Molecular interactions of cholinesterases inhibitors using in silico methods: current status and...

Review

New Biotechnology �Volume 25, Number 5 � June 2009 REVIEW

Molecular interactions of cholinesterasesinhibitors using in silico methods: currentstatus and future prospects

Mahmud Tareq Hassan Khan

Department of Pharmacology, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway1

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a low amount of acetylcholine

(ACh) in hippocampus and cortex. Acetylcholinesterase (AChE) is one of the most important enzymes in

many living organisms including human being and other vertebrates, insects like mosquitoes, among

others. Several reports have been published where it has been clearly shown that the genesis of amyloid

protein plaques associated with AD is connected to modifications of both AChE and

butyrylcholinesterase (BChE), since the plaque is significantly decreased in AD patients using

cholinesterase inhibitors (ChEIs). This review gives some examples of these inhibitors discovered during

past couple of years that have shown very prominent interactions at the active site triad of the proteins as

well as different other parts of the active site like, peripheral anionic site (PAS), oxyanionic hole, anionic

subsite or acyl binding pocket (ABP). Most of the inhibition and their interactions have been visualized

by X-ray crystallography, but some of the other inhibitors have been studied either by molecular docking

or molecular dynamic (MD) simulations or by both the in silico methods. Some of these prominent

studies have been crucially observed and reported here.

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331

Structural biology of cholinesterases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333

Active site and different subsites of AChE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341

Cholinesterase inhibitors: Some examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341

Molecular docking for the prediction of intermolecular interactions as well as affinity prediction: Identifying new leads . . . . . 341

Some case studies: docking studies of some inhibitors of cholinesterases (AChE, BChE, etc.). . . . . . . . . . . . . . . . . . . . . . . . . . . 342

Edrophonium-like ammonium salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342

Dehydroamino acid choline esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342

N-aryl derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343

Concluding remarks, Future prospects and challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343

IntroductionAlzheimer’s disease (AD) is the most common cause of dementia

characterized by progressive cognitive impairment in the elderly

E-mail address: Khan, M. T. H. ([email protected]), ([email protected]).1 Present address.

1871-6784/$ - see front matter � 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.nbt.2009.03.008

people. According to the World Health Organization (WHO), it is

estimated that 50% of people older than age 85 years are afflicted

with AD. Neurofibrillary tangles comprising hyperphosphorylated

tau proteins and neuritic amyloid plaques represent the core

neuropathologic features of AD [1,2]. This neurodegenerative

www.elsevier.com/locate/nbt 331

mailto:[email protected]

http://dx.doi.org/10.1016/j.nbt.2009.03.008

REVIEW New Biotechnology � Volume 25, Number 5 � June 2009

Review

disorder is often characterized by a low concentration of acetyl-

choline (ACh) in hippocampus and cortex [3]. In the past decade,

enormous efforts have been devoted to understand the genetics

and molecular pathogenesis of AD, which has been transferred

into extensive experimental approaches aimed at reversing disease

progression [2]. The disease is accompanied by dysfunctions in the

system of cholinergic neurotransmission of the central nervous

system (CNS) [4]. It is a chronic, slowly progressive neurodegen-

erative disorder [5–9]. The gradual loss of memory, decline in other

cognitive functions, and decrease in functional capacity result in

FIGURE 1

Alignment of primary structures of AChE sequences from Homo sapiens (PDB code 1

The Figure was created using the bioinformatic and sequence analysis software CL

alignment, the consensus, conservation, and the sequence logo also been shown

332 www.elsevier.com/locate/nbt

death approximately 8-10 years after the onset of symptoms

[10,11].

ACh was first synthesized in 1867 and detected in the adrenal

gland of human tissue in 1906 as a neurotransmitter [12]. The

fundamental role of the enzymes Acetylcholinesterase (AChE, EC

3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) at choliner-

gic synapses is to terminate neurotransmission by rapid hydrolysis

of the substrate, ACh, into choline (Ch) and acetic acid and thus

inactivated [13–17]. Thus these two enzymes acetylcholinesterase

(AChE) and butyrylcholinestarase (BChE) have been identified as

B41), Mus musculus (PDB code 1N5M) and Torpedo californica (PDB code 1EA5).

C Workbench (main) version 4.0 (www.clcbio.com). At the bottom part of the

.



FIGURE 2

The 3D structures of human acetylcholinesterase (HuAChE) complexed with

Fasciculin-II (Glycosylated Protein,) (PDB code 1B41) [32]. Fasciculin-II isshown in the figure as ‘stick’ format where as the protein is shown in 3D

‘cartoon’. The figure was created using PyMol (www.pymol.org) and rendered

with RayTrace.

FIGURE 3

2D structure of Galantamine (or Galanthamine, GAL).

Review

potential targets in the treatment of AD, myasthenia gravis, glau-

coma, and in the recovery of victims of nerve agent exposure [18].

AChE is a key component of cholinergic brain synapses and

neuromuscular junctions. The main role of this enzyme is the

termination of impulse transmission by rapid hydrolysis of the

cationic neurotransmitter acetylcholine [19]. BChE is produced in

the liver and enriched in the circulation. The exact physiological

role of BChE is still elusive, but it is generally viewed as a backup for

the homologous AChE [20,21].

It is reported that the genesis of amyloid protein plaques asso-

ciated with AD is connected to modifications of both AChE and

BChE, since the plaque is significantly decreased in AD patients

using ChE inhibitors (ChEIs) [22–25]. Consequently, it is not

surprising that ChEIs have shown more promising results in the

treatment of AD than any other strategy explored [26–29].

In this review some of the potential inhibitors of ChEs discovered

during past couple of years that have shown very prominent inter-

actionsat the active site triadof the proteins aswell asdifferentother

parts of the active site like, peripheral anionic site (PAS), oxyanionic

hole, anionic subsite or acyl binding pocket (ABP) have been criti-

cally reviewed. Most of the inhibitiors and their interactions have

been visualized by X-ray crystallography, but some of other inhi-

bitors have been studied by different in silico methods like docking,

molecular dynamic (MD) simulations, among others.

Structural biology of cholinesterasesAChE is one of the most important enzymes in many living

organisms, including humans and vertebrates, and is located in

the nervous system and in muscles [13,30,31]. It is one of the most

widely spread enzyme playing very important role in nerve signal

transmission. As AChE controls key processes, its inhibition leads

to the very fast death of an organism, including human being. This

feature is widely used for killing of unwanted organisms (insects

like mosquitoes, etc.). Then it is very important to know how

much do AChEs differ between species and to what extent.

Recently a theoretical report was published to identify the struc-

tural basis for such differences. Authors reported various primary

and tertiary alignments that showed AChEs are very evolutionary

conserved that could in fact lead to difficulties, for example, in the

search for specific inhibitors against particular species [30].

Authors found that the three-dimensional (3D) structure of AChE

is very evolutionary conserved (structural alignment is shown in

Figure 1), despite the lower conservation of the aminoacid

sequence [30]. The folding of the proteins also found to be similar

when the structures of AChE from Homo sapiens and Drosophila

melanogaster have been compared [30]. In the 3D structure of

Drosophila melanogaster AChE, various mutations of the active site

residues occur leading to differences in both steric and electrostatic

properties of the active site [30]. Most of the mutations were seen

at the peripheral anionic site (PAS). They have predicted the

structures of AChEs from Rattus norvegicus, Felis silvestris catus,

Oryctolagus cuniculus and Bos Taurus should be very similar to

the human structure and should have identical properties of the

active site [30]. These findings proved again that the specific

inhibitors of this enzyme are quite difficult and the inhibitors

might also affect human being.

The 3D structures of human AChE complexed with the snake-

venom toxin fasciculin II, a ‘three-finger’ 61 amino-acid polypep-

tide toxin purified from the venom of the eastern green mamba

(Dendroaspis angusticeps) [32], is shown in Figure 2. The toxin

fasciculin interacts predominantly with the peripheral anionic

site (PAS) without affecting the structure of the active centre

[33–35].

An alkaloid from the flower of the common snowdrop

(Galanthus nivalis), (�)-Galanthamine (GAL, also known as Galan-

tamine), showed potent anticholinesterase activity. The two-

dimensional (2D) structure of GAL is shown in Figure 2. This




FIGURE 4

Crystal structure of (�)-galantamine with Acetylcholinesterase at 2.3 A resolution (PDB code 1DX6) [36]. (A) shows GAL in orange ‘stick’ format with the TcAChE in

3D ‘cartoon’ format; and (B) shows the buried GAL at the active site gorge into the surface representation of the enzyme. The figure was created using PyMol(www.pymol.org) and rendered with RayTrace.

FIGURE 5

(A) 2D structure of bifunctional derivative of GAL; (B) the compound GAL (in ‘stick’ format) has been co-complexed with TcAChE (PDB code 1W4L) [37], where the

compound formed three hydrogen bonds (shown in green dotted line) with the water molecules inside the protein. The figure at B panel was created using the

Discovery Studio Visualizer version 1.5 (www.accelrys.com).

FIGURE 6

(A) 2D structure of tacrine (IUPAC name: 1,2,3,4-tetrahydroacridin-9-amine); (B) the tacrine, in ‘stick’ format, has been co-complexed with TcAChE (PDB code 1ACJ

[38]) at the active site gorge, where the compound formed hydrogen bonds (shown in green dotted line) with two water molecules. The figure at B panel was

created using the Discovery Studio Visualizer version 1.5 (www.accelrys.com).


Review





FIGURE 7

AChE complexed with the nootropic alkaloid, (�)-huperzine A (HupA) [39]. (A) 2D structure of HupA (IUPAC name: (5R,7R,9S,11E)-5-amino-11-ethylidene-7-methyl-

5,6,7,8,9,10-hexahydro-5,9-methanocycloocta b-pyridin-2(1H)-one); (B) the Huperzine, in ‘stick’ format, has been co-complexed with AChE (PDB code 1VOT). The

compound formed hydrogen bonds (shown in green dotted line) with two water molecules of the active site gorge. The figure at B panel was created using the

Discovery Studio Visualizer version 1.5 (www.accelrys.com).

Review

property has made GAL the target of research as to its effectiveness

in the treatment of Alzheimer’s disease. We have solved the X-ray

crystal structure of GAL bound in the active site of Torpedo cali-

fornica acetylcholinesterase (TcAChE) to 2.3 A resolution. The

FIGURE 8

The hydrophobic active site of AChE is subdivided into several subsites. Here negativ

corresponding amino acid residue numbers are also shown. The figure was created

inhibitor binds at the base of the active site gorge of TcAChE,

interacting with both the choline-binding site (Trp-84) and the

acyl-binding pocket (Phe-288, Phe-290). The tertiary amine group

of GAL does not interact closely with Trp-84; rather, the double

ely and positively charged areas are shown in RED and BLUE, respectively, and

using the ICM MolBrowser version 3.6.1b from Molsoft (www.molsoft.com).





Review

bond of its cyclohexene ring stacks against the indole ring. The

tertiary amine appears to make a non-conventional hydrogen

bond, via its N-methyl group, to Asp-72, near the top of the gorge.

The hydroxyl group of the inhibitor makes a strong hydrogen

bond (2.7 A) with Glu-199. The relatively tight binding of GAL to

TcAChE appears to arise from a number of moderate to weak

interactions with the protein, coupled to a low entropy cost for

binding due to the rigid nature of the inhibitor [36]. 2D molecular

structure (Figure 3) and crystal structure of GAL is shown in

Figure 4.

Greenblatt et al. (in 2004) designed and reported the bifunc-

tional derivatives (structure shown in A panel of Figure 5) of the

alkaloid GAL that interacted with both the active site of the AChE

and its peripheral cation binding (PCB) site. These have been

assayed with TcAChE, and the 3D structures (structure shown

in B panel of Figure 5) of their co-crystals with the enzyme have

been solved by X-ray crystallography and reported [37]. During

experimental studies there were some differences of the IC50 values

for TcAChE and those for Electrophorus electricus AChE (EeAChE).

Authors recognized these differences due to the sequence differ-

ences in one or two residues lining the active-site gorge of the

FIGURE 9

Different subsites of the active site of AChE; where catalytic triad (CT) consisting

subsite (AS) Trp86, Tyr133, Glu202, Gly448, Ile451; acyl binding pocket (ABP) Trp2

Ser125, Trp286, Tyr337, Tyr341; and the omega loop (OL) Thr83, Asn87, Pro88. Th

(www.molsoft.com).


enzyme [37]. The binding of one of the inhibitors disrupts the

native conformation of one wall of the gorge, formed by the loop

Trp279-Phe290. It was also proposed by the authors that flexibility

of this loop may permit the binding of inhibitors such as GAL,

which are too bulky to penetrate the narrow neck of the gorge

formed by Tyr121 and Phe330 as seen in the crystal structure [37].

The binding sites of TcAChE for quaternary ligands were inves-

tigated by X-ray crystallography and determined at 2.8 A resolu-

tion. In complex with edrophonium, the quaternary nitrogen of

the ligand interacts with the indole of Trp84, and its m-hydroxyl

displays bifurcated hydrogen bonding to two members of the

catalytic triad, Ser200 and His440. In a complex with tacrine

(shown in Figure 6), the acridine is stacked against the indole of

Trp84. The bisquaternary ligand decamethonium is oriented along

the narrow gorge leading to the active site; one quaternary group is

apposed to the indole of Trp84 and the other to that of Trp279,

near the top of the gorge. The only major conformational differ-

ence between the three complexes is in the orientation of the

phenyl ring of Phe330. The structural and chemical data, together,

show the important role of aromatic groups as binding sites for

quaternary ligands, and they provide complementary evidence

Ser203, His447, Glu334; oxyanion hole (OH) Gly121, Gly122, Ala204; anionic

36, Phe295, Phe297, Phe338; peripheral anionic subsite (PAS) Asp74, Tyr124,

e figure was created using the ICM MolBrowser version 3.6.1b from Molsoft



TABLE 1

Structural features of some molecules having potential inhibitory profiles against ChEs [63,71,72,79–81]


Review


TABLE 1 (Continued )

TABLE 2

X-ray crystallographic data from Protein Databank (http://www.pdb.org) [82,83]

PDB Protein Ligand name Res. (A) Method Year Refs

1cfj AChE Methylphosphonic Acid Ester Group 2.6 1999 [84]

1cfj AChE 2-(Acetylamino)-2-Deoxy-A-D-Glucopyranose 2.6 1999 [84]

1cfj AChE N-Acetyl-D-Glucosamine 2.6 1999 [84]

1dx6 AChE Tetraethylene Glycol 2.3 1999 [36,84]

1dx6 AChE (�)-Galanthamine 2.3 1999 [36,84]

1dx6 AChE N-Acetyl-D-Glucosamine 2.3 1999 [36,84]

1e66 AChE 3-Chloro-9-Ethyl-6,7,8,9,10,11-Hexahydro-7,11-Methanocycloocta[B]Quinolin-12-Amine

2.1 2002 [85,86]

1e66 AChE N-Acetyl-D-Glucosamine 2.1 2002 [85,86]

1ea5 AChE N-Acetyl-D-Glucosamine 1.8 TBP

1gpk AChE Huperaine A 2.1 2002 [85]

1gpn AChE Huperzine B 2.3 2002 [85]

1gpn AChE N-Acetyl-D-Glucosamine 2.3 2002 [85]

1hbj AChE 1-[3-({[(4-Amino-5-Fluoro-2-Methylquinolin-3-Yl)Methyl]Thio}Methyl)Phenyl]-2,2,2-Trifluoroethane-1,1-Diol

2.5 2001 [87]

1hbj AChE 2-(N-Morpholino)-Ethanesulfonic Acid 2.5 2001 [87]

1hbj AChE Tetraethylene Glycol 2.5 2001 [87]

1oce AChE Cis-2,6-Dimethylmorpholinooctylcarbamyleseroline 2.7 1999 [88]

1p0i BChE 2-(N-Morpholino)-Ethanesulfonic Acid 2.0 VDHD 2003 [89]


Review



TABLE 2 (Continued )

PDB Protein Ligand name Res. (A) Method Year Refs

1p0i BChE Butanoic Acid 2.0 VDHD 2002 [89]

1p0m BChE 2-(N-Morpholino)-Ethanesulfonic Acid 2.4 VDHD 2003 [89]

1p0m BChE 2-(N-Morpholino)-Ethanesulfonic Acid 2.4 VDHD 2002 [89]

1p0p BChE 2-(Butyrylsulfanyl)-N,N,N-Trimethylethanaminium 2.3 VDHD 2003 [89]

1p0p BChE Methylphosphonic Acid Ester Group 2.3 VDHD 2003 [89]

1p0p BChE 2-(Butyrylsulfanyl)-N,N,N-Trimethylethanaminium 2.3 VDHD 2002 [89]

1p0p BChE Methylphosphonic Acid Ester Group 2.3 VDHD 2002 [89]

1p0q BChE Methylphosphonic Acid Ester Group 2.4 VDHD 2003 [89]

1som AChE Methylphosphonic Acid Ester Group 2.2 1999 [84]

1vxo AChE Methylphosphonic Acid Ester Group 2.2 1999 [90]

1vxo AChE 2-(Acetylamino)-2-Deoxy-A-D-Glucopyranose 2.2 1999 [90]

1vxo AChE N-Acetyl-D-Glucosamine 2.2 1999 [90]

1vxr AChE 2-(Acetylamino)-2-Deoxy-A-D-Glucopyranose 2.2 1999 [90]

1vxr AChE O-Ethylmethylphosphonic Acid Ester Group 2.2 1999 [90]

1vxr AChE 2-(N-Morpholino)-Ethanesulfonic Acid 2.2 1999 [90]

1w4l AChE Galanthamine Derivative 2.2 1998 [37]

1w4l AChE N-Acetyl-D-Glucosamine 2.2 1998 [37]

1w6r AChE (�)-Galanthamine 2.0 1998 [37]

1w6r AChE N-Acetyl-D-Glucosamine 2.0 1998 [37]

1w75 AChE N-Acetyl-D-Glucosamine 2.4 2004 [37]

1w76 AChE (�)-Galanthamine 2.3 1998 [37]

1w76 AChE N-Acetyl-D-Glucosamine 2.3 1998 [37]

1xlu BChE N-Acetyl-D-Glucosamine 2.2 VDHD 2005 [91,92]

1xlu BChE Monoisopropyl Ester Phosphonic Acid 2.2 VDHD 2003 [91,92]

1xlv BChE N-Acetyl-D-Glucosamine 2.2 VDHD 2005 [91,92]

1xlv BChE Ethyl Dihydrogen Phosphate 2.2 VDHD 2005 [91,92]

1xlv BChE N-Acetyl-D-Glucosamine 2.2 VDHD 2002 [91,92]

1xlv BChE Ethyl Dihydrogen Phosphate 2.2 VDHD 2002 [91,92]

1xlw BChE Diethyl Phosphonate 2.1 VDHD 2005 [91,92]

1xlw BChE N-Acetyl-D-Glucosamine 2.1 VDHD 2005 [91,92]

2bag AChE 1s,3as,8as-Trimethyl-1-Oxido-1,2,3,3a,8,8a-Hexahydropyrrolo

[2,3-B]Indol-5-Yl 2-Ethylphenylcarbamate

2.4 VDHD 2002 [93]

2bag AChE 2-(N-Morpholino)-Ethanesulfonic Acid 2.4 VDHD 2002 [93]

2bag AChE N-Acetyl-D-Glucosamine 2.4 VDHD 1997 [93]

2bag AChE Pentaethylene Glycol 2.4 VDHD 1997 [93]

2bag AChE 1s,3as,8as-Trimethyl-1-Oxido-1,2,3,3a,8,8a-Hexahydropyrrolo

[2,3-B]Indol-5-Yl 2-Ethylphenylcarbamate

2.4 VDHD 1997 [93]

2bag AChE 2-(N-Morpholino)-Ethanesulfonic Acid 2.4 VDHD 1997 [93]

2ckm AChE N,N’-Di-1,2,3,4-Tetrahydroacridin-9-Ylheptane-1,7-Diamine 2.2 2006 [94]

2ckm AChE N-Acetyl-D-Glucosamine 2.2 2006 [94]

2cmf AChE N,N’-Di-1,2,3,4-Tetrahydroacridin-9-Ylpentane-1,5-Diamine 2.5 2006 [94]

2cmf AChE N-Acetyl-D-Glucosamine 2.5 2006 [94]

2j4c BChE 2-(N-Morpholino)-Ethanesulfonic Acid 2.8 TBP

2j4c BChE Butanoic Acid 2.8 TBP

2j4c BChE N-Acetyl-D-Glucosamine 2.8 TBP

2pm8 BChE N-Acetyl-D-Glucosamine 2.8 VDSD 2007 [95]

2pm8 BChE 2-(Acetylamino)-2-Deoxy-A-D-Glucopyranose 2.8 VDSD 2007 [95]

Notes: Here, ‘Proteins’ means whether it is acetyl-cholinesterase or butyryl-cholinesterase; ‘Method’ means methods of crystallization; ‘Year’ is the year of submission to PDB; ‘VDHD’

Vapour Diffusion, Hanging Drop; ‘VDSD’ Vapour Diffusion, Sitting Drop; ‘TBP’ is to be published, not yet published in paper form.


Review



Review


Review

assigning Trp84 and Phe330 to the AS of the active site and Trp279

to the PAS [38].

Raves and co-authors reported, during 1997, the 3D structure of

(�)-Huperzine A (HupA) complexed with AChE [39]. HupA

(Figure 7) was found in an extract from a club moss that has been

used for centuries in Chinese folk medicine. The action of HUP had

been attributed to its ability to strongly inhibit AChE. The crystal

structure of the complex of AChE with HupA at 2.5 A resolutions

showed an unexpected orientation for the inhibitor with surpris-

ingly few strong direct interactions with protein residues to explain

its high affinity [39]. The structure has been compared with the

native structure of AChE without any inhibitor at the same resolu-

tion. Analysis of the affinities of structural analogues of HupA,

correlated with their interactions with the protein, exhibited the

importance of individual hydrophobic interactions between HupA

and aromatic residues in the active-site gorge of AChE [39].

Active site and different subsites of AChEThe hydrophobic active site of AChE is subdivided into several

subsites (shown in Figs. 8 and 9), which can be distinguished in

AChE’s active site: esteratic subsite, also called the catalytic triad

(CT, Ser203, His447, Glu334), oxyanion hole (OH, Gly121,

Gly122, Ala204), anionic subsite (AS, Trp86, Tyr133, Glu202,

Gly448, Ile451), acyl binding pocket (ABP, Trp236, Phe295,

Phe297, Phe338), peripheral anionic subsite (PAS, Asp74,

Tyr124, Ser125, Trp286, Tyr337, Tyr341) and other residues of

the omega loop (OL, Thr83, Asn87, Pro88). The omega loop is a

disulphide-linked loop (Cys69–Cys96) that covers the active site of

AChE, which is buried at the bottom of a 20 A deep gorge approxi-

mately in the centre of the molecule [30].

Cholinesterase inhibitors: Some examplesIn recent years a large numbers of AChE inhibitors (AChEIs) have

been reported from natural sources to synthetic origin [2,40–45]. A

large numbers of natural alkaloids were found to have AChE and

BChE inhibitory activities [46–58]. Several classes of natural com-

pounds and extracts have also been reported as AChE and BChE

inhibitors (ChEIs) [47–50,52,54,55,58–73]. Some of the inhibitors

are found to affect directly to the mammalian memory [74] and

the most prominent inhibitors including galantamine (Raza-

dyne1), donepezil (Aricept1), rivastigmine (Exelon1), tacrine

(Cognex1), among others, have gone through different preclinical

and clinical studies [46,75–78].

Although not FDA-approved, AChEIs have also been evaluated

for use in vascular dementia, dementia with Lewy bodies, and

Parkinson’s-induced dementia.

A large number of molecules from natural sources have been

reported as potent AChE inhibitors and reviewed by many authors,

some of which exhibited potential for the development of ‘‘lead

molecule’’ for the treatment of AD [63,71,72,79,80]. Some of these

potential examples with their 2D molecular structures are given in

Table 1.

There are a large number of inhibitors (or ligands) of ChEs have

been co-crystallized and deposited at the Protein Databank

FIGURE 10

Flowchart of a successful VLS example [123]; where authors performed a structur

identified 22 top-scored ‘hit’ molecules from US-NCI compounds’ database; finally ainhibitor along with another eleven moderately potent compounds [123].

(www.pdb.org) in last couple of years. Some of these ligands are

mentioned in Table 2.

In the past couple of years several prospective candidates for the

drug development against AD have been studied using docking

calculations and other in silico studies have been reported targeting

AChE and BChE [17,27,45,46,96–113]. Some of the prominent

examples with their possible molecular interactions will be dis-

cussed in the later sections.

The currently approved ChE inhibitors, like donepezil, rivas-

tigmine, galantamine, among others, which are used for the

symptomatic treatment of mild to moderate AD [114]. Beside

the target organ brain, heart is also rich in ChEs and their inhibi-

tion may adversely affect cardiac function. These ChEIs are iden-

tified to raise blood pressure and slow the pulse rate through both

central and peripheral mechanisms; they also reduce cardiac beat-

by-beat fluctuations [115]. These drugs may also increase the

liability to falls in patients with AD and Lewy Body dementia,

who have an increased incidence of orthostatic hypotension and

carotid sinus hypersensitivity [116,117].

Molecular docking for the prediction of intermolecularinteractions as well as affinity prediction: Identifyingnew leadsDocking and scoring technology is applied at different stages of

the drug discovery process for three main purposes: (1) predicting

the binding mode of a known active ligand; (2) identifying new

ligands using virtual screening; (3) predicting the binding affinities

of related compounds from a known active series [118]. The

identification of novel lead compounds via traditional approaches

(like high-throughput screening) has been more fruitful compared

with the low hit rates observed with combinatorial methods

[119,120]. Practically ‘lead’ identification utilizing in silico rather

than via traditional approaches are faster and economical, as well

as easier to setup. Indeed, screening of large libraries has been used

in combination with (or in parallel to) or sometimes substituted by

virtual or in silico approaches [121].

Among the most commonly used virtual library/ligand screen-

ing (VLS) tools are docking methods, which have been success-

fully used to predict the binding modes and affinities of many

potent enzyme inhibitors as well as receptor antagonists. As a

result, many drugs developed partly by computer-aided structure-

based drug design methods are in late-stage clinical trials or have

now reached the market [122]. Speeding-up the drug discovery

process necessitate the predictive in silico procedures capable of

reducing or simplifying the synthetic and/or combinatorial chal-

lenge. Docking-based VLS methods have been developed and

successfully applied to a number of pharmaceutical targets

[121]. Figure 10 shows flowchart of a real life successful VLS

example [123].

Ultimately, docking and/or scoring programmes should be able

to identify novel potential ‘binders’ very accurately. Currently

other strategies, such as post-docking strategies or smart selection

of docked compounds, are used to reduce the number of false

positive and negatives [121].

e-based VLS, Thermolysin (a Zn-metalloproteinase) as target protein, and

highly potent compound has been experimentally identified as Thermolysin




FIGURE 11

2D molecular structures of edrophonium-like ammonium salts reported recently by Leonetti et al. (2008) [81].

FIGURE 12

Molecular structure of CBC-171-08-IIIf a a,b-dehydrophenylalanine cholineester reported by Grigoryan et al. during 2008 [97].

Review
Some case studies: docking studies of some inhibitors
of cholinesterases (AChE, BChE, etc.)Edrophonium-like ammonium saltsVery recently Leonetti et al. reported a number of mono-quatern-

ary and bis-quaternary ammonium salts, containing edropho-

nium-like and coumarin moieties tethered by an appropriate

linker, demonstrated to be highly potent and selective (over BChE)

dual binding inhibitors of AChE [81] (Figure 11).

Typical running docking studies are being carried out princi-

pally to investigate the effects on affinity of cation–p, p–p stack-

ing, and other non-bonded (like hydrophobic) interactions

involving charged and aromatic molecular moieties of our inhi-

bitors and the electron-rich W86 and W286 amino acid side chains

located in the catalytic and peripheral binding sites of AChE,

respectively [81].

Leonetti et al. performed docking studies first on the most active

AChE inhibitor BMC-08-12 (IC50 = 0.17 nM), while scaffold

match constraint was adopted to perform docking simulations

with the other selected inhibitors [81]. Top-scored docking pose of

BMC-08-12 (50.16 kJ/mol) displayed a cation–p interaction

between the trimethylammonium groups and the electron-rich

side chain of W86, a highly specific hydrogen bond between the

phenolic hydroxyl and an oxygen atom of the hydroxyl group of

S203 and a potential p–p stacking between the aromatic moiety of

the ligand and the aromatic ring(s) of W286 in the PAS. Similar

docking studies showed that the top-scored docking pose

(58.13 kJ/mol) of the most active hetero-bivalent inhibitor

BMC-08-14, displayed a binding pattern similar to that of

BMC-08-12. However, the p–p stacking interaction of the cou-

marin moiety was probably slightly weaker than the combined p–

p and cation–p interactions involving the phenyl-trimethylam-

monium moiety. Major interactions underlying the binding of the

strong inhibitors BMC-08-12 and BMC-08-14 took place at an

optimal distance assured by a four methylene linker. Their mole-

cular modelling results were found to be in full agreement with the

experimental affinities [81].

Dehydroamino acid choline estersRecently Grigoryan et al. during 2008 reported synthesis and

cholinesterase inhibitory profiles of number of dehydroamino

acid choline esters. Their affinity has been measured for the

inhibition of human red cell AChE and human plasma BChE

[97]. The most potent compound was a choline ester of dehydro-

phenylalanine where the amine group of the amino acid was


derivatized with a benzoyl group containing a methoxy in the

2-position (for structure see Figure 12).

Molecular docking studies of this compound (CBC-171-08-

IIIf) into the active site of the human BChE (PDB code 1p0i)

have been performed utilizing AutoDock 3.0.5 version and showed

that the two benzene rings of the lowest energy conformer

oriented towards Trp82 and Tyr332 whereas the positively charged

nitrogen group have been stabilized by Trp231. This orientation

placed the ester group 3.89 A from the active site Ser198, a distance

too far for covalent bonding, explaining why the esters are inhi-

bitors rather than substrates. The negatively charged carbonyl

oxygen in the peptide bond is stabilized by interaction with the

positively charged choline, which is a crown on the carbonyl

oxygen. A different structure is found when the compound is in

the active site of BChE or AChE [97].

Authors also docked the same compound into the structure of

human AChE. The orientation of CBC-171-08-IIIf in AChE was

completely different from its orientation in BChE. The linear

molecule extended from the peripheral anionic site to the bottom

of the gorge. The two benzene rings occupied the entrance of the

active site gorge, where one benzene ring interacted with Trp286

and Tyr72, while the other benzene ring interacted with Ser293

and Tyr341. The compound blocked access of substrate to the

active site of AChE, thus explaining why this is a competitive

inhibitor [97].


Review

This class of anticholinesterase agents has the potential for

therapeutic utility in the treatment of disorders of the cholinergic

system [97].

N-aryl derivativesCorrea-Basurto et al. in 2007, performed and reported molecular

docking studies and density functional theory (DFT) of 88 N-aryl

derivatives and for some AChE and BChE [17]. On the basis of the

results obtained from their modelling studies, some of compounds

have been synthesized and tested kinetically in vitro against AChE.

Some chemical properties of the N-aryl derivatives have been cal-

culated, like partition coefficient (p) and molecular electrostatic

potentials (MESPs) whereas their electronic effects (r) have been

derived from the literatures [17]. The results showed that all com-

pounds act inside the AChE gorge, making pep interactions and

hydrogen bonds with Trp86 and Ser203 and by high HOMO ener-

gies of Ser2003 and high LUMO energies of N-aryl derivatives. The

theoretical calculations for AChE are in agreement with the experi-

mental data, whereas such calculations for BChE do not show the

similar behaviours that could be due to the fact that in spite of both

ChEs displaying similar functional activities they do possess impor-

tant structural differences at their catalystic sites [17].

Their docking studies suggest that all the tested compounds

bind at the active site of both ChEs. This could be due to the fact

that they have an aromatic ring and a nitrogen atom, like other

ChE inhibitors [124,125]. However, there are several functional

groups that modify the electronic density on the aromatic ring and

the N atom, which might change the affinity between the ligands

and the enzymes. Molecular docking calculations allow predicting

the structure of all the complexes between the enzymes and the

ligands, thus suggesting the kind of interaction [17]. The p-p

interaction plays an important role, giving the ligand-AChE com-

plexes high stability and at the same time improving the recogni-

tion process between this enzyme and the compounds. The pep

interaction is formed between the aromatic ring of the ligands and

the aromatic ring from the Trp86 of AChE [30].

Concluding remarks, Future prospects and challengesToday, multiple novel ligands have been predicted and confirmed

by experiment, even to atomic resolution. Docking routinely

treats ligand flexibility and typically includes some receptor

plasticity, and scoring functions include most of the terms in

molecular mechanics force fields. Docking is now used by almost

every major pharmaceutical company. But it is also true that

docking seems to have reached a plateau and is waiting for an

important breakthrough [118]. In spite of the breathtaking

advancements in the field during the last decades and the wide-

spread application of docking methods, several downsides still

exist. In particular, protein flexibility—a crucial aspect for a

thorough understanding of the principles that guide ligand bind-

ing in proteins—is a major hurdle in current protein–ligand

docking efforts that needs to be more efficiently accounted for

[126].

Molecular modelling approaches, like molecular docking, mole-

cular dynamic (MD) simulations, linear interaction energies (LIE),

etc., allowed in depth analysis and interpretation of the structure–

affinity relationships and increased our understanding of the main

binding interactions taking place at the AChE binding sites.

Besides an expected important role played by cation–p

[127,128], p–p stacking, hydrophobic, and other non-bonded

interactions [129], the key role of a phenolic hydroxyl forming

a highly specific hydrogen bond with an oxygen atom of the

hydroxyl group of Ser 203, as already observed with edrophonium

[130], was confirmed [81].

Although ChEIs including tacrine, donepezil and galantamine

have been used for symptomatic treatment of patients with AD,

these conventional treatments fail to postpone the progression of

the disease [2,131–133]. The trend in future AD therapy has been

shifted from traditional anti-AChE treatment to multiple mechan-

isms-based treatments aiming amyloid plaques development and

amyloid peptides (Ab)-mediated cytotoxicity, and neurofibrillary

tangles generation [2].

The best treatments for the AD should be not only effectively

improving the dementia symptoms but also fundamentally redu-

cing the burden of senile plaques and neurofibrillary tangles and

thus protect the neurons from degeneration [2].

At present several molecules that either affect secretory amyloid

precursor degradation, or inhibit amyloid peptides aggregation or

block hyperphosphorylated tau protein formation are under inves-

tigation in preclinical trials [2].

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Review

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Molecular interactions of cholinesterases inhibitors using in silico methods: current status and...

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