On the intrinsic reactivity of highly potent trypanocidal cruzain inhibitors

Article abstract of DOI:10.1039/d0md00097c

The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. Hence, peptidomimetic cruzipain inhibitors having a reactive group (known as warhead) are subject to continuous studies

Full text of DOI:10.1039/d0md00097c

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RESEARCH ARTICLE
On the intrinsic reactivity of highly potent
trypanocidal cruzain inhibitors†
Cite this: DOI: 10.1039/
d0md00097c
Vinicius Bonatto,‡a Pedro Henrique Jatai Batista,‡a Lorenzo Cianni,a
Daniela De Vita,a Daniel G. Silva,a Rodrigo Cedron,a Daiane Y. Tezuka,ab
Sérgio de Albuquerque,b Carolina Borsoi Moraes,c Caio Haddad Franco,
c
ad
a
a and Carlos A. Montanari
*
*
Jerônimo Lameira,
Andrei Leitão
The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the
treatment of Chagas disease. Hence, peptidomimetic cruzipain inhibitors having a reactive group (known
as warhead) are subject to continuous studies to discover novel antichagasic compounds. Here, we
evaluated how different warheads for a set of structurally similar related compounds could inhibit the
activity of cruzipain and, ultimately, their trypanocidal effect. We first investigated in silico the intrinsic
reactivity of these compounds by applying the Fukui index to correlate it with the enzymatic affinity. Then,
we evaluated their potency against T. cruzi (Y and Tulahuen strains), which revealed the reversible cruzain
Y.strain
50
inhibitor Neq0656 as a better trypanocidal agent (EC
= 0.1 μM; SI = 58.4) than the current drug
Y.strain
50
Received 28th March 2020,
Accepted 3rd August 2020
benznidazole (EC
= 5.1 μM; SI > 19.6). We also measured the half-life time by HPLC analysis of three
lead compounds in the presence of glutathione and cysteine to experimentally assess their intrinsic
reactivity. Results clearly illustrated the reactivity trend for the warheads (azanitrile > aldehyde > nitrile),
where the aldehyde displayed an intermediate intrinsic reactivity. Therefore, the aldehyde bearing
peptidomimetic compounds should be subject for in-depth evaluation in the drug discovery process.
DOI: 10.1039/d0md00097c
rsc.li/medchem
has recently been approved for therapeutic use in children
under 12 in the USA. Nonetheless and beyond doubt, new
1. Introduction
Chagas disease, whose etiological agent is the protozoan
parasite Trypanosoma cruzi, is a serious health and social
problem for people living in Latin America and areas
previously considered non-endemic such as Japan, East
Europe and North America.1 More than 300 000 new cases are
reported every year in 21 countries around the world, with an
average of one million people currently infected with T.
cruzi.2–4 The only two existing drugs available for the
treatment of this unmet medical need, benznidazole and
nifurtimox, show many side effects and high inefficiency in
the chronic stage of the disease.5 Despite this, benznidazole
drugs that are safe and efficacious are therefore critically
needed. One approach consists in the discovery and
development of cruzain (Cz) inhibitors, which is the major T.
cruzi cysteine protease responsible for the survival and
propagation of the protozoan parasite.6 Recently, we have
reported different covalent reversible inhibitors of cruzain as
potent trypanocidal agents.7–10 Moreover, we explored the
effects on the affinity of cruzain inhibition by replacing a
nitrile group using alternative warheads.11
The active site of cruzain is V-shaped containing a Cys25,
His162, and Asn182 catalytic triad. In general, the catalytic
cysteine is deprotonated by the histidine, which is stabilized
by the Asn175 and a Trp184 residue shielding the thiolate–
imidazolium ion-pair from the solvent. The stabilized
negative charge renders the active site cysteine capable of
attacking the warhead of certain types of covalent
inhibitors.12 The same mechanism is present for different
mammalian and protozoan cysteine proteases. In general, 19
different types of warheads have been applied for cysteine
protease inhibition. However, just a few of them inhibit
cruzain.11,12 K777, the leader of the first generation of
irreversible Cz inhibitors, was withdrawn from the preclinical
phase due to substantial side effects caused by its irreversible
a Medicinal Chemistry Group, Institute of Chemistry of São Carlos, University of
São Paulo, Avenue Trabalhador Sancarlense, 400, 23566-590, São Carlos/SP,
Brazil. E-mail: andreileitao@iqsc.usp.br, carlos.montanari@usp.br
b Ribeirão Preto School of Pharmaceutical Sciences, University of São Paulo,
Ribeirão Preto, São Paulo, Brazil
c Laboratório Nacional de Biociências (LNBio), Centro Nacional de Pesquisa em
Energia e Materiais (CNPEM), Campinas, São Paulo, Brazil
d Laboratório de Planejamento e Desenvolvimento de Fármacos, Instituto de
Ciências Exatas e Naturais, Universidade Federal do Pará, Rua Augusto Corrêa 01,
CP 66075-110, Belém-PA, Brazil
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0md00097c
‡ These authors contributed equally to the work.
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mode of action.12–14 The main concern is to consider that
warheads require a balanced reactivity profile. They should
be sufficiently reactive to form a covalent bond with the
cysteine protease in the active site. Their inherent reactivity
should be reduced to a necessary minimum to prevent
nonspecific off-target labeling.15 As a result of the time-
dependent nature of covalent inhibition, even optimized
compounds with sufficient selectivity in screening panels
may exert significant off-target reactivity after extended
exposure times.12 The quantification of the half-life (t1/2) for
the reaction of compounds with glutathione (GSH) or
cysteine provides information about warhead electrophilicity
reactivity of compounds, through quantum mechanics (QM)
reactivity indices.21,22 These indices can help to reduce the
synthetic effort required during a drug discovery endeavor
while increasing knowledge of how substitutions affect
warhead reactivity. Herein, we have studied the intrinsic
reactivity of a set of Cz inhibitors and their trypanocidal
activities on two different T. cruzi strains resulting in four
trypanocidal agents equipotent to benznidazole and one
nanomolar T. cruzi killer acting on the Y strain. Also, we have
measured the t1/2 by HPLC for key compounds to evaluate
their intrinsic reactivity in water in the presence of the
cysteine nucleophilic thiol. We have found that the local
electrophilicity index correlates with the Cz affinity for a set
of selected pairs of compounds.
and liability toward the putative off-target reactivity.16
A
comparison of t1/2 data across a range of compounds with
different warheads provides useful information for the design
of new compounds within a desired reactivity range.17
Recently, Balogh and coworkers have used 137 chemical
probes with 36 different warheads in order to investigate the
impact of the warheads in the reactivity and specificity of a
given covalent fragment.18 In another contribution, Martin
and coworkers have demonstrated through an NMR based
assay that the reactivity of a covalent modifier is dependent
on the amino acid residue such as cysteine, serine, tyrosine,
and threonine as a nucleophile in aqueous solution.19
McGregor and coworkers have used experimental techniques
such as crystallography, hydrogen/deuterium exchange, and
differential scanning fluorimetry to study electrophiles that
are useful for targeting oncogenic K-Ras mutant proteins.20
Computational tools can also be employed to understand the
2. Results
2.1. Design, synthesis and kinetic characterization
In our previous study, we reported a dipeptidyl nitrile
compound, Neq0409, as a reversible covalent inhibitor of Cz.
The crystal structure (PDB: 4QH6) displayed the covalent
bond between the residue Cys25 at the nitrile group. Kinetic
characterization reveals a fast reversible mode of binding.7 In
the second step, we explored the effects on potency of cruzain
inhibition by replacing a nitrile group with alternative
warheads.11 Thanks to the knowledge gained in our recent
SARs, we have herein designed and synthesized compounds
Neq0673 and Neq0646. The other compounds present in this
study have already been reported in our previous work.11
Fig. 1 2D schematic representation of the compounds presented in this study. Blue color depicts the difference in warhead structures.
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the other modifications resulted in a decrement in the
reactivity profile for Cz inhibition.
2.2. Local electrophilicity index
One of the key aspects of the optimization process of
covalent drugs is the modulation of reactivity of the warhead.
Recently, Palazzesi and co-workers have used the
electrophilicity index to estimate the absolute covalent
warhead reactivity of acrylamides in aqueous solution.23 In
another contribution, Berteotti and coworkers have
investigated the mechanistic cysteine nucleophilic attack on
nitrile-carrying compounds using DFT calculations and
kinetic measurements in a water environment. Note that the
reactivity of the inhibitor can be influenced by the presence
of specific interactions in the active site of the protein.
However, in this work, the reactivity of compounds is
Fig. 2 Schematic representation of the warheads investigated using
electrophilicity indices. Carbon (in blue) reacts with the thiolate
nucleophile of cysteine. The Fukui index of electrophilicity was
calculated for the carbon represented in blue.
Neq0673 and Neq0646 bear isoxazole as a warhead, which
can be seen as prodrugs for the oxime group, which has
displayed an improvement in reactivity toward Cz compared
with nitrile-based inhibitors. Neq0673 and Neq0646 contain
Trp and Phe moieties in P2, respectively, which are privileged
substructures for inhibition of Cz and trypanocidal activity.7,9
Modifications in P3 were accomplished by exchanging a
carbamate (Cbz protecting group) for an amide bond with
benzoic acid to increase their metabolic stability (Fig. 1).
An overview of the synthesis of compounds Neq0646 and
Neq0673 is presented in Fig. 2. First, the 4-amine-isoxazole (2)
was synthesized by selective nitration of isoxazole followed by
reduction with SnCl2 under acidic conditions to give the desired
product. Then, the warhead (2) was coupled to the corresponding
compound (3 or 4) with EDC and HOBt to afford the desired
dipeptidyl isoxazole compounds. The final compounds were then
purified using a HPLC equipped with a chiral column to achieve
a purity higher than 95% (Fig. 1 and Scheme 1).
explored in
a water environment following the same
approach of previous studies.23,24 The advantage of this
approach is that QM calculations can estimate the reactivity
only based on the electrophilicity, without including protein–
inhibitor specific interaction effects, which would require
long computational times. Herein, the local electrophilicity
was used to predict the reactivity of warheads. We have used
the Fukui function, in order to estimate the electrophilic
character of the carbon (see Fig. 2) involved in the
nucleophilic attack of the Cys25. Overall, we make use of the
+
electrophilic Fukui function ( fc ), global electrophilicity (ω)
+
and local electrophilicity (ωc ) to estimate the covalent
inhibitor reactivity (see Table 1). Then, we have evaluated the
correlation of this parameter with the ligand binding affinity.
Other electronic parameters calculated for the compounds
can be found in the ESI.†
From the data shown in Table 3, we have found a strong
+
Cz inhibition was evaluated by fluorometric assays. The
results reported in Table 1 (see below) show a wide range of
affinity, spanning from high micromolar to one-digit
nanomolar inhibition. As already described in our previous
work,11 substitution of the nitrile warhead for the azanitrile
leads to a strong increase in potency (ΔpKi > 2.0) while all
correlation between the local electrophilicity (ωc ) and the
ligand binding affinity, as shown in Fig. 3. The correlation
between the other parameters and pKi can be found in the ESI.†
Fig. 3 shows that a higher value of the pKi against Cz is
+
related to a higher value of ωc . This result is interesting
because a single descriptor obtained in the aqueous phase
Scheme 1 Synthetic scheme for the synthesis of compounds Neq0646 and Neq0673. Conditions: (a) TFAA, NH4NO3, rt, 2 h; (b) 6 M HCl, SnCl2,
1.5 h, rt; (c) Trp-OH or Phe-OH, 1 M NaOH, 0 °C, 0.5 h; (d) EDC, HOBt, THF, rt, 18 h.
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Table
1 The Mulliken charge (Qc) of neutral and anionic forms,
the covalent bond can form different non-covalent interactions
with the target protein that also modulate the affinity (e.g.
difference between nitrile and ketoamide dipeptidyl nitriles for
cathepsin inhibition, PDB IDs: 3HHA and 1TU6).
electrophilic Fukui function (fc+), global electrophilicity (ω) and local
electrophilicity (ωc+) were computed for the compound set from which
the respective pKi against Cz was measured
Compound
(Neq)
Overall, the higher the value of local electrophilicity index
for the carbon to be attacked by cysteine, the greater the
stabilization of the addition of electrons; thus, this model
can also be used for choosing warheads or groups that can
influence the electronic properties of warheads in the design
of new compounds.
+
+
QC,Neutral QC,anion fc
ω (eV) ωc (eV) pKiCz
500
539
615
646
652
653
654
655
656
657
673
675
677
690
0.321
0.045
0.835
0.045
−0.027
0.081
−0.023
0.315
0.574
−0.036
0.045
0.125
0.515
0.518
0.332
0.050
0.833
0.049
−0.044
0.071
−0.026
0.333
0.580
−0.063
0.049
0.127
0.537
0.536
0.0108 0.183
0.0048 0.136
−0.0015 0.199
0.0040 0.130
−0.0179 0.131
−0.0105 0.135
−0.0025 0.148
0.0183 0.087
0.0051 0.177
−0.0270 0.164
0.0040 0.128
0.0030 0.238
0.0221 0.183
0.0181 0.189
0.0020 6.3
0.0006
−0.0003
5
4
0.0005 4.7
−0.0023 3.7
−0.0014 4.6
−0.0004 3.9
0.0016 5.2
0.0009 5.4
−0.0044 3.4
2.3. Biological activities
The trypanocidal activity was then assessed for all
compounds using two T. cruzi strains (Y and Tulahuen) as
reported in Table 2, along with the cytotoxicity against the
mammalian host cells (U2OS and LLC-MK2) and the
respective selectivity indices (SIs). In general, biological
activities show that Cz inhibitors are more potent against the
T. cruzi Y strain than the Tulahuen one. This effect could
arise from a different level of expression of Cz and its
isoforms for the different strains.29 The difference in potency
for different strains is already known, so it is vital to
emphasize the importance of testing the same set of
compounds in different strains.30 Neq0500, Neq0539, and
Neq0646, the dipeptidyl nitrile based compounds, appear to
be trypanocidal agents equipotent to Bz, showing a low
micromolar potency against the parasite (Y strain) and no
cytotoxicity to the host cell. On the other hand, these
dipeptidyl nitriles are ineffective against the Tulahuen strain,
as also underlined in our previous work.7
Compound Neq0655 has a higher cytotoxic profile and low
selectivity index when tested on the U2OS cell line.
Azadipeptidyl nitriles display the same trend being equipotent
to Bz as trypanocidal agents for both strains but with a
selectivity index lower than 10. Neq0675 bearing an isoxazole
as warhead is inactive for the Y strain of T. cruzi, while its
analogue (Neq0646) with a Phe in P2 shows mild potency and
no cytotoxicity to the same strain. Interestingly, compound
Neq0656 bearing an aldehyde as a warhead is a potent
trypanocidal agent for the Y strain with a high selectivity in
relation to that for the mammalian cell. Although the activity
of Neq0656 is not transferable to the Tulahuen strain, we
believe that this result is fundamental in the ongoing research
for small molecules as potent trypanocidal agents due to the
high infectivity of the Y strain.31 It is noteworthy that because
most of the reported compounds are covalent inhibitors of Cz,
their potency against the parasite is also time-dependent;
hence, future studies should be focused on the time-
dependency of the provided activity.
0.0005
0.0007
0.0041 8.7
0.0034 8.8
5
5
presented a good correlation with the inhibition of the
enzyme, as other authors have done in order to obtain a
descriptor correlated with the inhibition of cysteine
proteases,24,25 even knowing that it is difficult to find a single
property to describe the biochemical activity.21,26–28
Compounds Neq0539 (pKi = 5.0) and Neq0653 (pKi = 4.6) are
over an order of magnitude less potent than the reference
nitrile Neq0500 (pKi = 6.3). A ΔpKi value of 1.3 for the
transformation is sufficiently small to be consistent with
covalent bond formation. In the same direction, we observed a
loss in potency (ΔpKi = 2.3) when exchanging the nitrile
warhead to an amide group (Neq0615) which can be considered
as a negative control for the covalent bond formation. The
difference in potency for these compounds is nicely related to
the local electrophilicity. On the other hand, the functional
behaviour of warheads is sometimes assumed to be
determined entirely by electrophilicity; however, the product of
2.4. Study of the intrinsic reactivity
Covalent inhibitors binding normally involve a two-step
process in which an initial reversible binding event takes
place, followed by the covalent bond-forming reaction. In the
first stage, enzyme E and inhibitor I form an enzyme
Fig.
determination obtained through the linear correlation between ωc
and pKiCz
3 The putative linear equation and the coefficient of
+
.
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Table 2 Trypanocidal activity (EC50), cytotoxicity (CC50) and selectivity index (SI) for the set of compounds using the amastigote form of two strains (Y
and Tulahuen) of T. cruzi in the mammalian host cells (U2OS and LLC-MK2)
Neq ID
EC50 Tc Y (μM)
CC50 U2OS (μM)
SI
EC50 Tc Tula (μM)
CC50 LLC-MK2 (μM)
SI
409a
500
539
570a
615
646
652
653
654
655
656
657
673
675
677
690
Bz
—
—
—
Inact
Inact
25.8
Inact
Inact
ND
Inact
Inact
79.6
Inact
Inact
ND
ND
ND
21.7
8.4
4.3
>100
>100
>100
>100
>100
ND
>100
>100
>100
>100
>100
ND
ND
1.1
3.6
5.1
—
>100
>100
—
ND
>100
ND
ND
ND
6.0
3.0
>100
>100
>100
10.2
26.9
>100
>27.8
>19.6
—
ND
>16.6
ND
ND
ND
4.4
58.4
>8.2
>8.2
ND
3.2
3.2
>3.9
ND
ND
ND
ND
ND
>1.2
ND
ND
ND
ND
ND
1.9
ND
6.0
ND
ND
ND
1.0
0.1
12.1
22.4
Inact
3.2
8.4
5.1
ND
ND
42.0
26.9
3.2
>23.3
>19.6
>100
Standard deviations are lower than 15%. TC Y: T. cruzi Y strain. Tc Tula: T. cruzi Tulahuen strain. ND: not determined. Inact: inactive
a
compound (EC50 > 100 μM). Values retrieved from the recent literature.7
inhibitor complex (E⋯I) whose binding energy (ΔGbind
)
these compounds could be promiscuous electrophiles due to
the high reactivity. Hence, the intrinsic reactivity (given as
the half-life – t1/2) was assessed using a method described
recently,17 where cysteine and glutathione were the non-
specific nucleophiles. Glutathione is present in high
concentration (1–10 mM) in the cell.33 It works as an
important component of the intracellular redox machinery,
while cysteine is a building block amino acid for many
proteins, and it is the reactive amino acid in the active pocket
of cysteine proteases. We measured the half-life of four
compounds by HPLC using two different aqueous systems,
with the results displayed in Table 3. As a model for nitrile
warheads, we used Neq0409, whose crystallographic structure
was previously resolved, and Neq0570, for which the
reversible mode of action has also been investigated.7 In
general, we can identify a trend for the intrinsic reactivity for
both nucleophiles where the reactivity was azanitrile ≫
aldehyde > nitrile. In particular, the azanitrile compound
Neq0690 displayed such strong reactivity with both
glutathione and cysteine that it was not possible to quantify
it. This high intrinsic reactivity may explain the low selectivity
between the parasite and the host cell given by the low SI
depends only on non-covalent interactions and is related to
the inhibition constant Ki. The subsequent binding step
(kinact) involves the formation of a covalent bond between the
inhibitor and target. The latter step will be partly governed by
the intrinsic reactivity of the warhead.15,32 In our case, the
measured Ki values mostly reflect the binding free energy for
non-covalent interactions and the covalent bond formation.
It is difficult to define the contribution of the covalent bond
formation without the kinact values. Indeed, these values are
influenced by several factors other than just the chemical
nature of the warhead. Also, depending on the magnitude of
the non-covalent interactions with respect to the total
potency outcome, we should not expect a direct correlation
between the intrinsic reactivity, measured in the absence of
the target enzyme, and the Ki.
However, to better evaluate the promiscuity of the
different warheads independent of their biological target, the
+
local electrophilicity (ωc ) and intrinsic reactivity are still
pivotal parameters in drug discovery.
The correlation achieved between the local electrophilicity
+
(ωc ) and the enzyme inhibition led us to investigate whether
Table 3 Half-life measurement and decay constant by HPLC with glutathione and cysteine
Half-life with
glutathione
Half-life with
cysteine
Decay constant with
glutathione
Decay constant with
cysteine
Neq ID
pKi
Warhead
409
570
6.3a
6.6a
5.4
8.8
—
Methylene-nitrile
Cyclopropane-nitrile
Aldehyde
Aza-nitrile
—
>6000 min
>6000 min
40.0 min
<5 min
>5000 min
74.3 min
159.0 min
22.2 min
<5 min
—
—
9.33 × 10−3 min−1
4.36 × 10−3 min−1
3.12 × 10−2 min−1
—
656
1.73 × 10−2 min−1
690b
—
—
Nilvadipine
49.5 min
1.41 × 10−2 min−1
a
Values retrieved from the recent literature.7 b When the concentrations of the compound Neq0690 and the thiol are half of the initial values,
the kobs is 1.01 × 10−2 min−1 for the glutathione assay and 3.15 × 10−2 min−1 for the cysteine assay (t1/2 = 68.45 and 21.98 min respectively). The
standard deviation is lower than 10% for all experiments.
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values (Table 2). Nitrile based compounds have low intrinsic
reactivity, which was possible to measure when cysteine is
present in solution as the nucleophile. As expected, the
cyclopropane moiety (as in compound Neq0570) led to a
small decrease in reactivity when compared with the
methylene-nitrile pair (Neq0409). On the other hand, for the
Cz inhibition, we observed an opposite effect thanks to the
ability of the cyclopropane moiety to drive the warhead in the
active site.34 Also, dipeptidyl nitriles (Neq0409 and Neq0570)
are ten-times more potent inhibitors of Cz than the aldehyde
(Neq0656) but less reactive to nucleophilic attack in water
solution. This difference in reactivity/potency can arise from
the non-covalent interaction formed by the thiomidate with
the catalytic site of the enzyme.7,32
charge of atom k in the same geometry of the neutral
molecule, but in the anionic form. Since the covalent
inhibition of Cz involves the nucleophilic attack of the
negatively charged Cys25 (S atom) on the carbon atom of the
warhead (see Fig. 2), it is important to have a parameter
capable of estimating the reactivity of the warhead.
Therefore, using electrophilicity and the Fukui index, it is
possible to describe the electrophilic character of a reactive
site within a molecule, through the local electrophilicity
index41 ω(r):
ω(r) = ωfk+(r)
3.2. Synthetic chemistry
(3)
The aldehyde Neq0656 displays a balanced reactivity for
both systems under study. According to Macfaul,17 compounds
should have t1/2 at least equal to that of nilvadipine to be used
in further assays. Nonetheless, Neq0656, with half of the t1/2
value of nilvadipine, was still selective toward the parasite,
making it a good lead structure. These results are in agreement
with the cytotoxicity observed in the biological studies.
Therefore, we can assume that the substitution of the nitrile
warhead for the aldehyde can provide a balanced reactivity,
which leads to an increase in the trypanocidal activity with a
high selectivity index against the host cell.
Melting points were determined on a Büchi 510 oil bath
apparatus and are uncorrected. Infrared spectra were
obtained from a FT-IR Thermo Scientific Nicolet 380. The
reagents, starting materials and solvents were of commercial
quality and were used without further purification unless
otherwise stated. All syntheses started with enantiopure
amino acids. TLC analysis was carried out on Merck 60 F254
silica gel plates and visualized under UV light at 254 nm and
365 nm or by using a ninhydrin staining solution.
Purity was determined with an LC-MS instrument
(AmaZon SL ESI-MS, Shimadzu LC) with
a cellulose-2
Phenomenex column (250 × 4.6 mm, 5 μm) or a Daicel
column (IC-chiralpak, 250 × 4.6 mm, 5 μm). Isocratic elution
with MeCN and water was applied as specified (stop time 60
min, flow 0.5 mL min−1). NMR spectra were recorded on
Bruker Avance 400 MHz and Bruker Avance DRX 500 MHz
NMR spectrometers. Chemical shifts are reported in ppm
relative to TMS or the residual proton peak of the re-
protonated deuterated solvent, and the spectra were
calibrated against the residual proton peak of the used
deuterated solvent. The following symbols indicate spin
multiplicities: s (singlet), s br (broad singlet), d (doublet), dd
(doublet of doublet), t (triplet), tt (triplet of triplet), q
(quartet), sept (septet), and m (multiplet). HRMS spectra were
recorded on a Thermo Scientific LTQ Velos Orbitrap, in
electrospray ionization (ESI) mode by direct injection.
3. Material and methods
3.1. Computational details
Herein, all calculations were performed with Gaussian09.35
Geometry optimization and vibrational analysis were
performed at the density functional theory (DFT) level, using
the B3LYP functional36,37 and 6-311G(d,p) basis set38 in the gas
phase. After confirming that the structure was at the minimum
through frequency analysis, a single point calculation at the
perturbation theory of Møller–Plesset (MP2)39 level and the
diffuse basis set 6-311++G(d,p)38 was performed in the water
phase using the PCM40 for neutral, anionic and cationic
systems, keeping the external potential constant. Then, the
global electrophilicity index41 (ω) was obtained by:
μ2
Synthesis of 4-nitroisoxazole (1): isoxazole (15 mmol, 960
μL) was dissolved in TFAA (7.3 mL); then, NH4NO3 (22.5
mmol, 1.81 g) was added in 0.3 g portions each 15 min,
keeping the reaction mixture at 25–30 °C. After complete
addition, the mixture was kept at room temperature for 2 h.
After that ice water (30 mL) was poured and this aqueous
washing was extracted with CHCl3 (3 × 15 mL); the combined
organic extracts were dried over Na2SO4 and evaporated (bath
at room temperature) to give an oil that was triturated with
ω ¼
(1)
2η
where μ and η are the chemical potential and chemical
hardness, respectively.
+
In the present study, we have used the Fukui index42–45 ( fk )
as a parameter for the evaluation of electrophilicity of the
inhibitor. The Fukui index was obtained from the population
analysis46 and charges through NBO analysis47 at the MP2 level
using the diffuse basis set 6-311++G(d,p). The fk+ was calculated
through finite difference, as represented below:
1
n-hexane to give a yellow solid (50% yield). H-NMR (CDCl3) δ
= 9.29 (s, 1H), 8.83 (s, 1H) ppm.48
Synthesis of 4-aminoisoxazole (2): to a yellow solution of
4-nitroisoxazole (1, 160 mg, 1.4 mmol) in 6 M HCl (7 mL),
SnCl2 (1.327 g, 7 mmol) was added in one portion. After 1.5 h
at room temperature, the resulting orange solution was
treated with a saturated solution of Na2CO3 until the pH was
+
fk = qk(N + 1) − qk(N)
(2)
where qk(N) represents the Mulliken charge of atom k in the
neutral molecule, and qk(N + 1) corresponds to the Mulliken
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9. The formed solid was removed by filtration, and the
aqueous solution was extracted with ethylacetate (5 × 50 mL);
the organic phase, dried over MgSO4, was evaporated to give
a brown oil (Rf = 0.64, ethylacetate 100%/silica) and stored at
4 °C in an inert atmosphere (65% yield). H-NMR (DMSO-d6)
δ = 8.16 (s, 1H), 8.13 (s, 1H), 4.26 (s br, 2H) ppm.
HRMS (+) calc. for [C19H17N3O3]+ 335.12699, found:
336.12663 [M + H]+.
Synthesis of N-(3-(1H-indol-3-yl)-1-(isoxazol-4-ylamino)-1-
oxopropan-2-yl)benzamide (Neq0673): to a suspension of ( )-
2-benzamido-3-phenylpropanoic acid (216 mg, 0.70 mmol),
HOBt (124 mg, 0.91 mmol) and EDC (175 mg, 0.91 mmol) in
CH2Cl2 (8 mL) were added under argon at 0 °C. After stirring
for 1 hour at room temperature, the mixture was kept in an
ice-bath and a solution of 4-aminoisoxazole (235 mg, 2.80
mmol) in dry CH2Cl2 (2 mL) was added. The resulting
mixture was kept overnight at RT, then the solvent was
evaporated, and the residue was treated with AcOEt (30 mL)
and washed with H2O (2 × 20 mL) and brine (2 × 20 mL). The
organic phase was dried over MgSO4 and evaporated to give a
crude residue that was purified by column chromatography
on silica using CHCl3/AcOEt (1 : 1) as the mobile phase, to
give a solid (Rf = 0.4) crystallized from AcOEt (36% yield).
Secondary purification was carried out on a cellulose-2
Phenomenex column, in isocratic elution mode with a flow
rate of 2.36 mL min−1, at 32 °C; the mobile phase
composition was n-hexane/ethanol (70 : 30) (v/v) to give
1
Synthesis of 2-benzamido-3-phenylpropanoic acid (3) and
3-(1H-indol-3-yl)-2-(phenylformamido)propanoic acid (4): 2.75
mmol of the corresponding amino acid was dissolved in 1 M
NaOH (6 mL) in an ice-bath. Benzoyl chloride (261 μL, 2.25
mmol) was added. After 5 min, the reaction mixture was
allowed to stand at room temperature. After 20 min, the
solution was cooled in ice and 1 M KHSO4 (16 mL) was added
slowly. The obtained white solid was washed with 1 M KHSO4
(3 × 5 mL), H2O (10 × 3 mL), and 9 : 1 EtOH : H2O (3 × 3 mL)
and dried under vacuum on P2O5 (yield 88%). For compound
3: 1H NMR (500 MHz, DMSO-d6): 12.62 (s br, 1H, D2O
exchange), 10.75 (s, 1H, D2O exchange), 8.56 (m, 1H, D2O
exchange), 7.28 (m, 8H), 7.13 (m, 2H), 4.54 (m, 1H), 3.29 (m,
1H), 3.19 (m, 1H). For compound 4: 1H NMR (500 MHz,
DMSO-d6): 12.69 (s br, 1H, D2O exchange), 10.80 (s, 1H, D2O
exchange), 8.61 (d, J = 8.0 Hz, 1H, D2O exchange), 7.81 (d, J =
8.5 Hz, 2H), 7.59 (d, J = 8 Hz, 1H), 7.51 (t, J = 7.0 Hz, 1H),
7.44 (t, J = 7.5 Hz, 2H), 7.31 (d, J = 8.0 Hz, 1H), 7.20 (s, 1H),
7.05 (t, J = 7.5 Hz, 1H), 6.98 (t, J = 7.5 Hz, 1H), 4.65 (m, 1H),
3.30 (m, 1H, H2O overlapping), 3.19 (m, 1H).
1
Neq0673. H-NMR (500 MHz, DMSO-d6) δ = 10.80 (s br, 1H),
10.51 (s, 1H), 9.13 (s, 1H), 8.72 (d, J = 8.0 Hz, 1H), 8.63 (s,
1H), 7.84 (m, 2H), 7.69 (d, J = 8.0 Hz, 1H), 7.52 (tt, J = 7.5 Hz,
J = 1.5 Hz, 1H), 7.44 (m, 2H), 7.31 (dt, J = 8.0 Hz, J = 1.0 Hz,
1H), 7.23 (d, J = 2.0 Hz, 1H), 7.05 (m, 1H), 6.98 (m, 1H), 4.83
(qd, J = 9.5 Hz, J = 8.0 Hz, J = 5.0 Hz, 1H), 3.30 (dd, J = 14.5
Hz, J = 5.0 Hz, 1H), 3.22 (dd, J = 14.5 Hz, J = 9.5 Hz, 1H)
ppm. 13C NMR (DMSO-d6): 170.72, 166.90, 147.56, 144.85,
136.52, 134.23, 131.86, 128.63, 127.97, 127.59, 124.23, 121.41,
120.17, 118.89, 118.73, 111.83, 110.55, 54.89, 27.74 ppm.
HRMS (+) calc. for [C21H18N4O3]+ 374.13789, found:
375.13895 [M + H]+.
Synthesis
of
N-(1-(isoxazol-4-ylamino)-1-oxo-3-
phenylpropan-2-yl)benzamide (Neq0646): to a suspension of
( )-2-benzamido-3-phenylpropanoic acid (3, 216 mg, 0.70
mmol), HOBt (123 mg, 0.91 mmol) and EDC (175 mg, 0.91
mmol) in CH2Cl2 (8 mL) were added under argon at 0 °C.
After stirring for 1 hour at room temperature, the mixture
was kept in an ice-bath, and a solution of 4-aminoisoxazole
(235 mg, 2.80 mmol) in dry CH2Cl2 (2 mL) was added. The
resulting mixture was kept overnight at room temperature,
then the solvent was evaporated, and the residue was treated
with AcOEt (30 mL) and washed with H2O (2 × 20 mL) and
brine (2 × 20 mL). The organic phase was dried over MgSO4
and evaporated to give a crude residue which was purified by
silica column chromatography using CHCl3/AcOEt (1 : 1) as
the mobile phase, to give a solid (Rf = 0.4) crystallized from
AcOEt (36% yield).
3.3. Enzyme inhibition study
Recombinant cruzain, consisting of the catalytic domain of
cruzipain but excluding the carboxy-terminal extension, was
expressed and purified as previously described.11 The
inhibitors were assayed fluorometrically (Biotek® SynergyTM
HT), monitoring the hydrolysis rate of the fluorogenic
substrate Z-Phe-Arg-7-amido-4-methylcoumarin (Z-FR-AMC,
Sigma-Aldrich) by the enzyme cruzain with fluorescence
emission at 460 nm (excitation at 355 nm) and at 37 °C. The
reactions were followed over 5 min for all compounds (fast-
binding, irreversible and non-covalent inhibitors) excluding
Neq0690 and Neq0677 (slow-binding behaviour) for which
the reaction was followed over 30 min. Enzyme kinetic assays
were carried out in 200 mL of a solution containing 100 mM
Secondary purification was carried out on a cellulose-2
Phenomenex column, in isocratic elution mode with a flow
rate of 2.36 mL min−1, at 32 °C; the mobile phase
composition was n-hexane/ethanol (70 : 30) (v/v) to give
1
Neq0646. H-NMR (500 MHz, DMSO-d6) δ = 10.80 (s br, 1H),
10.51 (s, 1H), 9.13 (s, 1H), 8.72 (d, J = 8.0 Hz, 1H), 8.63 (s,
1H), 7.84 (m, 2H), 7.69 (d, J = 8.0 Hz, 1H), 7.52 (tt, J = 7.5 Hz,
J = 1.5 Hz, 1H), 7.44 (m, 2H), 7.31 (dt, J = 8.0 Hz, J = 1.0 Hz,
1H), 7.23 (d, J = 2.0 Hz, 1H), 7.05 (m, 1H), 6.98 (m, 1H), 4.83
(qd, J = 9.5 Hz, J = 8.0 Hz, J = 5.0 Hz, 1H), 3.30 (dd, J = 14.5
Hz, J = 5.0 Hz, 1H), 3.22 (dd, J = 14.5 Hz, J = 9.5 Hz, 1H)
ppm. 13C NMR (DMSO-d6): 170.72, 166.90, 147.56, 144.85,
136.52, 134.23, 131.86, 128.63, 127.97, 127.59, 124.23, 121.41,
120.17, 118.89, 118.73, 111.83, 110.55, 54.89, 27.74 ppm.
acetate buffer pH 5.5, 300 mM NaCl,
5 mM DTT
(dithiothreitol), 5% v/v DMSO (dimethyl sulfoxide), 0.01% v/v
Triton X-100 and 0.15 nM cruzain, using Corning® 96-well
black flat bottom microplates. The enzyme stock aliquot was
rapidly thawed at 37 °C and kept on ice until activation, in
which it was incubated for 20 min in the assay buffer (100
mM acetate pH 5.5, 300 mM NaCl, 5 mM DTT) followed by
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an additional 2 min with inhibitors before the reaction was
started by the addition of the substrate.
3.6. In vitro trypanocidal activity evaluation on intracellular
amastigote forms (Y strain)
Visual inspection and a pre-reading of plate wells were
performed to check respectively for possible precipitation
and background fluorescence. None of the compounds
displayed a significant fluorescence signal around 460 nm
(the emission wavelength used to monitor reaction kinetics).
The T. cruzi Y strain was donated by A. Avila (Instituto Carlos
Chagas, Fiocruz, Curitiba, Brazil). Trypomastigote forms were
obtained from the supernatant of LLC-MK2 tissue cultures
infected with the Trypanosoma cruzi Y. Infected cultures were
maintained in low glucose DMEM media (Vitrocell)
supplemented with 2% FBS, 100 U ml−1 penicillin and 100 μg
ml−1 streptomycin (all from Life Technologies), henceforth
described as “Low DMEM Media”. The experiment was
performed as previously described.11
3.4. Mammalian cytotoxicity assay
LLC-MK2 cells were cultured in 96-well plates at
a
concentration of 5 × 104 cells per mL. After 48 h, the plates
were washed twice with PBS, and 200 μL RPMI medium was
added with serial dilutions of the compounds and
benznidazole (1.95 μM to 250 μM) in triplicate. After 72 h at
37 °C, the cytotoxic activity of the compounds was
determined by the classical MTT [3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide] method. Briefly, 50 μL
MTT dissolved in PBS (2.0 mg mL−1) was added to each well,
and the plates were incubated for 4 h at 37 °C. The formed
formazan crystals were dissolved with DMSO (50 μL per well),
and the absorbance of the samples was measured using a
spectrophotometer at 570 nm after 30 min. The cytotoxicity
results (CC50) were calculated as a percentage by the formula
3.7. Intrinsic reactivity
Liquid chromatography assay measured the half-life in a
gradient mode (5–100% of B in 10 minutes) with multi-
channel PDA detection (210–400 nm). The assay medium was
0.05 M phosphate buffer (pH 7.4), with 1.0 mM EDTA, 5.0
mM cysteine solution and 5% acetonitrile. Initially, 80 μL of
inhibitor solution (2.5 mM) was added in the assay medium
and injected in the HPLC. After that, the final solution was
kept in thermal equilibrium at 37 °C. Each aliquot was taken
only at the time of injection. The half-life and K were
determined with GraphPad® software for a pseudo-first-order
reaction. The chromatographic analysis was performed with
a Shimadzu Prominence HPLC system. The HPLC system was
equipped with an LC-20AT/AD ternary pumping system, SIL-
20A autosampler, and CTO-20A column oven (Shimadzu
Corp). The column used was a Phenomenex Gemini® 5.0 μm
(4.6 × 150 mm, Phenomenex, Torrance, CA). The gradient
elution, used for separation, was performed with a mobile
phase composed of water as solvent A, and acetonitrile, as
solvent B, at a flow rate of 1.0 mL min−1. The gradient set is
as follows: 0.0–10.00 min phase B increased from 5% to
100%, 10.01–15.0 min phase B remained at 100%, 15.01 min
phase B decreased to 5%, and 15.01–25.0 min phase B
remained at 5%. The column oven was set at 37 °C, with the
injection of 5 μL aliquots. The cysteine employed was of the
levogyre form (L-cysteine), MW: 121.16 g mol−1 (C3H17NO2S)
sold by the brand Sigma-Aldrich (code C7352-25G), ≥98%
{[(ABScontrol
−
ABSsample)/ABScontrol]
×
100}, where
ABScontrol represents the mean absorbance of the untreated
control (viable cells) and ABSsample, the absorbance in each
cellular treatment.
The U2OS cells were kept in high glucose DMEM media, and
the culture conditions and assays were the same for LLC-MK2.
3.5. In vitro trypanocidal activity evaluation on intracellular
amastigote forms (Tulahuen strain)
Cells were evaluated in 96-well plates. LLC-MK2 cells were
plated at
a concentration of 5 ×
104 cells per mL.
Trypomastigote forms of the Tulahuen LacZ strain were
added at a concentration of 5 × 105 parasites per mL and
placed in an incubator at 37 °C with 5% CO2 for 24 h. After
the incubation period, the trypomastigote forms present were
removed by successive washes with PBS, remaining only as
intracellular amastigote forms. The compounds were added
at different concentrations (1.95 μM to 250 μM serial
dilutions) and incubated for 72 h. At the end of this period,
the substrate CPRG (chlorophenol red β-D-galactopyranoside,
400 μM in 0.3% Triton X-100, pH 7.4) was added. After 4 h of
from
a non-animal source. The glutathione used was
purchased from Sigma-Aldrich (PHR1359-690MG).
The glutathione and cysteine solutions were prepared with
a 50 mM final concentration in phosphate buffer with pH 7.4
and 50 mM concentration. Data have been processed with
GraphPad Prism® using the one phase decay model: Y = (Y0
− plateau) × exp(−K × X) + plateau. For each experiment, a
negative control without glutathione or cysteine was assessed
to measure the stability of the compounds and their
warheads in the time frame of the experiment.
incubation at 37 °C, the plates were analyzed on
a
spectrophotometer at 570 nm to obtain the effective
concentration (EC50) to reduce the parasitemia inside the
host cell. Benznidazole was used as a positive control at the
same concentrations as the substances, and DMSO as a
negative control. The compounds were solubilized in DMSO.
The selectivity index (SI) was calculated using the formula: SI
= EC50/CC50. All statistical analyses were done with the
program GraphPad Prism v.5.
4. Conclusions
In this study, we evaluated how the intrinsic reactivity of a set
of structurally similar related compounds influenced the
biological outcome of trypanocidal agents. Currently,
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computational methods are vital in the design of new chemical
entities, but there is still little effort to estimate the intrinsic
reactivity of warheads. Here, we report a successful approach to
using the Fukui index to correlate anti-T. cruzi action of
trypanocidal agents with different warheads. It is worth
Conselho Nacional de Desenvolvimento Científico
e
Tecnológico (CNPQ) and Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (CAPES) for their financial support.
JL also gives thanks for the access to the computational
resources of the Supercomputer Santos Dumont (SDumont)
provided by the Laboratório de Computação Científica (LNCC).
mentioning that we have discovered
a new compound,
Neq0656, which is 10 times more potent than the control drug,
benznidazole, as a trypanocidal agent. Neq0656 also shows a
higher selectivity index than benznidazole. This result
underlines that warhead replacement of nitrile to aldehyde is
an innovative strategy in the research of potent trypanocidal
agents against the Y strain of T. cruzi. Indeed, most of the
compounds here tested are also selective toward the Y strain,
in comparison with the Tulahuen strain.30 This discrepancy
may arise from the difference in the expression of Cz and its
isoforms. We also investigated the intrinsic reactivity by HPLC
to clearly illustrate that azanitrile warheads are highly reactive
moieties. The higher reactivity with respect to that of nitrile
can be explained by the presence of nitrogen in the alpha
position, as already mentioned by Gütschow and co-workers.49
Most of them, aldehydes, which have an intermediate intrinsic
reactivity between nitriles (low reactivity) and azanitriles (high
reactivity), display the best biological outcome against the
parasite. Although certain questions concerning the toxicity
and metabolic stability of the aldehyde moiety remained
unclear,50 this compound class was selected in the
optimization studies of different covalent inhibitors due to its
excellent potency and selectivity profile12,15 with low
cytotoxicity herein observed. Outstandingly, this warhead has
been selected in the structure-based design optimization
processes of covalent inhibitors of the SARS-CoV-2 main
protease (Mpro); the dipeptidyl aldehyde inhibitors displayed
good pharmacokinetic properties and low toxicity in vivo,
leading to promising drug candidates for the treatment of
COVID-19.51 However, aldehydes remain relatively unpopular
in drug discovery for their metabolic liability, as these chemical
functionalities are prone to reduction by aldo–keto reductases,
and oxidation to the corresponding acids by aldehyde
dehydrogenases.52
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