Varespladib

The synthetic varespladib molecule is a multi-functional inhibitor for PLA2and PLA2-like ophidic toxins

Guilherme H.M. Salvador a, Rafael J. Borges a, Bruno Lomonte b, Matthew R. Lewin c, Marcos R. M. Fontes a,*
a Departamento de Biofísica e Farmacologia, Instituto de Biociˆencias, Universidade Estadual Paulista (UNESP), Botucatu, SP, Brazil
b Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San Jos´e, Costa Rica
c Center for Exploration and Travel Health, California Academy of Sciences, San Francisco, CA 94118, USA

A B S T R A C T

Background: The treatment for snakebites is early administration of antivenom, which can be highly effective in inhibiting the systemic effects of snake venoms, but is less effective in the treatment of extra-circulatory and local effects. To complement standard-of-care treatments such as antibody-based antivenoms, natural and synthetic small molecules have been proposed for the inhibition of key venom components such as phospholipase A2 (PLA2) and PLA2-like toxins. Varespladib (compound LY315920) is a synthetic molecule developed and clinically tested aiming to block inflammatory cascades of several diseases associated with high PLA2s. Recent studies have demonstrated this molecule is able to potently inhibit snake venom catalytic PLA2 and PLA2-like toxins.
Methods: In vivo and in vitro techniques were used to evaluate the inhibitory effect of varespladib against MjTX-I. X-ray crystallography was used to reveal details of the interaction between these molecules. A new methodology that combines crystallography, mass spectroscopy and phylogenetic data was used to review its primary sequence.
Results: Varespladib was able to inhibit the myotoxic and cytotoxic effects of MjTX-I. Structural analysis revealed a particular inhibitory mechanism of MjTX-I when compared to other PLA2-like myotoxin, presenting an oligomeric-independent function.
Conclusion: Results suggest the effectiveness of varespladib for the inhibition of MjTX-I, in similarity with other PLA2 and PLA2-like toxins.
General significance: Varespladib appears to be a promissory molecule in the treatment of local effects led by PLA2 and PLA2-like toxins (oligomeric dependent and independent), indicating that this is a multifunctional or broadly specific inhibitor for different toxins within this superfamily.

Keywords:
Phospholipase A2 – like proteins Lys49-phospholipases A2 proteins Snake venom Myotoxicity inhibition Varespladib inhibitor Phospholipase A2 inhibitor

1. Introduction

Snakes from Bothrops genus are found throughout Latin America and are responsible for the highest number of snakebite envenomings in this region [1–3]. The venom of these snakes is composed of several proteins with great diversity of biological functions and deleterious effects to prey and unintended victims. Individuals stricken by snakes of this genus frequently suffer drastic local necrotic action which can lead to tissue loss, amputation of the affected limb and permanent disability of the victim [4,5]. Among the proteins present in bothropic venom, Phospholipase A2 (PLA2), PLA2-like toxins and proteinases are major components responsible for the rapid local myonecrosis, acting alone or synergistically [6–10].
Bothropic PLA2-like toxins display a homodimeric assembly, as demonstrated by several crystal structures and, also, by in solution studies [11–18]. These dimeric proteins present two distinct functional sites responsible for the membrane interaction and disruption called Membrane Docking Site (MDoS) and Membrane Disrupting Site (MDiS), respectively [11,19,20]. The MDoS is formed by positive amino acids from N-terminal (Lys16 and Lys20) and C-terminal (Lys115 and Arg118) portions, and the MDiS is composed by the hydrophobic residues Leu121 and Phe125 (Leu122 and Phe126 for MjTX-II due the Asn120 insertion) [11,18,19].
Myotoxin I (or MjTX-I) is a PLA2-like toxin isolated from B. moojeni venom that exhibits some particularities when compared to other PLA2- like toxins isolated from Bothrops venoms. The efficiency of MjTX-I to induce myotoxicity is relatively low when compared to other bothropic PLA2-like toxins [21,22]. This fact can be related to differences in its primary sequence, natural substitution of key amino acids, which re- flects in totally different quaternary conformation of this toxin [22,23], leading to the proposition of an oligomeric-independent myotoxic mechanism.
As previously reported, PLA2-like toxins from Bothrops snakes are inhibited by three main inhibitor classes that are related to the inter- action region in protein and to the induced structural modifications. The class 1 – inhibitors that block the access of fatty acids to the hydrophobic channel and also prevent the natural movement between the monomers of the dimeric structure; Class 2 – inhibitors that interacts with the functional sites of the proteins, blocking their MDoS and MDiS and; Class 3 – inhibitors that induce protein oligomerization, which may also share features of the first two classes [24].
As the neutralization of local effects caused by bothropic accidents is an important area of unmet need, the search for molecules that are efficient inhibitors for the initial in loco treatment and posterior com- plement of the antivenom therapy may be relevant to aid victims with poor access to health system [25–28]. Natural molecules isolated from plants generally used in folk medicine and synthetic compounds are frequently sought as candidates to specifically inhibit the biological ef- fects of ophidian venom components, such as inflammatory, myotoxic and hemorrhagic activities [11,12,18,24,25,29–40]. The search for efficient venom inhibitors derived from traditional sources can be refined, better understood and improved by strategies involving func- tional, biochemical, biophysical, structural and bioinformatics tools [25].
Varespladib (LY315920) is a synthetic compound that potently inhibits human secreted group IIA PLA2. It was previously and unsuc- cessfully developed for the prevention and mitigation of sPLA2-induced inflammation associated with conditions such as severe sepsis, rheu- matoid arthritis, ulcerative colitis as well as acute coronary and acute chest syndromes [41,42]. Since PLA2s found in snake venoms share functional homology to human group IIA PLA2, varespladib and, also, its orally bioavailable methyl-ester variant (LY333013) were tested against venoms from medically important snakes found in different countries of six continents, showing a potent inhibition capability [43–49]. Furthermore, catalytic PLA2s and PLA2-like toxins isolated from Bothrops snakes were tested, and varespladib appears to be a potent inhibitor of their catalytic and myotoxic activities [24,50].
Herein, we present the crystal structure of an oligomeric- independent PLA2-like toxin myotoxin I (MjTX-I) isolated from B. moojeni co-crystallized to varespladib (compound LY315920), a spe- cific inhibitor initially designed for catalytic PLA2s. The biological as- says revealed the inhibition of cytotoxic and myotoxic effects of MjTX-I by varespladib, as previously demonstrated for other catalytic PLA2s and non-catalytic PLA2-like proteins. The crystallographic structure analysis shows a tetrameric assembly, similar to the native crystal, but presenting one inhibitor molecule interacting to each protomer in the toxin’s hy- drophobic channel. In addition, we applied a new methodology, SEQUENCE SLIDER, that combines crystallographic, mass spectrometry and phylogenetic data, to review the primary sequence of MjTX-I, aiming to solve the discrepancies between the electron density map and the previous deposited sequence.

2. Experimental procedures

2.1. Isolation of MjTX-I and varespladib (LY315920)

The crude lyophilized venom from adult Bothrops moojeni specimens was purchased from Centro de Extraç˜ao de Toxinas Animais (CETA – Morungaba, Sa˜o Paulo). The MjTX-I was isolated in two steps of liquid chromatography, similar to protocols previously described [21–23]. The first step was carried out using 150 mg of whole venom diluted in 0.05 M ammonium bicarbonate (pH 8.0), centrifuged to remove debris and fractionated by a ion-exchange CM-FF (5 mL) column (GE healthcare) with a linear gradient from 0 to 100% of 1.0 M ammonium bicarbonate (pH 8.0). The second step was a reverse-phase HPLC on a C18 column (Shimadzu) at 1 mL/min by applying a linear gradient from 0.1% tri- fluoroacetic acid to 66.5% acetonitrile (containing 0.1% trifluoroacetic acid). The purity of samples was determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE at 13%) under reducing condition. Vares- pladib (compound LY315920) in free acid form was provided by Ophyrex Inc. (original source: ChemieTek, Indianapolis, Indiana – USA; 99.99% pure by NMR and HPLC).

2.2. Functional studies

2.2.1. In vitro cytotoxicity using C2C12 myotubes

The cytotoxicity assays were evaluated using murine C2C12 skeletal muscle myoblast cells (CRL-1772; American Type Culture Collection) as previously described [24,50,51]. The C2C12 cells cultures were main- tained as undifferentiated myoblasts at subconfluent levels in Dulbec- co’s modified Eagle’s medium (DMEM, Sigma) supplemented with 10% of fetal bovine serum (FBS), L-glutamine, penicillin and streptomycin.
Approximately 105/200 μL of cells were added to each well of a 96-well plate and incubated at 37 ◦C, with 7% CO2 humidified atmosphere. When cell cultures reached 80–90% of confluence, myoblasts were incubated under four experimental conditions: i) negative control (DMEM containing 4% DMSO (Dimethyl sulfoxide, 0% cytotoxicity), which does not result in a cytotoxic effect in the assay system, as pre- viously demonstrated [24]) and ii) positive control (DMEM containing 0.1% Triton X-100) (100% cytotoxicity), respectively; iii) toxin (30 μg) alone and iv) toxin (30 μg) pre-incubated with varespladib (400 μM) for 15 min at room temperature. After 3 h of cell exposure at 37 ◦C, an aliquot of 60 μL of each supernatant was removed to quantify the ac- tivity of lactate dehydrogenase (LDH) release from damaged cells, using a commercial assay (LDH-P UV AA – Wiener Lab). Assays were per- formed in triplicate cell cultures and results are presented as mean ± SD, with statistical significance of differences between means of two groups determined by the Student’s t-test, considering values of p < 0.05 as significant. 2.2.2. In vivo myotoxicity Four groups composed of four CD-1 mice (18–20 g body weight) with access to food and water ad libitum were used to evaluate the myotoxic activity assays [24,52]. The negative control groups were composed by: 1st control groups was injected in the gastrocnemius muscle with 100 μL of phosphate-buffered saline (PBS, pH 7.2; 0.12 M NaCl, 0.04 M sodium phosphate) and; 2nd was injected 100 μL of varespladib (400 mM) diluted in PBS (with 4% of DMSO). The positive control group was injected with 100 μL of PBS containing 50 μg of toxin. The treated group was injected with a mixture of 50 μg of toxin and 400 mM varespladib diluted in 4% DMSO in PBS. After 3 h, blood was collected from the tip of the tail into a hepa- rinized capillary and centrifuged to obtain the plasma. The plasmatic creatine kinase (CK) activity expressed in U/L was determined using a UV kinetic assay (CK-NAC UV, Wiener Lab). For comparison of mean values for more than two groups, ANOVA was used followed by Tukey- Kramer tests, considering statistical differences significance when p < 0.05. All the animal experiments were approved by “Comit´e Institucional para el Cuidado y Uso de los Animales” (CICUA, permit #084–17), Universidad de Costa Rica. 2.3. Crystallographic studies 2.3.1. Crystallization The co-crystallization of MjTX-I and varespladib was carried out using the conventional hanging-drop vapor-diffusion method [53]. The toxin sample was concentrated to 10 mg.mL—1 in 20 mM ammonium bicarbonate pH 8.0 and pre-incubated with varespladib (diluted in 100% DMSO) in a protein/ligand ratio of 1:10 respectively. Crystals were obtained after approximately 20 days at a constant temperature of 293 K from a mixture of 1 μL of toxin+ligand and 1 μL of reservoir so- lution, equilibrated against 500 μL of reservoir containing 32% w/v PEG4000, 0.1 M Tris HCl pH 8.5 and 0.1 M lithium sulfate.Crystals of the complex MjTX-I/varespladib were mounted in a nylon loop and flash-cooled in liquid nitrogen without cryoprotectant for diffraction experiments using synchrotron radiation at MX2 station, located at Laborato´rio Nacional de Luz Síncrotron (LNLS – Campinas, Brazil). 2.3.2. X-ray diffraction data collection, structural determination and refinement X-ray diffraction data were collected from a monocrystal of complex MjTX-I/varespladib using a wavelength of 1.425 Å at cryogenic tem- perature (100K) and a PILATUS 2 M detector (Dectris). The distance of crystal-to-detector used was 150 mm and oscillation range of 0.25◦, resulting in 1440 frames. The diffraction data were processed to 1.76 Å resolution using the XDS v.20180126 program [54] and the crystal structure was phased using the molecular replacement method with Phaser program [55] from the PHENIX package v.1.12 [56] and the monomer A from MjTX-I/ suramin crystal structure (PDB id: 6CE2) [23] as the search model. The insertion and adjustments of inhibitor, solvent, PEG4000 molecules and residues into electron density was performed manually using Coot v.0.8.9 [57]. Refinement of the crystal structure was performed by alternating cycles of manual rebuilding using Coot v.0.8.9 [57] and automated refinement with PHENIX v.1.12 [56]. To check the overall quality of the final model, Molprobity software [58] present in PHENIX v.1.12 was used. X-ray crystallographic structure coordinates were deposited in the Protein Data Bank (RCSB PDB – www.rcsb.org) under the identification code 7LYE. The crystallographic structures comparisons of the MjTX-I/ varespladib with other PLA2-like toxins were performed using Coot v.0.8.9 [57] and PyMOL v.1.8.6 [59] programs. All structural figures were generated using PyMOL. 2.4. Dynamic light scattering Dynamic Light Scattering (DLS) experiments were performed using the complex MjTX-I/varespladib (MjTX-I:varespladib molar ratio 1:10) in a protein concentration of 3 mg/mL (in 50 mM ammonium bicar- bonate pH 8.0) and a DynaPro Titan device (Wyatt Technology) at 291 K. Data was composed of 100 measurements acquired and analyzed using the DYNAMICS v.6.10 program (Wyatt Technology). 2.5. Mass spectrometry Tryptic digests of MjTX-I were solubilized in 0.1% (v/v) formic acid (solution A) and subjected to nano-ESI-LCMS/MS analysis, using an UltiMate 3000 HPLC (Dionex, Sunnyvale, CA, USA), coupled to a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific). Pep- tides were loaded on a trap column with nanoViper Fitting (P/N 164649, C18, 5 mm × 30 μm, Thermo Fisher Scientific) and eluted with a flow rate of 300 nL.min-1 using an isocratic gradient of 4% solution B (100% [v/v] acetonitrile containing 0.1% [v/v] formic acid) for 3 min. Thereafter, peptides were loaded on a C18 PicoChip column (Reprosil- Pur, C18-AQ, 3 μm, 120 Å, 105 mm, New Objective, Woburn, MA, USA) using a segmented concentration gradient from 4 to 55% B for 30 min, 55–90% B for 1 min, 90% B for 5 min, and then returning to 4% B for 20 min at a flow rate of 300 nL.min-1. Ion polarity was set to positive ionization mode using data-dependent acquisition (DDA) mode. Mass spectra were acquired with a scan range of m/z 200–2000, resolution of 70,000 and injection time of 100 ms. The fragmentation chamber was conditioned with collision energy between 29% and 35% with a reso- lution of 17,500, 50 ms of injection time, 4.0 m/z of isolation window, and dynamic exclusion of 10 s. The spectrometric data were acquired through the Thermo Xcalibur software v.4.0.27.19 (Thermo Fisher Scientific). Raw data were submitted to the PatternLab software v.4.0.0.84 [60] and searched against the NCBI database (https://www.ncbi.nlm.nih. gov/taxonomy) (Database: Bothrops – Taxonomy ID 8721, from April 02, 2020: 2179 sequences). The following search parameters were used: semi-tryptic cleavage products (two tryptic missed cleavages allowed), carbamidomethylation of cysteine as fixed modification, and oxidation of methionine as variable modification. Parent mass tolerance error was set to 40 ppm and fragment mass error to 0.02 ppm. Protein identifi- cations were considered with a minimum of one fragment ion per pep- tide, five fragment ions per protein, two peptides per protein and a false discovery identification rate set to 1%, estimated by a simultaneous search against a reversed database. 2.6. Bioinformatics 2.6.1. SEQUENCE SLIDER primary structure assignment The software SEQUENCE SLIDER was applied to assign the sequence of MjTX-I crystallographic model as some discrepancies of the electron density and the expected sequence was seen during the manual building. This method is written in Python 3 and distributed within ARCIMBOLDO (http://chango.ibmb.csic.es/) through the package installer for Python (pip) and CCP4 (Winn et al., 2011). Instead of aiding ARCIMBOLDO to phase structures with lower resolution than the usual 2.5 Å or larger than 600 residues by assembling most probable side chains [61], this implementation is adapted to assign sequence of natural purified pro- teins relying on electron density omit maps, mass spectrometry data and phylogenetic analysis. Rotamers for each one of the 20 possible amino acids were generated using the Coot v.0.8.9 [57] function “auto-fit-best rotamer” in each residue of the protein model. Atoms within 5 Å distance of a rotamer were refined using Coot’s “refine residues” [57]. Side chain atoms were selected and their real-space correlation coefficients (RSCC) were calculated using phenix.polder [62]. For each residue, amino acids pos- sessing RSCC of at least three points better than the rest were considered unique, with the exception of side chains having double occupancy. Amino acids with similar RSCC values were mutually considered to generate a theoretical database of sequences to be used against mass spectrometry data. Raw data were submitted to PatternLab software v.4.0.0.84 [60] and searched against this theoretical database. The same parameters described in the previous section were used. 2.6.2. Molecular dynamics simulations The molecular dynamics (MD) simulations of the complex MjTX-I/ varespladib were carried out using GROMACS (Groningen Machine for Chemical Simulation) v.5.0.5 [63] under the CHARMM36 force field [64]. The initial input parameters of MjTX-I and varespladib were generated by CHARMM-GUI web-server [65] and the protonation of the residues was set to pH 7.4, determined by PROPKA3 server [66]. The complex MjTX-I/varespladib was simulated using a cubic box with 5 Å from the farthest atoms, solvated with TIP3 water molecules and equilibrated with 100 mM of NaCl. The system was minimized until it reached energy below 100 kJ/mol/nm using the Steepest Descent algorithm and pos- teriorly, restraining the position of backbone and hydrogen atoms of the protein and varespladib, respectively. An 1-ns constant number, volume and temperature ensemble (NVT) was performed generating the veloc- ities randomly according to Maxwell-Boltzmann distribution at tem- perature of 300 K using the V-rescale thermostat [67] followed of an 1-ns constant number, pressure and temperature ensemble (NPT) with Berendsen barostat [68] at 1 bar. Subsequently, an unrestrained 100 ns NPT step was performed using the Nose-Hoover thermostat [69,70] and Parrinello-Rahman barostat [71]. Short-range cutoffs for electrostatic and Van der Waals interactions were set to 12 Å with a force-switch function from 10 to 12 Å and hydrogen bonds were constrained using LINCS al- gorithm [72]. The analysis of interaction of inhibitors to protein after molecular dynamics was performed using built-in tools provided by GROMACS. 3. Results and discussion 3.1. Inhibition of toxic effects of MjTX-I by varespladib The myotoxic effect of MjTX-I was evaluated by in vivo experiments after intramuscular injection of 50 μg of the toxin in gastrocnemius muscle from mice, which increases the levels of plasma creatine kinase activity indicating skeletal muscle necrosis. The pre-incubation of MjTX- I with varespladib (1:10) prevents in approximately 40% the myotox- icity (Fig. 1A), a statistically significant (P < 0.05) reduction. The cytotoxic effect of MjTX-I evaluated by in vitro experiments on C2C12 myoblasts was also significantly inhibited by pre-incubation with var- espladib (1:10), reducing the release of lactate dehydrogenase to the medium by approximately 85% in comparison to the effect of toxin alone (Fig. 1B). Despite of varespladib being able to inhibit both cytotoxic and myotoxic activities of MjTX-I and MjTX-II toxins, in vitro inhibition of cytotoxic effect is more efficient than in vivo myotoxic effect at the same concentration [24]. These differences in efficacy of varespladib are probably explained by the higher affinity of snake venom PLA2-like myotoxins for mature muscle in mouse gastrocnemius compared to the myoblast cell line in culture, as it has been shown that differentiation of the C2C12 myogenic cells increases their susceptibility to these myo- toxins [73]. PLA2-like myotoxins can be found in abundance in venom of snakes from Bothrops species, which can rapidly induce severe myonecrosis of the affected limb of human snakebite victims [4,74]. Particularly, the PLA2-like Myotoxin I (or MjTX-I) from B. moojeni also can express cytotoxic and myotoxic effects [21,22]. As previously reported, vares- pladib can inhibit the myotoxic effects caused by PLA2s proteins (including the catalytic PLA2s enzymes and non-catalytic PLA2-like toxins) from different species of snakes by pre-incubation before injec- tion, as well when delayed administered [24,44–46,48,50,75]. Two myotoxins from Bothrops snakes MT-I (B. asper) and MjTX-II (B. moojeni) were tested against varespladib [24,50]. MT-I is a basic PLA2 which can damage muscle tissue via a catalytic-dependent mechanism, which can be inhibited by varespladib [50]. Differently from MT-I, the MjTX-II myotoxicity and cytotoxic effects are expressed by a non-catalytic mechanism and were inhibited by pre-incubation with varespladib [24], similarly to the MjTX-I presented here. Therefore, varespladib appears to be a molecule with promising therapeutic potential due the inhibition of coagulopathic [75,76] and myotoxic effects caused by snake venom PLA2s and PLA2-like proteins. 3.2. Crystal structure of MjTX-I/varespladib and primary sequence assignment using SEQUENCE SLIDER MjTX-I/varespladib crystal revealed an asymmetric unit containing four protomers and C2 space group with cell constants a = 60.7, b = 128.6, c = 67.8 Å and β = 106.6◦. Each protomer present similar folding found in class-II PLA2s from snake venom, with an N-terminal α-helix, a “short” helix, a calcium binding loop (non-functional for PLA2-like toxins, as MjTX-I), two α-helix, two short strands of antiparallel β-sheet (called β-wing) and a C-terminal loop [77]. The refinement converged to a final Rcryst value of 19.5% (Rfree = 22.5%) for all data between 32.48 Å and 1.76 Å resolution. The tetrameric final model was completed with four varespladib molecules (one for each protomer shown in Fig. 2), one During the protein manual rebuilding, the high-resolution crystal structure obtained in this work allowed us to notice some discrepancies in electron density and the deposited sequence (UNIPROT database accession - P82114), leading us to review the protein sequence using the software SEQUENCE SLIDER applied to natural compounds [61]. This same approach has been recently used for the metalloproteinase of Bothrops moojeni [78]. Based on the electron density maps and real-space correlation coefficient of crystallographic data, 2,236,248 sequences hypotheses were generated and their peptides validated using mass spectrometry data. In the cases in which more than one amino acid for a given residue was observed in mass spectrometry results, the amino acid frequencies of phylogenetic analysis was used to aid distinction. The following substitution was found in respect to the deposited structures in the PDB (3T0R and 6CE2): Thr-Lys20, Ser-Asn76, Asn-Glu87, Ala-Pro89, Leu-Lys110, Asp-Gly111, Lys-Arg118, Asn-Val120, Ala-Gly130, Leu- Lys131 and Pro-Asp132 (Fig. 4). Several structural studies suggest the importance of the compact dimeric assembly for this class of proteins in the mechanism of expres- sion of their myotoxicity [11,16–19,79–81]. The maintenance and stabilization of the compact dimeric assembly for the PLA2-like toxins is established due the presence of hydrogen bond between the Tyr119 and electrostatic interactions between residues disposed of in the dimeric interface [17,80]. On the other hand, MjTX-I exhibits several natural substitutions that prevent the stabilization of the compact dimer but allow the formation of the dimeric extended assembly, or a tetramer formed by two extended dimers, being the latter seen in the asymmetric unit of native [22] and MjTX-I/varespladib crystals. The reviewed sequence obtained here showed that MjTX-I does not display the Tyr residue in position 119 and also is observed an insertion in position 120, leading to a final sequence composed of 122 amino acids, similarly to the PLA2-like MjTX-II, present in the same snake venom (Fig. 4). As observed in this work, the presence of varespladib and/or the alterations in the primary structure in MjTX-I do not affect the tetrameric quater- nary conformation when compared to its native crystal structure. 3.3. Structural basis for MjTX-I effects inhibited by varespladib The initial step of the proposed myotoxic mechanism for PLA2-like toxins is their activation by the interaction of fatty acids in their hy- drophobic channel, allowing the arrangement of two sites which are responsible for the next steps of myotoxic process. Once allosterically activated, the toxin can anchor to the membrane via the cationic Membrane Docking Site (MDoS) and the hydrophobic Membrane Dis- rupting Site (MDiS), provoking a non-controlled influx of ions, that can lead to cell death [11,18,19,81]. As discussed in the previous section, the crystal structure of the MjTX-I/varespladib complex presents the same tetrameric conformation (extended conformation) observed in the native structure in its asym- metric unit and, due to particular amino acid mutations, do not allow the formation of the compact assembly common in other PLA2-like toxins, that is essential for the proposed myotoxic mechanism [11,19,22]. However, in solution studies with native MjTX-I it was demonstrated that the toxin presents in monomeric conformation [22,23]. Those data corroborate with dynamic light scattering performed here using the complex MjTX-I/varespladib, that indicate the complex may present as a monomeric assembly in solution (2.2 nm of hydrodynamic radius with 11% polidispersity). Taking into account this structural information together other biophysical and bioinformatics studies, it was proposed a myotoxic mechanism for MjTX-I with a putative MDoS composed by amino acids from the N-terminal and the hydrophobic channel (Arg34, Lys49, Lys53, Lys69 and Lys70) and the same MDiS presented by PLA2- like toxins [22,23]. It is interesting to highlight that, in contrast with other PLA2-like toxins, this is an oligomeric-independent myotoxic activity. In this study, varespladib molecules were found in similar sites in crystal structures of MjTX-I and MjTX-II, interacting with residues in the hydrophobic channel, including His48 and Lys49 (Fig. 3). The crystal structure of MjTX-II presents the compact dimeric assembly, with one varespladib molecule bound to the hydrophobic channel of each pro- tomer and the inhibitor molecules buried by the dimeric assembly. On the other hand, due the quaternary extended assembly of MjTX-I in tetrameric form, the varespladib molecules are interacting with similar residues, but all of them are exposed to the solvent (Fig. 5). In order to investigate the stability of this interaction, we performed molecular dynamics simulations using the crystallographic complex. After 100 ns of simulation of the complex, the inhibitor molecule interactions to MjTX-I are preserved as expressed in the distance of center of mass from the inhibitor and the center of mass from the binding sites residues (Fig. 6). Previous studies with the MjTX-II/varespladib complex [24] suggested the inhibition of the cytotoxic and myotoxic effects by varespladib by two different ways: i) the interaction with the MDiS residues which physically prevents the interaction of the site to the target membrane and ii) blocking of fatty acids interaction with the toxin hydrophobic channel, which prevents the allosteric activation and consequent alignment of the functional MDoS and MDiS [24]. These mechanisms of inhibition for varespladib were described as class 1 and 2 [24]. The interaction of the varespladib molecules in the hydrophobic channel of the MjTX-II suggests a competitive inhibition, since the interaction of fatty acids molecules (responsible for the toxin activation) is prevented. While varespladib interacts with both MjTX-II and MjTX-I in similar regions, compared to MjTX-II, the presence of inhibitor in the hydrophobic channel of MjTX-I is not responsible for the observed toxin inhibition. This is supported by the fact that the myotoxic mechanism of MjTX-I is not related with the dimeric conformation change as in other PLA2-like toxins [19,23]. Thus, we suggest that the interaction of var- espladib with the MjTX-I N-terminus, the putative MDoS [23], is the responsible for the toxin inhibition (Fig. 7). Interestingly, suramin also interacts with the putative MDoS region of MjTX-I (Salvador et al., 2018) and interaction with the N-terminus has also been suggested as the reason for the toxin inhibition. Therefore, it appears that varespladib behaves only as class 2 inhibitor for MjTX-I according to the previously suggested classification [24]. 3.4. Evolutionary aspects and the relationship between primary sequence and function of MjTX-I Toxins are a group of proteins with one of the most rapid evolu- tionary divergence and variability [82], which can be observed within same species related to ontogeny [83], diet [84], seasonality [85], geographical location [86] and sex [87,88]. Venom toxin isoforms are often only differentiated by a small number of residues and their similar physicochemical properties hampers their separation. Therefore, recognition of new and important isoforms characterization can be hampered and sequences wrongly attributed to already available se- quences in a database. The joint results of crystallography, mass spec- trometry and phylogenetic analysis present an interesting strategy to avoid database bias while discovering new toxin properties and small, but critically important differences in sequence. In a phylogenetic tree analysis considering the evolutionary dis- tances of the PLA2-like toxins, MjTX-I presents the largest branch length of the tree, corresponding to the highest sequence difference from the common ancestor [22]. Based on these phylogenetic analyses, we sug- gest that MjTX-I can be evolutionary positioned between catalytic PLA2s and myotoxic PLA2-like toxins, since it presents features found in both protein classes. As example, the biological effects expressed by MjTX-I and catalytic PLA2s are related to the monomeric assembly and, on the other hand, the myotoxicity from MjTX-I is emerged from an MDoS and MDiS, as described for PLA2-like toxins. 4. Concluding remarks Several molecules derived from medicinal plant extracts and syn- thetic sources have demonstrated to be promising in the early treatments for ophidian accidents. Herein, we present functional and structural studies involving an oligomeric-independent PLA2-like myotoxin (MjTX- I) from B. moojeni snake and a synthetic molecule. Use of the varespladib has several potential and important advantages compared to previously described inhibitors. This molecule was developed and clinically tested for the purpose of blocking inflammatory cascades of several diseases associated with elevated sPLA2 levels, but was recently found to be a potent inhibitor of different classes of sPLA2 and sPLA2-like toxins found in abundance in the venoms of several snake genera. Thus, varespladib could be a promising molecule for development of initial snakebite envenoming and as an adjunct to antivenom therapies. Similar to several small molecule metalloproteinase inhibitors, small molecules like var- espladib already developed for human testing lowers the cost barrier and risk of evaluation and testing for snakebite. 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