The World Health Organization recently listed snakebite envenoming as a Neglected Tropical Disease, proposing strategies to significantly reduce the global burden of this complex pathology by 2030. In this context, effective adjuvant treatments to complement conventional antivenom therapy based on inhibitory molecules for specific venom toxins have gained renewed interest. Varespladib (LY315920) is a synthetic molecule clinically tested to block inflammatory cascades of several diseases associated with elevated levels of secreted phospholipase A₂ (sPLA₂). Most recently, Varespladib was tested against several whole snake venoms and isolated PLA₂ toxins, demonstrating potent inhibitory activity. Herein, we describe the first structural and functional study of the complex between Varespladib and a PLA₂-like snake venom toxin (MjTX-II). In vitro and in vivo experiments showed this compound’s capacity to inhibit the cytotoxic and myotoxic effects of MjTX-II from the medically important South American snake, Bothrops moojeni. Crystallographic and bioinformatics analyses revealed interactions of Varespladib with two specific regions of the toxin, suggesting inhibition occurs by physical blockage of its allosteric activation, preventing the alignment of its functional sites and, consequently, impairing its ability to disrupt membranes. Furthermore, based on the analysis of several crystallographic structures, a distinction between toxin activators and inhibitors is proposed.

Ophidian accidents are considered an important neglected tropical disease by the World Health Organization. Particularly in Latin America,Bothropssnakes are responsible for the majority of the snakebite envenomings that are not efficiently treated by conventional serum therapy. Thus, the search for simple and efficient inhibitors to complement this therapy is a promising research area, and a combination of functional and structural assays have been used to test candidate ligands against specific ophidian venom compounds. Herein, we tested a commercial drug (acetylsalicylic acid, ASA) and a plant compound with antiophidian properties (rosmarinic acid, RA) using myographic, crystallographic and bioinformatics experiments with a phospholipase A₂-like toxin, MjTX-II. MjTX-II/RA and MjTX-II/ASA crystal structures were solved at high resolution and revealed the presence of ligands bound to different regions of the toxin. However,in vitromyographic assays showed that only RA is able to prevent the myotoxic effects of MjTX-II. In agreement with functional results, molecular dynamics simulations showed that the RA molecule remains tightly bound to the toxin throughout the calculations, whereas ASA molecules tend to dissociate. This approach aids the design of effective inhibitors of PLA₂-like toxins and, eventually, may complement serum therapy.

Local myonecrosis is the main event resulting from snakebite envenomation by theBothropsgenus and, frequently, it is not efficiently neutralized by antivenom administration. Proteases, phospholipases A₂(PLA₂) and PLA₂-like toxins are found in venom related to muscle damage. Functional sites responsible for PLA₂-like toxins activity have been proposed recently; they consist of a membrane docking-site and a membrane rupture-site. Herein, a combination of functional, biophysical and crystallographic techniques was used to characterize the interaction between suramin and MjTX-I (a PLA₂-like toxin fromBothrops moojenivenom). Functionalin vitroneuromuscular assays were performed to study the biological effects of the protein-ligand interaction, demonstrating that suramin neutralizes the myotoxic effect of MjTX-I. Calorimetric assays showed two different binding events:(i)inhibitor-protein interactions and(ii)toxin oligomerization processes. These hypotheses were also corroborated with dynamic light and small angle X-ray scattering assays. The crystal structure of the MjTX-I/suramin showed a totally different interaction mode compared to other PLA₂-like/suramin complexes. Thus, we suggested a novel myotoxic mechanism for MjTX-I that may be inhibited by suramin. These results can further contribute to the search for inhibitors that will efficiently counteract local myonecrosis in order to be used as an adjuvant of conventional serum therapy.