Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 5, pp. 784-798 © Pleiades Publishing, Ltd., 2024.

784

REVIEW

Peptide-Based Inhibitors

of the Induced Signaling Protein Interactions:

Current State and Prospects

Vladimir Y. Toshchakov

Sirius University of Science and Technology, 354340 Sirius Federal Territory, Krasnodar Region, Russia

e-mail: toschakov.vy@talantiuspeh.ru

Received January 5, 2024

Revised February 29, 2024

Accepted March 12, 2024

Abstract— Formation of the transient protein complexes in response to activation of cellular receptors is a com-

mon mechanism by which cells respond to external stimuli. This article presents the concept of blocking interac-

tions of signaling proteins by the peptide inhibitors, and describes the progress achieved to date in the develop-

ment of signaling inhibitors that act by blocking the signal-dependent protein interactions.

DOI: 10.1134/S000629792405002X

Keywords: Toll-like receptors, TIR domains, signaling complexes, peptide inhibitors of protein interactions

Abbreviations: ACE2,angiotensin-converting enzyme2; AMP,antimicrobial peptide; BP,blocking peptide; BB-loop, the loop

between second β-strand and second α-helix; CPP,cell-permeating peptide; DD,death domain; IL,interleukin; LPS,lipopoly-

saccharide; MyD88,myeloid differentiation primary response protein88; PID,protein interaction domain; TIR,domain ho-

mologous in insect Toll proteins, human IL-1R, and plant disease-resistance protein; TIRAP,TIR-containing adapter protein,

also known as Mal; TLR,Toll-like receptor; TNF,tumor necrosis factor; TRAM,TRIF-related adapter molecule, also known

asTICAM-2; TRIF,TIR domain-containing adapter inducing IFN-β, also known as TICAM-1.

INTRODUCTION

Formation of multicomponent protein complexes

of a definite structure in response to activation of cel-

lular receptors is a common mechanism by which cells

translate external signals into a biologically relevant

response. The general principles of operation of such

complexes, often called signaling complexes, were

described at the end of the last century [1, 2]. This

conceptual understanding was complemented in the

recent two decades with achievements in structural

biology revealing the fine structure of many oligomer-

ic complexes consisting of signaling proteins or their

separate domains with resolution sufficient for visual-

izing localization of individual atoms[3]. This knowl-

edge significantly expanded our understanding of the

mechanisms of assembly of such complexes and their

functioning. At the same time, it has been recognized

that the data accumulated so far are fragmentary and

do not reflect dynamics of real complexes. Further-

more, considering that majority of the complexes have

been resolved using recombinant DNA technology,

there is a problem in validation of physiologically rel-

evant structures.

Formation of signaling complexes in the vast ma-

jority of cases is mediated by specialized protein do-

mains often called protein interaction domains (PIDs)

[2, 4]. Some of the PIDs, such as SH2, SH3, or bromo-

domains recognize specific features or short motifs

present in the protein primary structure, while other

domains such as TIR-domains [homology domain be-

tween insect Toll-protein, human IL-1R (interleukin 1

receptor), and plant disease-resistance proteins] or

death domains (DDs) do not have specific binding

motifs. PIDs of the last type can establish multiple

cooperative interactions with the domains of same

type either homo- or heterotypically [5, 6]. Currently

available structures of signaling complexes demon-

strate wide topological diversity and uniqueness of

the sites mediating individual binary interactions of

the components within the oligomeric complexes [3].

Structural diversity of signaling complexes includes

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linear double-stranded complexes formed by TIR do-

mains of activated Toll-like receptors (TLRs) and their

adapter proteins, and by members of the IL-1R family

[7,8], as well as helical complexes of different struc-

tures, including single- and double-stranded helices,

as represented by Myddosome [9], PIDDosome [10],

MDA5-MAVS[11], inflammasomes[12-14], and others

[3,15,16].

Cell signaling is an important pharmaceutical tar-

get. This notion is supported by more than 25 years of

clinical success of therapeutic antibodies designed to

block cellular receptors, as well as cytokines that acti-

vate these receptors [17-19]. Another indication of the

importance of signaling pathways as pharmacological

targets is the fact that small molecules that block bind-

ing of the G-protein-coupled receptors with ligands

represent a significant part of the existing drug pool

[20-23]. Considering that small molecules are not very

effective in blocking receptors activated by macro-

molecules (such as cytokines or exogenous molecules

from pathogens), as well as the fact that many signal-

ing pathways do not have circulating agonists, it seems

reasonable to assume that blocking of signaling path-

ways by disrupting assembly of signaling complexes

could be a promising approach for the development of

new pharmaceuticals. However, progress in the devel-

opment of new pharmaceuticals with such mechanism

of action is limited at present. At the same time, a large

amount of experimental data obtained both in  vitro

using cultivated cells and in various in  vivo models

indicates that targeted development of inhibitors of

signal transduction from the activated receptors by

blocking assembly of signaling complexes is possible.

Considering that the assembly of signaling complexes

in the majority of cases is mediated through interac-

tions between the PIDs[2], PIDs are among most pop-

ular targets of the inhibitors developed to date. This

article reviews current progress in the development of

signaling complex assembly inhibitors and discusses

the prospects and further progress in the field.

CONCEPT OF PEPTIDE-BASED

INHIBITORS OF THE TRANSIENT

PROTEIN INTERACTIONS

Many examples (some of which will be discussed

below) demonstrate that relatively short peptides cor-

responding to fragments of protein primary sequence

comprising functional protein–protein interaction sites

are capable of blocking protein function. Such pep-

tides, often called blocking peptides (BPs) or decoy

peptides, act through binding to the interaction sites

of their prototype protein with its protein partners,

thereby preventing formation of a functional complex

and, subsequently, inhibiting downstream functions

(Fig. 1). Because the assembly of signaling complexes

is an intracellular process, the segment mimicking the

interaction site must be complemented by the segment

facilitating permeation of the construct into the cell.

Peptide vectors, which are either derived from a natu-

ral protein or artificially obtained, are most often used

for facilitation of transmembrane transfer of blocking

peptides. Hence, the peptide-based blockers of interac-

tions between cytoplasmic proteins consist from two

functional parts: a specific part designed to mediate

binding with the target proteins and a peptide vector

required for intracellular permeation of the construct.

Below typical features of these two parts will be dis-

cussed separately.

Target-binding segment of blocking peptides.

Functional role of this peptide part is to block the pro-

totype protein interactions, causing the consequent

prevention of signaling complex formation (Fig. 1).

In the cases when the site of interaction in the pro-

tein is known, design of blocking peptides seems ob-

vious. However, results of screening of large peptide

libraries have demonstrated that a large fraction of the

peptides, which do correspond to the binding site, nev-

ertheless do not exhibit inhibitory activity in in vitro

or invivotests [24-26]. The most often factors account-

ing for the lack of functional activity in these cases

Fig. 1. Mechanisms of action of blocking peptides – preven-

tion of formation of the protein complex by competing with

the protein prototype for the functional binding site. a)Inter-

action of PID1 (blue shape) with PID2 (green sector) results

in formation of a functional binary complex. b)Peptide cor-

responding to the PID1 binding site of PID2 (blue triangle) re-

tains its ability to bind PID2. c) Binding of the peptide with

PID2 prevents formation of the PID1–PID2 complex.

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are nonspecific peptide binding by the components

of cell proteome, insufficient peptide solubility, neg-

ative effect of the target-binding part on the peptide

permeability, biological instability of the peptide, and

others [27]. In the cases, when boundaries of the bind-

ing site are unknown, inhibitory sequences are deter-

mined via screening of the peptide libraries or select-

ed empirically [5, 26, 28]. Typical size of binding sites

involved in the transient interactions of signaling pro-

teins should be taken into consideration in the case of

empirical selection. General characteristics of protein

interaction sites have been investigated in detail and

described in a number of publications [29-32]. Anal-

ysis of the known structures of signaling complexes

formed by PIDs confirms these general characteristics

and further specifies that the typical surface area of

binary interactions in signaling complexes (250-800 Å

)

is lower than the average size of protein interactions

in general [7-9, 33]. Taking into account that circulari-

ty of the interaction sites in globular proteins typically

is high (0.7, as an average [29]), upper estimate of a

binary interaction site in such complex could be mod-

elled by a circle of ~30 Å diameter. Alpha- or 3

-helices

of such length would correspond to the peptides con-

sisting of 20 or 15 aa, respectively, while the peptide of

same length in the β-strand conformation would con-

sist of 8-9 aa[34]. These simplified estimates correlate

quite well with the range of sizes of target-binding seg-

ments in the experimentally verified blocking peptides

[26,27].

Vectors facilitating intracellular penetration

of the blocking peptides. Peptide-based vectors are

frequently used to carry macromolecules across plas-

ma membrane. In the late 1980s, a natural capability

of Transcriptional transactivator (Tat) protein of Hu-

man immunodeficiency virus to translocate across

the plasma membrane was discovered [35, 36]. Soon

after this discovery, a similar capability to translocate

across plasma membranes was discovered for the

Drosophila Antennapedia homeodomain [37]. It was

shown that the permeating capability of Antennapedia

homeodomain is due to the presence of the 16 aa-long

segments of the primary sequence, which forms the

third α-helix of the protein [38]. Later, functionally

similar short sequences (often called cell-permeating

or cell-penetrating peptides, CPPs) have been found in

Tat and other proteins capable of permeating cell plas-

ma membranes [39-42]. Today, more than 1000 CPPs of

different types have been discovered [43, 44]. In addi-

tion to CPPs from natural proteins, artificially created

CPPs have been reported [40, 42,45]. Based on physi-

cochemical properties of peptides, the following types

of CPPs are recognized: cationic, hydrophobic, and

amphipathic consisting of cationic and hydrophobic

parts [45]. Permeating sequences of Tat(RKKRRQRRR)

and Antp(RQIKIWFQNRRMKWKK) proteins, as well as

other members of the family of transcription factors

that contain homeodomain, are examples of cation-

ic CPPs [42, 46]. Several artificial sequences, e.g., oli-

goarginines or oligolysines, also belong to this group

[45, 47]. Transportan, an artificially created combi-

nation of fragments of two natural proteins, is an ex-

ample of amphipathic CPP [48]. There are only a few

known hydrophobic CPPs, they are less sensitive to

amino acid substitutions [45]. Lower efficiency of hy-

drophobic CPPs in comparison with the CPPs of other

types has been noted.

CPPs internalization mechanisms remain a sub-

ject of discussion in the literature [42, 49]. It is recog-

nized that CPPs are internalized via either endocytosis

with subsequent release from endosomes, or via direct

interaction with the plasma membrane; moreover, in-

dividual CPPs could use multiple mechanism for pene-

tration with their importance differing for individual

peptides and cell lines. At the same time, CPPs do not

use protein transporters for cell penetration, which

explains versatility of their action with respect to dif-

ferent cell types. Length of the used CPPs varies in the

range 5-30 aa. In addition to proteins and peptides,

CPPs are capable of transporting macromolecules of

different nature to the cells including nucleic acids,

medicinal preparations, and contrast agents, as well as

complex supramolecular structures such as liposomes

and nanoparticles[45, 49,50]. Peptide vectors are effi-

cient for intracellular transport of various compounds

not only in a monolayer of cultivated cells. Numerous

studies demonstrated that CPPs also increase tissue

permeability, including permeability of blood-brain

barrier. In particular, it was shown in the early study

by Schwarzeetal.[51] that Tat-peptides facilitated pen-

etration of the intraperitoneally administered β-galac-

tosidase, a 120-kDa protein, into all tissues including

brain. Subsequent studies confirmed efficiency of CPPs

for intracellular transport of cargo of different nature

in vivo, as well wide distribution of cargo in the or-

gans, which was shown in the cases of both intraperi-

toneal and intravenous injections [52-55]. At the same

time, predominant accumulation of CPPs in liver and

kidneys was noted, while accumulation in brain and

muscles was significantly lower [53,54].

It is worth mentioning that typical working con-

centration of CPPs in cell culture are in a narrow range

varying from 1-5 µM to 50-100 µM for a wide variety of

CPPs, transported agents, and cell lines [26]. The most

plausible reason for existence of the lower limit of

effective concentration is low efficiency of transport

at lower concentrations of CPPs. This is confirmed by

significantly higher affinities of blocking peptides to

their protein targets demonstrated invitro using a re-

combinant protein, in comparison with the effective

peptide concentration in cell culture. One of the exam-

ples is the TLR-blocking peptide, 2R9, which exhibits

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a ~40 nM binding affinity to its target, TIR-domain

of the adaptor protein TIRAP, while the effective con-

centration of 2R9 in macrophage cell culture is almost

500-fold higher [56]. Existence of the upper limit of CPP

concentration used in cell is due to cytotoxicity often

observed, when concentration of BP is above 100 µM,

which is due to their membranotropic action and abili-

ty to form membrane pores at high concentrations.

Another manifestation of CPP membranotropic

activity is their antimicrobial properties, which are

due to similarity of their physicochemical properties

with the properties of antimicrobial peptides (AMPs)

[57, 58]. AMPs are plant and animal peptides that func-

tion as an element of antimicrobial defence effective

against various classes of pathogens, including bac-

teria, viruses, and fungi[59]. Both classes of the pep-

tides, CPPs and AMPs, are predominantly represented

by cationic peptides, which also contain a high num-

ber of hydrophobic amino acids [57, 60,61]. Both CPPs

and AMPs exhibit less cytotoxicity to animal cells in

comparison with their activity against prokaryotes.

Despite the discovery of many new CPPs, two vec-

tors discovered first, the Tat-peptide and the vector

based on Antp (also known as penetratin), are most

frequently used as CPPs. It must be mentioned that rel-

ative efficiency of these vector varies in different sys-

tems and depends on both the cell model and the na-

ture of molecular target: in some systems Tat has been

found to be more efficient, and in others– penetratin.

There is no unambiguous and verifiable explanation

for this fact in the literature, and primary selection of

CPP is performed by the researchers empirically. One

of the good features of penetratin is simplicity of quan-

tification of the penetratin-containing BPs. Penetratin

contains tryptophans, and, hence, its concentration can

be determined spectrophotometrically, while existence

of such option for the BPs containing Tat depends of

the presence of absorbing amino acid residues in the

blocking part of the peptide. It must be mentioned that

natural sequences mediating penetration of a protein

into the cell are evolutionary very well preserved.

Inparticular, penetrating sequence of the Drosophila

protein Antp is 100% preserved in the human homolog

[62]. The high degree of evolutionary conservation of

penetrating sequences reflects their biological signifi-

cance for the function of proteins, which contain such

sequences.

It is worth noting in conclusion of this section

that the concept of blocking peptides is based on the

often-observed ability of the peptides corresponding to

the fragment of the primary sequence of the protein

forming the site (or major part of the site) of the func-

tional protein–protein interaction to bind the protein

partner, and, as a result, to block this function of the

prototype protein. The concept of BPs is applicable

to intracellular targets if the peptide vectors capable

of transporting the specific blocking part of the pep-

tide inside the cell are used. The highest number of

the currently known examples of successful use of BPs

is related to the blocking of interactions mediated by

PIDs, protein domains specialized in the protein–pro-

tein interactions associated with signaling.

FUNCTIONAL CHARACTERISTICS

OF BLOCKING PEPTIDES

First successful attempts to modulate biological

functions with synthetic BPs have been reported in

1990s. The first targets of these studies were extracel-

lular protein interactions. In particular, different vari-

ants of the integrin-binding motif- (RGD) containing

peptides were used as agents to block cell adhesion in

attempts to develop new medicinal preparations [63].

Another early example of realization of the BP con-

cept is Akt protein kinase inhibition by peptides-pseu-

dosubstrates, as well as by the BP containing the βA

strand of TCL1 protein [64, 65]. Use of peptide-based

cell-permeating vectors in combination with the seg-

ments that provide the target binding specificity sig-

nificantly expanded applications of the blocking pep-

tide strategy. In particular, the peptides blocking NEMO

(nuclear factor kappa-B (NF-κB) essential modulator)

oligomerization, as well as peptides corresponding to

the NEMO-binding domain of IKK, inhibited activation

of NF-κB induced by tumor necrosis factor (TNF) or li-

popolysaccharide (LPS) [66-68]. The peptide containing

the 10 aa Tat sequence in combination with the 20  aa

JNK-binding motif of JNK interacting protein 1 was used

in the study by Borselloetal.[69]. Interestingly, both

L- and D-isomers of this peptide blocked the kinase ac-

tivity of JNK and exhibited neuroprotective effect in

the brain ischemia models [69]. Another example of

the protein segment blocking the mitogen-activated

protein kinases(MAP) activity is the MEK1-derived se-

quence (13 aa), which effectively inhibited the activity

of ERK kinases [70]. A peptide containing C-terminal

segment of Gα

protein linked with penetratin is an ex-

ample of successful realization of the BP concept with

respect to G-protein-coupled receptors. This peptide

specifically inhibited cAMP production stimulated by

adrenoreceptor agonists [71].

A number of studies used the BP concept for de-

velopment of TLR inhibitors. Horng et al. [72] were

the first to use synthetic cell-penetrating peptides for

inhibiting TLRs. The authors demonstrated that the

peptide consisting of the BB-loop (loop between sec-

ond β-strand and second α-helix of the TIR domain)

of adapter protein TIRAP linked to penetratin blocked

the LPS-induced activation of NF-κB and MAP-kinases

in a macrophage cell line. This peptide, however, did

not exhibit inhibitory activity when the cells were

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stimulated with a TLR9 agonist or IL-1[72]. TheTIRAP

peptide also selectively blocked production of the

TLR4-dependent cytokines and dendritic cell matura-

tion [72]. Another study compared the effects of BPs

containing the BB-loop of 4 adapter proteins that me-

diate the TLR signal transduction [73]. This and other

studies have demonstrated that the peptides contain-

ing the BB-loop of the TIR-containing adapter proteins

MyD88 (Myeloid differentiation primary response pro-

tein 88), TRIF (TIR domain-containing adapter induc-

ing IFN-β, also known as TICAM-1), and TRAM (TRIF-

related adapter molecule, also known as TICAM-2

inhibited TLR4 signaling [41, 73-76]. The inhibitory

efficiency of individual adapter peptides however dif-

fered, with the BP from TRAM been most potent [73].

A peptidomimetic imitating the central part of the

TRAM BP exhibited a cardioprotective effect in the

mouse model of myocardial infarction [77]. Authors

interpreted this observation as an indication of a sup-

pression of the TLR4-dependent inflammation by the

TRAM BP [77]. In the following study, the same group

confirmed that the dimeric peptidomimetic imitating

the TRAM BB loop inhibited the LPS-induced transcrip-

tion of IFN-β and CXCL10 in a dose-dependent manner

[78]. This agent, however, demonstrated only partial

selectivity as it also inhibited IFN-β induced by TLR8

and MDA5/RIG-I agonists [78]. TLR2 and TLR4 peptides

designed similarly to the adapter BB-peptides blocked

the activity of their respective prototype receptor and

exhibited some cross-reactivity, while the peptide

containing homologous sequence from TLR1 or TLR6

(these TLRs do not induce signaling as homodimers,

but only through heterodimerization with TLR2) did

not exhibit inhibitory activity [79].

The next step in the development of the blocking

peptide methodology was the screening of PID-derived

peptide libraries. This approach was based on then

emerging understanding of binding versatility of the

PIDs, together with the lack of information on the ex-

act positions of the PID interaction sites. The first com-

prehensive library representing the entire PID surface

was the library from the TIR of TLR4[5]. The library

comprised 11 peptides, each of which included a seg-

ment of TLR4 TIR primary sequence as the blocking

part. The segments were selected such that each repre-

sented a non-fragmented patch of the domain surface.

Penetratin was used as a penetrating sequence. Results

of this study validated the BP concept and indirectly

confirmed the assumption of multiple binding sites

present in TIR domains. Five peptides inhibited the

LPS-induced activation of MAP kinases and transcrip-

tion factors, and the expression of cytokine mRNA [5].

Inhibitory effect was exhibited by the peptides that

corresponded to the site connecting the TIR-domain

with the transmembrane portion of TLR, the AB and BB

loops, as well as α-helices B and D (Fig.2,a andb) [5].

A follow-up study performed a screening of the simi-

larly designed peptide library from the adapter TIRAP

[80]. The screening also identified 5 active peptides

derived from the following structural elements of the

TIR-domain: the AB loop, and the α-helices B, C, D,

and E [80]. Interestingly, activity of the BB loop pep-

tide used in this study, peptideTR4, was significantly

lower than the activity of the previously used TIRAP

BB-peptide, which differed from TR4 by one hydropho-

bic amino acid residue [72]. Peptide libraries from the

TIR-domains of adapter proteins of the MyD88-inde-

pendent pathway, TRAM and TRIF, were screened later

[55, 81]. Two active peptides were identified in each

of the libraries. In addition to the previously identi-

fied peptides derived from the BB loop of both adapt-

er proteins [73], the inhibitory activity was observed

for the peptide TM6 from the third helix in TRAM and

peptide TF5 from the second helix in TRIF [55, 81].

Using deletion analysis, the authors identified TM6-ΔC

and TF5-ΔC, which are fully active, truncated (9 aa)

versions of the corresponding parent peptides. Sub-

sequent studies that screened analogous peptide li-

braries from TIR-domains of TLR2, TLR2 co-receptors

(TLR1 and TLR6), TLR9, TLR7, and TLR5 identified in-

hibitory sequences in each of the screened libraries

[25, 26, 56, 82, 83]. Based on the analysis of positions

of the segments that represent the active BPs, it was

concluded that the active peptides originate from four

topologically conserved TIR domain regions responsi-

ble for the assembly of signaling complexes by activat-

ed TLRs (Fig. 2) [26, 83-85]. Two sites (S1andS4) are

located on the opposite sides of the TIR-domain near

the edge-forming strands of the β-sheet. In addition

to the amino acid residues forming the strand B, the

site S1 could include segments of the loops AB and/

or BB, and, as is the case for TLR2, TLR4, and TLR7

TIR, the segment connecting TIR with the transmem-

brane helix[5, 24,26]. The site S4 is represented by the

peptides corresponding to the strand E, as well as the

adjacent to the stand E α-helixE[24-26]. The sitesS2

andS3 are formed by three helical regions adjacent to

the convex side of the β-sheet i.e. helices B, C, andD.

The helicesB and/orC form the S2 site; the helixD–

siteS3(Fig.  2c)[26, 83]. It was proposed that the pri-

mary TLR signaling complexes assemble through mu-

tual interactions of site S1 with site S4, and of site S2

with siteS3 (Fig.2,d ande) [26,83]. This assumption

is also based on the results of structural analysis of

oligomeric complexes formed spontaneously in vitro

by the recombinant TIR-domains of the adaptor pro-

teins TIRAP and MyD88[7,8].

In addition to the BP design strategy mimicking

the eukaryotic protein interaction sites, other studies

used the approach based on the ability of some bacte-

rial and viral pathogens to block antimicrobial defenc-

es of higher animals by producing proteins capable

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Fig. 2. The structure of TIR-domains, location of sites corresponding to the inhibitory BPs, and suggested architecture of TLR

signaling complexes. a)Schematic representation of the secondary structure of TIR domains[84]. TIR domains belong to the

class of α/β protein domains with the secondary structure consisting of alternating β-strands and α-helices[84]. According to

the most popular nomenclature of the secondary structure elements TIR-domains are alphabetized starting from the N-termi-

nus. For example, βA and βE [shown in panel(a) by triangles] denote first and fifth strands, and αB and αD– second and fourth

helices (shown in the figure with brown circles). Loops are designated by two capital letters corresponding to the elements

of the secondary structure they connect. For example, the BB loop connects the second strand with the second helix, and the

DE-loop– fourth helix with fifth strand. b)Tertiary structure of the human TLR2 TIR domain [85]. A typical TIR domain consists

of 5strands arranged into a parallel β-sheet forming the domain core. α-Helices are located on both sides of the sheet– the first

and the last helices are located at the convex side. c)Four binding sites of TIR domains that mediate receptor TIR dimeriza-

tion and subsequent recruitment of adapter proteins are denoted as S1–S4. SitesS1 andS4 are located at the opposite sides of

the TIR domain near the edge strands in β-sheet. SitesS2 andS3 are located in one semi-sphere. SiteS2 is formed by helices B

and/orC; siteS3– by helix D together with adjacent loops [26,83]. d)TIR domain interactions in the signaling complex during

TLR9 activation in the presence of both adapters of the MyD88-dependent pathway, TIRAP and MyD88. SitesS1 and S4 in-

teract reciprocally and form links inside each strand of the two-stranded structure, while sites S2 and S3 form interchain

connections[83]. e)Interaction of TIR domains in the signaling complex during TLR9 activation in the absence of TIRAP[83].

The complex can grow unidirectionally.

ofbinding the components of immune system [86,87].

One example of such proteins is cowpox virus pro-

teins A46R and A52R that have the ability to bind TIR

domains of TLR4 and TLR4 adapter proteins, there-

by blocking the assembly of TLR signaling complexes

[88, 89]. These studies identified short peptides capa-

ble of inhibitingTLR in both viral proteins. In particu-

lar, the A52R-derived, polyarginine-linked peptide P13

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effectively inhibited cytokine production induced by

the TLR3, TLR4, and TLR7 agonists in cultured cells,

and reduced the lethality in the mouse model of sep-

tic shock induced by administration of LPS and D-ga-

lactosamine [90, 91]. Screening of the A46R peptide

library identified a potent TLR4 inhibitor effective in

cell culture at 1–5 µM concentrations [28]. This pep-

tide, named VIPER, was specific towards TLR4 and did

not inhibit TLR2, TLR3, or TLR9. The authors system-

atically investigated the effects of amino acid substi-

tutions on VIPER activity. Interestingly, substitution of

a single amino acid at any position except the central

leucine did not suppress the inhibitory activity of the

peptide [28].

Bacterial TIR domain-containing virulence factors

TcpB and TcpC are capable of inhibiting TLR signal-

ing due to MyD88 bindings [92]. Snyderetal.[93] ex-

amined the BB and DD loop peptides from the TcpC

TIR domain. Both peptides suppressed the LPS-in-

duced macrophage activation. By using immunopre-

cipitation, the authors demonstrated that the BB loop

peptide binds TLR4, whereas the DD peptide binds

MyD88[93]. Keetal.[94] screened the peptide library

of the TIR-domain from TcpB (TIR-containing protein

in Brucella). The screening identified two peptides,

TB-8 and TB-9, both of which inhibited response to

LPS in invitro and invivo models [94].

Blocking peptides also were used in attempts to

develop antiviral agents that block the entry of SARS-

CoV-2 virus into host cells [95]. The study examined

peptides derived from viral Spike protein and from

the human Spike receptor, angiotensin-converting

enzyme 2 (ACE2) [95]. Based on the structure of the

Spike–ACE2 complex, Loietal.[96] designed a series of

short peptides originating from different Spike-ACE2

interaction sites and demonstrated that these peptides

reduce the binding of virus to ACE2-expressing cells

when used separately or (more efficiently) in combi-

nation.

Thus, literature presented in this section demon-

strates that peptide-based blockers can target interac-

tions of extracellular proteins as well as interactions

of proteins located in the cytoplasm. In the latter case,

the blocking part must be supplemented with a pep-

tide vector to facilitate the BP transmembrane transfer.

Receptor signaling function could be suppressed via

blocking either receptor-receptor or adapter-adapter

interactions, or a combination of these. The screening

of peptide libraries created from PIDs is often more

productive due to multiple protein interaction sites

typically present in a PID. Another general observation

that could be made based on the analysis of available

publications is a relatively narrow range of effective

BP concentrations for suppression of the intracellular

targets. Effective BP concentration in a cell culture

typically is in the range of 5-40 µM, which could be

explained by the efficiency of transmembrane trans-

port by peptide vectors.

VERIFICATION OF THE MECHANISM

OF ACTION OF BLOCKING PEPTIDES

The blocking peptide concept is based on the as-

sumption that BPs retain, to a large extend, the spec-

ificity and affinity of interactions mediated by the

corresponding site of their full-size prototype protein.

However, in practice, BPs are identified based not on

the binding with their protein target in an invitro sys-

tem, but in functional tests using either screening or

existing knowledge of the location of the binding site

required for realization of the function that should be

inhibited. Obviously, verification of the mechanism of

action of BPs identified based on inhibition of the func-

tion should include confirmation of either direct bind-

ing of the BP with the target protein or/and blocking of

interaction of the target with the full-size protein-pro-

totype of the peptide. Currently there are data con-

firming this mechanism of action for the vast majority

of BPs. In particular, it was shown in one of the earlier

studies using immunoprecipitation technique that the

peptide corresponding to the BB loop of the TIR-do-

main in MyD88 prevents dimerization of MyD88[97].

Piao et al.  [56] demonstrated later using dot-blotting

technique that the peptide derived from the BB-loop in

the TIR-domain of MyD88 binds TIR-domains of TIRAP

and TLR9, in addition to MyD88. Another example of

multi-specific binding is the peptide7R11 correspond-

ing to the fifth helix of the TIR-domain of TLR7 (siteS4;

Fig. 2c). 7R11, but not a control peptide, was shown

to bind TIR-domains of both MyD88 and TIRAP; while

the peptide 7R9 from the 4th helix of the TIR-domain

from TLR7 (siteS3; Fig.2c) was shown to bind TIRAP,

but not MyD88 or the control protein[24]. It was re-

ported in the later studies that many BPs derived from

the TIR-domains demonstrate multi-specific binding

interacting with a specific subgroup of TIR-domain,

which correlates with the properties of full-size PIDs

[26]. These observations indicate that BPs could bind

several targets, which, in turn, define functional prop-

erties of each particular peptide.

At the same time, certain peculiarities in the spec-

ificity of binding of BPs derived from PIDs have been

noted. In particular, the BPs corresponding to the 4th

helix of the TIR-domain from TLR interacted with the

TIR-domains of adapter proteins, but not with the

TIR-domains of the receptors [26]. For example, the

peptide4R9 (D helix of TLR4) did not bind TIR-domains

of TLR4 and TLR2[5], but interacted with the TIR-do-

main of the TIRAP adapter [81]. The peptide2R9 (4th

helical region (D helix) of the TIR-domain from TLR2)

also predominately interacted with the TIR-domain of

NEW APPROACHES TO RECEPTOR INHIBITION 791

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

TIRAP, but not with the TIR-domains from TLR1, TLR4,

TLR6, or TLR9[56]. Similarly, the BPs from the 4th he-

lical region of the receptors TLR1 and TLR6 predom-

inantly interacted with the TIR-domains of adaptor

proteins, MyD88 and TIRAP, respectively, but not with

the TIR-domain of TLR2, which is a co-receptor of the

protein-prototypes of these BPs [82]. Another exam-

ple of specific interactions of BPs from TIR-domains is

binding exhibited by the peptides of the adapter pro-

tein TRIF belonging to the MyD88-independent signal-

ing pathway [81]. The peptide TF4 interacted with the

TIR-domain of the TLR4 receptor, and the TF5 peptide–

with the TIR-domains of both TLR4 and TRAM, but

none of the TRIF-peptides exhibited binding with the

adapter proteins of the MyD88-dependent pathway,

TIRAP and MyD88[81].

One of approaches that could confirm that bind-

ing of the protein with the peptide is indeed involved

in the inhibition of function of this protein, could be

matching of the apparent binding constant for the pro-

tein–BP pair measured in a cellular system with the

constant of inhibition of the protein function measured

also in the cellular system. In order to obtain such con-

firmation a system based on the Forster resonance

energy transfer was developed in our research group

to examine quantitatively binding of the peptides

with their protein targets directly in the cell. A panel

of plasmids was designed that encoded hybrid pro-

teins consisting of the TIR-domain conjugated with a

fluorescent label. To evaluate peptide finding with the

TIR-domains, the fluorescently labeled TIR-domains

considered as possible peptide targets were ectopically

expressed in the HeLa cells, and the cells were incu-

bated in the presence of blocking peptide labeled with

the fluorescent dye capable of quenching fluorescence

of the label on the TIR-domain[5,98]. Binding of the

peptide with the TIR-domain was evaluated from the

quenching of fluorescence of the protein label mani-

fested as a reduction of its fluorescence lifetime. Nu-

merous examples discussed in detail in the previously

published literature review [26], as well as in two pa-

pers published later[24,25] demonstrated practically

complete coincidence of the effective concentrations

required for blocking protein function by the cell-pen-

etrating BPs and for quenching fluorescence of the la-

belled TIR-domains. The apparent binding constants

measured in a cellular system for the inhibitory BPs

with the TIR-targets are in the range 1-20 µM for all ef-

ficiently binding BP-TIR pairs known at present [26].

Such relatively narrow range of the effective concen-

tration for a sufficiently large group of BPs identified

now is, likely, a consequence of the commonality of the

mechanism for BP penetration through the cell plas-

ma membrane. This hypothesis was confirmed by the

fact that BPs demonstrate significantly higher binding

constant in the in  vitro tests using recombinant protein

targets in comparison with the binding in the cellular

systems. For example, the peptide2R9 binds to the re-

combinant TIR-domain of the adapter protein TIRAP in

solution with K

 ~ 40 µM, which is significantly lower

than the apparent dissociation constant for this pair

in the cellular system [56]. Differences between the

binding constants measured in  vitro and in the cellular

system could be in part explained also by nonspecific

binding of the peptide with the extra- and intracellular

proteins. It was shown, in particular, that 2R9 binds to

the serum albumin with K

 ~ 1.5 µM [56]. The experi-

ments conducted with the help of surface plasmon res-

onance technique confirmed that high affinity of the

peptides to TIR-domain is due to the high rate of asso-

ciation and low rate of dissociation [56].

It could be stated in conclusion of the section that

the experimental data accumulated until now confirm

binding of the target proteins with the blocking pep-

tides as a main mechanism of BP action. And, although,

some BPs derived from PIDs, similar to their prototype

proteins, demonstrate ability to bind several proteins

representatives of the same class of proteins, many in-

teractions are selective. One of the examples of selec-

tivity of such interactions are interactions of the BPs

derived from α-helixD of the TIR-domains from TLR

with the TIR-domains of the adapter proteins, while

these peptides do not interact with the TIR-domains of

the receptors.

EFFICIENCY OF BLOCKING PEPTIDES

IN invivo APPLICATIONS

The peptide-based blockers of protein–protein in-

teractions identified in the experiments with cell cul-

tures were tested in the in  vivo experiments. It must

be mentioned, however, that at present the reported

examples of using BPs in  vivo are limited to the exper-

iments with small laboratory animals, and in the ma-

jority of cases involve testing of TLR inhibitors. In the

first attempts to modulate TLR functions in  vivo with

the help of BPs the peptides derived from the viral

proteins A46R and A52R were used. Tsung et al. [91]

demonstrated that the P13 peptide from the A52R pro-

tein decreased the level of circulating TNF induced

by administration of LPS to mice almost 2-fold. Simi-

lar effect was observed for the VIPER peptide in the

case of intravenous administration, which resulted

in the decrease of circulating IL-12p40 by ~50% [28].

Coutureetal.[80] were the first to test the TLR block-

ing peptides based on the mammalian proteins in

the mouse model. Two peptides identified during the

screening of the peptide library of the adapter pro-

tein TIRAP were tested; these were peptides from the

second and third helical site of the TIRAP sequence,

named, respectively, TR5 and TR6. The peptides

TOSHCHAKOV792

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

were injected intraperitoneally to mice at the dose

10 nmol/g 1  h prior to administration of sublethal dose

of LPS. Both peptides, but not the control cell-penetrat-

ing peptide of the similar length practically completely

blocked influx of TNF into circulation as a response to

LPS introduction, and also significantly decreased the

level of circulating IL-6[80].

In the following study, Piao et al. [55] evaluated

efficiency of BPs in the animal model in more detail.

Inhibitory peptides derived from the adapter protein

TRAM belonging to the MyD88-independent signaling

pathway were examined [99]. The experiments con-

firmed systemic inhibitory activity demonstrated by

the peptides administered to mice. The peptides TM4,

TM6, as well as the truncated peptide TM4-ΔC de-

creased the levels of circulating TNF and IL-6 by ~90%

of their peak levels [55]. The authors compared ac-

tivity of the peptides administered intraperitoneally

and intravenously. The BPs significantly decreased

systemic levels of the cytokines in both cases of ad-

ministration, however, inhibitory activity of the pep-

tides following intraperitoneal administration was

higher [55]. Efficiency of BPs was also investigated

in the case of so-called “therapeutic administration.”

Order of administration of BP and LPS was changed

in these experiments, BP was administered 30 min af-

ter administration of LPS, not one hour prior to that.

The experiments demonstrated significant decrease

of the levels of circulating cytokines already 1.5 h af-

ter “therapeutic administration” of BP[55]. In another

series of experiments from the same study the ability

of TRAM-peptides to prevent lethality due to adminis-

tration of LPS to mice was evaluated. Administration

of the TM4 and TM4-ΔC peptides at the dose 10 nmol/g

one hour prior to administration of LPS at the dose

17.5 µg/g prevented lethal outcome in 100% of cases,

while the peptides TM6 and TR6 were effective in ~65-

80% of cases [55]. Survival of mice after therapeutic

administration of the TM4-ΔC peptide (3 h after ad-

ministration of the lethal dose of LPS) decreased, as

expected, in comparison with the prophylactic admin-

istration, and was ~70% versus 100%-survival in the

case of the peptide administration 1 h prior to admin-

istration of LPS [55].

Use of several variants of the peptides derived

from the third helical fragment of the TIRAP sequence

has been reported in the literature. In particular,

Shahetal.[100] used the peptide MIP2, with eight of 12

amino acid residues in its sequence significantly over-

lapping with the sequence of TR6, inhibitory peptide

identified by Coutureetal.[80] in the original screen-

ing of the peptide library of the TIR-domain from

TIRAP. MIP2 demonstrated multi-specific effect with

respect to TLR inhibiting TLR2, TLR3, TLR4, TLR7, and

TLR9 [100]. This observation confirmed and expand-

ed the data presented in the Coutureetal. study[80],

in which effect of TR6 was evaluated only on TLR2

and TLR4. It was shown using the LPS-induced septic

shock model that MIP2 increased the 72-h survival

of mice from 0 to 20-25% [100]. Shah et al. [100] in-

vestigated in detail effects of MIP2 in the models of

chronic inflammatory diseases: psoriasis induced by

introduction of imiquimod, a TLR7 agonist; lupus (us-

ing the MRL/lpr mouse line spontaneously developing

this disease); as well as non-alcoholic fatty liver dis-

ease (NAFLD) induced by the diet with low content of

methionine and choline. The authors reported that the

6-day course of MIP2 had a significant anti-inflamma-

tory effect (similar to the effect of methotrexate) in

the psoriasis model, when the peptide was used at a

lower dose (1 nmol/g); the effect, however, decreased,

when the MIP2 peptide was used at high doses (10

and 20 nmol/g) [100]. In the mouse lupus model, the

20-day course of MIP2 significantly slowed progress

of inflammatory symptoms of the disease, while in

the model of NAFLD, a prolonged administration of

the peptide significantly decreased manifestations of

inflammation, although did not decrease the levels of

liver markers and histological signs of hepatic steatosis

[100]. Another example of the blocking peptide derived

from the third helical fragment of the TIRAP sequence

is TR667, which represents an evolutionary preserved

segment of the surface of TIR-domain differing from

MIP2 by the single- amino-acid shift towards the C-end

of TIRAP[25]. TR667, similarly to MIP2, exhibited multi-

specific inhibitory properties towards TLR and inhib-

ited TLR2, TLR4, TLR5, and TLR9; however, unlike the

MIP2 peptide, itdid not inhibit TLR7[25].

Efficiency of the peptide 2R9 with respect to sys-

temic production of cytokines induced by the TLR2

and TLR7 agonists, as well as for suppression of cyto-

kine response to a replication-capable pathogen (PR8

strain of the flu virus H1N1B was used) was evaluated

in the study by Piaoetal.[56]. The preliminary inves-

tigation showed that the peptide 2R9 identified during

screening of the peptide library of the TLR2 TIR-do-

main was multi-specific and blocked TLR2, TLR4,

TLR7, and TLR9 due to the binding of TIRAP, adapter

protein enhancing signal transduction from these re-

ceptors [56]. Administration of 2R9 to mice resulted

in significant inhibition of the cytokine response to

both Pam3Cys(S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-

N-palmitoyl-(R)-Cys-Ser-Lys

-OH), agonist of TLR2, and

to R848 (resiquimod), agonist of TLR7, as well as to

ODN1668, TLR9 agonist [56, 83]. 2R9 also blocked by

80-90% the MyD88-dependent secretion of TNF, IL-6,

and IFN-β by the cultivated peritoneal macrophages

infected with the flu virus [56]. Excessive secretion

of cytokines could be the cause of lethal outcome in

acute viral infections. Based on this consideration the

authors tested whether 2R9 could decrease lethality of

mice infected with the dose of flu virus (strain PR8)

NEW APPROACHES TO RECEPTOR INHIBITION 793

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

that causes ~90% lethality. The experiments showed

that the 5-day course of daily injections of the pep-

tide starting 48 h after infection with the virus signifi-

cantly reduced lethality [56]. However, it was shown

in another study that the effect of suppression of the

TLR-dependent immune response on the outcome of

disease significantly depends of the time of the start

of the therapy [101], very early administration of the

agents suppressing immune response could aggravate

the course of infection diseases.

The presented studies demonstrate efficiency

of BPs for both suppression of cytokine production

induced by introduction of synthetic agonists of spe-

cific receptors, as well as in more complex models of

chronic inflammation or inflammation induced by in-

fectious agents.

CONCLUSIONS

Studies of recent decades have led to a significant

progress in the discovery of new BPs and understand-

ing of mechanisms of their action. At present, tech-

niques for BP identification, evaluation of their bind-

ing specificity to targets invitro and in cellular models,

as well the methods for evaluation of their efficiency

in vivo have been established. High efficiency of the

methodology of blocking peptides for the development

of inhibitors of signaling pathways with mechanisms

of action based on blocking transient interaction of

signaling proteins realized through the specialized

protein domains has been demonstrated. Experiments

with small laboratory animals have demonstrated

that BPs are capable of suppressing the systemic re-

sponse to stimulation of certain receptors both in the

cases of intraperitoneal and intravenous injections of

the peptides. Moreover, there are examples of high

efficiency of BPs during prolonged administration

for suppression of chronic inflammatory processes.

It is recognized that the multi-specificity of binding

demonstrated by some BPs is not only important for

understanding of their biological effects, but also sig-

nificantly contributes to their efficacy in complex an-

imal models.

Nevertheless the large number of known BPs, ex-

act molecular determinants of their activity remain

to be established. This lack of understanding likely

stems from such factors as the diversity of inhibitory

sequences, the tolerance of BPs to amino acid substi-

tutions, multiplicity of binding sites of individual BPs,

as well as the lack of structural knowledge on the

BP-target complexes. Determination of high-resolution

3D-structures of BP complexes with their target pro-

teins should improve our understanding of signaling

protein recognition mechanisms, and suggest ways for

rational optimization of already known BPs. BPs’ effi-

cacy also could be improved via optimization of pene-

trating sequences for targeted delivery of the inhibitor

or via use of peptidomimetics to improve the biologi-

cal stability of the BPs.

Funding. This work was financially supported by

the Ministry of Science and Higher Education of the

Russian Federation (Agreement no. 075-10-2021-093;

project NIR-IMB-2102).

Ethics declarations. This review does not contain

original studies with human participants or animals

performed by the author. The author is co-owner of

the copyrights for commercial use of some of the pep-

tides described in the review.

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