Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 11, pp. 1757-1763 © The Author(s) 2025. This article is an open access publication.

Published in Russian in Biokhimiya, 2025, Vol. 90, No. 11, pp. 1879-1886.

1757

DISCUSSION

Cysteine Cathepsins and Drug Discovery:

Knowns and Unknowns

Andrey A. Zamyatnin, Jr.

1,2,3

Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119234 Moscow, Russia

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia

Department of Biological Chemistry, Sechenov First Moscow State Medical University, 119991 Moscow, Russia

e-mail: zamyat@belozersky.msu.ru

Received July 15, 2025

Revised September 10, 2025

Accepted September 11, 2025

Abstract— Cysteine cathepsins are a group of closely related proteolytic enzymes active at low pH. The most

well-studied function of these enzymes is protein degradation within lysosomes. However, accumulating evi-

dence suggests that cysteine cathepsins also function at physiological pH levels in other cellular compartments

outside lysosomes, as well as in the extracellular space. Many of these extra-lysosomal functions of cysteine

cathepsins are typically associated with pathological processes, contributing to conditions such as oncogenesis

and metastasis, neurodegenerative diseases, cardiovascular disorders, and autoimmune and inflammatory pro-

cesses. Consequently, cysteine cathepsins have been proposed as diagnostic and prognostic molecular markers,

as well as pharmacological targets. Notably, the pathological processes involving these enzymes often operate

independently of their classical lysosomal functions. This work aims to outline key questions, the answers

to which could enhance our understanding of the fundamental mechanisms governing the extra-lysosomal

functions of cysteine cathepsins. Addressing these questions is also critical for developing novel therapeutic

strategies to treat diseases in which cysteine cathepsins play a pathogenic role.

DOI: 10.1134/S0006297925602205

Keywords: cysteine cathepsins, papain-like cysteine proteases, pharmacological targets, drug development,

enzyme inhibitors

The term “cathepsin” was proposed in 1929 by

Richard Willstätter and Eugen Bamann [1]. It origi-

nates from the Ancient Greek compound word καθέψω

(from κατά, meaning “down,” and hépsō, meaning “to

boil”), which can be translated as “to boil down” or “to

digest”. Thus, the term reflects the most well-known

function of these proteases: as enzymes responsible

for the breakdown of proteins in lysosomes. The

proteolytic apparatus of human lysosomes includes

15 cathepsins. The majority belong to the cysteine

protease family (11 enzymes: cathepsins B, C, F, H,

K, L, O, S, V, W, and Z [also known as cathepsin  X]).

Additionally, two enzymes each belong to the ser-

ine (cathepsins  A and  G) and aspartate (cathepsins  D

and  E) protease families [2]. It is noteworthy that all

11 cysteine cathepsins belong to the papain-like C1A

subfamily, according to the MEROPS classification [3].

Among human cysteine cathepsins, there are endo-

peptidases with broad substrate specificity (cathepsins

F, K, L, O, S, V, and W), exopeptidases (cathepsins  C

and Z), as well as enzymes possessing both endo- and

exopeptidase activity (cathepsins B and H) [2]. Hence,

the optimal conditions for both the autocatalytic ac-

tivation of cysteine cathepsins and the manifestation

of their proteolytic activity are determined mostly by

an acidic pH, which is characteristic of the endolyso-

somal system in cells [2].

Cysteine cathepsins also perform a number of

important functions outside lysosomes. For example,

cathepsin  K is involved in degrading collagen and

other components of the bone matrix. To accomplish

this, the enzyme is secreted into the resorption lacuna

(the zone of contact between an osteoclast and bone),

where the local environment is acidified, facilitating

the expression of its proteolytic activity [4, 5]. It has

also been reported that cathepsins perform functions

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BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

in intracellular compartments where physiological pH

is maintained. In particular, cathepsin L regulates the

degradation of the transcription factor CDP/Cux in

the nucleus and is involved in cell proliferation and

DNA repair [6, 7]. Cathepsin S cleaves the pro-apop-

totic protein Bax in the cytoplasm, thereby partici-

pating in the regulation of apoptosis initiation [8]. In

the secretory vesicles of pancreatic β-cells, where the

pH ranges from 6.5 to 7.0, cathepsin L cleaves proen-

kephalin and proprotein convertases (PC1/PC2) [9, 10].

The list of physiological substrates for cysteine

cathepsins that are cleaved at neutral pH, as dis-

cussed above, is relatively short. Nevertheless, it

demonstrates the fundamental principle that these

enzymes can possess specific functions mediated by

their proteolytic activity at physiological pH. This list

of substrates could be significantly expanded to in-

clude proteins cleaved by cysteine cathepsins in var-

ious pathological states. For instance, cathepsin S has

been shown to cleave numerous substrates, thereby

promoting angiogenesis and tumor growth [11], and is

also involved in processing the chemokine CX3CL1 in

atherosclerosis [12]. During oncogenesis, E-cadherin is

a substrate for cathepsins B, L, S, and V, but not for

cathepsin C [13, 14]. Similarly, cathepsin V can cleave

N-cadherin and fibronectin [14]. Of particular note is

that identical substrates (e.g., collagen, elastin, E-cad-

herin) are cleaved by various cysteine cathepsins,

suggesting a degree of functional redundancy among

specific members of this enzyme group [15, 16].

The available data on the involvement of cys-

teine cathepsins in pathological processes have log-

ically stimulated the development of approaches to

use these enzymes and their genes as diagnostic

and prognostic molecular markers, as well as ther-

apeutic targets for pharmacological intervention [17,

18]. Initially, the focus was on the development and

pharmacological application of enzyme inhibitors. To

date, several clinical trials aimed at investigating the

effects of cathepsin K, S, C, and B inhibitors have

been completed or discontinued (Table  1; see also

review  [18]). These trials were designed to explore

the potential application of inhibitors for treating

osteoporosis, inflammatory diseases (including viral

and autoimmune conditions), and cancer. However,

despite the clearly observed physiological effects –

including those causing side effects – exerted by cys-

teine cathepsin inhibitors, none have been approved

as drugs, and their further development (with the

exception of a single inhibitor) has been discontin-

ued. This outcome can be attributed to several key

factors. For instance, the perception of cathepsins as

exclusively lysosomal enzymes still prevails, which

has led to insufficient attention being paid to studying

their extra-lysosomal functions in disease pathogene-

sis. Furthermore, the high degree of structural and

functional homology among members of the C1A sub-

family of cysteine proteases must be considered. This

homology results in potential functional redundancy

overlap and compensatory mechanisms, complicating

the development of specific inhibitors for individual

enzymes [2, 16, 22].

Collectively, these issues underscore the neces-

sity for a comprehensive approach to developing

therapeutic strategies. Such strategies should target a

specific cysteine cathepsin while also accounting for

potential off-target effects on other subfamily mem-

bers. This approach must consider all stages of the

biogenesis of these proteolytic enzymes. For example,

in addition to the conventional regulation of expres-

sion and sorting, cysteine cathepsins undergo a multi-

step activation process before becoming functional

enzymes [2]. Moreover, the determinants governing

their activity and substrate specificity remain incom-

pletely understood. These determinants likely differ

significantly between the acidic lysosomal milieu and

the neutral conditions at physiological pH [23].

The majority of cathepsins (B, H, L, C, X, F, O,

and V) are expressed in virtually all tissues. In con-

trast, cathepsins K, S, and W exhibit a more restricted,

tissue-specific distribution; for example, cathepsin K

is expressed predominantly in osteoclasts, cathepsinS

in immune cells, and cathepsinW in lymphocytes [2].

Furthermore, numerous studies indicate that the ex-

pression of certain cysteine cathepsin genes is upreg-

ulated during the development of various pathological

processes [24-26]. A recent comprehensive study pro-

filing the expression of all 11 human cysteine cathep-

sins detected their expression in both embryonic tis-

sue-derived cell cultures and cancer cell lines [27].

This study also revealed significant differences in cys-

teine cathepsin expression between cells of cancerous

and embryonic origin. Specifically, in renal carcinoma

cells, the expression of cathepsins V, B, L, and S was

3- to 9-fold higher than in embryonic kidney cells,

whereas the expression of cathepsins F and X was

significantly reduced [27].

The expression of cysteine cathepsins can be

regulated at both the transcriptional and post-tran-

scriptional levels [2]. Available data suggest the exis-

tence of complex signaling cascades that regulate the

transcription of cathepsin genes. For example, it has

been shown that increased expression of stefin A –

a natural inhibitor of cathepsins – also leads to en-

hanced expression of cathepsin B, and vice versa [28].

However, the mechanisms underlying this compensa-

tory regulation of cathepsin gene expression have not

yet been studied in detail. Therefore, the existence

of such regulatory mechanisms must be thorough-

ly investigated and subsequently taken into account

when developing agents for the pharmacological tar-

geting of cysteine cathepsins.

CYSTEINE CATHEPSINS AND DRUG DISCOVERY 1759

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

Table 1. Cysteine cathepsin inhibitors evaluated in clinical trials

Target

(cathepsin)

Inhibitor

(name/code)

Clinical

trial phase

Condition Brief description and status References

K ONO-5334 phase II osteoporosis

the inhibitor demonstrated efficacy

in reducing bone resorption

development was discontinued (2012)

due to hyperostosis

and cardiovascular risks

NCT01384188

NCT00532337

[19]

odanacatib

(MK-0822)

phase III osteoporosis

has been shown to reduce

the risk of fractures

development was discontinued (2016)

due to an increased risk of stroke

and atherosclerosis

NCT00729183

[20, 21]

balicatib

(AAE581)

phase II

osteoporosis,

knee

osteoarthritis

development was discontinued (2007)

due to scleroderma-like skin lesions

NCT00371670

K MIV-711 phase IIa

knee

osteoarthriti

has been shown to reduce bone/cartilage

degradation biomarkers but without

clinical improvement in symptoms

development was discontinued (2020)

NCT02705625

RO5459072

(RG-7625)

phase I/

Ib/II

– Sjögren’s

disease

– celiac disease

– rheumatoid

arthritis

– in a study on Sjögren’s disease,

it demonstrated a reduction

in cathepsin S activity but without

significant improvement in symptoms

(dryness, inflammation)

– for celiac disease, it did not meet

the primary endpoints (histological

improvement)

– in rheumatoid arthritis,

it did not demonstrate advantages

over standard therapy

development was discontinued (2021)

NCT02679014

NCT02701985

NCT02521610

S LY3000328 phase I solid tumors

development was discontinued (circa

2015) with no publication of results

NCT01515358

B VBY-376 phase I

hepatitis C

(proposed

indication)

a safety study in healthy volunteers

was completed in 2009

the results were not published

development was discontinued,

presumably due to a change

in company strategy

NCT00557583

ADZ7986

(brensocatib;

INS1007)

phase III

phase II

– bronchiectasis

– COVID-19

(severe cases)

– chronic

obstructive

pulmonary

disease

– primary endpoint: reduction

in the frequency of exacerbations;

phase II data demonstrated efficacy

(36% reduction)

– it was intended to suppress

the “cytokine storm” through

neutrophil inhibition

development was discontinued (2022)

– a reduction in inflammatory

biomarkers (p < 0.001) was observed,

but with no improvement in lung

function

development was discontinued (2020)

NCT04594369

NCT04817332

NCT03218917

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BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

The expression of cysteine cathepsins is also

regulated by various epigenetic mechanisms. For in-

stance, the expression of specific cathepsins is modu-

lated by the methylation of CpG islands within their

genes’ promoter regions [29, 30]. Furthermore, their

regulation via long non-coding RNAs [31, 32] and mi-

croRNAs [32-35] has been described. However, pub-

lished studies generally lack a systematic approach

to investigating this regulatory landscape. Specifical-

ly, the potential activation of compensatory mecha-

nisms mediated by the functional activity of other

cysteine cathepsin subfamily members has not been

explored.

The intracellular sorting and secretion of cysteine

cathepsins through the endoplasmic reticulum and

Golgi apparatus, followed by endosome formation,

have been described in detail [2]. For some cathepsins,

post-translational modifications – including glycosyla-

tion – are also known to occur during the sorting pro-

cess [2]. However, the mechanisms that transport cys-

teine cathepsins to other intracellular compartments

are not well understood. One proposed mechanism

suggests that some cathepsin molecules may enter the

cytoplasm upon lysosomal membrane permeabiliza-

tion [36]. Nevertheless, this does not account for the

nuclear localization observed for some enzymes in this

group [37, 38], as no canonical nuclear localization sig-

nals have been identified in their primary structure.

Thus, it can be concluded that the sorting mechanisms

of cysteine cathepsins – with the exception of their ly-

sosomal and extracellular localization – remain poorly

understood and require further investigation.

The expression of a cysteine cathepsin does not

necessarily mean that an active enzyme is present in

the cell, as a catalytic activation process must occur

after translation to produce the active form. The mat-

uration of papain-like cysteine proteases is a multi-

step process that typically involves the sequential

cleavage of the signal peptide and the prodomain.

This leads to the release of the active proteolytic do-

main, which constitutes the mature, active form of

the protease [39]. Intriguingly, expressing the proteo-

lytic domain separately from the prodomain in living

systems does not yield an active enzyme, suggesting

that proteolytic activation involves essential structural

rearrangements beyond simple cleavage for the matu-

ration of the active enzyme [40]. Cysteine cathepsins

are typically capable of autocatalytic activation under

acidic conditions [2, 39], a mechanism that satisfacto-

rily explains the accumulation of their active forms

within lysosomes. However, the presence of active cys-

teine cathepsins in other cellular compartments or the

extracellular space may arise from one of three mech-

anisms: (i)  the enzyme is autocatalytically activated

within lysosomes and subsequently transported out;

(ii)the autocatalytic activation is initiated by addition-

al factor(s) at physiological pH (e.g., it is known that

activation can be enhanced by polyanions, particular-

ly glycosaminoglycans [41]); or (iii)  their proteolytic

activation is mediated by another protease active at

physiological pH. Available literature data suggest that

all these scenarios are plausible, but the regulatory

details and fine-tuning of these processes remain to

be elucidated.

A substantial body of data on the enzymatic ac-

tivity of cysteine cathepsins has been accumulated

to date. Overall, the endopeptidase activity of these

enzymes is characterized by relatively broad sub-

strate specificity, with the hydrophobic S2 pocket of

the active site considered the primary determinant

of their substrate preferences [23,  42]. This under-

standing underpins modern strategies for developing

cathepsin inhibitors. It is important to note, however,

that most experimental data have been derived from

studies conducted in a lysosomal context – that is,

under the acidic pH conditions characteristic of these

organelles [43]. Concurrently, recent literature indi-

cates that at a neutral pH (representing physiologi-

cal conditions outside lysosomes), cysteine cathepsins

can exhibit heightened substrate specificity, selective-

ly cleaving particular proteins [23]. Nevertheless, the

molecular mechanisms governing substrate specificity

at physiological pH remain incompletely understood.

A detailed understanding of these mechanisms is cru-

cial for developing effective cathepsin inhibitors with

defined specificity profiles, including both broad- and

narrow-spectrum agents.

In summary, while a reasonably comprehensive

understanding of the classical lysosomal functions

of cysteine cathepsins has been established, their

roles outside the lysosome – under both normal and

pathological conditions – remain systemically poor-

ly understood. Consequently, while these enzymes

represent well-characterized pharmacological targets

for lysosomal pathologies [44], their extralysosomal

functions present a major knowledge gap. This gap

encompasses the regulation of their expression, mat-

uration, intracellular trafficking, substrate specific-

ity, and other activity-governing parameters. Their

partial functional redundancy is a particularly note-

worthy aspect. This lack of systemic knowledge ex-

plains the limited progress in drug development,

which thus far has been confined to enzyme inhib-

itors. However, it also reveals vast opportunities for

future research. Pursuing this agenda will yield not

only fundamental insights but also findings with high

translational potential. Successfully translating these

findings could lead to novel drugs targeting cysteine

cathepsins for a wide spectrum of human diseases,

including cancer, neurodegenerative, cardiovascular,

and autoimmune disorders, as well as other inflam-

matory conditions.

CYSTEINE CATHEPSINS AND DRUG DISCOVERY 1761

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

Acknowledgments

The author gratefully acknowledges Sophie Ellis Mole

for her assistance with the English proofreading of

this manuscript.

Funding

The study was conducted under the state assignment

of Lomonosov Moscow State University.

Ethics approval and consent to participate

This work does not contain any studies involving hu-

man and animal subjects.

Conflict of interest

The author of this work declares that he has no con-

flicts of interest.

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