REVIEW
DOI: 10.1002/cjoc.201400469
Tau Protein Associated Inhibitors in Alzheimer Disease†
Qian-Qian Li,a Ting-Ting Chu,a Yong-Xiang Chen,*,a and Yan-Mei Li*,a,b
a
Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department
of Chemistry, Tsinghua University, Beijing 100084, China
b
Beijing Institute for Brain Disorders, Beijing 100069, China
Neurofibrillary tangles composed of tau protein and senile plaque accumulated by amyloid-β (Aβ) are two
hallmarks in Alzheimer disease (AD). In the patients with AD, tau is abnormally hyperphosphorylated, mutated or
misfolding, which endows tau with stronger tendency to aggregate. Tau protein has the potency to mediate neuron
toxicity of Aβ. The reduction of tau level ameliorates degeneration of neuron axon. Therefore, many researches are
exploring new methods to regulate tau level. This review will mainly focus on small molecules that can directly inhibit tau fibrillation or control tau accumulation by regulating tau phosphorylation process.
Keywords tau protein, post-translational modification, Alzheimer disease, aggregation, inhibitors
Introduction
Scheme 1
Alzheimer disease (AD) is one of the most widely
distributed neurodegenerative diseases, and most of patients with AD are diagnosed with loss of synapses and
disability of memory and learning. What’s more,
microtubule associated protein tau (MAPT) has been
found to aggregate into paired helical filaments (PHFs)
and further accumulate into neurofibrillary tangles
(NFTs) inside the neuron (Scheme 1). Actually, in normal conditions, tau is soluble in cytosol with flexible
conformation.[1] Meanwhile, tau can undergo posttranslational modifications (PTMs), including phosphorylation,[2] glycosylation[3] and acetylation. The balance of different types of PTMs is determinant to the
physiological function of tau protein.[4,5] However, Tau
has been identified to be hyperphosphorylated at 5－9
amino acid sites in AD patients, and hyperphosphorylation of tau can lead to its aggregation into NFTs.[6]
NFTs has been proved to be related to AD, Picks disease, frontotemporal dementias and other tauopathies.[7]
Even though the mechanical link between tau and AD is
still indistinct, transgenic mouse or human tau protein in
mice mediates neurodegeneration induced by Aβ in the
central nervous system.[8] Further more, the reduction of
endogenous tau level in mice model that overexpresses
human amyloid precursor protein ameliorates behavioral
deficits and neuronal dysfunction.[9] Therefore, tau protein may be a promising therapeutic target for AD. According to the widely distribution of NFTs in tauopathies, tau aggregation inhibitors may be an effect
way.
Up to now, several potential treatments targeting tau
NFTs and senile plaque in neurons[10]
protein have been explored, for example, inhibiting tau
aggregation, promoting tau degradation, stabilizing the
interaction of tau protein with microtubule, decreasing
tau phosphorylation and further reducing the ability of
self-assembly and so on.
Structure and Functions of Tau Protein
Because of alternative mRNA splicing of exon 2, 3
and 10, there are six isoforms of tau in normal human
brains, and different isoforms contain 3 or 4 microtubule-binding repeats (3R or 4R) at the C-terminus. 4R
tau has stronger potency to aggregate. The ratio of
4R/3R in AD patients increases so that tau protein is
* E-mail: liym@mail.tsinghua.edu.cn, chen-yx@mail.tsinghua.edu.cn; Tel.: 0086-010-62794258
Received July 10, 2014; accepted September 26, 2014; published online October 8, 2014.
†
Dedicated to Professor Chengye Yuan and Professor Li-Xin Dai on the occasion of their 90th birthdays.
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© 2014 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2014, 32, 964—968
Tau Protein Associated Inhibitors in Alzheimer Disease
more inclined to accumulate.[1] Basically, tau protein is
comprised of three functional domains, N-terminus,
middle region and C-terminus. The N-terminus containing numerous acidic amino acids, such as aspartate and
glutamate, prefers to detach from microtubule and
stretch into the cytoplasm, which may facilitate the association of tau and other proteins. The middle region
has several target sites of proline-directed microtubule
associated protein kinases, and thus this region plays an
important role in tau phosphorylation. The C-terminus
with lots of basic residues can interact with microtubule
tightly and directly.[10,11] These microtubule binding
regions will stabilize microtubule, and it is significant in
keeping cell structure dynamic. In AD, hyperphosphorylated tau separates from microtubule and makes
microtubule network unstable, which may be one of
pathogenic mechanism for tauopathies.
Besides, tau protein impairs axon formation and
microtubule-dependent vesicle transport. In tau overexpressed neurons, axons are obviously degenerated, at the
same time, mitochondria and other membrane-coated
organelles also display an aberrant distribution and locate around the microtubule organizing center
(MTOC).[12] Maybe, it is because kinesin has more inclination to divorce from microtubule than dynein when
tau protein associates with microtubule.[13] For this reason, kinesin dependent plus-end transport can be impaired more severely than dynein dependent minus-end
transport. Through this way, tau protein can regulate the
direction of axon transport and determine the location of
organelles, which can further affect metabolic process of
neurons (Scheme 2).[14]
Scheme 2 Tau protein affects axon transport. (a) mitochondrial
misallocation around MTOC in AD model cells; (b) function of
tau in vesicle transport[12]
In physiological condition, tau protein can be
post-translationally modified, and different PTMs have
different influence on functions of tau. For instance,
hyperphosphorylated tau will easily detach from microtubule and accumulate into NFTs. O-GlcNAcylation of
tau can regulate tau phosphorylation in a manner of site
competition.[15] Moreover, acetylation on lysine residues
of tau by histone acetyltransferase or lysine acetyltransChin. J. Chem. 2014, 32, 964—968
ferase will occupy ubiquitination sites, and thus block
tau degradation through ubiquitin proteasome system
(UPS).[16] In summary, the homeostasis of tau modifications can regulate its functions.
Tau Protein Associated Inhibitors
Tau protein is soluble and unstructured in well-organized neurons. However, mutations, abnormal truncation or PTMs will change its conformation to form
β-sheet structure and finally insoluble fibrils. Beyond
that, the hydrophobic interaction between the
C-terminuses of tau proteins has been proved to be the
driving force of tau assembly.[17,18] In this review, we
will introduce three strategies for tau protein associated
treatments: directly inhibiting tau aggregation; inhibiting tau kinase or activating tau phosphatase; raising tau
glycosylation.
Aggregation inhibitors
Effective tau aggregation inhibitors have been found
through high throughput screening of small molecules
library with diverse structures in vitro. These inhibitors
could bind tau directly. Using Thioflavin S (ThS) fluorescence assay, tryptophan fluorescence assay and electron microscopy, Mandelkow and his cooperators[19]
found that several compounds can depolymerize tau
aggregation in vitro, including thioxothiazolidinones
(rhodanine), phenylthiazolylhydrazides, N-phenylamines, anthraquinones and phenylthiazolylhydrazides.
Rhodanine with 0.8 μmol/L IC50 has no obvious side
effect and becomes one of the most promising inhibitors
of tau accumulation. The structure of rhodanine is in
Figure 1a and the basic bone of rhodanine derivatives is
in Figure 1b. Rhodanine’s derivatives that substitute R1
and R2 with oxygen or nitrogen atom have less power to
prevent tau aggregation, which indicates that thioxo
group in rhodanine is indispensable.
Besides, phenylthiazolylhydrazides also have inhibitory ability of tau aggregation and their dissociation
constant (Kd) is about 62 μmol/L. The structural similarity of rhodanines and thiazolylhydrazides illustrates the
common structure and activity relationship (Figures 1c
and 1d): both of these two types of molecules have hydrogen bond acceptors and hydrophobic domains. These
two functional groups may contribute to their association with the similar site of tau protein.[19] Replacement
of R3 in phenylthiazolylhydrazides with a hydrogenbonding domain can improve the inhibitory potency,
such as BSc3094 (Figure 1e). Even though phenylthiazolylhydrazides are not so powerful in vitro as compared with rhodanine, these chemical compounds can
keep consistent activity in cells. It is perhaps because of
their excellent membrane penetrating ability.[21] Furthermore, the same structural analysis, namely two parts
of inhibitory domains: hydrogen bond acceptor and hydrophobic interaction respectively, also works on
N-phenylamines. For example, in B4D5 (Figure 1f), the
© 2014 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
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REVIEW
a
Li et al.
HO
N
S
O
O
Cl
O
e
c
O
O
R1
N
R
2
S
N
H
R1
N
N
R3
S
H
Phosphorylation inhibitors
H
N
N
NO2
S
BSc3094
NO2
f
H
N
R4
O
O
S
rhodanine
O
R2
N
N
N
H
O2N
H
N
N
O
2
OH
R
thiazolylhydrazides
d
R1
OO
R
1
N
H
g
N N
S
S
h
O
B4D5
O
H
N
O
values. At the same time, both of these two types of
molecules had no interference on tau binding with
microtubule and had ability to disturb aggregation of
Aβ.
R1
b
R3
S
R32
R
OH
O
OH
O
OH
Scheme 3
level
Small molecules for decreasing tau phosphorylation
HO
S2
R
S
O
S
PHF016
N
N
OH
HO
N744
Figure 1 Structures of several tau inhibitors. (a) Stucture of a
rhodanine-based inhibitor; (b) main bone structure of rhodanine’s
derivatives; (c) and (d) the structural comparison of rhodanines
and thiazolylhydrazides; (e) BSc 3094; (f) B4D5; (g) PHF016; (h)
N744.[19][20]
nitro group or carboxylic acid can form hydrogen bond
and aromatic cycle can bind with self-assembly region
of tau protein.[19] But N-phenylamines show much lower
inhibitory activity in vitro and higher cell toxicity.
Therefore, this type of compounds is rarely used.
Molecules derived from anthraquinones family, for
instance, PHF 016 (Figure 1g), can prevent tau assembly
with IC50 of 1－5 μmol/L and disassemble existed PHFs
at DC50 of 2－4 μmol/L in vitro. Anthraquinones do not
influence the stabilization of microtubule mediated by
tau protein but can ameliorate tau aggregation and concomitant cytotoxicity in cells.[20]
Besides, Necula et al.[21] found that benzothiazole-based tau inhibitors displayed nanomole value of
IC50 in vitro. In the case of N744 (Figure 1h),[20] it can
be speculated that cationic charge may promote the interaction of N744 with acidic amino acids in tau protein.
Actually, these molecules have been utilized for a long
time, for example, Thioflavin T and Thioflavin S are
widely used as probes to monitor β-sheet formation in
protein aggregation progress. But at high concentration,
these molecules tend to self-assemble and impair their
inhibitory potency.[19]
In addition, Hasegawa et al.[22] discovered that
polyphenols and porphyrins also displayed potency to
prevent tau protein accumulation with micromolar IC50
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Hyperphosphorylation of tau protein is a notable
characteristic of tauopathies.[23] Hyperphosphoryled tau
has a stronger tendency to fibrillate.[6] Therefore, it is
reasonable that reduction of phosphorylated tau by inhibiting the activity of tau kinases or activating phosphatases can at least partially rescue neuron impairment
caused by overexpressed tau protein (Scheme 3).[24]
www.cjc.wiley-vch.de
CDK5, GSK3β, MAPK and ERK2 have been reported to be main kinases responsible for tau phosphorylation. Recently, Meijer, L. found that bis-indole
indirubin, a traditional Chinese medicine could work as
an inhibitor of CDKs and GSK3β (IC50: 5－50 nmol/L)
in vitro and in vivo. Indirubin was associated with ATP
binding pocket of GSK3β and CDKs and blocked their
phosphorylated activity.[25] In addition, Lee’s work has
manifested that incubation of NT2N cells with insulin or
insulin-like growth factor-1 (IGF-1) for 5 min, could
lead to a significant reduction of phosphorylated tau
level. And further study implied that PI(3)K-PKB pathway was involved in insulin or IGF-1 mediated inhibition of GSK3β activity.[26] Duff, K. revealed that treatment of tau overexpressed transgenic mice with lithium
chloride, a GSK3β inhibitor, could reduce phosphorylated tau, decrease tau aggregation and alleviate neuron
degeneration.[27]
Moreover, the activity of tau phosphatase decreases
by about 30% in AD patients as compared with controls
at similar age.[24] Inhibiting phosphatase 2A selectively
can contribute to hyperphosphorylation and accumulation of tau.[28] It indicates that hyperphosphorylation of
tau may be caused by deficiency of tau phosphatase.
Therefore, molecules that can enhance the activity of
this phosphatase may alleviate neuron damage induced
by tau hyperphosphorylation. For example, Schweiger et
al.[29] declared that antidiabetic drug metformin could
upregulate PP2A activity and finally reduce tau phosphorylation in wild type and tau overexpressed marines.
In fact, metformin could prevent the interaction of the
catalytic subunit of PP2A with MID1-alpha 4 protein
complex that could adjust the degradation of PP2A
catalytic domain. In addition, Iqbal, K found that Memantine had capacity to inhibit the activity of I-2
(PP2A), an inhibitor of PP2A, in vitro, and this mole-
© 2014 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2014, 32, 964—968
Tau Protein Associated Inhibitors in Alzheimer Disease
cule can activate PP2A and then rescue dementia.[30]
These results lay a foundation on searching new chemicals that can increase the activity of phosphatase in the
future.
Increasing Glycosylation
It has been illustrated that O-GlcNAcylation of tau
can reversely regulate tau phosphorylation. And reduced
glucose uptake in AD patients can decrease tau
O-GlcNAcylation, as a result, raise the level of tau
phosphorylation in an animal model.[31] Hence, it seems
feasible that using mechanism-inspired O-GlcNAcase
inhibitor may increase the level of O-GlcNAcylation
and prevent hyperphosphorylation of tau. Actually, Vocadlo found a molecule, Thiamet-G, which displayed
such function of increasing tau O-GlcNAcylation
(Scheme 4).
Scheme 4
Thiamet-G inhibiting the activity of O-GlcNAcase[32]
Thiamet-G could inhibit human O-GlcNAcase with a
Ki value of 21 nmol/L and reduce phosphorylation on
tau pathological sites including Thr231 and Ser396 in
rat cortex and hippocampus.[32] In addition, Vocadlo’s
group[33] successfully used the recombinant protein
O-GlcNAcylated tau to map a new possible
O-GlcNAcylation site at Ser 409, 412 or 413 in vitro, It
is the third O-GlcNAcylation site except two other sites
Ser 400 and Thr 123 that have been already determined
by other groups. With the further study of basic mechanism, this original strategy gives us a new hope in
treatment of tauopathies.
Conclusions and Outlook
Reduction of tau aggregation or phosphorylated tau
level can significantly alleviate neuron dysfunction and
efforts in discovering such active molecules have made
progress. However, these existed molecules have their
own limitation in cell toxicity, membrane permeability,
poor solubility or specificity. Currently, only several
inhibitors of tau kinases and microtubule-stabilizing
compounds display expected effect in tau transgenic
mouse models. Moreover, tau-targeted drugs entering
into human clinical testing are just NAP, methylene blue
and LiCl.[19] Therefore, developing new tau inhibitors is
still in urgent need for treating tauopathies.
Chin. J. Chem. 2014, 32, 964—968
Acknowledgement
This work was supported by the Major State Basic
Research Development Program of China (Nos.
2013CB910700, 2012CB821600), the National Natural
Science Foundation of China (Nos. 21261130090,
91313301, 21102082 and 21472109) and the Research
Project of Chinese Ministry of Education (No.
113005A).
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Chin. J. Chem. 2014, 32, 964—968