Spike protein disulfide disruption as a potential treatment for SARS-CoV-2
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Abstract
The coronaviral pandemic is exerting a tremendously detrimental impact on global health, quality of life and the world economy, emphasizing the need for effective medications for current and future coronaviral outbreaks as a complementary approach to vaccines. The Spike protein, responsible for cell receptor binding and viral internalization, possesses multiple disulfide bonds raising the possibility that disulfide-reducing agents might disrupt Spike function, prevent viral entry and serve as effective drugs against SARS-CoV-2. Here we show the first experimental evidence that reagents capable of reducing disulfide bonds can inhibit viral infection in cell-based assays. Molecular dynamics simulations of the Spike receptor-binding domain (RBD) predict increased domain flexibility when the four disulfide bonds of the domain are reduced. This flexibility is particularly prominent for the surface loop, comprised of residues 456-490, which interacts with the Spike cell receptor ACE2. Consistent with this finding, the addition of exogenous disulfide bond reducing agents affects the RBD secondary structure, lowers its melting temperature from 52 to 36-39°C and decreases its binding affinity to ACE2 by two orders of magnitude at 37°C. Finally, the reducing agents dithiothreitol (DTT) and tris (2-carboxyethyl)phosphine (TCEP) inhibit viral replication at high µM – low mM levels with a negligible effect on cell viability at these concentrations. The antiviral effect of monothiol-based reductants N-Acetyl-L-cysteine (NAC) and reduced glutathione (GSH) was not observed due to decreases in cell viability. Our research demonstrates the clear potential for medications that disrupt Spike disulfides as broad-spectrum anticoronaviral agents and as a first-line defense against current and future outbreaks.
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SciScore for 10.1101/2021.01.02.425099: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources The proteins were expressed in Expi293 cells (Thermo Fisher Scientific) according to the manufacturer’s protocol. Expi293suggested: RRID:CVCL_D615)After 1 hour, the compounds were removed from the Vero’76 cells (cells-compound) and the pre-incubated virus-compound mixture was added to the cells and incubated for 1 hour at 37°C to allow for viral infection in the presence of the serially-diluted compounds. Vero’76suggested: None… SciScore for 10.1101/2021.01.02.425099: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources The proteins were expressed in Expi293 cells (Thermo Fisher Scientific) according to the manufacturer’s protocol. Expi293suggested: RRID:CVCL_D615)After 1 hour, the compounds were removed from the Vero’76 cells (cells-compound) and the pre-incubated virus-compound mixture was added to the cells and incubated for 1 hour at 37°C to allow for viral infection in the presence of the serially-diluted compounds. Vero’76suggested: NoneRecombinant DNA Sentences Resources The constructs of ACE2 and Spike protein of SARS-CoV-2 were PCR amplified from pCEP4-myc-ACE2, which was a gift from Erik Procko (Addgene plasmid # 141185; http://n2t.net/addgene:141185; RRID:Addgene_141185) and pcDNA3.1-SARS2-Spike -a gift from Fang Li (Addgene plasmid # 145032; http://n2t.net/addgene:145032; RRID:Addgene_145032). detected: RRID:Addgene_141185)Software and Algorithms Sentences Resources These results were plotted and IC90 and CC50 were calculated in GraphPad Prism 8 software, using non-linear four-parameter regression analysis. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)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 NCT04419025 Recruiting Efficacy of N-Acetylcysteine (NAC) in Preventing COVID-19 Fr… NCT04374461 Recruiting A Study of N-acetylcysteine in Patients With COVID-19 Infect… 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.
- No funding statement was detected.
- No protocol registration statement was detected.
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