Specific targeting of acetylcholinesterase and butyrylcholinesterase recognition sites. Rational design of novel, selective, and highly potent cholinesterase inhibitors

Article abstract of DOI:10.1021/jm0255668

Tacrine-based AChE and BuChE inhibitors were designed by investigating the topology of the active site gorge of the two enzymes. The homobivalent ligands characterized by a nitrogen-bridged atom at the tether level could be considered among the most poten

Full text of DOI:10.1021/jm0255668

© Copyright 2003 by the American Chemical Society
Volume 46, Number 1
J anuary 2, 2003
Letters
lation of cognitive functions in both normal and patho-
logical conditions has been widely reviewed.1 In the
central nervous system (CNS) the cholinergic system
is important in the regulation of memory and learning
processes, and there are two cholinesterases (hydro-
lases) that coexist ubiquitously in humans and contrib-
ute to the degradation of the neurotransmitter ACh:
acetylcholinesterase (E1.1.1.7; AChE) and butyrylcho-
linesterase (E1.1.1.8; BuChE).2 Functionally, both en-
zymes hydrolyze ACh efficiently but at different rates;
BuChE hydrolyzes butyrylcholine (BuCh) at rates faster
than ACh, while AChE degrades BuCh much more
slowly than ACh. Although highly homologous (50-
60%), these enzymes are encoded by different genes and
clearly differ in substrate specificity and sensitivity to
inhibitors, likely because of a larger void at the BuChE
active site gorge and/or structural differences in the
catalytic site.3,4 These differences supported the pres-
ence of a specific peripheral anionic site (PAS) at the
lip of the gorge of AChE, which is lacking in BuChE.5-7
Sp ecific Ta r getin g of
Acetylch olin ester a se a n d
Bu tyr ylch olin ester a se Recogn ition Sites.
Ra tion a l Design of Novel, Selective, a n d
High ly P oten t Ch olin ester a se In h ibitor s
Luisa Savini,† Alessandra Gaeta,†
Caterina Fattorusso,‡ Bruno Catalanotti,‡
Giuseppe Campiani,*,† Luisa Chiasserini,†
Cesare Pellerano,† Ettore Novellino,‡
Dawn McKissic,§ and Ashima Saxena§
Dipartimento Farmaco Chimico Tecnologico, Universita` di
Siena, via Aldo Moro, 53100 Siena, Italy, Dipartimento di
Chimica delle Sostanze Naturali e Dipartimento di Chimica
Farmaceutica e Tossicologica, Universita` di Napoli Federico
II, via D. Montesano 49, 80131 Napoli, Italy, and Division
of Biochemistry, Walter Reed Army Institute of Research,
Silver Spring, Maryland 20910
Received J uly 30, 2002
Abstr a ct: Tacrine-based AChE and BuChE inhibitors were
designed by investigating the topology of the active site gorge
of the two enzymes. The homobivalent ligands characterized
by a nitrogen-bridged atom at the tether level could be
considered among the most potent and selective cholinesterase
inhibitors described to date. The nitrogen-containing homo-
bivalent ligands 3e,g and the sulfur-containing 3h validated
the hypothesis of extra sites of interaction in the AChE and
BuChE active site gorges.
BuChE is widely distributed in the body of verte-
brates. It appears in serum, hemopoietic cells, liver,
lung, heart, and CNS (neurons, glial cells, and gliomas).8
Unlike AChE, its physiologic function in normal and
diseased humans is still unclear, and recently a role for
BuChE in development and neurobiological processes
was suggested. In particular, BuChE has been impli-
cated in neurogenesis and regulation of cell proliferation
and differentiation, as well as in changes in apoptotic
events.8 The BuChE gene is amplified or abnormally
expressed in tumorigenesis8 and some neuronal disor-
ders, suggesting that BuChE may be involved in cell
adhesion phenomena. Amplification of BuChE gene
could stimulate tumorigenic cells to proliferate more
rapidly, evading regular proliferation control. Further-
more, significant increases in the levels of BuChE and/
or AChE in various neurological disorders such as
dementias may be indicative of pathological conditions.
Thus, the development of selective BuChE inhibitors
may be of great interest to clarify the physiological role
of this enzyme and to provide novel therapeutics for
various diseases.
In tr od u ction . Neurodegenerative diseases involving
impairment of cognitive functions, such as dementia of
the Alzheimer’s type (AD), are characterized by a loss
of basal forebrain neurons and reduced cortical and
hippocampal levels of acetylcholine (ACh). AD is caused
by a progressive and rather specific degeneration of
certain neuron populations, with â-amyloid plaques
(extracellular deposit), neurofibrillary tangles (intracel-
lular deposit), and synaptic loss being neuropathological
hallmarks of the disease. The role of ACh in the regu-
* To whom correspondence should be addressed. Phone: 0039-0577-
234172. Fax: 0039-0577-234333. E-mail:campiani@unisi.it.
† Universita` degli Studi di Siena.
‡ Universita` degli Studi di Napoli “Federico II”.
§ Walter Reed Army Institute of Research.
10.1021/jm0255668 CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/07/2002

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J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Letters
Ch a r t 1. New Tacrine-Based Homo- and Heterodimers
Sch em e 1a
a
Reagents: (i) pentanol, N,N-bis(3-aminopropyl)acetamide, 160
°C, 12 h; (ii) 9-chloro-1,2,3,4-tetrahydroacridine or 6,8,9-trichloro-
1,2,3,4-tetrahydroacridine, pentanol, 160 °C, 12 h; (iii) KOH,
Br(CH2)nBr, CH3CN, room temp, 6 h; (iv) 1,2,3,4-tetrahydro-9-
mercaptoacridine, KOH, CH3CN, room temp, 2 h.
The use of reversible inhibitors of AChE is considered
to be one viable and attractive therapeutic approach to
amplify the action of ACh in the brain of AD patients.
Tacrine (THA, 1), a reversible nonselective inhibitor of
AChE and an even stronger inhibitor of BuChE, has
been marketed for use in the long-term palliative
treatment of AD since 1993. The widespread applicabil-
ity of this drug is limited by its severe liver toxicity as
well as side effects stemming from its low target
specificity. Since then, other potent AChE inhibitors
have been developed, such as huperzine A, E2020, and
rivastigmine, with the last two already approved for the
treatment of AD. Recently,7,9-11 the synthesis of several
bivalent tacrine-based AChE inhibitors was reported
whose increased activity and selectivity were the result
of the simultaneous binding of two units to the catalytic
and PAS of AChE. These tacrine-related homo- and
heterobivalent ligands could be promising drug candi-
dates for the treatment of AD, possessing pharmacologi-
cal properties superior to those of tacrine. The aim of
the present study was to develop, by the investigation
of the active site topology of AChE and BuChE, novel
tacrine-based homo- and heterobivalent ligands for
pharmacological evaluation against AChE and BuChE
and to provide molecular probes to get further insights
into the AChE/BuChE active site gorges. On the basis
of a rational approach, a series of potent tacrine-based
homo- and heterobivalent ligands were designed (3b-
i) and tested (Chart 1). The new subset was designed
to monitor the existence of extra sites of interaction into
the AChE and BuChE active site gorges. Indeed, by use
of molecular modeling techniques, a mid-gorge AChE
recognition site and a peripheral interaction site in
BuChE were hypothesized. The resulting affinity of the
newly designed compounds seems to validate these
hypotheses. The set of compounds included in this
molecular modeling study was synthesized using an
already described general procedure.11 The synthetic
pathways to obtain the most representative compounds
3e-i are reported in Scheme 1, while the tacrine
homodimer 3a , described earlier,6,7 was resynthesized
and tested under the same experimental conditions.
Molecu la r Mod elin g Stu d ies. Selected conformers
of compounds 3e,g and 3h were docked into both the
hAChE X-ray crystal structure (PDB code 1B41) and
the homology model of hBuChE (PDB code 1EHO),
using a protocol that included molecular mechanic,
Monte Carlo, and simulated annealing calculations
(Supporting Information). Molecular modeling studies
F igu r e 1. Compound 3e (colored by atom type) docked into
the active site of hAChE. Amino acids conserved in hBuChE
are orange, those replaced in hBuChE are cyan. van der Waals
volumes of W86 and W286 are displayed. Hydrogen bonds are
highlighted by green dashed lines. Hydrogens and water
molecules are omitted for clarity with the exception of those
involved in hydrogen bonds.
were performed using human enzymes, the final phar-
macological target for the development of new drugs,
although our pharmacological tests used fetal bovine
serum (FBS) AChE and equine (Eq) BuChE. Neverthe-
less, a comparison of their amino acid sequences with
those of the corresponding human enzymes revealed an
overall identity of 84% and 88%, respectively. No
differences were found in the amino acid composition
of the active sites of FBS and hAChE, while only two
differences were detected between human and Eq
BuChE (Figure 2).
Resu lts a n d Discu ssion . Previously,11 we reported
and discussed the ability of compounds 3a and 3d to
bind to Eq BuChE with good affinity (Table 1) and that
AChE/BuChE selectivity could be modulated by exploit-
ing the different amino acid compositions of the active
sites of the two enzymes. Indeed, the molecular model-
ing study herein described suggested that the AChE/
BuChE selectivity profiles of compounds 2, 3b, and 3c,
may be related to the only relevant difference in the
catalytic sites of the two enzymes: the substitution of
P446 in hAChE with the bulkier M437 in hBuChE
(Figures 1 and 2), which reduces the space to optimally

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
3
F igu r e 2. Compound 3h (colored by atom type) docked into
the active site of hBuChE. Conserved amino acids are cyan.
Those replaced in hAChE are orange. P285, replaced in Eq
BuChE by L313, is white. A277, replaced in hAChE by W286
and in Eq BuChE by V305, is red. van der Waals volumes of
W82 and F278 are displayed. Hydrogen bonds are highlighted
by green dashed lines. Hydrogens and water molecules are
omitted for clarity with the exception of those involved in
hydrogen bonds.
F igu r e 3. Compound 3e (colored by atom type) docked into
the active site of hAChE. Its interaction with the proposed mid-
gorge recognition site is shown. The interactions between the
protonatable amine function in the tether and amino acids in
the gorge are displayed using their van der Waals volumes.
The rest of the inhibitor is displayed as capped sticks, and its
hydrogens are omitted for clarity. Cyan residues are aliphatic
in hBuChE.
Ta ble 1. Dissociation Constants for the Inhibition of Fetal
Bovine Serum AChE and Equine BuChE by Analogues of
Tacrine
substrate, and the catalytic triad S203, E202, and H447
(hAChE numbering). On the other hand, 6 of the 14
aromatic residues (Y72, Y124, W286, F295, F297 and
Y337) in the active site gorge of hAChE are replaced
by aliphatic residues (N68, Q119, A277, L286, V288, and
A328) in hBuChE.2,4
a
a
tether
AChE Ki
BuChE Ki
compound
length (CH2)n
(nM)
(nM)
1, tacrine
40
7
2
1
50
On the basis of sequence analysis and molecular
modeling studies, we synthesized homo- and hetero-
bivalent ligands bearing different functional groups on
the tether (3e-g), designed to interact with specific
residues in the active site gorge. For example, to obtain
more potent and selective inhibitors of hAChE, the
central methylene unit of the alkyl tether of 3a was
replaced by a protonatable amine function (3e) capable
of providing additional specific interactions with aro-
matic residues in the active site gorge of hAChE
(Ki3a ) 1.3 nM vs Ki3e ) 60 pM). This is supported by
the comprehensive docking study of 3e in the active site
gorge of hAChE, shown in Figure 1. Interaction points
with the enzyme include (i) a hydrogen bond between
the carbonyl oxygen of H447 and the protonated tet-
rahydroacridine nitrogen atom, (ii) a hydrogen bond
between the amino group at C-9 of the tricyclic system
and a water molecule (643H), (iii) two π-π stacking
interactions involving the tetrahydroaminoacridine moi-
eties of 3e and W86 (catalytic site) and W286 (PAS),
(iv) a hydrogen bond between the protonated amino
nitrogen in the alkyl tether and the hydroxyl group of
Y124, and (v) cation-π interactions with a number of
aromatic side chains present in the hAChE gorge
(Figure 3). The specific interactions with the newly
discovered mid-gorge recognition site in the hAChE
active site gorge are likely responsible for the picomolar
binding affinity of 3e (three-point interaction, Ki ) 60
pM) and its AChE/BuChE affinity ratio of 0.01, with
an AChE inhibitory activity 650-fold higher than that
of tacrine (1). This increase in activity is much higher
than that reported by Carlier7,9 for the homobivalent
ligand 3a (two-point interaction) (under our experimen-
tal conditions: 1, Ki ) 40 nM vs 3a , Ki ) 1.3 nM; Table
1).
3a , THA-homo
7
7
7
7
7
7
7
8
5
1.3
150
6
340
0.06
3.7
1500
250
210
2.9
2
3b
>20000
180
35
3c
3d
3e
6
3f
11
3g
2
3h
0.4
10
3i
E2020
640
20c
120c
ethopropazineb
huprine Xd
173200
0.026e
a
Ki is the mean of at least three determinations. Standard
errors were all within 10% of the mean. Reference 4. c hBuChE.
b
Reference 13. e hAChE.
d
accommodate the chlorine atoms at C-6 and C-8 of
compounds 2, 3b, and 3c in hBuChE. On the other
hand, replacement of the amine group of a tacrine unit
by a sulfur atom provided compounds with increased
selectivity toward BuChE (3a vs 3d ),11 which can be
explained by the fact that W286 in hAChE, responsible
for cation-π interaction at the PAS, is replaced by an
aliphatic residue A277 in hBuChE (Figures 1 and 2).
These results provided the rational for an in-depth
molecular modeling analysis to find new and specific
sites of interaction in AChE and BuChE active sites that
can drive the design of new, potent and selective
inhibitors. Inspection of X-ray structures of AChEs from
various sources, alone or in complex with inhibitors,
revealed that the catalytic site is located at the bottom
of a narrow gorge whose striking feature is the presence
of 14 aromatic residues highly conserved through dif-
ferent species. The comparison of the amino acid
composition of hBuChE (no three-dimensional structure
is available to date for BuChEs) with that of hAChE
evidenced the same positioning of W86 responsible for
the binding of the quaternary ammonium group of the

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J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Letters
When a THA unit of 3e bears two chlorine atoms at
investigate the physiological role of BuChE and may
represent lead structures to generate inhibitors of
tumorigenesis. Starting from our molecular modeling
approach and the new leads, these studies may also
pave the way for future rational design of selective
AChE inhibitors as novel therapeutics for neurodegen-
erative diseases.
C6 and C8 (3f), the decreased binding affinity could be
due to the inability of different pharmacophore points
of 3f to assume the correct orientation in the AChE
active site. On the other hand, docking studies with 3g,
characterized by a bulkier, neutral amide group in the
alkyl tether, indicated that this compound could be
better accommodated in the larger hBuChE gorge (data
not shown). Accordingly, 3g showed a BuChE affinity
of 2 nM and an AChE/BuChE affinity ratio of 750 (Table
1). It is noteworthy that 3e was found to be more active
than E2020 (Table 1), and a comparison of their binding
mode is shown in Figure 4 (Supporting Information).
To design new selective inhibitors of hBuChE, we also
explored the possibility of varying the tether length of
3d, whose mercaptotetrahydroacridine moiety was found
to confer a certain degree of selectivity toward BuChE.11
While the 7-methylene tether represents a strict re-
quirement for high affinity binding to AChE, the pres-
ence of the aromatic residue F278 in hBuChE, located
at the rim of the gorge next to A277 (A277 is replaced
by W286 located at the PAS of hAChE), suggested that
a longer alkyl spacer (8-methylenes, 3h ) could provide
a selective BuChE inhibitor, optimally positioning the
S-tetrahydroacridine moiety to establish a π-π interac-
tion with F278 (Figure 2). Accordingly, 3h was found
to be a potent BuChE inhibitor and, although less
selective than ethopropazine, a prototype for the gen-
eration of highly selective BuChE bivalent ligands
(Ki ) 0.4 nM; AChE/BuChE affinity ratio of 625).
Figure 2 shows the results of our molecular modeling
studies with 3h docked into the active site of hBuChE.
The tacrine unit is placed in the catalytic site, reproduc-
ing a binding mode similar to that described for com-
pound 3e in the hAChE catalytic site. In addition, the
mercaptotetrahydroacridine moiety, placed at the rim
of the gorge, gives a face-to-edge π-π interaction with
F278 and a hydrogen bond between the sulfur atom and
a water molecule (672H) (Figure 2). The presence of a
specific site of interaction at the rim of BuChE gorge is
also supported by the binding affinities of compounds
3i, 3d , and 3h (Table 1), which are characterized by 5-,
7-, and 8-methylene tethers, respectively. Indeed, BuChE
affinities of compounds 3d and 3i are comparable and
could be due to the possible accommodation of the
S-tetrahydroacridine moiety in the large BuChE gorge.
In contrast, the subnanomolar affinity of 3h is consis-
tent with achievement of a new binding interaction with
F278 and supports the presence of a peripheral interac-
tion site in BuChE.
Ack n ow led gm en t. The authors thank Mr. J acob
Patterson for excellent technical assistance and are
grateful to Mrs. Stefania Contaldo and Mr. Alessandro
Schipani for their helpful contributions.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails for the new compounds 2 and 3b-i (chemistry, molecular
modeling, and pharmacology) and Figure 4. This material is
available free of charge via the Internet at http://pubs.acs.org.
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Con clu sion s. In summary, we disclosed the rational
design of novel and potent tacrine-based selective
inhibitors of AChE and BuChE. Their biological data
confirmed the existence of a peripheral site of interac-
tion in the active site gorge of BuChE and led to the
definition of an unprecedentedly described mid-gorge
interaction site in the active gorge of AChE. Together
with the recently described phenantridinium-based
heterobivalent ligand12 and huprine X,13 compound 3e
could be considered one of the most potent and selective
tacrine-based AChE inhibitors, while the novel, potent,
and highly selective bivalent BuChE inhibitors 3g,h
may be considered useful pharmacological tools to
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C.; Chiasserini, L.; Fedorko, J . M.; Saxena, A. Novel and Potent
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