Discovery of a Novel Inhibitor of Coronavirus 3CL Protease for the Potential Treatment of COVID-19

Abstract

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. The designed phosphate prodrug PF-07304814 is metabolized to PF-00835321 which is a potent inhibitor in vitro of the coronavirus family 3CL pro, with selectivity over human host protease targets. Furthermore, PF-00835231 exhibits potent in vitro antiviral activity against SARS-CoV-2 as a single agent and it is additive/synergistic in combination with remdesivir. We present the ADME, safety, in vitro , and in vivo antiviral activity data that supports the clinical evaluation of this compound as a potential COVID-19 treatment.

SciScore for 10.1101/2020.09.12.293498: (What is this?)

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

Table 1: Rigor

Institutional Review Board Statement	IACUC: In vivo efficacy studies in MA-SARS-CoV-1 mouse model: Animal treatment and subsequent observations of animal weight, and virus titration were conducted in the BSL-3 laboratory at University of Maryland College Park under IACUC approved protocols.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	Rat PK studies were done at Pfizer (Groton, CT) or BioDuro Pharmaceutical Product Development Inc. (Shanghai, PRC); Jugular vein-cannulated male Wistar-Hannover rats were purchased from Charles River Laboratories, Inc. (Wilmington, MA) or Vital River (Beijing, China) and were typically 7-10 weeks of age at the time of dosing.
Cell Line Authentication	not detected.

Table 2: Resources

Experimental Models: Cell Lines
Sentences	Resources
VeroE6 cells that are enriched for hACE2 expression were batched inoculated with SARS-CoV-2 (USA_WA1/2020) at a multiplicity of infection (MOI) of 0.002 in a BSL-3 lab (Southern Research Institute).	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
MRC-5 cells, seeded at a density of 20,000 cells/well were incubated overnight in MEM containing 5% FBS at 37oC and 5% CO2. (Wuxi AppTech).	MRC-5 suggested: None
Drug combination studies were performed using HeLa-ACE2 cells in a high content imaging assay.	HeLa-ACE2 suggested: None
For the inhibition studies, HEK293 cells were washed three times with warm transport buffer (Hanks’ balanced salt solution with 20mM 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid, pH 7.4) followed by incubation with test compounds containing probe substrates: 10 or 20 μM [14C]-metformin (OCT1, OCT2, MATE1, MATE2K), 0.5 μM [3H] para-aminohippuric acid (OAT1), 0.1 μM [3H] estrone-3-sulfate (OAT3) or 0.5 μM rosuvastatin (OATP1B1/1B3).	HEK293 suggested: None
Experimental Models: Organisms/Strains
Sentences	Resources
Rat PK studies were done at Pfizer (Groton, CT) or BioDuro Pharmaceutical Product Development Inc. (Shanghai, PRC); Jugular vein-cannulated male Wistar-Hannover rats were purchased from Charles River Laboratories, Inc. (Wilmington, MA) or Vital River (Beijing, China) and were typically 7-10 weeks of age at the time of dosing.	Wistar-Hannover suggested: None
Briefly, 8-week old male and female Sprague-Dawley rats (n=15 per sex per group) implanted with femoral catheters exteriorized between the scapulae were assigned to vehicle, or PF-07304814 dose groups (80, 360 and 1000mg/Kg).	Sprague-Dawley suggested: None
Software and Algorithms
Sentences	Resources
Images were analyzed using theMulti-Wavelength Cell Scoring Application Module (MetaXpress).	MetaXpress suggested: (MetaXpress, RRID:SCR_016654)
Analytes were quantified versus a standard curve using GraphPad Prism v8 (San Diego, CA) or Sciex Analyst software.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
Estimation of the TDI inactivation parameters was performed using Excel and GraphPad software.	Excel suggested: None GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
In vitro transporter inhibition studies using transporter-transfected cells: HEK293 cells, wild-type and stably transfected with OATP1B1, OATP1B3, OCT1, OCT2, MATE1 and MATE2K, were seeded at a density of 0.5-0.7 × 105 cells/well on BioCoat™ Poly-D-Lysine 96-well plates (Corning) and grown in Dulbecco’s modified Eagle’s medium containing 10% FBS, 1% sodium pyruvate, 1% GlutaMAX™, 1% Gentamicin and 1% nonessential amino acids for 48-72 hours at 37°C, 90% relative humidity, and 5% CO2.	BioCoat™ suggested: None Poly-D-Lysine suggested: None
Rat PK studies were done at Pfizer (Groton, CT) or BioDuro Pharmaceutical Product Development Inc. (Shanghai, PRC); Jugular vein-cannulated male Wistar-Hannover rats were purchased from Charles River Laboratories, Inc. (Wilmington, MA) or Vital River (Beijing, China) and were typically 7-10 weeks of age at the time of dosing.	Charles River Laboratories suggested: (Charles River Laboratories, RRID:SCR_003792)
For the prediction of DDIs of PF-00835231 with itraconazole, the vendor-verified compound files in Simcyp library were used, i.e., itraconazole (sv-itraconazole_fed capsule) with competitive Ki = 0.0013 μM and itraconazole metabolite (sv-OH-itraconazole) with competitive Ki = 0.0023 μM.	Simcyp suggested: (Simcyp, RRID:SCR_003944)

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
NCT04627532	Completed	Single Ascending Dose Study of Intravenous Infusion of PF 07…
NCT04535167	Recruiting	First-In-Human Study To Evaluate Safety, Tolerability, And P…

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.

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Discovery of a Novel Inhibitor of Coronavirus 3CL Protease for the Potential Treatment of COVID-19

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