Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 11, pp. 1536-1552 © Pleiades Publishing, Ltd., 2025.

Russian Text © The Author(s), 2025, published in Biokhimiya, 2025, Vol. 90, No. 11, pp. 1638-1656.

1536

REVIEW

New Aspects of Protein Biosynthesis Inhibition

by Proline-Rich Antimicrobial Peptides

Olga V. Shulenina

, Eugene A. Tolstyko

, Andrey L. Konevega

1,2,3,a

and Alena Paleskava

1,2,b

Petersburg Nuclear Physics Institute named by B.P. Konstantinov

of National Research Center “Kurchatov Institute”, 188300 Gatchina, Russia

Peter the Great St. Petersburg Polytechnic University, 195251 St. Petersburg, Russia

National Research Center “Kurchatov Institute”, 123098 Moscow, Russia

e-mail: konevega_al@pnpi.nrcki.ru

e-mail: polesskova_ev@pnpi.nrcki.ru

Received July 31, 2025

Revised October 30, 2025

Accepted November 3, 2025

Abstract— Proline-rich antimicrobial peptides (PrAMPs) are promising compounds for overcoming antibiotic

resistance, one of the global health threats, and stand out from other types of AMPs by their high safety

profile. The main cellular target of PrAMPs, like most modern antibiotics, is the conservative cellular struc-

ture – the ribosome. PrAMPs bind in the ribosomal tunnel, forming multiple interactions with nucleotides of

23SrRNA, and are divided into two classes depending on their mechanism of action: inhibition of elongation

or termination. The N-terminal part of the peptides, which is important for the activity of class I peptides,

extends into the A-site pocket, preventing the binding of aminoacyl-tRNA. A new family of PrAMPs, rumicidins,

was discovered using genomic search methods. Its representatives have the longest N-terminal part, as well

as a unique pair of amino acids Trp23 and Phe24 at the C-terminus. The Trp-Phe dyad forms a spacer at the

constriction site of the ribosomal tunnel, stabilizing the binding and leading to increased antibacterial activity.

New structural studies of the class I peptide Bac5 have demonstrated its ability to disrupt the correct position-

ing of the CCA-end of the P-site tRNA in the peptidyltransferase center of the ribosome, which can affect the

assembly of functional initiation complexes. Class II PrAMPs, according to new data, have additional binding

sites on the ribosome and have a complex effect on the bacterial cell: they disrupt the termination of pro-

tein synthesis, block the cellular ribosome release system, prevent the correct assembly of the 50S ribosomal

subunits, and, possibly, affect the first stage of translocation. Recent studies expand our understanding of the

antimicrobial activity of PrAMPs and contribute to the creation of future therapeutic drugs based on AMPs.

DOI: 10.1134/S0006297925602394

Keywords: ribosome, proline-rich antimicrobial peptides, PrAMPs, translation, antibiotics

* To whom correspondence should be addressed.

INTRODUCTION

Antimicrobial peptides (AMPs) are genetically en-

coded components of an organism’s innate immune

system [1]. Although AMPs are synthesized by the

representatives of all kingdoms of life, the primary

sources of known AMPs are animals (78%), bacteria

(12%), and plants (8%) (Fig.  1). According to their

name, most AMPs target bacteria and fungi, though

some also exhibit antiviral, antiparasitic, and anti-

tumor properties  [2]. For potential therapeutic appli-

cations, animal-derived AMPs are the most promis-

ing[3]. Pathogens develop resistance to AMPs far less

readily than to low-molecular-weight antibiotics, and

the genetic barriers are higher, impeding the hori-

zontal transfer of resistance gene between bacterial

species [4, 5].

AMPs are structurally diverse and target both

membranes, disrupting their barrier function, and

intracellular components, inhibiting vital processes

such as DNA repair, replication, transcription, trans-

lation, protein folding, and cell division [2]. A recent

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Fig. 1. Antimicrobial peptides (AMPs): sources, targets, and mechanisms of action (numerical values taken from the Anti-

microbial Peptide Database APD; https://aps.unmc.edu/ as of January 2025).

discovery has revealed the ability of polyproline

AMPs to disrupt bacterial biofilms [6]. The antibac-

terial mechanism of AMPs is often multifaceted. For

instance, amphibian AMPs can not only form pores

in bacterial membranes but also block membrane

transport by affecting the expression and assembly

of membrane proteins, inhibit efflux pump activity,

disrupt energy metabolism by targeting ATP synthase,

and inhibit function of FtsZ, a GTPase critical for cell

division that has no direct analog in eukaryotic cells

[7, 8]. Beyond direct elimination of infectious agents,

AMPs can also modulate the host immune response by

attracting and activating immune cells, which enhance

pathogen destruction and inflammation control [9].

Most AMPs function by compromising the integ-

rity of bacterial membranes, demonstrating activity

against both Gram-negative and Gram-positive bacte-

ria [10, 11]. However, these membranolytic properties

are often associated with a poor safety profile for

injectable substances, so they are primarily consid-

ered for topical use [12]. In contrast, certain classes

of AMPs can enter bacterial cells non-destructively

through porins to inhibit intracellular targets. This

mode of action makes them more promising as lead

compounds for systemic therapy [13, 14].

MAIN CHARACTERISTICS

OF PROLINE-RICH ANTIMICROBIAL PEPTIDES

Proline-rich antimicrobial peptides (PrAMPs) effi-

ciently penetrate bacterial cells without compromising

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Fig. 2. Proline-rich peptides: synthesis and mechanisms of penetration into bacterial cells. PrAMPs are synthesized on the

eukaryotic 80S ribosomes as inactive prepropeptides (pre-pro-AMP). The signal pre-sequence is cleaved off after directing

the immature propeptide (pro-AMP) to large granules. In the extracellular space, pro-AMP is activated by peptidases and

translocated into periplasmic space via the OmpF porin. Partial export of the peptide from the periplasmic space to the

external environment can be carried out by the MacAB-TolC efflux pump. Transport of PrAMPs through the inner membrane

into the cytoplasm is mediated by the proteins SbmA, YjiL/MdtM, and YgdD. Once inside the cytoplasm, PrAMPs interact

with the target, prokaryotic 70S ribosome.

membrane integrity. They are characterized by a pro-

line content exceeding 25%, a net positive charge, an-

timicrobial activity, and a mechanism of action that

involves binding intracellular targets like the DnaK

chaperone or 70S ribosome. Structurally, most PrAMPs

contain a characteristic PRP motif, though this is not

a strict requirement [15,  16]. Beyond their primary

antibacterial role, many PrAMPs also exhibit antifun-

gal activity [17]. Furthermore, some representatives of

this class demonstrate immunomodulatory functions,

such as enhancing chemotaxis and differentiation of

dendritic cells, activating adaptive immune respons-

es, and suppressing the release of pro-inflammatory

mediators [18, 19]. The functional repertoire of these

peptides may be even broader for those that incorpo-

rate additional domains alongside the proline-rich re-

gions, such as cysteine-rich or acidic amino acid-rich

domains, potentially leading to a wider spectrum of

biological activity [15].

PrAMPs are predominantly found in arthropods,

such as beetles, bees, wasps, flies and crabs, and in

mammals, like sheep, cows, pigs, goats, dolphins.

To date, only a single PrAMP has been identified in

a plant (rapeseed) [15, 20]. The range of methods

for discovering these peptides has expended signifi-

cantly. The first PrAMP, apidaecin (Api), was identi-

fied after injecting honeybees with a sublethal dose

of Escherichia coli and isolating the induced peptide

chromatographically [21]. In contrast, modern discov-

ery relies on bioinformatic analysis of whole-genome

sequencing data, an approach that has recently led

to the identification of new insect [22] and cetacean

PrAMPs [13], and an entire family of proline-rich

cathelicidins from ruminants, named rumicidins

(Rum) [23]. Notably, humans and other primates do

not produce PrAMPs [24], making the therapeutic

prospect for supplementing this gap in the innate

immune defense particularly attractive. Additionally,

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PrAMPs exhibit high stability against proteolytic deg-

radation, conferred by their increased proline con-

tent, and demonstrate no toxicity toward mammalian

cell lines [25].

In animals, PrAMPs are synthesized by eukary-

otic ribosomes as inactive precursors known as pre-

propeptides. The pre-region is a signal peptide that

directs the immature PrAMP to large granules and is

subsequently cleaved off [26, 27] (Fig.2). This biosyn-

thetic pathway ensures protection of the host’s own

ribosomes and ability to store antimicrobial compo-

nents, enabling a rapid response to bacterial attack.

The remaining propeptide consists of an inactivating

pro-sequence attached to the AMP. In mammalian

PrAMPs [28, 29] and in the fruit fly’s drosocin [30] the

pro-sequence is linked to a single antimicrobial pep-

tide. The propeptides of most insect PrAMPs contain

multiple, nearly identical AMP isoforms separated by

short, conserved oligopeptide spacers [21, 31]. Final

activation occurs through proteolytic cleavage, which

releases the mature PrAMP. The active peptide is then

transported to the extracellular space or into phago-

somes to exert its action on bacterial target [27, 32].

Many PrAMPs at their minimum inhibitory con-

centration (MIC) (Table  1), do not permeabilize the

bacterial membrane, but instead inhibit intracellu-

lar targets after passing through it [23, 33, 34]. Cur-

rent data support a multi-stage penetration model of

PrAMPs into the Gram-negative bacterial cells (Fig.2).

PrAMP electrostatically interacts with the outer mem-

brane. The peptide is then translocated into the peri-

plasmic space via the OmpF porin. Notably, a portion

of the peptide may be exported to the external envi-

ronment by the MacAB-TolC efflux pump [23]. Further

transport of the peptide through the inner membrane

into the cytoplasm is mainly carried out by the SbmA

protein [35, 36], a permease family protein mediating

the proton-dependent transfer of various antimicro-

bial agents [37, 38]. Subsequent research revealed an

alternative non-lytic uptake pathway in the E.  coli

ΔsbmA cells without permeabilization and significant

decrease in MIC for the PrAMPs such as several bac-

tenecin  7 (Bac7) variants, and cetacean peptides Bal1

and Lip1 [13, 39]. To date, at least two additional in-

ner membrane PrAMP transporters have been identi-

fied: YjiL/MdtM and YgdD. The role of YjiL/MdtM has

been confirmed in the internalization of natural ru-

minant peptides Bac7(1-35) and rumicidin  1 (Rum-1),

aphalin Tur1A, and the synthetically optimized onco-

cins (Onc) Onc18 and Onc112 [23, 37, 40]. YgdD me-

diates the uptake of the crab PrAMP arasin 1(1-23),

and the insect peptides apidaecin1b (Api-1b) and pyr-

rhocoricin (Pyr) [14, 41]. Functional characterization

using individual knockout strains of each transporter

established a clear hierarchy among these transport-

er systems. SbmA emerges as the primary importer,

while YgdD and YjiL/MdtM serve as less important

secondary transporters.

To date, two intracellular targets have been iden-

tified for PrAMPs, both of which ultimately disrupt the

production of functional bacterial proteins. In 2000s,

it was demonstrated that certain PrAMPs bind to the

substrate-binding pocket of Hsp70 chaperone DnaK,

thereby preventing proper protein folding [42,  43].

However, the observed sensitivity of DnaK-deficient

lines to PrAMPs pointed to the existence of addition-

al targets [44]. Subsequent studies revealed that the

main target for PrAMPs is the bacterial ribosome [45],

like for more than half of conventional low-molec-

ular-weight antibiotics. The ribosome’s central role

in translation process and its structural conservation

across bacterial species make it an ideal target for

various antimicrobial agents [46].

Based on their mechanism of action, PrAMPs

are categorized into two classes that target different

Table 1. Minimum inhibitory concentration (MIC) val-

ues of full-length and truncated PrAMPs against E.coli

strain BW25113

Peptide MIC, µM References

Bac7(1-35) 3 [40]

Bac7(1-16) 8 [39]

Bac5(1-25) 1 [62]

Bac5(1-15) 64 [63]

Bac5(3-25) 4 [63]

Onc72 7 [37]

Onc112 2 [37]

Rum-1 2 [23]

Rum-1(1-22) 16 [23]

Rum-1(9-29) 8 [23]

Rum-1(11-29) 8 [23]

Tur1A 1.2 [40]

Tur1A(1-16) 4 [40]

Bal1 1 [13]

Lip1 1 [13]

Api88 0.4 [37]

Api137 2 [37]

Api137(1-17) 29 [37]

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stages of the translation cycle. ClassI peptides, which

include oncocins and Bac7-like cathelicidins, bind to

the initiation ribosomal complex and act during the

elongation by sterically preventing binding of the first

aminoacyl-tRNA in the A-site. In contrast, classII pep-

tides, which include apidaecins and drosocin, bind to

the ribosome after release of the synthesized poly-

peptide chain. They prevent dissociation of release

factors RF1 and RF2 from the termination complex

[20,  47,  48]. Both classes of PrAMPs bind within the

nascent polypeptide exit tunnel (NPET) of the large

ribosomal subunit. The tunnel is primarily formed by

the negatively charged 23S rRNA, and peptide binding

is stabilized by a network of hydrogen bonds, stacking

interactions, and van der Waals forces [23, 40, 49-51].

This multi-point binding mechanism significantly re-

duces the risk of bacterial resistance developing

through single-point mutations in the 23S rRNA [52,

53]. Structural data demonstrate that the majority of

proline-rich peptides adopt a rigid, typeII polyproline

helix conformation [54]. This structure is critical for

their function: it allows efficient translocation through

the bacterial membranes without causing disruption

of the lipid structure [55] and facilitates penetration

into the NPET. Moreover, due to this conformation,

PrAMPs poorly bind to the active site of proteases,

which also contributes to their stability [56].

INTERACTION OF CLASS I PrAMPs

WITH THE RIBOSOME

Class I PrAMPs bind to the NPET in reverse orien-

tation relative to the growing polypeptide chain, i.e.,

N-terminus of the PrAMP is directed toward the pep-

tidyltransferase center (PTC), and C-terminus extends

deep into the NPET.

The N-terminal part of all class  I peptides pro-

trudes into the A-site tRNA binding pocket, howev-

er the number and composition of amino acids, as

well as contacts formed with 23S rRNA, significantly

vary between the arthropod and mammalian PrAMPs

(Fig. 3). The insect peptides (Onc, Pyr, metalnikowin)

are characterized by the short, conserved N-terminal

part consisting of 2 amino acids, valine and aspar-

tate, whose deletion disrupts binding to the bacterial

ribosome, indicating key role of the N-terminal regions

in antibacterial activity [49]. The N-terminal part of

mammalian peptides (Bac7, PR-39, Tur1A) is extend-

ed to 4-5 amino acids and consists mainly of argi-

nine residues. In  vitro studies have shown that two

N-terminal arginine residues are required for efficient

penetration into bacterial cells, and their removal

reduces antibacterial activity of the peptides [57].

Deletion of the first 4 amino acids almost complete-

ly inactivates the peptide, disrupting its interaction

with the main target – the ribosome [52, 58]. Despite

the increased length of N-terminus of the mammali-

an peptides, it does not extend deeper into the A-site

pocket than the insect peptides. High arginine content

in this region allows formation of a compact, posi-

tively charged loop, ideally suited in size and charge

for anchoring interactions with the groove formed by

the nucleotides C2452, A2453, and G2454 on one side

and nucleotides U2493 and G2494 of 23S rRNA on the

other, which is likely a characteristic feature of the

mammalian PrAMPs aimed at increasing the strength

of peptide-ribosome interaction [40,  52]. Thus, the

N-terminal parts of the peptides are fundamentally

important for antibacterial activity of all character-

ized representatives of class I PrAMPs, except for ru-

micidins, whose interaction with the ribosome will be

discussed in detail below.

Central part of the class I PrAMPs, corresponding

to the residues 3-11 of Onc and 6-14 of Bac7, is locat-

ed mainly in the A-site cleft but also occupies a small

area of the A-site pocket and the upper part of the

tunnel. This is a highly conserved so-called consensus

sequence (R/K)XX(R/Y)LPRPR containing the invariant

PRP motif. Conformation of the consensus part of all

structurally characterized peptides in the NPET of

70S ribosome complex is almost completely identical

[23, 40, 49-52].

The residues Lys3 and Arg9, common to the insect

PrAMPs (Onc112, metalnikowin, Pyr), form hydrogen

bonds with A2453 and U2584, respectively, and Arg9

additionally participates in stacking interactions with

C2610. The residues 5-7 of the insect PrAMPs corre-

spond to the residues 8-10 of the mammalian PrAMPs.

Tyr6 (Onc112, metalnikowin, Pyr) and the correspond-

ing residue Arg9 of Bac7 and Tyr9 of Tur1A form

stacking interactions with C2452 of 23S rRNA. The

mammalian PrAMPs contain more arginine residues

than the insect PrAMPs, establishing more stacking in-

teractions in the NPET: Arg15 and Arg16 of the Tur1A

peptide with U2586 and His69 of L4, respectively, and

Arg12, Arg14, and Arg16 of the Bac7 peptide with

C2610 (similar to Arg9 of insect PrAMPs), U2586, and

A2062, respectively [40, 49, 51, 52].

Replacement of any residue in the Arg9-Leu10-

Pro11 region of Bac7(1-16) drastically reduces its

potential to inhibit bacterial protein synthesis and

growth of E.  coli cells, while substitutions in other

regions have little effect on its activity [39]. Addition-

ally, the use of deep mutational scanning, which al-

lowed analysis of antibacterial potential of more than

600,000 variants of Bac7(1-23), demonstrated high im-

portance of preserving the native sequence of Bac7

at positions 6 and 9-14, with the most pronounced

invariance in the 9-11 segment [59].

The C-terminal residues of class I PrAMPs are

considered non-conserved and less important for

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Fig. 3. Alignment of amino acid sequences and spatial models of class I PrAMPs (Rum-2, PDB ID: 9D89; Bac7, PDB ID: 5HAU;

Onc112, PDB ID: 4Z8C; and Tur1A, PDB ID: 6FKR). The consensus region is indicated by the dashed line.

ribosome binding and inhibitory activity. In particu-

lar, the C-terminal region of Bac7 can be truncated

to form peptides of 35, 23, and even 16 amino ac-

ids with minimal loss of antibacterial activity [60].

Similarly, antimicrobial activity of Tur1A was only

slightly reduced when the peptide was truncated at

the C-terminus to 16 amino acids [40]. In both cases,

central part of the peptides, which ensures strong in-

teraction with the ribosome, remained intact. Further

truncation of Bac7 by just one amino acid to the 1-15

fragment [60], as well as deletion of 7 C-terminal res-

idues from the initially shorter insect peptide to form

the OncΔ7 peptide [51], resulted in complete loss of

peptide activity, likely due to the critical proximity of

the C-terminus to the consensus region. Additionally,

it is worth noting that in the majority of available

structures of the class I PrAMPs, C-terminal residues

are poorly resolved [40, 49-52], which could indicate

structural heterogeneity.

Against the background of uniformity of the se-

quences and structures of the above-described pep-

tides, it is interesting to focus on the recently char-

acterized representatives of the class I PrAMPs, which

have a number of unique features.

Fixation of rumicidins in the constriction area

of the nascent polypeptide exit tunnel. The search

for cathelicidin genes in the whole-genome sequenc-

ing database of Cetartiodactyla using a fragment of

the cathelicidin-like domain of the Bac7 precursor led

to the discovery of CATHL(3L2/8)-like genes encod-

ing an entire family of relatively short (28–30  ami-

no acids) ruminant PrAMPs, named rumicidins [23].

Despite the high degree of sequence conservation

among the rumicidins, the greatest variability was

found in the N-terminal region, which also dramat-

ically differed from the N-termini of the previously

described PrAMPs. The N-terminal region of rumici-

dins consists of 10 amino acids, exceeding the N-ter-

mini of the peptides from other mammals by at least

5 amino acids (Fig. 3). Although all rumicidins begin

with an arginine residue, unlike the previously char-

acterized mammalian peptides, none of the rumici-

din representatives have this residue in the second

position, which could indicate a somewhat different

mechanism of penetration into bacterial cell. Anoth-

er key difference between the rumicidins and other

class I PR-AMP representatives is the change in the

consensus sequence (R/K)XX(R/Y)LPRPR, which is nec-

essary for strong interaction with the NPET [13, 39]:

in all representatives of the new class, the key func-

tional site (Arg/Tyr)-Leu is replaced by His-Arg. Sev-

eral representatives of the rumicidins, demonstrating

the greatest differences in the N-terminal sequence,

were selected for structural and functional characteri-

zation: rumicidin 1 (Rum-1) from the Tibetan antelope

chiru Pantholops hodgsonii, rumicidin2 (Rum-2) from

the African antelope hirola Beatragus hunteri, and

rumicidin 3 (Rum-3) from the American pronghorn

Antilocapra americana [23].

The in vivo experiments demonstrated absence

of lytic activity of rumicidins against the bacteri-

al membrane and involvement of the porin- and

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Fig. 4. Interaction of rumicidin 2 (Rum-2) with the bacterial ribosome (PDB ID: 9D89). Rum-2 (shown in red) is located in

the NPET of the 70S initiation complex containing the initiator tRNA in the P-site (shown in green), sterically preventing

binding of tRNA in the A-site (tRNA model shown in purple). Rum-2 creates a “spacer” at the constriction site of the tunnel,

which is formed by the rRNA nucleotides and loops of the ribosomal proteins uL4 (shown in orange) and uL22 (shown

in light blue): Pro22 forms a hydrogen bond with Arg61 of the ribosomal protein uL4; Trp23 forms a stacking interaction

with the nucleotide A751 of 23S rRNA (shown in blue) and hydrogen bonds with Lys90 of the ribosomal protein uL22; a

CH–π- interaction occurs between Phe24 and Thr65 of ribosomal protein uL4.

SbmA-mediated transport for penetration into bacte-

rial cells, while the MacAB-TolC pump was involved

in the export of the peptide from the periplasmic

space (Fig.  2). The antibacterial effect applies to a

number of Gram-negative bacteria belonging to the

“ESKAPE” group of pathogens and some mycobacteria.

A panel of in  vitro studies showed that addition of

rumicidins led to ribosome stalling at the start codon,

prevented formation of the first peptide bond, and,

similar to Bac7(1-22), reduced the level of GFP pro-

tein fluorescence in the coupled transcription/trans-

lation system. Moreover, the structure of one of the

class representatives, Rum-2, bound to the 70S initi-

ation complex containing fMet-tRNA

fMet

in the P-site,

showed that rumicidins bind to the NPET in an orien-

tation opposite to the nascent peptide chain (Fig. 4).

Thus, it was convincingly shown that rumicidins, de-

spite their differences from the previously described

peptides, belong to the class I PrAMPs and inhibit

translation by preventing elongation [23].

Quality of the Coulomb potential map detect-

ed in the NPET allowed unambiguous fitting of the

rumicidin  2 residues 12-27. Density for the first 11

amino acids of the N-terminal part of Rum-2 was not

observed, likely due to the high mobility of this re-

gion [23]. However, analysis of the sequence of this

region of rumicidin suggests some spatial similarity

to the previously characterized mammalian peptides

[40, 49, 52]. The arginine-rich sequence of the first 6

residues can form a structure resembling the compact

N-terminal loop of Bac7 and Tur1A, but this struc-

ture is likely somewhat extended into the space of

the A-site pocket due to the presence of two prolines

at positions 7 and 8, found in all rumicidins.

Truncation of the N-terminus of Bac7 by just 4

amino acids (RRIR) led to the significant (32-fold and

more) decrease in antimicrobial activity against E.  coli

and Salmonella enterica strains [58]. For the most com-

parable in length to Bac7(5-35) truncated rumicidins,

Rum-1(9-29) and Rum-1(11-29), the MIC values against

E.  coli BW25113 increased only 4-fold (8µM) compared

to the full-length Rum-1 (2  µM) (Table  1). Truncation

of the N-terminus of Rum-1 by less than 9 amino ac-

ids had virtually no effect (a 2-fold increase in MIC

values compared to the full form of the peptide) [23].

Notably, the effect on protein synthesis inhibition in

the in vitro transcription/translation system differed

significantly for the tested variants of Rum-1(9-29) and

Rum-1(11-29). Disruption of the reporter protein pro-

duction was observed at 2  µM concentration of the

full-length peptide and 4  µM of Rum-1(9-29), while de-

letion of two additional amino acid residues increased

the concentration required to achieve a similar effect

for the truncated peptide (Rum-1(11-29)) by 16-fold

(32  µM), indicating a potentially important role of

amino acid residues at positions 9 and 10 of rumi-

cidins in inhibiting bacterial ribosomes. Meanwhile,

functional significance of the first 8 amino acids of

the N-terminus of rumicidins remains unclear, as their

deletion did not lead to pronounced disruption of the

peptide transport into the cell or significant inhibition

of protein biosynthesis. It can be assumed that the

unique elongated N-terminal region of rumicidins is

important at the stages preceding penetration of the

peptide into bacterial cell. Negative effect of N-termi-

nal deletions could also be compensated by the addi-

tional features of rumicidins that are absent in other

representatives of mammalian PrAMPs.

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All rumicidins have two amino acid substitu-

tions in the consensus fragment (R/K)XX(R/Y)LPRPR:

the highly conserved (Arg/Tyr)-Leu dyad is replaced

by the His-Arg pair, which is not found at these posi-

tions in other class I PrAMPs. Meanwhile, the central

part of Rum-2 (12-19 amino acids) aligns well with

the residues of the consensus sequence of the struc-

turally characterized mammalian peptides and forms

similar contacts with rRNA [40, 49, 52] (Fig. 3). Nota-

bly, correcting the consensus sequence of rumicidins

to the canonical form had no effect on the MIC val-

ues and efficiency of the protein synthesis inhibition

[23]. While several independent studies have shown

that the presence of the Arg-Leu dyad in the Bac7

sequence is crucial for antibacterial activity of the

peptide [39, 59].

A unique feature of the rumicidin 2 structure

was a clear density of the peptide in the upper

and the beginning of the middle part of the NPET,

allowing identification of the contacts of deeply lo-

cated amino acid residues in the NPET [23] (Fig. 4).

The most interesting are the contacts between the

peptide and constriction area of the NPET, which

is formed not only by rRNA nucleotides but also by

the loops of ribosomal proteins uL4 and uL22. Pro22

forms a hydrogen bond with Arg61 of the ribosomal

protein uL4, Trp23 forms a stacking interaction with

the nucleotide A751 of helix H34 of 23S rRNA and

hydrogen bonds with Lys90 of the ribosomal protein

uL22. A CH–π-interaction occurs between Phe24 and

Thr65 of the ribosomal protein uL4. Thus, Rum-2 cre-

ates a “spacer” at the constriction site of the NPET

[23]. Previously, a similar structure was shown at the

constriction site of the NPET for the class II PrAMP

Api137: Tyr7 formed an interaction with A751 of the

helix H34 of 23S rRNA, and Arg10 formed a hydrogen

bond with Arg61 of the ribosomal protein uL4 [53].

It is important to note that for the truncated vari-

ant of rumicidin Rum-1(1-22), concentration leading

to inhibition of the in  vitro transcription/translation

was 32  µM (16 times higher than for Rum-1), and

the MIC against E.  coli BW25113 was 16  µM (8 times

higher than for Rum-1). Meanwhile, the C-terminal

truncated form of Bac7, Bac7(1-16), in the similar

experiment demonstrated high inhibition efficien-

cy at concentration of 5  µM, and the MIC against

E.  coli BW25113 was 8  µM (2.7 times higher than for

Bac7(1-35)) [39]. The MIC values for Bac7(1-16) against

E.  coli and S.  enterica strains increased only 2-4 times

compared to the full-length form [58]. Thus, it can

be concluded that for maintaining activity of rumi-

cidins both in  vitro and in  vivo, the C-terminal part

and the N-terminal part starting from the 9th amino

acid (in the case of Rum-1) are necessary, while ac-

tivity of Bac7 is ensured by the presence of the full

N-terminal part.

Importance of the C-terminal part of rumicidins

is determined by the presence of a pair of conserved

amino acids Trp23 and Phe24 in all representatives

of this family, as demonstrated by the N-terminal

truncated peptide variant with modifications at these

positions. Concentration of Rum-1(9-29, W23A, F24A)

required to inhibit protein synthesis in the in  vitro

system was 16 times higher than that of Rum-1 and

8 times higher than of Rum-1(9-29). It is interesting to

note that aromatic amino acid residues at the C-ter-

minus are present in many PrAMPs: in the insect

peptides (Onc, Pyr), it is tyrosine; in the mammalian

peptides, it is one (Bac7, Tur1A) or two (RP-39) phe-

nylalanines; moreover, the Trp-Phe pair was found in

the cetacean PrAMP, Lip1 [13]. However, functional

role of the Trp-Phe dyad in Lip1, as well as other

C-terminal aromatic amino acid residues, has not yet

been determined, as most studies have focused on

substitutions of amino acid residues in the N-terminal

and central parts of the peptides [39, 41]. In this re-

gard, the results of deep mutational scanning studies

are extremely interesting, as they revealed that the

C-terminal part of Bac7 has great potential for optimi-

zation. The study showed that insertion of a tyrosine

residue is one of the most significant approaches for

increasing activity of the peptide, especially at posi-

tions 18 and 19 [59]. Given the presence of a phenyl-

alanine residue at position 20 of Bac7, the appearance

of tyrosine in the preceding positions may lead to for-

mation of an aromatic “spacer” that forms specific

contacts with amino acid residues and ribonucleotides

on the surface of the NPET in the constriction area,

similar to rumicidins.

Thus, representatives of the new rumicidin family

have an extremely long N-terminal part, differences

in the consensus region, and presence of an aromatic

“spacer” in the C-terminal region. It can be assumed

that fixation of the peptide in the constriction area

of the NPET increases the strength of its interaction

with the ribosome and can compensate for the partial

deletion of N-terminus, which, in the absence of key

interaction in the constriction area of the NPET, dra-

matically reduces antimicrobial effect of the peptide.

Non-canonical positioning of Bac5 in the ribo-

somal tunnel. Another unusual representative of

PrAMPs is Bac5. This peptide (43 amino acids long)

was isolated from the bovine neutrophils and con-

sists of an arginine-rich N-terminal region followed

by Arg-Pro-Pro-Ile/Phe repeats [26]. For manifestation

of antibacterial effect of the peptide, it was crucial to

preserve not only the length of the N-terminal region,

as truncation of the N-terminus of Bac5 by 4 amino

acids led to the loss of its inhibitory properties, but

also presence of arginine at the N-terminus or in its

immediate vicinity [61]. The variants of Bac5 with de-

letion of the C-terminal region retained antimicrobial

SHULENINA et al.1544

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

Fig. 5. Interaction of the class II PrAMPs with the termination ribosomal complex(a) (PDB ID: 8AKN, 5O2R), ribosome re-

lease complex(b) (PDB ID: 6YSU), and elongation ribosomal complex (c) (PDB ID: 8AM9). P-site tRNA (P-tRNA) is shown in

green; release factor RF1 is shown in blue; ribosome-rescue factor ArfB is shown in yellow; A-site tRNA (A-tRNA) is shown

in purple; PrAMPs Api137 and Dro1 are shown in orange.

activity, which is a consequence of translation inhibi-

tion [16, 62, 63]. Unique sequence of the peptide, con-

sisting of amino acid repeats, shows complete absence

of the consensus sequence and even of the previously

considered invariant PRP motif. Structural studies of

Bac5(1-17) in complex with a vacant bacterial ribo-

some determined that the peptide adopts an orienta-

tion opposite to that of the nascent polypeptide chain.

The conformation of Bac5(1-17) in the NPET differs

from that of class I (Bac7, Onc112, metalnikowin,

Pyr, Tur1A) and class II (Api, drosocin) PrAMPs [16].

Positioning of the N-terminus of the peptide in the

PTC suggests potential overlap not only with the CCA-

end of the A-site tRNA, as previously demonstrated

for all class I peptides, but also with the nucleotide

A76 of the initiator tRNA

fMet

. This suggests that Bac5

may have a different mechanism of action and, in

addition to disrupting accommodation of the A-site

tRNA, could interfere with the correct placement of

the initiator tRNA in the P-site of the PTC [16].

INTERACTION OF THE CLASS II PrAMPs

WITH THE RIBOSOME

Class II PrAMPs bind within the NPET in an

orientation corresponding to that of the synthesized

polypeptide chain: their C-terminus extends toward

the A-site, while their N-terminus is oriented toward

the tunnel exit [48, 53]. Apidaecin-like peptides associ-

ate with the ribosome immediately after hydrolysis of

the ester bond between the peptide and tRNA and the

release of the newly synthesized polypeptide chain

[48,53]. Once bound within the NPET, classII PrAMPs

obstruct the dissociation of release factors RF1 and

RF2. This stabilization of the termination complex

effectively halts the translation cycle by preventing

ribosome recycling [48, 53, 64] (Fig. 5). While the

overall stability of Api137, a synthetic derivative of

natural apidaecin 1b, is mediated by its interaction

with the NPET surface, the specific placement of its

C-terminal residues Arg17 and Leu18 into the A-site

cleft is crucial for translation inhibition [53, 65]. The

side chain of Arg17 is coordinated between the resi-

dues U2506, G2505, and C2452 of 23S rRNA and Gln235

of the GGQ motif of RF1. Simultaneously, Leu18 res-

idue of the peptide establishes interaction with A76

of the deacylated P-site tRNA [53]. The Api137 peptide

containing mutation at position 17, and the truncated

Api137(1-16) had significantly lower inhibitory activ-

ity compared to the values obtained for the original

peptide sequence, emphasizing the key role of the

C-terminal Arg17 [45]. Contacts in the constriction site

of the NPET are also important: Tyr7 forms a stacking

interaction with A751, and Arg10 forms a hydrogen

bond with Arg61 of the protein uL4 [53]. This binding

mode bears a notable resemblance to the engagement

of the aromatic Phe23-Trp24 dyad of rumicidin2 with

the NPET [23]. Additionally, screening of spontaneous

E.  coli mutants resistant to Api137 showed that mu-

tations in the proteins uL22, uL4, and substitutions

of the 23S rRNA nucleotides A2059, A2503 increase

resistance to Api137 [53].

Drosocin arrests translating ribosomes at the stop

codon by forming a water-mediated interaction be-

tween its Arg18 residue and Gln235 of the GGQ motif

of RF1 [48, 66], which resembles interaction between

Arg17 of Api137 and Gln235 of RF1 [53]. Notably, in

drosocin, the functionally critical amino acid residues

are distributed along the entire sequence, with some

located in the N-terminal part. Any substitution of

its key arginine residues (Arg9, Arg15, and Arg18),

INHIBITION OF PROTEIN BIOSYNTHESIS BY PEPTIDES 1545

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

aswell as Lys2 and Pro16, results in a significant de-

crease of antibacterial activity [48]. Drosocin’s high

potency is also dependent on a unique modification:

O-glycosylation at Thr11 [30, 67, 68], which establishes

multiple interactions with rRNA [48].

The class II PrAMPs can retain not only release

factors RF1/RF2 but also the ribosome-rescue factor

ArfB on the ribosome [69] (Fig.5). The N-terminal do-

main of ArfB contains a conserved GGQ motif neces-

sary for catalyzing hydrolysis of peptidyl-tRNA in the

P-site [70], and in vitro tests confirmed that Api137

traps ArfB after a single peptide hydrolysis round,

similar to what occurs with RF1/RF2 [69]. Structural

data of an ArfB-ribosome complex with Api137 in the

NPET and deacylated tRNA in the P-site confirm that,

as in the case of RF1/RF2, Api137 mimics the nascent

polypeptide chain. C-terminal Leu18 of Api137 inter-

acts with the ribose of A76 of the P-site tRNA, and the

guanidine group of Arg17 interacts with the carbonyl

group of the side chain of Gln28 in ArfB’s GGQ motif,

thereby stabilizing both the tRNA and ArfB [69].

Thus, apidaecin-like peptides disrupt protein syn-

thesis by imposing complex inhibition at the termina-

tion stage. Their direct action stalls a small fraction of

ribosomes bound to release factors RF1/RF2. However,

because the number of ribosomes in an E.  coli bacte-

rial cell far exceeds the number of available release

factors [71], this stall at stop codons has a profound

domino effect. It leads to the sequestration of the lim-

ited RF pool, causing ribosomes queuing behind the

stalled complexes on the mRNA [53,  72]. The release

factors pool depletion has two outcomes for the cell:

ribosomes that reach a stop codon either stall in the

pre-hydrolysis state, or they misread the stop codon

by incorporating a closely related aminoacyl-tRNA.

The latter results in the expression of the C-terminally

extended aberrant proteins [53]. By allowing transla-

tion to continue to the 3′-end of mRNA transcripts,

the apidaecin-like peptides activate the ribosome res-

cue system. However, the rescue system is rendered

ineffective due to a combination of the depleted ter-

mination factors pool, highly efficient blocking of

the stop codon of the arfA gene mRNA encoding the

release factor ArfA, as well as direct capture of the

RF-independent ribosome-rescue factor ArfB on the

ribosome [69, 72].

Unexpectedly, structural studies of the droso-

cin-bound ribosomal complexes revealed a small pop-

ulation of 70S ribosomes containing both a deacylated

initiator tRNA in the P-site and a dipeptidyl-tRNA in

the A-site [66] (Fig. 5). This finding suggests that the

classII PrAMPs may also play a role in inhibiting the

very first step of elongation. Although the density for

the dipeptide moiety of fMet-Leu-tRNA

Leu

was poorly

resolved, the overall configuration indicated a shift

of the dipeptide toward Arg18 due to the steric clash

of fMet and Val19 residue of drosocin. This configu-

ration could impair the first round of translocation

and prevent ribosome progression along the mRNA

[66]. This proposed effect of drosocin on early elon-

gation, is supported by in vivo data. Ribosome profil-

ing and proteomic analysis demonstrated that Api137

contributed to the ribosome stalling at the start codon

of some genes [72]. Among the most affected were

genes like zntA, srmB, cysS, tyrS, and yhbL. Notably,

the second codon in each of these mRNAs encodes

polar uncharged serine and threonine and hydropho-

bic leucine, alanine, and isoleucine, respectively [72].

These amino acids are similar in size and structure,

and Api137 may specifically stabilize complexes with

fMet-Ser/Thr/Leu/Ala/Ile-tRNA in the A-site, mirroring

the interaction observed between drosocin and the

ribosome in the presence of fMet-Leu dipeptide.

Additional binding sites of apidaecins on the

ribosome. Recent structural studies have revealed

that synthetic apidaecin 1b derivatives, Api137 and

Api88 [73], have two additional binding sites on the

50S ribosomal subunit: near the NPET exit pore (both

peptides) and inside domain III of 23S rRNA (only

Api88) [25] (Fig. 6). In the first case, the N-terminal

part of the peptides is located close to the ribosomal

proteins uL29 and uL23 and is attached to the sol-

vent-exposed nucleotides of 23S rRNA A1321, A507,

A508, A63, A92, and A93, while their C-terminus is

oriented into the NPET. In the second additional site

of apidaecin Api88 inside the domain III of 23S rRNA,

the N-terminus of the peptide is oriented toward the

NPET, and C-terminus points toward the 50S sub-

unit’s core. The central Pro9-Arg10-Pro11-Arg12 mo-

tif of Api88 is nestled between the helices H51 and

H49 of 23S rRNA, ensuring stable positioning of the

peptide [25]. The role of Api88’s binding inside do-

main III of 23S rRNA remains unclear, while location

of Api137 and Api88 near the exit pore may be crucial

for efficient penetration of apidaecin into the NPET.

To perform its function, apidaecin must bind to the

upper part of the NPET immediately after release of

the synthesized peptide but before dissociation of

the release factors RF1/RF2 or the ribosome-rescue

factor ArfB from the ribosome. Prepositioning near

the tunnel entrance likely increases the probability

of this molecular event. However, binding of apidae-

cins in the vestibule of the NPET may have a broader,

more strategic function. The NPET exit is formed by

ribosomal proteins uL29 and uL23, which serve as

a primary binding platform for the trigger factor –

a ribosome-associated molecular chaperone involved

in the early stages of folding of newly synthesized

peptides [74,  75], and the signal recognition particle,

which directs membrane proteins for insertion into

the plasma membrane [76,  77]. By occupying this re-

gion, PrAMPs, could form contacts with these essential

SHULENINA et al.1546

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

Fig. 6. Binding sites of apidaecins (using Api88 as an example) with the bacterial ribosome (PDB ID: 8RPZ, 8RQ0, 8RQ2).

Site 1 – canonical binding site of PrAMPs inside the nascent polypeptide exit tunnel. Site 2 – area near the exit pore

of the nascent polypeptide exit tunnel, N-terminal part of Api88 is located near the ribosomal proteins uL29 and uL23,

the C-terminus is oriented into the tunnel. Site 3 is located inside domain III of 23S rRNA, the central region of Api88

is located between the helices H51 and H49 of 23S rRNA, and the C-terminus points toward the 50S subunit’s core.

P-site tRNA (P-tRNA) is shown in green; Api88 is shown in blue.

factors or alter their interaction with the ribosome,

disrupting fine regulation of the functional protein

formation after biosynthesis [25].

Beyond their action on mature 70S ribosomes

and 50S ribosomal subunits, apidaecins interact with

precursors of 50S subunits, disrupting the ribosome

particle assembly process within the cell [78]. Treat-

ment of bacterial cells with Api137 leads to the ap-

pearance of an additional peak on the ribosomal pro-

file, which was attributed to the pre-50S state. This

effect was not observed with the structurally similar

Api88 or the class  I PrAMP Onc112 [79]. Structural

studies reveal that Api137 leads to the appearance

of four distinct pre-50S intermediates, differing in

structural completeness both in terms of proteins and

structural regions of 23S rRNA [78]. These intermedi-

ates are largely assembled but share a critical defect:

the functional core, including the PTC, located inside

the central protuberance of domain  V, and adjacent

rRNA elements, remain unstructured. In many precur-

sors, proteins uL22 and uL29 were absent, which led

to the change in the 50S assembly pathway. Notably,

Api137 does not bind within the NPET of these prema-

ture subunits, as apparently, the tunnel must be ful-

ly formed for peptide binding. However, Api137 was

found in the additional site [25], occupying a position

near the exit pore of the NPET in close proximity to

the sites of uL22 and uL29, creating steric hindrance

for the inclusion of these proteins [78].

STRENGTH OF PrAMP INTERACTION

WITH THE RIBOSOME

Class II PrAMPs interact with diverse ribosom-

al complexes, forming contacts only with the NPET

surface at the initial stages of elongation [66, 72] or

additionally with the ligands (P-site tRNA, RF1, ArfB)

during termination or ribosome rescue process [53,

66, 72]. The formation of complexes with different

peptide-binding modes was confirmed experimental-

ly using both ribosomal extracts containing additional

protein factors and purified ribosome preparations.

The dissociation constants (K

) values obtained us-

ing the fluorescence polarization method [80] using

5(6)-carboxyfluorescein-labeled Api137 and ribosomal

extract ranged from 0.34 to 0.62 µM [45, 81, 82], while

those determined using purified 50S and 70S prepa-

rations were 2.2  ±  0.1  µM and 4.7  ±  0.3  µM, respec-

tively (Table 2) [25]. The K

values for Api88 were

comparable in the ribosomal extract (0.7-1.31  µM) [45,

81, 82] and in the purified 50S and 70S preparations

(0.58  ±  0.05  µM and 1.82  ±  0.08  µM, respectively) [25].

The obtained values are consistent with the described

models of apidaecin-ribosome interactions [25, 53, 72].

Api137 is stabilized within the NPET by additional

factors, which explains its order-of-magnitude stron-

ger binding to ribosomes in the extract compared to

isolated 70S ribosomes. In contrast to Api137, Api88

has a weak effect on termination [25]. It appears

INHIBITION OF PROTEIN BIOSYNTHESIS BY PEPTIDES 1547

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

that additional factors do not contribute significantly

to its stabilization on the ribosome, so its K

values

in the extract and for purified 70S ribosomes are

similar and higher than those measured for Api137.

Notably, the additional binding sites for Api137 and

Api88 have been identified on the 50S subunit [25],

and therefore, the observed K

values represent a su-

perposition of affinities for each individual site. The

authors suggest the potential existence of an even

greater number of sites, which may differ between

the 50S and 70S particles. This may explain the 2-3-

fold differences in K

values observed between 50S

and 70S ribosome [25]. Further studies aimed at

characterizing the interactions of these peptides with

the trigger factor and signal recognition particle, as

well as investigating how PrAMPs binding alters the

affinity of these protein complexes to the ribosome

and impairs their function, will be crucial to deter-

mine whether binding to additional sites is essential

for their antibacterial activity. For such investigations,

50S subunits or 70S ribosomes lacking proteins uL22

and uL29, which appear to be necessary for PrAMPs

binding at the canonical site, could be employed [25].

In contrast to the apidaecin-like peptides, all

class I PrAMPs share a conserved binding mode, po-

sitioning their N-terminus within the A-site pocket of

the upper part of NPET. The remarkable consistency of

values for Onc112 determined using ribosomal ex-

tract (0.023-0.093  µM) [45, 81, 82] and purified 50S and

70S particles (0.050  ±  0.001  µM and 0.060  ±  0.005 µM,

respectively) [25], is likely due to the property of

Onc112 to bind to the ribosome without involvement

of additional stabilizing factors. This high affinity

of Onc112, which is comparable to that of conven-

tional NPET-binding antibiotics (for example, strep-

togramins B antibiotics quinupristin and linopristin

bind to the 70S ribosome with a K

of 0.04  ±  0.02  µM

and 0.040  ±  0.006  µM, respectively [83]), likely results

from extensive interactions within the tunnel. How-

ever, deletion of two N-terminal residues of oncocin

led to disruption of the peptide binding to the ribo-

some [49], indicating predominant importance of the

N-terminal region of the peptide. Structural studies

of mammalian peptides also revealed formation of a

compact, positively charged loop anchoring PrAMPs

in the A-site pocket region [40, 52]. Such non-specific

interaction could lead to the significant stabilization

of the peptide in the NPET due to formation of an

occluded state and could be more important than

the contacts within the tunnel. Along with the N-ter-

minal “anchor,” the aromatic “spacer” of rumicidins,

forming specific contacts with amino acid residues

of the proteins uL4 and uL22 and ribonucleotides on

the surface of the NPET in the constriction area [23],

could be a key element ensuring even stronger inter-

action of some class I PrAMPs members with ribo-

somal complexes. However, determining contribution

of these peptide elements requires further research.

CONCLUSION AND PERSPECTIVES

PrAMPs open up great perspectives as an alter-

native to traditional antibiotics. Their mechanism of

action based on disruption of bacterial translation –

one of the fundamental processes of life – empha-

sizes high therapeutic potential of these compounds.

The main advantages of PrAMPs are their broad

spectrum of antimicrobial action, low probability of

resistance development, and minimal toxicity to ani-

mal and human cells. However, there are several ob-

stacles to the clinical use of PrAMPs. These include

rapid peptide degradation in  vivo, low bioavailability

upon oral administration, complexity and high cost

of industrial synthesis, as well as insufficient clinical

data confirming their safety. At the current stage of

PrAMPs introduction into clinical practice, targeted

optimization of the peptide structures is necessary to

enhance their stability and boost antimicrobial activ-

ity. Along with mutational screening, expanding the

“library” of PrAMPs by discovering new members of

this class and conducting studies of the poorly in-

vestigated peptides, appear promising. Work in this

direction has led to the discovery of a unique fea-

ture of rumicidins – formation of a strong interaction

Table  2. K

values of PrAMPs. Preparations labeled

as 30S, 50S, 70S are purified ribosomal subunits and

ribosomes

Peptide K

, µM Preparation References

Onc112

0.023-0.093 70S extract [45, 81, 82]

0.060 ± 0.005 70S [25]

0.050 ± 0.001 50S [25]

1.14 ± 0.06 30S [25]

Api137

0.34-0.62 70S extract [45, 81, 82]

4.7 ± 0.3 70S [25]

2.2 ± 0.1 50S [25]

20 ± 8 30S [25]

Api88

0.7-1.31 70S extract [45, 82]

1.82 ± 0.08 70S [25]

0.58 ± 0.05 50S [25]

14 ± 3 30S [25]

SHULENINA et al.1548

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

with the ribosome due to location of the conserved

Trp23-Phe24 dyad in the constriction site of the exit

tunnel. This finding could serve as the foundation for

designing peptides that surpass natural ones in anti-

bacterial properties.

Thus, PrAMPs represent one of the most promis-

ing avenues in the development of new antibacterial

agents. Their further study and optimization offer ex-

tensive opportunities for transforming approaches to

treating infectious diseases.

Abbreviations

AMP antimicrobial peptide

MIC minimum inhibitory concentration

NPET nascent polypeptide exit tunnel

PrAMP proline-rich antimicrobial peptide

PTC peptidyltransferase center

Api apidaecin

Bac bactenecin

Onc oncocin

Pyr pyrrhocoricin

Rum rumicidin

Acknowledgments

We thank V. Paleskava for assistance in preparing il-

lustrations.

Contributions

O. V. Shulenina and A. Paleskava – writing the text;

E. A. Tolstyko – preparation of illustrations; A. L. Kone-

vega – editing the article text.

Funding

This work was supported by the Russian Science Foun-

dation (grant no.25-14-00253).

Ethics approval and consent to participate

This work does not contain any studies involving hu-

mans and animal subjects performed by any of the

authors.

Conflict of interest

The authors of this work declare that they have

noconflicts of interest.

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