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      Post-infection treatment with the E protein inhibitor BIT225 reduces disease severity and increases survival of K18-hACE2 transgenic mice infected with a lethal dose of SARS-CoV-2

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          Abstract

          The Coronavirus envelope (E) protein is a small structural protein with ion channel activity that plays an important role in virus assembly, budding, immunopathogenesis and disease severity. The viroporin E is also located in Golgi and ER membranes of infected cells and is associated with inflammasome activation and immune dysregulation. Here we evaluated in vitro antiviral activity, mechanism of action and in vivo efficacy of BIT225 for the treatment of SARS-CoV-2 infection. BIT225 showed broad-spectrum direct-acting antiviral activity against SARS-CoV-2 in Calu3 and Vero cells with similar potency across 6 different virus strains. BIT225 inhibited ion channel activity of E protein but did not inhibit endogenous currents or calcium-induced ion channel activity of TMEM16A in Xenopus oocytes. BIT225 administered by oral gavage for 12 days starting 12 hours before infection completely prevented body weight loss and mortality in SARS-CoV-2 infected K18 mice (100% survival, n = 12), while all vehicle-dosed animals reached a mortality endpoint by Day 9 across two studies (n = 12). When treatment started at 24 hours after infection, body weight loss, and mortality were also prevented (100% survival, n = 5), while 4 of 5 mice maintained and increased body weight and survived when treatment started 48 hours after infection. Treatment efficacy was dependent on BIT225 dose and was associated with significant reductions in lung viral load (3.5 log 10), virus titer (4000 pfu/ml) and lung and serum cytokine levels. These results validate viroporin E as a viable antiviral target and support the clinical study of BIT225 for treatment and prophylaxis of SARS-CoV-2 infection.

          Author summary

          Antiviral agents are highly important for the management of COVID-19. New antivirals are needed, because available drugs have drawbacks that limit their use and are threatened by drug resistance. This study demonstrates that the small molecule drug BIT225 is an inhibitor of an important viral ion channel (E protein). E protein is required for virus replication and is involved in eliciting inflammatory response to infection. Exacerbated inflammation is a hallmark of severe COVID-19 in mice and in humans. In a mouse model of severe SARS-CoV-2 infection, BIT225 treatment starting before or 24 hours after infection could protect all treated mice from developing disease, from experiencing weight loss and from death (100%, n = 17), while all untreated mice developed severe disease, started to lose body weight from Day 3 onwards and died within 9 days after infection. BIT225 treatment was associated with potent suppression of virus load, and reduced inflammation markers, consistent with effective clearance of the virus. These results are remarkable for the high efficacy achieved with a new mechanism of action. BIT225 is a clinical stage drug candidate with an established human safety profile. These results support clinical evaluation of BIT225 for the treatment of human SARS-CoV-2 infection.

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          Characteristics of SARS-CoV-2 and COVID-19

          Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible and pathogenic coronavirus that emerged in late 2019 and has caused a pandemic of acute respiratory disease, named ‘coronavirus disease 2019’ (COVID-19), which threatens human health and public safety. In this Review, we describe the basic virology of SARS-CoV-2, including genomic characteristics and receptor use, highlighting its key difference from previously known coronaviruses. We summarize current knowledge of clinical, epidemiological and pathological features of COVID-19, as well as recent progress in animal models and antiviral treatment approaches for SARS-CoV-2 infection. We also discuss the potential wildlife hosts and zoonotic origin of this emerging virus in detail.
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            Severe Acute Respiratory Syndrome Coronavirus Infection Causes Neuronal Death in the Absence of Encephalitis in Mice Transgenic for Human ACE2

            Journal of Virology, 82(15), 7264-7275
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              Dose-Response Analysis Using R

              Dose-response analysis can be carried out using multi-purpose commercial statistical software, but except for a few special cases the analysis easily becomes cumbersome as relevant, non-standard output requires manual programming. The extension package drc for the statistical environment R provides a flexible and versatile infrastructure for dose-response analyses in general. The present version of the package, reflecting extensions and modifications over the last decade, provides a user-friendly interface to specify the model assumptions about the dose-response relationship and comes with a number of extractors for summarizing fitted models and carrying out inference on derived parameters. The aim of the present paper is to provide an overview of state-of-the-art dose-response analysis, both in terms of general concepts that have evolved and matured over the years and by means of concrete examples.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: InvestigationRole: Methodology
                Role: Data curationRole: Formal analysisRole: InvestigationRole: Methodology
                Role: Investigation
                Role: Project administrationRole: Writing – original draft
                Role: ConceptualizationRole: MethodologyRole: Writing – original draftRole: Writing – review & editing
                Role: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: InvestigationRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: InvestigationRole: MethodologyRole: Project administration
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                PLOS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                7 August 2023
                August 2023
                : 19
                : 8
                : e1011328
                Affiliations
                [1 ] Biotron Limited, North Ryde, New South Wales, Australia
                [2 ] The Scripps Institute, Immunology and Microbiology, La Jolla, California, United States of America
                [3 ] University of Copenhagen, Department of Biomedical Sciences, Copenhagen, Denmark
                Duke University, UNITED STATES
                Author notes

                GE, AT and MM are employees of Biotron Limited; KK and SB are consultants of Biotron Limited. MMR is co-founder, member of the Board of Directors and minority shareholder of Synklino.

                Author information
                https://orcid.org/0000-0001-5919-8275
                Article
                PPATHOGENS-D-23-00564
                10.1371/journal.ppat.1011328
                10434922
                37549173
                71297e12-f7c0-455a-92d0-3b55d48f55cc
                © 2023 Ewart et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 30 March 2023
                : 6 July 2023
                Page count
                Figures: 4, Tables: 1, Pages: 20
                Funding
                Funded by: Biotron Limited
                Funded by: funder-id http://dx.doi.org/10.13039/100019180, HORIZON EUROPE European Research Council;
                Award ID: 682549
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100003554, Lundbeck Foundation;
                Award ID: lR242-2017-409
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100009708, Novo Nordisk Fonden;
                Award ID: NNF20OC0062899
                Award Recipient :
                The work was entirely funded by Biotron Limited, with the exception of the Xenopus oocyte experiments, which received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 682549 to MMR), the Lundbeck Foundation (lR242-2017-409 to MMR) and the NovoNordisk Foundation (NNF20OC0062899 to MMR). MM, GE and AT are full time employees of Biotron, and the study design, data analyses, publication decision and manuscript preparation were all undertaken in conjunction with the other authors.
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                2023-08-17
                The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.
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