A fluorescent switchable AIE probe for selective imaging of dipeptidyl peptidase-4 in vitro and in vivo and its application in screening DPP-4 inhibitors

Article abstract of DOI:10.1039/c5cc08921b

A novel fluorescent probe for in vitro and in vivo imaging of dipeptidyl peptidase-4 (DPP-4) was designed and synthesized. This probe is successfully utilized to screen DPP-4 inhibitors in living 3T3-L1 cells and zebrafish, which provides a novel approach

Full text of DOI:10.1039/c5cc08921b

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Fluorescent switchable AIE probe for selective
imaging of dipeptidyl peptidase-4 in vitro and in vivo
and its application in screening DPP-4 inhibitors
Cite this: DOI: 10.1039/x0xx00000x
Yi Wanga*, Xueli Wuc, Yiyu Chenga,b, Xiaoping Zhaoc*
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
www.rsc.org/
A novel fluorescent probe for in vitro and in vivo imaging of become highly emissive in the aggregate state, various AIE
dipeptidyl peptidase-4 DPP-4) was designed and molecules such as tetraphenylethene (TPE), 9,10-distyrylanthracene
(
synthesized. This probe is successfully utilized to screen (DSA), and silacyclopentadiene (silole) have been utilized as basic
DPP-4 inhibitors in living 3T3-L1 cells and zebrafish, which scaffolds for the design of novel probes.7,8,9 A few probes with high
provides a novel approach for the discovery of anti-diabetes selectivity have been applied in sensing Cu2+, Al3+, and other
drugs.
ions,10,11,12 monitoring physiopathological events,13,14,15 and
screening drug candidates.16,17,18
Type 2 diabetes mellitus (T2DM) is a common metabolic
disorder with growing morbidity worldwide. However,
previous therapies for T2DM including PPARγ agonists and
glucosidase inhibitors have limited efficacy and significant
mechanism-based side effects.1 In the past decade, dipeptidyl
peptidase-4 (DPP-4) has become one of the most promising and
remarkable targets for treating T2DM.2 DPP-4 is a type II
transmembrane glycoprotein widely distributed in tissues and
circulates. DPP-4 can cleave N-terminal amino acids from
glucagon-like peptide-1 (GLP-1) and glucose-dependent
insulinotropic polypeptide (GIP), which play important roles in
regulating glucose-induced insulin secretion.3 Oral DPP-4
inhibitors such as sitagliptin and alogliptin have exhibited
satisfied clinical outcomes.4 Thus, screening small-molecule
inhibitors of DPP-4 become a crucial issue in T2DM drug
discovery. Moreover, circulating level of DPP-4 is considered
as a potential biomarker for the prognosis of lymphoblastic
leukaemia and hyperglycemia,5 which also raise the demand of
sensitive assay to detect DPP-4 in biological samples.
Herein, we designed and synthesized a novel fluorescent
probe with AIE characteristics for DPP-4 assay and imaging
(Scheme 1). TPE was employed as the AIE fluorophore to
conjugate with a hydrophilic peptide Lys-Phe-Pro-Glu (KFPE)
that can be specifically cleaved by DPP-4. The probe is non-
emissive in solution, whilst after incubate with DPP-4,
fluorescent signal will be switched on due to the aggregation of
TPE-residue. As shown in Scheme 1, this probe can easily
penetrate the phospholipid layer of cell membrane, which
endows it with high sensitivity for in situ imaging of DPP-4
activity in living cells and organisms. Moreover, this probe can
be utilized to measure the reduction of fluorescent emission in
the presence of DPP-4 inhibitors, which facilitates screening
anti-diabetes drugs in vitro and in vivo
.
The synthesis of TPE-based probe was conveniently
achieved in four steps as illustrated in Scheme S1. The design
of peptide sequence was optimized to ensure that the probe
dissolved in aqueous solution is non-emissive and subsequently
can be recognized by DPP-4. It is clear that DPP-4
preferentially cleaves peptides with the amino acid proline in
position 2 of the N-terminus of the peptide chain to switch on
strong fluorescent signal due to the aggregation of TPE-based
residue (Fig.S1, ESI†). DPP-4 is unable to recognize peptides
KFPG and GPD connected to TPE-core due to the specificity of
substrate sequence.23 Thus, TPE-KFPE was finally selected as
Recently, florescent probes with aggregation-induced emission
(AIE) characteristics have attracted increasing interesting since Tang
et al. found the AIE phenomenon.6 Taking advantage of the distinct
feature of AIE molecules that they are non-emissive in solution but
a College of Pharmaceutical Sciences, Zhejiang University (Zijingang
Campus), Hangzhou, 310058, P. R. China. mysky@zju.edu.cn
1
b
the appropriate probe and characterized with HPLC, H NMR,
and 13C NMR spectroscopy (Fig.S2-S4, ESI†).
Collaborative Innovation Center for Diagnosis and Treatment of
Infectious Diseases, Zhejiang University, 310003 Hangzhou, China
c
College of Preclinical Medicine, Zhejiang Chinese Medical
University, Hangzhou 310053, PR China. zhaoxiaoping@zcmu.edu.cn
† Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
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DOI:10.1039/b000000x/
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DOI: 10.1039/C5CC08921B
Scheme 1 Schematic illustration of TPE-KFPE (A) for DPP-4
assay and inhibitor screening and living cell imaging (B).
The assay system for detecting DPP-4 activity in solution
was optimized. The acidified or alkalized solution may lead to
invalid assay, but solution with the pH range of 6.5 to 8.2 can
Fig. 1 LC-MS chromatograms of TPE-KFPE before (A) and
after incubated with DPP-4 (B). Corresponding fluorescent
spectra of TPE-KFPE before and after incubated with DPP-4 in
the presence or absence of diprotin A (C). λex = 320 nm.
produce satisfied fluorescence enhancement (Fig.S5, ESI
High ionic strength of HEPES buffer solution may reduce the
signal (Fig.S6, ESI ). We further used liquid chromatography
†).
†
coupled with mass spectrometery (LC-MS) to analyze our
probe and its hydrolysis products after incubation with DPP-4.
Kinetic experiments showed that the enzymatic reaction
was almost completed within 30 minutes, while none
fluorescence enhancement was observed in the absence of DPP-
4 (Fig.2A). The linear range of our probe for determining DPP-
4 activity was measured using a fluorescence microplate reader.
There is a good linear correlation (R = 0.9608) between the
relative fluorescence intensity and the amount of DPP-4 in the
range from 0.1 to 0.5 mU/mL with 10 ꢀM probe (Fig. 2B). We
determined the inhibitory rates of diprotin A with different
concentrations on DPP-4 activity by the proposed assay system.
As shown in Fig.2C, a dose-dependent curve was obtained with
a IC50 value of 4.15 ꢀM, which is in accordance to previous
reference.19 The probe was also applied in screening potential
DPP-4 inhibitors from natural products. An abundantly existed
catechin in green tea, (-)-epigallocatechin-3-gallate (EGCG),
was found to dose-dependently inhibit DPP-4 activity in the
concentration of 5 ꢀM to 25 ꢀM (Fig.S8, ESI†), which is
similar to a recent report.19 These results indicated that the
TPE-KFPE probe can be utilized as sensitive assay to measure
DPP-4 activity as well as to screen DPP-4 inhibitors.
After incubated with DPP-4 (5 mU/mL) for 30 min at 37
℃, a
new peak TPE-KF appeared in the LC-MS chromatogram
(Fig.1B), which is the hydrolysis product of our probe and leads
1
to the boost of fluorescent signal. By comparison of H NMR
and MS data of TPE-KFPE with the product of enzymatic
reaction after incubation with DPP-4, it clearly showed that two
amino acids were cleavaged from the probe (Fig.S2, S7, ESI†).
When incubated the probe with DPP-4 and diprotin A, a known
DPP-4 inhibitor, the probe is enough sensitive to monitor the
suppression of DPP-4 activity (Fig.1C).
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pretreated by DPP-4 inhibitor diprotin A (100 ꢀM) and above
mentioned active compound EGCG (100ꢀM) showed that only
weak fluorescent was detected. Quantitative analysis of those
images reveals that the relative fluorescent signal is
significantly decreased to approximately 65% compared to the
control (Fig.S11 , ESI†), which suggests that the probe can
also be used for in vitro screening of DPP-4 inhibitors.
Fig. 2 (A) Kinetic study of TPE-KFPE reacted with DPP-4
.
(B) Dose-dependent of TPE-KFPE in the presence of DPP-4.
(C) Dose-dependent inhibition of DPP-4 by diprotin A. (D)
Plot of (I-I0)/I0 versus different enzymes/proteins. TPE-KFPE
= 10 ꢀM, DPP-4 = 5 mU/mL, λex = 320 nm, λem = 450 nm.
The specificity and cytotoxicity of TPE-KFPE probe were
further exploited to ensure its capability for assessing DPP-4 in
physiological environment. As shown in Fig.2D, the probe
incubated with DPP-4 produced a much higher change in (I-
I0)/I0 compared to the signals induced by cytochrome C (CYC),
trypsin, collagenase I/II (Coll I/II), bovine serum albumin
(BSA), human serum albumin (HSA), proteinase K, α-
chymotrysin and lysozyme. Interestingly, DPP-8, another
dipeptidyl peptidase, also can not recognize the probe which
suggested satisfied specificity of TPE-KFPE on DPP-4.
Incubation of the probe (10, 30, 50 ꢀM) with 3T3-L1
preadipocyte cells for 24 h showed no significant effects on cell
survival (Fig.S9, ESI†). Because TPE-KFPE contains both
hydrophilic and hydrophobic regions that minimizes free
energy when interacted with the biological membrane, the
probe can easily penetrate the lipid bilayer of cells, Moreover,
the size of the probe is very small before aggregation, which is
also helpful for facile and minimally invasive cell penetration.
This feature is of great significance for the subsequent in vitro
and in vivo applications in molecular imaging.
Fig. 3 Bright-field (BF), fluorescence (FL), and overlay images
of 3T3-L1 cells stained by TPE-KFPE in the absence or
presence of diprotin A and EGCG. The images were captured
using Nikon A1R laser scanning confocal microscope equipped
with 405 nm laser (60 × lens). Excitation and emission
wavelength: 405 nm and 425-475 nm.
Next, we evaluated the in vivo tracking ability of TPE-KFPE in
zebrafish, a well-established model system for developmental
biology and high throughput screening. The zebrafish larvae are
transparent, which offers great convenience for optical and
fluorescence microscopic detection. Fig.4A and 4B show the images
of whole bodies of normal zebrafish larvae and larvae microinjected
with TPE-KFPE (2.5 mM, 10 nL) for 2 h. The zebrafish larvae
exposed to the probe exhibits significant fluorescence in yolk sac,
liver, pancreas, and other organs connected to gastrointestinal tract.
This can be explained that DPP-4 is highly expressed in liver,
exocrine pancreas, and small intestine.22 The accumulated
fluorescence in the yolk sac and in the intestine indicated that TPE-
KFPE entered into the digestive system and may be eliminated from
the body. A representative series of optical sections for in vivo
imaging of DPP-4 by our probe showed that distribution pattern of
DPP-4 chiefly located in yolk sac and blood vessel of zebrafish
(Fig.S12, ESI†). Further, we also stained the zebrafish pretreated
with diprotin A with the probe. As shown in Fig.4C, relative low
fluorescent signals can be observed. An approximate 50% decrease
of fluorescence intensity is observed in DPP-4 inhibitor treated
zebrafish (n=6, Fig.S13, ESI†). This suggests that TPE-KFPE can be
used for in vivo sensing of DPP-4 and screening the potential DPP-4
inhibitor in zebrafish.
DPP-4 widely exists in human body as an intracellular and
serum-soluble form, and is considered as an adipokine
produced by adipocytes.20 Previous method for measurement of
intracellular DPP-4 content needs tedious steps like cell
collection,
sample
preparation,
and
enzyme-linked
immunosorbent assay.21 Our probe exhibited its ability to
tracking DPP-4 activity in living cells by fluorescence
microscope. As shown in Fig.3, owing to the AIE character of
TPE-KFPE, high-contrast staining of intercellular region of
cells was achieved after incubated 50 ꢀM TPE-KFPE with 3T3-
L1 preadipocyte cells for 1 h. The dose-dependent relationship
of DPP-4 concentrations in 3T3-L1 cells with fluorescent
signals can be sensitively measured by the probe (Fig.S10,
ESI†). The probe was also successfully utilized to screen DPP-
4 inhibitors in vitro. Fluorescent images of 3T3-L1 cells
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DOI: 10.1039/C5CC08921B
(b)
A. A. Mehde, F. Yusof, W. A. Mehdi and J. A.
Zainulabdeen, Asian Pac J Cancer Prev, 2015, 16, 5059; (c)
T. Zheng, A. Baskota, Y. Gao, T. Chen, H. Tian and F.
Yang, Metabolism, 2015, 64, 498.
6
J. Luo, Z. Xie, J. W. Lam, L. Cheng, H. Chen, C. Qiu, H. S. Kwok, X.
Zhan, Y. Liu, D. Zhu and B. Z. Tang, Chem Commun (Camb),
2001, 1740.
7
8
9
X. Li, K. Ma, S. Zhu, S. Yao, Z. Liu, B. Xu, B. Yang and W. Tian, Anal
Chem, 2014, 86, 298.
M. Wang, D. Zhang, G. Zhang and D. Zhu, Chem Commun (Camb),
2008, 4469.
L. Zhang, W. Hu, L. Yu and Y. Wang, Chem Commun (Camb), 2015,
51, 4298.
10 S. Gui, Y. Huang, F. Hu, Y. Jin, G. Zhang, L. Yan, D. Zhang and R.
Zhao, Anal Chem, 2015, 87, 1470.
11 S. Samanta, S. Goswami, M. N. Hoque, A. Ramesh and G. Das, Chem
Commun (Camb), 2014, 50, 11833.
12 T. Han, X. Feng, B. Tong, J. Shi, L. Chen, J. Zhi and Y. Dong, Chem
Commun (Camb), 2012, 48, 416.
Fig. 4 Bright-field (BF), fluorescence (FL), and overlay images
of zebrafish. (A) Zebrafish without probe. (B) Zebrafish stained
by TPE-KFPE. (C) Zebrafish pre-incubated with diprotin A
stained by TPE-KFPE. The images were captured using a
fluorescent microscope equipped with Andor Zyla cMOS
camera and 5 × lens.
13 Y. Yuan, C. J. Zhang and B. Liu, Chem Commun (Camb), 2015, 51,
8626.
14 Q. Hu, M. Gao, G. Feng, X. Chen and B. Liu, ACS Appl Mater
Interfaces, 2015, 7, 4875.
15 M. Gao, C. K. Sim, C. W. Leung, Q. Hu, G. Feng, F. Xu, B. Z. Tang and
B. Liu, Chem Commun (Camb), 2014, 50, 8312.
In summary, we herein for the first time reported a probe with AIE
characteristics that exhibits excellent fluorescent switchable property
in the present of DPP-4. By the introduction of a specific peptide to
TPE core, the probe shows excellent sensitivity and linear range in
the detection of DPP-4 activity both in vitro and in vivo. The probe
is successfully applied to screen DPP-4 inhibitors in living cells and
zebrafish. The assay system demonstrated here provides a new
approach to enabling in vivo high-throughput screening of T2DM
drugs in zebrafish.
16 M. Wang, X. Gu, G. Zhang, D. Zhang and D. Zhu, Anal Chem, 2009, 81,
4444.
17 H. Wang, Y. Huang, X. Zhao, W. Gong, Y. Wang and Y. Cheng, Chem
Commun (Camb), 2014, 50, 15075.
18 Y. Wang, Y. Chen, H. Wang, Y. Cheng and X. Zhao, Anal Chem, 2015,
87, 5046.
19 J. Fan, M. H. Johnson, M. A. Lila, G. Yousef and E. G. de Mejia, Evid
Based Complement Alternat Med, 2013, 2013, 479505.
20 D. Lamers, S. Famulla, N. Wronkowitz, S. Hartwig, S. Lehr, D. M.
Ouwens, K. Eckardt, J. M. Kaufman, M. Ryden, S. Muller, F. G.
Hanisch, J. Ruige, P. Arner, H. Sell and J. Eckel, Diabetes, 2011,
60, 1917.
This study was supported by the National Key Scientific and
Technological Project of China (No. 2012ZX09304007).
21 S. S. Das, H. Hayashi, T. Sato, R. Yamada, M. Hiratsuka and N.
Hirasawa, Diabetes Metab Syndr Obes, 2014, 7, 185.
Notes and references
22 M. Itou, T. Kawaguchi, E. Taniguchi and M. Sata, World
Gastroenterol, 2013, 19, 2298.
J
1
D. E. Moller, Nature, 2001, 414, 821.
2(a)
R. E. Pratley and A. Salsali, Curr Med Res Opin, 2007, 23, 919;
(b)
J. A. Lovshin and D. J. Drucker, Nat Rev Endocrinol,
2009, 5, 262.
3
E. E. Mulvihill and D. J. Drucker, Endocr Rev, 2014, 35, 992.
G. A. Herman, A. Bergman, C. Stevens, P. Kotey, B. Yi, P. Zhao,
B. Dietrich, G. Golor, A. Schrodter, B. Keymeulen, K. C.
Lasseter, M. S. Kipnes, K. Snyder, D. Hilliard, M. Tanen, C.
Cilissen, M. De Smet, I. de Lepeleire, K. Van Dyck, A. Q. Wang,
W. Zeng, M. J. Davies, W. Tanaka, J. J. Holst, C. F. Deacon, K.
M. Gottesdiener and J. A. Wagner, J Clin Endocrinol Metab,
2006, 91, 4612; (b) N. Inagaki, H. Onouchi, H. Maezawa, S.
Kuroda and K. Kaku, Lancet Diabetes Endocrinol, 2015, 3, 191.
N. Tahara, S. Yamagishi, M. Takeuchi, A. Tahara, K. Kaifu, S.
Ueda, S. Okuda and T. Imaizumi, Clin Biochem, 2013, 46, 300;
4(a)
5(a)
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