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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_6035_Библиотеки_им_академика_М_И_Перельмана.pdf
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14.3.2 NPS FROM FUNGUS AS CATHEPSIN INHIBITORS
The investigation of fungal biodiversity represents a rich source of various biologically
active compounds that can serve as a substantial resource for discovering new NP inhibitors against cathepsins. One of the key NPs derived from fungi known for its inhibitory
effects on cathepsins is the epoxysucinyl peptide, specifically referred to as E-64 (8). For
example, E-64 is considered one of the most important NP inhibitors ever isolated in the
field of drug discovery (Figure 14.5). It was initially isolated from the culture extract of
Aspergillus japonicus
, obtained from freshly collected soil samples (Hanada et al., 1978).
The discovery of E-64 has greatly expanded our understanding and knowledge of cysteine
peptidases. Extensive evidence has conclusively demonstrated the effectiveness of E-64 as
an irreversible inhibitor against various CPs, including papain, Caths B, H, K, F, L, O, S,
V, and X, among others. However, E-64 is not a universal inhibitor of cysteine peptidases
(Powers et al., 2002). One of the advantages of E-64 is its high efficacy combined with low
toxicity (Sugita et al., 1980).
FIGURE 14.5 Chemical structure of E-64 first isolated from the culture of Aspergillus japonicas.
⏎
Crystal structures and binding modes of the papain–E-64 complex were determined
using X-ray diffraction (Varughese et al., 1988). Three primary binding modes have been
either observed or suggested. In the most commonly observed binding mode, the epoxysuccinate attaches to the S subsites. The carboxylate group at the C-2 position in E-64
positions the epoxide for alkylation by Cys25 through a hydrogen-bonding interaction
involving His159 and the oxyanion hole (Gln19). Consequently, E-64 forms a covalent
bond with the S subsites.
The mechanism of E-64 inhibition is the same for many CPs, involving irreversible
binding and the formation of a covalent bond (S–C) between the thiol group of the
cysteine residue and the carbon atom of the epoxide. The knowledge obtained from X-ray
crystal structure analysis of the papain–E-64 complex has enabled researchers to design
new drug candidates against cathepsins with improved specicity and/or potency. One
approach employed is the modication of the chemically reactive segment of the inhibitor,
specically the epoxide ring found in E-64, to synthesize novel drug candidates based on
a lead compound.
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Since the discovery of E-64, various new epoxysucinyl peptides (Figure 14.6)
have been discovered as NPs from different fungal strains (Goursalin et al., 1994).
Aspergillus oryzae O-1018, isolated from industrial koji for sake brewing, was found to
produce epoxysuccinate inhibitors (Yamada et al., 1998). Specically, compounds (9)–
(11) exhibited tenfold greater efcacy against Caths B and L compared to E-64, while
compounds (8) and (12) demonstrated approximately 100 times stronger inhibitory
activity than E-64 against Cath L.
FIGURE 14.6 Chemical structures of promising isolated trans-epoxysucinyl peptides (8)–(21) discovered as
NP of different fungal strain.
⏎
Similarly, the fungal strain Aphanoascus fulvescens was originally isolated from a
soil sample collected in Kanoya City, Kagoshima, Japan. Trans-epoxysuccinyl peptide
compounds (13) and (14) were isolated as a new type of peptide with a basic residue. These
peptides inhibited Caths B and L with IC
values of 8.4 and 66 nM, respectively . Likewise,
50
compound (14) inhibited Caths B and L with IC50 values of 13 and 72 nM, respectively.
These compounds showed higher effectiveness against Cath B (Otsuka et al., 2000).
325
Microascus longirostris was found to produce the trans-epoxysuccinyl peptide known
as cathestatins (Woo et al., 1995). Cathestatins (15)–(17) were identied as decarbamidoyl
analogs of estatins and exhibited specic inhibition against CPs. Cathestatin (17)
demonstrated potent and irreversible in-vitro inhibition against Cath B, with an IC50 value
of 8.8 nM. Cathestatins (15) and (16) showed moderate inhibitory activity against Cath
B, with IC50 values of 177.6 and 114.3 nM, respectively. Additionally, both cathestatins
exhibited potent inhibitory activity against Cath L, with IC50 values of 1.4 nM for
cathestatins (15), 3.0 nM for cathestatins (16), and 11.1 nM for cathestatins (17). Similarly,
cathestatin (15) were discovered in Penicillium citrinum (Yu et al., 1995). Cathestatins
(15) and (16) showed moderate inhibitory activities against different CPs, including Cath
B and L, papain, cin, and bromelain. However, they displayed lower inhibition against
serine proteases, a metalloprotease, and an aspartic protease, Cath D. Cathestatins were
specic inhibitors of CPs, similar to other epoxy succinyl peptides like estatin and E-64.
Cathestatins and estatins exhibited very similar potency and selectivity between Cath L and
B. Based on available information, E-64 does not show selectivity between Cath L and B.
It is suggested that the aromatic ring at the R2 position may be important for the selectivity
between cathepsins.
Compounds (18)–(21) were isolated from the fungus Gliocladium sp., which was
obtained from a rotten leaf of Phalaenopsis sp., a cultivated orchid, collected in W arabi-shi,
Saitama, Japan (Isshiki et al., 1998). The enzyme inhibitory activities of these compounds
were tested against various proteinases. They exhibited strong inhibition against cysteine
proteinases, particularly Cath L, with IC50 values of 13, 10, 10, and 6 nM, respectively.
Papain and Cath B showed moderate inhibitory activity against all compounds, while the
calpains were weakly inhibited.
On the other hand, serine proteinases, metalloproteinases, and aspartic proteinases were
not inhibited at a concentration of 100 μM. In comparison to the inhibitory activities of
compounds (18)–(21) against Caths L and B, the IC50 value for Cath B was approximately
20–50 times higher than that for Cath L. This observation indicates that these compounds
are selective inhibitors of Cath L.
A different structure of a trans-epoxysuccinyl-type peptide, compound (22), was
obtained from the culture mycelium of a fungus strain Colletotrichum sp. isolated from a
soil sample collected in Kanoya City , Kagoshima, Japan (Figure 14.7). WF14861 inhibited
human Cath Bs and L with IC50 values of 0.16 and 1.1 nM, respectively (Otsuka et al.,
1999). Compound (22) exhibited potent inhibitory effects on mouse crude bone cathepsin,
with an IC50 value of 0.4 nM using Z-Phe-Arg-AMC as the substrate. This assay system
is believed to reect the in-vivo bone resorption inhibitory activity triggered by proteases
such as Caths B, L, and K. On the other hand, epoxysuccinyl peptide (22) showed weak
inhibition toward calpain and papain compared to the tested cathepsins. Furthermore,
neither compound (22) nor E-64 showed inhibitory activity against serine proteases,
including bovine chymotrypsin, bovine trypsin, human Cath G, and human elastase. These
results suggest that compound (22) specically inhibits CPs. Compound (22) was extensively investigated by Otsuba et al. through various in-vitro and in-vivo assay systems. It
showed potential utility in targeting protease-related diseases, particularly those associated
with bone degradation (Otsuka et al., 1999). In the evaluation of compound (22) in the
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rat adjuvant arthritis model, Otsuba et al. demonstrated that it reduced cartilage and bone
destruction, as expected from its inhibition of Cath B and L. Additionally, compound (22)
inhibited both acute and chronic inammation. These results suggest that Caths B and L
likely participate in bone resorption in vivo. However, compound (22) was more effective
against Cath L.
FIGURE 14.7 Chemical structure of trans-epoxysuccinyl type peptide (22) isolated from Colletotrichum sp.
14.3.3 NPS FROM MARINE ORGANISM AS CATHEPSIN INHIBITORS
Marinostatin, the first bacterial protease inhibitor, was produced by bacteria isolated from
seawater. It exhibited inhibitory activity specifically against serine proteases (Imada et al.,
1986). Subsequently, monastatin and leupeptin (1) were identified. Monastatin exhibited
inhibitory activity against the protease produced by a fish-pathogenic bacterium (Imada
et al., 1985). On the other hand, leupeptin (1) demonstrated inhibitory activity against both
thiol and serine proteases (Hamato et al., 1992). The differentiation of all compounds was
based on amino acid sequencing.
In a distinct discovery, bioassay-guided separation led to the identication of a new
Cath B inhibitor named tokaramide A (23) from the marine sponge Theonella aff. mirabilis
(Figure 14.8). This inhibitor showed inhibitory activity with an IC50 value of 29 ng/mL
against Cath B (Fusetani et al., 1999). In another study, the marine blue sponge Theonella
aff. mirabilis was collected, and further examination of the extract led to the discovery of
another Cath B inhibitor named miraziridine A (24). It exhibited inhibition of Cath B with
an IC50 value of 2.9 nM (Nakao et al., 2000).
⏎
FIGURE 14.8 Tokaramide A (23) and miraziridine A (24) from marine sponge.
⏎
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The extract obtained from the marine sponge Asteropus simplex demonstrated potent
activity against Cath B. A new pteridine derivative named asteropterin (25) was isolated
(Figure 14.9). Asteropterin (25) exhibited inhibitory effects on Cath B, with an IC50 value
of 4.9 nM. The study also investigated the activity of various compounds with a lumazine
skeleton, as well as mixtures and isolated histamine. All these compounds showed inhi-
bition of Cath B. These ndings suggest that the connection between the lumazine and
histamine units is crucial for the inhibitory activity (Murayama et al., 2008).
FIGURE 14.9 Pteridine derivative named asteropterin (25) from the marine sponge Asteropus simplex.
⏎
Tasiamide F (26) and B (27) analogs were isolated from the marine cyanobacterium
Lyngbya sp. (Figure 14.10). The structural differences between (26) and (27) are the
replacement of amino acid residues in tasiamide B (27) with Ala to Gly, Leu to Ile, and Val
to Ile. Both compounds demonstrated relatively higher inhibitory activity against Caths
D and E. Tasiamide F (26) displayed IC50 values of 57 and 23 nM, respectively, while
Tasiamide B (27) exhibited IC50 values of 50 and 9 nM, respectively (Al-Awadhi et al.,
2016). Tasiamide B (27) is approximately sixfold more potent against Cath D. Similarly,
another aspartic protease inhibitor was isolated from a mixed cyanobacterial culture of
Symploca sp. and Lyngbya sp. A new N,N-dimethyl-terminated peptide named Symplocin
A (28) was identied. Symplocin A (28) exhibited potent activity as an inhibitor of Cath E
with an IC50 value of 300 pM (Molinski et al., 2012).
In 2009, Kwan et al. delved into marine cyanobacteria in search of new bioactive
substances, leading to the identication of a linear decadepsipeptide (Figure 14.11) named
grassystatins A (29), B (30), and C (31), all of which include a statine unit. These decadepsipeptides underwent screening for inhibitory activity against a panel of 59 proteases at
a concentration of 10 μM. Grassystatin A (29) selectively inhibited Caths D and E with
IC50 values of 26.5 nM and 886 pM, respectively. Grassystatin B (30) showed similar
potency and selectivity against Caths D and E, with IC50 values of 7.27 nM and 354 pM,
respectively. In comparison, the truncated peptide analog grassystatin C (31), which has
two fewer residues than A and B, was less potent against both enzymes but still exhibited
selectivity for Cath E, with an IC50 value of 1.62 nM, and for Cath D, with an IC50 value
of 42.9 nM. All grassystatins showed selectivity for Cath E over Cath D compared to
pepstatin A (2), with a selectivity range of approximately 20–38-fold.
Likewise, three other peptides, grassystatins D (32), E (33), and F (34), were found to
contain a statine unit responsible for their inhibitory activities against aspartic proteases
(Figure 14.11). Grassystatins D (32), E (33), and F (34) were isolated from the marine
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cyanobacteria L. confervoides. Grassystatin D (32), E (33), and F (34) inhibited the activity
of Cath D with IC50 values of 200, 900, and 50 nM, respectively, and Cath E with IC50
values of 30, 5, and 0.5 nM, respectively. Among these, grassystatin F (34) exhibited the
highest activity against both Caths D (32) and E (33). Additionally, grassystatin F (34)
inhibited Cath D and suppressed the cleavage of cystatin C and PAI-1 (plasminogen activator inhibitor). Moreover, grassystatin F inhibited the migration of MDA-MD-231 (triplenegative breast cancer cells) by activating downstream tPA (tissue plasminogen activator)
and cysteine cathepsins. These NPs serve as valuable tools to investigate the function of
Cath E (Kwan et al., 2009).
FIGURE 14.10 Tasiamide F (26), tasiamide B (27), and symplocin A (28) isolated from marine cyanobacterium.
Over 60 strains of cyanobacteria were fractionated, and their extracts were evaluated in
a biological screening against human Cath L. One particular fraction from the red-tipped
Schizothrix sp. collection showed 97% inhibition of Cath L at 3 μg/mL. The cyanobacterium
Schizothrix sp. was collected near Piedras Gallinas in the Portobelo National Marine Park,
Colon Province, on the North coast of Panama. Through bioassay-guided fractionation, a
new and highly functionalized linear peptide (Figure 14.12) named gallinamide A (34) was
⏎
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FIGURE 14.11 Chemical structures of linear decadepsipeptide grassystatins A (29), B (30), C (31), E (32), F
(33), and G (34) isolated from marine cyanobacteria.
⏎
obtained. Studies have revealed that gallinamide A (34) exhibits inhibitory activity against
human cathepsins (Linington et al., 2009). Gallinamide A (34) inhibited Cath L with an
IC50 of 47 nM. Notably, when gallinamide A and the enzyme were preincubated for 30
min before adding the substrate, the inhibitory activity showed increased potency, with an
IC50 of 5.0 nM (Miller et al., 2014). Time-dependent inhibition is a characteristic feature
of slow-binding inhibitors. Gallinamide A (34) was also tested for inhibitory activity
against the highly homologous CPs Caths V and B, as well as the cysteinyl exopeptidase
Cath H. IC50 values were determined with and without preincubation of the inhibitor
330
and enzyme. For Cath V, the IC50 values were 460 and 140 nM after 5 and 30 min of
reaction, respectively. Similarly, for Cath B, the IC50 values were 4.2 and 1.7 µM after 5
and 30 min of reaction, respectively. However, gallinamide A (34) exhibited no activity
against Cath H at the highest concentrations tested, and the IC50 values were greater than 30
µM. To assess selectivity, a selectivity index was calculated, indicating a 10-fold increase
in potency for Cath L compared to Cath V without preincubation, which increased to
28-fold after 30 min of incubation. Notably, the selectivity was signicantly higher for
Cath B, showing a 320-fold increase following 30 min of preincubation. Further molecular
docking and dynamics simulations revealed a specic binding pose of gallinamide A (34),
demonstrating high stability, a well-established hydrogen bond network, and the reactive
Michael acceptor enamide. Gallinamide A (34) exhibited irreversible inhibition of Cath L,
suggesting a proposed mechanism of covalent inhibition (Miller et al., 2014).
FIGURE 14.12 Chemical structure of gallinamide A (34) isolated from cyanobacterium Schizothrix sp.
⏎
Cath L is a key CP utilized by coronaviruses for cell entry and serves as a promising
drug target for novel antivirals against SARS-CoV-2. Recent studies have indicated that
inhibitors specically designed to target Cath L demonstrate inhibitory effects against
SARS-CoV-2 Mpro (Boudreau et al., 2019). Gallinamide A (34) was screened against
Caths L, B, V, K, and S, and it exhibited inhibitory activity against all tested cathepsins.
However, the IC50 values for the other cathepsins were generally 1–4 orders of magnitude
less potent than for Cath L. Consequently , Gallinamide A (34) demonstrates selectivity for
Cath L over the other cathepsins. It is a potent inhibitor of Cath L, with an IC50 value of
1.76 µM. When Cath L was incubated with 10 μM of the NP, its activity was effectively
inhibited, completely suppressing its function. However, under the same conditions, no
inhibition of the viral proteases Mpro or PLpro was observed. Additionally, gallinamide A
(34) exhibited no inhibitory effect on the activity of two crucial host proteases, furin and
TMPRSS2, which play a role in facilitating the entry of SARS-CoV-2 into cells, even at a
concentration of up to 50 μM. Gallinamide A (34) was shown to decrease viral load with an
IC90 of 88 nM. The addition of viral entry inhibitors resulted in a dose-dependent reduction
of cytopathic effects (CPE) or cell loss, characterized by sigmoidal inhibition curves. The
calculated EC
for these inhibitors was 28 nM. Remarkably, even at a low concentra-
50
tion of 625 nM, the inhibitors were able to completely prevent virus-induced CPE. When
VeroE6 cells were treated with native gallinamide A, covalent adducts of GalA–Cath L
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were identied in cell lysates using targeted MS/MS. Adducts were not detectable for other
cathepsins, likely due to a combination of the selectivity of gallinamide A (34) for Cath L
over other cathepsins and the higher abundance of Cath L in VeroE6 cells. These ndings
suggest that the antiviral activity of gallinamide A is primarily due to the inhibition of host
Cath L rather than targeting the viral proteases (Mpro and PLpro) or other host proteases.
Consequently, this research highlights the potential of Cath L as a promising target for
the development of new antiviral treatments against SARS-CoV-2 and other pathogenic
coronaviruses (Ashhurst et al., 2022).
Luesch’s group has been investigating Lyngbya confervoides, a marine cyanobac-
terium found in Florida waters. They have reported the isolation of a new cytotoxic
cyclic depsipeptide named grassypeptolide A (35). This cyclic depsipeptide consists of a
31-membered ring and contains interesting moieties such as 2-aminobutyric acid (Aba)
and 2-amino-3-methylbutyric acid (Maba), thiazoline rings, and an unusually high number
of D-amino acids (Kwan et al., 2008). At a concentration of 20 μM, grassypeptolide A
(35) displayed a signicant inhibition of 94% on the activity of Cath L. The cytotoxic
activity of grassypeptolide A was evaluated in four cell lines derived from human osteosarcoma (U2OS), cervical carcinoma (HeLa), colorectal adenocarcinoma (HT29), and
neuroblastoma (IMR-32) with IC50 values of 2.2, 1.0, 1.5, and 4.2 μM, respectively, which
denotes moderate broad-spectrum activity . Planktocyclin (36), a cyclooctapeptide protease
inhibitor produced by the freshwater cyanobacterium Planktothrix rubescens from Lake
Hallwilersee, Switzerland, was isolated (Figure 14.13). Planktocyclin (36) showed lower
inhibitory activity against Cath B (IC50 > 500 µM) (Baumann et al., 2007).
FIGURE 14.13 Cyclic depsipeptide grassypeptolide A (35) and planktocyclin (36) isolated from marine
cyanobacterium.
⏎
A sponge belonging to the Haplosclerida order was gathered in the vicinity of the “Blue
Hole” in the Republic of Palau. The extract underwent bioassay-guided fractionation,
resulting in the isolation of halitoxins, tryptamine (37), and a tryptamine-derived alkaloid
known as haploscleridamine (38) (Figure 14.14). Tryptamine (37), haploscleridamine (38),
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and tryptamine exhibited moderate inhibitory potency against Cath K, with IC50 values of
26 and 15 µM, respectively (Patil et al., 2002).
FIGURE 14.14 Chemical structures of tryptamine (37) and a tryptamine-derived alkaloid, haploscleridamine
(38) isolated from sponges.
⏎
Dibutyl phthalate (39) and di-(2-ethylhexyl) phthalate (40) are two inhibitors that have
been isolated from marine Pseudomonas sp. (Figure 14.15). Both phthalates signicantly
inhibited the activity of Cath B in a dose-dependent manner, with IC50 values of 0.42 and
0.38 mM, respectively. They exhibited noncompetitive inhibition with Ki values of 0.64
and 0.42 mM, respectively (Hoang et al., 2008). The release of Cath B varies signicantly
depending on the cell type and culture conditions. It has been observed that melanoma
B16 cells exhibit increased expression of Cath B in response to malignant progression.
The results suggested that phthalate was able to block pericellular Cath B activity in a
dose-dependent manner, with IC50 values of 0.23 and 0.14 mM, respectively. These results
highlight the potential of phthalate to block pericellular Cath B.
FIGURE 14.15 Chemical structure of dibutyl phthalate (39) and di-(2-ethylhexyl) phthalate (40) isolated
from marine Pseudomonas sp.
⏎
Polyketides named penicitrinol G (41), penicitrinol H (42), chrysophanol (43), and
(2,11-dihydroxy-1-methoxycarbonyl-9-carboxylxanthone (44) were isolated from a marinederived fungus Penicillium citrinum (Figure 14.16). Among these compounds, chrysophanol
(43) exhibited signicant inhibitory activity against Cath B with an IC50 value of 1.7 µM.