The authors have declared that no competing interests exist.
Conceived and designed the experiments: CAHF WGLC AMS MG MRMF. Performed the experiments: CAHF FFC WGLC AMS. Analyzed the data: CAHF FFC WGLC AMS MDP MG MRMF. Contributed reagents/materials/analysis tools: AMS MDP MG MRMF. Wrote the paper: CAHF FFC WGLC MG MRMF.
One of the main challenges in toxicology today is to develop therapeutic alternatives for the treatment of snake venom injuries that are not efficiently neutralized by conventional serum therapy. Venom phospholipases A2 (PLA2s) and PLA2-like proteins play a fundamental role in skeletal muscle necrosis, which can result in permanent sequelae and disability. This leads to economic and social problems, especially in developing countries. In this work, we performed structural and functional studies with Piratoxin-I, a Lys49-PLA2 from
In Asia, Africa and Latin America,approximately 98% of the world’s snakebites occur, with 421,000 envenomations and 20,000 deaths by ophidian accidents [
In Latin America, snakes of the
In folk medicine, especially in developing countries, several vegetal species are employed for the treatment of ophidian envenomations incommunities that lack prompt access to serum therapy [
Aristolochicacid (AA) and caffeic acid (CA) are plant compounds with anti-snake venom properties that are used in folk medicine [
In this work, we report functional (neuromuscular-blocking and muscle-damage activities) and crystallographicexperiments aimingto studythe possible inhibitory effects of AA and CA onPrTX-I, a Lys49-PLA2 isolated from
PrTX-I was isolated from
Institutional Animal Care and Use Committee (Institute of Biosciences–Sao Paulo State University–UNESP) approved this study under the number 033/05. Animal procedures were in accordance with the guidelines for animal care prepared by the Committee on Care and Use of Labor. Adult male mice weighing 25–30g were maintainedunder a 12 h light-dark cyclein atemperature-controlled environment (22±2°C) for at least 10 daysprior to the experiments, with food and water
Mice were euthanizedby cervical dislocation followed by exsanguination. The phrenic nerve-diaphragm muscle preparation was removed and mounted vertically in a conventional isolated organ-bath chamber containing 15 mL of Ringer’s physiological solution of the following composition (mM): NaCl, 135; KCl, 5; MgCl2, 1; CaCl2, 2; NaHCO3, 15; Na2HPO4, 1; glucose, 11. This solution was bubbled with carbogen (95% O2 and 5% CO2). The preparation was attached to an isometric force transducer (Grass, FT03) to record the twitch tension. The transducer signal output was amplified and recorded on a computer via a transducer signal conditioner (Gould, 13-6615-50) with an AcquireLab Data Acquisition System (Gould). The resting tension was 5 g. Indirect contractions were evoked by supramaximal pulses (0.2 Hz, 0.5 ms) delivered from an electronic stimulator (Grass, S88K) and applied to the phrenic nerve by means of a suction electrode.
The preparation was stabilized for 45 minutes before the addition of a single concentration of toxin. For inhibition experiments, a fixed amount of PrTX-I dissolved in Ringer’s physiological solution was mixed with AA and CAto obtain a 1:1 and 1:5 (w/w) toxin/inhibitor ratio. At molar ratio terms, it means 1:40 and 1:76 for 1:1 (w/w) toxin/inhibitor ratio for AA and CA, respectively, and 1:200 and 1:380 for 1:5 (w/w) ratio. The mixtures were incubated for 30 minutes at 35±2°C. The control experiments were performed in the absence of toxin or in the presence of inhibitors alone. The degree of protection offered by AA and CA after 90 minutes of contact with the preparation was expressed as a percentage of neuromuscular blockade observed in the presence of the mixture of toxin plus inhibitor relative to the blockade observed in the presence of toxin alone.
After the myographicstudy, the diaphragm muscle was removed from the bath and immersed in Bouin’s fixative, and then processed and embedded in Historesin (Kit Historesin Leica). Histological transverse sections (5 mm thick) were cut out in a microtome and stained with hematoxylin and eosin (HE) prior to examination by light microscopy [
The data are expressed as the mean ± S.E.M. The statistical analysis of the data was carried out using ANOVA complemented by the Tukey-Kramer test. Values of P<0.05 were considered significant.
Co-crystallization experiments were performed with PrTX-I at a concentration of 12 mg.mL-1. Crystals of the complexes were obtained by the hanging drop method [
The X-ray diffraction data for all crystals were collected at a wavelength of 1.45 Å using a synchrotron-radiation source (MX2 station, Laboratório Nacional de Luz Síncrotron, LNLS, Campinas, Brazil) and a MAR CCD imaging-plate detector (MAR Research). Crystals were mounted in nylon loops and flash-cooled in a stream of nitrogen gas at 100 K using no cryoprotectant. The data were processed using the HKL program package [
The crystal structures were determined by molecular replacement techniques implemented in the program MOLREP [
PrTX-I (1.0 μM) promoted a time-dependent blockade of indirectly evoked twitches in mice phrenic-diaphragm preparations. After 90 minutes, the twitch amplitudes were reduced to 89.4% (
The ordinate represents the percentage amplitude of twitches relative to the initial amplitude. The abscissa indicates the time from the beginning of each treatment in the organ bath. The points are the mean ± S.E. * indicates the point at which differences between PrTX-I treatments (alone and pre-incubated with CA and AA) and the control become significant. ** indicates the point at which differences between AA (68.5 μg/mL) and the control become significant.
Light microscopy showed that control and CA or AA-treated muscles were of normal appearance. Fibers were delimited by a thin layer of connective tissue (endomysium) and presented a polygonal shape, with an acidophilic sarcoplasmand peripheral nuclei(
Control muscle (A) and muscle exposed to caffeicacid (CA) and aristolochic acid (AA) (C, E, G and I) show fibers with normal appearance as evidenced by the polygonal aspect of fibers (PF) and endomysium (EN). A few fibers present loss of myofibrils in the muscle exposed to AA (G and I). (B) Muscle exposed to PrTX-I: edema (ED), round fibers (RF), some of which present loss of myofibrils (LM). (D, F, H and J) Muscle exposed to PrTX-I pre-incubated with CA and AA: The fibers have characteristics observed less frequently in the fibers treated with the PrTX-I alone, except in J, which occurred at similar frequencies.
The ordinate represents the percentage of damaged fibers relative to normal fibers and the abscissa indicates the experimental groups. The bars are expressed as the mean ± S.E. * indicates when differences between PrTX-I treatments (alone and pre-incubated with CA or AA) and their respective controls was significant. # indicates when there were significant differences between PrTX-I pre-incubated with inhibitors and PrTX-I alone treatments. + indicates significant differences between PrTX-I treatments pre-incubated with CA. ° indicates significant differences between the AA (68.5 μg/mL) treatment and the Control group.
Crystals of both complexesdiffracted at high resolution (
PrTX-I-Aristolochic Acid | PrTX-I-Caffeic Acid | |
---|---|---|
Unit Cell (Å) | ||
Space Group | P21212 | P21 |
Resolution (Å) | 25.61–1.96 (2.03–1.96) |
37.34–1.65 (1.70–1.65) |
Unique reflections | 15848 (1541) |
27814 (2724) |
Completeness (%) | 99.22 (98.59) |
94.47 (92.59) |
Rmerge |
6.3 (49.0) |
6.5 (39.5) |
Mean I/σ (I) | 14.33 (2.02) |
27.4(2.34) |
Rcryst |
17.30 | 18.23 |
Rfree |
23.52 | 22.87 |
Number of non-hydrogen atoms |
||
Protein | 1749 | 1849 |
Ligands | 60 | 108 |
Waters | 174 | 289 |
RMS (bonds) |
0.007 | 0.008 |
RMS (angles) |
1.14 | 1.18 |
Average B-factor (Å2) |
||
Protein | 29.60 | 32.10 |
Ligands | 54.40 | 56.40 |
Solvent | 37.10 | 40.60 |
Ramachandran favored (%) |
98 | 95 |
Ramachandran outliers (%) |
0 | 0 |
Clashscore |
4.77 | 11.37 |
MolProbity Overall Score |
1.54 | 1.78 |
a Numbers in parenthesis are for the highest resolution shell.
b Rmerge = ∑hkl(∑i(|Ihkl,i-<Ihkl>I))/∑hkl,i<Ihkl>, where Ihkl,i is the intensity of an individual measurement of thereflection with Miller indices h, k and l, and <Ihkl> is the mean intensity of that reflection. Calculated for I>-3σ (I).
c Rcryst = ∑hkl(||Fobshkl|-|Fcalchkl||)/|Fobshkl|, where |Fobshkl| and |Fcalchkl| are the observed and calculated structure factor amplitudes, respectively.
d Rfreeis equivalent to Rcryst but calculated with reflections (5%) omitted from the refinement.
e Calculated with Phenix [
f Calculated with MolProbity[
PEG molecules, sulfate ions,AA and CAare indicatedby sticks (in cyan, yellow, blue and green, respectively). In yellow sticks are also highlighted the aminoacids that compose MDiS (Leu121)andMDoS (Lys20, Lys155, Arg118) regions, which interact with AA and CA, respectively. (C) Cα superposition of apo-PrTX-I, PrTX-I/AA, PrTX-I/CA and PrTX-I complexed to rosmarinic acid (PrTX-I/RA) (monomers A and B, respectively) highlighting the most important structural deviations between them.
The inspection of the 2ǀFobsǀ-ǀFcalcǀ electronic density mapof the PrTX-I/CA structure revealed the presence of four CA molecules establishing hydrogen bonds in monomer A with Lys15 and Arg118 and by water molecules with Ile82 and Lys100 (
The distances (Å) between protein inhibitorsare shownas yellow dashes for bump distances and red dashes for hydrogen bond distances. Residues of monomers A and B in contact with AAand CAare represented by sticks. Omit electron density maps for AA and CA molecules (gray meshes) were calculated with the coefficients 2|Fobs|-|Fcalc| contoured at 1.0 standard deviation.
Drawn using Ligplot[
In addition,three PEG 4000 moleculeswere noted in the PrTX-I/CA structure,with two PEGs inside the hydrophobic channels from both monomersand the third PEG moleculeinteracting with Lys7 on monomer B. The PEG molecules found in these sites are found in other Lys49-PLA2s structures [
On the other hand, in the PrTX-I/AA structure just one AA molecule was observedwhich established hydrogen bonds with N-terminalresidues from monomer B (Gly15 and Asn17 by two water molecules)in close proximity to the C-terminal region from monomer A, especially Leu121 (
Superposition among the complexed structuresPrTX-I/AA,PrTX-I/CA and PrTX-I rosmarinic acid (PrTX-I/RA) [
apo-PrTX-I | |||
---|---|---|---|
Monomer A | Monomer B | ||
PrTX-I/AA | Monomer A | 0.82 | 0.41 |
Monomer B | 0.83 | 0.49 | |
PrTX-I/CA | Monomer A | 0.79 | 0.56 |
Monomer B | 0.83 | 0.58 | |
PrTX-I/RA | Monomer A | 1.04 | 0.55 |
Monomer B | 0.98 | 0.59 |
Regarding the quaternary structure, PrTX-Istructures complexed to AA CA or RA have two protomers in the asymmetric unit (
Several groups of indigenous people use specific plant extracts against snakebites, and the identification of their active compounds is an active field of study. Recently, some studies showed that several constituents of these plant extracts containanti-snake venom properties [
In this study, both caffeic (CA) and aristolochic acids (AA) partially neutralized the muscle damage promoted by PrTX-I. As CA is a RA precursor, it was expected that its inhibitory activities against PrTX-I effects would be similar in the phrenic-diaphragm preparation. However, CA was ineffective at inhibiting the
The paralyzing and muscle damage activities promoted by myotoxicLys49-PLA2s, represented here by PrTX-I, were due to their ability to alter the integrity of muscle cell membranes (for a review see [
Although the paralysis and the muscle fiber damage induced by Lys49-PLA2s are triggered by alterations in membrane permeability, the contractile process is more sensitive to such action because of its dependence on the cell excitability. Thus, ligands that partially inhibit the Lys49-PLA2 actions on cell membrane, such as CA and AA, promotes a partial protection of myotoxicity (up to 65% and 43%, respectively) with no positive interference on muscle paralysis. Corroborating this hypothesis, we have previously demonstrated that ligands able to promote a more effective binding to Lys49-PLA2, as rosmarinic acid, efficiently prevent both effects[
Recently, Fernandesand coworkers[
Interestingly, inthis study, we demonstrated that both ligands (AA and CA) are boundto residues related to MDoS and MDiS regions of the toxin and this fact may explain theirinhibitory characteristics. In the PrTX-I/CA crystal structure, four CA molecules interact with the protein. Two of them interact with Lys20, Lys115 and Arg118 residues for both monomers by hydrogen bonds and hydrophobic interactions (Figs
The crystal structure of PrTX-I/AAreveals an aristolochic acid molecule interacting by hydrogen bonds with Gly15(B) and via water molecules with Asn17(B) and Gln11(B) (
The crystal structure of a catalytic PLA2(an Asp49-PLA2)from
In conclusion, the data presented here demonstrate for the first timethe occurrence of two independent sites of interaction between a protein and a membrane target, whichcontributes to the validation of the proposed myotoxic mechanism [
One of the main challenges intoxicologytodayis to develop therapeutic alternatives to the treatment of snake venom injuries that are not efficiently neutralized by conventional serum therapy. In the case of Latin American snakes, the local myonecrosis caused by PLA2 and PLA2-like proteins is the main consequence of their envenomation[
In this work, we performed structural and functional approaches to provide substantial information about the inhibition of myotoxic Lys49-PLA2s using aristolochic and caffeic acids as molecular models. Taking into account the data presented here and previous studies reported in the literature with other ligands, we propose three different means to inhibit the myotoxicity caused by these proteins:
physical blocking forphospholipid binding at the hydrophobic channel of the toxin. There are two different ways to achieve this blocking: a)by binding to the putative "active site" region (His48 residue) (e.g., obstruction of the protein-membrane docking region (MDoS) (e.g., caffeic acid); obstruction of the protein region related to membrane destabilization (MDiS) (e.g., aristolochic acid).
AA and CA ligands provided relevant structural information about Lys49-PLA2s inhibition towards partial neutralization of the myotoxic activity of these proteins. These ligands experimentally highlighted the previously proposed mechanism of action of Lys49-PLA2s, binding with the two interaction sites of these proteins with the targetmembrane (MDoS and MDiS) [
PrTX-I/AA and PrTX-I/CA were superimposed using their Cα atoms. The region that composes the hydrophobic channel are highlighted in yellow on molecular surfaces. (C)Structural comparison of binding mode of AA, CA, and RA on the PrTX-I structure. PrTX-I/AA, PrTX-I/CA and PrTX-I/RA weresuperimposed using their Cα atoms. AA, CA, RA and PEG are highlighted in blue, green, cyan and magenta sticks, respectively. The aminoacidsthat compose MDiS (Leu 121, in orange sticks), MDoS (Lys20, Lys115 and Arg118, in yellow sticks) and hydrophobic channel (Phe3, Lys7, Gln11 and Gly15, in green olivesticks) which interact with AA, CA and RA, respectively, are also highlighted.
The results presentedhere can promote the design of more accurate structure-based inhibitors that, to cause a full inhibition of Lys49-PLA2s activity could obstruct both the hydrophobic channel and the MDoS and MDiS regions. The putative drug formed by different inhibitors could obstruct all of these regions simultaneously. Finally, it is important to note that other PLA2s-like proteins (e.g., Arg49 and Ser49-PLA2s) also have a hydrophobic channel and analogous MDoS and MDiS regions [
The coordinates were deposited in the Protein Data Bank (PDB) under the identification codes 4YZ7 (PrTX-I/AA) and 4YU7(PrTX-I/CA).
We also acknowledge the use of the Laboratório Nacional de Luz Síncrotron (LNLS, Brazil).