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.

Figure 1

Effects of the MjTX-II and the product of its pre-incubation with (a) rosmarinic acid (RA) and (b) acetylsalicylic acid (ASA) on indirectly evoked twitches in isolated mice phrenic diaphragm preparations. The ordinate indicates the percentage of twitches relative to its initial amplitude. The abscissa indicates the time (minutes) after addition of MjTX-II, (a) rosmarinic acid or (b) acetylsalicylic acid alone and the mixture of MjTX-II plus (a) rosmarinic acid (1:10, m/m) or (b) acetylsalicylic acid (1:20, m/m and 1:770, m/m) to the organ bath. The data are grouped as means ± SEM (P < 0.05) and all groups have N = 3. *Indicates the point from which there was a significant difference compared with control.

Figure 2

Crystal structures of MjTX-II/rosmarinic acid (RA) and MjTX-II/ acetylsalicylic acid (ASA) complexes. (a) The overall structure of the MjTX-II/RA complex is shown as a cartoon representation. The RA (yellow) molecule is illustrated as stick representation. (b) Omit electron density map (coefficients 2|F_obs| - |F_calc|) corresponding to RA is contoured at 1.0σ. (c) The overall structure of the MjTX-II/ASA complex is shown as a cartoon representation. ASA (yellow) molecules are illustrated as stick representation. (d) Omit electron density map (coefficients 2|F_obs| - |F_calc|) corresponding to the ASA molecules bound to monomer A and B, respectively, are contoured at 1.0σ.

Figure 3

Superposition of C_α atoms of one monomer from each of PLA₂s-like structures: MjTX-II/RA (green), MjTX-II/ASA (blue), PrTX-I/RA (magenta), MjTX-II/FA14 (cyan) and BthTX-I (yellow) and the relative disposition of other monomers of each structure.

Figure 4

Molecular dynamics simulations with MjTX-II complexes. (a) Backbone atoms RMSD for MjTX-II/RA and MjTX-II/ASA complexes (black line for MjTX-II/RA and red line for MjTX-II/ASA). (b) Non-H atoms RMSD for RA and ASA ligands (black line - RA and red line - ASA molecules). (c) Backbone atoms RMSD for unbound MjTX-II (green) and MjTX-II/FA (magenta) complexes. (d) Radius of gyration (R_g) for unbound MjTX-II (green) and MjTX-II/FA (magenta) complexes.

Figure 5

Superposition of the active MjTX-II crystal structure (MjTX-II/FA14, PDB id: 6B80) and structures from MD simulations. (a) Superposition between MjTX-II/FA14 crystal structure (cyan), the unbound MjTX-II model before (gray) and after (green) the MD simulation. (b) Superposition between MjTX-II/FA14 crystal structure (cyan), the MjTX-II/FA model before (gray) and after (magenta) the MD simulation.

Figure 6

Comparison between MjTX-II/RA and PrTX-I/RA crystal structures. (a) Crystal structure of the PrTX-I/RA (PDB id: 3QNL) is shown as cartoon representation and RA inhibitor (magenta) is illustrated in stick representation. (b) Interaction of RA molecule in the PrTX-I structure. The representation of the interactions of RA was depicted as polar contacts (broken lines) and hydrophobic contacts (arcs with radiating spokes). Water molecules are showed as cyan spheres. (c) Crystal structure of the MjTX-II/RA is shown as cartoon representation and RA inhibitor (dark blue) is illustrated in stick representation. (d) Interaction of RA molecule in the MjTX-II structure. The representation of the interactions of RA was depicted as polar contacts (broken lines) and hydrophobic contacts (arcs with radiating spokes).

Figure 7

Interaction of ASA molecules with MjTX-II. ASA molecule interacting with hydrophobic channel residues from MjTX-II - monomer A (a) and monomer B (b). The representation of the interactions was depicted as polar contacts (broken lines) and hydrophobic contacts (arcs with radiating spokes). Water molecules are showed as cyan spheres.

Figure 8

Schematic representation of structural changes caused by ligands. (a) In the MjTX-II/RA structure, the RA ligand is kept tight bound to the protein and blocks the interaction of fatty acids with the toxin, preventing the alignment of its functional sites. (b) In the MjTX-II/ASA structure, ASA ligands interact with low stability to the toxin, as observed in MD simulations, thus, fatty acids may bind with the toxin and, consequently, its functional sites may be exposed to the solvent.