Triterpenoids as novel natural inhibitors of human cathepsin L

Article abstract of DOI:10.1002/cbdv.201400065

Cathepsins L (catL) and B play an important role in tumor progression and have been considered promising therapeutic targets in the development of novel anticancer agents. Using a bioactivity-guided fractionation, a series of triterpenoids was identified as a new class of competitive inhibitors towards cathepsin L with affinity values in micromolar range. Among the 14 compounds evaluated, the most promising were 3-epiursolic acid (3), 3-(hydroxyimino)oleanolic acid (9), and 3-(hydroxyimino)masticadienoic acid (13) with IC₅₀ values of 6.5, 2.4, and 2.6 μM on catL, respectively. Most of the evaluated triterpenoids do not inhibit cathepsin B. Thus, the evaluated compounds exhibit a great potential to help in the design of new inhibitors with enhanced potency and affinity towards catL. Docking studies were performed in order to gain insight on the binding mode and SAR of these compounds.

Full text of DOI:10.1002/cbdv.201400065

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Triterpenoids as Novel Natural Inhibitors of Human Cathepsin L
a
a
a
b
by Suelem D. Ramalho ), Lorena R. F. De Sousa ), Liliane Nebo ), Stella H. Maganhi ), Ignez
b
a
c
d
Caracelli ), Julio Zukerman-Schpector ), Maria In eˆ s S. Lima ), Marcio F. M. Alves ), M. F a´ tima das
a
a
a
G. F. Da Silva ), Jo a˜ o B. Fernandes ), and Paulo C. Vieira* )
a) Department of Chemistry, Federal University of S a˜ o Carlos, 13565-905 S a˜ o Carlos, SP, Brazil
(
phone: þ55-16-33518061; fax: þ55-16-33518350; e-mail: dpcv@ufscar.br)
b) BioMat-Department of Physics, Federal University of S a˜ o Carlos, 13565-905 S a˜ o Carlos, SP, Brazil
c
)
Department of Botany, Federal University of S a˜ o Carlos, 13565-905 S a˜ o Carlos, SP, Brazil
d) Department of Biophysics, Federal University of S a˜ o Paulo, 04039–032 S a˜ o Paulo, SP, Brazil
Cathepsins L (catL) and B play an important role in tumor progression and have been considered
promising therapeutic targets in the development of novel anticancer agents. Using a bioactivity-guided
fractionation, a series of triterpenoids was identified as a new class of competitive inhibitors towards
cathepsin L with affinity values in micromolar range. Among the 14 compounds evaluated, the most
promising were 3-epiursolic acid (3), 3-(hydroxyimino)oleanolic acid (9), and 3-(hydroxyimino)mas-
ticadienoic acid (13) with IC50 values of 6.5, 2.4, and 2.6 mm on catL, respectively. Most of the evaluated
triterpenoids do not inhibit cathepsin B. Thus, the evaluated compounds exhibit a great potential to help
in the design of new inhibitors with enhanced potency and affinity towards catL. Docking studies were
performed in order to gain insight on the binding mode and SAR of these compounds.
Introduction. – Lysosomal cysteine proteases are characterized by having similar
amino acid sequences, and they share a common papain-like folding structure [1][2].
Cathepsin L (catL) is an enzyme active only as endopeptidase, containing two domains,
with Cys25, His163, and Asn187 forming the catalytic triad [3]. On the other hand,
cathepsin B (catB) is a carboxypeptidase, which can act, depending on the pH, both as
endopeptidase or exopeptidase. The main feature of catB is the occluding loop which
comprises the His110 and His111 residues [1][3].
Increases in cysteine protease expression and activity have been associated with
many types of human carcinomas such as colorectal [4], melanome [5], breast [6], and
brain tumors [7]. Recently reported as important targets in tumor progression, catB
and catL play an important role in angiogenesis, invasion, and metastasis [1][2][8][9]
which have been considered the major cause of death from cancer [1]. It has been
reported that cancer-associated proteases can be secreted by tumor cells acting on the
degradation of extracellular matrix (ECM) and basement membrane [2][8]. There-
fore, these cathepsins have been identified as promising therapeutic targets and are of
special interest in the development of novel anticancer agents.
Plants are one of the largest sources of active compounds, and advances in cancer
treatment require the continuing development of novel agents [10]. Over the past
years, natural products have played an important role in medicine, as a source not only
of potential chemotherapeutic agents, but also used as lead compounds in semisynthesis
or total synthesis of new drugs [11][12]. Triterpenoids are a widespread group of
ꢀ
2014 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
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natural products occurring in several types of higher plants and organisms. These
compounds present a broad range of pharmacological properties [13][14] such as
anticancer [14], antibacterial [15], anti-inflammatory [16], anti-AIDS [17], and
cytotoxic activities [18].
We have been interested in finding new cysteine protease inhibitors, and we
previously described acridone alkaloids isolated from Swinglea glutinosa (Bl.) Merr.
(
Rutaceae) as cathepsin V inhibitors [19]. Continuing the search for new enzyme
inhibitors, in the present study we have screened several natural triterpenes and some
of their derivatives against catB and catL. To the best of our knowledge, this is the first
report describing these triterpenoids as inhibitors of human catL.
Results and Discussion. – Chemistry. Bioactivity-guided fractionation of the hexane
active fraction from the stems of Myrcia lingua Berg. (Myrtaceae) resulted in the
isolation of active acidic triterpenes such as 3-O-acetylursolic acid (1) [20], ursolic acid
(2) [21], and 3-epiursolic acid (3; Fig. 1) [22]. To better investigate this class, some
other compounds previously isolated in the laboratory of natural products were also
evaluated against the proteases. 3-Oxoursolic acid (4) [23] was isolated from the stem
bark of Vochysia thyrsoidea (Vochysiaceae). Compound 1 was treated with CH N [24]
2
2
to give the corresponding methyl ester 5 [24]. Oleanolic acid (Aldrich O5504, ꢀ97%;
) was purchased from Aldrich Chemical Co. and Sigma, and some other derivatives
were obtained as indicated below. Compound 6 was acetylated with Ac O in pyridine
6
2
[
(
25], resulting in the 3-O-acetyloleanolic acid [25] (7). In addition, 3-oxooleanolic acid
8) [25] was obtained via oxidation of 6 with pyridinium chlorochromate (PCC) and
CH Cl [26]. Compound 8 was subjected to a reaction with NH OH·HCl in MeOH as
2
2
2
described in [27] to afford the 3-(hydroxyimino) derivative 9 [27]. Masticadienoic acid
(10) [28][29] and schinol (11) [29] were isolated from the fruit of Schinus
Fig. 1. Chemical structures of triterpenes and derivatives

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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
terebinthifolius (Anacardiaceae). Compound 10 was also treated with CH N [24] to
2
2
give the corresponding methyl ester 12 [28][29]. Compounds 10 and 12 were then
subjected to a reaction with NH OH·HCl in MeOH [27] to afford the 3-(hydroxy-
2
imino) derivatives 13 [28][29], 14 [28][29], respectively. All compounds (Fig. 1) were
isolated and identified by chromatographic and spectral methods, and all the obtained
data were compared with those in the literature.
Bioactivity-Guided Fractionation. Using bioactivity-guided fractionation to find
novel natural inhibitors of catB and catL, several native plants from the cerrado biome
were screened against these enzymes, leading to a group of active triterpenes isolated
from Myrcia lingua Berg. Among the obtained extracts from cerrado plants, the crude
extracts from M. lingua showed significant inhibitions on the evaluated enzymes and
were further selected to the isolation process. All extracts and fractions were evaluated
at a concentration of 125 mg/ml. The inhibition of EtOH extract was higher than 91%
on both enzymes, although the hexane fraction of the stems of M. lingua turned out to
be the most active with inhibition higher than 90% on catL and 75% on catB.
The isolation procedure of hexane fraction led to the known triterpenes 3-O-
acetylursolic acid (1), ursolic acid (2), and 3-epiursolic acid (3). The enzymatic assay
on cysteine proteases revealed that acidic triterpenes inhibited catL.
Biochemical Evaluation of Natural Products and Derivatives. As a general
procedure, the inhibition screen assay was first carried out at a concentration of
1
00 mm. To compare the potency and selectivity, we determined the IC values for both
50
cathepsins. The evaluated compounds showed good selectivity towards the enzymes.
Eqn. 1 was used to calculate the percentage of inhibition:
%
Inhibition¼100ꢁ(1ꢂV /V )
(1)
i
0
where V and V are initial velocities (enzyme activities) determined in the presence
i
0
and in the absence of the inhibitor, respectively.
Among the 14 compounds evaluated, the highest inhibitions were found for 3, 9,
and 13 with IC50 values of 6.5, 2.4, and 2.6 mm on catL, respectively (Table 1). The
majority of the evaluated triterpenoids did not inhibit catB.
It has been found that the more common structural modifications on triterpenes
were at C(3) (OH group) and C(28) (COO group), and the obtained derivatives
displayed enhanced pharmacological activities, especially anticancer properties [30].
Among the evaluated compounds, the presence of the C(28)OO group was important
for the inhibitory potency of triterpenoids towards catL. It was also noticed that the
presence of a hydroxyimino group at C(3) contributed significantly to the inhibition
improvement. Evaluating the obtained data, we recognized that both triterpene
skeletal types ursane and oleanane inhibited catL. Compounds 5, 12, and 14 completely
lost their activity towards catL, when the COO group was transformed into the
corresponding methyl ester, confirming the importance of a free C(28)OOH group for
the activity towards catL.
The results by Sporn et al. showed that the presence of methyl ester, amides and CN
groups at C(28) of compound 6 contribute to enhance the activity toward cancer cells
and also to the ability to decrease inflammation [31]. In contrast, our results on catL
demonstrated that the C(28)OOH is strongly required to inactivate the enzymes.

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
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Table 1. Inhibitory Activities of Triterpenoids towards CatL and CatB
Compound
IC50 [mm]a)
Cathepsin L
Cathepsin B
78.9ꢃ6.4
>250
>250
>250
>500
>250
>250
>250
>250
>250
1
2
3
4
5
6
7
8
9
12.3ꢃ1.6
39.5ꢃ4.7
6.5ꢃ0.9
8.3ꢃ0.5
>500
7.2ꢃ1.1
9.7ꢃ1.8
14.7ꢃ0.9
2.4ꢃ0.5
9.1ꢃ2.3
8.2ꢃ0.5
>250
1
1
1
1
1
0
1
2
3
4
20.4ꢃ2.0
>250
>250
>500
2.6ꢃ0.2
>500
b
E-64 )
0.03ꢃ0.004
0.04ꢃ0.004
a) The values represent means of three individual experiments ꢃSD. b) Positive control.
CatL and catB are the cysteine proteases strongly involved in metastasis,
angiogenesis, and tumor invasion [1][2][8][9]. Recent studies described compound 2
as inhibitor of matrix metalloproteinases (MMPs) on rat C-6 glioma cells, and the IC50
value was 20 mm for MMP-9 inhibition. MMPs are proteases also overexpressed in
tumor cells and related to cell invasion and degradation of ECM [30][32][33].
Interestingly, ursolic acid and its derivatives have been described as relatively nontoxic
agents, which further focused our interest on this class of compounds [30][32]. To
establish a correlation between anticancer and cathepsin activity, we found in the
literature that several triterpenoids with saponin, ursane, and oleanane skeletons are
being tested as anticancer agents, and some of them are already on clinical trials [30].
The most potent inhibitors were selected for further kinetic studies on catL to
determine the type of inhibition and dissociation constants (K ), although the kinetics
i
of those compounds on catB were not investigated due to their low activity.
Mechanism of Inhibition. Triterpenes 3, 6, 7, 9, and 13 were selected to determine
the type of inhibition towards catL. The inhibition kinetics analyzed by Line-
weaverꢂBurk double reciprocal plots [34] revealed the series of triterpenoids as
competitive inhibitors (Fig. 2); whereas the value of V
remained constant at all
max
inhibitor concentrations, the apparent value of K increased with increasing concen-
m
tration of the inhibitor.
The K values for inhibitors 3, 6, 7, 9, and 13 are compiled in Table 2, and they
i
confirm the evaluated triterpenoids as a new class of competitive inhibitors with affinity
values in a mm range. Thus, these compounds display a potential to aid the design of new
inhibitors with enhanced potencies and affinities towards catL.
Molecular-Docking Studies. To gain some insight on the experimental inhibition
data, docking studies were undertaken, and the results were compared to the poses in

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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Fig. 2. K Plots of compounds 3 (a), 6 (b), 7 (c), 9 (d), and 13 (e) on catL. Kinetics measurements were
i
conducted in the presence of increasing concentration of inhibitors. All data points are means of three
experiments ꢃSD.
two crystal structures of the compounds with inhibitory activities in the nm order
towards catL [35][36].

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
1359
Table 2. Inhibitory Effect of Triterpenoids on CatL
a
Compound
K [mm] )
Inhibition type
i
3
6
7
9
19.5
6.15
17.0
2.0
Competitive
Competitive
Competitive
Competitive
Competitive
1
3
9.23
a) The values represent means of three individual experiments ꢃSD.
In Table 3, the interactions of the different compounds with the amino acids of the
subsites of catL are collated. It can be seen that the compounds in the crystallographic
structures (pdb codes 2xu3 [35] and 2yj2 [36]) interact with amino acids of subsites S1,
where the catalytic cys25 is located, S2, and S3. There are also a few interactions with
amino acids of the S1’ and S2’ subsites (see Fig. 3).
The finding that compounds with four rings undergo only a few interactions with
amino acids of subsites S2 and S3 may explain their rather low inhibitory activity;
moreover, those with the lowest activity do not get close to the catalytic cys25 [37][38].
It should be noted that the five-membered ring compounds do not even interact with
amino acids of the S2’ subsite.
Due to the size and rigidity of the triterpenoid ring systems, the molecules resemble
in some way a cylinder that cannot pass through the narrow V-shaped channel that
leads to the active site, preventing them from interacting with amino acids of subsites S2
and S3, which are important for the inhibitory activity [38].
Fig. 3. View of compound 9 docked into the active site of catL. The residues are colored according to the
subsite they are located: S3, orange; S2, magenta; S1, blue; S1’, green; and S2’, purple (see Table 3).

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CatB-docking studies showed that the compounds have no interactions with the
catalytic residue Cys29. As previously reported by Caracelli et al., a potent inhibitor for
this protease should interact with Cys29 and residues of the S2’ subsite, in particular
with His110 and His111 of the occluding loop [40][41].
Conclusions. – A series of natural triterpenoids and their derivatives were screened
to evaluate their inhibitory activities towards cathepsins B and L. The promising results
revealed that the compounds in which the COOH group remained unchanged were
competitive inhibitors of catL. Docking studies on catL showed that the rigidity of the
ring system prevents the compounds to reach the S2 and S3 subsites which are
important for the inhibition. Finally, the achievement of the active compounds
confirmed the proper functioning of the bioactivity-guided study, and these insights are
important to aid the design of new inhibitors with enhanced potency and affinity
towards the target enzyme.
The authors acknowledge financial support # 2010/52326-9 and # 12/22524-9 (S. H. M.) from S a˜ o
Paulo Research Foundation (FAPESP) and the # 306121/2013-1 (I. C.) and # 305626/2013-2 (J. Z.-S.)
from National Council for Scientific and Technological Development (CNPq), Brazil, and CAPES (808/
2
009 to J. Z.-S. and I. C.).
Experimental Part
General. All commercially available chemicals and reagents were purchased from Aldrich Chemical
Co. and Sigma. Isolation procedures were carried out by anal. TLC on pre-coated aluminum silica gel 60
SiO ; Merck, 230–700 mesh). Solvents used in extracts preparation and chromatography fractionation
(
2
1
13
were purchased from Vetec. H- and C-NMR spectra: Bruker DRX-400 NMR spectrometer (400 and
1
00 MHz, resp.).
Enzyme Expression. The recombinant human catL was expressed in Pichia pastoris as described in
[
42]. CatB from human liver (Aldrich C8571) was purchased from Aldrich Chemical Co. and Sigma.
Extraction and Bioactivity-Guided Fractionation. The parts (leaves and stems) of seven different
species from cerrado biome were dried (408) for 10 d. The dried material was ground and extracted with
EtOH at r.t. in three extractions (each 200 g) of vegetable mass. The EtOH extracts were filtered and
concentrated under reduced pressure on a rotary evaporator, and then the dry extracts were evaluated on
catL and catB. All 14 extracts were subjected to liquidꢂliquid partition resulting in hexane, AcOEt, and
hydroalcoholic fractions.
The hexane fraction (MLCH; 117 g) from the stems of M. lingua Berg. showed good results on catL
and was subjected to CC (SiO 60 (4.0ꢁ40.0 cm); hexane/acetone 8 :2) to afford six fractions, which were
2
evaluated on catB and catL in order to assess the inhibitory potential of each one. Among them, the
fractions MLCH-B (440.0 mg) and MLCH-C (249.0 mg) showed 95% and 96.7% inhibition on catL, resp.
The fraction MLCH-B was separated by CC (SiO 60 (2.5ꢁ35.0 cm); hexane/acetone 8 :2) to give five
2
subfractions. The obtained active fraction MLCH-BE (94.7 mg) was subjected to CC (SiO 60 (1.5ꢁ
2
3
0.0 cm); hexane/AcOEt 9 :1) to afford the active triterpenes 1 (6.5 mg) and 3 (2.4 mg). MLCH-C was
purified by CC (SiO 60 (1.5ꢁ56.0 cm); hexane/acetone 8 :2) to yield six subfractions. Finally, the active
2
subfraction MLCH-CF (62.7 mg) was purified by CC (SiO 60 (2.5ꢁ35.0 cm); hexane/acetone 9 :1) to
2
furnish the active triterpene 2 (1.2 mg). The bioactivity-guided study was monitored with TLC, and all
1
13
pure compounds were fully characterized by NMR spectra ( H, C, and DEPT-135) and comparison with
literature data.
Plant Material. The stems of Myrcia lingua Berg. (Myrtaceae) were collected in May 2011 in S a˜ o
Carlos, S a˜ o Paulo state, and were identified by Dr. Maria In eˆ s Salgueiro Lima. A voucher specimen
(
8366) was deposited with the Herbarium of Botany Department (HUFSCar) at Federal University of
S a˜ o Carlos, Brazil.

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Kinectic Measurements. All commercially available chemicals and reagents were purchased from
Aldrich Chemical Co. and Sigma, and kinetic measurements were carried out in a fluorimeter Molecular
Devices Spectra MAX M3. Inhibitory activity was measured using the synthetic fluorometric substrate Z-
Phe-Arg-AMC (benzyloxycarbonyl-phenylalanyl-arginine 4-methyl-7-coumarylamide) at a concentra-
tion of 185 mm for catB (Km 123 mm [43]: Km 157.5 mm) and 10 mm for catL (Km 1.2 mm [44]: Km 2.4 mm))
[
44]. The molar concentrations of all active cathepsins were determined by titration using the irreversible
inhibitor E-64 [45]. CatB was assayed at 62 nm and catL was assayed at 55 nm. The enzyme was activated
during 5 min with DTE (1,4-dithioerythritol) and acetate buffer (pH 5.5) at 378, and then the mixture was
incubated during 5 min with the sample. The experiments were carried out in triplicate (in 96-well black
plates), and the final volume of the reaction mixture (200 ml) was kept under stirring (lexc 355 nm; lem
4
60 nm). All inhibitors were screened against catL and catB at an initial concentration of 100 mm. Control
assays were performed without inhibitor (negative control) and in the presence of the irreversible
inhibitor for cysteine peptidase, E-64 (positive control; 1-{N-[(l-3-trans-carboxyoxirane-2-carbonyl)-l-
leucyl]amino}-4-guanidinobutane), which can irreversibly inhibit a wide range of cysteine peptidases and
was first isolated and identified from Aspergillus japonicas in 1978 [46]. The IC50 values were determined
by rate measurements with at least seven inhibitor concentrations. The inhibition type and K values were
i
determined at the same experimental conditions. All kinetic parameters were determined by nonlinear
regression employing the SigmaPlot 12.0 enzyme kinetics module, and the type of inhibitor was
established by LineweaverꢂBurk plots of 1/V vs. 1/S at different inhibitor concentrations.
Molecular Docking. The three-dimensional structures of catL and catB were obtained from the
Protein Data Bank [47] (PDB code 2XU3 [35] and 1GMY [48], resp.). Docking studies were performed
using the GOLD program version 5.1 with the GoldScore function [49–51]. For the analysis of the
results, the graphical program Discovery Studioꢂ Visualizer 3.5 was used [52].
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Received February 5, 2014