A Vimentin-Targeting Oral Compound with Host-Directed Antiviral and Anti-Inflammatory Actions Addresses Multiple Features of COVID-19 and Related Diseases

Abstract

With the Delta variant currently fueling a resurgence of new infections in the fully vaccinated population, developing an effective therapeutic drug is especially critical and urgent in fighting COVID-19. In contrast to the many efforts to repurpose existing drugs or address only one aspect of COVID-19, we are developing a novel agent with first-in-class mechanisms of action that address both the viral infection and the overactive immune system in the pathogenesis of the disease.

SciScore for 10.1101/2021.08.26.457884: (What is this?)

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

Table 1: Rigor

Ethics	Field Sample Permit: Endocytosis and endosomal trafficking: HEK293T cells were purchased from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai); plasmid PMAX-GFP, from Lonza; transfection agent LipoMAX, from Invitrogen. Euthanasia Agents: Four days later, the mice were sacrificed with cervical dislocation, soaked in 75% alcohol for 3 min, The abdominal skin was cut open and made the peritoneum fully exposed. IACUC: The experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at KCI Biotech Inc.
Sex as a biological variable	Only data from 9 female animals (3 animals per time point) are presented.
Randomization	, Ten animals per group were randomly assigned in 1 mock-infected vehicle treatment group and 7 virus (103 PFU SARS-CoV-2 MA10)-infected groups treated p.o. via oral gavage respectively with vehicle and ALD-R491 at 3, 10 and 30 mg/kg in prophylactic protocol (dosing at −15, −5, +24, +48, +72, +96 h post infection) or therapeutic protocol (dosing at +24, +48, +96 h post infection).
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
The cells were incubated overnight at 4 °C with the anti-p47phox primary antibody (Santa Cruz, sc-17844) and the anti-vimentin primary antibody (Santa Cruz, sc-5565).	anti-p47phox suggested: None
Briefly, single-cell suspension from mouse spleen were incubated with anti-CD4-biotin antibody (Biolegend, 100508) then streptavidin-microBeads (Miltenyi, 130-048-102)	anti-CD4-biotin suggested: (Sigma-Aldrich Cat# SAB4700061, RRID:AB_10896068)
Then the CD4+ T cells were stained with anti-CD4-APC (Biolegend, 100412) and anti-CD25-PerCP-CY5.5 (BD, 551071) flow cytometry antibodies.	anti-CD4-APC suggested: (Sigma-Aldrich Cat# SAB4700557, RRID:AB_10896237) anti-CD25-PerCP-CY5.5 suggested: None
The permeabilized cells were blocked with 3% BSA for 1 h, incubated with anti-Vimentin primary antibody (Proteintech, 10366-1-AP, 1:200) overnight at 4°C.	anti-Vimentin suggested: (Proteintech Cat# 10366-1-AP, RRID:AB_2273020)
T cells were activated by coated-CD3 antibody (Biolegend, 100302) and diluted-CD28 antibody (Biolegend, 102102) with or without ALD-R491.	diluted-CD28 suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Endocytosis and endosomal trafficking: HEK293T cells were purchased from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai); plasmid PMAX-GFP, from Lonza; transfection agent LipoMAX, from Invitrogen.	HEK293T suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
Measurement of nanoparticle size, concentration, and distribution with nanoparticle tracking analysis (NTA): Overnight cultured Huh7NC12 at 70% confluence were treated for 2 days with media consisting of DMSO and the indicated concentrations of compounds, respectively.	Huh7NC12 suggested: None
Pseudoviral infection: HEK293T/hACE2 cells, Pseudovirus-2019-nCoV-GFP-IRES-LUC and control Pseudovirus-GFP-IRES LUC were purchased from FUBIO (Suzhou).	HEK293T/hACE2 suggested: RRID:CVCL_A7UK)
Cellular ROS measurement: Raw264.7 cells were seeded into a 12-well plate and cultured till 90% confluence, then added with ALD-R491 at different concentrations and incubated for another 2 h.	Raw264.7 suggested: None
Experimental Models: Organisms/Strains
Sentences	Resources
Treg activation: Preparation of CD4+CD25+/- cells: CD4+ T cells were isolated from spleen of Balb/c mice (6-8 weeks) by microbeads according to the manufacturer’s instructions.	Balb/c suggested: None
Tissue distribution in rats: Animals and housing: Sprague-Dawley rats of SPF grades at ages of 6 to 8 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd., with animal certificate No. of SCXK (Jing) 2016-0006 and with animal quality certificate No.1100112011030349 and No.1100112011030350.	Sprague-Dawley suggested: None
Recombinant DNA
Sentences	Resources
Endocytosis and endosomal trafficking: HEK293T cells were purchased from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai); plasmid PMAX-GFP, from Lonza; transfection agent LipoMAX, from Invitrogen.	PMAX-GFP suggested: RRID:Addgene_16007)
The cells were grown in 96-well plate to approximately 70% confluence after overnight culture and transfected in 6 replicates with pMAX-GPF plasmid DNA (0.6 μg per well) using LipoMAX according to the manufacture’s instruction.	pMAX-GPF suggested: None
Software and Algorithms
Sentences	Resources
The compound was synthesized at Bellen Chemistry Co, Ltd., analyzed at Porton Pharma Solutions, Ltd., and provided by Luoda Biosciences, Inc and Aluda Pharmaceuticals, Inc.	Luoda Biosciences suggested: None
FACS data were analyzed using FlowJo software (TreeStar).	FlowJo suggested: (FlowJo, RRID:SCR_008520)
Microsoft Office Excel (2007) was used for tissue concentration column graphs.	Microsoft Office Excel suggested: (Microsoft Excel, RRID:SCR_016137)

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: We found the following clinical trial numbers in your paper:

Identifier	Status	Title
NCT04468971	Active, not recruiting	REgulatory T Cell infuSion fOr Lung Injury Due to COVID-19 P…

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: Please consider improving the rainbow (“jet”) colormap(s) used on page 57. 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.
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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A Vimentin-Targeting Oral Compound with Host-Directed Antiviral and Anti-Inflammatory Actions Addresses Multiple Features of COVID-19 and Related Diseases

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