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DRUG DEVELOPMENT RESEARCH 50:573–583 (2000)
Research Overview
Structural Studies on Vertebrate and Invertebrate
Acetylcholinesterases and Their Complexes With
Functional Ligands
Harry M. Greenblatt,1 Israel Silman,2 and Joel L. Sussman1*
1
Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel
2
Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel
Strategy, Management and Health Policy
Venture Capital
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Enabling
Research
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Preclinical Development
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Phases I-III
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Phase IV
ABSTRACT
The structure of Torpedo californica acetylcholinesterase (AChE) was examined in complex with several inhibitors which are either in use or under development for treatment of Alzheimer’s
disease. The inhibitors vary greatly in their structures, offering differing starting points for future drug
design. The structure of T. californica AChE is also compared to the recently solved structure of Drosophila melanogaster AChE, the first invertebrate enzyme to have its structure determined. The structure of
D. melanogaster AChE complexed with a potential insecticide is described. Drug Dev. Res. 50:573–583,
2000. © 2000 Wiley-Liss, Inc.
Key words: cholinesterase; AChE; ligands
INTRODUCTION
Solution of the three-dimensional (3D) structure of
Torpedo californica acetylcholinesterase (TcAChE) in
1991 [Sussman et al., 1991] opened up new horizons in
research on an enzyme which was already the subject of
intensive investigation [Silman and Sussman, 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, recognition, and hydrolysis
[Botti et al., 1999]. This led to a series of theoretical and
experimental studies which took advantage of recent advances in theoretical techniques for the 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 align© 2000 Wiley-Liss, Inc.
ment and homology modeling were utilized [Cygler et
al., 1993]. The burgeoning number of members of this
new family resulted in the foundation of the ESTER database 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 which lacked one or more of the residues in the catalytic triad characteristic of the enzymes in the family, and
which had earlier been shown to be adhesion proteins,
suggested alternative functions for AChE. These, in turn,
Contract grant sponsor: the U.S. Army Medical Research Acquisition Activity; Contract grant number: DAMD17-97-2-7022;
Contract grant sponsors: the European Union IVth Framework in
Biotechnology, the Kimmelman Center for Biomolecular Structure
and Assembly, Israel, the Nella and Leon Benoziyo Center for Neurosciences.
*Correspondence to: Joel L. Sussman, Dept. of Structural Biology, Weizmann Institute of Science, Rehovot, Israel. E-mail:
[email protected]
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GREENBLATT ET AL.
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 structures of
complexes with a number of ligands [Harel et al., 1993],
provided the basis for a rational structure-based approach
to the design of drugs such as anti-Alzheimer’s medications [Fisher et al., 1998], to the treatment of organophosphate (OP) intoxication [Millard and Broomfield, 1995],
and to the development of new classes of insecticides
[Casida and Quistad, 1998], especially taken in conjunction with the recently solved 3D structures of human AChE
(hAChE) [Kryger et al., 1998, submitted], and of the Drosophila melanogaster enzyme (DmAChE) [Harel et al., 2000].
In the following, after a brief background, we analyze the binding modes of various noncovalent and covalent inhibitors with TcAChE. We then compare the
structure of the vertebrate enzyme to that of the insect
enzyme (DmAChE), and discuss binding of potential insecticides to the latter.
the serine proteases. However, the 3D structure displays
a number of unexpected features (Fig. 1). The active site
is deeply buried, 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,
since over 60% of its surface is lined by the rings of conserved aromatic residues, the aromatic gorge [Axelsen et
al., 1994; Sussman et al., 1991]. 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, 1996; Dougherty
and Stauffer, 1990], with the indole ring of one of the
conserved aromatic residues, Trp84. Another conserved
aromatic residue, Phe330, is also involved in the interaction [Harel et al., 1993].
THREE-DIMENSIONAL STRUCTURE
Crystal Structure
Functional Binding Sites
The 3D structure of TcAChE was solved in 1991
[Sussman et al., 1991], subsequent to solubilization, purification, and crystallization of the 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 unexpected structure of the active site itself,
and of the gorge leading to it, have been explored experimentally by two approaches: 1) crystallographic studies
of complexes of TcAChE with a repertoire of ligands
[Greenblatt et al., 1999; Harel et al., 1993, 1995, 1996;
Kryger et al., 1999; Raves et al., 1997]; 2) site-directed
mutagenesis [see Doctor et al., 1998].
Crystallographic studies, using suitable quaternary
Fig. 1. Ribbon diagram of the
3D structure of TcAChE. α-Helices are represented by helical
coils, and β-sheets by arrows. The
residues of the catalytic triad
(Ser200, His440, Glu327) are rendered as bold sticks; the arrow
indicates the entrance to the
gorge. N- and C-termini are labeled.
CHOLINESTERASE LIGAND COMPLEXES
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 suggested that the acyl pocket for the acetyl
group of ACh is provided by two more conserved aromatic residues, Phe288 and Phe290, as 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 bisquaternary ligand,
decamethonium, which was shown to bind along the active-site gorge, permitted identification of the “peripheral” anionic site at the entrance to the active-site gorge.
The “peripheral” site involves three more conserved aromatic residues, Tyr70, Tyr121, and Trp279 [Harel et al.,
1993], and is also the site of interaction with the snake
venom toxin, fasciculin, which thereby blocks the entrance to the gorge [Bourne et al., 1995; Harel et al., 1995].
Correlation With Site-Directed Mutagenesis Studies
Extensive site-directed mutagenesis studies, carried out primarily in the laboratories of Shafferman and
Taylor [see, e.g., Ordentlich et al., 1993; Shafferman et
al., 1994; Taylor and Radic, 1994] have supplemented the
structural data. A complete list of mutations is available
from the ESTHER server (see above). A few key issues
addressed are: 1) 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, normally 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 et al., 1993].
Radic et al. [1994] elegantly demonstrated the contribution of these residues to the binding of fasciculin. Thus,
the triple mutant in which the residues in mouse AChE
equivalent 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, consequently,
also the peripheral site [Harel et al., 1992]. Thus the sitedirected mutagenesis studies, in general, confirmed the
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structural assignments, highlighting the synergistic value
of utilizing these experimental approaches in tandem.
3D STRUCTURES OF COMPLEXES WITH DRUGS
The drugs and toxins which display AChE action
can be broadly divided into two classes, those which bind
noncovalently and reversibly, and those which form a
covalent bond, including the OPs and carbamates, 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, since they effectively
mimic the first stage of substrate hydrolysis [Silman et
al., 1999]. Thus, they must display the correct steric and
mechanistic characteristics for phosphorylating or
carbamylating the active site, with possible enhancement
of their affinity by recognition of suitable groups within
the substrate-binding site, such as those which recognize the acyl group and quaternary nitrogen of ACh. In
contrast, the ligands which inhibit ChEs reversibly are a
heterogeneous class of molecules, some of which bear
no obvious resemblance to ACh. Consequently, if one is
considering ligands of potential use as drugs or insecticides, structure-based design may be essential, since
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, we briefly review the structures of
conjugates with TcAChE anti-Alzheimer drugs, Exelon
[Bar-On et al., 1998], the physostigmine analog MF268
[Bartolucci et al., 1999], HupA [Raves et al., 1997] and it
closely related analog HupB, and E2020 [Kryger et al.,
1999]. We also examine the complex with the potential
Alzheimer drug GAL [Greenblatt et al., 1999]. This will
be followed by a comparison of DmAChE with TcAChE,
and a description of the complex of the insect enzyme with
a potential insecticide, ZAI [Harel et al., 2000] (see Fig. 2).
Conjugate With ENA-713 (Exelon™)
The anti-Alzheimer’s drug, (+)S-N-ethyl-3-[(1dimethylamino)ethyl]-N-methylphenylcarbamate (ENA713; Exelon™) (see Fig. 2), belongs to a series of miotine
derivatives, all of which display inhibitory action toward
AChE both in vitro and in vivo [Weinstock et al., 1986].
Compared to other clinically useful carbamates, it has a
longer duration of action in vivo, and preferentially inhibits AChE of the hippocampus and cortex [Enz et al.,
1993]. Kinetic studies revealed slow inhibition and a significant irreversible component in its interaction with
TcAChE [Bar-On et al., 1998], whereas the analog in
which the bulky leaving group, 3-[(1-dimethylamino)ethyl]phenol (NAP), had been replaced by chloride inhibited more rapidly and yielded a conjugate which
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GREENBLATT ET AL.
Fig. 2. Chemical structures of inhibitors discussed in this article. Note
that NAP is the leaving group resulting from the reaction of ENA-713 with
the active site serine of AChE.
reactivated fully and rapidly upon dilution. The crystal
structure revealed that the active-site serine, Ser200, was
ethylmethylcarbamylated, and that the leaving group,
NAP, remained bound noncovalently in the active site. It
was concluded that ENA-713 can inhibit both by
decarbamylation and by its action as a prodrug to deliver
the leaving group NAP, which is itself a good reversible
inhibitor of AChE.
Conjugate With the Physostigmine Analog MF268
8-(cis-2,6-dimethylmorpholino)Octylcarbamoyleseroline (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 inhibi-
tor [Perola et al., 1997]. Bartolucci et al. [1999] used Xray crystallography to investigate this phenomenon by
solving the crystal structure of the conjugate of MF268
with TcAChE. In the crystal structure, the dimethylmorpholinooctylcarbamic 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 blocking 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 existence of
an alternative route, viz. a “back door,” for its clearance.
Complex With Huperzine A
HupA (Fig. 2) is a nootropic alkaloid used in China
for centuries as a folk medicine [Liu et al., 1986]. It is a
CHOLINESTERASE LIGAND COMPLEXES
potent reversible inhibitor of AChE and is being used in
China for treatment of Alzheimer’s disease [Zhang et al.,
1991]. The structure of HupA reveals no obvious similarity to that of ACh. In fact, a number of studies, utilizing either computerized docking techniques and/or
site-directed mutagenesis [Ashani et al., 1994; Pang and
Kozikowski, 1994b; Saxena et al., 1994], predicted various possible orientations for HupA within the active site
of AChE. It seemed, therefore, desirable to solve the crystal structure of the HupA-TcAChE complex, thus establishing its correct orientation and providing the basis for
future structure-based drug design.
The crystal structure of the HupA-TcAChE complex
(Fig. 3) showed an unexpected orientation for the inhibitor, with surprisingly few strong direct interactions with
the protein to explain its high affinity [Raves et al., 1997].
Even though HupA has three potential hydrogen-bond
donor and acceptor sites, only one strong hydrogen bond
is seen, with Tyr130. The high affinity may be ascribed to
the cumulative effect of a large number of hydrophobic
contacts and of hydrogen bonds with water molecules
within the gorge which are, themselves, hydrogen-bonded
to other waters or to backbone or sidechain atoms of the
protein. Modeling Phe330 as tyrosine, the corresponding
residue in hAChE, permits formation of a 3.3 Å hydrogen
between the hydroxyl oxygen and the primary amino group
of HupA. This additional hydrogen bond, in addition to
π-cation interactions with both Trp84 and Phe(Tyr)330,
577
may explain why HupA binds to human AChE (hAChE)
5–10-fold more strongly than to TcAChE, and only weakly
to BChE, which lacks an aromatic residue at this position
[Ashani et al., 1994]. A consequence of the large number
of contacts mentioned above is that there does not appear
to be much room for additional substituents without causing clashes. Nevertheless, addition of a methyl group near
the amide group of HupA leads to an 8-fold increase in
affinity, probably due to extra hydrophobic contacts with
Trp84 [Kozikowski et al., 1996].
Huperzine B (HupB), a close analog of HupA, can
also be isolated from Huperzia serrata. Like HupA, this
molecule is also an anticholinesterase, with about 10-fold
lower affinity for AChE [Liu et al., 1999]. HupB binds to
TcAChE in a similar fashion to HupA (Dvir et al., in preparation). The main difference appears to be the loss of the
network of water molecules that interacts with the primary amine group of HupB. The possible interaction
between the main chain carbonyl group of His440 and
HupA is also lacking. Two new interactions between
HupB and the enzyme arise from the extra ring, and involve Trp84 and Phe330.
Complex With E2020 (Aricept™)
E2020 (Aricept™; Fig. 2), is a member of a large
family of N-benzylpiperidine-based AChE inhibitors
[Kawakami et al., 1996] and was approved for treatment
of Alzheimer’s disease by the FDA in 1996 [Nightingale,
Fig. 3. Detail of the HupA/
TcAChE complex, showing selected
residues in gorge. HupA is superimposed on the initial difference
electron density maps (4σ contour
level).
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GREENBLATT ET AL.
1997]. It shows high selectivity for AChE relative to
BChE [Sugimoto et al., 1995]. The earlier modeling studies attributed this differential specificity to differences
in geometry within the active sites of AChE and BChE
[Cardozo 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 [Kawakami et al.,
1996; Pang and Kozikowski, 1994a]. The 3D structure of
the complex of E2020-TcAChE fully confirms these latter assignments (Fig. 4) [Kryger et al., 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 Galanthamine
(–)-Galanthamine (GAL) is a naturally occurring alkaloid from the lily family of plants, in particular the common snowdrop (Galanthus nivalis). Since its discovery
more than 45 years ago, it has been tested in various
neurological applications [Bretagne and Valletta, 1965;
Bystrzanowska, 1969; Gujral, 1965; Harvey, 1995]. GAL
has an IC50 value of 0.35 µM for human erythrocyte AChE
[Thomsen and Kewitz, 1990] and an IC50 value of 0.65
µM for TcAChE (T. Lewis and C. Personeni, unpublished
results). It also acts as a nicotinic activator of both ganglionic and muscle receptors [Schrattenholz et al., 1996;
Storch et al., 1995] and of nicotinic receptors in the brain
[Pereira et al., 1993]. GAL has completed Phase III clinical trials in Europe, but has not yet been approved for
treatment of AD.
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, T.
Lewis, unpublished results) is not in agreement with the
experimentally determined 3D structure.
The crystal structure of the TcAChE/GAL complex
[Greenblatt et al., 1999] reveals that the inhibitor is bound
at the bottom of the gorge and interacts with both Trp84
and the acyl binding pocket (Fig. 5). Only one direct hydrogen bond is formed between the inhibitor and protein
sidechains: between the hydroxyl group of the inhibitor
and Glu199 Oε1 (2.7Å). A second interaction 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 Nε2 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 with
AChE presumably gains a significant contribution from
favorable entropy contributions owing to the rigid nature
of the inhibitor, and displacement of some bound water
molecules present in the native structure.
3D Structure of Drosophila melanogaster AChE
Fig. 4. Binding mode of E2020 to TcAChE. E2020 is displayed as a balland-stick model (chiral center marked with a black star). Water molecules
are represented as light gray balls; “standard” H-bonds as heavy dashed
lines; aromatic H-bonds, π-cation, and stacking interactions as light dashed
lines. Note the multiple water-mediated contacts of E2020 atoms with
the protein.
The structural studies on TcAChE [Sussman et al.,
1991] and hAChE [Kryger et al., 1998, 2000], as well as
studies on mouse [Bourne et al., 1999b] and Electrophorus AChEs [Bourne et al., 1999a] showed, as might be
expected from the high degree of sequence homology, that
they all possess a very similar 3D structure. It was obvious, however, both from the diversion in sequences and
from available biochemical data, that substantial structural
differences might be expected between the invertebrate
CHOLINESTERASE LIGAND COMPLEXES
579
insecticides and, thereby, reducing their toxicity toward
humans and other vertebrates.
The recently solved structure of native DmAChE,
and of two complexes with putative insecticides (ZIA and
ZA; Fig. 2), is the first structure of an invertebrate AChE
[Harel et al., 2000]. The structure of DmAChE is similar
to that of vertebrate AChEs in its overall fold, charge
distribution, and deep active-site gorge, but some surface loops deviate by up to 8 Å from their position in the
vertebrate structures, and the C-terminal helix is shifted
substantially. While the lower parts of the active-site
gorges of the insect enzyme and of the vertebrate enzyme are very similar, the upper part of the active-site
gorge of the insect enzyme is more constricted than that
of the vertebrate enzyme, due to the larger size of the
sidechains of certain amino acids lining it. Moreover, the
overall trajectories of the gorges differ by several angstroms (Fig. 6).
Comparison of the 3D structures of DmAChE and
the vertebrate AChE structures indeed suggests candidate residues which can be taken into account in attempting to achieve inhibitor selectivity. The primary
candidates for achieving this selectivity are eight residues clustered near the opening of the gorge, which difFig. 5. Binding of GAL in the active site of TcAChE. GAL is rendered as
a ball-and-stick model, the nitrogen colored dark gray, carbons colored
medium gray, and oxygens colored light gray. Protein atoms are rendered as stick models, colored light gray. The molecular surface of the
gorge is partially cut to reveal the interactions of GAL with Trp84, the acyl
binding pocket (Phe288, Phe 290), and Glu199. Two members of the
catalytic triad (Ser200 and His440) are shown.
and vertebrate enzymes. Solution of the 3D structure of
an insect enzyme would thus be expected to yield structural information which would be valuable for insecticide
design. Knowledge of differences in the 3D structures of
the human and insect enzymes would also, obviously, be
of great importance in the context of exposure of civilians
or military personnel to insecticides or to other pesticides
targeted toward AChE. Crop spraying with insecticides
exposes agricultural workers to high levels of pesticides,
and WHO has estimated that this results in 220,000 deaths
annually from acute exposure [Veil, 1992]. An insect structure would also be relevant in the context of recent findings concerning possible synergistic action of multiple
AChEs in relation to Gulf War syndrome [Abou-Donia et
al., 1996]. Taken in conjunction with knowledge of the
structures of the complexes of TcAChE with a variety of
AChE inhibitors [Harel et al., 1993, 1996], and the recently reported solution of the 3D structure of the complex of hAChE with the snake venom toxin, fasciculin
[Kryger et al., 1998, 2000], an insect structure could provide a rational basis for improving the selectivity of new
Fig. 6. Stereo views of the molecular surfaces in the gorges of DmAChE
and TcAChE. The surfaces are rendered semitransparent using the program DINO [Philippsen, 2000]. Residues that significantly influence the
trajectory and volume (see text) are shown as ball-and-stick. a: Activesite gorge of DmAChE. b: Active-site gorge of TcAChE. Residue Phe 330
is on the far side of the surface.
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GREENBLATT ET AL.
Fig. 7. a: DmAChE residues (light gray) interacting with ZAI (dark gray)
with distances <3.8 Å. b: Nine aromatic residues in native DmAChE
(black) change their conformation when ZAI (gray) shown with its van der
Waals surface, binds to DmAChE.
CHOLINESTERASE LIGAND COMPLEXES
581
fer in the insect and vertebrate sequences. The largest
differences, which are potential targets for inhibitor selectivity, are in Tyr71, Tyr73, Glu80, and Asp375
(DmAChE numbering), which are Asp, Gln, Ser, and Gly,
respectively, in vertebrates. However, differences in residues which do not line the gorge, but interact with gorge
residues (i.e., second shell), may play a role in influencing selectivity.
One known difference between vertebrate and insect AChEs is the latter’s ability to hydrolyze substrates
with larger acyl moieties, e.g., butyrylcholine [Gnagey et
al., 1987]. A possible reason for this difference is that in
the vertebrate acyl binding pocket, the residues equivalent to Leu328 and Phe371 in DmAChE (Phe288 and
Phe331 in TcAChE) are both phenylalanines, and form a
rigid π–π stacking pair. Since residue 328 in DmAChE is
a leucine, the rigid π–π stacking is lost (although the total aromatic nature of the pocket is maintained by the
“gain” of Phe440). The loss of rigid stacking renders
Phe371 more mobile, and may thus enable the insect acyl
binding pocket to accommodate larger moieties.
Four natural mutations have been identified in insect AChEs [Fournier et al., 1993; Yao et al., 1997] which
confer resistance to insecticides in both Drosophila and
in houseflies: Gly265Ala, Phe330Tyr, Ile161Val, and
Phe77Ser. The 3D structure has provided some possible
rationalizations for the observed resistance, as discussed
previously [Harel et al., 2000].
Ashani Y, Grunwald J, Kronman C, Velan B, Shafferman A. 1994. Role
of tyrosine 337 in the binding of huperzine A to the active site of
human acetylcholinesterase. Mol Pharmacol 45:555–560.
Binding of an Inhibitor to DmAChE
Bystrzanowska T. 1969. Trials in the treatment of facial nerve paralysis
using nivaline. Wiad Lek 22:1233–1239.
The synthetic compound 1,2,3,4-tetrahydro-N-(3iodophenylmethyl)-9-acridinamine (ZAI, see Fig. 2) is a
powerful anticholinesterase, with an apparent Ki=1.09
nM for the highly homologous Musca domestica (house
fly) AChE (C. Personeni and T. Lewis, unpublished data).
It makes intimate contacts with several aromatic residues
in the active-site gorge of DmAChE (Fig. 7a). The tacrine
(Fig. 2) moiety of ZAI occupies a position in the gorge
similar to that which it occupies in the TcAChE:tacrine
complex [Harel et al., 1993], and makes planar π–π interactions with Trp83 and Tyr370. Nine aromatic residues
in the active-site gorge change their conformation upon
insertion of the inhibitor. Thus, the sidechains of residues Tyr71, Trp83, Tyr324, Phe330, Tyr370, Phe371,
Tyr374, Trp472, and His480 all move so as to permit the
inhibitor to be accommodated (Fig. 7b).
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