Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_6035_Библиотеки_им_академика_М_И_Перельмана.pdf
313
development of various inherited diseases (Ketterer et al., 2017), such as pycnodysostosis,
Papillon–Lefèvre syndrome, myopia, and lysosomal storage diseases. Protease-targeted
therapeutics are being explored as potential treatments for these disorders, presenting
signicant diagnostic and therapeutic challenges. Small-compound drugs targeting
proteases are already available on the market, and a wide variety of structures are being
investigated in the drug discovery process.
NPs with biologically active pharmacophores exhibit inherent large-scale structural diversity . Research on NPs continues to yield a wide range of lead structures that serve as templates
for novel pharmaceutical compounds of novel pharmaceuticals sector. Nature represents an
innite eld of opportunity for the discovery of new molecules and has profoundly impacted
human health. It can be viewed as a “natural laboratory” for exploring a range of NPs.
14.2 CYSTEINE PROTEASES (CPS)
Cysteine proteases (CPs) are classified into five different proteolyzed enzymes: papain-like
enzymes, viral chymotrypsin-like, papain-like endopeptidases of RNA viruses, legumain-
type caspases and containing His, Glu/Asp, Gln, Cys residues in the catalytic cleft (Barrett,
1994). The most abundant proteins are members of the papain-like peptidases. CPs are
evolutionarily related to papain and share a common fold. Papain is a prototypical CPs
enzyme that was first characterized and is widely recognized as the best-known member of
the enzyme family. CPs are proteins of molecular mass ~21–30 kDa, except for tetrameric
Cath C with ~200 kDa (Turk et al., 2005).
CPs are involved in numerous biological conditions, encompassing both physiological
and pathological processes in a variety of plants, animals, and microorganisms (Turk et al.,
2012). Their fundamental functions include the catabolism and hydrolysis of peptide,
amide, ester, thiol ester, and thiono ester bonds (McGrath, 1999). The hallmark of CPs is
the presence of a cysteine residue in the enzyme’s active site mechanism (Vasiljeva et al.,
2007). CPs universally utilize a cysteine residue as the nucleophile and a histidine residue
as the general base for proton donation (Brömme, 2001).
CPs can be divided into exopeptidases and endopeptidases (Lecaille et al., 2002).
Exopeptidases, such as carboxypeptidases and cathepsins, act near the ends of polypeptide
chains. These enzymes cleave the peptide bond proximal to the amino or carboxy termini
of the substrate, liberating a single amino acid residue, a dipeptide, or a tripeptide. Endo-
peptidases, such as papain, bromelain, cain, and cathepsins, preferentially cleave peptide
bonds in the inner regions of peptide chains, hydrolyzing proteins. Cathepsins, which are an
important subgroup within the CPs family , can exhibit both endopeptidase and exopeptidase
activities. Cathepsins are also known as lysosomal CPs (Rawlings and Barrett, 2013).
14.2.1 CATHEPSIN
The protease cathepsin derives its name from the Greek word “kathepsein,” which
meaning “to digest.” Cathepsin was first used to characterize a protease identified in the
314
gastric mucosa. The enzyme exhibited activity in a slightly acidic environment (W illstätter
and Bamann, 1929). All mammalian lysosomal CPs are known as cathepsins, although the
reverse is not true. Subsequently, the term “cathepsin” was expanded to describe serine
proteases, aspartic proteases, and lysosomal cysteine cathepsins. Through bioinformatic
analysis and the sequencing of the human genome, it was revealed that there are 11 human
cathepsins, making them the largest family within the C1a family of the CA clan. The 11
human cathepsins are referred to as B, C (or J), F, H, K (or O2), L, O, S, V (or L2), W,
and X (also known as Z or P) (Turk et al., 2000, 2012; Biasizzo et al., 2022). However,
cathepsins are now classified into three groups based on their active site catalytic residue:
serine (Caths A and G), aspartic (Caths D and E), and CPs, with the latter group including
the 11 human cathepsins. Most cathepsins exhibit predominantly endopeptidase activity,
while Caths X and C function solely as exopeptidases. Caths H and B demonstrate both
endo- and exo-peptidase activity (Turk et al., 2012).
Cathepsins are primarily localized within the endolysosomal system and are expressed
ubiquitously in all living organisms (Turk et al., 2000). Cysteine cathepsins have traditionally been linked to protein degradation within lysosomes. Within the lysosomal system,
protein degradation takes place due to the combined action of various proteases, both
random and selective, which target intracellular and extracellular proteins.
Cysteine cathepsins have traditionally been associated with lysosomal protein degradation. In the lysosomal system, protein degradation occurs as a result of the combined
random and limited action of various proteases, which break down intracellular and
extracellular proteins. Cathepsins exhibit optimal activity in a slightly acidic and reducing
environment, such as that found in lysosomes. Moreover, they can remain functional in
the extracellular space and maintain activity outside of their optimal pH range (Bosea
et al., 2022). Enzyme action is not restricted to the lysosome and has also been observed
in other cellular compartments, such as the cytosol (Droga-Mazovec et al., 2008), nucleus
(Goulet et al., 2004), and secretory vesicles (Wartmann et al., 2010).
Cathepsins are currently recognized for their involvement in a range of distinct
physiological processes, such as apoptosis, adaptive immunity, prohormone activation,
extracellular tissue remodeling, and cell differentiation (Biasizzo et al., 2022). Dysregulation
of their control, whether stemming from heightened expression or diminished inhibition
by endogenous inhibitors, can contribute to the onset of various pathological conditions
(Turk et al., 2012). Cathepsins exhibit selective expression in specic tissues or cell
types. Cath K is predominantly expressed in osteoclasts, Cath V in the thymus, Cath S
in antigen-presenting cells, Cath F in macrophages, and Cath W in natural killer, and
cytotoxic T lymphocytes. However, signicant evidence supporting their physiological
signicance and their involvement in the development and progression of diseases has
emerged in the last decade. A detailed overview of the extracellular roles of cathepsins in
different pathologies can be found in Kramer et al. (2017), Patel et al. (2018), Petushkova
et al. (2019), Vidak et al. (2019), and Bosea et al. (2022). Table 14.1 provides a summary
overview of the classication of human cathepsins based on their specic proteolytic sites
and associated pathologies.
TABLE 14.1 Overview of Cathepsins, Their Peptidase-Specific Activities and Associated Diseases
Cathepsin Type
Protease
A Serine Carboxypeptidase Processing of endogenous
G Serine Endopeptidase Plays a crucial role in the clearance
D Aspartic Endopeptidase Mitogen and promotes
B Cysteine Carboxypeptidase/
C Cysteine Aminopeptidase Inflammation catalyzes the excision
E Aspartic Endopeptidase Antigen processing via the MHC
F Cysteine Endopeptidase N-glycosylation sites targeted
Mode of Substrate
Cleavage
Endopeptidase
Mechanism of Action Specify Functions Diseases
Inhibits the degradation of
bioactive peptides; Inhibit
autophagy
of intracellular pathogens, tissue
breakdown at inflammatory sites,
and contributes, as well as in antiinflammatory response
invasiveness
Cleaves ECM proteins
Promotes amyloid plaque; matrix
degradation and cell invasion;
enable virus entry into the cells
of dipeptides from the N-terminus
of protein and peptide substrates
class II pathway
to the endosomal/lysosomal
compartment via the mannose
6-phosphate receptor pathway
β-galactosidase and neuraminidase
within lysosomes; controls chaperone-
mediated autophagy by modulating the
degradation of lysosomal LAMP-2A
Inflammation and immune response
involve the migration of neutrophils,
monocytes, and antigen-presenting
cells (APCs); regulation of autoantigen
processing; activation of lymphocytes
Neuronal development, brain antigen
processing of antigens such as
α-synuclein, tau, amyloid β, and apoE;
degradation of hormones, proenzymes,
and growth factors
Growth factor processing including
EGF, IGF-1, and TGF-β; degradation
of amyloid-β; promotion of viral entry
into cells
Inflammatory responses; activation of
serine proteases
Carboxypeptidase A and IgE
processing
Invariant chain processing; MHC II
class responses
⏎
315
Muscular dystrophy,
galactosialidosis, cardiomyopathies,
arterial hypertension
Tuberculosis, rheumatoid
arthritis, coronary artery disease,
periodontitis, ischemic reperfusion
injury, autoimmune diseases
Breast cancer; neuronal ceroid
lipofuscinosis (NCL); Alzheimer’s,
Parkinson’s, and Huntington’s
diseases; ischemic heart disease;
sudden cardiac death
Alzheimer’s disease; acute
pancreatitis cancer: brain, skin,
breast, lung, gastric, bladder,
cervical, ovarian, colorectal,
hepatocellular carcinoma, pancreatic,
thyroid; liver fibrosis; angiogenesis
and leukocyte recruitment;
osteoarthritis and arthritis
Papillon–Lefèvre syndrome;
periodontitis; skin cancer;
rheumatoid arthritis; sepsis keratosis
Atopicdermatitis
Type B Kufs disease; cervical
cancer; neuronal ceroid
lipofuscinosis 13 (CLN13)
TABLE 14.1
Cathepsin Type
H Cysteine Aminopeptidase/
K Cysteine Endopeptidase Cleaves ECM protein collagen;
L Cysteine Endopeptidase Matrix degradation; cell invasion
O Cysteine Unknown Collagenolysis elastinolysis
S Cysteine Endopeptidase Antigen presentation; remodeling
(Continued)
Protease
Mode of Substrate
Cleavage
Endopeptidase
Mechanism of Action Specify Functions Diseases
Endopeptidase activity Eye development; immune regulation;
secretion by osteoclasts in bone
resorption
enable virus entry into the cells
osteoclastic bone resorption
of connective tissue and basement
membranes
Prostate tumors; diabetes mellitus
prohormone processing
Bone resorption; extracellular matrix
remodeling
Antigen presentation; cardiovascular
remodeling; adipogenesis and glucose
tolerance
Innate immunity Cardiovascular disease
Major histocompatibility complex
class II (MHC II) antigen presentation
type 1; myopia; osteoporosis;
Papillon–Lefèvre syndrome
Osteoporosis and osteoarthritis;
obesity atherosclerosis; schizophrenia; periodontitis; cancer: skin,
breast, lung, gastric, and prostate;
tuberculosis; pycnodysostosis;
angiogenesis/leukocyte; chronic
kidney disease; recruitment;
abdominal aortic aneurysm
Cancer; gingival overgrowth;
Parkinson’s disease; cardiac repair
and dilated cardiomyopathy; cancer:
brain, skin, breast, lung, gastric,
ovarian, colorectal, pancreatic;
peripheral arterial disease; abdominal
aortal aneurysm; type I diabetes;
angiogenesis/leukocyte recruitment;
kidney disease; osteoarthritis and
rheumatoid arthritis
Cancer: brain, breast, gastric, prostate,
colorectal, pancreatic, hepatocellular
carcinoma; rheumatoid arthritis;
lung fibrosis; cardiovascular and
kidney diseases; neuroinflammation
and hyperalgesia; atherosclerosis;
bronchial asthma; psoriasis;
wound healing; myasthenia gravis;
angiogenesis/leukocyte recruitment
316
TABLE 14.1
Cathepsin Type
V Cysteine Endopeptidase Production of enkephalin and
W Cysteine Unknown Cell-mediated cytotoxicity Role in NK cells; endoplasmic
X/Z Cysteine Carboxypeptidase Protein degradation Immune-cell proliferation, maturation,
(Continued)
Protease
Mode of Substrate
Cleavage
Mechanism of Action Specify Functions Diseases
Natural killer cell and CD8+ cytotoxic
neuropeptide Y
cell production
reticulum (ER) proteolytic machinery
migration, and adhesion
317
Keratoconus; thyroid cancer;
myasthenia gravis; atherosclerosis;
abdominal aortic aneurysm; chronic
kidney disease
Cancer malignancy; inflammation
Inflammatory bowel disease;
autoimmune gastritis; cancer:
gastric, prostate, colorectal,
hepatocellular carcinoma,
pancreatic; Helicobacter
pylori gastritis; aging and
neurodegeneration
318
14.2.2 STRUCTURE AND MECHANISM OF ACTION OF CATHEPSINS
Cathepsins share a common fold known as the papain-like structure. The papain-like fold
consists of two domains referred to as the left (L-) and right (R-) domains. The L-domain is
composed of three α-helices, while the R-domain adopts a β-barrel conformation with the
front strand(s) arranged in a coiled structure. An α-helix encloses the bottom of the barrel.
These domains separate at the top, forming a V-shaped active-site cleft. Within this cleft,
the catalytic site of the enzyme is formed by residues Cys25 and His159, with one residue
from each domain. Cys25 resides at the outset of the central helix within the L-domain.
As for His159, it is situated among the residues of the β-barrel within the R-domain. The
catalytic cleft showed Asn175 residue in a primordial position to direct the imidazolium
ring of the histidine (Figure 14.1) (Turk et al., 2000).
FIGURE 14.1 Representation of the three-dimensional structure of papain and V -shaped active site (extracted
from Protein Data Bank (1pb1) and personally designed in PyMOl).
⏎
Based on the kinetic and structural data presented, it has been proposed that substrate
binding is primarily inuenced by ve subsites (Turk et al., 1998).
The enzyme’s active thiol group must exist in its reduced state to facilitate catalytic
activity, as discussed by McGrath in 1999. Within this context, the imidazole group of
histidine serves to polarize the SH group of cysteine, allowing for deprotonation and the
creation of a highly nucleophilic thiolate/imidazolium ion pair. This concept has been
elaborated upon by Turk and GuncÏar (2003) and further explored by Grzonka et al. (2007).
To activate CPs, it is essential to provide a reducing and acidic environment. These
enzymes are known to establish an ion pair within the pH range of 3.5–8.0.
319
The reactive thiol group of the enzyme needs to be in the reduced form for catalytic
activity (McGrath, 1999). The imidazole group of the histidine polarizes the SH group of
the cysteine, enabling deprotonation and forming a thiolate/imidazolium ion pair, which is
highly nucleophilic (Turk and GuncÏar, 2003; Grzonka et al., 2007).
An S-acyl enzyme intermediate is produced when the thiolate group of the cysteine
residue undergoes a nucleophilic attack on the carbonyl group within the peptide bond of
interest. This process results in the generation of a tetrahedral intermediate during acylation.
The catalytic cycle involves the release of the N-terminal fragment during the deacylation
step, and regenerated the formation of the free enzyme (Figure 14.2).
14.3 NPS AS CATHEPSINS INHIBITORS
The interest in cathepsins as potential therapeutic targets for numerous diseases has
expanded in recent decades. The proteolytic activity of cathepsins is intricately connected
to numerous metabolic such as disease evolution and immune response (Berdowska,
2004). Cathepsin expression is typically increased in cancer progression and metastasis
(Sloane et al., 1981; Jedeszko and Sloane, 2004). It has long been recognized that the
higher concentration of cathepsin in tumors tissue was a critical motivation for prognosis
in melanoma, breast, glioblastoma, lung, prostate, colorectal, head, neck, and many other
cancers. Cathepsins inhibitors are thought to play as important role in the process of tumor
invasion and metastasis, and the reduced levels of cathepsin activity were observed in
different tumor cell lines (Nomura and Katunuma, 2005). Dysfunctions of cathepsins
within the lysosomal system are closely associated with mechanisms of neurodegenerative
diseases (Hook et al., 2020). Extensive evidence from Alzheimer’ s disease, traumatic brain
injury, and related brain disorders supports the involvement of cathepsins in cognitive and
behavioral deficits (Kim et al., 2021; Di Domenico et al., 2016). Cathepsin inhibitors have
demonstrated their importance as crucial pharmacological targets for the exploration and
advancement of innovative and novel therapeutic strategies. These findings lay a robust
groundwork for delving into cathepsin inhibitors as viable pharmacological targets in the
quest for uncovering and advancing innovative therapeutic approaches.
Cathepsin activities have also been implicated in diseases such as rheumatoid arthritis.
In cases of inammatory arthritis, these enzymes have the potential to induce bone and
cartilage degradation, subsequently prompting an immune response. Cathepsin inhibitors
show potential efcacy in slowing down or preventing articular cartilage degradation. In
recent decades, cathepsin K has been strongly associated with bone-related diseases such
as osteoporosis (Bossard et al., 1996; Salminen-Mankonen et al., 2007; Behl et al., 2022).
Cathepsin inhibitors have been shown to decrease or prevent articular cartilage degradation
(Lua et al., 2018).
There have been reported instances highlighting the signicance of cathepsins in viral
infections. Viruses have developed mechanisms to utilize cathepsins in order to enhance
cell infection. Studies demonstrated that cathepsins play a substantial role in the infection
of numerous viruses, including reovirus (Ebert et al., 2002; Chandran and Nibert, 2003),
henipaviruses, Ebola virus (EBOV) (Chandran et al., 2005; Sanchez, 2007; van der Linden
320
et al., 2016), Hendra virus (Pager and Dutch, 2005), Nipah virus (Diederich et al., 2012),
and SARS coronavirus. Spike protein activation in SARS-CoV and SARS-CoV-2 appears
to depend on Cath L (Simmons et al., 2005; Gomes et al., 2020; Utami et al., 2022). Consequently , lysosomal CPs like cathepsins emerge as promising tar gets for the development of
antiviral medications.
FIGURE 14.2 A schematic presentation of substrate binding active sites of CPs and mechanism of enzyme
hydrolysis. (1) Active form of enzyme ion pair Cys-S– (25)/His-ImH+ (159) and formed the first tetrahedral
intermediate by a noncovalent Michaelis complex formation. The stabilization is obtained by the oxyanion
hole formed by backbone NH of Cys25 and side chain NH of Gln19. (2) Acylation of the enzyme by rotation
of His159 and protonation of the leaving amine. (3) Hydrolysis reaction by nucleophilic attack of a water
molecule at the acyl enzyme and formed the second tetrahedral intermediate. (4) Deacylation and release the
product R–COOH. (5) Regeneration of the free enzyme.
⏎
321
Endogenous low-molecular-weight cathepsin inhibitors are part of the cystatins super-
family (Katunuma, 2010). Each cystatin exhibits different inhibitory specicity against
individual cathepsins. Researchers often draw inspiration from NPs isolated from fungi,
bacteria, marine organisms, and plants to explore potential drug candidates. To date,
several NPs from different organisms have been investigated for their ability to inhibit
the catalytic activity of cathepsins. It has consistently been observed that NP inhibitors
can effectively impede cathepsin function, offering potential preventive measures against
various pathologies.
NPs are widely recognized as valuable sources of lead compounds for drug discovery
(Newman and Cragg, 2020). NPs hold great promise for future advancements in discovering cathepsin inhibitors and developing new drugs. They constitute a valuable reservoir
of primary compounds in the eld of drug discovery and highlight promising directions for
the development of cathepsin inhibitors and new drugs.
14.3.1 NPS FROM BACTERIA AS CATHEPSIN INHIBITORS
Leupeptin (1) emerged as one of the earliest isolated NPs with cathepsin inhibitory
properties, having been discovered in 1969 from a Streptomyces strain (Vidal-Albalat
and González, 2016). Leupeptins are widely produced by actinomycetes from different
species and strains of Streptomyces. Leupeptin belongs to a group of NP peptides that also
includes pepstatin (2), antipain (3), and chymostatins (4). These inhibitors have been found
in cultures of various species of actinomycetes (Figure 14.3). These compounds share a
peptidyl aldehyde structure and exhibit inhibitory activity against Caths A, B, and D (Suda
et al., 1972). Leupeptin showed inhibitory activity against Caths A, B, and D with IC50
values of 4, 1, and 0.26 nM, respectively. Chymostatin exhibited IC50 values against Caths
A, B, and D of 100, 4.2, and 81 µM, respectively . On the other hand, antipain demonstrated
inhibitory activity against Caths A, B, and D with IC50 values of 2, 0.9, and 200 µM,
respectively.
Kruglyak et al. (2017) undertook a screening process involving extracts from 350 soil
and lichen-associated Streptomyces isolates gathered from various regions around the
world. Their aim was to discover inhibitors of human Cath K in vitro. Streptomyces strain
IS2-4, collected from soil under an olive tree in Parma, Italy , exhibited promising inhibition
of Cath K. Through bioassay-guided purication process of the extracts, the active component responsible for Cath K inhibition was identied as leupeptazin (5), a novel analog of
leupeptin (Figure 14.3). Leupeptazin competitively inhibits Cath K with a Ki value of 44
µM. In contrast, leupeptin (2) has a Ki value of 24 nM for Cath K. In leupeptin ( 2), the
peptide aldehyde binds covalently to the active site cysteine residue (Cys25), forming a
hemithioacetal with the deprotonated sulfur of Cys25. Leupeptazin (5) features a modi-
ed piperidinotriazine in place of the terminal aldehyde moiety. The signicant decrease
in afnity of leupeptazin for Cath K, around 1000-fold, in comparison to leupeptin, is
likely attributed to the absence of the aldehyde functionality and the increased bulk of
the terminal residue. Molecular modeling studies showed that both compounds efciently
dock in the active site of Cath K.
322
FIGURE 14.3 Chemical structures of NPs peptides leupeptins isolated from actinomycetes from different
species of Streptomyces sp.
⏎
Peptides YM-51084 (6) and YM-51085 (7) were produced by Streptomyces sp. and
their inhibitory effects against Cath L, B, and H were evaluated (Figure 14.4). Peptide (6)
exhibited the most potent inhibitory effects against Cath L activity, with IC50 values of 9.6
nM. Similarly, peptide (6) demonstrated inhibitory activity against Cath B, with an IC50
value of 350 nM. On the other hand, peptide (7) displayed IC50 values of 120 and 7300 nM
against Cath L and Cath B, respectively. Cath H, which is highly homologous to Caths L
and B, showed signicantly higher IC50 values, exceeding 100 µM (T eramura et al., 1996).
FIGURE 14.4 Chemical structures of peptides YM-51084 (6) and YM-51085 (7) isolated from Streptomyces sp.
⏎