PLOS NEGLECTED TROPICAL DISEASES
RESEARCH ARTICLE
Identification of a potent and selective LAPTc
inhibitor by RapidFire-Mass Spectrometry,
with antichagasic activity
Maikel Izquierdo1, De Lin2, Sandra O’Neill2, Lauren A. Webster2, Christy Paterson2,
John Thomas2, Mirtha Elisa Aguado1, Enrique Colina Araújo3, Daniel Alpı́zar-Pedraza4,
Halimatu Joji2, Lorna MacLean2, Anthony Hope2, David W. Gray2, Martin Zoltner5,6, Mark
C. Field7,8, Jorge González-Bacerio1,3*, Manu De Rycker ID2*
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1 Centre for Protein Studies, Faculty of Biology, University of Havana, La Habana, Cuba, 2 Drug Discovery
Unit, Wellcome Centre for Anti-Infectives Research, University of Dundee, Dundee, United Kingdom,
3 Department of Biochemistry, Faculty of Biology, University of Havana, La Habana, Cuba, 4 Centre for
Pharmaceuticals Research and Development, La Habana, Cuba, 5 Wellcome Centre for Anti-Infectives
Research, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of
Dundee, Dundee, United Kingdom, 6 Department of Parasitology, Faculty of Science, Charles University in
Prague, Biocev, Vestec, Czech Republic, 7 School of Life Sciences, University of Dundee, Dundee, United
Kingdom, 8 Biology Centre, Czech Academy of Sciences, Institute of Parasitology, České Budějovice, Czech
Republic
*
[email protected] (JGB);
[email protected] (MDR)
OPEN ACCESS
Citation: Izquierdo M, Lin D, O’Neill S, Webster LA,
Paterson C, Thomas J, et al. (2024) Identification
of a potent and selective LAPTc inhibitor by
RapidFire-Mass Spectrometry, with antichagasic
activity. PLoS Negl Trop Dis 18(2): e0011956.
https://doi.org/10.1371/journal.pntd.0011956
Editor: Igor C. Almeida, University of Texas at El
Paso, UNITED STATES
Received: July 27, 2023
Accepted: January 31, 2024
Published: February 15, 2024
Copyright: © 2024 Izquierdo 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.
Data Availability Statement: The authors confirm
that all data underlying the findings are fully
available without restriction. All relevant data are
within the paper and its Supporting Information
files.
Funding: This research was supported by the
following Wellcome awards (203134/Z/16/Z to
DWG, MDR, MCF; 204697/Z/16/Z to MCF and
224024/Z/21/Z to DWG, MDR). Wellcome URL:
https://wellcome.org/ The funder did not play a role
Abstract
Background
Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and leads to
~10,000 deaths each year. Nifurtimox and benznidazole are the only two drugs available but
have significant adverse effects and limited efficacy. New chemotherapeutic agents are
urgently required. Here we identified inhibitors of the acidic M17 leucyl-aminopeptidase
from T. cruzi (LAPTc) that show promise as novel starting points for Chagas disease drug
discovery.
Methodology/Principal findings
A RapidFire-MS screen with a protease-focused compound library identified novel LAPTc
inhibitors. Twenty-eight hits were progressed to the dose-response studies, from which 12
molecules inhibited LAPTc with IC50 < 34 μM. Of these, compound 4 was the most potent hit
and mode of inhibition studies indicate that compound 4 is a competitive LAPTc inhibitor,
with Ki 0.27 μM. Compound 4 is selective with respect to human LAP3, showing a selectivity
index of >500. Compound 4 exhibited sub-micromolar activity against intracellular T. cruzi
amastigotes, and while the selectivity-window against the host cells was narrow, no toxicity
was observed for un-infected HepG2 cells. In silico modelling of the LAPTc-compound 4
interaction is consistent with the competitive mode of inhibition. Molecular dynamics simulations reproduce the experimental binding strength (-8.95 kcal/mol), and indicate a binding
mode based mainly on hydrophobic interactions with active site residues without metal cation coordination.
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in the study design, data collection and analysis,
decision to publish, or preparation of the
manuscript.
Competing interests: The authors have declared
that no competing interests exist.
A potent and selective LAPTc inhibitor, antichagasic in vitro
Conclusions/Significance
Our data indicates that these new LAPTc inhibitors should be considered for further development as antiparasitic agents for the treatment of Chagas disease.
Author summary
Trypanosoma cruzi is a single cell eukaryotic parasite that infects humans and animals.
Infection can result in Chagas disease, a debilitating chronic disease that frequently affects
the heart. The major downside of the current treatments is their side-effects, which frequently prevent patients from completing their treatment course. New, safer medicines
are therefore urgently needed. Here we sought to enable the development of new medicines by identifying inhibitors of a key parasite enzyme; acidic M17 leucyl-aminopeptidase
(LAPTc). We identified 28 LAPTc inhibitors, among more than 3000 compounds tested.
The most potent inhibitor, compound 4, inhibited parasite growth and did not inhibit the
equivalent human aminopeptidase. Using computational tools, we predicted how this
compound binds to the enzyme. Our results indicate that the LAPTc inhibitors could be
considered for further development as antiparasitic agents for the treatment of Chagas
disease.
Introduction
Chagas disease is a neglected tropical disease caused by the protozoan kinetoplastid parasite
Trypanosoma cruzi, affecting mainly Latin America, but also present in migrant populations in
North America, Europe, Japan and Australia [1]. Approximately 6–7 million people are currently infected [2], with ~50,000 new cases and ~10,000 deaths annually. The parasite is transmitted between humans and local fauna by hematophagous triatomine insects [3].
Infection progresses through three phases: acute, indeterminate and chronic [4,5]. The
acute phase (4–8 weeks) is characterized by the presence of T. cruzi in blood and patients are
usually asymptomatic or have non-specific symptoms of infection (fever, anorexia, malaise,
lymphadenopathy, etc). The indeterminate phase is typified as silent, with no overt symptoms
[4,5], but 20–30% of infected people progress to the symptomatic chronic phase, manifested as
cardiomyopathy, neuropathy or gastrointestinal disorders [6,7].
Only two drugs are currently available: nifurtimox and benznidazole, but these nitroheterocyclic compounds are poorly tolerated and require protracted treatment regimens [8]. New
and effective chemotherapies are urgently required.
For infectious diseases, rational drug discovery is frequently based on the identification,
characterization and validation of molecular targets in the disease-causing agent. A key advantage of this strategy is to facilitate structure-guided compound design and rapid progress in
drug discovery [9]. Proteases have been successfully targeted in many diseases [10], including
infectious diseases [11] where they play key roles in microbial physiology [12,13].
All life-cycle stages of T. cruzi express an acidic M17 metallo-aminopeptidase (LAPTc),
responsible for the main leucyl aminopeptidase (LAP) activity in parasite extracts [14].
Although LAPTc has not been validated as a target, it may be involved in nutrient supply,
since the parasite lacks the biosynthetic pathway for leucine biosynthesis [14,15]. In agreement
with the critical functions proposed, the LAPTc inhibitor arphamenine A [16] inhibits in vitro
growth of T. brucei brucei, a parasite closely related to T. cruzi [17]. In addition, TbLAP1, an
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A potent and selective LAPTc inhibitor, antichagasic in vitro
M17 LAP from T. brucei, participates in kinetoplast DNA segregation and silencing causes a
delay in cytokinesis [18]. Interestingly, the classical metalo-aminopeptidase inhibitor bestatin
[19] inhibits LAPTc in epimastigotes, the insect parasite stage [20]. Therefore, LAPTc inhibition by bestatin-like molecules is a potential strategy to inhibit parasite growth and for development of anti-chagasic drugs.
M17 LAPs could also be exploited as targets in other parasites. The M17 LAP from Plasmodium falciparum (PfA-M17) is essential as it is involved in haemoglobin digestion [21] and
other housekeeping functions, as suggested by results obtained with a specific bestatin-derived
inhibitor [22]. Knockdown of M17 LAP in the parasite Acanthamoeba castellanii, or treatment
with EDTA, 1,10-phenanthroline (metallo-protease inhibitors) or bestatin, lead to cell wall
changes, closely related to inhibition of encystation [23]. Bestatin inhibition of Babesia bovis
growth has been attributed to inhibition of M17 LAP [24]. Two M17 LAPs from Schistosoma
mansoni could be involved in haemoglobin degradation, surface membrane remodelling and
egg hatching, as suggested by RNAi-mediated knockdown or treatment with bestatin [25].
Finally, knockout of Toxoplasma gondii M17 LAP inhibits the ability to invade cells in culture,
reduces replication and attenuates virulence in mice [26]. Therefore, these enzymes may be
important for developing therapeutic strategies for many parasitic diseases.
M17 LAP inhibitors are dipeptide-like compounds, with hydrophobic and bulky substituents. Only a few LAPTc inhibitors have been identified; bestatin [14], the bestatin-based peptidomimetic KBE009 [27] and arphamenine A [16] (Fig 1). PfA-M17 inhibitors have also been
reported. For example, bestatin (inhibition constant (Ki) = 25 nM [28]; Fig 1) and nitrobestatin
(Ki = 2.7 nM [21]; Fig 1). Both compounds have isobutyl and benzyl substituents (p-nitro-benzyl for nitrobestatin). In addition, PfA-M17 is inhibited by the bestatin-derived activity-based
probe Phe-Naphtyl (Ki = 29 nM [22]; Fig 1). The phosphinate dipeptide analogue Co4, with
two phenyl rings, inhibits also PfA-M17 (Ki = 13 nM [28]; Fig 1). Finally, PfA-M17 is inhibited
by the hydroxamates 13d (Ki = 28 nM; has pyrazole, phenyl and terbutyl groups [29]; Fig 1),
10o (Ki = 60 nM; two phenyl rings and the terbutyl group [30]; Fig 1) and 6k (Ki = 28.9 nM;
three hydrophobic and bulky rings in its structure [31]; Fig 1).
Here, we identified a potent LAPTc inhibitor through screening a small protease-focused
compound collection, using RapidFire-MS. The inhibitor is competitive for the LAPTc substrate, selective with respect to human LAP3 and has sub-micromolar potency against intracellular T. cruzi amastigotes. Molecular docking reveals a binding mode consistent with
competitive inhibition. Molecular dynamics simulations reproduce the experimental binding
strength (-8.95 kcal/mol) and indicate that binding is mainly driven by hydrophobic interactions rather than metal cation coordination. This molecule represents a valuable starting point
for development of a new antichagasic drug.
Methods
Compound library
The protease library screened contained 3,329 compounds that harbour common protease
inhibitor motifs. Compounds were selected and acquired from commercial sources. All compound structures are available in ChEMBL (see Data Availability statement).
Recombinant LAPTc production
Recombinant LAPTc production was described in [32]. Briefly, a LAPTc construct was
designed, codon optimized for expression in Escherichia coli, synthesized and cloned into the
vector pET-19b (Eurofins Genomics, Germany). Production of recombinant LAPTc in E. coli
BL21(DE3)pLysS was induced for 20 h at 25˚C with 1 mM IPTG and yielded soluble and active
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O
OH
N
OH
-
O
H2N
NH
H 2N
O
N
NH
O
O
O
O
OH
bestatin
NH2
H2N
+
H2N
nitrobestatin
OH
arphamenine A
OH
O
OH O
O
OH
N
H2N
NH2
NH
O
O
O
O
OH
O
NH
O P
tag
OH
NH2
KBE009
phe-naphtyl bestatin-derived probe
O
phosphinate dipeptide
analogue (Co4)
O
OH
NH
NH
OH
NH
NH
O
F
F
NH
O
OH
NH
N
F
F
O
N
O
F
F
6k
10o
(N-(2-(hydroxamino)-2-oxo- (N-(2-(hydroxamino)-2-oxo1-(3',4',5'-trifluoro-[1,1'1-(3',4',5'-trifluoro-[1,1'biphenyl]-4biphenyl]-4-yl)ethyl)yl)ethyl)adamantane-1pivalamide)
carboxamide)
13d
(N-(1-(4-(1H-pyrazol-1yl)phenyl)-2(hydroxamino)-2-oxoethyl)pivalamide)
Fig 1. Structures of previously described M17 LAP inhibitors [14,16,21,22,27–31].
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enzyme. The protein was purified in two steps by Immobilized Metal Cation Affinity Chromatography (IMAC) and Gel Filtration. For IMAC the nickel matrix was equilibrated with five
column volumes (CV) of cold binding buffer (50 mM Tris–HCl pH 8.0, 300 mM NaCl). After
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loading 10 mL of the protein extract, the column was washed with the same buffer until the
absorbance at 280 nm stabilized at the baseline. Next, the column was washed with 5 CV of
cold washing buffer [50 mM Tris–HCl pH 8.0, 300 mM NaCl, 50 mM imidazole (Sigma,
USA)]. Finally, the protein was eluted with 5 CV of cold elution buffer (50 mM Tris–HCl pH
8.0, 300 mM NaCl, 400 mM imidazole). The eluates were desalted by gel filtration chromatography, using a NAP-10 column (Sephadex G-25 Medium; Sigma, EUA) to eliminate the
imidazole.
Aminopeptidase activity assay by RapidFire-MS
This assay was previously described [16]. The assay was performed in 384-well clear F-bottom
polypropylene plates with a final reaction volume of 15 μL. The reaction mixture contained
7.5 μL LAPTc in 50 mM Tris-HCl, pH 7.5, 0.005% NP-40 and 7.5 μL LSTVIVR peptide substrate (Cambridge Research Biochemicals, Billingham, UK) in the same buffer was added to
start the reaction. The reaction was performed at room temperature for 40 min and then
stopped with 85 μL 1% formic acid containing 0.15 mg/mL STVIVR* internal standard (Cambridge Research Biochemicals, Billingham, UK).
High-throughput single point screening for LAPTc inhibitors by
RapidFire-MS
Single point screening of a library of 3329 compounds was performed in 384-well clear plates
(Greiner 781101) at room temperature. Test compounds (45 nL in DMSO) were transferred to
assay plates by an ECHO 550 acoustic dispenser (Labcyte). LAPTc was tested at 3 nM with
150 μM (approximately the value of the apparent Michaelis-Menten constant -appKM- [16])
LSTVIVR peptide substrate in a 40 min reaction. Before addition of substrate, LAPTc was preincubated with 45 nL compounds or DMSO for 15 min. Compounds, dissolved in DMSO,
were tested at 30 μM. Controls without compound (the same volume of DMSO, 0% inhibitory
effect) and without enzyme and compound (100% inhibitory effect) were prepared. The experiment was performed without replicates. The remaining experimental conditions were maintained as previously described [16]. Data were processed and analysed through ActivityBase
XE (IDBS). The selection criterion for hits in this single point screen was percent inhibition
larger than the mean plus three standard deviations. All primary screening data are available
in ChEMBL (see Data Availability statement).
Dose-response studies for LAPTc inhibition by RapidFire-MS
To generate half-maximum inhibitory concentration (IC50) data for LAPTc, 10-point doseresponse curves with 100 μM as highest concentration and 1:2 dilution in DMSO (0.195–
100 μM range) were prepared in 384 well plates. All other experimental conditions were as
described above. All IC50 curve fitting was performed by four-parameter logistic dose-response
curve fit using ActivityBase XE (IDBS). At least three replicates were generated for each hit
compound.
Mode of inhibition studies by RapidFire-MS
Five concentrations of the LSTVIVR peptide substrate (100–1600 μM range) were tested in the
presence of 3 nM LAPTc for 40 min. For each substrate concentration, compound 4 was tested
at 0, 0.1 and 0.2 μM. For each substrate and compound 4 concentration, a negative control
without enzyme was used. Before addition of substrate, LAPTc was preincubated with compound 4 for 15 min. Other experimental conditions were as previously described [16]. Ki was
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calculated by fitting the Morrison equation to the experimental data. Binding energy (ΔGb)
was calculated from Ki according to the equation ΔG = -RTlnKi.
Dose-response studies for human LAP3 inhibition by RapidFire-MS
Human LAP3 enzyme (Assay Genie, Ireland) was tested at 150 nM with 600 μM (~1 appKM)
LSTVIVR peptide substrate and 1 mM ZnCl2 in a 180 min reaction. All other experimental
conditions were as described above for dose-response assays with LAPTc.
Culture of Vero cells
Vero cells (African green monkey kidney cells, ECCAC 84113001) were maintained in culture
as previously described [33]. Briefly, these cells were maintained at 37˚C and 5% CO2 in MEM
supplemented with 10% FCS, sub-culturing every 2–3 days at a ratio of 1:5 after 5 min treatment with Trypsin-EDTA (Gibco).
T. cruzi in vitro culture
T. cruzi parasite, TcI strain Silvio X10/7 subclone A1 [34] was maintained in culture as previously described [33]. Parasites were maintained as amastigotes by passaging on a weekly basis
in Vero cells. Culture maintenance infections were carried out at an MOI (multiplicity of
infection) of 1.5.
T. cruzi intracellular assay
This assay was performed as described [35] with the only modification that treatment was 96 h
instead of 72 h. Briefly, Vero cells were infected overnight with tissue culture derived T. cruzi
trypomastigotes in T225 tissue culture flasks (MOI 5). Any remaining free trypomastigotes
were washed away with serum free MEM and the infected Vero cells were harvested by trypsinisation. Compounds (250 nL in DMSO) were dispensed using LabCyte ECHO (Beckman
Coulter Life Sciences, USA) into each well of Corning black flat bottomed 384-well plates
(Corning, USA). Ten-point potency curves were generated (1:3 dilutions in DMSO), with a
highest concentration of 50 μM. The infected Vero cells were then plated into the plates containing the compounds, at 4,000 cells per well in MEM media with 1% FCS. After 96 h incubation at 37˚C in presence of 5% CO2, the plates were fixed with 4% formaldehyde for 20 min at
room temperature and stained with 5 μg/mL Hoechst 33342. The plates were imaged on a Perkin Elmer Operetta high-content imaging system using a 20× objective. Images were analyzed
using the Columbus system (Perkin Elmer). The algorithm first identified the Vero nuclei followed by demarcation of the cytoplasm and identification of intracellular amastigotes. This
algorithm reported percent infected Vero cell and total number of Vero cells. All potency
determinations were carried out in at least three independent replicates and are reported as
pEC50 +/- standard deviation. pEC50 = -log(EC50[M]).
Cytotoxicity assay on human HepG2 cells
The assay was performed as reported [36]. Briefly, HepG2 cells were incubated for 72 h with
compounds, followed by a resazurin-based read-out (fluorescence, excitation 528 nm and
emission 590 nm) with a plate reader.
Docking studies
The 3D structure of compound 4 was generated with the graphical drawing interface Avogadro
version 1.2 [37]. All rotatable torsion angles of compound 4 were defined as flexible. There is
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only one LAPTc structure in the PDB database (PDB: 5NTG [15]). One of the Mn2+ atoms
(absent in the original structure) was manually added from superimposition of structures of
LAPTc and the acidic M17 LAP from T. brucei, TbLAP-A (PDB: 5NSM [15]). AutoDock
Tools v1.5.6 (ADT) [38] was used to prepare the protein and ligand (compound 4) for simulations, and UCSF Chimera v1.14 [39] to analyze the output.
All hydrogen atoms were added to the molecules, Gasteiger charges were calculated, nonpolar hydrogens were eliminated, and the AD4 atom-type assigned to each atom, following
Verma et al. [40]. A grid box of 26 Å × 26 Å × 26 Å and centred at the coordinates of X:
58.144, Y: 85.476 and Z: 90.384 was used to cover the entire enzyme active site. Docking
parameters were kept at default values, except the following: energy_range = 4 kcal,
num_modes = 20.
Docking simulations were performed with Autodock Vina [41] for a total of 100 models,
and conducted five times. LAPTc residues were considered rigid. Different conformers of the
compound 4-LAPTc complex were grouped using as criterion a root mean square deviation
(RMSD) value � 2 Å. In each group, the conformation with the lowest free energy of binding,
according to AutoDock Vina scoring function, was selected as the representative
conformation.
Optimization of the compound 4-LAPTc complex
To optimize the conformation of the compound 4:LAPTc complex, molecular dynamics simulations were performed, using NAMD v2.12 [42] and the force field CHARMM36 [43,44]. As
the starting structure, the best representative conformation of the compound 4:LAPTc complex, according to the binding energy value obtained from molecular docking, was used. The
parameters for compound 4 were obtained using the fftk (force field tool kit) plug-in [45]
implemented in VMD (Visualizer Molecular Dynamics) [46]. For non-protein components of
receptor, i.e. Mn2+, parameters were obtained from Won [47]. Vacuum molecular dynamics
simulations were performed, using a NVT ensemble, making flexible only amino acid residues
located at less than 10 Å from the ligand. Temperature was set to 310 K and was controlled
with the Langevin thermostat [48]. Time step was 2 fs and simulations were run for 1 ns, after
an energetic minimization of 1000 steps. Data were processed in VMD [46].
Stability analysis of the compound 4-LAPTc complex
As starting structure, the final structure from the vacuum molecular dynamics simulations,
selected as representative of the compound 4-LAPTc complex, was used. The in silico complex
was solvated and charge neutralized by adding Na+ and Cl- at 0.05 M, using the solvate and
autoionize plug-ins, respectively, from VMD [46]. A cubic solvation box of 20 Å3 was generated from the complex surface to the borderline of the box, using the explicit solvent model
TIP3 [49]. Simulations were carried out under periodic boundary conditions. Molecular
dynamics simulations were performed using NAMD v2.12 [42], and the CHARMM36 force
field [43,44]. An NPT ensemble was used, with temperature at 310 K and pressure at 1 bar,
controlled by the Langevin thermostat and barostat [50], respectively. All systems were subjected to 1000 minimization steps before runs. The simulations were performed during 100 ns
with intervals of 2 fs.
Analysis of interactions involved in complex stabilization
Hydrophobic interactions and hydrogen bonds of the final positions in compound 4-LAPTc
complex, selected by molecular dynamics simulations, were analyzed using LigPLot+ v2.1
[51]. Analysis of hydrogen bonds throughout the entire trajectory was performed using the
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hydrogen bond plug-in implemented in VMD [46]. The 3D structure of the representative
binding mode for the compound 4-LAPTc complex was visualised with Chimera v1.14 [39].
Energy calculation using LIE-D
Binding energy calculations for the compound 4-LAPTc complex was performed using LIE-D
methodology [52]. Briefly, this method considers the electrostatic (polar) and van der Waals
(non-polar) energies to calculate the binding free energies in protein-ligand complexes.
Results
High-throughput screen for LAPTc inhibitors
A selected and targeted set of 3,329 compounds that harbour common protease inhibitor
motifs was screened at 30 μM against LAPTc using a previously standardized RapidFire-MS
method [16]. For hit selection a threshold of 25.8 percent inhibition was selected (mean
response plus three standard deviations) (Fig 2), resulting in 30 putative active compounds or
hits (S1 File). Screen performance was good, with a Z’ robustness factor of 0.76 ± 0.05 and a
signal-to-noise ratio of 64 ± 7. The hits were progressed to 10-point dose-response studies.
Dose-response characterisation of hits by RapidFire-MS
To confirm the initial hits and assess their potency, single replicate dose-response studies
against LAPTc were performed using the RapidFire-MS method. As compounds 3 and 16
were not available, only 28 compounds were tested. Bestatin, a known LAPTc inhibitor, was
included as a reference. Dose-response profiling showed that 11 compounds were inactive,
five had a maximum effect below 50% (indicating partial inhibition of the enzyme) and 12
compounds had promising IC50 profiles (S2 File). For these 12 compounds we generated a further three dose-response curve replicates and the resulting average potency is shown in Fig 3
Fig 2. Primary screen. Percent inhibition of LAPTc for each compound in the screen. Compounds were tested in
single replicate at 30 μM using the RapidFire-MS enzymatic assay. Hit selection threshold is indicated by the black line,
hits are coloured green.
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Fig 3. Potency determination for LAPTc screening hits. Compound structures and pIC50 values against LAPTc as
determined by dose-response studies with the RapidFire-MS enzymatic assay. Data from four independent replicates. *
= one replicate was deemed inactive (maximum effect < 50%), average and standard deviation are for the three active
replicates.
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(see S2 File for data for all individual replicates). Potencies were generally in the low micromolar range. Compound 4 was the most potent, with a pIC50 of 6.36 (IC50 0.44 μM).
In vitro activity against intracellular T. cruzi amastigotes and cytotoxicity
Intracellular amastigotes are the most relevant form of T. cruzi for human disease as this is the
form that resides within host cells. To assess if the hit compounds possess antiparasitic effects,
we determined the potency of the 12 hits against intracellular T. cruzi amastigotes using in
vitro high-content imaging. As part of the analysis, cytotoxicity against the infected Vero host
cells was also measured. While most compounds were either inactive or lacked selectivity,
compounds 4 and 13 showed promising potency against intracellular amastigotes (pEC50 6.17
(EC50 0.7 μM) and 5.67 (EC50 2 μM), respectively) and exhibited some selectivity over host cell
toxicity (Fig 4, Table 1, and S3 File). Fig 4 shows potency curves for both compounds and representative images from the potency assay to illustrate the antiparasitic effects.
The potency of compounds 4 and 13 is comparable to that reported previously in this assay
for currently used antichagasic drugs, i.e. nifurtimox and benznidazole (pEC50 6.1 (EC50
0.8 μM) and 5.7 (EC50 2 μM), respectively) [33]. To further investigate potential host cell toxicity, we tested the effect of the compounds on uninfected HepG2 cells, a commonly used mammalian cytotoxicity model. Interestingly, all 12 hit compounds showed no or significantly less
toxicity against HepG2 cells compared to infected Vero cells (Fig 4, Table 1 and S4 File). The
effect of compound 4 was too small to allow determination of a half-maximal cytotoxic concentration (CC50) and compound 13 displayed a pCC50 of 4.36 (CC50 43 μM). Selectivity windows based on the HepG2 data were thus substantially larger at >166-fold and 20-fold
respectively, with the caveat that some toxicity was seen for compound 4 at 33 μM and
100 μM.
The structures of compounds 4 and 13 are shown in Table 1. Both are low-molecularweight compounds, with hydrophobic and voluminous functional groups, as is expected for
inhibitors of a M17 LAP. Both structures are nitroheterocycles; compound 4 with four rings
(two of them condensed) and compound 13 with three rings. Taking into account that compound 4 showed the best results at enzymatic and cellular level, it was selected for further
characterisation.
Determination of mode of inhibition for compound 4
Mode of inhibition studies for LAPTc enzyme were performed with compound 4 using the
RapidFire-MS method described above. Double-reciprocal Lineweaver-Burk analysis demonstrated that compound 4 is a competitive inhibitor with respect to the substrate peptide
LSTVIVR (Fig 5), with Ki of 0.27 μM, as determined by fitting the Morrison equation to the
experimental data.
Selectivity of compound 4 over human LAP3
To determine if compound 4 inhibition of LAPTc was selective, we determined the IC50 for
human LAP3, the closest human homologue of LAPTc. Concentrations up to 100 μM exhibited no inhibition of LAP3. Hence with an IC50 for human LAP3 is > 100 μM the selectivity
index of compound 4 for LAPTc is > 500. By contrast, bestatin has a pIC50 against LAP3 of
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Fig 4. Activity against intracellular T. cruzi amastigotes for compounds 4 and 13. Top panels: Dose response curves for compounds 4 and 13. Green and
blue curves are from the high-content imaging assay, green = percent of host cells infected with parasites (%INF; normalised to 100% effect (16 μM nifurtimox)
and 0% effect controls (DMSO)) and blue = percent inhibition of Vero host cells (normalised to 100% effect (no cells) and 0% effect control (DMSO). Data
points are average of four independent replicates, standard deviation is shown as error bars. Yellow curve is from the HepG2 assay (see methods). Data points
are average of two independent replicates, standard deviation is shown as error bars. Bottom panels: Fluorescence images from the high-content screening
assay. Hoechst 33342 was used to stain parasite and host cell DNA. Large structures represent host cell nuclei, small dots are parasite nuclei. Scale bar is 50 μm.
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6.60 +/- 0.05 (IC50 = 0.25 μM) and therefore has no selectivity between LAPTc and human
LAP3.
Modelling of LAPTc inhibition by compound 4 by molecular docking in
silico
LAPTc inhibition by compound 4 was modeled by molecular docking in silico. For this, the
only LAPTc 3D structure available in PDB (PDB: 5NTG [15]) was used to build a functional
dimer. Rotatable bonds of the ligand were considered flexible and LAPTc amino acid side
chains were taken as rigid. Seven conformers of the compound 4-LAPTc complex were
obtained, and we selected the conformation with the best binding energy value (-8.9 kcal/mol)
for energy optimization (Fig 6A). This value is consistent with the experimental value (-8.95
kcal/mol). Compound 4 binds the LAPTc active site in the substrate binding site (Fig 6A).
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Table 1. Potency against intracellular T. cruzi amastigotes, VERO host cells and HepG2 cells.
Compound
pEC50 (T. cruzi) +/- StDev
pCC50 (Vero) +/- StDev
2
<4.3
<4.3
4
6.17 +/- 0.07
5.65 +/- 0.29
5
<4.3
4.48 +/- 0.07
Selectivity index (fold window Tc/Vero)
pCC50 (HepG2)
3.3
<4
<4
<4
6
<4.3
<4.3
<4
7
*4.41 +/- 0.12
4.99 +/- 0.21
<4
8
<4.3
<4.3
<4
9
<4.3
<4.3
<4
12
5.15 +/- 0.03
6*
13
5.67 +/- 0.10
5.30 +/- 0.09
15
<4.3
4.42 +/- 0.09*
<4
24
4.83 +/- 0.20
4.94 +/- 0.07
<4
28
<4.3
<4.3
<4
<4
2.3
4.36
T. cruzi potency determinations were carried out in four independent experiments, HepG2 potency determinations in two independent experiments.
* indicates that compound was inactive in some replicates, in this case the average is calculated only for the active replicates.
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Therefore, the molecular docking result is also consistent with the competitive inhibition
mode experimentally determined.
Energy optimization of the compound 4-LAPTc complex
To optimize the conformation and minimize the energy, modelling of the compound
4-LAPTc complex was continued by vacuum molecular dynamics simulations for 1 ns. Fig 6B
Fig 5. Determination of the mode of inhibition of LAPTc by compound 4, with respect to the LSTVIVR peptide
substrate using the RapidFire-MS enzymatic assay. Double reciprocal Lineweaver-Burk plots are shown.
Determination coefficient values (R2) for the linear fittings are shown, and data are presented as means ± standard
deviations (n = 3). v0: initial velocity. AU: arbitrary units. appKM: apparent KM. appvmax: apparent maximal velocity.
[S]0: initial substrate concentration.
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Fig 6. Modelling of LAPTc inhibition by compound 4 by molecular docking in silico and molecular dynamic
simulations. (A) Structure of the selected conformer of the compound 4-LAPTc complex, according to its binding
energy value, obtained by molecular docking in silico (AutoDock Vina software). (B) Energy optimization of the
compound 4-LAPTc complex by vacuum molecular dynamics simulations. Compound 4 (in sticks) in its initial
conformation (beige) and after 1 ns of vacuum molecular dynamics simulations (magenta) is shown. The enzyme
active site is shown in cartoon (light grey), with the two Mn2+ atoms represented as purple spheres. (C) Temporal
course of RMSD variation in the solvated molecular dynamics simulations for the compound 4-LAPTc complex. 500
frames = 1 ns. (D) 2D representation of predicted stabilizing interactions between compound 4 and LAPTc after 100
ns of solvated molecular dynamics simulations. Compound 4 and R374 residue (carbons represented in black) are
shown in balls and sticks model. Hydrophobic interactions between amino acid residues and compound 4 are shown
with red curve lines, and the hydrogen bond with a green dashed line. In (A), (B) and (D) nitrogen atoms are
represented in blue, oxygens in red, sulfurs in yellow and hydrogen in white.
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shows the conformational and positional changes of compound 4 relative to its initial position.
´
The inhibitor hardly changed its position in the active site. Slight variations up to 2 Å were
observed, mainly associated with the rotation of the pyridyl ring. Based on these coordinates,
stability analysis was carried out for 100 ns in a solvated system.
Stability analysis of the compound 4-LAPTc complex
Solvated molecular dynamics simulations were performed for 100 ns to assess the stability of
the compound 4-LAPTc complex. RMSD variation analysis for the compound 4-LAPTc complex in a 100 ns simulation demonstrated that the inhibitor conformation remained stable
´
over time with very few fluctuations (Fig 6C). Compound 4 showed RMSD values below 4 Å.
The binding energy calculated for the complex by the LIE-D [52] was -8.9 kcal/mol, again consistent with the experimental value (-8.95 kcal/mol). Stability is a consequence of hydrophobic
interactions between compound 4 and the side chains of R310, D311, R374, T400, L401, T402, R460,
N461, S462, V463, N467 and A496 residues (Fig 6D). Only one hydrogen bond interaction was predicted, between R374 and the compound 4 thiazole ring. Notably, inhibitor coordination of
metal cations in the enzyme active site is not predicted.
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Discussion
Interest in metalo-aminopeptidases has increased recently, mainly promoted by involvement
in essential physiological processes and relevance to pathogenesis [12]. In parasites, aminopeptidases are involved in infection of mammalian cells, proliferation, differentiation, defence,
dissemination through host tissues and others [53]. Specifically, M17 metalo-aminopeptidases
have been identified as therapeutic targets; for example PfA-M17 from P. falciparum [22].
LAPTc is active in the three main parasite life cycle stages: epimastigote, trypomastigote
and amastigote [14]. Taking into account the absence of the biosynthetic pathway for leucine
[14,15], T. cruzi must acquire this amino acid through recycling and/or from the environment
[54]. Intracellular amastigotes are the key life-cycle stage for pathogenesis in Chagas disease
[3]. Inside the host cell amastigotes are exposed to low free amino acid concentrations, and
hence parasite LAP activity could play a major role in leucine supply through hydrolysis of
exogenous and endogenous peptides [14]. Therefore, LAPTc is an attractive target for identifying inhibitors with antichagasic activity. Specific LAPTc inhibition/inactivation may also clarify its roles in parasite survival/development in the human host.
In situ inhibition of LAPTc by bestatin in epimastigotes suggests that LAPTc can be inhibited by low-molecular-weight bestatin-like compounds in intact parasites [20]. Identification
of new inhibitors is particularly valuable in context of Chagas disease as a worldwide health
problem [2] and the lack of new drugs in the development pipeline.
To address this we performed a high-throughput screen for LAPTc inhibition by a methodology based on the identification and quantification of the enzymatic reaction product by
mass spectrometry. While RapidFire-MS has been used in high-throughput screens to identify
inhibitors for other enzymes, such as sphingosine kinase [55], arginase II [56], LRRK2 kinase
[57], demethylase-1 [58], monoacylglycerol acyltransferase [59], histone lysine demethylases
[60] and acetyl-coenzyme A carboxylase [61], this is the first report of applying this method to
identify inhibitors of an aminopeptidase. The assay performed well with a Z’ robustness coefficient of 0.76 ± 0.05 and signal-to-noise ratio of 64 ± 7, indicating suitability for high-throughput screening.
The confirmed LAPTc inhibitors exhibit structures consistent with typical M17 LAP inhibitors. All are low-molecular-weight compounds with hydrophobic and voluminous functional
groups, able to establish hydrophobic interactions with the enzyme active site pockets. These
structural requirements are common for M17 LAP inhibitors reported by other authors
[21,22,28–31]. The most potent inhibitor found here, compound 4 (pIC50 6.36, IC50 0.44 μM)
has a terminal pyridyl group linked to a thiazole ring (Table 1). At the other end, compound 4
has two condensed rings (furan and phenyl) and a methoxy group. Interestingly, compound
12, which shares the thiazole-pyridyl side of compound 4 but has an m-dimethoxyphenyl
group at the other end (Table 1) is 40-fold less active against LAPTc providing some initial
structure-activity relationship insight and supporting the importance of a double ring system
in compound 4 for high-affinity binding. It is also interesting that the four most potent inhibitors (compounds 4, 7, 28 and 5; Table 1) share the thiazole ring. These novel LAPTc inhibitors
complement the small cohort of previously described LAPTc inhibitors (including bestatin
[14], arphamenine A [16] and the bestatin-derivative peptidomimetic KBE009 [27]; Fig 1), and
provide new opportunities to explore this mechanism of action as a therapeutic target for Chagas disease as well as tool compounds to investigate basic biology. Compound 4 is highlighted
as a candidate for further progression and the only molecule with a submicromolar IC50 for
LAPTc inhibition.
When tested against intracellular amastigotes, compounds 4 and 13 demonstrate anti-parasitic activity. However, the selectivity window against Vero host cells was narrow (Fig 4), but
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both compounds showed less cytotoxicity against HepG2 cells, a common cell line used for
cell health experiments. The poor selectivity for compound 4 in infected Vero cells is not
driven by inhibition of the closest human homologue of LAPTc as no inhibition of LAP3 was
detected. It will be important to characterise the mechanism driving cellular toxicity, but it is
encouraging that less toxicity was seen in HepG2 cells compared to infected Vero cells. A
caveat is that compound exposure for uninfected HepG2 cells was 24 hours shorter than in
infected Vero cells. In addition, the toxicity in Vero cells may be compounded by the presence
of dying parasites. Thus, future work should explore effects of incubation time on cellular toxicity, in HepG2 cells and infected versus uninfected Vero cells. Additional studies are also
required to demonstrate that the compounds are active across multiple strains from different
DTUs, and to confirm that the antiparasitic activity of compounds 4 and 13 is mainly driven
through inhibition of LAPTc.
With respect to selectivity between LAPTc and human LAP3, optimised conditions were
used for each enzyme with substrate concentration ~1 appKM, enzyme concentration at 3 and
150 nM respectively, and over 40 and 180 minutes respectively [16]. Ultimately, confirmation
of the physiological level of selectivity should be obtained in relevant cellular models through
measurement of inhibition of substrate cleavage.
Compound 4 is a competitive inhibitor of LAPTc, with Ki 0.27 μM. The compound has a
good ligand efficiency (LE) of 0.33 (LE = -RT ln Ki / number of non-hydrogen atoms) and
shows high selectivity over the closest human homologue. Together, this makes compound 4
an excellent starting point for a LAPTc drug discovery campaign.
An in silico binding model for the compound 4-LAPTc complex was generated, and provides structural guidance for medicinal chemistry development of compound 4. The model is
consistent with compound 4 being a competitive inhibitor and the predicted binding energy
(-8.9 kcal/mol) is in line with the experimental value (-8.95 kcal/mol), providing validation for
the model. The main predicted interactions between compound 4 and LAPTc are hydrophobic, with hydrogen bonds less abundant (Fig 6D). This is consistent with the hydrophobicity of
the active site.
These enzymes have an S1 sub-site formed by hydrophobic and negative residues, and an
S1’ pocket mainly formed by hydrophobic and positive amino acids [62]. Although S1 and S1’
sub-sites have not been described for trypanosomatid M17 LAPs, they have been described for
PfA-M17. The narrow hydrophobic S1 pocket of PfA-M17 is formed by M392, M396, F398, T486,
G489, L492 and F583 residues [63]. Hydrophobic residues A460 and I547 are key residues of the
S1’ cavity [64].
Bestatin binds to the PfA-M17 active site through hydrophobic interactions. The benzyl
group in bestatin’s P1 position (Fig 1) interacts in the PfA-M17 S1 sub-site with the hydrophobic amino acids M392, M396, F398, G489 and A577. The isobutyl side chain in bestatin’s P1’ position (Fig 1) contacts the N457 and I547 residues [63]. In addition, bestatin coordinates the
divalent metal cations of PfA-M17 [63] and TbLAP-A [15]. For compound 4, interaction with
LAPTc’s metal cations is not predicted. A similar hydrophobicity-driven interaction has also
been modelled for the bestatin-based peptidomimetic KBE009 [27] (Fig 1), with hydrophobic
interactions between KBE009 and LAPTc residues K300, F304, D370, T402, G403, A496 and F497
(numbering differs in one residue). Two of these residues also participate in the predicted
interaction with compound 4 (T402 and A496).
A binding-mode based on hydrophobic interactions rather than metal cation coordination
could be favourable for inhibition selectivity [65], as binding would not be directed by the conserved and strong coordination with the metal cations, but by many, weaker individual interactions (mainly van der Waals interactions, with to a lesser extent ionic interactions and
hydrogen bonds). Such interactions could exploit the structural differences between the active
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sites of the parasite and human aminopeptidases, potentially allowing potent and specific inhibition of LAPTc. Importantly, LAPTc and human LAP3 have differences in their hydrophilicity/hydrophobicity at the entrance to their catalytic pockets and LAP3 also has a relatively
hydrophilic area close to the catalytic site. This is thought to drive the selectivity of KBE009, a
relatively hydrophobic compound, for LAPTc over LAP3 [27] (Fig 1). A similar effect may
explain the selectivity we observe for compound 4, which is also relatively hydrophobic.
Conclusions
We identified multiple novel LAPTc inhibitors through mass spectrometry-based highthroughput screening. Compound 4, in particular, is of interest as a potent, selective and competitive LAPTc inhibitor, with a submicromolar Ki. We propose a binding mode for this compound, based on docking and molecular dynamics evidence, that is in excellent agreement
with experimental data. Compound 4 exhibits in vitro antichagasic activity against T. cruzi
amastigotes, and while toxicity against the host cells needs to be explored further, the compound provides a valuable starting point for Chagas disease drug discovery going forward.
Supporting information
S1 File. RF LAPTc hits.xlsx: Structures, compounds identifier and percent inhibition from
primary screen for the 30 hits.
(XLSX)
S2 File. LAPTc pIC50 DRC.xlsx: Potency data for hits in RapidFire-MS assay. pIC50 =
-LOG(IC50[M]). Experiment column indicates if potency was obtained from initial potency
confirmation or subsequent replicate generation experiments. Hit confirmation column indicates if hits from primary single concentration screen confirmed in the potency experiment.
(XLSX)
S3 File. Tcruzi pEC50 DRC.xlsx: Potency data for intracellular parasites (T. cruzi) and
host cells (Vero). pEC50 = -LOG(EC50[M]).
(XLSX)
S4 File. HepG2 pEC50 DRC.xlsx: Potency data against HepG2 cells. pEC50 = -LOG
(EC50[M]).
(XLSX)
Acknowledgments
We thank Alan Fairlamb (University of Dundee) for the T. cruzi isolate. J.G.-B., M.I. and M.E.
A. are grateful to Susan Farrell (training manager, Wellcome Centre for Anti-Infectives
Research, University of Dundee) for the opportunity to participate in the WCAIR training
scheme. We also thank her for help with the logistics of their stay in Dundee and making them
feel so welcome. In addition, we acknowledge the computer resources and the technical support provided by “Empresa de Tecnologı́as de la Información ETI-BioCubaFarma”.
Author Contributions
Conceptualization: Daniel Alpı́zar-Pedraza, Anthony Hope, David W. Gray, Mark C. Field,
Jorge González-Bacerio, Manu De Rycker.
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Data curation: Maikel Izquierdo, De Lin, Christy Paterson, John Thomas, Mirtha Elisa
Aguado, Enrique Colina Araújo, Daniel Alpı́zar-Pedraza, Halimatu Joji, Lorna MacLean,
Jorge González-Bacerio, Manu De Rycker.
Formal analysis: Maikel Izquierdo, De Lin, Lauren A. Webster, Christy Paterson, John
Thomas, Mirtha Elisa Aguado, Enrique Colina Araújo, Daniel Alpı́zar-Pedraza, Halimatu
Joji, Jorge González-Bacerio, Manu De Rycker.
Funding acquisition: David W. Gray, Mark C. Field, Manu De Rycker.
Investigation: Maikel Izquierdo, De Lin, Sandra O’Neill, Christy Paterson, John Thomas,
Mirtha Elisa Aguado, Enrique Colina Araújo, Halimatu Joji, Martin Zoltner.
Methodology: De Lin, Sandra O’Neill, Anthony Hope, Martin Zoltner, Jorge GonzálezBacerio, Manu De Rycker.
Project administration: Lorna MacLean, David W. Gray, Mark C. Field, Manu De Rycker.
Resources: Lauren A. Webster.
Software: Enrique Colina Araújo.
Supervision: Lorna MacLean, David W. Gray, Mark C. Field, Jorge González-Bacerio, Manu
De Rycker.
Writing – original draft: Jorge González-Bacerio, Manu De Rycker.
Writing – review & editing: Mark C. Field, Jorge González-Bacerio, Manu De Rycker.
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