Volume 4, Number 4; 186-200, August 2013

Original Article

Functional Analysis and Molecular Docking studies of Medicinal Compounds for AChE and BChE in Alzheimer's Disease and Type 2 Diabetes Mellitus
Dowluru SVGK Kaladhar*, Nagendra Sastry Yarla and N. Anusha Department of Biochemistry/Bioinformatics, GIS, GITAM University, Visakhapatnam-530045, AP, India
[Received May 5, 2013; Revised June 13, 2013; Accepted June 14, 2013]

ABSTRACT: Acetylcholinesterase and Butyrylcholinesterase share unravelling link with components of metabolic syndromes that's characterised by low levels of HDL cholesterol, obesity, high fast aldohexose levels, hyper-trigliceridaemia and high blood pressure, by regulation of cholinergic transmission and therefore the enzyme activity within a living system. The phosphomotifs associated with amino acid and tyrosine binding motifs in AChE and BChE were known to be common. Phylogenetic tree was constructed to these proteins usinf UPGMA and Maximum Likelihood methods in MEGA software has shown interaction of AChE and BChE with ageing diseases like Alzheimer's disease and Diabetes. AChE has shown closely related to BChE, retinol dehydrogenase and -polypeptide. The present studies is also accomplished that AChE, BChE, COLQ, HAND1, APP, NLGN2 and NGF proteins has interactions with diseases such as Alzheimer's and D2M using Pathwaylinker and STRING. Medicinal compounds like Ortho-7, Dibucaine and HI-6 are predicted as good targets for modeled AChE and BChE proteins based on docking studies. Hence perceptive studies of cholinesterase structure and the biological mechanisms of inhibition are necessary for effective drug development.

Key words: AChE, BChE, Protein interactions, Phylogeny, Docking

Acetylcholinesterase (acetylhydrolase or AChE) could be a serine protease that hydrolyzes acetylcholine that acts because the neurochemical in varied species [1, 2]. AChE (Acetylcholinesterase) and BChE (Butyrylcholinesterase) belongs to carboxylesterase family of enzymes. AChE is principally found in several varieties of conducting tissue that serves to terminate junction transmission [3, 4, 5]. Biogenic amines will intervene many varieties of living thing communication in cellular organisms and somewhat evidence has indicated that biogenic amines produce intracellular responses aside from by triggering the protein production of second messengers [6].

AChE and BChE involve in control cholinergic transmission and also the proteinase activity and develops Alzheimers as a result of production of the amyloid macromolecule [7]. AChE catalysis the hydrolysis of acetylcholine of neurotransmitter to acetate and choline at cholinergic nerve terminals and terminates the action of AChE on postsynaptic receptors. BChE involves interactions inside the system in three different enzymatic activities in its structures like AChE, aryl acylamindase and proteinase [8]. Neuronic complications taking place in D2M (Diabetes Mellitus Type 2) is influenced as a result of AChE activity by membrane surface development characterised by morphological changes related to

*Correspondence should be addressed to: Dr. Dowluru SVGK Kaladhar. Department of Biochemistry/Bioinformatics, GIS, GITAM University, Visakhapatnam-530045, AP, India. E-mail: dr.dowluru@gmail.com ISSN: 2152-5250

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minimized motor and sensory conductivity velocities and is being corrected by hypoglycemic agent treatment [9]. The insertion of membrane proteins into the membrane core is also passionate about transmembrane potential that will have an effect on lipid-protein interaction [10] and these activities emerge in association with plaques and tangles in AD (Alzheimer's disease). This pathological cholinesterase with altered properties of AChE and BChE turn out additional severe cases in increasing variety of plant tissue and neurocortical amyloid made cerebral cortex amyloid made neurotic plaques and neurofibrillary tangles in manifestations of AD [11]. AChE is found exuberant in brain, muscle, and blood corpuscle membrane, whereas BChE has higher activity in liver, intestine, heart, excretory organ and respiratory organ. AChE and BChE contribute 65 percent aminoalkanoic acid sequence similarity and have connected molecular forms despite of being merchandise of various genes lying on human chromosomes 7(7q22) and 3(3q26) [12]. The active site within the structure of human BChE gap is schematized and therefore the peripheral ionic site (PAS) is found at the mouth of the gorge. Asp70 and Tyr332 residues of PAS are initial binding of charged substrates and have a bond that controls the operate design of the BChE situation gorge [13]. BChE could be a major detoxification protein of cocain in plasma with inactive metabolites made, like ecgonine methyl ester and benzoic acid, are rapidly excreted by kidney. A signal of cocain toxicity includes elevated vital sign, pathology grand-mal seizure and stroke. BChE protects from deadly effects of cocain by management of purified human serum [14]. AD and D2M occur with increasing frequency as age advances and disease development results in risk of onset of another disease. In the risk of AD and D2M, a range of mechanisms has been postulated like metabolic abnormalities of insulin resistance, dyslipidemia, high blood pressure, hypertension, hyperglycaemia, disturbing synaptic plasticity learning and memory [15]. BChE is related to interstitial tissue cells, epithelial tissue cells and neurons BChE which will be known from AChE in its kinetic response to concentration of acetylcholine. BChE hydrolysis high concentration of acetylcholine and is related to interstitial tissue cells [16]. In cell cultures and epidemiologic surveys, agents such as herbal extracts with inhibitor property and NSAID (nonsteroidal antiinflammatory drugs) showed protecting impact against AD pathology [17, 18, 19]. Exposure to organophosphate pesticides disrupts neurotransmission by inhibiting conjugation AChE resulting in acetylcholine accumulation within the junction and neural over stimulation result in death due

to cardiovascular and respiratory collapse [20]. Inhibition of AChE and BChE by organophosphorus compounds take in a very progressive manner and also the reactivation of phosphorylated accelerator are often done by treating with sturdy nucleophilic agents like oximes [21]. More than 10,000 molecules of acetylcholine are often cleaved per second in brain by AChE and BChE. AChE knockout mouse survives for many weeks in absence of AChE as a result of AChE is remunerated by BChE and provides backup and regulates cholinergic transmission that shows the precise cholinergic pathways regulated by BChE in brain of patients with AD [22]. Most of the proteins within the cell move with alternative protein molecules so a lot of essential for cellular method like cellular motion, signal transduction, transportation and most restrictive mechanism that are mediate by protein-protein interaction [23]. These proteins will have measurable effects in altering the kinetic properties of the cellular components. These transient altering are consequence of protein kinases, protein phosphotases, acyltransferases, glycosyl transferases, etc [24]. Experimental knowledge on physical protein-protein interactions are largely hold on and a STRING (Search Tool for Retrieval of INteracting Gene) info provides data on useful links between proteins [25]. Signalink info may be a uniformly curetted info of eight major signalling pathways [26]. HPRD (Human Protein Reference Database) is internet primarily based community[27, 28], a unique comprehensive protein data resource that depicts completely different options of proteins like domain structure, post-translational modifications, tissue expression, protein-protein interactions, enzymesubstrate relationships and molecular function. Protein-Protein interactions play a crucial role in identification of metabolic syndrome pathways, provides advancements from the past for understanding molecular options for vital network topologies in biological systems [29]. AChE and BChE are often found in blood and is related to options of the metabolic syndrome [30]. Central and involuntary nervous systems regulate immediate variations with AChE and BChE activity that's crucial with chemical change properties and general functions. Factor and haplotype variations within the enzyme genes were related to changes in blood. Both AChE and BChE activities brings changes in plasma lipid and lipoprotein concentrations, obesity and additional elements of the metabolic syndrome. The MetS (metabolic syndrome) is characterised by abdominal obesity, low levels of high-density lipoprotein (HDL) sterol, elevated fast aldohexose levels and hypertrigliceridaemia with cardiovascular disease [31, 32] during involvement of AChE and BChE in lipid

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metabolism. BChE, AChE and Neuroligin structurally belong to a family of alpha beta hydrolase fold and are inferred from the similar structural patterns [33]. Diabetes, Obesity and Neurological disturbances show co-occurrence by activity a comparative analysis with NLGN2 (Neuroligin) a protein belonging to the same family, alters the signal transduction of neurons, a possible reason for Neurological disturbances. The brain of mammals contains two major forms of cholinesterases, AChE and BChE have similar perform to enzymatic differences however show variations genetically, structurally and for and for their kinetics [34]. AChE activity decreases progressively whereas BChE activity shows some increase within the brain of AD patients. Cholinesterase inhibitors (ChEIs) are efficacious for mild to moderate AD exert 3 main actions: inhibit enzyme (ChE), increase extracellular levels of brain neurotransmitter (ACh) and improve psychological feature processes [35]. Severely and selectively damaged central cholinergic systems are concerned neurodegenerative diseases like Alzheimer's disease and dementia with Lewy bodies [36, 37]. MATERIALS AND METHODS AChE and BChE are interlinked with various proteins related to AD and D2M. To find out the protein interactions, an interaction profile studies and experimental graphs has been studied using STRING database. Sequence retrieval The National Centre for Biotechnology Information advances science and health by providing data access to biomedical and genomic information. The NCBI stores genome sequencing data in GenBank and an index of biomedical research analysis articles in PubMed and PubMed Central, in addition as alternative information relevant to biotechnology. All these databases are accessible on-line through the Entrez search engine. The interaction profile data from HPRD has presented good signaling profile and hence protein sequences related to these proteins has been retrieved for further studies. The molecular weight of AChE is 67376 Da present in gene map locus at 7q22. AChE interacts with various molecules such as Proline rich membrane anchor 1, Collagen-like tail subunit (single strand of homotrimer) of asymmetric acetylcholinesterase, HAND1, Ligatin, Laminin, alpha 1, Laminin beta 1, Collagen, type IV, alpha 1, Amyloid beta A4 protein. The sequence of AChE retrieved for modelling of receptor is:

>AChE sequence MRPPQCLLHT PSLASPLLLL LLWLLGGGVG AEGREDAELL VTVRGGRLRG IRLKTPGGPV SAFLGIPFAE PPMGPRRFLP PEPKQPWSGV VDATTFQSVC YQYVDTLYPG FEGTEMWNPN RELSEDCLYL NVWTPYPRPT SPTPVLVWIY GGGFYSGASS LDVYDGRFLV QAERTVLVSM NYRVGAFGFL ALPGSREAPG NVGLLDQRLA LQWVQENVAA FGGDPTSVTL FGESAGAASV GMHLLSPPSR GLFHRAVLQS GAPNGPWATV GMGEARRRAT QLAHLVGCPP GGTGGNDTEL VACLRTRPAQ VLVNHEWHVL PQESVFRFSF VPVVDGDFLS DTPEALINAG DFHGLQVLVG VVKDEGSYFL VYGAPGFSKD NESLISRAEF LAGVRVGVPQ VSDLAAEAVV LHYTDWLHPE DPARLREALS DVVGDHNVVC PVAQLAGRLA AQGARVYAYV FEHRASTLSW PLWMGVPHGY EIEFIFGIPL DPSRNYTAEE KIFAQRLMRY WANFARTGDP NEPRDPKAPQ WPPYTAGAQQ YVSLDLRPLE VRRGLRAQAC AFWNRFLPKL LSATASEAPS TCPGFTHGEA APRPGLPLPL LLLHQLLLLF LSHLRRL The molecular weight of BChE is 68418 Da and present at gene map locus of 3q26.1-q26.2. It expresses in Brain, plasma and Fetus. It interacts with proteins such as Collagen-like tail subunit (single strand of homotrimer) of asymmetric acetylcholinesterase. The sequence of BChE retrieved for modelling of receptor is: >BChE sequence MHSKVTIICI RFLFWFLLLC MLIGKSHTED DIIIATKNGK VRGMNLTVFG GTVTAFLGIP YAQPPLGRLR FKKPQSLTKW SDIWNATKYA NSCCQNIDQS FPGFHGSEMW NPNTDLSEDC LYLNVWIPAP KPKNATVLIW IYGGGFQTGT SSLHVYDGKF LARVERVIVV SMNYRVGALG FLALPGNPEA PGNMGLFDQQ LALQWVQKNI AAFGGNPKSV TLFGESAGAA SVSLHLLSPG SHSLFTRAIL QSGSFNAPWA VTSLYEARNR TLNLAKLTGC SRENETEIIK CLRNKDPQEI LLNEAFVVPY GTPLSVNFGP TVDGDFLTDM PDILLELGQF KKTQILVGVN KDEGTAFLVY GAPGFSKDNN SIITRKEFQE GLKIFFPGVS EFGKESILFH YTDWVDDQRP ENYREALGDV VGDYNFICPA LEFTKKFSEW GNNAFFYYFE HRSSKLPWPE WMGVMHGYEI EFVFGLPLER RDNYTKAEEI LSRSIVKRWA NFAKYGNPNE TQNNSTSWPV FKSTEQKYLT LNTESTRIMT KLRAQQCRFW TSFFPKVLEM TGNIDEAEWE WKAGFHRWNN YMMDWKNQFN DYTSKKESCV GL

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MEGA MEGA (Molecular Evolutionary Genetic Analysis) is an integrated tool used for conducting automatic and manual sequence alignment, inferring phylogenetic trees, mining web-based databases, estimating rates of molecular evolution, inferring ancestral sequences, and testing evolutionary hypotheses. ClustalW ClustalW is a widely used system for aligning any number of homologous nucleotide or protein sequences. ClustalW uses progressive alignment techniques for multi-sequence alignments that are useful to construct phylogenetic tree. Construction of phylogenetic tree Phylogenetic relationships of genes and the organisms usually are presented in a tree like form in cladistics. The sequences have shown homology with the sequences present in GenBank. The sequences of AChE (AAH94752), retinal dehydrogenase (AAC09250), polypeptide (AA126151), BChE (AAH08396) and tauprotein (AAA57264) has been retrieved from GenBank and constructed phylogeny in MEGA software using Maximum Likelihood and UPGMA Methods. Signalink PathwayLinker identifies and visualizes the first neighbour interactor network of the queried proteins, analyzes the signaling pathway of the proteins in subnet, and provides links to online web resources. Some of the biomedical research often focuses on altering the functions of selected proteins. PathwayLinker can assist experimental work by linking the queried proteins to signaling pathways through protein-protein and/or genetic interactions. Human Protein Reference Database (HPRD) The HPRD is a centralized tool that integrates data bearing on domain design, post-translational modifications, interaction networks and association for every macromolecule within the human protein. STRING (Search Tool for the retrieval of interacting Genes/Proteins) STRING is a well known database for predicting protein interactions and embrace physical (direct) and purposeful (indirect) associations between proteins.

STRING integrates interaction knowledge from the sources for an outsized range of organisms and transfers information between these organisms wherever applicable. Molecular modelling and Ligand design The sequences of AChE and BChE are retrieved from HPRD and modelled for evaluation of docking parameters with the ligands. The sequences have been submitted to Swiss-model and the protein molecules have been saved as .pdb format. Medicinal compounds like Huperzine, Rivastigmine, Ambenonium, Ortho-7, Donepezil, Pyridostigmine, Galanthamine, AChE inhibitor substrate, HI-6 have been selected from literature to test the activity against modelled AChE molecule. Medicinal compounds like Chlorpyrifosoxon, 1-anilino-8-naphthalene sulfonic acid, Dibucaine, Procainamide, Benzoylcholine, Propionyl thicholine, Acetyl thiocholine, Dialkylphenylphosphates, Tetra ethyl ammonium are selected to test the activity against modelled BChE molecule. These 2D ligands have been constructed using ChemSketch, optimised and saved as .mol format. The 2D ligands in .mol format has been opened in Hyperchem, and the 3D structures has been saved as .pdb format. Docking Docking is the process of bringing one molecule in vicinity with an another molecule. The present research work is conducted the drug molecules to be docked with the modelled receptors such as AChE and BChE. Docking is conducted using free softwares such as iGemdock v2.0 and Hex v6.0, and a commercial software such as Autodock v4.2. iGemdock v2.0 iGEMDOCK (iGeneric Evolutionary Method DOCKing) for molecular docking is a program for computing a ligand conformation and orientation relative to the active site of target protein. GEMDOCK has pharmacophorebased scoring function and the results performed based on the homology model. Autodock v4.2 AutoDock is a suite of automated and commercial docking tool designed to predict small molecules docking to a receptor (diseased or useful components of cell).

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Hex v6.0 Hex is an interactive protein docking and molecular superposition program. The instructions from Hex is

understands as protein or DNA structures in PDB format. It can also read small-molecule as SDF files.

Figure 1: Clade of AChE and BChE with Alzheimers and D2M (UPGMA Method)

RESULTS Sequencing studies of genome and proteome have provides an incredible landmark within the history of biology. A significant role for sequences of DNA is to encode the sequences of proteins that that are participating in essentially all processes. Proteins are flexible, vibrant and extremely complex molecules molecules in a very constant state of modification through initiating and driving interactions with several molecules leads to different cellular states. Phylogenetic tree was constructed to the proteins showing interaction with AChE and BChE involved in ageing diseases in MEGA. Phylogenetic tree is constructed using UGPMA from MEGA software

(UPGMA Method) and relatedness of the proteins was shown in Figure 1. The most challenging part of the phylogeny construction involves comparing the nearest relatives with specific domain content. Phylogenetic tree was constructed to the proteins showing interaction with AChE and BChE involved in agening diseases with Maximum Likelihood method in MEGA (Figure 2). The results predicted that AChE is closely related to retinol dehydrogenase and -polypeptide. AChE is also shown close relationship with BChE and tau-protein. The interaction between AChE and BChE with other proteins like COLQ, APP etc was retrieved from Pathway linker database by submitting the query in search box and the result is represented in Figure 3.

Figure 2: Phylogentic Tree Construction using MEGA (Maximum Likelihood Method)

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Figure 3: Pathway Linker Results for AChE and BChE

HPRD for AChE and BChE Protein interaction In HPRD, the information about the protein-protein interaction, post-translational modifications, enzymesubstrate relationships and disease association are retrieved and represented in Figure 4. The query protein related to AChE has been uploaded to HPRD Phosphomotif finder and results were retrieved and protein contains 152 Serine kinase / phosphatase motifs. The results are predicted that PhosphoMotifs of AChE is related to GSK-3, ERK1, ERK2, CDK5 substrate motif , G protein-coupled receptor kinase 1 substrate motif, X DNA dependent Protein kinase substrate motif, Casein Kinase I and II

substrate motifs GSK-3, ERK1, ERK2, CDK5 substrate motif, PKA kinase substrate motif, PKC kinase substrate motif, Calmodulindependent protein kinase II substrate motif, Growth associated histone HI kinase substrate motif, PP2C delta substrate motif, b-Adrenergic Receptor kinase substrate motif, Dual specificity protein phosphatase 6 substrate motif, MAPK 11, 13, 14 Kinase substrate motif, Pyruvate dehydrogenase kinase substrate motif, Pim1 kinase substrate sequence, LKB1 Kinase substrate motif, NIMA kinase substrate motif, AMPactivated protein kinase substrate motif, Chk1 kinase substrate motif.

Figure 4: Interaction report of Acetylcholinesterase

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Figure 5: Interaction report of Butyrylcholinesterase

In the query protein AChE there are 16 Serine binding motifs as WW domain binding motif, 14-3-3 domain binding motif, MDC1 BRCT domain binding motif and Plk1 PBD domain binding motif. For the given query BChE information about protein-protein interaction, disease association, post-translation modification, enzyme substrate relationships are retrieved using HPRD (Figure 5). The number of serine kinase/ phosphatase in the protein BChE is retrieved. There are 157 Serine kinase / phosphatase motifs such as AMP-activated protein

kinase substrate motif, Casein Kinase I and II substrate motif, PKA kinase substrate motif, PKC kinase substrate motif, Chk1 kinase substrate motif, Calmodulindependent protein kinase II substrate motif, G proteincoupled receptor kinase 1 substrate motif, PP2C delta substrate motif, Dual specificity protein phosphatase 6 substrate motif, b-Adrenergic Receptor kinase substrate motif, G protein-coupled receptor kinase 1 substrate motif, GSK-3, ERK1, ERK2, CDK5 substrate motif and CLK1 kinase substrate motif.

Figure 6: AChE and BChE Interactions shown in STRING Database

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Figure 7: Protein Interactions with genes responsible to metabolic syndrome

The query protein BChE was submitted in the phosphomotifs finder and the number of serine binding motifs was retrieved and there are 12 Serine binding motifs such as MDC1 BRCT domain binding motif, Plk1 PBD domain binding motif and WW domain binding motif. The proteins AChE and BChE were given in STRING database and its interaction with various proteins were retrieved where it shows interaction with some of the proteins like NGF, COLQ and HSPQ2 (Figure 6 and 7). The proteins AChE and BChE were given in STRING database and its interaction with various proteins were retrieved, where it shows direct interactions with some of the proteins like NGF (linked

to Alzheimer's disease), COLQ (associated with Congenital myasthenic syndromes (CMS)), NGF (cause of Alzheimers disease and atherosclerotic cardiovascular disease), NLGN2 ( associated with Alzheimers disease and autism) , etc (Figure 8). Docking is a mechanism used to increase enzyme substrate specificity and govern the binding of kinases and phosphatases to each other and other effectors that provides an understanding of proteinprotein interaction permitting fundamental insight to the researchers. Docking surfaces encode specific information about kinase or phosphatase interactions in the framework of many related peptide motifs. Docking results with some ligands has been presented in Figure 9.

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Figure 8: AChE and BChE interaction mapping

Table 1. Modelled AChE and its Ligands Docking (AutoDock)

Properties Hupe rzine -3.73 -0.21 1.83 Mm -3.73 -3.7 -0.04 0.0 0.0 0.0 0.0 38.75 None None ARG577 TYR581 GLY574 TRP578 Rivas tigm ine -3.9 -0.22 1.38 Mm -5.39 -5.33 -0.06 -0.17 1.49 -0.17 0.0 35.96 None None TRP571 GLY574 PHE575 ARG577 TRP578 Amben onium 0.96 0.03 -3.81 -3.79 -0.02 115.77 4.77 115.77 0.0 35.82 None None PHE575 TRP578 ARG577 TYR581 TRP571 Ortho-7 Donep ezil -4.58 -0.16 436.93 Um -6.37 -6.29 -0.08 -0.35 1.79 -0.35 0.0 33.66 None None PHE575 TRP578 ARG577 TYR581 TRP571 Pyrid Ostigm Ine -3.36 -0.26 3.44 Mm -3.96 -3.95 0.0 -0.29 0.6 -0.29 0.0 35.11 None None PHE575 TRP578 TRP571 Galan Tham ine -4.29 -0.15 711.8 Um -6.08 -5.97 -0.12 0.52 1.79 0.52 0.0 35.78 None None PHE575 TRP578 TYR581 TRP571 GLY574 AChE inhibitor substrate -2.12 -0.21 28.14 Mm -3.31 -3.28 -0.03 -0.11 1.19 -0.11 0.0 34.9 None None LYS572 TRP571 PHE575 HI-6

Binding- energy Ligandefficiency Inhib-constant Inhib-constant units Intermol-energy Vdw-hb-disolveenergy Electrostaticenergy Total-energy Torsional-energy Unbound-energy ClRMS RefRMS Rseed1 Rseed2 Active site of protein

-3.3 -0.13 3.79 Mm -6.58 -6.6 0.01 1.01 3.28 1.01 0.0 36.13 None None PHE575 TRP578 ARG577 TYR581 TRP571

-6.76 -0.32 11.12 Um -8.55 -8.51 -0.04 3.84 1.79 3.84 0.0 212.67 None None TYR72 VAL73 TYR124 SER125 ASP74 GLY120 SEN203 TRP86 GLU202 HIS447

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Table 2. Docking Results for Modelled BChE with ligands (AutoDock)

Properties Chlorp yrifosoxon 0.27 0.02 -0.32 -0.32 -0.01 0.23 0.6 -0.23 0.0 86.69 None None 1-anilino-8naphthalene sulfonic acid -0.49 -0.02 437.51 Mm 0.49 -0.47 -0.02 0.0 0.0 0.0 0.7 87.98 None None Dibucaine Procainamide Benzoy lcholine -5.31 -0.35 127.78 Um -5.31 -4.52 -0.8 0.0 0.0 0.0 0.0 102.11 None None Propionyl thicholine 0.23 0.02 0.23 0.26 0.49 0.0 0.0 0.0 0.0 97.06 None None Acetyl Thio choline -4.23 -0.42 794.59 Um -4.23 3.59 -0.64 0.0 0.0 0.0 0.0 104.0 None None Dialkylphenyl Phosphates 1.09 0.08 -0.41 -0.4 0.0 -0.49 1.49 -0.49 0.0 98.24 None None Tetra ethyl ammonium 0.82 0.09 0.22 -0.31 0.53 0.11 0.6 -0.11 0.0 86.94 None None

Binding- energy Ligand- efficiency Inhib-constant Inhib-constant units Intermol-energy Vdw-hb-disolveenergy Electrostaticenergy Total-energy Torsional-energy Unbound-energy ClRMS RefRMS Rseed1 Rseed2

0.51 0.02 0.21 0.29 0.5 0.07 0.3 0.07 0.0 94.47 None None

0.58 0.03 0.28 -0.25 0.53 -0.06 0.3 -0.06 0.0 92.5 None None

Homology modelled structure of BChE docking was performed with ligands using AutoDock and ligand Benzoylcholine with energy of -5.31 kcal/mol showed good binding affinity compared with other ligands (Table 2). Modelled AChE was docked with its ligands in iGemDock and the energy values, VDW, HBond and Electrostatic information was retrieved. Ortho-7 with energy -83.99 kcal mol-1 has shown good docking results (Table 3).
Table 3. Binding energies of Modelled AChE and Ligands in kcal mol-1 using iGEMDOCK
Compound Rivastigmine ACHE inhibitor substrate Ambenoniun Donepezil Galanthamine HI-6 Huperzine Ortho-7 Pyridostigmine-1 Energy -65.5849 -49.5339 -73.3879 -82.7563 -78.1548 -76.1653 -62.5742 -83.9951 -60.7988 VDW -58.2418 -49.5339 -67.907 -82.7563 -78.1548 -64.3835 -59.1771 -77.0341 -57.2988 HBond -7.34309 0 -5.48089 0 0 -11.7818 -3.39709 -6.96104 -3.5 Elec 0 0 0 0 0 0 0 0 0

results with binding energy value of -3764 kcal/mol and distance with 4 Angstroms (Table 5).
Table 4. Modelled BChE and its Ligands Docking Results in kcal mol-1 (using iGemDock)
Compound Acetylthiocholine 1-anilino-8naphthalenesulfo nicacid Benzoylcholine Chlorpyrifos Dialkylphenylph osphates Dibucaine Procainamide Tetraethylammo nium Propionylthiocho line Energy -35.022 -59.871 VDW -34.3761 -43.1287 HBond 0 -16.7423 Elec -0.645895 0

-49.1163 -54.4679 -50.2987 -65.3374 -48.351 -28.2795 -41.4

-44.3803 -40.8784 -35.1747 -58.3615 -42.5862 -28.2795 -31.13

-4.14502 -13.5895 -15.124 -6.97593 -5.76476 0 -10.3

-0.591029 0 0 0 0 0 0.03

Modelled BChE was docked with its ligand using the tool Hex. The energy values and R-values are retrieved and shown in Table 6. An energy value of 270.93 kcal mol-1 for Dibucaine showed good docking result with modelled BChE. DISCUSSION The functional properties of proteins are determined by their three-dimensional structures, self assemble into advanced structures is chargeable for their dominant role in biological functions. The range of life within the contemporary world has been generated by evolutionary processes functioning on these core processes through millions years. The generation of this diversity has fairly often resulted from the difference of existing

iGemDock is a tool used for docking using this tool, modelled BChE protein was docked with its ligands and results were retrieved which are shown in the Table 4 and the interaction of Dibucaine is shown in the Figure 9. Dibucaine showed better docking results with BChE with binding energy value of -65.3 kcal mol-1 compared with other tested compounds. Docking was performed for modelled AChE with nine ligands using Hex. Ortho-7 showed better docking

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biochemical components to new biological components instead of the event of essentially new biochemical

technology [38, 39]. BChE deficient individuals are typically healthy with no apparent signs of diseases [40].

Figure 9: Docking results

Table 5. Docking Result for Modelled AChE and its Ligand in kcal mol-1 (using Hex)
LIGAND Donepezil Galanthamine HI-6 Rivastigmine Huperzine Ortho-7 Pyridostigmine Ambenoniun AChE inhibitor substrate Etotal in kcal mol(-1) -235.98 -235.98 -229.82 -185.69 -166.63 -3764.00 -153.42 -247.22 -137.30 R-Value In A 17.6 17.6 19.2 12.0 7.2 4.0 6.4 8.0 7.2

There is lot of evidence that supports cholinergic mechanisms modulate learning and memory formation [41, 42]. The strength and influence of any neural system on behavioral output is modulated by internal secretion

standing by selective lesions of hippocampus, striatum, and also the amygdaloid nucleus [43]. AChE and BChE contains multiple potential substrate binding areas, is responsible for caparison and delivery of Benzoylcholine to the active site [44, 45]. Individuals with regular BChE and AChE have a high intravenous anesthetic range [46]. Obidoxime, trimedoxime, pralidoxime (2-PAM) and asoxime (HI-6) are commercial drugs containing the oxime group are able to split organophoshorus moiety from the the active site resulting in liberation and enzyme reactivation. All the chosen molecules have shown negative energies and therefore will target in control the AChE and BChE levels in humans. Rivastigmine and donepezil controls brain acetylcholine levels has shown by experimentation by Naik et al., in 2009 in acetylcholinesterase-deficient mice [47]. Huperzine and AChE inhibitor substrates from Huperzia cf chamaeleon and Huperzia reflexa, Lycopodium clavatum subsp. Clavatum showed

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AChE and BChE studies in Alzheimers and D2M

strong AChE inhibition [48, 49]. Galanthamine, an alkaloid derivative phytocompound isolated from snowdrop (Galanthus nivalis L.) [50] and Huperzine A, a naturally occurring sesquiterpene alkaloid compound isolated from Huperzia serrata (Thunb.) Trev., [50, 51] are the latest anticholinesterase drugs using against AD. Plant derived alkaloids like galanthamine, huperzine A and rivastigmine are known for their AChE inhibitory activity [52, 53]. Ambenonium, an old AChE inhibitor [54, 55], Ortho-7, a more efficient reactivator than HI-6 [56], Donepezil [57] and Pyridostigmine [56] has shown good activity against AChE. The present report has also been proved good with high binding affinity of AChE with Ortho-7.

Table 6. Docking Results for modelled BChE and its Ligand in kcal mol-1 (using Hex)
LIGANDS Chlorpyrifos-oxon 1-analino-8-naphtalene sulfonic acid Dibucaine Procainamide Benzoylcholine Propionyl thiocholine Acetylthiocholine Di alkyl phenyl phosphates Tetra ethyl ammonium Etotal in kcal mol(-1) -186.55 -192.87 -270.93 -214.37 -208.92 -172.31 -154.31 -174.23 -142.29 R-value In A 21.6 21.6 20.0 20.0 18.4 16.8 12.0 21.6 17.6

molecular disorders have been already studied using STRING and HPRD in MAPK pathway [65]. The AChE and BChE proteins [66, 67, 68] showed interaction with few other proteins like COLQ, NGF etc. The phosphomotifs for the protein AChE and BChE were also be retrieved and studied for better understanding of relationships in homologous sequences. AChE and BChE are regular targets of a large number of toxins and understanding of cholinesterase structure and the biological mechanisms of inhibition is necessary for novel drug development. Dibucaine, Ortho-7 and HI-6 are predicted as good targets for AChE and BChE proteins and can control metabolic syndromes in humans. The work concludes hat Cholinesterases play an important role in the human system. Humans in the present generation are exposing toxic products in body, in the form of pesticides and microbial toxins in foods. AchE and BChE are the proteins involved in ageing disease like Alzheimers and D2M. The present study shows the functional studies using phylogeny and protein interactions. The virtual molecular screening of drugs on AChE and BChE has been studied using docking methods. Acknowledgements Author would like to thank management and staff of GITAM University Visakhapatnam, India for their kind support in bringing out the above literature and providing lab facilities. Conflict of interest The authors do not have any conflict(s) of interest References
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