Insights into the Interactions of <i>Fasciola hepatica</i> Cathepsin L3 with a Substrate and Potential Novel Inhibitors through <i>In Silico</i> Approaches

<div><p>Background</p><p><i>Fasciola hepatica</i> is the causative agent of fascioliasis, a disease affecting grazing animals, causing economic losses in global agriculture and currently being an important human zoonosis. Overuse of chemotherapeutics against fascioliasis has increased the populations of drug resistant parasites. <i>F</i>. <i>hepatica</i> cathepsin L3 is a protease that plays important roles during the life cycle of fluke. Due to its particular collagenolytic activity it is considered an attractive target against the infective phase of <i>F</i>. <i>hepatica</i>.</p><p>Methodology/Principal Findings</p><p>Starting with a three dimensional model of FhCL3 we performed a structure-based design of novel inhibitors through a computational study that combined virtual screening, molecular dynamics simulations, and binding free energy (ΔG_bind) calculations. Virtual screening was carried out by docking inhibitors obtained from the MYBRIDGE-HitFinder database inside FhCL3 and human cathepsin L substrate-binding sites. On the basis of dock-scores, five compounds were predicted as selective inhibitors of FhCL3. Molecular dynamic simulations were performed and, subsequently, an end-point method was employed to predict ΔG_bind values. Two compounds with the best ΔG_bind values (-10.68 kcal/mol and -7.16 kcal/mol), comparable to that of the positive control (-10.55 kcal/mol), were identified. A similar approach was followed to structurally and energetically characterize the interface of FhCL3 in complex with a peptidic substrate. Finally, through pair-wise and per-residue free energy decomposition we identified residues that are critical for the substrate/ligand binding and for the enzyme specificity.</p><p>Conclusions/Significance</p><p>The present study is the first computer-aided drug design approach against <i>F</i>. <i>hepatica</i> cathepsins. Here we predict the principal determinants of binding of FhCL3 in complex with a natural substrate by detailed energetic characterization of protease interaction surface. We also propose novel compounds as FhCL3 inhibitors. Overall, these results will foster the future rational design of new inhibitors against FhCL3, as well as other <i>F</i>. <i>hepatica</i> cathepsins.</p></div

Time evolution of instantaneous ΔG_eff values for FhCL3 complexes.

<p>Effective binding free energies (green) are shown together with accumulated mean values (black). Dashed lines indicate the MD equilibration time of FhCL3 complexes. Every complex was labeled with the corresponding compound identifier.</p

Selected docking poses for the best five hits.

<p>FhCL3 in complex with HTS12701 (A), BTB03219 (B), SPB07884 (C), HTS11101 (D) and RH01594 (E). FhCL3 interacting residues as well as ligand atoms involved in possible hydrogen (blue dashed lines) and halogen (purple dashed lines) bonds are labeled. Protein surface is colored according to its polar (green) and non-polar (magenta) properties.</p

Pair-wise energy decomposition values for FhCL3-sustrate complex.^a

<p>^a All energies are in kcal/mol</p><p>Pair-wise energy decomposition values for FhCL3-sustrate complex.<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003759#t001fn001" target="_blank">^a</a></p

Binding free energies and its components for the studied systems.^a

<p>^a All energies are in kcal/mol</p><p>^b Standard Errors of mean</p><p>^c Effective free energy Δ<i>G</i>_<i>eff</i> = Δ<i>E</i>_<i>MM</i>+Δ<i>G</i>_<i>sol</i></p><p>^d Binding free energy Δ<i>G</i>_<i>bind</i> = Δ<i>G</i>_<i>eff</i>-TΔ<i>S</i></p><p>^e Inhibition constant obtained from Δ<i>G</i>_<i>bind</i> = -<i>RT</i>ln(<i>K</i>_<i>i</i>), where the temperature T is determined as 300K and values are given in mol/L.</p><p>Binding free energies and its components for the studied systems.<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003759#t003fn001" target="_blank">^a</a></p

Stability assessment and per-residue free energy decomposition for the FhCL3-peptide complex.

<p>(A) RMSD values relative to the initial structure calculated for the backbone atoms of the peptide (blue) and the FhCL3-substrate complex (red)during the simulation time. (B) Effective binding free energy (green) and accumulated mean (black) values versus simulation time. (C) Per-residue free energy values for key residues of the FhCL3-peptide complex. Bars are split into backbone, side chain, polar and non-polar contributions. Residue names are colored according to the enzyme’s subsite location, i.e. S1 in pink, S2 in blue and S3 in red. (D) Structural representation of FhCL3 hot-spots according to per-residue energy contribution values onto the average structure of the complex.</p

Nitrile re-docking into the substrate-binding site of HuCatL.

<p>(A) Nitrile chemical structure. (B) Superposition of nitrile best-re-docked pose (yellow) and crystal structure (blue) (RMSD = 3.4 Å). (C) Hydrogen bonds between the residues of HuCatL binding site and nitrile in both the crystal (blue) and the best re-docked (yellow) complex structures. Hydrogen bonds are shown as blue dotted lines, and HuCatL interacting residues (green) are depicted as sticks. Note that nitrile re-docking only takes into account its non-covalent interactions with the enzyme, therefore, it was treated as a non-covalent ligand.</p

Diagrams of protein-ligand interactions.

<p>FhCL3 residues interacting with Nitrile (A), HTS12701 (B), BTB03219 (C). Ligands (violet) and protein residues involved in polar interactions (brown) are depicted in ball and stick representation. Hydrogen (blue dashed lines) and halogen (purple dashed lines) bonds are shown together with their occupancy percent and distance values, respectively. Hydrogen donor and acceptor labels are shown in italic and bold styles, respectively. Residues establishing non-polar contacts are depicted as red semicircles.</p

Per-residue free energy decomposition for FhCL3 complexes.

<p>Bar graphs show the side chain, backbone, polar and non-polar contributions for each residue. Residue names are colored according to their location within S1 (pink), S2 (blue) and S3 (red) subsites. A structural representation of each complex interface is depicted as well. Interacting residues are colored according to energy value as shown in color scale. Hot/warm-spots are labeled in each case.</p