A fluorescent styrylquinoline with combined therapeutic and diagnostic activities against Alzheimer's and prion diseases

Article abstract of DOI:10.1021/ml3003605

(E)-6-Methyl-4′-amino-2-styrylquinoline (3) is a small molecule with the proper features to potentially diagnose, deliver therapy and monitor response to therapy in protein misfolding diseases. These features include compound fluorescent emission in the NIR region and its ability to interact with both Aβ and prion fibrils, staining them with high selectivity. Styrylquinoline 3 also inhibits Aβ self-aggregation in vitro and prion replication in the submicromolar range in a cellular context. Furthermore, it is not toxic and is able to cross the blood brain barrier in vitro (PAMPA test).

Full text of DOI:10.1021/ml3003605

Letter
pubs.acs.org/acsmedchemlett
A Fluorescent Styrylquinoline with Combined Therapeutic and
Diagnostic Activities against Alzheimer’s and Prion Diseases
Matteo Staderini,† Suzana Aulic,‡ Manuela Bartolini,§ Hoang Ngoc Ai Tran,‡ Víctor Gonzalez-Ruiz,∥
́
́
Daniel I. Perez,⊥ Nieves Cabezas,† Ana Martínez,⊥ M. Antonia Martín,∥ Vincenza Andrisano,§
́
Giuseppe Legname,‡ J. Carlos Menendez,† and Maria Laura Bolognesi*,§
́
∥
†Departamento de Química Organ
́
ica y Farmaceu
́
tica and Seccion
́
Departamental de Química Analítica, Facultad de Farmacia,
Universidad Complutense, 28040 Madrid, Spain
‡SISSA, Neuroscience Department, Via Bonomea 265, 34136 Trieste, Italy
§Department of Pharmacy and Biotechnology, Alma Mater Studiorum, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
⊥Instituto de Química Med
́
ica-CSIC, Juan de la Cierva 3, 28006 Madrid, Spain
S
* Supporting Information
ABSTRACT: (E)-6-Methyl-4′-amino-2-styrylquinoline (3) is a small molecule with
the proper features to potentially diagnose, deliver therapy and monitor response to
therapy in protein misfolding diseases. These features include compound fluorescent
emission in the NIR region and its ability to interact with both Aβ and prion fibrils,
staining them with high selectivity. Styrylquinoline 3 also inhibits Aβ self-aggregation
in vitro and prion replication in the submicromolar range in a cellular context.
Furthermore, it is not toxic and is able to cross the blood brain barrier in vitro
(PAMPA test).
KEYWORDS: Aggregation, protein misfolding diseases, amyloid, fibrillation inhibitors
available such as Congo Red (CR) and Thioflavin T (ThT) but
rotein misfolding diseases (PMD) include a broad range of
Phuman disorders characterized by a conformational change
of normally expressed proteins, that convert from a
physiological soluble monomeric form into oligomeric and
fibrillar forms, rich in stable β-sheet regions.1 These fibrillar
aggregates play a pivotal role in neuronal dysfunction and
survival, eventually leading to fatal disease. Alzheimer’s (AD)
and prion diseases (PrD) are prototypical examples of PMD. In
these maladies, amyloid-β protein (Aβ) and cellular prion
protein (PrPC), respectively, change their conformations into β-
sheet toxic isoforms. In the case of PrD, the toxic isoform
known as scrapie prion protein (PrPSc) is also infectious.2
In AD and PrD, the misfolded proteins have been regarded
both as neuropathological hallmarks for diagnosis and as
therapeutic targets.2 While the so-called “amyloid hypothesis”
in AD has been questioned, a recent interest has arisen in the
possibility that all proteins causing neurodegeneration are
prions.2 If confirmed, this hypothesis would profoundly
influence the development of diagnostic and therapeutic tools.2
Many techniques have been employed to detect fibrillar
aggregates, including positron emission tomography (PET)3
and fluorescence spectroscopy.4 PET is expensive and limited
by the narrow isotope availability of the required probes. On
the other hand, many fluorescent in vitro staining agents are
they cannot translate into clinical diagnostic and therapeutic
tools4 because of their low specificity, poor sensitivity, inability
to cross the blood brain barrier (BBB) and marked toxicity.5
Because fluorescence spectroscopy has proven suitable for
studying fibrillar aggregates also in vivo,6−9 there is an urgent
need for fluorescent sensors with improved properties.
With these concepts in mind, we focused our attention on
fluorescent compounds capable of staining Aβ and PrPSc fibrils
and, ideally, of simultaneously blocking their aggregation. To
this aim, ideal properties for a therapeutic and diagnostic small
molecule should include (1) ability to change fluorescence
properties upon binding to fibrils, (2) ability to absorb and emit
light in the far red/near-infrared (NIR) region (ca. 600−800
nm),7 where tissue scattering and absorption is lowest,10 (3)
large Stokes shift, (4) ability to modulate fibril aggregation, (5)
minimum interference from human serum albumin (HSA)
binding, (6) a small molecular size, which enables the small
molecule to cross the BBB, and (7) low toxicity.6−9,11
Received: October 22, 2012
Accepted: December 27, 2012
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
As a starting point, we noticed that several styryl derivatives,
designed by Li et al. to improve the pharmacokinetic properties
of CR and ThT, were employed as AD imaging agents in
vivo.12 Interestingly, some of them were also found to be active
against prion replication in infected cells.13 Furthermore,
Ishikawa et al. reported that a styrylbenzene derivative (BSB)
was useful to detect PrPSc aggregates ex vivo and showed
effectiveness in cellular assays.14 An isolated example of an (o-
hydroxystyryl)quinoline was described as a binder of both
amyloid and prion fibrils,15 and, very recently, Yang et al.
reported styryl-1H-indole and styrylquinoline derivatives as
SPECT imaging probe to detect Aβ plaques.16 These literature
data show that the styryl moiety is critical to the ability of small
molecules to label and interfere with pathological fibrillar
aggregates, in both PrD and AD.
Here we report a preliminary exploration of styrylquinolines
1−3 as Aβ/PrPSc diagnostic and therapeutic small molecules.
We focused on a quinoline moiety because it constitutes the
core of many antiprion compounds (i.e., chloroquine and
mefloquine),17 while displaying good pharmacokinetic proper-
ties and a high degree of druglikeness. An amino group was
purposely introduced (3) to have an electron-releasing
substituent conjugated with the quinoline nitrogen to shift
the fluorescence emission toward the far red/NIR spectral
region. In order to reinforce this effect by increasing the
electron-withdrawing character of the quinoline nitrogen, the
hydrochloride salt of 3 was also prepared.
Compounds 1 and 2 were synthesized via a vinylogous
variation of the Povarov reaction,18 while the amino derivative
3 was prepared by treating 1 with a mixture of fuming and
concentrated nitric acids followed by reduction of the crude
nitration product with iron powder in concentrated HCl. This
protocol afforded 3 in 68% yield after chromatography,
together with a small amount of the corresponding ortho
derivative (Scheme 1).
Figure 1. Fluorescence spectra for 3 (A) and 3·HCl (B) in solution,
and for 3·HCl in the solid state (C).
regard to the more polar water solvent. Since compound 3
would be adsorbed onto protein aggregates where its free
movement would be restricted, the fluorescence emission of
3·HCl in the solid state was also obtained (Figure 1C).
Interestingly, an emission maximum above 600 nm was
observed. On this basis we consider 3 as a starting lead
compound which can be easily manipulated by increasing the
electron-donating/accepting ability of the molecule to enhance
the spectral bathochromic shift, facilitating its use of this
compound as a NIR sensor.19 An additional advantageous
feature of 3 is its large Stokes shift (>150 nm), which minimizes
interference from the exciting radiation (Tables S1 and S2 in
the Supporting Information).
To assess the potential value of our compounds as CNS-
directed agents, the capability of 1−3 to cross the BBB was
studied by the parallel artificial membrane permeability assay
(PAMPA).20 Experimental data allowed us to predict optimal
BBB permeation for 2 and 3 (Table 1).
a
Scheme 1. Synthesis of Styrylquinolines 1−3
In vitro activities of compounds 1−3 on the target proteins
were then investigated. The inhibitory potency of 1−3 as
antiaggregating agents toward Aβ42 was determined by a ThT-
based fluorometric assay.21 When tested at a 1:1 ratio with
Aβ42, 1 and 2 did not significantly alter amyloid fibril formation,
a
Table 1. In Vitro Permeability (Pe) Values with Related
b
Predictive Penetration into the CNS and Antiprion Activity
c
and Cell Viability of 1−3 on ScGT1 Cells
Pe (10−6
EC
% of viable cells at
a
b
50d
d
compd
cm s−1
)
prediction
(μM)
EC50
a
quinacrine
0.4 0.1
1.5 0.1
0.5 0.1
0.7 0.1
0.5 0.1
100.0 4.3
93.4 7.3
90.5 6.8
74.9 4.1
94.6 4.9
Reagents and conditions: (i) CAN (15%), CH3CN, rt, 5 h; (ii) DDQ,
benzene, rt, 3 h; (iii) 65% HNO3/fuming HNO3 (2:8), rt, 14 h.; (iv)
Fe, 38% HCl, AcOH, EtOH, reflux, 4 h.
GN8
1
2
3
3.0 0.6
8.5 + 1.1
CNS +/−
CNS +
23.1 1.9
CNS +
We first investigated the native fluorescence of styrylquino-
lines 1−3 in several solvents, including phosphate buffered
saline (PBS) at physiological pH. In the case of 3, the
fluorescence emission greatly depended on solvent polarity
(Figure 1A,B and Tables S1 and S2 in the Supporting
Information). Its fluorescence emission in ethanolwhose
polarity mimics the protein environmentwas red-shifted with
a
b
Mean standard deviation (SD) of two experiments. CNS + means
Pe > 3.74 × 10−6 cm s−1, and CNS means Pe between 3.74 × 10−6
c
d
and 1.50 × 10−6 cm s−1. Mean SD of three experiments. Effect of
1−3 on inhibition of PrPSc replication and cell viability was measured
as reported in ref 25. Full details of the biological methods are given in
the Supporting Information.
B
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ACS Medicinal Chemistry Letters
Letter
while 3 decreased the fluorescence signal by 42.4 2.6%, thus
behaving as an inhibitor of fibrilization and an amyloid binder.
On the basis of data available in the literature, 3 can be ranked
as a moderate inhibitor, with potency slightly lower than that of
the well-known antiaggregating compound curcumin,22 which,
in the same assay conditions, was able to inhibit fibril formation
by 73%. No negative or positive interference in the fluorescence
signal of ThT was observed under the experimental conditions
(see Supporting Information and Figure 2C). In the light of
these findings, compound 3 was regarded sufficiently
interesting to warrant further study.
binding of 3 to the amyloid fibrils places it in a lower dielectric
constant environment. Indeed, adopting the β-sheet secondary
structure in the Aβ42 self-assembly process is likely to expose
hydrophobic regions that are accessible to 3. The spectral shift
observed for 3 is similar to that reported for the Aβ42
fluorescent sensor 1-anilinophthalene-8-sulfonic acid.23 Fur-
thermore, the fluorescence emission intensity at 450 nm
linearly correlated with the concentration of preaggregated Aβ42
(R2 = 0.9992) (Figure S1 in the Supporting Information).
Finally, to gain further insights into the binding mode of 3 to
Aβ, displacement studies in the presence of aggregated Aβ42
were carried out using a fixed concentration of Aβ42 and ThT
and an increasing concentration of 3. Overlaid excitation
spectra show that 3 binds amyloid fibrils with a high affinity. At
a low concentration (25 nM) and in the presence of Aβ42, 3
shows a significant emission signal (Figure 2B) without
affecting the ThT excitation and emission profile (Figure
2C). Conversely, at higher concentrations, 3 progressively
displaces ThT from its binding site(s)24 (Figure S2 in the
Supporting Information). This led us to hypothesize that 3 may
have a primary high affinity binding site distinct from ThT and
a secondary low-affinity binding site in common with ThT. To
discard the interference of HSA, a comparison between the
changes in the fluorescence intensity in the presence of HSA
and Aβ42 was carried out. It evidenced a stronger fluorescence
change for the latter peptide, as deduced from the slopes of the
titration plots (Figure S3 in the Supporting Information).
In addition, the capability to inhibit prion fibril formation in
vitro was studied, using a known amyloid seeding assay
(ASA).25 At a concentration of 50 μM, 1−3 delayed fibril
formation, extending the lag phase to ≥70 h (control: 59 h)
(Figure S4 in the Supporting Information). A similar profile
was found for GN8, an antiprion drug candidate, for which a
specific binding with PrP has been experimentally demon-
strated.26
Taken together, these results show that our candidate
molecule 3 is indeed able to recognize, stain and modulate Aβ42
and PrPSc fibril aggregation in vitro.
Next, the therapeutic antiprion potential (toxicity and
activity) of 1−3 was preliminarily assessed in a cell-based
model of PrD (ScGT1 cells). At a 1 μM concentration, 1 and 3
showed a very low toxicity, with cell viability above 90%, while
2 caused viability to decrease to 60%. Interestingly, at a 10 μM
concentration 3 still showed a tolerable toxicity, with a residual
60% cell viability not different from that of drug candidate GN8
(Figure S5 in the Supporting Information).
The three compounds were studied for their antiprion
activity in a nontoxic range of concentrations (Table 1). They
all showed remarkable activities (submicromolar EC50 values),
which make them more potent than GN8 and equipotent to
the gold standard quinacrine. Cell viability at the concentration
corresponding to EC50 was particularly high for 3 (94.6%).
To confirm the labeling of PrPSc aggregates in living cells,
fluorescent staining with 3 was carried out using the same
ScGT1 cell model (Figure 4). To corroborate the data an
additional cell line (ScN2a) was used (Figures S11−S15 in the
Supporting Information). We found that 0.025% of 3·HCl
(0.84 mM) was sufficient to observe many fluorescent spots in
the treated cells examined by fluorescent microscopy (Figure
3A,B). Importantly, no spots were observed in the uninfected
cells (Figure 3D−F), confirming a specific binding. Further-
more, the staining pattern was consistent with that observed
with 0.025% Thioflavin S (ThS), a common PrPSc dye (Figures
Figure 2. (A) Fluorescence emission spectra of 3 (5 μM) in aqueous
phosphate buffer (10 mM, pH 7.4) in the absence (dotted line) and in
the presence (solid line) of aggregated Aβ42 (3 μM). (B) Fluorescence
emission spectra of a solution containing aggregated Aβ42 protein and
3 [25 nM] in glycine−NaOH buffer (50 mM, pH 8.5) recorded at the
excitation maximum of ThT (λex = 446 nm, dotted line) and at the
excitation maximum of 3 (λexc = 375 nm, solid line). (C) Excitation
(λem = 490 nm) and emission (λexc = 446 nm) spectra of a solution
containing aggregated Aβ42 protein in 1.5 μM ThT buffered solution
(glycine−NaOH buffer, 50 mM pH 8.5) in the presence (dotted line)
and absence (solid line) of 3 [25 nM].
Due to its potential application as an amyloid sensor, the
emission spectra of compound 3 in the absence and presence of
aggregated Aβ42 were recorded. While the excitation maxima
for the bound and unbound forms of 3 did not significantly
differ, a strong hypsochromic shift of the emission maximum
(from 528 to 490 nm), concomitant with a hyperchromic effect
upon binding, was observed (Figure 2A). In agreement with
studies in solvents with decreasing polarity, this behavior seems
related to a change in the environmental conditions, as the
C
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ACS Medicinal Chemistry Letters
Letter
molecular weight than previous sensors,19 (2) a small-molecule
scaffold which is easily amenable to further manipulation to
improve fluorescence response and amyloid-binding properties.
Most importantly, with respect to the previously reported NIR
amyloid sensors,6−9,11 it offers the additional advantage of a
concomitant promising antifibrillar profile (in vitro and in a
cellular context), together with a low toxicity. If these peculiar
properties are confirmed in vivo, 3 is likely to become the first
purposely designed therapeutic and diagnostic tool (theranos-
tic) for PrD and AD.
ASSOCIATED CONTENT
* Supporting Information
■
S
Spectra, biological assay data and experimental procedures.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Phone: +39 051 209 9718. Fax: +39 051 209 9734. E-mail:
marialaura.bolognesi@unibo.it.
Figure 3. Detection of PrPSc aggregates with 3·HCl in ScGT1 cells by
intensity fluorescence (IF) before (A) and after (B) denaturation with
6 M guanidinium chloride (GndHCl). After PK digestion, PK resistant
PrP (PrPSc) was observed with 3·HCl (C). Uninfected GT1 cells with
or without denaturation step with 6 M GndHCl (D, E) and after PK
digestion (F) did not show staining in the presence of 3·HCl (0.84
mM). (G) Dose-dependent capacity of 1, 2 and 3 to decrease PrPSc
levels in comparison with GN8. Western blotting of ScGT1 cell lysates
depicting the presence or absence of prions (PrPSc) following
treatment with GN8 and 1, 2, 3 before (top) or after (bottom) PK
digestion.
Author Contributions
M.S. and S.A. contributed equally to this research. The
manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
Funding
Financial support from MICINN (Grants CTQ2009-12320-
BQU and SAF2009-13015-C02-01), UCM (Grupos de
Investigacion
truzione, dell’Universita
́
, Grant GR35/10-A-920234), Ministero dell’Is-
e della Ricerca under the program
̀
PRIN 2008 (Meccanismi Neurodegenerativi nelle Malattie da
Prioni: Studi Conformazionali, Fisiopatologia della Proteina
Prionica e Possibili Approcci Farmacologici), PRIN 2009 and
PRIN 2010 is gratefully acknowledged. D.I.P. acknowledges a
postdoctoral fellowship from CSIC (JAE program).
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
PMD, protein misfolding diseases; AD, Alzheimer’s disease;
PrD, prion diseases; PrC, PrSc, cellular and scrapie prion
protein; PBS, phosphate buffer saline; HSA, human serum
albumin; PET, positron emission tomography; CR, Congo
Red; ThT, Th S, Thioflavin T and S; BBB, blood brain barrier;
NIR, near-infrared; PAMPA, parallel artificial membrane
permeability assay; ASA, amyloid seeding assay; PK, proteinase
K; GndHCl, guanidium chloride; IF, intensity fluorescence
Figure 4. ScGT1 cell line treated with 6 M GndHCl and stained with
compound 3. (A) FITC filter. (B) ThS filter. Nuclei stained with
DAPI (blue). Scale bar 20 μm.
S8 and S12 in the Supporting Information). A further
experiment proved that 3·HCl (0.25 mM) distinguishes the
abnormal, aggregated and proteinase K (PK) resistant PrPSc
isoform from the normal, PK-sensitive PrPC isoform. Thus,
after eliminating PrPC through a PK digestion step, the previous
fluorescence-staining pattern was observed (Figure 3C). We
primarily used the FITC filter set for these studies, but we
confirmed the staining by employing the ThS one, which is
within the NIR optical window (Figure 4). At these emission
wavelengths, no interference from HSA was observed (Figure
S3 in the Supporting Information).
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