabha; Manasa Kumar Panda; Bryan R. Nelson; Akbar John; Hassan I. Sheikh; Bisnu Prasad Dash; Hisham A. Edinur; Siddhartha Pati

    doi:10.26434/chemrxiv.14112515.v2 Date: 2021-03-01 Source: ChemRxiv

    Corona viruses were first identified in 1931 and SARS-CoV-2 is the most recent. COVID-19 MESHD is a pandemic that put most of the world on lockdown and the search for therapeutic drugs is still on-going. Therefore, this study uses in silico screening to identify natural bioactive compounds from fruits, herbaceous plants and marine invertebrates that are able to inhibit protease activity in SARS-CoV-2(PDB: 6LU7). We have used various screening strategies such as drug likeliness, antiviral activity value prediction, molecular docking, ADME (absorption, distribution, metabolism, and excretion), molecular dynamics ( MD MESHD) simulation and MM/GBSA (molecular mechanics/generalized born and surface area continuum solvation). 17 compounds were shortlisted using Lipinski’s rule. 5 compounds revealed significantly good predicted antiviral activity values and out of them only 2 compounds, Macrolactin A and Stachyflin, showed good binding energy values of -9.22 and -8.00 kcal/mol within the binding pocket, catalytic residues ( HIS MESHD 41 and CYS 145) of Mpro PROTEIN. These two compounds were further analyzed for their ADME properties. The ADME evaluation of these 2 compounds suggested that they could be effective as therapeutic agents for developing drugs for clinical trials. MD simulations showed that protein-ligand complexes of Macrolactin A and Stachyflin were stable for 100 nano seconds. The MM/GBSA calculations of Mpro PROTEIN – Macrolactin A complex indicated higher binding free energy (-42.58 ± 6.35 kcal/mol) with Mpro PROTEIN protein target receptor (6LU7). DCCM and PCA analysis on the residual movement in the MD trajectories confirmed the good stability on Macrolactin A bound state of 6LU7. This signify the stable conformation of 6LU7 with high binding energy with Macrolactin A. Thus, this study showed that Macrolactin A could be an effective therapeutical agent for SARS-CoV-2protease MESHD (6LU7) inhibition. Additional in vitro and in vivo validations are needed to determine efficacy and dose of Macrolactin A in biological systems.

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MeSH Disease
HGNC Genes
SARS-CoV-2 Proteins

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