Protoporphyrin IX and verteporfin prevent SARS-CoV-2 infection in vitro and in a mouse model expressing human ACE2

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.

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: None
Vero-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-N suggested: None
The 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 IgG suggested: None
The wells were then washed with PBS and incubated with mouse anti-His antibody (1:1000 dilution, Abmart	anti-His suggested: (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-mouse suggested: None
HRP-goat anti-human Fc antibody (Abmart) was used for final signal detection.	anti-human Fc suggested: None
The 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 goat suggested: None IgG secondary antibody ( 1:5000 dilution , Invitrogen) suggested: None
To 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 IgG suggested: None
For 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 protein suggested: (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	HEK293T suggested: None
Then, the plasmid pAd5-hACE2 was linearized with restriction enzyme PacI and used to transfect HEK293 cells as described previously30.	HEK293 suggested: None
To 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-E6 suggested: None
Experimental 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/c suggested: 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)	GENEWIZ suggested: (GENEWIZ, RRID:SCR_003177)
Meanwhile, the structures of the compounds, protoporphyrin IX and verteporfin, were obtained from the EMBL-EBI and PubChem compound databases.	PubChem suggested: (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.	AutoDock suggested: (AutoDock, RRID:SCR_012746)
Then the three-dimension of the grid box was set in AutoDockTools to create the grid parameter file.	AutoDockTools suggested: None
Afterwards, AutoGrid was used to generate the grid maps and AutoDock was run for receptor-ligand docking.	AutoGrid suggested: (Autogrid, RRID:SCR_015982)
Statistical analysis: Data were analyzed using Prism 7 (GraphPad) and were presented as mean ± SEM.	GraphPad suggested: (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.	Prism suggested: (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:

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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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Protoporphyrin IX and verteporfin prevent SARS-CoV-2 infection in vitro and in a mouse model expressing human ACE2

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