Spike protein disulfide disruption as a potential treatment for SARS-CoV-2

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.

Use of Thiols in the Treatment of COVID-19: Current Evidence

“…It is plausible that NAC and erdosteine may cleave the two motifs that serve as a structural basis for the association between E and S; this would decrease SARS-CoV-2 infectivity [49]. However, thiols are likely to be ineffective against SARS-CoV-2 as a cell entry inhibitor as evidenced by the observation that even at NAC concentrations of 10 mM, it was impossible to observe a significant decrease in viral titre [57].…”

“…Furthermore, the ability of H 2 S to modulate the inflammatory response and proinflammatory cytokine cascade has been recognized in other settings [56]. H 2 S also contributes to the maintenance of elevated levels of GSH and normalizes the balance between oxidized and reduced GSH [glutathione disulphide (GSSG)/GSH ratio] that is increased by oxidative stress [61,62], preventing endothelial NO synthase (eNOS) uncoupling, which leads this enzyme to produce O 2 − instead of NO [57]. Furthermore, H 2 S increases the expression of ACE2 [62].…”

SARS-CoV-2 Detection in the Nasopharyngeal Swabs and Saliva of College Students using RT-qPCR and RT-LAMP

“…However, components of such inactivation buffers are often costly (tris(2-carboxyethyl)phosphine (TCEP) or thermolabile proteinase K) and have seen limited available during the COVID-19 pandemic. Chemical inactivation methods often function in a dual capacity to decrease viral infectivity 13 and to inactivate the abundant endonucleases that could rapidly degrade SARS-CoV-2 viral RNA (vRNA) and potentially inhibit downstream amplification during the RT-LAMP reaction 14 . The SLAMP method does not require the addition of any chemical agents, using instead a simple heat inactivation procedure that accomplishes viral inactivation.…”

Designing and Expression of Recombinant Chimeric Spike Protein from SARS-CoV-2 in Escherichia coli and Its Immunogenicity Assessment

“…Properly forming disulfide bonds guarantees a protein's accurate and efficient structure ( 40 ). The spike protein has multiple disulfide bonds ( 41 ). Therefore, the E. coli Rosetta-gami (DE3) was selected, as it is a proper host for the spike expression due to uncommon codons ( 42 ) and the formation of target protein disulfide bonds in the bacterial cytoplasm ( 36 ).…”