Targeting SARS‐CoV‐2 by synthetic dual‐acting thiol compounds that inhibit Spike/ACE2 interaction and viral protein production

Abstract

The SARS‐CoV‐2 life cycle is strictly dependent on the environmental redox state that influences both virus entry and replication. A reducing environment impairs the binding of the spike protein (S) to the angiotensin‐converting enzyme 2 receptor (ACE2), while a highly oxidizing environment is thought to favor S interaction with ACE2. Moreover, SARS‐CoV‐2 interferes with redox homeostasis in infected cells to promote the oxidative folding of its own proteins. Here we demonstrate that synthetic low molecular weight (LMW) monothiol and dithiol compounds induce a redox switch in the S protein receptor binding domain (RBD) toward a more reduced state. Reactive cysteine residue profiling revealed that all the disulfides present in RBD are targets of the thiol compounds. The reduction of disulfides in RBD decreases the binding to ACE2 in a cell‐free system as demonstrated by enzyme‐linked immunosorbent and surface plasmon resonance (SPR) assays. Moreover, LMW thiols interfere with protein oxidative folding and the production of newly synthesized polypeptides in HEK293 cells expressing the S1 and RBD domain, respectively. Based on these results, we hypothesize that these thiol compounds impair both the binding of S protein to its cellular receptor during the early stage of viral infection, as well as viral protein folding/maturation and thus the formation of new viral mature particles. Indeed, all the tested molecules, although at different concentrations, efficiently inhibit both SARS‐CoV‐2 entry and replication in Vero E6 cells. LMW thiols may represent innovative anti‐SARS‐CoV‐2 therapeutics acting directly on viral targets and indirectly by inhibiting cellular functions mandatory for viral replication.

NRF2 Antioxidant Response and Interferon-Stimulated Genes Are Differentially Expressed in Respiratory-Syncytial-Virus- and Rhinovirus-Infected Hospitalized Children

“…In conclusion, we report, for the first time, decreased NRF2 and HO1 levels and an inverse correlation with ISGs in RSV-infected, hospitalized children that shows higher disease severity than HRV-infected patients, thus, suggesting the contribution of NRF2 dysregulation to the severity of such viral infections. Interestingly, it has been reported that NRF2-activating molecules are able to inhibit inflammatory responses and the replication of respiratory viruses [29,43]. Our findings are consistent with the hypothesis that NRF2 antioxidant response may be effective as a critical control mechanism in reducing production of IFN-stimulated genes during RSV infections in the upper respiratory tract, and suggest the possible use of NRF2 activators to counteract viral replication and ameliorate the clinical course in RSV-infected children.…”

Anoxia Rapidly Induces Changes in Expression of a Large and Diverse Set of Genes in Endothelial Cells

“…Therefore, GSH levels in HUVEC were evaluated using HPLC at different time points (1, 4, and 8 h), as reported in [ 56 ]. GSH content decreased markedly after 1 h in anoxia and a similar profile was detected after 4 h. Only at 8 h were the cells able to restore their baseline levels, probably thanks to the activation of their antioxidant defense systems, as illustrated in Figure 5 .…”

The influence of redox modulation on hypoxic endothelial cell metabolic and proteomic profiles through a small thiol‐based compound tuning glutathione and thioredoxin systems

“…25% (v/v) Na 2 HPO 4 0.3 M and 10% (v/v) DTNB were added to the supernatants for thiol determination by HPLC through a BDS Hypersil™ C18 column (5 μm, 150 × 4.6 mm; Thermo Scientific). Separation and elution conditions were previously described elsewhere 34,35 . Following the detection at 330 nm, quantitative measurements were compared with known concentration standards and normalized to the protein content.…”

“…Separation and elution conditions were previously described elsewhere. 34,35 Following the detection at 330 nm, quantitative measurements were compared with known concentration standards and normalized to the protein content.…”