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

Abstract

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.

Conflict of interest statement

M.R.L. has an ownership stake in Ophirex, Inc, a Public Benefit Corporation. The other authors have declared that no competing interests exist.

Figures

Figure 1

Myotoxic and cytotoxic activities of MjTX-II in mice and cultured C2C12 myoblasts, respectively, and their inhibition by preincubation with Varespladib. (A) Mice were injected by intramuscular route with toxin alone (50 μg, in 100 μL of PBS), or preincubated for 15 min with Varespladib (VAR) at a final concentration of 400 μM. Control groups were injected with PBS alone or Varespladib alone, respectively. After 3 hr, blood was obtained and the plasma creatine kinase (CK) activity was determined, as described in Methods. Each bar represents the mean ± SD of 4–5 mice per group. (B) Cells were exposed to the toxin alone (20 μg, in 150 μL of medium), or preincubated for 15 min with Varespladib (VAR) at a final concentration of 400 μM. After incubating the cells for 3 hr at 37 °C, an aliquot of supernatant was assayed for lactate dehydrogenase (LDH) activity, as described in Methods. Cytotoxicity is expressed as percentage, considering the LDH activity of cells exposed to medium with 0.1% Triton X-100, or to medium alone, as 100% and 0%, respectively. Each bar represents the mean ± SD of three replicates. Statistically significant (p < 0.05) differences between values obtained with the toxin alone or the toxin preincubated with Varespladib are indicated by an asterisk.

Figure 2

Crystal structure of the complex MjTX-II/Varespladib. (A) The overall structure of the complex is depicted as cartoon representation (cyan) and the inhibitor molecules are represented as sticks (yellow). (B) Omit electron density map (coefficients 2|Fobs| − |Fcalc|) corresponding to Varespladib bound to monomer A and (C) Varespladib bound to monomer B. The maps corresponding to inhibitor molecules are contoured at 1.0 σ.

Figure 3

Molecular dynamic simulation of the MjTX-II/Varespladib complex. (A) RMSD for MjTX-II backbone atoms (black line) and Varespladib non-hydrogen atoms (red line). (B) Backbone atoms RMSF for MjTX-II chains A (dark blue line) and B (cyan line).

Figure 4

Schematic representation of the interaction of Varespladib molecules with MjTX-II. (A) Interaction of Varespladib with monomer A. (B) Contacts of Varespladib with monomer B. The figure was made using Lidia extension from Coot v.0.8.9.

Figure 5

Interaction of Varespladib and myristic acid (FA14) with side chain of residues from Helix-I, hydrophobic channel and MDiS residues. (A) Overall crystal structure of MjTX-II/Varespladib (cyan), zoomed region of contacts of inhibitor to side chain of aminoacids from protein and the same region after 90° of rotation. (B) Overall of crystal structure of MjTX-II/FA14 (green), zoomed region from the interaction of fatty acid molecule with the protein and the same region after 90° of rotation.

Figure 6

Different forms of inhibition of Lys49 PLA2-like toxins by inhibitors described in literature. The monomer A is showed as light gray and the monomer B is represented as dark gray (A) Class 1 inhibitors (yellow), which prevent the binding of fatty acids (yellow sticks - activator) to hydrophobic channel (cyan) of toxin. (B) Class 2 inhibitors (blue) which bind to functional MDoS (green) and MDiS (red) and prevent the interaction of toxin to membrane (orange). (C) Class 3 inhibitors (magenta) which induce protein oligomerization.