Design and activity of cationic fullerene derivatives as inhibitors of acetylcholinesterase

Article abstract of DOI:10.1039/b604361e

Four different regioisomers of cationic bis-N,N- dimethylfulleropyrrolidinium salts have been prepared and evaluated as inhibitors of the enzymatic activity of acetylcholinesterase. These fullerene-based derivatives were found to be noncompetitive inhibitors of acetylthiocholine hydrolysis. Molecular modelling was used to describe the possible interactions between the fullerene cage and the amino acids surrounding the cavity of the enzyme. The cationic C₆₀ derivatives used in this study represent a new class of molecules potentially able to modulate the enzymatic activity of acetylcholinesterase. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b604361e

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Design and activity of cationic fullerene derivatives as inhibitors of
acetylcholinesterase
Giorgia Pastorin,a Silvia Marchesan,b Johan Hoebeke,a Tatiana Da Ros,*b Laurence Ehret-Sabatier,c
Jean-Paul Briand,a Maurizio Pratob and Alberto Bianco*a
Received 24th March 2006, Accepted 8th May 2006
First published as an Advance Article on the web 25th May 2006
DOI: 10.1039/b604361e
Four different regioisomers of cationic bis-N,N-dimethylfulleropyrrolidinium salts have been prepared
and evaluated as inhibitors of the enzymatic activity of acetylcholinesterase. These fullerene-based
derivatives were found to be noncompetitive inhibitors of acetylthiocholine hydrolysis. Molecular
modelling was used to describe the possible interactions between the fullerene cage and the amino acids
surrounding the cavity of the enzyme. The cationic C60 derivatives used in this study represent a new
class of molecules potentially able to modulate the enzymatic activity of acetylcholinesterase.
by the rings of 14 conserved aromatic residues.17 The current
Introduction
inhibitors present different chemical structures, ranging from
Different classes of fullerene derivatives have shown interesting
simple cationic compounds to bis-quaternary salts able to interact
potential in biomedical applications.1 The biological proper-
ties of fullerenes include photocleavage,2 antiapoptotic activity,3
neuroprotection,4 drug and gene delivery,5 antioxidation,6
chemotaxis,7 antibacterial activity,8 and enzyme inhibition.9 Con-
cerning the development of enzyme inhibitors based on a C60
core, only a few examples have been reported.9 It has been
demonstrated that the structure of C60 is highly complementary
to the HIV protease (HIVP).9a,10 As a consequence, a series of
fullerene derivatives has been designed and found to bind to
the active site of HIVP, which is characterised by a hydrophobic
pocket. Similarly, an antibody raised against a series of fullerene
conjugates presented a non polar binding site highly specific for
C60.11
with both principal and peripheral binding sites.18,19 In particular,
the dual-site inhibitors are able to form an important cation–p
interaction with Trp279 and Trp84 at the top and the bottom
of the hydrophobic pocket, respectively. On the basis of these
chemico-physical characteristics of the enzyme cavity and the
search of efficient ligands, cationic fullerene derivatives may mimic
the action of bis-quaternary salts and become a new class of
potential inhibitors of AChE.
Results and discussion
Design and synthesis of AChE fulleropyrrolidine inhibitors
Another family of enzymes that contain a hydrophobic cavity
with the ideal dimensions to accommodate a fullerene moiety
is that of cholinesterases.12 Within these serine esterases, acetyl-
cholinesterase (AChE) plays an important role in the regulation
of functions of central and peripheral nervous systems.13 AChE
hydrolyses the cationic neurotransmitter acetylcholine (ACh).14
Dysfunctions on the level of ACh have a direct implication in the
development of neurodegenerative disorders such as Alzheimer’s
disease (AD).15 Therefore, selective ligands have been developed
over the years because of the pharmacological and toxicological
importance of AChE. The active site of AChE has been described
at the atomic resolution by X-ray crystallography of the protein
alone or complexed to different types of inhibitors.16 It comprises
the principal site, constituted by the catalytic triad Ser200,
Glu327 and His440, which is located at the bottom of a gorge
The most effective, past inhibitors of AChE were characterised
by the molecular structures bearing tertiary amines or quaternary
ammonium salts, which interacted with the catalytic triad and
Trp84 at the base of the gorge.18,20,21 After a demonstration that
the peripheral site at the rim of the cavity played also an important
role in the enzymatic activity, new generations of inhibitors able
to simultaneously block the two binding sites have been conceived
and developed.19,22 In fact, bivalent ligands enhance the affinity
for the target.21f For example, decamethonium is a symmetric
bis-quaternary ammonium salt that inhibits AChE by spanning
19a
˚
the distance of nearly 15 A between Trp84 and Trp279. The
˚
dimension of the C60 sphere (∼7.0 A diameter) and the possibility
of its double functionalization23 suggested the idea that fullerene
modified with the appropriate functionalities could accommodate
into the hydrophobic core of AChE and display an inhibitory ac-
tion. Indeed, cationic bis-N,N-dimethylfulleropyrrolidinium salts
1–4 (Fig. 1) can be considered very promising compounds because
the aromatic fullerene cage perfectly fits into the hydrophobic core
of AChE while the N,N-dimethylpyrrolidinium groups located
around the sphere may tackle both the main and secondary active
sites. The C60 cage would not only act as a spacer between the
quaternary ammonium moieties but might play an active role in
the target recognition process by establishing its own interaction
with the enzyme.
˚
approximately 20 A deep, and the peripheral site, near the edge
˚
of the cavity. The two sites, about 15 A apart, are surrounded
aInstitute of Molecular and Cellular Biology, UPR 9021 CNRS, 67084,
Strasbourg, France. E-mail: A.Bianco@ibmc.u-strasbg.fr; Fax: +33 388
610680; Tel: +33 388 417088
bDepartment of Pharmaceutical Sciences, University of Trieste, 34127,
Trieste, Italy. E-mail: daros@units.it
cEuropean School of Chemistry, Polymers and Materials, 67087, Strasbourg,
France
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the scope of this work, and the AChE inhibition experiments have
been performed using the racemic mixtures.
Study of the inhibition of AChE activity
The inhibitory activity of the four cationic fulleropyrrolidinium
regioisomers was evaluated on AChE from Electrophorus elec-
tricus.18 The determination of the type of inhibitory mechanism
can be directly correlated to the action of the inhibitor and its
interaction with the enzyme active site. The initial hydrolysis rates
(DA/Dt) were plotted versus the concentration of acetylthiocholine
iodide (ATCI), at different concentrations of inhibitors (Fig. 2).
The different curves were calculated using the typical equations
for the competitive and noncompetitive inhibition process by a
non linear regression method (Sigmaplot, Chicago, Il). The Ki
values and the corresponding square correlation coefficients (R2)
are reported in Table 1. The discrimination between competitive or
noncompetitive action of the inhibitors was assessed by comparing
the values of R2 using eqn (1) and (2) (see Experimental section).
We wanted first to test the enzyme specificity using edrophonium
chloride. This competitive inhibitor was employed in our study as
the reference compound.25 We found an inhibition constant of
0.054 lM which was in full agreement with the previous data
Fig. 1 Molecular structures of bis-N,N-dimethylfulleropyrrolidinium
salts 1–4. For simplicity, only one enantiomer of chiral trans-2 (1), trans-3
(2) and cis-3 (4) is illustrated.24b
Table 1 AChE enzymatic inhibition of bis-N,N-dimethylfullero-
pyrrolidinium salts
The synthesis of fullerene bis-adducts 1–4, using the 1,3-dipolar
cycloaddition reaction of azomethine ylides to C60, was previously
reported.24 C60 was reacted with sarcosine and paraformalde-
hyde in toluene. The mono-adduct and the different regioiso-
meric bis-adducts were separated by repeated medium-pressure
column chromatography, preparative TLC and semipreparative
HPLC. The most abundant bis-adducts were methylated with
methyl iodide, affording compounds 1–4 (Fig. 1), which were
used for the study of inhibition activity against AChE. The three
derivatives 1, 2 and 4 are chiral due to their double addition
pattern.24b The separation of the single enantiomers was beyond
Entry
K
M/lM
Ki/lMa
R2b
1 (trans-2)
76.9 9.4
64.4 4.7
80.9 10.6
76.3 11.0
68.9 12.3
15.6 1.7
31.4 1.4
27.8 2.8
17.7 1.8
0.054 0.009
0.950
0.988
0.959
0.963
0.967
2 (trans-3)
3 (trans-4)
4 (cis-3)
Edrophonium chloride
a The inhibition constants Ki are noncompetitive for 1–4 and competitive
for edrophonium chloride. b R2 corresponds to the square of the correlation
coefficient.
Fig. 2 Kinetics analyses of AChE inhibition by cationic bis-N,N-dimethylfulleropyrrolidinium salts 1–4. The initial hydrolysis rates are plotted against
the concentration of the substrate. The concentrations of each inhibitor are reported on the theoretical curves.
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reported in the literature (0.050 lM) (Table 1).25 All fulleropyrro-
lidinium salts 1–4 exhibited instead a noncompetitive inhibition
as clearly indicated by a decrease of Vmax values with increasing
amount of substrate, while the values of KM remained almost
unchanged (Table 1). This is a common behaviour for AChE
noncompetitive inhibitors that do not affect the affinity of the
enzyme towards the substrate.26 The fullerene derivatives appear
not to directly interact with the catalytic triad but partially block
the binding gorge. These fullerene-based ligands are probably
not able to enter the gorge as deep as other double cationic
inhibitors,18,22 and this could explain their noncompetitive activity.
Ki values for the four fulleropyrrolidinium salts are in the
micromolar range (Table 1). These values are similar to those
reported for other noncompetitive molecules.27 The comparison
between the affinity data of compounds 1–4 suggested that the
relative position of the N,N-dimethylpyrrolidinium moieties did
not produce a remarkable variation on the inhibition activity. In
the case of the trans isomers 1–3 the distance between the cationic
ligands were readily synthesised as racemates. The modelling
however required analysis of each enantiomer separately, because
the different isomers could interact differently with the chiral
environment of the AChE gorge. In the case of trans-2, the
calculation was done on both chiral regioisomers. Each derivative
was put manually into the active site of the enzyme close to
the catalytic triad. The gorge width calculated on the center of
27c
˚
mass of Trp279 and Gly335 has an average of 12.0 A. The
two quaternary pyrrolidinium groups were oriented towards the
principal and the peripheral binding site, respectively. After a first
minimization under fixation of the enzyme backbone, the contact
residues between the C60 ligand and the active site residues of the
˚
receptor within a distance of 5.0 A were determined. Subsequently,
dynamics and minimization using the frames corresponding to the
most stable lower energy conformation state were run, enabling the
fullerene derivatives to relax inside the enzyme active site. Water
molecules in the gorge were not taken into consideration during
the calculations.21c It is reasonable to assume that these molecules,
which present highly reduced hydrogen bond coordination, are
rapidly displaced.16a This phenomenon is also known as the
desolvation effect. Desolvation of hydrophobic surfaces was
shown to improve for example the binding affinity in the case of
fullerene inhibitors of HIV-1 protease.10a Any significant alteration
in the structure of the enzyme was observed after the minimization
analysis in comparison to the original crystal structure. Fig. 3
shows the molecular structures of the complexes between the
residues of the AChE active pocket (black) and the regioisomers 1,
3 and 4 (cyan). The catalytic triad is displayed in magenta while the
two key residues at the principal (Trp84) and secondary (Trp279)
binding sites are shown in blue. The C60 derivatives present a high
steric hindrance and a reduced conformational flexibility. The
elements introduced around the fullerene cage can be oriented
in a limited number of ways inside the AChE active channel. All
three isomers are characterised by close proximity between the
bottom dimethylpyrrolidinium group and Trp84 while the second
˚
nitrogens is comprised between 10.0 and 11.5 A (Table 2). The
best inhibitor is compound 1 (trans-2) where the functional groups
are further away. This spatial arrangement is likely in favour of a
better orientation towards Trp84 and Trp279, mimicking other bis-
quaternary salts, unless other residues affect this binding driving
the cationic groups in a different direction (vide infra). On cis
˚
isomer 4, the two charged groups are very close (7.45 A) and
located on the same side of the C60 cage. This conformation
does not permit the quaternary salts to orient towards both the
principal and secondary binding site. The inhibition value was
almost identical to that of compound 1, reinforcing the finding of
a noncompetitive mechanism of AChE inhibition displayed by this
new class of ligands. These results should be considered carefully,
but they seem to indicate that not all the cationic groups on C60
could completely reach the binding sites of the AChE gorge. To
explain this behaviour and to disclose the possible interactions
exerted by the bis-N,N-dimethylfulleropyrrolidinium salts inside
the hydrophobic cavity of AChE, we have subsequently carried
out a docking analysis.
˚
cationic group remains at a distance greater than 10 A from
Trp279 (Table 2). The lack of the second cation–p interaction,
typical of the bis-quaternary inhibitors,31 is compensated by a
series of salt bridges between the two ammonium groups and
various aspartic and glutamic residues. In particular, the bottom
pyrrolidine of the trans isomers is very close to Glu199 (4.01–
Docking inside AChE active site
The molecular modelling study was performed using the crystal
structure coordinates of AChE from Torpedo californica.20 This
acetylcholinesterase is structurally homologous to that from Elec-
trophorus electricus used for the determination of the fulleropyrro-
lidine inhibitory activity.28–30 The simulations were performed on
f,sC-trans-2 (1), trans-4 (3) and f,sA-cis-3 (4) isomers. The fullerene
˚
4.27 A) and that at the top is involved in bonding with Asp72 and
˚
Glu82 (4.44–4.91 A). The same interactions are less strong for the
cis isomer where the distance between the positive charges and the
˚
carboxylate side chains are comprised between 5.0 and 6.25 A.
We could also evidence stabilisation by a cation–p interaction
Table 2 AChE docking parameters of bis-N,N-dimethylfulleropyrrolidinium salts
+
+
a
+
van der Waals/kcal mol−1b
Electrostatic/kcal mol−1b
˚
˚
Entry
N (CH3)2 · · · N (CH3)2/A
W84 · · · N (CH3)2/A
1 (f,sC-trans-2)
1 (f,sA-trans-2)
3 (trans-4)
10.83
11.42
10.01
7.45
4.55
4.58
4.20
4.25
−107
−131
−113
−102
−613
−603
−613
−600
4 (f,sA-cis-3)
4 (f,sA-cis-3)
180◦ y-rotc
7.17
ndd
−120
−557
a The distance between the two pyrrolidine nitrogens was measured after the minimization of the ligand into the active site of AChE. b Energy of interaction
between the enzyme and the inhibitor (calculated as continuous evaluation energy). c rot = rotation. d nd = not determined.
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bridging the top pyrrolidinium nitrogen with Trp432. In addition,
the hydrophobic surface of the C60 is surrounded and stabilized by
a high number of p–p contacts with the aromatic side chains of
the residues located near the peripheral (Tyr70, Phe75, Tyr121,
Tyr334) and main (Phe288, Phe290, Phe330, Phe331) binding
domains. In fact, nearly 70% of the surface of the AChE cavity
is covered by aromatic rings.17 The favourable interactions by
van der Waals forces are also supported by the values of the
non covalent complex stabilization energy (Table 2). Simulation
using the second enantiomer of the other chiral trans-2 derivative
provided the same way of binding of its mirror image (not shown).
Molecular modelling clearly showed there were no deep differences
between the two enantiomers. The enzyme seemed to show an
inherent preference for either of the two molecules. It is worth
noting that the bis-N,N-dimethylpyrrolidinium moieties might not
be large enough to influence the stereospecific preference among
the two components of the racemate.32
Although enantiomeric selectivity is a key issue in the devel-
opment of new drugs and inhibition of racemates can be less
effective than inhibition of the preferred enantiomer, examples of
non selectivity between enantiomers have already been described
for other classes of AChE ligands.22a
Due to a difficulty in the bulky C60 sphere rotating and
reorienting into the enzyme cavity, we could not exclude that
the fulleropyrrolidinium salts might display alternative modes of
binding. In fact, the cis isomer could adopt different orientation
inside the binding pocket, stabilised by favourable interactions.
A simulation was repeated after rotation of 180◦ around the
y-axis. We found that the two quaternary ammonium groups
pointed towards Trp279 at an ideal distance for the formation
of a bidentate cation–p bond (Fig. 4). We measured a distance of
˚
4.31 and 5.07 A for the two pyrrolidinium nitrogens from the
indole ring of Trp279. The alternative stable complex derived
from this orientation supports the good affinity of the cis-3 C60
derivative in comparison to trans-3 and trans-4 despite the fact that
the ammonium groups are spatially located at a shorter distance
(Table 2).
Fig. 3 Ball and stick molecular model representation of the AChE hy-
drophobic pocket complexed with bis-N,N-dimethylfulleropyrrolidinium
salts f,sC-trans-2 (1) (A), trans-4 (3) (B), and f,sA-cis-3 (4) (C).
Fig. 4 Molecular model of f,sA-cis (4) inside the active site of AChE after
180◦ rotation of its minimized analogue around the y-axis.
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The prediction of the binding geometry and the analysis of the
ligand–receptor interactions permitted us to identify the possible
modes of binding of the fulleropyrrolidinium salts and to explain
the inhibition mechanism derived by the kinetics. The cationic bis-
fulleropyrrolidinium salts are probably not able to enter into the
hybrophobic channel as deep as decamethonium or other bivalent
ligands because they are sterically hindered.31 For this reason,
similar to other types of inhibitors, they are noncompetitive.27
However, the simulations are not conclusive and should be
considered as the initial step to provide possible structures of
the complex between AChE and the fullerene derivatives. As
other large quaternary dual inhibitors were able to crystallise into
the active cavity of the protein,19a we believe that this behaviour
could be exerted also by the fullerene derivatives. It has recently
been shown that a certain flexibility of the loops at the neck
of the gorge permitted the binding of bulky inhibitors such as
galanthamine-based derivatives.33 Indeed, enzymes are dynamic
molecular machines characterised by intrinsic motions which are
fundamental for their function.34
was defined as the amount of enzyme which hydrolyses 1 lmol
of ATCI per minute. We initially determined the concentration
of AChE necessary to hydrolyse 50% of the substrate in about 2
minutes. This value corresponded to 0.175 U ml−1. Self-hydrolysis
of ATCI was verified at concentrations of ATCI between 1·10−5
and 1·10−3 M in the presence of a constant amount of 5,5ꢀ-
dithiobis-2-nitrobenzoic acid (DTNB) (2.5·10−3 M). No ATCI
appreciable hydrolysis in the absence of the enzyme was observed
in this range. The inhibition activity was evaluated by preparing
a reaction mixture containing 10 lL of AChE solution (8.75 U
mL−1 of 0.1 M phosphate buffer, pH 7.2), 250 lL of a solution
of DTNB (stock solution 5·10−3 M in 0.1 M phosphate buffer,
pH 7.2), a variable volume of the buffer solution of ATCI (stock
solution 5·10−3 M in 0.1 M phosphate buffer, pH 7.2), and a
variable volume of the buffer solution of the inhibitor, namely
bis-N,N-dimethylfulleropyrrolidinium salts 1–4 and edrophonium
chloride. The final volume of the mixture was adjusted to 0.5 mL
with the buffer solution. The enzyme was added as the last
component. Edrophonium chloride, the competitive inhibitor that
was taken as a reference, was used in concentrations between
2·10−7 and 2·10−6 M, while the fullerene derivatives varied in the
range 5·10−6 and 5·10−5 M. Bis-N,N-dimethylfulleropyrrolidinium
salts 1–4 (about 1 mg) were initially dissolved in 50 lL of pure
DMSO. Subsequently, 25 lL were diluted in 975 lL of 0.1 M
phosphate buffer at pH 7.2 to obtain a final concentration of
about 4.5·10−4 M. This stock solution was further diluted for
each experiment according to the final concentration of required
inhibitor. The final concentration of DMSO was less then 0.5%.
This amount of DMSO did not alter the activity of AChE. The ki-
netics analyses were conducted for 4 minutes at 23 ◦C following the
variation of the absorbance (DA/Dt) at 412 nm. At this wavelength
the absorbance of fullerene derivatives at the highest inhibitor
concentration is negligible. The experiments were performed on a
UV–Vis Anthelie Advanced V2.5 spectrophotometer.
Finally, the description of the complex contacts may suggest
possible modifications on the surface of the fullerene that can
potentially improve the binding properties as well as modulate the
solubility and bioavailability of this new class of inhibitors.
Conclusions
In this study, we have designed a series of cationic bis-N,N-
dimethylfulleropyrrolidines and demonstrated that they were able
to inhibit the enzymatic activity of acetylcholinesterase. The
inhibition process was noncompetitive and the values of the
affinity constants were found in the micromolar range. The pos-
sible mechanism of ligand–receptor interaction was subsequently
analysed by molecular simulations. Each bis-quaternary salt was
docked into the active site of the enzyme and the models showed
that the aromatic fullerene core perfectly fits the hydrophobic
cavity of AChE, while the ammonium groups can interact with
the side chains of the key amino acid residues of the principal or
peripheral binding sites.
Data analysis
Initial rate (v0) corresponding to the variation of the absorbance
within the first 10 seconds was plotted against the concentration
of ATCI. Determination of the type of inhibition was based
on the Michaelis–Menten equation. Graphs were plotted using
Sigma Plot Multiple Function Non Linear Regression. Michaelis–
Menten parameters KM and Vmax were calculated from the reaction
rate as a function of the substrate concentration. Apparent
Ki constants were calculated by using eqn (1) and (2), that
describe a competitive and a noncompetitive inhibition process,
respectively.36
On the basis of our results, these new compounds might be
potential candidates for the modulation of acetylcholinesterase
activity.
Experimental
Chemistry
All reagents and solvents were obtained from commercial
suppliers and used without further purification. C60 was pur-
chased from Bucky-USA (Houston, TX). Cationic bis-N,N-
dimethylfulleropyrrolidinium salts 1–4 (Fig. 1) were prepared as
described in the literature.24
Vmax · [S]
ꢀ
ꢁ
v0 =
(1)
[I]
Ki
KM
·
1 +
+ [S]
Inhibition assays
Vmax · [S]
ꢀ
ꢁ
v0 =
(2)
Inhibition activity of AChE was determined using the standard
method of Ellman.35 AChE from Electrophorus electricus was
obtained from Sigma (V–S type, C-2888). Lyophilised AChE
was reconstituted in 0.1 M phosphate buffer at pH 7.2 (572 U
mL−1). Enzyme stock solution was prepared by taking 15.3 lL
and diluting in 1 mL of the same buffer. One enzymatic unit (U)
[I]
Ki
(KM + [S]) · 1 +
Vmax and KM were obtained in the absence of in-
hibitor, while Ki was calculated in the presence of the in-
hibitors. [I] and [S] represent the concentration of inhibitors
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(bis-N,N-dimethylfulleropyrrolidinium salts 1–4 and edropho-
nium chloride) and ATCI, respectively.
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Docking was performed using the crystal structure coordinates
1QTI from Torpedo californica.20 Fullerene derivatives 1, 3 and
4 were manually placed into the active site of the enzyme. After
a first minimization using a conjugate gradient under fixation of
˚
the enzyme backbone, the contact residues within 5.0 A between
the C60 derivatives and the amino acids of the active site were
determined. All residues outside the contact pocket and the
backbone (Ca, NH and CO) of the residues within a distance of
˚
5.0 A were fixed to perform 100 psec dynamics at 300 K after an
equilibration step of 10 psec. Conformations were collected every
1000 fsec. The frames corresponding to the minimum were then
selected for the minimization by a conjugate gradient algorithm
(Accelrys, San Diego, CA) enabling the fullerene derivative to relax
inside the enzyme active site.
Acknowledgements
This work was supported by CNRS, University of Trieste and
MIUR (PRIN 2004, prot. 2004035502). The authors thank Flo-
rence Dafoin for the excellent technical assistance and Christelle
Guillier (IBMC, Strasbourg) for the mass spectrometry measure-
ments. We are grateful to Marcel Hibert for helpful discussions on
these topics. Giorgia Pastorin is a recipient of a fellowship from
MNRT (GenHomme Network 2003).
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