The anti-HIV Drug Nelfinavir Mesylate (Viracept) is a Potent Inhibitor of Cell Fusion Caused by the SARS-CoV-2 Spike (S) Glycoprotein Warranting further Evaluation as an Antiviral against COVID-19 infections
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Abstract
Coronaviruses belong to a group of enveloped, positive-single stranded RNA viruses that are known to cause severe respiratory distress in animals and humans. The current SARS coronavirus-2 (SARS CoV-2) pandemic has caused more than 2,000,000 infections globally and nearly 200,000 deaths. Coronaviruses enter susceptible cells via fusion of the viral envelope with the plasma membrane and/or via fusion of the viral envelope with endosomal membranes after endocytosis of the virus into endosomes. Previous results with SARS and MERS CoV have shown that the Spike (S) glycoprotein is a major determinant of virus infectivity and immunogenicity. Herein, we show that expression of SARS CoV-2 S (S-n) glycoprotein after transient transfection of African green monkey kidney (Vero) cells caused extensive cell fusion in comparison to limited cell fusion caused by the SARS S (S-o) glycoprotein. S-n expression was detected intracellularly and on transfected Vero cell surfaces and caused the formation of very large multinucleated cells (syncytia) by 48 hours post transfection. These results are in agreement with published pathology observations of extensive syncytial formation in lung tissues of COVID-19 patients. This differential S-n versus S-o-mediated cell fusion suggests that SARS-CoV-2 is able to spread from cell-to-cell much more efficiently than SARS effectively avoiding extracellular spaces and neutralizing antibodies. A systematic screening of several drugs for ability to inhibit S-n and S-o cell fusion revealed that the FDA approved HIV-protease inhibitor, nelfinavir mesylate (Viracept) drastically inhibited S-n and S-o-mediated cell fusion in a dose-dependent manner. Complete inhibition of cell fusion was observed at a 10 micromolar concentration. Computational modeling and in silico docking experiments suggested the possibility that nelfinavir may bind inside the S trimer structure, proximal to the S2 amino terminus directly inhibiting S-n and S-o-mediated membrane fusion. Also, it is possible that nelfinavir mesylate acts on cellular processes to inhibit S proteolytic processing. These results warrant further investigations of the potential of nelfinavir mesylate as an antiviral drug, especially at early times after SARS-CoV-2 symptoms appear.
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SciScore for 10.1101/2020.04.24.060376: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The primary antibodies used were as follows: mouse anti-myc antibody (Abcam, MA, USA), mouse anti-FLAG antibody (Abcam, MA, USA). anti-mycsuggested: Noneanti-FLAGsuggested: (Abcam Cat# ab124462, RRID:AB_11000959)Goat anti-mouse antibody conjugated with HRP (Invitrogen, Inc. CA, USA) was used as a secondary antibody. anti-mousesuggested: NoneGoat anti-mouse antibody conjugated with alexa fluorophore 647 and goat anti-rabbit antibody conjugated with alexa … SciScore for 10.1101/2020.04.24.060376: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The primary antibodies used were as follows: mouse anti-myc antibody (Abcam, MA, USA), mouse anti-FLAG antibody (Abcam, MA, USA). anti-mycsuggested: Noneanti-FLAGsuggested: (Abcam Cat# ab124462, RRID:AB_11000959)Goat anti-mouse antibody conjugated with HRP (Invitrogen, Inc. CA, USA) was used as a secondary antibody. anti-mousesuggested: NoneGoat anti-mouse antibody conjugated with alexa fluorophore 647 and goat anti-rabbit antibody conjugated with alexa fluore 488 (Invitrogen, Inc. CA, USA) were used for immuno-fluorescence (IFA) assay. anti-rabbitsuggested: NoneExperimental Models: Cell Lines Sentences Resources Cell line: African green monkey kidney (Vero) cells were maintained in Dulbeco Modified Eagle’s Media (DMEM) with 10% fetal bovine serum (FBS) and 2% primocin (Invitrogen, Inc. CA, USA). Verosuggested: NoneSoftware and Algorithms Sentences Resources Computational Methods: Docking of the nelfinavir mesylate to the spike protein of SARS CoV-2 was performed using Autodock (35). Autodocksuggested: (AutoDock, RRID:SCR_012746)Structures within 2 kcal/mol from the lowest energy docked structures were represented as final possible docked structures using PyMol software (Schrodinger). PyMolsuggested: (PyMOL, RRID:SCR_000305)Olympus IX71 fluorescent microscope was used for live and phase contrast images using Cellsens software. Cellsenssuggested: NoneZeiss Axio Observer Z1 fluorescent microscope was used for fluorescent images using Zen software. Zensuggested: NoneResults from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 16, 13 and 15. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- No funding statement was detected.
- No protocol registration statement was detected.
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