Echis carinatus snake venom metalloprotease-induced toxicities in mice: Therapeutic intervention by a repurposed drug, Tetraethyl thiuram disulfide (Disulfiram)

Author summary India has highest incidence of deaths due to snakebite in the world. Echis carinatus (EC) is known as saw-scaled viper and its bite causes major mortality and morbidity in the Indian subcontinent. The abundant presence of zinc (Zn++) metalloproteases in Echis carinatus venom (ECV) is responsible for local tissue necrosis. An earlier report has shown that ECV activates neutrophils and leads to NETosis that blocks blood vessels leading to tissue necrosis. However, the toxin in ECV responsible for NETosis has not addressed. Here we investigated the Echis carinatus venom metalloproteases (ECVMPs) are responsible for NETosis and its associated tissue necrosis using Zn++ specific chelator, Tetraethyl thiuram disulfide (TTD). TTD inhibited ECVMPs-induced skin hemorrhage and footpad tissue necrosis by reduced expression of citrullinated H3 (citH3) and myeloperoxidase (MPO) in mice footpad tissue. TTD also neutralized ECV-induced systemic hemorrhage and conferred protection against lethality in mice. Moreover, TTD inhibited ECV-induced NETosis in human neutrophils and decreased the expression of p-ERK and NETosis markers. Further, we demonstrated that ECV-induced NETosis and tissue necrosis is mediated via PAR-1-ERK axis. Overall, our results provide an insight into SVMPs-induced toxicities and the promising protective efficacy of TTD can be extrapolated to treat severe tissue necrosis complementing anti-snake venom (ASV). Echis carinatus (EC) is known as saw-scaled viper and it is endemic to the Indian subcontinent. Envenoming by EC represents a major cause of snakebite mortality and morbidity in the Indian subcontinent. Zinc (Zn++) dependent snake venom metalloproteases (SVMPs) present in Echis carinatus venom is well known to cause systemic hemorrhage and coagulopathy in experimental animals. An earlier report has shown that Echis carinatus venom (ECV) activates neutrophils and releases neutrophil extracellular traps (NETs) that blocks blood vessels leading to severe tissue necrosis. However, the direct involvement of SVMPs in the release of NETs is not clear. Here, we investigated the direct involvement of EC SVMPs in observed pathological symptoms in a preclinical setup using specific Zn++ metal chelator, Tetraethyl thiuram disulfide (TTD)/disulfiram. TTD potently antagonizes the activity of SVMPs-mediated ECM protein degradation in vitro and skin hemorrhage in mice. In addition, TTD protected mice from ECV-induced footpad tissue necrosis by reduced expression of citrullinated H3 (citH3) and myeloperoxidase (MPO) in footpad tissue. TTD also neutralized ECV-induced systemic hemorrhage and conferred protection against lethality in mice. Moreover, TTD inhibited ECV-induced NETosis in human neutrophils and decreased the expression of peptidyl arginine deiminase (PAD) 4, citH3, MPO, and p-ERK. Further, we demonstrated that ECV-induced NETosis and tissue necrosis are mediated via PAR-1-ERK axis. Overall, our results provide an insight into SVMPs-induced toxicities and the promising protective efficacy of TTD can be extrapolated to treat severe tissue necrosis complementing anti-snake venom (ASV).

Automated summary

This study reveals that the metalloproteases in Echis carinatus venom are responsible for NETosis and tissue necrosis, and the use of the zinc-specific chelator TTD can inhibit this toxic effect and provide protection against severe tissue necrosis caused by snakebite.