Targeting Bothrops asper venom enzymes: Steroidal derivatives as potential inhibitors of phospholipase A2 and serine proteinases.

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 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 μM, 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 μM), prolonging plasma coagulation time by up to 300 s at the highest dose. For amidolytic activity (SVSP), compounds 2 and 4 showed inhibitory capacity, although with variability across concentrations. In contrast, compound 2 exhibited the highest inhibitory activity on the esterase activity of PLA2s using the monodisperse substrate 4-NOBA, whereas compound 3 showed the highest dose-dependent inhibitory activity on an aggregated substrate. Notably, none of the compounds inhibited the proteolytic activity of snake venom metalloproteinases (SVMPs). Although this negative result limits the therapeutic scope of these compounds against hemorrhagic manifestations, it reinforces the selectivity of ergostane derivatives for PLA₂ and SVSP and highlights the need for alternative chemical scaffolds to target snake venom metalloproteinases (SVMPs). 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 the experimental inhibitory activity, highlighting ergostane-based compounds as promising scaffolds for developing novel inhibitors targeting PLA2 and SVSP.