Protoporphyrin IX and verteporfin prevent SARS-CoV-2 infection in vitro and in a mouse model expressing human ACE2
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Abstract
The SARS-CoV-2 infection is spreading rapidly worldwide. Efficacious antiviral therapeutics against SARS-CoV-2 is urgently needed. Here, we discovered that protoporphyrin IX (PpIX) and verteporfin, two FDA-approved drugs, completely inhibited the cytopathic effect produced by SARS-CoV-2 infection at 1.25 μM and 0.31 μM respectively, and their EC50 values of reduction of viral RNA were at nanomolar concentrations. The selectivity indices of PpIX and verteporfin were 952.74 and 368.93, respectively, suggesting broad margin of safety. Importantly, PpIX and verteporfin prevented SARS-CoV-2 infection in mice adenovirally transduced with human ACE2. The compounds, sharing a porphyrin ring structure, were shown to bind viral receptor ACE2 and interfere with the interaction between ACE2 and the receptor-binding domain of viral S protein. Our study suggests that PpIX and verteporfin are potent antiviral agents against SARS-CoV-2 infection and sheds new light on developing novel chemoprophylaxis and chemotherapy against SARS-CoV-2.
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SciScore for 10.1101/2020.04.30.071290: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: The animal study protocol has been approved by the Animal Ethics Committee of School of Basic Medical Sciences, Fudan University. Randomization After a 4-hour co-cultivation period, five fields were randomly selected in each well and the number of fused and unfused cells in each field were counted directly under an inverted fluorescence microscope, based on much larger cell size of fused cells. Blinding not detected. Power Analysis not detected. Sex as a biological variable Transduction and infection of mice: Eight-week-old male mice (BALB/c) (SLAC Laboratory Animal, Shanghai, China) were raised in pathogen-free cages in the BSL-3 laboratory of Fudan University. Ce… SciScore for 10.1101/2020.04.30.071290: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: The animal study protocol has been approved by the Animal Ethics Committee of School of Basic Medical Sciences, Fudan University. Randomization After a 4-hour co-cultivation period, five fields were randomly selected in each well and the number of fused and unfused cells in each field were counted directly under an inverted fluorescence microscope, based on much larger cell size of fused cells. Blinding not detected. Power Analysis not detected. Sex as a biological variable Transduction and infection of mice: Eight-week-old male mice (BALB/c) (SLAC Laboratory Animal, Shanghai, China) were raised in pathogen-free cages in the BSL-3 laboratory of Fudan University. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Immunofluorescence analysis: To detect the viral nucleocapsid protein (N protein), anti-N polyclonal antibodies were generated using standard immunization of BALB/c mice with recombinant N protein derived from E. coli. viral nucleocapsid protein ( N protein)suggested: NoneVero-E6 cells grown in 96-well plate were fixed in 4% paraformaldehyde, permeabilized by 0.2% Triton X-100 (Thermo Fisher Scientific, Waltham, USA), blocked with 3% BSA, and stained overnight with the anti-N antibody (1:1000 dilution) at 4°C. anti-Nsuggested: NoneThe samples were then incubated with Alexa Fluor donkey anti-mouse IgG 488-labeled secondary antibody (1:1000 dilution, Thermo Fisher Scientific) for 1 hour at 37°C. anti-mouse IgGsuggested: NoneThe wells were then washed with PBS and incubated with mouse anti-His antibody (1:1000 dilution, Abmart anti-Hissuggested: (LSBio (LifeSpan Cat# LS-C129774-1000, RRID:AB_10832018), Berkeley Heights, USA) at 37°C for 1 hour, followed by incubation with Horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Abmart) at 37°C for 1 hour. anti-mousesuggested: NoneHRP-goat anti-human Fc antibody (Abmart) was used for final signal detection. anti-human Fcsuggested: NoneThe membranes were blocked with 3% bovine serum albumin (BSA) in PBST (PBS containing 0.05% Tween 20, pH7.0) and incubated with human ACE2 Rabbit Polyclonal antibody (1:100 dilution, Proteintech, Wuhan, China) followed by HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000 dilution, Invitrogen). HRP-conjugated goatsuggested: NoneIgG secondary antibody ( 1:5000 dilution , Invitrogen)suggested: NoneTo detect hACE2 expression, the sections were first incubated in blocking reagent and then with hACE2 Rabbit Polyclonal antibody (1:100 dilution, Proteintech) at 4 °C overnight, followed by incubation with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000 dilution, Invitrogen). anti-rabbit IgGsuggested: NoneFor viral antigen detection, the sections were sequentially incubated with mouse polyclonal antibody to SARS-CoV-2 N protein (1:500 dilution) and HRP-conjugated goat anti-mouse IgG secondary antibody (1:5000 dilution, Invitrogen). SARS-CoV-2 N proteinsuggested: (ABclonal Cat# A20021, RRID:AB_2862924)Experimental Models: Cell Lines Sentences Resources Cell line, virus, compounds and constructs: African green monkey kidney Vero-E6 cells and human embryonic kidney HEK293T cells were cultured at 37°C with 5% CO2 in Dulbecco’s modified Eagle medium (DMEM) (Gibco, Carlsbad, USA) containing 2 mmol/L L-glutamine, 50 U/mL penicillin, 100 mg/mL streptomycin, and 10% (vol/vol HEK293Tsuggested: NoneThen, the plasmid pAd5-hACE2 was linearized with restriction enzyme PacI and used to transfect HEK293 cells as described previously30. HEK293suggested: NoneTo evaluate the relationship between the timing of compound addition and the antiviral efficacy, Vero-E6 cells cultured in 96-well plate (4.0 x 104 cells/well) were treated with protoporphyrin IX (2.5 μM), verteporfin (1.25 μM) or DMSO at different timepoints relative to virus infection (Fig. 2a). Vero-E6suggested: NoneExperimental Models: Organisms/Strains Sentences Resources Transduction and infection of mice: Eight-week-old male mice (BALB/c) (SLAC Laboratory Animal, Shanghai, China) were raised in pathogen-free cages in the BSL-3 laboratory of Fudan University. BALB/csuggested: RRID:IMSR_ORNL:BALB/cRl)Software and Algorithms Sentences Resources pCAGGS-SARS-CoV-2-S that encodes the SARS-CoV-2 Spike gene was generated by GENEWIZ (Suzhou, China) GENEWIZsuggested: (GENEWIZ, RRID:SCR_003177)Meanwhile, the structures of the compounds, protoporphyrin IX and verteporfin, were obtained from the EMBL-EBI and PubChem compound databases. PubChemsuggested: (PubChem, RRID:SCR_004284)Firstly, the ligand and receptor coordinate files were prepared respectively to include the information needed by AutoDock and the PDBQT files were created. AutoDocksuggested: (AutoDock, RRID:SCR_012746)Then the three-dimension of the grid box was set in AutoDockTools to create the grid parameter file. AutoDockToolssuggested: NoneAfterwards, AutoGrid was used to generate the grid maps and AutoDock was run for receptor-ligand docking. AutoGridsuggested: (Autogrid, RRID:SCR_015982)Statistical analysis: Data were analyzed using Prism 7 (GraphPad) and were presented as mean ± SEM. GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)The dose response curves of viral RNA levels or cell viability versus the drug concentrations were plotted and evaluated by Prism 7. Prismsuggested: (PRISM, RRID:SCR_005375)Results 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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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