Computer-aided design of 1,4-naphthoquinone-based inhibitors targeting cruzain and rhodesain cysteine proteases

Article abstract of DOI:10.1016/j.bmc.2021.116213

Chagas disease and Human African Trypanosomiasis (HAT) are caused by Trypanosoma cruzi and T. brucei parasites, respectively. Cruzain (CRZ) and Rhodesain (RhD) are cysteine proteases that share 70% of identity and play vital functions in these parasites.

Full text of DOI:10.1016/j.bmc.2021.116213

Bioorg. Med. Chem. 41 (2021) 116213
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Computer-aided design of 1,4-naphthoquinone-based inhibitors targeting
cruzain and rhodesain cysteine proteases
,
b
a,
Leandro Rocha Silvaa , Ari Souza Guimaraes b, Jadiely do Nascimentob,
˜
a
c
c
´
Igor Jose do Santos Nascimento , Elany Barbosa da Silva , James H. McKerrow ,
Sílvia Helena Cardosob, Edeildo Ferreira da Silva-Júniora
,
*
a
˜
´
Chemistry and Biotechnology Institute, Federal University of Alagoas, Campus A.C. Simoes, Lourival Melo Mota Avenue, Maceio 57072-970, Brazil
b Laboratory of Organic and Medicinal Synthesis, Federal University of Alagoas, Campus Arapiraca, Manoel Severino Barbosa Avenue, Arapiraca 57309-005, Brazil
c Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA,
USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Chagas disease and Human African Trypanosomiasis (HAT) are caused by Trypanosoma cruzi and T. brucei
parasites, respectively. Cruzain (CRZ) and Rhodesain (RhD) are cysteine proteases that share 70% of identity and
play vital functions in these parasites. These macromolecules represent promising targets for designing new
inhibitors. In this context, 26 CRZ and 5 RhD 3D-structures were evaluated by molecular redocking to identify
the most accurate one to be utilized as a target. Posteriorly, a virtual screening of a library containing 120 small
natural and nature-based compounds was performed on both of them. In total, 14 naphthoquinone-based analogs
were identified, synthesized, and biologically evaluated. In total, five compounds were active against RhD, being
three of them also active on CRZ. A derivative of 1,4-naphthoquinonepyridin-2-ylsulfonamide was found to be
the most active molecule, exhibiting IC50 values of 6.3 and 1.8 µM for CRZ and RhD, respectively. Dynamic
simulations at 100 ns demonstrated good stability and do not alter the targets’ structures. MM-PBSA calculations
revealed that it presents a higher affinity for RhD (-25.3 Kcal molꢀ 1) than CRZ, in which van der Waals in-
teractions were more relevant. A mechanistic hypothesis (via C3-Michael-addition reaction) involving a covalent
mode of inhibition for this compound towards RhD was investigated by covalent molecular docking and DFT
B3LYP/6–31 + G* calculations, exhibiting a low activation energy (ΔG‡) and providing a stable product (ΔG),
with values of 7.78 and ꢀ 39.72 Kcal molꢀ 1, respectively; similar to data found in the literature. Nevertheless, a
reversibility assay by dilution revealed that JN-11 is a time-dependent and reversible inhibitor. Finally, this
study applies modern computer-aided techniques to identify promising inhibitors from a well-known chemical
class of natural products. Then, this work could inspire other future studies in the field, being useful for designing
potent naphthoquinones as RhD inhibitors.
Naphthoquinone
Drug design
Cysteine protease
MM-PBSA
DFT
1. Introduction
other millions are living at risk zones of infection, mostly in Latin
America. There are only two drugs to treat Chagas disease, nifurtimox
Trypanosoma cruzi and T. brucei are parasites that belong to the
Trypanosomatidae family responsible for Chagas and Human African
Trypanosomiasis - HAT (also named sleeping sickness disease), respec-
tively. Chagas disease is transmitted to humans through the bite of
infected blood-sucking Triatomine insects during their meal and blood
transfusion, while HAT is transmitted by tsetse flies.1–3 T. brucei presents
two subspecies, T. brucei rhodesiense and T. brucei gambiense, responsible
for different symptoms.4,5 Million people are infected worldwide, and
and benznidazole (Figure 1), both of these were introduced in the 1970
s, and these are associated with resistance cases and numerous side ef-
fects that make treatment difficult.6,7 For HAT, suramin and pentami-
dine (Figure 1) are prescribed for the early stage, presenting severe side
effects. Also, melarsoprol (Figure 1), a toxic derivative from arsenic, can
cause encephalopathies in the late stage of the disease.4,5 Besides, the
current treatments are highly inefficient due to the low effectiveness of
them.8 Additionally, fexinidazole and eflornithine (Figure 1) have been
* Corresponding author. Tel.: +55 82 9 9610 8311.
E-mail address: edeildo.junior@iqb.ufal.br (E.F. da Silva-Júnior).
https://doi.org/10.1016/j.bmc.2021.116213
Received 26 February 2021; Received in revised form 23 April 2021; Accepted 5 May 2021
Available online 11 May 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

L.R. Silva et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116213
used to treat infections caused by T. brucei gambiense in humans.9–12
Consequently, there is a crucial need for the development of new drugs
that would ideally be affordable, safe, and effective to treat these
Neglected Tropical Diseases (NTDs). In general, trypanosomiasis has
two clinic phases, being acute or chronic. The acute stage is asymp-
tomatic, although 10–50% of patients die during this phase. In the
chronic stage, 20% of patients develop digestive problems, heart dis-
eases, and even neurological disorders.13–15
including oxidative stress and direct interaction with cellular macro-
molecules.35–37 Notwithstanding these facts, this natural product class is
mainly known for interfering in the biological redox balance. In this
context, the redox cycle of quinones can be initiated by one-electron
reduction that leads to the formation of semiquinones.16,38–40 Then,
these compounds can covalently bind to nucleic acids and proteins,
inactivating them.41 Alternatively, another mechanism is reducing
quinones by two-electrons, mediated by DT-diaphorase, resulting in the
formation of hydroquinones. These molecules can disrupt the electron
transport chain, intercalate with DNA, uncouple oxidative phosphory-
lation, and produce reactive oxygen species (ROS).16,38–40
T. cruzi life cycle involves amastigote, trypomastigote, and epi-
mastigote forms. Trypomastigote forms that circulate in the host’s blood
are converted into epimastigote forms, after the ingestion of blood by
insect vectors. Subsequently, these forms proliferate and differentiate
into metacyclic forms, which are released in feces, being able to start a
new infection cycle.2,16 Similarly, T. brucei life cycle alternates between
a mammalian host, which presents slender, intermediate, and stumpy
forms, and tsetse fly when stumpy forms are converted into procyclic
forms, proliferate migrating to epimastigote forms, and developing in
metacyclic forms into salivary gland.17 For parasite’s survival, the major
cysteine proteases from T. cruzi and T. brucei, cruzain (CRZ) and rho-
desain (RhD), respectively; play essential functions involved in the host
invasion process and shares 70% identity, providing an attractive target
for the development of a broad-spectrum inhibitor for both disease.3,18
CRZ represents the largest cysteine protease from T. cruzi, and it is
associated with several biological processes into the parasite, such as
differentiation, invasion, and proliferation in host cells.19–21 Biochem-
ical studies involving animal models suggest that CRZ is the primary
target of infection control and parasite elimination.22,23 CRZ is a
cathepsin L-like protein from the papain family.24 Similarly, RhD (or
T. brucei cathepsin L) is the largest cysteine protease from T. brucei, and
it belongs to the papain-like subfamily, Clan CA family C1.25,26 More-
over, it is related to protein degradation and intracellular transport of
proteins between the insect and host cells.25 CRZ and RhD have four
main subsites for ligands (S1′, S1, S2, and S3), in which the S2 subsite is
the specific site with low solvent accessibility and responsible for in-
teractions with hydrophobic and basic groups.24 The active site is
Some studies have revealed that naphthoquinone analogs exhibit
diverse biological properties, including anticancer activity by inhibiting
topoisomerase II and telomerase through DNA alkylation or interaction,
and via inhibition of the heat shock protein HSP90.42 Also, naph-
thoquinones have been reported to inhibit the recombinant falcipain 2 in
vitro, a cysteine protease from Plasmodium falciparum.37 Among other
activities, 1,4-naphthoquinone-based compounds have been demon-
strated to act as trypanocidal agents. In this context, lapachol and
β-lapachone are naphthoquinone derivatives extracted from trees from
the Tabebuia genus, which indigenous people have used to treat many
parasitic infections. Nonetheless, these have not become drugs, but they
have been extensively explored in medicinal chemistry to seek new
trypanocidal compounds yet since these are considered privileged
scaffolds.43–45 Then, Salas et al.41 determined the activity of lapachol,
α
-lapachone, and β-lapachone (Figure 2) toward amastigotes and try-
´
pomastigotes of T. cruzi Tulahuen strain. Still, Bourguignon et al. verified
that β-lapachone reduced the activity (>65%) of T. cruzi proteic whole
extract of cysteine proteinase at 4.9 ± 1.0 µmol minꢀ 1 mg of protein-1
concentration. Zani et al.7 and Zani & Fairlamb46 identified that com-
pound (1) (Figure 2) presents a non-competitive inhibition mode upon
the recombinant trypanothione reductase (TR) from T. cruzi. Besides, it
was able to reduce the growth of epimastigotes. According to a study
performed by Jorqueira et al.,47 it was found that an oxyrane derivative
of α-lapachone (2) (Figure 2) has activity against epimastigote of T. cruzi
located between two domains, with the catalytic triad (Cys25, His159
,
Dm28C strain. Considering the previously reported results for β-lapa-
chone, Ferreira et al.48 performed a study that identified the compound
(3) as a trypanocidal agent against trypomastigotes of T. cruzi. Recently,
Klein et al.18 developed naphthoquinones containing a dipeptide
sequence H2N-L-Phe-L-Leu-OBn attached in their N-terminal regions
since RhD to hydrolyze compounds with this recognition unit. Then, the
authors identified analog (4) as a potent, time-dependent, and reversible
inhibitor of RhD.
and Asn175 for CRZ, and Cys25, His162, and Asn182 for RhD).24,27,28 The
nucleophilic residue Cys(S-) is capable of attacking carbonyl groups with
fissile amide bonds.29,30 Finally, both of these enzymes are present in all
stages of the parasite’s life cycle (epimastigotes, trypomastigotes, met-
acyclic trypomastigotes, and amastigotes), making them excellent tar-
gets to be explored in the discovery of drugs against these NTDs.24,31
In folk medicine, vegetal species containing 1,4-naphthoquinones
have been utilized for the treatment of many diseases,32 representing
a valuable source for new drugs.33,34 1,4-Naphthoquinones are present
in several plant families, including Bignoniaceae and Verbenaceae, and
even fungi and insects. 1,4-Naphthoquinone-based compounds are also
able to exert activity upon the different targets by diverse mechanisms,
Herein, we performed a virtual screening of a small in-house library
of 120 natural and nature-based compounds upon CRZ and RhD to
identify promising cysteine protease inhibitors. Fourteen 1,4-naphtho-
quinone-based compounds were found to be potentially active toward
these targets. Subsequently, these molecules were synthesized and
Fig. 1. Therapeutic arsenal used in the treatment of Chagas disease and Human African Trypanosomiasis (HAT).
2

L.R. Silva et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116213
Fig. 2. Lapachol- and β-Lapachone-based compounds with trypanocidal activity. TR: trypanothione reductase; km: Michaelis constant; ki: inhibitory constant; GI50
:
growth inhibition for 50%; a: concentration needed to decrease the growth constant (kc) by 50%; b: viability of infected Vero cells with trypomastigote of T. cruzi.
screened for their inhibitory activities. After structure–activity rela-
tionship (SAR) analyses, compound JN-11 was identified as a hit com-
pound in this study as a time-dependent inhibitor. Posteriorly, molecular
docking and dynamic simulations were performed to obtain data on its
interactions and complex stability, respectively. Furthermore, all dy-
namics’ trajectories were used to perform MM-PBSA calculations to
obtain essential physicochemical parameters on CRZ- and RhD-JN-11
complexes. Subsequently, we hypothesized a possible covalent mecha-
nism of inhibition for JN-11, which was investigated by covalent mo-
lecular docking and Density Functional Theory (DFT) calculations at an
atomistic level. Figure 3 summarizes the workflow followed for this
study. Further analyzes involving reversibility by dilution assays were
performed to evaluate our hypothesis. All methods and results described
in this study can be useful for other research teams worldwide,
Fig. 3. Rational design and study overview employed to identify 1,4-naphthoquinone-based compounds as inhibitors of cruzain and rhodesain cysteine
proteases. Initially, a dataset of cysteine proteases (CRZ and RhD) was used to perform molecular redocking simulations to identify the most accurate scoring function,
validated by heatmaps. Subsequently, the most accurate algorithm was used to virtually screening a small group of 120 in-house natural and nature-based compounds. Then,
substituted naphthoquinone-based compounds were identified as promising inhibitors, which were posteriorly synthesized and biologically evaluated against CRZ and RhD
enzymes. Finally, the hit compound was identified and explored by molecular dynamics (MM-PBSA), DFT calculations, time-dependence inhibition and reversibility assays to
investigate a possible covalent mechanism of inhibition.
3

L.R. Silva et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116213
increasing the chances to obtain promising drug candidates in the
future.
extracts of Tabebuia sp. bark, with 1–2% yield. In general, all compounds
were obtained by nucleophilic attack of the most basic nitrogenated
reagents directly into the carbonyl group (C1-addition) or in the carbon
from the 1,4-naphthoquinone core (C2-addition),55 with yields ranging
from 39 to 95%. Scheme 1 shows all compounds obtained in this study,
including their synthetic routes and reaction conditions. Finally, all
these compounds were evaluated in enzymatic assays against CRZ and
RhD proteases. These results will be discussed in the next section.
Fourier transform infrared spectroscopy (FTIR) data were used to
identify the major functional groups present in this series of compounds.
In general, FTIR spectra of products showed the deformation bands (δ) of
OH and NH groups in 3485–3305 cmꢀ 1 regions. Moreover, bands of
2. Results and discussion
2.1. Virtual Screening, Identification, and synthesis of Naphthoquinone-
based compounds
In our virtual screening protocol, 26 CRZ and 5 RhD structures
complexed with inhibitors were obtained from the PDB website. Sub-
sequently, molecular redocking simulations were performed using four
scoring functions, ChemPLP, GoldScore, ChemScore, and ASP, from the
GOLD® software. Thus, RMSD values were found ranging from 0.238 to
4.839 Å for CRZ, while from 0.383 to 3.105 Å for RhD (see Supple-
mentary Material, Table S1 and S2). Regarding redocking solutions,
RMSD values allow to categorize them as: (i) good solutions when RMSD
values are less than 2.0 Å; (ii) acceptable solutions when RMSD values
ranging from 2.0 to 3.0 Å; and (iii) bad solutions when RMSD values are
higher than 3.0 Å.49–52 ChemPLP showed the most accurate scoring
function for both cysteine proteases, presenting the lowest RMSD values
in redocking simulations. Additionally, the ChemPLP algorithm esti-
mated FitScore values of 50.03 and 52.74 toward CRZ and RhD,
respectively.
stretching vibrations (ν) for carbonyl groups (vC = O) were found from
1678 to 1591 cmꢀ 1 region, while (vC = S) was observed at 1678 cmꢀ 1 for
AS12/15. Still, (vC–N) vibration ranged from 1265 to 1140 cmꢀ 1 region,
while (vC–O) at 1043 cmꢀ 1 for JN-17. Furthermore, stretching vibra-
tions for SO2 groups were observed at 1142 and 1167 cmꢀ 1 for JN-11
1
and JN-13, respectively. H NMR spectra of the compounds exhibited
chemical shifts (δ) concerning the aromatic hydrogens from the naph-
thalene ring, ranging from δ 7.8 to 8.6 ppm as doublets and triplets. The
singlet corresponding to the hydrogen CH (C3) from the naphthalene
ring was observed from δ 5.4 to 6.4 ppm. Considering the iso-
nicotinoylhydrazide substituent present at IK-01, JN-22, and CR-70, the
four pyridyl protons were assigned ranging from δ 7.8 to 8.9 ppm. For
AS12/15, IK-01, JN-22, and CR-70, the singlet for the NH group was
displayed in the range of δ 8.1 and 11.0 ppm. The presence of the NH
peak great than 10.0 ppm was indicative of conformation in which there
CRZ and RhD structures (PDB id 1AIM53 and 6EXQ,54 respectively)
were selected based on their RMSD values. Posteriorly, a small in-house
dataset of 120 natural and nature-based compounds was investigated for
their potential activity upon CRZ and RhD. Among these compounds,
fourteen 1,4-naphthoquinone analogs (with FitScore values ≥ 53, see
Supplementary Material, Figure S1), including lapachol, were identi-
fied as promising inhibitors for both proteases. Then, some of them were
purchased, and others were synthesized. Lapachol was obtained from
55
–
is a hydrogen bonded to the carbonyl oxygen (C O). Also, this fact
–
was proved by DFT calculations (see Supplementary Material,
Figure S2), using JN-22 as example. The presence of a largely deshielded
signal in the range of δ 14.9 to 16.4 ppm in hydrazone analogs AS-12/
Scheme 1. Synthetic routes to obtain naphthoquinone-based compounds. Reaction conditions: (a) step 1- NaOH 0.1 M, thiosemicarbazide methanolic solution; step
2- HCl 10% solution. (b) Et3N 10% solution, isonicotinoyl hydrazide, acetic acid glacial. (c) Acetic acid glacial 80% solution, isonicotinoylhdrazide aqueous solution. (d) H2O
or EtOH, aromatic amine. (e) N-methylbenzylamine, DMF, 80 ◦C.
4

L.R. Silva et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116213
15, IK-01, and JN-22 was indicative of Z-conformation established by
and their influences on the activity is provided in the SAR analysis
section. Compound JN-17 displayed a very poor solubility at testing
conditions, preventing the determination of its biological activity. In
Figure 4 are shown IC50 curves for all active compounds against CRZ and
RhD.
55
–
the intramolecular NH∙∙∙O C six-membered hydrogen bonded ring.
–
–
In addition, our analysis of (Z)-stereochemistry for the C N imine
–
double bond in acylhydrazones derivatives (IK-01 and JN-22) or the
thiosemicarbazone (AS12/15) is corroborated by the results presented
by Campos et al.56 In this work, the authors defined the stereochemistry
1
based on their analysis of H NMR spectra and X-ray crystallographic
2.3. Structure-Activity relationship (SAR) analysis
data. 13C NMR spectra for these naphthoquinone derivatives revealed
–
the carbonyl groups (C O) ranging from δ 179.7 to 184.6 ppm, sug-
–
Regarding the biological results obtained from the enzymatic assays
performed on CRZ and RhD presented in Table 1, SAR analysis was
performed to identify the most promising molecular features found in
this small series of 1,4-naphthoquinone-based compounds. Initially,
lapachol was assumed as the starting point for such analysis. Subse-
quently, electron-withdrawing and electron-donating groups at position
gesting that the 1,4-naphthoquinone core was preserved and sub-
stitutions with nitrogenated substituents occurred at the C2 carbon (JN-
16, JN-11, JN-13, and CR-70). Aromatic carbon signals (CAr) for all
analogs were recorded in the range of δ 122.1–134.8 ppm. Finally, sig-
nals for C = NNH groups from IK to 01 and JN-22 were observed
–
varying from δ 127.5 to 129.5 ppm. Still, chemical shifts for C O from
–
2
were analyzed, followed by substituted aniline, iso-
the acylhydrazone group were computed from δ 155.5 to 170.3 ppm for
nicotinoylhydrazide, thiosemicarbazone, and sulfonyl groups, at posi-
tions 1 or 2. Furthermore, the inhibition results for those compounds on
CRZ at 100 µM concentration will be first discussed, and then RhD
results.
–
these compounds. C S peak from AS12/15 was observed at δ 179.5
–
ppm. HPLC analyzes revealed that these compounds present retention
time (RT) values ranging from 2.88 to 5.35 min, with purities varying
from 95.0 to 99.9%. Finally, all chemical data are in agreement with the
literature.55,57
Initially, screening was performed at 100 µM, lapachol displayed
low activity upon CRZ, with 35 ± 2% inhibition. However, when two
electron-withdrawing atoms, such as chlorine atoms, are inserted at
positions 2 and 3 from the 1,4-naphthoquinone core (2,3-Cl-NPQ), the
activity is slightly increased, showing 49 ± 3% inhibition. This activity is
still increased when a bromine atom is present at position 2 (and posi-
tion 3 remains unsubstituted), a compound with 51 ± 2% inhibition is
obtained (2-Br-NPQ). When this bromine atom is replaced with a strong
electron-donating group (2-OH-NPQ), the enzymatic activity is
improved, exhibiting 58 ± 2% inhibition.
2.2. Enzymatic activity on cruzain and rhodesain
All 1,4-naphthoquinone-based analogs were evaluated for their
inhibitory activity toward CRZ and RhD enzymes, following the protocol
described in the Material and Methods section. Results are presented in
Table 1. Compounds AS12/15, IK-01, and JN-11 were the most prom-
ising analogs against CRZ, showing IC50 values of 34 ± 1, 33 ± 2, and
6.3 ± 0.1 µM, respectively. Regarding the anti-RhD activity, 2-OH-NPQ,
lapachol, AS12/15, IK-01, and JN-11 were found to be the most active
compounds, exhibiting IC50 values of 33 ± 7, 58 ± 3, 28 ± 1, 20 ± 1, and
1.8 ± 0.1 µM, respectively. Surprisingly, lapachol and 2-OH-NPQ were
selective for RhD since these analogs did not demonstrate activity
against CRZ. In contrast, AS12/15, IK-01, and JN-11 could be consid-
ered broad-spectrum inhibitors since these molecules inhibited both of
these proteases. Finally, a complete discussion on chemical substituents
When it is substituted with a methoxyl group (2-OCH3-NPQ), a
reduction in the activity is about 1.8-fold. Concerning nitrogenated
substituents, it is verified that a 1-phenylethylamino group at position 2
(JN-16) significantly reduces the activity on CRZ, showing only 26 ± 2%
inhibition. In contrast, the replacement of this group with an aniline ring
generates a compound more active (JN-08), which demonstrates 48 ±
3% inhibition at 100 µM concentration. Unfortunately, when this aniline
ring is substituted at para-position with a methoxyl group, a compound
(JN-17) with poor solubility is obtained since it was impossible to test it.
Then, introducing an isonicotinoylhydrazide at position 2 (CR-70), a
compound less active than JN-08 is provided, exhibiting 37 ± 2% in-
hibition. Considering JN-08, when a sulfonamide-isoxazole group is
inserted at the para-position, the most poorly active compound is ob-
tained (JN-13), exhibiting only 6 ± 2% inhibition. In contrast, when the
isoxazole ring is replaced with a 2-pyridine ring, the best compound of
this series (JN-11) was generated, exhibiting 95 ± 1% inhibition,
resulting in an IC50 value of 6.3 ± 0.1 µM. Considering lapachol
structure, when the isonicotinoylhydrazide group is placed at position 1
(IK-01), its enzymatic activity is significantly improved, exhibiting 76 ±
2% inhibition, with an IC50 value of 33 ± 2 µM. Surprisingly, when the
isoprene unit at position 3 is removed is observed a slight reduction in
the activity, generating JN-22 with 66 ± 5% inhibition. Regarding IK-
01, when the nitrogenated group at position 1 is replaced with a thio-
semicarbazone substituent, an even more active compound is obtained
(AS12/15), displaying 83 ± 1% inhibition, resulting in an IC50 value of
34 ± 1 µM.
Table 1
Inhibitory activity of naphthoquinone-based compounds toward cruzain and
rhodesain proteases.
Compound
% Cruzain
inhibition (100
µM)a
IC50
% Rhodesain
inhibition (100
µM)a
IC50
cruzain
rhodesain
(µM)b
(µM)b
2,3-Cl-
NPQ
49.0 ± 3.0
ND
56.0 ± 4.0
ND
2-Br-NPQ
2-OH-NPQ
2-OCH3-
NPQ
51.0 ± 2.0
58.0 ± 2.0
32.0 ± 4.0
ND
ND
ND
50.0 ± 2.0
77.0 3.0
27.0 ± 0.0
ND
33.0 7.0
ND
Lapachol
AS12/15
IK-01
35.0 ± 2.0
83.0 1.0
76.0 2.0
48.0 ± 3.0
95.0 1.0
6.0 ± 2.0
26.0 ± 2.0
NT
ND
70.0 7.0
82.0 2.0
87.0 2.0
61.0 ± 2.0
93.0 0.0
1.0 ± 1.0
23.0 ± 3.0
NT
58.0 3.0
28.0 1.0
20.0 1.0
ND
34.0 1.0
33.0 2.0
ND
JN-08
JN-11
6.3 0.1
ND
1.8 0.1
ND
JN-13
JN-16
ND
ND
JN-17
NT
NT
Regarding the RhD inhibition, it was observed that lapachol was
more active upon RhD than CRZ, exhibiting 70 ± 7% inhibition, which
resulted in an IC50 value of 58 ± 3 µM. Compounds 2,3-Cl-NPQ and 2-
Br-NPQ demonstrated equivalent active profiles, with inhibition values
of 56 ± 4 and 50 ± 2%, respectively. It suggests that there are no
meaningful differences between these weak electron-withdrawing
groups. The strong electron-donating group, such as the hydroxyl sub-
stituent from the 2-OH-NPQ, improves the activity upon RhD than 2-
OCH3-NPQ. It demonstrated 77 ± 3% inhibition, resulting in an IC50
value of 33 ± 7 µM, while 2-OCH3-NPQ presented only 27 ± 0% inhi-
bition. Concerning the aminated substituents, JN-16 displayed a similar
JN-22
66.0 ± 5.0
37.0 ± 2.0
99.0 ± 1.0
ND
52.0 ± 2.0
37.0 ± 2.0
99.0 ± 2.0
ND
CR-70
E-64 (2
µM)
ND
ND
0.02 ± 0.1
0.0031 ± 0.0
a
Results represent the average and standard error of two independent ex-
periments in triplicate. Errors are given by the ratio between the standard de-
viation and the square root of the number of measurements. b: IC50 values
represent the average of two independent experiments determined based on at
least 8 compound concentrations in triplicate. Errors are given by the ratio be-
tween the standard deviation and the square root of the number of measure-
ments. ND: Not determined. NT: Not tested.
5

L.R. Silva et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116213
Fig. 4. Dose-response curves for cruzain and rhodesain inhibition. In A, Dose-response curves for cruzain compounds AS12/15, IK-01, and E64. In B, Dose-response
curves for rhodesain inhibition compounds 2-OH-NPQ, Lapachol, AS12/15, IK-01, and E64. IC50 curves represent two independent experiments, which were determined
using at least eight different concentrations of the compounds in triplicate.
inhibition profile for CRZ and RhD, showing 23 ± 3% inhibition.
Moreover, the 1-phenylethylamino group at position 2 reduces the ac-
tivity towards RhD in comparison with 2-OCH3-NPQ. When this sub-
stituent is replaced with an aniline ring (JN-08) occurs a modest
increase in the activity. In contrast, when this new group has a sulfon-
amide coupled to an isoxazole ring at the para-position results in the
inactivation of the analog (JN-13) since it presented 1 ± 1% inhibition.
When the isoxazole ring is replaced with a 2-pyridine ring, the most
active compound is obtained (JN-11), displaying 93 ± 0% inhibition,
which resulted in an IC50 value of 1.8 ± 0.1 µM. Like against CRZ, it was
found to be the hit compound towards RhD. Additionally, when this
chemical group at position 2 is replaced with an isonicotinoylhydrazide
(CR-70) is observed a significant reduction in its activity, showing only
37 ± 2% inhibition. When the lapachol structure is modified at position
1, introducing an isonicotinoylhydrazide group (IK-01), is observed a
significant increase in the activity, resulting in 87 ± 2% inhibition and
an IC50 value of 20 ± 1 µM. Still, when this group is replaced with a
thiosemicarbazone group, a compound with similar effects is obtained
(AS12/15), with inhibition and IC50 values of 82 ± 2% and 28 ± 1 µM,
respectively. However, when the isonicotinoylhydrazide group remains
at the same position and the isoprene unit is removed from position 3, it
is observed a meaningful reduction in the activity (JN-22), exhibiting
52 ± 2% inhibition. Finally, we would like to clarify that all SAR dis-
cussions focused on compounds with demonstrating only %inhibition
values were performed considering observations only at 100 µM con-
centration, obtained by two independent experiments in triplicates, we
did not consider trends in different concentrations in this SAR
discussion.
2.4. Molecular docking simulations on cruzain and rhodesain
As described in the previous section, initially, we discussed CRZ re-
sults and then RhD. For all compounds, their interactions can be found in
the Supplementary Material (Figures S3-S15). Additionally, none of
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L.R. Silva et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116213
–
IK-01, a π-σ interaction was seen involving its 4-pyridine ring and a C C
these 1,4-naphthoquinone compounds interacted with the complete
catalytic triad from both cysteine proteases. Among the interactions
sigma bond from the Ala138 residue. For lapachol, it was observed a
observed from CRZ-naphthoquinone complexes, van der Waals,
π-sulfur,
π-donor hydrogen-bonding interaction involving the carbonyl at C4 and
and
π
-alkyl interactions are frequently displayed, in which the most
Gly163, and benzene ring and Gly66 residue. Furthermore, only three
molecules present interactions with exclusive residues, Phe72 and Leu204
for IK-01, Asp117 for JN-08, and Ser24 and Val139 for lapachol. Lastly,
these docking studies revealed that active 1,4-naphthoquinone-based
frequent amino acid residues are Gly66, Met68, and Ala133. In general,
these 1,4-naphthoquinone analogs interact with several amino acids
from the CRZ binding site, in which the number of amino acids involved
ranges from 11 to 18 residues, being AS12/15 and IK-01 compounds
with the highest number of residues involved in their complexes’ for-
compounds frequently interact with Cys25, Trp26, Gly66, Leu67, Met68
,
Ala138, Leu160, Asp161, His162, and Gly163 residues, corroborating with
other previous studies.58,59
mation. Moreover, it was verified that Cys25, Trp26, Gly66, Leu67, Met68
,
Ala133, Leu157, Asp158, and Glu205 residues are present in all ligands’
interactions. All of these compose a pocket surrounding these analogs on
CRZ (Figure 5A). Then, these amino acids could be considered as char-
acteristics for this chemotype of natural and nature-based compounds.
According to Bourguignon et al.,58 this chemical class of compounds is
2.5. Molecular dynamics simulation on cruzain and rhodesain complexed
with JN-11
Initially, the stereochemical quality of the most stable poses of dy-
namic simulations for CRZ and RhD structures was analyzed using the
Ramachandran plot (see Supplementary Material, Figure S29 A and B).
For CRZ, 88% of residues are found in favored regions, 11.5% in allowed
regions; 0.5% in generously allowed regions; and 0.0% in not allowed
regions (outliers). For RhD, 82% of residues are found in favored re-
gions; 16.1% in allowed regions; 0.6% in generously allowed regions;
and 0.6% in not allowed regions (outliers). Finally, these results are in
accordance with reliable molecular models for performing virtual pro-
tocols.60,61 Then, these optimized models in native form were used to
explore the interactions of JN-11 and both of these targets. Molecular
dynamics evaluated the complexes’ structural stability at trajectories of
100 ns. Thus, the results of Cα RMSD (Figure 6A) show average values
ranging from 0.1 to 0.15 nm for both CRZ- and RhD-JN-11 complexes.
These complexes demonstrated to achieve the stabilization after 15 ns
simulation, remaining stable during all the simulation time (100 ns).
Still, it was verified that these complexes presented stable deviations
(less than0.3 nm).62 However, minor variations are observed for RhD-
JN-11 complex, suggesting high-stability for it. The RMSF plot
(Figure 6B) revealed low fluctuations for the residues and even minor
fluctuations for the catalytic triad. Thus, JN-11 at the active site does not
promote significant conformational changes in primary amino acid
residues. Additionally, the Rg plot (Figure 6C) shows conformational
changes varying from 1.62 to 1.63 nm, indicating high-rigidity and
compactness of the protein structure.63 Another crucial data to verify the
complexes’ stability was SASA analysis (Figure 6D). The results
demonstrate that during the simulation time, the area accessible to the
solvent did not present significant alterations, ranging from 93 to 103
nm2, suggesting that JN-11 does not modify the protein structure and it
typically placed into the CRZ S2 pocket, interacting with Ser64, Gly66
,
Leu67, Met68, Ala133, Leu157, Gly160, and Glu205, corroborating with our
results. For CRZ, all compounds are capable of interacting with Cys25
and His159 residues, except for JN-08, which interacts only with Cys25
.
The best CRZ inhibitors (AS12/15, IK-01, and JN-11) presented in-
teractions involving 18, 18, and 14 amino acid residues, respectively.
These analogs have exclusive interactions with some residues, being
Gln19 for AS12/15, Asp60 and Val134 for IK-01, and Gln115 for JN-11, in
which were observed van der Waals interactions between them. It sug-
gests that these amino acid residues could be associated with a signifi-
cant role in the CRZ inhibition by 1,4-naphthoquinone-based analogs.
Regarding the results of molecular docking toward RhD (see Sup-
plementary Material, Figure S15-S28), it was verified that these com-
pounds present several interactions varying from 12 (for 2,3-Cl-NPQ
and 2-Br-NPQ) to 17 (for lapachol and IK-01) residues. However, the
most active compounds on RhD (2-OH-NPQ, JN-11, AS12/15, IK-01,
and lapachol) also present interactions involving 15–17 residues. All
compounds interact with Cys25, Trp26, Gly66, Leu67, Met68, Ala138
,
Leu160, and His162 amino acid residues. All of these compose a pocket
surrounding these analogs on RhD (Figure 5B). Additionally, it was
observed that the most frequent chemical forces present in their com-
plexes are van der Waals, hydrogen bonding, and π-alkyl interactions.
Also, it was observed that none of these analogs are able to interact with
the complete catalytic triad since it was verified all of them interact only
with Cys25 and His162 residues. Surprisingly, among the most active
compounds, three of them present exclusive interactions. For AS12/15,
a sulfur-x interaction between its double bond (C2 = C3) from the 1,4-
naphthoquinone core and the sulfur atom at the Met68 are present. For
Fig. 5. Cluster of naphthoquinone derivatives into the binding site of cruzain (A) and rhodesain (B). PDB ids: 1AIM for cruzain and 6EXQ for rhodesain.
7

L.R. Silva et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116213
Fig. 6. Post-molecular dynamics analysis for 100 ns simulation time for cruzain (black line) and rhodesain (red line) complexed with JN-11. In (A), Cα
RMSD plot; (B): RMSF plot; (C): Radius of gyration (Rg). (D): Surface area solvent accessible (SASA). (E): Hydrogen-bonding interactions plot.
remains at the active site. It should be considered that increased SASA
values indicate greater exposure of the ligand to the environment
exposed to the solvent.64 Figure 6E shows that JN-11 performs up to
three hydrogen-bonding interactions during the simulation time, sug-
gesting that these could be stabilizing forces for these complexes. These
results revealed excellent complexes’ stabilities, which may indicate the
best activity observed for JN-11 against both CRZ and RhD.
believe that the most significant activity of JN-11 towards RhD is
possibly associated with its H-bonds performed at the active site.
2.6. MM-PBSA calculations for JN-11 in complex with cruzain and
rhodesain
Using trajectories from dynamic simulations, the determination of
free binding energy (ΔGbinding) values was done by MM-PBSA calcula-
tions (Table 2), performing a re-score of JN-11 and characterizing its
main interactions. As expected, JN-11 exhibited high-affinity for RhD
active site (ΔGbinding = -25.3 ± 3 Kcal molꢀ 1 ± SD) than for CRZ active
site (ΔGbinding = -12.6 ± 4 Kcal molꢀ 1 ± SD), corroborating with our
experimental results. Moreover, MM-PBSA calculations revealed that
van der Waals interactions are the main forces involved in the coupling
process for both proteases, with favorable energy values for CRZ (-21.7
± 5.2 Kcal molꢀ 1 ± SD) and RhD (-40.3 ± 2.3 Kcal molꢀ 1 ± SD) when
compared to electrostatic forces for CRZ and RhD (-2.7 ± 2.7 and ꢀ 4.8
± 2.2 Kcal molꢀ 1 ± SD, respectively). Interestingly, RhD-JN-11 complex
Evaluation of interactions post-dynamic simulations analysis for JN-
11 was performed to check if CRZ and RhD active sites are altered by
introducing chemical paramters. JN-11 showed similar interactions for
both proteases, such as
π
-alkyl with Leu67, Ala138, and Leu160
; π-sulfur
with Cys25 and Met68, and van der Waals with Trp26, Gly65, Gly66, and
His159/162 residues. On the other hand, JN-11 performed two hydrogen-
bonding interactions with Gly23 (3.47 Å) and Gly163 (3.08 Å) residues
(Figure 7A) at the CRZ active site, while three hydrogen-bonding in-
teractions were observed for RhD, involving Gly23 (3.93 Å), Cys63 (2.06
Å), and Gly163 (3.75 Å) residues (Figure 7B). In general, these in-
teractions are associated with the inhibition of these targets. Finally, we
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L.R. Silva et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116213
in different studies.18,36,65–67 Considering these data, we hypothesized
that JN-11 could be a potential covalent inhibitor and, then, we decided
to investigate our hypothesis by using covalent docking simulations and
DFT calculations. Posteriorly, we performed experimental assays to
verify our suppositions.
2.7. Our hypothesis of covalent inhibition for JN-11
2.7.1. Covalent molecular docking simulation
Using the most stable pose obtained from dynamic simulations at
100 ns, CRZ- and RhD-JN-11 complexes were investigated for a possible
covalent mechanism of inhibition. Then, covalent docking simulations
were used to predict if JN-11 could undergo a nucleophilic attack by
Cys25(S-) residue (also, named as thiolate anion) at the C3 atom from the
1,4-naphthoquinone core since this nucleophilic residue was located at
distances of 5.72 and 3.92 Å for CRZ and RhD, respectively. Romeiro et
al. suggested that these distance values should be lower than 4.65 Å to
result in a potential covalent inhibiton.68 It was observed that JN-11
exhibits a higher covalent score value for C3-Michael-addition by RhD
(9.78) than for CRZ value (6.05), suggesting that a higher probability of
JN-11 to be attacked by the thiolate anion from RhD protease. These
results may be associated with distances between the C3 atom and Cys25
residue. Additionally, C2-Michael-additions were also explored toward
both enzymes. Nevertheless, low covalent score values were obtained
(below 4.05) for both proteases. Recently, studies involving broadly
employed docking software concluded that none of them is capable of
predicting accurate Gibbs free energy values for covalent interactions
(ΔG) and their rate-limiting barriers of transition states (ΔG‡).69–72 In
contrast, quantum mechanics (QM) can be used for modeling biological
systems due to its ability to describe chemical systems with quantita-
tively accurate values of ΔG and ΔG‡.73–75 Notwithstanding these data, it
was decided to investigate a mechanistic hypothesis involving C3-
Michael-addition of JN-11 towards RhD, employing quantum chemis-
try calculations by DFT.
Fig. 7. Molecular interactions for JN-11 in complex with cruzain (A) and
2.7.2. Atomistic covalent mechanism for JN-11 by DFT calculations
In the literature is reported that the catalytic mechanism of cysteine
proteases depends on two residues at the active site, which typically is
composed of Cys and His amino acids. Besides, it is known that the
physiological environment favors that the imidazole group from the His
residue polarizes the SH group from the Cys residue, leading to the
highly nucleophilic Cys(S-)/His(H+) ion pair.76–79 This ion pair is
responsible for the high reactivity of these proteases toward electro-
philic groups, broadly used in the development of covalent inhib-
itors.76–79 Based on these facts, Density Functional Theory (DFT)
calculations at the 6–31 + G* basis set were employed to predict the
formation of a covalent complex involving JN-11 and RhD, describing
ΔG‡ and ΔG values.
rhodesain (B) after dynamic simulations at 100 ns.
Table 2
Binding energy from cruzain- and rhodesain-JN-11 complexes estimated by MM-
PBSA calculations.
Complex (Kcal molꢀ 1 ± SD)
Parameters
CRZ-JN-11
RhD-JN-11
Binding Energy (ΔGbinding
)
ꢀ 12.6 ± 4.0
ꢀ 2.5 ± 0.4
14.4 ± 5.5
ꢀ 2.7 ± 2.7
ꢀ 21.7 ± 5.2
ꢀ 25.3 ± 3.0
ꢀ 3.7 ± 0.2
23.6 ± 2.9
ꢀ 4.8 ± 2.2
ꢀ 40.3 ± 2.3
SASA Energy
Polar Solvation Energy
Electrostatic Energy
Van der Waals Energy
According to our hypothesis, JN-11 has a Michael-acceptor system
that could undergo a nucleophilic addition or substitution reaction at
presented lower SASA energy values (-3.7 ± 0.2 Kcal molꢀ 1 ± SD),
suggesting that JN-11 is placed into a hydrophobic environment at the
RhD active site, with less exposure to water molecules from the physi-
ological medium, when compared with CRZ active site (-2.5 ± 0.4 Kcal
molꢀ 1 ± SD). Finally, higher values of polar solvation energy for CRZ
and RhD (14.4 ± 5.5 and 23.6 ± 2.9 Kcal molꢀ 1 ± SD, respectively)
indicate that the solvation of proteases is significant for both complex
formations. Lastly, all these energetic data corroborate with the higher
affinity of JN-11 at the active site, corroborating with its IC50 value
towards RhD.
one of the carbon atoms from the α,β-unsaturated carbonyl group to-
wards the cysteine residue at the active site of RhD.18 In this context, we
generated the intrinsic reaction coordinate (IRC) for our hypothesis,
which revealed that the S1 atom from Cys25 residue (at a distance of
3.95 Å) could attack the C3 atom from JN-11, via a Michael-addition
mechanism (JN-11-IC) (Figure 8). Subsequently, S1––C3 connection
results in an electron transfer from C3 = C2 bond to the oxygen atom
(O1) at C1, providing an oxy-anion (
σ-complex) as a TS structure with a
ΔG‡ value of 7.78 kcal molꢀ 1 (JN-11-TS). This increased resonance ef-
fect is responsible for reducing the bond lengths for C3 ꢀ C2 ꢀ C1 bonds
from 1.42 to 1.40 Å for C3 ꢀ C2 bond, and from 1.44 to 1.42 Å for C2 ꢀ
C1 bond. It was observed that there is an approximation of Cys25(S1) to
the C3 atom (1.77 Å) to perform a covalent bond. Additionally, it was
verified that His162 residue has an essential role in stabilizing the TS
geometry since it performs three hydrogen-bonding interactions at dis-
tances of 1.95, 2.19, and 1.63 Å for H1∙∙∙O3 ꢀ Cys, H2∙∙∙O2 ꢀ JN-11,
It is known that the chemical reactivity of the 1,4-naphthoquinone
nucleus is associated with its
α,β-unsaturated system, which has an
ambident behavior when reacting with nucleophiles, being able to un-
dergo β-olefinic attack (1,4 addition) or direct addition to the carbonyl
(1,2-addition). Thus, the preferred position of the addition will depend
on the nature of the nucleophile and the reaction conditions.55 Their
chemical reactivities (via covalent mechanism) have been investigated
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L.R. Silva et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116213
Fig. 8. Intrinsic reaction coordinate (IRC) displaying relative Gibbs free energy values for the nucleophilic attack of Cys25 residue at the C3 atom from
compound JN-11. JN-11-IC: JN-11′s initial coordinates; JN-11-TS: JN-11′s transition state; JN-11-Cys25: JN-11′s complexed with Cys25 residue (product).
and H4∙∙∙N1 ꢀ JN-11, respectively. Then, a stable product was ob-
tained by regenerating the 1,4-naphthoquinone core, exhibiting a ΔG
value of ꢀ 39.72 Kcal molꢀ 1 (JN-11 ꢀ Cys25). It was verified a covalent
bond between Cys25(S1) and C3 atom from JN-11, with a bond length of
1.73 Å. Still, C3 ꢀ C2 and C2 ꢀ C1 bonds’ lengths were again estab-
lished, being 1.42 and 1.44 Å, respectively. Moreover, His162 residue
performs only one hydrogen-bonding interaction with the final product
via H4∙∙∙N1 ꢀ JN-11, at a distance of 1.75 Å. In general, this type of
mechanism is assumed to occur in a two-stage one-step mechanism.80
All these IRC results were supported by different studies found in the
literature, which focused on nucleophilic substitutions of 1,4-naphtho-
quinones toward nucleophiles (including cysteine proteases), making
our hypothesis more relevant. According to Delarmelina et al.,36 sulfur-
containing nucleophiles present TS values ranging from 4.7 to 5.06 Kcal
molꢀ 1 and, subsequently, their addition products exhibit ΔG values
ranging from ꢀ 34.63 to ꢀ 38.91 Kcal molꢀ 1. Additionally, Arafet et al.
developed studies involving cysteine proteases (falcipain-2 and CRZ)
and QM/MM calculations,76–78 which they verified that their catalytic
after 10 min pre-incubation with the compounds was compared to ac-
tivity without preincubation.83 RhD inhibition by compounds 2-OH-
NPQ, Lapachol, and JN-11 was greater upon compound pre-incubation
with the enzyme, consistent with a potential covalent mechanism of
inhibition. In contrast, compounds AS12/15 and IK-01 did not show
time-dependent inhibition. AS12/15 and IK-01 IC50 values were
equivalent for both conditions, no pre-incubation and 10 min pre-
incubation of these compounds with the target enzyme. In contrast,
the time-dependent inhibitors (2-OH-NPQ, Lapachol, and JN-11)
showed IC50 values, under no pre-incubation condition, higher than
three times (Table 3).
A dilution experiment was performed to check whether the com-
pounds were irreversible. We incubated the inhibitor and RhD at high
concentrations and then diluting the incubation mixture to the apparent
IC50 of the inhibitor.83,84 Once this is done with RhD and E-64, a known
irreversible cysteine protease inhibitor, the enzyme remained inhibited
upon dilution, as expected. However, when the same test was carried out
with the screening hits, full enzyme activity returned after dilution with
compound JN-11. Most of the RhD activity was also observed in the
presence of the other hits (Figure 9). This suggested that the inhibition
by these hits is reversible.
mechanisms occur with TS values ranging from 10 to 29.5 Kcal molꢀ 1
.
Still, activation barriers (TS values) higher than 40 Kcal molꢀ 1 were
considered as unreasonable steps.36,79 Valente et al. explored the
mechanism of addition on C3-unsubstituted and C2-substituted naph-
thoquinone analogs and verified that this chemotype can undergo a
nucleophilic attack at the C3-unsubstituted position.81 Bruno et al.
demonstrated also via kinetic and mass spectrometry studies that the
glyceraldehyde-3-phosphate dehydrogenase from T. brucei (TbGAPDH),
a cysteine protease, can covalently bond to the C3-unsubstituted atom
from a 1,4-naphthoquinone derivative, via a C3-Michael-addition
mechanism of reaction.82
Table 3
Time-dependence inhibition for the hit compounds.
%RhD inhibition (100 µM)a
IC50 RhD (µM)b
Compound
NI
I
NI
I
2-OH-NPQ
Lapachol
AS12/15
IK-01
27.0 ± 1.0
18.0 ± 1.0
79.0 ± 2.0
73.0 ± 2.0
48.0 ± 2.0
7.0 ± 2.0
77.0 ± 3.0
70.0 ± 7.0
82.0 ± 2.0
87.0 ± 2.0
93.0 ± 0.0
95.0 ± 1.0
> 100
33.0 ± 7.0
58.0 ± 3.0
28.0 ± 1.0
20.0 ± 1.0
1.8 ± 0.1
> 100
66.0 ± 3.0
29.0 ± 3.0
20.0 ± 3.0
N.D.
Even considering our IRC date presented here and all corroboration
with the studies aforementioned, it was decided to investigate insights
on this potential mechanism of inhibition by experimental analyzes
performing time-dependence inhibition and reversibility assays, in order
to evaluate our hypothesis. Furthermore, the hit compounds discovered
in this study were also biologically evaluated.
JN-11
E-64 (0.1 µM)
0.0031 ± 0.0
a
: Results represent the average and standard error of two independent ex-
periments in triplicate. Errors are given by the ration between the standard
deviation and the square root of the number of measurements. b: IC50 values
represent the average of two independent experiments which were determined
based on at least 8 compound concentrations in triplicate. Errors are given by the
ration between the standard deviation and the square root of the number of
measurements. NI: Enzyme inhibition without pre-incubation with the com-
pounds. I: Enzyme inhibition after 10 min pre-incubation with the compounds.
ND: Not determined.
2.7.3. Time-Dependence inhibition and reversibility assays
To gain more insights about the 1,4-naphthoquinone-based de-
rivatives, we evaluated whether the five RhD hits were time-dependent
inhibitors, a hallmark of covalent-acting molecules. Enzyme inhibition
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L.R. Silva et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116213
parasites with co-crystallized inhibitors were used, which were obtained
at the RCSB Protein Data Bank – PDB. In total, 26 CRZ (PDB ids: 1AIM,
1EWL, 1EWM, 1EWO, 1EWP, 1F2A, 1F2B, 1F2C, 1F29, 1ME3, 1ME4,
1U9Q, 2AIM, 2OZ2, 3HD3, 3I06, 3IUT, 3KKU, 3LXS, 4KLB, 4PI3, 4QH6,
4W5B, 4W5C, 4XUI, and 6UX6) and 5 RhD (PDB ids: 2P7U, 2P86, 6EX8,
6EXO, and 6EXQ) structures were selected for further analyses in our
virtual protocol. In this context, redocking of each co-crystallized in-
hibitor was performed to identify the most accurate scoring function
based on their RMSD values. Then, the best CRZ and RhD redocked poses
were selected for further analyses. Posteriorly, a small in-house library of
120 natural and nature-based compounds was virtually evaluated upon
CRZ and RhD enzymes by using molecular docking simulations. The best
ranked compounds were identified, synthesized, and then biologically
evaluated on enzymatic assays for their inhibitory effects. Subsequently,
the most significant molecular features were revealed by SAR analyses.
Compound JN-11 was identified as a hit compound towards both
cysteine proteases. Wherefore, molecular dynamics were performed in
order to obtain data concerning the CRZ- and RhD-JN-11 complexes’
stabilities and physicochemical parameters. Concerning their molecular
dynamic trajectories, molecular mechanics Poisson-Boltzmann surface
(MM-PBSA) calculations were performed to determine such physico-
chemical parameters, including ΔGnon-covalent. Finally, DFT calculations
were performed to investigate (at atomistic level) a hypothesis involving
a covalent mechanism of inhibition by JN-11, including the ΔG‡ (tran-
sition state) energy and conformation. Finally, all procedures and pro-
tocols adopted in this study are in accordance with other studies
previously published by our research team.52,59,85–87
Fig. 9. Reversibility assay. Following the dilution, product formation was
monitored for 30 min. Compound JN-11, the most potent naphthoquinone com-
pound (red), recovered the full enzyme activity, while compounds 2-OH-NPQ
(green) and Lapachol (cyan) reduced the enzymatic reaction rate by 50% compared
to vehicle control (black), while known irreversible inhibitor E-64 (blue) reduced
product formation by 90%.
3. Conclusion
In this work, we reported a rational virtual screening procedure
focusing on identifying the most accurate CRZ and RhD structures (for
our protocol) and, posteriorly, filtrating a small library of natural and
nature-based compounds targeting these proteases. 1,4-Naphthoqui-
none-based derivatives were identified as promising inhibitors. These
molecules were obtained in yields ranging from 39 to 95%, in which all
of them were chemically characterized by NMR, melting point, and FTIR
techniques. In total, five compounds (2-OH-NPQ, lapachol, AS12/15,
IK-01, and JN-11) exhibited activity against RhD, while three of them
(AS12/15, IK-01, and JN-11) were also active toward CRZ. SAR analysis
was discussed here. Among these compounds, JN-11 is highlighted since
it demonstrated the best inhibition results for both proteases. Addi-
tionally, this compound was further investigated by molecular dy-
namics, non– and covalent docking simulations. Thus, it was verified
that JN-11 presents good stability in complex with CRZ and RhD pro-
teases at 100 ns simulation. MM-PBSA calculations revealed that van der
Waals interactions are more significant forces to stabilize it. Also, it has
been seen that JN-11 presents a high-affinity for RhD, corroborating
with its low IC50 value. We hypothesized that JN-11 could undergo a
nucleophilic attack by Cys25 residue, via a C3-Michael-addition, verified
by covalent molecular docking and DFT calculations. Even all results
corroborating with literature data, we performed additional experi-
ments to better investigate our proposed mechanism. Thus, time-
dependent inhibition and reversibility assays by dilution suggested
that JN-11 is a time-dependent RhD inhibitor but it demonstrated to be a
reversible inhibitor. Furthermore, JN-11 emerges as a promising
candidate for the development of a new drug for the treatment of these
diseases. However, studies to evaluate effects of JN-11 in amastigotes
and trypomastigotes, as well as investigations into its mechanism of
action, are needed to guarantee its safety and efficacy.
4.3. Target selection and molecular docking simulations
Initially, 26 CRZ structures co-crystallized with inhibitors and 5 RhD
structures also containing inhibitors were obtained through the RCSB
PDB (http://www.rcsb.org/). For all of them, hydrogen atoms were
added, while water molecules and co-crystallized inhibitors were
removed. Subsequently, re-docking simulations were performed for
each inhibitor into their corresponding target. This procedure was per-
formed using the GOLD® v. 5.8.1 software (https://www.ccdc.cam.ac.
uk/solutions/csd-discovery/Component s/Gold/). For the re-docking
procedures were used Chemical Piecewise Linear Potential
(ChemPLP), GoldScore, ChemScore, and Astex Statistical Potential
(ASP) scoring functions. Then, the best binding poses were chosen, and
their Root-Mean-Square Deviation (RMSD) values were determined
using the PyMol® software (https://pymol.org/2/). Posteriorly, a
heatmap was generated in the Microsoft Excel® 2016, in which the
scoring function that resulted in the lowest RMSD value was considered
as the most accurate one. Thus, the best CRZ (PDB id: 1AIM) and RhD
(PDB id: 6EXQ) structures were identified and selected for further ana-
lyses in our protocol. Simultaneously, a small in-house library of natural
and nature-based compounds was drawn and converted into 3D-struc-
tures using the Argus Lab v. 4.0.1 (http://www.arguslab.
com/arguslab.com/ArgusLab.html). Also, the protonation state calcu-
lation at pH 5.5 was carried out on MarvinSketch® (https://chemaxon.
com/products/marvin) for all these ligands. Then, ten conformations
were generated for every ligand, resulting in the choice of the most
stable of them (those exhibiting the lowest energy values). Therefore, all
compounds were energetically minimized by semi-empirical calcula-
tions using PM3 (Parametric Model 3), also on the Argus Lab® v. 4.0.1
software. Finally, GOLD® software was used to perform all docking
simulations, in which a 6 Å region surrounding the co-crystallized ligand
was selected as a search box. Then, ten different binding poses were
generated for all compounds, and the highest FitScore value (≥53.0) was
used to select the most promising molecules to be synthesized and
evaluated on enzymatic assays. The hit compound was used for covalent
docking simulations to identify a potential covalent mechanism of ac-
tion. For this purpose, the sulfur atom from the Cys25 residue (nucleo-
phile) and electrophilic groups from ligands were selected as potential
4. Materials and methods
4.1. Computational details
The virtual protocol, including molecular docking and dynamics, and
DFT calculations were performed on the DELL® Workstation computer,
with Intel® Xeon E5-1660 processor, 3.3 GHz, 4 CPUs, NVIDIA®
GeForce RTX 2060 graphics card, RAM 8 GB, under the Linux® oper-
ating system.
4.2. Virtual screening and study overview
Initially, cysteine proteases’ structures from T. cruzi and T. brucei
11

L.R. Silva et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116213
interacting groups. Finally, this step was carried out in accordance with
the procedures mentioned above.
protein environment surrounding the active site were completely
ignored.36,59,90
4.4. Molecular dynamics simulation
4.7. Synthesis and characterization of Naphthoquinone-based compounds
All molecular dynamic simulations were performed using the GRO-
MACS® v. 2018.3 software (http://www.gromacs.org/). The CRZ- and
RhD-ligand complexes were obtained after molecular docking simula-
tions, previously described. Water molecules were removed, while all
charges and hydrogen atoms were added using the DockPrep module
from the Chimera® v. 1.15 software (https://www.cgl.ucsf.
edu/chimera/download.html). The CHARMM36 force field was
applied to the protein, followed by the TIP3P solvation method. To-
pologies of ligands were generated by SwissParam® web software
(http://www.swissparam.ch/). The protein–ligand complex was added
to a 1.0 nm triclinic box, including water molecules and ions at physi-
ological concentration. Subsequently, the system was initially mini-
mized in 10,000 steps by the conjugate gradient method, followed by the
system’s total minimization to 20,000 steps. NVT (constant number of
particles, volume, and temperature) and NPT (constant number of par-
ticles, pressure, and temperature) balances were performed at 300 K for
10 ns. With the system assembled, simulations were performed at 100
ns, with non– and complexed proteins. At the end of the simulation time,
the most stable conformation was chosen using the MD movie in
Chimera® software by performing cluster analyses. RMSD, root-mean-
square fluctuation (RMSF), the radius of gyration (Rg), and solvent-
accessible surface area (SASA) graphics were generated using the
Xmgrace® software (https://math.nyu.edu/aml/software/xmgrace.
html). Furthermore, the stereochemical quality of the most stable con-
formations was analyzed via Ramachandran plot, using the procheck
module from the SAVES® web software (https://saves.mbi.ucla.edu/).
Finally, covalent molecular docking simulations were performed
following the same aforementioned procedures, also using the GOLD® v.
5.8.1 software. In this context, both C3– and C2-Michael-addition were
investigated for their covalent score values.
Some naphthoquinones were purchased from Sigma-Aldrich (USA)
with high purity degree and were used without purification; these
included: 1,4-naphthoquinone core, 2,3-dichloro-1,4-naphthoquinone
(2,3-Cl-NPQ), 2-bromo-1,4-naphthoquinone (2-Br-NPQ), lawsone or
2-hydroxy-1,4-naphthoquinone (2-OH-NPQ), and 2-methoxy-1,4-naph-
thoquinone (2-OCH3-NPQ). In contrast, lapachol (2-hydroxy-3-(3-
methyl-2-butenyl)-1,4-naphthoquinone) was isolated from the bark of
Tabebuia sp. tree, collected in the Agreste Region of Alagoas State
(Brazil). Still, it was used as starting material for the synthesis of N’-(4-
hydroxy-3-(3-methylbut-2enyl)-2-oxonaphthalen-1(2H)-ylidene)thio-
semi-carbazide (AS12/15) and N’-(4-hydroxy-3-(3methylbut-2-enyl)-2-
oxonaphthalen-1(2H)-ylidene)isonicotinoylhydrazide (IK-01). The
lawsone (2-OH-NPQ) was used for the synthesis of N’-(4-hydroxy-
2oxonaphthalen-1(2H)-ylidene)isonicotinoylhydrazide (JN-22), N’-
(1,4-dihydro-1,4dioxonaphthalen-2-yl)isonicotinoyl hydrazide (CR-
70). Finally, 1,4-naphthoquinone core was utilized to prepare 2-(phe-
nylamino) naphthalene-1,4-dione (JN-08), 2-(4methoxyphenylamino)
naphthalene-1,4-dione (JN-17), 2-[N-(pyridin-2yl)sulfanilamide)]
naphthalene-1,4-dione (JN-11), 2-[N-(5-methylisoxazol-3yl)sulfanil-
amide)]naphthalene-1,4-dione (JN-13), and 2-(N-benzyl-N-methyl-
amino)naphthalene-1,4dione (JN-16).
Melting points were determined by the MQAPF-301® apparatus and
were uncorrected. Infrared spectra were obtained using the Bomem®
FTIR MB-102 spectrometer with KBr pellets. 1H NMR (400 MHz) and 13
C
NMR (100 MHz) spectra were recorded using the Bruker Advanced®
DPX spectrometer, utilizing CDCl3 or DMSO‑d6 as analytical solvents.
Column chromatography was performed using silica gel 60G 0.063–200
mm (70–230 mesh ASTM) Merck® and silica gel 60G 0.2–0.5 mm
VETEC®. TLC analyses were performed on precoated aluminum plates
of silica gel 60F 254 plates (0.25 mm, Merck®). Solvents were purified
and dried according to the standard procedure. For retention time (RT)
and purity degree (%) of all compounds, a Shimadzu® HPLC chro-
matograph, model SIL-20AHT, was used with a Luna® 5 µm C18(2) 100
Å column (250 × 4.6 mm) and wavelength (λ) of 254 nm (photo diode
array (PDA) detector). During all HPLC analyzes, a mixture of methanol/
trifluoroacetic acid HPLC degrees (≥99%) was utilized as a mobile phase
(v/v 99.9:0.1%). Furthermore, some parameters were established as (i)
sample concentration of 1 mg/mL, (ii) flow rate of 1 mL/min, (iii) run
time of 10 min, and (iv) injection volume of 5 µL. Lastly, RT and
absorbance values were computed in minutes (min) and milli-
absorbance unities (mAU). All spectra and chemical characterization
data can be found in the Supplementary Material of this manuscript.
Finally, all experimental procedures described in here are in accor-
dance with studies of previously published by our research
team.52,55,57,92
4.5. MM-PBSA calculations
The MM-PBSA (Molecular Mechanics Poisson-Boltzmann Surface)
method is used to calculate the energy values from interactions between
ligand and protein. This method calculates the binding free energy
(ΔGbinding) based on van der Waals and electrostatic interactions (un-
bound) between the ligand and its receptor during a molecular dynamics
simulation.88 MM-PBSA calculations were performed using trajectories
from molecular dynamics at 100 ns using the g_mmpbsa module from the
GROMACS® software.89 The ΔGbinding value was determined as the
average of the interaction and solvation free energy during the
simulation.88
4.6. Atomistic covalent mechanism for JN-11 into the active site from
rhodesain by DFT calculations
4.7.1. Extraction and characterization of lapachol
All geometry optimizations were carried out using the B3LYP func-
tional along with the 6–31 + G* basis set. Solvent effects were consid-
ered for optimization by using water as an implicit solvent using the
solvent model 8 (SM8) from Spartan v. 14 (https://www.wavefun.
com/spartan-latest-version). The intrinsic reaction coordinate (IRC)
calculations were performed to study, at the atomistic level, forward and
reverse reactions through the transition structure (TS), connecting the
reactants to the products and vice-versa.18,36 Final points obtained from
the IRC calculations were used as initial coordinates for optimizations of
local minima.18,36 Moreover, thermodynamic parameters at 298 K were
also useful to determine the zero-point energy (ZPE).36,59,90 Then, this
value was used as a correction factor for local minima.90,91 All relative
energy values are admitted as Gibbs free energy (ΔG), computed using
unscaled frequencies.36 Finally, the electrostatic and steric effects of the
Lapachol was isolated from wooden chips of Tabebuia sp. bark by
aqueous sodium carbonate extraction (10% w/v), followed by dilute
hydrochloric acid precipitation and then ethanol or ethyl acetate
recrystallization, leading to 1.0–1.5 g (1–2% yield) from the bark.
C
15H14O3; mp: 139–140 ◦C; Yellow solid; RT: 3.59 min; Purity: 99.9%;
1H NMR (400 MHz, CDCl3, ppm): δ 1.69 (3H, s), 1.79 (3H, s), 3.30 (1H,
d, J = 7.4 Hz), 5.21 (1H, m), 7.34 (1H, s, OH), 7.68 (1H, td, J = 7.6 and
1.3 Hz), 7.75 (1H, td, J = 7.6 and 1.3 Hz), 8.08 (1H, dd, J = 7.5 and 1.4
Hz), 8.12 (1H, dd, J = 7.6 and 1.3 Hz). 13C NMR (100 MHz, CDCl3, ppm):
δ 17.92 (CH3), 22.62 (CH), 25.79 (CH2), 119.63 (C and CH), 123.46 (C),
126.07 (CH) 126.77 (CH), 129.41 (C), 132.89 (CH), 133.88 (C), 134.87
(CH), 152.70 (C), 181.70 (C O), 184.61 (C O). FTIR (cmꢀ 1): 3354
–
–
–
–
(δOH), 2917 (CH2 and CH3), 1665 (vC = O), 1597 (vC = C), 724 (CHAr).
All these data are in accordance with Barbosa and Neto.93
12

L.R. Silva et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116213
4.7.2. Naphthoquinone-based analogs
2-N’-(phenylamino)naphthalene-1,4-dione (JN-08).
1-N’-(4-hydroxy-3-(3-methylbut-2-enyl)-2-oxonaphthalen-1(2H)-yli-
dene) thiosemicarbazide (AS12/15).
1,4-Naphthoquinone (474.1 mg, 3.0 mmol) was dissolved in 60.0 mL
of water, and after complete dissolution, it was added to 0.3 mL (325.9
mg, 3.5 mmol) of aniline solution. The mixture was stirred at reflux for
24 h when the TLC consumption of the starting material was observed.
Solids obtained were filtered, washed with cold water, dried, and
A suspension of 484.1 mg (2.0 mmol) lapachol in 20.0 mL of water
was added to 20.0 mL of 0.1 M NaOH, yielding a dark red solution. An
aqueous-methanolic (50%) solution of thiosemicarbazide (2.4 mmol;
10.0 mL) was added dropwise to the above solution with constant stir-
ring. The mixture was stirred for 54 h, after which time the solution was
neutralized with 10% HCl solution. The crude precipitated product was
filtered and washed with cold water. The resulting solid was purified by
column chromatography using ethyl acetate and n-hexane as eluent in
mixture with increasing polarity to give 283.6 mg (45% yield) the thi-
osemicarbazone AS12/15 and starting material lapachol. C16H17N3O2;
recrystallized with methanol to obtain 598.0 mg (69% yield) of JN-08.
◦
C
16H11N16NO2; mp: 156–160 C; Dark red solid; RT: 3.36 min; Purity:
98.9%; 1H NMR (400 MHz, CDCl3, ppm): δ 6.43 (1H, s), 7.23 (1H, t, J =
7.6 Hz), 7.27 (1H, br s), 7.29 (1H, br s), 7.43 (2H, t, J = 7.9 Hz), 7.68 (1H,
td, J = 7.5 and 1.4 Hz), 7.77 (1H, td, J = 7.5 and 1.4 Hz), 8.11 (2H, m).
13C NMR (100 MHz, CDCl3, ppm): δ 103.39 (CH), 122.62 (2CH), 125.64
(CH), 126.17 (CH), 126.55 (CH), 129.71 (2CH), 130.33 (C), 132.36
(CH), 133.20 (C), 134.93 (CH), 137.42 (C), 144.73 (C-NH), 182.06
mp: 174–175 C; Brown solid; RT: 5.35 min; Purity: 98.8%; 1H NMR
◦
(C O), 183.95 (C O). FTIR (cmꢀ 1): 3317 (δNH), 1670 and 1641 (vC =
–
–
–
(400 MHz, CDCl3, ppm): δ 1.62 (1H, s), 1.74 (1H, s), 3.40 (2H, br s), 5.09
(1H, m), 7.53 (2H, br s), 7.97 (1H, br s), 8.61 (1H, br s), 8.95 (1H, br s),
9.21 (1H, br s), 14.93 (1H, br s). 13C NMR (100 MHz, CDCl3, ppm): δ
17.87 (CH3), 21.13 (CH3), 25.44 (CH2), 117.36 (C), 121.98 (CH), 123.76
–
O
naphthoquinone), 1595 and 1446 (vC = CAr), 1244 (vC–N), 711 (CHAr). All
these data are in accordance with Cardoso et al.55 and Martinez et al.96
2-[N-(Pyridin-2-yl)sulfanilamide)]naphthalene-1,4-dione (JN-11).
–
–
(CH), 124.23 (CH), 127.04 (C), 128.80 (CH), 129.49 (C N), 129.92
1,4-Naphthoquinone (474.1 mg, 3.0 mmol) was dissolved in 20.0 mL
of ethanol and after complete dissolution or suspension formation, it was
added 498.1 mg (2.0 mmol) of sulfapyridine. The mixture was stirred at
reflux for 24 h when the TLC consumption of the starting material was
observed. Solids obtained were filtered, washed with cold water, dried,
and recrystallized with ethanol to obtain 765 mg (63% yield) of JN-11.
–
–
–
–
(CH), 131.13 (C), 131.25 (C), 155.38 (C), 179.48 (C S and C O). FTIR
(cmꢀ 1): 3485 (vNH2), 3355 (vNH), 3141 (CH2 and CH3), 1591 (vC = O),
1587 and 1435 (vC = C), 1286 (vC = S), 1172 (vC-N), 858 (vC = S), 761
(CHAr). All these data are in accordance with Souza et al.57 and Chikate
& Padhye.94
1-N’-(4-hydroxy-3-(3-methylbut-2-enyl)-2-oxonaphthalen-1(2H)-yli-
dene)isonicotinohydrazide (IK-01).
C
22H16N2O4S; mp: 259–260 ◦C; Orange solid; RT: 2.89 min; Purity:
95.1%; 1H NMR (400 MHz, CDCl3, ppm): δ 6.29 (1H, s), 6.87 (1H, t, J =
6.1 Hz), 7.17 (1H, d, J = 8.8 Hz), 7.54 (2H, d, J = 8.8 Hz), 7.73 (1H, m),
7.79 (1H, td, J = 7.5 and 1.4 Hz), 7.86 (2H, td, J = 7.5 and 1.5 Hz), 7.89
(1H, d, J = 8.7 Hz), 7.95 (1H, dd, J = 7.4 and 0.9 Hz), 7.99 (1H, d, J =
4.4 Hz), 8.05 (1H, dd, J = 7.5 and 0.9 Hz), 9.37 (1H, s). 13C NMR (100
MHz, CDCl3, ppm): δ 104.15 (CH), 112.61 (CH), 122.65 (CH), 125.47
(2CH), 126.37 (CH), 128.06 (2CH), 128.99 (C), 130.49 (C), 132.42
(CH), 133.07 (C), 135.09 (CH), 140.78 (C) 141.91 (CH), 145.17 (2C-
Lapachol (484.1 mg, 2.0 mmol) was dissolved in 20.0 mL of 10%
Et3N solution. To this solution, 5.0 mL of an aqueous solution of iso-
nicotinoylhydrazide (822.8 mg, 6.0 mmol) was added and was kept
under constant stirring at room temperature. After 48 h, TLC con-
sumption of the starting material was observed and the reaction was
treated with 4.0 mL of glacial acetic acid. A solid was obtained by the
precipitation of the medium using ice water; this was filtered and
recrystallized from ethanol to obtain 686.2 mg (95% yield) of IK-01.
NH), 181.41 (C O), 183.22 (C O). FTIR (cmꢀ 1): 3440 (vNH), 1676 and
–
–
–
–
C
22H20N3O3; mp: 164–167 ◦C; Orange solid; RT: 3.30 min; Purity:
1633 (vC = Onaphthoquinone), 1587 and 1522 (vC = CAr), 1302 (vS = O),
1142 (vC-N or SO2), 783 (CHAr). All these data are in accordance with
Lawrence et al.97
~95%; 1H NMR (400 MHz, DMSO‑d6, ppm): δ 1.62 (3H, s), 1.73 (3H, s),
3.24 (2H, d, J = 6.5 Hz), 5.08 (1H, m), 7.54 (2H, br s), 7.86 (2H, d, J =
5.5 Hz), 7.98 (1H, br s), 8.10 (1H, br s), 8.90 (2H, d, J = 5.5 Hz), 16.4
(1H, br s). 13C NMR (100 MHz, DMSO‑d6, ppm): δ 18.41 (CH3), 21.64
(CH2), 25.92 (CH3), 117.92 (C), 122.21 (CH), 122.77 (CH), 123.83 (CH),
2-[N-(5-Methylisoxazol-3-yl)sulfanilamide)]naphthalene-1,4-dione
(JN-13).
1,4-Naphthoquinone (474.1 mg, 3.0 mmol) was dissolved in 20.0 mL
of ethanol and after complete dissolution or suspension formation, it was
added 506.6 mg (2.0 mmol) of sulfamethoxazole. The mixture was
stirred at reflux for 48 h when the TLC consumption of the starting
material was observed. Solids obtained were filtered, washed with cold
water, dried, and recrystallized with ethanol to obtain 719 mg (59%
yield) of JN-13. C20H15N3O5S; mp: 189–191 ◦C; Orange solid; RT: 2.91
min; Purity: 97.8%; 1H NMR (400 MHz, DMSO‑d6, ppm): δ 2.30 (3H, s),
6.15 (1H, s), 6.38 (1H, s), 7.62 (1H, t, J = 8.8 Hz), 7.8 (1H, td, J = 7.5 and
1.0 Hz), 7.85–7.88 (3H, br s), 7.96 (1H, d, J = 7.7 Hz), 8.07 (1H, d, J =
7.3 Hz), 9.44 (1H, s), 11.4 (1H, s). 13C NMR (100 MHz, DMSO‑d6, ppm):
δ 12.53 (CH3), 95.90 (CH), 105.23 (CH), 122.95 (CH), 125.80 (CH),
126.72 (CH), 128.69 (CH), 130.86 (C), 132.71 (C), 133.43 (CH), 134.73
(C), 135.39 (CH), 143.51 (C), 145.20 (C-NH), 157.96 (C), 170.83 (C),
–
–
124.75 (CH), 128.43 (C N), 129.94 (CH), 130.88 (CH), 131.48 (CH),
131.79 (C), 140.79 (C), 150.68 (CH), 166.93 (C-OH and NHC = O),
180.17 (C O). FTIR (cmꢀ 1): 3438 (vNH), 2983 and 2917 (CH2 and
–
–
CH3), 1672 (vC = O), 1593 and 1429 (vC = CAr), 1240 (vC–N), 698
(CHAr). All these data are in accordance with Cardoso et al. (2018).55
2-N’-(1,4-dihydro-1,4-dioxonaphthalen-2-yl)isonicotinohydrazide (CR-
70).
Lawsone (2-OH-NPQ) (2.5 g, 14.4 mmol) was dissolved in 100.0 mL
of 80% glacial acetic acid solution. To this suspension, 1.6 g (11.7 mmol)
of isonicotinoyl hydrazide was added gradually. After the addition of the
hydrazide, a color change was observed from yellow to red. After 72 h of
constant stirring at room temperature, the solid obtained was filtered
off, washed with 80% acetic acid solution and water, dried, and
recrystallized from methanol to give 1.8 g (45% yield) of CR-70.
181.70 (C O), 183.59 (C O). FTIR (cmꢀ 1): 3305 (δNH), 1674 and
–
–
–
–
C
16H11N3O3; Degradation point: 222–224 ◦C; Orange solid; RT: 3.39
1612 (vC = Onaphthoquinone), 1591 and 1413 (vC = CAr), 1300 (vS = O),
1167 (vC-N or SO2), 694 (CHAr). All these data are in accordance with
Lawrence et al.97
min; Purity: 96.9%; 1H NMR (400 MHz, DMSO‑d6, ppm): δ 5.76 (1H, s),
7.76 (1H, td, J = 7.3 and 1.4 Hz), 7.85–7.83 (3H, m), 7.94 (2H, dd, J =
7.7 and 1.2 Hz), 8.03 (1H, dd, J = 7.4 and 1.2 Hz), 8.8 (2H, d, J = 5.9
Hz), 9.59 (1H, s), 11.05 (1H, br s). 13C NMR (100 MHz, DMSO‑d6, ppm):
δ 102.41 (CH), 121.86 (CH), 125.93 (CH), 126.34 (CH), 130.94 (C),
132.88 (C), 133.24 (CH), 135.48 (CH), 139.62 (C), 148.70 (C-NH),
2-(1-phenylethylamine)naphthalene-1,4-dione (JN-16).
1,4-Naphthoquinone (474.1 mg, 3.0 mmol) was dissolved in 5.0 mL
of dimethylformamide (DMF) and after complete dissolution, it was
added to 6.5 mmol of 1-phenylethanamine. The mixture was stirred at
80 ◦C for 24 h when the TLC consumption of the starting material was
observed. The crude was extracted with ethyl acetate and brine, organic
phases were evaporated and solids obtained were recrystallized with
ethanol to obtain 342.2 mg (39% yield) of JN-16. C18H15NO2; mp:
157–158 ◦C; Dark red crystal; RT: 3.40 min; Purity: 99.9%; 1H NMR
–
–
–
–
150.95 (CH), 164.43 (NHCO), 181.37 (C O), 182.74 (C O). FTIR
(cmꢀ 1): 3317 and 3249 (vNH), 1693 (vC = Oamide), 1674 and 1635 (vC =
O
naphthoquinone), 1527 and 1493 (vC = CAr), 1259 (vC–N), 983 (vN-N), 777
(CHAr). All these data are in accordance with Cardoso et al.55 and Rani
et al.95
13

L.R. Silva et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116213
(400 MHz, DMSO‑d6 ppm): δ 1.53 (3H, d, J = 6.8 Hz), 4.62 (1H, m), 5.48
(1H, s), 7.23 (1H, tt, J = 7.1 and 1.6 Hz), 7.33 (1H, t, J = 7.3 Hz), 7.41
(1H, d, J = 7.6 Hz), 7.71 (1H, td, J = 7.3 and 1.6 Hz), 7.74 (1H, br s), 7.79
(1H, td, J = 7.6 and 1.4 Hz), 7.86 (1H, dd, J = 7.6 and 0.9 Hz). 13C NMR
(100 MHz, DMSO‑d6, ppm): δ 23.05 (CH3), 51.84 (CH), 101.30 (CH),
125.25 (CH), 125.86 (CH), 126.04 (2CH), 127.07 (CH), 128.53 (2CH),
130.32 (C), 132.27 (CH), 132.77 (C), 134.77 (CH), 143.14 (C), 147.52
rates of reaction. Trans-Epoxysuccinyl-L-leucylamido(4-guanidino)
butane (E64) was used as a positive control. Compounds that inhib-
ited>70% enzyme activity had their IC50 determined. Dose-response
curves were determined in two independent experiments, each
involving at least eight compound concentrations in triplicates. Finally,
IC50 curves were determined by nonlinear regression analysis using
GraphPad
Prism®
v.
5.0
(https://www.graphpad.
(C-NH), 181.38 (C O), 181.50 (C O). FTIR (cmꢀ 1): 3350 (vNH), 3057
com/scientific-software/prism/).
–
–
–
–
(CHAr), 2973 and 2863 (CHaliph), 1674 and 1629 (vC = Onaphthoquinone),
1568 and 1502 (vC = CAr), 1246 (vC–N), 731 (CHAr).
4.9. Reversibility assay
2-N’-(4-methoxy-phenylamino)naphthalene-1,4-dione (JN-17).
1,4-Naphthoquinone (474.1 mg, 3.0 mmol) was dissolved in 20.0 mL
of ethanol and after complete dissolution, it was added 762.6 mg (6.2
mmol) of 4-methoxyaniline. The mixture was stirred for 24 h when the
TLC consumption of the starting material was observed. Solids obtained
were filtered, washed with cold water, dried, and recrystallized with
ethanol to obtain 468.9 mg (56% yield) of JN-17. C17H13NO3; mp:
157–158 ◦C; Dark red crystal; RT: 3.29 min; Purity: 99.9%; 1H NMR
(400 MHz, CDCl3, ppm): δ 3.84 (3H, s), 6.23 (1H, s), 6.96 (d, J = 8.8 Hz),
7.21 (d, J = 8.8 Hz), 8.11 (t, J = 6.4 Hz). 13C NMR (100 MHz, CDCl3,
ppm): δ 55.58 (CH3), 102.54 (CH), 114.94 (2CH), 124.86 (2CH), 126.15
(CH), 126.30 (CH), 130.04 (C), 130.44 (C), 132.20 (CH), 133.41 (C),
Rhodesain at 100-fold its final assay concentration was incubated
with the hits at 10-fold its respective IC50 value for 30 min in a volume of
2 μL. This mixture was diluted 100-fold with an assay buffer containing
2.5 μM Z-FR-AMC substrate to a final volume of 200 μL, resulting in a
standard concentration of enzyme and 0.1 times the IC50 value of hits.84
Irreversible inhibitor will maintain approximately 10% of enzymatic
activity, while a rapidly reversible inhibitor will dissociate from the
enzyme to restore approximately 90% of enzymatic activity following
the dilution event. Fluorescence intensities of 200 μL-wells were moni-
tored continuously for AMC hydrolysis on Synergy 2 (BioTek®) plate
reader for 30 min.
–
–
–
134.87 (CH), 145.70 (C-NH), 157.72 (C), 182.17 (C O), 183.75 (C O).
–
FTIR (cmꢀ 1): 3228 (δNH), 1678 and 1625 (vC = Onaphthoquinone), 1597
and 1506 (vC = CAr), 1296 (vC–O), 1232 (vC–N), 721 (CHAr). All these
data are in accordance with Martinez et al.96
Declaration of Competing Interest
The authors declared that there is no conflict of interest.
1-N’-(4-hydroxy-2-oxonaphthalen-1(2H)-ylidene)isonicotino-hydrazide
(JN-22).
Lawsone (2-OH-NPQ) (484.0 mg, 2.8 mmol) was dissolved in 20.0
mL of 10% Et3N solution. To this solution, 5.0 mL of an aqueous solution
of isonicotinoyl hydrazide (822.8 mg, 6.0 mmol) was added and was
kept under constant stirring at room temperature. After 54 h, TLC con-
sumption of the starting material was observed with the formation of
two majority compounds at CCD (eluent 9:1 dichloromethane/meth-
anol), one yellow and other purple. The reaction was treated with ice
water and 4.0 mL of glacial acetic acid. An orange solid was obtained by
the precipitation of the medium using ice water; this was filtered and
recrystallized from ethanol to obtain 489.1 mg (60% yield) of JN-22.
Acknowledgments
˜
The authors thank the Coordenaçao de Aperfeiçoamento de Pessoal
˜
`
de Nível Superior (CAPES), Fundaçao de Amparo a Pesquisa do Estado
de Alagoas (FAPEAL) and the National Council for Scientific and Tech-
nological Development (CNPq) for their support to the Brazilian Post-
Graduate Programs. Also, the authors thank to Prof. PhD. Thiago Men-
donça de Aquino and Prof. PhD. Edson de Souza Bento, both from the
Center of Analysis and Research in Nuclear Magnetic Resonance,
Chemistry and Biotechnology Institute, Federal University of Alagoas,
16H11N3O3; Degradation point: 222–224 ◦C; Orange solid; RT: 3.16
1
Maceio 57072-970, Brazil, for providing H and 13C NMR spectra for CR-
C
´
min; Purity: 96.4%; 1H NMR (400 MHz, DMSO‑d6, ppm): δ 5.93 (1H, s,
OH), 7.58 (1H, t, J = 7.3 Hz), 7.64 (1H, t, J = 7.3 Hz), 7.80 (1H, d, J =
6.0 Hz), 7.90 (2H, dd, J = 7.4 and 0.8 Hz), 8.12 (1H, br s), 8.87 (2H, d, J
= 6.0 Hz), 16.10 (1H, br s). 13C NMR (100 MHz, DMSO‑d6, ppm): δ
70, AS12/15, and Lapachol. Moreover, the authors also thank the
Research Collaboratory for Structural Bioinformatics-Protein Data Bank
for providing access to crystallographic structures of the target (avail-
able at https://www.rcsb.org/), which allowed us to elaborate illustra-
tions for this article.
–
–
104.68 (CH), 121.47 (CH), 123.61 (CH), 124.21 (CH), 127.04 (C N),
129.56 (CH), 131.30 (CH), 132.36 (2C), 139.54 (C), 150.58 (2CH),
169.82 (C-OH and NHCO), 181.66 (C O). FTIR (cmꢀ 1): 3442 (vOH or
–
–
Funding
NH), 1678 (vC = O), 1589 and 1446 (vC = CAr), 1265 (vC–N), 793 (CHAr).
This research was funded by the Brazilian Ministry of Health and
4.8. Assessment of activity upon cruzain and rhodesain
˜
`
Fundaçao de Amparo a Pesquisa do Estado de Alagoas/SUS Research
Program (FAPEAL/PPSUS), with the grant number: 60030000712/
2013.
Recombinant cruzain was expressed and purified as previously
described Silva et al. 2019.98 Recombinant rhodesain was generously
provided by Conor Caffrey (University of California San Diego). Pro-
teolytic activity was measured by monitoring the cleavage of the fluo-
rescent substrate Z-Phe-Arg-aminomethyl coumarin (Z-FR-AMC), in a
Synergy 2 (BioTek®) fluorimeter. All assays were performed in 96-well
Declaration of Interest
The authors have no relevant affiliations or financial involvement
with any organization or entity with a financial interest in or financial
conflict with the subject matter or materials discussed in the manuscript.
This includes employment, consultancies, honoraria, stock ownership or
options, expert testimony, grants or patents received or pending, or
royalties.
black plate format, in a final volume of 200 μL, in a buffer solution of 0.1
M sodium acetate pH 5.5 in the presence of 1 mM dithiothreitol, 0.01%
Triton X-100, 0.5 nM enzyme, and 2.5 µM of the substrate.99 The assays
were performed with 10 min of pre-incubation of the compounds with
the enzyme before the substrate addition. Hit compounds were also
tested without incubation to investigate whether they were time-
dependent inhibitors. The initial screen was performed with 100
μ
M
Appendix A. Supplementary data
of compounds. Two independent experiments were performed for each
assay, each in triplicates and monitored for 5 min. Enzymatic activities
were calculated based on comparison with a DMSO control, from initial
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116213.
14

L.R. Silva et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116213
27 Kerr ID, Wu P, Marion-Tsukamaki R, Mackey ZB, Brinen LS. Crystal Structures of
TbCatB and Rhodesain, Potential Chemotherapeutic Targets and Major Cysteine
Proteases of Trypanosoma brucei. Tschudi C, ed. PLoS Negl Trop Dis.. 2010;4(6):e701.
https://doi.org/10.1371/journal.pntd.0000701.
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