Journal of Molecular Neuroscience

Copyright © 2003 Humana Press Inc.

All rights of any nature whatsoever reserved.
ISSN0895-8696/03/20:369–383/$25.00

ALZHEIMER’S THERAPEUTICS: Cognitive Enhancement

Acetylcholinesterase
A Multifaceted Target for Structure-Based Drug Design of Anticholinesterase
Agents for the Treatment of Alzheimer’s Disease

Harry M. Greenblatt,1 Hay Dvir,1,2 Israel Silman,2 and Joel L. Sussman*,1

Departments of 1Structural Biology and 2Neurobiology, Weizmann Institute of Science, Rehovot, Israel
Received October 15, 2002; Accepted March 24, 2003

Abstract
The structure of Torpedo californica acetylcholinesterase is examined in complex with several inhibitors that
are either in use or under development for treating Alzheimer’s disease. The noncovalent inhibitors vary greatly
in their structures and bind to different sites of the enzyme, offering many different starting points for future
drug design.

Index Entries: Alzheimer’s disease; anticholinesterase; cholinergic deficiency; drug design; Torpedo.

Introduction
Solution of the three-dimensional (3D) structure
of Torpedo californica acetylcholinesterase (TcAChE)
in 1991 (Sussman et al.) opened up new horizons in
research on an enzyme that had already been the
subject of intensive investigation (Silman and Suss-
man, 1998).
The unanticipated structure of this extremely
rapid enzyme, in which the active site was found to
be buried at the bottom of a deep and narrow gorge,
lined by aromatic residues, led to a revision of the
views then held concerning substrate traffic, recog-
nition, and hydrolysis (Botti et al., 1999). This led to
a series of theoretical and experimental studies,
which took advantage of recent advances in theo-
retical techniques for treatment of proteins, such as
molecular dynamics and electrostatics, and of site-
directed mutagenesis, utilizing suitable expression
systems (Doctor et al., 1998).
The realization that AChE, together with several
other enzymes whose 3D structures were solved at
about the same time (Ollis et al., 1992), was a member
of a new family of proteins sharing a common fold,
the α/β hydrolase fold, likewise opened up a fertile
field of research in which state-of-the-art techniques
of sequence alignment and homology modeling were
utilized (Cygler et al., 1993). The burgeoning number
of members of this new family resulted in founda-
tion of the ESTHER data base at Montpellier (http://
www.montpellier.inra.fr:70/cholinesterase) (Cousin
et al., 1996), to collate and check the large amounts
of data accumulating, and to make them available
and accessible to a large body of users. The fact that
the α/β hydrolase-fold family included several
members that lacked one or more of the residues in
the catalytic triad characteristic of the enzymes in
the family and had earlier been shown to be adhe-
sion proteins suggested alternative functions for
AChE. These, in turn, necessitated fresh approaches
toward analysis of its structure (Botti et al., 1998).
Finally, at a more practical level, the 3D structure
itself, followed not long thereafter by the 3D struc-
tures of complexes with a number of ligands (Harel

*Author to whom all correspondence and reprint requests should be addressed. E-mail: Joel.Sussman@weizmann.ac.il

Journal of Molecular Neuroscience 369 Volume 20, 2003

370
et al., 1993), provided the basis for a rational struc-
ture-based approach to the design of drugs, such as
anti-Alzheimer’s disease (AD) medications (Fisher
et al., 1998), to the treatment of organophosphate
(OP) intoxication (Millard and Broomfield, 1995),
and to the development of new classes of insecti-
cides (Casida and Quistad, 1998), especially taken
in conjunction with the recently solved 3D structures
of human AChE (hAChE) (Kryger et al., 1998a, 2000)
and the Drosophila melanogaster enzyme (DmAChE)
(Harel et al., 2000).
In the following text, after giving a brief back-
ground, we will analyze the binding modes of vari-
ous noncovalent and covalent inhibitors with TcAChE.
Three-Dimensional Structure
Crystal Structure
The 3D structure of TcAChE was solved in 1991
(Sussman et al., 1991), subsequent to solubilization,
purification, and crystallization of the glycophos-
phatidylinositol (GPI)-anchored dimer present in
large amounts in Torpedo electric organ (Sussman et
al., 1988). Solution of the 3D structure indeed proved
that the ChEs contain a catalytic triad, albeit with a
glutamate in place of the aspartate found in the serine
proteases. However, the 3D structure displays a
number of unexpected features (Fig. 1). The active
site is deeply buried, being located almost 20 Å from
the surface of the catalytic subunit, at the bottom of
a long and narrow cavity. This cavity was named the
active-site gorge or, as >60% of its surface is lined by
the rings of conserved aromatic residues, the aro-
matic gorge (Sussman et al., 1991; Axelsen et al.,
1994). Despite the prediction that the anionic site
would contain several negative charges (Nolte et al.,
1980), in fact, only one negative charge is close to the
catalytic site, that of Glu199, adjacent to the active-
site serine, Ser200. Based both on docking of ACh
within the active site (Sussman et al., 1991) and on
affinity labeling (Weise et al., 1990), the quaternary
group of ACh appears to be interacting, via a cation-
π electron interaction (Dougherty and Stauffer, 1990;
Dougherty, 1996), with the indole ring of one of the
conserved aromatic residues, Trp84. Another con-
served aromatic residue, Phe330, is also involved in
the interaction (Harel et al., 1993).
Functional Binding Sites
The unexpected structure of the active site itself
and of the gorge leading to it have been explored
experimentally by two approaches: (1) crystallo-
Journal of Molecular Neuroscience
Greenblatt et al.
graphic studies of complexes of TcAChE with a reper-
toire of ligands (Harel et al., 1993, 1995, 1996; Raves
et al., 1997; Kryger et al., 1999); and (2) site-directed
mutagenesis (for literature, see Doctor et al., 1998).
Crystallographic studies, using suitable quater-
nary ligands (Harel et al., 1993, 1996), confirmed the
prediction mentioned above, made on the basis of
computerized docking of ACh, by clearly showing
that such ligands interact with Trp84 and, to a lesser
degree, with Phe330. Elucidation of the structure of
a complex with a powerful transition-state analog
(Harel et al., 1996) also bore out the prediction of a
three-pronged oxyanion hole (Sussman et al., 1991).
Modeling studies (Harel et al., 1992) strongly sug-
gested that the acyl pocket for the acetyl group of
ACh is provided by two more conserved aromatic
residues, Phe288 and Phe290, as was later confirmed
by inspection of the complex with a transition-state
analog (Harel et al., 1996). Finally, inspection of the
structure of a complex with the elongated bisqua-
ternary ligand, decamethonium, which was shown
to bind along the active-site gorge, permitted iden-
tification of the peripheral anionic site at the entrance
to the active-site gorge. The peripheral site involves
three more conserved aromatic residues, Tyr70,
Trp279, and Tyr121 (Harel et al., 1993).
Correlation with Site-Directed
Mutagenesis Studies
Extensive site-directed mutagenesis studies, car-
ried out primarily by the laboratories of Shafferman
and Taylor (see, for example, Ordentlich et al., 1993;
Taylor & Radic, 1994; Shafferman et al., 1994, 1995)
have supplemented the structural data. A complete
list of mutations is available from the ESTHER server
(see above). A few key issues addressed are as fol-
lows. Site-directed mutagenesis of Phe288 and
Phe290, the two aromatic residues shaping the acyl
pocket, was shown to broaden specificity, thus
enabling AChE to hydrolyze butyrylcholine, nor-
mally only hydrolyzed by butyrylcholinesterase
(BChE), which on the basis of sequence alignment
and molecular modeling, possesses a larger acyl
pocket in which these two aromatic residues are
replaced by aliphatic residues (Harel et al., 1992;
Ordentlich et al., 1993; Vellom et al., 1993). Mutation
of Trp84 to an aliphatic residue clearly demonstrated
the key role played by this residue in recognition of
the quaternary group of ACh (Ordentlich et al., 1993).
Mutation of the aromatic groups in the peripheral
site drastically reduced affinity for peripheral site
ligands, such as propidium (Harel et al., 1992; Radic
Volume 20, 2003
Acetylcholinesterase
Fig. 1. Ribbon diagram of the 3D structure of TcAChE. α-Helices are represented by helical coils, and β strands, by
arrows. The residues of the catalytic triad (Ser200, His440, Glu327) are rendered as bold sticks, and the arrow indicates
the entrance to the gorge. N and C termini are labeled.
et al., 1993). Radic and coworkers elegantly demon-
strated the contribution of these residues to the bind-
ing of fasciculin (Radic et al., 1994). Thus, the triple
mutant in which the residues in mouse AChE equiv-
alent to Tyr70, Trp84, and Tyr121 were all eliminated,
bound fasciculin 108-fold more weakly than the wild-
type enzyme, with an affinity equal to that of BChE,
which lacks all three of these aromatics and, conse-
quently, also the peripheral site (Harel et al., 1992).
Thus the site-directed mutagenesis studies, in gen-
eral, confirmed the structural assignments, high-
lighting the synergistic value of utilizing these
experimental approaches in tandem.
3D Structures of Complexes with Drugs
The drugs and toxins that display AChE action
can be broadly divided into two classes, those that
bind noncovalently and reversibly, and those that
form a covalent bond, including the OPs and car-
bamates, which either serve as slow substrates or
inhibit irreversibly (Aldridge and Reiner, 1972).
These latter reagents, which act by blocking the
active-site serine, comprise a rather homogeneous
class of inhibitors, as they effectively mimic the first
stage of substrate hydrolysis (Silman et al., 1999).
Thus, they must display the correct steric and
Journal of Molecular Neuroscience
371
mechanistic characteristics for phosphorylating or
carbamylating the active site, with possible
enhancement of their affinity by recognition of suit-
able groups within the substrate-binding site, such
as those that recognize the acyl group and quater-
nary nitrogen of ACh. In contrast, the ligands that
inhibit ChEs reversibly are a heterogeneous class of
molecules, some of which bear no obvious resem-
blance to ACh. Consequently, if one is considering
ligands of potential use as drugs or insecticides,
structure-based design may be essential, as QSAR
or computerized docking of the ligand with the
enzyme may not provide the correct orientation. This
was, indeed, the case for huperzine A(HupA), E2020,
and galanthamine (GAL), as will be discussed below.
In the following text, we will briefly review the
structures of conjugates with TcAChE of the anti-AD
drugs, Exelon™ (Bar-On et al., 1998), the physo-
stigmine analog, MF268 (Bartolucci et al., 1999),
(–)-HupA (Raves et al., 1997), its synthetic enan-
tiomer (+)-HupA, HupB (Dvir et al., 2002a), the
tacrine-huperzine hybrid, huprine X (Dvir et al.,
2002a), and E2020 (Kryger et al., 1999). We will also
present recent data on bifunctional compounds
derived from HupA (Wong et al., 2002), and exam-
ine the complex with the AD drug, GAL (Greenblatt
et al., 1999; Bartolucci et al., 2001; Pilger et al., 2001).
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372 Greenblatt et al.

Conjugate with Rivastigmine (Exelon™)

The anti-AD drug, (+)S-N-ethyl-3-[(1-dimethyl-
amino)ethyl]-N-methylphenylcarbamate (rivastig-
mine [ENA-713]; see Fig. 2), belongs to a series of
miotine derivatives, all of which display inhibitory
action towards AChE both in vitro and in vivo (Wein-
stock et al., 1986). Compared to other clinically useful
carbamates, it has a longer duration of action in vivo
and preferentially inhibits AChE of the hippocam-
pus and cortex (Enz et al., 1993). It is already in use
for treatment of AD under the trade name of Exelon.
Rivastigmine carbamylates TcAChE very slowly,
whereas the bimolecular rate constant for inhibition
of hAChE is >1600-fold higher. Spontaneous reacti-
vation of both conjugates, is however, extremely
slow, as compared to most carbamyl-ChE conjugates,
with <10% reactivation being observed for TcAChE
after 48 h. The crystal structure of the conjugate of
rivastigmine with TcAChE (Bar-On et al., 1998)
showed that the carbamyl moiety, as expected, is
linked to the active-site serine, with the leaving
group, (–)-S-3-[1-(dimethylamino)ethyl]phenol
(NAP), being retained in the anionic site (Fig. 3). A
significant movement was observed of the active-
site histidine (H440) away from its normal hydrogen-
bonding partner, E327, to form a hydrogen bond
with E199, adjacent to the active-site serine, S200,
thus disrupting the catalytic triad. This disruption
Fig. 2. Chemical structures of inhibitors discussed in this may provide an explanation for the unusually slow
article. Note that NAP is the leaving group resulting from the kinetics of reactivation. It is of interest that a simi-
reaction of rivastigmine (ENA-713) with the active-site serine. lar disruption of the catalytic triad is observed in the

Journal of Molecular Neuroscience Volume 20, 2003

Acetylcholinesterase 373

Fig. 3. Active site of TcAChE after inhibition with rivastigmine. Amino acid residues within the active site of AChE
that may interact with the inhibitor are shown. Rivastigmine is depicted with larger spheres and thicker lines for empha-
sis. The carbamyl portion of rivastigmine is positioned to make two H-bonds (dashed lines) with the amide nitrogens of
A201 and G119, as well as nonbonded contacts (dotted lines) with F288 and F290 in the acyl pocket. NAP, the aromatic
leaving group of rivastigmine, remains in the active site of crystalline AChE. NAP is within H-bonding distance of three
water molecules (large red spheres), as well as of the amide nitrogen of G118. Nonbonding contacts and π–π interac-
tions with W84 and F330 are depicted as dotted lines.

crystal structure of the conjugate of TcAChE with
the potent OP nerve agent, VX (Millard et al., 1999).
Conjugate with the Physostigmine Analog
MF268
8-(cis-2,6-dimethylmorpholino)octylcarbamoyle-
seroline (MF268; see Fig. 2) is a long-chain analog of
physostigmine. Whereas AChE inhibited by
physostigmine is reactivated quite rapidly, long-
chain physostigmine analogs form much more stable
conjugates; and MF268 behaves in vitro as an almost
irreversible inhibitor (Perola et al., 1997). Bartolucci
and coworkers used X-ray crystallography to inves-
tigate this phenomenon by solving the crystal struc-
ture of the conjugate of MF268 with TcAChE. In the
crystal structure, the dimethylmorpholinooctylcar-
bamic moiety of MF268 is covalently bound to the
active-site serine, Ser200. The alkyl group of the
inhibitor fills the upper part of the gorge, thus block-
ing the entrance to the active site. This prevents the
leaving group, eseroline, from exiting by this route.
Surprisingly, however, the bulky eseroline moiety is
not seen in the crystal structure, implying the exis-
tence of an alternative route, viz. a back door, for its
clearance.
Complexes with the Huperzine
Family of Inhibitors
(–)-HupA and (–)-HupB (Fig. 2) are two
Lycopodium alkaloids isolated from the herb Huperzia
serrata, which grows mainly in hilly regions in the
provinces of southern China (Liu et al., 1986). This
herb has been used in traditional Chinese medicine
for centuries to treat contusion, strain, swelling,
schizophrenia, and other ailments (Bai et al., 2000).
Both compounds are potent reversible inhibitors of
AChE (Wang et al., 1986; Kozikowski et al., 1991).
(–)-HupA, the most studied compound of the two,
has already been approved as a drug for the symp-
tomatic treatment of AD in China. A number of
studies, utilizing either computerized docking
techniques and/or site-directed mutagenesis
(Ashani et al., 1994; Pang and Kozikowski, 1994b;

Journal of Molecular Neuroscience Volume 20, 2003

374
Saxena et al., 1994), predicted various possible ori-
entations for (–)-HupAwithin the active site of AChE.
However, the crystal structure of TcAChE in com-
plex with (–)-HupA (Raves et al., 1997) showed an
unexpected orientation for the inhibitor, involving
few strong direct interactions with the protein.
The crystal structure of its analog, (–)-HupB, with
TcAChE (Dvir et al., 2002a) shows that the only sig-
nificant differences involve the interaction with the
extra ring of (–)-HupB and the ethylidene of (-)-HupA
with the enzyme. Although (–)-HupB appears to
make more intimate van der Waals or CH···π inter-
actions with Trp84 and Phe330, the latter utilizes its
ethylidene methyl group to make a C-H···O bond
with the main chain carbonyl of His440, which cannot
be formed with (–)-HupB. The twofold higher
potency of (–)-HupA for TcAChE (Dvir et al., 2002a)
was attributed to this difference.
The effects of (–)-HupA and its synthetic enan-
tiomer, (+)-HupA, on the NMDA receptor and in
protecting against β-amyloid toxicity were shown
to be similar. In contrast, the stereoselectivity of
HupA for AChE is substantial, the natural com-
pound, (–)-HupA, being almost 2 orders of magni-
tude more potent than the synthetic (+)-HupA
(McKinney et al., 1991; Saxena et al., 1994). Never-
theless, comparison of the crystal structures of the
complexes of TcAChE with both enantiomers (Dvir
et al., 2002a) shows that the α-pyridone moieties of
both molecules are oriented very similarly and prac-
tically overlap. To accomplish this, (+)-HupA must
be flipped relative to (–)-HupA, so that its ethyli-
dene group points away from the anionic site.
Instead, the three-carbon bridge of (+)-HupA binds
to the anionic site. This shows that the most power-
ful interactions made by HupA and its analogs can
be attributed to their α-pyridone group.
The most striking observation seen in the struc-
tures of all three Hup complexes is the flip of the
peptide bond between Gly117 and Gly118. This
conformational change appears to be induced by a
carbonyl-carbonyl repulsion (Fig. 4). The new
conformation is stabilized by Gly117O making
H-bonds with Gly119N and Ala201N, the other two
functional elements of the three-pronged oxyanion
hole. As a consequence, the former position of
Gly118N in the oxyanion hole is occupied by the car-
bonyl of Gly117. All three inhibitors would thus be
expected to abolish hydrolysis of all ester substrates,
even those that do not utilize the anionic site.
Journal of Molecular Neuroscience
Greenblatt et al.
It was proposed that the peptide flip itself is respon-
sible for the low on-rates observed for inhibition of
AChE by (–)-HupA (Raves et al., 1997; Dvir et al.,
2002a). Similarly it was hypothesized (Dvir et al.,
2002a) that its stabilization may contribute to the low
rates of dissociation observed (Ashani et al., 1992).
Finally, modeling a tyrosine residue in place of
Phe330 in TcAChE (Dvir et al., 2002b) revealed the
possibility of the primary amino group of (–)-HupA
hydrogen bonding to the tyrosine hydroxyl. The 5- to
40-fold higher affinity of (–)-HupA for mammalian
AChE was attributed to this possible interaction, which
does not exist in the corresponding TcAChE complex.
Complex of TcAChE with Huprine X
Superposition of the structures of TcAChE com-
plexed with the two Alzheimer’s drugs, tacrine and
(–)-HupA, shows that their binding sites are adja-
cent and overlap partially (Fig. 5). This suggested
that a combination of these two ligands might result
in a hybrid compound with binding properties
resembling those of both parents. Recently, the syn-
thesis and pharmacological evaluation of a series of
compounds that combine pharmacophores of
(–)-HupA and tacrine have been reported. Some of
these hybrids are potent inhibitors of AChE (Badia
et al., 1998; Carlier et al., 1999b). Indeed certain
hybrids, which contain the 4-aminoquinoline sub-
structure of tacrine and a carbobicyclic moiety resem-
bling that of (–)-HupAbut lack the ethylidene moiety
of HupA, display more powerful anticholinesterase
activity than either tacrine or (–)-HupA. These
hybrids were named huprines, and huprine X
(Fig. 2), the most powerful of the series, inhibited
hAChE with an inhibition constant of 26 pM, being
180-fold more potent than (–)-HupA and 1200-fold
more potent than tacrine (Camps et al., 2000). The 3D
structure of TcAChE in complex with huprine X (Dvir
et al., 2002b) shows that the inhibitor binds to the
anionic site and also hinders access to the esteratic
site. Its aromatic portion occupies the same binding
site as tacrine, stacking between the aromatic rings
of Trp84 and Phe330, whereas the carbobicyclic unit
occupies the same binding pocket as (–)-HupA
(Fig. 6). Its chlorine substituent was found to lie in a
hydrophobic pocket interacting with the rings of the
aromatic residues Trp432 and Phe330 and with the
methyl groups of Met436 and Ile439. Other steady-
state inhibition data (Dvir et al., 2002b) show that
huprine X binds to hAChE and TcAChE with Ki values
Volume 20, 2003
Acetylcholinesterase 375

Fig. 4. Effect of binding of (+)-HupA (light gray), (–)-HupA (dark gray), and (–)-HupB (medium gray) on the oxyanion
hole of TcAChE. The carbonyl group of all three huperzines binds in a similar position relative to the carbonyl of G117,
resulting in the close contact of ~2.8 Å shown (light gray dashed line), and presumably causes the observed conforma-
tional change.

of 0.67 and 0.13 nM, being 28- and 54-fold, respec-
tively, more potent than tacrine. This difference stems
from the fact that the aminoquinoline moiety of
huprine X makes interactions similar to those made
by tacrine, but additional interactions with the
enzyme are made by the huperzine-like substructure
and the chlorine atom. Furthermore, both tacrine and
huprine X bind more tightly to the Torpedo enzyme
than to hAChE, suggesting that their quinoline sub-
structures interact better with Phe330 than with
Tyr337, the corresponding residue in the hAChE
structure. Both (–)-HupAand huprine X display slow
binding properties, but only binding of the former
causes a peptide flip of Gly117. Based on this, it was
suggested that the relatively low association rate of
huprine X may be caused by its slow diffusion rate
down the active-site gorge owing to its bulky rigid
structure (Dvir et al., 2002b).
Complexes with Huperzine-Like
Bivalent Compounds
The fact that (–)-HupA is a natural product whose
chemical synthesis is relatively difficult, has dis-
couraged its development as a prescription drug for
treatment of AD. Simplified analogs of HupA, which
are more readily synthesized, have been used suc-
cessfully as building blocks for bis-acting compounds
(Carlier et al., 1999a, 1999b; Han et al., 1999) as had

Journal of Molecular Neuroscience Volume 20, 2003

376
Fig. 5. Structure of TcAChE/(–)-HupA complex (dark gray) superimposed on the structure of TcAChE/tacrine (light
gray), showing the partially overlapping binding sites of these ligands.
been shown previously for bis-tacrine compounds
(Pang and Kozikowski, 1994b). These compounds
are targeted to bind to both anionic sites (Trp84 and
Trp279) (Harel et al., 1993), with methylene linkers
of varying lengths joining the two functional groups
(Fig. 2) (Carlier et al., 2000). Despite the loss of bind-
ing affinity of the individual precursors because of
their simplification, linkage of these building blocks
results in compounds with greater affinity for AChE
than that of (–)-HupA. Thus, a linker of 10 methyl-
ene units yields E10E, which binds to TcAChE >170-
fold more tightly than (–)-HupA. A linker of 12 units
yields a less effective compound, E12E, which has
~25-fold greater affinity than (–)-HupA. In rat AChE,
however, the relative affinities are reversed; thus,
E12E has greater affinity than E10E. The recently
solved crystal structures of these compounds in
TcAChE provide insight into the underlying causes
of this species specificity (Wong et al., 2002).
Journal of Molecular Neuroscience
Greenblatt et al.
Complex with E2020 (Aricept™)
E2020 (Aricept™; Fig. 2), is a member of a large
family of N-benzylpiperidine-based AChE
inhibitors, developed, synthesized, and evaluated
by the Eisai Company in Japan (Kawakami et al.,
1996). It was approved for treatment of AD by the
FDA in 1996 (Nightingale, 1997) and shows high
selectivity for AChE relative to BChE (Sugimoto et
al., 1995). The earlier modeling studies attributed
this differential specificity to differences in geome-
try within the active sites of AChE and BChE (Car-
dozo et al., 1992). However, more recent studies,
carried out subsequent to determination of the 3D
structure of TcAChE, suggested that E2020 and other
Eisai compounds orient along the active-site gorge
and that the differential specificity can be attributed
to structural differences between AChE and BChE at
the top of the gorge, at the peripheral anionic site
Volume 20, 2003
 Fig. 6. Superposition of TcAChE/huprine X complex (dark gray) with TcAChE/tacrine complex (light gray) (A) and
TcAChE/(–)-HupA complex (light gray) (B).

Journal of Molecular Neuroscience Volume 20, 2003

378 Greenblatt et al.

Fig. 7. Binding mode of E2020 to TcAChE. E2020 is displayed as a ball-and-stick model. Water molecules are repre-
sented as light pink balls; standard H-bonds, as dotted orange lines; aromatic H-bonds, π-cation, and stacking interac-
tions, as dark lines. Note the multiple water-mediated contacts of E2020 atoms with the protein.

(Pang and Kozikowski, 1994a; Kawakami et al.,
1996). The 3D structure of the complex of E2020-
TcAChE fully confirms these latter assignments (Fig.
7) (Kryger et al., 1998b, 1999). It can be seen that
E2020 interacts with both the anionic subsite of the
active site, at the bottom of the gorge, and with the
peripheral anionic site, near its top, via aromatic
stacking interactions with conserved aromatic
residues. The piperidine nitrogen is also involved in
a cation-π electron interaction with the phenyl ring
of Phe330. E2020 does not, however, interact directly
with either the catalytic triad or the oxyanion hole,
but only indirectly, via solvent molecules. These loci,
and a finger-shaped void toward the acyl pocket,
provide spaces into which substituents could fit, thus
yielding analogs of E2020 with increased affinity and
selectivity.
Complex with GAL
(–)-Galanthamine (GAL) is a naturally occurring
alkaloid from the lily family of plants, in particular,
the common snowdrop (Galanthus nivalis). Since its

Journal of Molecular Neuroscience Volume 20, 2003

Acetylcholinesterase
Fig. 8. Initial difference electron density map from the TcAChE/GAL complex. GAL is rendered as a ball-and-stick
model, and protein atoms are rendered as stick models.
discovery >45 years ago, it has been tested in vari-
ous neurological applications (Bretagne and Valletta,
1965; Gujral, 1965; Bystrzanowska, 1969; Harvey,
1995). GAL has an IC50 value of 0.35 µM for human
erythrocyte AChE (Thomsen and Kewitz, 1990) and
an IC50 value of 0.652 µM for TcAChE (T. Lewis and
C. Personeni, unpublished results). It also acts as a
nicotinic activator of both ganglionic and muscle
receptors (Storch et al., 1995; Schrattenholz et al.,
1996), and of nicotinic receptors in the brain (Pereira
et al., 1993). In fact, evidence indicates that the effect
on the receptor also plays a role in cognitive improve-
ment (Santos et al., 2002). GAL is now in use for treat-
ment of AD in Europe and the United States.
Journal of Molecular Neuroscience
379
Theoretical predictions as to the binding mode of
GAL to TcAChE did not produce unambiguous
models; indeed, the most favored model (R. Fletcher,
R. Viner, and T. Lewis, unpublished results) is not
in agreement with the experimentally determined
3D structure.
The crystal structure of the TcAChE/GAL com-
plex (Greenblatt et al., 1999; Bartolucci et al., 2001)
reveals that the inhibitor is bound at the bottom of
the gorge and interacts with both Trp84 and the acyl
binding pocket (Fig. 8). Only one direct hydrogen
bond is formed between the inhibitor and protein
side chains: between the hydroxyl group of the
inhibitor and Glu199 Oε1 (2.7 Å). A second interac-
Volume 20, 2003
380
tion is possible between the hydrogen of Ser200 Oγ
and the O-methoxy group of GAL, but this would at
best be a bifurcated interaction, with the second
acceptor being His440 N ε2 of the catalytic triad.
The rest of the possible hydrogen bonds involve water
molecules bound to the enzyme. The reasonably tight
association observed for this inhibitor to AChE pre-
sumably gains a significant contribution from favor-
able entropy contributions owing to the rigid nature
of the inhibitor and displacement of some bound
water molecules present in the native structure.
Acknowledgments
This work was supported by the U.S. Army Med-
ical Research Acquisition Activity under contract no.
DAMD17-97-2-7022, the EC IVth and Vth Frame-
work Quality of Life Programs, the Kimmelman
Center for Biomolecular Structure and Assembly,
Israel, the Nella and Leon Benoziyo Center for
Neurosciences. I.S. is Bernstein-Mason Professor of
Neurochemistry. J.L.S. is the Morton and Gladys
Pickman Professor of Structural Biology.
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