Hydrazonophenol, a Food Vacuole-Targeted and Ferriprotoporphyrin IX-Interacting Chemotype Prevents Drug-Resistant Malaria

Article abstract of DOI:10.1021/acsinfecdis.8b00178

The rapid emergence of resistance against frontline antimalarial drugs essentially warrants the identification of new-generation antimalarials. Here, we describe the synthesis of (E)-2-isopropyl-5-methyl-4-((2-(pyridin-4-yl)hydrazono)methyl)phenol (18), w

Full text of DOI:10.1021/acsinfecdis.8b00178

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Article
Hydrazonophenol, a food vacuole targeted and ferriprotoporphyrin
IX-interacting chemotype prevents drug resistant malaria
Shubhra Jyoti Saha, Asim Azhar Siddiqui, Saikat Pramanik, DEBANJAN SAHA, Rudranil De, Somnath
Mazumder, Subhashis Debsharma, Shiladitya Nag, Chinmoy Banerjee, and Uday Bandyopadhyay
ACS Infect. Dis., Just Accepted Manuscript • Publication Date (Web): 25 Nov 2018
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Hydrazonophenol, a food vacuole targeted and ferriprotoporphyrin IX-
interacting chemotype prevents drug resistant malaria
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Shubhra Jyoti Saha, Asim Azhar Siddiqui, Saikat Pramanik, Debanjan Saha, Rudranil De,
Somnath Mazumder, Subhashis Debsharma, Shiladitya Nag, Chinmoy Banerjee, Uday
Bandyopadhyay*
Division of Infectious Diseases and Immunology, CSIR - Indian Institute of Chemical Biology, 4, Raja S.
C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India, Tel: 91-33-24995735, Fax: 91-33-
4730284
*To whom correspondence should be addressed: ubandyo_1964@yahoo.com, udayb@iicb.res.in.
Division of Infectious Diseases and Immunology, CSIR - Indian Institute of Chemical Biology, 4, Raja S.
C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India. Tel: 91-33-24995735, Fax: 91-33-
4730284. ORCID ID: Uday Bandyopadhyay; 0000-0002-5928-6790.
Keywords: Plasmodium, malaria, hemozoin, food vacuole, parasite metabolism
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Rapid emergence of resistance against the frontline antimalarial drugs essentially warrants for the
identification of new-generation antimalarials. Here, we describe the synthesis of (E)-2-isopropyl-5-
methyl-4-((2-(pyridin-4-yl) hydrazono) methyl) phenol (18) that binds ferriprotoporphyrin-IX
(FeIII-PPIX) (Kd = 33 nM) and offers antimalarial activity against chloroquine-resistant and
sensitive strains of Plasmodium falciparum in vitro. Structure-function analysis reveals that
compound 18 binds FeIII-PPIX through “-C=N-NH-” moiety and 2-pyridyl substitution at
hydrazine counterpart plays a critical role in antimalarial efficacy. Live cell confocal imaging using
fluorophore-tagged compound confirms its accumulation inside the acidic food vacuole (FV) of P.
falciparum. Furthermore, this compound concentration-dependently elevates the pH in FV
implicating a plausible interference with FeIII-PPIX crystallization (hemozoin formation) by dual-
function; through increasing the pH and by binding free FeIII-PPIX. Different “Off-target”
bioassays reduce the possibility of promiscuous nature of compound 18. Compound 18 also exhibits
potent in vivo antimalarial activity against chloroquine-resistant P. yoelii and P. berghei ANKA
(causing cerebral malaria) in mice with negligible toxicity.
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The adverse effect of malaria across the globe demands a serious attention due to increasing number of
1,2
reports of multi-drug resistant (MDR) strains posing a severe threat to human life and productivity
.
Reports of 216 million cases with 445,000 deaths in 2016, repeatedly accentuate the desideratum of new
3
antimalarial chemotherapeutics against MDR strains . Moreover, the emergence of drug resistance
against the artemisinin partner drugs like piperaquine and mefloquine has resulted in significant failure of
artemisinin combination therapy (ACT) in the Thai-Cambodian border, where chloroquine (CQ)
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resistance was developed almost fifty years ago . The spread of resistance therefore needs to be dealt
with the identification of new antimalarial chemotypes efficacious against multi-drug resistant malaria in
the most unfortified regions 1,5
.
Intraerythrocytic stages of P. falciparum are responsible for its clinical symptoms and during these
stages, the parasite digests hemoglobin in the acidic food vacuole (FV) and thereby releasing heme (FeIII-
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PPIX), a non-protein constituent of hemoglobin as a by-product 6,7. This free FeIII-PPIX, a well-known
pro-oxidant, may cause severe oxidative damage to the lipid bi-layers of the parasitic cell membrane
leading to membrane lesion 8–10. In order to evade the detrimental consequences of free FeIII-PPIX
accumulation, Plasmodium crystallizes it into non-toxic inert hemozoin (Hz) 11,12. It was found that the
development of CQ resistance in parasite was mainly because of multiple mutations in P. falciparum CQ
resistance transporter (PfCRT) that results in structure specific efflux of the drug from FV 13. Despite this
resistance, Hz crystallization pathway within parasite is still essential and thus it may be used as a
sustainable drug target 14. Thus, scaffolds which bind to free FeIII-PPIX and inhibit Hz crystallization can
be detrimental for parasite due to free FeIII-PPIX accumulation within the FV and hold merit as potent
antimalarials.
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In search for new antimalarial chemotypes, we focused our study on FeIII-PPIX binding moieties those
are capable of interacting with high affinity. A novel class of chiral gallium (III) complexes of amine-
phenol ligand and schiff-base phenol ligand were reported to possess decent efficacy against CQ sensitive
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and resistant strains . These cationic complexes have been proposed to inhibit the hemozoin formation
via the specific drug/FeIII-PPIX interaction. Different schiff-base phenol, amine phenol and schiff-base
naphthalene scaffolds containing the side chain of CQ has been reported as potent antimalarials in the
CQ-sensitive (HB3) and CQ-resistant (Dd2) strains 16. Moreover derivatives of schiff base hydrazones
containing oxalamide functionality, various acrydinylhydrazones, thiosemicarbazone having Fe(III)
17,18
chelating activity have been reported as promising antimalarials
.
The antioxidant phenolic
compounds extracted from stem barks of Parkia biglobosa elicited significant antiplasmodial activity
against the rodent malaria parasite 19. We took 2-isopropyl-5-methylphenol, a naturally occurring
phenolic antioxidant as the core pharmacophoric unit which exhibited various pharmacological activities
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including antimicrobial 20, antifungal 21, radical scavenging and anti-inflammatory property . We
optimized the scaffold with further chemical modification to derive a new class of FeIII-PPIX binder with
high affinity.
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Our primary aim was to develop new synthetic compound effective against CQ-resistant and multi-drug
resistant strains. In this paper, we report the synthesis of a new chemotype which prevents Hz formation
by inhibiting free FeIII-PPIX crystallization in FV of malaria parasite. Aiming heme detoxification
procedure as a drug target, we engineered a class of bioactive compounds that exhibits antimalarial
efficacy against CQ sensitive (3D7) and resistant (K1) strains of P. falciparum in vitro and also in vivo
against multi-drug resistant (MDR) parasites in mouse model.
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RESULTS AND DISCUSSION
Synthesis of Hydrazone Scaffolds (1-26). We commenced our study using 2-isopropyl-5-methylphenol
as a starting material for the synthesis of hydrazonophenol by para-formylation of commercially available
2-isopropyl-5-methylphenol, i followed by the reaction with aldehyde, ii with the hydrazine library.
H
CHO
N
H
NaOH/CHCl3
EtOH
MeOH
N
N
R
R
NH2
Na2SO4
HO
HO
HO
i
1-26
ii
1,
2,
3,
4,
5,
6,
7,
8,
9,
10,
11,
12,
13,
14,
R= 2-bromophenyl (81%)
R= 3-bromophenyl (89%)
R= 4-bromophenyl (92%)
R= 3-chlorophenyl (78%)
R= 4-chlorophenyl (87%)
R= 4-cyanophenyl (95%)
R= 2,5-diflurophenyl (71%)
R= pentafluorophenyl (74%)
R= 2,4-diflurophenyl (79%)
R= 3,5-bis(trifluoromethyl)phenyl (72%)
R= 4,5-dihydro-1H-imidazol-2-yl (80%)
R= benzoyl (82%)
R= picolinoyl (77%)
R= 2-pyridyl (81%)
15,
16,
17,
18,
19,
20,
21,
22,
23,
24,
25,
26,
R= 4-pyridyl (78%)
R= 2,2'-pyrimidyl (82%)
R= 4-carboxyphenyl (86%)
R= N-benzyl-N-methyl (89%)
R= benzyloxycarbonyl (75%)
R= 2-(1,3-dioxo-2,3-dihydro-1H-inden-2-yl) (72%)
R= 4-hydroxybenzoyl (74%)
R= 3,5-diflurophenyl (71%)
R= 1,2,4,5-tetraflurophenyl (81%)
R= 3-chloro-4-fluorophenyl (75%)
R= 2,6-dichloro-4-(trifluoromethyl)phenyl (68%)
R= 2-benzo[d]thiazolyl (83%)
Fig. 1. Synthesis of hydrazonophenols. Percentage in parenthesis represents yield.
The aldehyde (ii, 1.12 mmol, 1 eqv.) was suspended in anhydrous methanol (2 ml) and stirred to obtain
a clear solution. Respective hydrazines (1.1 eqv.) were added to the solution in portion wise and the
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reaction mixture was allowed to stir for 6 - 8 hours at room temperature to obtain desirable product
compound (1 - 6, 15 - 26) or at 0º C (7 - 14) (Figure 1). On completion, the solvent was evaporated under
reduced pressure and the crude reaction mixture was purified by silica gel column chromatography or by
crystallization. The structures of compounds (1-26) were confirmed by NMR (1H & 13C) and LC-MS
analysis.
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Identification of High Affinity FeIII-PPIX Interacting Compound. The molecules that can bind FeIII-
PPIX may inhibit hemozoin formation and lead to parasite death. Thus, we assessed the interaction of all
the synthesized hydrazonophenols with FeIII-PPIX in silico. Some molecules out of them showed
significant FeIII-PPIX binding (< -4 kcal mol-1) and among them compound 18 showed efficient FeIII-
PPIX binding affinity with binding energy of -5.50 kcal mol-1 (Table S1, Supporting Information). The
purity of the compound was confirmed by HPLC and X ray-crystallography (CCDC No. 1589463)
(Figure 2A and 2B; Table S2, Supporting Information). In silico studies indicated that compound 18
interacted with the FeIII-PPIX through non-bonding π-π interactions between two pairs of sp2 hybridized
N atom and pyrrole unit of FeIII-PPIX ring (Figure 2C and 2D). The binding property of the
hydrazonophenols was further evaluated by differential spectroscopy 24. Data indicated a strong FeIII-
PPIX binding of the lead molecule 18 with an equilibrium dissociation constant (Kd) of 33.06 ± 0.11 nM
(Figure 2E). The non-bonding π-π interaction can be attributed to high binding energy. The position of
electronegative nitrogen atoms present both in the heterocyclic ring and in the unsaturated carbon-
nitrogen double bond contributed significantly towards the π-interactions. Now, we were interested to
find the chemical motif responsible for such strong FeIII-PPIX binding of compound 18.
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Fig. 2. In silico analysis of the interaction between compound 18 and FeIII-PPIX. A. Compound 18.
B. X-ray Crystallographic structure of compound 18. C. Graphical representation of probable mode of
binding of the compound 18 to hematin ((FeIII-PPIX)2 π-π dimer). β-hematin tetramer was chosen as a
representative unit for Hz. D. Representative image for binding of compound 18 with FeIII-PPIX. E.
Binding of compound 18 with FeIII-PPIX. The differential spectroscopic study was performed using
compound 18 with increasing concentrations (10 nM to 800 nM; i, initial, 10 nM; f, final, 800 nM). Inset
shows 1/∆A versus 1/[S] plot that was used to calculate the dissociation constant (Kd) for the FeIII-PPIX-
18 complex.
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Identification of FeIII-PPIX binding Motif of Compound 18. In order to find out which moiety is
responsible for FeIII-PPIX binding, we trisected the molecule 18 into three parts and evaluated the FeIII-
PPIX binding capacity by differential spectroscopy using individual segment. Compound 18, expectedly
showed concentration-dependent FeIII-PPIX binding (Figure 3A), in contrast, the three parts; the phenol
(Figure 3B), the phenolic aldehyde and (Figure 3C) and the hydrazine (Figure 3D) did not exhibit any
significant FeIII-PPIX interactions. The study reflected that each of the phenol, aldehyde and hydrazine
scaffolds showed incompetent FeIII-PPIX binding. But, the hybridized molecule, the hydrazonophenol
(18) showed proficient binding with the macromolecule, FeIII-PPIX. Thus, it can be concluded that the “-
C=N-NH-” moiety in compound 18 was primarily required for the binding. Next, we assessed the
antiplasmodial activity of compounds in order to correlate with FeIII-PPIX binding.
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Fig. 3. Identification of FeIII-PPIX binding motif of compound 18. Binding analysis of different
structural components of compound 18 with FeIII-PPIX. A. Compound 18; B. Phenolic scaffold; C.
Phenolic aldehyde moiety and D. 2-phenyl hydrazine. All compounds were used at increasing
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concentrations (1 to 50 nM; i, initial, 1 nM; f, final, 50 nM) with fixed concentration of FeIII-PPIX (5
µM).
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In vitro Antiplasmodial Activity of Hydrazonophenols and SAR Studies. We screened the
functionalized hydrazonophenols for their in vitro antimalarial activity against the CQ-sensitive (3D7)
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and resistant (K1) strains of P. falciparum by SYBR green assay . The inhibitory concentration (IC50)
was quantitatively determined from the plot of compound concentrations against the percentage decrease
in SYBR green fluorescence at respective concentrations compared to DMSO control (Table 1). The pKa
and molecular polar surface areas (tPSA) were calculated using ChemBioDraw
Fig. 4. SAR studies for antiplasmodial efficacy of functionalized hydrazonophenols.
Ultra 14.0 (Table 1). Data indicated that the primary scaffold 2-isopropyl-5-methylphenol, i showed no
significant antimalarial activity (IC50 > 100 µM) but the aldehyde, ii derived simplest hydrazonophenol, iii
exerted a higher efficacy with an IC50 around 63 µM against the sensitive strain. The most active scaffolds
8 and 18 were chosen for structure-activity-relationship (SAR) analysis (Figure 4). The study revealed
that molecule with n = 1 and X, Y, Z = -CH (compound 16, 25) was highly non-effective and the –CN
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and -CO2H (electron with drawing functional groups) substitutions also did not show much improvement
(compound 6, 21). However, improved efficacy was found when it was n = 0 with X = N and Y, Z = H
(compound 18) having high pKa value comparable to CQ. But, when Y and/or Z = N, the efficacy of the
compounds was diminished or absent (compound 17, 19). Besides, when n = 0 and X, Y, Z = -CH, highly
electronegative group substitution (R = 4-F-3-Cl (12); 3, 5-di-CF3 (14); 1, 2, 3, 4, 5-penta-F (8)) was
found to be effective against parasite and the compounds showed high clogP value with increased
hydrophilicity. Compounds with fluorine substitution mainly at ortho- and para-positions showed a
greater degree of efficacy against P. falciparum strains (Table 1, entry 7, 9, 12). Among them, the penta-
fluoro derivative, compound 8 showed relatively lower IC50 (2.10 ± 0.27 µM) against 3D7 strain of P.
falciparum. Interestingly, the incorporation of heterocyclic moiety in the hydrazine counterpart resulted in
an increase in antiparasitic activity (Table 1, entry 15, 18, 19, 26). Although, compound 15 exhibited
antiplasmodial activity against CQ-sensitive 3D7 strain at low concentration, the diminished efficacy of
compound 15 against CQ-resistant K1 strain might be attributed to reduced uptake or efflux of the
molecule. Compound 18 containing 2-pyridyl moiety showed highest antiplasmodial activity against CQ
sensitive, 3D7 (IC50 = 1.49 ± 0.16 μM) and CQ resistant, K1 (IC50 = 2.56 ± 0.85 μM) strains. Compound
19 with a nitrogen atom substitution at para- position of the ring showed a decreased activity. However,
the replacement of pyridine moiety with a pyrimidine moiety (compound 20) resulted in loss of
antimalarial activity of the respective hydrazonophenol.
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The SAR study delineated the role of “-C=N-NH-” moiety in FeIII-PPIX binding which was thereby
correlated with antiplasmodial efficacy of hydrazonophenols. The calculated tPSA values (Table 1)
pointed towards high probability of the compounds to permeate the cell-membrane. Compounds 8 or 18
showed similar activities against 3D7 strain. However, the advantage of compound 18 with respect to
compound 8 was that the compound 18 showed similar behavior in sensitive and resistant strains of the
parasite. It was evident that structure of compound 18 provided a good spatial advantage for a suitable
non-bonding weak π-interaction between compound 18 and the two pyrrole moieties, as was deduced by
the in silico study. The participation of the 2-pyridine might stabilized the protonated form of compound
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18 probably through an intramolecular hydrogen bond. Previous studies showed that drug molecules with
planar conformation form heme-drug complex through weak π−π interactions 26–28. The absence of the
nitrogen atom in the structural analogue of 18 resulted in the disappearance of interactions with the FeIII-
PPIX unit that supported the experimental findings (Figure S1 and S2, Supporting Information). It
depicted the positional significance of nitrogen atom in the heterocyclic ring. The data showed moderate
correlation between the dissociation constant (Kd) for FeIII-PPIX binding of hydrazonophenol with in vitro
antiplasmodial activity (Table S3, Supporting Information). Moreover, because of the high pKa of
compound 18 (Table 1), it may get accumulated inside the acidic FV like CQ 29. Therefore, we were
interested to trace the cellular localization of compound 18 in P. falciparum.
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Table 1. In vitro screening of antimalarial activity of compounds 1 - 26.
IC50 (µM)
Mean ± SEM
3D7 strain
IC50 (µM)
Mean ± SEM
K1 strain
a
a
tPSAb
Entry
R
pKa
>100
44.62
9.09
NA
1
Br
Br
>100
NA
59.66 ± 3.42
25.11 ± 0.17
>100
44.62
44.62
44.62
9.91
9.91
9.91
2
3
4
Br
>100
NA
Cl
9.91
9.90
>100
>100
Cl
44.62
67.48
5
6
26.53 ± 0.66
10.17 ± 0.04
CN
F
3.07 ± 0.12
44.62
9.91
7
F
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F
F
F
F
44.62
44.62
2.10 ± 0.27
14.88 ± 0.83
10.64 ± 0.11
9.91
9.92
8
9
F
F
6.96 ± 0.60
>100
F
9
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18
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F
42.99 ± 0.40
12.88 ± 0.17
11.84 ± 0.09
44.62
44.62
44.62
9.91
9.91
9.91
10
11
12
F
F
F
F
10.77 ± 0.97
2.17 ± 0.21
F
Cl
F
Cl
Cl
CF3
44.62
>100
>100
9.89
13
CF3
CF3
44.62
69.01
61.69
9.91
9.87
>100
>100
>100
NA
14
15
H
N
3.46 ± 0.14
N
O
O
33.55 ± 1.93
>100
9.86
9.86
16
17
N
74.05
>100
1.49 ± 0.15
56.98
9.89
2.56 ± 0.85
18
N
N
12.30 ± 0.17
10.55 ± 0.39
56.98
9.90
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N
N
NA
NA
69.34
81.92
9.88
9.90
20
21
OH
O
41.68 ± 3.10
28.93 ± 4.63
73.94 ± 8.21
24.76 ± 0.70
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N
35.83
70.92
9.85
9.85
22
23
O
O
25.26 ± 2.87
24.54 ± 0.06
N
NA
69.97
81.92
9.72
9.87
>100
24
25
O
OH
50.45 ± 3.34
>100
S
N
31.29 ± 0.81
38.16 ± 6.53
63.13 ± 5.21
56.98
58.61
9.83
26
iii
10.05
NHNH2
n.d.
a In vitro antimalarial activity was presented as the IC50 (Mean ± SEM., wherever applicable) and values
were the average of at least two independent assays carried out in triplicate. Compounds were incubated
with parasites for 48 hours. b tPSA and pKa values were calculated using ChemBioDraw Ultra 14.0. NA:
Not active. n.d.: not determined.
Cellular Localization of Fluorophore Tagged Compound 18. To locate compound 18 inside the
parasite, we designed and synthesized a fluorophore tagged version of 18, compound 28 (Figure 5A).
Uranine, a well-known fluorophore was coupled with compound 18 via EDC-mediated coupling reaction
30. Parasite culture was incubated with LysoTracker (to stain FV) and Hoechst 33342 (to stain the
nucleus) 31. A set of non-infected RBCs incubated with compound 28 was used as control. Very selective
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uptake of the compound 28 was evident from its co-localization with LysoTracker Red, which localized
specifically in parasite FV, near Hz, the black pigment found only in FV of parasitized RBCs (Figure 5B).
SYBR green assay with compound 28 indicated no significant alteration of antiplasmodial activity of
compound 18 after fluorophore-tagging (IC50, 3D7 = 1.50 ± 0.06 µM; IC50, K1 = 2.59 ± 0.02 µM).
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Fig. 5. Confocal imaging of fluorophore labelled compound 18 (compound 28) A. Addition of
fluorophore signature to the compound 18 through two-step synthesis of oxygen coupled ester via DCC-
coupling. B. Live cell imaging of P. falciparum 3D7 cells after incubation with compound 28
(flurophore-tagged 18, green color, 5 µM), LysoTracker (magenta color) and Hoescht 33342 (blue color).
Scale bar measures 5 µm. Merge 1: LysoTracker + 28; Merge 2: LysoTracker + 28 + Hoescht. Merge 3:
LysoTracker + 28 + Hoescht + Differential Interference Contrast (DIC).
Live-cell confocal imaging was done to track the intracellular entrapment of the molecule. As it was
observed earlier that the hydrazone moiety and the nitrogen atom (in heterocyclic ring) was essential for
free heme binding, the confocal imaging study now corroborates to the phenomenon. ‘Merge 3’ segment
confirmed the co-localization of fluorophore tagged compound 18 (compound 28) with LysoTracker, a
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dye that accumulates inside FV, the acidic organelle of the parasite (Figure 5B). The biochemical reason
behind this might be the high pKa (~8) of compound 28 that caused its entrapment inside the FV. The
distributed pattern of LysoTracker and compound 28 might be due to the disruption of FV because of the
accumulation of free FeIII-PPIX that damaged FV. However, the leakage of FV might occur upon
illumination of live parasite under microscope 32.
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Restriction of FeIII-PPIX Crystallization (Hemozoin) by Compound 18. The localization of the
flurophore tagged version of compound 18 in the acidic vacuole of the parasite prompted us to investigate
its effect on free heme detoxification system in FV. Parasite was exposed to compound 18 at different
concentrations and the outcome was evaluated in terms of change in pH of the FV using an acidotropic
probe LysoSensor Green. The treated parasites were incubated with LysoSensor Green and fluorescence
intensity was measured. A gradual decrease in fluorescence intensity with increasing concentrations (10 -
100 µM) of compound 18 indicated the change in pH of FV due to the basic nature of the compound
(Figure 6A). Next, we incubated parasite with compound 18 and measured the Hz content inside the
parasite to follow the effect on hemozoin formation. The data clearly indicated that compound 18
concentration-dependently inhibited Hz formation in P. falciparum (Figure 6B). DMSO was used as
vehicle control. The stability of compound 18 was also assessed at food vacuolar pH of 5.2 as well as at
physiological pH of 7.4 by mass spectroscopy. This compound was found to be stable in both pH (Figure
S3, Supporting Information). The inhibitory effect of the compound in vitro on heme crystallization was
further confirmed by following inhibition of parasite lysate-mediated Hz formation at pH 5.2 33; a cell-
free system established by mimicking conditions like FV. The data showed that compound 18
concentration-dependently inhibited heme crystallization (Figure 6C). Moreover, the accumulation of free
FeIII-PPIX in solution as a consequence of inhibition of Hz formation by compound 18 was also
measured as described 34. Compound 18 concentration-dependently inhibited FeIII-PPIX crystallization
thereby leading to free FeIII-PPIX accumulation (Figure 6C).
LysoSensor Green is a pH sensitive fluorescent probe and gets accumulated in acidic compartments
where it becomes fluorescent upon protonation. The fluorescence intensity exerted by this probe increases
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with the lowering of pH of the solution. A gradual decrease of fluorescence intensity with increasing
concentration of compound 18 indicated a relative elevation of pH of the acidic organelle (Figure 6A).
This phenomenon can be attributed to the high pKa value of the compound. One of the major functions of
FV in parasites is the detoxification of pro-oxidant and toxic free FeIII-PPIX formed after hemoglobin
digestion by converting it into an inert polymer Hz and food vacuolar localization of compound 18
indicated its capability to affect free heme detoxification process.
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Fig. 6. Effect of compound 18 on the pH of parasite food vacuole (FV) and hemozoin formation. A.
Change of pH of parasite FV in presence of compound 18 (10 – 100 μM) measured by LysoSensor Green
DND-189. iRBC: infected RBC; CQ was used as a positive control. B. Inhibition of Hz formation in P.
falciparum by compound 18 (1 – 50 μM) in culture. Data represent Mean ± SEM. Data were collected
from at least two independent experiments; (**, P ≤ 0.01 compared to control). C. Inhibition of parasite
lysate - mediated Hz formation and quantification of free FeIII-PPIX in presence of compound 18 (5 - 100
µM). Data represent Mean ± SEM. D. Microscopic images of P. falciparum treated with compound 18 (5
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μM) and DMSO (vehicle control); Black arrow indicates the Hz formed inside healthy parasite and blue
arrow indicates the dead parasite. E. Visualization of Hz of parasite lysate collected from control and
treated (40 hours) parasite.
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Arrest of Intra-erythrocytic Stage Progression of P. falciparum. In vitro inhibition of Hz formation
and its impact on parasite growth were further validated by microscopic studies. To follow the
progression of stages of parasites in presence of compound 18, we treated the synchronized ring stage of
P. falciparum 3D7 with the compound. The morphology of parasite along with percentage parasitemia
was assessed at an interval of 10 hours. Microscopic view identified pyknotic morphology of parasite
after 20 hours of treatment with compound 18. The compound inhibited the parasite growth and resulted
in its death as was evident from its abnormal morphology. After 40 hours, untreated parasite grew to
become schizont while compound 18 treatment resulted in parasite death marked by the single blue spots
(Figure 6D). After 40 hours of treatment, very less amount of Hz was present in treated parasite lysate
compared to the untreated set. This confirmed that compound 18 effectively killed the parasite whereas
parasites in control set were able to grow and form Hz. The untreated lysate was red in color while the
treated lysate was found dark in color due to the presence of excess Hz (Figure 6E).The irregular and
abnormal shape of parasite nucleus was detected under the microscope. The inhibition of Hz
crystallization in presence of compound 18 causes the accumulation of free heme, which in turn generates
oxidative insult to the parasite leading to death.
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Non-promiscuous Nature of Compound 18. Compound 18 was assessed for its selectivity of action
and cellular toxicity. Keeping in knowledge, the potential safety issues of pan-assay interference
compounds (PAINs) in the arena of anti-infective drug innovation we performed assays with several non-
related drug targets 35. Still, even with promiscuous nature, compounds with exceptional effectiveness
should be cogitated for the invention of new therapeutic 36. We followed Soret spectroscopy of
hemoglobin (Hb) at physiological pH of 7.4 in presence of increasing concentrations of compound 18 to
follow the interaction of the compound with the heme (FeII-PPIX) present in hemoglobin. Data showed
no change in absorbance maxima of Hb which indicated that compound 18 did not interact with FeII-PPIX
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of hemoglobin (Figure 7A). The data also confirmed the specific interaction of compound 18 only with
FeIII-PPIX but not with FeII-PPIX in Hb. The hemolytic activity of Compound 18 (100 µM) was also
studied. Compound 18 did not exhibit any significant hemolytic activity when it was incubated with
human RBC for 12 hours (Figure 7B). Saponin (0.05%), which causes lysis of RBC, was used as positive
control. Non-promiscuous nature of the compound 18 was further verified by assessing its effects on non-
related proteins and cell system. Therefore, DNase I, alkaline phosphatase (ALP) and macrophage
migration inhibitory factor (MIF) tautomerase activities in presence of compound 18 were examined. The
inhibitory activity of compound 18 against DNase I was checked using a plasmid DNA (pBR322). Data
showed that DNase I completely cleaved plasmid DNA alone and in presence of compound 18 (0.1 – 20
µM) indicating compound 18 did not affect the DNase I activity (Figure 7C). However, EDTA which was
used as a positive control prevented the activity of DNase I (Figure 7C). Enzymatic activity of ALP
(Figure 7D) and tautomerase activity MIF (Figure 7E) were also remained unaltered in presence of
different concentrations compound 18. Slight decrease in optical density in set of positive control of ALP
may be attributed to the effect of DMSO. Effect of compound 18 on cell viability was assessed by 3-(4, 5-
dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction assay (Figure 7F). Negligible
loss of cell viability was observed as there was not much alteration seen in the amount of purple colored
formazan formed due to reduction of MTT by the functional dehydrogenases within the treated cells
compared with the control set. This data was well supported by the results obtained from flow cytometric
analysis (FACS) where insignificant apoptotic cell death was detected (Figure 7G).
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Presence of almost equal proportion of HepG2 cells in Q4 quadrant (apoptotic compartment) revealed
minimal toxicity of compound 18. Negligible effect of the compound on viability of HepG2 cell was
further evident from MTT reduction assay indicating a high IC50 value. The IC50 value was obtained from
the plot of percentage of viable cell against the concentration of treated compound. Intensity of formazan
absorbance was directly proportional to the viable cell density. The assessment of selectivity index using
mammalian HepG2 cell line indicated a high selectivity index (S.I. = 150.48) of the compound (Figure
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7H). Thus, the data obtained from these experiments established the non-promiscuous nature and
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Fig. 7. Evaluation of non-promiscuous nature of compound 18. A. Interaction of compound 18 with
hemoglobin (Hb). Compound did not bind to Hb (pH 7.4) Hb spectra were recorded with increasing
concentrations (10-50 µM) of compound 18. B. Non-hemolytic property of compound 18. Soret spectra
were recorded from the supernatant of RBCs exposed to compound 18 (100 µM) for 12 hours. Saponin
(0.05%) was used as positive control. C. Effect of compound 18 on DNase I activity; compound 18 (0.1 –
20 µM) was incubated with DNA and DNase I. Experimental sets were described along with lane number
of agarose gel. EDTA (20 mM) was used as positive control and DMSO was used as control blank. D.
Effect of compound 18 (0.1 – 20 µM) on alkaline phosphatase activity; Phosphatase inhibitory cocktail
(PIC) was used as positive control; data represent Mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***≤ P 0.001; ns:
not significant (P > 0.05). E. MIF tautomerase activity with increasing concentrations (1-50 µM) of
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compound 18. Ellagic acid (10 µM) was used as positive control. F. MTT reduction assay to follow the
effect of compound 18 on cell-viability. Experiment was performed in triplicate. Data represent Mean ±
SEM.; **P ≤ 0.01; ns: not significant (P > 0.05). G. FACS analysis to study HepG2 cell death in presence
of different concentrations (10 - 50 µM) of compound 18; values in the Q2 and Q4 quadrants depict
percentage of cells showing late apoptotic-necrotic and apoptotic cell death respectively; dot-plot
representations of FACS analysis are representative of a single experiment out of the independent
experiments performed in triplicate. H. Selectivity Index (S.I.) of compound 18. Details of the
methodology are described under materials and methods section in Supporting Information.
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In vivo Antimalarial Activity of Compound 18 against P. yoelii and P. berghei ANKA. In Peter’s four
day test, Swiss albino mice infected with lethal strains were treated with compound 18 intraperitoneally
once a day for four subsequent days, commencing four hours after the administration of parasitized
erythrocytes intraperitoneally. Mice were treated with compound 18 at different doses of 25 mg kg-1, 50
mg kg-1, 75 mg kg-1 and 100 mg kg-1 body weight wherein each of the experimental set contained 5 mice.
DMSO (vehicle) and antimalarial drug α/β-arteether were taken as control and positive control
respectively. The suppression of percentage parasitemia clearly indicated the antimalarial efficacy of the
compound 18 at a dose of 100 mg kg-1 which showed 100% suppression of parasitemia on Day 5 with
mean survival days (MSD) of 15.2 for the animals (Table 2). There was a significant drop in the parasite
load in the 100 mg kg-1 treatment set which was almost comparable to the α/β-arteether. Compound 18
showed almost complete suppression of the parasitemia in vivo and exerted improved survival rates with
increasing doses (Table 2 and Figure 8). Only two doses (50 mg kg-1 and 100 mg kg-1) of compound 18
were also selected to check the efficacy in the mice infected with P. berghei ANKA that causes cerebral
malaria 37. The activity of 18 against P. berghei was found to be similar to that of P. yoelii indicating
towards the efficacy and broad spectrum antimalarial activity of 18 against multiple species of
Plasmodium. Drug-likeliness of compound 18 was measured in silico using ‘Lipinski’s rule of five’
(Table S4, Supporting Information).
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Fig. 8. Compound 18 increases the survival rate of mice infected with MDR strain (P. yoelii).
Percentage survival of mice treated with different doses (0-100 mg kg-1 body weight) of compound 18
followed through the days after infection. Vehicle treated mice were taken as control. Five animals were
used for each set of dose. Mice were euthanized when on parasitemia reaching 60% in order to avoid
death otherwise.
Decrease in the parasitemia in animal showed the in vivo efficacy of the molecule. Moreover, the
molecule was found to be active against different species of P. falciparum. However the recurrence of
parasitemia in mice caused death after day 15 might be due to low bio-availability, low membrane
permeability or the difference in efficacy of the compound 18 against human and rodent malaria parasite.
Table 2. Peter’s 4-day test for parasitemia suppression by compound 18.
Parasitemia
Compound
Dose (mg kg-1)
suppression
MSDb
Survivalc
(%)a
Control
0.0 (P. yoelii)
0.0
0.0
4.3
4.0
0/5
0/5
5/5
5/5
0.0 (P. berghei)
α/β Arteether
4 × 25 (P. yoelii)
100.0
100.0
n.d.
n.d.
4 × 25 (P. berghei)
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Compound 18
4 × 25 (P. yoelii)
4 × 25 (P. berghei)
4 × 50 (P. yoelii)
4 × 50 (P. berghei)
4 × 75 (P. yoelii)
4 × 75 (P. berghei)
4 × 100 (P. yoelii)
4 × 100 (P. berghei)
59.0
n.d.
7.8
0/5
-
n.d.
78.1
79.8
81.8
n.d.
10.6
11.8
12.4
n.d.
1/5
1/5
3/5
-
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100.0
97.5
15.2
16.6
4/5
3/5
aParasitemia was determined on 5th day after the completion of treatment. Percent parasitemia suppression
was calculated as follows: [(C – P) / C] × 100, where C is the parasitemia in the control group and P is
the parasitemia in the treated group. bMean survival time in days (MSD). cNumber of healthy mice on day
15. n.d.: not determined.
Conclusions
Compound 18, a nanomolar binder of ferriprotoporphyrin IX (heme) was developed by rational
structure-based designing, which inhibited hemozoin formation targeting the food vacuole of P.
falciparum and offered antimalarial activity against drug-resistant parasite in vitro and in vivo. The
efficacy of the compound against different Plasmodium species further indicated towards the potential
activity of the compound against other species that cause malaria in human. The activity of the compound
at micromolar concentration in in vitro experiments might be because of its low solubility or
permeability; however, the target of the molecule was validated by confirmatory experiments. The scope
of this manuscript was the synthesis and validation of an active pharmacophore against malaria. The
active pharmacophore identified needs further optimization of the chemical scaffold which may improve
its antimalarial activity and bio-availability. Thus, compound 18 represented a new antimalarial
chemotype against resistant malaria.
Materials and methods
Materials and methods are descripted elaborately in the associated Supporting Information.
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All mice were received from the animal house of CSIR-Indian Institute of Chemical Biology, Kolkata.
We strictly followed procedures for handling animal and experiments in compliance with the regulations
of Institutional Animal Ethics Committee (IAEC) and Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA, Permit Number: 147/1999/CPCSEA).
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Conflict of Interests
The authors declare that they have no conflicts of interest with the contents of this article. The authors
also declare no competing financial interest.
ASSOCIATED CONTENT
Supporting Information
Supporting information includes general information, analysis of FeIII-PPIX binding with
hydrazonophenols, single crystal X-Ray structure of compound 18, macro-molecular interaction study of
structural analogues of compound 18, correlation between IC50 and equilibrium dissociation constant,
stability of compound 18, stability of compound 18 at food vacuolar low pH, evaluation of ADME
properties, materials and methods. It also includes analytical data, H and 13C NMR spectrum for
1
compounds ii, 1-20, 27 and 28.
AUTHOR INFORMATION
Corresponding Author e-mail: ubandyo_1964@yahoo.com, udayb@iicb.res.in
Division of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, 4, Raja
S. C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India. Tel: 91-33-24995735, Fax: 91-33-
4730284. ORCID: Uday Bandyopadhyay: 0000-0002-5928-6790.
ACKNOWLEDGMENT
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We gratefully acknowledge University Grant Commission for providing the fellowship to S. J. Saha.
We also acknowledge Council of Scientific and Industrial Research, India for providing fund (BEnD,
BSC0206) and DST (J. C. Bose Fellowship, SB/S2/JCB-54/2014), India. We are thankful to Saunak
Bhattacharya, technical officer, CSIR–IICB, Kolkata, for his support in the confocal studies, Ramdhan
Maji, Senior Technical Officer, CSIR–IICB and Kolkata for his guidance in HPLC analysis, Y. Singh for
his cordial support in wet-lab experiments and A. Mishra for his assistance during the crystallographic
data analysis.
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REFERENCES
(1)
(2)
WHO | World Malaria Report 2016. WHO 2017.
Davis, T. M. E.; Hung, T.-Y.; Sim, I.-K.; Karunajeewa, H. A.; Ilett, K. F. Piperaquine: A
Resurgent Antimalarial Drug. Drugs 2005, 65 (1), 75–87.
(3)
(4)
WHO | World Malaria Report 2017. WHO 2018.
Lu, F.; Culleton, R.; Zhang, M.; Ramaprasad, A.; von Seidlein, L.; Zhou, H.; Zhu, G.; Tang, J.;
Liu, Y.; Wang, W.; Cao, Y; Xu, S.; Gu, Y.; Li, J.; Zhang, C.; Gao, Q.; Menard, D.; Pain, A.;
Yang, H.; Zhang, Q; Cao, J. Emergence of Indigenous Artemisinin-Resistant Plasmodium
Falciparum in Africa. N. Engl. J. Med. 2017, 376 (10), 991–993. DOI: 10.1056/NEJMc1612765.
Lin, J. T.; Juliano, J. J.; Wongsrichanalai, C. Drug-Resistant Malaria: The Era of ACT. Curr.
Infect. Dis. Rep. 2010, 12 (3), 165–173. DOI: 10.1007/s11908-010-0099-y.
(5)
(6)
Verdier, F.; Le Bras, J.; Clavier, F.; Hatin, I.; Blayo, M. C. Chloroquine Uptake by Plasmodium
Falciparum-Infected Human Erythrocytes during in Vitro Culture and Its Relationship to
Chloroquine Resistance. Antimicrob. Agents Chemother. 1985, 27 (4), 561–564. DOI:
10.1128/AAC.27.4.561.
(7)
Fitch, C. D.; Chevli, R.; Banyal, H. S.; Phillips, G.; Pfaller, M. A.; Krogstad, D. J. Lysis of
Plasmodium Falciparum by Ferriprotoporphyrin IX and a Chloroquine-Ferriprotoporphyrin IX
Complex. Antimicrob. Agents Chemother. 1982, 21 (5), 819–822. DOI: 10.1128/AAC.21.5.819.
23
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Page 24 of 30
1
2
3
4
5
6
7
8
(8)
(9)
Pal, C.; Kundu, M. K.; Bandyopadhyay, U.; Adhikari, S. Synthesis of Novel Heme-Interacting
Acridone Derivatives to Prevent Free Heme-Mediated Protein Oxidation and Degradation.
Bioorganic Med. Chem. Lett. 2011, 21 (12), 3563–3567. DOI: 10.1016/j.bmcl.2011.04.127.
Pal, C.; Bandyopadhyay, U. Redox-Active Antiparasitic Drugs. Antioxid. Redox Signal. 2012, 17
(4), 555–582. DOI: 10.1089/ars.2011.4436.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(10) Dey, S.; Mazumder, S.; Siddiqui, A. A.; Iqbal, M. S.; Banerjee, C.; Sarkar, S.; De, R.; Goyal, M.;
Bindu, S.; Bandyopadhyay, U. Association of Heme Oxygenase 1 with the Restoration of Liver
Function after Damage in Murine Malaria by Plasmodium Yoelii. Infect. Immun. 2014, 82 (8),
3113–3126. DOI: 10.1128/IAI.01598-14.
(11) Egan, T. J.; Mavuso, W. W.; Ncokazi, K. K. The Mechanism of β-Hematin Formation in Acetate
Solution. Parallels between Hemozoin Formation and Biomineralization Processes. Biochemistry
2001, 40 (1), 204–213. DOI: 10.1021/bi0013501.
(12) Fitch, C. D.; Kanjananggulpan, P. The State of Ferriprotoporphyrin IX in Malaria Pigment. J. Biol.
Chem. 1987, 262 (32), 15552–15555.
(13) Fidock, D. A.; Nomura, T.; Talley, A. K.; Cooper, R. A.; Dzekunov, S. M.; Ferdig, M. T.; Ursos,
L. M. B.; Sidhu, A. B. S.; Naude, B.; Deitsch, K. W.; Su, X. Z.; Wootton, J. C.; Roepe, P. D.;
Wellems, T. E. Mutations in the P. Falciparum Digestive Vacuole Transmembrane Protein PfCRT
and Evidence for Their Role in Chloroquine Resistance. Mol. Cell 2000, 6 (4), 861–871. DOI:
10.1016/s1097-2765(05)00077-8.
(14) Kappe, S. H. I.; Vaughan, A. M.; Boddey, J. A.; Cowman, A. F. That Was Then But This Is Now:
Malaria Research in the Time of an Eradication Agenda. Science (80-. ). 2010, 328 (5980), 862–
866. DOI: 10.1126/science.1184785.
(15) Ocheskey, J. A.; Harpstrite, S. E.; Oksman, A.; Goldberg, D. E.; Sharma, V. Metalloantimalarials:
Synthesis and Characterization of a Novel Agent Possessing Activity against Plasmodium
Falciparum. Chem. Commun. 2005, 0 (12), 1622. DOI: 10.1039/b415771k.
(16) Harpstrite, S. E.; Collins, S. D.; Oksman, A.; Goldberg, D. E.; Sharma, V. Synthesis,
24
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1
2
3
4
Characterization, and Antimalarial Activity of Novel Schiff-Base-Phenol and Naphthalene-Amine
Ligands. Med. Chem. 2008, 4 (4), 392–395. DOI: 10.2174/157340608784872280.
5
6
7
8
(17) Sharma, M.; Chauhan, K.; Srivastava, R. K.; Singh, S. V.; Srivastava, K.; Saxena, J. K.; Puri, S.
K.; Chauhan, P. M. S. Design and Synthesis of a New Class of 4-Aminoquinolinyl- and 9-
Anilinoacridinyl Schiff Base Hydrazones as Potent Antimalarial Agents. Chem. Biol. Drug Des.
2014, 84 (2), 175–181. DOI: 10.1111/cbdd.12289.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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46
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48
49
50
51
52
53
54
55
56
57
58
59
60
(18) Walcourt, A.; Loyevsky, M.; Lovejoy, D. B.; Gordeuk, V. R.; Richardson, D. R. Novel
Aroylhydrazone and Thiosemicarbazone Iron Chelators with Anti-Malarial Activity against
Chloroquine-Resistant and -Sensitive Parasites. Int. J. Biochem. Cell Biol. 2004, 36 (3), 401–407.
DOI: 10.1016/S1357-2725(03)00248-6.
(19) Builders, M. .; Alemika, T.; Aguiyi, J. . Antimalarial Activity and Isolation of Phenolic Compound
from Parkia Biglobosa. J. Pharm. Biol. Sci. 2014, 9 (3), 78–85. DOI: 10.9790/3008-09347885.
(20) Tepe, B.; Daferera, D.; Sökmen, M.; Polissiou, M.; Sökmen, A. In Vitro Antimicrobial and
Antioxidant Activities of the Essential Oils and Various Extracts of Thymus Eigii M. Zohary et
P.H. Davis. J. Agric. Food Chem. 2004, 52 (5), 1132–1137. DOI: 10.1021/jf035094l.
(21) Podobnik, B.; Stojan, J.; Lah, L.; Kraševec, N.; Seliškar, M.; Rižner, T. L.; Rozman, D.; Komel,
R. CYP53A15 of Cochliobolus Lunatus, a Target for Natural Antifungal Compounds. J. Med.
Chem. 2008, 51 (12), 3480–3486. DOI: 10.1021/jm800030e.
(22) Aeschbach, R.; Löliger, J.; Scott, B. C.; Murcia, A.; Butler, J.; Halliwell, B.; Aruoma, O. I.
Antioxidant Actions of Thymol, Carvacrol, 6-Gingerol, Zingerone and Hydroxytyrosol. Food
Chem. Toxicol. 1994, 32 (1), 31–36. DOI: 10.1016/0278-6915(84)90033-4.
(23) Braga, P. C.; Dal Sasso, M.; Culici, M.; Bianchi, T.; Bordoni, L.; Marabini, L. Anti-Inflammatory
Activity of Thymol: Inhibitory Effect on the Release of Human Neutrophil Elastase.
Pharmacology 2006, 77 (3), 130–136. DOI: 10.1159/000093790.
(24) Ajdačić, V.; Senerovic, L.; Vranić, M.; Pekmezovic, M.; Arsic-Arsnijevic, V.; Veselinovic, A.;
Veselinovic, J.; Šolaja, B. A.; Nikodinovic-Runic, J.; Opsenica, I. M. Synthesis and Evaluation of
25
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5
6
Thiophene-Based Guanylhydrazones (Iminoguanidines) Efficient against Panel of Voriconazole-
Resistant Fungal Isolates. Bioorg. Med. Chem. 2016, 24 (6), 1277–1291. DOI:
10.1016/j.bmc.2016.01.058.
7
8
9
(25) Choubey, V.; Guha, M.; Maity, P.; Kumar, S.; Raghunandan, R.; Maulik, P. R.; Mitra, K.; Halder,
U. C.; Bandyopadhyay, U. Molecular Characterization and Localization of Plasmodium
Falciparum Choline Kinase. Biochim. Biophys. Acta - Gen. Subj. 2006, 1760 (7), 1027–1038.
DOI: 10.1016/j.bbagen.2006.03.003.
10
11
12
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14
15
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52
53
54
55
56
57
58
59
60
(26) Wicht, K. J.; Combrinck, J. M.; Smith, P. J.; Hunter, R.; Egan, T. J. Identification and SAR
Evaluation of Hemozoin-Inhibiting Benzamides Active against Plasmodium Falciparum.
2016,59(13), 6512-30. DOI: 10.1021/acs.jmedchem.6b00719.
(27) Okombo, J.; Singh, K.; Mayoka, G.; Ndubi, F.; Barnard, L.; Njogu, P. M.; Njoroge, M.; Gibhard,
L.; Brunschwig, C.; Vargas, M.; Keiser, J; Egan, T. J; Chibale, K. Antischistosomal Activity of
Pyrido[1,2-A]benzimidazole Derivatives and Correlation with Inhibition of β-Hematin Formation.
2017, 3(6), 411-420. DOI: 10.1021/acsinfecdis.6b00205.
(28) De Villiers, K. A.; Gildenhuys, J.; Le Roex, T. Iron(III) Protoporphyrin IX Complexes of the
Antimalarial Cinchona Alkaloids Quinine and Quinidine. 2012, 7 (4), 666–671. DOI:
10.1021/cb200528z.
(29) Yayon, A.; Cabantchik, Z. I.; Ginsburg, H. Identification of the Acidic Compartment of
Plasmodium Falciparum-Infected Human Erythrocytes as the Target of the Antimalarial Drug
Chloroquine. EMBO J. 1984, 3 (11), 2695–2700.
(30) Pal, C.; Bindu, S.; Dey, S.; Alam, A.; Goyal, M.; Iqbal, M. S.; Sarkar, S.; Kumar, R.; Halder, K.
K.; Debnath, M. C.; Adhikari, S.; Bandyopadhyay, U. Tryptamine-Gallic Acid Hybrid Prevents
Non-Steroidal Anti-Inflammatory Drug-Induced Gastropathy: Correction of Mitochondrial
Dysfunction and Inhibition of Apoptosis in Gastric Mucosal Cells. J. Biol. Chem. 2012, 287 (5),
3495–3509. DOI: 10.1074/jbc.M111.307199.
(31) Iqbal, M. S.; Siddiqui, A. A.; Banerjee, C.; Nag, S.; Mazumder, S.; De, R.; Saha, S. J.; Karri, S.
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K.; Bandyopadhyay, U. Detection of Retromer Assembly in Plasmodium Falciparum by
Immunosensing Coupled to Surface Plasmon Resonance. Biochim. Biophys. Acta - Proteins
Proteomics 2018, 1866 (5–6). DOI: 10.1016/j.pep.2015.12.004.
7
8
9
(32) Wissing, F.; Sanchez, C. P.; Rohrbach, P.; Ricken, S.; Lanzer, M. Illumination of the Malaria
Parasite Plasmodium Falciparum Alters Intracellular pH. Implications for Live Cell Imaging. J.
Biol. Chem. 2002, 277 (40), 37747–37755. DOI: 10.1074/jbc.M204845200.
10
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60
(33) Ncokazi, K. K.; Egan, T. J. A Colorimetric High-Throughput β-Hematin Inhibition Screening
Assay for Use in the Search for Antimalarial Compounds. Anal. Biochem. 2005, 338 (2), 306–319.
DOI: 10.1016/j.ab.2004.11.022.
(34) Fong, K. Y.; Sandlin, R. D.; Wright, D. W. Identification of β-Hematin Inhibitors in the MMV
Malaria Box. Int. J. Parasitol. Drugs Drug Resist. 2015,
5
(3), 84–91. DOI:
10.1016/j.ijpddr.2015.05.003.
(35) Panaceas, I. M. The Ecstasy and Agony of Assay Interference Compounds. 2017, 3 (3), 143–
147DOI: 10.1021/acscentsci.7b00069.
(36) Arrowsmith, C. H.; Audia, J. E.; Austin, C.; Baell, J.; Bennett, J.; Blagg, J.; Bountra, C.; Brennan,
P. E.; Brown, P. J.; Bunnage, M. E.; Buser-Doepner, C.; Campbell, R. M.; Carter, A. J.; Cohen, P.;
Copeland, R. A.; Cravatt, B.; Dahlin, J. L.; Dhanak, D.; Edwards, A. M.; Frederiksen, M.; Frye, S.
V.; Gray, N.; Grimshaw, C. E.; Hepworth, D.; Howe, T.; Huber, K. V.; Jin, J.; Knapp, S.; Kotz J.
D.; Kruger, R. G.; Lowe, D.; Mader, M. M.; Marsden, B.; Mueller-Fahrnow, A.; Müller, S.;
O'Hagan, R. C.; Overington, J. P.; Owen, D. R.; Rosenberg, S. H.; Roth, B.; Ross, R.; Schapira,
M.; Schreiber, S. L.; Shoichet, B.; Sundström, M.; Superti-Furga, G.; Taunton, J.; Toledo-
Sherman, L.; Walpole, C.; Walters, M. A.; Willson, T. M.; Workman, P.; Young, R. N., Zuercher,
W. The Promise and Peril of Chemical Probes. Nat. Chem. Biol. 2015, 11 (8), 536–541. DOI:
10.1038/nchembio.1867.
(37) Neill, A. L.; Hunt, N. H. Pathology of Fatal and Resolving Plasmodium Berghei Cerebral Malaria
in Mice. Parasitology 1992, 105 ( Pt 2), 165–175. DOI: 10.1017/S0031182000074072.
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