Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 12, pp. 1811-1848 © Pleiades Publishing, Ltd., 2025.

Russian Text © The Author(s), 2025, published in Biokhimiya, 2025, Vol. 90, No. 12, pp. 1941-1980.

1811

REVIEW

Classification of Mitochondrial Protonophoric

Uncouplers and their Modifications

in Biological Environment

Yuri N. Antonenko

1,a

*, Elena A. Kotova

, Vladimir S. Krasnov

1,2

and Roman S. Kirsanov

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University,

119991 Moscow, Russia

Faculty of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia

e-mail: antonen@belozersky.msu.ru

Received July 14, 2025

Revised October 7, 2025

Accepted October 8, 2025

Abstract— In this review, we analyze diversity of mitochondrial uncouplers, a class of compounds, which was

in the focus of Vladimir Skulachev’s attention throughout his scientific career, starting from the basics of bio-

energetics and validation of Mitchell’s chemiosmotic theory to the development of concepts of mild uncoupling

and its therapeutic role. The review is the first to put forward the idea of classifying uncouplers by the type

of a functional group that provides their protonophoric activity, i.e., the ability to transfer protons across

the membrane, causing its depolarization and thereby uncoupling the ATP synthesis from the operation of

proton pumps in the electron transport chain. In particular, it is shown that anionic and zwitterionic uncou-

plers can be divided into groups of OH-, NH-, SH-, and CH-acids. Of importance, here we consider metabolic

transformations of mitochondrial uncouplers determining tissue-specificity of their action.

DOI: 10.1134/S000629792560245X

Keywords: mitochondria, uncouplers, protonophores, oxidative phosphorylation, lipid membranes

* To whom correspondence should be addressed.

INTRODUCTION

Protonophores comprise a subgroup in a gener-

al group of ionophores. Ionophores are low molecu-

lar weight compounds of diverse chemical structure,

which are capable of forming complexes with ions

and transfer them across natural and artificial mem-

branes. In particular, the 12-membered cyclic peptide

valinomycin is capable of transporting potassium

cations across membranes with high potassium-sodi-

um selectivity [1]. In addition to electrogenic iono-

phores that transport ions together with their charge,

there are non-electrogenic ionophores that exchange

cations for protons or anions for hydroxide ions.

The best known among them is the polyether antibi-

otic nigericin [2] that exchanges potassium ions for

protons, and the dye of bacterial origin prodigiosin

[3] that exchanges chloride ions for hydroxide ions.

In this review we focus on the special type of

ionophores – electrogenic transporters of hydrogen

ions, which were coined protonophores. It was found

out that these compounds are able to uncouple both

oxidative and photosynthetic phosphorylation, i.e. to

disrupt coupling of proton pump functioning in elec-

tron transport chains of mitochondria, chloroplasts,

and bacteria with ATP synthesis on the energy-trans-

forming membranes. Under normal conditions, oxida-

tion of respiratory substrates in the inner mitochon-

drial membrane is coupled with the process of ADP

phosphorylation to ATP, which is a universal energy

“currency” in cells. Uncouplers disconnect these two

processes, so that mitochondrial respiration is not ac-

companied with ATP synthesis.

According to Mitchell’s theory, uncouplers per-

form their functions via transporting protons across

lipid parts of membranes, hence, they are protono-

phores [4, 5]. The simplest electric analogy of this

system is a battery (respiratory chain) and a lantern

ANTONENKO et al.1812

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

(ATP-synthase). If a shunt resistor is inserted between

them, the lantern is switched off. Opening of proton

channels formed by the special proton-translocating

proteins of the UCP family (Uncoupling Proteins) or

addition of special protonophoric uncouplers (com-

pounds that selectively transport hydrogen ions

across the membrane) could serve as such shunt re-

sistors. Among those, the uncouplers 2,4-dinitrophe-

nol (DNP) and carbonyl cyanide m-chlorophenylhy-

drazone (CCCP) are best known. They are weak acids

containing aromatic moieties, which help the mole-

cule to effectively cross the membrane even when

carrying negative charge despite the high energy bar-

rier for the anion to enter the hydrophobic region of

the membrane.

Due to disruptions in the cell bioenergetics

caused by protonophores they are considered as im-

portant compounds for biochemistry, cell biology, and

physiology. In addition to fundamental studies, they

are used in agriculture and forestry industry due to

their antimicrobial, insecticide, and herbicide (pesti-

cide) properties. In addition to the widely used DNP,

pentachlorophenol [6], 2-(1-methylpropyl)-4,6-dinitro-

phenol (dinoseb)[7,8], as well as diarylamine fluazi-

nam [9], and other compounds are used in industry.

However, not the industrial use of protonophores, but

their potential application in pharmacology is import-

ant for us. It is known that protonophores exhibit

protection against muliple significant pathologies in

animal models. They are cardio- [10], neuro- [11,  12],

nephro-  [13], and radio-  [14] protectors, and exert

antidiabetic effects [15-17]; and this list can be ex-

tended further. Potential of uncouplers as anticancer

preparations has been mentioned in many studies

[18]. Moreover, low doses of DNP were shown to sig-

nificantly extend lifespan of rats  [19], mice  [20], yeast

[21], and Drosophila flies [22].

Classic protonophores are organic acids with pK

close to physiological pH values, which have an ex-

tended system of π-electrons assumed to be capable of

delocalizing negative charge. This facilitates penetra-

tion of the anionic form of a protonophore (P

−

) across

the membrane in response to the applied voltage. Pro-

tonophores differ fundamentally from the penetrating

acids or bases, such as salicylic acid or 9-aminoacri-

dine, which are capable of crossing the membrane in

the neutral form, but cannot penetrate the membrane

in the charged form. As a result, they only can change

pH gradient on the membrane due to their concentra-

tion gradient, but cannot dissipate membrane potential

in mitochondria. Measurement of external concentra-

tion of salicylic acid could help to assess pH gradient

on mitochondrial and bacterial membranes [23, 24],

which is usually around 0.5-1 pH units [25].

In the recent study by Bertholet et al. [26], an

electric current through an individual mitoplast at-

tached to a glass micropipette was measured using

the patch-clamp technique. The addition of 50  µM

DNP resulted in a significant increase in the current

through a fragment of the inner mitochondrial mem-

brane, while in the case of plasma membrane of the

same surface area no increase in the current was ob-

served at 50  µM DNP. However, higher concentration

of DNP (500  µM and 1  mM) increased the current

through the plasma membrane. The DNP-induced

current through mitoplast was suppressed by the ad-

dition of the specific inhibitor of adenine nucleotide

translocator  1 (ANT1) carboxyatractyloside (CATR).

Similar results were obtained by another method in

the earlier studies conducted in Skulachev’s laborato-

ry demonstrating that the uncoupling effect of DNP

on mitochondria is partially mediated by interaction

with ANT1 [27]. In addition to the effect of CATR, Ber-

tholet et al. investigated [26] mitoplasts derived from

the mice with knockout of the ANT1 gene. It was

shown that the effect of other well-known uncou-

plers, CCCP and carbonyl cyanide p-trifluoromethoxy-

phenylhydrazone (FCCP), also is partially mediated by

the interaction with ANT1 [26, 28]. These results are

in agreement with the results of studies conducted

in 1960s-1970s reporting ability of uncouplers to bind

to proteins [29] and the existence of specific binding

sites for uncouplers on the membranes [30, 31].

CHEMICAL STRUCTURES

OF PROTONOPHORES, THEIR DIVERSITY

AND CLASSIFICATION

Several reviews have been published in recent

years that summarize the data of numerous studies

on various uncouplers [32-36]. However, no classifi-

cation of uncouplers has been discussed in these re-

views. According to the type of the charged form of

the molecules, which participate in proton transfer

across membranes, protonophores could be divided

into three main classes: anionic, cationic, and zwit-

terionic (Fig. 1). We suggest this classification in the

present review.

In the earlier studies, protonophores were often

divided into two types: type  1 included those com-

pounds that transport protons as monomers, and

type  2– transport as dimers [37,38]. The dimer-based

mechanism was postulated for DNP [39], tetrachlo-

rotrifluoromethyl benzimidazole (TTFB) [40], 5,6-di-

chloro-2-trifluoromethylbenzimidazole (DTFB) [41],

and pentachlorophenol  [42] based on measuring

electric current through planar bilayer lipid mem-

brane (BLM). The current across BLMs induced by

these protonophores increased quadratically with the

increase in their concentration under certain con-

ditions. On the other hand, the uncoupling activity

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Fig. 1. Operation cycles of anionic protonophore (a), cationic protonophore (b), zwitterionic protonophore in cationic

charged form (c), and zwitterionic protonophore in anionic charged form (d).

in isolated mitochondria depended linearly on the

concentration of these compounds [43, 44]. Of note,

the formation of dimers in the case of pentachloro-

phenol depended significantly on the solvent and was

suppressed upon the addition of water to organic sol-

vent [45]. The formation of dimers of uncouplers was

also reduced in the case of BLM formed from the lip-

ids dissolved in chlorodecane but not from the lipids

dissolved in the commonly used decane [44]. It could

be suggested that unlike the case of planar BLM, the

uncoupling activity of these compounds in mitochon-

dria proceeds via the monomeric mechanism due to

the presence of a large number of proteins in these

membranes, which affect physicochemical properties

of the membrane. Hence, the previously suggested

classification of protonophores into two types could

not correspond to their properties in biological en-

vironment.

Anionic protonophores. At present, anionic pro-

tonophores comprise the most numerous group of

compounds that includes the well-known uncouplers

such as DNP and CCCP. Such protonophores could

exhibit very high rate of operation, because anions

penetrate through membranes much more effectively

than cations due to the presence of the dipole poten-

tial on lipid membranes [46, 47]. The dipole poten-

tial is formed by the oriented dipoles of phospholipid

heads and a layer of tightly bound water, with large

contribution provided by carbonyls of ester bonds be-

tween fatty acids and glycerol [48]. In turn, anionic

protonophores could be divided into classes according

to the type of a group from which proton is cleaved:

OH-acids, NH-acids, SH-acids, and CH-acids.

Based on the results of investigation of uncou-

plers on BLM, a classic scheme of operation of an-

ionic protonophores has been suggested: an anionic

form of a protonophore (P

−

) crosses the membrane in

response to the applied potential, then it is protonat-

ed (transforming into the PH-form), diffuses in a neu-

tral form to the opposite side along the concentration

gradient, and, finally, is deprotonated, thus complet-

ing the full cycle of protonophore operation (Fig.  1a).

Such a carousel-like proton cycling may occur with

proton release into water and without it. The latter

variant was termed ‘small carousel’ by Markin and

Chizmadzhev  [49]. In this model, transport of the

anionic form of a protonophore P

−

is determined by

electrogenicity and voltage dependence. Proton se-

lectivity of protonophore functioning is usually eval-

uated from the potential of the open circuit in the

presence of pH gradient, while comparison with the

Nernst equation allows one to quantitatively estimate

the selectivity. High permeability of the neutral (PH)

form of a protonophore, resulting in its equal con-

centrations at the opposite sides of the membrane

[PH]

 = [PH]

, determines the proton selectivity of a

protonophore. Provided that protonation/deprotona-

tion reactions at the membrane are at equilibrium,

we have got [PH]  =  [P

−

]∙[H

]/K

at both sides of the

membrane. It follows from this equation that the ra-

tio of [P

−

]

to[P

−

]

is equal to the ratio [H

]

to [H

]

It means that the pH gradient leads to formation

ANTONENKO et al.1814

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

of the gradient of anionic forms of the protonophore

−

at two sides of the membrane. In this case, in-

variability of the near-membrane pH, which is pro-

vided by high buffer capacity of aqueous solution, is

an important issue. The question arises: at what pH

value the highest permeability of protonophore will

be achieved? Theoretical estimates indicate that this

pH value corresponds to the value of pK

plus half

of the logarithm of the ratio of permeabilities of the

neutral and anionic forms of the protonophore (see

equation 3.8 in the book by Markin and Chizmadzhev

[49]) [50]. Correspondingly, in the case of equal per-

meabilities of these two forms, we would obtain max-

imum permeability of the protonophore at the pH

value equal to pK

Above, we presented a kinetic model of protono-

phore operation. To have a possibility of predicting

the protonophoric activity of new potential drugs,

several authors have made attempts to create theo-

retical models based on the structure of compounds

[51-56]. Most of these models suggest that the key

factors determining the uncoupling activity are hy-

drophobicity of the compound and its pK

. In addi-

tion, the importance of the permeability not only of

the neutral form, but also of the deprotonated anion

has been noted. It has been reported in the review

by Kotova and Antonenko [34] that validity of such

models is limited due to contribution of the proteins

in the inner mitochondrial membrane to the uncou-

pling activity of protonophores.

The question arises: how closely associated are

the protonophore properties on BLM and their un-

coupling capacity in mitochondria? Correlation be-

tween these parameters has been shown to be rather

strong [57-59], but there are striking exceptions for

certain compounds [34, 60]. Correlation between pro-

tonophoric activity in mitochondria and in artificial

membranes is significantly better in the case of using

liposomes instead of BLMs [58]. Even better correla-

tion has been observed if the protonophoric activity

in mitochondria is assessed from their swelling under

certain conditions [61] or from the pH change in the

suspension of mitochondria in the presence of vali-

nomycin [38].

Uncouplers based on OH-acids. All synthetic phe-

nols uncouple mitochondria to varying degrees. Var-

ious substituted phenols have been analyzed previ-

ously in a number of reviews [30, 37, 38], and it has

been concluded that their protonophoric activity is

determined by their lipophilicity and the pK

value.

In addition to these two parameters, the activity is

affected by certain other factors. In particular, 3,5-di-

bromo-4-hydroxybenzonitrile has almost the same

lipophilicity and pK

, as DNP, but is 6-9-fold more

active in mitochondria [30, 62]. How could we ex-

plain this? Two main scenarios could be considered.

(1)  Translocation across the membrane is a complex

process associated with adaptation of the molecule

to the local environment. (2)  Another possible cause

could be interaction of substituted phenols with pro-

teins as described in the case of DNP [63].

The uncoupling action of halogen-substituted

phenols has been thoroughly investigated in the ear-

lier studies [51, 59, 64]. Pentachlorophenol, which

uncouples mitochondria at submicromolar concentra-

tions, has been found to be the most active among

them [43]. Its strong protonophoric activity has been

demonstrated on BLM [42, 58, 60, 65]. However, pen-

tachlorophenol has been found to inhibit respiratory

complex II with high efficiency [66]. Pentachloro-

phenol is widely used in the forestry industry as a

wood preservative, as well as pesticide in agriculture

[6]. The compound itself and its esters are also used

in peptide synthesis as reagents activating carboxyl

group. Extensive use of pentachlorophenol caused

significant contamination of the environment with

this compound, which is toxic to humans and, in par-

ticular, is a carcinogen.

The problem of environmental contamination is

even more significant in the case of using another

phenol – triclosan (5-chloro-2-(2′,4′-dichlorophenoxy)

phenol (Fig.  2). This compound exerts strong pro-

tonophoric action on the model lipid membranes,

while its uncoupling effect in mitochondria is rather

weak [67]. Actually, triclosan induces proton current

through BLM more effectively than CCCP, while in

mitochondria it should be added at a concentration

100-fold higher to induce the same effect as CCCP.

The reason for such discrepancy has not been eluci-

dated yet. The weak uncoupling activity of triclosan

could be associated with its inability to interact with

mitochondrial proteins that facilitate proton transfer

by uncouplers  [34]. Triclosan is well known as an

antibacterial agent, which has been widely used in

personal hygiene products. Currently, its use is legally

limited due to harmful effects on water ecosystems

via contaminated wastewater.

The best-known uncoupling phenol is DNP

(Fig. 2), which for some time was used as a drug

for stimulating metabolism in obesity treatment.

High interest in this compound arose during and

after World War I, when the toxic effect of DNP to

the workers, which participated in its production for

the war needs, was reported. Studies of the effects

of low doses of DNP revealed its ability to reduce

the amount of excessive fat both in animals and hu-

mans. This led to the use of DNP, as well as dinitro-

cresol, for obesity treatment. In the period from 1931

to 1934 this over-the-counter medicine was used by

~100,000 individuals [68]. Its effectiveness in terms of

weight loss was confirmed in more than 80% of the

cases. However, significant side effects were observed.

MITOCHONDRIAL UNCOUPLERS 1815

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Fig.  2. Chemical structures of DNP, triclosan, SF6847, HU6, niclosamide, nitazoxanide, tizoxanide, fluorescein, 7-hydroxycou-

marin, 1799, phloretin, NPA, chlorfenapyr and its metabolite AC-303268.

ANTONENKO et al.1816

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Some individuals had problems with liver, skin ulcers

were formed, and cases of cataract development were

reported. These side effects were observed even with

some patients taking recommended doses, not due to

the use of higher doses. Unfortunately, the cause of

enhanced toxicity of DNP for some individuals had not

been established. Monitoring the workers at chemical

factories producing DNP revealed that high toxicity

is exhibited in the individuals with high urine con-

centration of 2-amino-4-nitrophenol, a product of DNP

degradation [68]. Development of cataract was report-

ed in approximately 1% of the patients taking DNP,

and this occurred months after completion of the obe-

sity treatment. Search for therapeutics to treat such

cataract was unsuccessful, however, surgical interven-

tion helped in the majority of cases. Finally, in 1934

the use of DNP as a drug was prohibited. However, up

until now the use of DNP as an effective weight-loss

medicine has been reported in some fitness communi-

ties [69-71]. Interestingly enough, labels on the pack-

ages of DNP in pharmacies in 1930s stated that the

excessive effect of DNP could be suppressed by taking

baking soda [70]. It was indeed shown in the recent

study by Khailova et al. [72] that uncoupling of rat

liver mitochondria (RLM) by DNP could be effectively

inhibited by millimolar concentrations of bicarbonate.

Unfortunately, the mechanism of this effect has not

been elucidated. Further, we will consider in detail

modern approaches for decreasing side effects of DNP

and development of therapeutics on its basis.

After DNP was banned for use as a medicine due

to its high toxicity for humans, researchers started

to search for its analogues that would not cause side

effects and would be less toxic. In particular, it was

suggested to use methyl- or ethyl ethers of DNP, which

could be hydrolyzed in an organism producing DNP

[73]. This idea was developed in later studies [11,

15, 74]. At present, the preparation containing ethyl

ether of DNP is being examined in clinical trails as

a drug for treatment of Alzheimer’s and Parkinson’s

diseases. The fact is that DNP has manifested itself

as a neuroprotector in mouse models [12, 75,  76], as

well as cardioprotector [77,  78] and nephroprotec-

tor [13]. Another example of DNP ethers is methyl

ether of DNP (DNPME, 1-methoxy-2,4-dinitroben-

zene), which was investigated in Shulman’s laborato-

ry [74] in the USA. The authors demonstrated that

DNPME effectively protected mice from development

of non-alcoholic fatty liver disease (NAFLD), as well

as from insulin tolerance development. The authors

mentioned that DNPME hydrolysis and release of DNP

occurs predominantly in liver, hence, this preparation

is more suitable for treatment of liver disorders than

DNP itself. However, particular biochemical path-

ways mediating DNPME hydrolysis in liver cells were

not identified in this study.

Etherification of DNP with (1-methyl-2-nitro-

1H-imidazol-5-yl)methanol results in formation of

the compound HU6 (5-[(2′,4′-dinitrophenoxy)methyl]-

1-methyl-2-nitroimidazole) shown in Fig.  2. HU6

was tested in humans as an anti-obesity agent, as

well as in the patients with developed NAFLD [79].

It was concluded that HU6 could become a promising

pharmaceutical for treating patients with obesity and

NAFLD, as well as with their metabolic complications

[79]. According to the results of the study by Kitz-

manetal.[80], this agent could be also promising for

treating heart failure associated with obesity. It was

suggested in these studies that HU6 is a precursor

of DNP in an organism, which, in turn, mediates its

pharmacological effect. However, no details of HU6

metabolism were actually presented.

At present, DNP together with CCCP (Fig.  3) re-

main the most used uncouplers in the laboratory

practice, especially working with isolated mitochon-

dria. Itis the opinion of a number of researchers that

there is still significant therapeutic potential in using

DNP, which could be further increased by improv-

ing pharmacokinetics of DNP delivery to target cells.

C8-alkyloxy-substituted derivatives of FCCP and DNP

were used in the study by Ng et al. [81] for targeted

delivery of uncouplers to adipose tissues. The obtained

compounds containing ester bond (carbonyl cyanide

4-octyloxyphenylhydrazone and 2,6-dinitro-4-octyloxy-

phenol) accumulated in adipose tissues and were

more effective in acceleration of cellular metabolism

than FCCP and DNP; however, the esters were shown

to be unstable in the organism of mice, which was

considered as a drawback in this study. Several lab-

oratories have synthesized compounds that could

accumulate in mitochondria and release DNP upon

illumination or addition of hydrogen sulfide. Thus,

the light- and H

S-activated protonophores based on

4-hydroxybenzylidenepropanedinitrile (AG10) [82]

and DNP [83-86] were obtained. Scientists hope that

mitochondrial targeting of such forms of uncouplers

could ensure reduction of their acting concentrations

and initiate local uncoupling upon illumination or

in the presence of H

S. It was shown in our recent

study that 2-azido-4-nitrophenol, on the contrary, ex-

hibits properties of a protonophore that could be in-

activated by light [87], which creates a possibility of

local control of mitochondrial uncoupling.

One of the strongest mitochondrial uncouplers

is SF6847 ([(3,5-di-tert-butyl-4-hydroxyphenyl)methyl-

idene]propanedinitrile) (Fig.  2). This substituted phe-

nol uncouples mitochondria at nanomolar concen-

trations [88, 89]. The authors explain the enhanced

activity by high stability of the anionic form of

SF6847 in the membrane. According to calculations,

the anion has a planar structure, which facilitates

effective delocalization of the charge involving the

MITOCHONDRIAL UNCOUPLERS 1817

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Fig. 3. Chemical structures of CCCP, FCCP, fluazinam, BAM15, pentachlorothiophenol (PCBT), SR4, and nonyl 3-picolinoyl-

carbazate (PDTC-9).

malononitrile group [90]. Low acting concentration of

SF6847  [89] could indicate that the uncoupler itself

rather than its complex with a protein operates as a

protonophore. However, it has been suggested in the

study by Grivennikova et al. [91] that the uncoupling

action of SF6847 could be partially mediated by its

binding to respiratory Complex I. It has been discov-

ered by Starkov et al. [92,  93] that the uncoupling

activity of SF6847 and other strong protonophores is

suppressed by 6-ketocholestanol, known as a modifi-

er of the membrane dipole potential [94]. Therefore,

the association of the recoupling with the action of

6-ketocholestanol on the lipid part of the membrane

cannot be ruled out [93,  95]. SF6847 is also known

as an inhibitor of tyrosine kinase [96,  97] and a po-

tential anticancer agent, usually named tyrphostin or

malonoben [96,  97].

Another strong uncoupler, niclosamide, belongs

to the group of salicylanilides and is the best-known

representative of this numerous group of com-

pounds (Fig.  2). Despite its high uncoupling activity,

this agent exhibits low cytotoxicity and is used as an

anthelmintic drug. Based on the approval of the Food

and Drug Administration of USA (FDA), at present,

niclosamide is tested for fighting many other diseases

including COVID-19 [98]. The fact is that one of the

critical stages of virus entry into cytoplasm is acidi-

fication of endosomes, which could be suppressed by

uncouplers [99,  100]. That is why many uncouplers

exhibit antiviral activity. With regards to the mech-

anism of action, it is known that niclosamide and

other salicylanilides induce proton current through

BLM [101,  102], however, more detailed investiga-

tion of the proton-transporting properties of salic-

ylanilides were conducted using the compound S13

(3-tert-butyl-4′-nitro-2′,5-dichlorosalicylanilide) as an

example. In the experiments with BLM it was shown

that classical monomeric model of proton transport

described above for CCCP is fully applicable for S13

[103]. The only difference consisted in the fact that

permeabilities of the protonated and deprotonat-

ed forms of S13 were approximately 10-fold higher

ANTONENKO et al.1818

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

than those of CCCP. Due to poor solubility, it is dif-

ficult to measure pK

of S13, but in the presence of

liposomes the pK

value was 7.0-7.3 [103]. S13 is a

strong uncoupler of oxidative phosphorylation be-

ing second only to SF6847 [38]. Hydroxyl serves as

a proton-donor group in salicylanilides; for example,

its methylation in niclosamide results in the loss of

the uncoupling activity [104]. Of note, salicylanilides

contain amino group in the form of CO–NH-linker

between two aryl groups, so that the amino group

proton could participate in formation of a hydrogen

bond with the hydroxyl oxygen atom in the salicylic

residue, which could facilitate delocalization of the

negative charge with formation of additional 6-mem-

bered cycle, thus increasing permeability of the com-

pounds through lipid membranes [105, 106].

Nitazoxanide has been also approved by FDA

as an anthelmintic drug. In a human organism it

is rapidly deacetylated and transformed into tizox-

anide (Fig.  2), an analogue of the above-described

niclosamide, and exhibits uncoupling properties both

in mitochondria and whole cells [107,  108]. Unlike

niclosamide, nitazoxanide has good bioavailability

and can easily penetrate into blood circulation in

the case of peroral administration. Based on the data

that nitazoxanide inhibits formation of atheroscle-

rotic plaques in the ApoE

−/−

mice with liver steatosis

fed according to the so-called ‘Western diet’ [108],

its use as a new antiatherosclerosis agent with high

clinical potential was suggested. As shown in another

study [109], administration of nitazoxanide through

a tube caused therapeutic effect in liver steatosis

induced by high-fat-diet in the C57BL/6J mice. This

study also confirmed that nitazoxanide is a promising

therapeutic agent against liver pathologies.

It is known that many natural phenols uncou-

ple oxidative phosphorylation in mitochondria [110].

Among those phloretin [111] (Fig.  2), as well as quer-

cetin [112] and hyperforin [113] should be men-

tioned. Many plant-derived metabolites of phenolic

nature exert mitochondrial uncoupling [112] and in-

crease electric current through BLM [110]. At micro-

molar concentrations, phloretin is known as a strong

modifier of dipole potential in BLM [114,115] (Fig.2).

Phloretin molecule has a large dipole moment and is

adsorbed at the membrane-water interface in an ori-

ented manner, which leads to suppression of proton

current mediated by anionic uncouplers and stimu-

lation of current mediated by cationic protonophores

[116]. Interestingly, natural phenols could be bromi-

nated, as, for example, (4,5,6-tribromo-2-(2′,4′-dibro-

mophenoxy)phenol also known as P01F08 [117]. This

compound exhibits high uncoupling capacity and

hasbeen investigated as a potential anticancer agent.

It is well-known that not only substituted phe-

nols could be uncouplers and anionic protonophores

of the OH-series. Fluorescein also has a suitable hy-

droxyl with pK

 (~7) in its more complex aromatic

core; however, fluorescein itself does not exhibit un-

coupling properties (Fig. 2). This could be explained

by the presence of a free carboxyl group in the fluo-

rescein molecule. Nevertheless, butyl ester of fluores-

cein is also inactive, while the octyl ester of fluores-

cein exhibits uncoupling properties at submicromolar

concentrations [118]. Thus, it could be concluded that

fluorescein does not possess enough lipophilicity for

protonophoric activity and acquires it upon pending

a lipophilic tail. Lipophilic fluorescein derivatives

could be of interest as antibacterial agents [119].

Similar phenomenon, namely, the emergence

of protonophoric activity based on proton-donating

property of a hydroxyl substituent in the aromatic

system, caused by the addition of optimal-length alkyl

substituents to this system, has been demonstrated in

our study of 7-hydroxycoumarin derivatives (Fig.  2).

Coumarins (derivatives of 2H-1-benzopyran-2-one)

comprise a large class of natural heterocyclic com-

pounds exhibiting a wide range of therapeutic effects

[120]. Uncoupling activity of ostruthin [121] and some

other 7-hydroxycoumarin (umbelliferone) derivatives

[122] with hydroxyl pK

of 7.5 [123] has been re-

ported previously. It was shown in our recent study

that the introduction of hydrophobic substituents to

7-hydroxycoumarin results in creation of effective un-

couplers acting at micromolar or even submicromo-

lar concentrations [124, 125]. Detailed investigation

of the uncoupling activity of octyl and decyl esters

of 7-hydroxycoumarin-3-carboxylic acid on RLM re-

vealed a very interesting feature, namely, sponta-

neous decay of the activity occurring due to the loss

of alkyl substituents [125] as a result of enzymatic

hydrolysis of the esters. Surprisingly, the uncoupling

remains constant in the case of fluorescein octyl ester

[118]. The phenomenon of time-limited uncoupling

and metabolism of uncouplers will be discussed in

more detail further in the review.

All examples of anionic protonophores of the OH-

type described above are compounds with aromatic

hydroxyl. However, it is known that uncouplers could

be found among the compounds with aliphatic hy-

droxyl, such as, for example compound 1799 (2,6-di-

hydroxy-2,6-bis(trifluoromethyl)-1,1,1,7,7,7-hexafluoro-

heptane-4-one) (Fig. 2), which is a strong uncoupler

and protonophore [126]. It is known that the com-

pound 1799 increases permeability of BLM [58,  60].

However, the mechanism of its action is unknown, and

can be only assumed. Protonophoric action of 1799

indicates that aromaticity is not needed for effective

penetration of anionic form of protonophore across

the membrane. The absence of the effect of aroma-

ticity on the membrane permeability has been also

noted for tricyclohexylphosphonium derivatives[127].

MITOCHONDRIAL UNCOUPLERS 1819

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Another variant of non-aromatic hydroxyls with

suitable pK

are fatty acids. It has been known for

a long time that fatty acids uncouple mitochondria

at concentrations of tens of micromoles. At the same

time, they exhibit very weak ability to increase con-

ductance of planar BLMs: noticeable currents have

been observed only for the membranes formed from

liposomes [128] according to the method of Montal

and Mueller [129]. Moreover, it was shown that the

uncoupling effect of fatty acids in mitochondria is

partially mediated by the proteins – ADP/ATP-anti-

porter and UCP [27, 63, 128, 130-137]. Interestingly

enough, dicarboxylic fatty acids are also capable of

uncoupling mitochondria; more precisely they stim-

ulate respiration, but not dissipate mitochondrial

membrane potential [138]. This rather complicated

phenomenon was explained by the interaction of

such compounds with respiratory complex II in mi-

tochondria. Dicarboxylic fatty acids do not increase

current in BLM, but penetrate across the BLM in a

neutral form [139]. It should be noted that carbox-

yl groups in the vicinity of aromatic ring usually do

not mediate uncoupling as can be demonstrated with

salicylic acid as an example [140]. But in some cases,

the compounds with such structure could function as

weak uncouplers, as in the case of BT2 (3,6-dichlo-

robenzo[b]thiophene-2-carboxylic acid), which uncou-

ples mitochondria at hundreds of micromoles and

exhibits a cardioprotective effect [141]. Azido-aryl-de-

rivatives of various physiologically active compounds

are often used for searching target proteins [142].

Upon illumination with near ultraviolet light, the

azido group loses N

and is transformed into the

highly reactive nitrene, which is capable of reacting

with various groups in proteins, in particular, amino

groups. 2-Azido-4-nitrophenol (NPA; Fig.  2) was syn-

thesized as the closest analogue of DNP and was used

for investigation of interactions of uncouplers with

mitochondrial proteins [143]. NPA was shown to be

even more active as an uncoupler than DNP (active

concentration of NPA is approximately 3-4-fold lower

than that of DNP) [87, 144]. Based on the studies of

isolated mitochondria, NPA has specific binding sites

on unidentified mitochondrial proteins; moreover,

this affinity depends significantly on the presence

of other known uncouplers such as DNP and CCCP

[30, 143]. Among these proteins, a band, correspond-

ing to the molecular mass ~30,000  Da, was found.

In the case of azido-derivatives of fatty acids ex-

posed to UV illumination, researchers observed cova-

lent binding to the protein with molecular mass of

~30  kDa, which was identified as ANT1 [145].

It has been shown recently that the antibiotic

pyrrolomycin is also, most likely, an anionic proto-

nophore of OH-acid type [146, 147]. The pH-depen-

dence of pyrrolomycin  D activity in BLM exhibited

an optimum at pH 9 [147]. Unlike synthetic protono-

phores, pyrrolomycins are produced by the bacteria

Streptomyces vitaminophilus, Streptomyces sp., and

Streptomyces fumanus. These compounds, having a

large number of chlorine atoms in the molecule, un-

couple mitochondria and submitochondrial particles

(SMPs) at subnanomolar concentrations [147]. These

concentrations are extraordinary low, because usual-

ly acting concentrations of uncouplers in SMPs are

significantly higher than those in mitochondria. The

question on the site of deprotonation in the pyrro-

lomycin molecule remains open, because in addition

to phenolic hydroxyl, deprotonation may also occur

at the nitrogen atom in the pyrrole ring. However,

antibacterial and protonophoric activities of pyrrolo-

mycinsI andJ, having methylated phenolic hydroxyl,

are significantly reduced [146].

Based on the natural antibiotic – derivative of

halogenated pyrrole dioxapyrrolomycin [7], the in-

secticide chlorfenapyr was developed. Its toxicity is

too high for crop treatment, but it is used for treat-

ment of home gardens and forests where there are

no edible plants. Chlorfenapyr itself does not exert

the uncoupling effect on RLM; however, in insect

cells N-dealkylation occurs resulting in the forma-

tion of free pyrrole, presumably with participation

of cytochromes P450 (CYP450) [148]. Structures of

chlorfenapyr and the product of its metabolism with

uncoupling properties are presented in Fig.  2. In fact,

the chlorfenapyr metabolite, although related to pyr-

rolomycins, performs the mitochondrial uncoupling

as NH-acid.

Uncouplers based on NH-acids. This numerous

group includes compounds that contain nitrogen

both within and outside the aromatic ring. The best

known uncoupler CCCP, as well as its close analogue

FCCP (Fig.  3), both introduced by Heytler at the be-

ginning of 1960s[126, 149,150], belong to this group.

Despite the fact that these compounds are unstable

in the presence of thiol-reagents [150-155], CCCP

became very popular, especially in the tests involv-

ing mitochondrial uncoupling in cells. Most likely,

this is associated with its low acting concentrations

(hundreds of nanomoles for isolated mitochondria

and several micromoles for intact cells), as well as

with its rather low toxicity [14, 156, 157]. Another

advantage of CCCP is its weak inhibiting effect on

mitochondrial respiratory pumps, which is observed

only at concentrations tens-fold higher than the un-

coupling concentrations. With regard to the mecha-

nism of the protonophoric action, CCCP is a typical

anionic protonophore with pK

  ≈  6 and maximum

protonophoric activity at pH around 8 [158-160]. The

difference between pK

andpH-optimum of the activ-

ity is explained by a large difference in permeability

for the anionic and neutral forms of the uncoupler.

ANTONENKO et al.1820

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Similar to DNP, CCCP was tested in cells and animal

physiological models of various pathologies, where it

was proved to have properties of neuroprotector [161]

and cardioprotector [78]. Similar to the case of DNP,

azido-CCCP was synthesized (N

CCP, carbonyl cyanide

2-nitro-4-azidophenyl hydrazone) and its interaction

with mitochondria in the dark and under illumina-

tion with UV light was investigated; conjugation with

some mitochondrial proteins was observed under

illumination [31, 162]. Unfortunately, these proteins

have not been yet identified, the label was observed

in the band corresponding to the protein with mo-

lecular weight of 10,000-15,000  Da. Azido-CCCP was

poorly investigated as an uncoupler: there are even

no data on comparison of its acting concentrations

with those for CCCP in mitochondria and no data at

all on its protonophoric activity in BLM.

It was mentioned above that CCCP and its more

active analogue FCCP could react with thiols in a

chemical system in the absence of mitochondria. How-

ever, the issue of the possibility of interaction of CCCP

and FCCP with natural thiols in cells, and primarily

with glutathione (GSH), remains unsolved. This prob-

lem becomes especially important because, according

to our data, CCCP and FCCP can interact with N-acetyl

cysteine (NAC) in a chemical system [155]. The fact

is that NAC as an antioxidant is often used in the

experiments with cells (as well as in  vivo) together

with CCCP and FCCP [163-169]. In particular, it was

shown that cardioprotective effect of FCCP could be

completely eliminated by the addition of NAC [163],

which was explained as an effect of FCCP on the level

of reactive oxygen species (ROS) in heart cells. How-

ever, another possibility exists, that involves simple

chemical modification of FCCP upon the addition of

NAC to this system, which could block the uncoupling

action.

Another synthetic anionic protonophore of the

NH-acid series – BAM15 (N

-bis(2-fluorophenyl)

[2,1,3]oxadiazolo[4,5-b]pyrazine-5,6-diamine) (Fig.  3),

introduced by Santos’s research group in 2013 [170],

could be considered as currently the most investigat-

ed from the physiological point of view. BAM15 ex-

hibits strong hepatoprotective properties [171] and is

effective in murine obesity models [172]. The chem-

ical structure of BAM15 is more complex than that

of CCCP, hence, the issue of its protonation-depro-

tonation is not so trivial. By using the capillary elec-

trophoresis method, it was shown that BAM15 could

be deprotonated in two steps with pK

6.44 and 7.99

forming mono- and di- anions, respectively [50]. The

proton transfer across BLM mediated by BAM15

could be suppressed by the addition of phloretin,

which confirms the anionic nature of this protono-

phore [50]. It has been stated previously that BAM15

has significant advantage compared to CCCP, because

it does not change permeability and electrical activity

of the plasma membrane [170], however, this conclu-

sion was challenged in the study by Firsovet al. [50].

In addition to the direct protonophoric action, BAM15

is capable of inducing proton transport across the in-

ner mitochondrial membrane via its interaction with

the ANT1 protein [26, 28, 50].

Fluazinam is also a very strong uncoupler of the

NH-type, which belongs to the group of diarylamines

(Fig. 3). Noteworthy, diarylamines comprise a large

group of compounds synthesized with the primary

goal to create effective pesticides [7]. The protono-

phoric action of fluazinam on BLM, demonstrated by

Khailova et al. [155], appeared to be close to that of

FCCP. The uncoupling concentrations of fluazinam

were shown to be subnanomolar, i.e., even less than

those of SF6847 [173]. However, fluazinam is rapid-

ly metabolized in RLM with formation of conjugates

with glutathione facilitated by glutathione-S-transfer-

ase (GST) [155, 173-175].

Similar metabolic transformation was observed

in our recent study [176] on another group of NH-

type uncouplers – derivatives of N-phenylthiophen-

2- amine [177]. Details of the transformation will be

discussed further in the section devoted to metab-

olism of uncouplers. The protonophore, discovered

almost 10 year ago – endosidin-9 (ES9, 5-bromo- N-

(4-nitrophenyl)thiophene-2-sulfonamide), which is

N-aryl thiophenesulfonamide – compound related to

anilinothiophenes, also belongs to the NH-acid type

of protonophores [178].

Uncouplers of the NH-type also include deriva-

tives of the well-known fluorescent dye NBD (7-nitro-

benz-2-oxa-1,3-diazole), modified by introduction of

amino group at position 4 of the benzene ring, which

could be deprotonated with pK

~10 [179]. A series of

4-alkylamino-7-nitrobenz-2-oxa-1,3-diazoles was syn-

thesized, for which it was shown that starting with

octyl the uncoupling and protonophoric properties

have been observed [180]. The maximum uncoupling

effect on mitochondria was observed precisely for

the N-octyl derivative with acting concentration be-

ing tens of micromoles. The N-dodecyl-derivative was

shown to be a significantly stronger protonophore

than the octyl analogue in experiments on BLM [180].

Such difference between the activities in mitochon-

dria and BLM could be associated with the ability of

7-nitro-4-octylaminobenz-2-oxa-1,3-diazole to interact

with ANT1, thereby promoting additional proton flow

with participation of this protein [180, 181].

All the above-mentioned uncouplers of the

NH-acid type are synthetic compounds. However, it

was shown recently that the natural toxin aetok-

thonotoxin, which is produced by the cyanobacteria

Aetokthonos hydrillicola that causes poisoning in

fishes and birds, is a strong uncoupler of oxidative

MITOCHONDRIAL UNCOUPLERS 1821

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

phosphorylation, and this uncoupling is likely the

main mechanism of the toxicity [182]. This toxin

contains an unusual bi-indole group and has five

bromine atoms as substituents. Methylation of the ni-

trogen atom in the indole ring results in the loss of

the uncoupling activity in animal cells at micromolar

concentrations of this toxin. It was shown that at the

same concentrations aetokthonotoxin induces electric

current through BLM at neutral pH.

Compounds based on diphenylurea such as SR4

(N,N′-bis(3,5-dichlorophenyl)urea) (Fig.  3) and ana-

logues could be also assigned with a certain caution

to protonophores of the NH-acid type [183]. Some au-

thors, on the contrary, associate protonophoric prop-

erties of SR4 with its ability to bind hydroxide ions

as well as other inorganic anions [184]. N,N′-diphenyl

urea is a natural compound – a plant hormone, which

controls, in particular, the formation of flowers. SR4

was shown to dissipate membrane potential and stim-

ulate respiration at submicromolar concentrations

[183]. Furthermore, derivatives of diphenylurea in-

creased proton permeability of liposome membranes

[185], which indicates their ability to act as classical

protonophores. These compounds were investigated

during the search for effective anticancer agents

[186]. For example, Sorafenib, which is a deriva-

tive of diphenylurea with a picolinamide fragment,

is used as an anticancer agent, and its main mech-

anism is inhibition of kinases [187]. On the other

hand, it was shown that Sorafenib uncouples mito-

chondria, and this property could be significant for

the anticancer effect [188, 189]. Interestingly enough,

similar compounds also could transport chloride ions

and other anions across model and natural mem-

branes [184].

Uncouplers based on CH-acids. C–H bond has, as

a rule, a negligibly weak capacity for dissociation, es-

pecially in an aqueous environment. However, in the

case of carboranes it was possible to observe disso-

ciation in different protic and aprotic solvents [190].

Moreover, decachloro-o-carborane has pK

  ~  7, and its

uncoupling action was demonstrated in the study by

Libermanetal.[191] already in 1970. The uncoupling

activity of ortho-carborane, unlike meta- and pa-

ra-carborane, in line with higher acidity of ortho-iso-

mers as compared to others [190], was confirmed in

the study by Rokitskaya et al. [192]. The uncoupling

effect on mitochondria correlated with the protono-

phoric activity in BLM and liposomes [192]. To the

best of our knowledge, these compounds have not

been tested in physiological experiments with ani-

mals. However, there are many studies on application

of carboranes as pharmacophores; a large number of

compounds – conjugates with carboranes were syn-

thesized [193]. Part of them exhibited the uncoupling

activity and was tested in tissue cultures [194].

Uncouplers based on SH-acids. Uncoupling action

of pentachlorothiophenol (PCBT; Fig. 3) in mitochon-

dria stimulating their respiration in the state 4 was

investigated in the early study by Wilson et  al. [43].

In particular, it was shown that PCBT uncouples mi-

tochondria at submicromolar concentrations, while it

practically does not induce current through BLM [43].

In the later (1983) study by Smejtek et al. [195] it

was clarified that in the study by Wilson et al. [43]

in 1971, photosensitivity and poor water solubility of

PCBT was not taken into account, and in fact perme-

ability induced by PCBT in BLM was approximately

10-fold higher, than the one at the same concentra-

tion of pentachlorophenol [195]. The pH-dependence

of BLM permeability in induced by PCBT had max-

imum at pH  ~  6; and pK

of PCBT was estimated

as 4.3 [43], while pK

of pentachlorophenol was ~4.7.

All these data imply that uncouplers based on SH-

acids have high activity and their application is lim-

ited only by their low chemical stability in biological

environment.

This conclusion has been supported by the results

of another study on the uncoupling activity of alkyl

acyldithiocarbazates, and, in particular, of nonyl 3-pi-

colinoyldithiocarbazate (PDTC-9) [196], which uncou-

ples mitochondria and induces proton permeability of

liposomes at micromolar concentrations. In aqueous

solutions PDTC-9 exists as two tautomers, with one

of them being thione, and another – thiol (Fig.  3).

Methylation of the nitrogen atom N2 in thione or the

sulfur atom in thiol results in complete loss of the

uncoupling activity. Considering that one of the tau-

tomers is a NH-acid, and another – SH-acid, PDTC-9

could be assigned both to NH- and SH-protonophores.

According to the data reported by Teradaet al. [196],

PDTC-9 exhibits weak reactivity with such natural thi-

ols as cysteine and GSH. Several structural analogues

of PDTC-9 were tested, and it was shown, in partic-

ular, that their activity increases with an increase

in the length of alkyl substituent reaching maximum

in the case of nonyl; further increase in the alkyl

substituent length leads to a decrease in the uncou-

pling activity [196].

Cationic protonophores. This group of uncou-

plers is not numerous in comparison with the group

of anionic protonophores. These compounds exist in

protonated cationic and deprotonated neutral forms.

It is essential that the protonated form in this group

of compounds is a charged molecule and not a zwit-

terion. Schematic representation of functioning of

these protonophores is shown in Fig.  1b. The cation-

ic charged form penetrates across the membranes

significantly less effective than the anionic one due

to the membrane dipole potential, hence, cationic

protonophores usually are effective at higher con-

centrations than the anionic ones. Another feature

ANTONENKO et al.1822

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Fig. 4. Chemical structures of ellipticine, C4R1, Nile Blue, bupivacaine, and F16.

of cationic protonophores is their ability to accu-

mulate in the mitochondrial matrix, because mito-

chondrial membrane potential is negative inside.

Ellipticine is the best-known uncoupler of this type

[197] (Fig. 4). These compounds are practically not

used in the laboratory practice as uncouplers, because

they produce effect at very high concentrations and

the mechanism of their action is poorly understood.

However, ellipticine and related compounds exhibit

anticancer activity, and in this relation were investi-

gated in great detail [198]. The rhodamine derivative

C4R1 [199, 200] and the Nile Blue dye [201] could also

be assigned to cationic uncouplers.

Ellipticine is an anticancer alkaloid isolated from

the plants Ochrosia elliptica. It was shown that it

uncouples mitochondria at concentrations of tens of

micromoles with maximum at ~100  µM [197]. Ellipti-

cine contains a pyridine ring (Fig.  4), in which meth-

ylation of nitrogen atom results in almost complete

loss of the uncoupling activity. This observation indi-

cates that protonation-deprotonation of the nitrogen

atom is responsible for the protonophoric action of

ellipticine. The measured value of pK

of this nitro-

gen atom is close to 7.0 [202]. It was shown that in

cancer cells ellipticine is accumulated in nuclei and

mitochondria, furthermore, fluorescence of ellipticine

in mitochondria depends on the pH gradient on the

membrane [203].

As reported in 1984, rhodamine 6G, rhodamine

19 ethyl ester, uncouples mitochondria at micromo-

lar concentrations [204]. It was shown in the fol-

lowing studies that the butyl ester (C4R1; Fig. 4) ex-

hibits the highest uncoupling activity in the series

of rhodamine derivatives with alkyl substituents of

varying length [199]. Surprisingly, attempts to detect

the process of deprotonation in rhodamine 6G failed.

It seems that there was an error in the study by

Duvvurietal.[205]: in fact, there is no pH dependence

of the rhodamine 6G spectrum. It was shown with the

help of capillary electrophoresis that rhodamine 6G

does not exhibit pK

in the physiological range of

pH [206], while this method was successful in reli-

able determination of pK

for many other uncouplers.

It was also shown that the derivatives of rhodamine

19 increase current across BLM and behave as proto-

nophores [207]. This current is stimulated by phlore-

tin, i.e., it increases with a decrease in membrane

dipole potential [116]. At the same time, the ATPase

enzyme is apparently involved in the uncoupling ef-

fect of C4R1 on mitochondria [200]. Weak toxicity

of C4R1 was demonstrated in the experiments with

mice; in addition, this rhodamine stimulated weight

loss in rats maintained on a high fat diet [208].

Alkyl-rhodamines also exhibited antibacterial proper-

ties [209, 210].

Other dyes, such as Nile Blue (Fig. 4), pyronin Y,

and acridine orange also show properties of cation-

ic uncouplers [201]. However, Nile Blue is an inhibi-

tor of ATP synthase, and it has been suggested that,

similar to C4R1, it operates as an uncoupler in mito-

chondria with participation of this protein. This dye

was not investigated on BLM, however, its structur-

ally close derivative increased proton current across

a lipid membrane [211].

Bupivacaine (Fig. 4) is a local anesthetic with

piperidine heterocycle, which can be protonated at

MITOCHONDRIAL UNCOUPLERS 1823

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

neutral pH. The use of bupivacaine was approved

by FDA. It was shown that, similarly to some other

anesthetics [212], it decreases mitochondrial mem-

brane potential and stimulates their respiration at

millimolar concentrations, i.e. it functions as an un-

coupler [213, 214]. Evaluation of pK

of protonation

of the piperidine ring in bupivacaine gave the value

of 8.2[215]. Investigation of bupivacaine in liposomes

showed its protonophoric properties, however, the

authors suggest that the mechanism of the uncou-

pling effect of this anesthetic involves the formation

of selective proton channels by its aggregates [216],

which is in agreement with the data on the ability of

bupivacaine to form defects in the lipid membrane,

obtained by Terada et al. [214] with planar BLMs.

Screening a wide range of synthetic compounds

for anticancer effects revealed the cationic compound

F16 ((E)-4-(3-indolylvinyl)-N-methylpyridinium iodide)

with high antitumor activity [217] (Fig.  4). The au-

thors suggested that this anticancer effect is associat-

ed with accumulation of this cation in mitochondria

of tumor cells, causing induction of a mitochondrial

pore, release of cytochrome  c, and finally the apop-

tosis. It was shown both in the first study by Fan-

tin et al. [217], and in the later study [218] that F16

stimulates respiration of mitochondria and behaves

as an uncoupler. The authors of the later study by

Wang et al. [218] concluded that the uncoupling ac-

tivity of F16 is precisely responsible for its antican-

cer effects. Isomers of F16 were synthesized (o-F16,

(E)-2-(3-indolylvinyl)-N-methylpyridinium iodide and

m-F16, (E)-3-(3-indolylvinyl)-N-methylpyridinium io-

dide), which differ only by orientation of indole and

pyridine rings of the molecule [219]. It was found

that o-F16 exhibits properties of a strong uncoupler,

while m-F16 is practically inactive [219]. The authors

associate this property with lower acidity of the me-

ta-isomer. It should be mentioned that the mechanism

of deprotonation of F-16 and o-F16 is poorly under-

stood. Dissociation of proton from these compounds

could result in the formation of both neutral mole-

cules without charged groups, and zwitterions, as

was postulated in the study by Xu et  al. [219]. The

completely neutral deprotonated forms of F16 and

o-F16 were previously isolated and characterized

with the help of IR- and mass-spectroscopy [220]

(scheme in Fig. 4). We believe that formation of the

less hydrophobic zwitterion is more probable in the

case of deprotonation of m-F16. This fact could also

explain the reduced activity of m-F16. Of note, if in-

deed the deprotonated form of F16 is a zwitterion,

this uncoupler should be assigned to the next group

in our classification, namely the group of zwitteri-

onic uncouplers. To the best of our knowledge, the

protonophoric action of F16 on BLM has not been

investigated. In recent years, works on synthesis of

the conjugates of F16 with other anticancer agents

were published. In particular, the conjugate of F16

with betulinic acid exhibited significantly higher cyto-

toxicity towards tumor cells than individual betulinic

acid or F16 [221]. In later studies, the list of anti-

cancer preparations based on F16 was significantly

expanded [222-224].

Zwitterionic protonophores. Relatively recently

zwitterionic protonophores have been discovered, i.e.,

compounds with deprotonated form carrying simulta-

neously positive and negative charges comprising a

zwitterion [225,226]. Mechanism of their functioning

is presented in Fig. 1c. It is essential that zwitterion-

ic and cationic forms of such protonophores should

exhibit high permeability across the membrane. One

could imagine the process, when the zwitterionic

form of protonophores could be combined with an-

ionic form as a pair for proton transport (Fig.  1d),

however, such compounds have not been observed

experimentally. The majority of the confirmed zwit-

terionic protonophores are conjugates of triphenyl-

phosphonium with anionic protonophores (Fig.  5).

It was shown that these protonophores initiate pro-

ton current through BLM, which is stimulated by de-

creasing dipole potential upon the addition of phlore-

tin or including certain lipids in the composition of

BLMs [116], i.e. in this regard they behave as cation-

ic protonophores. Furthermore, these compounds are

accumulated in the cell mitochondria, which allows

considering them as mitochondria-targeted protono-

phores [225]. It must be noted that similar to anion-

ic protonophores, zwitterionic protonophores could

be classified according to the type of proton-donor

group: OH-, NH-, and CH-acids.

Conjugates of anionic uncouplers with triphenyl-

phosphonium cations. The first attempt to synthesize

a mitochondria-targeted protonophore was made in

2006 in the research group of Murphy and Smith

[227] by conjugating triphenylphosphonium with

DNP. However, the obtained compound termed mi-

toDNP (3-(4′-hydroxy-3′,5′-dinitrophenyl)propyltriph-

enylphosphonium methanesulfonate) (Fig.  5) did not

exhibit uncoupling properties in mitochondria, which

made questionable the initial assumption about the

existence of zwitterionic protonophores. However, in

2014 a conjugate of triphenylphosphonium and fluo-

rescein was synthesized, which was named mitoFluo

(10-[(2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoyl)oxy]

decyltriphenylphosphonium bromide) (Fig.  5); this

compound appeared to be more advantageous [225].

It was shown that mitoFluo uncouples mitochondria

at submicromolar concentrations, and is accumu-

lated in mitochondria, which is easily detected due

to the bright fluorescence of this compound. The

weak uncoupling effect of mitoDNP could be ex-

plained by: (1) the weaker uncoupling effect of DNP

ANTONENKO et al.1824

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Fig. 5. Chemical structures of mitoDNP, mitoFluo, mitoCCCP, SF-C5-TPP, PP6, CMTPP-Cn (precursors of phosphonium ylides).

in comparison with alkyl-fluorescein and (2) by the

too short trimethylene linker in mitoDNP. The mito-

Fluo compound contained the decyl linker, and the

uncoupling effect was significantly reduced in the

case of a similar compound with the butyl linker

[228]. MitoFluo induced electric current through BLM,

which was selective for protons [225]. It was shown

that in vivo mitoFluo exerts neuro- and nephro-pro-

tective effects in the models of brain trauma and

rat kidney ischemia-reperfusion, respectively [228].

MitoFluo, as well as mitoNBD – the conjugate of

decyltriphenylphosphonium with NBD (7-nitrobenz-

2-oxa-1,3-diazole) also exhibited strong antibacterial

properties especially towards Gram-positive bacteria

[119, 229].

The success with mitoFluo stimulated research

in this direction: conjugates of triphenylphosphoni-

um with CCCP (mitoCCCP, 10-([4′-(dicyanomethylene)

hydrazinyl-2′-chlorophenyl]oxy)decyltriphenylphos-

phonium bromide) [230] and with the strongest

uncoupler SF6847 (mitoSF, covalent conjugates of

[(3,5-di-tert-butyl-4-hydroxyphenyl)methylidene]pro-

panedinitrile with alkyltriphenylphosphonium) were

synthesized [231]. It should be noted that attachment

MITOCHONDRIAL UNCOUPLERS 1825

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

of triphenylphosphonium to an anionic protono-

phore does not change its proton-donor group, hence,

mitoDNP, mitoFluo, and mitoSF could be considered

as OH-acids, similar to corresponding anionic pro-

tonophores, while mitoNBD and mitoCCCP – as NH-

acids. MitoCCCP exhibited very weak uncoupling

effect at submicromolar concentrations, while the

conjugate with SF6847 uncoupled mitochondria at

submicromolar concentrations. Among the deriva-

tives of SF6847, the conjugate with the pentyl linker

(SF-C5-TPP, (6-[3′,5′-di-tert-butyl-4′-hydroxyphenyl]-

7,7-dicyanohept-6-en-1-yl)triphenylphosphonium bro-

mide) exhibited the highest activity, while the uncou-

pling activity was significantly reduced in the case of

butyl- and decyl- linkers. It was also shown that the

compound with butyl linker is accumulated in mito-

chondria in response to their energization. Inisolated

RLM, SF-C5-TPP exhibited significantly lower uncou-

pling activity as compared to SF6847 itself (approxi-

mately by 2 orders of magnitude), however, it effec-

tively decreased membrane potential in mitochondria

in cell culture, which could be associated with the

ability of SF-C5-TPP to accumulate in cell mitochon-

dria. Furthermore, it induced proton current across

BLM, which was stimulated by the addition of phlore-

tin. This indicates that transmembrane diffusion of

the protonated cation is the rate-limiting step in the

protonophoric cycle of SF-C5-TPP. Of note, mitoCCCP

was found to be a strong uncoupler in the system

of inverted submitochondrial particles, as well as in

subbacterial particles, while this compound was a

weak uncoupler in isolated mitochondria [230].

In another series of studies, derivatives of [(E)-

2-(5-chloro-2-hydroxyphenyl)-2-phenylethenyl]phos-

phonium with substituents of varying length at the

phosphorus atom, which could be considered as

conjugates of phosphonium with para-chlorophenol

(PP6; Fig. 5) were investigated [226, 232, 233]. The

most active compounds in this series exhibited the

uncoupling effect in mitochondria at micromolar con-

centrations, suppressed growth of Bacillus subtilis

bacteria, and also induced proton current in BLMs.

Compounds with higher hydrophobicity were able to

create nonspecific defects in lipid membranes and

even cause hemolysis of erythrocytes.

Precursors of phosphonium ylides. Phosphonium

ylides are often used in the Wittig reaction. They com-

prise zwitterions with the positive charge at the phos-

phorus atom and the negative charge at the closest

carbon atom [234]. It was shown in our recent study

[235] that the cations of (alkyloxycarbonylmethyl)

triphenylphosphonium (Fig.  5) could release protons

of the methylene group and, in this way, to partici-

pate in the proton transfer across lipid membranes.

It was found out that the precursor of the phospho-

nium ylide CMTPP-C8 ((octyloxycarbonylmethyl)triph-

enylphosphonium bromide) stabilized by the ester

bond, is able to uncouple mitochondria at micromo-

lar concentrations and induce proton current across

BLM. Hence, the precursors of stabilized ylides, as

well as the carboranes described above, are protono-

phoric uncoulplers based on CH-acids. CMTPP-C8 and

its analogues exhibited moderate cytotoxicity in cell

culture and caused a decrease in the mitochondrial

membrane potential in cells at concentrations that

did not cause a significant decrease in cell survival.

CMTPP-C12 dissipated pH gradient in thylakoid mem-

branes of chloroplasts and exhibited antimicrobial

activity. It was also shown that methylation of phe-

nyl residues significantly increased the protonophoric

activity of these compounds, so the uncoupling con-

centrations in mitochondria decreased to the level of

tens of nanomoles [236].

Uncouplers not characterized in model sys-

tems. At present not all described uncouplers can be

classified. The data available in the literature demon-

strate uncoupling activity of certain compounds,

which were investigated only in biological systems

(cell cultures and mice), while their physicochemi-

cal properties and mechanisms of uncoupling were

not explored. In particular, the compound CZ5 (ethyl

(2E)-5-(4-chlorophenyl)-2-[(3,5-dibromo-4-hydroxyphe-

nyl)methylidene]-7-methyl-3-oxo-2,3-dihydro-5H-[1,3]

thiazolo[3,2-a]pyrimidine-6-carboxylate), described

in the study by Fu et  al. [237], most likely operates

as an anionic uncoupler, because it is a derivative

of 2,6-dibromophenol. The same could be said about

OPC-163493, a triazole-containing compound (4-(5-

methyl-2-(4-(trifluoromethyl)phenyl)-1,3-thiazol-4-yl)-

1H-1,2,3-triazole-5-carbonitrile), which most likely

could be deprotonated at alkaline pH [17]. However,

classification of these and some other protonophores

described in the literature requires further investi-

gation.

Cation-dependent protonophores. It has been

noted above in this review that the transporter of po-

tassium ions nigericin (Fig. 6a) is a non-electrogenic

ionophore and cannot uncouple mitochondria at low

concentrations; its main function is the induction of

potassium/H

-exchange. However, it was shown that

at high concentrations nigericin is capable of mito-

chondrial uncoupling, i.e. accelerating mitochondri-

al respiration [238]. Moreover, such well-known un-

coupler of oxidative phosphorylation as usnic acid

(UA) (Fig.  6a) [239,  240] is also an ionophore trans-

porting divalent cations [241]. Below the mechanism

of their protonophoric function will be discussed

in detail.

It was shown previously that nigericin at con-

centrations of tens of micromoles is able to stimulate

mitochondrial respiration and reduce the efficiency

of oxidative phosphorylation [238]. Acceleration of

ANTONENKO et al.1826

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Fig. 6. Chemical structure of nigericin and usnic acid (UA)(a). Scheme of functioning of cation-dependent protonophores(b).

Scheme of functioning of complexes of fatty acids with lipophilic cations (c). BLM, bilayer lipid membrane.

respiration was clearly pronounced in the case of us-

ing succinate as a substrate of respiratory complexII,

while in the case of using substrates of respiratory

complex I (glutamate and malate) nigericin inhibit-

ed respiration of mitochondria [242]. Both stimula-

tion of respiration and its inhibition were observed

only in the presence of potassium ions in the me-

dium, while no effect was observed in presence of

other cations. This is in accordance with the cation

selectivity of nigericin as an ionophore. Inhibition of

respiration in the case of using glutamate and ma-

late could be possibly associated with inhibition of

transporters of these compounds in the mitochondrial

membrane.

Nigericin at micromolar concentrations increased

permeability of BLM proportionally to the square of

concentration [243]. The authors hypothesized that

this pattern of membrane permeability could be ex-

plained by formation of nigericin dimers in complex

with potassium ions and proton, in particular, pro-

ton is transported across BLM in the form of a neu-

tral complex with nigericin, while potassium cation

is transported in the charged form by the dimer of

protonated nigericin molecules.

Selectivity of ion transport mediated by nigeri-

cin was examined in the study by Markin and Soko-

lov[244] based on potential of the open circuit in the

presence of ion concentration gradient on the mem-

brane. This parameter usually varies in the range

from zero to the value calculated from the Nernst

equation, i.e. approximately 59  mV per 10-fold con-

centration gradient of monovalent ions. It was un-

expectedly observed by Markin and Sokolov that

in the case of nigericin a potential of opposite sign

is generated in the presence of the pH gradient,

while in the presence of the potassium ion gradient

MITOCHONDRIAL UNCOUPLERS 1827

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

a ‘super-Nernst’ potential is generated, i.e., potential

higher than 59 mV. The authors developed a theoret-

ical model, which described adequately the obtained

data [244]. According to their suggestion, the process

does not involve establishing of interfacial equilibri-

um, but equilibrium between the penetrating compo-

nents. In the case of complex co-transport of several

ions, as in the case of nigericin, its cessation could

occur without equalization of electrochemical poten-

tials of these ions, hence, the potential could differ

from the Nernst value. The obtained experimental

data are consistent with the model, implying that

transport stoichiometry of the ions is 2K

/1H

Usnic acid (Fig.  6a) is a secondary metabolite of

certain lichens, used as a supplement in the weight-

loss diets. It has been known for a long time that

UA uncouples mitochondria at micromolar concentra-

tions [239,245]. According to our data [241], the pres-

ence of calcium or magnesium ions in the medium

is necessary for induction of electric current across

BLM by UA; in the presence of the chelating agent

EDTA the current becomes zero. The BLM current is

proportional to the square of usnic acid concentra-

tion, which indicates the formation of UA dimers in

the course of the ion transport across BLM. Investi-

gation of selectivity of the UA-mediated ion transport

revealed that potentials in the case of pH gradient

were approximately 2-fold higher than the Nernst po-

tentials [246]. Use of the model suggested by Markin

and Sokolov for description of the UA-mediated ion

transport led to the conclusion that the main process

of this transport is electrogenic exchange of three

protons for calcium or magnesium ion (see scheme

in Fig. 6b).

The pentadecapeptide gramicidin A produced by

Bacillus brevis has the following amino acid sequence:

HCO-L-Val

-Gly

-L-Ala

-D-Leu

-L-Ala

-D-Val

-L-Val

-D-

Val

-L-Trp

-D-Leu

-L-Trp

-D-Leu

-L-Trp

-D-Leu

-L-

Trp

-NHCH

OH; it is known as a channel-former

selective for monovalent cations [247-249]. It is often

used for dissipation of membrane potential in vari-

ous bioenergetic systems such as mitochondria [250],

bacteria [251], submitochondrial particles [252], and

chloroplasts [253]. As was shown, gramicidin  A also

can transport protons; and proton transport is 2 or-

ders of magnitude more effective than transport of

potassium ions [254]. However, in biological systems

gramicidin  A primarily functions as a potassium and

sodium ionophore, because amount of protons in

cells is approximately 7 orders of magnitude lower

than that of potassium ions. From this point of view,

it formally could be assigned to the group of cat-

ion-dependent protonophores. However, in reality,

the mechanism is fundamentally different involving

the formation of an ionic channel and transport of

protons along the hydrophilic wall of the channel

inside the membrane. Wide use of gramicidin  A as

an uncoupler is limited due to its high cytotoxicity

associated with equalization of potassium and sodi-

um ion gradients on the plasma membrane of cells

[255, 256], as well as by its low permeability across

cellular and subcellular membranes [253, 257]. Some

derivatives of gramicidin  A exhibit significantly lower

cytotoxicity, but, at the same time, maintain high pro-

tonophoric activity [255, 258]. It was shown that one

of such analogues with replacement of valine at the

first position with glutamic acid exhibits neuroprotec-

tive and nephroprotective properties, similar toother

uncouplers [259].

Complexes of fatty acids with hydrophobic

cations (and other transporters) as protonophores.

Certain amounts of fatty acids are always present in

biological membranes, which themselves cannot be

considered as protonophores due to low efficiency

of penetration of fatty acid anionic form across lipid

membranes. However, there are compounds that form

complexes with anions of fatty acids and to certain

extent facilitate their translocation across the mem-

brane. Among those relatively popular compounds

there are conjugates of decyltriphenylphosphonium

with plastoquinone or ubiquinone, which were named

SkQ1 (plastoquinoyldecyltriphenylphosphonium bro-

mide) and mitoQ (ubiquinonyldecyltriphenylphospho-

nium methanesulfonate), respectively. It was shown

that at micromolar concentrations SkQ1 (or dodecyl-

triphenylphosphonium C

TPP) uncouples mitochon-

dria due to interaction with endogenous fatty acids

[260]. In the review by Childressetal.[32] mitoQ and

TPP are described as usual protonophores, which

is not precisely correct. Below the mechanism of ac-

tion of these and similar compounds exhibiting the

uncoupling effect due to interaction with fatty acids

will be considered in more detail.

Complexes of fatty acids with lipophilic cations.

It was shown previously that fatty acids are capable

of forming complexes with lipophilic cations in lip-

id membranes [260, 261]. These complexes facilitate

diffusion of fatty acid anions across the membranes,

which results in protonophoric activity due to high

membrane permeability of the neutral form of fatty

acid. Scheme of this process is presented in Fig.  6c. De-

rivatives of alkyltriphenylphosphonium, rhodamine19,

rhodamine  B, and berberine could serve as lipophilic

cations [260-263], as well as some local anesthetics of

a cationic nature [264]. In general, it has been known

for a long time that hydrophobic cations form com-

plexes with hydrophobic anions in lipid membranes

[46, 264,265]. Interestingly enough, anions of classical

anionic uncouplers can play a role of such hydropho-

bic anions, and the addition of lipophilic cations re-

sults in significant acceleration of proton transfer by

CCCP and DNP [266-268].

ANTONENKO et al.1828

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Derivatives of urea and thiourea. It was shown

using liposomes loaded with the pH indicator pyra-

nine that lipophilic cations in combination with

thiourea-containing compounds could transport hy-

drogen ions [269]. Previously, it was reported that

such compounds are transporters of inorganic anions,

including chloride ions [184]. Complexes of fatty acid

carboxyls with a thiourea group are assembled via

formation of several hydrogen bonds between them.

The uncoupling effect of one of the thiourea deriva-

tives, NT-1505 (N-allyl-N′,N′-dibenzyl-S-ethylthioisourea

hydroiodide) was demonstrated in the study by An-

tonenkoet al. [270] both in isolated mitochondria and

in the neuron culture. It was also shown that NT-1505

increases proton current across BLM in the presence

of palmitate. According to the studies by York et  al.

[186, 271], the ability of transporting fatty acid an-

ions is characteristic not only for the derivatives of

thiourea, but also for other urea derivatives such

as, for example, N,N′-bis(3,5-dichlorophenyl)urea also

known as compound SR4. Protonophoric properties of

SR4 have been described above in the section devot-

ed to NH-acids, i.e. these compounds could serve as

protonophores even without fatty acids.

Uncoupling via formation of defects in the

membrane. The process of uncoupling in mitochon-

dria implies preservation of membrane integrity

and formation of selective pathway for hydrogen

ions in the presence of the added proton transport-

er. However, the main signs of uncoupling, namely

stimulation of respiration and depolarization of the

mitochondrial membrane, could be also caused by

compounds inducing formation of non-selective pores

in lipid membranes [272-276], or even through di-

rect membrane disruption [277-279]. In some cases,

the true uncoupling and formation of non-selective

leakage is difficult to distinguish. For example, in

the case of anesthetic bupivacaine described above

it is not clear whether this compound is a cationic

protonophore, or it induces defects in the membrane.

Below this quite contradictory issue will be discussed

in more detail.

Detergent action and uncoupling. Classical action

of detergents occurs at concentrations above the crit-

ical micelle concentration (CMC) and results in com-

plete solubilization of membrane components (pro-

teins and lipids). However, the uncoupling effect of

classical detergents could be observed at concentra-

tions significantly lower than CMC [277-279]. This is

in line with the fact that detergents induce formation

of non-specific channels in BLMs at concentrations,

which are several orders of magnitude lower than

the concentration of micelle formation [280]. Under

these conditions, BLMs maintain their integrity and

are not disrupted when regular potentials are ap-

plied. Changes in spontaneous curvature of monolay-

ers in the bilayer due to non-lamellar structure of the

majority of detergents could play a significant role

in formation of such transient defects by lowering

the energy of formation of hydrophilic cavities and

transient channels in the membrane, which defines

its ionic permeability  [281]. As examples, the action

of minocycline  [282] and SkQ1  [283] could be consid-

ered, which cause leakage in both mitochondrial and

artificial membranes. Terada and colleagues [284] de-

scribed the induction of ionic transport through the

mitochondrial membrane and BLM by the dicationic

cyanine dye triS-C4(5) (2,2′-[5-(3-butyl-4-methyl-1,3-

thiazol-2-ylidene)penta-1,3-diene-1,3-diyl]bis[3-butyl-

4-methyl-1,3-thiazolium] diiodide), which depended

on the presence of phosphate in the medium. The

authors suggested that it is exactly the complex of

triS-C4(5) with phosphate that is capable of induc-

ing transient defects in the lipid membrane, which

manifested themselves in mitochondria as uncoupling

of oxidative phosphorylation.

Interaction of lauryl sulfate with UCP1 and ANT1

proteins. It was noted earlier in this review that an-

ionic uncouplers could interact with ANT1 and ac-

tivate proton leakage through this protein. It was

shown that this protein could also interact with sodi-

um lauryl (dodecyl) sulfate (SDS) causing uncoupling

in mitochondria, which is inhibited by the specific

inhibitor of ANT1 carboxyatractyloside [27]. Further-

more, it was shown that SDS stimulates proton leak-

age via the UCP1 protein [133]. In this experiment

the used concentrations of SDS (micromoles) were

significantly lower than CMC (~8  mM).

Nonprotonophoric uncoupling. Quite a few

agents are known that provide the pattern of un-

coupling (stimulation of respiration, cessation of ATP

synthesis, and drop of membrane potential) without

relation to the described above proton uncoupling.

A classic example is an effect of arsenate [285, 286]

Arsenate is a substrate of ATP synthase; ADP-arse-

nate, formed as a result of this reaction, is unstable

in water and decomposes rapidly to initial arsenate

and ADP. This causes stimulation of respiration in mi-

tochondria in the presence of ADP, which, however,

is not accompanied by ATP synthesis. Phenomeno-

logically, in the presence of arsenate, mitochondria

are transformed into the continuous ‘third’ state.

The Ca

-exchanger A23187 (calcimycin) could be

another example, which causes the uncoupling be-

cause the electrogenic calcium uniporter is present

in the mitochondrial membrane [287]. The list of

such examples could be continued, but they are not

the subject of this review. It should be also men-

tioned that freezing-thawing cycles transform mito-

chondria into the completely uncoupled state due to

the formation of multiple defects in its inner mem-

brane [288].

MITOCHONDRIAL UNCOUPLERS 1829

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

METABOLIC TRANSFORMATIONS

OF PROTONOPHORES IN CELLS

It is obvious that protonophores, similar to the

majority of xenobiotics, are subjected to metabolic

transformations in organisms. Chemical modifica-

tions of xenobiotics, including drugs, environmental

contaminants, or food supplements, which are com-

pounds foreign for cells, predominantly occur via en-

zymatic processes classified as phase  I and phase  II

reactions. These metabolic pathways are directed

towards increasing hydrophilicity of xenobiotics,

which facilitates their excretion from an organism by

phase  III transporters. Phase  I reactions include func-

tionalization of a compound via oxidation, reduction,

or hydrolysis, which makes the molecule more reac-

tive in the subsequent conjugation during phase  II.

The key player in oxidation is the family of cyto-

chrome P450 enzymes, which introduce polar groups

such as hydroxyl. For example, oxidation of benzene

derivatives to phenol mediated by CYP450 increases

their solubility. The reactions of reduction are less

common; they involve transformation of nitro groups

into amines. Hydrolytic enzymes, such as esterases,

cleave ester or amide bonds, as occurs with aspirin,

which is transformed into salicylic acid [289]. It is

remarkable that in phase  I super-reactive intermedi-

ate products, such as epoxides, sometimes could be

formed, which could exert toxic effects, if they are

not detoxified effectively.

Phase II reactions include conjugation of wa-

ter-soluble fragments with initial compounds or with

compounds modified during phase I. Glucuronization

catalyzed by uridine diphosphate (UDP)-glucurono-

syltransferase (UGT) attaches glucuronic acid from

UDP-glucuronic acid to substrates [290] such as mor-

phine forming highly soluble, excretable glucuronides

[291]. Sulfotransferases (SULT) transfer sulfate groups

from 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to

phenols and alcohols [292], as in the case of sulfa-

tion of paracetamol. Glutathione-S-transferases (GST)

link glutathione with electrophilic centers [293], thus

neutralizing metabolites formed, for example, in the

case of acetaminophen overdose. N-acetyltransfer-

ases (NAT) catalyze the transfer of an acetyl group

from acetyl-CoA to aromatic amines [294], while

methyltransferases, such as catechol-O-methyltrans-

ferase (COMT), catalyze the transfer of a methyl

group from S-adenosylmethionine (SAM) to substrates

such as catecholamines [295]. Conjugation with ami-

no acids, although less common, links xenobiotics

with glycine or taurine; one of the examples of such

processes is excretion of benzoic acid with formation

of hippuric acid.

Concerning metabolic transformations of proto-

nophoric uncouplers, the following reactions could

be of interest: phase  I reactions involving hydrolysis

of ester bonds catalyzed, in particular, by esterases;

phase II reactions involving glutathionylation. Among

the enzymes with esterase activity, in this review we

will consider only enzymes that are primarily in-

volved in detoxification and metabolism of xenobi-

otics via hydrolysis of ester bonds. Carboxylesterases

(CES), members of the superfamily of serine hydro-

lases, are representatives of this group. CES1 and

CES2, main isoforms in humans, hydrolyze substances

containing esters (such as clopidogrel, irinotecan) and

toxic compounds from the environment. They belong

to the α/β-hydrolase-fold family with the catalytic

triad (Ser-His-Glu/Asp) and conserved oxyanion hole

(for stabilization of negative charge at the deprotonat-

ed oxygen in the intermediate state), which provides

broad substrate specificity. CES enzymes are localized

in endoplasmic reticulum and plasma, where they

process lipophilic esters into hydrophilic metabolites

for excretion. There are also data on CES activity as-

sociated with mitochondria [296, 297].

Glutathionylation involved in xenobiotic metabo-

lism is associated primarily with conjugation of GSH

with electrophilic xenobiotics, which represents a

critical mechanism of detoxification mediated by glu-

tatione-S-transferases. This process, a part of the mer-

capturate pathway, neutralizes harmful compounds

by increasing their solubility in water, facilitating

excretion through urine or bile [298]. The reaction

involves a nucleophilic attack of the thiole group in

GSH on electrophilic centers of xenobiotics forming

glutathione-S-conjugates. These conjugates are further

processed by enzymes such as γ-glutamyl transferase

and dipeptidase for the production of cysteine S-con-

jugates, which are further acetylated with formation

of mercapturic acids (conjugates of N-acetylcysteine)

for elimination [298]. Glutathione-S-transferases com-

prise a superfamily of enzymes playing a central role

in metabolism of phaseII via conjugation of the GSH

tripeptide (γ-glutamyl-cysteinyl-glycine) with electro-

philic xenobiotics and endogenous toxins. The conju-

gation increases solubility of these compounds facili-

tating their excretion via phase III transporters, such

as proteins associated with multidrug resistance.

Ester derivatives of 7-hydroxycoumarin and

esterases. Structure and uncoupling properties of

7-hydroxycoumarin ester derivatives have been de-

scribed above. In particular, rapid hydrolysis of the

compounds of this series in RLM and the observed

tissue specificity of the inactivation was mentioned

before. 7-Hydroxycoumarin is a natural pH-dependent

fluorophore, which is derived from medicinal plants

of the Umbelliferae family (hence, the second name –

umbelliferone) and some others. We have synthesized

two series of esters: with umbelliferone-3-carbox-

ylic acid and umbelliferone-4-acetic acid [124, 125].

ANTONENKO et al.1830

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Compounds of both series exhibited pronounced un-

coupling activity in mitochondria. Surprisingly, this

activity disappeared within minutes, unlike the case

of classical uncouplers DNP and CCCP. Taking into

account that protonophoric activity of the esters in

model membranes (BLMs and liposomes) did not

change with time, it was suggested that the uncou-

pling activity in mitochondria disappeared due to

enzymatic hydrolysis of the esters. Indeed, thin lay-

er chromatography (TLC) showed that the umbellif-

erone-containing esters, incubated in the presence

of isolated RLM, were almost completely converted

into initial acids within minutes, while no hydrolysis

was observed in the case of fluorescein octyl ester

[118]. We hypothesized that the mitochondrial alde-

hyde dehydrogenase ALDH2 could be a good candi-

date for the role of an enzyme catalyzing hydrolysis

of the umbelliferone-containing esters, because this

enzyme exhibits high esterase activity [299]. More-

over, this activity is sensitive to the same inhibitors

as the main activity of this enzyme – oxidation of

acetaldehyde. The known inhibitor of aldehyde de-

hydrogenases disulfiram suppressed the drop in the

uncoupling activity of the umbelliferone esters [124].

An alternative candidate of such enzymatic activity is

the carboxyl esterase CES1, which plays a significant

role in metabolism of xenobiotics. Its activity is also

inhibited by disulfiram [300, 301]. Certain isoforms

of carboxyl esterases are located predominantly in

liver, while only low amounts of these enzymes are

detected in heart and kidney, which could explain

the tissue specificity of the mitochondrial uncoupling

by the umbelliferone derivatives, namely: the sponta-

neous disappearance of the uncoupling activity of the

umbelliferone esters found in RLM was not observed

in rat heart and kidney mitochondria [125]. Hence,

the umbelliferone esters could be considered as tis-

sue-specific uncouplers.

Diarylamines and glutathione-S-transferases.

Above we have already discussed diarylamines, a

group of compounds with some representatives exhib-

iting properties of anionic uncouplers due to an abili-

ty of their NH group to lose a proton. The best-known

compound among them is fluazinam (Fig.  3), which

uncouples mitochondria at nanomolar concentrations

[155, 174, 175]. This compound is a broad-spectrum

fungicide widely used in agriculture. In the pioneer

study published in 1991 it was shown that the time

course of oxygen consumption by isolated RLM after

the addition of fluazinam is biphasic, namely: stim-

ulation of mitochondrial respiration by fluazinam is

followed byreversal of the respiration rate to the ini-

tial level on the minute time scale [174]. Efficiency

of the reversal depended significantly on the level of

glutathione (GSH) in mitochondria, and in the pres-

ence of GSH-depleting agents the reversal practically

disappeared. The author suggested that the resto-

ration of the respiration rate is associated with conju-

gation of fluazinam with GSH, which is catalyzed by

mitochondrial glutathione-S-transferase. The analog of

fluazinam B-3 (N-[2,4-dinitro-6-trifluoromethyl-3-chlo-

rophenyl]-5-(trifluoromethyl)pyridine-2-amine), dis-

covered in the study by Brandt et  al.  [175], also ex-

hibited very high uncoupling activity, but, unlike

fluazinam itself, was not metabolized by RLM due to

the absence of electrophilic substituent in the third

position of the benzene ring. According to the data

reported by Clarke et  al.  [9], fluazinam was found to

be the most reactive substrate of glutathione-S-trans-

ferase among the very wide range of agrochemical

compounds. In our study [173], the addition of GSH

to RLM enhanced the fluazinam inactivation. The

formation of the conjugates of fluazinam and GSH

was detected experimentally using methods of TLC

and chromato-mass spectrometry (LC-MS): catalytic

replacement of chlorine and one of the nitro groups

with glutathione occurred in the process. It is worth

noting that the phase of fluazinam inactivation is ab-

sent in the case of rat heart mitochondria, i.e. fluazi-

nam exhibits the properties of a tissue-specific un-

coupler [173].

N-phenylthiophenamines and glutathione-S-

transferases. N-phenylthiophenamines comprise a

class of organic compounds having a thiophene ring

(5-membered aromatic heterocycle containing sulfur

atom) attached through an amino group to a phenyl

ring. Interestingly enough, in 1970 the paper was pub-

lished by Buechel and Schaefer [302] describing ef-

fects of a large series of anilinothiophene derivatives

on isolated RLM. The study mainly concentrated on

three groups of compounds: 2-anilino-3,5-dinitrothio-

phenes, 2-anilino-5-halo-3,4-dinitrothiophenes and de-

rivatives of 2-hydroxy-3,5-dinitrothiophene. Buechel

and Schaefer[302] revealed the relationship between

the structure and activity, demonstrating the manda-

tory requirement of NH group for the uncoupling ac-

tivity: the absence of this group resulted in the com-

plete loss of activity. Electron acceptor substituents in

the phenyl ring, such as nitro (-NO

), trifluoromethyl

(-CF

), and chlorine, enhanced the activity likely due

to lowering of pK

. It is worth mentioning that some

of these compounds exhibited properties similar to

those described later for fluazinam [174, 177], name-

ly, the ability to stimulate mitochondrial respiration,

which disappeared within minutes. However, the au-

thors explained this phenomenon not by chemical

modification of anilinothiophenes, but by their inter-

action with non-identified components of the mito-

chondrial respiratory chain. Noteworthy, the study by

Buechel and Schaefer [302] (1970) was conducted in

the era, when the modern ideas of bioenergetics just

started to emerge. The biphasic uncoupling action of

MITOCHONDRIAL UNCOUPLERS 1831

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Fig. 7. Chemical structures of BDNT and its adduct with glutathione.

5-bromo-3,4-dinitro-N-(4-nitrophenyl)thiophen-2-amine

(BDNT) in RLM was investigated in great detail in our

recent study [176]. The restoration of the membrane

potential level after the BDNT-induced decrease be-

came more pronounced upon the addition of GSH

to incubation medium and practically disappeared

upon complete depletion of intramitochondrial GSH

pool. In the case of rat heart mitochondria (RHM),

the restoration of the decreased membrane poten-

tial did not occur. By using capillary electrophoresis

and LC-MS, the formation of BDNT conjugates with

glutathione (Fig. 7) was found upon incubation with

RLM, and these conjugates were absent in the case

of incubation with RHM. In was concluded that BDNT

is a substrate of the glutathione-S-transferase (GST)

enzyme, which catalyzed the formation of the BDNT

conjugate with GSH: similar to the case of fluazinam,

the nitro group of thiophene is replaced with gluta-

thione. Higher expression of GST or predominance of

another isoform in liver as compared to heart could

be responsible for the tissue-specific action of BDNT.

It should be mentioned that BDNT caused depolariza-

tion of mitochondria in the culture of fibroblasts, but

not in liver cells (HepG2). BDNT induced proton-selec-

tive current through planar BLM. The current inten-

sity decreased upon the addition of phloretin, which

indicated the anionic nature of the protonophore. The

value of BDNT was found to be 7.38.

CONCLUSIONS

By classifying mitochondrial uncouplers, we

were able for the first time to systematize numerous

examples of uncouplers present in the literature. This

allowed us to review the chemical diversity of uncou-

plers, as well as to identify gaps in the table of chem-

ical structures. In particular, among the known zwit-

terionic uncouplers there are many examples with

a proton cycle performed by the pair zwitterion-cat-

ion, while there are yet no examples with a proton

cycle performed by the pair zwitterion- anion in the

literature (Fig.  1). Such a compound could consist of

triphenylborate and a fragment containing amino

group capable of protonating-deprotonating under

physiological pH. It should be examined in future,

how effective this molecule (or the similar one) is in

transporting hydrogen ions across lipid membranes.

Such ‘anionic’ zwitterionic protonophores could be

much more active than ‘cationic’ zwitterionic proto-

nophores, because anions are transported significant-

ly more effectively across lipid membranes as com-

pared to cations of the similar structure due to the

presence of membrane dipole potential.

A goal in the search for the most active uncou-

plers is usually their selective action in the uncou-

pling of oxidative phosphorylation without affecting

other processes vital for the cell functioning. Pene-

tration of these compounds across the plasma mem-

brane and their predominant accumulation in mito-

chondria are essential issues. In this regard, cationic

uncouplers as well as zwitterionic uncouplers that

accumulate in mitochondria due to the inner mem-

brane potential of approximately −180  mV should

have a certain advantage. However, this advantage is

offset by the weak protonophoric activity of cationic

uncouplers; likely, this is the reason why most of the

commonly used uncouplers belong to the class of an-

ionic protonophores.

On the other hand, as has been mentioned pre-

viously [33], a therapeutic effect of uncouplers could

be significantly improved by using the compounds

that have advantages in certain tissues. In particu-

lar, the use of DNP ethers for treating non-alcohol-

ic steatohepatitis (NASH) was demonstrated to be

successful [15]. Ethyl and methyl ethers of DNP are

easily absorbed from intestine to blood and are de-

livered to liver, where they are rapidly transformed

ANTONENKO et al.1832

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

into DNP by hydrolases. Hence, in this case the un-

coupling agents emerge locally, which should reduce

side effects of DNP. This means that application of

tissue-specific uncouplers is of great interest in med-

icine. Ideally, such uncouplers could actively work in

one tissue type, while exhibiting low activity (ideally

no activity at all) in other tissues. In the series of

studies on the uncoupling activity of fluazinam and

anilinothiophenes, it was demonstrated that these

compounds are subjected to rapid glutathionylation

in liver, while in heart and kidney their activity is

preserved [173,  176]. In another series of studies,

the tissue-specific activity was demonstrated for the

derivatives of 7-hydroxycoumarin [125]. Hence, tis-

sue-specific inactivation together with formation of

an active uncoupler in the specific tissue could be

also used for achieving partial or complete tissue

specificity of the corresponding uncouplers.

Another approach in the search for tissue-spe-

cific uncouplers is the use of ability of certain com-

pounds to mediate proton transfer across membranes

via interaction with the proteins of the inner mi-

tochondrial membrane, primarily with the protein

transporters of the SLC25 family. In particular, it was

shown that DNP uncouples mitochondria partially via

interaction with the adenine nucleotide translocator

ANT1 [26, 27, 63]. Considering that the patterns of

ANT1 expression differ significantly in different tis-

sues, the ability of DNP to uncouple mitochondria

could vary significantly: it is shown that the uncou-

pling activity of DNP is significantly higher in heart

mitochondria than in liver mitochondria [63]. Hence,

even such widely known and used uncoupler as DNP

could be considered as a tissue-specific uncoupler. In

this regard, fatty acids could be also considered as

uncouplers specific for brown adipose tissue due to

participation of the UCP1 protein in their uncoupling

activity. Further studies should reveal uncouplers

with higher tissue specificity than DNP, moreover,

the protein partner of such an uncoupler should not

be necessary ANT1. In particular, it was shown that

the uncoupling effect of the cationic uncoupler C4R1

involves the proton ATPase of mitochondria [200].

Vladimir Petrovich Skulachev has strongly boost-

ed studies of uncouplers by suggesting the concept

of ‘mild uncoupling’ [286, 303]. This concept is based

on the steep dependence of the ROS formation rate

on mitochondrial membrane potential. Considering

that organism aging is often associated with oxida-

tive stress and accumulation of oxidation products,

uncouplers could cause an increase in life span.

Indeed, it was shown that low doses of DNP reliably

increase life span of rats [19], mice [20], yeast [21],

and Drosophila flies [22]. Investigation of life span of

animals is a rather complex experimental task; how-

ever, fortunately, uncouplers exhibit therapeutic prop-

erties, which are easier to investigate, with many of

them mentioned above. One could hope that all the

facts taken together would make this review useful

for readers and inspire researchers to further inves-

tigate new uncouplers and elucidate mechanisms of

their action.

Abbreviations

ANT1 adenine nucleotide translocator 1 (ATP/

ADP-antiporter)

BDNT 5-bromo-3,4-dinitro-N-(4-nitrophenyl)

thiophen-2-amine

C4R1 butyl ester of Rhodamine 19

CCCP carbonyl cyanide 3 chlorophenylhydra-

zone

CES carboxylesterases

DNP 2,4-dinitrophenol

DNPME 1-methoxy-2,4-dinitrobenzene

o-F16 F16, and m-F16, (E)-2-, (E)-3-, and

(E)-4-(3-indolylvinyl)-N-methylpyridini-

um iodide, respectively

FCCP carbonyl cyanide p-trifluoromethoxy-

phenylhydrazone

GSH glutathione

HU6 5-[(2′,4′-dinitrophenoxy)methyl]-1-meth-

yl-2-nitroimidazole

BLM bilayer lipid membrane

mitoCCCP 10-([4′-(dicyanomethylene)hydraz-

inyl-2′-chlorophenyl]oxy)decyltriphen-

ylphosphonium bromide

mitoDNP 3-(4′-hydroxy-3′,5′-dinitrophenyl)prop-

yltriphenylphosphonium methanesul-

fonate

mitoFluo 10-[(2-(6-hydroxy-3-oxo-3H-xanthen-

9-yl)benzoyl)oxy]decyltriphenylphos-

phonium bromide

NAC N-acetyl cysteine

PCBT pentachlorothiophenol

PDTC-9 nonyl 3-picolinoyldithiocarbazate

S13 3-tert-butyl-4′-nitro-2′,5-dichlorosalicy-

lanilide

SF6847 [(3,5-di-tert-butyl-4-hydroxyphenyl)

methylidene]propanedinitrile

SF-C5-TPP (6-[3′,5′-di-tert-butyl-4′-hydroxyphenyl]-

7,7-dicyanohept-6-en-1-yl)triphenyl-

phosphonium bromide

SkQ1 10-(6′-plastoquinonyl)decyltriphenyl-

phosphonium

SR4 N,N′-bis(3,5-dichlorophenyl)urea

UA usnic acid

UCP uncoupling proteins

Contributions

Y. N. Antonenko – concept and supervision of the

work; Y. N.Antonenko, E. A.Kotova, V. S.Krasnov, and

R. S.Kirsanov – writing and editing of the paper.

MITOCHONDRIAL UNCOUPLERS 1833

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Funding

The study was conducted under the state assign-

ment of Lomonosov Moscow State University (no.

AAAA-A19-119031390114-5).

Ethics approval and consent to participate

This work does not contain any studies involving hu-

man and animal subjects.

Conflict of interest

The authors of this work declare that they have

noconflicts of interest.

REFERENCES

1. Ovchinnikov, Yu. A., Ivanov, V. T., and Shkrob, A. M.,

Membrane active complexons [in Russian], Moscow:

Nauka, 1974.

2. Henderson, P. J. F., McGivan, J. D., and Chappell,

J. B. (1969) The action of certain antibiotics on mi-

tochondrial, erythrocyte and artificial phospholipid

membranes. The role of induced proton permeabil-

ity, Biochem. J., 111, 521-535, https://doi.org/10.1042/

bj1110521.

3. Sato,T., Konno,H., Tanaka,Y., Kataoka, T., Nagai, K.,

Wasserman, H. H., and Ohkuma, S. (1998) Prodigios-

ins as a new group of H

/Cl

–

symporters that uncouple

proton translocators, J.Biol. Chem., 273, 21455-21462,

https://doi.org/10.1074/jbc.273.34.21455.

4. Mitchell, P., and Moyle, J. (1967) Acid-base titration

across the membrane system of rat-liver mitochon-

dria. Catalysis by uncouplers, Biochem. J., 104, 588-

600, https://doi.org/10.1042/bj1040588.

5. Skulachev, V. P. (1970) Electric fields in coupling

membranes, FEBS Lett., 11, 301-308, https://doi.org/

10.1016/0014-5793(70)80554-3.

6. Ranga Rao, K. (1978) Pentachlorophenol: Chemistry,

pharmacology, and environmental toxicology, Plenum

Press, New York.

7. Hollingworth, R.M., and Gadelhak, G.G. (1998) Mech-

anisms of action and toxicity of new pesticides that

disrupt oxidative phosphorylation, Rev. Toxicol., 2,

253-266.

8. Palmeira, C. M., Moreno, A. J., and Madeira, V. M.

(1994) Interactions of herbicides 2,4-D and dinoseb

with liver mitochondrial bioenergetics, Toxicol.

Appl. Pharmacol., 127, 50-57, https://doi.org/10.1006/

taap.1994.1138.

9. Clarke, E. D., Greenhow, D. T., and Adams, D.

(1998) Metabolism-related assays and their appli-

cation to agrochemical research: reactivity of pes-

ticides with glutathione and glutathione transferas-

es, Pestic. Sci., 54, 385-393, https://doi.org/10.1002/

(SICI)1096-9063(199812)54:4<385::AID-PS842>

3.0.CO;2-C.

10. Cadenas, S. (2018) Mitochondrial uncoupling, ROS

generation and cardioprotection, Biochim. Biophys.

Acta Bioenerg., 1859, 940-950, https://doi.org/10.1016/j.

bbabio.2018.05.019.

11. Geisler, J. G. (2019) 2,4 Dinitrophenol as medicine,

Cells, 8, E280, https://doi.org/10.3390/cells8030280.

12. Zorov, D. B., Andrianova, N. V., Babenko, V. A.,

Pevzner, I.B., Popkov, V.A., Zorov, S.D., Zorova, L.D.,

Plotnikov, E. Y., Sukhikh, G. T., and Silachev, D. N.

(2021) Neuroprotective potential of mild uncoupling

in mitochondria. Pros and Cons, Brain Sci., 11, 1050,

https://doi.org/10.3390/brainsci11081050.

13. Plotnikov, E. Y., Silachev, D. N., Jankauskas, S. S.,

Rokitskaya, T. I., Chupyrkina, A. A., Pevzner, I. B.,

Zorova, L.D., Isaev, N.K., Antonenko, Y.N., Skulachev,

V.P., and Zorov, D.B. (2012) Mild uncoupling of respi-

ration and phosphorylation as a mechanism provid-

ing nephro- and neuroprotective effects of penetrating

cations of the SkQ family, Biochemistry (Moscow), 77,

1029-1037, https://doi.org/10.1134/S0006297912090106.

14. Rai,Y., Anita, Kumari,N., Singh,S., Kalra,N., Soni,R.,

and Bhatt, A. N. (2021) Mild mitochondrial uncou-

pling protects from ionizing radiation induced cell

death by attenuating oxidative stress and mitochon-

drial damage, Biochim. Biophys. Acta Bioenerg., 1862,

148325, https://doi.org/10.1016/j.bbabio.2020.148325.

15. Perry, R. J., Zhang, D., Zhang, X. M., Boyer, J. L., and

Shulman, G.I. (2015) Controlled-release mitochondrial

protonophore reverses diabetes and steatohepatitis in

rats, Science, 347, 1253-1256, https://doi.org/10.1126/

science.aaa0672.

16. Tao,H., Zhang,Y., Zeng,X., Shulman, G.I., and Jin,S.

(2014) Niclosamide ethanolamine-induced mild mito-

chondrial uncoupling improves diabetic symptoms

in mice, Nat. Med., 20, 1263-1269, https://doi.org/

10.1038/nm.3699.

17. Kanemoto, N., Okamoto, T., Tanabe, K., Shimada, T.,

Minoshima, H., Hidoh, Y., Aoyama, M., Ban, T.,

Kobayashi, Y., Ando, H., Inoue, Y., Itotani, M., and

Sato,S. (2019) Antidiabetic and cardiovascular bene-

ficial effects of a liver-localized mitochondrial uncou-

pler, Nat. Commun., 10, 2172, https://doi.org/10.1038/

s41467-019-09911-6.

18. Shrestha, R., Johnson, E., and Byrne, F. L. (2021) Ex-

ploring the therapeutic potential of mitochondrial

uncouplers in cancer, Mol. Metab., 51, 101222, https://

doi.org/10.1016/j.molmet.2021.101222.

19. Tainter, M. L. (1938) Growth, life-span and food

intake of white rats fed dinitrophenol through-

out life, J.Pharmacol. Exp. Ther., 63, 51-57, https://

doi.org/10.1016/S0022-3565(25)08958-X.

20. Caldeira da Silva, C.C., Cerqueira, F.M., Barbosa, L.F.,

Medeiros, M.H., and Kowaltowski, A.J. (2008) Mild mi-

tochondrial uncoupling in mice affects energy metab-

olism, redox balance and longevity, Aging Cell, 7, 552-

560, https://doi.org/10.1111/j.1474-9726.2008.00407.x.

ANTONENKO et al.1834

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

21. Barros, M. H., Bandy, B., Tahara, E. B., and Kowal-

towski, A. J. (2004) Higher respiratory activity de-

creases mitochondrial reactive oxygen release and in-

creases life span in Saccharomyces cerevisiae, J.Biol.

Chem., 279, 49883-49888, https://doi.org/10.1074/

jbc.M408918200.

22. Padalko, V.I. (2005) Uncoupler of oxidative phosphor-

ylation prolongs the lifespan of Drosophila, Biochem-

istry (Moscow), 70, 986-989, https://doi.org/10.1007/

s10541-005-0213-1.

23. Setty, O. H., Hendler, R. W., and Shrager, R. I. (1983)

Simultaneous measurements of proton motive force,

delta pH, membrane potential, and H

/O ratios in in-

tact Escherichia coli, Biophys. J., 43, 371-381, https://

doi.org/10.1016/S0006-3495(83)84360-4.

24. Westerhoff, H. V., Juretic, D., Hendler, R. W., and

Zasloff, M. (1989) Magainins and the disruption of

membrane-linkedfree-energy transduction, Proc.

Natl. Acad. Sci. USA, 86, 6597-6601, https://doi.org/

10.1073/pnas.86.17.6597.

25. Jung, D. W., Davis, M. H., and Brierley, G. P. (1989)

Estimation of matrix pH in isolated heart mitochon-

dria using a fluorescent probe, Anal. Biochem., 178,

348-354, https://doi.org/10.1016/0003-2697(89)90651-9.

26. Bertholet, A.M., Natale, A.M., Bisignano,P., Suzuki,J.,

Fedorenko,A., Hamilton,J., Brustovetsky,T., Kazak,L.,

Garrity, R., Chouchani, E. T., Brustovetsky, N.,

Grabe,M., and Kirichok, Y. (2022) Mitochondrial un-

couplers induce proton leak by activating AAC and

UCP1, Nature, 606, 180-187, https://doi.org/10.1038/

s41586-022-04747-5.

27. Andreyev, A. Y., Bondareva, T. O., Dedukhova, V. I.,

Mokhova, E. N., Skulachev, V. P., Tsofina, L. M.,

Volkov, N. I., and Vygodina, T. V. (1989) The ATP/

ADP-antiporter is involved in the uncoupling effect

of fatty acids on mitochondria, Eur. J. Biochem.,

182, 585-592, https://doi.org/10.1111/j.1432-1033.1989.

tb14867.x.

28. Khailova, L. S., Kirsanov, R. S., Rokitskaya, T. I.,

Krasnov, V. S., Korshunova, G. A., Kotova, E. A., and

Antonenko, Y. N. (2024) Mitochondrial uncoupling

caused by a wide variety of protonophores is differ-

ently sensitive to carboxyatractyloside in rat heart

and liver mitochondria, Biochim. Biophys. Acta Bio-

energ., 1865, 149506, https://doi.org/10.1016/j.bbabio.

2024.149506.

29. Weinbach, E. C., and Garbus, J. (1969) Mechanism

of action of reagents that uncouple oxidative phos-

phorylation, Nature, 221, 1016-1018, https://doi.org/

10.1038/2211016a0.

30. Hanstein, W.G. (1976) Uncoupling of oxidative phos-

phorylation, Biochim. Biophys. Acta, 456, 129-148,

https://doi.org/10.1016/0304-4173(76)90010-0.

31. Katre, N. V., and Wilson, D. F. (1978) Interaction of

uncouplers with the mitochondrial membrane: iden-

tification of the high affinity binding site, Arch. Bio-

chem. Biophys., 191, 647-656, https://doi.org/10.1016/

0003-9861(78)90403-4.

32. Childress, E. S., Alexopoulos, S. J., Hoehn, K. L., and

Santos, W. L. (2018) Small molecule mitochondrial

uncouplers and their therapeutic potential, J. Med.

Chem., 61, 4641-4655, https://doi.org/10.1021/acs.jmed-

chem.7b01182.

33. Goedeke, L., and Shulman, G. I. (2021) Therapeutic

potential of mitochondrial uncouplers for the treat-

ment of metabolic associated fatty liver disease and

NASH, Mol. Metab., 46, 101178, https://doi.org/10.1016/

j.molmet.2021.101178.

34. Kotova, E.A., and Antonenko, Y.N. (2022) Fifty years

of research on protonophores: mitochondrial uncou-

pling as a basis for therapeutic action, Acta Naturae,

14, 4-13, https://doi.org/10.32607/actanaturae.11610.

35. Dos Santos, B. G., Brisnovali, N. F., and Goedeke, L.

(2024) Biochemical basis and therapeutic poten-

tial of mitochondrial uncoupling in cardiometabol-

ic syndrome, Biochem. J., 481, 1831-1854, https://

doi.org/10.1042/BCJ20240005.

36. Samartsev, V.N., Semenova, A.A., Belosludtsev, K.N.,

and Dubinin, M. V. (2023) Modulators reducing the

efficiency of oxidative ATP synthesis in mitochon-

dria: protonophore uncouplers, cyclic redox agents,

and decouplers, Biophys. Rev., 15, 851-857, https://

doi.org/10.1007/s12551-023-01160-8.

37. McLaughlin, S. G., and Dilger, J. P. (1980) Transport

of protons across membranes by weak acids, Physi-

ol. Rev., 60, 825-863, https://doi.org/10.1152/phys-

rev.1980.60.3.825.

38. Terada, H. (1981) The interaction of highly active

uncouplers with mitochondria, Biochim. Biophys.

Acta, 639, 225-242, https://doi.org/10.1016/0304-4173

(81)90011-2.

39. McLaughlin, S. (1972) The mechanism of action of

DNP on phospholipid bilayer membranes, J. Membr.

Biol., 9, 361-372, https://doi.org/10.1007/BF01868062.

40. Borisova, M. P., Ermishkin, L. N., Liberman, E. A.,

Silberstein, A. Y., and Trofimov, E. M. (1974) Mech-

anism of conductivity of bimolecular lipid mem-

branes in the presence of tetrachlorotrifluorometh-

ylbenzimidazole, J. Membr. Biol., 18, 243-261, https://

doi.org/10.1007/BF01870115.

41. Foster, M., and McLaughlin, S. (1974) Complexes

between uncouplers of oxidative phosphorylation,

J. Membr. Biol., 17, 155-180, https://doi.org/10.1007/

BF01870177.

42. Smejtek,P., Hsu,K., and Perman, W.H. (1976) Electri-

cal conductivity in lipid bilayer membranes induced

by pentachlorphenol, Biophys.J., 16, 319-336, https://

doi.org/10.1016/S0006-3495(76)85691-3.

43. Wilson, D.F., Ting, H.P., and Koppelman, M.S. (1971)

Mechanism of action of uncouplers of oxidative

phosphorylation, Biochemistry, 10, 2897-2902, https://

doi.org/10.1021/bi00791a016.

MITOCHONDRIAL UNCOUPLERS 1835

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

44. Dilger,J., and McLaughlin,S. (1979) Proton transport

through membranes induced by weak acids: a study

of two substituted benzimidazoles, J.Membr. Biol., 46,

359-384, https://doi.org/10.1007/BF01868755.

45. Barstad, A. W., Peyton, D. H., and Smejtek, P. (1993)

AHA – heterodimer of a class-2 uncoupler – penta-

chlorophenol, Biochim. Biophys. Acta, 1140, 262-270,

https://doi.org/10.1016/0005-2728(93)90065-N.

46. Liberman, E.A., and Topaly, V.P. (1969) Permeability

of biomolecular phospholipid membranes for fat-sol-

uble ions [in Russian], Biofizika, 14, 452-461.

47. Brockman, H. (1994) Dipole potential of lipid mem-

branes, Chem. Phys. Lipids, 73, 57-79, https://

doi.org/10.1016/0009-3084(94)90174-0.

48. Gawrisch, K., Ruston, D., Zimmerberg, J., Parsegian,

V.A., Rand, R. P., and Fuller,N. (1992) Membrane di-

pole potentials, hydration forces, and the ordering of

water at membrane surfaces, Biophys. J., 61, 1213-

1223, https://doi.org/10.1016/S0006-3495(92)81931-8.

49. Markin, V. S., and Chizmadzhev, Yu. A. (1974) Induced

ion transport [in Russian], Nauka, Moscow.

50. Firsov, A. M., Popova, L. B., Khailova, L. S., Nazarov,

P.A., Kotova, E.A., and Antonenko, Y. N. (2021) Pro-

tonophoric action of BAM15 on planar bilayers, li-

posomes, mitochondria, bacteria and neurons, Bio-

electrochemistry, 137, 107673, https://doi.org/10.1016/

j.bioelechem.2020.107673.

51. Miyoshi,H., Tsujishita,H., Tokutake,N., and Fujita,T.

(1990) Quantitative analysis of uncoupling activi-

ty of substituted phenols with a physicochemical

substituent and molecular parameters, Biochim.

Biophys. Acta, 1016, 99-106, https://doi.org/10.1016/

0005-2728(90)90011-R.

52. Spycher, S., Escher, B. I., and Gasteiger, J. (2005)

A quantitative structure–activity relationship model

for the intrinsic activity of uncouplers of oxidative

phosphorylation, Chem. Res. Toxicol., 18, 1858-1867,

https://doi.org/10.1021/tx050166j.

53. Spycher, S., Smejtek, P., Netzeva, T. I., and Escher,

B. I. (2008) Toward a class-independent quantitative

structure–activity relationship model for uncouplers

of oxidative phosphorylation, Chem Res. Toxicol.,

21, 911-927, https://doi.org/10.1021/tx700391f.

54. Martineau, L. C. (2012) Simple thermodynamic mod-

el of unassisted proton shuttle uncoupling and pre-

diction of activity from calculated speciation, lipo-

philicity, and molecular geometry, J.Theor. Biol., 303,

33-61, https://doi.org/10.1016/j.jtbi.2012.02.032.

55. Naven, R. T., Swiss, R., Klug-McLeod, J., Will, Y., and

Greene,N. (2013) Thedevelopment of structure-activ-

ity relationships for mitochondrial dysfunction: un-

coupling of oxidative phosphorylation, Toxicol. Sci.,

131, 271-278, https://doi.org/10.1093/toxsci/kfs279.

56. Ebert,A., and Goss, K.-U. (2020) Predicting uncoupling

toxicity of organic acids based on their molecular

structure using a biophysical model, Chem. Res. Tox-

icol., 33, 1835-1844, https://doi.org/10.1021/acs.chem-

restox.0c00063.

57. Liberman, E. A., Topaly, V. P., Tsofina, L. M., Jasaitis,

A. A., and Skulachev, V. P. (1969) Mechanism of cou-

pling of oxidative phosphorylation and the mem-

brane potential of mitochondria, Nature, 222, 1076-

1078, https://doi.org/10.1038/2221076a0.

58. Bakker, E.P., van den Heuvel, E.J., Wiechmann, A.H.,

and van Dam, K. (1973) A comparison between the

effectiveness of uncouplers of oxidative phosphoryla-

tion in mitochondria and in different artificial mem-

brane systems, Biochim. Biophys. Acta, 292, 78-87,

https://doi.org/10.1016/0005-2728(73)90252-1.

59. Miyoshi, H., Nishioka, T., and Fujita, T. (1987) Quan-

titative relationship between protonophoric and un-

coupling activities of substituted phenols, Biochim.

Biophys. Acta, 891, 194-204, https://doi.org/10.1016/

0005-2728(87)90011-9.

60. Ting, H. P., Wilson, D. F., and Chance, B. (1970) Ef-

fects of uncouplers of oxidative phosphorylation on

the specific conductance of bimolecular lipid mem-

branes, Arch. Biochem. Biophys., 141, 141-146, https://

doi.org/10.1016/0003-9861(70)90116-5.

61. Cunarro, J., and Weiner, M. W. (1975) Mechanism of

action of agents which uncouple oxidative phosphor-

ylation: direct correlation between proton-carrying

and respiratory-releasing properties using rat liver

mitochondria, Biochim. Biophys. Acta, 387, 234-240,

https://doi.org/10.1016/0005-2728(75)90106-1.

62. Parker, V.H. (1965) Uncouplers of rat-liver mitochon-

drial oxidative phosphorylation, Biochem.J., 97, 658-

662, https://doi.org/10.1042/bj0970658.

63. Zuna, K., Jovanovic, O., Khailova, L. S., Skulj, S.,

Brkljaca, Z., Kreiter, J., Kotova, E. A., Vazdar, M.,

Antonenko, Y. N., and Pohl, E. E. (2021) Mitochon-

drial uncoupling proteins (UCP1-UCP3) and adenine

nucleotide translocase (ANT1) enhance the protono-

phoric action of 2,4-dinitrophenol in mitochondria

and planar bilayer membranes, Biomolecules, 11,

1178, https://doi.org/10.3390/biom11081178.

64. Yaguzhinskii, L. S., Ratnikova, L. A., and Kolesova,

G.M. (1973) Quantitative relation between the value

of the dissociation constant of uncoupling agents and

their efficiency in mitochondrial membranes [in Rus-

sian], Biofizika, 18, 460-465.

65. Liberman, E. A., and Topaly, V. P. (1968) Selective

transport of ions through bimolecular phospholipid

membranes, Biochim. Biophys. Acta, 163, 125-136,

https://doi.org/10.1016/0005-2736(68)90089-8.

66. Afanas’eva, E. V., and Kostyrko, V. A. (1986) Penta-

chlorophenol inhibition of succinate oxidation by

the respiratory chain in submitochondrial parti-

cles from the bovine heart [in Russian], Biokhimiia,

51, 823-829.

67. Popova, L.B., Nosikova, E.S., Kotova, E.A., Tarasova,

E. O., Nazarov, P. A., Khailova, L. S., Balezina, O. P.,

ANTONENKO et al.1836

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

and Antonenko, Y. N. (2018) Protonophoric action of

triclosan causes calcium efflux from mitochondria,

plasma membrane depolarization and bursts of min-

iature end-plate potentials, Biochim. Biophys. Acta

Biomembr., 1860, 1000-1007, https://doi.org/10.1016/

j.bbamem.2018.01.008.

68. Horner, W.D. (1941) A study of dinitrophenol and its

relation to cataract formation, Trans. Am. Ophthal-

mol. Soc., 39, 405-437.

69. Miranda, E. J., McIntyre, I. M., Parker, D. R., Gary,

R. D., and Logan, B. K. (2006) Two deaths attributed

to the use of 2,4-dinitrophenol, J. Anal. Toxicol., 30,

219-222, https://doi.org/10.1093/jat/30.3.219.

70. Colman,E. (2007) Dinitrophenol and obesity: an ear-

ly twentieth-century regulatory dilemma, Regul. Tox-

icol. Pharmacol., 48, 115-117, https://doi.org/10.1016/

j.yrtph.2007.03.006.

71. Grundlingh, J., Dargan, P. I., El-Zanfaly, M., and

Wood, D.M. (2011) 2,4-dinitrophenol (DNP): a weight

loss agent with significant acute toxicity and risk

of death, J. Med. Toxicol., 7, 205-212, https://doi.org/

10.1007/s13181-011-0162-6.

72. Khailova, L. S., Vygodina, T. V., Lomakina, G. Y.,

Kotova, E. A., and Antonenko, Y. N. (2020) Bicarbon-

ate suppresses mitochondrial membrane depolariza-

tion induced by conventional uncouplers, Biochem.

Biophys. Res. Commun., 530, 29-34, https://doi.org/

10.1016/j.bbrc.2020.06.131.

73. Wesson, L.G. (1940) Alkyl ethers of 2,4-dinitrophenol

as stimulants of the metabolic rate, J.Am. Chem. Soc.,

62, 3466-3468, https://doi.org/10.1021/ja01869a049.

74. Perry, R.J., Kim,T., Zhang, X.M., Lee, H.Y., Pesta,D.,

Popov, V.B., Zhang,D., Rahimi,Y., Jurczak, M.J., Cline,

G. W., Spiegel, D. A., and Shulman, G. I. (2013) Re-

versal of hypertriglyceridemia, fatty liver disease,

and insulin resistance by a liver-targeted mitochon-

drial uncoupler, Cell Metab., 18, 740-748, https://

doi.org/10.1016/j.cmet.2013.10.004.

75. De Felice, F. G., and Ferreira, S. T. (2006) Novel neu-

roprotective, neuritogenic and anti-amyloidogen-

ic properties of 2,4-dinitrophenol: the gentle face

of Janus, IUBMB Life, 58, 185-191, https://doi.org/

10.1080/15216540600702198.

76. Sebollela,A., Freitas-Correa,L., Oliveira, F.F., Mendes,

C.T., Wasilewska-Sampaio, A. P., Camacho-Pereira, J.,

Galina, A., Brentani, H., Passetti, F., De Felice, F. G.,

Dias-Neto, E., and Ferreira, S. T. (2010) Expression

profile of rat hippocampal neurons treated with the

neuroprotective compound 2,4-dinitrophenol: up-reg-

ulation of cAMP signaling genes, Neurotox. Res., 18,

112-123, https://doi.org/10.1007/s12640-009-9133-y.

77. Holmuhamedov, E. L., Jahangir, A., Oberlin, A.,

Komarov,A., Colombini,M., and Terzic,A. (2004) Potas-

sium channel openers are uncoupling protonophores:

implication in cardioprotection, FEBS Lett., 568,

167-170, https://doi.org/10.1016/j.febslet.2004.05.031.

78. Modriansky, M., and Gabrielova, E. (2009) Uncou-

ple my heart: the benefits of inefficiency, J. Bioen-

erg. Biomembr., 41, 133-136, https://doi.org/10.1007/

s10863-009-9212-z.

79. Noureddin, M., Khan, S., Portell, F., Jorkasky, D.,

Dennis, J., Khanb, O., Johansson, L., Johansson, E.,

and Sanyal, A. J. (2023) Safety and efficacy of once-

daily HU6 versus placebo in people with non-alco-

holic fatty liver disease and high BMI: a randomised,

double-blind, placebo-controlled, phase 2a trial,

Lancet Gastroenterol. Hepatol., 8, 1094-1105, https://

doi.org/10.1016/S2468-1253(23)00198-X.

80. Kitzman, D.W., Lewis, G.D., Pandey,A., Borlaug, B.A.,

Sauer, A. J., Litwin, S. E., Sharma, K., Jorkasky, D. K.,

Khan, S., and Shah, S. J. (2024) A novel controlled

metabolic accelerator for the treatment of obesity-re-

lated heart failure with preserved ejection fraction:

rationale and design of the Phase 2a HuMAIN tri-

al, Eur. J. Heart Fail., 26, 2013-2024, https://doi.org/

10.1002/ejhf.3305.

81. Ng, M. Y., Song, Z. J., Venkatesan, G., Rodriguez-

Cuenca,S., West, J.A., Yang,S., Tan, C.H., Ho, P.C.L.,

Griffin, J.L., Vidal-Puig,A., Bassetto,M., and Hagen,T.

(2024) Conjugating uncoupler compounds with hy-

drophobic hydrocarbon chains to achieve adipose

tissue selective drug accumulation, Sci. Rep., 14,

4932, https://doi.org/10.1038/s41598-024-54466-2.

82. Avlonitis, N., Chalmers, S., McDougall, C., Stanton-

Humphreys, M.N., Brown, C.T., McCarron, J.G., and

Conway, S. J. (2009) Caged AG10: new tools for spa-

tially predefined mitochondrial uncoupling, Mol. Bio-

syst., 5, 450-457, https://doi.org/10.1039/b820415m.

83. Chalmers, S., Caldwell, S. T., Quin, C., Prime, T. A.,

James, A.M., Cairns, A. G., Murphy, M. P., McCarron,

J. G., and Hartley, R. C. (2012) Selective uncoupling

of individual mitochondria within a cell using a mi-

tochondria-targeted photoactivated protonophore,

J.Am. Chem. Soc., 134, 758-761, https://doi.org/10.1021/

ja2077922.

84. Bobba, K. N., Binoy, A., Koo, S., Nedungadi, D.,

Podder, A., Sharma, A., Mishra, N., Kim, J. S., and

Bhuniya, S. (2019) Direct readout protonophore in-

duced selective uncoupling and dysfunction of in-

dividual mitochondria within cancer cells, Chem.

Commun., 55, 6429-6432, https://doi.org/10.1039/

C9CC01483G.

85. Kand, D., Pizarro, L., Angel, I., Avni, A., Friedmann-

Morvinski, D., and Weinstain, R. (2019) Organelle-

targeted BODIPY photocages: visible-light-mediated

subcellular photorelease, Angew. Chem. Int. Ed. Engl.,

58, 4659-4663, https://doi.org/10.1002/anie.201900850.

86. Lopez-Corrales,M., Rovira,A., Gandioso,A., Nonell,S.,

Bosch, M., and Marchan, V. (2023) Mitochondria-

targeted COUPY photocages: synthesis and visi-

ble-light photoactivation in living cells, J.Org. Chem.,

88, 7128-7140, https://doi.org/10.1021/acs.joc.3c00387.

MITOCHONDRIAL UNCOUPLERS 1837

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

87. Firsov, A. M., Khailova, L. S., Nazarov, P. A., Ukrain-

tsev, M., Kirsanov, R. S., Kovalchuk, S. I., Lyamzaev,

K. G., Panteleeva, A. A., Korshunova, G. A., Kotova,

E. A., and Antonenko, Y. N. (2025) 2-Azido-4-nitro-

phenol is a light-sensitive antibacterial protono-

phore, Bioelectrochemistry, 167, 109055, https://

doi.org/10.1016/j.bioelechem.2025.109055.

88. Muraoka, S., and Terada, H. (1972) 3,5-Di-tert-butyl-

4-hydroxybenzylidenemalononitrile, a new powerful

uncoupler of respiratory-chain phosphorylation, Bio-

chim. Biophys. Acta Bioenerg., 275, 271-275, https://

doi.org/10.1016/0005-2728(72)90047-3.

89. Terada, H., and van Dam, K. (1975) On the stoichi-

ometry between uncouplers of oxidative phosphor-

ylation and respiratory chains. The catalytic action

of SF 6847 (3,5-di-tert-butyl-4-hydroxybenzylidene-

malononitrile), Biochim. Biophys. Acta Bioenerg., 387,

507-518, https://doi.org/10.1016/0005-2728(75)90089-4.

90. Terada,H. (1990) Uncouplers of oxidative phosphory-

lation, Environ. Health Perspect., 87, 213-218, https://

doi.org/10.1289/ehp.9087213.

91. Grivennikova, V. G., Khailova, L. S., Zharova, T. V.,

Kotova, E. A., and Antonenko, Y. N. (2022) Inhibi-

tion of respiratory complex I by 6-ketocholestanol:

relevance to recoupling action in mitochondria, Bio-

chim. Biophys. Acta Bioenerg., 1863, 148594, https://

doi.org/10.1016/j.bbabio.2022.148594.

92. Starkov, A. A., Dedukhova, V. I., and Skulachev, V. P.

(1994) 6-ketocholestanol abolishes the effect of the

most potent uncouplers of oxidative phosphorylation

in mitochondria, FEBS Lett., 355, 305-308, https://

doi.org/10.1016/0014-5793(94)01211-3.

93. Starkov, A.A., Bloch, D.A., Chernyak, B.V., Dedukhova,

V.I., Mansurova, S.E., Severina, I.I., Simonyan, R.A.,

Vygodina, T. V., and Skulachev, V. P. (1997) 6-Keto-

cholestanol is a recoupler for mitochondria, chro-

matophores and cytochrome oxidase proteolipo-

somes, Biochim. Biophys. Acta, 1318, 159-172, https://

doi.org/10.1016/S0005-2728(96)00134-X.

94. Franklin, J.C., and Cafiso, D.S. (1993) Internal electro-

static potentials in bilayers - measuring and controlling

dipole potentials in lipid vesicles, Biophys.J., 65, 289-

299, https://doi.org/10.1016/S0006-3495(93)81051-8.

95. Cuellar, A., Ramirez,J., Infante, V. M., and Chavez,E.

(1997) Further studies on the recoupling effect of

6-ketocholestanol upon oxidative phosphorylation in

uncoupled liver mitochondria, FEBS Lett., 411, 365-

368, https://doi.org/10.1016/S0014-5793(97)00741-2.

96. Gazit, A., Yaish, P., Gilon, C., and Levitzki, A. (1989)

Tyrphostins I: synthesis and biological activity of

protein tyrosine kinase inhibitors, J. Med. Chem., 32,

2344-2352, https://doi.org/10.1021/jm00130a020.

97. Levitzki, A., and Gilon, C. (1991) Tyrphostins as

molecular tools and potential antiproliferative

drugs, Trends Pharmacol. Sci., 12, 171-174, https://

doi.org/10.1016/0165-6147(91)90538-4.

98. Needham, D. (2024) Niclosamide: a career build-

er, J.Control Release, 369, 786-856, https://doi.org/

10.1016/j.jconrel.2023.07.016.

99. Jurgeit, A., Mcdowell, R., Moese, S., Meldrum, E.,

Schwendener,R., and Greber, U.F. (2012) Niclosamide

is a proton carrier and targets acidic endosomes with

broad antiviral effects, PLoS Pathog., 8, e1002976,

https://doi.org/10.1371/journal.ppat.1002976.

100. Jung, E., Nam, S., Oh, H., Jun, S., Ro, H. J., Kim, B.,

Kim, M., and Go, Y. Y. (2019) Neutralization of acid-

ic intracellular vesicles by niclosamide inhibits mul-

tiple steps of the dengue virus life cycle in vitro,

Sci. Rep., 9, 8682, https://doi.org/10.1038/s41598-019-

45095-1.

101. Kozhokaru, A. F., Liberman, E. A., Topaly, V. P., and

Topaly, E. E. (1976) Study of the mechanism of ac-

tion of fasciolocides using bimolecular phospholipid

membranes [in Russian], Biofizika, 21, 766-767.

102. Kozhokaru, A.F., G’bev, E. E., Vladimirov, Iu.A., and

Akoev, I. G. (1981) Action of new class of substitut-

ed salicylanilides on ion conductivity of lipid mem-

branes [in Russian], Biofizika, 26, 995-998.

103. Kasianowicz, J., Benz, R., and Mclaughlin, S. (1987)

How do protons cross the membrane solution in-

terface? kinetic studies on bilayer membranes ex-

posed to the protonophore s-13 (5-chloro-3-tert-bu-

tyl-2-chloro-4nitrosalicylanilide), J. Membrane Biol.,

95, 73-89, https://doi.org/10.1007/BF01869632.

104. Ng, M.Y., Song, Z.J., Tan, C.H., Bassetto,M., and Ha-

gen, T. (2024) Structural investigations on the mito-

chondrial uncouplers niclosamide and FCCP, FEBS

Open Bio., 14, 1057-1071, https://doi.org/10.1002/

2211-5463.13817.

105. Terada, H., Goto, S., Yamamoto, K., Takeuchi, I.,

Hamada, Y., and Miyake, K. (1988) Structural re-

quirements of salicylanilides for uncoupling activ-

ity in mitochondria: quantitative analysis of struc-

ture-uncoupling relationships, Biochim. Biophys.

Acta, 936, 504-512, https://doi.org/10.1016/0005-2728

(88)90027-8.

106. Storey, B. T., Wilson, D. F., Bracey, A., Rosen, S. L.,

and Stephenson, S. (1975) Steric and electronic ef-

fects on the uncoupling activity of substituted 3,5 di-

chlorosalicylanilides, FEBS Lett., 49, 338-341, https://

doi.org/10.1016/0014-5793(75)80780-0.

107. Amireddy, N., Puttapaka, S. N., Vinnakota, R. L.,

Ravuri, H. G., Thonda, S., and Kalivendi, S. V. (2017)

The unintended mitochondrial uncoupling effects

of the FDA-approved anti-helminth drug nitazoxa-

nide mitigates experimental parkinsonism in mice,

J.Biol. Chem., 292, 15731-15743, https://doi.org/

10.1074/jbc.M117.791863.

108. Ma, M.-H., Li, F.-F., Li, W.-F., Zhao,H., Jiang,M., Yu, Y.-

Y., Dong, Y.-C., Zhang, Y.-X., Li, P., Bu, W.-J., Sun, Z.-J.,

and Dong, D.-L. (2023) Repurposing nitazoxanide as

a novel anti-atherosclerotic drug based on mitochon-

ANTONENKO et al.1838

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

drial uncoupling mechanisms, Br.J. Pharmacol., 180,

62-79, https://doi.org/10.1111/bph.15949.

109. Li,F., Jiang,M., Ma,M., Chen,X., Zhang,Y., Zhang,Y.,

Yu,Y., Cui,Y., Chen,J., Zhao,H., Sun,Z., and Dong,D.

(2022) Anthelmintics nitazoxanide protects against

experimental hyperlipidemia and hepatic steato-

sis in hamsters and mice, Acta Pharm. Sin. B, 12,

1322-1338, https://doi.org/10.1016/j.apsb.2021.09.009.

110. Kozhokaru, A. F., Ruzieva, R. Kh., Topaly, V. P., and

Topaly, E. E. (1978) Protonophoric and uncoupling

activity of water-soluble pea flavonols [in Russian],

Biofizika, 23, 716-717.

111. De Jonge, P. C., Wieringa, T., Van Putten, J. P., Krans,

H. M., and van Dam, K. (1983) Phloretin – an un-

coupler and an inhibitor of mitochondrial oxi-

dative phosphorylation, Biochim. Biophys. Acta,

722, 219-225, https://doi.org/10.1016/0005-2728(83)

90177-9.

112. Trumbeckaite, S., Bernatoniene, J., Majiene, D.,

Jakstas,V., Savickas,A., and Toleikis,A. (2006) Theef-

fect of flavonoids on rat heart mitochondrial func-

tion, Biomed. Pharmacother., 60, 245-248, https://

doi.org/10.1016/j.biopha.2006.04.003.

113. Sell, T. S., Belkacemi, T., Flockerzi, V., and Beck, A.

(2014) Protonophore properties of hyperforin are es-

sential for its pharmacological activity, Sci. Rep., 4,

7500, https://doi.org/10.1038/srep07500.

114. Andersen, O.S., Finkelstein, A., Katz, I., and Cass, A.

(1976) Effect of phloretin on the permeability of thin

lipid membranes, J.Gen. Physiol., 67, 749-771, https://

doi.org/10.1085/jgp.67.6.749.

115. Pohl, P., Rokitskaya, T. I., Pohl, E. E., and Saparov,

S. M. (1997) Permeation of phloretin across bilayer

lipid membranes monitored by dipole potential and

microelectrode measurements, Biochim. Biophys.

Acta, 1323, 163-172, https://doi.org/10.1016/S0005-

2736(96)00185-X.

116. Rokitskaya, T. I., Ilyasova, T. M., Severina, I. I.,

Antonenko, Y. N., and Skulachev, V. P. (2013) Elec-

trogenic proton transport across lipid bilayer mem-

branes mediated by cationic derivatives of rhodamine

19: comparison with anionic protonophores, Eur. Bio-

phys. J., 42, 477-485, https://doi.org/10.1007/s00249-

013-0898-9.

117. Schmitt, L., Krings, K. S., Wolsing, A., Buque, X.,

Zimmermann, M., Flores-Romero, H., Lenz, T.,

Lechtenberg, I., Peter, C., Stork, B., Teusch, N.,

Proksch, P., Stuehler, K., Garcia-Saez, A. J., Reichert,

A. S., Aspichueta, P., Bhatia, S., and Wesselborg, S.

(2024) Targeting mitochondrial metabolism by the

mitotoxin bromoxib in leukemia and lymphoma

cells, Cell Commun. Signal., 22, 541, https://doi.org/

10.1186/s12964-024-01913-2.

118. Shchepinova, M. M., Denisov, S. S., Kotova, E. A.,

Khailova, L. S., Knorre, D. A., Korshunova, G. A.,

Tashlitsky, V. N., Severin, F. F., and Antonenko, Y. N.

(2014) Dodecyl and octyl esters of fluorescein as pro-

tonophores and uncouplers of oxidative phosphory-

lation in mitochondria at submicromolar concentra-

tions, Biochim. Biophys. Acta Bioenerg., 1837, 149-158,

https://doi.org/10.1016/j.bbabio.2013.09.011.

119. Nazarov, P.A., Kirsanov, R.S., Denisov, S.S., Khailova,

L.S., Karakozova, M.V., Lyamzaev, K.G., Korshunova,

G.A., Lukyanov, K.A., Kotova, E.A., and Antonenko,

Y. N. (2020) Fluorescein derivatives as antibacterial

agents acting via membrane depolarization, Biomole-

cules, 10, 309, https://doi.org/10.3390/biom10020309.

120. Venugopala, K. N., Rashmi, V., and Odhav, B. (2013)

Review on natural coumarin lead compounds for

their pharmacological activity, Biomed. Res. Int.,

2013, 963248, https://doi.org/10.1155/2013/963248.

121. Dadak,V., Zboril,P., Sanek,J., and Svoboda,A. (1980)

Mode of effect of ostruthin, a phenolic coumarin on

respiration and oxidative phosphorylation of rat liver

mitochondria, Collect. Czechoslovak Chem. Commun.,

45, 653-666, https://doi.org/10.1135/cccc19800653.

122. Du, L., Mahdi, F., Jekabsons, M. B., Nagle, D. G., and

Zhou, Y. D. (2010) Mammea E/BB, an isoprenylated

dihydroxycoumarin protonophore that potently un-

couples mitochondrial electron transport, disrupts

hypoxic signaling in tumor cells, J. Nat. Prod., 73,

1868-1872, https://doi.org/10.1021/np100501n.

123. Nowak, P. M., Sagan, F., and Mitoraj, M. P. (2017)

Origin of remarkably different acidity of hydroxy-

coumarins – joint experimental and theoretical

studies, J. Phys. Chem. B, 121, 4554-4561, https://

doi.org/10.1021/acs.jpcb.7b01849.

124. Krasnov, V. S., Kirsanov, R.S., Khailova, L. S., Firsov,

A. M., Nazarov, P. A., Tashlitsky, V. N., Korshunova,

G.A., Kotova, E.A., and Antonenko, Y.N. (2022) Alkyl

esters of umbelliferone-4-acetic acid as protonophores

in bilayer lipid membranes and ALDH2-dependent

soft uncouplers in rat liver mitochondria, Bioelec-

trochemistry, 145, 108081, https://doi.org/10.1016/

j.bioelechem.2022.108081.

125. Krasnov, V.S., Kirsanov, R.S., Khailova, L.S., Popova,

L. B., Lyamzaev, K. G., Firsov, A. M., Korshunova,

G. A., Kotova, E. A., and Antonenko, Y. N. (2022) Al-

kyl esters of 7-hydroxycoumarin-3-carboxylic acid as

potent tissue-specific uncouplers of oxidative phos-

phorylation: involvement of ATP/ADP translocase

in mitochondrial uncoupling, Arch. Biochem. Bio-

phys., 728, 109366, https://doi.org/10.1016/j.abb.2022.

109366.

126. Heytler, P. G. (1979) Uncouplers of oxidative phos-

phorylation, Methods Enzymol., 55, 462-472, https://

doi.org/10.1016/0076-6879(79)55060-5.

127. Rokitskaya, T.I., Kotova, E.A., Luzhkov, V.B., Kirsanov,

R.S., Aleksandrova, E.V., Korshunova, G.A., Tashlitsky,

V.N., and Antonenko, Y.N. (2021) Lipophilic ion aro-

maticity is not important for permeability across

lipid membranes, Biochim. Biophys. Acta Biomem-

MITOCHONDRIAL UNCOUPLERS 1839

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

br., 1863, 183483, https://doi.org/10.1016/j.bbamem.

2020.183483.

128. Rupprecht,A., Sokolenko, E.A., Beck,V., Ninnemann,O.,

Jaburek, M., Trimbuch, T., Klishin, S. S., Jezek, P.,

Skulachev, V. P., and Pohl, E. E. (2010) Role of the

transmembrane potential in the membrane pro-

ton leak, Biophys. J., 98, 1503-1511, https://doi.org/

10.1016/j.bpj.2009.12.4301.

129. Montal, M., and Mueller, P. (1972) Formation of bi-

molecular membranes from lipid monolayers and

a study of their electrical properties, Proc. Natl.

Acad. Sci. USA, 69, 3561-3566, https://doi.org/10.1073/

pnas.69.12.3561.

130. Andreyev, A. Y., Bondareva, T. O., Dedukhova, V. I.,

Mokhova, E. N., Skulachev, V. P., and Volkov, N. I.

(1988) Carboxyatractylate inhibits the uncoupling

effect of free fatty acids, FEBS Lett., 226, 265-269,

https://doi.org/10.1016/0014-5793(88)81436-4.

131. Urbankova,E., Voltchenko, A., Pohl, P., Jezek, P., and

Pohl, E. E. (2003) Transport kinetics of uncoupling

proteins. Analysis of UCP1 reconstituted in planar

lipid bilayers, J.Biol. Chem., 278, 32497-32500, https://

doi.org/10.1074/jbc.M303721200.

132. Beck, V., Jaburek, M., Demina, T., Rupprecht, A.,

Porter, R. K., Jezek, P., and Pohl, E. E. (2007) Poly-

unsaturated fatty acids activate human uncou-

pling proteins 1 and 2 in planar lipid bilayers,

FASEB J., 21, 1137-1144, https://doi.org/10.1096/

fj.06-7489com.

133. Fedorenko, A., Lishko, P. V., and Kirichok, Y. (2012)

Mechanism of fatty-acid-dependent UCP1 uncoupling

in brown fat mitochondria, Cell, 151, 400-413, https://

doi.org/10.1016/j.cell.2012.09.010.

134. Bertholet, A.M., Chouchani, E.T., Kazak,L., Angelin,A.,

Fedorenko, A., Long, J. Z., Vidoni, S., Garrity, R.,

Cho, J., Terada, N., Wallace, D. C., Spiegelman, B. M.,

and Kirichok, Y. (2019) H

transport is an integral

function of the mitochondrial ADP/ATP carrier,

Nature, 571, 515-520, https://doi.org/10.1038/s41586-

019-1400-3.

135. Kreiter, J., Rupprecht, A., Skulj, S., Brkljaca, Z.,

Žuna,K., Knyazev, D.G., Bardakji,S., Vazdar,M., and

Pohl, E.E. (2021) ANT1 Activation and inhibition pat-

terns support the fatty acid cycling mechanism for

proton transport, Int.J. Mol. Sci., 22, 2490, https://

doi.org/10.3390/ijms22052490.

136. Kreiter,J., Skulj,S., Brkljaca,Z., Bardakji,S., Vazdar,M.,

and Pohl, E. E. (2023) FA sliding as the mechanism

for the ANT1-mediated fatty acid anion transport

in lipid bilayers, Int.J. Mol. Sci., 24, 13701, https://

doi.org/10.3390/ijms241813701.

137. Pohl, E. E., Vazdar, M., and Kreiter, J. (2025) Explor-

ing the proton transport mechanism of the mito-

chondrial ADP/ATP carrier: FA-cycling hypothesis

and beyond, Prot. Sci., 34, e70047, https://doi.org/

10.1002/pro.70047.

138. Semenova, A.A., Samartsev, V.N., and Dubinin, M.V.

(2021) The stimulation of succinate-fueled respira-

tion of rat liver mitochondria in state 4 by α,ω-hexa-

decanedioic acid without induction of proton con-

ductivity of the inner membrane. Intrinsic uncou-

pling of the bc1 complex, Biochimie, 181, 215-225,

https://doi.org/10.1016/j.biochi.2020.12.021.

139. Evtodienko, V. Y., Bondarenko, D. I., and Antonenko,

Y. N. (1999) Permeation of dicarboxylic acids with

different terminal position of two carboxylic groups

through planar bilayer lipid membranes, Biochim.

Biophys. Acta, 1420, 95-103, https://doi.org/10.1016/

S0005-2736(99)00095-4.

140. Radde, B. N., Alizadeh-Rad, N., Price, S. M., Schultz,

D. J., and Klinge, C. M. (2016) Anacardic acid, sal-

icylic acid, and oleic acid differentially alter cel-

lular bioenergetic function in breast cancer cells,

J. Cell Biochem., 117, 2521-2532, https://doi.org/

10.1002/jcb.25544.

141. Acevedo, A., Jones, A. E., Danna, B. T., Turner, R.,

Montales, K. P., Beninca, C., Reue, K., Shirihai, O. S.,

Stiles,L., Wallace, M., Wang, Y., Bertholet, A. M., and

Divakaruni, A. S. (2024) The BCKDK inhibitor BT2

is a chemical uncoupler that lowers mitochondrial

ROS production and de novo lipogenesis, J. Biol.

Chem., 300, 105702, https://doi.org/10.1016/j.jbc.

2024.105702.

142. Zhang, Y., Tan, J., and Chen, Y. (2023) Visible-light-

induced protein labeling in live cells with aryl

azides, Chem. Commun., 59, 2413-2420, https://doi.org/

10.1039/D2CC06987C.

143. Hanstein, W. G., and Hatefi, Y. (1974) Characteriza-

tion and localization of mitochondrial uncoupler

binding sites with an uncoupler capable of photoaf-

finity labeling, J.Biol. Chem., 249, 1356-1362, https://

doi.org/10.1016/S0021-9258(19)42889-5.

144. Hatefi,Y. (1975) Energy conservation and uncoupling

in mitochondria, J.Supramol. Struct., 3, 201-213,

https://doi.org/10.1002/jss.400030302.

145. Schonfeld, P., Jezek, P., Belyaeva, E. A., Borecky, J.,

Slyshenkov, V.S., Wieckowski, M.R., and Wojtczak,L.

(1996) Photomodification of mitochondrial proteins

by azido fatty acids and its effect on mitochondri-

al energetics. Further evidence for the role of the

ADP/ATP carrier in fatty-acid-mediated uncoupling,

Eur. J. Biochem., 240, 387-393, https://doi.org/10.1111/

j.1432-1033.1996.0387h.x.

146. Valderrama,K., Pradel,E., Firsov, A.M., Drobecq,H.,

Bauderlique-le Roy, H., Villemagne, B., Antonenko,

Y. N., and Hartkoorn, R. C. (2019) Pyrrolomycins are

potent natural protonophores, Antimicrob. Agents

Chemother., 63, e01450-19, https://doi.org/10.1128/

AAC.01450-19.

147. Firsov, A.M., Khailova, L.S., Rokitskaya, T.I., Kotova,

E. A., and Antonenko, Y. N. (2022) Antibiotic pyrro-

lomycin as an efficient mitochondrial uncoupler,

ANTONENKO et al.1840

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Biochemistry (Moscow), 87, 812-822, https://doi.org/

10.1134/S0006297922080120.

148. Treacy, M., Miller, T., Black, B., Gard, I., Hunt, D.,

and Hollingworth, R. M. (1994) Uncoupling activity

and pesticidal properties of pyrroles, Biochem. Soc.

Trans., 22, 244-247, https://doi.org/10.1042/bst0220244.

149. Heytler, P.G., and Prichard, W.W. (1962) Anew class

of uncoupling agents – carbonyl cyanide phenylhy-

drazones, Biochem. Biophys. Res. Commun., 7, 272-

275, https://doi.org/10.1016/0006-291X(62)90189-4.

150. Heytler, P. G. (1963) Uncoupling of oxidative phos-

phorylation by carbonyl cyanide phenylhydrazones.

I. Some characteristics of m-Cl-CCP action on mito-

chondria and chloroplasts, Biochemistry, 2, 357-361,

https://doi.org/10.1021/bi00902a031.

151. Blum, J.J., and Sanadi, D.R. (1964) Activation of my-

osin adenosine triphosphatase by carbonyl cyanide

p-chlorophenylhydrazone, J.Biol. Chem., 239, 452-454,

https://doi.org/10.1016/S0021-9258(18)51700-2.

152. Drobnica, L., and Sturdik, E. (1979) The reaction of

carbonyl cyanide phenylhydrazones with thiols, Bio-

chim. Biophys. Acta, 585, 462-476, https://doi.org/

10.1016/0304-4165(79)90091-6.

153. Toninello, A., and Siliprandi, N. (1982) Restoration

of membrane potential in mitochondria deener-

gized with carbonyl cyanide p-trifluoromethoxy-

phenylhydrazone (FCCP), Biochim. Biophys. Acta,

682, 289-292, https://doi.org/10.1016/0005-2728(82)

90110-4.

154. Mlejnek, P., and Dolezel, P. (2015) Loss of mitochon-

drial transmembrane potential and glutathione de-

pletion are not sufficient to account for induction of

apoptosis by carbonyl cyanide 4-(trifluoromethoxy)

phenylhydrazone in human leukemia K562 cells,

Chem. Biol. Interact., 239, 110, https://doi.org/10.1016/

j.cbi.2015.06.033.

155. Khailova, L. S., Firsov, A. M., Kotova, E. A., and

Antonenko, Y. N. (2019) Interaction of potent mito-

chondrial uncouplers with thiol-containing antiox-

idants, Antioxidants, 8, 194, https://doi.org/10.3390/

antiox8060194.

156. Doczi, J., Karnok, N., Bui, D., Azarov, V., Pallag, G.,

Nazarian, S., Czumbel, B., Seyfried, T. N., and

Chinopoulos, C. (2023) Viability of MCF cells is not

correlated with mitochondrial bioenergetics, Sci.

Rep., 13, 10822, https://doi.org/10.1038/s41598-023-

37677-x.

157. Demine, S., Renard, P., and Arnould, T. (2019) Mito-

chondrial uncoupling: a key controller of biological

processes in physiology and diseases, Cells, 8, E795,

https://doi.org/10.3390/cells8080795.

158. LeBlanc, O. H. (1971) The effect of uncouplers of

oxidative phosphorylation on lipid bilayer mem-

branes: carbonylcyanide m-chloropenylhydrazone,

J. Membr. Biol., 4, 227-251, https://doi.org/10.1007/

BF02431973.

159. O’Shaughnessy, K., and Hladky, S. B. (1983) Tran-

sient currents carried by the uncoupler, carbonyl

cyanide m-chlorophenylhydrazone, Biochim. Bio-

phys. Acta, 724, 381-387, https://doi.org/10.1016/

0005-2728(83)90097-X.

160. Benz, R., and McLaughlin, S. (1983) The molecu-

lar mechanism of action of the proton ionophore

FCCP (carbonylcyanide p-trifluoromethoxyphenyl-

hydrazone), Biophys. J., 41, 381-398, https://doi.org/

10.1016/S0006-3495(83)84449-X.

161. Cho, I., Song, H.-O., and Cho, J. H. (2017) Mitochon-

drial uncoupling attenuates age-dependent neuro-

degeneration in C. elegans, Mol. Cells, 40, 864-870,

https://doi.org/10.14348/molcells.2017.0172.

162. Katre, N. V., and Wilson, D. F. (1977) Interaction of

uncouplers with the mitochondrial membrane: a

high-affinity binding site, Arch. Biochem. Biophys.,

184, 578-585, https://doi.org/10.1016/0003-9861(77)

90468-4.

163. Brennan, J.P., Southworth,R., Medina, R.A., Davidson,

S. M., Duchen, M. R., and Shattock, M. J. (2006) Mi-

tochondrial uncoupling, with low concentration

FCCP, induces ROS-dependent cardioprotection in-

dependent of KATP channel activation, Cardiovasc.

Res., 72, 313-321, https://doi.org/10.1016/j.cardiores.

2006.07.019.

164. Chaudhari, A.A., Seol, J.W., Kim, S.J., Lee, Y.J., Kang,

H.S., Kim, I. S., Kim, N. S., and Park, S.Y. (2007) Re-

active oxygen species regulate Bax translocation and

mitochondrial transmembrane potential, a possible

mechanism for enhanced TRAIL-induced apoptosis

by CCCP, Oncol. Rep., 18, 71-76, https://doi.org/10.3892/

or.18.1.71.

165. Han, Y.H., and Park, W.H. (2011) Intracellular gluta-

thione levels are involved in carbonyl cyanide p-(tri-

fluoromethoxy) phenylhydrazone-induced apoptosis

in As4. 1 juxtaglomerular cells, Int. J. Mol. Med., 27,

575-581, https://doi.org/10.3892/ijmm.2011.604.

166. Lee, J.E., Kang, J.S., Shin, I.C., Lee, S.J., Hyun, D.H.,

Lee, K. S., and Koh, H. C. (2011) Fluazinam-induced

apoptosis of SH-SY5Y cells is mediated by p53 and

Bcl-2 family proteins, NeuroToxicol., 32, 702-710,

https://doi.org/10.1016/j.neuro.2011.08.004.

167. Jing, K., Shin, S., Jeong, S., Kim, S., Song, K.-S., Park,

J.-H., Heo, J.-Y., Seo, K.-S., Park, S.-K., Kweon, G.-R.,

Wu,T., Park, J.-I., and Lim,K. (2014) Docosahexaeno-

ic acid induces the degradation of HPV E6/E7 oncop-

roteins by activating the ubiquitin–proteasome sys-

tem, Cell Death Dis., 5, e1524, https://doi.org/10.1038/

cddis.2014.477.

168. Zhang, X., Cheng, X., Yu, L., Yang, J., Calvo, R.,

Patnaik, S., Hu, X., Gao, Q., Yang, M., Lawas, M.,

Delling,M., Marugan,J., Ferrer,M., and Xu,H. (2016)

MCOLN1 is a ROS sensor in lysosomes that regulates

autophagy, Nat. Commun., 7, 12109, https://doi.org/

10.1038/ncomms12109.

MITOCHONDRIAL UNCOUPLERS 1841

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

169. Xiao, B., Goh, J.-Y., Xiao, L., Xian, H., Lim, K.-L., and

Liou, Y.-C. (2017) Reactive oxygen species trigger

Parkin/PINK1 pathway-dependent mitophagy by in-

ducing mitochondrial recruitment of Parkin, J. Biol.

Chem., 292, 16697-16708, https://doi.org/10.1074/

jbc.M117.787739.

170. Kenwood, B. M., Weaver, J. L., Bajwa, A., Poon, I. K.,

Byrne, F.L., Murrow, B.A., Calderone, J.A., Huang,L.,

Divakaruni, A. S., Tomsig, J. L., Okabe, K., Lo, R. H.,

Coleman, G. C., Columbus, L., Yan, Z., Saucerman,

J.J., Smith, J.S., Holmes, J.W., Lynch, K.R., Ravichan-

dran, K.S., Uchiyama,S., Santos, W.L., Rogers, G.W.,

Okusa, M. D., Bayliss, D. A., and Hoehn, K. L. (2013)

Identification of a novel mitochondrial uncoupler

that does not depolarize the plasma membrane, Mol.

Metab., 3, 114-123, https://doi.org/10.1016/j.molmet.

2013.11.005.

171. Childress, E. S., Salamoun, J. M., Hargett, S. R.,

Alexopoulos, S. J., Chen, S.-Y., Shah, D. P., Santiago-

Rivera, J., Garcia, C. J., Dai, Y., Tucker, S. P., Hoehn,

K.L., and Santos, W.L. (2020) [1,2,5]Oxadiazolo[3,4-b]

pyrazine-5,6-diamine derivatives as mitochondrial

uncouplers for the potential treatment of nonalco-

holic steatohepatitis, J. Med. Chem., 63, 2511-2526,

https://doi.org/10.1021/acs.jmedchem.9b01440.

172. Alexopoulos, S. J., Chen, S.-Y., Brandon, A. E.,

Salamoun, J.M., Byrne, F.L., Garcia, C.J., Beretta,M.,

Olzomer, E.M., Shah, D.P., Philp, A.M., Hargett, S.R.,

Lawrence, R. T., Lee, B., Sligar, J., Carrive, P., Tucker,

S. P., Philp, A., Lackner, C., Turner, N., Cooney, G. J.,

Santos, W. L., and Hoehn, K. L. (2020) Mitochondri-

al uncoupler BAM15 reverses diet-induced obesity

and insulin resistance in mice, Nat. Commun., 11,

2397, https://doi.org/10.1038/s41467-020-16298-2.

173. Khailova, L. S., Krasnov, V. S., Kirsanov, R. S., Pop-

ova, L. B., Tashlitsky, V. N., Kotova, E. A., and An-

tonenko, Y. N. (2023) The transient character of mi-

tochondrial uncoupling by the popular fungicide

fluazinam is specific for liver, Arch. Biochem. Bio-

phys., 746, 109735, https://doi.org/10.1016/j.abb.2023.

109735.

174. Guo,Z., Miyoshi,H., Komyoji,T., Haga,T., and Fujita,T.

(1991) Uncoupling activity of a newly developed

fungicide, fluazinam [3-chloro-N-(3-chloro-2,6-dini-

tro-4-trifluoromethylphenyl)-5-trifluoromethyl-2-pyr-

idinamine], Biochim. Biophys. Acta, 1056, 89-92,

https://doi.org/10.1016/S0005-2728(05)80077-5.

175. Brandt, U., Schubert, J., Geck, P., and von Jagow, G.

(1992) Uncoupling activity and physicochemical

properties of derivatives of fluazinam, Biochim.

Biophys. Acta, 1101, 41-47, https://doi.org/10.1016/

0167-4838(92)90464-O.

176. Kirsanov, R. S., Khailova, L. S., Krasnov, V. S., Firsov,

A.M., Lyamzaev, K.G., Panteleeva, A.A., Popova, L.B.,

Nazarov, P. A., Tashlitsky, V. N., Korshunova, G. A.,

Kotova, E.A., and Antonenko, Y.N. (2024) Spontaneous

reversal of small molecule-induced mitochondrial

uncoupling: the case of anilinothiophenes, FEBS J.,

291, 5523-5539, https://doi.org/10.1111/febs.17329.

177. Schaefer, G., and Buechel, K. H. (1970) Cyclic re-

sponse of energy linked functions of liver mito-

chondria to uncoupling thiophene derivatives, FEBS

Lett., 6, 217-220, https://doi.org/10.1016/0014-5793(70)

80061-8.

178. Dejonghe, W., Kuenen, S., Mylle, E., Vasileva, M.,

Keech, O., Viotti, C., Swerts, J., Fendrych, M., Ortiz-

Morea, F.A., Mishev,K., Delang,S., Scholl,S., Zarza,X.,

Heilmann, M., Kourelis, J., Kasprowicz, J., Nguyen,

L. S. L., Drozdzecki, A., Van Houtte, I., Szatmari,

A.-M., Majda,M., Baisa,G., Bednarek, S.Y., Robert,S.,

Audenaert, D., Testerink, C., Munnik, T., Van

Damme, D., Schumacher, K., Winne, J., Friml, J.,

Verstreken, P., and Russinova, E. (2016) Mitochon-

drial uncouplers inhibit clathrin-mediated endo-

cytosis largely through cytoplasmic acidification,

Nat. Commun., 7, 11710, https://doi.org/10.1038/

ncomms11710.

179. Cardoso, R.M.S., Filipe, H.A.L., Gomes,F., Moreira,

N. D., Vaz, W. L. C., and Moreno, M. J. (2010) Chain

length effect on the binding of amphiphiles to se-

rum albumin and to POPC bilayers, J.Phys. Chem. B,

114, 16337-16346, https://doi.org/10.1021/jp105163k.

180. Denisov, S. S., Kotova, E. A., Khailova, L. S.,

Korshunova, G. A., and Antonenko, Y. N. (2014)

Tuning the hydrophobicity overcomes unfavorable

deprotonation making octylamino-substituted 7-ni-

trobenz-2-oxa-1,3-diazole (n-octylamino-NBD) a pro-

tonophore and uncoupler of oxidative phosphoryla-

tion in mitochondria, Bioelectrochemistry, 98, 30-38,

https://doi.org/10.1016/j.bioelechem.2014.02.002.

181. Antonenko, Y. N., Denisov, S. S., Khailova, L. S.,

Nazarov, P. A., Rokitskaya, T. I., Tashlitsky, V. N.,

Firsov, A. M., Korshunova, G. A., and Kotova, E. A.

(2017) Alkyl-substituted phenylamino derivatives of

7-nitrobenz-2-oxa-1,3-diazole as uncouplers of ox-

idative phosphorylation and antibacterial agents:

involvement of membrane proteins in the uncou-

pling action, Biochim. Biophys. Acta Biomembr.,

1859, 377-387, https://doi.org/10.1016/j.bbamem.2016.

12.014.

182. Rebhahn, V. I. C., Saoud, M., Winterhalter, M.,

Schanbacher, F., Jobst, M., Rulz, R., Sonntag, A.,

Kollatz,J., Sprengel,R., Donovan, S.F., Del Favero,G.,

Rennert, R., and Niedermeyer, T. H. J. (2025) Aetok-

thonotoxin, the causative agent of vacuolar my-

elinopathy, uncouples oxidative phosphorylation

due to protonophore activity, Chem. Res. Toxicol.,

38, 1495-1508, https://doi.org/10.1021/acs.chemrestox.

5c00147.

183. Figarola, J. L., Singhal, J., Tompkins, J. D., Rogers,

G. W., Warden, C., Horne, D., Riggs, A. D., Awas-

thi, S., and Singhal, S. S. (2015) SR4 uncouples mito-

ANTONENKO et al.1842

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

chondrial oxidative phosphorylation, modulates

AMP- dependent kinase (AMPK)-mammalian target of

rapamycin (mTOR) signaling, and inhibits prolifera-

tion of HepG2 hepatocarcinoma cells, J.Biol. Chem.,

290, 30321-30341, https://doi.org/10.1074/jbc.M115.

686352.

184. Wu,X., Judd, L.W., Howe, E.N.W., Withecombe, A.M.,

Soto-Cerrato, V., Li, H., Busschaert, N., Valkenier, H.,

Perez-Tomas, R., Sheppard, D. N., Jiang, Y.-B., Davis,

A. P., and Gale, P. A. (2016) Nonprotonophoric elec-

trogenic Cl transport mediated by valinomycin-

like carriers, Chem., 1, 127-146, https://doi.org/

10.1016/j.chempr.2016.04.002.

185. Routaboul, J.-M., Taillandier, G., Ravanel, P., and

Tissut, M. (1994) Potent non-protonophore uncou-

plers acting on natural and artificial membranes,

Phytochemistry, 35, 553-559, https://doi.org/10.1016/

S0031-9422(00)90560-0.

186. York,E., McNaughton, D.A., Roseblade,A., Cranfield,

C. G., Gale, P. A., and Rawling, T. (2022) Structure-

activity relationship and mechanistic studies of bis-

aryl urea anticancer agents indicate mitochondrial

uncoupling by a fatty acid-activated mechanism, ACS

Chem. Biol., 17, 2065-2073, https://doi.org/10.1021/

acschembio.1c00807.

187. Wilhelm, S.M., Adnane,L., Newell,P., Villanueva,A.,

Llovet, J. M., and Lynch, M. (2008) Preclinical over-

view of sorafenib, a multikinase inhibitor that tar-

gets both Raf and VEGF and PDGF receptor tyrosine

kinase signaling, Mol. Cancer Ther., 7, 3129-3140,

https://doi.org/10.1158/1535-7163.MCT-08-0013.

188. Jian,C., Fu,J., Cheng,X., Shen, L.-J., Ji, Y.-X., Wang,X.,

Pan, S., Tian, H., Tian, S., Liao, R., Song, K., Wang,

H.-P., Zhang, X., Wang, Y., Huang, Z., She, Z.-G.,

Zhang, X.-J., Zhu, L., and Hongliang, L. (2020) Low-

dose sorafenib acts as a mitochondrial uncoupler

and ameliorates nonalcoholic steatohepatitis, Cell

Metab., 31, 892-908, https://doi.org/10.1016/j.cmet.

2020.04.011.

189. Ek,F., Blom,K., Selvin,T., Rudfeldt, J., Andersson,C.,

Senkowski, W., Brechot, C., Nygren, P., Larsson, R.,

Jarvius, M., and Fryknaes, M. (2022) Sorafenib and

nitazoxanide disrupt mitochondrial function and in-

hibit regrowth capacity in three-dimensional mod-

els of hepatocellular and colorectal carcinoma, Sci.

Rep., 12, 8943, https://doi.org/10.1038/s41598-022-

12519-4.

190. Fox, M. A., and Hughes, A. K. (2004) Cage C–H⋯X

interactions in solid-state structures of icosahedral

carboranes, Coord. Chem. Rev., 248, 457-476, https://

doi.org/10.1016/j.ccr.2003.10.002.

191. Liberman, E. A., Topaly, V. P., and Silberstein, A. Y.

(1970) Charged and neutral ion carriers through

bimolecular phospholipid membranes, Biochim.

Biophys. Acta, 196, 221-234, https://doi.org/10.1016/

0005-2736(70)90010-6.

192. Rokitskaya, T. I., Khailova, L. S., Makarenkov, A. V.,

Ol’shevskaya, V. A., Kalinin, V. N., and Antonenko,

Y. N. (2016) Weak C–H acids as protonophores can

carry hydrogen ions through lipid membranes and

mitochondria: a case of o-carborane, Phys. Chem.

Chem. Phys., 18, 16476-16482, https://doi.org/10.1039/

C6CP02581A.

193. Bregadze, V. I., Sivaev, I. B., and Glazun, S. A.

(2006) Polyhedral boron compounds as poten-

tial diagnostic and therapeutic antitumor agents,

Anticancer Agents Med. Chem., 6, 75-109, https://

doi.org/10.2174/187152006776119180.

194. Rokitskaya, T. I., Khailova, L. S., Makarenkov, A. V.,

Shunaev, A. V., Tatarskiy, V. V., Shtil, A. A., Ol'shev-

skaya, V. A., and Antonenko, Y. N. (2019) Carborane

derivatives of 1,2,3-triazole depolarize mitochon-

dria by transferring protons through the lipid part

of membranes, Biochim. Biophys. Acta Biomem-

br., 1861, 573-583, https://doi.org/10.1016/j.bbamem.

2018.12.009.

195. Smejtek, P., Jayaweera, A. R., and Hsu, K. (1983)

Electrical conductivity, transfer of hydrogen ions

in lipid bilayer membranes and uncoupling effect

induced by pentachlorobenzenethiol (pentachlo-

rothiophenol), J. Membr. Biol., 76, 227-234, https://

doi.org/10.1007/BF01870365.

196. Terada, H., Uda, M., Kametani, F., and Kubota, S.

(1978) Structural requirements of alkyl acyldithio-

carbazates for the uncoupling of oxidative phos-

phorylation in mitochondria, Biochim. Biophys. Acta,

504, 237-247, https://doi.org/10.1016/0005-2728(78)

90172-X.

197. Schwaller, M.A., Allard,B., Lescot,E., and Moreau,F.

(1995) Protonophoric activity of ellipticine and iso-

mers across the energy-transducing membrane of

mitochondria, J.Biol. Chem., 270, 22709-22713, https://

doi.org/10.1074/jbc.270.39.22709.

198. Auclair, C. (1987) Multimodal action of antitumor

agents on DNA: the ellipticine series, Arch. Bio-

chem. Biophys., 259, 1-14, https://doi.org/10.1016/

0003-9861(87)90463-2.

199. Khailova, L. S., Silachev, D. N., Rokitskaya, T. I.,

Avetisyan, A. V., Lyamzaev, K. G., Severina, I. I.,

Il’yasova, T. M., Gulyaev, M. V., Dedukhova, V. I.,

Trendeleva, T.A., Plotnikov, E.Y., Zvyagilskaya, R.A.,

Chernyak, B. V., Zorov, D. B., Antonenko, Y. N., and

Skulachev, V. P. (2014) A short-chain alkyl deriva-

tive of Rhodamine 19 acts as a mild uncoupler of

mitochondria and a neuroprotector, Biochim. Bio-

phys. Acta, 1837, 1739-1747, https://doi.org/10.1016/

j.bbabio.2014.07.006.

200. Zorova, L. D., Pevzner, I. B., Khailova, L. S.,

Korshunova, G. A., Kovaleva, M. A., Kovalev, L. I.,

Serebryakova, M. V., Silachev, D. N., Sudakov, R. V.,

Zorov, S. D., Rokitskaya, T. I., Popkov, V. A., Plotnikov,

E. Y., Antonenko, Y. N., and Zorov, D. B. (2023) Mito-

MITOCHONDRIAL UNCOUPLERS 1843

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

chondrial ATP synthase and mild uncoupling by butyl

ester of rhodamine 19, C4R1, Antioxidants, 12, 646,

https://doi.org/10.3390/antiox12030646.

201. Mai, M. S., and Allison, W. S. (1983) Inhibition of an

oligomycin-sensitive ATPase by cationic dyes, some

of which are atypical uncouplers of intact mitochon-

dria, Arch. Biochem. Biophys., 221, 467-476, https://

doi.org/10.1016/0003-9861(83)90165-0.

202. Schwaller, M.A., Allard,B., Sureau,F., and Moreau,F.

(1994) Protonation equilibrium of ellipticine bound

to the energy-transducing membrane of mitochon-

dria, J.Phys. Chem., 98, 4209-4211, https://doi.org/

10.1021/j100067a002.

203. Sureau, F., Moreau, F., Millot, J. M., Manfait, M.,

Allard, B., Aubard, J., and Schwaller, M. A. (1993)

Microspectrofluorometry of the protonation state

of ellipticine, an antitumor alkaloid, in single cells,

Biophys. J., 65, 1767-1774, https://doi.org/10.1016/

S0006-3495(93)81273-6.

204. Gear, A. R. (1974) Rhodamine 6G. A potent inhib-

itor of mitochondrial oxidative phosphorylation,

J.Biol. Chem., 249, 3628-3637, https://doi.org/10.1016/

S0021-9258(19)42620-3.

205. Duvvuri, M., Gong, Y., Chatterji, D., and Krise, J. P.

(2004) Weak base permeability characteristics influ-

ence the intracellular sequestration site in the multi-

drug-resistant human leukemic cell line HL-60, J.Biol.

Chem., 279, 32367-32372, https://doi.org/10.1074/

jbc.M400735200.

206. Milanova, D., Chambers, R. D., Bahga, S. S., and

Santiago, J. G. (2011) Electrophoretic mobility mea-

surements of fluorescent dyes using on-chip capil-

lary electrophoresis, Electrophoresis, 32, 3286-3294,

https://doi.org/10.1002/elps.201100210.

207. Antonenko, Y.N., Avetisyan, A. V., Cherepanov, D.A.,

Knorre, D. A., Korshunova, G. A., Markova, O. V.,

Ojovan, S. M., Perevoshchikova, I. V., Pustovidko,

A. V., Rokitskaya, T. I., Severina, I. I., Simonyan,

R.A., Smirnova, E.A., Sobko, A.A., Sumbatyan, N.V.,

Severin, F. F., and Skulachev, V. P. (2011) Derivatives

of rhodamine 19 as mild mitochondria-targeted cat-

ionic uncouplers, J.Biol. Chem., 286, 17831-17840,

https://doi.org/10.1074/jbc.M110.212837.

208. Kalinovich, A.V., and Shabalina, I.G. (2015) Novel mi-

tochondrial cationic uncoupler C4R1 is an effective

treatment for combating obesity in mice, Biochem-

istry (Moscow), 80, 620-628, https://doi.org/10.1134/

S0006297915050156.

209. Luo,X., Qian,L., Xiao,Y., Tang,Y., Zhao,Y., Wang,X.,

Gu,L., Lei, Z., Bao, J., Wu,J., He, T., Hu,F., Zheng, J.,

Li, H., Zhu, W., Shao, L., Dong, X., Chen, D., Qian, X.,

and Yang, Y. (2019) A diversity-oriented rhodamine

library for wide-spectrum bactericidal agents with

low inducible resistance against resistant patho-

gens, Nat. Commun., 10, 258, https://doi.org/10.1038/

s41467-018-08241-3.

210. Nazarov, P.A., Maximov, V.S., Firsov, A.M., Karakozova,

M. V., Panfilova, V., Kotova, E. A., Skulachev, M. V.,

and Antonenko, Y. N. (2024) Rhodamine 19 alkyl es-

ters as effective antibacterial agents, Int.J. Mol. Sci.,

25, 6137, https://doi.org/10.3390/ijms25116137.

211. Pfeffermann, J., Yadav, R., Glasnov, T., Thorn-

Seshold,O., and Pohl,P. (2025) Phototriggered proton-

selective carrier currents through photoswitch-

able membranes, bioRxiv, https://doi.org/10.1101/

2025.01.13.632814.

212. Rottenberg, H. (1983) Uncoupling of oxidative phos-

phorylation in rat liver mitochondria by general an-

esthetics, Proc. Natl. Acad. Sci. USA, 80, 3313-3317,

https://doi.org/10.1073/pnas.80.11.3313.

213. Dabadie, P., Bendriss, P., Erny, P., and Mazat, J.-P.

(1987) Uncoupling effects of local anesthetics on

rat liver mitochondria, FEBS Lett., 226, 77-82,

https://doi.org/10.1016/0014-5793(87)80554-9.

214. Terada, H., Shima, O., Yoshida, K., and Shinohara, Y.

(1990) Effects of the local anesthetic bupivacaine on

oxidative phosphorylation in mitochondria. Change

from decoupling to uncoupling by formation of a

leakage type ion pathway specific for H

in coop-

eration with hydrophobic anions, J.Biol. Chem.,

265, 7837-7842, https://doi.org/10.1016/S0021-9258

(19)39006-4.

215. Strichartz, G.R., Sanchez,V., Arthur, G.R., Chafetz,R.,

and Martin, D. (1990) Fundamental properties of lo-

cal anesthetics. II. Measured octanol:buffer parti-

tion coefficients and pKa values of clinically used

drugs, Anesth. Analg., 71, 158-170, https://doi.org/

10.1213/00000539-199008000-00008.

216. Sun, X., and Garlid, K. D. (1992) On the mechanism

by which bupivacaine conducts protons across the

membranes of mitochondria and liposomes, J. Biol.

Chem., 267, 19147-19154, https://doi.org/10.1016/

S0021-9258(18)41754-1.

217. Fantin, V. R., Berardi, M. J., Scorrano, L., Korsmeyer,

S. J., and Leder, P. (2002) A novel mitochondriotox-

ic small molecule that selectively inhibits tumor

cell growth, Cancer Cell, 2, 29-42, https://doi.org/

10.1016/S1535-6108(02)00082-X.

218. Wang, J., He, H., Xiang, C., Fan, X.-Y., Yang, L.-Y.,

Yuan,L., Jiang, F.-L., and Liu,Y. (2018) Uncoupling ef-

fect of F16 is responsible for its mitochondrial toxic-

ity and anticancer activity, Toxicol. Sci., 161, 431-442,

https://doi.org/10.1093/toxsci/kfx218.

219. Xu, J., He, H., Zhou, L. J., Liu, Y. Z., Li, D. W., Jiang,

F. L., and Liu, Y. (2018) Pyridinium and indole ori-

entation determines the mitochondrial uncou-

pling and anti-cancer efficiency of F16, Eur. J. Med.

Chem., 154, 305-313, https://doi.org/10.1016/j.ejmech.

2018.05.036.

220. Stupnikova, T. V., Kalafat, V. N., Klyuev, N. A., Mar-

shtupa, V. P., and Sagitullin, R. S. (1980) Vinylogous

anhydro bases of pyridylindoles, Chem. Heterocycl.

ANTONENKO et al.1844

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Compd., 16, 1031-1035, https://doi.org/10.1007/

BF00496604.

221. Dubinin, M. V., Nedopekina, D. A., Ilzorkina, A. I.,

Semenova, A. A., Sharapov, V. A., Davletshin, E. V.,

Mikina, N. V., Belsky, Y. P., Spivak, A. Yu., Akatov,

V. S., Belosludtseva, N. V., Liu, J., and Belosludtsev,

K. N. (2023) Conjugation of triterpenic acids of ur-

sane and oleanane types with mitochondria-target-

ing cation F16 synergistically enhanced their cyto-

toxicity against tumor cells, Membranes, 13, 563,

https://doi.org/10.3390/membranes13060563.

222. Heise, N. V., Heisig, J., Meier, K., Csuk, R., and

Mueller, T. (2024) F16 Hybrids derived from steviol

or isosteviol are accumulated in the mitochondria of

tumor cells and overcome drug resistance, Molecules,

29, 381, https://doi.org/10.3390/molecules29020381.

223. Xing, W., Liu, G., Zhang, Y., Zhang, T., Lou, H., and

Fan, P. (2024) Selective antitumor effect and lower

toxicity of mitochondrion-targeting derivatization

of triptolide, J. Med. Chem., 67, 1093-1114, https://

doi.org/10.1021/acs.jmedchem.3c01508.

224. Miao, H., Cui, W., Zhang, T., Zhang, Y., Zhang, J.,

Lou, H., and Fan, P. (2024) Mitochondrial targeting

derivatives of honokiol enhanced selective antitu-

mor activity in NCI-H446 cells and decreased invivo

toxicity in Caenorhabditis elegans, Eur. J. Med.

Chem., 264, 115996, https://doi.org/10.1016/j.ejmech.

2023.115996.

225. Denisov, S.S., Kotova, E.A., Plotnikov, E.Y., Tikhonov,

A.A., Zorov, D.B., Korshunova, G.A., and Antonenko,

Y. N. (2014) A mitochondria-targeted protono-

phoric uncoupler derived from fluorescein, Chem.

Commun., 50, 15366-15369, https://doi.org/10.1039/

C4CC04996A.

226. Rokitskaya, T. I., Terekhova, N. V., Khailova, L. S.,

Kotova, E.A., Plotnikov, E.Y., Zorov, D.B., Tatarinov,

D. A., and Antonenko, Y. N. (2019) Zwitterionic pro-

tonophore derived from 2-(2-hydroxyaryl)alkenyl-

phosphonium as an uncoupler of oxidative phos-

phorylation, Bioconjug. Chem., 30, 2435-2443,

https://doi.org/10.1021/acs.bioconjchem.9b00516.

227. Blaikie, F.H., Brown, S.E., Samuelsson, L.M., Brand,

M. D., Smith, R. A., and Murphy, M. P. (2006) Target-

ing dinitrophenol to mitochondria: limitations to the

development of a self-limiting mitochondrial pro-

tonophore, Biosci. Rep., 26, 231-243, https://doi.org/

10.1007/s10540-006-9018-8.

228. Antonenko, Y. N., Denisov, S. S., Silachev, D. N.,

Khailova, L. S., Jankauskas, S. S., Rokitskaya, T. I.,

Danilina, T. I., Kotova, E. A., Korshunova, G. A.,

Plotnikov, E. Y., and Zorov, D.B. (2016) A long-linker

conjugate of fluorescein and triphenylphosphoni-

um as mitochondria-targeted uncoupler and fluo-

rescent neuro- and nephroprotector, Biochim. Bio-

phys. Acta, 1860, 2463-2473, https://doi.org/10.1016/

j.bbagen.2016.07.014.

229. Iaubasarova, I. R., Khailova, L. S., Nazarov, P. A.,

Rokitskaya, T. I., Silachev, D. N., Danilina, T. I., Plot-

nikov, E.Y., Denisov, S.S., Kirsanov, R.S., Korshunova,

G.A., Kotova, E.A., Zorov, D.B., and Antonenko, Y.N.

(2020) Linking 7-nitrobenzo-2-oxa-1,3-diazole (NBD)

to triphenylphosphonium yields mitochondria-target-

ed protonophore and antibacterial agent, Biochemis-

try (Moscow), 85, 1578-1590, https://doi.org/10.1134/

S000629792012010X.

230. Iaubasarova, I. R., Khailova, L. S., Firsov, A. M.,

Grivennikova, V. G., Kirsanov, R. S., Korshunova,

G. A., Kotova, E. A., and Antonenko, Y. N. (2020)

Themitochondria-targeted derivative of the classical

uncoupler of oxidative phosphorylation carbonyl cy-

anide m-chlorophenylhydrazone is an effective mito-

chondrial recoupler, PLoS One, 15, e0244499, https://

doi.org/10.1371/journal.pone.0244499.

231. Kirsanov, R. S., Khailova, L. S., Rokitskaya, T. I.,

Lyamzaev, K. G., Panteleeva, A. A., Nazarov, P. A.,

Firsov, A. M., Iaubasarova, I. R., Korshunova, G. A.,

Kotova, E. A., and Antonenko, Y. N. (2024) Synthe-

sis of triphenylphosphonium-linked derivative of

3,5-ditert-butyl-4-hydroxybenzylidene-malononitrile

(SF6847) via knoevenagel reaction yields an effec-

tive mitochondria-targeted protonophoric uncoupler,

ACS Omega, 9, 11551-11561, https://doi.org/10.1021/

acsomega.3c08621.

232. Terekhova, N. V., Khailova, L. S., Rokitskaya, T. I.,

Nazarov, P.A., Islamov, D.R., Usachev, K.S., Tatarinov,

D. A., Mironov, V. F., Kotova, E. A., and Antonenko,

Y. N. (2021) Trialkyl(vinyl)phosphonium chlorophe-

nol derivatives as potent mitochondrial uncouplers

and antibacterial agents, ACS Omega, 6, 20676-20685,

https://doi.org/10.1021/acsomega.1c02909.

233. Rokitskaya, T.I., Khailova, L.S., Strelnik, A.G., Kotova,

E.A., Mironov, V.F., Tatarinov, D.A., and Antonenko,

Y. N. (2025) Replacement of phenyl substituents at

phosphorus by hexyl ones in 2-(2-hydroxyaryl)vin-

ylphosphonium salts can tune the nature of the in-

duced proton transport through lipid membranes,

Biochim. Biophys. Acta Biomembr., 1867, 184417,

https://doi.org/10.1016/j.bbamem.2025.184417.

234. Listvan, V. N., and Listvan, V. V. (2003) Reactions of

phosphorus ylides with acyl chlorides: pathways and

preparative potential, Russ. Chem. Rev., 72, 705-713,

https://doi.org/10.1070/RC2003v072n08ABEH000800.

235. Kirsanov, R. S., Khailova, L. S., Rokitskaya, T. I.,

Iaubasarova, I. R., Nazarov, P. A., Panteleeva, A. A.,

Lyamzaev, K. G., Popova, L. B., Korshunova, G. A.,

Kotova, E. A., and Antonenko, Y. N. (2023) Ester-sta-

bilized phosphorus ylides as protonophores on bilay-

er lipid membranes, mitochondria and chloroplasts,

Bioelectrochemistry, 150, 108369, https://doi.org/

10.1016/j.bioelechem.2023.108369.

236. Rokitskaya, T. I., Kirsanov, R. S., Khailova, L. S.,

Panteleeva, A.A., Lyamzaev, K.G., Korshunova, G.A.,

MITOCHONDRIAL UNCOUPLERS 1845

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Kotova, E. A., and Antonenko, Y. N. (2024) Methyla-

tion of phenyl rings in ester-stabilized phosphorus

ylides vastly enhances their protonophoric activi-

ty, Chembiochem., 25, e202300848, https://doi.org/

10.1002/cbic.202300848.

237. Fu, Y.Y., Zhang,M., Turner,N., Zhang, L.N., Dong, T.C.,

Gu,M., Leslie, S.J., Li, J.Y., Nan, F.J., and Li,J. (2013)

Anovel chemical uncoupler ameliorates obesity and

related phenotypes in mice with diet-induced obesity

by modulating energy expenditure and food intake,

Diabetologia, 56, 2297-2307, https://doi.org/10.1007/

s00125-013-2987-9.

238. Ferguson, S. M. F., Estrada-O, S., and Lardy, H. A.

(1971) Potassium-specific uncoupling by nigericin,

J.Biol. Chem., 246, 5645-5652, https://doi.org/10.1016/

S0021-9258(18)61855-1.

239. Johnson, R. B., Feldott, G., and Lardy, H. A. (1950)

Themode of action of the antibiotic, usnic acid, Arch.

Biochem., 28, 317-323.

240. Abo-Khatwa, A. N., Al-Robai, A. A., and Al-Jawhari,

D. A. (1996) Lichen acids as uncouplers of oxidative

phosphorylation of mouse-liver mitochondria, Nat.

Toxins, 4, 96-102, https://doi.org/10.1002/19960402NT7.

241. Antonenko, Y. N., Khailova, L. S., Rokitskaya,

T. I., Nosikova, E. S., Nazarov, P. A., Luzina, O. A.,

Salakhutdinov, N. F., and Kotova, E. A. (2019) Mech-

anism of action of an old antibiotic revisited: role

of calcium ions in protonophoric activity of usnic

acid, Biochim. Biophys. Acta Bioenerg., 1860, 310-316,

https://doi.org/10.1016/j.bbabio.2019.01.005.

242. Graven, S. N., Estrada-O, S., and Lardy, H. A. (1966)

Alkali metal cation release and respiratory inhi-

bition induced by nigericin in rat liver mitochon-

dria, Proc. Natl. Acad. Sci. USA, 56, 654-658, https://

doi.org/10.1073/pnas.56.2.654.

243. Toro, M., Gomez-Lojero, C., Montal, M., and

Estrada-O, S. (1976) Charge transfer mediated by

nigericin in black lipid membranes, J. Bioenerg., 8,

19-26, https://doi.org/10.1007/BF01559387.

244. Markin, V. S., and Sokolov, V. S. (1990) A new con-

cept of electrochemical membrane equlibrium cou-

pled transport and membrane potential, Bioelec-

trochem. Bioenerg., 23, 1-16, https://doi.org/10.1016/

0302-4598(90)80001-Y.

245. Whitehouse, M.W., and Dean, P.D.G. (1965) Biochem-

ical properties of anti-inflammatory drugs-V: uncou-

pling of oxidative phosphorylation by some γ-resor-

cyl and other dihydroxybenzol compounds, Biochem.

Pharmacol., 14, 557-567, https://doi.org/10.1016/

0006-2952(65)90228-5.

246. Rokitskaya, T. I., Kotova, E. A., and Antonenko,

Y. N. (2021) Anomalous potentials on bilayer lipid

membranes in the presence of usnic acid: Markin–

Sokolov versus Nernst–Donnan equilibrium, Bioelec-

trochemistry, 141, 107825, https://doi.org/10.1016/

j.bioelechem.2021.107825.

247. Andersen, O. S. (1984) Gramicidin channels, Annu.

Rev. Physiol., 46, 531-548, https://doi.org/10.1146/an-

nurev.ph.46.030184.002531.

248. Woolley, G. A., and Wallace, B. A. (1992) Model ion

channels– gramicidin and alamethicin, J.Membrane

Biol., 129, 109-136, https://doi.org/10.1007/BF00219508.

249. Andersen, O. S., and Koeppe, R. E. (1992) Molecu-

lar determinants of channel function, Physiol Rev.,

72, S89-S158, https://doi.org/10.1152/physrev.1992.72.

suppl_4.S89.

250. Mitchell, P., and Moyle, J. (1967) Respiration-driv-

en proton translocation in rat liver mitochondria,

Biochem. J., 105, 1147-1162, https://doi.org/10.1042/

bj1051147.

251. Grinius, L. L., Daugelabichius, R. I., and Al’kimavi-

chius, G. A. (1980) Study of membrane potential of

Bacillus subtilis and Escherichia coli cells by the pen-

etration ions methods [in Russian], Biokhimiia, 45,

1609-1618.

252. Matsuno-Yagi, A., and Hatefi, Y. (1989) Uncoupling

of oxidative phosphorylation: different effects of li-

pophilic weak acids and electrogenic ionophores

on the kinetics of ATP synthesis, Biochemistry, 28,

4367-4374, https://doi.org/10.1021/bi00436a037.

253. Nishio, J. N., and Whitmarsh, J. (1991) Dissipation

of the proton electrochemical potential in intact

and lysed chloroplasts: I. The electrical potential,

Plant Physiol., 95, 522-528, https://doi.org/10.1104/

pp.95.2.522.

254. Myers, V. B., and Haydon, D. A. (1972) Ion transfer

across lipid membranes in the presence of gram-

icidin A. II. The ion selectivity, Biochim. Biophys.

Acta, 274, 313-322, https://doi.org/10.1016/0005-

2736(72)90179-4.

255. Sorochkina, A. I., Plotnikov, E. Y., Rokitskaya, T. I.,

Kovalchuk, S. I., Kotova, E. A., Sychev, S. V., Zorov,

D. B., and Antonenko, Y. N. (2012) N-terminally glu-

tamate-substituted analogue of gramicidin A as pro-

tonophore and selective mitochondrial uncoupler,

PLoS One, 7, e41919, https://doi.org/10.1371/journal.

pone.0041919.

256. David, J. M., and Rajasekaran, A. K. (2015) Grami-

cidin A: a new mission for an old antibiotic, J. Kid-

ney Cancer VHL, 2, 15-24, https://doi.org/10.15586/

jkcvhl.2015.21.

257. Khailova, L. S., Rokitskaya, T. I., Kovalchuk, S. I.,

Kotova, E.A., Sorochkina, A. I., and Antonenko, Y.N.

(2019) Role of mitochondrial outer membrane in the

uncoupling activity of N-terminally glutamate-substi-

tuted gramicidin A, Biochim. Biophys. Acta Biomem-

br., 1861, 281-287, https://doi.org/10.1016/j.bbamem.

2018.06.013.

258. Haoyang, W.-W., Xiao, Q., Ye, Z., Fu, Y., Zhang, D.-W.,

Li, J., Xiao, L., Li, Z.-T., and Hou, J.-L. (2021) Grami-

cidin A-based unimolecular channel: cancer cell-tar-

geting behavior and ion transport-induced apoptosis,

ANTONENKO et al.1846

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Chem. Commun., 57, 1097-1100, https://doi.org/10.1039/

D0CC08073J.

259. Silachev, D.N., Khailova, L.S., Babenko, V.A., Gulyaev,

M. V., Kovalchuk, S. I., Zorova, L. D., Plotnikov, E. Y.,

Antonenko, Y.N., and Zorov, D.B. (2014) Neuroprotec-

tive effect of glutamate-substituted analog of gramici-

dinA is mediated by the uncoupling of mitochondria,

Biochim. Biophys. Acta Gen. Subject, 1840, 3434-3442,

https://doi.org/10.1016/j.bbagen.2014.09.002.

260. Severin, F. F., Severina, I. I., Antonenko, Y. N.,

Rokitskaya, T. I., Cherepanov, D. A., Mokhova, E. N.,

Vyssokikh, M. Y., Pustovidko, A. V., Markova, O. V.,

Yaguzhinsky, L. S., Korshunova, G. A., Sumbatyan,

N.V., Skulachev, M.V., and Skulachev, V.P. (2010) Pene-

trating cation/fatty acid anion pair as a mitochondria-

targeted protonophore, Proc. Natl. Acad. Sci.

USA, 107, 663-668, https://doi.org/10.1073/pnas.

0910216107.

261. Rokitskaya, T. I., Sumbatyan, N. V., Tashlitsky, V. N.,

Korshunova, G. A., Antonenko, Y. N., and Skulachev,

V. P. (2010) Mitochondria-targeted penetrating cat-

ions as carriers of hydrophobic anions through lipid

membranes, Biochim. Biophys. Acta, 1798, 1698-1706,

https://doi.org/10.1016/j.bbamem.2010.05.018.

262. Lyamzaev, K. G., Pustovidko, A. V., Simonyan, R. A.,

Rokitskaya, T. I., Domnina, L. V., Ivanova, O. Yu.,

Severina, I. I., Sumbatyan, N. V., Korshunova, G. A.,

Tashlitsky, V. N., Roginsky, V. A., Antonenko, Y. N.,

Skulachev, M. V., Chernyak, B. V., and Skulachev,

V. P. (2011) Novel mitochondria-targeted antioxi-

dants: plastoquinone conjugated with cationic plant

alkaloids berberine and palmatine, Pharm. Res., 28,

2883-2895, https://doi.org/10.1007/s11095-011-0504-8.

263. Chernyak, B. V., Antonenko, Y. N., Domnina, L. V.,

Ivanova, O. Yu., Lyamzaev, K. G., Pustovidko, A. V.,

Rokitskaya, T.I., Severina, I.I., Simonyan, R.A., Tren-

deleva, T. A., and Zvyagilskaya, R. A. (2013) Novel

penetrating cations for targeting mitochondria, Curr.

Pharm. Des., 19, 2795-2806, https://doi.org/10.2174/

1381612811319150015.

264. Garlid, K.D., and Nakashima, R.A. (1983) Studies on

the mechanism of uncoupling by amine local anes-

thetics. Evidence for mitochondrial proton trans-

port mediated by lipophilic ion pairs, J.Biol. Chem.,

258, 7974-7980, https://doi.org/10.1016/S0021-9258

(20)82015-8.

265. Stark, G. (1980) Negative hydrophobic ions as trans-

port-mediators for positive ions. Evidence for a car-

rier mechanism, Biochim. Biophys. Acta Biomembr.,

600, 233-237, https://doi.org/10.1016/0005-2736(80)

90428-9.

266. Ahmed, I., and Krishnamoorthy, G. (1990) En-

hancement of transmembrane proton conductivi-

ty of protonophores by membrane-permeant cat-

ions, Biochim. Biophys. Acta, 1024, 298-306, https://

doi.org/10.1016/0005-2736(90)90358-U.

267. Kolajova,M., Antalik,M., and Sturdik,E. (1993) Study

of the complex formation between amine local anes-

thetics and uncouplers of oxidative phosphorylation

carbonyl cyanide phenylhydrazones, Gen. Physiol.

Biophys., 12, 213-229.

268. Antonenko, Y. N., Khailova, L. S., Knorre, D. A., Mar-

kova, O.V., Rokitskaya, T.I., Ilyasova, T.M., Severina,

I. I., Kotova, E.A., Karavaeva, Y. E., Prikhodko, A.S.,

Severin, F.F., and Skulachev, V. P. (2013) Penetrating

cations enhance uncoupling activity of anionic pro-

tonophores in mitochondria, PLoS One, 8, e61902,

https://doi.org/10.1371/journal.pone.0061902.

269. Wu, X., and Gale, P. A. (2016) Small-molecule un-

coupling protein mimics: synthetic anion receptors

as fatty acid-activated proton transporters, J. Am.

Chem. Soc., 138, 16508-16514, https://doi.org/10.1021/

jacs.6b10615.

270. Antonenko, Y. N., Veselov, I. M., Rokitskaya, T. I.,

Vinogradova, D. V., Khailova, L. S., Kotova, E. A.,

Maltsev, A. V., Bachurin, S. O., and Shevtsova, E. F.

(2024) Neuroprotective thiourea derivative uncou-

ples mitochondria and exerts weak protonophoric

action on lipid membranes, Chem. Biol. Interact., 402,

111190, https://doi.org/10.1016/j.cbi.2024.111190.

271. York,E., McNaughton, D.A., Duman, M.-N., Gale, P.A.,

and Rawling, T. (2023) Fatty acid-activated proton

transport by bisaryl anion transporters depolaris-

es mitochondria and reduces the viability of MDA-

MB-231 breast cancer cells, Biomolecules, 13, 1202,

https://doi.org/10.3390/biom13081202.

272. Shkinev, A. V., Gagelgans, A. I., Tashmukhamedov,

B.A., and Miroshnikov, A.I. (1978) Effect of melittin

from bee venom on mitochondrial functions [in Rus-

sian], Biokhimiia, 43, 1452-1457.

273. Sholtz, K.F., Aliverdieva, D.A., and Kotelnikova, A.V.

(1985) Action of alamethicin on the coupling mem-

brane of mitochondria [in Russian], Dokl. Akad. Nauk

SSSR, 283, 723-727.

274. Hugosson,M., Andreu,D., Boman, H.G., and Glaser,E.

(1994) Antibacterial peptides and mitochondrial

presequences affect mitochondrial coupling, res-

piration and protein import, Eur. J. Biochem., 223,

1027-1033, https://doi.org/10.1111/j.1432-1033.1994.

tb19081.x.

275. Diaz-Achirica, P., Prieto, S., Ubach, J., Andreu, D.,

Rial, E., and Rivas, L. (1994) Permeabilization of

the mitochondrial inner membrane by short ce-

cropin-A-melittin hybrid peptides, Eur. J. Biochem.,

224, 257-263, https://doi.org/10.1111/j.1432-1033.

1994.tb20019.x.

276. Aliverdieva, D., Mamaev, D., Snezhkova, L., and

Sholtz, C. (2012) Evaluation of molecularity of

rate-limiting step of pore formation by antimicro-

bial peptides studied using mitochondria as a bio-

sensor, Toxicol In Vitro, 26, 939-949, https://doi.org/

10.1016/j.tiv.2012.04.006.

MITOCHONDRIAL UNCOUPLERS 1847

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

277. Lees, H. (1967) The effects of laurylamine on res-

piration and related reactions of liver mitochon-

dria in vitro, Biochim. Biophys. Acta, 131, 310-316,

https://doi.org/10.1016/0005-2728(67)90144-2.

278. Bragadin,M., and Dell’Antone,P. (1996) Mitochondri-

al bioenergetics as affected by cationic detergents,

Arch. Environ. Contam. Toxicol., 30, 280-284, https://

doi.org/10.1007/BF00215809.

279. Bragadin, M., Moret, I., Piazza, R., Grasso, M., and

Manente, S. (2002) The interactions of cetyltrimeth-

ylammonium with mitochondria: an uncoupler or a

detergent? Chemosphere, 46, 219-223, https://doi.org/

10.1016/S0045-6535(01)00095-9.

280. Bangham, J. A., and Lea, E. J. (1978) The interaction

of detergents with bilayer lipid membranes, Biochim.

Biophys. Acta, 511, 388-396, https://doi.org/10.1016/

0005-2736(78)90275-4.

281. Kondrashov, O.V., and Akimov, S.A. (2023) Alteration

of average thickness of lipid bilayer by membrane-

deforming inclusions, Biomolecules, 13, 1731, https://

doi.org/10.3390/biom13121731.

282. Antonenko, Y. N., Rokitskaya, T. I., Cooper, A. J. L.,

and Krasnikov, B.F. (2010) Minocycline chelatesCa

binds to membranes, and depolarizes mitochondria

by formation of Ca

-dependent ion channels, J. Bio-

energ. Biomembr., 42, 151-163, https://doi.org/10.1007/

s10863-010-9271-1.

283. Sokolov, S., Zyrina, A., Akimov, S., Knorre, D., and

Severin, F. (2023) Toxic effects of penetrating cat-

ions, Membranes, 13, 841, https://doi.org/10.3390/

membranes13100841.

284. Terada,H., Nagamune,H., Osaki,Y., and Yoshikawa,K.

(1981) Specific requirement for inorganic phosphate

for induction of bilayer membrane conductance by

the cationic uncoupler carbocyanine dye, Biochim.

Biophys. Acta, 646, 488-490, https://doi.org/10.1016/

0005-2736(81)90320-5.

285. Ter Welle, H. F., and Slater, E. C. (1967) Uncoupling

of respiratory-chain phosphorylation by arsenate,

Biochim. Biophys. Acta, 143, 1-17, https://doi.org/

10.1016/0005-2728(67)90104-1.

286. Skulachev, V. P. (1998) Uncoupling: new approaches

to an old problem of bioenergetics, Biochim. Bio-

phys. Acta, 1363, 100-124, https://doi.org/10.1016/

S0005-2728(97)00091-1.

287. Wallace, K. B., and Starkov, A. A. (2000) Mitochon-

drial targets of drug toxicity, Annu. Rev. Pharmacol.

Toxicol., 40, 353-388, https://doi.org/10.1146/annurev.

pharmtox.40.1.353.

288. Sherman, J. K. (2025) Correlation in ultrastructural

cryoinjury of mitochondria with aspects of their re-

spiratory function, Exp. Cell Res., 66, 378-384, https://

doi.org/10.1016/0014-4827(71)90691-4.

289. Guengerich, F. P. (2008) Cytochrome p450 and chem-

ical toxicology, Chem. Res. Toxicol., 21, 70-83, https://

doi.org/10.1021/tx700079z.

290. Rowland, A., Miners, J. O., and Mackenzie, P. I.

(2013) The UDP-glucuronosyltransferases: their role

in drug metabolism and detoxification, Int. J. Bio-

chem. Cell Biol., 45, 1121-1132, https://doi.org/10.1016/

j.biocel.2013.02.019.

291. Bock, K. W. (2014) Homeostatic control of xeno- and

endobiotics in the drug-metabolizing enzyme sys-

tem, Biochem. Pharmacol., 90, 1-6, https://doi.org/

10.1016/j.bcp.2014.04.009.

292. Gamage, N., Barnett, A., Hempel, N., Duggleby, R. G.,

Windmill, K. F., Martin, J. L., and McManus, M. E.

(2006) Human sulfotransferases and their role in

chemical metabolism, Toxicol. Sci., 90, 5-22, https://

doi.org/10.1093/toxsci/kfj061.

293. Josephy, P.D. (2010) Genetic variations in human glu-

tathione transferase enzymes: significance for phar-

macology and toxicology, Hum. Genomics Proteomics,

2010, 876940, https://doi.org/10.4061/2010/876940.

294. Sim, E., Westwood, I., and Fullam, E. (2007) Aryl-

amine N-acetyltransferases, Expert. Opin. Drug

Metab. Toxicol., 3, 169-184, https://doi.org/10.1517/

17425255.3.2.169.

295. Mato, J.M., Martinez-Chantar, M.L., and Lu, S.C. (2013)

S-adenosylmethionine metabolism and liver disease,

Ann. Hepatol., 12, 183-189, https://doi.org/10.1016/

S1665-2681(19)31355-9.

296. Jiang, A., Chen, G., Xu, J., Liu, Y., Zhao, G., Liu, Z.,

Chen, T., Li, Y., and James, T. D. (2019) Ratiometric

two-photon fluorescent probe for in situ imaging of

carboxylesterase (CE)-mediated mitochondrial acid-

ification during medication, Chem. Commun., 55,

11358-11361, https://doi.org/10.1039/C9CC05759E.

297. Miwa,S., Treumann,A., Bell,A., Vistoli,G., Nelson,G.,

Hay, S., and von Zglinicki, T. (2016) Carboxylester-

ase converts Amplex red to resorufin: implications

for mitochondrial H

release assays, Free Rad-

ic. Biol. Med., 90, 173-183, https://doi.org/10.1016/

j.freeradbiomed.2015.11.011.

298. Cooper, A. J. L., and Hanigan, M. H. (2018) Metab-

olism of glutathione S-conjugates: multiple path-

ways, Comprehens. Toxicol., 10, 406, https://doi.org/

10.1016/B978-0-12-801238-3.01973-5.

299. Sidhu, R.S., and Blair, A.H. (1975) Human liver alde-

hyde dehydrogenase. Esterase activity, J.Biol. Chem.,

250, 7894-7898, https://doi.org/10.1016/S0021-9258

(19)40899-5.

300. Zemaitis, M. A., and Greene, F. E. (1976) Impairment

of hepatic microsomal and plasma esterases of the

rat by disulfiram and diethyldithiocarbamate, Bio-

chem. Pharmacol., 25, 453-459, https://doi.org/10.1016/

0006-2952(76)90349-X.

301. Nousiainen, U., and Torronen, R. (1984) Differentia-

tion of microsomal and cytosolic carboxylesterases

in the rat liver by in vivo and in vitro inhibition,

Gen. Pharmacol., 15, 223-227, https://doi.org/10.1016/

0306-3623(84)90163-0.

ANTONENKO et al.1848

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

302. Buechel, K. H., and Schaefer, G. (1970) 2-Anilinothio-

phenes as uncouplers of oxidative phosphorylation

in mitochondria [in German], Z. Naturforsch., 25 b,

1465-1474, https://doi.org/10.1515/znb-1970-1232.

303. Skulachev, V. P., Vyssokikh, M. Y., Chernyak, B. V.,

Mulkidjanian, A.Y., Skulachev, M.V., Shilovsky, G.A.,

Lyamzaev, K. G., Borisov, V. B., Severin, F. F., and

Sadovnichii, V.A. (2023) Six functions of respiration:

isn’t it time to take control over ROS production in

mitochondria, and aging along with it? Int. J. Mol.

Sci., 24, 12540, https://doi.org/10.3390/ijms241612540.

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