Synthetic compounds from an in house library as inhibitors of falcipain-2 from Plasmodium falciparum

Article abstract of DOI:10.3109/14756366.2014.920839

Falcipain-2 (FP-2) is a key cysteine protease from the malaria parasite Plasmodium falciparum. Many previous studies have identified FP-2 inhibitors; however, none has yet met the criteria for an antimalarial drug candidate. In this work, we assayed an in-house library of non-peptidic organic compounds, including (E)-chalcones, (E)-N'-benzylidene-benzohydrazides and alkyl-esters of gallic acid, and assessed the activity toward FP-2 and their mechanisms of inhibition. The (E)-chalcones 48, 54 and 66 showed the lowest IC50 values (8.5±0.8μM, 9.5±0.2μM and 4.9±1.3μM, respectively). The best inhibitor (compound 66) demonstrated non-competitive inhibition, and using mass spectrometry and fluorescence spectroscopy assays, we suggest a potential allosteric site for the interaction of this compound, located between the catalytic site and the hemoglobin binding arm in FP-2. We combined structural biology tools and mass spectrometry to characterize the inhibition mechanisms of novel compounds targeting FP-2.

Full text of DOI:10.3109/14756366.2014.920839

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–9
2014 Informa UK Ltd. DOI: 10.3109/14756366.2014.920839
!
ORIGINAL ARTICLE
Synthetic compounds from an in house library as inhibitors of falcipain-2
from Plasmodium falciparum
2
1
Jean Borges Bertoldo1, Louise Domeneghini Chiaradia-Delatorre1,2, Alessandra Mascarello , Paulo Cesar Leal ,
´
Marlon Norberto Sechini Cordeiro , Ricardo Jose Nunes , Emir Salas Sarduy , Philip Jon Rosenthal , and
´
Hernan Terenzi
2
2
3
4
´
1
1
2
Centro de Biologia Molecular Estrutural (CEBIME) and Laboratorio Estrutura e Atividade (LEAT), Universidade Federal de Santa Catarina, Campus
´
3
Trindade, Florianopolis – SC, Brasil, Facultad de Biologıa, Centro de Estudio de Proteınas, Universidad de la Habana, Habana, Cuba, and
4Department of Medicine, San Francisco General Hospital, University of California, San Francisco, CA, USA
´
´
´
Abstract
Keywords
Falcipain-2 (FP-2) is a key cysteine protease from the malaria parasite Plasmodium falciparum.
Many previous studies have identified FP-2 inhibitors; however, none has yet met the criteria
for an antimalarial drug candidate. In this work, we assayed an in-house library of non-peptidic
organic compounds, including (E)-chalcones, (E)-N’-benzylidene-benzohydrazides and alkyl-
esters of gallic acid, and assessed the activity toward FP-2 and their mechanisms of
inhibition. The (E)-chalcones 48, 54 and 66 showed the lowest IC50 values (8.5 0.8 mM,
9.5 0.2 mM and 4.9 1.3 mM, respectively). The best inhibitor (compound 66) demonstrated
non-competitive inhibition, and using mass spectrometry and fluorescence spectroscopy
assays, we suggest a potential allosteric site for the interaction of this compound, located
between the catalytic site and the hemoglobin binding arm in FP-2. We combined structural
biology tools and mass spectrometry to characterize the inhibition mechanisms of novel
compounds targeting FP-2.
Chalcones, falcipain-2 inhibitors, malaria,
Plasmodium falciparum
History
Received 14 March 2014
Revised 8 April 2014
Accepted 12 April 2014
Published online 20 June 2014
Introduction
peptides, peptidomimetics, isoquinolines, thiosemicarbazones and
chalcones9, with both reversible and irreversible mechanisms of
action. Despite the potency of some described inhibitors such as
peptidyl vinyl sulfones10 and pyrimidine nitriles11, no compound
has progressed through the drug discovery pipeline to the
candidate stage. Nonetheless, FP-2 remains a promising target
for antimalarial chemotherapy.
Malaria, a disease caused by Plasmodium falciparum, is one of
the most serious infectious disease in the world, leading to about
627 000 deaths in 20121. A key factor contributing to the inability
to control this disease is the resistance to nearly all available
antimalarial drugs1.
Erythrocytic P. falciparum parasites take up hemoglobin from
erythrocyte cytosol and transport it to an acidic food vacuole,
where it is hydrolyzed, and used as a source of amino acids2.
Among the P. falciparum proteases that hydrolyze hemoglobin is
the cysteine-protease falcipain-2 (FP-2)3, which was shown to be
responsible for 93% of the total hemoglobin proteolysis encoun-
tered in soluble trophozoite extracts3,4, being a key enzyme for the
successful establishment of a P. falciparum infection5. Another
P. falciparum cysteine protease, falcipain-3, is also a key
hemoglobinase that is biochemically similar to FP-2, but it is
active later than FP-2 along the life cycle6.
In this context, chalcones, benzohydrazides and alkyl-esters of
gallic acid are of particular interest since these classes of
compounds present a variety of biological activities. Chalcones
are intermediate compounds in flavonoid biosynthesis presenting
a benzalacetophenone fundamental core12, and benzohydrazides
are compounds structurally related to them having the propanone
group replaced by a hydrazide group. The alkyl esters of gallic
acid are derived from the natural triphenol flavonoid gallic acid
and are widely used in food and pharmaceutical industries as
antioxidants13. Several reports have documented the properties of
natural or synthesized chalcones, benzohydrazides and alkyl-
esters of gallic acid, which include antitumoral, antiinflammatory,
Several studies have focused on FP-2 as a promising target for
antimalarial chemotherapy7,8, and several classes of organic
compounds have been identified as FP-2 inhibitors, including
antimicrobial and antiviral, among other biological activities14–19
.
Chalcones and benzohydrazides had already been studied as
antimalarial compounds14–16 and as FP-2 inhibitors9.
In this work, we assayed an in-house library of non-peptidic
organic compounds, including 82 (E)-chalcones, 17 (E)-N0-
benzylidene-benzohydrazides and 11 alkyl-esters of gallic acid
(Figure 1), and assessed their activity toward FP-2 inhibition and
their mechanism of action.
´
Address for correspondence: Prof. Dr. Hernan Terenzi, Centro de
Biologia Molecular Estrutural, Universidade Federal de Santa Catarina,
´
Campus Trindade, CEP: 88040–900, Florianopolis – SC, Brasil. Tel: +55
48 3721 6426. Fax: +55 48 3721 9672. E-mail: hterenzi@ccb.ufsc.br

2
J. B. Bertoldo et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
H5), 7.15 (d, 1H, J ¼ 8.0 Hz, H6), 7.19 (s, 1H, H2), 7.36 (d, 1H,
J ¼ 15.2 Hz, Ha), 7.70 (m, 1H, H50), 7.81 (d, 1H, J ¼ 15.2 Hz,
Hb), 8.33 (d, 1H, J ¼ 8.0 Hz, H60), 8.43 (d, 1H, J ¼ 8.0 Hz, H40),
8.81 (s, 1H, H20). 13C NMR (CDCl3) d 102.1 (–OCH2O–), 107.0
(C2), 109.0 (C5), 118.8 (C6), 123.4 (Ca), 126.2 (C60), 127.2 (C1),
129.0 (C40), 130.1 (C50), 134.3 (C20), 139.9 (C10), 146.9 (Cb),
148.6 (C30), 148.8-150.8 (C3, C4), 188.1 (C¼O). IR ꢀmax/cm–1
1661, 1211 (C¼O), 1588 (C¼C), 1248, 1036 (C–O), 1527, 1347,
850 (N¼O), 3040, 2900, 1609, 1503, 1489, 1446, 1102, 927, 808,
700 (Ar) (KBr). Anal. Calcd for C16H11NO5: C 64.65; H 3.73;
N 4.71; found: C 64.70; H 3.80; N 4.99. Yield ¼ 40%.
54 – (2E)-1-(30-nitrophenyl)-3-(2,4,5-trimethoxyphenyl)prop-
2-en-1-one. Dark yellow solid, m.p. 161–162 ꢀC; 1H NMR
(400 MHz, DMSO-d6) d 3.83 (s, 3H, o-OCH3), 3.89 (s, 3H,
m-OCH3), 3.92 (s, 3H, p-OCH3), 6.77 (s, 1H, H3), 7.58 (s, 1H,
H6), 7.84 (d, 1H, J ¼ 16.0 Hz, Ha), 7.88 (t, 1H, J ¼ 8.0 Hz, H50),
8.16 (d, 1H, J ¼ 16.0 Hz, Hb), 8.49 (dd, 1H, J ¼ 8.0/1.0 Hz, H60),
8.61 (dd, 1H, J ¼ 8.0/1.0 Hz, H40), 8.77 (m, 1H, H20); 13C NMR
(100 MHz, DMSO-d6) d 55.8 (o-OCH3), 56.3 (m,p-OCH3), 97.3
(C3), 111.0 (C1), 113.9 (C6), 117.7 (C20), 122.5 (Ca), 126.9
(C40), 130.4 (C50), 134.5 (C60), 139.2 (C10), 139.9 (Cb), 143.0
(C2), 148.1 (C30), 153.4 (C5), 154.7 (C4), 187.1 (C¼O). Anal.
Calcd for C18H17NO6: C 62.97, H 4.99, N 4.08. Found: C 63.15,
H 5.03, N 4.06. Yield ¼ 83%.
Figure 1. Generic chemical structure of the three classes of compounds
assayed in this work: (E)-chalcones 1–82, (E)-N0-benzylidene-benzohy-
drazides 83–99, and alkyl-esters of gallic acid 100–110.
66
–
(2E)-1-(20,40,50-trimethoxyphenyl)-3-[5-(2-chloro-5-tri-
Materials and methods
fluoromethyl-phenyl)-furan-2-yl]-2-propen-1-one. Gold
1
yellow solid, m.p. 134–136 ꢀC; H NMR (CDCl3) d 3.91 (s, 3H,
o-OCH3), 3.96 (s, 3H, m-OCH3), 3.99 (s, 3H, p-OCH3), 6.57 (s,
Synthesis and purification of the compounds
The 110 assayed synthetic compounds (Table S1, Supplementary 1H, H30), 6.78 (d, 1H, J ¼ 4.0 Hz, H5), 7.33 (d, 1H, J ¼ 4.0 Hz,
information), were prepared as described in our previous H4), 7.46 (s, 1H, H60), 7.47 (dd, 1H, J ¼ 8.0/1.0 Hz, H300), 7.54 (d,
reports19–29. Reagents were obtained from Sigma-AldrichÕ 1H, J ¼ 16.0 Hz, Ha), 7.59 (d, 1H, J ¼ 8.0 Hz, H400), 7.81 (d, 1H,
(St. Louis, MO) and solvents from Vetec (Duque de Caxias, RJ, J ¼ 16.0 Hz, Hb), 8.25 (s, 1H, H600). 13C NMR (CDCl3) d 56.2 (o-
Brazil).
OCH3), 56.3 (m- and p-OCH3), 96.7 (C30), 113.2 (C60), 114.8
The (E)-chalcones 1–82 were prepared by aldol condensation (C5), 117.0 (C4), 119.9 (C10), 123.7 (CF3), 124.8 (C400), 124.9
using methanol as solvent under basic conditions (KOH 50% w/v) (C600), 126.1 (Ca), 127.4 (C300, C500), 129.2 (C200), 131.6 (Cb),
at room temperature for 24 h. Distilled water and 10% hydro- 133.8 (C100), 143.4 (C50), 149.9 (C1), 152.2 (C3), 154.0 (C20),
chloric acid were added to the reaction for total precipitation of 155.3 (C40), 188.5 (C¼O). IR ꢀmax/cm–1 1649, 1224 (C¼O), 1585
the compounds, which were then obtained by vacuum filtration (C¼C), 1141 (C–Cl), 1265, 1027 (C–O), 1164 (C–F), 2954, 2844,
and later recrystallized in dichloromethane and hexane20–29
.
1610, 1515, 1466, 1402, 1333, 1110, 986, 899, 791, 678 (Ar)
The
(E)-N0-benzylidene-benzohydrazides
83–99 were (KBr). Anal. Calcd for C23H18ClF3O5: C 59.17, H 3.89. Found: C
synthesized by condensation of the benzohydrazide (2 mmol) 58.63, H 3.21. Yield ¼ 92%.
or 3,4,5-trimethoxy-benzohydrazide (2 mmol), with the appro-
priate aldehyde (2 mmol) in methanol (15 mL) and refluxed for Plasmid for expression of FP-2
2 h. After cooling, the crude product was collected by
Plasmid pTrcHis2A-35proFP2 containing the 875 bp FP-2 gene is
filtration, washed and recrystallized from hot ethanol to give
white solids20–29
To obtain the alkyl-esters of gallic acid 100–110, gallic acid
(5 mmol) and the corresponding alcohol (15 mmol) were mixed.
The mixture was dissolved in toluene (70 mL) and sulfuric acid
concentration (0.4 mL), heated for 8–12 h in reflux using Dean-
described in the original report for FP-2 expression3, the construct
was designed to include the catalytic domain of FP-2, 35 residues
from N-terminal pro-domain and a 6xhis-tag30.
.
Expression, purification and refolding of FP-2
Stark, and the solvent was removed under reduced pressure. A BL21-DE3 Escherichia coli colony harboring the pTrcHis2A-
Alternatively, the mixture was dissolved in dioxane (10 mL) and 35proFP2 plasmid was transferred to a 10 mL Luria-Bertani (LB)
p-toluenesulfonic acid (0.3 mL), and heated for 2–4 h in bath oil medium tube containing 100 mg/mL ampicillin and grown
under vacuum. All products were purified by column overnight at 37 ꢀC. Thereafter, 10 mL medium were transferred
chromatography19.
to a 250-mL flask containing LB medium with the same antibiotic
The purity of the compounds was confirmed by melting point concentration and grown at 37 ꢀC until OD600 ¼ 0.8 was reached.
measurement (with a MGAPF-301 apparatus), infrared spectros- Isopropyl b-D-thiogalactoside 1 mM was used to induce bacterial
copy (in an Abb Bomen FTLA 2000 spectrometer on KBr disks; expression, and maximal expression was achieved after four hours
1
Zurich, Switzerland) and H and 13C nuclear magnetic resonance at 37 ꢀC30. Cells were harvested by centrifugation (5000 ꢁ g, 4 ꢀC,
spectroscopy (on a Varian Oxford AS-400 400 MHz instrument; 20 min), resuspended in cold buffer (100 mM Tris-HCl, EDTA
Agilent Technologies, Santa Clara, CA), as well as elementary 10 mM, pH 7.4) and disrupted by sonication (12 cycles, 10 s, 10 s
analysis (carried out using a CHNS EA 1110). The chemical rest). The homogenate was then centrifuged at 12 000 ꢁ g, 4 ꢀC
characterization of the three more active compounds is shown.
for 30 min. The pellet was washed once with cold buffer A (2.5 M
48
–
(2E)-1-(30-nitrophenyl)-3-(1,3-benzodioxol-5-yl)-2- urea, 20 mM Tris-HCl, 2.5% Triton X-100, pH 8.0), centrifuged at
propen-1-one. Brown solid, m.p. 144–146 ꢀC; 1H NMR 17 000 ꢁ g, 4 ꢀC for 30 min and solubilized in buffer B (6 M
(CDCl3) d 6.05 (s, 2H, –OCH2O–), 6.86 (d, 1H, J ¼ 8.0 Hz, guanidine HCl, 20 mM Tris-HCl, 250 mM NaCl, 20 mM

DOI: 10.3109/14756366.2014.920839
An in house library of falcipain-2 inhibitors
3
imidazole, pH 8.0) for 60 min. The suspension was centrifuged at 200 Hz) using an extracting voltage of 20 kV, ion gate 400 m/z,
27 000 ꢁ g 4 ꢀC for 30 min to remove all insoluble content. The 4000 laser shots over a mass range from 800 to 4000 m/z. All
supernatant was loaded on a Ni2+-NTA resin (Qiagen, Venlo, experiments were done at least three times in the automated
Netherlands) previously equilibrated with buffer (8 M urea, mode, and the spectra were processed using FlexAnalysis 3.3
30 mM imidazole, 20 mM Tris-HCl, pH 8.0). The resin was sofware (Bruker Daltonics).
washed sequentially with the same buffer, containing 30, 60 and
100 mM imidazole. Finally, FP-2 was eluted with imidazole 1 M, Fluorescence analyses
and reduced with dithiothreitol (DTT) 10 mM at 37 ꢀC for 45 min.
FP-2 (1 mM) was incubated in the presence of 100 mM of the
Refolding was carried out in a buffer (100 mM Tris-HCl, 1 mM
selected inhibitors for 5 min in 100 mM NaOAc, 10 mM DTT, pH
EDTA, 250 mM L-arginine, 30% glycerol, 1 mM reduced
5.5 at room temperature, in a quartz cuvette. The measurements
glutathione (GSH), 1 mM oxidized glutathione (GSSG), pH 9.0)
were obtained in a JASCO J815 Spectropolarimeter (JASCO,
with a 100-fold dilution of reduced FP-2 into 200 mL of the ice-
Easton, MD) equipped with a fluorescence detection unit (FMO-
cold buffer. Complete processing of the active form of FP-2 was
427). Fluorescence excitation was set to 295 nm in order to excite
achieved as previously described31.
only tryptophans, and the emission was scanned over the range
from 320 to 500 nm. The assays were done in triplicate.
Enzymatic assay for FP-2 inhibition
Stock solutions of the 110 synthesized compounds were prepared at
10 mM in 100% DMSO. Fluorimetric assays were performed in a
buffer (100 mM NaOAc, 10 mM DTT, pH 5.5) using Z-FR-AMC
Molecular modeling
FP-2 models were constructed with the PyMOL Software version
1.5 (Delano Scientific LLC, Philadelphia, PA) using the PDB file
(7-amino-4-methylcoumarin, N-CBZ-L-phenylalanyl-L-arginine
amide) as a substrate. FP-2 (70 nM) was incubated for five minutes
2GHU33.
at room temperature with 50 mM of each of the 110 compounds
before substrate addition. The AMC release after hydrolysis was
monitored for one minute at room temperature in a fluorimeter
(Cary Eclipse, Agilentß, Agilent Technologies, Santa Clara, CA)
with excitation set to 355 nm and emission at 460 nm. All scans
Results and discussion
Inhibition of FP-2 activity
An in-house library of 110 compounds, including 82 (E)-
chalcones, 17 (E)-N0-benzylidene-benzohydrazides and 11 alkyl-
esters of gallic acid, were screened against recombinant FP-2. In
were corrected from the corresponding blanks, and controls
(FP-2 + DMSO 2%, Z-FR-AMC + DMSO 2% and FP-2 + Z-FR-
initial screening, 17 compounds inhibited FP-2 activity ꢂ85%,
AMC + DMSO 2%). Compounds capable of inhibiting at least 85%
of the FP-2 activity in the initial screening were selected to
determine IC50 values. The experiments were done four times, and
IC50 values were derived from plots of activity over at least six
compound concentrations.
Table 1. IC50 values of compounds active against recombinant falcipain-2.
Compound
Chemical structure
IC50 value (mM)
2
O
12.0 2.1
Kinetic assays
O N
2
Compounds with IC50 values below 10 mM were selected to
discriminate the mechanism of inhibition. Ki values were obtained
from replots of KMapp or Vi over inhibitor concentration. At least
three different concentrations of the selected inhibitors and seven
concentrations of the substrate (1–64 mM) were used in the assays,
which were repeated at least five times.
O
10
23
420
420
O N
2
O
Cl
Mass spectrometry
Cl
Inhibitors with IC50 values below 10 mM were selected for mass
spectrometry analyses. The experiments were done following
the previously published methodology32 with minor changes. In
a final reaction volume of 10 mL, approximately 1 mM FP-2 was
incubated in the presence of the selected inhibitors (100 or
300 mM) in 100 mM NaOAc, 10 mM DTT, pH 5.5 for 5 min at
room temperature (final DMSO concentration 2%). FP-2 was
also incubated in the presence of the substrate and used as a
control. Finally, 40 mL of 25 mM NH4HCO3, pH 8.0 were
added to the reaction, followed by trypsin (10 ng/mL with a
protease:protein ratio of 0.5:1.0 (w/w)) (Promega, Madison, 39
WI). The reactions containing FP-2, selected inhibitors and
trypsin were incubated at 37 ꢀC for three hours with gentle
stirring. After digestion, about 1 mL of each reaction was mixed
with 9 mL of the matrix a-cyano-4-hydroxycinnamic acid (5 mg/
O
24
34
19.1 1.5
11.2 0.3
NO
2
O
O
O
O
CH
3
OCH
O
3
420
O
O
OCH
3
OCH
3
mL) dissolved in 50% acetonitrile and 3% trifluoracetic acid,
O
43
14.8 1.4
then, 1 mL of this mixture was spotted in triplicate in a MALDI
MTP 384 target polished steel plate (Bruker Daltonics, Bremen,
Germany). MS analysis was performed on a matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF)/TOF spectrometer model Autoflex III (Bruker
Daltonics) equipped with a 337 nm nitrogen laser (SmartbeamÕ
NO
O
2
O
(continued )

4
J. B. Bertoldo et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
Table 1. Continued
Compound
Chemical structure
IC50 value (mM)
O
48
8.5 0.8
O N
2
O
O
O
OCH
OCH
54
57
66
3
9.5 0.2
O N
2
OCH
3
3
OCH O
3
420
Cl
H CO
3
OCH
3
OCH O
Cl
3
4.9 1.3
O
H CO
3
CF
3
OCH
3
O
72
76
420
420
H CO
3
O
CH
3
H CO
3
OCH
3
O
H CO
S
3
H CO
3
OCH
3
OCH O
79
3
420
420
Cl
Cl
OCH
H CO
3
3
O
104
HO
HO
O
OH
O
105
106
420
420
HO
HO
O
Figure 2. Lineweaver–Burk plots of the kinetic behavior of inhibitors 48,
54 and 66. The plots represent an average of at least five different
experiments.
OH
O
HO
HO
O
Among the (E)-chalcones, inhibitor 66 (IC50 ¼ 4.9 1.3 mM)
has a 2,4,5-trimethoxyphenyl group at A-ring position, and a 2-(2-
chloro-5-(trifluoromethyl)phenyl)furan group as its B-ring.
Switching the A-ring to a 2,5-dimethoxyphenyl group (compound
81) or a 3,4-methylenedioxyphenyl group (compound 44) led
to a decrease in inhibitory activity (data not shown). Replacing
the B-ring of compound 66 with a 3-chlorophenyl ring (com-
pound 57, IC50420 mM) also decreased inhibitory activity at
least 4-fold.
Both the (E)-chalcones 48 (IC50 ¼ 8.5 0.8 mM) and 54
(IC50 ¼ 9.5 0.2 mM) showed similar inhibitory activity, and
contain a 3-nitrophenyl group as the A-ring, and a 3,4-
methylenedioxyphenyl or 2,4,5-trimethoxyphenyl group as the
B-ring. The inhibitory activity was slightly reduced when the
B-ring was changed to the 1-naphthyl group (compound 2,
OH
including 14 (E)-chalcones (2, 10, 23, 24, 34, 39, 43, 48, 54, 57,
66, 72, 76 and 79) and three alkyl-esters of gallic acid (104, 105
and 106) (Table S1, Supplementary information). These 17
compounds were selected to determine the IC50 values (Table 1).
The three alkyl-esters of gallic acid (104, 105 and 106) and
(E)-chalcones (10, 23, 39, 57, 72, 76 and 79) slightly inhibited
FP-2, with IC50420 mM. Compounds 2, 24, 34 and 43 had modest
activity (IC50 ¼ 10–20 mM), and interestingly the (E)-chalcones
48, 54 and 66 were the most active compounds, with IC50 values
below 10 mM.

DOI: 10.3109/14756366.2014.920839
An in house library of falcipain-2 inhibitors
5
IC50 ¼ 12.0 2.1 mM) and reduced 2-fold when the B-ring was the Determination of the mechanism of FP-2 inhibition by
2-naphthyl group (compound 10, IC50420 mM). When the rings selected compounds
of compound 48 (IC50 ¼ 8.5 0.8 mM) were inverted, forming
The three most active compounds, the (E)-chalcones 48, 54 and
66, were used in kinetic analyses for elucidation of the mechanism
of inhibition of FP-2, using different concentrations of Z-FR-
AMC as substrate. Three different concentrations of each inhibitor
were used to calculate the Ki values (10, 20 and 40 mM for
compounds 48 and 54, and 5, 10 and 20 mM for compound 66).
Interestingly, the Lineweaver–Burk double-reciprocal plots for
these three compounds (Figure 2) displayed three different kinetic
behaviors.
compound 43 (IC50 ¼ 14.8 1.4 mM) with a 3-nitrophenyl group
as B-ring, the inhibitory activity was reduced almost 50%.
Similarly, the inversion of the rings of compound 54
(IC50 ¼ 9.5 0.2 mM), forming compound 64 with a 3-nitrophenyl
group as B-ring, reduced the inhibitory activity considerably
(data not shown). The inversion of the rings of compound 10
(IC50420 mM), resulting in compound 24 (IC50 ¼ 19.5 1.5 mM)
with a 3-nitrophenyl group as B-ring, did not change the activity.
These data allow us to suggest that the 3-nitrophenyl group is well
tolerated at the A-ring, and in the B-ring reduces the inhibitory
activity.
When the 3,4-methylenedioxyphenyl group is the A-ring,
compounds with a 3-nitrophenyl group (34, IC50 ¼ 11.2 0.3 mM)
or a substituted furan (43, IC50 ¼ 14.8 1.4 mM) as B-ring
showed decreased inhibition of FP-2. When the B-ring was
replaced by a 2,4,5-trimethoxyphenyl group, the inhibitory
activity was further reduced (39, IC50420 mM). Still, compounds
with the 3,4,5-trimethoxyphenyl group as A-ring showed slight
inhibition of FP-2 (72 and 76, both with IC50420 mM), even when
the B-ring is a substituted furan (72) as that present in compound
34 (IC50 ¼ 11.2 0.3 mM). Compounds with chlorine atoms at the
B-ring also showed low inhibitory activity when the A-ring was a
2,4,6-trimethoxyphenyl group (79, IC50420 mM) or a 2-naphthyl
group (23, IC50420 mM).
In the plot of compound 66 (Figure 2), all lines converged at
the x-axis (–1/KMapp), whereas the y-axis intercepts (1/Vmax app)
varied as a function of the inhibitor concentration. The constant
value of KMapp and the decreased values of Vmax proportional to
increasing inhibitor concentrations indicated that this compound
is a non-competitive inhibitor (a ¼ 1), with a Ki value of
7.0 2.2 mM.
In the plot of compound 54 (Figure 2), all lines converged at
the y-axis (1/Vmax), so Vmax remained constant. The slope
(–KMapp/Vmax) and x-axis intercepts (–1/KMapp) varied with
inhibitor concentration, and the Kmapp values increased with
increasing inhibitor concentrations. These observations are con-
sistent with a competitive mechanism, where the inhibitor
competes with the substrate for binding to the free enzyme
active site. The Ki value determined for compound 54 was
12.0 1.8 mM.
Figure 3. Peptide mass fingerprint of falcipain-2 with a coverage of 73%. Inset: peak m/z 2263.324 corresponds to the peptide-containing the catalytic
Cys42. The spectrum represents a sum of 4000 lasers shots subtracted from the baseline.

6
J. B. Bertoldo et al.
J Enzyme Inhib Med Chem, Early Online: 1–9

DOI: 10.3109/14756366.2014.920839
An in house library of falcipain-2 inhibitors
7
Figure 5. Peptide mass fingerprint of falcipain-2. The peak corresponding to the peptide 204-NSWGQQWGER-213 is labeled (m/z 1247.719, arrows).
The panels show falcipain-2 in the absence of the inhibitors, in the presence of the non-competitive inhibitor 66, and in the presence of the competitive
inhibitor 54. The spectra represents a sum of 4000 lasers shots subtracted from the baseline. Stars represent the peaks m/z 1928.771 and m/z 2056.878,
which are not affected by inhibitor 66.
NCGSCWAFSSIGSVESQYAIR-58 (m/z 2263.324), which con-
In the plot of compound 48 (Figure 2), the lines converged
tains the catalytic Cys42. This peptide covers a large part of the
below the x-axis. Vmax app and KMapp decreased as a function of
catalytic pocket (Figure S2, Supplementary information).
inhibitor concentration, but to different degrees. This mechanism
The PMF of FP-2 in the presence of the substrate Z-FR-AMC
is recognized as non-competitive (a51). The Ki value determined
was used as a control (Figure S3, Supplementary information). In
for compound 48 was 44.9 5.95 mM.
the presence of the substrate, the peak m/z ¼ 2263.324, which
corresponds to the peptide-containing catalytic Cys42, was not
Interaction of compounds 48, 54 and 66 with FP-2
found. This might be explained by the substrate occupying the
analyzed by mass spectrometry
catalytic pocket and, therefore protecting it from trypsin digestion.
In order to investigate the mechanisms of FP-2 inhibition Spectra of all four replicates showed similar profiles.
observed in the FP-2 kinetic experiments, further analysis of the
We analyzed the PMF spectra of FP-2 in the presence of the
interaction between FP-2 and compounds 48, 54 and 66 was done most active inhibitors at the same experimental conditions
by MALDI-TOF/TOF mass spectrometry and trypsin protection (Figure 4). Interestingly, the best inhibitor 66 (IC50 ¼ 4.94 mM),
assays. The peptide mass fingerprint (PMF) of FP-2 in the which in the kinetics evaluations showed a non-competitive
presence of the selected inhibitors at different concentrations pattern (Figure 2), failed to protect the catalytic pocket, as seen by
was compared to the PMF without inhibitors, as well as in the the presence of the peptide-containing Cys42 (m/z 2263.376) in
presence of 50 mM substrate.
the spectrum (Figure 4, panel B). Most likely inhibitor 66
About 73% of the FP-2 primary structure was covered in interacts with another region of the FP-2 structure, consistent with
the PMF (Figure S1, Supplementary information). Figure 3 its demonstrated non-competitive mechanism of inhibition.
shows a series of ions that correspond to peptides from FP-2
The PMF spectrum of 54 (IC50 ¼ 9.47 mM) (Figure 4, panel C)
cleaved by trypsin. The inset indicates the peptide 38- was very similar to that of FP-2 in the presence of the substrate

8
J. B. Bertoldo et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
Figure 6. Intrinsic tryptophan fluorescence of falcipain-2 in the absence (solid line) and in the presence (dashed line) of the three best inhibitors:
compound 48 (A), compound 54 (B) and compound 66 (C). Spectra represent a mean of three scans from two different experiments.
Z-FR-AMC (Figure S3, Supplementary information). The pep- spectrometry, we were able to depict a potential allosteric site
tide-containing Cys42 was absent, indicating that this inhibitor where compound 66 binds, located between the catalytic site, and
might interact with the catalytic pocket, consistent with its the hemoglobin binding arm in FP-2. Fluorescence data also
demonstrated competitive mechanism of inhibition (Figure 2).
revealed that compound 66 was responsible for modifications in a
Inhibitor 48 (IC50 ¼ 8.45 mM) exhibited a mixed-type inhib- particular tryptophan microenvironment leading to a conform-
ition in the kinetics experiments (Figure 2). Its PMF spectra ational change, which seems consistent with an allosteric inhibitor
(Figure 4, panel D) displayed a similar profile to that of interaction. We used a combined enzyme kinetic analysis,
compound 54 (Figure 4, panel C).
fluorescence spectroscopy and mass spectrometry to determine
We also analyzed other peaks found in the PMF of inhibitors inhibition mechanisms for novel FP-2 inhibitors. The (E)-
54 and 66, compared with the control (PMF of FP-2; Figure 3). chalcones 66, 54 and 48, containing a new chemical scaffold for
Figure 5 shows that peaks at m/z 1928.771 and 2056.878 were FP-2 inhibition, are attractive lead compounds for further
unaltered in the presence of the selected inhibitors. However, the optimization.
peak which correspond to the peptide 204-NSWGQQWGER-213
(1247.719 m/z) decreased proportionally with increasing concen-
tration of inhibitor 66, but it was not affected even with the
highest concentrations of inhibitor 54 (300 mM) (Figure 5, lower
panel). This peptide is the first indication of a possible site of
interaction for the inhibitor 66 with FP-2. This region is relatively
far from both the catalytic site and the hemoglobin binding site
(Figure S4, Supplementary information). Moreover, this peptide
contains two tryptophan residues (206 and 210), which may be
used to get insights about FP-2 tertiary structure, for example, by
fluorescence analysis.
Acknowledgements
We thank Taisa Regina Stumpf for technical support in some of the
chemical synthesis.
Declaration of interest
We thank CNPq and CAPES for financial support.
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Supplementary material available online
Supplementary Table 1 and Figures S1–S4.