Pyridinium and indole orientation determines the mitochondrial uncoupling and anti-cancer efficiency of F16

Article abstract of DOI:10.1016/j.ejmech.2018.05.036

F16 is a mitochondria-targeted, broad-spectrum anticancer agent in the pre-clinic cancer therapy. Here we developed two fluorescent isomers of F16 (o-F16 and m-F16) with entirely different photophysical properties, uncoupling activity, and cytotoxicity by

Full text of DOI:10.1016/j.ejmech.2018.05.036

Accepted Manuscript
Pyridinium and indole orientation determines the mitochondrial uncoupling and anti-
cancer efficiency of F16
Juan Xu, Huan He, Lian-Jiao Zhou, Yu-Zhu Liu, Dong-Wei Li, Feng-Lei Jiang, Yi Liu
PII:
S0223-5234(18)30449-5
10.1016/j.ejmech.2018.05.036
EJMECH 10447
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 February 2018
Revised Date: 21 May 2018
Accepted Date: 22 May 2018
Please cite this article as: J. Xu, H. He, L.-J. Zhou, Y.-Z. Liu, D.-W. Li, F.-L. Jiang, Y. Liu, Pyridinium and
indole orientation determines the mitochondrial uncoupling and anti-cancer efficiency of F16, European
Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.05.036.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
1
2
3
Pyridinium and indole orientation determines the
mitochondrial uncoupling and anti-cancer efficiency
of F16
4
5
a,†
a,†
a
a
a
Juan Xu , Huan He , Lian-Jiao Zhou , Yu-Zhu Liu , Dong-Wei Li ,
a,*
a,b,c,*
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
Feng-Lei Jiang and Yi Liu
a.
State Key Laboratory of Virology & Key Laboratory of Analytical
Chemistry for Biology and Medicine (MOE), College of Chemistry and
Molecular Sciences, Wuhan University, Wuhan 430072, P. R. China
1
1
1
1
1
1
1
1
1
1
2
2
2
b.
College of Chemistry and Material Sciences, Guangxi Teachers
Education University, Nanning 530001, P. R. China
c.
Key Laboratory of Coal Conversion and Carbon Materials of Hubei
Province, College of Chemistry and Chemical Engineering, Wuhan
University of Science and Technology, Wuhan 430081, P. R. China.
†
Theses authors contribute equally to this work.
*Corresponding Author. Tel.: 86-27-68756667. Fax: 86-27-68754067.
Email address: yiliuchem@whu.edu.cn, fljiang@whu.edu.cn.
1

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
3
3
3
3
3
3
3
4
5
6
7
8
9
0
1
2
3
4
5
Abstract F16 is a mitochondria-targeted, broad-spectrum anticancer
agent in the pre-clinic cancer therapy. Here we developed two fluorescent
isomers of F16 (o-F16 and m-F16) with entirely different photophysical
properties, uncoupling activity, and cytotoxicity by merely modifying the
linking orientation of pyridinium and indole units. Individually, o-F16
acted as a strong uncoupler to reduce the mitochondrial respiration
efficiency, while m-F16 could hardly uncouple the mitochondrial
respiration due to its poor proton dissociation capability. Owing to their
intrinsic fluorescence, o-F16 and m-F16 could specifically image
mitochondria in the green and red channel, respectively. This work could
provide useful information for the development of uncouplers and design
of mitochondrial-targeted drugs.
3
3
3
3
4
4
4
4
4
6
7
8
9
0
1
2
3
4
Introduction
Mitochondria have long been considered as the cell’s growth energy
1
factory . Recent studies reveal that mitochondria also play an essential
2
role in the control of cell death . Compared with normal cells,
mitochondria in cancer cells exhibit lower oxidative phosphorylation,
higher trans-membrane potential, increased reactive oxygen species (ROS)
3
and deregulated mitochondrial apoptotic pathway . These features make
mitochondrial-targeted anticancer drugs a very promising strategy for the
3
, 4
malignancy treatments .
2

ACCEPTED MANUSCRIPT
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
F16 (Scheme 1) is a kind of delocalized lipophilic cations (DLC)
5
screened from approximately 16,000 small molecules . F16 selectively
accumulates in mitochondrial matrix driven by the negative
transmembrane potential and induces a strong inhibition of tumour
6
growth . Our previous studies indicated that the F16-triggered cell death
was closely associated with its uncoupling effect on mitochondrial
7
respiration . Treatment of cancer cells with F16 resulted in a significant
decrease in intracellular ATP level and subsequent energetic breakdown.
These features suggested that F16 may serve as an uncoupler on
mitochondria. We have also previous proved that F16 could be utilized as
8
a targeting moiety for selective delivering of fluorescent probes or
9
anticancer drugs into mitochondria. Based on our investigations, F16
could act as a promising fluorescent and toxic substitute for the most
1
0,11,12
commonly used tetraphenylphosphonium (TPP)
. However, there
still exists room for improving the biological activities of F16 such as
fluorescence and toxicity. This requires a detailed investigation on the
structure-activity relationship (SAR) of F16. For example, it has been
reported that incorporation of fluorine on indole ring of F16 could
1
3
enhance its cellular uptake and cytotoxicity . In this work, we developed
two fluorescent F16 isomers (o-F16 and m-F16, Scheme 1) by simply
modifying the linking orientation of pyridinium and indole moiety.
Cellular and sub-cellular investigations suggested the two isomers
3

ACCEPTED MANUSCRIPT
6
6
7
8
significantly varied in fluorescence and toxicity.
6
7
7
7
7
7
7
7
7
7
7
8
8
8
8
8
8
8
8
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
Experimental
Chemicals and reagents Gramine, para-pyridnecarboxaldehyde,
rhodamine123 (Rh123), dinitrophenol (DNP), carbonyl cyanide
p-trifluo-methoxyphenylhydrazone
diacetate (DCFH-DA), 5,5‘,6,6‘-Tetrachloro-1,1‘,3,3‘-tetraethyl-
imidacarbocyanine iodide (JC-1) and rotenone were obtained from Sigma
Aldrich (St. Louis, USA). 3-(4, 5-dimethylthiazol-2-yl)-2,
(FCCP),
2,7-dichlorfluorescein
5-diphenyltetrazolium bromide (MTT) was obtained from Amresco
(Solon, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal
bovine serum (FBS) were purchased from Gibco (MA, USA). Modified
DMEM (no Glucose) were purchased from Thermofisher Scientific (MA,
USA). Annexin V-PE/7-AAD apoptosis kit was purchased from
MultiSciences (Hangzhou, China). Tyrpsin-EDTA solution was purchased
from Beyotime Technology (Shanghai, China). Antibodies were
purchased from Cell Signaling Technology (Boston, USA). All other
common reagents were of analytical reagent grade from Shenshi (Wuhan,
China) and used without further purification.
Synthesis A mixture of para-pyridnecarboxaldehyde (0.30 mL, 3.0
mmol), N-butyl phosphorus (0.74 mL, 3.0 mmol), gramine (348 mg, 2.0
mmol), and 5.0 mL acetonitrile was stirred at 80 °C for 24 h. The mixture
4

ACCEPTED MANUSCRIPT
8
9
9
9
9
9
9
9
9
9
9
9
0
1
2
3
4
5
6
7
8
9
was concentrated under vacuum, and the oil product was purified by
silica chromatography with hexanes/EtOAc (1:1 V/V) to give
non-charged precursor (yield 42.5%) for F16. The non-charged precursor
(10 mg, 0.54 mmol) were added to 3 mL dry DMF at room temperature
and stirred overnight. DMF was removed and the residues were
recrystallized in CHCl (0.3 mL) and n-hexane to obtain F16 (9.74 mg,
3
yield 51.7%). 1H NMR (400 MHz, DMSO-d6): δ=4.169 (s, 3H),
7.250-7.266 (m, 2H), 7.318(s, 1H), 7.526 (d, 1H),7.963(s, 1H),
8.110-8.126(m, 2H), 8.133-8.258(m, 2H), 8.669-8.691(d, 2H), 11.939(s,
1H); ESI-MS (m/z): calcd: 235; found: 235.2. o-F16 and m-F16 were
synthesized from ortho- or meta- pyridnecarboxaldehyde using the same
1
1
1
1
1
1
1
1
1
1
1
00 method. The yields were 35.0% and 54.3%, respectively. 1H NMR for
01 o-F16 (400 MHz, DMSO-d6): δ=4.336 (s, 3H), 7.223-7.518 (m, 3H),
02 7.540 (d, 1H), 7.772(s, 1H), 8.110-8.113(m, 2H), 8.245-8.386(m, 2H),
03 8.523(d, 1H), 8.770 (d, 1H), 12.041(s, 1H); ESI-MS (m/z): calcd: 235;
04 found: 235.2. 1H NMR for m-F16 (400 MHz, DMSO-d6): δ=4.334 (s,
05 3H), 7.187-7.237 (m, 3H), 7.449 (d, 1H),7.885(s, 1H), 8.040-8.092(m,
06 2H), 8.688-8.711(d, 2H), 9.208(s, 1H), 12.041(s, 1H); ESI-MS (m/z):
07 calcd: 235; found: 235.2.
08 Cytotoxicity assay Human gastric carcinoma (SGC-7901) cells, human
09 breast adenocarcinoma cells (MCF-7) and human alveolar basal epithelial
10 cells (A549) were cultured in DMEM medium containing 10% fetal
5

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
11 bovine serum (FBS, Gibco) in a humidified atmosphere containing 5%
4
12 CO at 37 °C. The cells were seeded onto 96-well plates at 10 cells per
2
13 well and incubated for 24 h. F16 isomers with varying concentrations
14 were respectively added to the cells followed by further culture of 24h or
15 48h, and the culture media were discarded. MTT solutions in PBS were
16 added to each well followed by incubation for 4 h. The supernatant was
17 discarded and 150 µL DMSO was added into each well. The absorbance
18 value at 570 nm at each well was recorded by a microplate reader. The
19 survival curves plotted as a function of the concentration of F16 isomers
20 and cell viability rate (%).
21 Confocal laser scanning microscopy measurements SGC-7901 cells
22 were seeded onto 35 mm dishes and incubated for 24 h for attachment.
23 Cells were then treated with o-F16 or m-F16 (10 µM) for 4 h. Mitotracker
24 Red (0.2 µM) or Mitotracker Green (0.2 µM) were used to stain the cells
25 for 30 min. The cells were then examined by confocal laser scanning
26 microscopy. Fluorescence images were obtained by a PerkinElmer
27 UltraVIEW VoX confocal system. The 488 laser was used to excite o-F16
28 or Mitotracker Green, m-F16, and o-F16, and 561 laser was used to excite
29 Mitotracker Red. The fluorescence emission signals were separated by
30 using a 525/25 nm filter (green channel) and a 605/20 nm filter (red
31 channel), respectively.
32 Measurement of mitochondrial membrane potential The SGC-7901
6

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
33 cells were seeded onto 6-well plates overnight. After incubation with F16
34 isomers (1.0 and 5.0 µM) for 4h, stained with JC-1 for 1 h. FCCP (2.0 µM)
35 was set as positive control. The cells were washed 3 times with PBS,
36 dissociated with tyrpsin-EDTA solution, resuspended in PBS, then
37 analyzed with BD ACCURI C6 flow cytometer by detecting the
38 fluorescence of JC-1 at channel 1 and 2.
39 Determination of intracellular ATP The SGC-7901 cells were grown in
40 DMEM medium without or with F16 isomers (5.0 µM) for 48 h.
41 Intracellular ATP levels were determined by a luciferin-luciferase
7
42 method . Then the cells were harvested and lysed according to the
43 manufacturer’s procedure. The intracellular ATP level in SGC-7901 cells
44 cultured in glucose-free DMEM medium (supplemented with 10 mM
45 D-galactose) was also determined using the same method.
46 Annexin V-PE/7-AAD double staining assay SGC-7901 cells were
47 incubated in DMEM medium with 5.0 µM of F16, m-F16 or o-F16 for 48
48 h. Then the cells were harvested, centrifugated, and resuspended in 500
49 µL staining buffer containing both Annexin V-PE and 7-AAD, to a final
50 concentration of 50 nM. After 10 min, the cells were analyzed with BD
51 ACCURI C6 flow cytometer to record the fluorescence in channel 3 and
52 4.
53 Western blot analysis SGC-7901 cells were treated with F16 isomers for
54 24h. The whole cell pellets were then lysed with RIPA lysis and
7

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
55 extraction buffer (25 mM Tris·HCl, 150 mM NaCl, 1% NP-40, 1%
56 sodium deoxycholate, 1% SDS, pH=7.6) for 30 min. After 13000 rpm
57 centrifugation for 10 min, the supernatant was determined by BCA
58 protein assay kit (Beyotime, Wuhan, China). An equal amount of the total
59 protein was loaded onto an SDS-PAGE minigel and electro-transffered
60 onto PVDF membranes. Proteins were detected using primary antibodies,
61 followed by HRP-conjugated secondary antibody and visualized using
62 ECL substrate. Immunoblot quantification was performed by ImageJ
63 software.
64 Isolation of rat liver mitochondria Wistar rats (200-250 g) were
65 purchased from Hubei Research Centre of Experimental Animals (Wuhan,
66 China). Rat liver mitochondria were isolated by standard differential
14
67 centrifugation . The concentration of mitochondria protein was measured
68 by the Biuret method. All operations were performed at 0-4 ºC.
69 Measurement of mitochondrial respiration Mitochondrial respiration
70 rates of isolated mitochondria were measured with a Clark-type oxygen
71 electrode in a 1 mL thermostated, water-jacketed, closed chamber with
72 magnetic stirring. Mitochondria (1 mg/protein) were incubated in 1 mL
73 medium containing 250 mM sucrose, 20 mM KCl, 5 mM K HPO4, 10
2
74 mM HEPES, and 2 mM MgCl , 1 µM rotenone, for 3 min before
2
75 energization with 5 mM succinate (state 4). State 3 respiration were
76 initiated by adding ADP (100 µM).
8

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
77 Measurement of mitochondrial membrane potential of isolated liver
78 mitochondria Changes in mitochondrial membrane potential (∆Ψ ) of
m
79 isolated liver mitochondria were indicated by the accumulation of Rh123
80 (250 µM) as monitored by the changes in fluorescence emission intensity
1
5
81 at 25 ºC (λ = 488 nm, λ = 525 nm) .
ex
em
+
82 Measurement of mitochondrial H permeabilization Mitochondrial
83 swelling was measured spectrophotometrically by monitoring the
84 absorbance at 540 nm over 10 min at 25 ºC by UNICO 4802 UV-Vis
85 Double Beam Spectrophotometer. Mitochondria were suspended in 2 mL
86 buffer containing 135 mM K-acetate, 5 mM HEPES, 0.1 mM EGTA, 0.2
87 mM EDTA and 2 µM rotenone.
88 Measurement of mitochondrial membrane fluidity Mitochondrial
89 membrane fluidity was measured by the fluorescence anisotropy changes
90 of hematoporphyrin (HP)-labeled mitochondria at 626 nm (λ = 520 nm)
ex
91 by an LS-55 fluorophotometer (Perkin-Elmer, Norwalk, CT).
92 Observation of mitochondrial ultrastructure Observations of
93 mitochondrial ultrastructure were performed with a JEM-100CX II
94 transmission electron microscope (JEOL, Tokyo, Japan). Mitochondria
95 were incubated in the presence of 50 µM of F16, o-F16 or m-F16 at 25 ºC
96 for 30 min, and then fixed for 30 min at 4 ºC using glutaraldehyde at a
97 final concentration of 2.5% (v/v) in PBS buffer, then the micropellets
98 were postfixed with 1% (w/v) osmium tetroxide and dehydrated.
9

ACCEPTED MANUSCRIPT
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
99 Molecular dynamics simulations We used NAMD 2.6 for running MD
15
00 simulations and the Adaptive Biasing Force (ABF) method for
01 modeling the potentials of mean force (PMF). The temperature was
16
02 maintained with the Berendsen thermostat using a coupling parameter
-1
03 of 5 ps . The pressure was maintained at 1 atm by the Langevin piston
1
7
04 method with piston mass of 100 atomic mass unit and Langevin
-1
05 collision frequency of 50 ps . Non-bond interactions were computed
06 using the particle mesh Ewald method with 10 Å cutoff for the
07 electrostatic interactions and the switching function between 10 and 12 Å
08 for the van der Walls interactions. The non-bond interaction list was
09 constructed using a cutoff of 14 Å, update every 50 steps. The covalent
10 bonds involving hydrogen atoms were constrained using the SHAKE
11 algorithm. MD integration step size was set to 2 fs.
12 Molecular structure of hydrated palmitoyloleylphosphocholine (POPC)
1
8
13 bilayer was constructed In VMD software . The hydrated POPC bilayer
14 consisted of 100 lipid unit with an aqueous phase surrounded by 5450
15 water molecules. The system was large enough to ensure that the F16
16 isomers in the middle of the membrane did not perturb the entire
17 membrane structure. Molecular force field parameterization for F16
18 isomers was obtained by the combination of the standard CHARMM27
19 parameters with parameters determined by ab initio calculations using the
20 GAUSSIAN 09 program. The equilibrium bond lengths and angles were
1
0

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
21 obtained from DFT calculations with B3LYP functional and Pople
22 6-31G(d) basis set. Partial charges of atoms were calculated by
23 electrostatic potential fitting. At the starting conformation, the F16
24 molecule (or o-F16, m-F16) was 25 Å away from the upper of the lipid
25 membrane.
26 MD simulations were performed in the NPT ensemble at T = 320 K and
27 pressure 1 atm. After initial 25 ns equilibration, the ABF calculations in
28 the NAMD implementation were initiated by dragging the N atom on
29 pyridine moiety from the bulk toward the positive half of the z-axis
30 toward the bilayer. The dragging force was determined within the
31 classical thermodynamic integration formalism. The free energy
32 derivative was estimated locally as the simulation progresses, thereby
33 providing a continuous update of the biasing force. When applied to the
34 system, this bias generates a Hamiltonian exempt of a net average force
35 along z. MD simulations for each F16 isomer continued for 160 ns.
36
2
2
2
2
2
2
37 Results and Discussion
38
F16, o-F16, and m-F16 were first synthesized in two steps from
39 gramine and para-, ortho- or meta-pyridinecarboxaldehyde, respectively
40 (Scheme 1). The absorption and emission spectra of the three compounds
41 are shown in Fig. S1. Surprisingly, these isomers were entirely different
42 in absorption and emission spectra despite their high similarity in
1
1

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
43 chemical structures. F16 and o-F16 gave green fluorescence with
44 emission maxima at 523 and 509 nm respectively, while m-F16 gave red
45 fluorescence with an emission maximum at 602 nm. The difference in
46 emission maximum of m-F16 came from its significant lower energy gap
47 between HOMO and LUMO (Fig. S1c). Especially, the configuration of
48 LUMO of m-F16 was entirely different from F16 and m-F16, while their
49 HOMO configurations were similar.
2
2
50
51
52
Scheme 1 Synthetic route of F16, o-F16 and m-F16.
2
2
2
2
2
2
2
2
We then turned to investigate the subcellular localization of o-F16 and
53 m-F16 in human gastric carcinoma (SGC-7903) cell line by their intrinsic
54 fluorescence. Cells were incubated with 10 µM of o-F16 or m-F16 for 4
55 hours, and confocal microscopic imaging experiments were then
56 conducted as shown in Fig. 1 and Fig. S3. It can be observed that the
57 green fluorescence of o-F16 was perfectly overlapped with the red
58 fluorescence of Mito Tracker Red (Pearson’s Correlation = 0.943), while
59 the red fluorescence of m-F16 was perfectly overlapped with the green
1
2

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
60 fluorescence of Mito Tracker Green (Pearson’s Correlation = 0.970),
61 indicating that both of o-F16 and m-F16 could be accumulated in
62 mitochondria of SGC-7901 cells. This might arise from the excellent
63 dispersity of positive charge of the three compounds (Fig. S2). To
64 preclude cell line-specific artifacts, localization experiment was also
65 performed on human breast adenocarcinoma cell line (Fig. S4).
2
2
66
67 Fig. 1 Confocal fluorescence imaging of o-F16 (a-c) and m-F16 (d-e) in
2
2
2
2
2
2
68 SGC-7901 cells. (a) Fluorescent image of o-F16 in SGC-7901 cells (10
69 µM, λ = 488 nm. (b) Fluorescent image of Mitotracker Red (0.2 µM, λex
ex
70 = 561 nm). (c) Merged image of a and b. (d) Fluorescent image of
71 Mitotracker Green (0.2 µM, λ = 488 nm). (e) Fluorescent image of
ex
72 o-F16 (10 µM, λ = 488 nm). (f) Merged images of d and e. The inserted
ex
73 bar indicates 20 µm.
1
3

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
74
The cytotoxicity of the three compounds against three different tumor
75 cell lines were then investigated by a standard MTT [(3-(4,
76 5)-dimethylthiazol)-2,5-diphenyltetrazolium bromide] method (Fig. 2a,
77 Table S1). In agreement with previous reports, F16 isomers displayed
78 strong cytotoxicity on SGC-7901 cells (IC = 3.5 ± 0.3 µM). However,
5
0
79 the cases were entirely different for its two isomers. As shown in Fig. 2a,
80 o-F16 exhibited inhibitory effects on viability and growth of SGC-7901
81 cells (IC = 5.7 ± 0.7 µM), while the inhibition effects of m-F16 (IC >
50
50
82 50 µM) were feeble. The inhibition rate of SGC-7901 cells treated with
83 20 µM of m-F16 was less than 15%. We further investigated the effects of
84 the three compounds on the physiological properties of mitochondria.
85 Changes in mitochondrial membrane potential ∆Ψ were indicated by the
m
19
86 ratio of red and green fluorescence of JC-1 . We can observe that F16
87 and o-F16 significantly induced the dissipation of ∆Ψ after 4 h
m
88 incubation, while the apoptosis of the cell has not been initiated (Fig. S5)
89 The effect of m-F16 on ∆Ψ dissipation was very insufficient. In
m
90 mitochondria, ATP synthesis was closely related to ∆Ψ . As shown in Fig.
m
91 2c, the intracellular level of ATP in SGC-7901 cells decreased about
92 47.2%, 42.1% and 5.1% after the treatment of 5 µM F16, o-F16 and
93 m-F16. The results of ∆Ψ dissipation and ATP synthesis blockage
m
94 indicated that m-F16 was less mitochondriotoxic than F16 and o-F16.
95 The strong inhibition effects of F16 and o-F16 on mitochondria finally
1
4

ACCEPTED MANUSCRIPT
2
2
2
96 resulted in apoptosis in SGC-7901 cells, as revealed by a typical Annexin
2
0
97 V-PE/7-AAD double staining assay , while the effect of m-F16 on
98 apoptosis was not significant.
2
3
99
00 Fig. 2 (a) Cell viability of SGC-7901 cells incubated with different
3
3
3
3
3
3
3
01 dosage of F16 isomers for 48h. (b) The collapse of mitochondrial
02 membrane potential (∆Ψ ). SGC-7901 cells were incubated with F16
m
03 isomers for 4h, followed by JC-1 staining. The JC-1 red/green ratios are
04 presented as mean ± SD of three independent samples. Greater red/green
05 ratio values reflect more polarized ∆Ψ . (c) The change of intracellular
m
06 ATP level. SGC-7901 cells were treated with F16 isomers for 48h, and
07 ATP level was determined. (d) The results of apoptosis of SGC-7901 cells
1
5

ACCEPTED MANUSCRIPT
08 by Annexin-PE/7-AAD double staining assay. Cells were treated by F16
09 isomers (5.0 µM) for 48h. Experiments were all performed in three
10 replicates, and the error bars represent standard deviations.
11
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
12
Cells cultured in DMEM medium could compensate for the loss of
13 mitochondrial ATP by glycolysis. The intracellular ATP and apoptosis
14 assay was also conducted on SGC-7901 cells incubated in glucose-free
15 medium containing 10 mM galactose. As shown in Fig. S6, we can find
16 the ATP level significantly decreased after the addition of F16 isomers in
17 galactose supplemented medium. Notably, m-F16, which could hardly
18 impair the ATP production in glucose-containing medium, reduce the ATP
19 level by ~42%. The percentage of apoptosis cells also increased as the
20 ATP amount decreased.
21
To character the apoptotic pathways involved in o-F16 and m-F16
22 induced lethality, we determined the expression level of Bcl-2 and Bax,
23 two critical apoptosis-related proteins localized to the outer membrane of
24 mitochondria. As shown in Fig. 3, o-F16 up-regulated Bcl-2 and
25 down-regulated Bax, while the effect of m-F16 was relatively weaker.
26 These results supported o-F16 and F16 induced cell death in
27 mitochondrial apoptosis pathway. As Caspase3 is known as the apoptotic
28 executor, we also monitored the expression level of cleaved-Caspase3.
29 We observed that o-F16 significant increased the expressions of
1
6

ACCEPTED MANUSCRIPT
3
3
30 cleaved-Caspase3, while the effects of m-F16 were minor. These results
31 were in good agreements with the cell death experiments.
3
3
32
33 Fig. 3 F16 isomers induce apoptosis through regulation of Bcl-2/Bax and
3
3
3
3
3
3
3
34 activation of Caspase3. After treatment with m-F16 and o-F16 for 48h,
35 the expression level of Bcl-2, Bax, and cleaved-Caspase3 were
36 determined by western blot analysis. GADPH was measured as a loading
37 control. a, control; b, 1 µM m-F16; c, 5 µM m-F16; d, 1 µM o-F16; e, 5
38 µM o-F16. Experiments were all performed in three replicates, and the
39 error bars represent standard deviations.
40
1
7

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
41
The substantial effects on ∆Ψ collapse and ATP degradation indicated
m
21
42 that F16 and o-F16 might act as a mitochondrial uncoupler . Isolated
43 mitochondria have been validated as an efficient model to investigate the
2
2, 23
44 direct toxic effects of mitochondrial-targeted drugs or poisons
. Rat
45 liver mitochondria were isolated by differential centrifugation method as
22
46 previously described . It has to be mentioned that there exist differences
47 between mitochondria of cancerous and non-cancerous tissue, but the
48 isolated mitochondrial could serve as useful tools for the investigation of
49 mitochondrial respiration and uncoupling. Effects of the three compounds
50 on respiration of isolated mitochondria were measured using a standard
51 polarographic technique with a Clark-type oxygen electrode. As shown in
52 Fig. 4a, the state 3 (ADP-stimulated) respiration rates were slightly
53 reduced with the addition of F16, o-F16 or m-F16, while the state 4 (non
54 ADP-stimulated) respiration increased in a dose-dependent manner. At a
55 concentration of 50 µM, the state 4 respiration rates of mitochondria
56 treated with F16 and o-F16 increased about two folds, while that of
57 m-F16 increased about 50%. These results indicated that F16 and o-F16
58 induced a stronger uncoupling effect than m-F16. The efficiency of
59 oxidative phosphorylation was assessed in Fig. 4b by calculating the
24
60 respiratory control ratio (RCR), defined as the rate of state 3 and state 4
61 respiration. We can find in Fig. 4b that the respiration efficiency
62 decreased after treatment of F16 isomers. Notably, the uncoupling
1
8

ACCEPTED MANUSCRIPT
3
3
3
63 efficiency of F16 and o-F16 at 50 µM was equivalent to 2,
64 4-dinitrophenol (DNP) at the same concentration, which was widely
65 regarded as a strong mitochondrial uncoupler.
3
3
66
67 Fig. 4 Uncoupling effects of F16, o-F16 and m-F16. (a) Effects of F16
3
3
3
3
3
3
3
68 isomers on state 3 and 4 respiration rates. The O consumption represents
2
69 the averaged consumption speed in incubation time of 5 minutes, and
70 error bars represent standard deviations. (b) Effects on respiration control
71 ratio (RCR) at a concentration of 50 µM. DNP (50 µM) was used as a
72 positive control. (c) F16 isomers (50 µM) increased the IMM permeation
+
+
73 to H . IMM permeation to H was monitored by the 540 nm absorbance
74 of mitochondrial suspension in medium containing K-Acetate. (d)
1
9

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
75 Protective effects of 6-Kch, CAtr, and GDP on F16 induced
76 mitochondrial membrane potential (∆Ψ ) collapse. ∆Ψ was recorded by
m
m
77 the fluorescence intensity of Rh123.
78
79
To further examine the mechanism for uncoupling action of F16
80 isomers, we investigated their effects on mitochondrial swelling in
2
5
81 hyperosmotic potassium acetate medium . Protonated acetate can cross
82 the mitochondrial inner membrane (MIM) and dissociate to acetate anion
+
83 and H , producing a proton gradient. Matrix swelling occurred when the
84 proton gradient was dissipated. We can find in Fig. 4c that absorbance
85 decreased rapidly in the presence of F16 isomers, indicating a quick
86 dissipation on proton gradient across the MIM. These result represented
87 that the three compounds induced the proton leak from the mitochondrial
88 matrix into the inter-membrane space, that is to say, F16 and its two
14
89 isomers acted as protonophores to affect the respiration . The direct
90 influence of uncoupling of mitochondrial respiration induced by F16 and
91 o-F16 was the collapse of ∆Ψ (Fig. 4d), as indicated by the increase of
m
1
5
92 fluorescence intensity of Rhodamine 123 (Rh123). In accordance with
93 the measurement in SGC-7901 cells, the ∆Ψ collapse induced by m-F16
m
94 was significantly weaker than F16 and o-F16. The dissipation in ∆Ψm
95 induced by F16 isomers could be partially reserved by the adding of
96 6-ketocholestanol (recoupler for FCCP), CAtr (an inhibitor of adenine
2
0

ACCEPTED MANUSCRIPT
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
97 nucleotide translocator) and GDP (an inhibitor for uncoupling proteins),
98 indicating the uncoupling effects of the three isomers were related with
1
6
99 various transporters located in the MIM .
00
Recent studies have reported that dissipation of ∆Ψ was always
m
01 accompanied by the occurrence of mitochondrial membrane permeation
29
02 transition (MPT) . We then turned to investigate the effects of F16
03 isomers on MPT from the observation of mitochondrial ultrastructure. As
04 shown in Fig. 5(a), mitochondrial extracted from rat liver maintained
05 their integrity, with classical ultrastructure containing well defined outer
06 membrane, narrow intermembrane space, and dense cristae. After
07 incubation with F16 (b) and o-F16 (c) for 30 minutes, an increase of
08 mitochondrial volume was observed, this indicated that mitochondrial
09 swelling occurred. The mitochondria appeared to maintain a globular
10 configuration with decreased matrix electron density, clustered cristae,
11 enlarged intermembrane space and expanded volume, which was the most
12 direct evidence of membrane permeability transition (MPT) occurrence.
13 As for m-F16 (Fig. 5d), no visible matrix swelling was observed. In good
14 agreement with its weaker uncoupling activity, effects of m-F16 on
15 structure and function of mitochondria was minor, which could be
16 revealed from the monitoring of matrix swelling (Fig. S7) and
17 measurement of inner membrane fluidity (Fig. S8).
2
1

ACCEPTED MANUSCRIPT
4
4
18
19 Fig. 5 Effects of F16 isomers on mitochondrial ultrastructure observed by
20 the transmission electron microscopy (TEM). (a) Fresh liver
21 mitochondrial were incubated in medium containing 200 mM sucrose, 10
22 mM Tris, 10 mM Mops, 1 mM Na PO , 10 µM EGTA-Tris, 0.5 µg/mL
4
4
4
4
4
4
4
4
3
4
23 oligomycin, 5 mM succinate and 2 µM rotenone; pH = 7.4. (b)
24 Mitochondria were incubated in the same medium with the addition of 50
25 µM F16 for 30 min. (c) 50 µM o-F16 for 30 min. (d) 50 µM m-F16 for 30
26 min. The inserted bar represents 0.5 µm.
27
2
2

ACCEPTED MANUSCRIPT
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
28
We then turned to investigate the reason for the discrepancy in
29 uncoupling efficiency of the three compounds. It has been widely
30 accepted that the uncoupling activity for an uncoupler was determined by
31 its proton dissociation capability and translocation barrier across the
16
32 MIM (Fig. 6a). Molecular dynamic (MD) simulations approach was
33 then applied to stimulate the movement of F16 isomers through a bilayer
30
34 planar phospholipid membrane (BLM). Adaptive biasing force (ABF)
35 was applied to the nitrogen atom on pyridine moiety. The free energy
36 profile was constructed by integrating the mean force applied to the
37 molecule along the membrane plane. We can find in Fig. 6b that the
38 transmembrane barriers (∆G ) for F16 isomers were very low due to their
p
39 planar and hydrophilic molecular structure. More importantly, the
40 transmembrane barrier for the three isomers was very similar in value.
41 The capability of the three compounds to dissociate proton was also
42 investigated by theoretic methods. The total energy of F16 isomers
43 (Epronated) and their dissociated forms (Edissociated) were calculated using
44 density functional theory (DFT) method with 6-31G(d) basis set. The
31
45 dissociation energies (∆G ) for F16 or its isomers were then calculated
d
46 as (Epronated - Edissociated). As shown in Fig. 6c, we can observe that the
47 dissociation energy for m-F16 was relatively higher than that of F16 and
48 o-F16, indicating m-F16 was more difficult to dissociate proton. The
49 difference in proton dissociation cloud also be reflected from the
2
3

ACCEPTED MANUSCRIPT
4
4
4
4
4
4
4
50 measurement of dissociation constant (pK ) for the compounds. In the
a
51 cellular mitochondrial matrix, the pH was in the range of 7.8 to 8.1
3
2
52 depending on the cell types . The high pK value for m-F16 (pK = 8.83)
a
a
53 than F16 and o-F16 (7.96 and 8.15, respectively) indicated that m-F16
54 might be difficult to dissociate protons in the mitochondrial matrix. These
55 results were in good agreements with the significant lower uncoupling
56 efficiency.
4
4
57
58 Fig. 6 (a) Scheme of F16 isomers-mediated proton transport across the
4
4
4
59 MIM. (b) Free energy profiles of F16 isomers translocation across the
60 membrane calculated from MD simulations. (c) Dissociation energies for
61 F16 isomers.
2
4

ACCEPTED MANUSCRIPT
4
62
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
63 Conclusion
64
In conclusion, we presented here two isomers of F16 (o-F16 and
65 m-F16) with different fluorescence by merely modifying the linking
66 orientation of pyridinium and indole. Fluorescent images of live cells and
67 MD simulations indicated the two isomers could readily diffuse across
68 the biomembranes and accumulate into mitochondria. Toxicological data
69 on SGC-7901 cell and isolated mitochondria indicated that o-F16 acted as
70 a strong uncoupler to reduce the respiration efficiency and subsequently
71 induced mitochondrial membrane permeation transition and cellular
72 apoptosis. Differently, m-F16 could hardly uncouple the mitochondrial
73 respiration due to its poor proton dissociation capability. Importantly,
74 m-F16 was red-emitting and low toxic, making it a very promising
75 fluorescent substitute for TPP to delivery specific probes or sensors into
76 mitochondria. This work may open a new door for the development of
77 mitochondrial uncouplers and design of novel mitochondrial-targeted
78 drugs.
79
4
4
4
80 Conflicts of interest
81 There are no conflicts to declare.
82
4
83 Acknowledgements
2
5

ACCEPTED MANUSCRIPT
4
4
4
84 This work was supported by the National Natural Science Foundation of
85 China (21673166, 21473125), Guangxi Science and Technology Project
86 (GuiKeAD17195081), and Bagui Scholar Program of Guangxi Province
4
4
87 (2016).
88
4
89 References
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
1 N. Raimundo, Trends Mol. Med., 2014, 20, 282-292.
2 S. W. Tait and D. R. Green, Nat. Rev. Mol. Cell Biol., 2010, 11, 621-632.
3 S. Fulda, L. Galluzzi and G. Kroemer, Nat. Rev. Drug Discov., 2010,
447-464.
9
,
4 A. Wongrakpanich, S. M. Geary, M. L. Joiner, M. E. Anderson and A. K.
Salem, Nanomedicine, 2014, , 2531-2543.
5 V. R. Fantin, M. J. Berardi, L. Scorrano, S. J. Korsmeyer and P. Leder, Cancer
Cell, 2002, , 29-42.
9
2
6 V. R. Fantin and P. Leder, Cancer Res., 2004, 64, 329-336.
7 J. Wang, H. He, C. Xiang, X. Yang Fan, L. Y. Yang, L. Yuan, F. L. Jiang and
Y. Liu, Toxicol. Sci., 2017. DOI: 10.1093/toxsci/kfx218.
8 H. He, D. W. Li, L. Y. Yang, L. Fu, X. J. Zhu, W. K. Wong, F. L. Jiang and Y.
Liu, Sci. Rep., 2015,
9 J. Wang, X.-Y. Fan, L.-Y. Yang, H. He, R. Huang, F.-L. Jiang and Y. Liu,
MedChemComm, 2016, , 2016-2019.
5, 13543.
7
10K. Rohlenova, K. Sachaphibulkij, J. Stursa, A. Bezawork-Geleta, J. Blecha, B.
Endaya, L. Werner, J. Cerny, R. Zobalova, J. Goodwin, T. Spacek, E. Alizadeh
Pesdar, B. Yan, M. N. Nguyen, M. Vondrusova, M. Sobol, P. Jezek, P. Hozak,
J. Truksa, J. Rohlena, L. F. Dong and J. Neuzil, Antioxid. Redox Sign., 2017,
26, 84-103.
2
6

ACCEPTED MANUSCRIPT
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
11G. Cheng, J. Zielonka, O. Ouari, M. Lopez, D. McAllister, K. Boyle, C. S.
Barrios, J. J. Weber, B. D. Johnson, M. Hardy, M. B. Dwinell and B.
Kalyanaraman, Cancer Res., 2016, 76, 3904-3915.
12J. Rohlena, L.-F. Dong, K. Kluckova, R. Zobalova, J. Goodwin, D. Tilly, J.
Stursa, A. Pecinova, A. Philimonenko, P. Hozak, J. Banerjee, M. Ledvina, C.
K. Sen, J. Houstek, M. J. Coster and J. Neuzil, Antioxid. Redox Sign., 2011, 15
,
2923-2935.
13L. Ma, J.-X. Dong, C. Wu, X.-Y. Li, J. Chen, H. Zhang, Y. Liu, J. Mem. Biol.
,
2017, 250, 195-204.
14D.-W. Li, H. He, B. Lin, Z.-Q. Xu, F.-L. Jiang, Y. Liu, RSC Adv., 2014, 4,
3913-3919.
15J. C. Philips, R. Braun, W. Wang, J. Gumbart, E. Tajkhorshid, E. Villa, C.
Chipot, R. D. Skeel, L. Kale, K. Schulten, J. Comput. Chem., 2005, 26,
1781-1802.
16S. J. A. Koh, H. P. Lee, C. Lu, Q.-H. Cheng, Physic. Rev. B, 2005, 72, 085414.
17H. He, J. Xu, D.-Y. Chen, F.-L. Jiang, Y. Liu, J. Phys. Chem. B, 2017, 121,
1211-1221.
18W. Humphrey, A. Dalke, K. Schulten, J. Mol. Graph. Model., 1996, 14, 33-38.
19W. Zhou, X. Wang, M. Hu, C. Zhu and Z. Guo, Chem. Sci., 2014,
5,
2761-2770.
20X. Y. Fan, Y. J. Liu, K. Chen, F. L. Jiang, Y. J. Hu, D. Liu, Y. Liu and Y. S.
Ge, Eur. J. Med. Chem. 2017. DOI: 10.1016/j.ejmech.2017.05.022
21D. N. Silachev, L. S. Khailova, V. A. Babenko, M. V. Gulyaev, S. I.
Kovalchuk, L. D. Zorova, E. Y. Plotnikov, Y. N. Antonenko and D. B. Zorov,
Biochim. Biophys. Acta - Gen. Sub., 2014, 1840, 3434-3442.
22J. Li, Y. Zhang, Q. Xiao, F. Tian, X. Liu, R. Li, G. Zhao, F. Jiang and Y. Liu, J.
Hazard. Mater., 2011, 194, 440-444.
23Y. N. Antonenko, S. S. Denisov, D. N. Silachev, L. S. Khailova, S. S.
Jankauskas, T. I. Rokitskaya, T. I. Danilina, E. A. Kotova, G. A. Korshunova,
2
7

ACCEPTED MANUSCRIPT
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
E. Y. Plotnikov and D. B. Zorov, Biochim. Biophys. Acta - Gen. Sub., 2016,
1860, 2463-2473.
24C. Porter, N. M. Hurren, M. V. Cotter, N. Bhattarai, P. T. Reidy, E. L. Dillon,
W. J. Durham, D. Tuvdendorj, M. Sheffield-Moore, E. Volpi, L. S. Sidossis, B.
B. Rasmussen and E. Borsheim, Am. J. Physiol. Endoc. M., 2015, 309
,
E224-232.
25M. A. Fernandes, J. B. Custodio, M. S. Santos, A. J. Moreno and J. A. Vicente,
Mitochondrion, 2006, , 176-185.
26S. S. Denisov, E. A. Kotova, E. Y. Plotnikov, A. A. Tikhonov, D. B. Zorov, G.
6
A. Korshunova and Y. N. Antonenko, Chem. Commun., 2014, 50
,
15366-15369.
27Y. Jang, S. Kim, W. K. Oh, C. Kim, I. Lee and J. Jang, Chem. Commun., 2014,
50, 15345-15347.
28Y. N. Antonenko, S. S. Denisov, L. S. Khailova, P. A. Nazarov, T. Rokitskaya,
V. N. Tashlitsky, A. M. Firsov, G. A. Korshunova and E. A. Kotova, Biochim.
Biophys. Acta - Biomembranes, 2017, 1859, 377-387.
29F. Sedlic, A. Sepac, D. Pravdic, A. K. S. Camara, M. Bienengraeber, A. K.
Brzezinska, T. Wakatsuki, Z. J. Bosnjak, Amer. J. Physiol.-Cell Physiol., 2010,
299, 506-515.
30F. F. Severin, Severina, II, Y. N. Antonenko, T. I. Rokitskaya, D. A.
Cherepanov, E. N. Mokhova, M. Y. Vyssokikh, A. V. Pustovidko, O. V.
Markova, L. S. Yaguzhinsky, G. A. Korshunova, N. V. Sumbatyan, M. V.
Skulachev and V. P. Skulachev, Proc. Natl. Acad. Sci. USA, 2010, 107
663-668.
,
31S. Zhang, J. Comput. Chem., 2012, 33, 2469-2482.
32A. Wiederkehr, Islets, 2009, 1, 154-156.
5
65
2
8