Synthetic Heparan Sulfate Mimetic Pixatimod (PG545) Potently Inhibits SARS-CoV-2 by Disrupting the Spike–ACE2 Interaction

Version published to 10.1021/acscentsci.1c01293

Mar 29, 2022

SciScore for 10.1101/2020.06.24.169334: (What is this?)

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

Table 1: Rigor

Institutional Review Board Statement	IACUC: K18-hACE2 mouse experiments: Mouse experiments are approved by the QIMR Berghofer MRI Biosafety Committee and Animal Ethics Committee (project P3600), and are conducted in accordance with the “Australian Code for the care and use of animals for scientific purposes” as defined by the National Health and Medical Research Council of Australia.
Randomization	Pixatimod was randomly placed in the box.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	Female 6-8 week old K18 hACE2 mice (n=4 per group) were treated once with 16 mg/kg of pixatimod in 200 µl saline i.p. on the day before infection.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Binding of His-tagged S1-RBD was detected with Alexa Fluor 488 anti-his tag antibody (clone J095G46, Biolegend, UK) 1:5000 in 25 μL PBST + 0.1% BSA per well.	anti-his tag suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Briefly, 100 mL of HEK293-6E cells were seeded at a cell density of 0.5 × 106 cells/ml 24hr before transfection with polyethyleneimine (PEI).	HEK293-6E suggested: RRID:CVCL_HF20)
The virus-compound mixture was transferred onto the wells of a washed 24-well plate that had been seeded with Vero E6 cells [ECACC 85020206] the previous day at 1.5 × 105 cells/well.	Vero E6 suggested: None
A cytopathic effect assay was performed with the SARS-CoV-2 DE-Gbg20 isolate and Vero cells (ATCC) plated at 2 × 104 per well in 96-well plates the day prior to the experiment.	Vero suggested: RRID:CVCL_ZW93)
BCi-NS1.1 cells were grown at an Air Liquid Interface (ALI) in PneumaCult-ALI Basal Medium supplemented with Pneumacult ALI supplement hydrocortisone, PneumaCult ALI maintenance supplement, heparin, nystatin and penicillin–streptomycin (all from Stemcell Technologies) at 37°C in 5% CO2.	BCi-NS1.1 suggested: RRID:CVCL_T029)
Experimental Models: Organisms/Strains
Sentences	Resources
Age and gender matched C57BL/6J mice (n=4) were included as a drug toxicity control and received 16 mg/kg of pixatimod in 200 µl saline i.p., but were not infected with virus. hCoV-19/Australia/QLD02/2020 (QLD02) (GISAID Accession ID; EPI_ISL_407896) virus was used to inoculate K18 hACE2 mice (n=4 per group) intranasally with 104 CCID50 of virus in 50 μl medium while under light anaesthesia; 3% isoflurane (Piramal Enterprises Ltd., Andhra Pradesh, India) delivered using The Stinger, Rodent Anesthesia System (Advanced Anaesthesia Specialists/Darvall, Gladesville, NSW, Australia).	C57BL/6J suggested: None
Software and Algorithms
Sentences	Resources
Collected data were analysed with Spectral Manager II software prior to processing with GraphPad Prism 7, using second order polynomial smoothing through 21 neighbours.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
Tm values were calculated using MatLab softaware (R20018a, MathWorks) and ΔTm values determined from the difference between the Tm of RBD alone or in the presence of heparin or pixatimod.	MatLab suggested: (MATLAB, RRID:SCR_001622)
Cells were maintained at 50-75% confluence in DMEM supplemented with 10% foetal bovine serum, 20 mM L-glutamine, 100 U/mL penicillin-G and 100 U/mL streptomycin sulfate (all purchased from Gibco/ThermoFisher, UK).	Gibco/ThermoFisher suggested: None
Statistical analyses were performed using analysis of a two-tailed Student’s t test with GraphPad Prism (GraphPad Software) unless otherwise noted.	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
Statistical analysis for the K18-hACE2 mouse work was performed using IBM SPSS Statistics for Windows, Version 19.0 (IBM Corp., Armonk, NY, USA).	SPSS suggested: (SPSS, RRID:SCR_002865)

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
NCT02042781	Completed	Study of the Safety and Tolerability of IV Infused PG545 in …

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 pages 28 and 5. 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.

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Synthetic Heparan Sulfate Mimetic Pixatimod (PG545) Potently Inhibits SARS-CoV-2 by Disrupting the Spike–ACE2 Interaction

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