Nature-Inspired Multifunctional Ligands: Focusing on Amyloid-Based Molecular Mechanisms of Alzheimer's Disease

Article abstract of DOI:10.1002/cmdc.201500422

The amyloidogenic pathway is a prominent feature of Alzheimer's disease (AD). However, growing evidence suggests that a linear disease model based on β-amyloid peptide (Aβ) alone is not likely to be realistic, which therefore calls for further investigati

Full text of DOI:10.1002/cmdc.201500422

DOI: 10.1002/cmdc.201500422
Full Papers
Nature-Inspired Multifunctional Ligands: Focusing on
Amyloid-Based Molecular Mechanisms of Alzheimer’s
Disease
Elena Simoni,[a] Melania M. Serafini,[b] Manuela Bartolini,[a] Roberta Caporaso,[a]
Antonella Pinto,[b] Daniela Necchi,[b] Jessica Fiori,[a] Vincenza Andrisano,[c] Anna Minarini,[a]
Cristina Lanni,*[b] and Michela Rosini*[a]
The amyloidogenic pathway is a prominent feature of Alzheim-
er’s disease (AD). However, growing evidence suggests that
a linear disease model based on b-amyloid peptide (Ab) alone
is not likely to be realistic, which therefore calls for further in-
vestigations on the other actors involved in the play. The pro-
oxidant environment induced by Ab in AD pathology is well
established, and a correlation among Ab, oxidative stress, and
conformational changes in p53 has been suggested. In this
study, we applied a multifunctional approach to identify allyl
thioesters of variously substituted trans-cinnamic acids for
which the pharmacological profile was strategically tuned by
hydroxy substituents on the aromatic moiety. Indeed, only cat-
echol derivative 3 [(S)-allyl (E)-3-(3,4-dihydroxyphenyl)prop-2-
enethioate] inhibited Ab fibrilization. Conversely, albeit to dif-
ferent extents, all compounds were able to decrease the for-
mation of reactive oxygen species in SH-SY5Y neuroblastoma
cells and to prevent alterations in the conformation of p53 and
its activity mediated by soluble sub-lethal concentrations of
Ab. This may support an involvement of oxidative stress in Ab
function, with p53 emerging as a potential mediator of their
functional interplay.
Introduction
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder, with a complex interplay of genetic and biochemical
factors contributing to pathological decline. Progression of the
disease involves misfolding and aggregation of b-amyloid pep-
tide (Ab) from soluble nontoxic monomers into insoluble fi-
brils. The most toxic form of Ab is believed to be soluble oligo-
mers, which are potent mediators of synaptotoxicity.[1] In AD
drug development, programs based on the Ab cascade hy-
pothesis have dominated research for the past 20 years and
still play a major role in pharmaceutical product pipelines.
However, Ab-centric approaches have not yet resulted in clini-
cally effective drugs. This has raised a degree of skepticism,
which has in turn led to a review of the science underpinning
the Ab model.[2] Besides the consolidated evidence that Ab
might trigger the disease process, intertwined correlations be-
tween Ab and the other main players of the disease have been
identified.[3] This has prompted researchers to develop multi-
functional anti-amyloid agents[4] that, by acting simultaneously
on several AD targets instead of the amyloidogenic pathway
alone, are intended to trigger a synergistic response with supe-
rior efficacy and safety profile.[5] Further, we think that mole-
cules endowed with a multifaceted pharmacology have great
potential in exploring the Ab partnership with other crucial AD
features. A deeper comprehension of amyloid-based disease
mechanisms might offer the chance for the repositioning of
Ab in the disease network, which would be of help in bridging
the gap between basic and translational research. In particular,
the etiopathogenic loop generated by Ab and oxidative stress
offers a new key for reading the causative role of Ab.[6] Oxida-
tive stress is known to trigger the amyloidogenic pathway and
to promote Ab toxicity.[7] On the other hand, several lines of
evidence indicate that Ab exacerbates oxidative stress, with
other cellular pathways emerging as determining mediators of
this vicious cycle.[8] In this respect, regulation of the conforma-
tion and function of p53 may represent a crucial feature of this
puzzling scenario.
[a] Dr. E. Simoni, Prof. Dr. M. Bartolini, R. Caporaso, Dr. J. Fiori,
Prof. Dr. A. Minarini, Prof. Dr. M. Rosini
Department of Pharmacy and Biotechnology
Alma Mater Studiorum, University of Bologna
Via Belmeloro 6, 40126 Bologna (Italy)
E-mail: michela.rosini@unibo.it
[b] M. M. Serafini, A. Pinto, Dr. D. Necchi, Dr. C. Lanni
Department of Drug Sciences (Pharmacology Section)
University of Pavia, V.le Taramelli 14, 27100 Pavia (Italy)
E-mail: cristina.lanni@unipv.it
[c] Prof. Dr. V. Andrisano
p53 is a tumor-suppressor protein primarily involved in
cancer biology. However, recent observations have showed
that p53 may also play a central role in aging and in neurode-
generative disorders.[9] Conformational changes and functional
alterations of p53 have been found in patients with AD.[10] Un-
folded p53 is not able to exert its pro-apoptotic activity in AD
Department for Life Quality Studies
Alma Mater Studiorum, University of Bologna
Corso d’Augusto 237, 47921 Rimini (Italy)
This article is part of a Special Issue on Polypharmacology and
Multitarget Drugs. To view the complete issue, visit:
http://onlinelibrary.wiley.com/doi/10.1002/cmdc.v11.12/issuetoc.
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cells, which leads to aberrant cell
cycle progression[11] and to the
accumulation of aging-associat-
ed abnormalities. p53 is an in-
trinsically unstable protein, the
conformation and DNA binding
domain of which can be modu-
lated by metal chelators and
redox status.[12] In particular, an
alteration in oxidative homeosta-
sis, resulting in sub-toxic and
chronic exposure to reactive
oxygen species (ROS), impairs
the tertiary structure of wild-
type p53, and this induces
a switch toward the nonfunc-
tional unfolded form of p53.[13]
Alteration of the physiological
Figure 1. Design strategy for compounds 1–3. Curc: curcumin, Coum: coumarin, FA: ferulic acid, RA: rosmarinic
acid, DAS: diallyl sulfide, DADS: diallyl disulfide, DATS: diallyl trisulfide.
functions of p53 can also result from exposure to soluble, non-
toxic Ab and has been shown to be related to the ability of Ab
to interfere with two key proteins, that is, zyxin and the home-
odomain-interacting protein kinase 2 (HIPK2).[14] Zyxin is an
adaptor protein identified as a regulator of HIPK2–p53 signal-
ing in response to DNA damage.[15] The activity of HIPK2 is in
turn fundamental in maintaining the function of wild-type p53,
as it controls the destiny of cells upon exposure to DNA-dam-
aging agents. In particular, soluble Ab peptides downregulate
zyxin expression, which is fundamental in maintaining the sta-
bility of HIPK2 and in turn the activity of p53.[14b] This Ab-medi-
ated downregulation may be responsible for early pathological
changes that precede the amyloidogenic pathway in the neu-
rodegenerative cascade. Therefore, the induction of the unfold-
ed state of p53 by leading to the accumulation of dysfunction-
al neurons in the central nervous system, is emerging as
a novel amyloid-based mechanism of AD pathogenesis.
As part of our ongoing work aimed at deepening our insight
into the cross-talk between Ab function and oxidative stress in
AD, we envisioned nature as a structural “muse”. Natural prod-
ucts offer great chemical diversity[16] and have already proven
to be a rich source of therapeutics. Polyphenols are widely dif-
fused in nature. They have been shown to modulate several
AD pathways, including oxidative injuries and Ab aggrega-
tion.[17] Interestingly, many of them present a hydroxycinnamoyl
function as a recurring motif. On the other hand, diallyl sulfides
are garlic-derived organosulfur compounds carrying allyl mer-
captan moieties. They counteract oxidative stress through anti-
oxidant enzyme expression.[18] Herein, we combined these priv-
ileged molecular fragments in new chemical entities to afford
hybrids 1–3 (Figure 1).
Curcumin was herein the reference compound. On the basis of
its pleiotropic nature, curcumin is a consolidated prototype for
AD studies, and it has already provided an outstanding plat-
form for numerous biologically active ligands.[19]
Results and Discussion
Chemistry
The synthesis of 1–3 was performed in a linear fashion, as de-
picted in Scheme 1. tert-Butyldimethylsilyl (TBDMS) protection
of the alcohol followed by coupling with N,N’-dicyclohexylcar-
bodiimide (DCC) in the presence of 4-(N,N-dimethylamino)pyri-
dine (DMAP) gave intermediates 10–12. Finally, treatment of
10–12 with tetrabutylammonium fluoride (TBAF) effected desi-
lylation to give final compounds 1–3.
The synthesized molecules were characterized by NMR spec-
1
troscopy and ESI mass spectrometry. The H NMR spectra show
that all compounds have the E configuration, as indicated by
The synthesized compounds were first tested in vitro to
assess their anti-aggregating properties toward Ab42, the most
amyloidogenic isoform of Ab. They were then assayed in neu-
roblastoma cells to explore their ability to counteract oxidative
stress and to exert a neuroprotective effect against Ab42-in-
duced toxicity.
The efficacy of 1–3 in modulating an Ab-induced conforma-
tional state alteration of the p53 protein was also investigated.
Scheme 1. Reagents and conditions: a) DMF, imidazole, N2, overnight, RT;
b) DCC, DMAP, CH2Cl2, N2, overnight, 08C!RT; c) TBAF, THF, N2, 30min, RT.
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the large coupling constants (~16 Hz) of the a-H and b-H pro-
tons on the double bonds.
10 mm);[22] furthermore, 3 is only fivefold less potent than the
flavonoid myricetin [IC50 =(2.60Æ0.33) mm].[23]
To explore the possibility of tuning the anti-aggregating pro-
file of 3, a detailed structure–activity relationship study is in
progress, and the results will be published in due course.
Biological assays
Inhibition of Ab42 self-aggregation (thioflavin T-based assay)
Inhibition of Ab42 self-aggregation (mass spectrometry assay)
We fostered the development of nature-inspired multifunction-
al ligands as an attractive opportunity to gain insight into the
cross-talk between oxidative damage and Ab pathways. There-
fore, the synthesized compounds were first tested to evaluate
their possible anti-aggregating properties by means of a thiofla-
vin T (ThT)-based fluorimetric assay. ThT dye shows a character-
istic redshift in the excitation/emission spectrum and an in-
crease in the quantum yield upon binding to fibrillar b-sheet
structures.[20] The ThT-based assay is commonly used to moni-
tor Ab fibrilization and its inhibition.
Motivated by the promising results, we sought to gain
a deeper understanding of the mode of action of 3 at a molec-
ular level by using an orthogonal method, that is, electrospray
ionization ion trap mass spectrometry (ESI-IT-MS) in flow injec-
tion mode, which allows the monomeric form of Ab42 to be de-
tected and quantitated.[23] Amyloid aggregation was monitored
by evaluating the decrease in the amount of the Ab monomer
after 24 h incubation in the presence and absence of the
tested inhibitor by using reserpine as the internal standard (IS).
Under the experimental conditions, in the absence of any in-
hibitor a progressive decrease in the content of the monomer,
expressed as the sum of the native (Ab42 Native) and oxidized
forms (Ab42 Ox) of Ab42, was observed within 24 h owing to in-
clusion of the Ab monomers into growing stable oligomers.[24]
In agreement with this trend, upon incubating Ab42 alone,
a dramatic decrease (83%) in the content of the monomer was
observed after incubating for 24 h (Figure 3).
The evaluation of 1–3 clearly highlights a strong influence of
the aryl decoration on the ability to prevent the Ab42 self-as-
sembly process. Interestingly, the catechol moiety (compound
3) turned out to be essential for activity. Compound 3, in a 1:1
ratio with Ab42, almost completely inhibited the self-aggrega-
tion of Ab42 (inhibition>90%), and it was even more effective
than curcumin (inhibition=73.7%). Notably, under the same
experimental conditions, a complete loss of the anti-aggregat-
ing efficacy was observed for 1 and 2, which lack the m- and
p-hydroxy function, respectively (Figure 2).
Conversely, upon treating Ab42 with 3 in a peptide/inhibitor
ratio of 1:1, after incubating for 24 h a high monomer content
was detected; consequently, 3 strongly inhibited inclusion of
the monomer into growing amyloid oligomers (Figure 3).
Indeed, the residual percentage of the Ab42 monomer at 24 h
was only 17% in the absence of any inhibitor and 78% in the
presence of 3. Curcumin, tested under the same conditions,
was a much weaker inhibitor of early-phase Ab42 aggregation
(residual percentage of monomer after 24 h incubation: 36%).
This striking result points to the catechol moiety as a key
recognition fragment in amyloid binding. The inhibitory effect
exerted by 3 was found to be concentration dependent with
an IC50 value of (12.5Æ0.9) mm. On the basis of this value, 3
can be considered a good inhibitor of the self-aggregation of
Ab42, as its inhibitory potency is similar to that of the well-
known multipotent compound bis(7)tacrine [IC50 =(8.4Æ
1.4) mm][21] and similar to that of derivative D737 (IC50 ~
Figure 2. Inhibition of Ab42 aggregation by 1–3 or curcumin (Curc), as deter-
mined by a ThT-based assay. ThT-related fluorescence intensity of Ab42
(50 mm) samples after a 24 h incubation period in the absence (Ctrl) or in
the presence of the indicated test compounds (all at 50 mm). Values are the
meanÆSEM of two independent measurements each performed in dupli-
cate.
Figure 3. Inhibition of Ab42 aggregation by 3 and curcumin (Curc), both at
50 mm, as determined by ESI-IT-MS. The total Ab42 monomer (Ab42m) content
in the absence (Ctrl) and in the presence of inhibitor is displayed as the sum
of the native (Ab42 Native) and oxidized (Ab42 Ox) forms of Ab42. IS: internal
standard (reserpine); **p<0.01, ***p<0.001 versus Ctrl 24 h; Dunnett’s mul-
tiple comparison test.
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These results, other than confirming the anti-aggregating ac-
tivity resulting from the ThT-based assay, also showed that 3
was able to strongly retard the overall assembly process of Ab
by acting at the monomer level in the early stage of amyloid
aggregation and by strongly preventing the formation of
stable soluble oligomers. This is of utmost importance because
of the cytotoxic effects exerted by soluble aggregation inter-
mediates.[25] The overall inhibition percentage was (74.5Æ
6.5)%, in agreement with the data obtained from the ThT fluo-
rimetric assay. On the other hand, curcumin showed an inhibi-
tion of (22Æ7.6)%.
Previous studies performed on the natural polyphenol myri-
cetin showed pro-oxidant properties toward Ab42 peptide.[24]
These properties can be explained by the well-accepted atti-
tude of polyphenols to act as either antioxidant or pro-oxidant
agents.[26] The oxidized form of Ab42 (Ab42 Ox) was shown to be
less prone to aggregate than the native one (Ab42 Native),
which thus explains the slower aggregation rate.[27] With these
concepts in mind, we sought to verify whether 3, bearing a cat-
echol moiety, could partially exert its inhibitory activity
through an oxidation-based mechanism. On the basis of their
different molecular weights, both the native and oxidized
forms of Ab42 can be detected by MS analysis. A small percent-
age of Ab42 Ox is always present in commercial samples of
Ab42 (~15%, detectable at t=0), and in agreement with the
lower inclination of Ab42 Ox to aggregate, the initial content of
the oxidized Ab species just slightly decreases after incubating
for 24 h (Figure 3).[24]
Figure 4. Effect of curcumin (Curc) and compound 3 on Ab42-mediated cyto-
toxicity in neuroblastoma cells. SH-SY5Y cells were pretreated for 24 h with
curcumin or compound 3 at 5 or 10 mm and then incubated for an addition-
al 24 h with Ab42 at 10 mm. Cell viability was determined by MTT assay. Data
are expressed as percentage cell viability versus control; **p<0.01 versus
Ab42; Dunnett’s multiple comparison test.
Antioxidant effect on H2O2-induced damage
To determine the potential interest of thioesters 1–3 as antioxi-
dants, we investigated their protective effects against H2O2-in-
duced oxidative damage. The scavenging effect of ROS was
evaluated in neuroblastoma cells by using the fluorescent
probe dichlorofluorescein diacetate (DCF-DA) as a specific
marker for the quantitative intracellular formation of ROS. In
comparison with untreated neuroblastoma cells (dashed line,
Figure 5), the intracellular DCF fluorescence intensity in H2O2-
treated cells significantly increased (gray line, Figure 5). Treat-
ment with curcumin and compounds 1–3 significantly sup-
pressed the production of H2O2-induced intracellular ROS
(Figure 5), and 2 was strongly more effective in counteracting
the formation of ROS.
Upon treating Ab42 samples with 3 in a peptide/inhibitor
ratio of 1:1, only a slightly increase in the oxidized species
after 24 h with respect to the initial content was observed,
which thus excludes a significant oxidation-mediated mode of
inhibition (Figure 3). Hence, on the basis of these results, stabi-
lization of the Ab42 monomeric form and inhibition of its inclu-
sion into growing oligomers, which greatly retards the overall
assembly process of Ab, can be postulated.
Protective effect of 3 on Ab42-induced toxicity in SH-SY5Y
Effect on the zyxin–HIPK2–p53 signaling pathway
neuroblastoma cells
The pro-oxidant environment induced by Ab is well estab-
lished in AD pathology, and a correlation among Ab, oxidative
stress, and conformational changes of p53 has already been
suggested.[13,29]
To determine whether 3 may exert any neuroprotective effect
against Ab42-induced toxicity, a cell-viability study in SH-SY5Y
human neuroblastoma cells was performed by using the 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
In this context, our experimental setting was based on data
from the literature indicating that sub-lethal concentrations of
Ab modulate oxidative stress by inducing high levels of oxida-
tive markers, such as 4-hydroxy-2-nonenal Michael adducts
and 3-nitrotyrosine, and by altering the conformation of p53
mainly as a result of nitration of its tyrosine residues.[30] Howev-
er, it is notable that Ab may also act as an antioxidant under
specific conditions, and this ability seems to be dependent on
the concentration of the peptide.[31]
(MTT) assay. Incubation of SH-SY5Y cells with 10 mm Ab42 re-
sulted in a decrease in cell viability by approximately 25%, and
this can be ascribed to the formation of oligomeric species.[28]
Nontoxic concentrations of 3 and curcumin (5 and 10 mm)
were then co-incubated with Ab42. The results depicted in
Figure 4 clearly show that 3 is able to exert a dose-dependent
protective effect. Indeed, whereas at a concentration of 5 mm
of 3 could not prevent Ab42 cytotoxicity, a strong protective
effect was observed if 3 was used at a concentration of 10 mm.
At this concentration, 3 almost completely prevented Ab-in-
duced cell death. In the same assay, curcumin was not able to
counteract Ab toxicity even at a concentration of 10 mm.
The mechanisms by which Ab induces the deregulation of
zyxin and HIPK2 and consequent conformational changes in
p53 may therefore be related to the capability of the peptide
to alter oxidative homeostasis. If this is the case, compounds
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with antioxidant activity should decrease conformational
changes in p53 mediated by Ab.
To substantiate this hypothesis, compounds 1–3 were fur-
ther investigated in a neuroblastoma cell line to verify whether
they may affect alterations in the zyxin–HIPK2–p53 pathway
mediated by soluble sub-lethal concentrations of Ab. For this
experimental setting, we diluted Ab in dimethyl sulfoxide, as
evidence from the literature indicates that diluted solutions of
Ab peptides in this solvent are quite stable and are less prone
to fibrilization at near-physiologic concentrations.[32]
We first characterized SH-SY5Y neuroblastoma cells in terms
of HIPK2 and zyxin expression and the conformational status
of p53. In agreement with our previous data,[14b] a sub-lethal
concentration of Ab42 (10 nm) significantly decreased the pro-
tein levels of HIPK2 and zyxin (Figure 6a).
The conformational status of p53 was analyzed by immuno-
precipitation by using two conformation-specific antibodies,
that is, PAb1620 and PAb240, which discriminate between the
folded and unfolded tertiary structures of p53, respectively.[33]
As previously verified with other cell lines, in neuroblastoma
cells Ab42 also induced the expression of unfolded p53, as rec-
ognized by the PAb240 antibody (Figure 6b).
Figure 5. Compounds 1–3 reverse ROS-formation-induced oxidative stress.
Cells were pretreated with curcumin (Curc) and compounds 1–3 (5 mm) for
24 h and then loaded with 25 mm DCF-DA for 45 min. DCF-DA was removed,
and cells were then exposed to 300 mm H2O2. Intracellular ROS levels were
determined on the basis of DCF fluorescence by using a fluorescent micro-
plate. The graph shows the intracellular fluorescence intensity of DCFÆSD
at various time treatments. Fluorescence intensity for curcumin and com-
pounds 1–3 at any time is significant, with p<0.001 versus H2O2; Dunnett’s
multiple comparison test.
On this basis, neuroblastoma cells were then treated with
10 nm Ab42 in the presence and absence of compounds 1–3 at
Figure 6. Compounds 1–3 positively modulate alterations in the zyxin–HIPK2–p53 pathway mediated by soluble sub-lethal levels of Ab42. a) Total cell extracts
of SH-SY5Y cells treated with 10 nm Ab42 for 48 h were analyzed for zyxin and HIPK2 expression. Anti-tubulin was used as the protein loading control. b) SH-
SY5Y cell lysates were immunoprecipitated with PAb240 or PAb1620 antibody. Immunoprecipitates were analyzed by western blot with the CM1 polyclonal
anti-p53 antibody. c) Total cell extracts of SH-SY5Y cells incubated for 48 h with 10 nm Ab42 and then treated with 5 mm compounds 1–3 for 24 h were ana-
lyzed for the conformational state of p53. Cell lysates were immunoprecipitated with PAb240 or PAb1620 antibody. Immunoprecipitates were analyzed by
western blot with the CM1 polyclonal anti-p53 antibody. After densitometric analysis, data were expressed as integrated density of the ratio of PAb240/
PAb1620 antibodies signal and represent the meanÆSEM of at least three independent experiments; *p<0.05, ***p<0.001 versus Ab treatment; Tukey’s
multiple comparison test. d) SH-SY5Y cells were incubated with 10 nm Ab42 for 24 h and then treated for an additional 24 h with compounds 1–3 at 5 mm.
Cells were then resuspended in fresh medium and finally exposed to 0.5 mm doxorubicin for 24 h. Cell viability was determined by MTT assay. Data are ex-
pressed as percentage cell viability versus control; *p<0.05, ***p<0.001 versus control; Bonferroni multiple comparison test.
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a concentration of 5 mm. Upon adding compounds 1–3 to the
Ab-pretreated cells, the level of unfolded p53 was significantly
lowered, as the intensity of the PAb240-positive band was
lower than that obtained for cells treated with Ab42 alone. The
ratio between the intensities of the bands immunoreactive to
PAb240 and PAb1620 was similar to that observed for the con-
trol cells (Figure 6c), for which 2 was significantly more effec-
tive. These data show that pretreatment of neuroblastoma
cells, in particular with compound 2, for which marked antioxi-
dant properties are not accompanied by any anti-aggregating
activity, prevented Ab-induced conformational changes in p53.
This finding may support the involvement of oxidative stress in
Ab function.
volvement of radical species in the loss of p53 conformation
and function induced by sub-toxic levels of Ab. Most impor-
tantly, the multifunctional ligand (S)-allyl (E)-3-(3,4-dihydroxy-
phenyl)prop-2-enethioate, together with compounds (S)-allyl
(E)-3-(4-hydroxyphenyl)prop-2-enethioate and (S)-allyl (E)-3-(3-
hydroxyphenyl)prop-2-enethioate, in which only the anti-ag-
gregating activity was switched off, emerge as promising phar-
macologic instruments that can be used to deepen our insight
into the molecular mechanisms potentially involved in chronic
Ab injuries.
Experimental Section
Chemistry
Loss of the wild-type conformation and function of p53 in-
duced by soluble nontoxic Ab has been shown to contribute
to the accumulation of cell damage, which makes cells unable
to activate the proper apoptotic program if exposed to nox-
ae.[11a,14a,b] In light of this evidence, we sought to study cell sen-
sitivity to doxorubicin, a genotoxic agent able to induce apop-
tosis in a p53-dependent manner,[34] following treatment with
10 nm Ab42 in the presence and absence of 5 mm 1–3. Notably,
cells treated with 1–3 and Ab42 were more vulnerable to dox-
orubicin than cells treated with Ab42 alone. Doxorubicin in-
duced a decrease in cell viability by approximately 30% in Ab-
treated cells, whereas the decrease in cell viability was approxi-
mately 50% in the presence of Ab42 and each tested com-
pound (Figure 6d). The obtained results indicate that com-
pounds 1–3 may prevent the production of the unfolded iso-
form of p53 induced by Ab, which makes the cells more sensi-
tive and able to respond to an insult.
General methods
Chemical reagents were purchased from Sigma–Aldrich, Fluka, and
Lancaster (Italy). The reactions were monitored by thin-layer chro-
matography (TLC) on 0.20 mm silica gel 60 F254 plates (Merck, Ger-
many), which were visualized with a UV lamp. NMR spectra were
1
recorded at 400 MHz for H and 100 MHz for 13C with a Varian VXR
400 spectrometer. Chemical shifts are reported in parts per millions
(ppm) relative to tetramethylsilane, and spin multiplicities are
given as s (singlet), brs (broad singlet), d (doublet), t (triplet), q
(quartet), and m (multiplet). Direct infusion ESI-MS mass spectra
were recorded with a Waters ZQ 4000 apparatus. Final compounds
1–3 were >95% pure, as determined by HPLC analyses. The analy-
ses were performed under reversed-phase conditions by using
a Phenomenex Jupiter C18 (150”4.6 mm I.D.) column with the use
of a binary mixture (A/B) of H2O/CH3CN (60:40 v/v) as the mobile
phase, UV detection at l=302 nm, and a flow rate of 0.7 mLminÀ1
.
Liquid chromatography was performed by using a Jasco Corpora-
tion (Tokyo, Japan) model PU-1585 UV equipped with a 20 mL loop
valve.
Conclusions
The amyloidogenic pathway is thought to be crucial to the
complex nature of Alzheimer’s disease. However, Ab-centric
drug programs have had limited success in AD clinical trials so
far. Yet, growing evidence suggests that merely hitting Ab pro-
duction or aggregation will not be enough to undermine the
architecture of AD, which calls for a deeper understanding of
the functions of Ab. To this aim, we synthesized three nature-
inspired compounds to investigate the connection between
Ab and oxidative stress, and p53 emerged as a possible media-
tor of this functional interplay.
Synthesis
General procedure for the synthesis of intermediates 7–9:
TBDMSCl (2–3 equiv) and imidazole (5 equiv) were added to a solu-
tion of appropriate trans-cinnamic acid 4–6 (1 equiv) in dry DMF
(5 mL) under a nitrogen atmosphere. The mixture was left at room
temperature overnight, and then the mixture was concentrated to
dryness and the residue was purified by column chromatography
on silica gel to yield intermediate 7–9. Compounds 4–6 are com-
mercially available or can be synthesized as described in the litera-
ture for the synthesis of trans-cinnamic acid through the Knoeve-
nagel–Doebner reaction.[35]
Interestingly, the hydroxy substituents on the aromatic
moiety allowed strategic tuning of the pharmacological pro-
files of the compounds. Notably, of the three synthesized de-
rivatives, only the catechol derivative (S)-allyl (E)-3-(3,4-dihy-
droxyphenyl)prop-2-enethioate inhibited the formation of Ab
fibrils, which underlines the importance of the catechol moiety.
By acting at the early stage of amyloid aggregation, this cate-
chol derivative strongly prevented the formation of cytotoxic
stable oligomeric intermediates. Conversely, although to differ-
ent extents, all hybrids were able to decrease the formation of
ROS and to inhibit Ab-induced conformational changes in p53,
and the stronger antioxidant (S)-allyl (E)-3-(3-hydroxyphenyl)-
prop-2-enethioate, which lacks anti-aggregating properties,
was significantly more effective. These findings suggest the in-
(E)-3-{4-[(tert-Butyldimethylsilyl)oxy]phenyl}acrylic acid (7): Syn-
thesized from 4 (500 mg, 3.04 mmol). Elution with petroleum ether
(PE)/EtOAc (6:4) afforded 7 as a waxy solid; yield: 466 mg (55%);
1H NMR (400 MHz, CDCl3): d=7.71 (d, J=16.0 Hz, 1H), 7.45 (d, J=
8.0 Hz, 2H), 6.85 (d, J=8.0 Hz, 2H), 6.31 (d, J=16.0 Hz, 1H), 0.99 (s,
9H), 0.23 ppm (s, 6H).
(E)-3-{3-[(tert-butyldimethylsilyl)oxy]phenyl}acrylic acid (8): Syn-
thesized from 5 (500 mg, 3.04 mmol). Elution with PE/EtOAc (7:3)
afforded 8 as a waxy solid; yield: 370 mg (44%); H NMR (400 MHz,
CDCl3): d=7.72 (d, J=16.0 Hz, 1H), 7.24 (t, J=8.0 Hz, 1H), 7.13 (d,
J=8.0 Hz, 1H), 7.00 (s, 1H), 6.87 (d, J=8.0 Hz, 1H), 6.40 (d, J=
16.0 Hz, 1H), 0.98 (s, 9H), 0.20 ppm (s, 6H).
1
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(E)-3-{3,4-Bis[(tert-butyldimethylsilyl)oxy]phenyl}acrylic acid (9):
Synthesized from commercially available 6 (500 mg, 2.78 mmol).
Elution with PE/EtOAc (8:2) afforded 9 as a waxy solid; yield:
318 mg (28%); 1H NMR (400 MHz, CDCl3): d=7.64 (d, J=16.0 Hz,
1H), 7.02 (d, J=8.0 Hz, 1H), 7.01 (s, 1H), 6.82 (d, J=8.0 Hz, 1H),
6.22 (d, J=16.0 Hz, 1H), 0.96 (s, 18H), 0.19 ppm (s, 12H).
2H); 13C NMR (100 MHz,CDCl3): d=190.33, 156.20, 140.85, 135.43,
132.75, 130.22, 124.89, 121.12, 118.31, 118.05, 114.91, 31.94 ppm;
MS (ESIÀ): m/z: 219 [MÀH]À.
(S)-Allyl (E)-3-(3,4-dihydroxyphenyl)prop-2-enethioate (3): Syn-
thesized from 12 (160 mg, 0.344 mmol). Elution with PE/EtOAc
(5:5) afforded 3 as a waxy solid; yield: 50 mg (62%); 1H NMR
(400 MHz, CD3OD): d=7.54 (d, J=16.0 Hz, 1H), 7.10 (s, 1H), 7.02 (d,
J=8.0 Hz, 1H), 6.84 (d, J=8.0 Hz, 1H), 6.63 (d, J=16.0 Hz, 1H),
5.94–5.87 (m, 1H), 5.31 (d, J=16.8 Hz, 1H), 5.14 (d, J=10.1 Hz, 1H),
3.68 ppm (d, J=6.4 Hz, 2H); 13C NMR (100 MHz, CD3OD): d=
189.34, 148.56, 145.47, 141.37, 133.43, 125.91, 122.14, 120.93,
116.56, 115.18, 113.93, 30.94 ppm; MS (ESI+): m/z: 259 [M+Na]+.
General procedure for the synthesis of intermediates 10–12:
DCC (1.1 equiv) and DMAP (cat.) were added to an ice-cooled solu-
tion of appropriate acid 7–9 (1 equiv) in dry CH2Cl2 (4 mL). The
mixture was stirred for 10 min, which was followed by the addition
of 2-propene-1-thiol (3 equiv). Stirring was continued at room tem-
perature overnight; the mixture was filtered and concentrated. The
crude material was purified by chromatography on silica gel.
(S)-Allyl (E)-3-{4-[(tert-butyldimethylsilyl)oxy]phenyl}prop-2-ene-
thioate (10): Synthesized from 7 (160 mg, 0.575 mmol). Elution
with PE/EtOAc (9.8:0.2) afforded 10 as a waxy solid; yield: 100 mg
Biological methods
Sample preparation for Ab42 self-aggregation: 1,1,1,3,3,3-Hexafluoro-
2-propanol (HFIP)-pretreated Ab42 samples (Bachem AG, Switzer-
land) were resolubilized with a CH3CN/0.3 mm Na2CO3/250 mm
NaOH (48.4:48.4:3.2) mixture to have a stable stock solution
([Ab42]=500 mm).[36] Tested inhibitors were dissolved in MeOH and
diluted in the assay buffer. Experiments were performed by incu-
bating the peptide diluted in 10 mm phosphate buffer (pH 8.0)
containing 10 mm NaCl at 308C (Thermomixer Comfort, Eppendorf,
Italy) for 24 h (final Ab concentration=50 mm) with and without in-
hibitor.
1
(52%); H NMR (400 MHz, CDCl3): d=7.57 (d, J=15.6 Hz, 1H), 7.43
(d, J=8.0 Hz, 2H), 6.84 (d, J=8.0 Hz, 2H), 6.59 (d, J=15.6 Hz, 1H),
5.88–5.83 (m, 1H), 5.28 (d, J=17.0 Hz, 1H), 5.13 (d, J=10.0 Hz, 1H),
3.66 (d, J=6.8, 2H), 0.98 (s, 9H), 0.22 ppm (s, 6H).
(S)-Allyl (E)-3-{3-[(tert-butyldimethylsilyl)oxy]phenyl}prop-2-ene-
thioate (11): Synthesized from 8 (370 mg, 1.33 mmol). Elution with
PE/EtOAc (9.8:0.2) afforded 11 as a waxy solid; yield: 260 mg
1
(58%); H NMR (400 MHz, CDCl3): d=7.57 (d, J=15.6 Hz, 1H), 7.15
(t, J=8 Hz, 1H), 7.13 (s, 1H), 6.88 (d, J=8.0 Hz, 1H), 6.70 (d, J=
8.0 Hz, 1H), 6.66 (d, J=16.0 Hz, 1H), 5.88–5.83 (m, 1H), 5.28 (d, J=
16.0 Hz, 1H), 5.13 (d, J=10.0 Hz 1H), 3.66 (d, J=6.4 Hz, 2H), 0.97
(s, 9H), 0.20 ppm (s, 6H).
Inhibition of Ab42 self-aggregation as determined by the ThT assay:
Inhibition studies were performed by incubating Ab42 samples
under the assay conditions reported above, with and without
tested inhibitors. Inhibitors were first screened at 50 mm in a 1:1
ratio with Ab42. To quantify amyloid fibril formation, the ThT fluo-
rescence method was used.[20b,37] After incubation, samples were
diluted to a final volume of 2.0 mL with 50 mm glycine–NaOH
buffer (pH 8.5) containing 1.5 mm ThT. A 300 s time scan of fluores-
cence intensity was performed (lexc =446 nm; lem =490 nm), and
values at plateau were averaged after subtracting the background
fluorescence of 1.5 mm ThT solution. Blanks containing inhibitor
and ThT were also prepared and evaluated to account for quench-
ing and fluorescence properties. The fluorescence intensities were
compared and the percentage inhibition was calculated. For com-
pound 3, the IC50 value was also determined. To this aim, four in-
creasing concentrations were tested. The IC50 value was obtained
from the plot of percentage inhibition versus log(concentration of
inhibitor).
(S)-Allyl (E)-3-{3,4-bis[(tert-butyldimethylsilyl)oxy]phenyl}prop-2-
enethioate (12): Synthesized from 9 (200 mg, 0.500 mmol). Elution
with PE/EtOAc (9.5:0.5) afforded 12 as a waxy solid; yield: 160 mg
(70%); 1H NMR (400 MHz, CDCl3): d=7.49 (d, J=16 Hz, 1H), 7.00
(d, J=8.0 Hz, 1H), 6.80 (d, J=8.0 Hz, 1H), 6.72 (s, 1H), 6.50 (d, J=
16.0 Hz, 1H), 5.76–5.65 (m, 1H), 5.25 (d, J=16.8 Hz, 1H), 5.09 (d,
J=10.1 Hz, 1H), 3.56 (d, J=6.8 Hz, 2H), 0.97 (s, 9H), 0.96 (s, 9H),
0.19 (s, 6H), 0.18 ppm (s, 6H).
General procedure for the synthesis of 1–3: TBAF (4 equiv) was
added to a solution of appropriate organosilane intermediate 10–
12 (1 equiv) in THF (5 mL) and stirring was continued at room tem-
perature. After 20–30 min, the reaction was quenched by the addi-
tion of a saturated aqueous NH4Cl solution; the aqueous phase
was extracted with EtOAc (3”10 mL), and the combined organic
layer was dried (Na2SO4). Following evaporation of the solvent, the
residue was purified by column chromatography on silica gel.
Inhibition of Ab42 self-aggregation by 3, as determined by flow injec-
tion ESI-MS: Inhibition studies were performed by incubating Ab42
samples under the assay conditions reported above, with and with-
out tested inhibitor 3 or curcumin. At t=0 and t=24 h, aliquots
with and without inhibitor were analyzed by flow injection (FIA)
ESI-IT-MS. FIA–MS analyses were performed, as described by Fiori
et al.[24] Briefly, the Ab42 samples were analyzed by 10 mL loop injec-
tion after previous addition of reserpine as internal standard. ESI-
IT-MS analyses were performed with a Jasco PU-1585 Liquid Chro-
matograph (Jasco, Tokyo, Japan) interfaced with a LCQ Duo Mass
Spectrometer (Thermo Finnigan, San Jose, CA, USA) equipped with
an ESI source and operating with an ion-trap analyzer. The mobile
phase consisted of 0.1% (v/v) formic acid in CH3CN/H2O (30:70).
The ESI system employed a 4.5 kV spray voltage and a capillary
temperature of 2008C. Mass spectra were operated in positive po-
larity, in the scan range of m/z=200 to 2000 at the scan rate of
three microscans per second. Single-ion monitoring (SIM) chroma-
tograms for the quantitative analysis were reconstructed at the
base peaks corresponding to the differently charged amyloid mo-
(S)-Allyl (E)-3-(4-hydroxyphenyl)prop-2-enethioate (1): Synthe-
sized from 10 (100 mg, 0.299 mmol). Elution with PE/EtOAc (7:3) af-
forded 1 as a waxy solid; yield: 30 mg (46%); 1H NMR (400 MHz,
CDCl3): d=7.56 (d, J=16.0 Hz, 1H), 7.42 (d, J=8.0 Hz, 2H), 6.84 (d,
J=8.0 Hz, 2H), 6.58 (d, J=16.0 Hz, 1H), 5.88–5.81 (m, 1H), 5.29–
5.25 (d, J=17.0 Hz, 1H), 5.13–5.10 (d, J=10.0 Hz, 1H), 3.65 ppm (d,
J=8.0 Hz, 2H); 13C NMR (100 MHz, CDCl3): d=189.97, 158.23,
140.83, 133.05, 130.44, 126.66, 122.29, 118.02, 116.03, 31.80 ppm;
MS (ESI+): m/z: 243 [M+Na]+.
(S)-Allyl (E)-3-(3-hydroxyphenyl)prop-2-enethioate (2): Synthe-
sized from 11 (210 mg, 0.63 mmol). Elution with CH2Cl2/MeOH
1
(9.7:0.3) afforded 2 as a waxy solid; yield: 110 mg (79%); H NMR
(400 MHz, CDCl3): d=7.54 (d, J=16.0 Hz, 1H), 7.24 (t, J=8.0 Hz,
1H), 7.08 (d, J=8.0 Hz, 1H), 7.02 (s, 1H), 6.90 (d, J=8.0 Hz, 1H),
6.58 (d, J=16.0 Hz, 1H), 6.09 (brs, 1H), 5.88–5.81 (m, 1H), 5.28 (d,
J=16.0 Hz, 1H), 5.13 (d, J=10 Hz, 1H), 3.65 ppm (d, J=6.4 Hz,
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nomer ions (Native, N) and oxidized ions (Ox). The ratio between
the total monomer area and the IS area was used for Ab42 mono-
mer determination. The areatotal monomer/areaIS ratio at t=0 was con-
sidered 100% of the monomer content. The results are expressed
as meanÆSD of three independent experiments, and p<0.05 was
considered statistically significant (Dunnett’s multiple comparison
test).
(62.5 mm Tris·HCl pH 6.8, 2% SDS, 10% glycerol, 50 mm dithiothrei-
tol, 0.1% bromophenol blue) and subjected to western blot analy-
sis. Proteins were subjected to SDS-PAGE (8%) and then transferred
onto a polyvinylidene fluoride (PVDF) membrane 0.45 mm (Immobi-
lion, Millipore Corp, Bedford, MA, USA). The membrane was
blocked for 1 h with 5% nonfat dry milk in Tris-buffered saline con-
taining 0.1% Tween 20 (TBST). Membranes were immunoblotted
with the rabbit antihuman zyxin or HIPK2 polyclonal antibody (at
1:1000 dilution in 5% nonfat dry milk, from Cell Signaling Technol-
ogy, EuroClone, Milan, Italy). Detection was performed by incuba-
tion with horseradish peroxidase conjugated goat anti-rabbit IgG
(1:5000 dilution in 5% nonfat dry milk, from Pierce, Rockford, IL,
USA) for 1 h. The blots were then washed extensively and the pro-
teins of interest were visualized by using an enhanced chemilumi-
nescent method (Pierce, Rockford, IL, USA). Tubulin was also per-
formed as a normal control of proteins.
Reagents for cellular experiments: All culture media, supplements
and fetal bovine serum (FBS) were obtained from Euroclone (Life
Science Division, Milan, Italy). Electrophoresis reagents were ob-
tained from Bio-Rad (Hercules, CA, USA). All other reagents were of
the highest grade available and were purchased from Sigma
Chemical Co. (St. Louis, MO, USA) unless otherwise indicated. Ab42
was solubilized in DMSO at a concentration of 100 mm and frozen
in stock aliquots that were diluted at the final concentration of
10 nm prior to use. For each experimental setting, one aliquot of
the stock was thawed out and diluted at the final concentration of
10 nm to minimize peptide damage as a result of repeated freeze
and thaw. The Ab42 concentration was chosen following dose–re-
sponse experiments (data not shown), for which maximal modula-
tion of the p53 structure and its transcriptional activity[38] were ob-
tained at 10 nm. All of the experiments performed with Ab42 were
made in 1% serum. H2O2 was diluted to working concentration
(1 mm) in phosphate-buffered saline (PBS) at the moment of use.
Mouse monoclonal anti a-tubulin was purchased from Sigma–Al-
drich (St. Louis, MO, USA). Host-specific peroxidase conjugated IgG
secondary antibodies were obtained from Pierce (Rockford, IL,
USA).
p53 conformational immunoprecipitation: The conformational state
of p53 was analyzed by immunoprecipitation as detailed previous-
ly.[10a] Briefly, cells were lysed in immunoprecipitation buffer
(10 mm Tris, pH 7.6, 140 mm NaCl, and 0.5% NP40 including pro-
tease inhibitors); 100 mg of total cell extracts was used for immu-
noprecipitation experiments performed in a volume of 500 mL with
1 mg of the conformation-specific antibodies PAb1620 (wild-type
specific) or PAb240 (mutant specific; Neomarkers, CA, USA). Immu-
nocomplexes were separated by 10% SDS-PAGE and immunoblot-
ting was performed with rabbit anti-p53 antibody (FL393; Santa
Cruz, CA, USA). Immunoreactivity was detected with the ECL-chem-
iluminescence reaction kit (Amersham, Little Chalfont, UK).
Cell cultures: Human neuroblastoma SH-SY5Y cells from European
Collection of Cell Cultures (ECACC No. 94030304) were cultured in
medium with equal amount of Eagle’s minimum essential medium
and Nutrient Mixture Ham’s F-12, supplemented with 10% FBS,
glutamine (2 mm), penicillin/streptomycin, nonessential amino
acids at 378C in 5% CO2/95% air.
Densitometry and statistics
All experiments, unless specified, were performed at least three
times. Following acquisition of the western blot image through an
AGFA scanner and analysis by means of the Image 1.47 program
(Wayne Rasband, NIH, Research Services Branch, NIMH, Bethesda,
MD, USA), the relative densities of the bands were analyzed as de-
scribed previously.[39] The data were analyzed by analysis of var-
iance (ANOVA) followed, if significant, by an appropriate post hoc
comparison test, as indicated in the figure legends. The reported
data are expressed as meanÆSD of at least three independent ex-
periments. Values p<0.05 were considered statistically significant.
Cell viability: The mitochondrial dehydrogenase activity that re-
duces 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT; Sigma, St. Louis, MO, USA) was used to determine cellular vi-
ability, in a quantitative colorimetric assay. At day 0, SH-SY5Y cells
were plated at a density of 2”104 viable cells per well in 96-well
plates. After treatment, according to the experimental setting, cells
were exposed to MTT solution in PBS (1 mgmLÀ1). Following 4 h in-
cubation with MTT and treatment with sodium dodecyl sulfate
(SDS) for 24 h, cell viability reduction was quantified by using
a BIO-RAD microplate reader (Model 550; Hercules, CA, USA).
Acknowledgements
The authors thank the University of Bologna (grants from the
RFO), the University of Pavia (grants from the FAR–Fondo Ateneo
Ricerca), and Unirimini for financial support.
Measurement of intracellular ROS: 2’,7’-Dichlorofluorescin diacetate
(DCF-DA; Sigma–Aldrich) was used to estimate intracellular ROS.
Briefly, cells (2”104 cells per well) were pretreated with reference
curcumin and compounds 1–3 (5 mm) for 24 h and then loaded
with 25 mm DCF-DA at 378C for 45 min. DCF-DA was removed after
centrifuge and cells were resuspended in PBS and then exposed to
300 mm H2O2. The results were visualized by using a Synergy HT mi-
croplate reader (BioTek) with excitation and emission wavelengths
of 485 and 530 nm, respectively.
Keywords: Alzheimer’s disease · antioxidants · amyloid-beta
peptide · p53 · multifunctional ligands
[1] a) D. J. Selkoe, Behav. Brain Res. 2008, 192, 106–113; b) J. LaurØn, D. A.
Gimbel, H. B. Nygaard, J. W. Gilbert, S. M. Strittmatter, Nature 2009, 457,
1128–1132.
[2] a) E. Karran, M. Mercken, B. De Strooper, Nat. Rev. Drug Discovery 2011,
10, 698–712; b) K. Herrup, Nat. Neurosci. 2015, 18, 794–799.
[3] a) E. Mura, F. Pistoia, M. Sara, S. Sacco, A. Carolei, S. Govoni, Curr. Pharm.
Des. 2014, 20, 4121–4139; b) L. M. Ittner, J. Gçtz, Nat. Rev. Neurosci.
2011, 12, 65–72; c) Z. Esposito, L. Belli, S. Toniolo, G. Sancesario, C. Bian-
coni, A. Martorana, CNS Neurosci. Ther. 2013, 19, 549–555.
Immunodetection of zyxin and HIPK2: Cell monolayers were washed
with ice-cold PBS (2”5 mL), lysed on the tissue culture dish by ad-
dition of ice-cold lysis buffer [50 mm Tris·HCl pH 7.4, 150 mm NaCl,
50 mm EDTA, 0.2 mm 4-(2-aminoethyl)benzenesulfonylfluoride hy-
drochloride (AEBSF), 20 mgmLÀ1 leupeptin, 25 mgmLÀ1 aprotinin,
0,5 mgmLÀ1 pepstatin A, and 1% Triton X-100], and an aliquot was
used for protein analysis with the Pierce Bicinchoninic Acid kit, for
protein quantification. Cell lysates were diluted in sample buffer
[4] a) E. Viayna, I. Sola, M. Bartolini, A. De Simone, C. Tapia-Rojas, F. G. Serra-
no, R. SabatØ, J. Juµrez-JimØnez, B. PØrez, F. J. Luque, V. Andrisano, M. V.
ChemMedChem 2016, 11, 1309 – 1317
www.chemmedchem.org
1316
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Clos, N. C. Inestrosa, D. MuÇoz-Torrero, J. Med. Chem. 2014, 57, 2549–
2567; b) E. Nepovimova, E. Uliassi, J. Korabecny, L. E. PeÇa-Altamira, S.
Samez, A. Pesaresi, G. E. Garcia, M. Bartolini, V. Andrisano, C. Bergamini,
R. Fato, D. Lamba, M. Roberti, K. Kuca, B. Monti, M. L. Bolognesi, J. Med.
Chem. 2014, 57, 8576–8589; c) M. Rosini, E. Simoni, M. Bartolini, E. Sor-
iano, J. Marco-Contelles, V. Andrisano, B. Monti, M. Windisch, B. Hutter-
Paier, D. W. McClymont, I. R. Mellor, M. L. Bolognesi, ChemMedChem
2013, 8, 1276–1281; d) M. Rosini, V. Andrisano, M. Bartolini, M. L. Bolog-
nesi, P. Hrelia, A. Minarini, A. Tarozzi, C. Melchiorre, J. Med. Chem. 2005,
48, 360–363; e) M. Rosini, E. Simoni, M. Bartolini, A. Cavalli, L. Ceccarini,
N. Pascu, D. W. McClymont, A. Tarozzi, M. L. Bolognesi, A. Minarini, V. Tu-
miatti, V. Andrisano, I. R. Mellor, C. Melchiorre, J. Med. Chem. 2008, 51,
4381–4384.
ni, A. Cavalli, M. L. Bolognesi, A. Minarini, P. Hrelia, G. Lenaz, M. Rosini,
C. Melchiorre, J. Med. Chem. 2010, 53, 7264–7268.
[20] a) M. Groenning, L. Olsen, M. van de Weert, J. M. Flink, S. Frokjaer, F. S.
Jørgensen, J. Struct. Biol. 2007, 158, 358–369; b) H. Naiki, K. Higuchi, M.
Hosokawa, T. Takeda, Anal. Biochem. 1989, 177, 244–249.
[21] A. Minarini, A. Milelli, V. Tumiatti, M. Rosini, E. Simoni, M. L. Bolognesi, V.
Andrisano, M. Bartolini, E. Motori, C. Angeloni, S. Hrelia, Neuropharma-
cology 2012, 62, 997–1003.
[22] A. F. McKoy, J. Chen, T. Schupbach, M. H. Hecht, J. Biol. Chem. 2012, 287,
38992–39000.
[23] M. Bartolini, M. Naldi, J. Fiori, F. Valle, F. Biscarini, D. V. Nicolau, V. Andri-
sano, Anal. Biochem. 2011, 414, 215–225.
[24] J. Fiori, M. Naldi, M. Bartolini, V. Andrisano, Electrophoresis 2012, 33,
3380–3386.
[5] a) A. Cavalli, M. L. Bolognesi, A. Minarini, M. Rosini, V. Tumiatti, M. Reca-
natini, C. Melchiorre, J. Med. Chem. 2008, 51, 347–372; b) M. Rosini,
Future Med. Chem. 2014, 6, 485–487; c) A. Anighoro, J. Bajorath, G. Ras-
telli, J. Med. Chem. 2014, 57, 7874–7887.
[6] M. Rosini, E. Simoni, A. Milelli, A. Minarini, C. Melchiorre, J. Med. Chem.
2014, 57, 2821–2831.
[7] M. Coma, F. X. Guix, G. Ill-Raga, I. Uribesalgo, F. Alameda, M. A. Valverde,
F. J. MuÇoz, Neurobiol. Aging 2008, 29, 969–980.
[8] A. M. Swomley, S. Fçrster, J. T. Keeney, J. Triplett, Z. Zhang, R. Sultana,
D. A. Butterfield, Biochim. Biophys. Acta, Gen. Subj. 2014, 1842, 1248–
1257.
[9] a) S. Salvioli, M. Capri, L. Bucci, C. Lanni, M. Racchi, D. Uberti, M. Memo,
D. Mari, S. Govoni, C. Franceschi, Cancer Immunol. Immunother. 2009,
58, 1909–1917; b) W. Chatoo, M. Abdouh, G. Bernier, Antioxid. Redox
Signaling 2011, 15, 1729–1737; c) A. Salminen, K. Kaarniranta, Cell. Sig-
nalling 2011, 23, 747–752.
[10] a) D. Uberti, C. Lanni, T. Carsana, S. Francisconi, C. Missale, M. Racchi, S.
Govoni, M. Memo, Neurobiol. Aging 2006, 27, 1193–1201; b) C. Lanni,
M. Racchi, G. Mazzini, A. Ranzenigo, R. Polotti, E. Sinforiani, L. Olivari, M.
Barcikowska, M. Styczynska, J. Kuznicki, A. Szybinska, S. Govoni, M.
Memo, D. Uberti, Mol. Psychiatry 2008, 13, 641–647; c) X. Zhou, J. Jia,
Neurosci. Lett. 2010, 468, 320–325.
[11] a) D. Uberti, T. Carsana, E. Bernardi, L. Rodella, P. Grigolato, C. Lanni, M.
Racchi, S. Govoni, M. Memo, J. Cell Sci. 2002, 115, 3131–3138; b) Y.
Yang, D. S. Geldmacher, K. Herrup, J. Neurosci. 2001, 21, 2661–2668.
[12] P. Hainaut, J. Milner, Cancer Res. 1993, 53, 4469–4473.
[13] C. Lanni, M. Racchi, M. Memo, S. Govoni, D. Uberti, Free Radical Biol.
Med. 2012, 52, 1727–1733.
[14] a) C. Lanni, L. Nardinocchi, R. Puca, S. Stanga, D. Uberti, M. Memo, S.
Govoni, G. D’Orazi, M. Racchi, PLoS One 2010, 5, e10171; b) C. Lanni, D.
Necchi, A. Pinto, E. Buoso, L. Buizza, M. Memo, D. Uberti, S. Govoni, M.
Racchi, J. Neurochem. 2013, 125, 790–799; c) E. Mura, S. Zappettini, S.
Preda, F. Biundo, C. Lanni, M. Grilli, A. Cavallero, G. Olivero, A. Salamone,
S. Govoni, M. Marchi, PLoS One 2012, 7, e29661.
[25] a) C. G. Glabe, R. Kayed, Neurology 2006, 66, S74–78; b) M. P. Lambert,
A. K. Barlow, B. A. Chromy, C. Edwards, R. Freed, M. Liosatos, T. E.
Morgan, I. Rozovsky, B. Trommer, K. L. Viola, P. Wals, C. Zhang, C. E.
Finch, G. A. Krafft, W. L. Klein, Proc. Natl. Acad. Sci. USA 1998, 95, 6448–
6453; c) D. M. Walsh, I. Klyubin, J. V. Fadeeva, W. K. Cullen, R. Anwyl,
M. S. Wolfe, M. J. Rowan, D. J. Selkoe, Nature 2002, 416, 535–539.
[26] S. C. Forester, J. D. Lambert, Mol. Nutr. Food Res. 2011, 55, 844–854.
[27] L. Hou, I. Kang, R. E. Marchant, M. G. Zagorski, J. Biol. Chem. 2002, 277,
40173–40176.
[28] S. Sabella, M. Quaglia, C. Lanni, M. Racchi, S. Govoni, G. Caccialanza, A.
Calligaro, V. Bellotti, E. De Lorenzi, Electrophoresis 2004, 25, 3186–3194.
[29] L. R. Borza, Rev. Med.-Chir. Soc. Med. Nat. Iasi 2014, 118, 19–27.
[30] a) L. Buizza, C. Prandelli, S. A. Bonini, A. Delbarba, G. Cenini, C. Lanni, E.
Buoso, M. Racchi, S. Govoni, M. Memo, D. Uberti, Cell Death Dis. 2013,
4, e484; b) L. Buizza, G. Cenini, C. Lanni, G. Ferrari-Toninelli, C. Prandelli,
S. Govoni, E. Buoso, M. Racchi, M. Barcikowska, M. Styczynska, A. Szybin-
ska, D. A. Butterfield, M. Memo, D. Uberti, PLoS One 2012, 7, e29789;
c) G. Cenini, G. Maccarinelli, C. Lanni, S. A. Bonini, G. Ferrari-Toninelli, S.
Govoni, M. Racchi, D. A. Butterfield, M. Memo, D. Uberti, Amino Acids
2010, 39, 271–283.
[31] D. G. Smith, R. Cappai, K. J. Barnham, Biochim. Biophys. Acta 2007, 1768,
1976–1990.
[32] H. Levine III, Anal. Biochem. 2004, 335, 81–90.
[33] C. MØplan, M. J. Richard, P. Hainaut, Biochem. Pharmacol. 2000, 59, 25–
33.
[34] a) K. Hayakawa, M. Hasegawa, M. Kawashima, Y. Nakamura, M. Matsuura,
H. Toda, N. Mitsuhashi, H. Niibe, Oncol. Rep. 2000, 7, 267–270; b) E. Lor-
enzo, C. Ruiz-Ruiz, A. J. Quesada, G. Hernµndez, A. Rodríguez, A. López-
Rivas, J. M. Redondo, J Biol. Chem. 2002, 277, 10883–10892; c) S. Wang,
E. A. Konorev, S. Kotamraju, J. Joseph, S. Kalivendi, B. Kalyanaraman, J.
Biol. Chem. 2004, 279, 25535–25543.
[35] C. N. Xia, H. B. Li, F. Liu, W. X. Hu, Bioorg. Med. Chem. Lett. 2008, 18,
6553–6557.
[36] M. Bartolini, C. Bertucci, M. L. Bolognesi, A. Cavalli, C. Melchiorre, V. An-
drisano, ChemBioChem 2007, 8, 2152–2161.
[15] J. Crone, C. Glas, K. Schultheiss, J. Moehlenbrink, E. Krieghoff-Henning,
T. G. Hofmann, Cancer Res. 2011, 71, 2350–2359.
[16] S. Rizzo, H. Waldmann, Chem. Rev. 2014, 114, 4621–4639.
[17] a) C. B. Pocernich, M. L. Lange, R. Sultana, D. A. Butterfield, Curr. Alzheim-
er Res. 2011, 8, 452–469; b) M. Stefani, S. Rigacci, Int. J. Mol. Sci. 2013,
14, 12411–12457.
[37] H. Levine III, Protein Sci. 1993, 2, 404–410.
[38] D. Uberti, G. Cenini, L. Olivari, G. Ferrari-Toninelli, E. Porrello, C. Cecchi,
A. Pensalfini, A. Pensafini, G. Liguri, S. Govoni, M. Racchi, M. Maurizio, J.
Neurochem. 2007, 103, 322–333.
[39] C. Lanni, M. Mazzucchelli, E. Porrello, S. Govoni, M. Racchi, Eur. J. Bio-
chem. 2004, 271, 3068–3075.
[18] S. Kim, H. G. Lee, S. A. Park, J. K. Kundu, Y. S. Keum, Y. N. Cha, H. K. Na,
Y. J. Surh, PLoS One 2014, 9, e85984.
[19] a) G. Gerenu, K. Liu, J. E. Chojnacki, J. M. Saathoff, P. Martinez-Martin, G.
Perry, X. Zhu, H. G. Lee, S. Zhang, ACS Chem. Neurosci. 2015, 6, 1393–
1399; b) E. Simoni, C. Bergamini, R. Fato, A. Tarozzi, S. Bains, R. Motterli-
Received: September 15, 2015
Published online on October 26, 2015
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