A long-linker conjugate of fluorescein and triphenylphosphonium as mitochondria-targeted uncoupler and fluorescent neuro- and nephroprotector

Article abstract of DOI:10.1016/j.bbagen.2016.07.014

Background Limited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory models of diseases. Linking a triphenyl-phosphonium cation to fluorescein through a decyl (C₁₀) spacer yields a fluorescent uncoupler, co

Full text of DOI:10.1016/j.bbagen.2016.07.014

BBAGEN-28554; No. of pages: 11; 4C: 2, 5, 6, 7, 8, 9
Biochimica et Biophysica Acta xxx (2016) xxx–xxx
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Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbagen
A long-linker conjugate of fluorescein and triphenylphosphonium as
mitochondria-targeted uncoupler and fluorescent neuro-
and nephroprotector
a,
a,b
a
a
Yuri N. Antonenko ⁎, Stepan S. Denisov , Denis N. Silachev , Ljudmila S. Khailova ,
a
a
a
a
Stanislovas S. Jankauskas , Tatyana I. Rokitskaya , Tatyana I. Danilina , Elena A. Kotova ,
Galina A. Korshunova , Egor Y. Plotnikov , Dmitry B. Zorov
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia
Faculty of Chemistry, Lomonosov Moscow State University, Moscow 119991, Russia
a
a
a
a
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Background: Limited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory
models of diseases. Linking a triphenyl-phosphonium cation to fluorescein through a decyl (C10) spacer yields
a fluorescent uncoupler, coined mitoFluo, that selectively accumulates in energized mitochondria (Denisov
et al., Chem.Commun. 2014).
Methods: Proton-transport activity of mitoFluo was tested in liposomes reconstituted with bacteriorhodopsin.
To examine the uncoupling action on mitochondria, we monitored mitochondrial membrane potential in parallel
with oxygen consumption. Neuro- and nephroprotecting activity was detected by a limb-placing test and a
kidney ischemia/reperfusion protocol, respectively.
Received 22 April 2016
Received in revised form 16 June 2016
Accepted 16 July 2016
Available online xxxx
Keywords:
Mitochondria
Mild uncoupler
Protonophore
Fluorescein
Results: We compared mitoFluo properties with those of its newly synthesized analog having a short (butyl)
spacer (C
resulting in stimulation of mitochondrial respiration. The dramatic difference in the uncoupling activity of
mitoFluo and C -mitoFluo was in line with the difference in their protonophoric activity on a lipid membrane.
The accumulation of mitoFluo in mitochondria was more pronounced than that of C -mitoFluo. MitoFluo
4 4
-mitoFluo). MitoFluo, but not C -mitoFluo, caused collapse of mitochondrial membrane potential
Membrane potential
4
4
decreased the rate of ROS production in mitochondria. MitoFluo was effective in preventing consequences of
brain trauma in rats: it suppressed trauma-induced brain swelling and reduced a neurological deficit. Besides,
mitoFluo attenuated acute kidney injury after ischemia/reperfusion in rats.
Conclusions: A long alkyl linker was proved mandatory for mitoFluo to be a mitochondria- targeted uncoupler.
MitoFluo showed high protective efficacy in certain models of oxidative stress-related diseases.
General significance: MitoFluo is a candidate for developing therapeutic and fluorescence imaging agents to treat
brain and kidney pathologies.
©
2016 Published by Elsevier B.V.
Abbreviations: Δψ, transmembrane electric potential difference; BLM, bilayer lipid
1. Introduction
membrane; DPhPC, diphytanoylphosphatidylcholine; C
mitoFluo (or 10-mitoFluo), 10-[2-(3-hydroxy-6-oxo-xanthen-9-yl)benzoyl]oxydecyl-
triphenyl-phosphonium bromide; -mitoFluo, 4-[2-(3-hydroxy-6-oxo-xanthen-9-
yl)benzoyl]oxybutyl-triphenyl-phosphonium bromide; C -FL, fluorescein n-octyl ester;
4
R1, rhodamine 19 n-butyl ester;
C
Uncouplers of oxidative phosphorylation of protonophoric type
dissipate the electrochemical potential of hydrogen ions ðΔμ Þ on the
C
4
þ
8
H
Amplex Red, N-acetyl-3,7-dihydroxyphenoxazine; TMRE, tetramethylrhodamine ethyl ester;
CCCP, carbonyl cyanide m-chlorophenylhydrazone; DNP, 2,4-dinitrophenol; MitoDNP, 3-(3,5-
dinitro-4-hydroxyphenyl)propyltriphenylphosphonium methanesulfonate; FCCP, carbonyl
cyanide-p-trifluoromethoxyphenylhydrazone; DiS-C3-(5), 3,3′-dipropylthiodicarbocyanine io-
dide; BHT, butylated hydroxytoluene; HRP, horseradish peroxidase; FCS, fluorescence correla-
tion spectroscopy; MR, magnetic resonance; MRI, magnetic resonance imaging; PBS,
phosphate buffered saline; PIA, peak intensity analysis; ROS, reactive oxygen species; I/R, ische-
mia/reperfusion; TBI, traumatic brain injury; BUN, blood urea nitrogen; i/n, intranasal.
inner mitochondrial membrane which is known to couple respiratory
pumps with ATP synthesis. The most popular uncoupler 2,4-dinitrophe-
nol (DNP) was used to fight obesity at the beginning of the 20th century
[1]. Recently, DNP and other uncouplers have been shown to exert ef-
fective protection in diabetic models in mice and rats [2–4], besides
they display neuroprotective and cardioprotective activities in different
models [5]. Since mitochondria is a major source of reactive oxygen
species (ROS) playing important role in the development of oxidative
stress-related pathologies [6–8], the beneficial action of uncouplers
⁎
Corresponding author at: Belozersky Institute of Physico-Chemical Biology, Moscow
State University, Moscow 119991, Russia.
E-mail address: antonen@genebee.msu.ru (Y.N. Antonenko).
http://dx.doi.org/10.1016/j.bbagen.2016.07.014
0304-4165/© 2016 Published by Elsevier B.V.
Please cite this article as: Y.N. Antonenko, et al., A long-linker conjugate of fluorescein and triphenylphosphonium as mitochondria-targeted
uncoupler and fluorescent neuro- and neph..., Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbagen.2016.07.014

2
Y.N. Antonenko et al. / Biochimica et Biophysica Acta xxx (2016) xxx–xxx
could be related to the steep dependence of the ROS generation
on ΔμH [9,10]. Of relevance to this point, it has been hypothesized
þ
that one of the key functions of mitochondrial uncoupling pro-
teins (UCPs) is to lower the mitochondrial membrane potential
if it exceeds a certain threshold, thus regulating ROS production
[
11,12].
High concentrations of uncouplers exert deleterious action obviously
through the impairment of mitochondrial energetics and a drop in ATP
concentration. In fact, uncouplers are used as pesticides [13] and are
promising candidates for antibiotics [14]. Unfortunately, overdosage of
DNP caused several deaths in humans prior to the ban of its use as a
drug [15]. Thus, it is of importance to find a “mild uncoupler” having a
broad window between concentrations starting to decrease the
membrane potential and concentrations blocking ATP synthesis. The
well-known antioxidant butylated hydroxytoluene (BHT) has been
found to exhibit the uncoupling activity with the broad concentration
window [16]. However, BHT is unable to fully stimulate mitochondrial
respiration even at high concentrations. Besides, it is unknown whether
BHT is able to fight obesity similar to DNP or the other frequently used
uncoupler usnic acid [17,18].
The toxic action of uncouplers is generally assumed to result from
critical depolarization of the inner mitochondrial membrane leading
to complete uncoupling of oxidative phosphorylation. However,
this important issue has not been confirmed in direct experiments.
Moreover, poor correlation has been observed between the effect of
DNP and its derivatives on the respiration of isolated mitochondria
and the toxic action on mice [13]. In particular, LD50 for toxicity of
DNP and 6-cyclohexyl-DNP differs 1.5-fold, while uncoupling concen-
trations differ more than 30-fold. The data on the inhibition of
the growth of yeast cells by DNP and FCCP support the idea of non-
mitochondrial toxicity of these conventional uncouplers [19]. The possi-
ble non-mitochondrial target of uncouplers could be the cytoplasmic
membrane potential [20,21]. Besides, DNP has been found to bind to
several important non-mitochondrial proteins [22,23]. FCCP has been
shown to be susceptible to the attack of sulfhydryl reagents [24]
pointing to a possibility of the interaction with cystein residues of
cytoplasmic or membrane proteins [25].
This consideration suggests that toxic action of uncouplers can
be diminished by affecting their pharmacokinetics. In fact, it has
been shown that the use of DNP methyl ester instead of DNP im-
proves the therapeutic action of the uncoupler in diabetic models
in mice [26]. Besides, DNP incorporated in nanoparticles has ap-
peared to effectively ameliorate hypertriglyceridemia, insulin re-
sistance, hepatic steatosis, and diabetes in rats [2]. In line with
these findings, the design of mitochondria-targeted uncouplers
may dramatically reduce the side effects because of their
selective accumulation in mitochondria. Unfortunately, conjugation of
the mitochondrial cationic tag triphenylphosphonium (TPP) to DNP
Fig. 1. A. Chemical structures of mitoFluo (10-[2-(3-hydroxy-6-oxo-xanthen-9-
yl)benzoyl]oxydecyl-triphenyl-phosphonium bromide, or C10-mitoFluo), C -mitoFluo
4-[2-(3-hydroxy-6-oxo-xanthen-9-yl)benzoyl]oxybutyl-triphenyl-phosphonium
bromide), and C -FL (fluorescein n-octyl ester). B. Scheme of mitoFluo-mediated
8
proton transport through the inner mitochondrial membrane.
4
(
(
MitoDNP) has produced an inactive uncoupler probably due to inhibi-
1 μM of mitoFluo stimulates mitochondrial respiration similar to
FCCP, thus exhibiting properties of a true uncoupler. These results
prompted us to conduct further research on the properties of this
interesting compound displaying bright fluorescence in mito-
chondria and liposomes. The present study, aimed at elucidating
tissue-protective mechanisms of the mitochondria-specific
protonophore mitoFluo, i) addressed structural requirements
for mitochondria-specific uncoupling (structure-activity rela-
tion of the efflux of deprotonated MitoDNP [27]. On the other hand, con-
jugation of TPP to certain polyphenols led to compounds exhibiting
uncoupling action on isolated mitochondria [28]. It has been shown
recently in our laboratory that the cationic compound rhodamine 19
n-dodecyl ester (C12R1) accumulates in mitochondria and uncouples ox-
idative phosphorylation [29]. More hydrophilic rhodamine 19 n-butyl
ester (C
protonophoric action on artificial membranes is weaker than that of
12R1 [30]. According to [30], this paradox can be explained by involve-
ment of certain mitochondrial proteins in the uncoupling action of C R1.
Importantly, C R1 has been used successfully for combating obesity in
mice at concentrations less than those of DNP [31].
4
R1) is even more effective in mitochondria, although its
tionship between mitoFluo and its two analogues: C
a short linker between fluorescein and the cationic TPP group, and C
4
-mitoFluo, having
-FL,
C
8
4
lacking a TPP moiety (Fig. 1) [33]) and ii) directly demonstrated protec-
tive effects on animal tissue following acute injury. In contrast to
mitoFluo, the more hydrophilic C -mitoFluo neither caused dissipation
4
4
In our previous work [32], we have synthesized and tested a
conjugate of TPP with a derivative of fluorescein, which exhibits
the uncoupling action of the protonophoric type (mitoFluo or
of mitochondrial membrane potential, nor stimulated mitochondria res-
piration. Moreover, the short-linker conjugate exhibited poor accumula-
tion in mitochondria upon energization. Importantly, mitoFluo exhibited
neuroprotective and nephroprotective action on the models of brain
trauma and kidney ischemia/reperfusion in rats.
C
10-mitoFluo, structure shown in Fig. 1). Importantly, it accumu-
lates in isolated mitochondria upon their energization. Besides,
Please cite this article as: Y.N. Antonenko, et al., A long-linker conjugate of fluorescein and triphenylphosphonium as mitochondria-targeted
uncoupler and fluorescent neuro- and neph..., Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbagen.2016.07.014

Y.N. Antonenko et al. / Biochimica et Biophysica Acta xxx (2016) xxx–xxx
3
2
. Materials and methods
2.5. Proton transport in liposomes reconstituted with bacteriorhodopsin
2
.1. Materials
Bacteriorhodopsin (bR), isolated from Halobacterium salinarum cells,
was a generous gift of Dr. Sergey Sychev (Shemyakin and Ovchinnikov
Institute of Bioorganic Chemistry, Moscow). The reconstitution of
bR into proteoliposomes containing Type II-S phospholipid from soybean
(Sigma-Aldrich) was performed as described in [34]. 10 μl of
proteoliposome dispersion (0.25 mg/ml of bR) was added to 1 ml of
150 mM KCl, 10 mM Tris, 10 mM MES, 10 μM oxonol VI, and pH was
adjusted to 7.4. The measurements were conducted in the thermostated
cell at 15 °C with a Panorama Fluorat 02 spectrofluorimeter (Lumex,
Russia) adjusting excitation at 590 nm and emission at 620 nm.
8
MitoFluo and C -FL were synthesized as described in [32,33]. Sucrose
was from ICN. Rhodamine 6G was from Fluka. Tetramethylrhodamine
ethyl ester (TMRE), and Amplex Red reagent were from Invitrogen.
Total E. coli lipid was from Avanti Polar Lipids. Other chemicals were
from Sigma-Aldrich.
2
.2. Synthesis of 4-brombutyl-triphenyl-phosphonium bromide
The synthesis was similar to that described in [32]. Namely, a
solution of 1,4-dibrombutyl (1.1 ml, 12 mmol) and triphenylphosphine
2.62 g, 10 mmol) in benzene (0.5 ml) was heated at 80 °C during 20 h
2
.6. Isolation of rat liver mitochondria
(
Rat liver mitochondria (RLM) were isolated by differential centrifu-
in a tightly closed flask. Then the reaction mass was cooled to 20 °C, di-
luted with dichloromethane and evaporated to dryness. The residue
was dissolved in a minimal volume of dichloromethane, then an excess
of diethyl ester was added and the suspension formed was kept at 4 °C
until the solution became clear. Then the liquid phase was decanted, the
residue was dissolved again in dichloromethane and treated with
diethyl ether to complete precipitation. This procedure was repeated
three times. Finally the residue was dissolved in a minimal volume of
the solvent system ethanol-dichloromethane (1:5) and applied to a
chromatographic silica gel column (MN Kieselgel 60, 240–400 mesh)
in the same solvent system as an eluent. Detection was carried out
using TLC by UV-absorbance and Dragendorf reaction. Fractions with
the same chromatographic mobilities were combined and evaporated
gation [35] in medium containing 250 mM sucrose, 5 mM MOPS,
mM EGTA, and bovine serum albumin (0.5 mg/ml), pH 7.4. The final
1
washing was performed in medium of the same composition. Protein
concentration was determined using the Biuret method. Handling of
animals and experimental procedures with them were conducted in
accordance with the international guidelines for animal care and use
and were approved by the Institutional Ethics Committee of Belozersky
Institute of Physico-Chemical Biology at Moscow State University.
2.7. Mitochondrial respiration
Respiration of isolated mitochondria was measured using a standard
polarographic technique with
Strathkelvin Instruments, UK) at 25 °C using the 782 system software.
The incubation medium contained 250 mM sucrose, 5 mM MOPS, and
a Clark-type oxygen electrode
in vacuo (yield 1,8 g, 40%). ESI-MS: calculated for C22
H
23PBr 398.3,
(
+
observed 399.2 (M + H) .
1
0
mM EGTA, pH 7.4. The mitochondrial protein concentration was
.8 mg/ml. Oxygen uptake was expressed as nmol/min·mg protein.
2
.3. Synthesis of 4-[2-(3-hydroxy-6-oxo-xanthen-9-yl)benzoyl]oxybutyl-
triphenyl-phosphonium bromide (C -mitoFluo)
4
2.8. Membrane potential (ΔΨ) measurement in isolated mitochondria
A solution of fluorescein (1 g, 3.0 mmol) and sodium carbonate
0.5 g) in DMF (50 ml) was heated to 60 °C. The solution of 1,4-
ΔΨ was estimated using the safranine O dye [36]. The difference in
(
the absorbance between at 555 and 523 nm (ΔA) was recorded with
an Aminco DW-2000 spectrophotometer in the dual wavelength
mode. The following incubation medium was used: 250 mM sucrose,
mM MOPS, 1 mM EGTA, 2 μM rotenone, 5 mM succinate (pH 7.4),
5 μM safranine O. The mitochondrial protein content was 0.6–0.9 mg
brombutyltriphenylphosphonium bromide (1 g, 2.2 mmol) in a mini-
mal volume of dichloromethane was added dropwise and the reaction
was carried out for 3 h under stirring at 60 °C. Then the reaction mass
was cooled and diluted with 100 ml of dichloromethane. The organic
solution was washed twice with 5% hydrochloric acid and dried over
calcium chloride. The organic phase was filtered and evaporated in
vacuo. The residue was dissolved in a minimal volume of the solvent
mixture ethanol-dichloromethane (1:5) and applied to a silica gel
column (MN Kieselgel 60, 240–400 mesh) in the same solvent system
used as an eluent for chromatographic purification. Fractions were
detected by direct visualization of colored zones and additionally ana-
lyzed using TLC, those having equal chromatographic mobilities were
combined. The solvents were removed and the residues were dried in
vacuo and analyzed by LC-MS. The resulting compound was eluted as
an orange band and after evaporation of solvents was obtained as
5
1
protein/ml, the temperature was 26 °C.
2
.9. Mitochondrial ROS measurements by Amplex Red fluorescence assay
Hydrogen peroxide production was measured fluorometrically
employing the dye Amplex Red (Molecular Probes, Eugene, OR, USA)
in combination with horseradish peroxidase (HRP) [37]. In these
experiments, the incubation medium was supplemented with 1 μM
Amplex Red reagent and 5 U/ml HRP. Amplex Red (N-acetyl-3,7-
dihydroxyphenoxazine) is a non-fluorescent molecule that when
oxidized by H
fluorescent compound with an emission peak at about 585 nm. The
detection of H in mitochondrial suspensions was recorded as an
2 2
O in the presence of HRP produces resorufin, a highly
orange powder (320 mg, 20%). ESI-MS: calculated for C42
34 5
H O P 649.6;
observed 649.4.
2 2
O
increase in fluorescence at 585 nm with the excitation wavelength set
at 550 nm. The fluorescence response was calibrated by sequential
additions of known amounts of hydrogen peroxide solution.
2
.4. Planar phospholipid bilayers
Bilayer lipid membrane (BLM) was formed from 2% decane solution
of E. coli total lipid on a 0.6-mm aperture in a Teflon septum separating
2.10. FCS experimental setup
the experimental chamber into two compartments of equal size
(
volumes, 3 ml). Electrical parameters were measured with two AgCl
Uptake of fluorescein derivatives by isolated mitochondria was mea-
sured by fluorescence correlation spectroscopy (FCS). Peak intensity
analysis (PIA) was applied to fluorescence time traces of suspensions
of dye-doped mitochondria, representing sequences of peaks of differ-
ent intensity, which reflected their random walk through the confocal
electrodes placed into the solutions on the two sides of the BLM via
agar bridges, using a Keithley 6517 amplifier (Cleveland, Ohio, USA).
The incubation mixture contained 10 mM MES, 10 mM β-alanine,
1
00 mM KCl, pH 4.
Please cite this article as: Y.N. Antonenko, et al., A long-linker conjugate of fluorescein and triphenylphosphonium as mitochondria-targeted
uncoupler and fluorescent neuro- and neph..., Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbagen.2016.07.014

4
Y.N. Antonenko et al. / Biochimica et Biophysica Acta xxx (2016) xxx–xxx
volume [38]. The experimental data were obtained under stirring,
which increased the number of events by about three orders of magni-
tude, thus substantially enhancing the resolution of the method. The
setup of our own construction was described previously [38]. Briefly,
fluorescence excitation and detection were provided by a Nd:YAG
solid state laser with a 532-nm beam, attached to an Olympus IMT-2
epifluorescence inverted microscope equipped with a 40×, NA 1.2
water immersion objective (Carl Zeiss, Jena, Germany). The fluorescence
passed through an appropriate dichroic beam splitter and a long-pass fil-
ter and was imaged onto a 50-μm core fiber coupled to an avalanche
photodiode (SPCM-AQR-13-FC, Perkin Elmer Optoelectronics, Vaudreuil,
Quebec, Canada). The output signal F(t) was sent to a personal computer,
using a fast interface card (Flex02-01D/C, Correlator.com, Bridgewater,
NJ). The signal was measured in Hz, i.e. number of photons per second.
The data acquisition time T was 30 s. The card generated the autocorre-
lation function of the signal G(τ) defined as
2.13. Kidney ischemia/reperfusion (I/R) protocol
Rats were randomly divided by 3 groups as follows: Sham + Saline
(n = 5), I/R + Saline (n = 8), I/R + mitoFluo (n = 5). Rats in
“Sham + Saline” group were subjected to the right side nephrectomy.
In “I/R + Saline” group animals were subjected to 40-min warm ische-
mia of the left kidney and right side nephrectomy. In “I/R + mitoFluo”
animals were treated with mitoFluo in addition to left kidney I/R and
right kidney removal. Briefly, unilateral renal arteries were clamped
by a microvascular clip for 40 min, and then circulation was restored
by removing the clip. During operation, the body temperature of the
rat was maintained at 37 ± 0.5 °C. On the second day after ischemia
blood samples were taken to determine blood urea nitrogen (BUN).
The therapeutic protocol of mitoFluo treatment: i/p injection of
500 nmoles/kg mitoFluo 3 h before I/R, 1 h after I/R, and subsequent in-
jections at 13, 25 and 37 h; in total, each animal received 2500 nmoles/kg
mitoFluo.
Z
T
1
2.14. Analysis of mitoFluo accumulation in kidney
GðτÞ ¼ T
FðtÞFðt þ τÞdt ¼bFðt þ τÞFðtÞN
0
Rats were injected i/p with 2 μmol/kg mitoFluo, kidneys were ex-
cised after 3 h and sliced using a VibroSlice microtome (World Precision
Instruments, USA) into 100 mm thick sections. Slices were imaged with
a LSM510 inverted confocal microscope (Carl Zeiss Inc, Jena, Germany)
with excitation at 488 nm and emission collected at 505–530 nm. As a
negative control kidneys from untreated animals were used.
2
.11. Traumatic brain injury model (TBI)
The animal protocols used in this work were evaluated and ap-
proved by the institutional animal ethics committee in accordance
with FELASA guidelines. The experiments were performed on outbred
white male rats (350–400 g). The animals had unlimited access to
food and water and were kept in cages with a temperature controlled
environment (20 ± 2 °C) with light on from 9 AM to 9 PM. For all surgi-
cal procedures rats were anesthetized with i/p injections of 300 mg/kg
2.15. Statistics
Statistical analyses were performed using STATISTICA 7.0 for
Windows (StatSoft, Inc.). All data are presented as means ± standard
error of means (SEM). The neurological deficit scores are expressed as
median ± interquartile ranges, the 25th to 75th percentile being shown
in parentheses. Variance homogeneity was assessed with Levene's test.
Statistical differences between groups in the data of brain damage volume
and brain swelling were analyzed using one-way ANOVA with Tukey's
post-hoc test. Statistical differences in the limb-placing tests between
groups were analyzed using the Kruskal–Wallis test with the Mann–
Whitney U-test (the Bonferroni post-hoc correction was applied). Values
for p b 0.05 were assumed to be statistically significant.
(
12%) chloral hydrate. A feedback-controlled heating pad maintained
the core temperature (37.0 ± 0.5 °C) during ischemia supplemented
with an infrared lamp until awake.
In the present work, we employed our own modification of the
earlier used model of focal open severe brain trauma in rats [39,40].
To create the trauma, the left frontal part of the skull was trepanized
above the sensorimotor cortex zone, and a movable Teflon piston
4
mm in diameter with depth of insertion of 2.5 mm was placed into
it; this piston was struck from the height of 10 cm with a 50 g load
sliding along a directing rail. For localization of the sensorimotor cortex
zone we used following stereotaxic coordinates; +4 to −3 mm anteri-
or and posterior from bregma and +1 to +4.5 mm lateral from the
midline. In sham-operated rats, the experiments have been done
using the same protocol except that trauma was excluded. The rats
were treated by intranasal (i/n) administration of mitoFluo immediate-
ly after induction of TBI. Saline (40 μl) containing mitoFluo at a dose
3. Results and discussion
To study the uncoupling activity of mitoFluo and C -mitoFluo, we
measured the mitochondrial membrane potential (the electrical
4
potential difference across the inner mitochondrial membrane) via the
absorbance changes of safranine O. As seen in Fig. 2A, the addition of
mitoFluo to energized mitochondria led to a drop in the mitochondrial
membrane potential, which was enhanced with increasing the mitoFluo
concentration (black curve). On the contrary, no decrease in the mem-
brane potential was observed upon the addition of the short-linker
5
00 nmol/kg was administered i/n as drops released from a small
pipette every 5 min into both sides of the nasal cavity, followed by
μl for the last dose (for a total of 20 min). Rats were randomly divided
in 3 groups as follows: (1) Sham + Saline (n = 10), (2) TBI + Saline
n = 10), (3) TBI + mitoFluo (n = 11). Volume of the damage was
5
conjugate C -mitoFluo (red curve). Another fluorescein analog lacking
4
(
the TPP group, C -FL, decreased the membrane potential at concentra-
8
quantified by analyzing brain magnetic resonance (MR)-images obtain-
ed 14 day after the TBI as described previously [41].
tions similar to those of mitoFluo (blue curve). Absorbance measure-
ments in control experiments without safranine showed that the
4
additions of mitoFluo and C -mitoFluo did not contribute to the absor-
bance at 555 nm and 523 nm measured in the presence of 10 μM safra-
2
.12. Limb-placing test
nine (data not shown). The inability of C -mitoFluo to uncouple
4
mitochondria also manifested itself in the failure of this compound to
A modified version of the limb-placing test, consisting of seven tasks,
increase the respiration rate, in contrast to the marked acceleration of
mitochondrial respiration by mitoFluo (insert in Fig. 2A, see also [32]).
These results were further supported by comparing effects of mitoFluo
was used to assess forelimb and hindlimb responses to tactile and pro-
prioceptive stimulation [42]. The rats were habituated for handling and
tested before operation and at 1st, 2nd, 4th, 7th and 14th post-injury
days. For each task, the following scores were used: 2 points, normal
response; 1 point, delayed and/or incomplete response; 0 points, no
response. The total score over seven tasks was evaluated.
and C -mitoFluo on the light-induced generation of membrane potential
4
on liposomal membranes with reconstituted bacteriorhodopsin, as mea-
sured by oxonol VI fluorescence. It is seen in Fig. 2B that the oxonol re-
sponse to illumination with green light (marked ON) was substantially
Please cite this article as: Y.N. Antonenko, et al., A long-linker conjugate of fluorescein and triphenylphosphonium as mitochondria-targeted
uncoupler and fluorescent neuro- and neph..., Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbagen.2016.07.014

Y.N. Antonenko et al. / Biochimica et Biophysica Acta xxx (2016) xxx–xxx
5
Fig. 2. A. Effect of mitoFluo, C
succinate, 5 mM; rotenone, 1 μM. For other conditions, see Materials and methods. Insert: Dependence of the mitochondrial respiration on the concentration of mitoFluo, C
mitoFluo. B–D. Effect of mitoFluo and C -mitoFluo on the membrane potential of proteoliposomes with reconstituted bacteriorhodopsin assessed by oxonol VI fluorescence.
Concentrations: oxonol VI, 10 μM; lipid, 100 μg/ml. For other conditions, see Materials and methods.
8 4
-FL and C -mitoFluo on the membrane potential of rat liver mitochondria assessed by safranine O absorbance changes. Concentrations: safranine O, 15 μM;
8
-FL and C -
4
4
reduced upon the addition of 2 nM mitoFluo, but was practically unal-
tered at the same concentration of C -mitoFluo (Fig. 2C). The effective
between the mitochondria-targeting TPP group and a proton-carrying
moiety for uncoupling activity. The inactivity of MitoDNP as an uncou-
pler [27] was most likely also associated with the short (propyl) linker.
According to [10,43,44], the action of several popular uncouplers is
mediated at least partially by their interaction with certain protein
transporters located in the inner mitochondrial membrane. In particu-
lar, the action of DNP was partially inhibited by carboxyatractyloside
(CAtr), a specific inhibitor of the ADP/ATP carrier [45], while the action
of FCCP was blocked by 6-ketocholestanol [46,47]. It has been shown
4
concentrations of the conjugates differed by about two orders of magni-
tude (panel D in Fig. 2). Interestingly, in bacteriorhodopsin proteolipo-
somes, mitoFluo appeared to be much more effective than FCCP, in
contrast to the situation in mitochondria. In fact, about 100 nM of FCCP
was required to reduce the oxonol VI response halftime (data not shown).
The striking difference in the behavior of mitoFluo and C
4
-mitoFluo in
the experiments depicted in Fig. 2 highlighted the role of the alkyl linker
Please cite this article as: Y.N. Antonenko, et al., A long-linker conjugate of fluorescein and triphenylphosphonium as mitochondria-targeted
uncoupler and fluorescent neuro- and neph..., Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbagen.2016.07.014

6
Y.N. Antonenko et al. / Biochimica et Biophysica Acta xxx (2016) xxx–xxx
recently that the essential compound of the plant extract from Verbesina
persicifolia, an eudesman derivative, uncouples mitochondria via inter-
action with the terminal proton pump cytochrome oxidase [48]. To
gain insight into the possible interaction of mitoFluo with mitochondrial
membrane proteins, we examined the sensitivity of its uncoupling
activity to these recouplers. Neither 6-ketocholestanol nor CAtr
8
inhibited the uncoupling action of mitoFluo or C -FL as judged from
the membrane potential of rat liver mitochondria (black and blue
curves in Fig. 3). Control experiments showed substantial sensitivity
of FCCP action to 6-ketocholestanol and insensitivity to CAtr, while
the opposite effects were observed with palmitate in agreement with
the literature data (red and green curves in Fig. 3). These data pointed
to the plain protonophoric activity of both mitoFluo and C -FL as a
8
mechanism of their uncoupling action on mitochondria, although
mitoFluo-mediated proton current across a planar BLM at neutral pH
was substantially lower compared to that of C
To further examine the mechanism of the mitoFluo uncoupling ac-
tion, we compared its protonophoric activity with that of C -mitoFluo
8
-FL [32,33].
4
Fig. 4. MitoFluo was more effective than C
potential on planar bilayer lipid membrane made from total lipid of E. coli. Concentrations
of mitoFluo and C -mitoFluo were 0.25 μM. The solution was 10 mM MES, 10 mM
β-alanine, 100 mM KCl, pH 4. Formation of ΔpH was performed by shifting pH to the
value of 5 at one side of the BLM leading to positive potential at this side.
-mitoFluo in the induction of H+-diffusion
4
by measuring proton diffusion potential on a planar BLM separating
two solutions with different pH values. In such a system, the addition
4
+
of a protonophore (e.g. FCCP) resulted in a transmembrane H flux gen-
erating an electrical potential difference across the BLM, the more acidic
compartment being negatively charged. It was found (Fig. 4) that
mitoFluo (black curve), but not C
generated the proton diffusion potential. In the case of mitoFluo, the
potential was close to the theoretical limit (about 60 mV), while in the
case of C -mitoFluo, it was about three times lower, suggesting poor
4
protonophoric activity exhibited by this short-linker analog in agree-
ment with our uncoupling data in mitochondria (Fig. 2).
Fig. 1B shows a scheme of protonophoric action of mitoFluo on a lipid
membrane. In contrast to other known protonophores (both anionic and
cationic), two forms of mitoFluo responsible for proton transport are
charged, one form being cationic, while another being zwitterionic. It is
generally assumed that lipid membranes are impermeable for zwitterion-
ic compounds, because even the presence of a single charge on a com-
pound decreases its permeability by several orders of magnitude [49]. It
has been shown, however, that the presence of an alkyl linker of the
appropriate length makes TPP-based dications permeable through artifi-
cial lipid membranes and mitochondrial membranes [50,51]. Keeping in
mind larger permeability of anions compared to cations of similar size
due to the presence of the so-called dipole potential on the water-
4
-mitoFluo (red curve), effectively
membrane interface of lipid membranes, it can be assumed that the
permeability of the zwitterionic form of mitoFluo is not impossible. Of
note, according to a recent study [49], the anticancer drug topotecan
can permeate through lipid membranes in a zwitterionic form, showing
the permeability close to that of the neutral form. Therefore, the scheme
in Fig. 1B, which includes the step of the transmembrane diffusion of
the zwitterionic form of mitoFluo, is justified and does not contradict
the data available. This scheme assumes that the protonophoric action
of mitoFluo proceeds predominately via shuttling from one membrane in-
terface to the other rather than from one aqueous phase to the other. The
4
poor activity of C -mitoFluo can be assigned to its weak lipophilicity
preventing its binding to the membrane surface. An increase in the linker
chain length by 6 carbon atoms should lead to about 1000-fold difference
in the octanol-water partition coefficients [52,53].
Fig. 5. Time-resolved count rates of 20 nM mitoFluo (black curve), 20 nM C
4
-mitoFluo (red
Fig. 3. Effect of 6-ketocholestanol (100 μM) and carboxyatractyloside (CAtr, 1 μM) on the
curve), and 20 nM C -FL (blue curve) in the presence of rat liver mitochondria (0.1 mg/ml)
8
8
uncoupling action of mitoFluo (black curve, 40 nM), C -FL (blue curve, 50 nM), FCCP (red
in the incubation buffer. The signal was recorded under stirring condition in solution:
curve, 20 nM), and palmitate (green curve, 15 μM) in rat liver mitochondria estimated by
the mitochondrial membrane potential measurements with safranine O (15 μM). For
other conditions, see Materials and methods.
250 mM sucrose, 20 mM MOPS, 1 mM EGTA, pH 7.4. B. Statistical analysis in the form of
corresponding dependences of n(F N F ) on F derived as described in the Materials and
0 0
methods.
Please cite this article as: Y.N. Antonenko, et al., A long-linker conjugate of fluorescein and triphenylphosphonium as mitochondria-targeted
uncoupler and fluorescent neuro- and neph..., Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbagen.2016.07.014

Y.N. Antonenko et al. / Biochimica et Biophysica Acta xxx (2016) xxx–xxx
7
To assess the role of the linker for mitochondrial targeting, we com-
pared mitochondrial accumulation of mitoFluo and C -mitoFluo by the
4
FCS technique, successfully used in our previous studies for TMRE and
other rhodamine derivatives [38,54]. FCS is based on recording fluores-
cence fluctuations arising from the Brownian motion of single particles
inside a small laser-illuminated volume (confocal volume) of about
−
15
~
10
red curve) displayed only a small number of bright peaks, which
corresponded to the passage of mitochondrial particles carrying C
4
l [55]. The FCS recording obtained with C -mitoFluo (Fig. 5A,
4
-
mitoFluo across the confocal volume. In the case of mitoFluo, the num-
ber of the peaks was substantially higher (black curve in Fig. 5A, similar
to the recording presented in [32]). These measurements were carried
out in the presence of succinate, which provided generation of the mito-
chondrial membrane potential driving the permeating cations into mi-
tochondria. The recording made in the presence of C
curve) showed similar number of peaks as in the case of C
8
-FL (Fig. 5A, blue
-mitoFluo.
4
The experimental data were obtained under stirring conditions which
increased substantially the number of events, thereby enhancing the
resolution of the method [38]. Panel B of Fig. 5 presented statistics of
Fig. 7. Effect of K+-diffusion potential in liposomes on the fluorescence of various
fluorescein derivatives. Traces of fluorescence at 520 nm (excitation 490 nm) normalized
to the signal prior to the addition of valinomycin are shown in a medium: 100 mM
choline chloride, 3 mM Tris, pH 7.5. Additions: 10 nM valinomycin, 50 mM of KCl (green
curve). Liposomes, 10 μg/ml DPhPC. Concentrations of mitoFluo, C -FL, and C -mitoFluo
8 4
were 20 nM.
the number of peaks (n) with an amplitude exceeding the certain
value F (i.e. n(F N F )) during the recording of 30 s. The analysis con-
firmed our conclusion that the number of peaks in the case of mitoFluo
was substantially higher than in the case of C -mitoFluo or C -FL (about
–5 times depending on F ). Therefore, C -mitoFluo accumulation in
0
0
4
8
3
0
4
mitochondria (associated with its lower membrane permeability) was
weaker than that of mitoFluo possibly because of its lower hydrophobic-
8
ity. Limited accumulation of C -FL was in agreement with our previous
measurements [33].
Fig. 6. A. Effect of mitochondrial energization on the fluorescence of different fluorescein
derivatives. The fluorescence was normalized to the initial level measured prior to the
addition of mitochondria. Traces of fluorescence at 520 nm (excitation 490 nm) are
shown in a medium: 250 mM sucrose, 10 mM MOPS, 3 mM MgCl , 2 mM KH PO (grey
2 2 4
curve was without phosphate), 1 mM EGTA, pH 7.4. Additions where indicated: 0.2 mg/ml
rat liver mitochondria (RLM), 1 μM rotenone, 5 mM succinate, 100 nM of FCCP.
Fig. 8. Effect of mitoFluo (black curve) or FCCP (red curve) on H
mitochondria. Incubation medium: 250 mM sucrose, 20 mM MOPS, 5 mM KH
MgCl , 1 mM EGTA, 1 μM Amplex Red, and 5 U/ml horseradish peroxidase, pH 7.4.
Succinate, 3 mM, mitochondrial protein, 0.5 mg/ml.
2
O
2
production in rat liver
8 4
Concentrations of mitoFluo, C -FL, and C -mitoFluo were 20 nM. B. Dose dependence of
2
PO4, 3 mM
the effect of succinate on the fluorescence of mitoFluo. Shown is the percent of the signal
decrease with respect to the initial level of the mitoFluo fluorescence.
2
Please cite this article as: Y.N. Antonenko, et al., A long-linker conjugate of fluorescein and triphenylphosphonium as mitochondria-targeted
uncoupler and fluorescent neuro- and neph..., Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbagen.2016.07.014

8
Y.N. Antonenko et al. / Biochimica et Biophysica Acta xxx (2016) xxx–xxx
Fluorescence of mitoFluo in mitochondria could be sensitive to a mi-
was about an order of magnitude lower than its uncoupling concentra-
tions (about 200 nM). The addition of rotenone increased the fluores-
cence signal, while succinate decreased the signal to the initial level,
and subsequent addition of FCCP increased the signal up to the level
observed with rotenone. It may be concluded that the changes in the
mitoFluo fluorescence were reciprocal to corresponding changes in the
mitochondrial membrane potential reported previously (see, for exam-
ple, Fig. 9 in [56]), namely: mitochondria generated membrane potential,
when respiring with endogenous substrates, rotenone depolarized the
mitochondria by inhibiting respiratory complex I, the complex II substrate
tochondrial functional state, because the fluorescence of fluorescein is
strongly pH dependent (de-protonated form possesses bright fluores-
cence) and exhibits self-quenching. Energization of mitochondria should
lead to an increase in the mitoFluo fluorescence due to alkalinization of
the matrix pH. On the other hand, energization of mitochondria should
decrease the mitoFluo fluorescence due to its accumulation inside the
matrix resulting in an increase in its local concentration. Fig. 6A shows
the time course of the mitoFluo fluorescence added to a suspension of
rat liver mitochondria. Here, the concentration of mitoFluo (20 nM)
Fig. 9. Protection against traumatic brain injury by the mitoFluo treatment starting immediately after trauma. (A) Representative T2-weighted MR-images from coronal brain sections
0.8 mm thick, from rostral (top) towards caudal (bottom)) obtained on the 14th day after reperfusion. Light regions refer to ischemic areas. (B) Damage volume was evaluated by using
(
MRI with analysis of T2-weighted images. (D) Neurological deficit scores estimated in the limb-placing test. *Denotes significant difference from the TBI + Saline or TBI + mitoFluo
groups (p b 0.05) (One-way ANOVA, followed by Tukey's post-hoc analysis).
Please cite this article as: Y.N. Antonenko, et al., A long-linker conjugate of fluorescein and triphenylphosphonium as mitochondria-targeted
uncoupler and fluorescent neuro- and neph..., Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbagen.2016.07.014

Y.N. Antonenko et al. / Biochimica et Biophysica Acta xxx (2016) xxx–xxx
9
succinate restored the membrane potential, and the protonophore FCCP
depolarized the mitochondria once again. With C -mitoFluo, the addition
in the matrix. In view of the available data on the fluorescence behavior of
a number of cationic dyes such as DiS-C3-(5) [57], safranine O [36], or
TMRE [58], the self-quenching seems more justified. Consistent with
this assumption, the degree of the mitoFluo quenching upon energization
increased with raising mitoFluo concentration from 5 nM to 200 nM
(Fig. 6B). Further elevation of the mitoFluo concentrations led to a relative
decrease in the effect of energization due to the uncoupling action of
mitoFluo.
4
of mitochondria led to a substantially smaller decrease in the fluores-
cence, than with mitoFluo, and subsequent energization-deenergization
did not change the level of the signal (Fig. 6A, red curve). The addition
of 2 mM phosphate to the medium did not change the levels of the
mitoFluo fluorescence before and after mitochondrial energization, sug-
gesting that the alkalinization of the mitochondrial matrix upon energiza-
tion did not contribute to changes in the mitoFluo fluorescence (Fig. 6A,
We also tested potential-sensitive changes of mitoFluo fluorescence
+
grey curve). With C
did not depend on the mitochondrial energization (Fig. 6A, blue curve),
similar to the case of C -mitoFluo.
8
-FL lacking the TPP group, the fluorescence signal
in a system of liposomes with the K diffusion potential generated in
the presence of valinomycin. It was shown previously that the fluores-
cence of cationic DiS-C3-(5) decreased in this system upon the addition
of valinomycin due to concentration-dependent quenching of the dye
[59]. Fig. 7 shows similar experiments with mitoFluo (black curve),
4
The decrease in the mitoFluo fluorescence upon mitochondrial
energization could result from either a total increase of the protonation
level of the compound or fluorescence self-quenching upon accumulation
4 8
C -mitoFluo (red curve) and C -FL (blue curve). In the case of mitoFluo,
Fig. 10. MitoFluo prevents acute kidney injury after renal ischemia/reperfusion. A: Experimental design. Male rats were subjected to 40-min warm ischemia of the left kidney and right
kidney nephrectomy. Blood samples were obtained 48 h after I/R to determine BUN. MitoFluo (500 nmoles/kg) was injected i/p 3 h before I/R, 1 h after I/R, and subsequent injections
at 18, 30 and 42 h. B: Renal function estimated by BUN concentration 48 h after I/R. C: MitoFluo accumulates in kidney tubules after i/p injection. Confocal microscopy of kidney
tissues slices 3 h after i/p injection of 2 μmol/kg mitoFluo showing its retention and distribution over kidney compartments (right panel). As a negative control, kidney from untreated
animals was displayed (left panel). Bar, 100 μm.
Please cite this article as: Y.N. Antonenko, et al., A long-linker conjugate of fluorescein and triphenylphosphonium as mitochondria-targeted
uncoupler and fluorescent neuro- and neph..., Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbagen.2016.07.014

10
Y.N. Antonenko et al. / Biochimica et Biophysica Acta xxx (2016) xxx–xxx
the addition of valinomycin (at t = 110 s) led to a decrease in the
leak to molecular oxygen, thereby leading to a drop of mitochondrial
ROS production to a more safe level. According to our data (Fig. 8),
mitoFluo exhibited this ability in isolated mitochondria. This consider-
ation explains mitoFluo action both in brain trauma model (Fig. 9) and
the kidney ischemia/reperfusion model (Fig. 10). Importantly, the
uncoupling concentration of mitoFluo in different tissues in vivo was
lower than its cytotoxic concentration: in particular, the IC50 of
mitoFluo in cells of transformed fibroblasts was found to be 2 μM [32].
Thus, our in vivo data showed the therapeutic potential of mitoFluo as a
neuroprotector and a nephroprotector. We believe that the tissue protec-
tion experiments (Figs. 9, 10) can be considered as a proof of concept
experiments for mitochondria-targeted uncouplers and further work is
required to develop an uncoupler exhibiting maximum protection with
minimum side-effect properties. Further optimization of the linker chain
length is in progress, while the choice of fluorescein as a protonophoric
unit is under consideration.
fluorescence signal (about 50%, black curve), while there was no fluores-
+
cence decrease in the absence of the K gradient on liposomes (about 5%,
green curve). In agreement with the data on mitochondria, fluorescence
of C -mitoFluo and C -FL did not respond to changes in membrane
4 8
potential of liposomes (red and blue curves in Fig. 7). Based on these re-
sults, mitoFluo might be proposed as the mitochondrial membrane po-
tential fluorescent probe in a certain concentration range. However,
strong energy-independent binding, the narrow dynamic range and
confounding uncoupling properties make it rather unsuitable.
As it was pointed out in the Introduction, uncouplers exhibited pro-
tective action in several pathologies, associated with the oxidative
stress, possibly as a result of their ability to reduce ROS generation by
mitochondria [5,60]. However, this phenomenon was shown for classi-
cal anionic uncouplers which may act by the mechanism differing from
that of mitoFluo. To this end, we measured the effect of mitoFluo on the
generation of hydrogen peroxide by mitochondria using an Amplex
Red/HRP system. Fig. 8 demonstrates that mitoFluo decreased the
Transparency document
H O
2 2
production of rat liver mitochondria in the presence of succinate
as a substrate. Grey curve is a recording of H production during
0 min, red curve displays an experiment with FCCP. Black curve
2 2
O
The Transparency document associated with this article can be
found, in online version.
1
shows that the addition of 150 nM mitoFluo led to a jump of the fluores-
cence, measured at 620 nm, due to fluorescent properties of mitoFluo,
however the slope of the curve after the addition of mitoFluo was sub-
Acknowledgements
2 2
stantially smaller, thereby demonstrating the suppressed rate of H O
The authors are grateful to Professor Vladimir Skulachev for valuable
discussions. This work was financially supported by the Russian Science
Foundation grant 16-14-10025.
production, as measured by fluorescence of the Amplex Red oxidation
product resorufin. Thus, mitoFluo could be used as an inhibitor of ROS
production similar to other anionic uncouplers.
Keeping in mind neuroprotective properties of uncouplers in stroke
[30,61,62] or in brain trauma models [63,64], we examined the protec-
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day after TBI rats scored only 1.7 ± 0.2. The treatment with mitoFluo re-
stored the neurological status to 5.7 ± 0.5 and 7.3 ± 0.9 scores
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Please cite this article as: Y.N. Antonenko, et al., A long-linker conjugate of fluorescein and triphenylphosphonium as mitochondria-targeted
uncoupler and fluorescent neuro- and neph..., Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbagen.2016.07.014