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 (ECV) is well known to cause systemic hemorrhage and coagulopathy in experimental animals. An earlier report has shown that 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).

Snakebite envenoming causes more than 100,000 deaths annually, with India bearing nearly half of this burden. Current equine plasma–derived antivenoms suffer from variable efficacy, safety concerns, and limited cross-neutralization of geographically diverse venoms. Here, we report a rationally designed recombinant antivenom composed of five nanobodies (V _H Hs) that target the α-neurotoxins, cytotoxins, and phospholipases A ₂ prevalent in the venoms of cobra and king cobra species from India. These V _H Hs were previously identified and functionally validated against homologous toxin families from African elapids. In murine preincubation and rescue models of snakebite envenoming, the cocktail prevented mortality after injection with venom from the monocled cobra ( Naja kaouthia ), both king cobra species ( Ophiophagus kaalinga and Ophiophagus hannah ) from the Western Ghats and Northeast India, and pan-Indian populations of spectacled cobra ( Naja naja ). These findings demonstrate the feasibility of developing broad-spectrum nanobody-based recombinant antivenoms with continent-wide coverage as a safe and regionally adaptable biologic for snakebite envenoming therapy.

Snakebite envenoming is a major yet neglected tropical disease in sub-Saharan Africa, where antivenom efficacy is critically limited by intraspecific venom variation shaped by local ecological pressures. Nigeria’s sharply contrasting Sudan Savanna (North) and Lowland Rainforest (South) provide an ideal natural system to investigate this variation, yet a comparative analysis of its medically important snakes has been lacking. We conducted an integrated proteomic and functional characterization of venoms from the puff adder (Bitis arietans) and black-necked spitting cobra (Naja nigricollis) collected in Kaduna (North) and Ibadan (South). Using high-resolution LC-MS/MS, SDS-PAGE, and biochemical assays (phospholipase A₂, protease, fibrinogenolytic, hemolytic, and coagulation activities), we mapped region-specific venom compositions and characterized their functional activities. Bitis arietans displayed region-associated divergence: southern venom was enriched in serine proteases, whereas northern venom was dominated by lectins and distinct snake venom metalloproteinase isoforms. Naja nigricollis showed a conserved phospholipase A₂/three-finger toxins backbone, yet southern venoms exhibited elevated snake venom metalloproteinase III and L-amino acid oxidase. These molecular differences manifested functionally, with southern B. arietans venom showing higher protease activity than northern B. arietans, whereas southern and northern N. nigricollis venom exhibited similar protease activity but enhanced phospholipase A₂ activity in southern N. nigricollis. This work provides the first integrated proteomic and functional comparison of venoms from northern and southern Nigerian venom sample of B. arietans and N. nigricollis. While based on a limited number of individuals, the observed differences should be considered preliminary and indicative of potential regional trends rather than population-level characteristics.