A Redox-Active Fluorescent pH Indicator for Detecting Plasmodium falciparum Strains with Reduced Responsiveness to Quinoline Antimalarial Drugs

Article abstract of DOI:10.1021/acsinfecdis.5b00141

Mutational changes in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) have been associated with differential responses to a wide spectrum of biologically active compounds including current and former quinoline and quinoline-like antimalarial drugs. PfCRT confers altered drug responsiveness by acting as a transport system, expelling drugs from the parasite's digestive vacuole where these drugs exert, at least part of, their antiplasmodial activity. To preserve the efficacy of these invaluable drugs, novel functional tools are required for epidemiological surveys of parasite strains carrying mutant PfCRT variants and for drug development programs aimed at inhibiting or circumventing the action of PfCRT. Here we report the synthesis and characterization of a pH-sensitive fluorescent chloroquine analogue consisting of 7-chloro-N-{2-[(propan-2-yl)amino]ethyl}quinolin-4-amine functionalized with the fluorochrome 7-nitrobenzofurazan (NBD) (henceforth termed Fluo-CQ). In the parasite, Fluo-CQ accumulates in the digestive vacuole, giving rise to a strong fluorescence signal but only in parasites carrying the wild type PfCRT. In parasites carrying the mutant PfCRT, Fluo-CQ does not accumulate. The differential handling of the fluorescent probe, combined with live cell imaging, provides a diagnostic tool for quick detection of those P. falciparum strains that carry a PfCRT variant associated with altered responsiveness to quinoline and quinoline-like antimalarial drugs. In contrast to the accumulation studies, chloroquine (CQ)-resistant parasites were observed cross-resistant to Fluo-CQ when the chemical probe was tested in various CQ-sensitive and -resistant parasite strains. NBD derivatives were found to act as redox cyclers of two essential targets, using a coupled assay based on methemoglobin and the NADPH-dependent glutathione reductase (GRs) from P. falciparum. This redox activity is proposed to contribute to the dual action of Fluo-CQ on redox equilibrium and methemoglobin reduction via PfCRT-mediated drug efflux in the cytosol and then continuous redox-dependent shuttling between food vacuole and cytosol. Taking into account these physicochemical characteristics, a model was proposed to explain Fluo-CQ antimalarial effects involving the contribution of PfCRT-mediated transport, methemoglobin reduction, hematin binding, and NBD reduction activity catalyzed by PfGR in CQ-resistant versus CQ-sensitive parasites. Therefore, introduction of NBD fluorophore in drugs is not inert and should be taken into account in drug transport and imaging studies.

Full text of DOI:10.1021/acsinfecdis.5b00141

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Article
A redox-active fluorescent pH indicator for detecting P. falciparum
strains with reduced responsiveness to quinoline antimalarial drugs
Mouhamad Jida, Cecilia Sanchez, Karène Urgin, Katharina Ehrhardt, Saravanan
Mounien, Aurelia Geyer, Mourad Elhabiri, Michael Lanzer, and Elisabeth Davioud-Charvet
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.5b00141 • Publication Date (Web): 23 Nov 2016
Downloaded from http://pubs.acs.org on November 25, 2016
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A Redox-Active Fluorescent pH Indicator for Detecting P.
falciparum Strains with Reduced Responsiveness to Quinoline
Antimalarial Drugs
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Mouhamad Jida,#,a Cecilia P. Sanchez, #,b Karène Urgin,a Katharina Ehrhardt,a,b Saravanan
Mounien,a Aurelia Geyer,a Mourad Elhabiri,a Michael Lanzer,*,b Elisabeth Davioud-Charvet*,a
aUMR 7509 Centre National de la Recherche Scientifique and University of Strasbourg, European School of Chemistry,
Polymers and Materials (ECPM), 25, rue Becquerel, F-67087 Strasbourg, France.
bZentrum für Infektiologie, Parasitologie, Universität Heidelberg, Im Neuenheimer Feld 324, 69120 Heidelberg,
Germany.
Supporting Information
ABSTRACT: Mutational changes in the Plasmodium falciparum chloroquine resistance transporter
(PfCRT) have been associated with differential responses to a wide spectrum of biologically active
compounds including current and former quinoline and quinoline-like antimalarial drugs. PfCRT
confers altered drug responsiveness by acting as a transport system, expelling drugs from the
parasite’s digestive vacuole where these drugs exert, at least part, of their antiplasmodial activity.
To preserve the efficacy of these invaluable drugs, novel functional tools are required for
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epidemiological surveys of parasite strains carrying mutant PfCRT variants and for drug
development programs aimed at inhibiting or circumventing the action of PfCRT. Here we report
the synthesis and characterization of a pH-sensitive fluorescent chloroquine analogue consisting of
7-chloro-N-{2-[(propan-2-yl)amino]ethyl}quinolin-4-amine functionalized with the fluorochrome
7-nitrobenzofurazan, henceforth termed Fluo-CQ. In the parasite, Fluo-CQ accumulates in the
digestive vacuole, giving rise to a strong fluorescence signal but only in parasites carrying the wild
type PfCRT. In parasites carrying the mutant PfCRT, Fluo-CQ does not accumulate. The
differential handling of the fluorescent probe, combined with live cell imaging, provides a
diagnostic tool for quick detection of those P. falciparum strains that carry a PfCRT variant
associated with altered responsiveness to quinoline and quinoline-like antimalarial drugs. In
contrast to the accumulation studies, chloroquine (CQ)-resistant parasites were observed cross-
resistant to Fluo-CQ when the chemical probe was tested in various CQ-sensitive and –resistant
parasite strains. NBD derivatives were found to act as redox cyclers of two essential targets, using a
coupled assay using methemoglobin and the NADPH-dependent glutathione reductase (GRs) from
Plasmodium falciparum. This redox activity is proposed to contribute to the dual action of Fluo-CQ
on redox equilibrium and methemoglobin reduction via PfCRT-mediated drug efflux in the cytosol
and then continuous redox-dependent shuttling between food vacuole and cytosol. Taking into
account these physicochemical characteristics, a model was proposed to explain Fluo-CQ
antimalarial effects involving the contribution of PfCRT-mediated transport, methemoglobin
reduction, hematin binding, and NBD reduction activity catalysed by PfGR in CQ-resistant versus
CQ-sensitive parasites. Therefore, introduction of NBD fluorophore in drugs is not inert and should
be taken into account in drug transport and imaging studies.
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KEYWORDS: chloroquine resistance, fluorescent pH indicator, malaria, PfCRT transporter,
Plasmodium falciparum, redox
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Malaria continues to impose a heavy socio-economic and health burden on many nations.
Particularly Sub-Saharan Africa and parts of Southeast Asia where the vast majority of the
estimated 198 million malaria-related disease episodes and 584,000 deaths occur ache under the
yoke of this infectious disease.1 Relief is not in sight. The world malaria situation will remain tense
not least because human malaria parasites have emerged and spread that are resistant to many
currently used antimalarial drugs.2 Of particular concern is the reduced responsiveness of
Plasmodium falciparum to artemisinin, quinoline, and quinoline-like derivatives,3,4,5,6,7,8,9,10 which
as artemisinin combination therapy carry the brunt of malaria chemotherapy.11 In order to combat
resistance and preserve the efficacy of these indispensable drugs, better tools are required for
diagnosis of resistant strains and for identifying novel chemotherapeutic intervention strategies that
prevent or circumvent established drug resistance mechanisms. Currently the surveillance of drug
resistant P. falciparum strains relies on clinical observations, time-consuming and labor-intensive
genotype analysis, and in vitro drug response testing. None of these approaches are particularly
suitable for bedside diagnostics, although rapid and easy-to-perform tests to monitor drug resistant
parasites would benefit patient care, particularly in areas of high prevalence of drug and multi-drug
resistance.
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Our understanding of the molecular mechanisms underpinning drug resistance in P. falciparum has
substantially advanced in recent years. For instance, reduced susceptibility to quinoline antimalarial
drugs, such as chloroquine (CQ), quinine, and amodiaquine, and quinoline-like drugs, such as
lumefantrine and halofantrine, is associated, amongst other factors, with polymorphisms in a gene
called the P. falciparum chloroquine resistance transporter gene (pfcrt).12,13 This gene encodes a
protein of 424 amino acids that is localized at the membrane of the parasite’s digestive vacuole. The
P. falciparum chloroquine resistance transporter protein (PfCRT) shares structural features with the
drug/metabolite carrier family and was shown to transport the quinoline antimalarial drugs CQ,
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quinine, and quinidine.14,15 However, PfCRT only alters drug transport when it contains the key
mutational changes associated with quinoline resistance, such as the K76T substitution, whereas the
wild type form does not act on these drugs.16 PfCRT-mediated drug transport is directed out of the
digestive vacuole. Due to their acidotropic nature, quinoline antimalarial drugs accumulate in the
acid digestive vacuole where they interfere with endogenous heme detoxification mechanisms,
although they also might exploit other molecular targets. The digestive vacuole serves as the main
proteolytic organelle for digestion of hemoglobin, which the parasite takes up from its host cell
during intraerythrocytic development to meet its nutrient requirements. The large amounts of
cytotoxic heme released as a consequence of hemoglobin degradation are biomineralized to inert
hemozoin. Quinoline antimalarial drugs interfere with heme biomineralization, resulting in a build-
up of toxic heme and heme:drug adducts. Thus, a key feature of quinoline resistant parasites is to
reduce the intra-digestive vacuolar drug concentration via PfCRT-mediated efflux below levels
required to inhibit heme biomineralization.17,18,19
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The information regarding the role of PfCRT in reduced quinoline responsiveness has inspired the
search for, and development of, fluorescent CQ derivatives that differentiate between wild type
strains and those mutant strains carrying PfCRT variants associated with reduced quinoline
susceptibility.20,21,22,23 However, most of the currently available probes are compromise in one way
or another either because they require expensive building blocks, are difficult to synthesize, have
poor spectral properties, are not a validated substrate of PfCRT, or do not allow interactions with
PfCRT to be studied directly without confounding effects such as the binding to heme.
Here we report a novel fluorescent reporter, called Fluo-CQ, based on a short CQ analogue (CQ1),
functionalized at the side chain with 7-nitrobenzofurazan (NBD). The probe is easy to synthesize; it
has improved physicochemical properties; and it serves as a fluorescence pH indicator. The probe
appears to be a substrate of the P. falciparum Dd2 strain PfCRT (PfCRTDd2) variant and may
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therefore allows the transport activity of this protein to be monitored in living parasites in real time
using a fluorescence microscopic set-up. As the nitroaromatic NBD moiety is known to act as
redox-cycler and substrate of many reductases a comprehensive study was performed to interrogate
the interaction of the redox-active Fluo-CQ with four targets: PfGR-catalysed NBD reduction,
methemoglobin reduction, hematin binding, and PfCRT-mediated transport. The
physico(bio)chemical features of the new fluorophore Fluo-CQ were shown to contribute both to
the antimalarial and fluorescent reporter activities of the chemical probe discriminating CQ-
resistant versus CQ-sensitive parasites. The probe offers novel opportunities for diagnostic
purposes, studies probing the mechanism of quinoline resistance, and efforts aimed at developing
inhibitors of PfCRT.
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RESULTS
Design and synthesis of the fluorescent NBD-functionalized chloroquine analogue (Fluo-CQ).
We have previously described the synthesis of a family of chloroquine derivatives with altered
amino alkyl side chains.24 From these compounds we selected 7-chloro-N-{2-[(propan-2-
yl)amino]ethyl}quinolin-4-amine (compound 2) as a scaffold for functionalization with a
fluorescent group. The reasons for choosing this compound were as follows: i) the compound is
easy to synthesize in high yield;24 ii) it is an amphiphilic diprotic weak base with pKa values of 7.7
± 0.2 and 9.6 ± 0.2 (Table S1, Supporting information) allowing it to cross membranes by passive
diffusion as the free base and to accumulate in acidic compartments, such as the parasite’s digestive
vacuole; and iii) the compound is a substrate of PfCRT.24
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Scheme 1. Structural formula of chloroquine (A) and synthetic route to prepare Fluo-CQ (6).
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Scheme 1 shows the synthesis strategy to produce compound 6 functionalized with the fluorescent
group 7-nitrobenzofurazan (NBD), henceforth called Fluo-CQ. As an initial building block we used
N-(7-chloroquinolin-4-yl)ethyl-1,2-diamine (1), which reacted in a one-step reductive amination
with propan-2-one to compound 2 in the presence of titanium isopropylate (IV) and sodium
borohydride as reductive reagent (yield 92%). The subsequent step involved a peptide coupling
reaction between compound 2 and N-(tert-butoxycarbonyl) glycine in the presence of N,N,N’,N’-
tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophos-phate (HBTU) and 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (EDC) as coupling reagents (yield 90%). The amide group of
the resulting compound 3 was subsequently reduced to the amine using borane dimethyl sulfide
complex (BH3-Me2S). BH3-Me2S was preferred over other reducing agents because of the better
yield of the resulting product (4) (60% versus 0%, 16%, 25% when the reduction reaction was
performed using sodium bis(2-methoxyethoxy) aluminium hydride (Red-Al), lithium aluminium
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hydride (LAH), or borane tetrahydrofuran complex (BH3-THF) with 1 M in tetrahydrofuran,
respectively). Finally, the desired compound Fluo-CQ (6) was easily obtained in 60% yield (over 2
steps) by removal of the Boc protective group in the presence of HCl (6N), followed by a peptide
coupling reaction between the intermediary product 5 and NBD-6-aminohexanoic acid (7) in the
presence of hydroxybenzotriazole (HOBt) and excess of EDC as coupling reagents. For this
purpose, the NBD-6-aminohexanoic acid precursor 7 was prepared in 77% yield by a nucleophilic
substitution reaction between 6-aminohexanoic acid and NBD-chloride in the presence of excess of
sodium bicarbonate.25
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Physicochemical properties of Fluo-CQ (6). We next investigated the physicochemical properties
of Fluo-CQ and assessed the influence of pH on the emission properties (Figure S1-S11, Supporting
information). Coupled absorption spectrophotometric (or spectrofluorimetric) and potentiometric
titrations allowed us to calculate the pKa values of the bifunctional molecule (Table S1, Supporting
information). It was found that Fluo-CQ is a weak base with pKa values of 6.4 ± 0.6 and 7.8 ± 0.4
close to those measured for its synthetic precursor compound 4 (pKa1 = 6.9 ± 0.3 and pKa2 = 8.5 ±
av
0.2). Although Fluo-CQ (pKa = 7.1) was less basic than CQ (pKa values of 8.4 ± 0.2 and 10.6 ±
0.2; pKaav = 9.5) and compound 2 (pKa values of 7.7 ± 0.2 and 9.6 ± 0.2; pKaav = 8.65), it possesses,
2+
2+
however, an enhanced lysosomotropic activity as indicated by the calculated LH2 (pH=5)/LH2
(pH=7.4) ratio (L = CQ, 2, or Fluo-CQ). The ratios were measured to be 14.4, 1.49, and 1.1 for
Fluo-CQ, compound 2, and CQ, respectively. This finding suggests an enhanced theoretical
accumulation of the diprotic form of Fluo-CQ in the parasite’s digestive vacuole as compared to CQ
and compound 2.
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1.0
0.5
0.0
100
1.0
0.5
0.0
LH22+
A
B
378 nm
550 nm
L
50
0
LH+
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350 400 450 500 550 600 650
4
6
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10
 (nm)
pH
Figure 1. Spectral properties of Fluo-CQ. Panel A: Reconstituted relative fluorescence spectra of
Fluo-CQ free base (L, red), mono-protonated (LH+, blue), and di-protonated (LH2+, black). F,
relative fluorescence signal. Panel B: Distribution diagram showing the different forms of Fluo-CQ
as a function of pH (color code as above). Overlaid is a graph showing the relative fluorescence
signal (F) of quinoline and NBD at excitation and emission wavelengths of exc. = 265 nm, em.
=
378 nm and  = 550 nm, respectively, as a function of pH. em. = 378 nm relates to the quinoline
emission while  = 550 nm is characteristics of the NBD emission. Solvent: H2O/DMSO (1/1 v/v); I
= 0.1 M NaClO4, T = 25.0°C; l = 1 cm; [Fluo-CQ]tot = 1.16 x 10-5 M; excitation and emission slit
widths = 5.5 nm; filter at 290 nm.
A pH-dependent spectrofluorimetric analysis of Fluo-CQ and its related analogues confirmed the
pKa values and revealed a strong pH effect on the fluorescence emission spectrum. To exclude any
possibility of the self-folding of the two chromophores that are borne by Fluo-CQ (i.e. 4-
aminoquinoline and NBD), its absorption and emission characteristics were compared to those of
the model compounds (CQ, CQ1SPACboc, NBD). These studies established that the two
chromophores that belong to Fluo-CQ are not self-interacting in solution meaning that the two
chromophores retained their intrinsic properties within the Fluo-CQ dyad. At an acidic pH (<6),
Fluo-CQ emits a strong fluorescence signal at the NBD-centred wavelengths (max = 543 nm, abs
=
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2.3% in PBS pH 7.5; max = 518 nm, abs = 14% and  = 3.56 ns in CH2Cl2). This peak is attenuated
4 to 5-fold at neutral and alkaline pH values. At an alkaline pH, a new fluorescence peak arising
from the 4-aminoquinoline subunit emerged at lower wavelengths (max = 378 nm, Figure 1A). The
distribution of the different forms of Fluo-CQ as a function of the pH is shown in Figure 1B. The
complex pH-dependent emission spectrum might be explained as follows: Fluo-CQ contains two
fluorescently active chromophores, the 4-amino-quinoline and the NBD moiety, which emit
fluorescence light at approximately 373 nm and 535 nm, respectively (Figure 1B).26,27 As shown in
Figure 1B, the emission from the NBD unit is the highest at an acidic pH and then declines as the
pH becomes more alkaline. Conversely, fluorescence emission by the quinoline unit increases with
increasing pH. At an acidic pH, fluorescence emission by the quinoline chromophore may be
disfavoured upon protonation of the 4-amino group due to an intramolecular charge transfer within
the quinoline unit. This suppressive effect is removed at an alkaline pH. On the other hand, the
fluorescence signal emitted by the NBD moiety is inhibited at alkaline pH values possibly by
photoinduced electron transfer between the deprotonated tertiary amine present in the spacer region
and the fluorescence emitter. These data show that Fluo-CQ is a fluorescent pH indicator, with its
maximal fluorescence emission peak shifting from 543 nm to 374 nm as the pH changes from acid
to basic and vice versa.
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Hematin-binding properties of Fluo-CQ (6) and related 4-aminoquinolines. Up to now, 4-
aminoquinolines are considered as antimalarial agents, targeting specific hemoglobin digestion
pathways by interacting with hematin and thereby inhibiting the biocristallisation process leading to
hemozoin. For example, chloroquine accumulates in the acidic digestive vacuole of the parasite,28,29
and was proposed to interact with the µ-oxo dimer of oxidized heme (FeIIIPPIX), thereby preventing
hemozoin formation.30 CQ strongly binds to FeIIIPPIX leading to a 1:1 complex with association
constants log KA ~ 5-6 (KD ~ 2-15 µM) at pH 7.5.31 The π-π interaction between CQ and the
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conjugated π-system of hematin is believed to govern and stabilize the formation of adducts. In
order to assess the capacity of the studied compounds, including Fluo-CQ, to interact with hematin,
an ESI-MS approach coupled to collision induced dissociation (CID)32 was applied to reach the 1:1
hematin:drug stoichiometry of the complexes and to determine the dissociation voltage at which
50% of the complexes were dissociated (DV50) (Table 1).
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Table 1. Hematin binding and calculated DV50 evaluated from stability responses of the
[hematin.drug]+ complexes shown in the ESI-CID spectra (drug = CQ, CQ1 2, CQ1-SPAC-Boc
4, and CQ1-SPAC-NBD 6).
DV50 (V)[a]
Compound
(m/z) of the
KD (µM)[b]
pKa values[c]
[drug:hematin]+ species
261 (935.5)
0.85
8.4 ± 0.2
CQ
10.6 ± 0.2
236 (879.5)
0.41
7.71 ± 0.2
9.57 ± 0.2
2 (CQ1)
267 (1022.65)
309 (1198.7)
nd
6.94 ± 0.3
8.46 ± 0.2
4 (CQ1-SPAC-Boc)
6 (Fluo-CQ)
nd
6.39 ± 0.6
(= CQ1-SPAC-NBD)
7.82 ± 0.5[d]
[a] Stability responses and calculated DV50 of the [hematin.drug]+ complexes were
obtained from an ESI-CID study with chloroquine (CQ) used as gold standard. ESI-MS+-
CID test conditions: 10 µM hematin + 10 µM drug in CH3CN/H2O (1/1 v/v) + 0.1%
formic acid; fragmentor voltage ranges from 20 V to 400 V with 20 V increments; see ref.
(32). [b] 0.2 M sodium Hepes buffer pH 7.5, see ref. (31). [c] Absorption titrations; I = 0.1
M NaClO4, T = 25.0°C; Error = 1 with  = standard deviation. [d] water/DMSO (1:1
v/v); I = 0.1 M NaClO4.
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The DV50 sequence of the drugs:hematin species (7:hematin << 2:hematin (CQ1) < CQ:hematin <
4:hematin (CQ1-SPAC-Boc) < 6:hematin (Fluo-CQ, CQ1-SPAC-NBD)) shown in Table 1 (and
Table S2, Supporting Information) demonstrates the ability of 4-aminoquinolines to interact with
hematin directly, with the strongest interaction found for Fluo-CQ.
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Antimalarial Activities of Fluo-CQ (6) and related 4-aminoquinolines. The antimalarial
activities of Fluo-CQ (6) and related 4-aminoquinolines 2, 4, and chloroquine as drug
reference, were evaluated in a SYBRgreen-based cell proliferation screening assay using red
blood cells parasitized with the P. falciparum laboratory strains, the CQ-sensitive HB3 or
the CQ-resistant Dd2.33 In comparison to CQ, there is a trend in the half maximal inhibition
(IC50) values attesting for the higher antiplasmodial activities exerted by CQ1 (
2) and Fluo-
CQ ( ) against the CQ-resistant strain Dd2 versus the CQ-sensitive strain HB3 (Table S3,
6
Supporting information). These results with their hematin binding properties indicate that the
primary target of 4-aminoquinolines CQ1 (2) and Fluo-CQ (6), is likely heme biocristallisation in
the CQ-sensitive strain. The synthetic intermediate CQ1-SPAC-Boc (4) displayed the lowest
antimalarial activity, whatever the wild-type and CQ-resistant P. falciparum strains HB3 and Dd2
used, with comparable IC50 of 95 nM and 77 nM, respectively. This latter observation suggested
that the NBD moiety in Fluo-CQ might contribute per se to the antimalarial activity of the hybrid
molecule (6).
In order to confirm that CQ-resistant parasites are cross-resistant to Fluo-CQ the strain
susceptibilities of two additional strains, 3D7 (CQS) and K1 (CQR), to both compounds CQ1 (2),
and Fluo-CQ (6) were also measured in comparison to CQ (Table 2). First, the IC50 values clearly
indicated that both compounds showed decreased antiplasmodial activities against the CQ-resistant
parasite strains Dd2 and K1, but to a lesser extent than CQ. These findings suggested that i) CQ-
resistant parasites are cross-resistant to Fluo-CQ and that Fluo-CQ and CQ share at least one
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primary molecular target (hemozoin formation); ii) the most parsimonious explanation of the
reduced activity of Fluo-CQ against the CQ-resistant parasites is one that would evoke Fluo-CQ
being effluxed by PfCRT-Dd2 from the vacuole into the cytosol, where it can no longer target
hemozoin formation. Therefore, the antimalarial action of Fluo-CQ 6 deserved to be discussed with
cautious considerations. The potent antimalarial activities of 2 and Fluo-CQ in the CQ-resistant
strains led us to perform a detailed investigation of their interactions with potential targets, beside
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the PfCRT variants. The strongest antiplasmodial effects of CQ1 (2) and Fluo-CQ (6) against the
CQ-resistant strain Dd2 compared to CQ suggested that the difference is at least partially
attributed to the higher ability of 2 and 6 to cross the membranes of infected cells as a consequence
of their modified physicochemical properties. These latter might led to a higher lipophilicity value,
lower flexibility of the side chain, or lower ionization constant (pKa) of the amine groups, resulting
in improved antimalarial effects, as confirmed in the physicochemical study. Indeed, as reported
above, introduction of the lipophilic ethylenediamine chain, and the NBD hexanoic acid moiety
modified the physicochemical properties of the 4-aminoquinolines, as attested by the calculated
clogP values for the final compounds 2 and 6 being equal to 3.76 and 6.89 versus 5.06 for CQ,
respectively (calculated from ChemBioDraw Ultra 14.0).
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Table 2. Averaged IC50 values (nM) for CQ, CQ1 and Fluo-CQ determined from growth inhibition
assays with Plasmodium falciparum strain HB3 (CQS), 3D7 (CQS) and Dd2 K1 (CQR) and K1
(CQR).[a]
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IC50 ± SEM (nM)
Compound
2 (CQ1)
6 (Fluo-CQ)
CQ
Strain
HB3 (CQS)
7 ± 2
6 ± 2
5 ± 2
7 ± 1
25 ± 8
21 ± 8
3D7 (CQS)
Dd2 (CQR)
K1 (CQR)
150 ± 30
160 ± 25
46 ± 7
40 ± 9
70 ± 18
81 ± 23
[a]
[b]
Test conditions: synchronous cultures, SYBRgreen assay, 72 h incubation.
Values are means of at least two independent determinations in triplicate. The IC50
value of the antimalarial drug chloroquine (CQ) is indicated as a reference.
Fluo-CQ inhibits PfCRT-Dd2 mediated CQ uptake in X. laevis oocytes.
We
further
investigated the interactions between PfCRT and Fluo-CQ by performing direct measurements of
CQ transport in the PfCRTHB3 or PfCRTDd2 expressing Xenopus laevis oocyte system.15 We
observed that Fluo-CQ interacts with PfCRT and inhibits PfCRT in a concentration dependent
manner (Figure 2). Preliminary attempts to detect the accumulation of Fluo-CQ in the PfCRT-
expressing oocytes were positive but not confirmed because Fluo-CQ intercalates into the oocyte
membrane resulting in equally high fluorescence signals in water-injected oocytes and in oocytes
expressing PfCRTHB3 or PfCRTDd2. In spite of numerous trials we were not able to demonstrate a
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highly reproducible transport of Fluo-CQ via PfCRT hampering the determination of a Km value for
Fluo-CQ nor inhibition parameters of Fluo-CQ uptake by verapamil. However, it was possible to
use the native fluorescence of Fluo-CQ to visualize the accumulation of Fluo-CQ in PfCRTHB3 or
PfCRTDd2 expressing P. falciparum-infected erythrocytes.
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2.0
1.5
1.0
0.5
0.0
0
10
50 100 250 500
Fluo-CQ (µM)
Figure 2. Fluo-CQ inhibits the transport of radiolabeled CQ in PfCRTDd2 expressing Xenopus laevis
oocytes. PfCRTDd2-expressing oocytes were incubated for 60 min in buffer containing 40 nM
[3H]CQ, 10 µM cold CQ, and Fluo-CQ at the concentrations indicated. Water-injected oocytes were
analysed in parallel. PfCRT-mediated CQ uptake was calculated by subtracting the amount of CQ
taken up by water-injected oocytes from that of PfCRTDd2 expressing oocytes. The means ± SEM
are shown for at least three independent replications, with each single measurement being made
from 10 oocytes.
Fluo-CQ as a reporter for PfCRT-mediated CQ flux. We next examined Fluo-CQ accumulation
in P. falciparum-infected erythrocytes. Fluo-CQ accumulated in the digestive vacuole of the CQ-
sensitive strain HB3, as indicated by a strong food vacuolar fluorescence signal (Figure 3A).
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Accumulation of Fluo-CQ in the digestive vacuole of strain HB3 did not occur in glucose starved
cells. In the absence of metabolic energy, a diffuse fluorescence signal was observed throughout the
parasite’s cytoplasm (Figure 3A). This finding is explained by the requirement of an energy input to
acidify the digestive vacuole via the combined action of a H(+)-ATPase and a H(+)-
pyrophosphatase,34 which in turn is a prerequisite for accumulation and trapping of Fluo-CQ in this
organelle. Also note that the green Fluo-CQ’s fluorescence signal is the strongest at an acidic pH
(Figure 1). Accumulation of Fluo-CQ in the digestive vacuole was independent of verapamil (30
µM), a chemosensitizer of CQ resistance and a partial mixed type inhibitor of PfCRT.15,35
Differential fluorescence images were observed for erythrocytes infected with the CQ-resistant
strain Dd2 (Figure 3B). The food vacuolar Fluo-CQ fluorescence signal that was very prominent in
vital HB3 parasites was only observed in Dd2 following treatment with verapamil (30 µM) causing
inhibition of PfCRT. In untreated cells and in glucose starved cells, the Fluo-CQ fluorescence was
weak and distributed throughout the parasite’s cytoplasm. Quantification of the fluorescence signals
confirmed the differential partitioning behaviour of Fluo-CQ in the CQ-sensitive and CQ-resistant
P. falciparum strain (Figure 3C). These findings are consistent with PfCRT from the CQ-resistant
strain being able to transport Fluo-CQ out of the digestive vacuole, whereas PfCRT from the CQ-
sensitive strain does not act on this fluorochrome - and hence, the differential accumulation and
fluorescence phenotypes. Therefore, Fluo-CQ might accurately reflect CQ transport via PfCRT
without confounding effects such as the binding to heme because the time frame of the antimalarial
potency experiments, i.e. drug incubation with synchronized ring stage parasite cultures for 72 h,
excludes to be confounded with the interactions with PfCRT studied directly by fluorescence of
Fluo-CQ in very short time, i.e. following 1 h incubation with the parasitized erythrocytes.
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A
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B
C
HB3
Dd2
2.5
2.0
1.5
1.0
0.5
0.0
**
**
glu
VP
+
-
-
-
+
+
+
-
-
-
+
+
Figure 3. Fluo-CQ live cell imaging in the CQ-sensitive P. falciparum strain HB3 (Panel A) and
the CQ-resistant strain Dd2 (Panel B). P. falciparum-infected erythrocytes at the trophozoite stage
were incubated with 0.5 µM Fluo-CQ for 20 min at 37oC before images were taken using a confocal
fluorescence microscope. Where indicated, cells were glucose starved (-glucose), treated with 30
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µM verapamil (+VP), or incubated without Fluo-CQ (-Fluo-CQ). DNA was stained using Hoechst
33342 (blue channel). (Panel C) Ratio of the digestive vacuolar and total parasite fluorescence
signals (Fvacuole / Fparasite). The means ± SEM of over 20 independent determinations are shown. ** P
< 0.01 compared to Fluo-CQ treated vital cells, as determined using the Holm-Sidack ANOVA test.
Bar, 1 µm.
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P. falciparum GR-catalyzed 7-nitrobenzofurazan reductase Activity. The ability of P.
falciparum GR to reduce the nitroaromatic group of the 7-nitrobenzofurazan (NBD) derivatives was
then investigated in the methemoglobin (metHb) reduction assay coupled to the PfGR/NADPH
system in vitro under quasi-physiological conditions (equations 1-4). This assay was recently
established as a relevant in vitro model in our laboratory and allowed to assess redox characteristics
of the P. falciparum GR-catalyzed nitroreduction kinetics.31,36,37 The UV-visible absorption
spectrum of metHb(FeIII) between 300 nm and 700 nm is characterized by an intense Soret band of
the Fe(III) heme whose maximum absorbance is centred at 405 nm and was used as a valuable
spectroscopic probe. Upon metHb(FeIII) reduction by the enzymatically-generated reduced species
of the redox-active compounds, the occurrence of the oxyHb(FeII) species is associated to a
bathochromic shift of the maximum of absorption from 405 nm to ~ 410 nm (now corresponding to
a low spin hexacoordinated ferrous ion), as well as the formation of two new less intense
absorptions lying at ~ 536 nm and ~ 576 nm, respectively (Figure 4, Figure S12-S13, Supporting
information). The nitroaromatic reductase activity of P. falciparum GR was observed as long as
NADPH is present in the cuvette and compared to the intrinsic NADPH oxidation activity of the
enzyme in the absence of the NBD derivative (equation 2). As an example, Figure 4 depicts the
spectral absorption variation recorded for Fluo-CQ (Panels A-B), and 7 (Panel C) under these
experimental conditions. The Table in Figure 4 reports on the determined MetHb(FeIII) reduction
kinetic parameters determined in the presence of the compounds, 4-aminoquinolines (2 and 4), and
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the NBD derivatives (Fluo-CQ, 6 and the NBD-hexanoic acid, 7). The kinetic analysis clearly
evidenced the presence of two rate-limiting steps. The first one did not induce any classical shift of
the Soret band usually observed for metHb reduction, and the kinetic rates are seemingly
independent of the nature of the compounds within the experimental errors. The second one is
clearly associated with the metHb reduction as evidenced by the bathochromic shift of the Soret
band. Marked effects can be seen on the values of these second pseudo-first order rate constants
depending on the nature of the system. With the most efficient redox-cycling activity to convert
metHb to oxyHb, we confirmed the ability of the NBD-hexanoic acid (7) to behave as an efficient
substrate of PfGR in the direct enzymic assay using the NADPH-dependent P. falciparum GR. The
nitroreductase activity of P. falciparum GR was compared to the intrinsic NADPH oxidation
activity of the enzyme in the absence of the NBD derivative (7) (equation 2). The flavoenzyme
displayed a 2.9-fold higher NADPH oxidation activity in the presence of 150 µM the NBD-
hexanoic acid (7) (equations 1 or 2); the observed NADPH oxidation rates were 0.013 U/mg protein
in the absence of the NBD derivative and 0.347 U/mg protein in the presence of 150 µM NBD-
hexanoic acid (7).
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-•
NADPH + H+ + 2 O2  NADP+ + 2 O2
(eq. 1) enzymatic
•-
NADPH + H+ + 2 Ar-NO2 (NBDox)  NADP+ + 2H+ + 2 Ar-NO2
(eq. 2) enzymatic
•-
•-
Ar-NO2 + O2  Ar-NO2 + O2
(eq. 3) non enzymatic
(eq. 4) non enzymatic
MetHb(Fe3+) + Ar-NO2 (NBDred)  OxyHb(Fe2+) + Ar-NO2
•-
The Km and kcat values, 508.7 µM and 14 ± 0.004 s-1 corresponding to Vmax = 14.629 ± 6.52 U/mg
protein were derived from two measurements at seven different substrate concentrations in
duplicate (Figure 4D). When following NADPH consumption, the NBD-hexanoic acid (7) was
reduced by P. falciparum GR with a catalytic efficiency kcat/Km of 27.5 x 103 M-1 s-1, a half-value
only when compared to that determined for methylene blue (kcat/Km = 50.0 x 103 M-1 s-1).38 These
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data are also in good agreement with previous observations on various nitroaromatics reported by
4
5
Cenas et al.,39,40,41 Blumenstiel et al.,42 and Biot et al.43
6
7
8
9
0.76
A
B
0.72
0.68
0.64
0.60
0.56
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0.10
0.08
0.06
0.04
0.02
0.00
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1
0
2000
4000
Time (s)
6000
2
2
1
550
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400
 (nm)
 (nm)
0.76
0.72
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0.60
D
C
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Time (s)
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 (nm)
400
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[7] (µM)
Compound
kobs1 (x 10-3 s-1) (±)
1.39 ± 0.04
kobs2 (x 10-5 s-1) (±)
5.2 ± 0.5
2 (CQ1)
4 (CQ1-SPAC-Boc)
1.44 ± 0.05
11.6 ± 0.4
6 (Fluo-CQ, = CQ1-SPAC-NBD)
7 (NBD-hexanoic acid)
0.98 ± 0.02
3.3 ± 0.11
2.5 ± 0.4
48 ± 0.4
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Figure 4. Kinetics of metHb(FeIII) reduction in an established coupled assay with PfGR/NADPH in
the presence of the NBD derivatives, Fluo-CQ (6) (Panels A and B) and NBD-hexanoic acid (7)
(Panel C), and rate constants of metHb(FeIII) reduction kinetics observed for the NBD derivatives
(Table). Initial velocities of the nitroreductase activity of the NADPH-dependent PfGR in the
presence of NBD-hexanoic acid (7) as substrate (Panel D).
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Panels A and B: UV-visible absorption spectra recorded as a function of time in the coupled assay
in the presence of Fluo-CQ (6); Panel A inset: Absorbance variation at 405 nm as a function of
time. Panel C: UV-visible absorption spectra recorded as a function of time in the coupled assay in
the presence of the NBD-hexanoic acid (7); Panel C inset: Absorbance variation at 405 nm as a
function of time. Solvent: water (47 mM phosphate buffer pH 6.9 + 1 mM EDTA + 200 mM KCl);
T = 25.0°C; 120 µM NADPH + 1.72 nM PfGR + 8 µM metHb + 20 µM GSSG + 40 µM Fluo-CQ
(6) or NBD-hexanoic acid (7); (1) t = 0; (2) t = 120 min. Table: Kinetic parameters determined in
the coupled MetHb(FeIII) reduction assay in the presence of the compounds, 4-aminoquinolines (2
and 4), and the NBD derivatives (Fluo-CQ, 6 and the NBD-hexanoic acid, 7). Panel D:
Nitroreductase Activity of P. falciparum Glutathione Reductase. The nitroreductase activity of P.
falciparum GR (Panel D) in the presence of the NBD-hexanoic acid, 7 (—●—) was tested by
monitoring the oxidation of NADPH at 340 nm, at 25 °C, and pH 6.9, under steady-state conditions.
The NBD-hexanoic acid was dissolved in DMSO and the NADPH-oxidation activity was measured
at seven different concentrations in duplicate (0 – 150 µM) in the presence of 1% DMSO. The
initial rate for aerobic NADPH oxidation activity of PfGR was subtracted from the rates measured
in the presence of the nitroaromatic derivative. For the determination of Km and Vmax values, the
steady-state rates were fitted by using nonlinear regression analysis software (Kaleidagraph) to the
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Michaelis-Menten equation,44 and the turnover number kcat and the catalytic efficiency kcat/Km were
4
5
therefore calculated.
6
7
8
9
In our work, the NBD-moiety from Fluo-CQ 6 showed a GR-catalyzed 7-nitrobenzofurazan
reductase activity per se, i.e. a redox activity with a validated target in P. falciparum, which has
been shown in several reports to be relevant to explain the antimalarial activity of redox-active
agents.31,45,46,47,48 a 29-fold higher value compared to that of the carboxylic acid counterpart M5
(kcat/Km = 945.0 M-1 s-1).43 In previous works, the combination of various nitroaromatics41,49 and
quinones,24,50 such as the carboxylic acid M5, a menadione alkylated in C-2 by the hexanoic acid
chain, and 4-aminoquinolines resulted in hybrid drugs, called Trojan horse drugs, displaying a
higher antimalarial activity than both starting materials, probably through a dual action in the
parasite. This effect seems to be specifically pronounced in infected red blood cells since
cytotoxicity against hMRC-5 cells was observed neither for Fluo-CQ (data not shown), nor for the
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menadione-hexanoic acid M5 and the NBD-hexanoic acid 7 (data not shown).
DISCUSSION
The malaria parasite P. falciparum degrades most of host cell hemoglobin to provide aminoacids
essential for its intraerythrocytic growth. Endocytosis is a fundamental process of eukaryotic cells
and fulfils numerous functions, most notably, that of macromolecular nutrient uptake. Malaria
parasites invade red blood cells and during their intracellular development endocytose large
amounts of erythrocyte cytosol for digestion in a specialized lysosomal compartment, the food
vacuole, via a complex structure constituted by the cytostome (an invagination in the
parasitophorous vacuolar membrane) and transport vesicles. The food vacuole contains a number of
proteolytic enzymes identified in the process of hemoglobin digestion as potential drug targets,
including the aspartic acid proteases (plasmepsins) that initiate the cleavage of hemoglobin,
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cysteine proteases (falcipains), the metalloprotease falcilysin and a histoaspartic protease that
catalyze further degradation into small peptides. These enzymes are active under the acidic
conditions prevailing in the food vacuole, which was observed to have a pH between 5.0 and 5.5. In
this context, it has been known for long that the antimalarial drug CQ, a dicationic weak base
belonging to the 4-aminoquinoline series, penetrates and accumulates in the food vacuole of
plasmodial parasites through its lysosomotropic properties, and exerts its action by interfering with
heme metabolism. Hemoglobin digestion is accompanied by intensive pulses of reactive oxygen
species (ROS), which are important cell signalling molecules. The redox homeostasis results from a
subtle interplay between the thiol network controlled by various redox enzymes, heme
biocrystallisation, and transport of redox-active components. CQ-resistance phenotype associated to
reduced susceptibility of the strains to CQ has been corroborated with the presence of mutations in
the food vacuole membrane protein PfCRT, and with lower levels of glutathione and hemozoin. A
novel role for mutated PfCRT in CQ-resistant parasites has been recently discovered with strong
data supporting a dual mechanism for PfCRT facilitating both influx of glutathione in the food
vacuole and efflux of CQ from the food vacuole.51As mutated PfCRT has also been associated with
transport of redox-active agents (e.g. methylene blue)52 we investigated in this study the possible
contribution of PfCRT-mediated transport of the redox-active nitroaromatic component of Fluo-CQ,
which discriminates CQ-sensitive and -resistant parasites.
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Well-known redox-cyclers (or subversive substrates of various members of the disulfide reductase
family) are nitrofuran derivatives displaying notable antiparasitic activities. The molecular basis of
nitroaromatics toxicity is the enzyme-catalyzed reduction to nitroradicals which then reduce oxygen
to superoxide anion radicals thereby regenerating the redox-cycler (eq. 2).53 This futile redox
cycling and oxygen activation lead to increased levels of hydrogen peroxide (eq. 8) and GSSG.
Electrochemical studies have demonstrated that the kinetics of the reaction with molecular oxygen
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is very fast, thus privileging the reaction pathway (eq. 5).54 The electrochemical potential remains
one of the relevant descriptor that has to be taken into consideration in any attempts to design
redox-active nitroaromatics, including the differently functionalized NBD derivatives.55
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Redox cycling is known to occur when a redox-active agent (e.g. phen(othi)azines, pyocyanin,
naphthoquinones, nitroaromatics) undergoes alternating reduction and oxidation reactions, resulting
in the transfer of electrons from the reductant (e.g. NAD(P)H) to the oxidant (e.g. oxygen).56,57
Redox-cyclers, such as phenazines, can cross cell membranes, enabling the transfer of reducing
equivalents from inside the cell to the extracellular environment.58 In the case of P. falciparum-
infected red blood cells the compartment where are present the powerful oxidants (metHb, oxygen)
is the food vacuole. The antimalarial methylene blue/pyocyanin/plasmodione, in the oxidized form
behave as oxidants accepting the electrons produced upon NADPH-dependent GR-catalyzed
reduction in the cytosol along with concomitant NADPH oxidation to NADP+. The generated
reduced form of the redox-active mediators (leucomethylene blue, reduced pyocyanin, reduced
benzylmenadiones, reduced nitroaromatics) might also be membrane-permeable and diffuse away
from the cytosol – possibly via iron complexation – to the food vacuole where, the reduced species
is then re-oxidized in the course of the redox-cycling activity of methemoglobin(FeIII) into
oxyhemoglobin(FeII). The leakage of the reduced species out of the cytosol was exemplified by the
antimalarial redox-active indolone N-oxide derivatives.59 The oxidized mediator is then ready to
repeat the redox-dependent shuttling between the parasitized erythrocytic cytosols and the food
vacuole. This cycling continually drains off metabolic reducing power from P. falciparum-infected
erythrocytes to give “electrical power at the electrodes” (Figure 5, panel B).46 Thus, the antimalarial
nitroaromatics (chinifur, NBD) derivatives are proposed to act catalytically as redox-active
mediators that are shuttled – via mutated PfCRT – in and out the food vacuole in parasitized red
blood cells, thereby oxidizing major intracellular reductants (like NADPH in the cytosol) and
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subsequently reducing the oxidants (oxygen, hematin and metHb).
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Coming back to the redox-active fluorescent pH indicator and likely substrate of PfCRT, the
observations might be rationalized as followed. In both the CQ-sensitive and -resistant parasite HB3
and Dd2 strains Fluo-CQ is proposed to be internalized in the food vacuole of parasitized red blood
cells. The antimalarial activity of Fluo-CQ against the CQ-sensitive strain corroborates with the
vizualisation of the pH-dependent fluorophore concentrating in the food vacuole where the 4-
aminoquinoline moiety interacts with hematin (Figure 5, Panel A). The efflux is not supposed to
occur since the PfCRT gene is not mutated in CQ-sensitive parasites. In the CQ-resistant parasite
Dd2 strain Fluo-CQ might rapidly be extruded by the mutated PfCRT transporter to the cytosol
where the NBD moiety may be reduced by the NADPH-dependent PfGR. The reduced NBD
species is proposed to leave the cytosol to penetrate the acidic compartment to transfer electrons to
oxidants. Thereby, in agreement with the images (Figure 3) Fluo-CQ is more visualized out of the
food vacuole in the P. falciparum Dd2 strain with reduced responsiveness to the quinoline
antimalarial drug (Figure 5, Panel B). However, the mechanism of the antimalarial action of Fluo-
CQ might result from its dual action, i.e. inhibition of hemozoin crystallisation upon 1e-transfer to
(FeIII) species and disturbance of the redox equilibrium in the infected red blood cells.
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With respect to the literature, the redox-active pH indicator acridine orange (AO) has also been used
as a fluorescent dye for the diagnosis of malaria60 and as a probe for estimating the differential
accumulation of, and susceptibility to, CQ between CQ-sensitive and CQ-resistant parasites.61,62,63
However, while the observed difference in the accumulation of AO has been linked to a lower pH
of the food vacuole in CQ-resistant parasites versus their CQ-sensitive counterparts the
interpretation has alternatively been suggested to be due to its export from the food vacuole via
mutant PfCRT.64 Furthermore, recent studies broadened the known substrate range of mutant
PfCRT to include tricyclic compounds and established that the PfCRT-mediated efflux of
quinacrine and methylene blue from the food vacuole decreases the parasite’s susceptibility to these
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molecules, adding therefore further support to the hypothesis that PfCRT behaves as a multidrug
resistance carrier.52 The behaviour of redox-active pH-dependent fluorophores should be considered
when used to measure changes in the pH of acidic compartments containing oxidants since PfCRT
seems to be a transporter responsible for efflux of many redox-active agents.
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Figure 5. Models for how the antimalarial Fluo-CQ affects the growth of CQ-sensitive and -
resistant P. falciparum parasites.
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Panel A: In the intraerythrocytic cycle P. falciparum parasites digest host hemoglobin in the food vacuole.
Fluo-CQ is proposed to be internalized in the food vacuole through a gradient of pH. Then, Fluo-CQ
interacts with oxidants (heme and MetHb). The final result is an inhibition of hemozoin formation and the
arrest of parasite development.
Panel B: Similarly to the two first steps of the mechanism described in Panel A, after penetration in the
food vacuole Fluo-CQ is extruded by active PfCRT-induced efflux from the food vacuole to the cytosol
where the redox homeostasis is maintained by the NADPH-dependent glutathione reductase (GR). Fluo-CQ
is proposed to be reduced at the nitroaromatic NBD moiety by PfGR in the cytosol. The 1e--reduced species
are assumed to be transported into the food vacuole where the electrons are transferred to oxidants (heme
and MetHb). Subsequently, in oxidized form, Fluo-CQ might be transported by PfCRT again into the
cytosol where it is reduced in a continuous redox cycle. The final result is an inhibition of hemozoin
formation through MetHb reduction and the arrest of parasite development.
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Conclusions. Fluo-CQ extends the repertoire of tools available to study altered responsiveness of P.
falciparum to quinoline and quinoline-like drugs. Fluo-CQ is a probable substrate of PfCRT and
thus a likely competitor of PfCRT’s drug transport activity. Fluo-CQ could be useful as a pH-
dependent fluorescent probe for functional studies on PfCRT in the parasite. While it is very
difficult to ascertain the essential requirement of the redox component of the chemical probe in the
applications to discriminate CQ-sensitive from CQ-resistant parasites our work allowed us to
measure the NBD derivatives as redox cyclers interacting with four essential targets: i) in
metHb(FeIII) reduction, ii) in hematin crystallisation, iii) in mutated PfCRT-mediated drug
transport, and iv) in NADPH-dependent PfGR reduction. We provide here data that suggests that
the fluorescent NBD moiety may contribute to altered redox equilibrium, PfCRT-mediated efflux of
Fluo-CQ, and reduction of (FeIII)species (metHb, heme) via continuous redox-dependent shuttling
of the fluorescent chemical probe between food vacuole and cytosol. Taking into account these
characteristics, a model was proposed to explain the Fluo-CQ antimalarial effects involving the
contribution of PfCRT-mediated transport, the hematin binding, the NBD reduction activity
catalysed by GR in CQ-resistant versus CQ-sensitive parasites. Introduction of a NBD fluorophore
in drugs is therefore not innocent and should be taken into account in drug transport and imaging
studies. In this study, a robust fluorescent probe was designed and characterized, with several lines
of evidence suggesting that it is a substrate of PfCRT-Dd2. Despite being redox active, this
compound could be used to visually detect P. falciparum strains with reduced susceptibilities to
quinoline antimalarial drugs. More work will be now developed to set up an in cellulo diagnostic
test that might be advantageous in large screens, such as drug development projects and
epidemiological surveys.
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Enzymes. Recombinant Plasmodium falciparum glutathione reductases (GR, E.C.1.6.4.2) was
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purified as previously reported.65,66 One unit of GR activity is defined as the consumption of 1 µmol
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NADPH per min (340
= 6.22 mM-1 cm-1) under conditions of substrate saturation. The enzyme
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nm
(P. falciparum GR) stock solutions used for kinetic determinations were > 98% pure as judged from
silver stained SDS-PAGE and had specific activities of 155 U/mg. All other reagents were of the
highest available purity and were purchased from Biomol, Boehringer, and Sigma.
P. falciparum GR-catalyzed 7-nitrobenzofurazan reductase activity. The ability of P.
falciparum GR to reduce the nitroaromatic derivative 7 was assayed by monitoring the oxidation of
NADPH at 340 nm (340
nm
= 6.22 mM-1 cm-1). The assay mixtures, in a total volume of 1 mL,
contained 100 mM phosphate buffer pH 6.9, 200 mM KCl, 100 µM NADPH, and 24.2 nM P.
falciparum GR (0.22 units/assay in the cuvette). The nitroaromatic derivative 7 was dissolved in
DMSO and the NADPH-oxidation activity was measured at seven different inhibitor concentrations
(0 - 150 µM) in the presence of 1% DMSO. For the determination of Km and Vmax values, the
steady-state rates were graphically fitted by using nonlinear regression analysis software
(Kaleidagraph) to the Michaelis-Menten equation,44 and the turnover number kcat and the catalytic
efficiency kcat/Km were calculated. The initial rate for intrinsic NADPH oxidation activity of P.
falciparum GR was not subtracted from the rates measured in the presence of the nitroaromatic
derivative when it proved negligible in comparison to GR-catalyzed nitroreductase activity.
Antimalarial activities of Fluo-CQ (6) and related 4-aminoquinolines. The antimalarial
activities were evaluated in the SYBRgreen assays33 using parasitized red blood cells in
culture. To assess this, the 50% inhibitory concentration (IC50) for each compound was
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determined in the presence of two P. falciparum laboratory strains, the CQ-sensitive HB3 or
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the CQ-resistant Dd2.
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Drug Transport Assays. X. laevis adult females were purchased from Xenopus Express and kept
as described.67 Anestesia was adminstered by immersion in ice water containing 0.1% ethyl-3-
aminobenzoate methanesulfonate salt. Ovarian fragments were surgically removed and incubated in
Ca2+-free medium (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES; pH 7.6). Oocytes
follicular envelopes were removed by enzymatic treatment with 1mg/ml (0.2-0.3 U/mg) collagenase
A (Sigma-Aldrich) for 30-40 minutes. Oocytes (Dumont stage V-VI) were incubated in ND96
solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES; pH 7.5)
supplemented with 0.1 mg/ml gentamicin and 2.5 mM sodium pyruvate. Expression of PfCRTDd2 at
the plasma membrane of Xenopus oocytes was achieved as described previously (Martin et al.
Science 325:1680-1682).14 Briefly, cRNA was prepared by in vitro transcription from the SP6
promoter (mMessage mMachine in vitro transcription kit, Ambion). 30 ng cRNA (in 40 nl water)
were injected into each X. laevis oocyte, using a pneumatic microinjector (5246, Eppendorf-
Netheler-Hinz). Oocytes injected with 40 nl water served as controls. After injection of the cRNA,
the oocytes were incubated in ND96 medium for 3 days at 18°C before uptake studies were
performed. For inhibition studies, oocytes expressing PfCRTDd2 and water-injected oocytes were
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incubated for 60 min at 25 C in ND96 buffer pH 6.0 containing 40 nM [3H]CQ, 10 µM cold CQ,
and Fluo-CQ at concentrations ranging from 0 to 500 µM. The uptake assays were terminated by
removing the reaction medium and washing the oocytes three times with 3 ml of ice-cold ND96
buffer. Each oocyte was transferred to a separate scintillation vial and lysed by the addition of 5%
sodium dodecyl sulfate (200 μl). The radioactivity in the sample solution was measured using a β-
scintillation counter. PfCRTDd2-mediated drug transport was calculated by subtracting the uptake
measured in water-injected oocytes from that in oocytes expressing PfCRTDd2. At least three
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