Anti-trypanosomal activity of non-peptidic nitrile-based cysteine protease inhibitors

Article abstract of DOI:10.1371/journal.pntd.0005343

The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. Anti-trypanosomal activity against the CL Brener strain of T. cruzi was observed in the 0.1 μM to 1 μM range for three nitrile-based cysteine protease inhibitors based on two scaffolds known to be associated with cathepsin K inhibition. The two compounds showing the greatest potency against the trypanosome were characterized by EC₅₀values (0.12 μM and 0.25 μM) that were an order of magnitude lower than the corresponding K_ivalues measured against cruzain, a recombinant form of cruzipain, in an enzyme inhibition assay. This implies that the anti-trypanosomal activity of these two compounds may not be explained only by the inhibition of the cruzain enzyme, thereby triggering a putative polypharmacological profile towards cysteine proteases.

Full text of DOI:10.1371/journal.pntd.0005343

RESEARCH ARTICLE
Anti-trypanosomal activity of non-peptidic
nitrile-based cysteine protease inhibitors
1
Antonio C. B. Burtoloso , Sergio de Albuquerque , Mark Furber , Juliana C. Gomes ,
2
3
1
´
Cristiana Gonc¸alez2, Peter W. Kenny4*, Andrei Leitão1,4, Carlos A. Montanari1,4*, Jose
´
4
Carlos Quilles, Junior , Jean F. R. Ribeiro , Josmar R. Rocha
4
4
´
´
1 Instituto de Quımica de São Carlos, Universidade de São Paulo, São Carlos, São Paulo, Brasil,
2 Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São
¨
´
Paulo, Brazil, 3 AstraZeneca, Molndal, Sweden, 4 Grupo de Estudos em Quımica Medicinal – NEQUIMED,
´
Instituto de Quımica de São Carlos – Universidade de São Paulo, São Carlos, São Paulo, Brazil
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
* Carlos.Montanari@usp.br (CAM); pwkenny@iqsc.usp.br (PWK)
Abstract
The cysteine protease cruzipain is considered to be a validated target for therapeutic inter-
vention in the treatment of Chagas disease. Anti-trypanosomal activity against the CL
Brener strain of T. cruzi was observed in the 0.1 μM to 1 μM range for three nitrile-based
cysteine protease inhibitors based on two scaffolds known to be associated with cathepsin
K inhibition. The two compounds showing the greatest potency against the trypanosome
were characterized by EC50 values (0.12 μM and 0.25 μM) that were an order of magnitude
lower than the corresponding Ki values measured against cruzain, a recombinant form of
cruzipain, in an enzyme inhibition assay. This implies that the anti-trypanosomal activity of
these two compounds may not be explained only by the inhibition of the cruzain enzyme,
thereby triggering a putative polypharmacological profile towards cysteine proteases.
OPEN ACCESS
Citation: Burtoloso ACB, de Albuquerque S, Furber
M, Gomes JC, Gonc¸alez C, Kenny PW, et al. (2017)
Anti-trypanosomal activity of non-peptidic nitrile-
based cysteine protease inhibitors. PLoS Negl Trop
Dis 11(2): e0005343. doi:10.1371/journal.
pntd.0005343
Editor: Abhay R Satoskar, Ohio State University,
UNITED STATES
Received: July 8, 2016
Accepted: January 21, 2017
Published: February 21, 2017
Author summary
Copyright: © 2017 Burtoloso et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Chagas disease is still considered a neglected tropical disease (NTD). There are more than
5 million people infected worldwide of which 99% are located in the Americas. Chagas
disease has huge social and economic consequences for countries with significant propor-
tions of their populations living in poverty. Chagas disease causes approximately seven
thousand deaths per year and half a million people live with disabilities caused by the dis-
ease. Based on disability-adjusted life-years (DALYs), the disease burden of Chagas dis-
ease is five times greater than malaria and is approximately one-fifth of HIV/AIDS in the
Latin American and Caribbean region. Despite being characterized over a century ago by
Carlos Chagas who identified Trypanosoma cruzi as the causative agent, treatment of the
disease is restricted to just two drugs (benznidazole and nifurtimox) that are effective only
in the acute phase of the disease. Failure to rapidly diagnose T. cruzi infections and a poor
side effect profile that causes many patients to abandon treatment both limit the effective-
ness of treatment in the acute phase Chagas disease and many patients ultimately progress
to the chronic phase. In this study, we have identified three compounds with anti-trypa-
nosomal effects on the infective T. cruzi CL Brener form prevalent in various regions of
Data Availability Statement: All data collected and
contained in the manuscript and in the Supporting
Information are available to the public with no
restriction.
Funding: Fundacao de Amparo a Pesquisa do
Estado de Sao Paulo (FAPESP) through the
granting award number 2013/18009-4, which can
be accessed at http://internet.aquila.fapesp.br/
agilis/publico/, provided funding to carry out this
work. We are also indebted to Conselho Nacional
de Desenvolvimento Cientifico e Tecnologico
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Cruzain inhibitors with trypanocidal activity
(CNPq) for providing the grant from project
number 301614/2010-5 (www.cnpq.br). In
addition, CAPES and CNPq are also acknowledged
for providing scholarships. The sponsors had no
role in study design, data collection and analysis,
decision to publish, or preparation of the
manuscript.
the Americas, with potency in the 0.1 μM to 1 μM range and minimal cytotoxicity, even at
128 μM. Additionally, two of these compounds are significantly more potent against the
parasite than against the recombinant form of the cysteine protease cruzipain which is
generally considered to be a valid target for therapeutic intervention in the treatment of
Chagas disease. These observations raise questions about the relevance of cruzain inhibi-
tion as a predictor of anti-trypanosomal activity and reinforce the case for using pheno-
typic assays in the search for new antichagasic agents. Correlation between enzyme
inhibition and activity in cell-based assays is a general issue in drug discovery and we dis-
cuss the importance of intracellular unbound concentration in this context. We believe
that this study is of significant interest both on account of the potent anti-trypanosomal
activity observed for three of the compounds studied and the weak link between this activ-
ity and cruzain inhibition.
Competing interests: The authors have declared
that no competing interests exist. Mark Furber is
employed by the commercial company
AstraZeneca, Pepparedsleden 1, Mo¨lndal 431 83,
Sweden and has also declared that no competing
interests exist. None of the AZ compounds that we
have used are subject to IP.
Introduction
Chagas disease, also known as American trypanosomiasis, is a significant public health prob-
lem in Latin America [1–3]. Although considered to be a neglected tropical disease (NTD),
Chagas disease is becoming more prevalent outside Latin America due to increased migration
[4]. Chagas disease is caused by the protozoan parasite T. cruzi, which is transmitted by blood-
sucking reduviid bugs of the subfamily Triatominae [1–3]. Current therapy is limited to benz-
nidazole (1; chemical structures for compounds discussed in this study are presented in Fig 1)
or nifurtimox, both of which are associated with side effect profiles that may impair the ther-
apy and are only effective in the acute phase of the disease [2,5,6]. Recently, the antifungal
drug posaconazole showed limited curative potential in a randomized clinical trial against
chronic T. cruzi infection [7] and this has stimulated debate [8,9] about the extent to which the
result might have been predicted using in vivo imaging. The paucity of therapeutic options for
chronic Chagas disease has fueled interest in the discovery of new macromolecular targets and
led to collaborative efforts worldwide, including initiatives such as Drugs for Neglected Dis-
eases (DNDi) [10].
Cruzipain, also known as GP57/51, is the major cysteine protease of T. cruzi and is consid-
ered to be a validated target for therapeutic intervention in the treatment of Chagas disease
[11–16]. The oral activity observed [13,15] for the cysteine protease inhibitor K777 (2) in ani-
mal disease models provides some of the basis for target validation. The natural enzyme is
expressed as a mixture of isoforms, which differ in substrate preference and susceptibility to
inhibitors, and consists of a catalytic domain linked to a carboxy-terminal extension which is
retained in the mature protein [14,17]. Cruzipain is differentially expressed in the main stages
of the parasite’s life cycle and is subject to extensive post-translational modification, mainly at
sites in the carboxy-terminal extension [14]. Asn 33 in the catalytic domain of the mature
enzyme is reported to be always glycosylated in vivo but this glycosylation site is absent in the
cruzipain 2 isoform [14].
The activity [18] in a murine Chagas disease model of 3, a structurally-elaborated analog
of the cathepsin K inhibitor odanacatib (4) [19], the clinical development of which was
recently discontinued in Phase III, provided the motivation for the current study. One objec-
tive was to gain a greater understanding of structure-activity relationships (SAR) for com-
pounds based on the odanacatib scaffold. To this end, cruzain inhibition and anti-
trypanosomal activity were characterized for odanacatib (4) and two of its analogues that
were synthesized as part of this study to build on the extensive structure activity relationships
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Cruzain inhibitors with trypanocidal activity
Fig 1. Known antichagasic agents (1–3) and cysteine protease inhibitors evaluated in the current
study (4–12).
doi:10.1371/journal.pntd.0005343.g001
reported for this scaffold [20]. Another objective was to evaluate cathepsin K inhibitors
based on a cyclohexane dicarboxamide scaffold with respect to both cruzain inhibition and
anti-trypanosomal activity.
During the course of this study, it was observed that the anti-trypanosomal activity of two
of the compounds was significantly greater than what would be expected on the basis of their
cruzain inhibitory activity. This leads us to pinpoint that reduction in potency when going
from enzyme to cell inhibition is not observed for these compounds, and demonstrates that it
is not the result of non-specific cytotoxicity.
Materials and methods
Ethics statement
The Ethics Committee on Animal Experimentation of the Faculty of Pharmacy of Ribeirao
Preto–University of Sao Paulo, approved the cytotoxicity assays (approval no. 010263/2014).
This Committee adheres to Conselho Nacional de Controle de Experimentac¸ão Animal–
CONCEA, created by Brazilian Law number 11794 of 8 October 2008. Assays were run
according to the guidelines of the Ministry of Science, Technology and Innovation of Brazil.
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Cruzain inhibitors with trypanocidal activity
The Biosafety Committee of the Faculty of Pharmacy of Ribeirao Preto–University of Sao
Paulo, also approved the use of genetic modified organisms (approval no. 0019–17).
Enzyme kinetic assays
Recombinant cruzain, consisting of the catalytic domain of cruzipain but excluding the car-
boxy-terminal extension, was expressed and purified as previously described [21]. Enzyme
kinetic assays for cruzain were carried out at 37˚C in 200 μL of a solution containing 100 mM
acetate buffer pH 5.5, 300 mM NaCl, 5 mM dithiothreitol (DTT), 5% v/v dimethyl sulfoxide
(DMSO), 0.01% v/v Triton X-100 and 0.15 nM cruzain, using Corning1 96-well black flat bot-
tom microplates. (S)-N-(1-((cyanomethyl)amino)-1-oxo-3-phenylpropan-2-yl)benzamide was
used as a positive control. The rate of the reaction was monitored using a Biotek Synergy HT
plate reader through the fluorescence emission at 460 nm (excitation at 355 nm) due to the
hydrolysis of the substrate Z-Phe-Arg-7-amido-4-methylcoumarin (Z-FR-AMC, Sigma-
Aldrich). The enzyme stock aliquot was rapidly thawed at 37˚C and kept on ice until activa-
tion, in which it was incubated for 20 min. in the assay buffer (100 mM acetate pH 5.5 and 5
mM DTT) followed by additional 2 min. incubation with inhibitors before the reaction was
started by the addition of the substrate.
Visual inspection and a pre-reading of plate wells were performed to check for possible pre-
cipitation and background fluorescence, respectively. Fluorescence emission spectra were also
recorded for all inhibitors, using the same excitation wavelength (355 nm) as for the fluoro-
metric assay. None of the compounds displayed a significant fluorescence signal around 460
nm, the emission wavelength used to monitor the reaction kinetic. Thus, potential inner-filter
effects did not have to be taken into account in our experiments. The reaction was started by
the addition of varying concentrations of the substrate and the wells monitored for a total of 5
minutes of reaction. The initial velocities of the substrate hydrolysis under first-order reaction
were calculated by Gen5 Biotek software based on the linear-regression coefficient from the
data fitting of Relative Fluorescence Unit (mRFU) as a function of time (min).
Each experiment was performed in duplicates for each compound being tested. A control
measurement in absence of inhibitor (KM) was carried out for each setup plate and the kinetic
affinity constants (Ki) were calculated from non-linear fit of Michaelis-Menten curves to the
data of initial velocities as a function of eight different concentrations of the substrate
Z-FR-AMC from 30.0–0.8 μM. All inhibitors were evaluated in two different concentrations,
which were chosen based on a previous screening (percentage of inhibition) at 1.7 μM sub-
strate (~KM). SigmaPlot (v. 10.0) was employed for the non-linear fit and the kinetic parame-
ters determination.
Enzyme kinetic assays for cathepsin L were performed in a similar manner to the cruzain
assays at 37˚C in 200 μL of a solution containing 100 mM acetate buffer pH 5.5, 300 mM
NaCl, 1 mM dithiothreitol (DTT), 1 mM EDTA, 5% v/v dimethyl sulfoxide (DMSO), 0.01%
v/v Triton X-100 and 0.30 nM cathepsin L, using Corning1 96-well black flat bottom micro-
plates. Using the QFRET technology, compounds 9–12 were tested for inhibition of cathepsin
L-mediated cleavage of a synthetic peptide Z-Phe-Arg-AMC (10 μM final concentration)
using 0.1 nM [final] human Cat L (prepared in-house) [24]. A potassium phosphate buffer was
used to maintain pH at 6.4.
Assays for anti-trypanosomal activity and cytotoxicity
Assays were performed against amastigote forms of T. cruzi CL Brener strain, which expresses
β-galactosidase derived from Escherichia coli [22], infecting monkey kidney (LLC-MK2) cells
as described [21]. Previously, LLC-MK2 host cells were cultivated in 96-wells microplates (103
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Cruzain inhibitors with trypanocidal activity
cells/well). After 12 hours, the trypomastigote form of CL Brener (B5 clone) strain was added
in a 1:10 ratio and incubated for another 2 hours. Then, the wells were washed with PBS to
remove the extracellular parasites and the tested compounds were added in the final concen-
trations of 0.031, 0.125, 0.5, 2.0, 8.0, 32.0, 128 and 512 μM. After this, the microplates were
incubated at 37˚C, in CO2 atmosphere (5%), for 5 days. Then, 10 μL of FluoReporter lacZ/
Galactosidase Quantitation Kit (Molecular Probes, ThermoFisher Scientific) were added and
after 30 minutes the fluorometric reaction was read in a microplate reader (Synergy H1, Bio-
tek) at 386 nm excitation and 448 nm emission. The metabolism of the substrate is directly
correlated with the amount of β-galactosidase activity and, therefore, the number amastigotes.
The percentage of activity for each compound was determined from the following formula: %
activity = 100 –{[(X-PC)/(NC-PC)]x100}, where the optical density values of the samples (X),
the positive controls (PC) and negative controls (NC) were used. Culture medium alone was
used as positive control (PC) and cells with medium and 0.6% DMSO (v/v) as negative control
(NC). All assays were performed in triplicate with two independent experiments.
All compounds were subjected to the MTT colorimetric assay using Balb/C 3T3 clone A31
cells (mouse fibroblast cells) acquired from the Rio de Janeiro Cell Bank (BCRJ code 0047).
Inhibition values were determined in the cell-based assay as previously described [23]. Briefly,
cells were cultured at 37˚C in an atmosphere of 5% CO2 using DMEM medium (Cultilab,
Campinas-SP, Brazil) supplemented with 3.5 g glucose (Sigma-Aldrich), 1% penicillin/strepto-
mycin solution and 10% FBS (Cultilab). Cells were plated at a concentration of 105 cell/mL in
96-well plates and incubated for 24 h. All compounds were freshly diluted from 50 mM
DMSO stock solutions to obtain the final concentration of 250 μM and added to each well by
replacing the medium. The cell viability was assessed during 24, 48 and 72 h using the MTT
(Sigma-Aldrich) reagent, with an incubation time of 3 h. Formazan crystals were dissolved
using a solubility reagent composed of DMSO, glacial acetic acid and extran for 1 h. The read-
out was obtained using Biotek Synergy HT plate reader at 570 nm. Benznidazole was used as
control. This assay was done in quadruplicate in two independent experiments. Statistical anal-
yses were made in GraphPad Prism 5. Concentration-response curves were fitted for the best
compounds and the statistical parameters are provided with the graphs in S9 Fig in the supple-
mental material.
Synthetic chemistry
General synthetic procedures. Commercially available reactants and solvents were used
as received, without further purification, unless otherwise stated. All solvents were dried and
distilled prior to use by standard procedures. Reaction progress was monitored by thin layer
chromatography on silica gel (aluminum foils) and spotted under UV light (254 nm), followed
by staining with ethanolic 25% phosphomolybdic acid solution or with aqueous potassium
permanganate.
Purification by column chromatography was carried out on silica gel (Merck 60, particle
size 0.040–0.063 mm). Melting points were determined in a Kofler apparatus. Infrared spectra
were obtained using FTIR at 4.0 cm-1 resolution and are reported in wavenumbers. 1H-NMR
spectra were recorded at 400 MHz and the 13C-NMR spectra at 100 MHz, in CDCl3 or d6-
DMSO at room temperature, in a Varian Mercury 400. Chemical shifts (δ) were reported
in ppm (relative to TMS) and the coupling constants (J) in Hertz (Hz). 1H-NMR spectra were
referenced to DMSO (δ = 2.54 ppm) or CDCl3 (δ = 7.23 ppm) as internal standard, and
13C-NMR were referenced to the central signal of the d6-DMSO multiplet (δ = 40.45 ppm) or
CDCl3 triplet (δ = 77.00 ppm). Signal multiplicity was assigned as singlet (s), doublet (d), dou-
ble doublet (dd), triplet (t), double triplet (dt), quartet (q), quintuplet (qt), multiplet (m) and
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Cruzain inhibitors with trypanocidal activity
broad (br). The high resolution mass spectra (HRMS) were recorded using a Q-TOF Micro-
mass equipment (Waters, UK) by means of ESI-MS techniques.
Synthesis of compound 5. (S)-methyl 2-amino-3-phenylpropanoate. L-Phenylalanine
(18.2 mmol, 3.0 g, 1 equiv,) was suspended in methyl alcohol (21 ml), and cooled to 0˚C. Thio-
nyl chloride (20.0 mmol, 1.45 ml, 1.1 equiv.) was added slowly, over a period of 20 min and the
reaction mixture was then heated on a steam bath for 1h and afterward at ambient temperature
overnight. Dry ether was added to the solution until turbidity appeared. The mixture was then
refrigerated for several hours, during which time the methyl ester crystallized. The product
was collected by filtration to afford the aminoester in 88% yield. White solid, Mp 156–160˚C.
FT-IR (KBr, ʋmax): 3439, 2847, 2623, 1747, 1583, 1497, 1242, 1146, 742 cm-1. 1H NMR (400
MHz, CDCl3): δ = 3.10 (dd, 7.69 and 13.93Hz, 1H), 3.24 (dd, J = 5.51 and 13.94Hz, 1H), 3.63
(s, 3H), 4.19 (dd, J = 5.59 and 7.62Hz, 1H), 7.23–7.36 (m, 5H) 8.86 (s, 2H) ppm. 13C NMR (100
MHz, CDCl3): δ = 36.2, 52.9, 53.7, 127.7, 129.0, 129.8, 135.2, 169.7 ppm. HRMS (ESI): m/z
calcd for C10H14NO2 [M + H]+: 180.10191; found: 180.10178.
(2S)-3-phenyl-2-((2,2,2-trifluoro-1-phenylethyl)amino)propanoic acid. To an (S)-methyl
2-amino-3-phenylpropanoate solution in dry methanol (0.47 M), under argon atmosphere,
potassium carbonate (5.2 mmol, 727 mg, 0.94 equiv.) and freshly prepared (ref 2) 2,2,2-Tri-
fluoroacetophenone (6.1 mmol, 0.86 mL, 1.1 equiv.) were added. The mixture was stirred at
50˚C for 18 h and then the reaction was filtered and the solvent was removed in vacuum. The
collected oil was subsequently washed with tert-butyl methyl ether (3 x 10 mL) and dried
under vacuum until constant mass. The products isolated in this way were essentially pure to
be used in the next step without chromatographic purification. The potassium (S,Z)-3-phenyl-
2-((2,2,2-trifluoro-1-phenylethylidene) amino)propanoate (1.4 mmol, 500 mg, 1 equiv.) was
dissolved in dry THF (10mL), stirred at 0˚C and then, NaBH4 (5.6mmol, 210 mg, 4 equiv.) was
added in one batch. After stirring for 4h, at room temperature, the system was cooled to 0˚C
and then a 2 M HCl solution was added dropwise until stopping the gas release. Next, the sol-
vent was evaporated under vacuum and the crude extracted with ethyl acetate (3 x 10 mL). The
combined organic layers were then dried over Na2SO4, filtered, and evaporated in rotary evap-
orator. Purification by flash column chromatography (30% AcOEt/Hexane) afforded (2S)-
3-phenyl-2-((2,2,2-trifluoro-1-phenylethyl)amino)propanoic acid in 67% yield. White solid.
Mp 160–163˚C. FT-IR (KBr, ʋmax): 3306, 3242, 2927, 2530, 1720, 1470, 1261, 1221, 894, 619
cm-1. 1H NMR (400 MHz, CDCl3): δ = 2.81(dd, J = 8.10 and 13.43 Hz, 1H), 2.89 (dd, J = 5.40
and 13.44 Hz, 1H), 3.06–3.09 (m, 1H), 4.41 (q, J = 7.67 Hz, 1H), 7.12–7.41 (m, 10H) ppm. 13
C
NMR (100 MHz, CDCl3): δ = 39.0, 59.6, 62.1 (q, J = 27.94 Hz, 1C), 122.4, 124.6, 126.8, 126.9,
128.4, 128.8, 129.3, 129.4, 129.8, 133.7, 138.2, 138.3, 174.7, 174.8 ppm. HRMS (ESI): m/z calcd
for C17H17F3NO2 [M + H]+: 324.12059; found: 324.12067.
(S)-N-(1-cyanocyclopropyl)-3-phenyl-2-(((R)-2,2,2-trifluoro-1-phenyl-ethyl)amino)propana-
mide (5) (2S)-3-phenyl-2-((2,2,2-trifluoro-1-phenylethyl)amino)propanoic acid (0.31 mmol,
100 mg, 1 equiv.), 1- [Bis(dimethylamino)methylene]-1H-1,2,3-triazol[4,5-b]pyridinium-
3-oxido hexafluorophosphate (HATU, 0.46 mmol, 176 mg, 1.5 equiv) and 2-cyclopropylami-
nonitrile (0.34 mmol, 40 mg, 1.1 equiv.) were sequentially introduced into a 10 mL round-bot-
tomed reaction flask, provided with magnetic stirring and under argon atmosphere. Next, dry
DMF (1mL) and N,N-Diisopropylethylamine (0.46 mmol, 80μL, 1.5 equiv.) were added into
the flask. The reaction was stirred at room temperature overnight. The disappearance of the
starting trifluoromethyl amino acid was confirmed by TLC monitoring. When the reaction
was complete, ethyl acetate (10 ml) was added to the reaction and the organic layer was washed
with saturated aqueous NaCl solution (10x10 ml). The organic layer was then dried over
Na2SO4, filtered, and evaporated in rotary evaporator. Purification by flash column chroma-
tography (30% AcOEt/Hexane) afforded the mixture of 5 and its diastereomer in 90% yield
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Cruzain inhibitors with trypanocidal activity
(trans:cis ratio = 95:5). The diasteromers were separated by HPLC (Phenomenex celulose II,
25cm x 4.6μm, Acetonitrile/Water: 60:40). 92% Yield. Colorless oil. [α]D24 = -59.57˚ (MeOH,
c = 1,17x 10−3). FT-IR (KBr, ʋmax): 3329, 3246, 3032, 2237, 1649, 1539, 1499, 1456, 1263, 1171,
1126, 850, 742, 567 cm-1. 1H NMR (400 MHz, CDCl3): δ = 1.16–1.22 (m, 1H), 1.26–1.32 (m,
1H), 1.55–1.65 (m, 2H), 2.13 (s, 1H), 2.68 (dd, J = 9.87 and 13.86Hz, 1H), 3.14 (dd, J = 4.25
and 13.87Hz, 1H), 3.24 (dd, J = 4.25 and 9.85Hz, 1H), 3.74 (q, J = 7.73Hz, 1H), 6.81 (d,
J = 7.52Hz, 2H), 7.19–7.33 (m, 6H), 7.71 (s, 1H) ppm. 13C NMR (100 MHz, CDCl3): δ = 16.4,
17.0, 20.1, 39.2, 63.6 (q, J = 63.59Hz), 119.7, 123.7, 125.9, 127.4, 127.8, 128.8, 129.0, 129.1,
129.2, 132.2, 135.7, 173.6 ppm. HRMS (ESI): m/z calcd for C21H21F3N3O [M + H]+: 388.16312;
found: 388.16379. HPLC: Phenomenex celulose II, 25cm x 4.6μm, Acetonitrile/Water: 50:50,
rt: 21.115 min, 206nm. Additional analytical data is provided as supplemental information: S1
and S2 Figs for 1H NMR, 13C NMR spectra; S5 and S6 Figs and S1 and S2 Tables for chromato-
grams of compound 5.
Synthesis of compound 6. Compound 6 was prepared in an analogous manner to 5
S)-N-(1-cyanocyclopropyl)-3-phenyl-2-(((R)-2,2,2-trifluoro-1-(4-methoxy-phenyl)ethyl)
amino)propanamide (6):
Colorless oil. [α]D24 = -5.68˚ (MeOH, c = 1,76x 10−3). FT-IR (KBr, ʋmax): 3273, 3038, 2237,
1672, 1610, 1533, 1512, 1265, 1145, 1039, 873, 794, 561 cm-1. 1H NMR (400 MHz, CDCl3): δ =
1.17–1.32 (m, 2H), 1.55–1.64 (m, 2H), 2.08 (s, 1H), 2.63 (dd, J = 9.75 and 14.03 Hz, 1H), 3.07
(dd, J = 4.27 and 14.02 Hz, 1H), 3.20 (dd, J = 4.27 and 9.73 Hz, 1H), 3.73 (q, J = 7.70Hz, 1H),
3.83 (s, 3H), 6.75–6.95 (m, 6H), 7.21–7.24 (m, 2H), 7.31–7.34 (m, 1H) ppm. 13C NMR (100
MHz, CDCl3): δ = 16.4, 17.0, 20.1, 38.3, 55.3, 63.66 (q, J = 29.20Hz), 114.4, 119.7, 123.7, 125.9,
127.5, 127.8, 128.8, 129.2, 130.0, 132.3, 158.9, 173.7 ppm. HRMS (ESI): m/z calcd for
C22H23F3N3O2 [M + H]+: 418.17369; found: 418.17435. HPLC: Phenomenex celulose II, 25cm
x 4.6μm, Acetonitrile/Water: 50:50, rt: 25.261 min, 206nm. Additional analytical data is pro-
vided as supplemental information: S3 and S4 Figs for 1H NMR, 13C NMR spectra; S7 and S8
Figs and S3 and S4 Tables for chromatograms of compound 6.
Results
A number of the compounds in this study are known to be potent inhibitors of cathepsin K.
Compound 4 (odanacatib [19]) is a cathepsin K inhibitor that was recently discontinued
from advanced clinical development. Compound 3 was the result of optimization [18,20] of
4 with respect to cruzain inhibition although neither this nor the anti-trypanosomal activity
of 4 appear to have been reported previously. Compounds 5 and 6, in which the S3 substitu-
ent of 4 has been deconstructed, were synthesized (Fig 2) during the course of this study.
Compounds 7–12 are published [24,25] cathepsin K inhibitors based on a cyclohexane dicar-
boxamide scaffold. This set of six probes was selected to include both structural prototypes
(e.g. 7 and 8) [23] and compounds (e.g. 10, 11 and 12) [25] for which pharmacokinetic data
are available. Measured data including IC50 values against a number of cathepsins, aqueous
solubility, logD7.4 and microsomal stability have been published for all six compounds
[24,25].
Inhibition of cruzain and a number of cathepsins are reported in Table 1 (see Fig 1 for
structures). We report enzyme inhibitory activity as pKi or pIC50 which facilitates SAR percep-
tion and provides a more appropriate representation of measurement precision than do Ki or
IC50. With the exception of odanacatib (4), none of the compounds show sub-micromolar
potency against cruzain. Compounds 5 and 6 are slightly more potent against cathepsin L than
cruzain and for the other compounds this trend is reversed. Compounds 4 and 7–12 are all
two to three orders of magnitude more potent against cathepsin K than against cruzain.
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Cruzain inhibitors with trypanocidal activity
Fig 2. Synthesis of compounds 5 and 6. Reagents and conditions: (i) SOCl2, MeOH, r.t., 2.5 h, reflux 30
min, r.t. 4 h, 88%; (ii) PhC (= O)CF3, K2CO3, MeOH, 50˚C, 18 h, R = H 66%, R = OCH3 58%; (iii) THF, NaBH4,
R = H 67%, R = OCH3 56%; (iv) HATU, 1-amino-1-cyclopropanecarbonitrile, DIPEA, DMF, r.t., 24 h, R = H
92%, R = OCH3 90%.
doi:10.1371/journal.pntd.0005343.g002
Anti-trypanosomal activity (as pEC50 values) and cytotoxicity are presented in Table 2 for
the compounds and data for benznidazole (1) are also included for comparison. It was not pos-
sible to derive meaningful EC50 values for the less potent compounds and % anti-trypanosomal
activity in the concentration range 0.125 μM to 8 μM is also listed in order to allow compari-
sons to be made with the less potent compounds. Concentration-responses for anti-trypanoso-
mal activity are shown in Fig 3 for the two most potent compounds (5 and 11). It was not
possible to determine meaningful CC50 values for any of the compounds because only low lev-
els of cytotoxicity were observed for LLC-MK2 and Balb/C 3T3 clone A31 cells at the highest
concentration (128 μM) in the assays.
Discussion
The published [18] efficacy of 3 and structural analogs in a murine Chagas disease model pro-
vided a motivation for the current study. Previously, we reported [21] that potent dipeptidyl
Table 1. Inhibition of cruzain and cathepsins by nitrile-based cysteine protease inhibitors.
a,b
a,b
c
c
c
c
Compound Cruzain pKi
Cathepsin L pKi
5.2 (0.07)
Cathepsin L pIC50
5.5 d
Cathepsin K pIC50
9.7 d
Cathepsin B pIC50
6.0 d
Cathepsin S pIC50
7.2 d
4
6.9 (0.09)
5.6 (0.07)
5.1 (0.05)
5.1 (0.02)
5.3 (0.02)
5.3 (0.09)
5.9 (0.06)
5.6 (0.06)
5.5 (0.07)
5
6.1 (0.04)
6
5.8 (0.04)
7
< 4 e
< 4 e
< 5 f
< 5 f
< 5 f
< 5 f
7.1 e
8.1 e
7.7 g
8.4 g
8.5 g
8.0 g
5.1 e
6.3 e
5.6 g
5.7 g
5.9 g
6.1 g
5.7 e
6.0 e
5.7 g
5.8 g
5.9 g
5.9 g
8
9
10
11
12
a pKi = −log10(Ki/M)
b Uncertainty shown in parentheses
c pIC50 = −log10(IC50/M)
d Reference [19]
e Reference [24]
f Determined using the Cat L inhibition assay method described in ref. [24]
g Reference[25]
doi:10.1371/journal.pntd.0005343.t001
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Cruzain inhibitors with trypanocidal activity
Table 2. Anti-trypanosomal activity of cysteine protease inhibitors.
b
Compound
%activity a
at 8 μM
%activity a
at 2 μM
%activity a
at 0.5 μM
%activity a
at 0.125 μM
pEC50
Cytotox
LLC-MK2 c
Cytotox
Balb/C 3TC d
1 e
4
59 (3)
44 (2)
70 (6)
22 (2)
12 (4)
35 (2)
33 (5)
28 (5)
83 (5)
50 (4)
33 (3)
36 (5)
62 (3)
17 (4)
9 (1)
7 (3)
3 (2)
5.6 (0.04)
5.6 (0.08)
6.9 (0.08)
4.9 (0.08)
N/A
41 (8)
31 (4)
0 (0)
0 (0)
27 (15)
1 (7)
19 (6)
2 (7)
0 (0)
3 (7)
0 (0)
0 (0)
0 (0)
28 (2)
55 (4)
1 (2)
22 (4)
44 (2)
0 (0)
5
6
0 (0)
7
2 (1)
0(0)
4 (3)
8
21 (3)
30 (4)
26 (6)
76 (5)
45 (3)
7 (3)
1 (0.4)
6(2)
N/A
29 (8)
0 (0)
9
15 (3)
22 (3)
62 (4)
40 (5)
N/A
10
11
12
6(1)
N/A
15(3)
36 (7)
8 (2)
33 (3)
20 (2)
6.6 (0.04)
5.9 (0.06)
a %activity; standard deviation (N = 6) in parentheses
b pEC50 = −log10(EC50/M)
c % Cytotoxicity in LLC-MK2 cells at 128 μM; standard deviation (N = 6) in parentheses
d % Cytotoxicity in Balb/C 3T3 clone A31 cells at 128 μM; standard deviation (N = 8) in parentheses
e Anti-trypanosomal activity of the antichagasic drug benznidazole is included for comparison purposes
doi:10.1371/journal.pntd.0005343.t002
nitrile inhibitors of cruzain did not show significant anti-trypanosomal activity and questioned
the relevance of potency in this enzyme kinetic assay as a predictor for anti-trypanosomal
activity. In searching for alternatives to the dipeptidyl scaffold, we observed that cathepsin K
inhibitors based on cyclohexane dicarboxamide and odanacatib scaffolds bound to their target
in a similar manner (Fig 4). These both differ from the dipeptidyl scaffold used in our previous
study in that they lack the secondary amide linking the S3 substituent to the scaffold and are
arguably less peptidic. The scaffold on which odanacatib is based allows easier access to the S2
region of the binding site than does the cyclohexane dicarboxamide scaffold.
Compound 4 (pKi = 6.9; pEC50 = 5.6) was the most potent cruzain inhibitor of the com-
pounds assayed and its pEC50 value is the same as that measured for benznidazole (1; pEC50
5.6). The anti-trypanosomal activity of 5 (pKi = 5.6; pEC50 = 6.9), 11 (pKi = 5.6; pEC50 = 6.6)
=
and 12 (pKi = 5.5; pEC50 = 5.9) exceeds the acceptability threshold recommended [28] for hits
Fig 3. Concentration-response of anti-trypanosomal activity for compounds 5 and 11 (c = concentration of compound; M = mol/dm3).
doi:10.1371/journal.pntd.0005343.g003
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Cruzain inhibitors with trypanocidal activity
Fig 4. Overlay of cathepsin K inhibitors based on cyclohexane dicarboxamide (green) and odanacatib
(red) scaffolds generated by alignment of Protein Data Bank (www.rcsb.org) [26] entries 1VSN [27]
and 4DMX [24]. Cysteine sulfur with which the nitrile carbon forms a covalent bond is shown as a yellow
sphere.
doi:10.1371/journal.pntd.0005343.g004
by one to two orders of magnitude even though these compounds are all less potent cruzain
inhibitors than 4. Compounds 5 (pEC50 − pKi = 1.3), 11 (pEC50 − pKi = 1.0) and 12 (pEC50
− pKi = 0.4) all show higher levels of anti-trypanosomal activity than might be expected on the
basis of their cruzain inhibition. While it was not possible to determine meaningful pEC50 val-
ues for every compound, structure-activity relationships (SAR) for anti-trypanosomal activity
can still be discerned. For example, 5 is the most potent of the compounds based on the odana-
catib scaffold despite having the lowest molecular weight. Potency tends to increase with
molecular size for compounds in structural series so this observation is noteworthy. The con-
trasting profiles of 4 (pKi = 6.9; pEC50 = 5.6) and 5 (pKi = 5.6; pEC50 = 6.9) may also reflect ste-
reochemical differences since the configuration of the S3 substituent of the former is S while
that of the latter is R. In contrast, the lower molecular weight compounds based on the cyclo-
hexane dicarboxamide scaffold show lower levels of anti-trypanosomal activity than 11 (pKi =
5.6; pEC50 = 6.6) or 12 (pKi = 5.5; pEC50 = 5.9).
Although failure of potency against the enzyme to translate to potency against cells would
usually be seen as a problem to be solved rather than a phenomenon to be explained, it is
important to first articulate why a compound might be less potent in an enzyme inhibition
assay than in a cell-based assay. In drug discovery programs, failure of potent enzyme inhibi-
tion to translate to cell-based potency is typically attributed to poor permeability. Slow diffu-
sion of a drug into cells means that the intracellular unbound concentration of drug will be
lower than its unbound concentration in assay buffer. It can be helpful to think of drug action
as being driven by its Target Engagement Potential [29], one measure of which can be derived
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Cruzain inhibitors with trypanocidal activity
by scaling unbound concentration (Cu) [30–32] by the dissociation constant (KD) for the com-
plex between drug and target:
mdrug ¼ log10ðCu=KDÞ
In this framework, poor permeability can be thought of as preventing μdrug from attaining
levels required for cellular activity. Even when passively permeable, compounds may be sub-
strates for transporters and subject to efflux [31,33,34,35]. However, a compound may also fail
to show activity in a cell-based assay because the enzyme has a relatively high affinity for its
substrate(s) and/or cofactor or that the latter species may be present at higher concentrations
than in the enzyme kinetic assay. In these situations, a higher value of μdrug is required for tar-
get engagement in the cell than in the enzyme kinetic assay and this is particularly relevant for
kinase targets which are all exposed to the same intracellular concentration of adenosine tri-
phosphate. Although intracellular unbound concentration of compounds can be measured
[30–32] in vitro in some situations, in vivo measurement is not currently feasible and phase I
clinical trials are typically incomplete when targets are intracellular [36]. This is a factor that
needs to be considered when interpreting poor efficacy in a phase II trial as evidence that the
target is not relevant to human disease. When the infectious agent itself is intracellular, as is
the case for Chagas disease, leishmaniasis, malaria and tuberculosis, the uncertainty in physio-
logical concentration is correspondingly greater and permeability requirements are likely to be
even more stringent. In one scenario, transporter-driven efflux might lead to significant differ-
ences between drug concentration in the parasite and in the host cell (where off-target effects
may lead to toxicity). Perturbation of host cell transporter expression by intracellular infec-
tious agents should be anticipated as a parasite defense mechanism.
An increase in potency going from enzyme to cell assay, such as was observed for 5 and 11 is
less commonly encountered in drug discovery projects. The primary inference that may be
drawn from the observation that 5 and 11 are both less potent against the enzyme than against
the parasite is that anti-trypanosomal activity of these compounds cannot be explained by their
cruzain inhibition. The possibility of non-specific cytotoxicity is always a concern when com-
pounds are more potent in a cell-based assay than in the corresponding enzyme inhibition
assay. However, only low levels of cytotoxicity in two cell lines were observed for the com-
pounds in this study at 128 μM (top concentration used in the cytotoxicity assay), which is
almost three orders of magnitude greater than EC50 values for the two most potent compounds.
It should be stressed that the primary purpose of the cytoxicity assay in a study like this is to
show that the activity of compounds is not due to non-specific cytoxicity rather than to provide
evidence that compounds are safe for dosing in vivo. It is also relevant that anti-trypanosomal
activity is observed for compounds based on two distinct scaffolds and that the SAR is not ‘flat’.
As noted previously, uncertainty in unbound concentration [30–32] at the site of action must
always be considered when interpreting results of cell-based assay results, especially when the
site of action is within an intracellular parasite. However, neither 5 nor 11 has a basic center
that could cause it to concentrate [37] within acidic compartments in the trypanosomes and
one would need to invoke transporter-mediated influx [31] in order to explain elevated free
concentrations at the site(s) of action. It must be stressed that neither poor permeability nor
substrate concentration/affinity can be invoked as an explanation for compounds showing
greater potency in a cell-based assay than when assayed against the isolated enzyme.
The observation that 5 and 11 are an order of magnitude more potent in the assay for anti-
trypanosomal activity than in the cruzain inhibition assay points to other plausible scenarios
that are relevant to the search for effective antichagasic agents. The original validation of cruzi-
pain as a target (and cruzain as a model for cruzipain) was performed using irreversible
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Cruzain inhibitors with trypanocidal activity
cysteine protease inhibitors such as peptidyl diazomethanes [11], peptide-fluoromethyl
ketones [12] and vinyl sulfones such as 2 [13] that do not inhibit cruzain selectively. Com-
pound 2 even shows efficacy in a murine model of schistosomiasis [38]. One interpretation of
the results of the current study is that the anti-trypanosomal effects of the most potent com-
pounds are due to inhibition of one or more cysteine proteases (including isoforms, such as
cruzipain-2 for instance) that have not yet been identified as potential targets for therapeutic
intervention. There is a possibility that compounds 5 and 11 may show a polypharmacological
profile, targeting isoforms of cruzipain or other cysteine proteases present in the parasite. For
example, the study of cruzipain-2 [39] highlights the necessity of a deeper understanding of
the implications of a putative family-driven inhibitor instead of a highly selectivity one in
terms of T. cruzi activity.
The cysteine protease inhibition profile (Table 1) suggests that a putative new antichagasic
target may well share molecular recognition characteristics with cathepsin K and that pheno-
typic screening libraries for discovery of antichagasic agents should specifically include potent
inhibitors of this enzyme.
Another possibility is that the unglycosylated catalytic domain of a single isoform used in
the enzyme kinetic assay and for virtual screening [40,41] may, in some situations, be an inap-
propriate model for the trypanosomal enzyme, which is expressed as a mixture of isoforms
and is subject to extensive post-translational modification. While ‘false positives’ are recog-
nized [40] as a problem when screening against cruzain, a physiologically inappropriate target
model can also lead to ‘false negatives’ which, by their nature, are less easily recognized. Even
when a target has been validated, there may still be advantages in using a phenotypic [42,43]
assay for discovery of compounds that show activity against the target and to assess SAR.
Conclusions
We have demonstrated that compounds 5 (pEC50 = 6.9; EC50 = 0.12 μM), 11 (pEC50 = 6.6;
EC50 = 0.25 μM) and 12 (pEC50 = 5.9; EC50 = 1.3 μM) are potent anti-trypanosomal agents.
Given the current dearth of therapeutic options for this NTD, the anti-trypanosomal activity
of these compounds is of considerable interest in its own right. However, the results also have
broader relevance for antichagasic drug design because compounds 5 and 11 are an order of
magnitude more potent against T. cruzi than against the recombinant form of cruzipain,
which is generally considered to be a valid target. The results highlight two potential scenarios
relevant to the discovery of new antichagasic agents. First, the anti-trypanosomal effects of the
compounds could be due to engagement of one or more targets other than cruzipain. Sec-
ondly, the molecular recognition characteristics of cruzipain may either differ from cruzain or
be altered by post-translational modification, which would highlight the need for new struc-
tural models for cruzipain. Knowledge of the activity of 5, 11 and 12 could also be used to
develop tools for the exploration of each scenario. The results of this study reinforce the case
for using phenotypic assays in the search for new antichagasic agents even when the target is
‘known’.
Supporting information
S1 Fig. 1H NMR spectrum (400 MHz, CDCl3) for 5.
(DOCX)
S2 Fig. 13C NMR spectrum (100 MHz, CDCl3) for 5.
(DOCX)
PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005343 February 21, 2017
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Cruzain inhibitors with trypanocidal activity
S3 Fig. 1H NMR spectrum (400 MHz, CDCl3) for 6.
(DOCX)
S4 Fig. 13C NMR spectrum (100 MHz, CDCl3) for 6.
(DOCX)
S5 Fig. Chromatogram of mixture of 5 and its diasteromer.
(DOCX)
S6 Fig. Chromatogram after HPLC separation to compound 5.
(DOCX)
S7 Fig. Chromatogram of mixture 6 and its diastereomers.
(DOCX)
S8 Fig. Chromatogram after HPLC separation to compound 6.
(DOCX)
S9 Fig. Concentration response for trypanocidal activity.
(DOCX)
S1 Table. Chromatogram data for S5 Fig.
(DOCX)
S2 Table. Chromatogram data for S6 Fig.
(DOCX)
S3 Table. Chromatogram data for S7 Fig.
(DOCX)
S4 Table. Chromatogram data for S8 Fig.
(DOCX)
Acknowledgments
We thank the three anonymous reviewers of the manuscript for their constructive and insight-
ful comments.
Author Contributions
Conceived and designed the experiments: PWK AL CAM.
Performed the experiments: MF ACBB JCG JFRR JRR SdA CG JCQJ.
Analyzed the data: ACBB SdA PWK AL CAM.
Contributed reagents/materials/analysis tools: MF ACBB SdA PWK AL CAM.
Wrote the paper: PWK AL CAM.
Compound selections: MF Chemical Synthesis: ACBB JCG Biochemical Assays: JFRR JRR
Trypanocidal assays: SdA CG Cytotoxicity assays: JCQJ.
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