Thiol-based chemical probes exhibit antiviral activity against SARS-CoV-2 via allosteric disulfide disruption in the spike glycoprotein

Abstract

The development of small-molecules targeting different components of SARS-CoV-2 is a key strategy to complement antibody-based treatments and vaccination campaigns in managing the COVID-19 pandemic. Here, we show that two thiol-based chemical probes that act as reducing agents, P2119 and P2165, inhibit infection by human coronaviruses, including SARS-CoV-2, and decrease the binding of spike glycoprotein to its receptor, the angiotensin-converting enzyme 2 (ACE2). Proteomics and reactive cysteine profiling link the antiviral activity to the reduction of key disulfides, specifically by disruption of the Cys379–Cys432 and Cys391–Cys525 pairs distal to the receptor binding motif in the receptor binding domain (RBD) of the spike glycoprotein. Computational analyses provide insight into conformation changes that occur when these disulfides break or form, consistent with an allosteric role, and indicate that P2119/P2165 target a conserved hydrophobic binding pocket in the RBD with the benzyl thiol-reducing moiety pointed directly toward Cys432. These collective findings establish the vulnerability of human coronaviruses to thiol-based chemical probes and lay the groundwork for developing compounds of this class, as a strategy to inhibit the SARS-CoV-2 infection by shifting the spike glycoprotein redox scaffold.

SARS-CoV-2 ORF8 reshapes the ER through forming mixed disulfides with ER oxidoreductases

“…Recently, it has been reported that SARS-CoV-2 infection interferes with the metabolism and redox of cellular thiols in the host cell, and antivirals and NAC can prevent such defect [ 44 ]. Reducing compounds can also disrupt key disulfides in S protein and decrease the binding to ACE2 and infectivity of SARS-CoV-2 [ 45 ]. Thus, future drug development based on small molecule reducing compounds could kill two birds with one stone, by targeting the disulfides of both S and ORF8 proteins.…”

Characterization and Utilization of Disulfide-Bonded SARS-CoV-2 Receptor Binding Domain of Spike Protein Synthesized by Wheat Germ Cell-Free Production System

“…When subjected to the AlphaScreen assay, all the point mutants showed a significant reduction in luminescence, with some of the mutants showing a drastic reduction similar to the negative control ( Figure 3 E and Figure S1B ). The mutations on Cys379-Cys432 and Cys480-488, which are essential for ACE2 interaction [ 5 , 29 ], exhibited remarkably lower signals. These results confirm the importance of S-S in the RBD–ACE2 interaction and imply that the AlphaScreen assay can be a sensitive and pliable tool to identify this interaction.…”

“…Cysteine residues in RBD are conserved among SARS-CoV-2 variants and SARS-related coronaviruses and are responsible for the stabilization of the RBD structure and modulating immunogenicity [ 5 , 36 ]. Previous studies have reported that unfolded RBD or the short form of RBD (318–510 aa; lack of Cys521) induce lower titers of neutralizing antibodies [ 37 , 38 ].…”

“…One of the key factors involved in the RBD–ACE2 interaction is the cysteine–cysteine disulfide bonds (S-S), which are responsible for the proper configuration of the RBD quaternary structure that facilitates its interaction with ACE2 [ 4 ]. Disruption of S-S on the RBD impairs its interaction with ACE2, thereby impairing viral entry [ 5 , 6 , 7 ]. In order to mimic biological processes, the S-S of RBD must be kept intact while the protein is produced in in vitro systems for diagnostic, therapeutic and research applications.…”

Redox stress in COVID-19: Implications for hematologic disorders

“…In addition to covalently labeling the catalytic cysteine of MPro, the oxidation statuses of cysteines in the mature virion have been targeted with specific probes. Chemical probes to selectively reduce the allosteric disulfides of the S protein have been published by the Carroll laboratory [ 99 ]. Specifically, they found that the thiol-based reducing agents P2119 and P2165 target Cys379-Cys432 and Cys391-Cys525 in the RBD domain and prevents the S protein from recognition by ACE2.…”