Functional Analysis and Molecular Docking studies of Medicinal Compounds for AChE and BChE in Alzheimer’s Disease and Type 2 Diabetes Mellitus

*Correspondence should be addressed to: Dr. Dowluru SVGK Kaladhar. Department of Biochemistry/Bioinformatics, GIS,

GITAM University, Visakhapatnam-530045, AP, India. E-mail: [email protected]

ISSN: 2152-5250 186

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

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

D. S. K. Kaladhar et al AChE and BChE studies in Alzheimers and D2M

Aging and Disease Volume 4, Number 4, August 2013 187

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 anti-

inflammatory 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, enzyme-

substrate 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 hyper-

trigliceridaemia with cardiovascular disease [31, 32]

during involvement of AChE and BChE in lipid



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



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 tau-

protein (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 pharmacophore-

based 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).



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)



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, enzyme-

substrate 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, AMP-

activated protein kinase substrate motif, Chk1 kinase

substrate motif.

Figure 4: Interaction report of Acetylcholinesterase



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, Calmodulin-

dependent protein kinase II substrate motif, G protein-

coupled 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



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 enzymesubstrate 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.



Figure 8: AChE and BChE interaction mapping

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

Properties Hupe

rzine

Rivas

tigm

ine

Amben

onium

Ortho-7 Donep

ezil

Pyrid

Ostigm

Ine

Galan

Tham

ine

AChE

inhibitor

substrate

HI-6

Binding- energy -3.73 -3.9 0.96 -3.3 -4.58 -3.36 -4.29 -2.12 -6.76

Ligand-

efficiency

-0.21 -0.22 0.03 -0.13 -0.16 -0.26 -0.15 -0.21 -0.32

Inhib-constant 1.83 1.38 - 3.79 436.93 3.44 711.8 28.14 11.12

Inhib-constant units

Mm Mm - Mm Um Mm Um Mm Um

Intermol-energy -3.73 -5.39 -3.81 -6.58 -6.37 -3.96 -6.08 -3.31 -8.55

Vdw-hb-disolve-

energy

-3.7 -5.33 -3.79 -6.6 -6.29 -3.95 -5.97 -3.28 -8.51

Electrostatic-

energy

-0.04 -0.06 -0.02 0.01 -0.08 0.0 -0.12 -0.03 -0.04

Total-energy 0.0 -0.17 115.77 1.01 -0.35 -0.29 0.52 -0.11 3.84

Torsional-energy 0.0 1.49 4.77 3.28 1.79 0.6 1.79 1.19 1.79

Unbound-energy 0.0 -0.17 115.77 1.01 -0.35 -0.29 0.52 -0.11 3.84

ClRMS 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

RefRMS 38.75 35.96 35.82 36.13 33.66 35.11 35.78 34.9 212.67

Rseed1 None None None None None None None None None


Active site of

protein

ARG577

TYR581

GLY574

TRP578

TRP571

GLY574

PHE575

ARG577

TRP578

PHE575

TRP578

ARG577

TYR581

TRP571

PHE575

TRP578

ARG577

TYR581

TRP571

PHE575

TRP578

ARG577

TYR581

TRP571

PHE575

TRP578

TRP571

PHE575

TRP578

TYR581

TRP571

GLY574

LYS572

TRP571

PHE575

TYR72

VAL73

TYR124

SER125

ASP74

GLY120

SEN203

TRP86

GLU202

HIS447



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

Properties Chlorp

yrifos-

oxon

1-anilino-8-

naphthalene

sulfonic acid

Dibucaine Procainamide Benzoy

lcholine

Propionyl

thicholine

Acetyl

Thio

choline

Dialkylphenyl

Phosphates

Tetra ethyl

ammonium

Binding- energy 0.27 -0.49 0.51 0.58 -5.31 0.23 -4.23 1.09 0.82

Ligand- efficiency 0.02 -0.02 0.02 0.03 -0.35 0.02 -0.42 0.08 0.09

Inhib-constant - 437.51 - - 127.78 - 794.59 - -

Inhib-constant units

- Mm - - Um - Um - -

Intermol-energy -0.32 0.49 0.21 0.28 -5.31 0.23 -4.23 -0.41 0.22

Vdw-hb-disolve-

energy

-0.32 -0.47 0.29 -0.25 -4.52 0.26 3.59 -0.4 -0.31

Electrostatic-

energy

-0.01 -0.02 0.5 0.53 -0.8 0.49 -0.64 0.0 0.53

Total-energy 0.23 0.0 0.07 -0.06 0.0 0.0 0.0 -0.49 0.11

Torsional-energy 0.6 0.0 0.3 0.3 0.0 0.0 0.0 1.49 0.6

Unbound-energy -0.23 0.0 0.07 -0.06 0.0 0.0 0.0 -0.49 -0.11

ClRMS 0.0 0.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0

RefRMS 86.69 87.98 94.47 92.5 102.11 97.06 104.0 98.24 86.94



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 Energy VDW HBond Elec

Rivastigmine -65.5849 -58.2418 -7.34309 0

ACHE inhibitor

substrate

-49.5339 -49.5339 0 0

Ambenoniun -73.3879 -67.907 -5.48089 0

Donepezil -82.7563 -82.7563 0 0

Galanthamine -78.1548 -78.1548 0 0

HI-6 -76.1653 -64.3835 -11.7818 0

Huperzine -62.5742 -59.1771 -3.39709 0

Ortho-7 -83.9951 -77.0341 -6.96104 0

Pyridostigmine-1 -60.7988 -57.2988 -3.5 0

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

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 Energy VDW HBond Elec

Acetylthiocholine -35.022 -34.3761 0 -0.645895

1-anilino-8-

naphthalenesulfo

nicacid

-59.871 -43.1287 -16.7423 0

Benzoylcholine -49.1163 -44.3803 -4.14502 -0.591029

Chlorpyrifos -54.4679 -40.8784 -13.5895 0

Dialkylphenylph

osphates

-50.2987 -35.1747 -15.124 0

Dibucaine -65.3374 -58.3615 -6.97593 0

Procainamide -48.351 -42.5862 -5.76476 0

Tetraethylammo

nium

-28.2795 -28.2795 0 0

Propionylthiocho

line

-41.4 -31.13 -10.3 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



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

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

Table 5. Docking Result for Modelled AChE and its

Ligand in kcal mol-1 (using Hex)

LIGAND Etotal in kcal mol(-1) R-Value In A

Donepezil -235.98 17.6

Galanthamine -235.98 17.6

HI-6 -229.82 19.2

Rivastigmine -185.69 12.0

Huperzine -166.63 7.2

Ortho-7 -3764.00 4.0

Pyridostigmine -153.42 6.4

Ambenoniun -247.22 8.0

AChE

inhibitor

substrate

-137.30 7.2



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 Etotal

in kcal mol(-1)

R-value

In A

Chlorpyrifos-oxon -186.55 21.6

1-analino-8-naphtalene sulfonic acid -192.87 21.6

Dibucaine -270.93 20.0

Procainamide -214.37 20.0

Benzoylcholine -208.92 18.4

Propionyl thiocholine -172.31 16.8

Acetylthiocholine -154.31 12.0

Di alkyl phenyl phosphates -174.23 21.6

Tetra ethyl ammonium -142.29 17.6

People with BChE deficiency are likely to be

intolerant of standard doses of the anti-Alzheimer's drugs

such as huperzine A and donepezil [58]. Chlorpyrifos

oxon [59], 1-anilino-8-naphthalene sulfonic acid [60],

Dibucaine, Procainamide, Benzoylcholine, Propionyl

thicholine, Acetyl thiocholine, Dialkylphenylphosphates,

Tetra ethyl ammonium are virtually screened and

selected for the activity against BChE [61. 62]. The

present report has also been proved good with high

binding affinity of BChE with Dibucaine as drug to AD

and D2M.

BChE, resembling AChE, is typically associated

with catalyzing the hydrolysis of the neurotransmitter

acetylcholine (ACh) and yield choline and acetic acid.

BChE has a clear role in neural functions in the co-

regulation of cholinergic and non-cholinergic

neurotransmission [63]. Cerebrovascular disease is

thought to decrease the threshold at which amyloid

deposition causes the manifestations of AD that starts

with memory impairment, reaching the threshold of mild

cognitive impairment, and eventually dementia [64].

Protein interactions between AChE and BChE with

other proteins were retrieved using STRING, HPRD and

Pathway linker database. Protein-Protein interactions in

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

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Functional Analysis and Molecular Docking studies of Medicinal Compounds for AChE and BChE in Alzheimer’s Disease and Type 2 Diabetes Mellitus

Documents

ache acetylcholinesterase

interaction of ache

modeled ache

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result of ache activity

bche proteins

bche butyrylcholinesterase

alzheimers disease