In terms of redox-modified cysteines, 12 viral peptides were identified as covalently altered by P2119, whereas no stable covalent adducts with P2165 were recognized. region of the ACE2 binding surface, is not susceptible to alkylation during live cell infection, establishing the stability of this disulfide in a native setting. In contrast, Cys432 and Cys525, which form disulfides with Cys379 and Cys391, respectively, were identified as hyperreactive cysteines that form semistable disulfides. Molecular docking analysis provides insight into the targeting of Cys432 by both reducing brokers. Molecular dynamics (MD) simulations predict that reduction of these three disulfides controls ACE2 binding by triggering conformational changes in the RBD. The latter obtaining suggests that Cys379CCys432 and Cys391CCys525 bonds are allosteric, a unique and rare category of disulfide, unique from structural and catalytic functions. This work establishes the vulnerability of human coronaviruses to two new thiol-based reducing brokers, laying the groundwork to advance the field of thiol-based chemical probes for SARS-CoV-2 in the realms of target selection and site-specific redox assessment. Results Thiol-Based Reducing Brokers Have Antiviral Activity against Human Coronaviruses. The antiviral activities of P2119 and P2165 were first evaluated in a recombinant infectious clone of SARS-CoV-2 computer virus produced in human nasal epithelial (HNE) cells (Fig. 1 0.05; **** 0.0001. (and and and and Dataset S1). In terms of redox-modified cysteines, 12 viral peptides were identified as covalently altered by P2119, whereas no stable covalent adducts with P2165 Antazoline HCl were identified. In addition, we recognized redox-sensitive and -altered cysteines from your human proteome (and and and and and and and and em SI Appendix /em , Fig. S7). Docking poses obtained for both reducing brokers were comparable, with only minor differences in the hydroxylated carbon tail conformations. Utilizing RBD structures derived from the native-state MD simulations, in which the pocket has a smaller cavity volume, yielded similar results (Fig. 6). Small differences in benzyl orientations were observed, Antazoline HCl but close distances were consistently maintained between corresponding sulfur atoms ( em SI Appendix /em , Table S1). It is worth noting that binding of these reducing ARHGEF11 agents, as well as other hydrophobic ligands, may rely on the spike open/partially open conformational state, as you will find no large channels or cavities connecting this pocket with the protein surface Antazoline HCl in the closed state of the spike glycoprotein. Conversation Although several thiol-containing compounds have been shown to inhibit viral receptor binding in?vitro (44, 45), they lack potency (e.g., NAC or glutathione [GSH]), or Antazoline HCl are cytotoxic (e.g., DTT or TCEP). In this study, we characterized more recently synthesized P2119 and P2165 compounds as prototypic thiol-reductants for activity against human coronaviruses, including SARS-CoV-2, which exhibited activities comparable to or greater than reported for neutralizing antibodies (NAbs) (46, 47). Despite being administered at millimolar concentrations to maintain a necessary redox potential, these compounds have established security profiles through aerosol administration in animals at doses that bracket those used in this study (22, 48), confirming that these concentrations can be pharmacologically relevant. Their virucidal effects allowed comparisons of the intrinsic reducing ability of the thiol group and the ability to form stable covalent linkages. Furthermore, their ability to impair ACE2 binding correlates to the virucidal effect, consistent with what has been shown in NAbs (49). Based on their restricted location of action (i.e., the extracellular compartment), the ability of P2119 and P2165 to inhibit RBD-ACE2 binding in?vitro, and qualitative proteomic cysteine site-reactivity mapping in SARS-CoV-2, the simplest explanation for the observed antiviral activity of these brokers is that they efficiently reduce key disulfides in SARS-CoV-2 required for infectivity. However, there is certainly off-target reduction of host proteins in parallel. Whether these off-target effects are tolerable to the host must be assessed from the therapeutic index of these compounds, which will be formally quantitated in future pharmacology and inhaled toxicity studies. Only four proteins (Dataset S1) from the entire SARS-CoV-2 proteome were reactive to reductive treatments. These proteins included the spike (S) and membrane (M) proteins, which are the most protruding or abundant proteins in the virion envelope (E), respectively (the molar ratio of E:S:M in the lipid bilayer is usually 1:20:300) (50); and two accessory proteins ORF8 and ORF7a, which are expressed by infected cells as transmembrane or secreted proteins, respectively (51). The observation that most mapped cysteine modifications ( 65%) occurred around the spike protein supports our proposed mechanism of action for P2119 and P2165 to block infectivity. Of particular importance is the reduction of two RBD disulfides, Cys379CCys432 and Cys391CCys525, which were identified as redox-sensitive in both native spike protein and recombinant RBD. Despite the susceptibility of the Cys480CCys488 pair to reduction in recombinant RBD, this disulfide was not in-play in native computer virus during.