Biocompatible G-Quadruplex/BODIPY assembly for cancer cell imaging and the attenuation of mitochondria

Article abstract of DOI:10.1016/j.bmcl.2019.05.043

The G-quadruplex aptamer is a high-order structure formed by folding of guanine-rich DNA or RNA. The recognition and assembly of G-quadruplex and compounds are important to find biocompatible drugs. Herein, triphenylamine conjugated 4, 4-difluoro-4-bora-3

Full text of DOI:10.1016/j.bmcl.2019.05.043

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Biocompatible G-Quadruplex/BODIPY assembly for cancer cell imaging and
the attenuation of mitochondria
a
a
a,⁎
b
b
Peng-Li Zhang , Zhuo-Kai Wang , Qiu-Yun Chen , Xia Du , Jing Gao
b
a
School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013, PR China
School of Pharmacy, Jiangsu University, Zhenjiang 212013, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
BODIPY
The G-quadruplex aptamer is a high-order structure formed by folding of guanine-rich DNA or RNA. The re-
cognition and assembly of G-quadruplex and compounds are important to find biocompatible drugs. Herein,
triphenylamine conjugated 4, 4-difluoro-4-bora-3a, 4a-diaza-s-indacene (BODIPY) compound (BPTPA) was
synthesized, and the interaction of BPTPA with G4 DNA was studied. It is found that BPTPA selectively binds
Cancer
G4-aptamer
Mitochondria
with G
tochondria and inhibit the expression of TrxR
membrane potential of mitochondria and inhibit the proliferation of BGC-823 cancer cells. Therefore, BPTPA-
can be the biocompatible attenuator of mitochondria for cancer image and chemotherapy.
3
T
3
G4 DNA forming a water-compatible nanocomplex (BPTPA-G
3
T
3
). BPTPA-G
3
T
3
can image mi-
2
. Cytotoxicity results indicate BPTPA-G
T
3 3
can decrease the
G
T
3 3
Mitochondrial is the central link of all metabolic reactions in cells.
They play an important role in the glucose metabolism, amino acid
nanocomplex and targeting mitochondrial selectively. The G-quad-
ruplex DNA is a high-order structure formed by folding DNA or RNA
1
10
metabolism and fat metabolism of cancer cells. Tumor cells are sen-
rich in tandem repeat guanine (G). This type of structure is ubiquitous
sitive to disturbances of mitochondrial function. Attenuating mi-
tochondrial dysfunction has been a strategy for cancer diagnostic and
in the telomere end, the oncogene promoter region and the 5′UTR of
1
1
mRNA, and is involved in the regulation of gene transcription. Its
structure is closely related to the occurrence and development of
cancer. Moreover, study on the recognition and assembly of G-quad-
ruplex with compounds would be helpful to the development of new
nanodrugs or carriers for cancer diagnosis and therapy.
2
chemotherapy. Mammalian thioredoxin reductase (TrxR) enzymes
are overexpressed and related to the drug-resistance in cancer che-
motherapy. Thus, TrxR has been reported as potential target for
3
cancer chemotherapy. Mitochondria targeting inhibitor of TrxR
2
can
4
also be applied in the treatment of Parkinson’s disease. Liang et al
To develop mitochondria targeting attenuator of TrxR , tripheny-
2
reported a mitochondria targeting inhibitor of TrxR
2
for mitochon-
lamine conjugated 4, 4-difluoro-4-bora-3a, 4a-diaza-s-indacene
4
c
drial apoptosis of HeLa cancer cells. . Therefore, regulating the
function of mitochondria in tumor cells and targeting inhibition of
(BODIPY) compound (BPTPA) was prepared. Furthermore, the re-
cognition of BPTPA with G‑Quadruplex aptamer (G
3
T
3
) makes G
3
T be
3
TrxR
tumor cells.
It is well known that 4, 4-difluoro-4-bora-3a, 4a-diaza-s-indacene
2
in mitochondria will be a new method for clinical treatment of
a good carrier of BPTPA with the formation of water-compatible na-
nodyes (BPTPA-G , Scheme 1). Moreover, we found that BPTPA-G
could target mitochondria, decrease the membrane potential of mi-
tochondria and inhibit the expression of TrxR . Study on the recogni-
tion and assembly of fluorescent compounds with G‑Quadruplex apta-
T
3 3
T
3 3
(
BODIPY) derivatives can be as good bio-probes and biomaterials due to
2
5
,6
its bright fluorescence emission. Moreover, the BODIPY core can be
7
modified at the pyrrole ring positions, and the meso-position. Tri-
mers provides a new method for the transfer of hydrophobic
phenylamine (TPA) was commonly used as an electron donor in the
compounds into water-compatible supermolecules.
8
design of photomaterials. The combinations of TPA with conjugated π-
spacer could construct compounds with good hole-transporting ability.
Moreover, TPA group also was found to interact with the G-quadruple
Results and discussion
9
aptamers. Therefore, it is possible for triphenylamine modified
The triphenylamine was introduced at the 3-sites of BODIPY re-
sulting fluorescence BODIPY derivatives (BPTPA, Scheme S1). The
BODIPY bind to G-quadruple aptamer forming water compatible
⁎
Corresponding author.
E-mail address: chenqy@ujs.edu.cn (Q.-Y. Chen).
https://doi.org/10.1016/j.bmcl.2019.05.043
Received 2 February 2019; Received in revised form 20 May 2019; Accepted 20 May 2019
0960-894X/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Peng-Li Zhang, et al., Bioorganic & Medicinal Chemistry Letters, https://doi.org/10.1016/j.bmcl.2019.05.043

P.-L. Zhang, et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
−1
−1
−1
1
182 cm (CeC stretch), 1080 cm (CeO stretch) and 966 cm (Ar-
H bending vibrations) were observed for BPTPA. As shown in Fig. S4,
BPTPA had the highest absorbance in CH
2
Cl
2
. The maximum UV–vis
absorption of BPTPA is 618 nm in CH
2
Cl
2
. The BPTPA is red shift due to
*
the π-π transition. The maximum emission wavelength of BPTPA is
6
99 nm (Fig. S5). Therefore, BPTPA is a near-infrared fluorescent
compound.
It is reported that aptamers (such as, G T , HRAS, HTG-21, et al see
3
3
Table S1) can form G-quadruple structures in the presence of 10 mM
1
2
Tris-HCl (pH 7.4, 100 mM KCl/NaCl) buffer. In order to select a G-
quadruplex aptamer that binds to BPTPA, we determined the change of
absorbance of BPTPA after addition of various G-quadruplexes (Fig. 1).
It was found that BPTPA selectively binds well to G
3
T with G-quad-
3
ruplex structure, while it show weak interaction with other aptamers,
single strand G (ssG ), linear duplex (ctDNA), and self-com-
plementary duplex strands (polyd(A-T), polyd(G-C)). To investigate the
binding of BPTPA with G , we determined the UV titration spectra of
to BPTPA and calculated its binding constant (Fig. 2a and b). The
results indicated that the absorbance of BPTPA decreases with the in-
creasing amount of G . Moreover, BPTPA exhibited higher binding
affinity to G than other G-quadruple DNA in Table 1. The effect of
3
T
3
3 3
T
T
3 3
G
T
3 3
Scheme 1. Schematic illustration of BPTPA-G
3
T nanocomplex.
3
3 3
T
T
3 3
BPTPA on the conformation of G
3
T G4-DNA was investigated by using
3
circular dichroism (CD) assessments. As shown in Fig. 2c, in the pre-
+
sence of 10 mM K ions, G
3
T was of the typical anti-parallel G-quad-
3
ruplex structure, with a major positive band at 289 nm and a negative
1
3,14
peak at ∼240 nm
tion, there was no significant change in the structure of G
It indicates that BPTPA binds to G G4-DNA without changing the
conformation of G G4-DNA. To get more details about the interac-
tion of BPTPA with G
. Upon the addition of BPTPA to the G
3
T
3
solu-
T
3 3
G4-DNA.
T
3 3
3 3
T
3
T , molecular docking was performed to obtain
3
detection models and binding free energies of BPTPA by Poisson-
Boltzmann surface area (MM/PBSA) calculations (Fig. 3a). From the
BPTPA-G
T
3 3
molecular model, the binding pattern of BPTPA to G
3 3
T -
G4 DNA was the groove binding mode. In addition, BPTPA exhibited a
superior binding energy (-88.3264 kcal/mol). Spectroscopic titration
and docking calculation results demonstrate that BPTPA and G
3
T G4-
3
DNA can be well combined. TEM shows the formation of BPTPA-G
nanosheet (Fig. 3b). Therefore, BPTPA was specifically combined with
G4-DNA resulting water compatible BPTPA-G nanocomplex
based on their self-assembly.
3
T
3
G
T
3 3
T
3 3
When BGC-823 cells treated with BPTPA and BPTPA-G3T3, the
strong cellular fluorescence was observed (Fig. 4b). Electron micro-
graphs of the cells provided direct evidence that BPTPA-G3T3 can
enter BGC-823 cells. Next, the mitochondrial target imaging of
BPTPA-G3T3 was evaluated by comparison with the mitochondria
targeting dye, Mito Tracker Green FM. The yellow overlapped images
of Mito Tracker Green FM and BPTPA-G3T3 in the cell demonstrate
that BPTPA-G3T3 can enter mitochondria. The observed clear image
and cell shapes indicate that BPTPA-G3T3 is a good mitochondrial
targeting probe. In contrast, unclear images and precipitations were
observed when the BPTPA was added into cells because of its low
Fig. 1. The change of absorbance for BPTPA with various G4 DNAs and non-G4
DNAs in 10 mM Tris-HCl (pH 7.4, 100 mM KCl/NaCl) buffer (A
0
: initial ab-
sorbance, A: final absorbance).
structure of BPTPA was characterized by 1H NMR spectra, C NMR
spectra, electrospray mass spectrometry (ESI-MS) and IR spectra (Figs.
13
+
+
S1–S3). MS (ESI , CH
3
CN): Calcd for C47
H
43BF
2
N
4
OS [M] m/
+
+
1
z = 759.75, found m/z = 786.84 [(M-F) +CH
3
CN]
.
H NMR
(
400 MHz CDCl
3
). δ = 7.48–7.43 (m, 4H), 7.39–7.34 (t, J = 20 Hz, 2H),
7
.30–7.28 (d, J = 8 Hz, 4H), 7.13–7.11 (m, 6H), 6.93–6.91 (d,
water solubility. To further investigate the effect of BPTPA-G
3
3
T on
J = 8 Hz, 2H), 6.89–6.85 (m, 4H), 6.57 (s, 1H), 6.01 (s, 1H), 3.82 (s,
mitochondria, we monitored the mitochondrial membrane potential
6
3
1
1
1
H), 3.76–3.74 (t, J = 8 Hz, 4H), 2.61 (s, 6H), 2.49–2.47 (t, J = 8 Hz,
of BPTPA-G
3
T
3
treated BGC-823 cells using a fluorescent and vol-
1
3
15
H). C NMR (100 MHz CDCl
3
) δ = 152.01, 147.76, 147.38, 146.16,
tage-sensitive dye JC-1. As shown in Fig. 5a, the change in red/
41.47, 140.89, 133.78, 129.90, 129.75, 129.39, 129.07, 128.92,
28.71, 127.93, 126.66, 124.71, 123.42, 123.32, 123.22, 122.84,
18.00, 117.87, 66.82, 63.00, 53.47, 22.23, 14.63. The characteristic
green fluorescence ratio directly reflects changes in mitochondrial
membrane potential. Different concentrations of BPTPA-G
3
T were
3
added to BGC-823 cells for 24 h. As the concentration of BPTPA-G
T
3 3
−
1
−1
peaks of BPTPA at 2924 cm (CeH stretching vibrations), 1465 cm
increased, JC-1 dye exhibited progressively enhanced green fluor-
−
1
(
C]C stretching vibrations), 1375 cm
(CeN stretching vibrations),
escence intensity and decreased red fluorescence intensity indicating
2

P.-L. Zhang, et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Fig. 2. (a) UV–vis spectra of BPTPA in buffer (10 mM Tris–HCl, pH 7.4, 100 mM KCl) in the presence of increasing amounts of G
3
T
3
G4-DNA. BPTPA = 10 μM,
[
G
3
T
3
] = 0–1 μM from top to bottom. (b) The plot of [DNA]/(ε
a
-ε
f
) versus [DNA] for the titration. (c) CD spectra of G
3
T
3
G4-DNA (1 μM) and BPTPA-G
T
3 3
(
[BPTPA] = 10 μM, [G
T
3 3
] = 1 μM) in buffer (10 mM Tris-HCl, pH 7.4, 100 mM KCl).
Table 1
Apparent binding constants (Ka) of G-quadruplex DNA,
determined from Ultraviolet titrations.
G-quadruplex DNA
Ka [106 M−1
]
2
2AG
0.087
0.246
0.158
0.069
0.077
0.068
0.143
0.915
CM22
Cmyc
Ckit
Ckit
1
3
HTG-21
HRAS
3 3
G T
that the mitochondrial potential was decreased. This result demon-
strates that BPTPA-G is the attenuator of the mitochondrial
3 3
T
functions. The rapid metabolism of cancer cells makes them more
1
6
susceptible to mitochondrial perturbations. The mitochondria of
cancer cells are different from normal cells in structures and func-
tions, such as the molecular composition of the mitochondrial inner
2
Fig. 3. (a) The complex model of G
BPTPA-G
3
T G4-DNA with BPTPA. (b) TEM images of
3
membrane and mitochondrial membrane penetration. Mitochon-
3 3
T .
drial dysfunction is involved in the apoptotic process and energy
1
7
metabolism of malignant cells. The cytotoxicity of BPTPA-G
3 3
T
was determined by MTT method to study its inhibition to the growth
of cancer cells (BGC-823, Patu 8988 and MCF-7 cell lines, Figs. 4a
and MCF-7. So we selected BGC-823 gastric cancer cells for next
experiments.” The IC50 value of BPTPA-G to BGC-823 cells was
31.5 ± 0.4 μmol/L. Therefore, BPTPA-G can be a mitochondrial
attenuator and has a good inhibitory effect on cancer cell growth.
3 3
T
and S6). Results show that BPTPA-G
3
T
3
has a concentration-depen-
3 3
T
dent cytotoxicity on BGC-823 cells, but has little effect on Patu 8988
3

P.-L. Zhang, et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Fig. 4. (a) Inhibition activities of BPTPA-G3T3 on the proliferation of BGC-823 cells. [G3T3] = 0 μM, 1.25 μΜ, 2.5 μΜ, 5 μΜ, 10 μΜ. (b) Fluorescence image in BGC-
23 cells. (a1-2) Sample imaging (2 μM) [G3T3] = 0.2 μM, 0.4 μM; (b1-2) Mito Tracker Green FM imaging; (c1-3) overlapped imaging of sample and Mito Tracker
Green FM.
8
Thioredoxin reductase (TrxR) is a member of the family of anti-
further shows that BPTPA-G
3
T
3
can selectively target subcellular mi-
oxidant systems widely distributed in organisms. It is responsible for
the regulation of enzymes and transcription factors and redox states at
the cellular level, and is involved in cell growth, proliferation and
tochondria and induce mitochondrial dysfunction by attenuating the
mitochondrial potential and inhibiting the expression of TrxR .
2
In conclusion, the triphenylamino group conjugated BODIPY de-
rivatives (BPTPA) was synthesized and assayed for its interactions
with G-quadruple aptamers. It is found that BPTPA can bind to G-
1
8
apoptosis. There are three isozymes in TrxR, of which TrxR
2
2
is mainly
is over-
distributed in mitochondria. Studies have found that TrxR
1
9,20
expressed in cancer tissues promoting cancer cell proliferation.
To
quadruple aptamer (G
3
T ) forming water-compatible nanosheet
3
study the effect of the mitochondria targeting BPTPA on the expression
(BPTPA-G
3
T
3
). Live cell imaging showed that BPTPA-G
3
3
T could carry
of TrxR
cubated with BPTPA-G
a similar BODIPY derivative, structure and toxicity were shown in
2
, the expression of TrxR
1
and TrxR
2
in BGC-823 cells after in-
BPTPA to mitochondria and decrease the membrane potential of mi-
tochondria. Mitochondria play an important role in tumorigenesis.
T
3 3
were carried out and compared with BDP-M
(
BPTPA-G
3
T can attenuate mitochondrial function and inhibit the
3
2
1
Scheme S1 and Fig. S7, respectively). The high expression of TrxR
and TrxR in BGC-823 cells can be detected after incubated with BDP-M
indicating BODIPY structure has less contribution to the inhibition; in
contrast, BPTPA-G shows good inhibition on the expression of mi-
tochondrial TrxR (Figs. 5b and S8). It indicates that the triphenylamino
group of the BPTPA plays key role to the inhibition of TrxR . This
1
expression of mitochondrial TrxR
2
. Therefore, BPTPA-G
3
T
3
can inhibit
2
cancer cell growth. The recognition and assembly of fluorescent
compound BPTPA and G‑Quadruplex aptamer G
T
3 3
provide a new
3
T
3
method for the transfer of hydrophobic compounds into mitochondrial
targeting water-compatible supermolecules for cancer diagnostic and
attenuation in future.
2
2
4

P.-L. Zhang, et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Fig. 5. (a) Fluorescence imaging of mitochondrial membrane potential in the presence different concentrations of BPTPA-G
3
T
3
. [G
3
T ] = 0 μM, 0.1 μΜ, 0.2 μΜ,
3
0
.4 μΜ, 0.8 μΜ. (b) The expression of TrxR and TrxR in BGC-823 cells after incubated with BPTPA-G for 24 h. [G ] = 0 μM, 2 μΜ, 4 μΜ.
1
2
3
T
3
T
3 3
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