Unravelling the mechanistic and regulatory aspects of redox-sensitive cell responses modulated by low molecular weight (LMW) synthetic thiol compounds in physiological and pathological conditions

The redox state plays a crucial role in numerous biological processes, making its modulation essential for their regulation. Low molecular weight (LMW) thiol compounds, such as I-152, NACMEAA, and C4-GSH, that are a co-drug of N-acetylcysteine (NAC) and cysteamine (MEA), its deacetylated metabolite, and a hydrophobic GSH derivative respectively, have been studied for their ability to modulate redox states across biological systems and their therapeutic potential in contexts like viral infections, inflammation, hypoxia, and cellular metabolism. The life cycle of respiratory viruses, including SARS-CoV-2, is highly dependent on redox balance, that influences both viral entry and replication. A reducing environment hampers the spike (S) protein's binding to the ACE2 receptor, while an oxidizing environment facilitates this interaction. Additionally, SARS-CoV-2 disrupts the redox balance in infected cells to assist viral protein folding. Our studies demonstrated that LMW thiols shift the redox state of the receptor-binding domain (RBD) of the S protein to a more reduced form, reducing its ACE2 binding affinity and impairing viral entry. These compounds also interfere with viral protein folding and maturation, inhibiting viral replication. SARS-CoV-2 and other respiratory virus infections as well as other stress responses promote inflammation; thus, another study explored how redox modulation using the thiol-based compound I-152 could affect pro-inflammatory cytokine expression in lipopolysaccharide (LPS)-stimulated macrophages (MΦ). The results revealed the ability of I-152 to attenuate inflammatory cytokine gene expression and secretion through the downregulation of c-Jun/AP-1 or NF-kB pathways depending on the dose. It also affected NLRP3 inflammasome priming/activation, leading to decreased IL-1β and IL-18 release and inhibition of pyroptosis. Furthermore, co-culturing redox-modulated MΦ with endothelial cells (ECs) revealed that I-152 reduced inflammation in both cell types. Delving into the immunomodulatory effects of I-152 in a murine model infected with the LP-BM5 retrovirus, we found that the molecule promoted immunoproteasome upregulation, potentially boosting innate cell-mediated immune responses. Another aspect of this research focused on redox modulation in hypoxic human umbilical vein ECs (HUVECs); I-152 increased GSH levels, modulated redox-related metabolic pathways, reduced HIF-1α and VEGF gene expression, and quenched ROS. In parallel, on HUVECs under physiological conditions, I-152 boosted mitochondrial ATP production, without inducing ROS production, and altered metabolomic profiles, enhancing oxidative phosphorylation. In I-152 treated-HUVECs co-cultured with LPS-stimulated MΦ the compound sustained GSH and Cys levels and downregulated adhesion molecule transcription. In vivo preliminary studies in mice confirmed that I-152 modulates redox content in various organs, particularly in muscles. In conclusion: 1) LMW thiols hold promise as therapeutic agents against respiratory viruses, including SARS-CoV-2, by targeting both viral components and cellular functions necessary for virus replication; 2) redox modulation by LMW thiols can influence the secretory profile of stimulated MΦ and their crosstalk with ECs, reducing both oxidative stress and inflammation; and 3) LMW compounds may contribute to new thiol-based therapies for diseased tissues without harming surrounding healthy tissues.