Search for efficient inhibitors of myotoxic activity induced by ophidian phospholipase A2-like proteins using functional, structural and bioinformatics approaches

Abstract

Ophidian accidents are considered an important neglected tropical disease by the World Health Organization. Particularly in Latin America, Bothrops snakes 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 vitro myographic 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.

Structural basis for phospholipase A2-like toxin inhibition by the synthetic compound Varespladib (LY315920)

“…The superposition between dimeric proteins reveals a high distortion in their dimeric assembly, expressed by the high value of the Euler roll angle 21 and RMSD values (Table 3). When compared to other PLA 2 -like toxins, the distorted conformation observed in the crystal structure of MjTX-II/Varespladib is also observed in the crystal structure of MjTX-II/rosmarinic acid (MjTXII/RA) and MjTX-II/acetylsalicylic acid (MjTX-II/ASA) 50 , suggesting that this structural conformation may be related to the inactive structure of MjTX-II and also shedding light on how the inhibitors can influence the structure of the toxin.

Figure 4Schematic representation of the interaction of Varespladib molecules with MjTX-II. ( A ) Interaction of Varespladib with monomer A.

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“…Ideally, these novel antidotes could be used in the field rapidly after the onset of envenoming, hence halting the deleterious action of venom toxins in the tissues. In order to understand how these inhibitors block the action of toxins, protein crystallography has been employed as a powerful tool to understand the inhibitory mechanisms of a variety of small ligands toward PLA 2 toxins 6,21,41,44,45,47,49,50 .…”

The allosteric activation mechanism of a phospholipase A2-like toxin from Bothrops jararacussu venom: a dynamic description

“…4B). The MDiS is a major region of PLA 2 -like toxins related with their activity 18 , supported by mutagenesis 22,25,49 , synthetic peptides 12,21 and inhibitory experiments 19,30,42,50 .…”

“…Indeed, studies of the molecular mechanisms for the inhibition of PLA 2 -like toxins with different molecules have confirmed the proposal of myotoxic sites. It has been observed that these molecules inhibit PLA 2 -like proteins in different ways, including: (i) binding to Helix-I/MDiS region, physically blocking access to the hydrophobic channel (e.g., rosmarinic 48 , 50 , chicoric 30 , and aristolochic acids 4 ); (ii) binding to the MDoS (e.g., caffeic acid 4 and suramin 20 ); (iii) binding in the hydrophobic channel (e.g., caftaric acid 42 , p-bromophenacyl bromide (BPB) 9 ; Varespladib 51 and zinc ions 26 ); (iv) binding to the MDiS (e.g., aristolochic 4 , chicoric acid 30 , zinc ions 26 , suramin 19 , 20 and Varespladib 51 ); or (v) induction of the toxin’s oligomerization (e.g., suramin 19 ). Although the binding regions of the inhibitors are well described, the structural importance of an allosteric activator is only related to the dimer reorientation 14 , 26 .…”

Amplification of Snake Venom Toxicity by Endogenous Signaling Pathways

“…sPLA 2 s are intimately involved in the peripheral neuro-myotoxicity caused by envenoming bites of many dangerous snakes and because both s-and cPLA 2 are implicated in inflammatory and degenerative disease of the nervous system, roles that are discussed below. PLA 2 can also mediate cell-based toxicity, for example in Bothrops PLA 2 myotoxicity [61]. PLA 2 s can be the dominant venom component in some species.…”