LAUSR

Significance Some coronaviruses utilize angiotensin-converting enzyme 2 (ACE2) for entry into host cells. Although reducing agents, such as N-acetylcysteine, disrupt viral binding to ACE2 in general, these compounds are cytotoxic, have low potency, and because of their membrane permeability, have undefined mechanism of action. With qualitative chemoproteomic mapping to delineate cysteine thiol/disulfide reactivity in native spike and recombinant receptor binding domain (RBD), we report nontoxic, cell-impermeable thiol-based chemical probes that significantly decrease the ACE2 binding and infectivity of SARS-CoV-2. We map the reactive cysteines and show the dynamic consequences of breaking allosteric disulfide bonds in the RBD. Altogether, our work underscores a clear redox-based mechanism of antiviral activity in which reducing compounds disrupt key RBD disulfides specifically in extracellular spaces. 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.