Acetylcholinesterase inhibitors with photoswitchable inhibition of β-amyloid aggregation

Article abstract of DOI:10.1021/cn500016p

Photochromic cholinesterase inhibitors were obtained from cis-1,2-α-dithienylethene-based compounds by incorporating one or two aminopolymethylene tacrine groups. All target compounds are potent acetyl- (AChE) and butyrylcholinesterase (BChE) inhibitors in the nanomolar concentration range. Compound 11b bearing an octylene linker exhibited interactions with both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. Yet upon irradiation with light, the mechanism of interaction varied from one photochromic form to another, which was investigated by kinetic studies and proved "photoswitchable". The AChE-induced β-amyloid (Aβ) aggregation assay gave further experimental support to this finding: Aβ_1-40 aggregation catalyzed by the PAS of AChE might be inhibited by compound 11b in a concentration-dependent manner and seems to occur only with one photochromic form. Computational docking studies provided potential binding modes of the compound. Docking studies and molecular dynamics (MD) simulations for the ring-open and -closed form indicate a difference in binding. Although both forms can interact with the PAS, more stable interactions are observed for the ring-open form based upon stabilization of a water molecule network within the enzyme, whereas the ring-closed form lacks the required conformational flexibility for an analogous binding mode. The photoswitchable inhibitor identified might serve as valuable molecular tool to investigate the different biological properties of AChE as well as its role in pathogenesis of AD in in vitro assays.

Full text of DOI:10.1021/cn500016p

Research Article
pubs.acs.org/chemneuro
Acetylcholinesterase Inhibitors with Photoswitchable Inhibition of
β‑Amyloid Aggregation
Xinyu Chen,†,§ Sarah Wehle,§ Natascha Kuzmanovic,‡ Benjamin Merget,§ Ulrike Holzgrabe,§
Burkhard Konig,‡ Christoph A. Sotriffer,§ and Michael Decker*,†,§
̈
†Institut fur Pharmazie, ‡Institut fur Organische Chemie, Universitat Regensburg, Universitatsstraße 31, 93053 Regensburg, Germany
̈
̈
̈
̈
§Institut fur Pharmazie und Lebensmittelchemie, Julius-Maximilians-Universitat Wurzburg, Am Hubland, 97074 Wurzburg, Germany
̈
̈
̈
̈
S
* Supporting Information
ABSTRACT: Photochromic cholinesterase inhibitors were obtained from cis-1,2-α-dithienylethene-based compounds by
incorporating one or two aminopolymethylene tacrine groups. All target compounds are potent acetyl- (AChE) and
butyrylcholinesterase (BChE) inhibitors in the nanomolar concentration range. Compound 11b bearing an octylene linker
exhibited interactions with both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. Yet upon
irradiation with light, the mechanism of interaction varied from one photochromic form to another, which was investigated by
kinetic studies and proved “photoswitchable”. The AChE-induced β-amyloid (Aβ) aggregation assay gave further experimental
support to this finding: Aβ1−40 aggregation catalyzed by the PAS of AChE might be inhibited by compound 11b in a
concentration-dependent manner and seems to occur only with one photochromic form. Computational docking studies
provided potential binding modes of the compound. Docking studies and molecular dynamics (MD) simulations for the ring-
open and -closed form indicate a difference in binding. Although both forms can interact with the PAS, more stable interactions
are observed for the ring-open form based upon stabilization of a water molecule network within the enzyme, whereas the ring-
closed form lacks the required conformational flexibility for an analogous binding mode. The photoswitchable inhibitor identified
might serve as valuable molecular tool to investigate the different biological properties of AChE as well as its role in pathogenesis
of AD in in vitro assays.
KEYWORDS: Alzheimer’s disease, beta-amyloid aggregation, cholinesterase inhibitors, tacrine, photochromism, 1,2-dithienylethene
lzheimer’s disease (AD) is the most common cause of
catalytic active site (CAS) that is located at the bottom of a 20
Adementia and is affecting tens of millions of people Å gorge responsible for neurotransmitter hydrolysis, and the
peripheral anionic site (PAS) that is rich in aromatic residues
located at the rim of the gorge on the surface of the enzyme.4
The noncholinergic functions of AChE are directly related to
the PAS of the enzyme. However, even after many studies, the
precise function and pathological mechanism of PAS inhibition
still remains unclear, especially its role in the progress of Aβ
formation. The PAS may not only stimulate the formation and
aggregation of Aβ, but also increase the neurotoxicity of
amyloid fibrils. Besides, there may be a positive feedback loop
between AChE activity and tau hyperphosphorylation and Aβ
formation, respectively.3,5 As a result, proper specific
pharmacological testing models are needed as well as suitable
tool drugs to decipher more details about inhibition of the PAS
and resulting pathophysiological consequences.6
(normally aged over 65) worldwide, especially in developed
countries due to the higher life expectancy.1 Though research is
also focusing on β-secretase 1 (BACE-1) and tau protein
hyperphosphorylation, cholinesterase inhibitors (ChEIs), that
is, the AD drugs tacrine, rivastigmine, donepezil, and
galantamine, are still the only established approved therapy
for treatment of AD, apart from the N-methyl-D-aspartate
(NMDA) receptor antagonist memantine. Until now, the use of
ChEIs is only indicated for symptomatic treatment of AD, since
they are not able to prevent the progress of the disease.
However, in the last years, ChEIs have again come into the
focus of drug development efforts because acetylcholinesterase
(AChE) might play a role in the formation of β-amyloid (Aβ)
deposits.2,3 Aβ is deposited in extracellular toxic plaques in
Alzheimer patients’ brains.3 It is noteworthy that the interaction
of AChE with Aβ and its correlation with tau hyper-
phosphorylation are not related to effects in cholinergic
neurotransmission. AChE possesses two binding sites: the
Received: January 31, 2014
Revised: March 3, 2014
Published: March 14, 2014
© 2014 American Chemical Society
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Tacrine, the first anti-AD drug that had been approved by the
United States Food and Drug Administration (FDA) two
decades ago, still receives a lot of attention despite its dose-
dependent hepatotoxicity due to its simple chemical structure
and its very high inhibitory activity at both AChE and BChE.
Tacrine-based bivalent or hybrid compounds were developed
possessing multiple pharmacological actions,7 such as anti-
oxidant,8,9 NO-donor,10 PAS inhibitor,11,12 and so forth. Such
versatile compounds were recently reviewed by Tumiatti et al.12
The results are very encouraging, especially from the blockade
of the PAS disease-modifying therapeutic strategies for AD
might emerge.12−14
Photochromic (or “photoswitchable”) inhibitors can rever-
sibly control biological functions such as the activity of an
enzyme. Examples include azobenzene-based ligands and
thiophenfulgide derivatives.15 Compounds based on the 1,2-
dithienylethene (DTE) scaffold have the advantage of better
thermal stability (of both photoisomers) and quantitative rates
of photoisomerization compared to azobenzene derivatives.16
DTEs can be reversibly toggled between their photoisomers via
a 6-π electrocyclic reaction by UV or visible light (Figure 1).
that are able to interact with the two binding sites of AChE,
which are both being discussed for their therapeutic value.12
A
photoswitchable AChE inhibitor, whose activity can be
controlled by light in a spatiotemporal manner, would be a
valuable tool to investigate AChE’s roles in the AD disease state
and would allow to further explore AChE’s potential for drug
development beyond symptomatic treatment via prevention of
ACh hydrolysis.
RESULTS AND DISCUSSION
■
Chemistry. Photochromic tacrine-DTE-derivatives were
synthesized in a convergent way by amide formation of cis-
1,2-α-dithienylcyclopentene carboxylic acid 5 (first path) with
9-aminoalkylamino-1,2,3,4-tetrahydroacridine 9 (second path)
using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI)
and 1-hydroxybenzotriazole (HOBt) as coupling reagents
(Scheme 1). 1,2-α-Dithienylcyclopentene carboxylic acid 5 for
the first path was synthesized according to the method
described in the literature (Scheme 1):21 Friedel−Crafts
acylation followed by McMurry ring closure reaction gave the
dichloro diarylcyclopentene 4, which was converted by
chlorine/lithium exchange and reaction with gaseous carbon
dioxide to form the corresponding carboxylic acid 5. With
regard to the second path, tacrine derivatives 9 were prepared
by reacting ethylenediamine or octane-1,8-diamine with 9-
chlorotetrahydroacridine 8 using previously described proce-
dures.8,22 The synthesis of bivalent compounds (11a, b) with
two tacrine moieties used a 1:2 ratio of DTE dicarboxylic acid
and the tacrine derivative, whereas for the synthesis of univalent
compounds (10a, b) a 1:1 ratio was applied (Scheme 1). In
each case, two different spacer lengths were incorporated. All
Figure 1. Light-induced switching of a DTE derivative between its
ring-open and -closed isomer.
Photochromic compounds are applied as modulators in
optoelectronic devices and even as regulators of biological
1
target compounds were characterized by H NMR, 13C NMR,
processes.17 Konig et al. used the DTE scaffold to successfully
and HRMS, and their purities were determined by HPLC. This
also applies to both photoisomeric forms of 11b.
̈
develop a photoswitchable inhibitor of the enzyme human
carbonic anhydrase I, in which the DTE was connected with a
4-amino-sulfonamide, a low-affinity enzyme inhibitor, and an
imidazole-binding copper complex.18 Similarly, azo-propofol
compounds containing an azobenzene unit were applied at γ-
aminobutyric acid A (GABAA) and α-amino-3-hydroxy-5-
methyl-4-isoxazolepropionic acid (AMPA) receptors, impres-
sively enabling the control of the neural system in a light-
dependent way.19 In another example, the azobenzene motif
was used as a spacer to connect chromone structures that
inhibit the activation of mast cells in allergy diseases. In the
latter case, the target compound not only shows higher potency
than the lead compound, but also exhibits photoswitchability
during inhibition.20 It has been shown in very recent times that
photoswitchable compounds can be successfully used to study
biological mechanisms in an innovative manner; the prereq-
uisite is the identification of photoisomers with a pronounced
difference in biological activities.19,20
Physicochemical Properties. The photochemical proper-
ties of all tacrine-DTE-derivatives were investigated by UV/vis
spectroscopy. The absorbance spectra of all compounds are
presented in the Supporting Information. All target molecules
showed reversible photoswitchable behavior. Absorbance
spectra were recorded starting with the ring-opened photo-
isomers and irradiating with 312 nm light in periods of 5 s (e.g.,
11b, Figure 2). For all compounds, the absorbances around 270
nm decreased upon UV-irradiation, whereas the absorbance
band at 340 nm increased, and a new absorbance band
maximum at 525 nm evolved (cf. Supporting Information
Figures 1−7) due to the formation of the benzodithiophene
conjugation system.16,18 We especially investigated and
characterized the structure of the ring-closed form of these
compounds, exemplified by compound 11b: the changing of
the retention time in HPLC (cf. Supporting Information
1
Figures 8−9) and the shifting of corresponding signals in H
Herein we report the synthesis, physicochemical character-
ization and several biological studies (ChEs inhibitory activities,
enzyme kinetics, and their influence on inhibiting AChE-
induced Aβ aggregation, respectively) of molecules constructed
from the photochromic diarylethene scaffold combined with
aminopolymethylene tacrine moieties. Additionally, computa-
tional studies (docking as well as molecular dynamics
simulations) were performed to find an explanation on the
molecular level for the biological data observed. In this work,
we used the photochromic diarylethene scaffold as the
photoswitchable component due to its stability of both
photoisomers to incorporate it into the well-known bis-tacrines
NMR (cf. Supporting Information Figure 10). At a
concentration of 100 μM and after 30 s of irradiation, more
than 92% of the ring-closed form is detected by HPLC (cf.
Supporting Information Figure 9).
We investigated the photostability of the target compounds
by successive ring-closing-/ring-opening cycles applying 312
nm and wavelengths ≥ 420 nm, respectively. All target
molecules showed a degradation of 20−30% after seven cycles
as exemplarily presented for compound 11b in Figure 3. The
photodecomposition is also reported for other dithienylcyclo-
pentene systems, but the exact mechanism remains unknown.23
Kept in the dark, the ring-closed photoisomer 11b was stable
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Scheme 1. Synthesis of Photochromic Uni- and Bivalent ChE
a
Inhibitors
Figure 2. UV−vis absorbance spectra evolution of compound 11b (50
μM in buffer solution with 10% ethanol) by irradiation with 312 nm
light. Arrows indicate the changes of the absorption maxima with
irradiation periods of 5 s.
Figure 3. Cycle performance of compound 11b, changes in absorption
at 525 nm during an alternated irradiation of a solution (50 μM in pH
= 8.0 buffer with 10% ethanol) with 312 nm light for 30 s and ≥420
nm light for 5 min. 80% of the compound remained photoactive after
seven cycles of on- and off-switching.
exhibiting moderately higher inhibitory activities at BChE
(Table 1).
The bivalent inhibitor 11a with a short ethylene unit
represents a potent two-digit nanomolar inhibitor in its ring-
closed form being only slightly less potent than tacrine, and also
the ring-opened form of 11a inhibits AChE in the same
concentration range with slightly lower activities. Surprisingly,
the octylene-bridged bivalent inhibitor 11b with a sterically
demanding DTE group represents (in both the ring-opened
and -closed form) a one-digit nanomolar inhibitor being 3- to
4-fold more potent at AChE than even tacrine itself. The
respective octylene-bridged univalent inhibitor 10b is also a
very active inhibitor (21 nM for ring-opened form, 20 nM for
the ring-closed form) when compared to the ethylene-bridged
univalent inhibitor 10a with only submicromolar inhibition
(153 and 117 nM, respectively). Taken together, both bivalent
inhibitors are potent AChE and BChE inhibitors, irrespective of
the fact whether the compounds are inhibiting in their ring-
opened or -closed forms.
a
Reagents and conditions: (i) AlCl3, CH3NO2, 0 °C to r.t., 71%; (ii)
Zn, TiCl4, dry THF, 40 °C, 58%; (iii) nBuLi, gaseous CO2, dry THF,
r.t., 98%; (iv) POCl3, reflux, 60%; (v) ethylenediamine or 1,8-
diaminooctane, 1-hexanol, 150 °C, 12 h, 66−73%; (vi) EDCI, HOBt,
dry DMF, r.t., 12 h, 22−32%.
for several months.18 Photochemical stability was therefore
given for the application of the photoisomers under the assay
conditions applied and described below, since the ring-opening
process demands long exposure (>5 min) and intense light
(150 W), whereas during the testing the vial with the testing
solution would only be exposed to light for a few seconds.
Cholinesterase Inhibition. All tacrine-DTE derivatives are
very active inhibitors with IC50 values (AChE) ranging from 1.8
nM (bivalent compound 11b) to 153 nM (univalent compound
10a) (note that the terms “univalent” and “bivalent” only refer
to the constitution of the compounds with either one or two
tacrine moieties, respectively, regardless of the actual binding
mode). In most cases, the compounds show similar activities at
both AChE and BChE with the ethylene-bridged compounds
Interestingly, in some cases, the inhibition curves of the ring-
opened and -closed forms reveal significant differences between
the two photoisomers with regard to their Hill slopes, best
exemplified by compound 11b (Table 2, Figure 4). A Hill slope
of 1 indicates one site interaction between inhibitor and
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Table 1. Inhibition Results of Target Compounds at AChE and BChE and Resulting Selectivities Expressed as the Ratio of IC50
Values
a
b
c
Values are means of at least three determinations, pIC50 = −log IC50. AChE from electric eel. BChE from equine serum.
Table 2. Inhibition of AChE from Electric Eel (eeAChE) and Human (recombinant, hAChE) by Tacrine and Both
Photochromic Forms of Compound 11b, with Short and Long Incubation Time
4.5 min incubation
IC50, nM (pIC50 SEM)
eeAChE hAChE
15.6 95.9
1 h incubation
IC50, nM (pIC50 SEM)
eeAChE hAChE
23.5 84.8
Hill slopes
Hill slopes
compd
tacrine
eeAChE
hAChE
eeAChE
hAChE
0.9
2.1
1.2
1.0
1.0
2.1
1.0
1.1
(7.81 0.05)
4.3
(7.02 0.06)
49.6
(7.63 0.01)
14.0
(7.07 0.04)
41.4
11b opened
11b closed
1.6
1.2
1.6
1.0
(8.36 0.03)
1.8
(7.30 0.05)
19.1
(7.85 0.03)
12.9
(7.38 0.06)
14.2
(8.74 0.02)
(7.72 0.06)
(7.89 0.05)
(7.85 0.07)
enzyme, normally with the active center, that is, the CAS of
AChE. A steeper Hill slope indicates positive cooperativity, in
the case of AChE plausibly due to concomitant interaction with
a second binding site.24 For bivalent and hybrid inhibitors, the
hydrophobic PAS represents the favored second binding site,
the inhibition of which slows down the AChE-induced
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concentration of 11b, the Vmax value (i.e., the reciprocal of the
Y-intercept) decreases, whereas the Km value (i.e., the negative
reciprocal of the X-intercept) remains the same. Therefore,
compound 11b in its ring-opened form represents a non-
competitive inhibitor of AChE.8,25,26 In contrast, in its ring-
closed form, a different Lineweaver−Burk curve illustrates the
difference in Km and Vmax values. The Km values differ with
different 11b concentrations, while the Vmax values remain
essentially unchanged; that is, this photoisomer of 11b is a
competitive inhibitor of AChE and therefore presumably might
only inhibit the CAS of the enzyme. The Lineweaver−Burk
plots for AChE clearly show reversible and noncompetitive
inhibition by 11b as ring-opened form, yet competitive
inhibition as ring-closed form. These results from the kinetic
studies are in accordance with the initial assumptions gained
from Hill slopes. Based upon the analyses of the chemical
structures of the target compounds as well as of the 3D model
of AChE,27 we expect that the more flexible ring-opened form
is able to interact with both the CAS and the PAS of AChE,
whereas for the ring-closed compound formed after irradiation
the rigid backbone of DTE limits this interaction, most likely
with the PAS (cf. computational studies described below). The
change of interaction keeps the potent inhibition of AChE
intact, but binding interaction with the PAS might be different
or even lost in 11b’s ring-closed form.
The fact that compound 11b acts as a potent inhibitor of
AChE with equal potency for both photoisomers, but is able to
interact differently with a second binding site, classifies it as a
“photoswitchable” or photochromic PAS-inhibitor. If this is the
case, it should also be possible to “switch on or off” AChE’s
catalytic effect on Aβ aggregation. Consequently, both
photoisomeric forms of compound 11b were incubated
together with AChE and their abilities to interfere with the
extent of Aβ aggregation as a result of the inhibition of AChE’s
PAS were quantified.
AChE Induced β-Amyloid Aggregation. In order to
further prove our hypothesis, compound 11b was tested for its
influence on AChE-induced β-amyloid aggregation, since the
different inhibitory manner of 11b as ring-opened and -closed
forms might translate into different abilities to modulate β-
amyloid aggregation despite their almost identical IC50 values
for AChE inhibition.
Figure 4. Inhibition curves of compound 11b at eeAChE as both ring-
opened (blue solid curve) and -closed (red dashed curve) forms after 1
h incubation. A clear switching of the Hill slope from ring-opened
form (nH = 2.1) to ring-closed form (nH = 1.0) after the irradiation
with light is observed (for detailed data, cf. Figure 11 in the Supporting
Information).
aggregation of Aβ, a core process in AD pathology.2,6 To
confirm this result, we tested the compound 11b further on
human AChE, with both usual (4.5 min) and prolonged (1 h)
incubation times. The latter was used to ensure that the
interaction between the compound and the enzyme has
reached equilibrium. The results indicate repeatability of this
switch in the Hill slope at AChE from both species at different
incubation times (Table 2), with the effect being less
pronounced at hAChE.
We therefore further investigated the mechanistic details of
the mode of inhibition of compound 11b in both its ring-
opened and -closed forms by kinetic studies. The mechanism of
inhibition was analyzed by recording substrate-velocity curves
(Michaelis−Menten) in the presence of different concen-
trations of compound 11b (cf. Figure 12 in the Supporting
Information). Substrate (i.e., acetylthiocholine) concentration
was varied between 28.2 and 452 μM. For the ring-opened
form, 15, 20, 25, and 30 nM compound 11b were applied, and
for the ring-closed form 25, 30, 35, 40, and 50 nM were used.
Vmax and Km values were calculated directly from substrate
velocity curves using GraphPad Prism. Lineweaver−Burk plots
(Figure 5) are shown for better illustration and clearness, that
is, reciprocal rates versus reciprocal substrate concentrations for
the different inhibitor concentrations, as both ring-opened and
-closed forms. For the ring-opened form, when increasing the
Figure 5. Lineweaver−Burk plots of AChE activity with different substrate concentration (28.2−452 μM) in the presence of suitable concentrations
of compound 11b (15−50 nM). The graph on the left was tested with 11b in its ring-opened form, whereas the one on the right was tested in its
ring-closed form.
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An AChE-induced Aβ1−40 aggregation assay was established
using the thioflavin T fluorescence method, which was slightly
modified from those reported in the literature in order to suit
our purpose.2,14,28,29 First, a phosphate buffer containing NaCl
was chosen, because of its ability to enhance the ionic strength
and consequently accelerate the speed of aggregation.28
Second, the pH value of the buffer was set to 8 where AChE
shows the highest activity with regard to substrate hydrolysis.14
Third, only AChE-induced aggregation was presented without
considering the influence of self-aggregation due to the fact that
under such conditions the rate of self-induced Aβ aggregation
should be much slower than the AChE-induced one.2 Lastly,
AChE from electric eel instead of the human recombinant one
was used to keep the results consistent with the ones obtained
in the enzyme inhibition and kinetic studies.
Table 3. Inhibition of AChE-Induced Aβ1‑40 Aggregation by
Compound 11b in Both Photoforms
percent inhibition with compound after
a
compound
concentration
5 h incubation
b
11b ring-opened
1 μM
100%
0.2 μM
53.8−60.2%
11b ring-closed
1 μM
0−43.0%
0%
c
0.2 μM
a
All the experiments were performed in duplicate; therefore, the data
b
here are given as a range instead of mean SEM. Experimentally, a
slight decrease in fluorescence was observed, due to complete
c
inhibition of Aβ aggregation. Experimentally, a slight increase in
fluorescence was observed compared to the blank value; that is, no
standard error of means can be provided due to complete lack of
inhibition.
The resulting curves show different inhibitory activities for
ring-opened and -closed forms of 11b (Figure 6): As predicted
very well due to the inhibition of self-induced Aβ1−40
aggregation (data not shown). Nevertheless, there is a clear
dose-dependent effect on reduction of AChE-induced Aβ1−40
aggregation for the ring-open form of 11b (green lines). The
inhibition of Aβ1−40 aggregation by compound 11b in both
photoforms after 5 h is also quantitatively summarized in Table
3. At the same concentration, the aggregation of Aβ1−40
inhibited by compound 11b in its ring-opened form is always
significantly higher than for the ring-closed form under
corresponding conditions (Table 3).
Computational Binding Mode Analysis. Docking studies
were carried out to investigate potential binding modes of
compound 11b in both its ring-opened and -closed photoforms.
Despite the ligands’ large number of conformational degrees of
freedom, the docking procedure provided reasonable poses on
the top ranks. The partially saturated (cyclohexen-like) ring of
the tacrine moiety was generally handled in the twisted half-
chair conformation, except for 11b closed, where better results
were obtained with a half-chair conformation of the tacrine
group located in the PAS (cf. binding-mode description in the
following paragraph). Due to the complementarity of
interactions, the positions obtained at rank 2 for both 11b
open and 11b closed appeared most feasible upon visual
inspection. This impression was confirmed by rescoring the
docking poses obtained from GOLD with DrugScoreX
(DSX):30 With values of −253 (11b opened) and −241 (11b
closed) DSX assigned the clearly best score to the poses ranked
second in GOLD (see Table 1 in the Supporting Information
for the detailed scores of the top five ranks). Accordingly, these
poses were selected for further analysis.
Both photoforms of 11b extend from the CAS to the PAS,
reminiscent of the binding mode of the 7-carbon-linker bis-
tacrine of structure 2CKM.31 The first tacrine moiety is placed
in the CAS (Ser200, Glu327, His440), with its aromatic
nitrogen atom in close hydrogen-bonding distance to the
backbone oxygen of His440. The ring system is located
between Trp84 and Phe330, showing good π-stacking
interactions with the Trp84 side chain of the “anionic” site.
For both inhibitors, the second tacrine group is sandwiched
between Tyr70 and Trp279 of the PAS. The orientation,
however, is different (Figure 7): The photochromic units of the
two inhibitors are located at the entrance of the binding gorge
in an almost perpendicular orientation relative to each other,
resulting in different access of the tacrine groups to the PAS.
The tacrine moiety of the ring-opened form of 11b enters the
PAS from the side of the binding gorge, whereas in the ring-
Figure 6. Inhibition curves of AChE-induced Aβ1−40 aggregation by
compound 11b in a concentration dependent manner for both its ring-
opened and -closed forms (measured in duplicate). The blue lines
represent compound 11b in the ring-closed form, whereas the green
lines represent the opened form. The quantitative summary of part of
the graph is also presented below as a table.
from kinetic studies, all the curves for ring-closed 11b show
higher values of aggregation (i.e., lower inhibition) than the
curves with corresponding concentrations as ring-opened form.
The ring-closed form of 11b does not interfere with the
accelerating effect of AChE on aggregation rate resulting in
higher fluorescence. As a result, after a certain time, the
fluorescence increase (blue lines in Figure 6) should be closer
to the blank value (black line in Figure 6). Low concentrations
of 11b (0.2 and 1 μM) in the ring-closed form show no or little
inhibition of AChE-induced aggregation of Aβ1−40 (Table 3).
The effects are above or around the blank value (black line). At
a high concentration (5 μM) of 11b in its ring-closed form, a
lowered aggregation can be observed which may be due to the
inhibitory activity of the compound to prevent self-induced
aggregation of Aβ1−40
.
In contrast, the ring-opened form of 11b seems to interfere
with the PAS and consequently inhibits the Aβ aggregation in a
concentration-dependent manner resulting in lower fluores-
cence (green lines in Figure 6). At low concentrations of 11b in
the ring-opened form (0.2 and 1 μM), fluorescence emission
remained in the same range as in the beginning, i.e. the increase
is around zero for both concentrations (green lines in Figure 6)
reflecting pronounced inhibition of aggregation in contrast to
the accelerated Aβ aggregation observed for the blank (Table
3). The reason for an additional decrease of fluorescence
emission at a concentration of 5 μM of ring-opened 11b may
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Figure 7. Docked poses of ring-opened 11b (left, colored pink) and ring-closed 11b (right, colored turquoise). The residues of the CAS (Ser200,
His440, Glu327) are shown in magenta, Trp84 of the catalytic “anionic” site in orange, and the residues of the PAS (Tyr70, Tyr121, Trp279) in light
green.
closed 11b the methylene linker wraps around Trp279 to
enable access from the more distal site. Presumably, this is
required by the restricted conformational freedom of the closed
form. In comparison to the 7-carbon-linker bis-tacrine of
2CKM, the much longer dimension of the 11b compounds
prevents a direct extension from the CAS to the PAS. Instead,
only the alkyl chain between the CAS-tacrine and the
photochromic unit is located entirely in the gorge for both
photoisoforms, whereas the linker between the photochromic
unit and the PAS-tacrine is (necessarily) rather solvent exposed.
To investigate whether the docking poses are reliable and
form stable complexes with AChE, molecular dynamics (MD)
simulations of 18 ns were performed for both complexes.
Regarding the interactions in the CAS, the trajectories reveal
significantly smaller distances to the His440 backbone for the
ring-opened 11b than for the ring-closed 11b, indicating a more
stable interaction of the opened form throughout the trajectory
(cf. Figure 13 in the Supporting Information). Moreover, the
aromatic interaction of the tacrine moiety of 11b (ring-opened)
with Trp84 exhibits a smaller average distance of 3.73 Å,
compared to 3.94 Å of the ring-closed 11b (statistically
significant based on Mann−Whitney U test, p < 10−5). Within
the PAS, the most stable π-stacking is observed between ring-
opened 11b and Tyr70, underlining the ability of ring-opened
Figure 8. Cumulative occupancies for hydrogen bonds between the
11b to successfully inhibit the PAS. Unlike ring-closed 11b,
ring-opened 11b is also further stabilized in the PAS by a
complex network of water molecules. This network is formed in
a cavity near the PAS as a consequence of a temporary (1 ns)
opening of Trp279 toward a water channel. Thereby, the PAS-
tacrine moiety of ring-opened 11b is stabilized by hydrogen
bonding to water molecules (48% occupancy, Figure 8),
whereas similar interactions are only rarely observed for ring-
closed 11b (11% occupancy, Figure 8). This stabilization of
ring-opened 11b in the PAS is also reflected by representative
snapshots obtained from the last conformational family of each
trajectory (Figure 9): For ring-opened 11b, each tacrine group
shows a virtually identical binding mode as in the docking pose.
In contrast, larger shifts (as well as higher fluctuations along the
trajectory) are observed for the ring-closed 11b tacrines. These
findings indicate that the PAS-tacrine in the ring-closed form is
more flexible, compared to the ring-opened form, and might
PAS-tacrines and water molecules over the entire 18 ns trajectory for
ring-closed 11b (left, 11.3%) and ring-opend 11b (right, 47.8%).
diffuse more easily from the PAS. The PAS interaction is
presumably weaker for the ring-closed form compared to the
PAS-tacrine of the open form, which is stabilized as part of a
hydrogen-bond network. The binding mode of the photo-
switchable compound 11b resembles the binding mode of
other dual binding AChE inhibitors.10,12,13 However, due to the
size and complexity of the compound, the pronounced
difference of the photoisomers with regard to PAS inhibition
and, therefore, Aβ aggregation is only understandable applying
MD simulations. Whereas the ring-opened form is stably fixed
in the PAS, the ring-closed form is more loosely bound and
retains considerable flexibility. Possibly, this difference trans-
lates into the different inhibition behavior of the two isomers
with respect to β-amyloid-aggregation. The similar IC50 values,
383
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ACS Chemical Neuroscience
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Figure 9. Comparison of the docking pose and a representative MD snapshot for ring-opened 11b (left) and ring-closed 11b (right). The docking
poses are colored as in Figure 7, whereas the MD snapshots are shown in dark red (left) and cyan (right). The MD snapshots were obtained in each
case from the last conformational family observed in a 2D RMSD analysis of the 18 ns trajectory by averaging all members of this conformational
family and selecting the snapshot closest to this average structure as a representative. Figures 7 and 9 were generated with PyMOL (The PyMOL
Molecular Graphics System, version 1.5.0.5, Schrodinger, LLC, 2012).
̈
on the other hand, might be due to enthalpy−entropy
compensation: While the ring-opened form shows good
enthalpic interactions (also with water molecules) at the cost
of an entropy loss, the residual flexibility of the ring-closed form
reduces the entropy loss, but gains less enthalpy. Obviously,
this is only a qualitative interpretation of the observations made
in the simulation studies. We are fully aware of their limitations,
as well as of the possibility that alternative explanations might
exist for the experimental data obtained in this study. A
crystallographic determination of the binding mode and
calorimetric information regarding enthalpic and entropic
contributions would be helpful to support or disprove the
model. The nature of the ligands, however, precludes a
straightforward access to such experimental data.
inhibition by light may be useful in further investigating the
physiological functions of AChE and its role in AD; therefore,
these compounds might serve as useful molecular tools to
investigate the different biological properties of AChE in in
vitro assays.
METHODS
■
Chemistry. General Methods for Synthesis. Melting points are
uncorrected and were measured in open capillary tubes, using a
Barnstead Electrothermal IA9100 melting point apparatus. H NMR
1
and 13C NMR spectral data were obtained from a Bruker Advance
spectrometer (300 and 75 MHz, respectively). TLC was performed on
silica gel on aluminum foils with fluorescent indicator 254 nm (Fluka)
or aluminum oxide on TLC-PET foils with fluorescent indicator 254
nm (Fluka). For detection, iodine vapor or UV light (254 nm),
respectively, were used. ESI-MS samples were analyzed using
electrospray ionization ion-trap mass spectrometry in nanospray
mode using a Thermo Finnigan LCQ Deca. For column
chromatography, silica gel 60, 230−400 mesh (Merck) was used. In
addition to mass spectrometry and NMR, the purity of the target
compound was evaluated by high-resolution mass following HPLC
system (confirming purity ≥ 95%).
CONCLUSION
■
In summary, photochromic uni- and bivalent tacrine-based
AChE inhibitors were synthesized and evaluated, whereby the
bivalent compounds show potent and almost identical
inhibitory activities (with similar or even higher activity than
tacrine) as both photoisomers at AChE (and BChE).
Surprisingly, even with a sterically demanding 1,2-α-dithienyl-
ethene group in the middle of the linker, bivalent compounds
still remain nanomolar inhibitors at both ChEs. Computational
docking studies suggested that 11b might bind to the CAS and
the PAS of AChE in both opened and closed photoforms.
However, significant local differences in the binding of the two
forms are observed within the 18 ns MD simulation, as only for
the opened form a stabilization of the interactions in the CAS
and, especially, in the PAS is seen, with the latter one mediated
by a water hydrogen-bond network. The mechanism of AChE
inhibition by compound 11b might be controlled by light:
Upon irradiation with UV−vis light, the binding mode of the
inhibitor (CAS and PAS inhibition) can be switched, hinting at
the change of the Hill slope in inhibition studies, and proved by
kinetic studies. It could also be shown that inhibition of the
PAS of AChE might be translated into a reduced Aβ
aggregation. The ability to control the mode of AChE
Analytical HPLC using a VWR HITACHI L-2130 pump coupled to
VWR HITACHI column oven L-2350 and L-2455 diode array
detector: The solvents were as follows: (A) water +0.05% trifluoro-
acetic acid and (B) acetonitrile +0.05% trifluoroacetic acid; flow, 0.4
mL/min. Column Hibra 125-4 Purospher STAR RP-18e (3 μm) at 20
°C, detecting at 249 nm; solvent A from 90 to 10% for 30 min, then
10% for 15 min, from 10% to 90% for 10 min, 90% for 5 min.
4,4′-(Cyclopent-1-ene-1,2-diyl)bis(5-methyl-N-(2-((1,2,3,4-tetra-
hydroacridin-9-yl)amino)ethyl)thiophene-2-carboxamide) (11a).
To the solution of dithiophene carboxylic acid 5 (150 mg, 0.43
mmol) in tetrahydrofuran (10 mL) was added 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 182 mg,
0.95 mmol) and 1-hydroxybenzotriazole (HOBt, 15 mg, 0.1 mmol),
and the resulting solution was stirred at room temperature for 1 h. The
solution of aminoalkyl tacrine 9a (230 mg, 0.95 mmol) in
tetrahydrofuran (5 mL) was added to the above solution, and
agitation continued overnight. The target compound precipitated from
the solution and was collected by filtration as yellow solid (50 mg,
22%). MP 192−195 °C. ESI-MS 398.2 m/z [M+2H]2+, 795.3 m/z [M
384
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Research Article
1
+H]+. H NMR (MeOH-d4, 300 MHz) δ: 1.79−1.88 (m, 14H, 2 ×
(tacrine), 2 × CH3), 2.03−2.08 (m, 2H, CH2CH2CH2 (cyclo-
pentene)), 2.66−2.69 (m, 4H, 2 × CH2(CH2)2CH2CN (tacrine)),
2.77−2.82 (t, 4H, CH2CH2CH2 (cyclopentene), J = 7.48 Hz), 2.96−
2.98 (m, 4H, 2 × CH2(CH2)2CH2CN (tacrine)), 3.23−3.28 (t, 4H,
2 × CONHCH2(CH2)7NH, J = 7.00 Hz), 3.71−3.76 (t, 4H, 2 ×
CONH(CH2)7CH2NH, J = 7.26 Hz), 7.42−7.48 (m, 4H, arom
(tacrine), 2 × CH (thiophene)), 7.65−7.75 (m, 4H, arom), 8.21−8.24
(d, 2H, arom, J = 8.67 Hz) ppm. 13C NMR (MeOH-d4, 75 MHz) δ:
14.76 (CH3), 22.66 (CH2CH2CH2 (cycloheptene)), 23.50
(CH2(CH2)2CH2 (tacrine)), 23.92, 25.44, 27.72, 27.87, 30.21, 30.53,
31.35, 31.82 (CH2(CH2)3CN (tacrine), (CH2)3CH2CN (ta-
crine), NHCH2(CH2)6CH2NHCO), 39.52 (CH2CH2CH2 (cyclo-
pentene)), 40.78 (NHCH2 (CH2 )6 CH2 NHCO), 49.43
(NHCH2(CH2)6CH2NHCO), 114.44 (arom), 118.80 (arom),
123.36 (CH (tacrine)), 125.69 (2 × CH (tacrine)), 130.63 (CH
(tacrine)), 132.35 (CH (thiophene)), 136.24 (arom), 137.90 (arom),
141.39 (arom), 143.13 (arom), 154.69 (arom), 156.07 (arom), 164.18
(CONH) ppm. ESI-HRMS: C59H75N6O2S2 calcd 482.2730 m/z [M
+2H]2+, 963.5387 m/z [M+H]+, anal. 482.2735 m/z [M+2H]2+,
963.5377 m/z [M+H]+. HPLC purity 95.4%, retention time 21.26
min.
CH2(CH2)2CH2 (tacrine), 2 × CH3), 2.02−2.07 (m, 2H,
CH2CH2CH2 (cyclopentene)), 2.67−2.79 (m, 8H,
2 ×
CH2(CH2)2CH2CN (tacrine), CH2CH2CH2 (cyclopentene)),
2.92−2.94 (m, 4H, 2 × CH2(CH2)2CH2CN (tacrine)), 3.64−3.68
(t, 4H, 2 × CONHCH2CH2NH, J = 4.97 Hz), 4.09−4.12 (t, 4H, 2 ×
CONHCH2CH2NH, J = 5.31 Hz), 7.34 (s, 2H, 2 × CH (thiophene)),
7.49−7.53 (t, 2H, arom, J = 8.23 Hz), 7.66−7.79 (m, 4H, arom),
8.39−8.41 (d, 2H, arom, J = 8.59 Hz) ppm. 13C NMR (MeOH-d4, 75
MHz) δ: 15.42 (CH3), 22.05 (CH2CH2CH2 (cyclopentene)), 23.21
(CH2(CH2)2CH2 (tacrine)), 25.24 (CH2(CH2)3CN (tacrine)),
29.60 (CH2)3CH2CN (tacrine)), 39.79 (CH2CH2CH2 (cyclo-
pentene)), 41.11 (NHCH2CH2NHCO), 51.17 (NHCH2CH2NHCO),
113.36 (arom), 117.36 (arom), 120.42 (CH (tacrine)), 126.64 (CH
(tacrine)), 126.66 (CH (tacrine)), 131.81 (CH (tacrine)), 134.15 (CH
(thiophene)), 134.91 (arom), 136.37 (arom), 138.18 (arom), 139.97
(arom), 142.49 (arom), 151.71 (arom), 158.24 (arom), 166.00
(CONH) ppm. ESI-HRMS: C47H52N6O2S2 calcd 398.1791 m/z [M
+2H]2+, 795.3509 m/z [M+H]+, anal. 398.1797 m/z [M+2H]2+,
795.3509 m/z [M+H]+. HPLC purity 95.5%, retention time 23 min.
5-Methyl-4-(2-(2-methyl-5-((2-((1,2,3,4-tetrahydroacridin-9-yl)-
amino)ethyl)carbamoyl)thiophen-3-yl)cyclopent-1-en-1-yl)-
thiophene-2-carboxylic Acid (10a). The mixture of dithiophene
carboxylic acid 5 (100 mg, 0.29 mmol), EDCI (56 mg, 0.29), and
HOBt (15 mg, 0.1 mmol) in DMF (5 mL) was agitated for 30 min at
room temperature. The solution of aminoalkyl tacrine 9a (69 mg, 0.29
mmol) in DMF (3 mL) was added to the above solution, and agitation
was continued overnight. Water (30 mL) was added to the reaction
solution, and the aqueous phase was extracted with dichloromethane/
methanol mixture (10:1, 3 × 30 mL). The combined organic phases
were washed with brine (2 × 20 mL), dried over anhydrous sodium
sulfate, and concentrated under vacuum. The residue was purified via
column chromatography with dichloromethane/:methanol/ammonia
5:1:0.1 as eluent system. The target compound was obtained as yellow
solid (40 mg, 24%). MP > 300 °C (decomp.). ESI-MS 482.2 m/z [M
+2H]2+, 963.6 m/z [M+H]+. 1H NMR (MeOH-d4 + CDCl3, 300
MHz) δ: 1.87−1.98 (m, 10H, CH2(CH2)2CH2 (tacrine), 2 × CH3),
2.01−2.06 (m, 2H, CH2CH2CH2 (cyclopentene)), 2.72−2.96 (m, 8H,
CH2(CH2)2CH2 (tacrine), CH2CH2CH2 (cyclopentene), 3.68−3.72
(t, 2H, J = 5.48 Hz, CONHCH2CH2NH), 4.14−4.17 (t, 2H, J = 5.35
Hz, CONHCH2CH2NH), 7.22 (s, 1H, CH (thiophene)), 7.26 (s, 1H,
CH (thiophene)), 7.52−7.57 (m, 1H, arom), 7.73−7.80 (m, 2H,
arom), 7.95 (s, 1H, CONH), 8.36−8.39 (d, 1H, J = 8.66 Hz, arom)
ppm. 13C NMR (MeOH-d4, 75 MHz) δ: 15.16 (CH3), 15.35 (CH3),
21.95 (CH2CH2CH2 (cyclopentene)), 23.12, 23.97 (CH2(CH2)2CH2
(tacrine)), 25.14 (CH2(CH2)3CN (tacrine)), 29.45 (CH2)3CH2C
N (tacrine)), 39.54, 39.64 (CH2CH2CH2 (cyclopentene)), 41.09
(NHCH2CH2NHCO), 51.15 (NHCH2CH2NHCO), 113.34 (arom),
117.30 (arom), 120.24 (CH (tacrine)), 126.59 (CH (tacrine)), 126.65
(CH (tacrine)), 131.53 (CH (tacrine)), 134.19 (CH (tacrine)), 134.93
(CH (tacrine)), 135.87 (CH (thiophene)), 136.27 (arom), 138.07
(arom), 138.14 (arom), 142.57 (arom), 144.21 (arom), 158.46
(arom), 159.42 (COOH), 165.96 (CONH) ppm. ESI-HRMS:
C32H34N3O3S2 calcd 572.2036 m/z [M+H]+, anal. 572.2037 m/z [M
+H]+. HPLC purity 95.0%, retention time 25 min.
9a,9b-Dimethyl-N2,N8-bis(8-((1,2,3,4-tetrahydroacridin-9-yl)-
amino)octyl)-5,6,9a,9b-tetrahydro-4H-indeno[5,4-b:6,7-b′]-
dithiophene-2,8-dicarboxamide (11b Ring-Closed form). ESI-MS
1
482.2 m/z [M+2H]2+, 963.6 m/z [M+H]+. H NMR (MeOH-d4, 400
MHz) δ: 1.29−1.44 (m, 16H, 2 × NHCH2CH2(CH2)4CH2CH2-
NHCO), 1.51−1.58 (m, 4H, 2 × NH(CH2)6CH2CH2NHCO), 1.80−
1.88 (m, 6H, 2 × NHCH2CH2(CH2)6NHCO, CH2CH2CH2 (cyclo-
pentene)), 1.94 (s, 6H, 2 × CH3), 1.95−1.99 (m, 8H, 2 ×
CH2(CH2)2CH2 (tacrine)), 2.44−2.48 (t, 4H, CH2CH2CH2 (cyclo-
pentene), J = 2.46 Hz), 2.69−2.72 (m, 4H, 2 × CH2(CH2)2CH2CN
(tacrine)), 3.01−3.03 (m, 4H, 2 × CH2(CH2)2CH2CN (tacrine)),
3.23−3.27 (t, 4H, 2 × CONHCH2(CH2)7NH, J = 3.25 Hz), 3.94−
3.97 (t, 4H, 2 × CONH(CH2)7CH2NH, J = 3.95 Hz), 6.63 (s, 2H, 2 ×
CH (thiophene)), 7.57−7.61 (m, 4H, arom (tacrine)), 7.75−7.87 (m,
4H, arom), 8.38−8.41 (d, 2H, arom, J = 8.39 Hz) ppm. HPLC purity
95.4%, retention time 22.22 min.
5-Methyl-4-(2-(2-methyl-5-((8-((1,2,3,4-tetrahydroacridin-9-yl)-
amino)octyl)carbamoyl)thiophen-3-yl)cyclopent-1-en-1-yl)-
thiophene-2-carboxylic Acid (10b). This compound was synthesized
analogously as 11a, using compound 5 (64 mg, 0.18 mmol),
aminoalkyl tacrine 9b (60 mg, 0.18 mmol), EDCI (35 mg, 0.18
mmol), triethylamine (28 μL, 0.2 mmol) and a catalytic amount of
HOBt (10 mg, 0.07 mmol) in DMF (10 mL). The crude compound
was purified via column chromatography with dichloromethane/
methanol/ammonia 10:1:0.1 as eluent system. The target compound
was obtained as yellow solid (30 mg, 25%). MP 195−200 °C. ESI-MS:
655.3 m/z [M+H]+. ESI-HRMS: 328.1612 m/z [M+2H]2+, 655.3147
m/z [M+H]+. 1H NMR (MeOH-d4, 300 MHz) δ: 1.15−1.20 (m, 8H,
NHCH2CH2(CH2)4CH2CH2NHCO), 1.46−1.56 (m, 2H, NH-
(CH2)6CH2CH2NHCO), 1.65−1.70 (m, 2H, NHCH2CH2(CH2)6-
NHCO), 1.89−1.93 (m, 10H, CH2(CH2)2CH2 (tacrine), 2 × CH3),
2.03−2.13 (m, 2H, CH2CH2CH2 (cyclopentene)), 2.72−2.85 (m, 6H,
CH2(CH2)2CH2CN (tacrine)), CH2CH2CH2 (cyclopentene)),
2.97−2.99 (m, 2H, CH2(CH2)2CH2CN (tacrine)), 3.23−3.28 (t,
2H, NH(CH2)7CH2NHCO, J = 7.09 Hz), 3.63−3.69 (t, 2H,
NHCH2(CH2)7NHCO, J = 7.22 Hz), 7.40−7.47 (m, 3H, arom
(tacrine), 2 × CH (thiophene)), 7.61−7.66 (m, 1H, arom (tacrine)),
7.73−7.76 (m, 1H, arom (tacrine)), 8.17−8.20 (d, 1H, arom (tacrine),
J = 8.77 Hz) ppm. 13C NMR (MeOH-d4, 75 MHz) δ: 14.65 (CH3),
14.68 (CH3), 21.80, 22.99 (CH2(CH2)2CH2 (tacrine)), 23.87
(CH2CH2CH2 (cyclopentene)), 24.83 (CH2(CH2)2CH2CN (ta-
crine)), 27.54, 27.76, 29.29, 30.07, 30.08, 30.45, 31.41
(NHCH2(CH2)6CH2NHCO, CH2(CH2)2CH2CN (tacrine)),
39.41, 39.46 (CH2CH2CH2 (cyclopentene)), 40.66 (NHCH2(CH2)6-
CH2NHCO), 49.62 (NHCH2(CH2)6CH2NHCO), 112.87 (arom),
117.08 (arom), 120.05 (CH (tacrine)), 126.34 (CH (tacrine)), 126.54
(CH (tacrine)), 130.55 (CH (thiophene)), 131.77 (CH (thiophene)),
134.14 (CH (tacrine)), 135.63 (arom), 136.14 (arom), 136.20 (arom),
136.25 (arom), 137.86 (arom), 137.97 (arom), 139.80 (arom), 141.42
4,4′-(Cyclopent-1-ene-1,2-diyl)bis(5-methyl-N-(8-((1,2,3,4-tetra-
hydroacridin-9-yl)amino)octyl)thiophene-2-carboxamide) (11b
Ring-Opened Form). This compound was synthesized analogously
as 11a, using compound 5 (91 mg, 0.26 mmol), aminoalkyl tacrine 9b
(170 mg, 0.53 mmol), EDCI (105 mg, 0.55 mmol), triethylamine (77
μL, 0.55 mmol), and a catalytic amount of HOBt (10 mg, 0.07 mmol)
in DMF (10 mL). The crude compound was purified via column
chromatography with dichloromethane/methanol/ammonia 10:1:0.1
as eluent system. The target compound was obtained as yellow solid
(80 mg, 32%). MP 145−148 °C. ESI-MS 482.2 m/z [M+2H]2+, 963.6
m/z [M+H]+. ESI-HRMS: 482.2735 m/z [M+2H]2+, 965.5377 m/z
[M+H]+. 1H NMR (MeOH-d4, 300 MHz) δ: 1.28−1.35 (m, 16H, 2 ×
NHCH2CH2(CH2)4CH2CH2NHCO), 1.50−1.54 (m, 4H, 2 ×
NH(CH2)6CH2CH2NHCO), 1.67−1.74 (m, 4H, 2 × NHCH2-
CH2(CH2)6NHCO), 1.86−1.92 (m, 14H, 2 × CH2(CH2)2CH2
385
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Research Article
(arom), 142.19 (arom), 158.09 (arom), 164.20 (COOH), 166.43
(CONH) ppm. ESI-HRMS: C38H47N4O2S2 calcd 655.3135 m/z [M
+H]+, anal. 328.1612 m/z [M+2H]2+, 655.3147 m/z [M+H]+. HPLC
purity 95.1%, retention time 26 min.
remained, following with drying under high vacuum for 2 h. The
residue was then dissolved in DMSO to form a 2 mM stock solution,
which was subsequently stored frozen at −20 °C. The testing
compound 11b was dissolved in DMSO to form 500 μM stock
solution and subsequently diluted to 100, 20, and 4 μM solutions in
DMSO (corresponding to 5, 1, and 0.2 μM in final testing), which
were stored in the freezer at −20 °C.
For the thioflavin T-based fluorometric assay, analyses were
performed with a Varian Cary Eclipse fluorescence spectrophotometer
using 96-well fluorescence microtiter plates (Nunc, Denmark).
Fluorescence was monitored at 450 nm (λexc) and 482 nm (λem)
with excitation and emission slits of 5 nm bandwidth using Cary
Eclipse Kinetics program. The fluorescence emission spectrum was
recorded as a scan of the average intensity values around 5 min after
subtracting the background fluorescence from thioflavin T (2.5 μM)
and AChE (1 U/mL) solution with or without the corresponding
inhibitor. Each assay was run in duplicate.
To the mixed solution of AChE (40 μL, final concentration 1U/
mL), buffer (50 μL), and inhibitor (5 μL as either ring-opened or
closed form, final concentration 5, 1, and 0.2 μM) was added the
solution of Aβ1−40 (5 μL, final concentration 100 μM). The resulting
solution (100 μL volume) was incubated at 25 °C for 1, 2, and 5 h
with or without the presence of inhibitor. Thioflavin T solution (20
μL, final concentration 2.5 μM) was added to the corresponding well
at the specific time spot, that is, 1, 2, and 5 h, and the fluorescence was
measured according to the method described above.
Experimental Procedures for the Pharmacological Inves-
tigations. Cholinesterase Inhibition and Kinetic Studies. AChE
(E.C.3.1.1.7, Type VI-S, from electric eel), hAChE (recombinant,
expressed in HEK 293 cells), and BChE (E.C.3.1.1.8, from equine
serum) were purchased from Sigma-Aldrich (Germany). 5,5′-
Dithiobis(2-nitrobenzoic acid) (DTNB, Ellman’s reagent), acetyl-
thiocholine (ATC), and butyrylthiocholine (BTC) iodides were also
obtained from Sigma-Aldrich (Germany). UV measurements were
performed on Varian Cary 50 Scan UV−vis spectrometer, and 4 mL
disposable cuvettes were used for the testing. As reference for ChE
inhibition, the established drug tacrine hydrochloride was used, and
purchased from Sigma-Aldrich (UK).
Ellman’s assay was performed as described in the following
procedure: stock solutions of the test compounds were prepared in
ethanol, 100 μL of which gave a final concentration of 10−3 M when
diluted to the final volume of 3.32 mL. The highest concentration of
the test compounds applied in the assay was 10−4 M (10% EtOH in
the stock solution did not influence enzyme activity). The rest of the
testing concentrations were diluted from stock solution using the
buffer below. In order to obtain an inhibition curve, at least five
different concentrations (normally 10−4−10−9 M) of the test
compound were measured at 20 °C and 412 nm, each concentration
in triplicate.9,22
All the data were processed on GraphPad Prism 5 software (version
For buffer preparation, 1.36 g of potassium dihydrogen phosphate
(10 mmol) was dissolved in 100 mL of water and adjusted with NaOH
to pH = 8.0 0.1. Enzyme solutions were prepared to give 2.5 units/
mL in 1.4 mL aliquots. Furthermore, 0.01 M DTNB solution, 0.075 M
ATC solution, and 0.075 M BTC solution were used. A cuvette
containing 3.0 mL of phosphate buffer, 100 μL of the respective
enzyme, 100 μL of DTNB, and 100 μL of the test compound solution
was allowed to stand at 25 °C for 4.5 min (or 1 h in the case of
prolonged incubation). The reaction was started by addition of 20 μL
of the substrate solution (ATC/BTC). The solution was mixed
immediately, and exactly 2.5 min after substrate addition the
absorption was measured. For the reference value, 100 μL of water
replaced the test compound solution. For determining the blank value,
additionally 100 μL of water replaced the enzyme solution. The
inhibition curve was obtained by plotting the percentage enzyme
activity (100% for the reference) versus the logarithm of test
compound concentration using GraphPad Prism software (version
5.0).
5.0) to generate the aggregation curves.
Docking and Molecular Dynamics Simulations. To reveal
potential binding modes of compound 11b, docking studies were
carried out with the open and the closed form of the inhibitor. The
AChE crystal structure with PDB code 2CKM from the RCSB Protein
Data Bank was selected for this purpose.31,33 It corresponds to the
structure of Torpedo californica AChE (TcAChE) in complex with a
bis-tacrine linked by a seven-carbon spacer. The selection was based
on the good resolution of the structure (2.15 Å) and the similarity of
the bound ligand with the compounds investigated here. Moreover,
TcAChE shows a high sequence similarity (58% identity and 73%
similarity) and a conserved binding site with respect to Electrophorus
electricus AChE (eeAChE) used for the biochemical/pharmacological
studies. The identity and similarity calculations were performed by the
pairwise sequence alignment of TcAChE (2CKM) and EeAChE
(1C2O) with the Needle program, using the EBLOSUM62 matrix, of
EMBOSS v.6.3.1.34
Docking was carried out with GOLD 5.1.35 The protein was set up
for docking by removing all water molecules and nonprotein atoms
and protonating the amino acids with MOE 2011.10 using Protonate
3D at pH 7.36 The search region was focused on the binding area
comprising the CAS, the binding gorge, and the PAS. The binding site
was defined by the following residues: Tyr70, Asp72, Trp84, Asn85,
Gly117, Gly118, Gly119, Tyr121, Glu199, Ser200, Trp279, Phe288,
Phe290, Glu327, Phe330, Phe331, Tyr334, Trp432, Met436, Ile439,
His440, and Tyr442. The ligand structures were either built in MOE
(compounds 11b opened and closed) or extracted from the crystal
structure (ligands for redocking experiments, cf. below). The
protonation was set according to the expected ionization state at pH
7; for 11b, this implied protonation at the tacrine nitrogen atoms,
consistent with their pKa values between 8.7 and 9.6, as calculated with
MoKa.37 The structures were energy minimized in MOE using the
MMFF94x force field and an rms-gradient of 0.001 kcal/(mol·Å).
Using default settings in GOLD, the best-suited scoring function
was selected based on redocking experiments with the seven-carbon
spacer bis-tacrine of crystal structure 2CKM. Fifty docking runs carried
out with each of the four scoring functions available in GOLD (ASP,
CHEMPLP, ChemScore, GoldScore) showed that best results are
obtained with the Astex Statistical Potential (ASP),38 with good
clustering on the top ranks and rmsd values of 1.07 Å (rank 1) and
0.74 Å (rank 2) of the docked pose with respect to the native pose in
the crystal structure. Similar results were obtained in redocking
For human AChE, the reaction time after the addition of ATC was
slightly prolonged to 5 min.
For the kinetic measurements the following substrate concen-
trations were used: 28.2, 56.5, 113, 226, and 452 μM. In contrast to
the above-described affinity measurements, the reaction time was
extended to 4 min before measurement of the absorption. Vmax and Km
values of the Michaelis−Menten kinetics were calculated by nonlinear
regression out of the substrate velocity curves. Linear regression
(double reciprocal) was used for calculating the Lineweaver−Burk
plots for reasons of illustration. All kinetic studies were also processed
using GraphPad Prism 5 software (version 5.0).32
AChE-Induced β-Amyloid Aggregation Testing. Thioflavin T
(Basic Yellow 1) was purchased from Acros (Belgium). AChE (from
electric eel), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), and absolute
DMSO (BioReagent) were purchased from Sigma-Aldrich (Germany).
Deionized water was used to prepare the buffer solution. Amyloid β-
protein (1−40) trifluoroacetate was purchased from Bachem AG
(Burbendorf, Switzerland). Buffers and other chemicals were of
analytical grade. The stock solutions of thioflavin T and AChE were
prepared using the same buffer solution (25 mM NaH2PO4, 100 mM
NaCl and final pH 8.0) to reach final concentration at 15 μM and 2.5
U/mL, respectively.
Aβ1−40 was suspended in HFIP and left to stay at room
temperature for 4 h until a clear solution (20 mg/mL) was formed.
The solvent was removed under a stream of nitrogen until a clear film
386
dx.doi.org/10.1021/cn500016p | ACS Chem. Neurosci. 2014, 5, 377−389

ACS Chemical Neuroscience
Research Article
experiments with the five-carbon spacer bis-tacrine to crystal structure
2CMF.31
Author Contributions
M.D. was responsible for the supervision and development of
the whole project in collaboration with B.K. N.K. under the
supervision of B.K. performed the synthesis of compound 5 and
the testing of physicochemical properties of the target
compounds. X.C. performed the syntheses of the target
compounds, enzyme inhibition, and kinetic studies as well as
AChE-induced β-amyloid inhibition testing with the help from
U.H. S.W. conducted the computaional docking studies and
B.M. helped with the molecular dynamics study, both under the
supervision of C.A.S.
Accordingly, docking of the 11b inhibitors was carried out with the
ASP function. Due to the higher conformational flexibility of the
inhibitors in comparison to the bis-tacrines of 2CKM and 2CMF, the
number of operations in the GOLD GA settings was increased to
4 000 000. Furthermore, to improve the convergence, a scaffold match
constraint was applied to place one tacrine moiety in the CAS, using
the corresponding moiety of the bis-tacrine crystal structure 2CMF as
reference for the least-squares fit (with a weight set to 5.0 in GOLD).
Molecular dynamics (MD) simulations were carried out to analyze
the stability and plausibility of the binding modes suggested by
docking. The ligands were setup for simulation with modules of
Amber 12.39 RESP charges were calculated for all ligands based on
HF/6-31G electrostatic potentials obtained with Gaussian 03.40,41 The
General Amber Force Field (GAFF) was used for the ligands,
assigning atom and bond types with antechamber and missing force
field parameters with parmchk.42,43 For the protein, the Amber ff99SB
force field was used. All simulations were based on protein structure
2CKM. The missing amino acids (His486, Ser487, Gln488, and
Asn489) between Pro485 and Ser490 were inserted using Modeller
9.11 with rigid protein during model refinement.44 Three disulfide
bonds were identified and retained in the MD setup (between
cysteines 67−94, 254−265, and 402−521, respectively). All histidines
were protonated at the ε-nitrogen, except His398 and His440, which
were both protonated at the δ-nitrogen to ensure a proper hydrogen-
bonding network. Water molecules present in the crystal structure
within a radius of 4 Å of the seven pocket residues (Tyr70, Trp84,
Tyr121, Ser200, Trp279, Glu327, and His440) were included in the
setup.
To relieve initial strains, very short energy minimizations (30 steps)
of the docked ligands within the rigid proteins were performed using a
generalized Born implicit solvent model as implemented in the Amber
module sander.45,46 Subsequently, the entire complexes were
minimized for 200 steps. After minimization, the complexes were
solvated with tleap in rectangular TIP3P water boxes,47 resulting in box
sizes of approximately 85 Å × 83 Å × 82 Å. For equilibration, water
molecules were allowed to move freely (keeping protein and ligand
rigid) in the constant-volume box while increasing the temperature
from 100 to 300 K over 20 ps, followed by cooling to 100 K over 5 ps.
The temperature was adjusted by means of the Berendsen weak
coupling algorithm with a time constant of 0.5 ps. Subsequently, the
complete systems were treated without constraints and gradually
heated to 300 K over a time period of 25 ps. The systems thus
obtained were used as input for MD production runs with NAMD.48
Using a time step of 2 fs, trajectories of 18 ns were generated for each
system. The systems were simulated under NPT periodic boundary
Funding
Part of this work was funded by the German Research
Foundation (Deutsche Forschungsgemeinschaft under DFG
DE1546/6-1).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Michaela Prinz for her kind help on
establishing the Aβ aggregation assay.
ABBREVIATIONS
■
Aβ, β-amyloid protein; AChE, acetylcholinesterase; AD,
Alzheimer’s disease; BACE-1, beta-site amyloid precursor
protein cleaving enzyme 1, or beta-secretase 1; BChE,
butyrylcholinesterase; CAS, catalytic active site; ChE(I),
cholinesterase (inhibitor); DTE, 1,2-α-dithienylethene; EDCI,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochlor-
ide; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; HOBt, hydrox-
ybenzotriazole; NMDA, N-methyl-D-aspartate; PAS, peripheral
anionic site; SARs, structure−activity relationships; TFA,
trifluoroacetic acid
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ASSOCIATED CONTENT
■
S
* Supporting Information
Spectral data of target compounds; HPLC chromatography for
compound 11b; figures showing results of pharmacological
tests; figures showing results of computational studies. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
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Corresponding Author
*E-mail: michael.decker@uni-wuerzburg.de. Tel: 0049-931-31-
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