A highly sensitive fluorescent probe that quantifies transthyretin in human plasma as an early diagnostic tool of Alzheimer's disease

Article abstract of DOI:10.1039/c9cc04172a

The development of sensitive and reliable fluorescent probes for the early diagnosis of Alzheimer's disease (AD) is highly challenging and plays an important role in achieving effective treatments. Herein, we designed and synthesized an indole-based fluorophore for TTR in human plasma, an important hallmark of AD pathogenesis. This robust and simple fluorescent method allows quantification of TTR in the complex biological matrix.

Full text of DOI:10.1039/c9cc04172a

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DOI: 10.1039/C9CC04172A.
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A highly sensitive fluorescent probe that quantifies transthyretin
in human plasma as an early diagnostic tool of Alzheimer’s
disease
Received 00th January 20xx,
Accepted 00th January 20xx
Hye Rim Lim,a,d Seo Yun Kim,a,d Eun Hee Jeon,a Yun Lan Kim,a Yu Mi Shin,a Tae-Sung Koo,a Sung
DOI: 10.1039/x0xx00000x
Jean Park,c Ki Bum Hong, b,* Sungwook Choi a,*
The development of sensitive and reliable fluorescent probes and cerebrospinal fluid (CSF). TTR is independently synthesized
for the early diagnosis of Alzheimer’s disease (AD) is highly in the liver and choroid plexus, after which is secreted into the
challenging and plays an important role in achieving effective blood (3-7 µM) and CSF (0.1-0.4 µ
M), respectively.4 While CSF
treatments. Herein, we designed and synthesized an indole-based TTR is the primary carrier of thyroid hormone thyroxine (T4),
fluorophore for TTR in human plasma, an important hallmark of AD serum TTR transports mainly the holo-retinol binding protein
pathogenesis. This robust and simple fluorescent method allows (RBP), with more than 99% of its T4 binding sites being
quantification of TTR in the complex biological matrix.
unoccupied.4a Interestingly, although TTR is known to be one of
the human amyloidogenic proteins representative of
pathological conditions, TTR has also been implicated in the
neuroprotection of AD. Evidence for the latter emerged from
Alzheimer’s disease (AD) is an age-associated
neurodegenerative disorder and the most common form of
dementia, affecting more than 50 million people worldwide. AD
is characterized by progressive loss of memory and cognitive
function, eventually leading to death. The major
neuropathological hallmarks of the disease are the progressive
accumulation of extracellular senile plaques, consisting of
the following results: (1) TTR is one of the major A
proteins in human CSF, suppressing A
aggregation5 and is
involved in brain A
efflux6 and peripheral clearance through
β-binding
β
β
proteolytic functions;7 (2) overexpression of WT-TTR in an
APP23 transgenic mouse model showed improved cognitive
functions;8 (3) TTR levels are reduced both in the CSF and the
blood of AD patients, compared with age-matched controls.9
Therefore, investigations on diagnostic tool and therapeutic
drug developments using TTR's unique biological role are
actively underway.
amyloid
neurofibrillary tangles constituted by hyperphosphorylated
forms of tau protein.1 A
peptides are produced by sequential
proteolytic cleavage of the amyloid precursor protein (APP) by
– and
–secretases.2 Under abnormal conditions, this
eventually leads to the accumulation of A
oligomers, and fibrillary aggregates,
β peptide (Aβ) aggregates and intraneuronal
β
β
γ
β
monomers,
triggering
Various methods have been employed to determine TTR
levels in the CSF and blood, including mostly immunoassays and
2D gel electrophoresis−
mass spectrometry.9a-c, 10 Most methods
neurodegeneration. The underlying mechanisms for AD remain
elusive but substantial genetic, pathological and biochemical
evidence suggest that the ultimate therapy would involve
suffer from a lack of consistency and reliability due to the
difficulty of standardizing detection methods. However,
fluorescence methods present as powerful tools by virtue of
their excellent sensitivity, simplicity, rapid response, and cost-
effective instrumentation.11 More importantly, TTR biomarkers
that can be detected in the plasma have greater diagnostic
value and are easier to access than CSF-based biomarkers. In
this study, we designed and synthesized indole-based
fluorescent probes for early detection of AD through
measurements of blood concentrations of TTR because indole
possess desirable photophysical properties and high sensitivity
to its environment.12
lowering A
between A
β
β
peptide levels through regulating the balance
production and clearance.3
Transthyretin (TTR) is a homotetrameric protein composed
of four 15 kDa, -sheet rich subunits and circulates in the blood
β
a.Graduate School of New Drug Discovery and Development, Chungnam National
University, Daejon, 305-764, Republic of Korea. E-mail: swchoi2010@cnu.ac.kr;
Fax: +82 42-821-8927; Tel: +82 42-821-8625
b.New Drug Development Center (NDDC), Daegu-Gyeongbuk Medical Innovation
Foundation (DGMIF), 80 Cheombok-ro, Dong-gu, Daegu 701-310, Republic of
Korea. E-mail: kbhong@dgmif.re.kr; Fax (+82)-53-790-5219; Tel: +82 53-790-5272
c. College of Pharmacy, Gachon University, 534-2 Yeonsu 3-dong, Yeonsu-gu,
Incheon 406-799, Republic of Korea
Indole-based compounds 1-10 were synthesized following
d.These authors contributed equally to this work
the synthetic route depicted in Scheme 1. Indole derivatives
were obtained from direct nitration and reduction of
A
†
Electronic Supplementary Information (ESI) available: General synthetic
procedures, spectral data and other supplementary information. See DOI:
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µM) in PBS showed 920-, 210- and 111-foldViienwcArretiaclseeOsnliinne
(2
NHNH2
R
fluorescence intensity, compared to eacDhOcI:o1m0.1p0o3u9/nCd9CaClo0n41e72inA
PBS, respectively. A blue shift in emission maximum was also
observed. The quantum yields were also measured using
coumarin 503 and quinine sulfate as the reference compounds.
i
N
R
A
H
O
Probes 2, 5, and 10 display significant values (0.178, 0.252, and
ii
R
OH
0.379) in the quantum yield relative to other probes. A large
stokes shift is a beneficial photophysical property for imaging
and sensing applications because these probes can show a low
possibility of self-quenching.13 From this perspective, almost all
designed indole probes in this study may be promising TTR
sensing probes as they exhibit large stokes shifts (∆λ = 100 ~
120 nm), whereas typical fluorophore dyes such as fluorescein
dyes, rhodamine, and BODIPY all have small stokes shift (∆λ ≤
70 nm).14 Therefore, based on their relative fluorescence
intensity, stokes shift, and quantum yield, probe 10 was
comprehensively selected for further examination and
assessment as a TTR sensing probe.
In order to confirm that the probe selectively binds to the
two T4 binding sites of WT-TTR, probe 10 was incubated with a
double mutant TTR (F87M, L110M) that adopts a monomeric
structure (M-TTR).15 and measured for its fluorescence
spectrum (Fig. 1). The fluorescence intensity of probe 10
changed from 4 to 476, with a 111-fold increase in the presence
N
R = COOH
R = CONH
R = COOMe
OH
1
3
5
2
4
R = OH
R
2
B
R = NH
+
2
N
O
R
A
H
ii, iii
N
N
NH
N
N
7
R = COOMe
R = OMe
R = CONH
R = OH
SEM
6
2
8
10
9
C
R = NH
2
Scheme 1. Synthesis of the probes 1-10. (i) 3-methyl-2-butanone, AcOH,
118 oC; (ii) HCl, EtOH, 80 oC; (iii) 4 M HCl in dioxane, 60 oC
unsubstituted indole or the reaction of substituted
phenylhydrazine with 3-methyl-2-butanone. Compounds 1-5
were synthesized by reacting the HCl salt of substituted indoles
A
with compounds
B
. Compounds 6-10 were synthesized by
with compounds C,
of 2 µM WT-TTR, whereas the same concentration of M-TTR did
reacting the HCl salt of substituted indole
A
not induce any significant changes, similarly to the results with
probe 10 alone in PBS. These observations indicated that probe
10 detects only the tetramerically folded TTR. Furthermore, Job
plot experiments were carried out to determine the binding
stoichiometry of the WT-TTR–probe 10 complexes (Fig. 1); the
results suggest that the formation of the TTR–probe 10 complex
followed a 1:1.42 stoichiometry.
followed by the deprotection of their SEM group using 4 M HCl
in dioxane.
The absorption and fluorescence properties of
and in the presence of WT-TTR in PBS were assessed as shown
in Table 1, Figure 1 and Figure S1 (see ESI). Probes and 10
1-10 in PBS
2, 4
exhibited a strong increase in fluorescence intensity (turn-on
response) upon binding to WT-TTR in PBS relative to other
The medium effect in different organic solvents was then
examined to gain a better insight into the environmental
sensitivity of probe 10’s fluorescence (Fig. 2a). While slight
fluorescence changes in aprotic solvents (toluene, THF, ACN,
DCM) were observed, a significant bathochromic shift of its
emission spectrum was observed in protic solvents (EtOH and
water). The environmental sensitivity of probe 10 was observed
in which the fluorescence intensity of probe 10 in a relatively
less polar solvent (acetonitrile) was gradually decreased by an
probes. Incubation of probes 2, 4, and 10 (4 µM) with WT-TTR
Table 1 Spectroscopic properties of probes 1-10 (4 µM) upon binding
to WT-TTR (2
µM) in PBS
Stokes
shift
(nm)
λex
(nm)
λem
(nm)
Folda
RFI b
Φf
Comp
1
2
3
4
5
6
7
8
336
460
459
389
388
329
324
334
480
566
558
513
513
450
452
457
5
920
210
11
13
3
144
106
99
0.04
0.35
0.21
0.14
0.62
0.01
0.14
0.03
0.178c
0.024c
0.057c
0.267c
124
125
121
128
123
2
4
9
382
357
468
476
26
86
0.23
1.00
0.065d
0.379d
10
111
119
a Fold increase of fluorescence intensity after incubation with WT-TTR
in PBS, compared to probe alone in PBS.
Relative fluorescence intensity ratio was defined as the ratio of the
b
Fig. 1 Fluorescence emission spectra of probe 10 (4
incubation with WT-TTR (2 M, red trace) and M-TTR (8
purple trace) in PBS (10 mM sodium phosphate, 100 mM KCl, 1 mM
EDTA, pH 7.0). The inset shows a Job plot analysis of TTR–probe 10
complexes recorded by the mixture of TTR and probe 10 at different
µ
M) after
µ
µM, solid
fluorescence intensity of probes over that of 10
.
c
Coumarin 503 was used as reference for quantum yield
measurements.
d Quinine sulfate used as reference for quantum yield measurements.
ratios, at a constant total concentration of 4.5 µM.
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Fig. 3 The fluorescence intensity of probe 10 in the presence of various
biological species, including glutathione (GSH, 10 M), homocystein
(Hcy, 10 M), -globulins (10 mg/L), transferrin (10 mg/L), -1 acid
glycoprotein (AGP, 10 mg/L), ribonuclease A (Ribo, 10 mg/L), trypsin (10
mg/L), human serum albumin (HSA, 2000 mg/L), and TTR (1 M).
(b)
µ
µ
γ
α
µ
changes in the fluorescence intensity of the WT-TTR−10
complexes and the responses of the probe toward the pH
changes from 5 to 8 were inconsequential. All of these findings
suggest that probe 10 could selectively bind to WT-TTR and
generate strong fluorescence responses.
Next, we investigated the plasma stability of probe 10 to
evaluate any susceptibility to degradation and modification by
enzymes, particularly hydrolases and esterase. Probe 10
exhibited high plasma stability in freshly obtained rat plasma
(93.3%) and commercially available human plasma (96.5%) after
1hr of incubation at 37oC (Fig S4, see ESI).17
To demonstrate the practical usefulness of the studied
system for TTR detection in real biological samples, we
evaluated the sensing potential of probe 10 for measuring the
exact concentration of TTR in two types of samples (human
male AB plasma and human male AB clotted serum, Sigma-
aldrich). Samples were analyzed by SDS-PAGE, followed by
western-blotting using anti-TTR antibody as a control method
(Fig. 4 and Fig. S5, see ESI). Human serum albumin is the most
abundant blood protein (ca. 0.63 mM) and carrier for many
neutral and weak acidic endogenous and exogenous
molecules,18 whose properties may affect the output of
fluorescence signals. Therefore, there is a need to develop a
practical fluorescence method for detecting TTR in a complex
Fig. 2 (a) Normalized emission spectra of probe 10 (4
solvents. (b) The fluorescence spectra of probe 10 (4
presence of different TTR concentrations (0-1 M) in PBS and 18-fold
diluted human plasma (dotted line). Insert shows the linear
relationship between fluorescence enhancement of probe 10 and TTR
concentrations in PBS. λex = 357 nm
µ
M) in various
µ
M) in the
µ
increase of polar solvent (water) ratio along with bathochromic
shifts (54 nm, Fig S2a, see ESI). In addition, a similar
phenomenon was observed in the nonpolar methylene chloride
and the polar methanol system (Fig. S2b, see ESI). Collectively,
these observations indicate that a dramatic increase in the
fluorescence intensity and the strong dependence of emission
maximum of probe 10 upon binding to WT-TTR in PBS results
from the intramolecular charge transfer16 and occupancy of
hydrophobic T4-binding pockets on WT-TTR.
To examine our accuracy, the linearity of fluorescence
change in response to TTR concentration was examined. As
shown in Figure 2b and S2c, fluorescence intensity gradually
increased with TTR concentrations, obtaining a good linearity
relationship (R2 = 0.997). Considering that the average
concentration of TTR in human blood is around 4
for TTR in PBS showed a low detection limit (limit of detection =
0.007 M) based on the 3 /k rule, indicating that the
µM, probe 10
µ
σ
quantification of TTR was quite sensitive and highly reliable.
Prior to the complex biological application, we examined the
selectivity of probe 10 in the presence of a series of biological
samples existing in human serum plasma. Only slight
fluorescent enhancement was observed upon addition of highly
concentrated albumin, whereas no substantial fluorescence
increase was observed when probe 10 was incubated with other
biological species (Fig. 3). As shown in Fig. S3, we tested the
anti-interference of the probe toward various inorganic
substances (Na+, K+, Cu2+, Zn2+, and Fe3+) and pH changes.
Various metal ions in the serum plasma led to negligible
Fig. 4 Quantitative detection of TTR in human plasma- and human
serum-employing immunoassay method (white bar, control method), a
turn-on fluorescence method after subtracting the fluorescence
intensity resulted from human serum albumin (black bar), and a turn-
on fluorescence method of human albumin deleted samples (gray bar).
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M. P. Vitek, S. Lyubski, W. J. Strittmatter, J. J. Enghilde, R. Bhasin
biological environment that can avoid interference from
albumin. For our fluorescence detection method, excess probe
10 (4 µM) was incubated with 18-fold diluted human plasma
sample in buffer medium (25 mM Tris, 192 mM Glycine, 0.1%
SDS, 1 mM DTT, pH 8.6). We monitored the changes in emission
intensity and the normalized fluorescence intensity was
obtained by subtracting the fluorescence intensity resulted
from 0.63 mM of human serum albumin. Then, the TTR
concentration in this sample was estimated by using a standard
calibration plot (Fig. 2b and Fig. S6, see ESI). As shown in Figure
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, J. Silverman, K. H. Weisgraber, P. K. Coyle, M. G. Zagorski, J. Tal
DOI: 10.1039/C9CC04172A
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1
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moiety for detection of TTR, which serves as a potential
biomarker for early diagnosis of AD. Spectroscopic and stability
studies demonstrated that probe 10 exhibited the desired
photo-physical properties (large stokes shifts, high quantum
yield, and low limit of detection) and high plasma stability,
which are beneficial for its biological application. The simple
and sensitive fluorescence method developed according to the
TTR level of human plasma and serum is highly sensitive and
reliable, and can further enhance AD's diagnostic ability through
composite analysis of different biomolecules (A
neuroimaging studies.
We are grateful for the support of the National Research
Foundation of Korea, funded by the Ministry of Education,
Science and Technology (NRF-2018R1D1A3B07046990) and the
Ministry of Science and ICT (NRF-2015M3A9B5053643).
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