Alkyl-substituted phenylamino derivatives of 7-nitrobenz-2-oxa-1,3-diazole as uncouplers of oxidative phosphorylation and antibacterial agents: involvement of membrane proteins in the uncoupling action

Article abstract of DOI:10.1016/j.bbamem.2016.12.014

In search for new effective uncouplers of oxidative phosphorylation, we studied 4-aryl amino derivatives of a fluorescent group 7-nitrobenz-2-oxa-1,3-diazol (NBD). In our recent work (Denisov et al., Bioelectrochemistry, 2014), NBD-conjugated alkyl amines

Full text of DOI:10.1016/j.bbamem.2016.12.014

Alkyl-substituted phenyl-amino derivatives of 7-nitrobenz-2-oxa-1,3-diazole
as uncouplers of oxidative phosphorylation and antibacterial agents: involve-
ment of membrane proteins in the uncoupling action
Yuri N. Antonenko, Stepan S. Denisov, Ljudmila S. Khailova, Pavel A.
Nazarov, Tatyana Rokitskaya, Vadim N. Tashlitsky, Alexander M. Firsov,
Galina A. Korshunova, Elena A. Kotova
PII:
DOI:
Reference:
S0005-2736(16)30407-2
doi:10.1016/j.bbamem.2016.12.014
BBAMEM 82377
To appear in:
BBA - Biomembranes
Received date:
Revised date:
Accepted date:
22 August 2016
15 December 2016
19 December 2016
Please cite this article as: Yuri N. Antonenko, Stepan S. Denisov, Ljudmila S. Khailova,
Pavel A. Nazarov, Tatyana Rokitskaya, Vadim N. Tashlitsky, Alexander M. Firsov,
Galina A. Korshunova, Elena A. Kotova, Alkyl-substituted phenyl-amino derivatives of 7-
nitrobenz-2-oxa-1,3-diazole as uncouplers of oxidative phosphorylation and antibacterial
agents: involvement of membrane proteins in the uncoupling action, BBA - Biomembranes
(2016), doi:10.1016/j.bbamem.2016.12.014
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
1
Alkyl-substituted phenyl-amino derivatives of 7-nitrobenz-2-oxa-1,3-diazole as uncouplers
of oxidative phosphorylation and antibacterial agents: involvement of membrane proteins
in the uncoupling action
Yuri N. Antonenko1*, Stepan S. Denisov1,2, Ljudmila S. Khailova1, Pavel A. Nazarov1, Tatyana
Rokitskaya1, Vadim N. Tashlitsky2, Alexander M. Firsov1, Galina A. Korshunova1, Elena A.
Kotova1*
1Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, and
2Faculty of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1, Moscow
119991, Russia
Address correspondence to: Yuri Antonenko, Belozersky Institute of Physico-
Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia.
Fax:+(7-495)-939-31-81, E-mail: antonen@genebee.msu.ru
Elena A. Kotova, Belozersky Institute of Physico-Chemical Biology, Lomonosov
Moscow State University, Moscow 119991, Russia; Fax:+(7-495)-939-31-81, e-mail:
kotova@genebee.msu.ru
ABSTRACT
In search for new effective uncouplers of oxidative phosphorylation, we studied 4-aryl
amino derivatives of a fluorescent group 7-nitrobenz-2-oxa-1,3-diazol (NBD). In our recent work
(Denisov et al., Bioelectrochemistry, 2014), NBD-conjugated alkyl amines (NBD-Cn) were
shown to exhibit uncoupling activity. It was concluded that despite a pKa value being about 10,
the expected hindering of the uncoupling activity could be overcome by insertion of an alkyl
chain. There is evidence in the literature that the introduction of an aryl substituent in the 4-
amino NBD group shifts the pKa to neutral values. Here we report the data on the properties of a
number of 4-arylamino derivatives of NBD, namely alkylphenyl-amino-NBD (Cn-phenyl-NBD)
with varying alkyl chain Cn. By measuring the electrical current across planar bilayer lipid
membrane, the protonophoric activity of Cn-phenyl-NBD at neutral pH grew monotonously from
C1- to C6-phenyl-NBD. All of these compounds increased the respiration rate and reduced the
membrane potential of isolated rat liver mitochondria. Importantly, the uncoupling action of C6-
and C4-phenyl-NBD was partially reversed by glutamate, diethyl pyrocarbonate (DEPC), 6-
ketocholestanol and carboxyatractyloside, thus pointing to the involvement of membrane
proteins in the uncoupling activity of Cn-phenyl-NBD in mitochondria. The pronounced

ACCEPTED MANUSCRIPT
2
recoupling effect of DEPC, an inhibitor of an aspartate-glutamate carrier (AGC), and that of its
substrates for the first time highlighted AGC participation in the action of potent uncouplers on
mitochondria. C6-phenyl-NBD produced strong antimicrobial effect on Bacillus subtilis, which
manifested itself in cell membrane depolarization and suppression of bacterial growth at
submicromolar concentrations.
Key words: mitochondria, bilayer lipid membrane, uncoupler, protonophore, NBD, membrane
potential, oxidative phosphorylation
Abbreviations: NBD, 7-nitrobenz-2-oxa-1,3-diazole; NBD-Cl, 4-chloro-7-nitrobenz-2-oxa-1,3-
diazole; alkyl-NBD, 4-alkylamino derivatives of 7-nitrobenz-2-oxa-1,3-diazole; NBD-Cn, 4-
alkylamino derivatives of 7-nitrobenz-2-oxa-1,3-diazole with n hydrocarbon units; NBD-C8, 4-
octylamino-7-nitrobenz-2-oxa-1,3-diazole; aryl-NBD, 4-arylamino derivatives of 7-nitrobenz-2-
oxa-1,3-diazole; phenyl-NBD, 4-phenylamino-7-nitrobenz-2-oxa-1,3-diazole; C1-phenyl-NBD,
4-(p-tolyl)amino-7-nitrobenz-2-oxa-1,3-diazole; C4-phenyl-NBD, 4-(p-butylphenyl)amino-7-
nitrobenz-2-oxa-1,3-diazole; C6-phenyl-NBD, 4-(p-hexylphenyl) amino-7-nitrobenz-2-oxa-1,3-
diazole; Cn-phenyl-NBD, 4-(p-alkylphenyl)amino derivatives of 7-nitrobenz-2-oxa-1,3-diazole
with n hydrocarbon units; NBD-bis-C4, 4-dibutylamino-7-nitrobenz-2-oxa-1,3-diazole; mitoFluo,
10-[2-(3-hydroxy-6-oxo-xanthen-9-yl)benzoyl]oxydecyl-triphenyl-phosphonium bromide; CAtr,
carboxyatractyloside; DEPC, diethylpyrocarbonate; DisC3(5), 3,3′-dipropylthiadicarbocyanine
iodide; DNP, 2,4-dinitrophenol; DPhPC, diphytanoylphosphatidylcholine; FCCP, carbonyl
cyanide-p-trifluoromethoxyphenylhydrazone; CCCP, carbonyl cyanide m-chlorophenyl
hydrazone; BLM, bilayer lipid membrane; IMM, inner mitochondrial membrane; PMF, proton
motive force; ANT, adenine nucleotide translocator; AGC, aspartate-glutamate carrier.
Highlights
Cn-phenylamino 7-nitrobenz-2-oxa-1,3-diazole (Cn-phenyl-NBD) varied in Cn alkyl chain
Cn-phenyl-NBD with n=1, 4, 6 carried protons across planar BLM with increasing efficacy
C6-phenyl-NBD accelerated respiration and lowered membrane potential of mitochondria
Uncoupling action of C6-phenyl-NBD was reversed by glutamate and diethyl pyrocarbonate
C6- and C4-phenyl-NBD suppressed membrane potential and growth of Bacillus subtilis

ACCEPTED MANUSCRIPT
3
1. Introduction
An electrochemical gradient of protons across a bacterial cytoplasmic membrane called
proton motive force (PMF) is a unique store of energy in bacterial cells, which is indispensable
for maintaining critical cellular functions, either directly or indirectly, e.g., via coupling to ATP
synthesis. The energy-dependent functions comprise multitude metabolic reactions, cell division,
flagellar motility and a plethora of active transport processes. Of these, operation of energy-
coupled efflux systems causing antimicrobial multidrug resistance (MDR) has become a subject
of intensive studies due to the importance of MDR as a serious health-threatening problem.
Intensive research efforts have been aimed in recent years at search for inhibitors of efflux
pumps. Alternatively, drug efflux could be avoided by using uncouplers of oxidative
phosphorylation, i.e. compounds capable of decreasing the electrochemical proton gradient on
mitochondrial and bacterial membranes, thereby stimulating the electron transport in respiratory
chains. Decreasing PMF would impede numerous metabolic processes and inhibit operation of
proton-dependent multidrug efflux systems [1]. Therefore, targeting the PMF can be a promising
approach to combat bacterial infections [2-10], thus prompting a search for new effective
uncouplers. On the other hand, the so-called mild uncoupling of mammalian mitochondria [11],
i.e. a limited decrease in PMF to a level sufficient for maintaining ATP synthesis, has been
proved beneficial for preventing a variety of oxidative stress-related diseases [12] due to steep
dependence of reactive oxygen species generation on PMF [13-16]. Recently, the ability of
uncouplers to decrease biomass growth has attracted increasing attention in the environmental
research due to the possibility of employing potential uncouplers in biological waste water
treatment systems [17].
Two structural requirements are of major importance for the uncoupling activity: a
certain (close to neutral) pKa value of a proton-dissociable group, enabling the compound to
readily bind and release protons under physiological conditions, and appropriate lipophilicity,
needed for effective translocation of a protonophore across a membrane [18-22]. Recently we
have found that alkylamino derivatives of the polarity sensitive optical probe 7-nitrobenz-2-oxa-
1,3-diazole (NBD) [23] exhibit protonophoric and uncoupling activity. We believed that the
effect of an unfavorable pKa value (about 10) [24] of their amino group, being very far from the
physiological one, was compensated by optimal lipophilicity. While further analyzing literature
on NBD analogues, we encountered the data on the pKa value of arylamino NBD derivatives
being close to neutral (i.e. 7.40 [25] or 7.68 [26] for phenyl-NBD and 7.87 for C1-phenyl-NBD
[26]), which encouraged us to test compounds of this type for the uncoupling activity. With
unsubstituted phenyl-NBD, no uncoupling activity was detected. By contrast, alkyl-substituted

ACCEPTED MANUSCRIPT
4
phenylamino-NBD (Fig. 1), although being nearly non-fluorescent [27,28], appeared to be of
significant interest. In particular, these NBD derivatives exhibited higher uncoupling activity in
rat liver mitochondria than NBD-C8 (the most effective uncoupler of the alkylamino-NBD
(NBD-Cn) series [23]) and demonstrated high antibacterial potency. Moreover, the uncoupling
effect of the arylamino-NBD analogues described here was found to be sensitive to both
glutamate and aspartate, as well as to the inhibitor of aspartate-glutamate carriers (AGC) diethyl
pyrocarbonate (DEPC), which for the first time suggested AGC to be seriously involved in the
uncoupling action other than that of free fatty acids [29,30].
2. Materials and methods
2.1. Materials
Diphytanoylphosphatidylcholine (DPhPC) was from Avanti Polar Lipids (Alabaster,
Alabama). 4-Chloro-7-nitrobenz-2-oxa-1,3-diazole (NBD-Cl), rotenone, carbonyl cyanide 4-
(trifluoromethoxy)phenylhydrazone (FCCP), 2,4-dinitrophenol (DNP), carboxyatractyloside
(CAtr), 6-ketocholestanol and 3,3′-dipropylthiadicarbocyanine iodide (DisC3(5)) were from
Sigma. NBD-C8 was gifted by Prof. Maria J. Moreno (Departamento de Química, Faculdade de
Ciencias e Tecnologia, Universidade de Coimbra, Portugal). DEPC was from AMRESCO.
2.2. Synthesis
Phenyl-NBD, C1-phenyl-NBD, C4-phenyl-NBD and C6-phenyl-NBD were synthesized
via a reaction of NBD-Cl with the corresponding primary arylamines (aniline, 4-toluidine , 4-
n- butylaniline, 4-n-hexylaniline) at the conditions similar to those described earlier [23]. Briefly,
sodium carbonate (2 mmol) was added to the solution of arylamine (1 mmol) in anhydrous
chloroform, then the solution of NBD-Cl (0.9 mmol) in anhydrous chloroform was added by
portions with stirring at room temperature in the dark. After 72 h the reaction mixture was
diluted with chloroform and the organic phase was washed sequentially with 5% hydrochloric
acid solution and water, and then dried over sodium sulfate. The residue obtained by evaporation
was purified on a silica gel column in an appropriate solvent system. The yields of the
compounds were 50-60%. The structures were confirmed by mass spectra LC MS: phenyl-NBD
found [M+H] 257,29, calculated for C12H8N4O3 M=256.22; C1-phenyl-NBD, found [M+H]
271,20, calculated for C13H10N4O3 M=270.24; C4-phenyl-NBD, found [M+H] 313,19, calculated
for C16H16N4O3 M=312.32; C6-phenyl-NBD, found [M+H] 341,27, calculated for C18H20N4O3
M=340.38. The LC MS mass-spectra, as well as
are presented in the supporting information.
NMR spectra and chromatograms

ACCEPTED MANUSCRIPT
5
2.3. Planar bilayers
Bilayer lipid membrane (BLM) was formed by the brush technique [31] from a 2%
decane solution of DPhPC on a 0.6-mm aperture in a Teflon septum separating the experimental
chamber into two compartments of equal 3-ml volume. Electrical parameters were measured
with two AgCl electrodes placed into the solutions on the two sides of the BLM via agar bridges,
using a Keithley 428 amplifier (Cleveland, Ohio, USA).
2.4. Octanol-water partition coefficient
The octanol–water partition coefficient (Pow) of the NBD derivatives was determined
using a method based on correlation of this parameter with the retention time in a reverse-phase
high performance liquid chromatography (HPLC) column [32]. It was previously shown for
related compounds that this method estimates the coefficient with satisfactory accuracy [33]. The
mobile phase consisted of 0.1 % trifluoroacetic acid in water and a gradient of acetonitrile from
40 to 95% (11 min; a flow rate of 1.5 ml/min). The logarithm of the partition coefficient logPow
was calculated from the equation:
log P  4.26log

tR t0  7.95

ow
where t0 is the dead time, while tR has the usual meaning of retention time for an analyte. All the
chromatographic runs were performed on an Agilent 1100 chromatograph (Agilent, USA) at
25°C. The HPLC columns used were Luna C18(2), 5 µm, 4.6 x 150 mm.
2.5. Isolation of rat liver mitochondria
Rat liver mitochondria were isolated by differential centrifugation [34] in a medium
containing 250 mM sucrose, 10 mM MOPS, 1 mM EGTA, pH 7.4. The final washing was
performed in the medium additionally containing bovine serum albumin (0.1 mg/ml). Protein
concentration was determined using the Biuret method. Handling of animals and experimental
procedures were conducted in accordance with the international guidelines for animal care and
use and were approved by the Institutional Ethics Committee of A.N. Belozersky Institute of
Physico-Chemical Biology at the Moscow State University.
2.6. Mitochondria respiration
Respiration of isolated mitochondria was measured using a standard polarographic
technique with a Clark-type oxygen electrode (Strathkelvin Instruments, UK) at 25°C using the

ACCEPTED MANUSCRIPT
6
782 system software. The incubation medium contained 250 mM sucrose, 5 mM MOPS, and 1
mM EGTA, pH 7.4. The mitochondrial protein concentration was 0.8 mg/ml. Oxygen uptake is
expressed as nmol/min·mg protein.
2.7. Membrane potential () measurement in isolated mitochondria
 was estimated using the safranine O dye [35]. The difference in the absorbance 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, 5 mM MOPS,
1 mM EGTA, 2 μM rotenone, 5 mM succinate (pH 7.4), 1 μg/ml oligomycin, 15 μM safranine O.
The mitochondrial protein content was 0.6 – 0.9 mg protein/ml, the temperature was 26°C.
2.8. Bacterial growth
The standard laboratory strain Bacillus subtilis subs. subtilis Cohn 1872, stain BR151
(trpC2 lys-3 metB10) was used. Overnight bacterial cells cultures were diluted in fresh LB
media. 200 µl of bacterial cell cultures (5*105 cells/ml) were inoculated into 96-well plates
(Eppendorf AG, Hamburg, Germany) and the NBD derivatives were added to reach appropriate
concentrations. Cells were left to grow for 21 hours at 37°C. Optical densities at 620 nm and
kinetic growth curves were measured by using a Thermo Scientific Multiskan FC plate reader
with an incubator (Thermo Fisher Scientific, USA). All experiments were performed at least in
triplicates.
2.9. Measurement of B. subtilis membrane potential
Membrane potential in B. subtilis was measured by estimating fluorescence of the
potential-dependent probe DiSC3(5) [36]. B. subtilis from the overnight culture were seeded into
fresh LB medium followed by growth until reaching the optical density 0.8 at 620 nm. Then the
bacteria were diluted 20-fold in a buffer containing 100 mM KCl, 10 mM Tris, pH 7.4.
Fluorescence intensity at 670 nm (excitation at 640 nm) was measured by using a Fluorat-02-
Panorama fluorimeter.
3. Results
3.1. Cn-phenyl-NBD induced proton current on BLM
According to our previous work [23], NBD-C8 induced electrical current across the planar
BLM system, although the effect of NBD-C10 was stronger. To test the idea that C6-phenyl-NBD
can also induce proton permeability in BLM, the electrical current was measured under voltage-

ACCEPTED MANUSCRIPT
7
clamp conditions in this model system. A typical trace of the BLM current after the addition of
C6-phenyl-NBD is shown in Fig.2A. It is seen that 5.5 μM C6-phenyl-NBD caused a substantial
increase in the current up to about 60 pA. C1-phenyl-NBD or phenyl-NBD did not cause an
increase in the current up to 20 µM, while C4-phenyl-NBD exhibited low activity. The series of
activity C6-phenyl-NBD > C4-phenyl-NBD > C1-phenyl-NBD > phenyl-NBD (Fig. 2B) was in
line with the gradual decrease in octanol-water partition coefficient estimated from reverse-phase
column chromatography, namely: 7800, 3400, and 530 for C6-phenyl-NBD, C4-phenyl-NBD and
C1-phenyl-NBD, respectively. Control experiment showed that the addition of glutamate was of
no effect on the C6-phenyl-NBD-mediated current across BLM (Fig. 2A).
To determine ion selectivity of C6-phenyl-NBD-mediated permeability, I-V curves were
measured under asymmetrical conditions of different pH at the opposite sides of the membrane
(pHcis=7.5, pHtrans=8.5, Fig. 2C, open circles). The value of a zero-current voltage (Vzero) under
these asymmetrical conditions was -33 mV, having “minus” sign at the side with lower pH. The
RT
Vzero value corresponded to substantial proton selectivity of the conductance (Vmax
=
 pH
F
=58 mV at ΔpH=1.0). This analysis relied on the Nernst equation following [37].
3.2. Mitochondrial respiration and membrane potential
C6-phenyl-NBD, C4-phenyl-NBD and C1-phenyl-NBD stimulated respiration of rat liver
mitochondria at submicromolar concentrations (Fig. 3A). Succinate was used as a substrate and
rotenone was added to prevent inhibition by oxaloacetate. C6-phenyl-NBD exhibited an
increased activity, as compared to C4-phenyl-NBD and C1-phenyl-NBD, in agreement with the
data on planar BLM. Substantially higher concentrations of phenyl-NBD (Fig. 3A, insert) were
required for stimulation of respiration (about 30-fold), even higher than the concentrations of
NBD-C8 studied in our previous work.
The stimulation of respiration by C6-phenyl-NBD and C4-phenyl-NBD was accompanied
by a substantial decrease in membrane potential, as measured by the potential-sensitive dye
safranine O (Fig. 3B, black and cyan curves), with C6-phenyl-NBD being the most effective. The
activity of other compounds in this assay correlated well with their ability to stimulate
respiration (Fig. 3A,B). Of note, the maximum acceleration of respiration caused by C6-phenyl-
NBD (at about 1 µM) was almost equivalent to that induced by a high concentration of DNP (Fig.
3A), while the reduction of membrane potential by C6-phenyl-NBD (at about 1 µM) was far
from maximal (Fig. 3B). This result is consistent with earlier data on conventional uncouplers
[38] and could be associated with nonlinear potential dependence of the activity of respiratory
proton pumps.

ACCEPTED MANUSCRIPT
8
Interestingly, the effect of C6-phenyl-NBD both on the respiration rate (Fig. 4A) and
mitochondrial membrane potential (Fig. 4B) was substantially reversed by glutamate and
aspartate (red and blue curves), substrates of the mitochondrial AGC, which assumed the
involvement of this family of transporters [39] in the uncoupling action of the NBD derivatives.
This assumption was supported by the sensitivity of this action to DEPC (Fig. 4A,B, pink
curves), an inhibitor of AGC [29,40]. Of note, it has been shown in the early work [41] that
interaction of NBD-Cl with AGC causes suppression of its transport activity. Consistent with
this, glutamate also caused partial reversal of the uncoupling effect of NBD-C8, but not that of
gramicidin A (data not shown). Importantly, the absence of the glutamate effect on the
protonophoric activity of C6-phenyl-NBD on the planar BLM (Fig. 2A) excluded any
contribution of the direct interaction of glutamate with C6-phenyl-NBD to the effect on its
uncoupling activity.
The assumed involvement of AGC, as evidenced by partial reversal by glutamate and
aspartate of the C6-phenyl-NBD-induced uncoupling, did not exclude possible participation of
other carriers of the inner mitochondrial membrane (IMM) in the uncoupling action of the NBD
derivatives. Actually, the uncoupling effect of C6-phenyl-NBD was also partially reversed by
both 6-ketocholestanol, the so-called recoupler for FCCP [42], and CAtr, an inhibitor of the
adenine nucleotide translocator ANT (Fig. 4B, green and cyan curves). Both 6-ketocholestanol
and CAtr decreased the rate of respiration which was stimulated by C6-phenyl-NBD (data not
shown). The significant recoupling effect of DEPC (Fig. 4A,B, pink curves) might be partially
due to involvement of tricarboxylate [43] and phosphate [44] transporters, along with AGC.
3.3. Bacterial membrane potential and growth
Fig. 5A displays the effect of C6-phenyl-NBD on the membrane potential of Bacillus
subtilis, as measured by the fluorescence intensity of the potential-sensitive dye DiSC3(5). It is
seen that submicromolar concentrations of C6-phenyl-NBD induced dequenching of the
fluorescence of the probe with the lag-phase decreasing with the increase in the dose. According
to the data shown in Fig. 5B, C4-phenyl-NBD was also effective at submicromolar
concentrations, while C1-phenyl-NBD required the addition of micromoles to decrease the
potential in the minutes time scale. The addition of glutamate (Fig. 5C, black curve) partially
reversed the depolarizing effects of the NBD derivatives, thereby pointing to the involvement of
a glutamate transporter [45] in their uncoupling action. Glutamate was of no effect in case of
uncoupling by low concentrations of gramicidin A (Fig.5C, red curve).
Protonophoric uncouplers are known to suppress bacterial viability via inhibition of
bacterial bioenergetics, in particular, the ATP synthesis [46,47]. In the present work we studied

ACCEPTED MANUSCRIPT
9
the effect of C6-phenyl-NBD, C4-phenyl-NBD and C1-phenyl-NBD, as well as that of NBD-C8
on the growth of B. subtilis. As seen from Fig. 6, C6-phenyl-NBD and C4-phenyl-NBD at a
concentration of 10 µM completely inhibited the growth of B. subtilis, while C1-phenyl-NBD
and NBD-C8 were almost ineffective at this concentration. Noteworthy, C6-phenyl-NBD and C4-
phenyl-NBD caused a drop in membrane potential of B. subtilis at much lower concentrations
than those needed to suppress the growth (Fig. 5). Obviously, bacteria could switch their
metabolism to glycolysis pathway in order to survive in the presence of moderate concentrations
of uncouplers, whereas complete suppression of their growth, observed at tens of micromoles of
C6-phenyl-NBD and C4-phenyl-NBD, could result from damage to other cellular targets than
PMF.
4. Discussion
The uncoupling activity has been found for a large number of compounds of diverse
structure comprising weak acids, weak bases, zwitterions, fatty acids, penetrating cations (in
combination with fatty acids [48]). The majority of uncouplers represent protonophores, i.e.
agents that carry protons across a membrane [21,37,49,50]. However, fatty acids exert moderate
uncoupling action on mitochondria [51], despite lacking the protonophoric activity on planar
BLM [52]. It has appeared that fatty acids behave as cycling protonophores through interaction
with mitochondrial uncoupling proteins [53-56] or other IMM carriers, such as ANT [11,50,57-
60], AGC [29] and the dicarboxylate carrier [61]. Based on the sensitivity to CAtr and 6-
ketocholestanol [16,42,57,58,62-64], the action of the conventional uncouplers DNP, FCCP,
CCCP and SF6847 was suggested to be partially mediated by interaction with certain IMM
proteins, such as ANT and possibly cytochrome c oxidase [11,65,66]. The sensitivity to CAtr
was found not only with DNP, but also with some other uncouplers, such as diazoxide, pinacidil
[67], butylated hydroxytoluene [68], quercetin [69] and asiatic acid [70], while the recoupling
effect of 6-ketocholestanol was observed with uncoupling curcumin derivatives [71], eudesman
[72], SR4 [73] and a bicyclic hydroquinone [74], besides the most potent uncouplers FCCP,
CCCP and SF6847 [42,65,75].
So far, there have been found a rather large number of compounds that exert uncoupling
of oxidative and photosynthetic phosphorylation in mitochondria, chloroplasts and bacteria.
Because of continuously increasing resistance to conventional antibiotics, search for uncouplers
that are effective against pathogenic bacteria, but nontoxic to humans, offers a promising way to
combat infectious diseases. Albeit these two requirements seem to be incompatible, a striking
difference has been described recently [7] between the uncoupling efficacies of a series of

ACCEPTED MANUSCRIPT
10
tuberculosis drug leads in bacterial and mitochondrial membranes. In particular, the new
antitubercular drug SQ109 caused depolarization of bacterial membranes at a concentration of
about 100 nM, while it induced membrane potential collapse in porcine mitochondria at a
concentration of about 100 µM [7]. This difference could be associated with participation of
bacterial membrane proteins in the uncoupling action. Such a mechanism would open the
possibility to find selective antibacterial uncoupling agents. On the other hand, there has been a
long story of using typical uncouplers, such as DNP and usnic acid, to treat obesity, which is
illegal now because of adverse effects of these compounds [76,77]. The toxicity of uncouplers
might be associated either with plasma membrane depolarization [78] or with inhibition of
respiratory proton pumps [79]. Therefore, another criterion to obtain more therapeutically useful
uncouplers consists in widening the difference between therapeutic and toxic concentrations [68].
Here, we found that Cn-phenyl-NBD compounds exhibit protonophoric activity, resulting
in the mitochondrial uncoupling and bacterial growth suppression. Proton transfer apparently
proceeds via cyclic mechanism implying proton gradient-driven translocation of the protonated
Cn-phenyl-NBD from one side of the membrane to another and the backward translocation of the
deprotonated compound (a scheme at the bottom of Fig. 1). Similar to NBD-Cn, the deprotonated
Cn-phenyl-NBD is formed as a result of proton dissociation from the amino group of the
compound. According to [23], NBD-bis-C4, the methylated form of NBD-C4, with the protons of
the amino group replaced by methyl groups, was ineffective in the induction of ion current
through BLM, obviously because this tertiary amine could not be deprotonated. Similar to other
anionic uncouplers [19,37,80,81], translocation of a deprotonated anion across a lipid bilayer is
considered as the rate-limiting step in the proton transfer cycle. Based on the recoupling effects
of glutamate, DEPC, 6-ketocholestanol and CAtr observed here (Fig. 4), it can be concluded that
the mitochondrial uncoupling by the NBD derivatives represents not only a pure proton transfer
through the lipid bilayer by protonophore molecules, but at least partially proceeds with the aid
of proteins, e.g. AGC and ANT. Scheme in Fig.1 shows the binding of an anionic form of an
NBD derivative to these proteins at the matrix side and its translocation into the intermembrane
space. The transmembrane diffusion of an anionic form of a protonophore is generally thought to
be a rate-limiting step. The glutamate-induced partial reversal of the depolarizing effect of the
NBD derivatives, found with B. subtilis (Fig. 5C), for the first time indicated involvement of a
glutamate transporter in the uncoupling action on bacterial membranes. The fact that the
uncoupling activity of the NBD derivatives might be mediated by different proteinaceous
carriers in bacterial and mitochondrial membranes indicates a possibility of finding the
conditions for selective cytotoxic action of these uncouplers on bacteria. On the other hand,
qualitative correlation of the data on the protonophoric activity of the series of arylamino

ACCEPTED MANUSCRIPT
11
derivatives of NBD on planar BLM (Fig. 2B) with those on their uncoupling activity in isolated
mitochondria (Fig. 3) argues in favor of classical protonophoric mechanism of uncoupling by Cn-
phenyl-NBD compounds.
Acknowledgements
This work was financially supported by the Russian Science Foundation grant 16-14-10025
(Y.N.A).

ACCEPTED MANUSCRIPT
12
References
[1] I.T.Paulsen, M.H.Brown, R.A.Skurray, Proton-dependent multidrug efflux systems,
Microbiol.Rev. 60 (1996) 575-608.
[2] J.Behr, R.F.Vogel, Mechanisms of hop inhibition: hop ionophores, J.Agric.Food Chem.
57 (2009) 6074-6081.
[3] L.Ejim, M.A.Farha, S.B.Falconer, J.Wildenhain, B.K.Coombes, M.Tyers, E.D.Brown,
G.D.Wright, Combinations of antibiotics and nonantibiotic drugs enhance antimicrobial
efficacy, Nat.Chem.Biol. 7 (2011) 348-350.
[4] M.A.Farha, C.P.Verschoor, D.Bowdish, E.D.Brown, Collapsing the proton motive force
to identify synergistic combinations against Staphylococcus aureus, Chem.Biol. 20
(2013) 1168-1178.
[5] K.Goldberg, H.Sarig, F.Zaknoon, R.F.Epand, R.M.Epand, A.Mor, Sensitization of gram-
negative bacteria by targeting the membrane potential, FASEB J. 27 (2013) 3818-3826.
[6] K.Li, L.A.Schurig-Briccio, X.Feng, A.Upadhyay, V.Pujari, B.Lechartier, F.L.Fontes,
H.Yang, G.Rao, W.Zhu, A.Gulati, J.H.No, G.Cintra, S.Bogue, Y.L.Liu, K.Molohon,
P.Orlean, D.A.Mitchell, L.Freitas-Junior, F.Ren, H.Sun, T.Jiang, Y.Li, R.T.Guo,
S.T.Cole, R.B.Gennis, D.C.Crick, E.Oldfield, Multitarget drug discovery for tuberculosis
and other infectious diseases, J.Med.Chem. 57 (2014) 3126-3139.
[7] X.Feng, W.Zhu, L.A.Schurig-Briccio, S.Lindert, C.Shoen, R.Hitchings, J.Li, Y.Wang,
N.Baig, T.Zhou, B.K.Kim, D.C.Crick, M.Cynamon, J.A.McCammon, R.B.Gennis,
E.Oldfield, Antiinfectives targeting enzymes and the proton motive force,
Proc.Natl.Acad.Sci.U.S.A 112 (2015) E7073-E7082.
[8] B.C.Schurr, H.Hahne, B.Kuster, J.Behr, R.F.Vogel, Molecular mechanisms behind the
antimicrobial activity of hop iso-alpha-acids in Lactobacillus brevis, Food Microbiol. 46
(2015) 553-563.
[9] G.Kaneti, O.Meir, A.Mor, Controlling bacterial infections by inhibiting proton-dependent
processes, Biochim.Biophys.Acta 1858 (2016) 995-1003.
[10] M.H.Foss, S.Pou, P.M.Davidson, J.L.Dunaj, R.W.Winter, S.Pou, M.H.Licon, J.K.Doh,
Y.Li, J.X.Kelly, R.A.Dodean, D.R.Koop, M.K.Riscoe, G.E.Purdy, Diphenylether-
modified 1,2-diamines with improved drug properties for development against
Mycobacterium tuberculosis, ACS Infect.Dis. 2 (2016) 500-508.
[11] V.P.Skulachev, Uncoupling: new approaches to an old problem of bioenergetics,
Biochim.Biophys.Acta 1363 (1998) 100-124.
[12] D.B.Zorov, Mitochondrial damage as a source of diseases and aging: a strategy of how to
fight these, Biochim.Biophys.Acta 1275 (1996) 10-15.
[13] A.Boveris, Mitochondrial production of superoxide radical and hydrogen peroxide,
Adv.Exp.Med.Biol. 78 (1977) 67-82.
[14] S.S.Liu, Generating, partitioning, targeting and functioning of superoxide in
mitochondria, Biosci.Rep. 17 (1997) 259-272.

ACCEPTED MANUSCRIPT
13
[15] V.P.Skulachev, Role of uncoupled and non-coupled oxidations in maintenance of safely
low levels of oxygen and its one-electron reductants, Q.Rev.Biophys. 29 (1996) 169-202.
[16] S.S.Korshunov, V.P.Skulachev, A.A.Starkov, High protonic potential actuates a
mechanism of production of reactive oxygen species in mitochondria, FEBS Lett. 416
(1997) 15-18.
[17] S.Detchanamurthy, P.A.Gostomski, Metabolic uncouplers in environmental research: a
critical review, Rev.Chem.Eng. 28 (2012) 309-317.
[18] H.Miyoshi, H.Tsujishita, N.Tokutake, T.Fujita, Quantitative analysis of uncoupling
activity of substituted phenols with a physicochemical substituent and molecular
parameters, Biochim.Biophys.Acta 1016 (1990) 99-106.
[19] S.Spycher, P.Smejtek, T.I.Netzeva, B.I.Escher, Toward a class-independent quantitative
structure--activity relationship model for uncouplers of oxidative phosphorylation, Chem.
Res.Toxicol. 21 (2008) 911-927.
[20] L.C.Martineau, Simple thermodynamic model of unassisted proton shuttle uncoupling
and prediction of activity from calculated speciation, lipophilicity, and molecular
geometry, J.Theor.Biol. 303 (2012) 33-61.
[21] R.T.Naven, R.Swiss, J.Klug-McLeod, Y.Will, N.Greene, The development of structure-
activity relationships for mitochondrial dysfunction: uncoupling of oxidative
phosphorylation, Toxicol.Sci. 131 (2013) 271-278.
[22] M.M.Shchepinova,
S.S.Denisov,
E.A.Kotova,
L.S.Khailova,
D.A.Knorre,
G.A.Korshunova, V.N.Tashlitsky, F.F.Severin, Y.N.Antonenko, Dodecyl and octyl esters
of fluorescein as protonophores and uncouplers of oxidative phosphorylation in
mitochondria at submicromolar concentrations, Biochim.Biophys.Acta 1837 (2014) 149-
158.
[23] S.S.Denisov, E.A.Kotova, L.S.Khailova, G.A.Korshunova, Y.N.Antonenko, Tuning the
hydrophobicity overcomes unfavorable deprotonation making octylamino-substituted 7-
nitrobenz-2-oxa-1,3-diazole (n-octylamino-NBD) a protonophore and uncoupler of
oxidative phosphorylation in mitochondria, Bioelectrochemistry. 98 (2014) 30-38.
[24] A.A.Aboderin, R.E.Semakula, E.Boedefeld, R.A.Kenner, Base-Promoted Cleavage of
Alpha-N-Nbd-Peptides, FEBS Lett. 34 (1973) 90-94.
[25] H.Heberer, H.Kersting, H.Matschiner, Derivatisierung von Aminen mit 4-substituierten
7-Nitrobenz-2,1,3-oxadiazolen, J.Prakt.Chem. 327 (1985) 487-504.
[26] J.C.Halle, M.Mokhtari, P.Soulie, M.J.Pouet, Evaluation of the electron-withdrawing
character of the 4-nitrobenzofurazanyl moiety: Ionization of 7-anilino-4-
nitrobenzofurazans, Can.J.Chem. 75 (1997) 1240-1247.
[27] P.B.Ghosh, M.W.Whitehouse, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole:
a
new
fluorigenic reagent for amino acids and other amines, Biochem.J. 108 (1968) 155-156.
[28] S.Uchiyama, T.Santa, T.Fukushima, H.Homma, K.Imai, Effects of the substituent groups
at the 4- and 7-positions on the fluorescence characteristics of benzofurazan compounds,
J.Chem.Soc.-Perkin Trans. 2 (1998) 2165-2173.

ACCEPTED MANUSCRIPT
14
[29] V.N.Samartsev, A.V.Smirnov, I.P.Zeldi, O.V.Markova, E.N.Mokhova, V.P.Skulachev,
Involvement of aspartate/glutamate antiporter in fatty acid-induced uncoupling of liver
mitochondria, Biochim.Biophys.Acta 1319 (1997) 251-257.
[30] V.N.Samartsev, E.N.Mokhova, V.P.Skulachev, The pH-dependent reciprocal changes in
contributions of ADP/ATP antiporter and aspartate/glutamate antiporter to the fatty acid-
induced uncoupling, FEBS Lett. 412 (1997) 179-182.
[31] P.Mueller, D.O.Rudin, H.T.Tien, W.C.Wescott, Methods for the formation of single
bimolecular lipid membranes in aqueous solution, J.Phys.Chem. 67 (1963) 534-535.
[32] F.Lombardo, M.Y.Shalaeva, K.A.Tupper, F.Gao, ElogD(oct): a tool for lipophilicity
determination in drug discovery. 2. Basic and neutral compounds, J.Med.Chem. 44
(2001) 2490-2497.
[33] A.A.Martyushin, D.A.Tsarev, M.A.Grigorenko, I.I.Fedorov, G.V.Ramenskaya,
V.N.Tashlitsky, V.P.Skulachev, Estimation of lipophilicity of new generation of
antioxidants, Pharmacia 5 (2008) 23-29.
[34] Y.Avnir, Y.Barenholz, pH determination by pyranine: medium-related artifacts and their
correction, Anal.Biochem. 347 (2005) 34-41.
[35] K.E.Akerman, M.K.Wikstrom, Safranine as a probe of the mitochondrial membrane
potential, FEBS Lett. 68 (1976) 191-197.
[36] J.B.Miller, D.E.Koshland, Jr., Sensory electrophysiology of bacteria: relationship of the
membrane potential to motility and chemotaxis in Bacillus subtilis,
Proc.Natl.Acad.Sci.U.S.A 74 (1977) 4752-4756.
[37] S.G.McLaughlin, J.P.Dilger, Transport of protons across membranes by weak acids,
Physiol Rev. 60 (1980) 825-863.
[38] S.Luvisetto, D.Pietrobon, G.F.Azzone, Uncoupling of oxidative phosphorylation. 1.
Protonophoric effects account only partially for uncoupling, Biochemistry 26 (1987)
7332-7338.
[39] N.D.Amoedo, G.Punzi, E.Obre, D.Lacombe, A.De Grassi, C.L.Pierri, R.Rossignol,
AGC1/2, the mitochondrial aspartate-glutamate carriers, Biochim.Biophys.Acta 1863
(2016) 2394-2412.
[40] T.Dierks, R.Stappen, A.Salentin, R.Kramer, Probing the active site of the reconstituted
aspartate/glutamate carrier from bovine heart mitochondria: carbodiimide-catalyzed
acylation of a functional lysine residue, Biochim.Biophys.Acta 1103 (1992) 13-24.
[41] R.Stappen, T.Dierks, A.Broer, R.Kramer, Probing the active site of the reconstituted
aspartate/glutamate carrier from mitochondria. Structure/function relationship involving
one lysine and two cysteine residues, Eur.J.Biochem. 210 (1992) 269-277.
[42] A.A.Starkov, V.I.Dedukhova, V.P.Skulachev, 6-ketocholestanol abolishes the effect of
the most potent uncouplers of oxidative phosphorylation in mitochondria, FEBS Lett. 355
(1994) 305-308.

ACCEPTED MANUSCRIPT
15
[43] R.S.Kaplan, J.A.Mayor, N.Johnston, D.L.Oliveira, Purification and characterization of
the reconstitutively active tricarboxylate transporter from rat liver mitochondria,
J.Biol.Chem. 265 (1990) 13379-13385.
[44] R.S.Kaplan, R.D.Pratt, P.L.Pedersen, Purification and characterization of the
reconstitutively active phosphate transporter from rat liver mitochondria, J.Biol.Chem.
261 (1986) 12767-12773.
[45] B.Tolner, T.Ubbink-Kok, B.Poolman, W.N.Konings, Characterization of the
proton/glutamate symport protein of Bacillus subtilis and its functional expression in
Escherichia coli, J.Bacteriol. 177 (1995) 2863-2869.
[46] F.M.Harold, Conservation and transformation of energy by bacterial membranes,
Bacteriol.Rev. 36 (1972) 172-230.
[47] F.M.Harold, Gleanings of a chemiosmotic eye, Bioessays 23 (2001) 848-855.
[48] F.F.Severin,
I.I.Severina,
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, V.P.Skulachev, Penetrating
cation/fatty acid anion pair as
a
mitochondria-targeted protonophore,
Proc.Natl.Acad.Sci.U.S.A 107 (2010) 663-668.
[49] H.Terada, Uncouplers of oxidative phosphorylation, Environ.Health Perspect. 87 (1990)
213-218.
[50] K.B.Wallace,
A.A.Starkov,
Mitochondrial
targets
of
drug
toxicity,
Annu.Rev.Pharmacol.Toxicol. 40 (2000) 353-388.
[51] H.A.Lardy, B.C.Pressman, Effect of surface active agents on the latent ATPase of
mitochondria, Biochim.Biophys.Acta 21 (1956) 458-466.
[52] J.Gutknecht, Proton conductance caused by long-chain fatty acids in phospholipid bilayer
membranes, J.Membrane Biol. 106 (1988) 83-93.
[53] P.J.Strieleman, K.L.Schalinske, E.Shrago, Fatty acid activation of the reconstituted
brown adipose tissue mitochondria uncoupling protein, J.Biol.Chem 260 (1985) 13402-
13405.
[54] K.D.Garlid, D.E.Orosz, M.Modriansky, S.Vassanelli, P.Jezek, On the mechanism of fatty
acid-induced proton transport by mitochondrial uncoupling protein, J.Biol.Chem. 271
(1996) 2615-2620.
[55] V.Beck, M.Jaburek, T.Demina, A.Rupprecht, R.K.Porter, P.Jezek, E.E.Pohl,
Polyunsaturated fatty acids activate human uncoupling proteins 1 and 2 in planar lipid
bilayers, FASEB J. 21 (2007) 1137-1144.
[56] A.Rupprecht, E.A.Sokolenko, V.Beck, O.Ninnemann, M.Jaburek, T.Trimbuch,
S.S.Klishin, P.Jezek, V.P.Skulachev, E.E.Pohl, Role of the transmembrane potential in
the membrane proton leak, Biophys.J. 98 (2010) 1503-1511.
[57] A.Y.Andreyev, T.O.Bondareva, V.I.Dedukhova, E.N.Mokhova, V.P.Skulachev,
N.I.Volkov, Carboxyatractylate inhibits the uncoupling effect of free fatty acids, FEBS
Lett. 226 (1988) 265-269.

ACCEPTED MANUSCRIPT
16
[58] A.Y.Andreyev, T.O.Bondareva, V.I.Dedukhova, E.N.Mokhova, V.P.Skulachev,
L.M.Tsofina, N.I.Volkov, T.V.Vygodina, The ATP/ADP-antiporter is involved in the
uncoupling effect of fatty acids on mitochondria, Eur.J.Biochem. 182 (1989) 585-592.
[59] P.Schonfeld, Does the function of adenine nucleotide translocase in fatty acid uncoupling
depend on the type of mitochondria?, FEBS Lett. 264 (1990) 246-248.
[60] V.P.Skulachev, Anion carriers in fatty acid-mediated physiological uncoupling,
J.Bioenerg.Biomembr. 31 (1999) 431-445.
[61] M.R.Wieckowski, L.Wojtczak, Involvement of the dicarboxylate carrier in the
protonophoric
action
of
long-chain
fatty
acids
in
mitochondria,
Biochem.Biophys.Res.Commun. 232 (1997) 414-417.
[62] A.Vianello, F.Macri, E.Braidot, E.N.Mokhova, Effect of 6-ketocholestanol on FCCP-
and DNP-induced uncoupling in plant mitochondria, FEBS Lett. 365 (1995) 7-9.
[63] E.Chavez, R.Moreno-Sanchez, C.Zazueta, A.Cuellar, J.Ramirez, H.Reyes-Vivas,
C.Bravo, S.Rodriguez-Enriquez, On the mechanism by which 6-ketocholestanol protects
mitochondria against uncoupling-induced Ca2+ efflux, FEBS Lett. 379 (1996) 305-308.
[64] A.A.Starkov, D.A.Bloch, B.V.Chernyak, V.I.Dedukhova, S.E.Mansurova, I.I.Severina,
R.A.Simonyan, T.V.Vygodina, V.P.Skulachev, 6-Ketocholestanol is a recoupler for
mitochondria,
chromatophores
and
cytochrome
oxidase
proteoliposomes,
Biochim.Biophys.Acta 1318 (1997) 159-172.
[65] A.A.Starkov, "Mild" uncoupling of mitochondria, Biosci.Rep. 17 (1997) 273-279.
[66] A.A.Starkov, Protein-mediated energy-dissipating pathways in mitochondria,
Chem.Biol.Interact. 163 (2006) 133-144.
[67] D.M.Kopustinskiene, J.Jovaisiene, J.Liobikas, A.Toleikis, Diazoxide and pinacidil
uncouple pyruvate-malate-induced mitochondrial respiration, J.Bioenerg.Biomembr. 34
(2002) 49-53.
[68] P.H.Lou, B.S.Hansen, P.H.Olsen, S.Tullin, M.P.Murphy, M.D.Brand, Mitochondrial
uncouplers with an extraordinary dynamic range, Biochem.J. 407 (2007) 129-140.
[69] R.Ortega, N.Garcia, The flavonoid quercetin induces changes in mitochondrial
permeability by inhibiting adenine nucleotide translocase, J.Bioenerg.Biomembr. 41
(2009) 41-47.
[70] Y.Lu, S.Liu, Y.Wang, D.Wang, J.Gao, L.Zhu, Asiatic acid uncouples respiration in
isolated mouse liver mitochondria and induces HepG2 cells death, Eur.J.Pharmacol. 786
(2016) 212-223.
[71] H.Ligeret, S.Barthelemy, D.G.Bouchard, P.A.Carrupt, J.P.Tillement, S.Labidalle,
D.Morin, Fluoride curcumin derivatives: new mitochondrial uncoupling agents, FEBS
Lett. 569 (2004) 37-42.
[72] V.L.Dalla, A.N.Garcia-Argaez, A.Braga, M.Martinez-Vazquez, S.Grancara, P.Martinis,
E.Agostinelli, A.Toninello, An eudesman derivative from Verbesina persicifolia D.C. as
a natural mild uncoupler in liver mitochondria. A new potential anti-obesity agent?,
Curr.Pharm.Des 20 (2014) 253-261.

ACCEPTED MANUSCRIPT
17
[73] J.L.Figarola, J.Singhal, J.D.Tompkins, G.W.Rogers, C.Warden, D.Horne, A.D.Riggs,
S.Awasthi, S.S.Singhal, SR4 uncouples mitochondrial oxidative phosphorylation,
modulates AMP-dependent kinase (AMPK)-mammalian target of rapamycin (mTOR)
signaling, and inhibits proliferation of HepG2 hepatocarcinoma cells, J.Biol.Chem 290
(2015) 30321-30341.
[74] F.A.Urra, M.Cordova-Delgado, M.Lapier, A.Orellana-Manzano, L.Acevedo-Arevalo,
H.Pessoa-Mahana, J.M.Gonzalez-Vivanco, M.Martinez-Cifuentes, O.Ramirez-Rodriguez,
J.P.Millas-Vargas, B.Weiss-Lopez, M.Pavani, J.Ferreira, R.Araya-Maturana, Small
structural changes on a hydroquinone scaffold determine the complex I inhibition or
uncoupling of tumoral oxidative phosphorylation, Toxicol.Appl.Pharmacol. 291 (2016)
46-57.
[75] C.von Ballmoos, O.Biner, T.Nilsson, P.Brzezinski, Mimicking respiratory
phosphorylation using purified enzymes, Biochim.Biophys.Acta 1857 (2016) 321-331.
[76] J.Grundlingh, P.I.Dargan, M.El-Zanfaly, D.M.Wood, 2,4-dinitrophenol (DNP): a weight
loss agent with significant acute toxicity and risk of death, J.Med.Toxicol. 7 (2011) 205-
212.
[77] C.T.Moreira, A.L.Oliveira, J.F.Comar, R.M.Peralta, A.Bracht, Harmful effects of usnic
acid on hepatic metabolism, Chem.Biol.Interact. 203 (2013) 502-511.
[78] D.T.Poole, The effect of uncoupling agents on the pH gradient across the plasma
membrane of the Ehrlich ascites tumor cell, Biochem.Biophys.Res.Commun. 32 (1968)
403-407.
[79] V.P.Skulachev, A.A.Sharaf, L.S.Yagujzinsky, A.A.Jasaitis, E.A.Liberman, V.P.Topali,
The effect of uncouplers on mitochondria, respiratory enzyme complexes and artificial
phospholipid membranes, Curr.Mod.Biol. 2 (1968) 98-105.
[80] J.Kasianowicz, R.Benz, S.Mclaughlin, The kinetic mechanism by which CCCP (carbonyl
cyanide m- chlorophenylhydrazone) transports protons across membranes, J.Membr.Biol.
82 (1984) 179-190.
[81] S.M.Saparov, Y.N.Antonenko, P.Pohl, A new model of weak acid permeation through
membranes revisited: does Overton still rule?, Biophys.J. 90 (2006) L86-L88.

ACCEPTED MANUSCRIPT
18
Figure legends
Fig.1. Chemical structures of 7-nitrobenz-2-oxa-1,3-diazole (NBD) derivatives used in the
present work and a scheme depicting their protonophoric activity in mitochondria.
Fig. 2. A. C6-phenyl-NBD induced electrical current through planar BLM made from DPhPC.
The solution was 10 mM Tris, 10 mM MES, 10 mM beta-alanine, 10 mM KCl, pH 7.5. BLM
voltage 50 mV. B. Mean±S.E. of electrical current through the BLM induced by 10 µM C6-
phenyl-NBD, C4-phenyl-NBD, C1-phenyl-NBD and phenyl-NBD. NBD-C10 (10 µM) was also
tested for comparison. Experimental conditions were the same as described in panel A. C.
Current-voltage (I-V) curve for 5 µM C6-phenyl-NBD under symmetrical (closed circles, pH cis
and trans were either 7.5 (black) or 8.5 (red)) and asymmetrical conditions (open circles, pH cis
was 7.5, pH trans was 8.5). The solution was 10 mM Tris, 10 mM MES, 10 mM β-alanine, 10
mM KCl.
Fig. 3. Effect of NBD derivatives on rat liver mitochondria respiration (A) and mitochondrial
membrane potential (B) with succinate as a substrate. The incubation medium was 250 mM
sucrose, 5 mM MOPS, and 1 mM EGTA, pH 7.4; 1 µg/ml oligomycin. Succinate, 5 mM,
Rotenone, 2 µM. Membrane potential was assayed by measuring the difference of the
absorbance of safranine O at 555 and 523 nm. 0.5 µM of the compounds was added at 180 s, 300
s, and 450 s; FCCP (1 µM) at 580 s. For other conditions see “Materials and Methods”. Points
and error bars represent Mean±S.E. with minimum three independent experiments.
Fig. 4. A. Effect of glutamate (2 mM, green curve), aspartate (2 mM, blue curve), and
diethylpyrocarbonate (DEPC, 100 µM, pink curve) on the stimulation of respiration of rat liver
mitochondria by C6-phenyl-NBD (0.6 µM). In samples with succinate, the medium was
supplemented with 2 μM rotenone. Additions where indicated: 5 mM succinate, 40 μM DNP. B.
Effect of glutamate (2 mM), diethylpyrocarbonate (DEPC, 100 µM), carboxyatractyloside (CAtr,
1 µM), and 6-ketocholestanol (100 µM) on the membrane potential of mitochondria in the
presence of C6-phenyl-NBD (0.6 µM). The experimental conditions were as in Fig.3B.
Fig. 5. A. Dose-dependent effect of C6-phenyl-NBD on the kinetics of the membrane potential of
Bacillus subtilis cells assessed by DiSC3(5) fluorescence. B. Effect of various NBD derivatives
on the membrane potential of Bacillus subtilis cells, as estimated by the time needed for 50 %
increase in the DiSC3(5) fluorescence. C. Recoupling effect of glutamate (2 mM) on the

ACCEPTED MANUSCRIPT
19
membrane potential of Bacillus subtilis cells in the presence of C6-phenyl-NBD (black curve, 50
nM), C1-phenyl-NBD (blue curve, 1 µM) or low concentration of gramicidin A (red curve, 5
ng/ml), as measured by fluorescence of DiSC3(5). For other conditions see Materials and
Methods.
Fig. 6. A. Dose-dependent effect of C1-phenyl-NBD on the growth kinetics of Bacillus subtilis
cells assessed by the absorbance at 620 nm. B. Effect of different concentrations of NBD
derivatives on the growth of B. subtilis evaluated by the absorbance at 620 nm after 21-hour
incubation at 370C by a 96-well Multiskan FC Microplate Reader. The data points represent
meanSD of at least three experiments.

ACCEPTED MANUSCRIPT
20

ACCEPTED MANUSCRIPT
21

ACCEPTED MANUSCRIPT
22

ACCEPTED MANUSCRIPT
23

ACCEPTED MANUSCRIPT
24

ACCEPTED MANUSCRIPT
25

ACCEPTED MANUSCRIPT
26
Graphical abstract

ACCEPTED MANUSCRIPT
27
Highlights
Cn-phenylamino 7-nitrobenz-2-oxa-1,3-diazole (Cn-phenyl-NBD) varied in Cn alkyl chain
Cn-phenyl-NBD with n=1, 4, 6 carried protons across planar BLM with increasing efficacy
C6-phenyl-NBD accelerated respiration and lowered membrane potential of mitochondria
Uncoupling action of C6-phenyl-NBD was reversed by glutamate and diethyl pyrocarbonate
C6- and C4-phenyl-NBD suppressed membrane potential and growth of Bacillus subtilis