Inhibiting Aβ toxicity in Alzheimer's disease by a pyridine amine derivative

Article abstract of DOI:10.1016/j.ejmech.2019.02.052

Alzheimer's disease (AD) is a neurodegenerative disorder with no radical therapy. Aggregation of amyloid β-peptide (Aβ) induced by various factors is associated with pathogenesis of AD. A pyridine amine derivative, 3-bis(pyridin-2-ylmethyl)aminomethyl-5-h

Full text of DOI:10.1016/j.ejmech.2019.02.052

Accepted Manuscript
Inhibiting Aβ toxicity in Alzheimer's disease by a pyridine amine derivative
Zhenzhu Zhu, Tao Yang, Lei Zhang, Lulu Liu, Enmao Yin, Changli Zhang, Zijian Guo,
Chen Xu, Xiaoyong Wang
PII:
S0223-5234(19)30166-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.02.052
EJMECH 11144
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 December 2018
Revised Date: 7 February 2019
Accepted Date: 17 February 2019
Please cite this article as: Z. Zhu, T. Yang, L. Zhang, L. Liu, E. Yin, C. Zhang, Z. Guo, C. Xu, X. Wang,
Inhibiting Aβ toxicity in Alzheimer's disease by a pyridine amine derivative, European Journal of
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.02.052.
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Graphical abstract
Inhibiting Aβ Toxicity in Alzheimer’s Disease by a Pyridine Amine
Derivative
Zhenzhu Zhu a, 1, Tao Yang a, 1, Lei Zhanga, Lulu Liua, EnmaoYina, Changli Zhangb,
Zijian Guo c, Chen Xu a, *, and Xiaoyong Wang a, *
aState Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University,
Nanjing 210023, P. R. China
bState Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering,
Nanjing University, Nanjing 210023, P. R. China
cDepartment of Chemistry, Nanjing Xiaozhuang College, Nanjing 210017, P. R. China
* Corresponding author. State Key Laboratory of Pharmaceutical Biotechnology, School of Life
Sciences, Nanjing University, Nanjing 210023, P. R. China.
E-mail addresses: boxwxy@nju.edu.cn
(X. Y. Wang), xuchn@nju.edu.cn (C. Xu)
1These authors contributed equally to this work.

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Inhibiting Aβ toxicity in Alzheimer’s disease by a
pyridine amine derivative
Zhenzhu Zhu a,#, Tao Yang a,#, Lei Zhanga, Lulu Liua, Enmao Yina, Changli Zhangb,
Zijian Guo c, Chen Xu a,*, and Xiaoyong Wang a,*
a State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences,
Nanjing University, Nanjing 210023, P. R. China
b
Department of Chemistry, Nanjing Xiaozhuang College, Nanjing 210017, P. R.
China
c State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical
Engineering, Nanjing University, Nanjing 210023, P. R. China
* Corresponding author. E-mail addresses: boxwxy@nju.edu.cn (X. Y. Wang),
xuchn@nju.edu.cn (C. Xu)
# These authors contributed equally to this work.
Abstract
Alzheimer’s disease (AD) is a neurodegenerative disorder with no radical
therapy. Aggregation of amyloid β-peptide (Aβ) induced by various factors is
associated with pathogenesis of AD.
A
pyridine amine derivative,
3-bis(pyridin-2-ylmethyl)aminomethyl-5-hydroxybenzyltriphenylphosphonium
bromide (PAT), is synthesized. The inhibition of self- and metal-induced Aβ
aggregation by PAT is confirmed by thioflavine T fluorescence, circular dichroism
spectroscopy, and TEM. Western blot, RT-PCR and fluorescence imaging indicate
that PAT can alleviate the Aβ-induced paralysis, reduce the production of ROS, and
protect the mitochondrial function in transgenic C. elegans. Genetic analyses indicate
that heat shock protein is involved in the alleviation of Aβ toxicity. PAT also inhibits
the activity of acetylcholinesterase in C. elegans. Morris water maze test shows that
the memory and cognitive ability of APP/PS1 AD model mice are significantly
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improved by PAT. Both in vitro and in vivo studies demonstrate that PAT is effective
in counteracting Aβ toxicity and ameliorating cognitive functions in AD mice, and
therefore a potential lead compound of anti-AD drugs.
Keywords: Alzheimer’s disease • amyloid-β peptide • anti-AD drug • heat shock
protein • Caenorhabditis elegans
1. Introduction
Alzheimer’s disease (AD) is a devastating neurodegenerative disorder
encountered frequently by elderly people in the world [1, 2]. Although some small
molecules, such as inhibitors of γ-secretase, β-secretase, tau aggregation or microglial
activation, and immunotherapeutic agents targeting at amyloid-β peptide (Aβ) and
p-tau clearance are in clinical trials [3], only five drugs are approved by the FDA for
the treatment of AD [4]. Even so, no disease-modifying treatment that can halt or
slow the progression of AD is available up to now [5, 6].
The exact molecular mechanism leading to AD remains unclear; however, a
growing body of evidence implicates that AD is caused by multiple factors, such as
Aβ aggregation, tau-protein hyperphosphorylation, oxidative stress and acetylcholine
breakdown [7]. Among them, the Aβ aggregation plays a major role in neural loss and
cognitive impairment by forming senile plaques; it also relates to the increased
oxidative stress responsible for the neuronal injury and death [8]. The high level of
Aβ in the AD brain came from the abnormal cleavage of amyloid precursor protein
(APP) by β- and γ-secretases, which gives Aβ40 and Aβ42 containing 40 and 42 amino
acid residues respectively [9]. These Aβ species form unbranched fibrils consisting of
parallel and orderly β-sheet structures [10]. Aβ, especially Aβ42, is prone to form toxic
aggregates [11]; among them, soluble Aβ oligomers are the main cytotoxic species
[12]. Therefore, preventing Aβ aggregation is a major strategy against AD; beyond
this, reducing oxidative stress, or activating disease-modifying pathways could also
relieve the onset of AD [13].
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On the other side, AD is recognized to be linked to the function and status of
metal ions, such as Cu2+, Zn2+ or Fe2+ [14]. These ions are present at high
concentrations in Aβ plaques (Cu2+ ∼400 µM, Zn2+ ∼1 mM, Fe2+ ∼1 mM), which
contribute to the stability of Aβ aggregates [15], the production of reactive oxygen
species (ROS), and the loss of neuronal function [16]. Thus, rebalancing the metal
ions and relieving oxidative stress in AD pathogenesis is becoming a potential
strategy for the therapy of AD [17, 18].
Transgenic caenorhabditis elegans (C. elegans) that expresses human Aβ gene
has been used as a model system to mimic AD in order to probe the molecular
mechanism of Aβ toxicity [19] and screen therapeutic agents [20], because it has a
short lifespan, well-characterized genome and considerable homology with human
genomes [21]. Transgenic worms, such as CL4176 and GMC101, were genetically
engineered to express the human Aβ42 gene in muscle cells [22]. The accumulation of
Aβ species in C. elegans muscle cells results in either a progressive or a rapid
paralytic phenotype, providing a simple biological readout of Aβ toxicity [23]. It was
reported that overexpression of heat shock protein or factors could suppress Aβ
toxicity by interacting directly with Aβ and altering its oligomerization pathways,
thereby reducing the formation of toxic species in C. elegans [24, 25].
Multifunctional compounds containing metal chelating moieties, such as
8-hydroxyquinoline, polyphenols, flavonoids and pyridine amine derivatives, have
been reported as potential anti-AD agents [26]. For example, pyridine amine
derivative in Fig. 1 forms stable metal complexes by incorporating N,N,O donor
atoms; moreover, it shows anti-oxidative activity due to the presence of phenolic
group [27]. These compounds interact with Cu2+ and Zn2+ to disaggregate the
metal-associated Aβ aggregates. On the other hand, lipophilic cations such as
triphenylphosphonium (TPP+) derivatives are rapidly and extensively taken up by
mitochondria due to large mitochondrial membrane potential (MMP) [28]. Covalent
attachment of TPP+ has been proven to be a robust method to target small and
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bioactive molecules to mitochondria [29]. For example, mitoquinone (MitoQ) is a
mitochondrion-targeted antioxidant designed to protect against oxidative damage
within mitochondria and has undergone clinical trials in humans [30].
Considering the advantages of multifunctional compounds, a pyridine amine
derivative,
3-bis(pyridin-2-ylmethyl)aminomethyl-5-hydroxybenzyltriphenyl-
phosphonium bromide (PAT), was designed in this study (Fig. 1). The presence of
phenolic group and mitochondrion-targeted TPP moiety in PAT may protect against
the oxidative damage in mitochondria. Cell-free studies showed that PAT could
inhibit the self-assembly of Aβ fibrils and Zn2+/Cu2+-induced Aβ aggregation; in
Aβ-transgenic C. elegans, it dramatically delayed the progression of body paralysis,
reduced the amount of Aβ species and oxidative activity. More importantly, PAT
played a prominent role in protecting against Aβ toxicity and maintaining healthy
proteostasis in AD worms. As a result, the cognitive and memory abilities of
APP/PS1 AD mice were significantly improved after PAT treatment.
Fig. 1. Structures of PAT and its parents.
2. Results and discussion
2.1. Design and characterization
The synthetic route to PAT was shown in Scheme 1. The compound was
characterized by 1H, 31P NMR and ESI-MS spectroscopy (Fig. S1).
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Bis(2-pyridylmethyl)amine was selected as the chelating moiety because its binding
ability to Cu2+ and Zn2+ (logKCu = 9.3; logKZn = 7.6) [31] is moderately higher than
that for Aβ40 (logKCu = 5–9; logKZn = 3–7) [32, 33]. Lipophilicity is an important
parameter influencing a compound to penetrate the cellular membrane and
blood-brain barrier (BBB). Thus, lipophilic triphenylphosphonium cation (TPP+) was
used to improve the lipophilicity of PAT and facilitate its accumulation in
mitochondria owing to the characteristic of MMP (180–200 mV, negative inside) [34].
In view of the potential anti-oxidative activity, 3,5-bis(bromomethyl)phenol was
selected to link the two functional moieties. We suppose that PAT may have several
properties favorable for AD treatment: (i) inhibiting metal-induced Aβ aggregation
through the chelating moiety; (ii) enhancing the BBB-penetrating power via the TPP
lipophilic cation; and (iii) reducing the oxidative stress by the phenolic group.
Although the molecular weight (580.69, without Br‒) and ClogP (7.51) are larger than
those suggested by the Lipinski’ rules (Table S1) [35], we still expect that PAT could
cross the BBB due to its poor water solubility. To ensure the BBB penetration of PAT,
dimethylsulfoxide (DMSO) was used in the in vivo studies to realize the
solvent-mediated disruption to the membrane [36].
Scheme 1. Synthetic route to PAT. Reagents and conditions: (a) pyridine, rt, 48 h; (b)
NBS, BPO, CCl4; (c) triphenylphosphine, CCl4, rt, overnight; (d)
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bis(2-pyridylmethyl)amine, DIEA, CHCl3, rt, 48 h; (e) NaOH, HCl, CH3OH/H2O.
2.2. Inhibition of Aβ aggregation
Metal ions such as Zn2+ and Cu2+ can interact with Aβ and facilitate its
aggregation, producing toxic oligomeric peptide species [14]. The effect of PAT on
the Aβ aggregation was investigated using thioflavine T (ThT) because it can
specifically bind to the aggregated β-sheet fibrils common to amyloid structures and
emit enhanced fluorescence [37]. As presented in Fig. 2A, the fluorescence intensity
of ThT decreases dramatically when PAT is added to the solution of Zn2+- or
Cu2+-Aβ40 aggregates. The most potent effect of PAT on the Zn2+-induced Aβ42
aggregation was observed at the half maximal inhibitory concentration (IC50) of 27.42
± 1.52 µM (Fig. 2B). In the progression of AD, part of Aβ switches from a nontoxic
α-helix to a toxic β-sheet conformation, forming self-assembled aggregates and
leading to neurotoxicity [38]. ThT assay indicates that the IC50 value of PAT against
the self-aggregation of Aβ42 is 12.53 ± 1.35 µM (Fig. 2B), and the efficacy is
dependent on both its concentration and reaction time (Fig. S2). This property is
similar to the activity of many reported modulators such as curcumin and resveratrol
that affect the fibrillogenesis of Aβ through blocking hydrophobic interactions [39].
The fluorescence intensity of ThT decreased significantly after Aβ42 was incubated
with PAT, suggesting that the total amount of Aβ fibers was reduced.
Circular dichroism spectroscopy was used to investigate the conformational
transition of Aβ42, including secondary structure α-helix, β-sheets and random coils, in
the process of fibrillation [40]. As shown in Fig. 2C, a strong negative peak and a
weak positive peak appeared at 217 and 203 nm after Aβ42 was incubated at 37 °C for
24 h, suggesting that β-sheets were formed in the process of self-aggregation and
fibrillation. The ellipticity for the positive band increased and that for the negative
band decreased after Aβ42 was co-incubated with Zn2+, suggesting that less β-sheets
were formed in the Zn2+-induced Aβ aggregation compared to those formed in the
self-aggregation, because the Zn2+-induced Aβ aggregates contain both β-sheets and
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random coils. After incubation with PAT for 24 h, the ellipticity for the negative band
decreased dramatically and shifted to around 230 nm, and a broad positive band
appeared, which suggest that PAT can inhibit the formation of β-sheets and modulate
the conformation of Aβ42. The morphology of Aβ aggregates were visualized by
transmission electron microscope (TEM). As shown in Fig. 2D, large amounts of Aβ
fibrils are formed after addition of Zn2+ or Cu2+ to Aβ40, which are similar to our
previous observations [41]; however, in the presence of PAT, the Zn2+- or
Cu2+-induced Aβ40 aggregates or fibrils are disaggregated into granule-like species or
small soluble fragments. All the results presented in Fig. 2 show that PAT can
significantly inhibit the formation of aggregated β-sheet fibrils induced by Aβ
self-aggregation and Zn2+ or Cu2+ in vitro.
Fig. 2. (A) ThT fluorescence intensity (λex = 450 nm, λem = 485 nm) of Zn2+- or Cu2+
(20 µM)-induced Aβ40 (20 µM) aggregates after incubation with or without PAT (20
µM) at 37 °C for 24 h; (B) dose-depend inhibition of Aβ42 (20 µM) aggregation by
PAT in the presence or absence of Zn2+ (20 µM) after incubation at 37 °C for 24 h in
Tris-HCl buffer (pH 7.4); (C) conformational transition of Aβ42 (40 µM) before or
after treatment with PAT at 37 °C for 24 h in the presence of Zn2+, [Aβ42]:[metal
ion]:[chelator] = 1:1:2; (D) TEM images of Zn2+- or Cu2+-induced Aβ40 aggregates
incubated with or without PAT.
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2.3. Neurotoxicity
The rat adrenal pheochromocytoma cells (PC12) can easily differentiate into
neuron-like cells when treated with nerve growth factor (NGF), releasing
neurotransmitter by vesicles. This makes PC12 cells useful as a model system for
neuronal differentiation and neurosecretion [42]. The cell viability of PC12 cells in
the presence of PAT was evaluated by the MTT assay. PAT has little toxicity to PC12
cells, in that the cell viability is 80.50 ± 1.86% even at 100 µM (Fig. S3).
2.4. Alleviation of Aβ-induced paralysis in C. elegans
Transgenic C. elegans strains CL4176 and GMC101 expressing Aβ42 gene in the
body wall muscle cells were used to determine the effects of PAT on Aβ-induced
toxicity. When temperature is up-shifted from 15 to 25 °C, the deposition of Aβ42
aggregates would cause paralytic phenotype in CL4176 and GMC101 [23]. As shown
in Fig. 3, the onset of paralysis was just started for the PAT-treated group after 33 h;
meantime 80% CL4176 worms were paralyzed in the control group. The percentage
of paralyzed worms were 74%, 66%, and 35% after treatment with 50, 100, and 200
µM of PAT, respectively, for 49 h, while that of the control is 87% (Fig. 3A). The
onset of paralysis in CL4176 strains was faster than in GMC101 strains, in that the
percentages of the paralyzed GMC101 strains after 92 h were similar to those of
CL4176 after 49 h (Fig. 3B). The results show that PAT can alleviate the Aβ-induced
paralysis in both CL4176 and GMC101 strains. Under the microscope, the onset of
paralysis in CL4176 strains was observed after the upshift of temperature. The worms
did not move or only moved the head without PAT; after incubation with PAT, many
worms still moved as a roller, which were indicated by red arrows (Fig. 3C). GMC101
strains were further tested by fluorescence imaging because Aβ42 and green
fluorescent protein (GFP) are co-expressed in the body wall muscle (Fig. 3D). ImageJ
analysis showed that the green fluorescence intensity was decreased to 43% and 20%
after treatment with 50 and 100 µM of PAT, respectively, relative to the control worms.
Similarly, the fluorescence intensity was decreased in the PAT-treated CL4176 strains,
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which were stained with ThT dye (Fig. S4). These results suggest that PAT could
delay the onset of paralysis and reduce the Aβ deposits.
Fig. 3. Effect of PAT on the Aβ-induced paralysis in transgenic C. elegans strain
CL4176 (A) and GMC101 (B), data were expressed as percentage based on three
experiments (n = 120); photos of CL4176 with or without PAT treatment at 25 °C for
48 h (C), and fluorescence confocal images of GMC101 (λex = 488 nm) (D), data were
obtained from two experiments with 15 worms in each group.
2.5. Expression of Aβ species in C. elegans
The effect of PAT on Aβ deposits in transgenic CL4176 was tested by the
western blot assay. As shown in Fig. 4A, after treatment with PAT, the immunoblot
showed that Aβ species (< 55 KDa), including those with low molecular weight (12,
16 KDa), were reduced significantly compared with that of the control strains.
Different from the observations reported for Metformin [43] and Liuwei Dihuang [44],
monomeric Aβ was not observed in the PAT-treated strains.
Although the C. elegans genome encodes an APP orthologue, apl-1, it lacks high
sequence identity with Aβ, because the products of β-secretase activity could not be
detected in C. elegans [19]. Thus, real-time PCR was performed to quantify the
mRNA expression level of human Aβ transgene (amy-1). The results indicate that PAT
could reduce the mRNA expression level of amy-1 gene compared with the control
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(Fig. 4B).
Aβ distribution, which can be upregulated in larval animals by temperature, was
further detected by fluorescence confocal imaging. Strains that stained with
rhodamine-phalloidin (red) were used to visualize the actin filaments in muscle cells
[45], and green fluorescence signals appeared after immunostaining with anti-Aβ
antibody 6E10 (Fig. 4C). As compared with the control staining, the green
fluorescence intensity was decreased evidently after treatment with PAT. These results
indicate that PAT could downregulate the expression of Aβ at the mRNA level and
reduce the Aβ deposits in transgenic C. elegans.
Fig. 4. (A) Expression of Aβ species in CL4176 worms after treatment with PAT (50
µM) at 25 °C for 48 h, and (B) expression of gene (amy-1) in CL4176 worms after
treatment with PAT (50, 100 µM) at 25 °C for 48 h quantified by RT-PCR (2−∆∆Ct
method). The gene ama-1 was used as the internal control; data represent an average
from three independent experiments, ** p < 0.01. (C) Immunolocalization of anti-Aβ
antibody 6E10 (green) and rhodamine-phalloidin (red) in the presence or absence of
PAT in CL4176 worms.
2.6. Antioxidant activity in C. elegans
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Aβ plaques can induce ROS accumulation and lead to oxidative stress, which can
further aggravate the AD pathological progresss [46]. Therefore, the ROS level in C.
elegans was investigated using fluorescence probe H2DCF-DA, which reflects the
ROS production by fluorescence intensity. Juglone is a superoxide-generating
compound used for acute assays of oxidative stress [47]. As shown in Fig. 5, PAT
(100 µM) suppressed the production of ROS to 21.72%, which is less than that of
control (33.68%). For the juglone-stimulated group, the relative fluorescence intensity
of the control group increased to 74.23%; while that of the PAT-treated (50 µM, 100
µM) group decreased to 48.97% and 28.14%, respectively. These results suggest that
the ROS level was significantly inhibited in the nematodes fed with PAT, and PAT can
protect CL4176 against oxidative stress. The delayed onset of Aβ-related paralysis in
CL4176 worms by PAT might be partly due to its antioxidant activity.
Fig. 5. ROS level in CL4176 worms treated with or without juglone (300 µM) and
PAT at 48 h after upshift of temperature measured by using H2DCF-DA fluorescence
probe. Results are expressed as relative fluorescence intensity, normalized by protein
concentration. Data represent mean ± S. D., 180 worms per well, 5 wells per group.
2.7. Expression of stress-responsive genes in C. elegans
Several regulators of stress-responsive markers, including stress-induced
transcription factor (skn-1), superoxide dismutase 3 (sod-3), heat shock factor (hsf-1),
and small heat shock proteins (hsp-16.2, hsp-60), play important roles in regulating
Aβ aggregation and thereby protecting C. elegans from Aβ toxicity [48, 49]. Thus,
real time-PCR was performed using CL4176 worms to measure the gene expression
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of skn-1, sod-3, hsf-1, hsp-16.2 and hsp-60, respectively. No significant difference
was observed in the expressions of skn-1 and sod-3; however, the expressions of hsf-1,
hsp-16.2 and hsp-60 genes were upregulated 1.55-, 2.15- and 1.59-fold, respectively,
in CL4176 worms supplemented with PAT (Fig. 6). Heat shock protein (HSP) was
reported to alleviate Aβ toxicity by disaggregating and degrading large Aβ aggregates
into peptides or amino acids [50]. Surprisingly, we observed that PAT can
significantly stimulate the overexpression of HSP, which may offer a protection
against Aβ toxicity in vivo by modulating oligomerization of Aβ in C. elegans.
Fig. 6. Expression of stress-responsive genes in CL4176 worms after treatment with
PAT quantified by real-time PCR (2−∆∆Ct method). Gene ama-1 was used as the
internal control; data represent an average of three independent experiments, ns = not
significant, **p < 0.01, ****p < 0.0001.
2.8. Mitochondrial function
ROS accumulation could lead to oxidative damage to the mitochondria, which
would reduce the viability of neuron in the AD pathological progress [51]. Thus, the
effect of PAT on the oxygen consumption rate (OCR) in CL4176 worms was
determined by the Seahorse XFe24 Cell Bioanalyzer to evaluate the mitochondrial
function. The basal respiration of the PAT-treated group is similar to that of the control
group (Fig. 7). After stimulation by oligomycin (an ATP synthase inhibitor), the OCR
of the control group decreased, while that of the PAT-treated group remained at the
level similar to the basal respiration. The results indicate that PAT could resist
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oligomycin-induced stress in the mitochondria, which ensure the level of ATP
production in CL4176 strain. Therefore, PAT not only attenuates mitochondrial
dysfunction, but also protects mitochondria from oxidative damage by producing less
ROS in CL4176 strains.
Fig. 7. OCR of CL4176 worms in the presence or absence of PAT before or after
stimulation by oligomycin (1 µM) at 25 °C for 48 h (50 worms per well, 5 wells per
group).
2.9. Acetylcholinesterase (AChE) activity in C. elegans
AChE is one of the most crucial enzymes for nerve response and function, which
degrades the neurotransmitter acetylcholine into choline and acetic acid [52]. Aβ can
potently inhibit several steps of acetylcholine (ACh) synthesis and release, and
decrease the availability of acetylCoA, which is also related to mitochondrial
dysfunction [53]. We thus determined the activity of AChE in CL4176 strains in the
presence of PAT. As shown in Table 1, the activity of AChE was reduced from 272.59
to 134.76 Units L‒1. However, the mRNA level of the AChE gene ace-1 was not
changed in Aβ transgenic CL4176 by PAT (Data not shown). The results suggest that
PAT could inhibit the AChE activity in CL4176 strains, and thereby recovering the
ACh processing, which is conducive to the alleviation of AD symptoms.
Table 1. The effect of PAT (µM) on AChE activity (Units L‒1) in CL4176 strains after
treatment at 25 °C for 48 h. Data represent mean ± S.D., normalized by protein
concentration.
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[PAT]
Control
25
50
100
AChE 272.59 ± 14.60
247.68 ± 11.75 166.40 ± 8.51 134.76 ± 5.95
2.10. Cognition and memory of APP/PS1 AD mice
AD is the most common type of neurodegenerative disease that correlates with
memory deficiency and cognitive dysfunction [54]. The in vivo therapeutic efficacy of
PAT was tested using APP/presenilin protein 1 (PS1) double-transgenic mice
(9-month-old, male) as an animal model of AD. The APP/PS1 model produces
elevated levels of human Aβ by expressing human APP and PS1 mutant from 5
months of age [55]. The memory and cognitive abilities were assessed by Morris
water maze test [56]. The learning ability of AD mice after intravenously
administration of PAT or saline for 3 months were evaluated. Age-matched WT mice
(9-month-old, male) were also subjected to behavioral tests as control group. The
escape latency of the PAT-treated group (34.25 ± 21.80 s, p < 0.1) is significantly
shorter than that of the saline-treated group (50.08 ± 17.95 s) on day 5 (Fig. 8A). In
the probe trial on day 6, the platform was removed out of the target quadrant II; the
PAT-treated mice spent 17.16 ± 4.38 s in the target quadrant, while the saline-treated
mice almost swim around the water pool and spent 11.77 ± 5.28 s in the target
quadrant (Fig. 8B, Table S3). For the PAT-treated mice, the total number of crossing
the hidden escape platform in the pool also increased. The results demonstrate that the
cognition and memory of AD mice was effectively improved by PAT.
Fig. 8. Effects of PAT (1 mg kg‒1, 15% DMSO) on double-transgenic (APP/PS1) AD
mice evaluated by the Morris water maze test. (A) Escape latency time recorded daily
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during training trials; data are presented as the mean ± S. D., n = 6 each group, *p <
0.1; (B) Swimming paths (red lines) of APP/PS1 mice (n = 6 each group) with or
without PAT treatment on day 6 (green circle represents the location of the platform).
3. Conclusions
Multifunctional small molecules could be potential drug candidates for the
treatment of multifactorial diseases like AD. Based on this belief, we designed and
synthesized a pyridine amine derivative (PAT) in the present study. Multifarious
assays demonstrate that PAT can inhibit the self- and metal-induced Aβ aggregation,
modify the conformation of Aβ, and reduce the toxicity of Aβ aggregates against
neuro-like cells. In vivo studies on transgenic C. elegans carrying human Aβ gene
show that PAT can down-regulate the mRNA level of human Aβ gene, reduce the
expression of Aβ42 species, alleviate the Aβ-induced paralysis, and inhibit the AChE
activity; it can also reduce the ROS level in the worms, thereby protecting them from
oxidative stress and mitochondrial oxidative damage. Gene screening analyses reveal
that PAT can significantly increase the mRNA level of hsp-16.2, hsp-60, and hsf-1
genes. Particularly, induction of heat shock protein hsp-16 expression represents a
protective response to the accumulation of abnormal proteins, such as promoting Aβ
sequestration, degradation, or refolding [24], which is important for mediating the
tolerance to Aβ toxicity in the nematode. Last but not least, PAT can improve the
cognition and memory of the APP/PS1 model mice, thus showing a promising
prospect for the treatment of AD.
4. Experimental Section
4.1. Materials and methods
Chemical reagents, including 3,5-dimethylphenol, aceticanhydride, pyridine,
N-bromosuccinimide,
benzoyl
peroxide,
triphenylphosphine,
N,N-diisopropylethylamine, bis(2-pyridylmethyl)amine, thioflavin T (ThT), CuCl2,
ZnCl2, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
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were
purchased
from
J&K
Scientific
(Beijing,
China).
and
Aβ40
Aβ42
(DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV)
(DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA) were obtained
from Nanjing Taiye Company. Biological reagents, such as agar, peptone, cholesterol,
yeast extract, tryptone, 5-fluoro-2ʹ-deoxy-β-uridine (FUdR), 2,7-dichlorofluorescein
diacetate (H2DCF-DA), 5-hydroxy-1,4-naphthalenedione (Juglone), the primary
antibody of β-actin, oligomycin, and AChE activity kit were purchased from Sigma.
The primary antibody 6E10 was purchased from Abcam (USA). HRP-conjugated goat
anti-mouse antibody was purchased from Biomart (China). Alexa Fluor 488 goat
anti-mouse was purchased from Molecular Probes (USA). Paraformaldehyde (4%)
and triton X-100 (1%, 125 mM Tris, pH 7.4) were purchased from Byotime (China).
Rhodamine-phalloidin was purchased from Cytoskeleton, Inc.. The primers used for
the qRT-PCR were purchased from Genscript (China). Trizol agent was purchased
from Invitrogen (USA). The iScriptTM cDNA synthesis kit was purchased from
Bio-Rad (USA). XFe24 extracellular flux assay kit was purchased from Seahorse
Bioscience (USA).
1H NMR spectra were acquired on a Bruker DRX-500 spectrometer at 298 K.
Electrospray ionization mass spectra (ESI-MS) were obtained using an LCQ
spectrometer (Finnigan). Fluorescence intensity was recorded using a multifunctional
microplate test system (Varioskan Flash, Thermo Fisher Scientific, USA). The CD
spectra were determined using a Jasco-810-150S spectropolarimeter (JASCO, Tokyo
Japan). A quartz cell with a 1.0 cm optical path was used. TEM images were taken by
using a JEOL JEM-2100 LaB6 (HR) transmission electron microscope (200 kV,
25000× magnification). C. elegans was observed by Stereo Microscope, Motic.
Fluorescence confocal imaging was carried out on a laser scanning confocal imaging
system (Olympus TH4-200) consisting of ZEISS Laser Scanning Microscope (LSM
710) and a 20 mW-output 488 nm argon ion laser. Fluorescence photographs were
recorded by fluorescent microscopy (Ningbo Sunny instruments Co. Ltd.). Western
blotting was carried on the Bio-Rad mini-PROTEAN tetra system and Bio-Rad
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Powerpack Universal. Images were captured using a Chemiscope 3400 mini (Clinx
science instrument co. Ltd). Quantitative real-time reverse transcription-polymerase
chain reaction (qRT-PCR) was performed on the CFX96 real-time PCR detection
system (Bio-Rad). Oxygen consumption rate was determined using the Seahorse
XFe24 instrument (Seahorse Bioscience). Morris water maze test was recorded by a
video camera-based Ethovision System (Nodulus, The Netherlands).
Data were expressed as means ± standard deviation (S. D.). Two-way Analysis of
Variance (ANOVA) was used to ascertain significant differences between the controls
and PAT-treated groups by the GraphPad Primer 7.00 software. Differences at the p ≤
0.05 level were considered as significant.
4.2. Synthesis
3,5-Bis(bromomethyl)phenyl acetate (2) was prepared according to the literature
method with some modifications [57]. Briefly, 3,5-dimethylphenol (5.00 g, 40.93
mmol) in aceticanhydride/pyridine (1:1, 16 mL) was stirred at room temperature for
48 h, and 3,5-dimethylphenyl acetate (1) was obtained after removal of the solvent.
Compound 1 (5.00 g, 30.45 mmol) was brominated with N-bromosuccinimide (NBS,
10.90 g, 61.24 mmol) and benzoyl peroxide (BPO, 0.28 g) in CCl4 (150 mL). The
exothermic reaction completed automatically after the initial heating. The mixture was
filtered, and the solvent was removed to give a crude product, which was purified by
SiO2 column chromatography (petroleum ether/ethyl acetate, 10:1, v/v), and white
1
solid was obtained as compound 2. H NMR (CDCl3, 500 MHz) δ/ppm: 2.32 (s, 3H,
-CH3), 4.45 (s, 4H, -CH2), 7.10 (d, 2H, Ph-H), 7.42–7.43 (d, 1H, Ph-H).
3-Acetoxy-5-(bromomethyl)benzyltriphenylphosphonium bromide (3) was
prepared by dissolving 2 (1.60 g, 5.00 mmol) and triphenylphosphine (1.31 g, 5.00
mmol) in CCl4 (15 mL) and stirring at room temperature overnight. The mixture was
filtered and white powder product was obtained. 1H NMR (CDCl3, 500 MHz) δ/ppm:
2.20 (s, 3H, -CH3), 4.22 (s, 2H, -CH2), 5.53–5.58 (d, 2H, -CH2), 6.85–7.00 (m, 3H,
Ph-H), 7.62–7.67 (m, 6H, Ph-H), 7.74–7.79 (m, 9H, Ph-H). ESI-MS (CH3CN,
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positive mode, m/z): [3–Br]+, found (calcd) 504.28 (504.38).
PAT was prepared as follows. N,N-Diisopropylethylamine (DIEA, 0.50g) and
bis(2-pyridylmethyl)amine (BPA, 0.90 g, 4.50 mmol) were added into 3 (2.63 g, 4.5
mmol, 10 mL CHCl3) successively. The mixture was stirred for 2 days and the
resulting DIEA salt was eliminated by filtration. The solvent in the filtrate was
evaporated. The resulting oily compound 4 was stirred with methanol/water (10
mL/10 mL) and NaOH (25 mL, 40%) for 12 h. The mixture was neutralized with HCl
(37%) and then extracted with CHCl3. The extraction was dried with anhydrous
Na2SO4, the solvent was removed, and the crude product was purified by SiO2 column
chromatography (CHCl3/CH3OH, 10:1, v/v, 1% NH3·H2O). PAT was obtained as a
1
brown powder. Yield: 60%. H NMR (CDCl3, 500 MHz) δ/ppm: 3.23 (s, 2H,-CH2-),
3.54 (s, 4H,-CH2-), 4.64–4.69 (d, 2H,-CH2-), 6.27 (s, 1H, Ph-H), 6.88 (s, 1H, Ph-H),
7.04 (s, 1H, Ph-H), 7.10–7.14 (m, 2H, py-H), 7.46–7.71 (m, 15H, Ph-H, 4H, py-H),
8.44–8.46 (d, 2H, py-H). 31P NMR (CDCl3, 202 MHz) δ/ppm: 22.51. ESI-MS
(CH3OH, positive mode, m/z): [PAT–Br]+, found (calcd) 580.58 (580.69).
4.3. Inhibition of Aβ40/42 aggregation
The stock solution of Aβ (200 µM) was prepared by Tris-HCl buffer (50 mM Tris,
150 mM NaCl, pH 7.4). The stock solution was dissolved with 10% NH3·H2O, diluted
with ddH2O, and adjusted to pH 7.4 by HCl (1%, v/v). The concentration of Aβ was
determined by Bradford assay.
Aβ40 (20 µM) was incubated with CuCl2 or ZnCl2 (20 µM) for 5 min at room
temperature; PAT (20 µM) was added and incubated at 37 °C for 24 h. Each sample
was transferred to a 96-well plate, and ThT (5 µM) was added. The fluorescence
intensity was recorded by a multifunctional microplate reader (λex = 450 nm, λem
=
485 nm). Data were expressed as mean ± standard deviations (S. D.) of at least three
independent experiments.
Aβ42 (20 µM) was mixed with different concentrations of PAT (0.1‒100 µM) in
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the presence or absence of Zn2+ (20 µM) and incubated at 37 °C for 24 h. Each sample
was transferred to a 96-well plate to a final volume of 200 µL containing ThT (5 µM).
Samples for testing Aβ42 fibril formation kinetics were prepared by adding Aβ42
solution (200 µL, 20 µM) in 96-well plates. PAT was added to the samples to obtain a
final concentration of 10, 20, 40 and 60 µM, respectively, and ThT (5 µM) was then
added. The fluorescence intensity of these samples was monitored at 0–720 min.
Aβ42 (40 µM) was incubated with ZnCl2 or CuCl2 (40 µM) for 5 min at room
temperature; PAT (80 µM) was added to the solution and incubated at 37 °C for 24 h.
CD spectra were determined using a Jasco-810-150S spectropolarimeter at 190–270
nm (bandwidth = 0.1 nm, response time =10 s).
TEM samples were prepared in the same way as in the ThT assay. An aliquot of
each solution (5 µL) was spotted on the 300-mesh carbon-coated copper grids for 2
min at room temperature and excess sample was removed. Each grid was stained with
uranyl acetate (1%, w/v, 5 µL) for 1 min. Uranyl acetate was blotted up and the grids
were dried for 20 min at room temperature. TEM images were recorded.
4.4. Cytotoxicity
The rat adrenal pheochromocytoma (PC12) cells were purchased from American
Type Culture Collection (ATCC). The cells were treated with nerve growth factor
(NGF, Sigma) to stop dividing and promote differentiating. Highly differentiated
PC12 cells were cultured over-night in RPMI-1640 medium supplement [10%
heat-inactivated fetal bovine serum (v/v), 2 mM glutamine, 100 U mL‒1 penicillin,
and 100 µg mL‒1 streptomycin] and a highly humidified atmosphere of 95% air with 5%
CO2 at 37 °C. Cell viability was measured by the MTT assay as we described
previously [58]. The amount of MTT formazan was determined using a
multifunctional microplate reader at 570 nm. The optical density (OD) was used to
calculate the percentage of cell viability relative to the untreated control values, that is,
(ODtest – ODblank)/(ODcontrol – ODblank) × 100%, and the mean of three replicates was
taken as the final result.
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4.5. Maintance of C. elegans
Transgenic model strains CL4176 and GMC101 were obtained from the
Caenorhabditis Genetics Center. The genotype of CL4176 is smg-1(cc546) I; dvIs27
[myo-3p::Aβ1-42::let-851 3’UTR + rol-6(su1006)] X. The genotype of GMC101 is
dvIs100 [unc-54p::Aβ1−42::unc-54 3’UTR + mtl-2p:GFP]. The strains were cultured
on the standard Nematode Growth Media (NGM) plates spotted with E. coli strain
OP50 grown in Luria Broth (LB) medium. NGM (12.0 g agar, 1.5 g peptone, 1.8 g
NaCl, 590 mL H2O) was autoclaved. CaCl2 (0.6 mL, 1 M), cholesterol (0.6 mL, 5 mg
mL‒1 in ethanol), MgSO4 (0.6 mL, 1 M) and KPO4 buffer (15.0 mL, 1 M; 17.8 g
KH2PO4, 54.2 g K2HPO4, 500 mL, pH 6.0) were added to NGM. An aliquot (5.0 mL)
of NGM was transferred into each petri dish (φ35 mm) and allowed to solidify
overnight. LB medium was prepared by dissolving NaCl (10.0 g), yeast extract (5.0 g)
and tryptone (10.0 g) in double distilled water (1.0 L). E. coli strain OP50 was grown
in LB medium by shaking overnight. Each plate containing solidified NGM was
spotted with 100 µL of OP50 and allowed to dry for 4 h; it was then cultured in 37 °C
overnight, cooled to room temperature and stored in 4 °C for later use.
4.6. Paralysis assay
Age-synchronous worm populations of CL4176 or GMC101 were started with a
synchronized parental generation, using a platinum worm picker to transfer 10 gravid
adults onto several NGM plates (φ35 mm) spread with OP50 for laying eggs at 15 °C.
The gravid adults were removed and the progenies were allowed to reach the third
larval (L3); 120 worms were transferred to new plates spotted with FUdR (50 µL, 0.5
mM). NGM plates containing PAT (50, 100 and 200 µM) were prepared. The plates
were incubated at 25 °C, a temperature at which Aβ42 production increases, and noted
as day 0. Paralyzed worms were counted at 20 h after the initiation of temperature
upshift. Nematodes that did not move or only moved the head, or failed to complete
full body movement under a gentle touch with the worm picker were regarded as in
paralysis. The movement of worms was observed by Stereo Microscope at the same
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time. The counting continued for several hours until all worms on each plate were
paralyzed. To reduce any misjudgment, the number of paralyzed worms was
double-checked by two investigators independently. The results were reported as the
mean value with S. D. based on the data from three independent experiments.
4.7. Fluorescence imaging and ThT stainning
Transgenic C. elegans GMC101 strains (L3) were transferred to new plates
containing PAT (50, 100 µM) and then cultured at 25 °C for 2 days; 20 of them were
picked into 2% agarose pads under a glass slide. Fluorescence confocal imaging (λex =
488 nm, λem = 500–530 nm) was carried out directly.
CL4176 strains (L3) were transferred to new plates containing PAT (50, 100 and
200 µM) and then cultured at 25 °C for 2 days. Imaging was performed in triplicate
with 15–20 worms per group. Worms were stained with ThT (10 µM) for 5 min and
then picked into fresh NGM plate without E. coli OP50. The samples of CL4176 were
picked and mounted on 2% agarose pads under a glass slide after incubation for 2 h.
Fluorescence photographs (λex = 488 nm, λem = 500–530 nm) were taken by
fluorescent microscopy (Ningbo Sunny instruments Co. Ltd.), with 1× objective and
10× magnification.
4.8. Western blotting and immunofluorescence
CL4176 strains were synchronized and their eggs were allowed to hatch and
develop to the L3 stage on NGM plates containing PAT (50 µM) at 15 °C. The
temperature was raised to 25 °C and maintained for 2 days. The worms were collected
from the plates with M9 buffer and washed twice to eliminate bacteria. The final
worm pellet was resuspended in phosphate buffer (200 µL, 0.1 M), sonicated on ice
for 10 s 5 times, and centrifuged at 10000 rpm for 5 min to remove insoluble debris.
Total protein in the supernatant was quantified using Bradford reagent (BioRad). The
protein was boiled with loading buffer at 95 °C for 5 min before being loaded into the
gel. Equal amount of protein (16 µg) was added into the lane of the 12%
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Tris-Glycine-SDS-PAGE gel. Protein marker (10‒180 kDa, Thermo Scientific) was
used as indicator of molecular weight. Samples were run at 80 V for 30 min on
stacking gel, and 120 V for 60 min on separating gel. The gel was transferred to
PVDF membrane (0.22 µm, Immobilon P, Millipore) using 20% methanol transfer
buffer at 200 mA for 2 h. Blots were blocked in PBS-Tween + 5% milk for 1 h. Aβ
species were detected with the primary antibody 6E10 (1:1000, Abcam), using β-actin
(1:5000, Sigma) as a control. HRP-conjugated goat anti-mouse antibody (1:8000,
Biomart) was used as the second antibody. Images were captured using a Chemiscope
3400 mini.
CL4176 strains (L3) were cultured on NGM plates containing PAT (50,100 µM)
at 25 °C for 2 days, collected, rinsed and fixed in 4% paraformaldehyde overnight.
After fixation, worms were rinsed twice with PBS and permeabilized by 1% Triton
X-100 (125 mM Tris, pH 7.4). Aβ species was detected with the primary monoclonal
antibody 6E10, and maintained by the Alexa Fluor 488 goat anti-mouse (Molecular
Probes) secondary antibody. Samples were also stained with rhodamine-phalloidin
(1:4000) for the visualization of actin filaments in muscle cells. The animals were
mounted on 2% agarose pads, covered with a cover slip and sealed. The samples were
photographed by a ZEISS Laser Scanning Microscope (LSM 710) at λex = 488 nm,
λem = 500–530 nm (Alexa Fluor 488); or at λex = 543 nm, λem = 565–605 nm
(rhodamine).
4.9. RNA Extraction and qRT-PCR
RNA was prepared using Trizol reagent (Invitrogen) and stored at −80 °C.
Complementary DNA was prepared by using iScriptTM cDNA synthesis kit (Bio-Rad).
The qRT-PCR was performed using SsoFast EvaGreen Supermix (Bio-Rad) and
according to the protocol suggested by the manufacturer: 30 s at 95 °C, followed by
40 cycles of 5 s at 95 °C, and 5 s at 60 °C. Ama-1 was used as an internal control to
normalize the expression level of target transcripts. Relative fold changes for
transcripts were calculated using the comparative CT (2−∆∆CT) method. Each qRT-PCR
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experiment was repeated three times by independent RNA/cDNA preparations. Aβ
transgene (amy-1), stress induced transcription factor (skn-1), superoxide dismutase 3
(sod-3), small heat shock protein (hsp-16.2 and hsp-60), and heat shock factor (hsf-1)
were subjected to qRT-PCR, and the transcript quantity was normalized using the
actin genes (ama-1) [59]. Samples were run in triplicate (n = 3), and the primers used
for the qRT-PCR were shown in Table S2.
4.10. Measurement of ROS in C. elegans
Intracellular ROS in CL4176 strains was measured using H2DCF-DA. Freshly
laid CL4176 eggs (100 eggs per plate) were transferred to NGM plates containing
PAT (0, 25, 50, 100 µM) and incubated at 15 °C. The temperature was raised to 25 °C
for 48 h. The worms were collected by M9 buffer, washed twice with PBS to remove
E. coli OP50, transferred to a 96-well plate (Costar) with 200 mL of PBS containing
Tween 20 (0.01%), and H2DCF-DA (100 µM) was added. In the parallel experiment
for determining the oxidative stress tolerance, juglone (300 µM) was added, and the
fluorescence intensity (λex = 485 nm, λem = 530 nm) was quantified by a microplate
reader after 6 h. Data were represented as mean ± S.D., n = 3.
4.11. Oxygen consumption rate (OCR)
CL4176 strains were synchronized, treated with or without PAT (100 µM) at
25 °C for 48 h. The worms were picked to new NGM plates with no bacteria, washed
with M9 and put into the XF 24-well plates 50 worms per well. Experiments were
repeated three times with 5 wells under each condition. Before measurement on the
Seahorse XFe24 instrument, the culture medium was changed with XFe24 extracellular
flux assay medium. OCR was read using the following settings: 5 cycles of mixing
(30 s), waiting (30 s) and measuring (3 min). Oligomycin (1 µM) was added after the
detection of basal respiration. Each experiment was repeated three times.
4.12. Activity of acetylcholinesterase
Fresh lysates from the CL4176 worms after treatment with PAT (0, 25, 50, 100
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µM) at 25 °C for 48 h were prepared by sonication in phosphate buffer (0.1 M, pH
7.4), followed by centrifugation at 13000 rpm for 5 min, and collection of the
supernatants. AChE activity kit (Sigma) was used to quantify the thiocholine
produced in the hydrolysis of acetylthiocholine by AChE in worm extracts. The
absorption of DTNB adduct was used to measure the amount of thiocholine, which is
proportional to the AChE activity. The kit provides a colorimetric one-step assay to
detect 10–600 units/L AChE. The signal was read by an absorbance microplate reader
at 412 nm.
4.13. Animal studies
Double transgenic AD mice, B6C3-Tg (APPswe, PSEN1dE9)85Dbo/J (male, 3
months old, 30−45 g, n = 12), were purchased from the Guangdong Medical
Laboratory Animal Center. The APP/PS1 mice were randomly allocated into two
groups (n = 6 for each group) when they were 6-month old, and intravenously
administered with PAT (1 mg kg−1, 15% DMSO) or saline containing 15% DMSO
every three days, lasted for 3 months. Wildtype C57BL/6 mice (male, 8 months old,
20−25 g, n = 6) were purchased from the Model Animal Research Center of Nanjing
University. The mice were fed in individual cages at 23 °C, with ad libitum access to
sterile food and water. All experimental procedures on animals were approved by the
Model Animal Research Center of Nanjing University.
4.14. Morris water maze test
The learning and cognitive abilities of AD mice (9-month old) were assessed by
the Morris water maze test following the procedures reported in the literature [56].
The test consisted of 4 platform trials per day for consecutive 5 days, and followed by
a probe trial on the 6th day. In platform trials, the distance of path and escape latency
were measured. After training, the mice were tested to search for the platform in the
Morris water maze apparatus, in which they were permitted to swim freely for 60 s. In
probe trials, the swimming path were recorded by a video camera-based Ethovision
System (Nodulus, the Netherlands). The daily test data were analyzed statistically.
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The data for escape latency, the traveled distances, and the number of times for
crossing platform region were recorded in Table S3.
Acknowledgements
This work was supported by the National Natural Science Foundation of China
(Grants 31700714, 21877059, 81671390), the National Postdoctoral Program for
Innovative Talents (BX201600069), the China Postdoctoral Science Foundation
(2017M611777), and the State Key Laboratory of Pharmaceutical Biotechnology of
Nanjing University (grant number: 02ZZYJ-201302).
Appendix A. Supplementary data
Supplementary data to this article can be found XXX
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