Assessing the Interactions between Snake Venom Metalloproteinases and Hydroxamate Inhibitors Using Kinetic and ITC Assays, Molecular Dynamics Simulations and MM/PBSA-Based Scoring Functions

<i>Bothrops</i> species are the main cause of snake bites in rural communities of tropical developing countries of Central and South America. Envenomation by <i>Bothrops</i> snakes is characterized by prominent local inflammation, hemorrhage and necrosis as well as systemic hemostatic disturbances. These pathological effects are mainly caused by the major toxins of the viperidae venoms, the snake venom metalloproteinases (SVMPs). Despite the antivenom therapy efficiency to block the main toxic effects on bite victims, this treatment shows limited efficacy to prevent tissue necrosis. Thus, drug-like inhibitors of these toxins have the potential to aid serum therapy of accidents inflicted by viper snakes. Broad-spectrum metalloprotease inhibitors bearing a hydroxamate zinc-binding group are potential candidates to improve snake bites therapy and could also be used to study toxin-ligand interactions. Therefore, in this work, we used both docking calculations and molecular dynamics simulations to assess the interactions between six hydroxamate inhibitors and two P-I SVMPs selected as models: Atroxlysin-I (hemorrhagic) from <i>Bothrops atrox</i>, and Leucurolysin-a (nonhemorrhagic) from <i>Bothrops leucurus</i>. We also employed a large variety of end-point free energy methods in combination with entropic terms to produce scoring functions of the relative affinities of the inhibitors for the toxins. Then we identified the scoring functions that best correlated with experimental data obtained from kinetic activity assays. In addition, to the characterization of these six molecules as inhibitors of the toxins, this study sheds light on the main enzyme-inhibitor interactions, explaining the broad-spectrum behavior of the inhibitors, and identifies the energetic and entropic terms that improve the performance of the scoring functions.