Synthesis and in vitro characterization of some benzothiazole- and benzofuranone-derivatives for quantification of fibrillar aggregates and inhibition of amyloid-mediated peroxidase activity

Article abstract of DOI:10.1007/s00044-012-0012-3

Neurodegenerative diseases are characterized by amyloid deposition. Thioflavin T (ThT) is one of the molecules considered for detection of amyloid deposits; however, its lipophilicity is too low to cross the blood-brain barrier. Therefore, there is a strong motivation to develop suitable compounds for in vitro fibril quantification as well as for in vivo amyloid imaging. Moreover, oxidative stress (particularly, uncontrolled peroxidase activity) has frequently been reported to play a critical role in the onset/progression of some neurodegenerative disorders. In this study, we describe the synthesis of some benzothiazole and benzofuranone compounds and examine their peroxidase inhibitory properties. Furthermore, to establish the potential binding of synthesized compounds to amyloid aggregates, their in vitro binding to some non-disease related amyloidogenic proteins were characterized. Analyses of the in vitro binding studies indicated that compounds 2 and 4 bind to the amyloid structures successfully while compounds 1 and 3 showed a low affinity in binding to fibrils. Furthermore, compounds 3 and 4 were observed to inhibit amyloid-mediated peroxidase activity in a reversible un-competitive manner.

Full text of DOI:10.1007/s00044-012-0012-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:115–126
DOI 10.1007/s00044-012-0012-3
ORIGINAL RESEARCH
Synthesis and in vitro characterization of some
benzothiazole- and benzofuranone-derivatives for quantification
of fibrillar aggregates and inhibition of amyloid-mediated
peroxidase activity
•
Seyyed Abolghasem Ghadami Zahra Hossein-pour
•
•
Reza Khodarahmi Sirous Ghobadi
•
Hadi Adibi
Received: 12 December 2011 / Accepted: 27 February 2012 / Published online: 20 March 2012
Ó Springer Science+Business Media, LLC 2012
Abstract Neurodegenerative diseases are characterized
by amyloid deposition. Thioflavin T (ThT) is one of the
molecules considered for detection of amyloid deposits;
however, its lipophilicity is too low to cross the blood–
brain barrier. Therefore, there is a strong motivation to
develop suitable compounds for in vitro fibril quantifica-
tion as well as for in vivo amyloid imaging. Moreover,
oxidative stress (particularly, uncontrolled peroxidase
activity) has frequently been reported to play a critical role
in the onset/progression of some neurodegenerative disor-
ders. In this study, we describe the synthesis of some
benzothiazole and benzofuranone compounds and examine
their peroxidase inhibitory properties. Furthermore, to
establish the potential binding of synthesized compounds
to amyloid aggregates, their in vitro binding to some non-
disease related amyloidogenic proteins were characterized.
Analyses of the in vitro binding studies indicated that
compounds 2 and 4 bind to the amyloid structures suc-
cessfully while compounds 1 and 3 showed a low affinity
in binding to fibrils. Furthermore, compounds 3 and 4 were
observed to inhibit amyloid-mediated peroxidase activity
in a reversible un-competitive manner.
Keywords Amyloid determination ꢀ a-Chymotrypsin ꢀ
Peroxidase activity
Abbreviations
PMSF Phenylmethylsulphonyl fluoride
DMSO Dimethyl sulphoxide
TFE
ThT
TMB
AD
2,2,2-Trifluoroethanol
Thioflavin T
3,30,5,50-Tetramethylbenzidine
Alzheimer’s disease
Parkinson’s disease
PD
Ab
Amyloid-b
Central nervous system
CNS
BBB
TEM
AFM
HRP
Blood–brain barrier
Transmission electron microscopy
Atomic force microscopy
Horseradish peroxidase
DPPH 1,1-Biphenyl-2-picrylhydrazyl
FRAP Ferric reducing antioxidant power
ORAC Oxygen radical absorbance capacity
S. A. Ghadami ꢀ Z. Hossein-pour ꢀ R. Khodarahmi
Medical Biology Research Center, Kermanshah University of
Medical Sciences, Kermanshah, Iran
Z. Hossein-pour ꢀ R. Khodarahmi (&) ꢀ H. Adibi (&)
Faculty of Pharmacy, Kermanshah University of Medical
Sciences, Kermanshah, Iran
Introduction
e-mail: rkhodarahmi@kums.ac.ir; rkhodarahmi@mbrc.ac.ir
Amyloidosis defines a condition in which insoluble fibrillar
protein aggregates are formed from innocuous soluble
proteins; it is considered a common pathological feature of
various neurodegenerative disorders, such as Alzheimer’s,
Parkinson’s, Huntington’s, and Prion diseases (Bhak et al.,
2009). For example, the characteristic signature of
H. Adibi
e-mail: hadibi@kums.ac.ir
S. A. Ghadami ꢀ S. Ghobadi
Department of Biology, Faculty of Science, Razi University,
Kermanshah, Iran
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Med Chem Res (2013) 22:115–126
Alzheimer’s disease (AD) is the deposition of amyloid-b
(Ab) plaques and neurofibrillary tangles in the patient’s
brain (Gorman and Chakrabartty, 2001), which parallel the
disease but can only be diagnosed with certainty after death
on autopsy. Amyloid structures and fibril formation (con-
tinuous b-sheet with the strands perpendicular to the lon-
gitudinal axis of fibril) (Jimenez et al., 1999; Sumner
Makin and Serpell, 2004) have attracted a great deal of
attention to reveal possible molecular reasons for the
cytotoxicity, although their cause-or-effect relationship
with the disorders is still elusive (Joseph et al., 2001;
Robinson and Bishop, 2002). It has been suggested that
protofibrillar intermediates are responsible for cell death by
creating possible pore formation on cellular membranes,
including the mitochondrial membrane (Mirzabekov et al.,
1996; Lashuel et al., 2002; Murphy, 2007; Domanov and
Kinnunen, 2008; Engel, 2009). Alternatively, amyloids
may also provide a depot for redox-active metals that act as
a production center for reactive oxygen species eventually
affecting cell viability (Aliev et al., 2002; Takuma et al.,
2005). Although known pathogenic proteins, such as Ab
are, typically involved in several neurodegenerative dis-
eases, various non-disease-associated proteins can also be
converted into amyloid fibrils (Darghal et al., 2006;
Ghadami et al., 2011).
binding fluorescence has been most widely employed to
detect amyloids in vitro as well as ex vivo, the molecular
mechanism of the dye binding to fibrils is not well estab-
lished. Recently, it was suggested that enhanced ThT
fluorescence originate from both dye-amyloid interaction
and formation of ThT micelles on the fibrillar structures
(Khurana et al., 2005). The binding fluorescence at
482 nm, on the other hand, would be quenched by unbound
ThT present in excess during the amyloid assay.
The need for new analytical tools is highlighted with
regard to the fact that a comprehensive understanding of
molecular details of amyloidosis is essential for developing
strategies to control amyloid formation and consequently,
neurodegenerative disorders, and this requires a more
reliable probe for identifying amyloids. Since compounds
without a permanent positive charge are mainly capable of
crossing the blood–brain barrier (BBB) (Darghal et al.,
2006), in this study, we synthesized and employed ben-
zothiazole and benzofuranone derivatives (including neu-
tral ThT analogues), both as fluorescent probes to
quantitatively determine the amyloid fibrils made of chy-
motrypsin (Khodarahmi et al., 2009b) and crystallin
(Khodarahmi et al., 2010) and as potential inhibitors for
peroxidase activity. The resulting data may be useful in
providing mechanistic insights to develop potential diag-
nostic, curative, and/or preventive strategies in vivo against
amyloid-related neurodegenerative disorders.
On the other hand, several researcher groups have
recently proposed that Ab and even other non-disease-
related amyloid fibrils can potentially bind heme in vitro,
and therefore, may induce oxidative stress in vivo. It is well
accepted that oxidative stress to the central nervous system
(CNS) manifests predominantly as lipid peroxidation as
well as H2O2 formation. There are considerable amounts of
intracellular peroxide species, free heme and amyloid
fibrils present in the intracellular space; thus, the initiation
of uncontrollable H2O2-dependent peroxidase activity of
‘‘heme-amyloid’’ system under the pathological conditions
may damage intracellular DNA, neurotransmitters, and
vital enzymes (as potential substrates) as a result of com-
bined action of peroxides and peroxidase systems (Atamna
and Frey, 2004; Atamna and Boyle, 2006; Atamna, 2006;
Khodarahmi et al., 2009a, 2010). Also, since some anti-
oxidant compounds display strong anti-fibrillogenic activ-
ity, a specific interconnection between oxidative stress
(especially peroxide formation) and fibrillogenesis/neuro-
degeneration can be expected (Pratico et al., 1998; Ono and
Yamada, 2006).
Materials and methods
Materials and equipments
3,30,5,50-tetramethylbenzidine (TMB), 30 % H2O2, PMSF,
bovine a-chymotrypsin, and ThT were obtained from
Sigma chemical company (St. Louis, MO, USA). TFE was
purchased from Merck (Darmstadt, Germany). All other
chemicals were of the highest analytical grade of purity
available and were used as obtained from suppliers. All
solutions were prepared with double distilled water. Unless
otherwise stated, all solutions were made in 20 mM sodium
phosphate buffer (pH 7.4). A stock solution of ThT (1 mM)
was freshly prepared in water and stored in dark at 4 °C.
A Cary-100 Bio (VARIAN) spectrophotometer was used
for protein determination and peroxidase assay. All fluo-
rescence measurements were performed in the ratio mode
using a 1 cm cell in a Cary Eclipse (VARIAN) fluores-
cence spectrophotometer, equipped with a 150 W xenon
lamp and a thermostated cell holder, at room temperature
or as stated. Appropriate vehicle controls were run in all
experiments. All the reported results are averages of 2–3
separate experiments whenever the coefficients of variation
were less than 5 %.
Amyloid formation has been monitored with various
methods, including light scattering, Thioflavin T (ThT),
Congo red, transmission electron microscopy (TEM), and
atomic force microscopy (AFM) (Nilsson, 2004). Devel-
oping molecules to be used as markers of b-amyloid
deposits in AD has been a goal of researchers for many
years (Sumner Makin and Serpell, 2004). Although ThT
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Med Chem Res (2013) 22:115–126
Chemistry
117
and the extract dried with anhydrous MgSO4. The filtrate
was evaporated and the corresponding benzothiazole was
obtained as the only product (Bahrami et al., 2008).
(E)-6-hydroxy-2-(2-hydroxy-3-methoxybenzylidene)
benzofuran-3(2H)-one (compound 3) and (E)-2-(2,4-di-
hydroxybenzylidene)-6-hydroxybenzofuran-3(2H)-one,
(compound 4).
All chemicals were purchased from Merck and Fluka
chemical companies. The products were characterized by
comparing the physical data with those of known samples
or by their spectral data. Column chromatography was
performed using silica gel 60 (230–400 mesh). All yields
refer to isolated yield. The chemical structures of test
compounds are summarized in Fig. 1. The structure of
In order to synthesize (E)-6-hydroxy-2-(2-hydroxy-3-
methoxybenzylidene)benzofuran-3(2H)-one (compound 3)
and (E)-2-(2,4-dihydroxybenzylidene)-6-hydroxybenzofu-
ran-3(2H)-one, (compound 4, see Fig. 1), as new compounds,
the starting 6-hydroxy-3-coumaranone B (see Scheme 1) was
prepared by dissolving resorcinol (1 mmol) and bromoacetyl
bromide (1 mmol) in acetonitrile (3 mL) with 400 mg silica
sulfuric acid added as catalyst. The reaction mixture was
heatedat80 °Cfor 3 h. Aftercoolingtoroomtemperatureand
filtering the reaction mixture, the compoundA was purified by
column chromatography. Subsequently, the compound A was
cyclized using NaOH 2 M to produce 6-hydroxy-3-coumar-
anone B in 75 % yield after 1 h. mp = 243–246 °C; 1H NMR
(CDCl3) d 7.411 (d, 1H, H5), 6.542–6.568 (d, 1H, H4), 6.664
(s, 1H, H7), 4.602 (s, 2H, CH2), 4.580 (s, 1H, OH); IR (KBr,
cm-1) t 1745 (C=O), 3452 (OH).
To a solution of 6-hydroxy-3-coumaranone B (1 mmol),
and appropriate aryl aldehyde (1 mmol) in ethanol (3 mL)
was added piperazine (0.1 mmol) as the basic catalyst. The
reaction mixture was heated in 80 °C for 2 h. The reaction
mixture was allowed to stand at room temperature for
24 h. The precipitate was filtered, dried, and crystallized
from acetic acid to afford pure (E)-6-hydroxy-2-(2-hydroxy-
3-methoxybenzylidene) benzofuran-3(2H)-one 3 and (E)-2-
(2,4-dihydroxybenzylidene)-6-hydroxybenzofuran-3(2H)-one
compounds was characterized by IR, H NMR, and 13C
1
NMR spectra. The melting point was taken on a Kofler hot
stage apparatus and is uncorrected. IR spectrum was
recorded on a Shimadzu 470 spectrophotometer (KBr
disk). 1H NMR spectrum was recorded on a Bruker FT-200
NMR spectrophotometer using CDCl3 as solvent and TMS
as an internal standard. The purity of the compound was
monitored by thin layer chromatography.
Typical procedure for synthesis of (2-(4-methoxy-
phenyl)benzo[d]thiazole) (compound 1) and (4-(benzo[d]
thiazol-2-yl)-N,N-dimethylaniline) (compound 2) as known
compounds (see ‘‘Bahrami et al., 2008’’ and also Fig. 1).
In a round-bottomed flask (50 mL) equipped with
a magnetic stirrer, a solution of o-aminothiophenol
(1 mmol), 4-methoxybenzaldehyde, and/or 4-N,N-dime-
thylaminobenzaldehyde (1 mmol) in MeCN (3 mL) was
prepared. H2O2 (30 %, 4 mmol, 0.4 mL) and NH4Ce(NO2)6
(0.1 mmol, 0.0548 g) were added and the mixture was
stirred at room temperature for 1 h. The progress of the
reaction was monitored by TLC (eluent: n-hexane/ethyl
acetate: 6/4). When the starting materials had completely
disappeared, the reaction mixture was quenched by adding
water (10 mL), extracted with ethyl acetate (4 9 10 mL)
CH3
N
S
N
S
N
(H3C)2N
H3CO
(H3C)2N
S
+
Cl-
4-(benzo[d]thiazol-2-yl)-N ,N-dimethylaniline
2-(4-methoxyphenyl)benzo[d]thiazole
H3C
Compound 2
Compound 1
Thioflavin-T
H
H
OH
O
O
HO
HO
O
HO
OMe
O
OH
OH
OH
OH
(E)-2-(2,4-dihydroxybenzylidene)-6-
hydroxybenzofuran-3(2H)-one
(E)-6-hydroxy-2-(2-hydroxy-3-methoxybenzylidene)
benzofuran-3(2H)-one
Compound 3
trans-resverartol
Compound 4
Fig. 1 Chemical structures of synthesized compounds (2-(4-methoxy-
phenyl)benzo[d]thiazole, 1, 4-(benzo[d]thiazol-2-yl)-N,N-dimethylani-
line, 2, (E)-6-hydroxy-2-(2-hydroxy-3-methoxybenzylidene)benzofuran-
3(2H)-one, 3 and (E)-2-(2,4-dihydroxybenzylidene)-6-hydroxybenzofu-
ran-3(2H)-one, 4. See also ThT and resveratrol structures (center) for
comparison
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Med Chem Res (2013) 22:115–126
O
O
SSA (400 mg)
CH3CN
Br
NaOH
+
Br
HO
OH
HO
OH
Br
A
SSA = Silica Sulfuric acid
O
O
O
H
Piperazine
+
H
R
O
R
EtOH (3 ml)
HO
O
HO
6-hydroxy-3-coumaranone
B
6-hydroxy-2-benzylidene-3-coumaranones
R = 2-OH-3-MeO
R = 2,4-(OH)2
3
4
Scheme 1 Synthesis of compounds 3 and 4: reagents and conditions
4 in 70 and 65 % yields, respectively (Scheme 1). Structural
assignments of the products are based on their IR, 1H NMR,
13C NMR spectra, and melting point.
fibrillar proteins. The fibrils used in all experiments came
from the same batch.
Compound 3: (E)-6-hydroxy-2-(2-hydroxy-3-methox-
ybenzylidene) benzofuran-3(2H)-one 3: yield 70 %; mp =
Preparation of working solutions
1
305–306 °C; H NMR (CDCl3) d 7.71 (d, 1H, H4), 7.62
Working solutions of free dyes at 5 9 10-6 M were pre-
pared by dilution of the dye stock solution in 20 mM
sodium phosphate buffer (pH 7.4). Working solutions of
dye–protein complexes were prepared by mixing an aliquot
of the dye stock solution (2.0 lL) with an aliquot (1, 2, and
3 lL) of native or fibrillar proteins in buffer. Concentra-
tions of the proteins in working solution were 0.03, 0.05,
0.07, 0.10, and 0.30 mg/ml, respectively for both native
and aggregated proteins. Measurements were performed, in
the presence of 10 lL of protein, only for the most efficient
dyes which demonstrated significant fibrillar proteins
binding preference values as compared with native protein.
All working solutions were prepared immediately before
the experiment.
0
0
(s, 1H, C=CH), 7.11–7.17 (m, 2H, H5 , H6 ), 7.04 (s, 1H,
H7), 6.91 (s, 1H, H7), 6.73 (d, 1H, H‘4), 6.50 (d, 1H, H5),
5.22 (s, 2H, OH), 3.84 (s, 3H, OCH3); 13C NMR (CDCl3)
d 182.1, 167.5, 165.2, 151.2, 148.4, 146.7, 128.0, 122.6,
121.4, 117.4, 115.2, 111.8, 105.3, 101.3, 56.4; IR (KBr,
cm-1) t 1730 (C=O), 3250 (OH).
Compound 4: (E)-2-(2,4-dihydroxybenzylidene)-6-hy-
droxybenzofuran-3(2H)-one 4: yield 65 %; mp = 330–
1
0
332 °C; H NMR (CDCl3) d 7.96–7.90 (m, 2H, H6 , H4),
7.35 (s, 1H, C=CH), 6.70 (s, 1H, H7), 6.55 (d, 1H, H5),
0
0
6.25 (d, 1H, H5 ), 6.15 (s, 1H, H3 ), 3.49–3.42 (s, 3H, OH);
13C NMR (CDCl3) d 182.1, 167.0, 165.2, 159.3, 157.3,
146.7, 131.3, 128.3, 115.2, 112.3, 108.3, 105.7, 103.5, IR
(KBr, cm-1) t 1735 (C=O), 3455 (OH).
Spectroscopic measurements
Sample preparation and fibrillogenesis
Fluorescence excitation and emission spectra were col-
lected on a Cary Eclipse fluorescence spectrophotometer
(Varian, Australia). Fluorescence spectra were measured
with excitation and emission slit widths set to 10 nm. The
correction of the obtained spectra for the wavelength
dependence on the excitation source intensity was per-
formed by the Cary Eclipse fluorescence spectrophotome-
ter. At the same time, fluorescence excitation and emission
spectra were not corrected for the sensitivity of the Cary
Eclipse detection system. However, the primary aim of the
work was to study the fluorescent response of each dye in
the presence of native and aggregated proteins. Since for
each dye the positions of the corresponding spectra are
very similar in free form and in the presence of native and
Amyloid made of a-chymotrypsin was monitored with the
dyes binding fluorescence. The a-chymotrypsin, was con-
verted to amyloid-like fibrils in the presence of 32 % TFE
(Khodarahmi et al., 2009b, Nilsson, 2004).
Preparation of stock solutions of dyes and biological
molecules
1 9 10-3 M dye stock solutions were prepared by dis-
solving the dye in DMSO. The concentrations of proteins
in stock solutions were 2 mg/mL for both native and
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Med Chem Res (2013) 22:115–126
119
aggregated proteins, the absence of correction for the
detection system sensitivity does not influence the results
of our study. In vitro binding studies were conducted at
least in triplicate by the procedures described in detail by
Groenning et al. (2007). Spectroscopic measurements were
performed in standard quartz cells (1 9 1 cm). All mea-
surements were carried out at room temperature. All
measurements were made at the respective excitation
maxima of each dye.
amyloid aggregates, however, the fluorescence intensity of
the test compounds did not undergo a significant change
upon interaction with the soluble (monomeric) form of
a-chymotrypsin. As is evident from the curve slopes
(Fig. 3), the increase in dye fluorescence was less signifi-
cant in the presence of native protein. Such mild and
steadily enhanced emission intensities are attributed to the
less effective (or nonspecific) interaction between com-
pounds and amyloid fibrils. Therefore, it may be inferred
that all tested compounds, especially X and Y (with a
specific configuration in aqueous solution) might experi-
ence structural transition to new chromophores at the
amyloid aggregate-bound state. Overall, these compounds
appear to be reliable fluorescent probes to determine the
amyloids quantitatively. In addition, the binding fluores-
cence of test compounds was demonstrated to be highly
specific for amyloids as binding fluorescence was hardly
observed with other proteins such as globin (a protein with
8 consecutive a-helical segments, (Kavanaugh et al., 1993,
see also Fig. 3) and native crystalline solution (and amor-
phous protein aggregates made of chymotrypsin, data not
shown). As demonstrated by the curve slopes (Fig. 3), the
increase in fluorescence intensities was much less signifi-
cant compared to those observed for dye–amyloid inter-
action. These steadily decreasing emission intensities may
be attributed to generation of microenvironments with low
dielectric constant (within or between soluble protein
molecules and) upon amorphous protein aggregation.
Selective binding to amyloid-containing aggregates may
make the test compound possibly applicable in imaging
and in situ staining of amyloid fibrils.
Peroxidase activity of the horseradish peroxidase
The peroxidase activity of horseradish peroxidase (HRP)
was measured by oxidation of TMB by H2O2 by following
the increase in absorbance at 652 nm, which allows con-
tinuous monitoring of the TMB oxidation product. The
peroxidase activity of horseradish peroxidase (0.07 lg/mL)
was tested in the concentration range of 0.3–5.0 mM of the
TMB for 60 s at room temperature. The effect of potential
inhibitor compounds on peroxidase activity was measured
by TMB assay. The extent of TMB oxidation by horse-
radish peroxidase in the absence or presences of com-
pounds were tested in the kinetic mode for 60 s at room
temperature and the values of DA/Dt were determined.
Results and discussion
Amyloid formation has been monitored with various
spectroscopic methods, using ThT and congo red (CR). In
addition, the resveratrol (the major phenolic constituent of
red wine) binding fluorescence was recently demonstrated
to be highly specific for the amyloids (Ahn et al., 2007).
Thus, there is a possibility that other phenolic and or thi-
azole compounds show selectivity in binding to amyloid
fibrils and are possibly applicable in both in vivo amyloid
imaging techniques and in situ staining of amyloid plaques
in tissue section.
Scaria et al. (1986) studied the effect of dye binding on
the conformation of poly L-lysine polypeptides. They
observed that dye binding leads to a conformational tran-
sition of poly L-lysine from the a-helix to the b-sheet, the
structure with further susceptibility for binding to dye
molecules. Therefore, it can be concluded that strong
interaction between dyes molecules and b-sheet structures
is more likely to be due to geometric considerations. In
addition, considering the presence of extended b-con-
formers in amyloid structures (see scheme 2), further
binding of dye molecules may be anticipated. Thus, we
observed a large difference in dye fluorescence character-
istics between ‘‘dye-native crystalline’’ (a b-rich protein,
see Fig. 3) and ‘‘dye-amyloid aggregate’’ incubation mix-
tures (Fig. 3).
Binding of compounds 1–4 to the a-chymotrypsin
amyloid aggregate was examined by observing fluores-
cence emission spectra between 300 and 600 nm using
specific excitation wavelengths (360, 400, 398, and 408 nm
for compounds 1–4, respectively) and at a fixed concen-
tration (40 lM) of compounds. Also, the excitation
wavelength for ThT was set at 442 nm. As indicated in
Fig. 2, the fluorescence intensities (emissions at 400, 430,
500, and 500 nm, respectively) increased significantly
upon interaction with amyloid aggregates. Moreover, as the
amount of amyloid aggregates (corresponding to the con-
centration of native a-chymotrypsin solution) increased,
the fluorescence intensities were augmented proportionally
so that the maximum fluorescence emissions increased
linearly as a function of amyloid concentration. Unlike the
Although the exact mechanism of dye binding to amy-
loid fibrils remains unclear and, putting aside the report of
(Eisert et al., 2006), most of the binding models described
in the literature state that binding of amyloid-specific dyes
(such as ThT and Congo red) occurs along their long
molecular axis parallel to the fibril axis (Krebs et al., 2005;
Cooper, 1974; Jin et al., 2003). As the side chains on each
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Med Chem Res (2013) 22:115–126
Fig. 2 Binding fluorescence spectra of test compounds in the
presence of a-chymotrypsin amyloid aggregates. Compounds 1–4
binding fluorescence spectra between 300 and 600 nm were obtained
with excitation at 360, 400, 398, and 408 nm, respectively, in the
presence of various amounts (0, 0.03, 0.05, 0.07, 0.1, and 0.32 mg/ml)
of the a-chymotrypsin aggregates (open circle) and were plotted
separately with various amounts of the native protein (open square).
The fluorescence intensities were plotted with a fixed amount of
compounds at 40 lM. Data shown are representative example of
three independent experiments and standard deviations were approx-
imately within 5 % of the experimental values. Further details are
given in the ‘‘Material and methods’’ section
side of beta-sheet form neat rows (so called ‘‘channels’’,
see also scheme 2) running along the fibril (and therefore
perpendicular to the strands), it seems likely that the dye
molecule can insert itself into these channels. Thus, the dye
molecule must be flat and thin enough to enter such a
binding channel (Krebs et al., 2005). Based on this data, it
may be expected that bulky substituents in the dye struc-
tures will complicate dye insertion in this channel. On the
other hand, different fluorescent dyes used for amyloid
detection are proposed to bind to fibrils via diverse binding
sites (Volkova et al., 2007). Due to the mentioned reasons,
binding mechanism of dyes specific for amyloid fibrils
requires further investigation.
concentrations. In this study, a-chymotrypsin amyloido-
genesis was assayed under the effect of various concen-
trations of compounds and the results were compared with
the amyloid binding properties of ThT. The amyloid stock
was prepared by incubating 2 mg/mL of a-chymotrypsin,
in the presence of 32 % TFE. The fluorescence of various
amounts of ThT (at 482 nm) was evaluated in the presence
of a fixed concentration (0.1 mg/mL) of amyloid aggre-
gates. As indicated by Fig. 4, the binding fluorescence was
maximal at 80 lM of ThT and gradually decreased to
200 lM of the dye. This fact reflects that the free ThT
molecules might quench the binding fluorescence.
Also, the dye concentration responsible for the maximal
binding fluorescence shifted to higher values as the amount
of amyloids increased (for instance, see Fig. 4, compound 3),
indicating that the optimal dye concentration actually
ThT binding fluorescence of amyloids has been sug-
gested to vary depending on the dye concentration so
that its binding fluorescence is quenched at high dye
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Med Chem Res (2013) 22:115–126
121
Fig. 3 Fluorescence characteristics of test compounds under the
effect of various amounts of a-chymotrypsin amyloid aggregates
(filled triangle), native a-crystallin (open square), and heme-free
globin (open triangle). The protein concentrations were 0, 0.03, 0.05,
0.07, 0.10, and 0.32 mg/mL in 50 mM phosphate buffer (pH 7.4); in
the presence of fixed amount (10 mM) of compounds. The binding
fluorescences were monitored with the emission scan between 300
and 600 nm following excitations at 360, 400, 398, and 408 nm,
respectively. Ribbon representation of human oxyhemoglobin (left)
and bovine AlphaA crystallin (right) were obtained from the Protein
Data Bank (ID code 1HHO and 3L1E, respectively). The structures
visualization and figures generation were performed with PyMOL
version 0.99 beta06 (http://www.pymol.org) (Fendri et al., 2007).
Data shown are representative example of three independent exper-
iments and standard deviations were approximately within 5 % of the
experimental values. Further details are given in the ‘‘Material and
methods’’ section
depends on the quantity of amyloids analyzed. In other
words, an amyloid/dye ratio would be critical to obtaining
an appropriate and optimal binding fluorescence signal.
This would limit universal use of ThT for amyloid detec-
tion. On the other hand, dye binding to the amyloid
aggregates has been shown to exhibit an ideal property of
saturation for a ligand interaction at the fixed concentration
of the protein (Fig. 4). Since strong binding of the dye
molecules is required for exact amyloid determination,
there is a possibility that fluorescence characteristic of each
compound is controlled by the binding constants. To
evaluate this possibility, we calculated dissociation con-
stants using linear regression. According to Fig. 5, which
compares the Kd and Bmax for compound as well as ThT,
Scatchard analyses for the binding of compounds 1–4
showed apparent Kd = 35.2, 2.6, 16.9, 3.0 (lM) and
Bmax = 59.9, 30.6, 93.2, 12.1 (lM dye/lM amyloid),
respectively. The calculated dissociation constants for
dye–amyloid complexes are in the following order:
1 [ 3 [ 4 [ 2. Furthermore, the initial slopes of the sat-
uration curves, which might reflect dependence of the
compounds binding fluorescence on the amount of com-
pounds, increased linearly as the amyloid level rose
(Fig. 5). This indicates that the binding fluorescence is
proportional to not only the compound concentration but
also the amount of amyloids. Therefore, it may be con-
cluded that a relatively wide range of dye concentrations
may be employed to detect amyloids, whereas the amount
of ThT for an amyloid assay needs to be selected within a
rather short range on the basis of the amyloid/dye ratio.
Taking the binding data into account, it can be concluded
that compounds 2, 4 may be considered as potential fluo-
rescent probes to quantify amyloid formation.
The development of molecules to be used as markers/
probes of b-amyloid deposits in amyloid diseases has been
a goal of researchers for several years (Klunk, 1998). ThT
123

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Med Chem Res (2013) 22:115–126
Scheme 2 The common
structure of amyloid fibrils and a
structural rationale for fibril–
dye interactions. Top cross-b
structure of amyloid fibrils,
formed from layers of laminated
b-sheets. Bottom ‘‘Channel’’
model of dye binding to fibril-
like b-sheets. Dye is proposed to
bind along surface side-chain
grooves running parallel to the
long axis of the b-sheet (See
Khodarahmi et al., 2010 and
reference [38] within. Also, for
better interpretation, the reader
is referred to the web version of
this article)
is one of the (candidate) molecules of choice in attempts
made at detection of amyloid deposits in ex vivo and in
vitro systems. This compound is a histologic benzothiazole
dye (see Fig. 1) capable of staining amyloid structures
selectively. It has been frequently reported that ThT is
unable to cross BBB in vivo or to cross it in sufficient
amounts for acceptable sensitivity; this is the result of its
low lipophilicity and importantly, the presence of a per-
manent positive charge (Klunk et al., 2001; Mathis et al.,
2002; Nesterov et al., 2005). Uncharged analogues of ThT
managed to permeate through the BBB in substantial
amounts (see compounds 1 and 2 structures in current
study). Therefore, ThT derivatives without permanent
positive charge and with better lipophilicity (but not
excessively, as this will lead to its aggregation and accu-
mulation on blood proteins and within red blood cell
membranes) have been synthesized and they were report-
edly able to cross BBB in vivo (Mathis et al., 2002;
Nesterov et al., 2005). For example, two ThT uncharged
analogues, compounds 1 and 2 may be highly efficient both
in crossing the BBB and in selective binding to amyloid
aggregates. For general use, the binding of these potential
compounds to amyloids needs to be further evaluated in
terms of its b-sheet interaction and a possible formation of
new chromophore on its amyloid binding. In addition,
other practical applications of the candidate compounds,
such as the ability to detect amyloids within tissues, are
worth investigating.
Despite extensive efforts around the world, development
of an effective treatment for neurodegenerative diseases
such as AD remains elusive. Current therapeutic strategies
are limited to those that attenuate AD symptomology
without deterring the progress of the disease itself, and thus
only postpone the inevitable deterioration of the affected
individual. Notably, as oxidative stress is perhaps the ear-
liest feature of an AD brain (Nunomura et al., 2001; Zhu
et al., 2001, 2004), successful neuronal protection from
oxidative damage will potentially prevent the disease
altogether, if appropriately administered. In this regard,
efforts focused on elucidation of the molecular mecha-
nisms involved in the oxidative stress within the cell have
yielded significant results and will hopefully succeed
through clinical trials for efficacy. Recently, it has been
proposed that Ab, a pathogenic peptide in AD, can
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Med Chem Res (2013) 22:115–126
123
Fig. 4 Changes in the dye binding fluorescence (a.u.) of a-chymo-
trypsin-derived amyloid aggregates under the effect of the concentra-
tion of (ThT and) compounds. The a-chymotrypsin amyloids at three
different concentrations of 0.02 (open square), 0.05 (open circle), and
0.1 (open triangle) mg/mL were treated with various concentrations of
either ThT or compounds. The enhanced dye binding fluorescence
intensities obtained at the corresponding maximum emission wave-
lengths and were plotted versus the dye concentrations
potentially bind heme, forming an Ab-heme complex, in
vitro (Howlett et al., 1997; Atamna and Boyle, 2006). In
this case, the binding of heme to protein creates heme
deficiency, mitochondrial dysfunction and increased pro-
duction of oxidants such as H2O2 (Atamna and Boyle,
2006; Atamna and Frey, 2004). Increasing evidence
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Med Chem Res (2013) 22:115–126
Fig. 5 Scatchard plots of binding of the synthesized compounds to
a-chymotrypsin amyloid aggregates. Data shown are representative
example of three independent experiments and standard deviations
were approximately within 5 % of the experimental values. Further
details are given in the paper text
suggests that hydrogen peroxide and possibly other
hydroperoxide species are generated during the very early
stages of amyloid formation and that oxidative stress plays
an important role in the pathogenesis of this type of
neurodegenerative disorder (Khodarahmi et al., 2009a;
Turnbull et al., 2003). Oxidative stress to the CNS (and to
the other organs) manifests predominantly as lipid perox-
idation due to the susceptibility of unsaturated fatty acids to
oxidation (Butterfield et al., 2002). Despite extensive
experimental work conducted in this field, the mechanism
involved in the peroxide release is still not completely
understood. Furthermore, there is discrepancy between
different reports on chronological precedence of amyloid
aggregation and oxidative reactions. On the other hand,
Atamna (2006) and Khodarahmi et al. (2010) reported the
H2O2-dependent peroxidase activity of ‘‘heme-amyloido-
genic protein’’ complex using in vitro experimental sys-
tems. Due to the presence of high amounts of peroxide
species other than H2O2, in some amyloid diseases (Pratico
et al., 1998), there is a possibility that the generated per-
oxide molecules serve as oxidant substrate for peroxidase
systems. Also, since anti-oxidant compounds have potent
anti-fibrillogenic and fibril destabilizing effects in a dose–
dependent manner (Ono and Yamada, 2006), a specific
interconnection between peroxide formation and fibrillo-
genesis can be expected. Since the basic and clinical
aspects of heme binding to (structural intermediates of)
amyloid fibrils as well as observed peroxidase activity
(Pramanik and Dey, 2011; Yuan et al., 2012) have yet to be
identified, additional data on this field may provide insight
to manage amyloid-related processes/diseases. Considering
the potential role of peroxidase activity of ‘‘heme-amyloid
fibril’’ complex in onset/progression of neurodegenerative
disorders, as well as the fact that peroxidase-mediated
oxidative stress can play at least a partially causative rather
than merely by-stander role in the neurofibrillary pathology
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Med Chem Res (2013) 22:115–126
125
Fig. 6 The un-competitive inhibition of HRP activity. Lineweaver–
Burk plot for the inhibition of peroxidase activity by compounds 3
(left) and 4 (right). The enzyme concentration in the assay buffer
(potassium phosphate 20 mM, pH 7.2) was 0.7 9 10-4 mg/mL. The
units of Y and X axes are (min.Abs-1) and (mM-1), respectively
Conflict of interest The authors do not declare any conflict of
interest.
in AD/PD in vivo, and may affect nucleic acids, enzymes,
and neurotransmitters (Yuan et al., 2012), ongoing clinical
trials should therefore focus on simultaneous application of
fibril stabilizers, aggregation inhibitors and peroxidase
inhibitors as a new generation of amyloid-based thera-
peutics. In the current study, since phenolic compounds 3
and 4 showed significant anti-oxidant activity using ferric
reducing antioxidant power (FRAP), oxygen radical
absorbance capacity (ORAC), and 1,1-biphenyl-2-picryl-
hydrazyl (DPPH) methods (data not shown), they were
considered as potential inhibitors of peroxidase activity.
The peroxidase activity of HRP, at different TMB con-
centrations, either in the presence or absence of compounds
3 and 4 were assessed and the results were appraised using
Lineweaver–Burk plots. TMB, a classic substrate for per-
oxidases, was rapidly oxidized by H2O2 when enzyme was
added. Clearly, peroxidase activity follows a linear kinetic
pattern with no significant lag period as demonstrated by
the increase in absorbance, at 652 nm (data not shown).
Then, enzyme activity was followed in the presence of 0,
50, and 150 mM of compounds 3, 4 (and 2.6 mM H2O2).
As indicated in Fig. 6, it was found that both compounds
inhibit the HRP peroxidase activity through an un-com-
petitive mechanism. It is noteworthy that due to the het-
erogeneous nature and inherent turbidity of insoluble
amyloid aggregates, changes in the peroxidase activity in
the presence of the compounds, was recorded using native
peroxidase enzyme (HRP). Previous studies (Atamna and
Frey, 2004; Atamna and Boyle, 2006; Khodarahmi et al.,
2010) have indicated the similar catalytic behavior of
‘‘amyloid-heme’’ complex and the native peroxidase.
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