Pyronaridine Protects Against SARS-CoV-2 in Mouse

Abstract

There are currently relatively few small-molecule antiviral drugs that are either approved or emergency approved for use against SARS-CoV-2. One of these is remdesivir, which was originally repurposed from its use against Ebola and functions by causing early RNA chain termination. We used this as justification to evaluate three molecules we had previously identified computationally with antiviral activity against Ebola and Marburg. Out of these we previously identified pyronaridine, which inhibited the SARS-CoV-2 replication in A549-ACE2 cells. Herein, the in vivo efficacy of pyronaridine has now been assessed in a K18-hACE transgenic mouse model of COVID-19. Pyronaridine treatment demonstrated a statistically significant reduction of viral load in the lungs of SARS CoV-2 infected mice. Furthermore, the pyronaridine treated group reduced lung pathology, which was also associated with significant reduction in the levels of pro-inflammatory cytokines/chemokine and cell infiltration. Notably, pyronaridine inhibited the viral PL ^pro activity in vitro (IC ₅₀ of 1.8 µM) without any effect on M ^pro , indicating a possible molecular mechanism involved in its ability to inhibit SARS-CoV-2 replication. Interestingly, pyronaridine also selectively inhibits the host kinase CAMK1 (IC ₅₀ of 2.4 µM). We have also generated several pyronaridine analogs to assist in understanding the structure activity relationship for PL ^pro inhibition. Our results indicate that pyronaridine is a potential therapeutic candidate for COVID-19.

One sentence summary

There is currently intense interest in discovering small molecules with direct antiviral activity against the severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). Pyronaridine, an antiviral drug with in vitro activity against Ebola, Marburg and SARS-CoV-2 has now statistically significantly reduced the viral load in mice along with IL-6, TNF-α, and IFN-β ultimately demonstrating a protective effect against lung damage by infection to provide a new potential treatment for testing clinically.

SciScore for 10.1101/2021.09.30.462449: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	IRB: Mouse studies Ethical approval: All the experimental procedures were performed in accordance with the guide for the use of laboratory animals of the University of Sao Paulo and approved by the institutional ethics committee under the protocol number 105/2021.
Sex as a biological variable	SARS-CoV-2 experimental infection and treatments: Female K18-hACE2 mice, 8-week-old, were infected with 2×104 PFU of SARS-CoV-2 (in 40 µL) by intranasal route.
Randomization	A total of 10 photomicrographs in 40X magnification per animal were randomly obtained using a microscope Novel (Novel L3000 LED, China) coupled to a HDI camera for images capture.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Experimental Models: Cell Lines
Sentences	Resources
The virus was propagated and titrated in Vero E6 cells in a biosafety level 3 laboratory (BSL3) at the Ribeirão Preto Medical school	Vero E6 suggested: RRID:CVCL_XD71)
The viral inoculum was added to Vero cells in DMEM 2% FBS and incubated at 37 °C with 5% CO2 for 48 h.	Vero suggested: None
For expression, BL21 cells transformed with plasmid were grown in ZYM-5052 to an OD600 of 0.6-0.8 at 37°C and 200 RPM.	BL21 suggested: RRID:CVCL_M639)
Experimental Models: Organisms/Strains
Sentences	Resources
K18-hACE2 mice: To evaluate the effects of Pyronaridine in vivo, we infected the K18-hACE2 humanized mice (B6.Cg-Tg(K18-ACE2)2Prlmn/J)(21, 65, 66).	K18-hACE2 suggested: RRID:IMSR_GPT:T037657) B6.Cg-Tg(K18-ACE2)2Prlmn/J suggested: RRID:IMSR_JAX:034860)
Recombinant DNA
Sentences	Resources
PCR product was digested with NcoI and XhoI and cloned into pET28a (Novagen) in frame with a C-terminal his-tag coding sequence E. coli BL21 transformed with plasmids were grown in LB to an optical density (OD600) of 0.6 at 37 °C and 200 RPM.	pET28a suggested: RRID:Addgene_114156)
Software and Algorithms
Sentences	Resources
The total septal area and total area were analyzed with the aid of the Pro Plus 7 software (Media Cybernetics, Inc., MD, USA).	Pro Plus suggested: (Image-Pro Plus, RRID:SCR_016879)
Prior to docking, ligand and protein were prepared with LigPrep (Schrödinger, 2017, USA) and Protein Preparation Wizard (Schrödinger, 2017, USA) using default parameters.	LigPrep suggested: (Ligprep, RRID:SCR_016746)
Figures were generated with ChimeraX.	ChimeraX suggested: (UCSF ChimeraX, RRID:SCR_015872)

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 did not find any issues relating to the usage of bar graphs.

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.

Results from scite Reference Check: We found no unreliable references.

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Pyronaridine Protects Against SARS-CoV-2 in Mouse

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