Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 2, pp. 212-222 © Pleiades Publishing, Ltd., 2024.

212

Relationship of Cytotoxic and Antimicrobial Effects

of Triphenylphosphonium Conjugates

with Various Quinone Derivatives

Pavel A. Nazarov

1,a

*, Lyudmila A. Zinovkina

, Anna A. Brezgunova

1,2

Konstantin G. Lyamzaev

1,3

, Andrei V. Golovin

1,2

, Marina V. Karakozova

Elena A. Kotova

, Egor Yu. Plotnikov

, Roman A. Zinovkin

1,3

Maxim V. Skulachev

1,4

, and Yuri N. Antonenko

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

119991Moscow, Russia

Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University,

119991Moscow, Russia

Russian Clinical Research Center for Gerontology of the Ministry of Healthcare of the Russian Federation,

Pirogov Russian National Research Medical University, 129226Moscow, Russia

Institute of Mitoengineering, Lomonosov Moscow State University, 119991Moscow, Russia

e-mail: nazarovpa@gmail.com

Received December 4, 2023

Revised January 30, 2024

Accepted February 21, 2024

Abstract— Quinone derivatives of triphenylphosphonium have proven themselves to be effective geroprotec-

tors and antioxidants that prevent oxidation of cell components with participation of active free radicals– per-

oxide(RO

·), alkoxy (RO·), and alkyl (R·) radicals, as well as reactive oxygen species (superoxide anion, singlet

oxygen). Their most studied representatives are derivatives of plastoquinone (SkQ1) and ubiquinone (MitoQ),

which in addition to antioxidant properties also have a strong antibacterial effect. In this study, we investigated

antibacterial properties of other quinone derivatives based on decyltriphenylphosphonium (SkQ3, SkQT, and

SkQThy). We have shown that they, just like SkQ1, inhibit growth of various Gram-positive bacteria at micromolar

concentrations, while being less effective against Gram-negative bacteria, which is associated with recognition

of the triphenylphosphonium derivatives by the main multidrug resistance (MDR) pump of Gram-negative bac-

teria, AcrAB-TolC. Antibacterial action of SkQ1 itself was found to be dependent on the number of bacterial cells.

It is important to note that the cytotoxic effect of SkQ1 on mammalian cells was observed at higher concentrations

than the antibacterial action, which can be explained by (i) the presence of a large number of membrane organelles,

(ii) lower membrane potential, (iii) spatial separation of the processes of energy generation and transport, and

(iv) differences in the composition of MDR pumps. Differences in the cytotoxic effects on different types of eukary-

otic cells may be associated with the degree of membrane organelle development, energy status of the cell, and level

of the MDR pump expression.

DOI: 10.1134/S0006297924020032

Keywords: antioxidants, SkQ1, MDR pumps, AcrAB-TolC, bacteria, mammalian cell cultures, cytotoxicity, antibiotic,

mitochondria

Abbreviations: CFU,colony-forming unit; MDR,multidrug resistance; MIC,minimum inhibitory concentration; MTAs,mito-

chondria-targeted antioxidants; SkQs,“Skulachev ions” used in the study; SkQ1,10-(6-plastoquinonyl)decyl triphenylphos-

phonium; SkQ3,10-(6′-methylplastoquinonyl) decyl triphenylphosphonium; SkQThy,10-(2-isopropyl-5-methyl-1,4-benzoqui-

nonyl-6) decyltriphenylphosphonium; SkQT,a mixture of SkQT-para and 10-(5-toluquinonyl) decyltriphenylphosphonium;

SkQT-para,10-(6-toluquinonyl) decyltriphenylphosphonium; TPP,triphenylphosphonium.

* To whom correspondence should be addressed.

ANTIBACTERIAL PROPERTIES OF SkQs 213

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

INTRODUCTION

Bacteria and mitochondria have much in common.

Mitochondria are cellular powerhouses that generate

cellular energy in the form of adenosine triphosphate

and, like bacterial cells, have a negative membrane po-

tential (–180mV) on their inner membrane[1]. There-

fore, the positively charged compounds accumulate

in the mitochondrial matrix and within bacterial cells

against their concentration gradient.

In 1970s, the research team of Vladimir Petrovich

Skulachev[2-4] proposed the use of triphenylphospho-

nium (TPP) derivatives as mitochondria-targeted sub-

stances in which TPP serves as a “locomotive” for their

transport into mitochondria. Two decades later, Michael

Murphy etal.[5-7] applied this approach to the deliv-

ery of an antioxidant moiety into mitochondria, which

prompted creation of a variety of molecules based on

the triphenylphosphonium derivatives[8], including a

series of “Skulachev ions”, mitochondria-targeted anti-

oxidants (MTAs) created within the framework of the

“megaproject” on anti-aging penetrating ions[9].

MTAs based on quinone derivatives have become

widespread both in the studies of the role of mito-

chondria in various physiological processes and as

therapeutic agents[10, 11]. It was shown that in addi-

tion to their antioxidant action, these compounds also

exhibit an uncoupling effect on mitochondria, which

manifests itself as stimulated respiration and drop

in the mitochondrial membrane potential. Moreover,

this uncoupling effect is not necessarily toxic for the

organism, since partial mitochondrial uncoupling was

shown to be protective in the case of pathologies as-

sociated with oxidative stress [12-15], which may be

linked to the dependence of reactive oxygen species

(ROS) generation on the membrane potential of mito-

chondria[16]. The proposed mechanism of this uncou-

pling effect of SkQ1 (10-(6-plastoquinonyl)decyl triph-

enylphosphonium) on the mitochondrial membrane is

based on its ability to interact with the endogenous

fatty acids and facilitate fatty acid diffusion across the

membrane by electrostatic interaction of fatty acid

anions and SkQ1 cations [17]. The protonated forms

of fatty acids penetrate well through the membrane,

ensuring cyclic transmembrane proton transfer simi-

lar to the functioning of conventional protonophores,

such as 2,4-dinitrophenol.

Despite the relative similarity between mito-

chondria and bacteria, MTAs such as SkQ1 were long

thought to lack antibacterial properties[18], however,

alkyltriphenylphosphonium cations (CnTPPs) and SkQ1

were subsequently shown to exhibit a potent antibac-

terial effect on Gram-positive and Gram-negative bac-

teria [19, 20]. It turned out that the observed lack of

antibacterial action on the Escherichia coli bacterium

is due to operation of the main multidrug resistance

(MDR) pump AcrAB-TolC [20, 21], which is the only

known bacterial MDR pump capable of pumping out

SkQ1[22].

Bactericidal effect of SkQ1 makes it a promising

compound for use in clinical practice, in particular, to

combat infections caused by Gram-positive bacteria,

such as Staphylococcus aureus, Streptococcus mutans,

or Mycobacterium tuberculosis[22-24].

Other triphenylphosphonium-based substances,

such as fluorescein ester MitoFluo (10-[2-(3-hydroxy-6-

oxoxanthen-9-yl)benzoyl] oxydecyl-triphenylphospho-

nium) [25], or chloramphenicol-triphenylphosphoni-

um conjugate CAM-C10-TPP (N-{[(1R,2R)-dihydroxy-1-

(4-nitrophenyl)propan-2-yl]amino}-11-oxoundecyl triph-

enylphosphonium) also exhibited antibacterial ef-

fects [26]. At the same time, chimeric molecules re-

tained the properties of their components. For exam-

ple, the CAM-C10-TPP molecule inherited the ability of

chloramphenicol to inhibit protein synthesis on ribo-

somes and the ability of alkyltriphenylphosphonium

to reduce membrane potential on the bacterial mem-

branes. The conjugates based on quinones, which are

similar to SkQ1[20], can exhibit both antioxidant and

antibacterial properties, which makes them promising

research objects.

The aim of the present study was to compare the

effects of several quinone derivatives on eukaryotic

and prokaryotic cells. In particular, almost all deriv-

atives were shown to exhibit antibacterial activity

against Gram-positive bacteria, while Gram-negative

E. coli bacteria were resistant, which is the result of

operation of the AcrAB-TolC MDR pump. With regard

to their toxic effect on mammalian cells, such quinone

derivatives as SkQ3 (10-(6′-methylplastoquinonyl) de-

cyl triphenylphosphonium), SkQT (a mixture of SkQT-

para (10-(6-toluquinonyl) decyltriphenylphosphonium),

10-(5- toluquinonyl) decyltriphenylphosphonium), and

SkQThy (10-(2-isopropyl-5-methyl-1,4-benzoquinonyl-6)

decyltriphenylphosphonium) differ slightly from SkQ1,

while their toxic effect depends on the cell type, which

may be due to different levels of MDR pump expression.

MATERIALS AND METHODS

Materials. Penetrating cations (Fig.1) were kind-

ly provided by the Institute of Mitoengineering, Lo-

monosov Moscow State University, and their synthesis

was carried out according to the previously published

methods [27, 28]. All other reagents, unless otherwise

noted, were from Sigma-Aldrich, USA.

Bacterial strains. Standard laboratory strains of

Bacillus subtilis subs. subtilis Cohn 1872, strain BR151

(trpC2 lys-3 metB10), and Escherichia coli Castellani

and Chalmers 1919, strain MG1655 (F-lambda-ilvG-

rfb-50 rph-1), were used in our study. Staphyllococcus

NAZAROV et al.214

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Fig. 1. Structural formulas of the “Skulachev ions” (SkQs), the

cations used in the present study. The structures with arrows

indicate a mixture of isomers, the arrows demonstrate posi-

tion of decyltriphenylphosphonium. Asterisk (*) marks posi-

tion of decyltriphenylphosphonium for the SkQT-para com-

pound.

aureus Rosenbach 1884 (no. 144) and Mycobacterium

smegmatis Lehmann and Neumann 1899 (no.377) bac-

teria were obtained from the collection of microor-

ganisms of Lomonosov Moscow State University.

Deletion strains ECK3026 (∆tolC), ECK0456 (acrB),

ECK2465 (∆acrD), ECK3253 (∆acrF), ECK2071 (∆mdtB),

ECK3498 (∆mdtF), ECK0870 (∆macB), ECK2680 (∆emrB)

and ECK2363 (∆emrY) were kindly provided by

Dr. H. Niki (National Institute of Genetics, Japan)[29].

Cultivation of microorganisms. Bacteria were

grown in LB medium overnight at 30 or 37°C on a

shaker at 200 rpm until optical density of 1.5 at 600 nm

was achieved. Optical density at 600 nm was measured

with an Ultrospec1100 pro spectrophotometer (Amer-

sham Biosciences, UK).

Measurements of minimum inhibitory concen-

trations. Minimum inhibitory concentrations were

measured by double dilution method according to the

protocol [30] recommended by the Clinical and Labo-

ratory Standards Institute (CLSI) in a liquid Mueller–

Hinton medium.

TolC screening. Screening of a panel of deletion

mutants of TolC-containing transporters was carried

out according to the previously published works [20,

21, 31] in LB medium in 96-well plates (Citotest, China).

Preselected concentrations of SkQs (5, 30, and 50 μM)

were added to each mutant, which was allowed to

grow for 21h at 37°C. After that, optical density was

measured at 620 nm by using a Multiskan FC plate

spectrophotometer (Thermo Fisher Scientific, USA).

Analysis of the dependence of antibacterial

action on the number of cells. Various volumes of

B. subtilis culture were added to fresh LB medium con-

taining 1 µM SkQ1 and incubated for 3 h at 37°C and

220 rpm. Optical density was measured at 600 nm by

using an Ultrospec 1100 pro spectrophotometer.

Analysis of the effect of volume of the incuba-

tion SkQ1-containing medium on survival. Anover-

night culture of S. aureus bacteria was diluted to

~15,000 CFU (colony-forming units)/ml and incubated

for 3 h at 37°C and 220 rpm in various volumes of sa-

line solution of glucose (0.9% NaCl, 5 mM D-glucose)

with 1 μM SkQ1, after which the bacteria were plated

on LB agar to determine CFU.

Analysis of the protective effect of dead cells.

Killed S. aureus cells were obtained by heating the bac-

teria for 90 min at 65°C; loss of bacterial viability was

confirmed by plating on LB agar. A mixture of various

proportions of live and dead S. aureus cells was incu-

bated for 3 h at 37°C and 220 rpm with 0.25 μM SkQ1 in

saline with glucose, after which CFU for the survived

bacteria was determined.

Molecular modeling and docking. Molecular

docking was performed by using the QuickVina2 pro-

gram [32] as part of the ODDT software modules [33]

for Python. Lack of information about the binding site

and size of the protein make it impossible to effectively

scan the entire volume at once in a single experiment.

To solve this problem, the entire volume was divided

into 10 intersecting cells measuring 40×40×40 Å with

a center shifted by 15 Å with each step. Docking was

performed with redundant space scanning with ex-

haustiveness = 128 for two TolC states: closed (PDB ID:

1ek9) and open (PDB ID: 2×mn). Independent calcula-

tions were made on all three axes to fully cover the

volume of TolC. The docking results were visualized in

the PyMol program[34].

Experiments with human RKO and MRC5-SV40

cell lines. Human colon carcinoma cell line RKO

(ATCC CRL-2577) and MRC5-SV40 fibroblasts (EcACC

Cat.no.84100401) were cultured in a modified DMEM

supplemented with 10% fetal bovine serum (FBS), strep-

tomycin (100 units/ml) and penicillin (100 units/ml).

Cell viability was analyzed using a CellTiter-Blue re-

agent (Promega, USA). The cells were seeded into

96-well plates at 10,000 cells per well and cultured for

24 h at 37°C. The cells were treated with SkQ1 for 17h,

ANTIBACTERIAL PROPERTIES OF SkQs 215

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

then a Cell Titer-Blue reagent (20μl per well) was add-

ed, after which the cells were incubated for 1 h before

fluorescence was measured (ex = 560 nm; em = 590 nm)

by using a Fluoroskan Ascent Microplate Fluorimeter

(Thermo Fisher Scientific).

Experiments with cultures of renal tubular ep-

ithelial cells. Primary cultures of renal tubular cells

were obtained from the kidneys of 5-7-day-old male

Wistar rats. The protocols for working with animals

were approved by the ethical committee of animal re-

search of the Belozersky Institute of Physico-Chemical

Biology, Lomonosov Moscow State University (Proto-

col3/19 of 18 March 2019). The kidneys were sterilely

isolated, cut into small pieces and incubated with a

0.125% solution of type II collagenase (Gibco, Thermo

Fisher Scientific) in DMEM/F12 without bicarbonate at

37°C for 15min.

After incubation, the kidney pieces in the collage-

nase solution were dispersed by pipetting for several

minutes, and the resulting suspension was centrifuged

for 5 min at 400g to sediment the tubule fraction.

Theresulting pellet was resuspended in a complete cul-

ture medium consisting of DMEM/F-12 (1/1) containing

10% FBS, 2% amino acids, 1% vitamins, and 1% L-glu-

tamine, and seeded in a 96-well plate. Cells were cul-

tured in an incubator with 5% CO

. After 24 h of cell

seeding, the culture medium was replaced to remove

cell debris. After 24h, SkQT, SkQ3 and SkQ1 were add-

ed at concentrations of 0.125, 0.25, 0.5, 1, 2, 4, 8, 16,

and 32µM and the renal tubular epithelial cells were

incubated with these substances for 24 h in a complete

nutrient medium. Control cells were incubated in the

same medium without addition of SkQT, SkQ3, or SkQ1.

Cell viability was assessed by the standard MTT test,

for which the culture medium was replaced with a

DMEM/F-12 without bicarbonate containing 5 mg/ml of

MTT reagent and incubated for 1 h. Then the medium

was removed and 50 µl of DMSO were added to each

well. Formazan absorbance was measured at 540 nm

with a Zenyth 3100 plate spectrofluorimeter (Anthos

Labtec, Austria).

RESULTS

Antibacterial action of SkQs. It was previous-

ly shown that addition of the micromolar concentra-

tions of SkQ1 or C

TPP to bacteria results in inhibi-

tion of their growth and bactericidal effect [20, 24].

Table 1 shows measured minimum inhibitory concen-

trations (MICs) for three Gram-positive (B. subtilis,

S. aureus, M. smegmatis) and one Gram-negative bac-

terium (E. coli). For all the SkQs we studied, MIC val-

ues comparable to those of SkQ1 were obtained for

Gram-positive bacteria as well as for Gram-negative

E. coli. Just as in the case of SkQ1, all the SkQs we stud-

ied demonstrated antibacterial action only against the

deletion mutants deficient in the tolC and acrB genes,

while against other deletion mutants deficient in the

acrD, acrF, mdtB, mdtF, macB, emrB, and emrY genes,

antibacterial activity was comparable to that exhibited

against the wild-type E. coli. The MIC values for all dele-

tion mutants (except ∆tolC and ∆acrB) were similar for

all SkQs, indicating that all other SkQs, just like SkQ1,

are pumped out only by the AcrAB-TolC MDR pump.

SkQ1 docking in TolC. Our data demonstrate

important role of the AcrAB-TolC MDR pump in bac-

terial resistance to SkQ1 [20-22]. We started the work

on modeling the process of SkQ1 interaction with this

pump. In the first step, molecular docking calculations

were performed for the TolC pump component. The re-

sults (Fig. 2) allow us to conclude that no obvious bind-

ing pockets for SkQ1 are present on the inner surface

of TolC in both of its states (closed and open). How-

ever, an interesting clustering of sites appears at the

entrance of the pump in the closed state, where the

phosphonium group interacts with the abundant GLU

and ASP residues, the aromatic rings stack with three

tyrosine residues, and ASP371 forms hydrogen bonds

with the quinone. Unfortunately, scoring functions for

ranking the docking results do not allow estimating

affinity of such binding even approximately. It can be

concluded that the most specific binding site of SkQ1

in TolC is the entrance of the pump in the closed state.

Table 1. Minimum inhibitory concentrations (µM)

SkQ B. subtilis S. aureus M. smegmatis E. coli ∆tolC ∆acrB other*

SkQ1 1 1 1 35 1 1 35

SkQ3 1 1 1 35 1 1 35

SkQT 2 2 2 35 2 2 35

SkQT-para 1 1 1 35 1 1 35

SkQThy 1 1 1 35 1 1 35

Note. Asterisk (*) denotes deletion mutants deficient in other TolC-containing pump proteins (AcrD, AcrF, MdtB, MdtF, MacB,

EmrB and EmrY).

NAZAROV et al.216

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Fig. 2. Results of modeling SkQ1 docking to the internal cavity of TolC in its closed state. Structure of TolC is shown in gray,

demonstrating secondary structure elements. SkQ1 positions are shown in different colors to display covalent bonds.

Antibacterial activity depends on the amount

of cells. Although SkQ1 can be considered as an an-

tibacterial agent or as an antibiotic, its properties still

differ from those of the traditional antibiotics. Unlike

for other antibiotics, activity of SkQ1 depends on the

number of cells, which is explained by its lipophilici-

ty and protonophore-like properties, allowing it to re-

duce bacterial membrane potential. Fig. 3a shows the

6-h growth curves for the samples containing different

amounts of B. subtilis cells cultured overnight in the

medium with the same concentration of SkQ1. The ob-

tained results indicate that antibacterial action of the

lipophilic cation SkQ1 decreases with the increase in

the number of cells and, consequently, the amount of

cellular membranes/lipids. Thus, despite the fact that

the protonophore-like effect of SkQ1 is mediated by

free fatty acids, addition of exogenous fatty acids may

result not in the increase in the protonophoric effect,

but in the protective effect due to competition of fatty

acid micelles with bacterial cells for SkQ1 [35]. Such

protective mechanism was previously described for

S. aureus, which was resistant to daptomycin due to

decrease in the activity of this antibiotic through the

release of membrane phospholipids into the external

environment with subsequent incorporation of the

antibiotic in phospholipid micelles [36]. In the case of

addition of exogenous fatty acids, we observed a pro-

tective effect (Table 2): MIC increased 2-4-fold, which

further confirms dependence of the antibacterial ac-

tion on the amount of membranes/lipids or micelles.

Itshould be noted that addition of exogenous fatty ac-

ids did not have a negative effect on the growth of bac-

terial cells without the addition of SkQ1.

Protective effect of dead cell population against

the action of SkQ1. Addition of the heat-killed bacte-

ria to the live S. aureus cells prevented the SkQ1-me-

diated death of live S. aureus bacteria, and the more

killed bacteria were present, the stronger the protec-

tive effect was (Fig.3b). Interestingly, with a 1000-fold

excess of dead bacteria, growth rate of live cells in-

creased approximately 2-fold relative to the control,

which can be explained not only by the SkQ1 sorption

Table 2. Increase in the minimum inhibitory concentration of SkQ1 upon addition of exogenous fatty acids (µM)

Acids Formula Fatty acid, µM SkQ1 MIC, µM

Myristic acid C

0.5 2-4

Palmitic acid C

0.5 2-4

Stearic acid C

0.5 2-4

Linoleic acid C

0.5 2-4

Without any additives – 0 1

Note. Addition of fatty acids did not affect growth rate of the B.subtilis population.

ANTIBACTERIAL PROPERTIES OF SkQs 217

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Fig. 3. Antibacterial action depends on the number of cells. a)Dependence of antibacterial action of 0.5µM SkQ1 on the num-

ber of B. subtilis cells. b)Effect of dead S. aureus cells on bacterial survival under the action of SkQ1. Bacteria at concentration

of ~60,000 CFU per ml were incubated for 3 h at 37°C in a saline solution with glucose (0.9% NaCl, 5mM D-glucose, 1μM SkQ1).

Control cells(c) were incubated without SkQ1. Relative results of bacterial survival (CFU) when incubated with 0.5μM SkQ1 in

the presence of aliquots of dead bacteria (1 to 1; 1 to 10; 1 to 100; 1 to 1000) are presented. Mean ± SEM values are given, n = 4.

*p < 0.01 when compared with the “C” sample by unpaired Student’s t-test.

on the dead cells and prevention of its toxic effect, but

also by the presence of metabolites from the dead cells

in the incubation medium. The dead cells are appar-

ently used by the live bacteria for growth and repro-

duction (necrotrophic growth)[37].

Volume of the incubation medium affects bac-

terial survival under the action of SkQ1. When

 aureus was incubated with 1 μM SkQ1, it was found

that increase in the volume of the incubation medium

leads to the significant decrease in bacterial survival

(Fig.4), which apparently indicates the ability of bacte-

ria to accumulate SkQ1 from the incubation medium.

Effect of SkQs on eukaryotic cells. Although ac-

cording to the theory [27] mitochondria of eukaryotic

cells should accumulate an order of magnitude more

SkQs than prokaryotic cells, experiments on various

cell cultures show that cytotoxic effect is observed at

higher concentrations than for prokaryotic cells.

SkQT, SkQ3, and SkQ1 had a pronounced cytotoxic

effect on the cells of the primary culture of rat renal

tubules only at concentration of 32 μM (Fig. 5a), which

is more than 2 orders of magnitude higher than the

MIC for Gram-positive bacteria and is comparable to

the MIC for E. coli.

Primary renal tubular cell culture treated with

SkQT at concentrations of 0.125-16 μM did not show

decrease in viability compared to the control group.

Moreover, at concentration of 1 μM, which is the MIC

for Gram-positive bacteria, SkQT increased the cell

survival. When incubated with SkQ3, cell viability

exhibited a tendency to increase up to concentration

of 2 μM. However, at concentration of 4 μM or more,

SkQ3 was already causing a slight decrease in viability.

SkQ1 at concentration of 0.125-4 μM did not reduce cell

viability in the primary renal tubules cell culture, and

concentrations of 0.5 and 1 μM improved cell survival.

Increasing concentration to above 8 μM led to the de-

crease in cell viability. Thus, toxicity for the primary

cell culture was comparable with what we have previ-

ously observed for immortalized HeLa cancer cells[20].

A completely different situation was observed

for the human colon carcinoma RKO cell line and

MRC5-SV40 fibroblasts (Fig. 5b). Human lung fibroblast

Fig. 4. Survival of S. aureus in various volumes of incuba-

tion medium with 1 µM SkQ1. Bacteria at concentration of

~15,000 CFU per ml were incubated for 3 h at 37°C in var-

ious volumes of the medium (0.9% NaCl, 5 mM D-glucose,

1 μM SkQ1). Results of bacterial survival (CFU) are presented.

Mean ± SEM values are provided, n = 4. * p < 0.01 when com-

pared with the control (–SkQ1) by unpaired Student’s t-test.

NAZAROV et al.218

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Fig. 5. Evaluation of mammalian cell viability upon addition of SkQs. a)Cell viability of the primary culture of rat renal tubule

cells after addition of SkQs. The cells were incubated for 24h. Cell viability was determined by using MTT test. b)Viability of

human colon carcinoma RKO cells and MRC5-SV40 fibroblasts after addition of SkQ1. The cells were incubated for 17h. Cell via-

bility was determined by using CellTiter-Blue Reagent (Promega, USA).

MRC5-SV40 cells were also not affected by the SkQ1

cytotoxicity at concentration of 1μM, but a significant

decrease in their viability was observed at 10 μM of

SkQ1. For the RKO carcinoma cells, 1 μM concentration

of SkQ1 was already causing noticeable cytotoxicity.

At the concentration of 10µM, survival was only 10%.

Thus, toxicity was significantly higher than what we

had previously observed for the HeLa cells[20], which

apparently indicates a difference in metabolism and

gene expression in these immortalized cells.

DISCUSSION

Despite the significant progress in the study of

MTAs, the mechanisms of their action on prokary-

otic and eukaryotic cells remain not fully understood.

Just like SkQ1, a well-studied MTA, other quinone

derivatives, such as SkQT [38, 39], SkQThy [40, 41],

and SkQ3 [9, 42] also exhibited pronounced antioxi-

dant properties, however, antibacterial activity has not

been demonstrated experimentally for any of them.

All living cellular organisms without exception

contain MDR pumps localized on their cell membrane.

Bacteria have six classes of these pumps divided into

two large groups: ATP-dependent pumps and H

/Na

gradient-dependent pumps [43], so it was very like-

ly that one of them could recognize SkQs and start

pumping them out, thereby increasing bacterial resis-

tance. All the more surprising is the observed fact that

all of the SkQs we have studied are pumped out by the

single AcrAB-TolC pump.

Thus, if only the AcrAB-TolC pump is capable of

pumping out SkQs, then many Gram-positive bacteria

would be sensitive to SkQs since they simply cannot

possess such pumps (with the possible exception of

Negativicutes). In the case of Gram-negative bacteria,

in which existence of such pumps is possible, resis-

tance would depend on the presence of the AcrAB-TolC

pump or a similar one, structure of which is close to

that of the E. coli AcrAB-TolC pump [22]. In the case of

eukaryotes, such as Saccharomyces cerevisiae yeast, the

SkQ1 pumping occurs due to operation of at least sev-

eral ATP-dependent pumps, including Pdr5[44].

Moreover, the main MDR pumps in eukaryotes are

ATP-dependent, since the process of energy genera-

tion is separated from the process of substance trans-

port and occurs within mitochondria and not on the

plasma membrane. Thus, decrease in the membrane

potential due to the SkQ1 protonophore-like cycle

leads to the shutdown of the H

/Na

gradient-depen-

dent pumps (such as AcrAB-TolC) in prokaryotes, but

does not result in a shutdown of the ATP-dependent

pumps in eukaryotes.

Unlike in the case of prokaryotes where the

mechanism of cytotoxic action of SkQ1 and protection

against it is sufficiently clear, no such clarity exists for

eukaryotes. Indeed, eukaryotic mitochondria should

theoretically pump out an order of magnitude more

SkQs than bacterial cells. The difference in electric

potentials between the extracellular environment and

the mitochondrial matrix in eukaryotes is approxi-

mately –240 mV (~ –60 mV on the plasma membrane

and –180 mV on the inner mitochondrial membrane),

whereas potential on the bacterial membrane is only

–180 mV. Since the eukaryotic plasma membrane has a

lower electric potential than the bacterial cell mem-

brane, accumulation of SkQs in the cytoplasm of eu-

karyotic cells should be less efficient than in bac-

terial cells. It should be noted that operation of the

AcrAB-TolC pump in the Gram-negative bacteria only

reduces the rate of SkQs accumulation on the inner

membrane of bacteria, giving the Gram-negative bac-

teria a chance to increase their biomass and, thereby,

ANTIBACTERIAL PROPERTIES OF SkQs 219

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Fig. 6. Scheme illustrating accumulation of SkQs in eukaryotic and prokaryotic cells and protective effect of the membrane-en-

closed intracellular organelles, lipid/membrane micelles, dead cells (due to SkQs deposition) and live cells (due to decrease

inthe SkQs/membrane ratio) ([27], with modifications).

reduce the ratio of SkQs to membrane fractions due to

the cell division. Results of our experiments studying

dependence of the antibacterial action of SkQ1 on the

number of cells and on the presence of dead cells con-

firm this conclusion.

Another mechanism protecting eukaryotic cells

from the toxic effects of SkQs is the presence of mem-

brane organelles (intracellular vacuoles, Golgi appa-

ratus, endoplasmic reticulum, lysosomes, endosomes,

etc.), in which lipophilic SkQs molecules could be tem-

porarily deposited, which also reduces the rate of SkQs

accumulation at the inner mitochondrial membrane.

Results of our experiments on the effect of addition

of exogenous fatty acids on the antibacterial action of

SkQ1 confirm this conclusion. Figure6 shows a scheme

of the distribution of the penetrating SkQ cation out-

side and inside eukaryotic (left) and prokaryotic (right)

cells. To be specific, for this diagram, we assume that

SkQ concentration outside of the cells is 1 pM, with

the external space serving as an infinite source of this

compound. Then, as a result of its potential-dependent

accumulation in the cytoplasm of eukaryotic cells, SkQ

concentration will be 10 pM, and ~100 nM within the

plasma membrane (due to the high membrane-water

distribution coefficient). In the case of prokaryotic

cells, SkQ concentration in the cytoplasm should be

1000 pM = 1 nM, and ~10 nM SkQ should accumulate

in the mitochondrial matrix of eukaryotic cells. When

the outside source of SkQ is limited, which should be

the case of a real-life situation, concentration of SkQ

inside the mitochondria should be below 10 nM due

to the presence of other membrane organelles in the

cells, which should effectively accumulate hydropho-

bic SkQ. Similarly, dead cells and lipid micelles outside

of the cell will effectively accumulate hydrophobic

SkQ and reduce its concentration within the live cells

in a real-life situation.

The Δψ values on the plasma membrane and the

inner mitochondrial membrane are taken to be –60 and

–180 mV, respectively. The membrane/water distribu-

tion coefficient for SkQ is assumed to be 10,000 : 1[27].

CONCLUSION

The obtained results allow us to conclude that an-

tibacterial activity of SkQs depends on the amount of

lipid components of the membranes or micelles (Fig.6).

This allows us to formulate several major reasons that

determine the increased resistance of eukaryotic cells,

namely: (i) presence of a large number of membrane

organelles (endoplasmic reticulum, Golgi apparatus,

etc.) which deposit a certain amount of SkQs within;

(ii) difference in the cell membrane potential in pro-

karyotes (~180 mV) and eukaryotes (~60 mV), which

determines slower penetration of SkQs into eukaryotic

cell; (iii) spatial separation of the processes of energy

generation (mitochondria) and substance transport

(cell membrane) in eukaryotes, in contrast to com-

bination of these processes on the cell membrane

of prokaryotes; (iv) difference in the composition of

MDR pumps on the membranes of eukaryotes (main-

ly ATP-dependent pumps) and prokaryotes (mainly

/Na

gradient-dependent pumps). Together, all four

of these major factors determine the increased resis-

tance of eukaryotic cells compared to their theoreti-

NAZAROV et al.220

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

cally expected sensitivity. Difference in the sensitivity

of different eukaryotic cell types is apparently deter-

mined by: (i) the degree of membrane organelle de-

velopment (endosome system, Golgi apparatus, etc.),

(ii) energy status of the cell [45], and (iii) level of the

MDR pump expression in them.

Acknowledgments. The authors wish to express

their gratitude to N. V. Sumbatyan for assistance in the

study and fruitful discussion of its results. The authors

are grateful to A. I. Sorochkina for help in translating

the article.

Contributions. P.A.N., Yu.N.A., M.V.S., R.A.Z., and

E.Yu.P. study concept and management; P.A.N., L.A.Z.,

A.A.B., K.G.L., and A.V.G. conducting experiments; P.A.N.,

Yu.N.A., M.V.S., R.A.Z., E.Yu.P., M.V.K., A.V.G., and K.G.L.

discussion of the results; P.A.N., M.V.S., A.V.G., E.A.K.,

R.A.Z., E.Yu.P., M.V.K., and Yu.N.A. writing the man-

uscript; P.A.N., E.A.K., E.Yu.P., and M.V.K. editing the

manuscript.

Funding. The present study was carried out with

financial support of the Russian Science Foundation

(grant no.22-15-00099, P. A. Nazarov). The experiments

on human cell lines were carried out with financial

support of the Russian Science Foundation (grant

23-14-00061, K. G. Lyamzaev). Molecular docking was

performed with support of the Moscow State Uni-

versity Interdisciplinary Scientific and Educational

School “Brain, Cognitive Systems, Artificial Intelli-

gence” (A. V. Golovin).

Ethics declarations. All the applicable interna-

tional, national and/or institutional guidelines for

the care and use of animals were followed. The pro-

tocols for working with animals were approved by

the ethical committee of animal research of the Be-

lozersky Institute of Physico-Chemical Biology, Lo-

monosov Moscow State University (Protocol 3/19

of March 18, 2019).

Conflict of interests. M. V. Skulachev is the direc-

tor of the Mitotech company which develops and com-

mercializes drugs based on SkQ-type MTAs. The au-

thors of this work declare that they have no conflicts

of interest.

REFERENCES

1. Zorova, L.D., Popkov, V.A., Plotnikov, E.Y., Silachev,

D.N., Pevzner, I.B., Jankauskas, S.S., Babenko, V.A.,

Zorov, S. D., Balakireva, A. V., Juhaszova, M., Sollott,

S.J., and Zorov, D.B. (2018) Mitochondrial membrane

potential, Anal. Biochem., 552, 50-59, doi: 10.1016/j.

ab.2017.07.009.

2. 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 membrane

potential of mitochondria, Nature, 222, 1076-1078,

doi:10.1038/2221076a0.

3. Grinius, L.L., Jasaitis, A.A., Kadziauskas, Y.P., Liber-

man, E.A., Skulachev, V.P., Topali, V.P., Tsofina, L.M.,

and Vladimirova, M.A. (1970) Conversion of biomem-

brane-produced energy into electric form. I.Submito-

chondrial particles, Biochim. Biophys. Acta, 216, 1-12,

doi:10.1016/0005-2728(70)90153-2.

4. Liberman, E.A., and Skulachev, V.P. (1970) Conversion

of biomembrane-produced energy into electric form.

IV. General discussion, Biochim. Biophys. Acta, 216,

30-42, doi:10.1016/0005-2728(70)90156-8.

5. Burns, R. J., Smith, R. A., and Murphy, M. P. (1995)

Synthesis and characterization of thiobutyltriphenyl-

phosphonium bromide, a novel thiol reagent targeted

to the mitochondrial matrix, Arch. Biochem. Biophys.,

322, 60-68, doi:10.1006/abbi.1995.

6. Murphy, M. P. (1997) Selective targeting of bioac-

tive compounds to mitochondria, Trends Biotechnol.,

15, 326-330, doi:10.1016/S0167-7799(97)01068-8.

7. Smith, R. A., Porteous, C. M., Coulter, C. V., and Mur-

phy, M. P. (1999) Selective targeting of an antioxi-

dant to mitochondria, Eur. J. Biochem., 263, 709-716,

doi:10.1046/j.1432-1327.1999.00543.x.

8. Zielonka,J., Joseph,J., Sikora,A., Hardy,M., Ouari,O.,

Vasquez-Vivar,J., Cheng,G., Lopez,M., and Kalyanara-

man, B. (2017) Mitochondria-targeted triphenylphos-

phonium-based compounds: syntheses, mechanisms

of action, and therapeutic and diagnostic applica-

tions, Chem. Rev., 117, 10043-10120, doi: 10.1021/acs.

chemrev.7b00042.

9. Skulachev, V.P. (2007) A biochemical approach to the

problem of aging: “megaproject” on membrane-pen-

etrating ions. The first results and prospects, Bio-

chemistry (Moscow), 72, 1385-1396, doi: 10.1134/

s0006297907120139.

10. Skulachev, V. P., Antonenko, Y. N., Cherepanov, D. A.,

Chernyak, B.V., Izyumov, D.S., Khailova, L.S., Klishin,

S. S., Korshunova, G. A., Lyamzaev, K. G., Pletjush-

kina, O. Y., Roginsky, V. A., Rokitskaya, T. I., Severin,

F.F., Severina, I.I., Simonyan, R.A., Skulachev, M.V.,

Sumbatyan, N. V., Sukhanova, E. I., Tashlitsky, V. N.,

Trendeleva, T. A., Vyssokikh, M. Y., and Zvyagilska-

ya, R. A. (2010) Prevention of cardiolipin oxidation

and fatty acid cycling as two antioxidant mecha-

nisms of cationic derivatives of plastoquinone (SkQs),

Biochim. Biophys. Acta, 1797, 878-889, doi: 10.1016/

j.bbabio.2010.03.015.

11. Murphy, M.P., and Smith, R.J.A. (2007) Targeting anti-

oxidants to mitochondria by conjugation to lipophilic

cations, Annu. Rev. Pharmacol. Toxicol., 47, 629-656,

doi:10.1146/annurev.pharmtox.47.120505.

12. Cunha, F.M., Caldeira da Silva, C.C., Cerqueira, F.M.,

and Kowaltowski, A.J. (2011) Mild mitochondrial un-

coupling as a therapeutic strategy, Curr. Drug Targets,

12, 783-789, doi:10.2174/138945011795528778.

ANTIBACTERIAL PROPERTIES OF SkQs 221

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

13. Zorov, D.B., Andrianova, N. V., Babenko, V. A., Pevz-

ner, 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,

doi:10.3390/brainsci11081050.

14. Shabalina, I. G., and Nedergaard, J. (2011) Mitochon-

drial (‘mild’) uncoupling and ROS production: phys-

iologically relevant or not? Biochem Soc Trans., 39,

1305-1309, doi:10.1042/BST0391305.

15. Starkov, A. A. (1997) “Mild” uncoupling of mito-

chondria, Biosci. Rep., 17, 273-279, doi: 10.1023/

a:1027380527769.

16. Korshunov, S. S., Skulachev, V. P., and Starkov, A. A.

(1997) High protonic potential actuates a mecha-

nism of production of reactive oxygen species in

mitochondria, FEBS Lett., 416, 15-18, doi: 10.1016/

s0014-5793(97)01159-9.

17. Severin, F.F., Severina, I.I., Antonenko, Y.N., Rokitska-

ya, 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) Penetrating cation/

fatty acid anion pair as a mitochondria-targeted pro-

tonophore, Proc. Natl. Acad. Sci. USA, 107, 663-668,

doi:10.1073/pnas.0910216107.

18. Anisimov, V.N., Egorov, M.V., Krasilshchikova, M.S.,

Lyamzaev, K.G., Manskikh, V.N., Moshkin, M.P., No-

vikov, E.A., Popovich, I.G., Rogovin, K.A., Shabalina,

I.G., Shekarova, O.N., Skulachev, M. V., Titova, T. V.,

Vygodin, V.A., Vyssokikh, M.Y., Yurova, M. N., Zabe-

zhinsky, M. A., and Skulachev, V. P. (2011) Effects of

the mitochondria-targeted antioxidant SkQ1 on lifes-

pan of rodents, Aging, 3, 1110-1119, doi: 10.18632/

aging.100404.

19. Khailova, L.S., Nazarov, P.A., Sumbatyan, N.V., Kor-

shunova, G. A., Rokitskaya, T. I., Dedukhova, V. I.,

Antonenko, Y. N., and Skulachev, V. P. (2015) Uncou-

pling and toxic action of alkyltriphenylphospho-

nium cations on mitochondria and the bacterium

Bacillus subtilis as a function of alkyl chain length,

Biochemistry (Moscow), 80, 1589-1597, doi: 10.1134/

S000629791512007X.

20. Nazarov, P.A., Osterman, I.A., Tokarchuk, A.V., Kara-

kozova, M. V., Korshunova, G. A., Lyamzaev, K. G.,

Skulachev, M. V., Kotova, E. A., Skulachev, V. P., and

Antonenko, Y.N. (2017) Mitochondria-targeted antiox-

idants as highly effective antibiotics, Sci. Rep., 7, 1394,

doi:10.1038/s41598-017-00802-8.

21. Nazarov, P.A., Kotova, E.A., Skulachev, V.P., and An-

tonenko, Y. N. (2019) Genetic variability of the Acr-

AB-TolC multidrug efflux pump underlies SkQ1 re-

sistance in gram-negative bacteria, Acta Naturae, 11,

93-98, doi:10.32607/20758251-2019-11-4-93-98.

22. Nazarov, P.A., Sorochkina, A.I., and Karakozova, M.V.

(2020) New functional criterion for evaluation of ho-

mologous MDR pumps, Front. Microbiol., 11, 592283,

doi:10.3389/fmicb.2020.592283.

23. Churilov, M. N., Denisenko, Y. V., Batyushin, M. M.,

Bren, A. B., and Chistyakov, V. A. (2018) Prospects of

SkQ1 (10-(6’-plastoquinoyl) decyltriphenylphosphoni-

um) application for prevention of oral cavity diseases,

Rasayan J. Chem., 11, 1594-1603.

24. Nazarov, P.A., Majorov, K.B., Apt, A.S., and Skulachev,

M.V. (2023) Penetration of triphenylphosphonium de-

rivatives through the cell envelope of bacteria of My-

cobacteriales order, Pharmaceuticals (Basel), 16, 688,

doi:10.3390/ph16050688.

25. 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, doi:10.3390/biom10020309.

26. Pavlova, J. A., Khairullina, Z. Z., Tereshchenkov,

A.G., Nazarov, P.A., Lukianov, D.A., Volynkina, I.A.,

Skvortsov, D.A., Makarov, G.I., Abad,E., Murayama,

S.Y., Kajiwara,S., Paleskava,A., Konevega, A.L., An-

tonenko, Y.N., Lyakhovich,A., Osterman, I.A., Bogdan-

ov, A.A., and Sumbatyan, N.V. (2021) Triphenilphos-

phonium analogs of chloramphenicol as dual-acting

antimicrobial and antiproliferating agents, Antibiotics

(Basel), 10, 489, doi:10.3390/antibiotics10050489.

27. Antonenko, Y. N., Avetisyan, A. V., Bakeeva, L. E.,

Chernyak, B.V., Chertkov, V.A., Domnina, L.V., Ivano-

va, O.Y., Izyumov, D.S., Khailova, L.S., Klishin, S.S.,

Korshunova, G. A., Lyamzaev, K. G., Muntyan, M. S.,

Nepryakhina, O. K., Pashkovskaya, A. A., Pletjushki-

na, O.Y., Pustovidko, A.V., Roginsky, V.A., Rokitskaya,

T. I., Ruuge, E. K., Saprunova, V.B., Severina, I.I., Si-

monyan, R.A., Skulachev, I.V., Skulachev, M.V., Sum-

batyan, N.V., Sviryaeva, I.V., Tashlitsky, V.N., Vassiliev,

J.M., Vyssokikh, M.Y., Yaguzhinsky, L.S., Zamyatnin,

A.A.,Jr., and Skulachev, V.P. (2008) Mitochondria-tar-

geted plastoquinone derivatives as tools to interrupt

execution of the aging program. 1. Cationic plas-

toquinone derivatives: synthesis and invitro studies,

Biochemistry (Moscow), 73, 1273-1287, doi: 10.1134/

s0006297908120018.

28. Korshunova, G. A., Shishkina, A. V., and Skulachev,

M. V. (2017) Design, synthesis, and some aspects

of the biological activity of mitochondria-targeted

antioxidants, Biochemistry (Moscow), 82, 760-777,

doi:10.1134/S0006297917070021.

29. Baba,T., Ara,T., Hasegawa,M., Takai,Y., Okumura,Y.,

Baba, M., Datsenko, K. A., Tomita, M., Wanner, B. L.,

and Mori, H. (2006) Construction of Escherichia coli

K-12 in-frame, single-gene knockout mutants: the Keio

collection, Mol. Syst. Biol., 2, 2006.0008, doi: 10.1038/

msb4100050.

30. Clinical and Laboratory Standards Institute (CLSI)

(2012) Methods for Dilution Antimicrobial Suscepti-

NAZAROV et al.222

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

bility Tests for Bacteria That Grow Aerobically, 9th

ed.; CLSI Document M07-A9, Approved Standard;

CLSI: Wayne, PA, USA; Vol. 32, URL: https://clsi.org/

media/1928/m07ed11_sample.pdf.

31. Eshboev, F., Karakozova, M., Abdurakhmanov, J., Bo-

bakulov, K., Dolimov, K., Abdurashidov, A., Baymir-

zaev, A., Makhnyov, A., Terenteva, E., Sasmakov, S.,

Piyakina, G., Egamberdieva, D., Nazarov, P. A., and

Azimova, S. (2023) Antimicrobial and cytotoxic ac-

tivities of the secondary metabolites of endophytic

fungi isolated from the medicinal plant Hyssopus of-

ficinalis, Antibiotics (Basel), 12, 1201, doi: 10.3390/

antibiotics12071201.

32. Alhossary,A., Handoko, S.D., Mu,Y., and Kwoh, C.K.

(2015) Fast, accurate, and reliable molecular dock-

ing with QuickVina 2, Bioinformatics, 31, 2214-2216,

doi:10.1093/bioinformatics/btv082.

33. Wójcikowski, M., Zielenkiewicz, P., and Siedlec-

ki, P. (2015) Open Drug Discovery Toolkit (ODDT): a

new open-source player in the drug discovery field,

J.Cheminform., 7, 26, doi:10.1186/s13321-015-0078-2.

34. The PyMOL Molecular Graphics System, Version 2.0

Schrödinger, LLC, URL: https://pymol.org/2/.

35. Nazarov, P.A. (2018) Alternatives to antibiotics: phage

lytic enzymes and phage therapy, Bull. Russ. State

Med. Univ., 1, 5-15, doi:10.24075/brsmu.2018.002.

36. Pader,V., Hakim,S., Painter, K.L., Wigneshweraraj,S.,

Clarke, T. B., and Edwards, A. M. (2016) Staphylo-

coccus aureus inactivates daptomycin by releasing

membrane phospholipids, Nat. Microbiol., 2, 16194,

doi:10.1038/nmicrobiol.2016.194.

37. Temmerman, R., Vervaeren, H., Noseda, B., Boon, N.,

and Verstraete,W. (2006) Necrotrophic growth of Le-

gionella pneumophila, Appl. Environ. Microbiol., 72,

4323-4328, doi:10.1128/AEM.00070-06.

38. Genrikhs, E.E., Stelmashook, E.V., Popova, O.V., Kapay,

N.A., Korshunova, G.A., Sumbatyan, N.V., Skrebitsky,

V.G., Skulachev, V.P., and Isaev, N.K. (2015) Mitochon-

dria-targeted antioxidant SkQT1 decreases trauma-

induced neurological deficit in rat and prevents am-

yloid-β-induced impairment of long-term potentiation

in rat hippocampal slices, J.Drug Target, 23, 347-352,

doi:10.3109/1061186X.2014.997736.

39. Rogov, A.G., Goleva, T.N., Trendeleva, T.A., Ovchen-

kova, A.P., Aliverdieva, D.A., and Zvyagilskaya, R.A.

(2018) New data on effects of SkQ1 and SkQT1 on rat

liver mitochondria and yeast cells, Biochemistry (Mos-

cow), 83, 552-561, doi:10.1134/S0006297918050085.

40. Goleva, T.N., Rogov, A.G., Korshunova, G.A., Trende-

leva, T.A., Mamaev, D.V., Aliverdieva, D.A., and Zvy-

agilskaya, R.A. (2019) SkQThy, a novel and promising

mitochondria-targeted antioxidant, Mitochondrion,

49, 206-216, doi:10.1016/j.mito.2019.09.001.

41. Epremyan, K.K., Rogov, A.G., Goleva, T.N., Lavrushki-

na, S.V., Zinovkin, R.A., and Zvyagilskaya, R.A. (2023)

Altered mitochondrial morphology and Bioenergetics

in a new yeast model expressing Aβ42, Int.J. Mol. Sci.,

24, 900, doi:10.3390/ijms24020900.

42. Eremeev, S.A., Motovilov, K.A., Volkov, E.M., and Yagu-

zhinsky, L.S. (2011) SkQ3: The new member of the class

of membranotropic uncouplers, Biochem. Moscow Sup-

pl. Ser.A,

5, 310-315, doi:10.1134/S1990747811050047.

43. Nazarov, P.A. (2022) MDR pumps as crossroads of re-

sistance: antibiotics and bacteriophages, Antibiotics

(Basel), 11, 734, doi:10.3390/antibiotics11060734.

44. Knorre, D.A., Markova, O.V., Smirnova, E.A., Karavae-

va, I.E., Sokolov, S.S., and Severin, F.F. (2014) Dodec-

yltriphenylphosphonium inhibits multiple drug resis-

tance in the yeast Saccharomyces cerevisiae, Biochem.

Biophys. Res. Commun., 450, 1481-1484, doi: 10.1016/

j.bbrc.2014.07.017.

45. Nazarov, P.A., Khrulnova, S.A., Kessenikh, A.G., No-

voyatlova, U. S., Kuznetsova, S. B., Bazhenov, S. V.,

Sorochkina, A. I., Karakozova, M. V., and Manukhov,

I.V. (2023) Observation of cytotoxicity of phosphoni-

um derivatives is explained: metabolism inhibition

and adhesion alteration, Antibiotics (Basel), 12, 720,

doi:10.3390/antibiotics12040720.

Publisher’s Note. Pleiades Publishing remains

neutral with regard to jurisdictional claims in pub-

lished maps and institutional affiliations.