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Cite this article: Patil N, Ranjan A, Chauhan A, Jindal T (2018) Mechanistic of Organophosphate Mediated Inhibition of Human Acetylcholinesterase by Mo-lecular Docking. J Bioinform, Genomics, Proteomics 3(2): 1032.

*Corresponding authorAnuj Ranjan, Amity Institute of Environmental Toxicology, Safety and Management, Amity University, Noida, Sector-125, Uttar Pradesh, India, Tel: 91-9990907571; Email :

Submitted: 28 March 2018

Accepted: 28 June 2018

Published: 30 June 2018

ISSN: 2576-1102

Copyright© 2018 Ranjan et al.

OPEN ACCESS

Short Communication

Mechanistic of Organophosphate Mediated Inhibition of Human Acetylcholinesterase by Molecular DockingNeha Patil1, Anuj Ranjan2*, Abhishek Chauhan2 and Tanu Jindal2

1Amity Institute of Environmental Sciences, Amity University, India 2Amity Institute of Environmental Toxicology, Safety and Management, Amity University, India

Abstract

Organophosphates (OP) are broad-spectrum pesticide (also referred as nerve agents), it inhibits the acetylcholinesterase (AChE) enzyme in the synaptic gap of the nervous system leading the accumulation of Acetylcholine and neurotoxicity. Mechanistic insight of AChE inhibition has been broadlyon model organisms. Crystal structure of OP with human AChE is rarely reported in protein data bank therefore we are forced to reply upon the information generated using model organism. In this research, we have studied inhibition of OP with human AChE using Argus lab as molecular docking tools. We screened 80OPwith human AChE using Argus lab tool. Best dock score (over all binding energy) was exhibited by Phoxim (-10.7562 kcal/mol), followed by Azinphos Ethyl (-10.4978 kcal/mol) and Fonofos (-10.4752 kcal/mol). Analyzing all OP-human AChE complex individually highlights the basic insight of the interaction of OP-human AChE. OP interacts with human AChE by H-bonding (largely contributed by Ser203 and Tyr124) and Π interactions (contributed by Trp86, Phe295, Tyr337 and Tyr341). Amino acids like Trp86, Phe295 and Tyr337 are helpful in stabilizing the OP-human AChE interaction through Π-interactions. Validation studies done by Glide docking reveals Trp86 involvement in maximum Pi-Cation interaction on anionic subsite of human AChE other than Ser203 (Catalytic site). With extra precision glide docking Phoxim Ethyl Phosphonate (PEP) tops among 80OPbased on glide docking score and interacted with Trp86, Gly121 and Ser203 whereas MM-GBSA score shows less binding affinity than heptenophos and dichlorovos. Trp86 has been found closest to the OP where as Tyr337 and Tyr341 are farthest. Phe295 mostly been noticed in intramolecular Π-Π interaction which is insignificant in terms of OP-AChE interaction. Study infers the importance of Tyr124, Ser203, and Trp86 which helps to hold the OP in binding cavity; however Tyr337 and Tyr341 are at the farthest.

INTRODUCTIONOrganophosphate (OP) are synthetic pesticide [1], generally

esters and mostly liquid at different vapor pressure. Among most of the insecticides, OP is found to be highly toxic to human as well as for the wildlife. It can be absorbed by all modes – dermal, inhalation, ingestion, eyes. Every year hundreds of thousands of individuals suffer OP poisoning from dietary, household, accidental or occupational exposure [2].

Acetylcholine is the neurotransmitterwhich transfers signal from nerve to muscle cell. To pass the new signal, the previous signal should be demolished, so that the new and old signal would not get mix together and start accumulating in the cell, which causes difficulty in transmission. AChE hydrolyzes neurotransmitter acetylcholine (into two components-choline and acetic acid) at the synaptic gap after the completion of one signal, so that next message can be transmitted without any hindrance [3].

OP inhibits the AChE enzyme in the synaptic gap of the nervous system which results the accumulation of acetylcholine.

It causes continuous trigger of signaling and neurotoxicity which ultimately leads to muscle paralysis, seizure and death if effective and appropriate prophylaxis is not done.

AChE enzyme is present in all type of conducting tissue from nerve cell to sensory fibers. It has three distinct residues on its binding site, the triad of Ser203, Glu334, and His447, which act as charge relay system. Binding site is composed of two different pockets connected by a narrow gorge, which is lined with 14 conserved aromatic amino acids enzyme activity [4].

AChE is very catalytic in nature and being coded by AChE gene on chromosome 7 at 7q22 [5]. The polypeptide has 614 amino acid length of sequence which bears signal peptide of first thirty-one amino acids. OP exerts its toxicological effects through non-reversible phosphorylation of esterases in the central nervous system. The acute toxic effects are related to irreversible inactivation of AChE. OP is substrate analogues to acetylcholine, and like natural substrate enter the active site covalently binding to serine –OH group. During acetylation, OP splits and phosphate radicals of OP bind covalently to the active sites of the cholinesterase, converting them into enzymatically

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inactive proteins [6]. Dephosphorylation of AChE is very slow (on the order of days), and phosphorylated enzyme cannot hydrolyze the neurotransmitter [7].

Persistency of OP is less than that of organochlorine. They have shorter half-lives and reported to undergo degradation in sunlight and UV light [8,9]. However natural products are nowadays being used as substituents of OP to combat persistency of synthetic and chemical pesticides [10-13].

Ser203 is one of the key residue in phosphorylation/ inactivation of AChE whereas Trp86 helps guard the inhibitor on the bottle neck on the binding gorge [12-14]. Trp86 is one of the key residue which is responsible for the activity of the enzyme. A mutation/change of this residue makes AChE an enzymatically inert protein [15,16].

METHODMaterials and tools

Model of human AChE structure was retrieved from Protein Data Bank (PDB ID- 5FPQ), model of OP moleculeswas retrieved from PubChem library of NCBI. SWISS-PDB Viewer [17] was used for energy minimization.Structure Analysis and Verification Server, University of California Log Angels (SAVES, UCLA) was used to check and validate protein structure, Argus lab (Planaria Software LLC) [18], and Glide module of Schrodinger were used to run docking program which allows flexible as well as rigid docking.Discovery studio visualizer 4.0 (acelrys.com/collaborative-science/biovia-discovery-studio/visualization) was used for editing the molecules and to analyze ligand-human AChE complexes.

Preparation of target structure (human AChE)

Human AChE model was retrieve from Protein Data Bank. The structure was co-crystalized withtwo other ligands in the retrieved structure. To use the model for our studycrystal associated ligands were removed and hydrogen was added. Swiss PDB viewer was used to optimizing the model by energy minimization. Seven rounds of the minimization were carried out and quality of the structure was checked after every round of the minimization on SAVES server [19]. Model with best structural attributes were used for the further study.

Preparation of ligands structure

OP selected for this study was chosen on the basis of the ecological effects, environmental fate and effect on non-target organism. After the selection of OP, the molecules were converted into pdb format to make them compatible for using in Arguslab. The geometry of the ligands was optimized by providing UFF force field. A ligand group was made from the selected ligand.

Defining binding site and docking

Experimental facts about the active site of human AChE points out the essential residues such as Ser203, Glu334 and His447. Human AChE model was imported in to Argus lab. The active site was defined using 29 residues. These residues include: Gln71-Tyr-Val-Asp-Thr-Leu76, Gly82-Thr83-Glu84, Trp86- Asn87-Pro88, Leu130, Tyr133, Glu202-Ser203-Ala204, Trp286, Phe295, Phe297, Glu334, Tyr337-Phe338, Tyr341, Trp439, His447-Gly448-Tyr449, and Ile451.

Arguslab provides a platform to dock two molecules. Argus Dock engine was used for docking, calculation type was opted as dock and flexible ligand docking was allowed. At 0.4 Å of grid resolution and 150 poses of maximum numbers with high precision flexible ligand docking OP were simulated to bind with human AChE on predefined binding site. Coordinates of the binding site was X=23.575, Y=26.837, Z=23.957 Å. Aqueous medium was not defined.

Validation studies

The preliminary screening of OP-human AChE was done using Argus lab, however, the study was corelated with Glide module of Schrodinger suite.

Preparation of protein target structure

The human AChE (PDB:5FPQ), target protein was obtained from the Protein Data Bank in pdb format. Structure was recovered from the model by removing crystal associated hetero atoms, ligands and water molecules. Structure was imported to Schrodinger suit, Maestro v9.5. Protein Preparation Wizard [20] tool was used to optimize the structure. It comprised biological unit and assigned bond orders, zero order bonds to metals, formed disulfide bonds, deleted water molecules beyond 5 Å from hetero groups, generated metal binding states, added missing hydrogens, completed any missing side chains and loops and Protein Preparation Wizard. Protein Preparation Wizard has a refine function, which helped in optimization of H-bond network to fix the overlapping hydrogens. pH range was established to 7.0 and the structure was minimized by applying OPLS 2005 force field [21]. Restrained minimization was used until the average root mean square deviation (RMSD) of the non-hydrogen atoms converged to 0.3Å.

Receptor grid preparation

Binding site was defined as per the method discussed in section 2.3. Grid generation was performed using OPLS_2005.

Evaluation of docking method

The virtual screening protocol was validated by enrichment factor and by Receiver operating curve (ROC) analysis. Enrichment factor expresses the number of active compounds found by employing a certain virtual screening strategy. It is a widely used validation tool for assessing the quality of virtual screening protocol. Conceptually the enrichment factor metric is purely the measure of how many more actives we find within a defined “early recognition” fraction of the ordered list relative to a random distribution.

ROC curve analysis is considered as one of the best approaches for the performance characterization of virtual screening protocols so far. The ROC is represented equivalently by plotting the fraction of true positives (TPR: true positive rate) versus the fraction of false positives (FPR: false positive rate).

3D structure of OP compounds was obtained from PubChem database. 80 unique active compounds were used for the enrichment analysis. These compounds were converted into 3D structures and generated their protonation states (biological pH), canonical tautomer and corrected their geometric configuration using LigPrep tool.

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The OP and decoy sets from the Directory of Useful Decoys, Enhanced (DUD-E) are used for enrichment analysis. The decoys also prepared for their proper ionization states (pH of 7.2), tautomeric forms, using LigPrep default settings. 80 OP and 3892 decoys were mixed together and exported as structure data files to glide module of Schrodinger suite. The docking method was evaluated by the receiver operating characteristic (ROC), enrichment factors (EF) (Figure 1,5).

Sensitivity (S) is defined as true positive rate (TPR), specificity (SP) is true negative rate (TNR) and accuracy is the overall correct prediction rate [(TP + TN)/N]. The ROC is [S/(1- SP)], i.e. TPR over FPR. EF is [TP (subset)/n(subset)]/[P(total)/n(total)], i.e. the ratio of true positives detected in the subset divided by the fraction of overall (total) positives. The OP inhibitors were clustered by maximum common substructure using Clustering script of Schrodinger. Correlation coefficients were computed for aggregate docking scores versus median activity for all clusters.

Preparation of OP ligands

Commonly used OP compounds were obtained from Pub chem library (https://pubchem.ncbi.nlm.nih.gov/). Over 80 different variant structures were used for screening. Prior to screening, preparation of OP structures was done using LigPrep module of Schrodinger suite. Ligands were imported into Schrodinger workspace and each structure was neutralized, checked for any metal binding states, desalted, generated tautomers and 32 stereoisomers per ligand was allowed. Keeping in view of the flexibility of the rings present in each ligand and their possibility to change conformations during docking calculations, we have specified to generate 1 low energy ring conformation per ligand. Finally, each ligand was energetically minimized using OPLS 2005 force field.

Virtual screening and docking

The virtual screening was performed using Virtual Screening Workflow (VSW) module of the Schrödinger Suite 2014. This workflow includes LigPrep for ligand preparation, QikProp (QikProp, version 4.0, Schrödinger 2014) to filter out ligands based on properties, and Glide docking [22] at the three precision levels, High Throughput Virtual Screening (HTVS), Standard Precision (SP), and Extra Precision (XP). HTVS and SP modes were used for a large set of ligands and XP docking is more accurate than the above two methods. After ensuring that the protein and ligands were in the correct form for docking, the previously defined binding site by receptor-grid was used which includes Ser203, Glu334, and His447 forming a catalytic triad. Glide generates conformations internally and passes these through a series of filters. This first places the ligand center at various grid positions of a 1 Å grid and rotates it around the three Euler angles. In the first stage, basic score and geometrical filters eliminate unlikely binding approaches.

Second filter involves a grid-based force field evaluation and refinement of docking solutions including torsional and rigid body movements of the ligand. The final energy estimation is done with Glide Score, and a single best pose is generated as the output for a particular ligand. Glide module of the XP visualizer analyses the specific interactions. Glide includes ligand-protein

interaction energies, hydrophobic interactions, hydrogen bonds, internal energy, π-π stacking interactions and root mean square deviation (RMSD) and desolvation.

G Score = a* vdW + b* Coul + Lipo + Hbond + Metal + RotB + Site

Where, vdW => van der Waal energy;

Coul => Coulomb energy;

Lipo => lipophilic contact term;

HBond => hydrogen-bonding term;

Metal => metal-binding term;

RotB => penalty for freezing rotatable bonds;

Site => polar interactions at the active site; and the coefficients of vdW and Coul are: a =0.065, b = 0.130.

All the Glide docking runs were performed on i3 Processor CPU @ 2.60 GHz, with 4 GB DDR RAM. Glide was compiled and run under Linux CentOS 6.5 operating system. The output from Glide calculations were exported to .pdb format and studied for their detailed interactions at atomic level and all the images were rendered using Schrodinger’s maestro interface v9.6 and Accelry’s® Discovery Studio Visualizer v 4.0 [23].

RESULT AND DISCUSSIONThe structure of human AChE was optimized after removal

of co-crystal ligands. Seven round of energy minimization were performed in Swiss-PDB viewer until energy of the system was stabilized. After each round of minimization structure assessment was carried out. Best optimized structure of human AChE model was obtained at-29723.834 Kcal/mol with quality of crystal structure by 96.4 % residues found in favored region, 3.0 % of the residues in allowed region and 0.6 % of residues were outliers on Ramachandran plot. This structure was used for the docking process. Binding of OP with human AChE was simulated individually using Argus lab.

Top three rank OP are Phoxim (-10.7562 kcal/ mol), followed by Azinphos Ethyl (-10.4978 kcal/mol) and Fonofos (-10.4752 kcal/mol). Dock score of each OP is listed below in Table 1. Phoxim was interacting with AChE by making two H-bonds and a π-cation. No π-sigma bond was formed. Tyr124 formed hydrogen bond by interacting with O5, N6 at 1.35 and 1.57Å. Tyr341 made π-cation bond at 6.88Å with P2 which is very farthest (Figure 1).

Azinphos Ethyl was observed interacting with AChE by making three types of bonds- hydrogen bond, π-cation, and π-sigma. Ser203 formed H-bond with S2 at 2.35 Å. Π- cation bond was formed by Tyr337 with P3 at 6.33 Å and Trp86 made two bonds with P3 at 4.95 and 4.40 Å. Trp86 made π-sigma bond with H33 at 2.56Å. In this case Ty337 is farthest where as Trp86 is closest aromatic residue (Figure 2). Fonofos interacted with AChE by making two types of bond- hydrogen and π-cation. No π-sigma bond was made. Gly121 with O4 made hydrogen bond at 2.41 Å. Trp86 formed two Π-cation bonds with P3 at 5.61 and 5.77 Å. Pattern of OP interaction with human AChE for the rest of the OP are depicted in 2D and 3D (Figure 3).

Molecular interaction between a protein and a ligand always

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requires a co-crystal structure. In case of OP and human AChE the co-crystal structure is very rarely reported in protein data bank. The purpose of this study was to explore binding mechanistic of different OP with humanAChE.

Validation studies

Virtual screening and docking: Screening of over 80 OP using Glide module in three different levels of docking and scoring processes were used for this study starting with HTVS, followed by Standard Precision (SP) and with Extra Precision (XP). SP based on Glide score criteria value allowed to pass only 13 molecules. The final docking with XP qualified only one molecule that is Phoxim Ethyl Phosphate.

Top three OP based of glide docking score ranks are Phosxim Ethyl Phosphonate (-6.247 Kcal/mol), Methamediphos (-5.871 Kcal/mol) and Heptenophos (-5.664 Kcal/mol). Phoxim Ethyl Phosphate tops in ranking in Extra Precision Glide docking (-6.247 kcal/mol) however MM-GBSA binding score shows its lesser affinity (-39.923 Kcal/mol) in docked pose towards human AChE than that of Heptenophos (-53.017 Kcal/mol) and

Dichlorovos (-41.297 Kcal/mol). Glide Docking score and MM-GBSA Binding score in recorded in Table 2.

Docked files were observed in 3D space to study the interaction pattern. The objective during the observation was to see the involvement of Trp-86 of human AChE while binding with OP. At standard precision Trp86 had shown involvement while binding with Pyridaphenthion, leptophos, Phoxim and Phoxim Ethyl Phosphonate by Π interactions and hydrophobic interaction of Trp86 with Trichlorofon, Vamidothion, Dichlorovos and Dimefox.

At Extra precision docking, Phoxim Ethyl Phosphate qualified among OP and binds with human AChE in which Trp-86 contributes the Π-Π interaction to benzene group of the ligand (Figure 6), Trp86 is also involved in binding with Leptophos and Phoxim (Figure 7). Role of Trp86 was majorly found in stabilizing with ring containing OP by Π interaction and it was also conspicuous that smaller OP which is not bearing any ring structure with them, Trp86 interacted with them by hydrophobic interactions. Figure 8 shows the interaction of Dichlorovos, Dimefox and Diemethoxon with human AChE.

In case of Pyridafenthion (Figure 9) with human AChE, A Π-Π bonding is shared by Tyr-124 to benzene moiety of this ligand and Ser-203 to oxygen of diethoxyphosphinothioyloxy group. In this binding, Trp86 is partially incorporated through hydrophobic interaction.

By analyzing all OP- AChE interaction, results conclude that OP binds with human AChE by H-bonding (contributed by Ser203 and Tyr124) and stabilized by Π interactions (contributed by Trp86, Phe295, Tyr337 and Tyr341). Since binding cavity of human AChE is lined with 14 conserved aromatic amino acids, so amino acids like Trp86, Phe295 and Tyr337 are helpful in stabilizing the OP-human AChE complex through Π interactions. Trp86 has been found closer to the OP where as Tyr337 and Tyr341 are farthest. Phe295 was also noticed in making intramolecular Π-Π interaction which is insignificant in terms of OP-AChE interaction. Phosphate group of OP is having two or more oxygen atoms attached with central phosphorus. Electronegative nature of oxygen atoms generates partial positive charges on central phosphorus atom, which is helpful in making π-cation bonds with electron rich aromatic amino acids there by stabilizing the complex along with H-bonds supported by Ser203 and other accessory amino acids.

Study infers that these amino acids are actively stabilizing Π interactions. Anionic surface of aromatic ring of these amino acids help in contributing Π interaction. A majority of the OP interacting with human AChE have Π-cation interaction led by Trp86. The face of rings of aromatic rings has partial negative charge owing to the Π electrons. Positive charge deficit of central atom of OP and partial negative charge on rings of aromatic amino acids are possibly helpful in creating electrostatic potential by stabilizing Π -cation interaction.Role of Trp86 in all the interaction was very prominent (Figure 4).

The study was also supported by Saxena et al., 2003 who studied MoAChE using site-specific mutants of the implicated residue for the differences in the reactivity of E2020 toward AChE. Studies suggested that residues at the peripheral anionic site

Table 1: Dock scores (in Kcal/mol), represent the lowest binding energy of the organophosphate ligands with human AChE enzyme model (PDB ID-5FPQ). Dock score of a ligand is determined by selecting best binding pose with minimum energy.ORGANOPHOSPHATES DOCK SCORE (in kcal/mol)

Phoxim -10.7562

Azinphos ethyl -10.4978

Fonofos -10.4752

Phoxim ethyl phosphonate -9.9770

Chorpyrifos -8.9425

Phorate -8.8077

Ethion -8.6957

Famphur -8.2152

Dichorvos -7.9852

Acephate -7.2348

Table 2: OP binding with human AChE at standard precision docking in Glide, Top three docking score and MM-GBSA has been mentioned in bold.Organophos-phates

MMGBSA-dG Binding (Kcal/mol)

Docking score (Kcal/mol)

Phoxim ethyl phos-phonate -39.923 -6.247

Methamidophos -33.207 -5.871heptenophos -53.017 -5.664Fospirate -34.948 -5.087Dichlorovos -41.297 -4.474Pyridaphenthion -33.207 -4.394Dimefox -38.427 -4.143Cyanophos -32.454 -3.969baythion -39.288 -3.637Coroxon -34.732 -3.348Vamidathion -38.248 -3.108Trichlorofon -33.207 -2.301

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Figure 1 AChE and Phoxim (left) 2D Structure and (right) 3D Structure: No π-sigma bond was formed. Tyr124 formed hydrogen bond by interacting with O5, N6 at 1.35 and 1.57 Å. Tyr341 made π-cation bond at 6.88 Å with P2. a) AChE –Phoxim .

Figure 2 AChE-Azinphos Ethyl (left) 2D and (right) 3D structure: Ser 203 formed H-bond with S2 at 2.35Å. Π- Cation bond was formed by Tyr337 with P3 at 6.33 Å and Trp86 made two bonds with P3 at 4.95 and 4.40 Å. Trp86 made π-sigma bond with H33 at 2.56 Å.

Figure 3 AChE- Fonofos (left) 2D and (right) 3D structure: No π-sigma bond seen. Gly121 with O4 made h-bond at 2.41Å. Trp86 made two π-cation bonds with P3 at 5.61 and 5.77Å.

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Figure 4 Pareto Chart for the H-bonds and all π-interactions. Bonding frequency of contributing amino acids are plotted in descending orders.

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Figure 5 ROC area under curve illustrates the enrichment of OP and decoy set against human AChE (1B41).

Figure 6 Phoxim Ethyl Phosphate interacting with human AChE.

Figure 7 Leptophos interaction with human AChE (left) Benzene moiety of Leptophos is being interacted with two aromatic amino acids Trp86 and Tyr337. Phoxim interacting with human AChE(right) Benzene moiety is interacting with Trp86.

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Figure 8 Binding of (a) Dichlorovos, (b) Dimefox and (c) Dimethoxon with human AChE.

Figure 9 Pyridaphenthion interacting with human AChE.

such as Asp74 (72), Tyr72 (70), Tyr124 (121), and Trp286 (279) in mammalian AChE may be important in the binding of E2020 to AChE. Molecular modeling studies suggested that E2020 interacts with the active-site and the peripheral anionic site in AChE, as the gorge is larger, E2020 cannot simultaneously interact at both sites. Ashani et al 1995 studied active site of AChE by substituting the key amino acid residues at the entrance to the active-site gorge (Trp-286, Tyr-124, Tyr-72, and Asp-74) influence the reactivation kinetics of the bisquaternary reactivator HI-6 compared with the monoquaternary reactivator P2S. Replacement of Phe-295 by Leu enhanced reactivation by HI-6 but not by P2S. Of residues forming the choline-binding subsite, the E202Q mutation had a dominant influence where reactivation by both oximes was decreased 16- to 33-fold. Residues Trp-86 and Tyr-337 in this subsite showed little involvement in reactivation. These kinetic findings, together with energy minimization of the oxime complex with the phosphonylated enzyme, provide a model for differences in the reactivation potencies of P2S and HI-6. The two kinetic components of oxime reactivation of MEPQ-inhibited AChE

arise from the chirality of O-ethyl methylphosphonyl moieties conjugated with Ser-203 and may be attributable to the relative stability of the phosphonyl oxygen of the two enantiomers in the oxyanion hole.

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Patil N, Ranjan A, Chauhan A, Jindal T (2018) Mechanistic of Organophosphate Mediated Inhibition of Human Acetylcholinesterase by Molecular Docking. J Bio-inform, Genomics, Proteomics 3(2): 1032.

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