Article

ISSN 0006-2979, Biochemistry (Moscow), 2026, Vol. 91, No. 1, pp. S118-S132 © Pleiades Publishing, Ltd., 2026.

Russian Text © The Author(s), 2026, published in Uspekhi Biologicheskoi Khimii, 2026, Vol. 66, pp. 159-178.

S118

REVIEW

Targeted Protein Degradation: Methods and Prospects

Ilya V. Sklyar

1,a

*, Aleksandra M. Rozhkova

1,2

, Elena G. Kondratyeva

1,2

and Arkadiy P. Sinitsyn

1,2

Fundamentals of Biotechnology Federal Research Centre, Russian Academy of Sciences,

119071 Moscow, Russia

Faculty of Chemistry, Lomonosov Moscow State University, 119992 Moscow, Russia

e-mail: h8love@gmail.com

Received August 19, 2025

Revised September 23, 2025

Accepted October 9, 2025

Abstract— In recent years, targeted proteolysis systems have emerged as powerful tools for directed deg-

radation of pathogenic proteins, offering novel therapeutic strategies for cancer, neurodegenerative disor-

ders, and infectious diseases. This review systematizes key mechanisms and recent advances in inducible

targeted proteolysis, including targeted proteasomal degradation (PROTACs, AbTACs, molecular glues), lyso-

some-mediated degradation (LYTACs, AUTACs, ATTECs) via endocytosis or autophagy, and targeted proteolysis

in bacteria (BacPROTACs), which extends degradation technologies to prokaryotic systems. The structural

features, advantages, and limitations of each platform are discussed in detail, along with key publications

demonstrating their preclinical and clinical efficacy. Special attention is given to the prospects for translating

these technologies into therapeutics, including overcoming challenges such as selectivity and invivo delivery.

DOI: 10.1134/S0006297925604034

Keywords: PROTAC, LYTAC, AUTAC, ATTEC, BacPROTAC, AbTAC, Homo-BacPROTAC, AUTOTAC, GlueTAC

* To whom correspondence should be addressed.

INTRODUCTION

Proteolytic mechanisms are natural regulators

of cellular homeostasis. In recent years, these mech-

anisms have been deliberately harnessed for thera-

peutic purposes, particularly in cancer treatment.

Targeted protein degradation is based on selective

tagging of proteins of interest, recruitment of cellular

degradation machinery (such as proteasomes), and

acceleration of proteolysis through specialized small

molecules that bind the target protein. Over the past

25 years, several classes of molecules and associat-

ed technologies have been developed to induce in-

tracellular protein degradation, including PROteolysis

TArgeting Chimeras (PROTACs), antibody-based pro-

teolysis targeting chimeras (AbPROTACs), and bacte-

rial proteolysis targeting chimeras (BACPROTACs), as

well as so-called molecular glues. Collectively, these

approaches not only significantly expanded the ap-

plication of targeted protein degradation, but have

opened new avenues in drug discovery. The aim of

this review was to examine the development of these

targeted proteolysis strategies and to compare and

discuss their therapeutic applications.

Protein degradation and proteolysis are integral

components of proteostasis, i.e., the cellular protein

homeostasis that regulates the composition and qual-

ity of the proteome [1]. Proteolysis is the cleavage of

proteins and peptides into smaller peptides or amino

acids via hydrolysis of the polypeptide backbone by

proteolytic enzymes, also known as proteases, pro-

teinases, or peptidases. In living cells, proteolysis is

highly regulated and selective, with specific proteins

targeted for degradation. In eukaryotic cells, protein

degradation is primarily mediated by proteasomes

and lysosomes [2,3]. Proteasomes are mainly respon-

sible for the degradation of soluble, short-lived, and

misfolded proteins, whereas lysosomes degrade long-

lived proteins, as well as protein complexes, macro-

molecules, and entire organelles [4]. In bacterial cells,

proteolysis is carried out by ATP-dependent protease

complexes, such as ClpP and Lon proteases, as well as

by bacterial 20S proteasome, which together ensure

the removal of damaged or unnecessary proteins [5].

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Fig. 1. Mechanism of PROTAC action. A PROTAC molecule consists of a ligand binding E3 ligase, a linker, and a ligand

interacting withPOI. Simultaneous binding of PROTAC with E3 ubiquitin ligase and POI induces polyubiquitination of POI

and its degradation by the proteasome. The molecular glue also induces the interaction between the POI and the E3 ubiq-

uitin ligase through binding with them, but compared to PROTACs, molecular glues contain no linker and have a lower

molecular weight. AbPROTACs and ligand-based PROTACs (LiPROTACs) penetrate into cells via endocytosis and then into

the cytoplasm through the endocytic release.

TARGETED PROTEIN DEGRADATION

BY PROTEASOMES

Targeted proteolysis is based on selective tagging

of defective proteins leading to their degradation.

Ineukaryotic cells, this is most commonly the covalent

attachment of ubiquitin molecules. Importantly, ubiq-

uitination not only serves as a signal for proteasomal

degradation but can also mark the protein for other

cellular pathways, including endocytosis and autoph-

agy [6]. Proteasomes are components of the ubiqui-

tin–proteasome system, which also includes ubiquitin

ligases and deubiquitinating enzymes. Ubiquitination

is carried out by three enzymes: ubiquitin-activating

enzyme  E1, ubiquitin-conjugating enzyme  E2, and

ubiquitin ligase  E3. First, E1 binds ubiquitin in an

ATP-dependent manner and transfers it to E2. Next,

E3 catalyzes the transfer of ubiquitin from E2 to the

target substrate. The repeated action of these three

enzymes results in the substrate polyubiquitination.

Ubiquitin molecule has seven lysine residues (K6,

K11, K27, K29, K33, K48, and K63) and the amino

group of the N-terminal methionine, which act as at-

tachment points for forming different types of poly-

ubiquitin chains, each directing proteins to distinct

cellular fate. The K48 and K63 chains are most com-

mon (about 80%) polyubiquitin chains in mammalian

cells. Proteins with K48 chains are primarily targeted

for proteasomal degradation, whereas proteins with

K36 chains play a key role in the regulation of lyso-

somal functions and inflammatory response [7-9]. The

principles of the ubiquitin–proteasome system form

the foundation for the function of PROTACs.

PROTACs. PROTAC molecules consist of two li-

gands connected by a linker: one ligand binds to E3

ligase; the other binds the protein of interest (POI)

targeted for ubiquitination. Simultaneous binding

of the E3 ligase and the POI induces ubiquitination

of the latter, leading to its proteasomal degradation,

whereas the PROTAC molecule itself is not degraded

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during this process (Fig.  1). In contrast to small-mol-

ecule inhibitors, PROTACs are recyclable and act in

multiple catalytic cycles; therefore, they are expected

to be effective at lower doses. Theoretically, PROTACs

can be designed to target virtually any intracellular

or transmembrane protein, including proteins previ-

ously considered resistant to targeted cleavage, such

as nonenzymatic proteins [10].

Modern PROTAC research has undergone signifi-

cant changes since the emergence of the PROTAC con-

cept in 2001. PROTAC1 employed the E3 ligase and

SCF complex consisting of SKP1 (S  phase kinase-asso-

ciated protein1), cullin  1, and F-box protein and was

initially tested in Xenopus laevis egg extracts [11].

Subsequent advances transformed PROTACs from ear-

ly peptide-based constructs into fully synthetic small

molecules capable of using alternative E3 ligase com-

plexes to induce proteasomal degradation [12,  13].

However, only 13 of the approximately 600 known

E3 ligases have been successfully employed for the

PROTAC design, highlighting an opportunity for fur-

ther innovation [14]. PROTACs possess inherent lim-

itations, including relatively high molecular weights

(0.6-1.3  kDa) compared to traditional small-molecule

inhibitors (<0.5  kDa) and large polar surface areas,

which negatively affect their solubility and mem-

brane permeability, thus limiting the overall bioavail-

ability of these molecules in clinical applications and

upon oral administration. Moreover, because PROTACs

rely on the ubiquitin–proteasome system, their activi-

ty is largely restricted to intracellular and transmem-

brane proteins, leaving secreted proteins and certain

monotypic membrane proteins inaccessible to con-

ventional PROTAC strategies[15]. Despite these limita-

tions, more than 20 PROTAC-based therapeutics have

entered phase  I and  II clinical trials for both solid

tumors (e.g., ARV-766, KT-333) and hematologic ma-

lignancies (e.g., NX-2127, DT2216) [16]. PROTACs tar-

geting the androgen receptor (ARV-110; NCT03888612)

and estrogen receptor (ARV-471; NCT05654623) have

progressed to phase  II and  III clinical trials for pros-

tate and breast cancers, respectively, emphasizing the

growing clinical promise of PROTACs as a novel anti-

cancer therapeutic strategy.

One of the most attractive features of PROTACs

as therapeutic agents is their ability to target histor-

ically “undruggable” oncoproteins, such as MYC and

signal transducer and activator of transcription 3

(STAT3). PROTACs offer other advantages, including

enhanced targeting specificity and sustained deg-

radation of target proteins, even in cells that have

developed resistance to conventional inhibitors [17].

Thus, a KRAS

G12C

-targeting PROTAC (LC-2) has been

developed as an alternative to the small-molecule

inhibitor MRTX849. LC-2 selectively targeted onco-

genic KRAS

G12C

and enhanced its degradation, even

in inhibitor-resistant cell lines. A distinctive feature

of LC-2 is its high selectivity for the mutant KRAS,

with no interaction with the wild-type protein [18,

19]. Consequently, PROTACs that target mutant on-

coproteins are expected to exert limited side effects

on normal cells. Furthermore, the recyclable na-

ture of PROTACs enables sustained protein degrada-

tion at relatively low effective doses compared with

traditional small-molecule inhibitors. As a result,

PROTAC-based therapies may minimize the adverse

effects commonly associated with conventional che-

motherapeutic agents

Another critical challenge in oncology is the de-

velopment of therapeutic resistance. PROTACs offer a

promising strategy to address this problem by target-

ing and eliminating proteins involved in resistance

mechanisms. Importantly, PROTACs may be applied

preventively to suppress the pathways underlying

drug resistance. This approach is particularly relevant

for malignancies with poor prognoses, such as met-

astatic colorectal cancer resistant to BRAF V600E in-

hibitors, for which median survival is often less than

one year [20]. For instance, PROTAC-mediated degra-

dation of bromodomain-containing protein  4 (BRD4)

implicated in resistance to doxorubicin, has been

shown to enhance doxorubicin efficacy in both can-

cer cell lines and mouse models [21]. Although these

findings are still at an early stage of development,

they indicate the potential of PROTACs to prevent or

overcome resistance to existing therapies and provide

compelling evidence for their capacity to restore drug

sensitivity across diverse oncological contexts.

Molecules capable of simultaneously targeting

multiple proteins are also under active development.

Due to their enhanced specificity and effectiveness

at lower concentrations compared with conventional

inhibitors, PROTACs represent promising therapeutic

tools for difficult-to-treat tumor cell subpopulations.

For example, a dual-target PROTAC (PROTAC 753B) ca-

pable of degrading both B-cell lymphoma extra-large

(BCL-XL) and B-cell lymphoma  2 (BCL-2) proteins has

been developed to alleviate chemotherapy-induced

cellular senescence in acute myeloid leukemia [22].

Beyond dual-target chimeras, multifunctional PROTACs

are emerging. One such example is Y-PROTAC, which

targets anaplastic lymphoma kinase (ALK) and is

linked via a glutathione-cleavable disulfide bond to

a cyclin-dependent kinase  4/6 (CDK4/6) inhibitor [23].

This molecule exhibits the antitumor and antiprolif-

erative activities in vitro not only through ALK deg-

radation and CDK4/6 inhibition, but also through the

simultaneous degradation of both ALK and CDK4.

Although many currently known PROTACs are

highly effective protein-degrading agents, they gener-

ally lack tissue specificity because of their reliance

on E3 ligases with broad expression profiles. A major

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BIOCHEMISTRY (Moscow) Vol. 91 Suppl. 1 2026

obstacle to the clinical translation of PROTAC tech-

nology is the lack of methods for efficient and se-

lective delivery to the desired tissues and cell types.

Their unfavorable biophysical properties of PROTACs,

including relatively high molecular weight and po-

larity, limit their cellular permeability. Moreover,

nonspecific distribution and activity of PROTACs may

lead to adverse effects, particularly when wild-type

oncogenic proteins are targeted [24]. Depending on

the POI, other deleterious side effects are possible,

including formation of malignant neoplasms, as aber-

rant expression levels of certain genes, such as tumor

protein p53 (TP53) and RAD51 recombinase (RAD51),

are themselves pathogenic [25, 26].

Several strategies have been developed to address

these limitations, including AbPROTACs, LiPROTACs

(Fig.  1), and nanoparticle-encapsulated PROTACs

(nanoPROTACs) [24,  27]. AbPROTACs are aimed to

achieve cell type-specific delivery via antibody/anti-

gen-mediated endocytosis, followed by endosomal es-

cape to avoid premature lysosomal degradation. This

strategy requires the presence of a cancer-specific

surface antigen and a corresponding high-affinity an-

tibody. For example, trastuzumab (Herceptin), which

targets HER2 tyrosine kinase receptor, has been suc-

cessfully incorporated into PROTAC constructs, re-

sulting in improved delivery to HER2-positive breast

cancer cells [28]. However, AbPROTACs suffer from

significant drawbacks, including large molecular size

and limited stability upon systemic administration,

which constrain their clinical applicability. One more

interesting approach to enhance the tissue specificity

is conjugating PROTACs with small-molecule ligands

whose receptors are overexpressed in tumor cells. For

example, folate receptor alpha (FOLR1) is frequently

upregulated in multiple cancer types. The conjuga-

tion of folate group with a ligand of ubiquitin ligase

VHL E3 increased the specificity of PROTAC, while

reducing its off-target activity [29]. However, despite

an enhanced efficacy, such modifications increase the

molecular weight of already large PROTAC molecules,

which may have a negative effect on their pharmaco-

kinetic properties and limit their clinical applications.

To overcome the limitations associated with large an-

tibody or ligand conjugates, smaller peptide simulat-

ing antibody–ligand recognition sites have emerged

as an attractive alternative. In particular, cross-linked

(stapled) peptides, which contain covalent constraints

such as hydrocarbon bridges, can maintain α-helical

structure and exhibit enhanced stability compared to

unmodified peptides. Incorporating cross-linked pep-

tides that selectively bind either the target protein or

the E3 ligase may reduce the overall size of PROTAC

constructs while preserving their specificity and ef-

ficacy [30]. Although the application of cross-linked

peptides in PROTAC design is still under active inves-

tigation, this approach holds promise for the develop-

ment of more stable, selectively delivered PROTACs.

The nanoparticle technology has attracted consid-

erable interest due to its potential to enhance thera-

peutic efficiency and specificity, as well as to simpli-

fy molecular delivery. Based on their cellular uptake

mechanisms, nanoparticles can be classified as pas-

sive or active. Passive targeted nanoparticles exploit

the hypoxic and highly angiogenic environment of

malignant tumors, which increases vascular permea-

bility and facilitates diffusion or endocytic uptake by

cancer cells. In contrast, active targeted nanoparticles

rely on the receptor-mediated endocytosis, analogous

to PROTACs conjugated with antibodies or ligands

[27]. The first nanoPROTACs, developed in 2022, were

targeted at a Lewis lung carcinoma model in  vivo.

Specifically, the BRD4-targeting PROTAC dBET6 was

encapsulated in nanoparticles composed of a pH/glu-

tathione-responsive polymer (DS-PLGA). To further

enhance the specificity, two additional molecules

were generated, one of which included the macro-

phage-specific CRV peptide, to enable the targeting of

tumor-associated macrophages. All three nanoPROTAC

formulations demonstrated prolonged circulation

times in vivo and induced significantly greater tu-

mor volume reduction (~75-90%) compared with the

same PROTAC administered without nanoparticle en-

capsulation (~50%). Analysis of the nanoparticle bio-

distribution revealed that membrane-bound PROTACs

accumulated predominantly in tumor cells; however,

non-targeted accumulation in the liver and spleen

was also observed [31].

Despite these advances, the development of resis-

tance to PROTACs remains a concern. Evidence sug-

gests that resistance to PROTACs is primarily driven

by alterations in the recruited E3 ubiquitin ligase

rather than by changes in the target protein itself

[32,  33].

Molecular glues. Molecular glues promote the

dimerization or colocalization of two proteins, re-

sulting in the formation of a ternary complex [34].

Through this mechanism, they can regulate a wide

range of biological processes, including transcription,

chromatin remodeling, protein folding, localization,

and degradation. The earliest examples of molecular

glues were the immunosuppressants cyclosporin  A

(CsA) and FK506. CsA induces the formation of a cy-

clophilin–CsA–calcineurin complex, whereas FK506

promotes the assembly of the FKBP12 (FK506-binding

protein12)–FK506–calcineurin complex. These discov-

eries laid the foundation for the term molecular glues

[35]. Later, another immunosuppressant, rapamycin,

was identified, which stabilizes the FKBP12–rapamy-

cin–FRB (mTOR) complex. In addition to their im-

munosuppressive effects, rapamycin and its analogs

display the antifungal and antitumor activities [36].

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Molecular glues used for targeted proteolysis

induce interactions between an E3 ubiquitin ligase

and a target protein, leading to ubiquitination and

subsequent proteasomal degradation [37] (Fig.  1).

Like PROTACs, molecular glues exploit the ubiqui-

tin–proteasome system; however, there are several

important distinctions between these two systems.

PROTACs are heterobifunctional molecules that si-

multaneously bind an E3 ligase and a target protein,

whereas molecular glues typically bind to only one

component – most often the ligase – and promote or

stabilize its interaction with the substrate. Moreover,

molecular glues generally have a lower molecular

weight because they lack a linker, which contributes

to improved oral bioavailability and enhanced cell

permeability compared with PROTACs. Despite these

advantages, molecular glues are more challenging to

develop, although rational design strategies are now

beginning to emerge.

Notable examples of molecular glues employed

in targeted proteolysis include thalidomide, lenalido-

mide, and pomalidomide. These compounds had been

approved by the FDA for the treatment of various

malignancies long before their mechanisms of action

were fully understood. Years later, it was discovered

that their antitumor activity arises from their func-

tion as molecular glues: they promote interactions be-

tween the cereblon E3 ligase (a substrate-recognition

subunit of the E3 ligase complex) and transcription

factors IKZF1 and IKZF3, leading to their ubiquitina-

tion and degradation. Given their drug-like proper-

ties, molecular glues are expected to attract an in-

creasing interest from both the scientific community

and the pharmaceutical industry [38].

TARGETED PROTEOLYSIS

IN LYSOSOMES

Lysosomes mediate intracellular degradation of

proteins and organelles through three major path-

ways: endocytosis, phagocytosis, and autophagy [39].

During endocytosis, cells internalize extracellular

materials or membrane-associated proteins. Phago-

cytosis involves the recognition and engulfment of

large particles, such as viruses and bacteria. Auto-

phagy is a cellular process responsible for the re-

moval of misfolded or aggregated proteins, damaged

organelles, and intracellular pathogens. Autophagy

occurs through three distinct routes: macroautoph-

agy, microautophagy, and chaperone-mediated auto-

phagy (CMA). In macroautophagy, defective proteins

or organelles are selectively recognized by autoph-

agy receptors and sequestered into vesicles known

as autophagosomes [40]. Autophagosomes fuse with

lysosomes, where their contents are degraded. In mi-

croautophagy, lysosomes directly engulf cytosolic car-

go, leading to its degradation [41]. In contrast, CMA

involves selective recognition of substrate proteins by

cytosolic chaperones, followed by their direct trans-

location across the lysosomal membrane for degra-

dation. CMA possesses two distinguishing features:

first, it selectively degrades soluble proteins rather

than organelles; second, it does not require autopha-

gosome formation [42]. Several strategies for target-

ed proteolysis in lysosomes exploit these pathways.

Unlike proteasome-based targeted degradation, which

is largely restricted to specific intracellular proteins,

lysosome-based targeted proteolysis enables clearance

of protein aggregates, damaged or excess organelles,

membranes, and extracellular proteins.

Lysosomal proteolysis of proteins. LYTACs

and their analogs. LYTACs (LYsosome-TArgeting Chi-

meras) induce the degradation of extracellular and

membrane proteins through the endosomal–lysosom-

al pathway. Because extracellular and membrane pro-

teins account for approximately 40% of all encoded

proteins and play key roles in neurodegenerative, au-

toimmune, and cancer diseases, the LYTAC approach

represents an important complement to the PROTAC-

based strategies. LYTAC molecules are designed to

simultaneously bind an extracellular domain of a

membrane protein or a soluble extracellular pro-

tein and a lysosome-targeting receptor (LTR), such

as cation-independent mannose 6-phosphate receptor

(CI-MPR), expressed on the cell surface. Formation of

this ternary complex triggers receptor-mediated endo-

cytosis, directing the target protein to lysosomes for

degradation [43]. In late endosomes, the acidic envi-

ronment promotes dissociation of the targeted pro-

tein from CI-MPR followed by its degradation, while

CI-MPR is recycled back to the Golgi apparatus and

then to the cell surface. The first reported LYTAC

molecules were based on CI-MPR, also known as in-

sulin-like growth factor  2 receptor (IGF2R). These con-

structs consisted of a small molecule or an antibody

conjugated to a synthetic glycopeptide ligand that

binds CI-MPR (Fig.  2)[43]. This strategy demonstrated

strong potential for the degradation of multiple ther-

apeutically relevant proteins. For example, a LYTAC

generated by covalent conjugation of a CI-MPR ligand

to the anti-EGFR antibody Cetuximab was shown to

selectively degrade EGFR in various cell lines [43].

The bispecific aptamer chimera composed of a

DNA aptamer targeting CI-MPR and a transmembrane

target protein can be regarded to as a LYTAC analog.

Such aptamer chimeras can direct membrane pro-

teins, including receptor tyrosine kinases MET and

PTK-7, to lysosomes for degradation, while exerting

minimal effects on non-target proteins. Overall, this

strategy represents a powerful, efficient, and broadly

applicable platform for inducing selective degradation

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Fig.  2. Lysosomal proteolysis of proteins mediated by LYTACs and their analogs. LYTAC consists of a small molecule or an

antibody  (A) conjugated with a ligand that binds to the lysosome-targeting receptors (LTR) such as CI-MPR  (B). CI-MPRs

are internalized by endocytosis together with the LYTAC molecules and the POI. Whereas the POI is degraded in the ly-

sosomes, the ligand is returned to the plasma membrane for reuse. The bispecific aptamer chimera uses a DNA aptamer

for targeting the POI  (A) to the LTR  (B). AbTAC uses a recombinant bispecific antibody to attract the membrane POI (A)

and the membrane-bound E3 ligase RNF43 (B). The POI is degraded in the lysosomes (and not by proteasomes). GlueTAC

consists of a covalently modified nanoparticle  (A), a cell-penetrating peptide (CPP)  (B), and a lysosomal sorting sequence  (C).

The nanoparticle is responsible for binding the POI, while CPP induces endocytosis of the GlueTAC–POI complex, followed

bylysosomal degradation.

of membrane proteins. Nucleic acid aptamers offer

several advantages over antibodies, such as ease of

synthesis, high structural precision, and superior sta-

bility [44] (Fig. 2).

Unlike antibody-based approaches, GlueTACs em-

ploy nanoparticles conjugated with a cell-penetrating

peptide (CPP) and a lysosome sorting sequence (LSS).

The nanoparticles enhance the cellular uptake, while

the CPP-LSS promotes lysosomal trafficking and sub-

sequent degradation. To overcome a relatively low

binding affinity and to reduce off-target effects, the

nanoparticles and antigens are linked by covalent

bonds. For instance, a designed GlueTAC molecule ef-

fectively reduced PD-L1 levels in cells and suppressed

tumor growth in immunodeficient mice, outperform-

ing the FDA-approved anti-PD-L1 antibody atezoli-

zumab [45] (Fig.  2).

Noncanonical bispecific AbTACs also exploit the

lysosomal degradation pathways. Unlike convention-

al PROTACs, AbTACs are capable of targeting mem-

brane proteins, thereby substantially expanding the

repertoire of substrates amenable to modern target-

ed proteolysis strategies [46]. Although classified as

PROTACs, AbTACs are more closely related to LYTACs.

AbTACs use bispecific antibodies, with one arm rec-

ognizing a cell-surface POI and the other engag-

ing a transmembrane E3 ubiquitin ligase, such as

RNF43 [47]. After AbTAC binding, the resulting ter-

nary complex is internalized and trafficked to lyso-

somes, where the target protein undergoes degrada-

tion. However, the mechanism of action of AbTACs

remains less well understood than that of LYTACs.

In particular, it is unclear whether ubiquitination of

the intracellular domain of the target protein occurs

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Fig.  3. Targeted protein degradation via autophagy. AUTAC molecule consists of a ligand targeting the POI, a linker, and

a cGMP-based degradation tag. The degradation tag recruits autophagosomes for the degradation of cytoplasmic proteins

and cell organelles. ATTEC molecule simultaneously binds LC3 and POI, while AUTOTAC molecule binds p62 and POI.

Thebinding induces autophagosome formation, and the subsequent fusion of autophagosomes with lysosomes leads to the

POI degradation.

prior to endocytosis and, if so, how this ubiquitina-

tion facilitates lysosomal trafficking. Moreover, it re-

mains unknown whether RNF43 is recycled and re-

used following degradation, as observed for LYTAC

receptors and other endocytic receptors (Fig.  2).

Protein degradation via autophagy. A molecule

of autophagy-targeting chimera (AUTAC) (Fig.  3) con-

sists of three elements: a cGMP-based degradation

tag, a linker, and a small ligand molecule that binds

either a target protein or a specific organelle. AUTACs

induce K63 polyubiquitination, thereby triggering se-

lective autophagy and subsequent lysosomal degrada-

tion. This mechanism contrasts with that of PROTACs,

which promote K48 polyubiquitination and proteaso-

mal degradation. Notably, AUTACs can target not only

cytosolic proteins but also entire cellular organelles,

such as mitochondria. For example, AUTAC4 contains

a ligand that binds a transporter located on the out-

er mitochondrial membrane, enabling selective mito-

phagy. Treatment with AUTAC4 has been shown to

restore mitochondrial membrane potential and ATP

production [48].

Similar to AUTACs, AuTophagy-TEthering Com-

pounds (ATTECs) (Fig.  3) promote selective protein

degradation by using the autophagosomal machinery.

Whereas AUTACs recruit autophagosomes indirectly,

ATTECs directly bind microtubule-associated protein

light chain  3 (LC3), a key component of autopha-

gosomes. For example, small molecules have been

developed that simultaneously bind LC3 and patho-

genic mutant huntingtin protein (mHTT) containing

expanded polyglutamine tract that causes the Hun-

tington’s disease. Notably, these ATTECs selectively

recognize mHTT while sparing the wild-type protein,

despite the two differing only in the polyglutamine

tract length. This high specificity opens up new op-

portunities for treating the Huntington’s disease [49].

AUTOTAC molecules (Fig.  3) consist of two func-

tional modules: one that binds the ZZ domain of the

autophagy receptor p62 and another that recognizes

the POI [50]. By linking the target protein to p62 in-

dependently of ubiquitination, AUTOTACs induce p62

oligomerization and activation, thereby promoting

degradation via the autophagy. AUTOTACs enable the

targeted degradation of both monomeric and aggre-

gation-prone proteins. In mouse models overexpress-

ing pathological human tau, AUTOTAC treatment ef-

fectively cleared misfolded tau protein. In contrast,

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proteasome-based strategies such as PROTACs and

molecular glues, are generally ineffective against pro-

tein aggregates. Beyond tau, AUTOTACs effectively re-

moved various oncoproteins, including the androgen

receptor (AR) [50].

Chaperon-mediated autophagy. In CMA, heat

shock protein 70 (HSC70) recognizes soluble pro-

teins containing the pentapeptide KFERQ. This motif

functions as a degradation signal, analogous to ubiq-

uitination, and may become exposed on the protein

surface as a result of unfolding, post-translational

modifications (e.g., acetylation or phosphorylation), or

other structural perturbations. The HSC70–substrate

complex binds to the lysosome-associated membrane

protein  2A (LAMP2A) on the lysosomal membrane,

enabling translocation of the protein substrate into

the lysosomal lumen for degradation. Based on this

mechanism, chimeric peptides incorporating the

KFERQ motif together with the sequence that binds

a target protein can be used to induce selective deg-

radation of pathogenic or misfolded proteins. Chime-

ric molecules targeting proteins for CMA consist of

three functional domains: a cell-penetrating sequence,

a target protein-binding sequence, and a CMA-target-

ing motif. Upon cellular uptake, these molecules bind

the target protein and direct it to lysosomes for deg-

radation [51]. However, compared with PROTACs and

LYTACs, CMA-based chimeras exhibit limited stability

and inefficient intracellular delivery. Consequently,

this strategy has not yet led to effective therapeutic

applications.

TARGETED PROTEOLYSIS IN BACTERIA

Protein degradation in bacteria is carried out by

proteases such as Clp and Lon, as well as by bac-

terial 20S proteasome, all of which contain AAA+

ATPase domains. As a general principle, proteolytic

complexes consist of an ATPase that unfolds polypep-

tide substrates and a protease that catalyzes hydro-

lysis of peptide bonds. In addition, bacteria possess

numerous other proteases with specialized functions

and diverse intracellular or extracellular localizations

[52]. The gene encoding the caseinolytic protease

ClpP is present in most bacterial genomes, with the

exception of mycoplasmas [53]. ClpP has also been

found in eukaryotes, primarily in chloroplasts and

mitochondria. It is an ATP-dependent serine protease

that associates with AAA+ chaperones. Structurally,

ClpP assembles into a tetradecameric, barrel-shaped

complex composed of two stacked heptameric rings.

In some bacteria harboring two paralogous clpP1 and

clpP2 genes (e.g., Mycobacteriaceae, Listeriaceae, and

Pseudomonaceae), ClpP1 and ClpP2 form separate ho-

moheptameric rings that stack on top of each other

to generate a functional protease complex. The active

sites of the 14 subunits face inward toward the cen-

tral proteolytic chamber. Because of the small diame-

ter of the entrance pore, ClpP alone can degrade only

unstructured proteins and short peptides. Efficient

degradation of folded proteins requires cooperation

with AAA+ chaperones, which recognize substrates

and actively unfold them prior to the translocation

into the proteolytic chamber. In general, the subunits

of processive proteases exhibit low substrate specific-

ity; therefore, substrate selection is mediated by short

recognition sequences known as degrons. Degrons

are recognized by AAA+ subunits either directly or

via adaptor proteins that facilitate substrate deliv-

ery to the proteolytic subunits. Both C-terminal and

N-terminal degrons exist in bacteria. Incompletely

synthesized proteins stalled on ribosomes are typical-

ly tagged with C-terminal degrons, such as the ssrA

peptide, and are subsequently degraded by the ClpXP

protease complex[54]. Most often, bacterial N-degrons

are generated through endoproteolytic processing or

by the attachment of primary destabilizing residues

to specific N-terminal amino acids through the action

of amino acid transferases [55].

There are very few known examples of targeted

proteolysis in bacteria, causing a substantial meth-

odological gap between bacterial and eukaryotic sys-

tems. This disparity largely arises from the absence of

the ubiquitin–proteasome system in bacteria, which

serves as the central mechanism for targeted protein

degradation in human cells. Nevertheless, the diver-

sity of proteases and degradation pathways discussed

in this review highlights the potential for developing

strategies to target specific bacterial proteins toward

controlled degradation.

Bacterial PROTACs (BacPROTACs). The first ex-

ample of a small molecule causing specific protein

degradation in bacteria through protein destabiliza-

tion was pyrazinamide. This compound inactivates

aspartate 1-decarboxylase PanD (pantothenate biosyn-

thesis gene  D), an enzyme essential for coenzyme  A

(CoA) biosynthesis in Mycobacterium tuberculosis.

Pyrazinamide was originally classified as a conven-

tional enzyme inhibitor; however, recent studies have

demonstrated that it promotes PanD degradation by

ClpC1P [56]. Pyrazinamide exposes the C-terminal de-

gron of PanD and alters the multimeric assembly of

the PanD complex, thereby triggering its proteolysis.

Pyrazinamide represents the first antimicrobial agent

shown to exploit targeted protein degradation as its

primary mechanism of action.

In addition, so-called Homo-BacPROTACs (homobi-

functional BacPROTACs) have been developed. These

molecules consist of two cyclic heptapeptides derived

from cyclomarins (biologically active marine cyclopep-

tides), that bind to the mycobacterial unfoldaseClpC1.

SKLYAR et al.S126

BIOCHEMISTRY (Moscow) Vol. 91 Suppl. 1 2026

By dimerizing ClpC1, Homo-BacPROTACs redirect the

Clp protease machinery to degrade this regulato-

ry subunit, effectively removing the cell’s degra-

dation machinery and leading to bacterial death.

Homo-BacPROTACs have been shown to induce deg-

radation of the ClpC1 N-terminal domain in  vitro, as

well as endogenous full-length ClpC1 in Mycobacteri-

um smegmatis cells. Notably, compared with mono-

meric analogues, Homo-BacPROTACs display enhanced

antibacterial activity against wild-type M.  tuberculo-

sis, intracellular bacteria residing in macrophages,

and drug-resistant strains[57].

The application of the PROTAC technology to bac-

teria could, in principle, enable the development of

more potent antibiotics with novel mechanisms of ac-

tion. However, bacteria lack the E3 ligase–proteasome

system, which prevents the direct application of this

strategy for antibiotic discovery [5].

The first antibiotic functioning through the tar-

geted proteolysis was discovered serendipitously.

Pyrazinamide, a cornerstone of first-line tuberculosis

therapy for more than six decades, is a prodrug that

is converted by bacterial pyrazinamidase into its ac-

tive form, pyrazinoic acid (POA)[58]. Remarkably, the

mechanism of action of POA had remained elusive

for many years. The studies of pyrazinamide- and

POA-resistant M.  tuberculosis strains in  vitro and in a

specialized mouse model have identified mutations in

PanD [59,  60]. These findings demonstrated that POA

suppresses CoA biosynthesis by binding to PanD [59,

61], but does not directly inhibit its enzymatic activi-

ty. Further investigations revealed mutations in ClpC,

a component of the Clp complex [62]. The studies in

reporter strain showed that PanD contains a C-ter-

minal degradation tag and represents a substrate of

the ClpCPprotease complex, indicating that PanD lev-

els are regulated post-translationally by Clp complex.

The binding of POA to PanD induces conformational

changes that promote recognition of the C-terminal

degradation tag by the ClpC1 complex, leading to the

targeted proteolysis of PanD [56].

Later, BacPROTAC-1 and BacPROTAC-3 were devel-

oped. BacPROTAC-1 consists of a biotin moiety linked

to a polyarginine motif, which functions as a ligand

for monomeric streptavidin selected as the model

POI. BacPROTAC-1 successfully mediated the biochem-

ical degradation of monomeric streptavidin by a puri-

fied ClpCP protease complex derived from B.  subtilis

[63,64]. In 2022, the first fully functional BacPROTAC

platform was reported, demonstrating highly specific

retargeting of the ClpCP protease toward new sub-

strates. ClpCP is found in Gram-positive bacteria and

mycobacteria, where it recognizes phosphorylated

arginine residues as degradation signals. Engineered

BacPROTAC molecules consist of three components:

a ligand for the target protein, a chemical linker,

and an anchor that binds the N-terminal domain of

ClpC (ClpCNTD) (Fig.  4). Originally, this anchor was a

phosphorylated peptide derivative that mimicked the

native bacterial degradation tag. This BacPROTAC ef-

ficiently induced the degradation of four distinct en-

gineered proteins, with the highest activity observed

for the substrate with the least structured C-terminal

region. The experiments were conducted in B.  subtilis

and M. smegmatis. Detailed structural analysis of

the ClpCP complex revealed that BacPROTAC binding

triggered a reorganization of ClpCP from its inactive

decameric state into the active assembly of four hex-

americ rings. This finding suggests that BacPROTACs

not only recruit substrates to the protease but also

promote conformational activation of ClpCP, thereby

facilitating proteolysis [65].

Structural analysis of the ClpCP complex from

B. subtilis demonstrated that the BacPROTAC-1 bind-

ing induces a conformational transition of ClpC from

its inactive decameric state to an active higher-order

oligomer capable of forming a functional complex

with ClpP. This observation suggests that BacPROTAC

molecules directly promote substrate engagement by

triggering ClpCP activation. BacPROTAC-1 induced

the degradation of monomeric streptavidin (POI)

by the purified ClpCP complex from M.  smegmatis,

indicating that the BacPROTAC strategy may be ap-

plicable to other bacterial species as well [65]. De-

spite these promising results, the polyarginine motif

of BacPROTAC-1 limits its cellular permeability, and

the molecule itself is chemically unstable. Moreover,

validation of BacPROTAC activity in intact cells using

the biotin–streptavidin system is complicated by com-

petition of intracellular biotin for binding with mo-

nomeric streptavidin. To overcome these limitations,

the authors redesigned both the protease-recruiting

element and the POI-targeting ligand, leading to the

development of BacPROTAC-3 (BacPROTAC-2 displayed

activity similar to BacPROTAC-1 and did not represent

a substantial improvement). BacPROTAC-3 consists of

the JQ1 ligand, which binds the protein of interest

BRDTBD1 (bromodomain-1 of the bromodomain tes-

tis-specific protein) and a protease-recruiting motif

derived from the antibiotic cyclomarin  A (CymA),

known to bind ClpC in mycobacteria [66, 67].

Interestingly, BacPROTAC-3 induces the ClpCP-de-

pendent degradation of POI both in  vitro and in

M.  smegmatis cells expressing the target protein.

Proteomic analysis confirmed that treatment with

BacPROTAC-3 resulted in the selective degradation of

the target protein [65]. These findings indicate that

CymA-based BacPROTACs can mediate highly selec-

tive protein degradation in intact mycobacterial cells.

Although current BacPROTACs have been designed

primarily to engage ClpC, future BacPROTACs may be

developed to recruit alternative bacterial proteases,

TARGETED PROTEIN DEGRADATION S127

BIOCHEMISTRY (Moscow) Vol. 91 Suppl. 1 2026

Fig.  4. The mechanism of action of BacPROTACs and Homo-BacPROTACs. BacPROTACs bind the POI, while the polyarginine

region is recognized by ClPC1, targeting the POI to the protease complex. Homo-BacPROTACs induce the degradation of

ClPC1, which in this case is the POI.

such as the serine protease Lon or metalloprotease

FtsH.

The previously synthesized polypeptide deoxycy-

clomarin-C (dCym) was utilized in the next genera-

tion of Homo-BacPROTAC molecules. CymA is a potent

inhibitor of ClpC1 [66], and its analog dCym signifi-

cantly disrupts the mycobacterial proteome, inducing

a ~600-fold increase in the ClpC2 levels [68]. ClpC2,

which shares structural similarity with the receptor

domain of ClpC1, can bind dCym and thereby com-

pete with ClpC1 for ligand binding. This competition

reduces the cytotoxicity of dCym approximately four-

fold. In M. smegmatis, ClpC2, like the recently de-

scribed ClpC3, functions as a regulatory component of

the Clp protease, possessing the same ligand-binding

site as ClpC1. Through competition for substrate bind-

ing, these paralogs act to limit excessive proteolysis

and thereby protect the cell [68, 69].

Cyclic peptide dimers designed and synthesized

to disrupt the “protective” effect against dCym- and

CymA-mediated inhibition through competitive bind-

ing to ClpC2/ClpC3 were named Homo-BacPROTACs.

They feature the dCym motifs at both ends, connect-

ed via linkers of varying lengths in order to simul-

taneously target ClpC1, ClpC2, and the ClpC1P1P2

complex. Using M.  smegmatis as a model system,

Homo-BacPROTACs were shown to reduce ClpC1 and

ClpC2 levels to approximately 40% and 45-60%, re-

spectively, compared to monomeric dCym after 24  h

of treatment. The antimicrobial activity was assessed

in M.  tuberculosis H37Rv, where the minimum inhib-

itory concentrations (MICs) of HBP-6 and HBP-7 were

0.34  µM and 0.26  µM, respectively, compared to 39  µM

for dCym. Both compounds effectively reduced ClpC1

and ClpC2 levels and demonstrated activity against

dormant M.  tuberculosis cells [68] (Fig.  4).

The accidental discovery that pyrazinamide,

a first-line antituberculosis drug, exerts its effect

through targeted proteolysis has highlighted the po-

tential of this approach for antibacterial drug discov-

ery. However, this finding alone has not enabled the

development of a rational, broadly applicable strat-

egy to direct any POI to degradation. Achieving this

requires bifunctional molecules capable of simulta-

neously engaging the target protein and the cellular

degradation machinery. Since bacteria lack the E3

ligase–proteasome system, the conventional PROT-

AC approach cannot be directly applied to bacterial

cells. Recent studies have addressed this challenge

by inducing proximity between the POI and bacterial

SKLYAR et al.S128

BIOCHEMISTRY (Moscow) Vol. 91 Suppl. 1 2026

proteasome using bifunctional molecules known as

BacPROTACs. This strategy, guided by structural and

biochemical insights, has successfully demonstrated

the possibility of targeted protein degradation in bac-

terial systems.

CONCLUSIONS

Molecules designed for targeted proteolysis rep-

resent a unique class of compounds with a pseudo-

catalytic mechanism of action. Although they are not

catalysts in the classical sense, their ability to trigger

multiple cycles of target protein degradation (through

the recycling of E3 ligases or lysosomal receptors)

makes them exceptionally efficient tools for precise

modulation of the proteome.

A defining feature of these molecules is their

dual functionality: one region binds specifically to the

POI, while the other links it to the cellular machinery

responsible for degradation, such as the proteasome,

lysosome, chaperones, or bacterial enzymatic com-

plexes. Once bound, the molecule effectively tags the

target protein for degradation and facilitates its deliv-

ery to the appropriate proteolytic system. One of the

most important advantages of this approach is the

“recycling” capability of these compounds. Most mol-

ecules remain active throughout multiple biochemical

cycles, returning to their original state after each deg-

radation event. This property allows drugs based on

targeted proteolysis to maintain efficacy at low doses

and sustain long-lasting therapeutic effects.

Early generations of PROTACs faced challeng-

es such as low membrane permeability and limited

bioavailability. Recent advances, particularly in small

molecule-based PROTACs, have improved the phar-

macokinetics and enabled activity against both mem-

brane-bound and intracellular targets. The emergence

of heterobifunctional platforms – such as LYTACs,

AUTACs, and ATTECs – has broadened the spectrum

of degradable targets to include extracellular proteins,

protein aggregates, and even organelles. In bacterial

systems, BacPROTACs have adapted the principles of

targeted proteolysis to prokaryotic degradation mech-

anisms, for example, by recruiting ClpXP proteases,

thus paving the way for next-generation antibiotics.

In oncology, PROTACs have already shown effica-

cy against traditionally “undruggable” targets, includ-

ing BRD4 and ERα, while LYTACs address the chal-

lenge of degrading ligand–receptor complexes, such

as PD-L1.

Systems targeting tau protein and α-synuclein

(e.g., ATTECs) hold promise for the management of

neurodegenerative diseases.

Despite their high specificity, these technologies

can still exhibit off-target effects, e.g., unintended

activation of the immune response by LYTACs, high-

lighting the need for further optimization. Overcom-

ing the blood–brain barrier for neurodegenerative

applications and achieving tissue-specific targeting

remain critical for clinical translation. Additionally,

cases of resistance to PROTACs have been reported,

often due to mutations in E3 ligases or in target pro-

teins themselves.

Future directions in this field include integrating

targeted proteolysis with complementary approaches,

such as CRISPR-based target validation or nanocar-

rier-mediated delivery, and developing multispecific

degraders capable of simultaneously modulating mul-

tiple oncogenes. Even now, targeted protein degrada-

tion represents a transformative paradigm in thera-

peutic development, seamlessly combining chemistry,

structural biology, and cell biology.

Abbreviations

AbPROTAC antigen-based proteolysis targeting

chimera

ATTEC autophagy-tethering compound

AUTAC autophagy-targeting chimera

BacPROTAC bacterial proteolysis targeting

chimera

CPP cell-penetrating peptide

LYTAC lysosome-targeting chimera

PROTAC proteolysis targeting chimera

Contributions

I.V.S. conducted the literature search, analyzed the

materials, and wrote the text of the article; A.M.R.,

A.P.S., and E.G.K. developed the study concept and

edited the manuscript.

Funding

This study was supported by the Ministry of Science

and Higher Education of the Russian Federation.

Ethics approval and consent to participate

This work does not contain any studies involving hu-

man or animal subjects.

Conflict of interest

The authors of this work declare that they have no

conflicts of interest.

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