Targeting Bothrops asper Venom Enzymes: Steroidal Derivatives as potential Inhibitors of Phospholipase A2, Serine proteinases, and metalloproteinases.

Abstract

Snakebite envenoming is a neglected tropical disease responsible for an estimated 1.8-2.7 million envenomings and 80,000-140,000 deaths annually, with Bothrops asper accounting for 66.7% of cases and 73.2% of deaths in Colombia. The inhibitory activity of three semi-synthetic ergosterol-derived compounds (2, 3, and 4) was evaluated against the major enzymes of Bothrops asper venom--snake venom metalloproteinases (SVMPs), phospholipases A2 (PLA2s), and serine proteinases (SVSPs)--through in vitro and in silico studies, aiming to identify potential adjuvants for the treatment of local damage. In vitro assays were developed to assess the inhibition of procoagulant, amidolytic, proteolytic, phospholipase A2, and esterase activities using compound concentrations ranging from 62.5 to 500 M, along with molecular docking studies to predict enzyme-ligand interactions. Compound 4 was the most effective inhibitor of coagulant activity (SVSP), showing a significant dose-dependent effect (p < 0.001) at all tested concentrations (62.5-500 M), prolonging plasma clotting time by up to 300 s at the highest doses. For amidolytic activity (SVSP), compounds 2 and 4 showed inhibitory capacity, although with variability across concentrations. In contrast, compounds 2 and 3 were the most potent inhibitors of PLA2 activity inhibitors, exhibiting a significant dose-dependent effect. Notably, none of the compounds inhibited SVMP proteolytic activity. Molecular docking and molecular dynamics simulations were performed to investigate the binding mechanisms of the selected compounds with PLA2 and SVSPs. Compound 2 was analyzed in complex with PLA2, and compounds 3 and 4 were evaluated against SVSP. The results revealed that ligand binding was primarily driven by hydrophobic interactions, supported by key electrostatic contributions, leading to stable ligand-receptor complexes throughout the simulations. MM-GBSA calculations showed favorable binding free energies consistent with experimental inhibitory activity, highlighting ergostane-based compounds as promising scaffolds for the development of novel inhibitors targeting PLA2 and SVSP. Author summaryEvery year, hundreds of thousands of people are bitten by snakes, most of them farmers or children living in rural areas far from hospitals. Many suffer permanent damage or do not survive. Snakebite is a serious global health problem that rarely receives the attention it deserves. In Colombia, Bothrops asper -- known locally as mapana or terciopelo -- is responsible for most of these cases. Its venom acts quickly, destroying tissue, causing bleeding, and disrupting the bloods ability to clot. Although treatments exist, they often cannot prevent the severe damage that occurs within the first minutes after a bite. With this in mind, we explored whether molecules derived from ergosterol, a natural compound found in mushrooms, could help block some of the most harmful effects of the venom. Through laboratory experiments and computer simulations, we found that some of these molecules were able to reduce venom activity linked to tissue damage and clotting disorders, although they did not block all of its effects. We hope these findings represent a step toward developing complementary treatments that are simpler and more accessible, ultimately improving care for the people who need it most.