Emetine and Indirubin- 3- monoxime interaction with human brain acetylcholinesterase: A computational and statistical analysis

Cellular and Molecular Biology, 2021, 67(4): 106-114 Emetine and Indirubin3monoxime interaction with human brain acetylcholinesterase: A computational and statistical analysis Syed Sayeed Ahmad, Haroon Khan*, Mohammad Khalid , Abdulraheem SA Almalki Department of Bioengineering, Faculty of Engineering, Integral University, Lucknow-226026, India; (S.S) Department of Pharmacy, Abdul Wali Khan University, Mardan-23200, Pakistan; (H.K.) College of Pharmacy, Department of Pharmacognosy, Prince Sattam Bin Abdul Aziz University, Alkharj 16278, Riyadh, Saudi Arabia. Department of Chemistry, Faculty of Science, Taif University, Taif 21974, Saudi Arabia


Introduction
Neurological disorders (ND) are one of the first reasons for death, especially in a developed nation and Alzheimer's disease (AD) is at the top of the list (1). AD is a dynamic and irreversible ND and the most widely recognized foundation of dementia in the older population > 60 years and the seventh leading cause of death throughout the world (2,3). In the US alone, an increasing number of individuals are being diagnosed with AD. Statistics released data by the 2018 AD, details, and statistics report indicates that AD financial records for approximately 60-70% of all types of dementia cases and 5.5 million individuals of varying ages in the US have been evaluated to have AD. Additionally, this report indicates that around 47.5 million individuals worldwide are living with dementia actuated by this disease and it is evaluated that by 2050, over 115 million individuals will have dementia (4,5).
In AD brains, the cholinergic network is the most drastically affected, demonstrating restoration of acetylcholine (ACh) and different markers of cholinergic activity (6). In light of this perception, the medications galantamine (7), donepezil (8), and rivastigmine (9) have been affirmed by the US Food and Drug Administration (FDA) and presently promoted for the symptomatic treatment of AD (10). Cholinesterase is a family of enzymes that catalyze the neurotransmitter ACh through hydrolysis into choline and acetic acid. In AD, acetylcholinesterase (AChE) inhibitors act by preventing ACh breakdown, which is the basis of their use in treating this disease. The mechanism of action of most FDA-approved drugs for AD, which is based on the cholinergic hypothesis, involves the enhancement of ACh levels in the diseased brain. Therefore, inhibition of AChE plays a significant role in enhancing cholinergic transmission in the diseased brain (11,12). AChE is found in high concentrations principally in the red blood cells in addition to the brain at neuromuscular junctions and cholinergic synapses (13). Currently, cholinesterase inhibitors have been proven to be the most effective treatment for AD owing to their ability to affect cognition and function in AD (14). Experimental evidence indicates that general cholinesterase inhibitors targeting AChE have potential remedial advantages in the treatment of AD and other related dementia. Cholinesterase inhibitors improve cholinergic action by restraining AChE that hydrolyze ACh following synaptic discharge and, hence, prolongs the activity of ACh (15,16).
Emetine is an alkaloid obtained from ipecac species that act as a blocking agent in the early S phase of DNA replication (17). It is a ribosomal and mitochondrial protein combination inhibitor, which hinders the synthesis of DNA and RNA. Emetine ties to the 40S ribosomal subunit to hinder the protein blend (18). Structure-movement relationship (SAR) contemplates have demonstrated that the N-2′position of emetine is pivotal to its hindrance to protein synthesis and must be an optional amine (19,20). It was reported to be one of the most potent inducers of PPARG coactivator 1 (PGC)-1α expression. Increasing PGC-1α movement has been proposed to help control muscular dystrophy, diabetes, and ND (21,22). It was additionally appeared to be an antagonist of dopamine (D1 and D3) receptors, substance P, and neurokinin NK3 (23).
In this study, we elucidated the molecular enzymatic inhibition of human brain AChE by emetine and I3M using a state-of-the-art molecular docking approach. Based on the better free energy of binding emetine was well elaborated in this study and further analyzed by statistical analysis using an analysis of variance (ANOVA) of all conformers used with validation of the findings.

Materials and methods Preparation of Receptor
The recombinant structure of human AChE was taken from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB, ID: 3LII) (http://www.rcsb.org/pdb/home/home.do). RCSB is a solitary, worldwide archive for data about the 3D structures of macromolecules, as determined by X-ray crystallography, NMR spectroscopy, and cryoelectron microscopy (24). The PDB record was cleaned and the heteroatoms of the receptor were removed since these are non-standard stores of the protein.

Preparation of Ligand and Lipinski's Rule of Five
The canonical Simplified Molecular Input Line Entry Specification (SMILES) details of the emetine and I3M were obtained from the PubChem database (http://www.ncbi.nlm.nih.gov/pccompound). The 3D structure was built using the online exhibition of CORINA (http://www.molecularnetworks.com/products/corina). To check the properties of selected ligands like hydrogen donor/ acceptors, LogP, and molecular weight, Lipinski's rule of five was used (24).

ADMET Prediction
Pre-ADMET software was used for the investigation of solubility, plasma protein binding ability, intestinal absorption, blood-brain-barrier (BBB) penetration. Leads can be recognized by utilizing high-throughput screening approaches. To diminish the cost and clinical inefficacy of new medications, the compound dataset is adequately screened in the early stages for ADMET, since the available technologies for their assessment are advancing and becoming more sophisticated and reliable than previous methods are. These criteria all have an expected effect on the pharmacological and pharmacokinetic properties of drugs (25 , 26).

Docking Simulations and Interaction Study
Polar H-atoms, Kollman joined particle, and atom compose parameters were included in the analysis, furthermore, non-polar H-atoms were merged in the protein pdbqt. In the ligand pdbqt record, polar Hatoms were included, non-polar H-atoms were joined, and the number of torsions and rotatable bonds was portrayed. A cubic volume of 60 × 60 × 60 Å 3 with 0.408 Å grid point spacing and center axes coordinates as X: 90.81, Y: 83.98, and Z: -8.04 were set to cover the whole active site and enable ligands to move uninhibitedly 27 . The Lamarckian genetic algorithm was used for the receptor and ligand-flexible docking calculations. The conformer with the lowest ΔG was selected for further examination (28,29).

Statistical Analysis
The SPSS version-20 was utilized to establish the relationship among the analyzed data. Results and discussion Identification of phenolic compounds through HPLC-UV technique

Results and discussion
The 3D structure of AChE empowers with two hemispheres that sandwich the synergist focus between the acyl-and omega-circles. These circles are considered as sidewalls of the active site gorge that is around 300 Å 3 . The selected ligand was docked at the catalytic site where the quaternary ammonium of the choline of ACh interacted (30). It has been reported that cholinergic neurotransmitter levels are significantly reduced under conditions such as AD (31,32), AChE is typically targeted in terms of enzyme inhibition since this strategy temporarily increases ACh levels. The thought that cholinergic shortfalls are centrally connected with the pathogenesis of neurodegenerative conditions, for example, AD particularly centers around the loss of cholinergic neurons and the following decrease of synapse levels (33).
In our study, data using PreADMET showed that human intestinal absorption of emetine and I3M was 96.595% and 88.66% respectively, which suggests that it is well absorbed through the intestine. Further, we found that emetine and I3M were moderately permeable since the absorption value through Caco-2 cells was 56.697 and 19.99 respectively. Subsequently, the plasma protein binding and BBB penetrability for emetine were 63.098 and 0.8754, while for I3M it was 99.81 and 4.79 respectively. The docking simulation system was performed using the AutoDock 4.2 program with a natural plant-derived compound. The compound was docked into the active site of the chosen target. The lowest energy of the docked conformation of the best group was chosen for further evaluation. It merits referencing that the ligands and protein side chains were held versatile by the docking programming all through the examination.
In the present examination, the active site of human brain AChE was found to cooperate with emetine through the 10 amino acid residues: Asp74, Trp86, Tyr124, Tyr133, Ser203, Phe295, Phe297, Tyr337, Phe338, and His447 ( Figure 1). The free energy of binding (∆G) and estimated inhibition constant (Ki) for the emetine-AChE complex interaction were -9.72kcal/mol and 75.07nM, respectively. The vital role of two H-bonds was established for the precise placing of the ligand, UNK1:H63-Tyr124:OH and Tyr133:HH-UNK1:O34 in the active site of AChE, with H-bond distances of 2.03938 and 2.16778 Å, respectively. Furthermore, the OH molecule of Tyr124 and HH group of Tyr133were found to interact with H63 and O34 of the ligand, respectively. Seven carbon atoms of ligand, C3, C13, C24, C25, C26, C27, and C28 were observed to be engaged with hydrophobic collaborations with amino acid deposits of the compound in which C3, C13, C24, C25, and C28 interacted with Trp86 and while C26 and C27 were attached to Phe297 and Phe295, respectively. In particular, one oxygen atom of the ligand, O3, formed a polar bond that included one amino acid residue Asp74 of the AChE enzyme. Cation piinteractions were additionally observed in this interaction in which atoms O4, C28, and C29 interacted with Asp74 while atoms O3, C24, and C25 interacted with Asp86. Atoms such as C3 and C4 were observed to connect with the Tyr133 of the enzyme. In the Pi-Pi interaction, H29 and H30 of the selected ligand were associated with the Tyr337 and His447 residues of the enzyme. The entire interacting surface area for the generated complex was 873.79 Å 2 . Van der Waals, hydrogen bonds, and desolvation energy components collectively contributed -10.22 kcal/mol for this interaction. The electrostatic energy component was -1.59 kcal/mol in this interaction. The total internal energy and intermolecular energies were -1.25 and -11.81 kcal/mol for the complex of the emetine and AChE, respectively. The findings of this interacting complex are summarized in Table1. I3M was found to interact with AChE through 20 amino acid-like, Gln71, Tyr72, Asp74, Trp86, Asn87, Gly120, Gly121, Gly122, Tyr124, Ser125, Gly126, Tyr133, Glu202, Ser203, Phe297, Tyr337, Phe338, His447, Gly448, and Ile451. ∆G and Ki for the I3M-AChE complex were found to be -7.09kcal/mol and 6.49 µM, respectively. Two H-bonds were established in this interaction, TRP86:NE1 -:UNK1:O10 and TYR337:OH -:UNK1:O10 in the active site of AChE, with H-bond distances of 3.1877and 2.98454, respectively. The donor atom NE1 of Trp86 was found to interact with the O10 of I3M, while OH of Tyr337 was found to interact with O10 of I3M.
Van der Waals, hydrogen bonds, and desolvation energy components collectively contributed -7.56 kcal/mol for this interaction. The electrostatic energy component was -0.08 kcal/mol in this interaction. The intermolecular energy was -7.64 kcal/mol for the complex of the I3M and enzyme, respectively.

Thermodynamics Analysis
For a better understanding of the sub-atomic recognition between a protein and its ligand, it is important to comprehend the physicochemical components underlying the protein-ligand interaction. In this section, the fundamental thermodynamics, interaction for ligand-protein binding, binding driving forces, and enthalpy-entropy compensation (34) are presented. The possibility of a protein-ligand association is constrained by the negative ΔG, which can be considered to decide the dependability of any protein-ligand complex or the coupling affinity of a ligand to a particular acceptor. It should be noticed that the free energy is a component of the condition of a framework and ΔG is portrayed by the underlying and last thermodynamic states (35,36). The standard ΔG, which alludes to the free energy change assessed under 1 atm. pressure, at a temperature of 298 K, and the incredible reactant (protein and ligand) with 1 M, is identified with the binding consistent Kb by the Gibbs relationship as shown in the following equation: Where R is the gas constant (1.987 cal·K -1 ·mol -1 ) and temperature (T) in Kelvin. The ΔG at any time during the involvement is calculated as: Where, Q is the reaction quotient, which is characterized as the extent of the grouping of the protein-ligand complex to the result of the centralizations of the free protein and free ligand at any minute in time. ΔG can be characterized as its enthalpy and entropic through the resulting condition: ΔG = ΔH -TΔS [3] Where ΔH and ΔS are changing in enthalpy and entropy of the framework following ligand binding, respectively and T is the temperature in Kelvin. Enthalpy is the proportion of the aggregate energy of a thermodynamic system and it is negative for exothermic conditions (37,38). The value of the partition function (Q) for the complexes emetine and I3M with AChE was found to be the same as 10.08. The values of the free and internal energy for the target with emetine complex were -1369.20 and -5.00 kcal/mol, while for I3M with the target was -1371.31 and -7.07 kcal/mol respectively. The predicted value of entropy for both ligands with target has been found 4.58 kcal·mol -1 ·K -1 . However, if enthalpy (-∆H) and entropy (∆S) are considered for the complex, the major contribution to free energy (-∆G) comes from entropy (∆S or -T∆S). The increase in entropy (i.e., positive value) indicated that the interaction was entropy-driven and the complex consisted of dominant hydrophobic interactions (39). All the above data were acquired at a temperature of 298.15 K.

Statistical Analysis
In this study, R 2 = 1, indicating that 100% of the variation in the intermolecular energy was due to van der Waals forces and electrostatic energy as shown in Table 2. The functional relationship between the dependent and independent variables was identified as the relationship shown in Table 3.
An ANOVA was performed to determine whether the regression line was significant and the constructed relationship model was significant as shown in Table  4. The graphical representation of intermolecular energy and binding energy is shown in Figure 2. The calculated R 2 = 0.999, which indicated a linear relationship. The binding energy and intermolecular energy were plotted on the X and Y axes, respectively. The relationship between van der Waals forces and intermolecular energy was similarly linear with an R 2 = 0.994, shown in Figure 3. The value of intermolecular energy and Van der Waals force was taken on X and Y-axis respectively. The final relationship between all the related energy Van der Waals, intermolecular energy, and electrostatic energy was shown in Figure 4.

Conclusions
This study examines the effective molecular interaction of the enzyme AChE with the emetine and I3M. The value of ΔG for emetine and I3M was -9.72 and -7.09 kcal/mol, respectively, while the remaining contribution to the free energy was entropy since the entropy was shown to increase during the interaction. Hence, we established that the interaction was entropy-driven with a prevalent hydrophobic interaction. This binding affinity was shown by the formation of different bonds such as H-bonds, polar bonds, cation-pi, Pi-Pi interactions, and hydrophobic interactions. Presently, the docked complex was validated statistically using an ANOVA for all the conformers used. This study confirmed that the ligand may prove to be a promising inhibitor of AChE for the management of AD. The scope of subsequent studies would focus on corroborating the 3D structure of the depicted complexes using X-ray crystallography to confirm the present findings.

Author Contributions
Conceptualization