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Article

Antiplasmodial Activity of Nitroaromatic Compounds: Correlation with Their Reduction Potential and Inhibitory Action on Plasmodium falciparum Glutathione Reductase

by
Audronė Marozienė
1,
Mindaugas Lesanavičius
1,
Elisabeth Davioud-Charvet
2,
Alessandro Aliverti
3,
Philippe Grellier
4,
Jonas Šarlauskas
1 and
Narimantas Čėnas
1,*
1
Department of Xenobiotics Biochemistry, Institute of Biochemistry of Vilnius University, Saulėtekio 7, LT-10257 Vilnius, Lithuania
2
UMR7042 CNRS-Unistra-UHA, Laboratoire d’Innovation Moléculaire et Applications (LIMA), Bioorganic and Medicinal Chemistry Team, European School of Chemistry, Polymers and Materials, 25 rue Becquerel, F-67087 Strasbourg, France
3
Department of Biosciences, Universita degli Studi di Milano, via Celoria 26, I-20133 Milano, Italy
4
MCAM, UMR7245, Museum National d’Histoire Naturelle, CNRS, 61 rue Buffon, F-75231 Paris CEDEX 05, France
*
Author to whom correspondence should be addressed.
Molecules 2019, 24(24), 4509; https://doi.org/10.3390/molecules24244509
Submission received: 25 October 2019 / Revised: 21 November 2019 / Accepted: 4 December 2019 / Published: 10 December 2019
(This article belongs to the Special Issue COST CM1307: Targeted Chemotherapy towards Diseases Caused by Endoparasites—Proceedings in Medicinal and Natural Product Chemistry)
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Abstract

:
With the aim to clarify the mechanism(s) of action of nitroaromatic compounds against the malaria parasite Plasmodium falciparum, we examined the single-electron reduction by P. falciparum ferredoxin:NADP+ oxidoreductase (PfFNR) of a series of nitrofurans and nitrobenzenes (n = 23), and their ability to inhibit P. falciparum glutathione reductase (PfGR). The reactivity of nitroaromatics in PfFNR-catalyzed reactions increased with their single-electron reduction midpoint potential (E17). Nitroaromatic compounds acted as non- or uncompetitive inhibitors towards PfGR with respect to NADPH and glutathione substrates. Using multiparameter regression analysis, we found that the in vitro activity of these compounds against P. falciparum strain FcB1 increased with their E17 values, octanol/water distribution coefficients at pH 7.0 (log D), and their activity as PfGR inhibitors. Our data demonstrate that both factors, the ease of reductive activation and the inhibition of PfGR, are important in the antiplasmodial in vitro activity of nitroaromatics. To the best of our knowledge, this is the first quantitative demonstration of this kind of relationship. No correlation between antiplasmodial activity and ability to inhibit human erythrocyte GR was detected in tested nitroaromatics. Our data suggest that the efficacy of prooxidant antiparasitic agents may be achieved through their combined action, namely inhibition of antioxidant NADPH:disulfide reductases, and the rapid reduction by single-electron transferring dehydrogenases-electrontransferases.

1. Introduction

The emergence of the resistance of the malaria parasite Plasmodium falciparum to available drugs (e.g., chloroquine or artemisinin [1]) has resulted in the demand for new antimalarial agents and in a better understanding of their mechanisms of action. P. falciparum is particularly vulnerable to oxidative stress, that is, to enhanced generation of reactive oxygen species (ROS), which may be caused by the absence of the antioxidant enzymes catalase and glutathione peroxidase [2].
The antibacterial and antiparasitic activity of nitroaromatic compounds (ArNO2) is well known. In addition to a number of nitroheterocyclic drugs such as nifurtimox and benznidazole that have been used against Chagas disease and sleeping sickness since the 1970s, a new 5-nitroimidazole derivative, fexinidazole, has recently been approved for a treatment against sleeping sickness [3]. Frequently, the therapeutic action of ArNO2 is attributed to single-electron reduction into their anion radicals (ArNO2−), which in turn undergo redox cycling with the formation of ROS, or to their two/four-electron reduction into hydroxylamines (ArNHOH), able to modify DNA [4,5,6]. The single-electron reduction of ArNO2 is commonly performed by flavoenzymes dehydrogenases- electrontransferases, which possess natural single-electron acceptors, such as heme- or FeS-proteins [7,8,9,10]. However, there is a relative lack of information about the enzymes responsible for these reactions in parasites. Another point of view is that in trypanosomatids and Leishmania spp., a possible mode of ArNO2 action is the inhibition of the antioxidant flavoenzyme trypanothione reductase (TR) [11,12,13,14,15,16]. In this case, nitroaromatics also undergo TR-catalyzed redox cycling. In schistosomatids, a possible target of ArNO2 and other aromatic electron-deficient compounds is thioredoxin glutathione reductase [17,18].
A number of nitrofurans, nitrobenzenes, nitroimidazoles, and 4-nitrobenzothiadiazole were shown to possess in vitro antiplasmodial activity at micromolar or lower concentrations [19,20,21,22,23]; however, the mechanisms of their action remain poorly understood. The activity of a series of nitrobenzenes and nitrofurans roughly increased with their single-electron reduction midpoint potential (redox potential of ArNO2/ArNO2− couple, E17) [19], thus demonstrating a possible relationship between the compound’s ease of reductive activation and antiplasmodial activity. The antimalarial activity of nitrothiophenes was also attributed to the formation of ROS [24]. On the other hand, nitroaromatic compounds inhibit antioxidant flavoenzyme glutathione reductase from various sources [19,23,25,26]. Since P. falciparum glutathione reductase (PfGR) plays a key role in the antioxidant defense of the parasite [2,27,28], it is believed that its inhibitors may act as efficient antiplasmodial agents. PfGR is a 2 × 55 kD homodimer containing FAD and catalytic disulfide in each subunit, which catalyzes the reduction of glutathione (GSSG) at the expense of NADPH [29]. Human erythrocyte host glutathione reductase (HGR) possesses 45% amino acid sequence identity with PfGR and its role in the parasite survival is a matter of debate. Both PfGR and HGR are inhibited by aromatic electron-deficient compounds which were observed to bind at the dimer interface [27,28]. Our previous study demonstrated the absence of relationship between the antiplasmodial activity of nitroaromatic compounds and their efficacy as HGR inhibitors [19]. However, the relationship between the PfGR inhibition and the antiplasmodial activity of nitroaromatics has not been studied so far.
Extending our previous studies [19,30], here we demonstrate that the in vitro antiplasmodial activity of nitroaromatic compounds partly correlates with their efficacy as PfGR inhibitors, and partly with their reactivity with single-electron transferring P. falciparum ferredoxin:NADP+ oxidoreductase (PfFNR). Given the data currently available, PfFNR may act as the most efficient generator of ArNO2 free radicals in Plasmodium.

2. Results

2.1. Relationship between Antiplasmodial Activity of Nitroaromatic Compounds and Their Single-Electron Reduction Midpoint Potential

The toxicity of nitroaromatic compounds against mammalian cells and bacteria often increases with their single-electron reduction midpoint potential (redox potential of ArNO2/ArNO2− couple, E17). The relationship ∆log IC50/∆E17 ~ −10 V−1, where IC50 is the compound concentration for 50% cell survival or, in the case of bacteria or parasites, for 50% growth inhibition, indicates that the main factor of cytotoxicity is redox cycling and oxidative stress [4,8,31]. Indeed, the rates of single-electron reduction of ArNO2 by flavoenzymes dehydrogenases-electrontransferases, such as NADPH:cytochrome P-450 reductase, ferredoxin:NADP+ oxidoreductase, and NO-synthase that initiate their redox cycling, increase with E17 of oxidants, and are relatively insensitive to their structure [7,8,9,10].
In this work, we used a series of nitroaromatic compounds (Figure 1, Table 1) with available E17 values. For a major part of them, the IC50 values against the chloroquine-resistant P. falciparum strain FcB1 and the inhibition efficacy against HGR were characterized in a previous work [19]. Among the examined compounds, the representatives of vinylquinoline-substituted nitrofurans (IIIa–IIIh, Figure 1) possess well-promising diverse properties such as inhibition of trypanothione reductase, that is, the potential trypanocidal activity [12] as well as bactericidal and antitumor in vitro activity [32,33], the latter property gaining increasing interest [34]. Nitrobenzenes, nitrofurantoin, and nifuroxime (compounds 1–9,12,14,23, Table 1) were used as model compounds. Table 1 reports the IC50 values of compounds against P. falciparum strain FcB1, their E17 values, and their calculated octanol/water distribution coefficients (log D). Importantly, the IC50 values for several nitroaromatic compounds obtained in separate studies were sufficiently close (Table 1). For quantitative analysis, when available, data obtained within the current work were used. The regression analysis of the log IC50 versus E17 relationship yielded a ratio ∆log IC50/∆E17 = −8.37 ± 1.25 V−1 (r2 = 0.6802) (Figure S1, Supplementary Materials). The dependence of log IC50 on log D is poorly expressed (r2 = 0.2913, Figure S2 Supplementary Materials). However, an introduction of compound log D as a second independent variable resulted in some improvement of the correlation:
log IC50 = −(0.65 ± 0.44) – (7.39 ± 1.34) E17 – (0.12 ± 0.07) log D, (r2 = 0.7193)
This shows that the oxidant potency of nitroaromatics and, to some extent, their lipophilicity play definite roles in their antiplasmodial activity.

2.2. Single-Electron Reduction of Nitroaromatics by PfFNR and PfGR

To the best of our knowledge, the pathways of reduction of nitroaromatic compounds in P. falciparum are not yet well understood. Among flavoenzymes dehydrogenases-electrontransferases that can initiate redox cycling of ArNO2, a potential candidate is ferredoxin:NADP+ oxidoreductase localized in the apicoplast of the parasites [36,37]. This enzyme plays a significant role in parasite survival, because the functional analysis of P. falciparum genome revealed a high fitness cost of disruption of its gene [38]. Table 1 lists the bimolecular reduction rate constants of ArNO2 by PfFNR (kcat/Km). The kcat values of the reactions were not determined because of a nearly linear dependence of reaction rate on ArNO2 concentration except for the most reactive oxidant tetryl (kcat = 27.5 ± 2.0 s−1).
Figure 2 shows the linear relationship between log kcat/Km and E17 of nitroaromatics characterized by the ratio ∆log (kcat/Km)/∆E17 = 11.69 ± 0.73 V−1 (r2 = 0.9408), which mirrors to some extent the relationship between log IC50 and E17 (Equation (1)). PfFNR catalyzes a single-electron reduction of ArNO2, as demonstrated by the observation that in the presence of compounds 5,12,14, and 23 (Table 1), the reduction of added cytochrome c takes place at rates that are 140–195% those of NADPH oxidation. Moreover, the reduction of cytochrome c is 15–25% inhibited by 100 U/mL superoxide dismutase. The redox cycling of ArNO2 is also evident from the consumption of excess O2 over ArNO2 during the reaction (Figure 3A). However, it is also important to note that PfFNR-catalyzed formation of stable products of ArNO2 reduction does not start after complete O2 exhaustion, but takes place at [O2] = 40–50 µM after an initial lag time (in the case of N-methylpicramide or nitrofurantoin), or starts even without delay (in the case of tetryl or p-dinitrobenzene) (Figure 3B).
Both yeast and erythrocyte GR catalyze the single-electron reduction of nitroaromatics, although at a low rate [19,25]. Since the formed ArNO2− undergoes further redox cycling, nitroaromatics are considered as “subversive substrates“ for GR. Moreover, there is some evidence that redox-active xenobiotics may be reduced at the NADP(H) binding site of GR [39,40]. It is supposed that the same reactions catalyzed by trypanothione reductase are at least partly responsible for the trypanocidal activity of nitrofurans [11]. Among the examined compounds, tetryl oxidized PfGR most efficiently (kcat = 5.9 ± 0.5 s−1 and kcat/Km = 7.6 ± 0.8 × 103 M−1·s−1), with parameters similar to those of HGR oxidation (kcat ≥ 5.0 s−1, kcat/Km = 2.0 × 103 M−1·s−1 [37]). When present, cytochrome c was reduced at rates that are 170–180% those of NADPH oxidation, through a process partly inhibited by superoxide dismutase. 2,4,6-Trinitrotoluene (TNT) (kcat = 0.2 ± 0.05 s−1, kcat/Km = 150 ± 40 M−1·s−1), 1,4-dinitrobenzene (kcat = 0.3 ± 0.07 s−1, kcat/Km = 300 ± 60 M−1·s−1), and nifuroxime (kcat ≤ 0.06 s−1, kcat/Km ≤ 110 M−1·s−1) oxidized PfGR much more slowly. Other nitroaromatic compounds were even less efficient oxidants of PfGR. At their saturating concentrations, the rate of NADPH oxidation was almost indistinguishable from the intrinsic NADPH-oxidase activity of the enzyme, 0.07 s−1. This is in line with the previously reported properties of yeast and erythrocyte GR [19,25]. In conclusion, PfGR-catalyzed redox cycling of nitroaromatics proceeds with much lower rates than in the analogous reaction of PfFNR.

2.3. Inhibition of P. falciparum Glutathione Reductase by Nitroaromatic Compounds

Next, we analyzed the inhibition of P. falciparum GR by ArNO2. PfGR acts through a “ping-pong“ reaction mechanism with separate reductive and oxidative half-reactions [29]. At saturating concentrations of substrates, 100 μM NADPH and 1.0 mM GSSG, the catalytic constant (kcat) of PfGR was 138 ± 4.0 s−1. Nitroaromatic compounds acted on PfGR as non- or uncompetitive inhibitors with respect to GSSG at fixed NADPH concentration (Figure 4A,B). Similarly, nitroaromatics acted as uncompetitive inhibitors with respect to NADPH at fixed GSSG concentration (Figure 5).
These findings are in line with the well-characterized inhibition of yeast and human erythrocyte GR by nitroaromatics [19,25], and PfGR and HGR by arylisoalloxazines and quinones [27,30,41]. The latter compounds may bind at the interface domain of two subunits of PfGR in the vicinity of Val-56,56′ and Asp-58,58′, which correspond to His-75,75′ and Phe-78,78′ in HGR [27]. This site is distant from both the NADP(H)- and GSSG-binding regions. The Ki values of nitroaromatic compounds of PfGR determined using GSSG as a variable substrate (Figure 4A,B), are given in Table 2. The table also contains Ki values of nitroaromatic compounds towards HGR determined previously [19] and partly within the present work. Importantly, the Ki values for several nitroaromatic compounds obtained in both studies were sufficiently close (Table 2). It should be noted that significant differences exist between HGR and PfGR inhibition constants, and that their log values are poorly related (r2 = 0.3840). This may be attributed to differences in the structure, shape, and flexibility of the intersubunit regions of PfGR and HGR [24,25]. However, the analysis of the inhibition profile of the two enzymes is beyond the scope of the present work.
The analysis of antiplasmodial activity of nitroaromatics (Table 1 and Table 2) shows that the dependence of log IC50 on log Ki is poorly expressed in the case of PfGR, being characterized by r2 = 0.5878 (Figure S3, Supplementary Materials). An introduction of log D as a second variable improved the correlation:
log IC50 = (0.59 ± 0.23) + (0.61 ± 0.11) log Ki – (0.21 ± 0.07) log D, (r2 = 0.7253)
Although the log IC50 of nitroaromatics also increased with their log Ki for HGR (Table 2), this dependence was poorly expressed (r2 = 0.3323). An introduction of log D as a second variable improved it up to r2 = 0.4543. Finally, the antiplasmodial activity of nitroaromatics was best described by a regression using E17, log Ki for PfGR, and log D as independent variables:
log IC50 = −(0.27 ± 0.43) – (4.16 ± 1.78) E17 + (0.36 ± 0.14) log Ki – (0.15 ± 0.07) log D, (r2 = 0.7866)
On the other hand, the use of log Ki for HGR as a variable resulted in a lower regression coefficient, and in an uncertain relationship between log IC50 and log Ki:
log IC50 = −(0.70 ± 0.46) – (8.24 ± 1.90) E17 − (0.10 ± 0.15) log Ki – (0.13 ± 0.08) log D, (r2 = 0.7252)

3. Discussion

Our study resolves the debated problem about the mechanisms of antiplasmodial activity of nitroaromatics [19,23,26], and demonstrates that their activity increases both with the ease of their bioreductive activation, expressed as the value of E17, and the efficiency of inhibition of PfGR (Equation (3)). These data complement our previous findings on the role of inhibition of PfGR in the activity of quinones against the same strain [30]. In both cases, the relationship between the activity of compounds and their efficiency as PfGR inhibitors is revealed using multiparameter regression analysis. On the analogy with Equation (3), the activity of quinones was described by the relationships with ∆log IC50/∆log Ki = 0.633 – 0.763 [30]. These observations point to the importance of PfGR as potential target for antiplasmodial agents. Apart from protection against the oxidative stress, PfGR supplies GSH for the glyoxalase system, for the degradation of uncrystallized ferriprotoporphyrin IX, and as a source of reducing equivalents for ribonucleotide synthesis and thioredoxin-dependent antioxidant system [43,44,45,46]. Although no PfGR knockout data are available in the case of P. falciparum, the functional analysis of its genome revealed a high fitness cost of disruption of the PfGR gene [38]. On the other hand, the comparison between Equations (3) and (4) points to an insignificant role of HGR inhibition in the antiplasmodial action of nitroaromatics. Although the role of HGR in the survival of P. falciparum is a matter of debate [2,47], our data are in favor of its minor importance for parasite killing. However, this does not discard its role in the protection against an oxidative environment which limits the parasite infection rate, as it is in the case of glucose-6-phospate dehydrogenase deficiency [40]. In this context, one may note that the relationship ∆log IC50/∆log Ki (Equation (3)) is significantly lower than unity. It may point to a limited role of inhibition of PfGR in the antiplasmodial activity of nitroaromatics. On the other hand, the disturbance of GSSG/GSH homeostasis under the oxidative or alkylative stress in certain cases may enhance the expression of GR as a compensation mechanism. This phenomenon has been observed in yeast, plants, and mammalian cells [48,49,50,51].
In this context, two other issues related to the mechanism of antiplasmodial activity of ArNO2 may be discussed. First is the dependence of their IC50 values on their E17 (Equation (1)), the latter being associated with their redox cycling activity. However, because P. falciparum, during its intraerythrocyte stage, adopts microaerophilic metabolism and relies mainly on anaerobic processes [52,53], the role of the ROS-promoted parasite death should be interpreted with caution. On the other hand, Equation (1) may equally well reflect the rates of formation of DNA-damaging hydroxylamines under the action of single-electron transferring enzymes such as PfFNR (Figure 3A,B) which may take place under partly anaerobic conditions. Another point is the role of PfFNR in the formation of free radicals and/or other reduced forms of ArNO2 in plasmodia. To the best of our knowledge, nitroreductase activity of P. falciparum flavoenzymes has not been previously evaluated. Some conclusions may be drawn from the enzyme reactivity with a model compound menadione (2-methyl-1,4-naphthoquinone, E17 = −0.20 V) (Table 3), because, as a rule, flavoenzymes reduce quinones much faster than nitroaromatics, or, in exceptional cases, with similar rates ([7,53], and references therein). Thus, given the data currently available (Table 3), PfFNR may act as the most efficient generator of ArNO2 free radicals in plasmodia. P. falciparum thioredoxin reductase may be next to it according to menadione reductase activity (Table 3). On the other hand, the nitroreductase activity of mitochondrial type II NADH dehydrogenase is expected to be very low because menadione is slowly reducible. To the best of our knowledge, nitroaromatic compounds and soluble quinones have not been previously studied as oxidants of other enzymes of the mitochondrial respiratory chain, namely dihydroorotate dehydrogenase, succinate dehydrogenase, and malate:quinone oxidoreductase.
A more general conclusion following from our study is that the efficacy of redox active antiparasitic agents such as quinones, nitroaromatic compounds, isoalloxazines, and aromatic N-oxides [6] may be achieved through two separate types of action, namely inhibition of antioxidant NADPH:disulfide reductases and rapid reduction by flavoenzymes dehydrogenases- electrontransferases. Currently, these groups of compounds are mainly considered as “subversive substrates” of disulfide reductases such as glutathione reductase, trypanothione reductase, or thioredoxin reductase [11,30,40,54,56]. However, the single-electron reduction of the above compounds by dehydrogenases-electrontransferases of different origin (e.g., mammalian NADPH: cytochrome P-450 reductase, NO-synthase, NADH:ubiquinone reductase, algal FNR, and bacterial flavohemoglobin) is usually faster [7,8,9,10]. Thus, more attention should be given to the studies of these reactions catalyzed by parasite enzymes of this group.

4. Materials and Methods

4.1. Materials

Recombinant PfGR and P. falciparum ferredoxin:NADP+ oxidoreductase were prepared as previously described [29,36], and their concentrations were determined spectrophotometrically according to ε461 = 11.7 mM−1·cm−1 and ε461 = 10.1 mM−1 cm−1, respectively. Recombinant HGR was obtained from Sigma-Aldrich (St. Louis, MO, USA), and its concentration was determined according to ε464 = 11 mM−1·cm−1.
Nitrobenzene derivatives 1,2,4–9,12 and nitrofurans 10,11 (Table 1) were obtained from Sigma-Aldrich (St. Louis, MO, USA) and used as received. TNT, 2,4,6-trinitrophenyl-N- methylnitramine (tetryl) (Figure 1), and N-methylpicramide were synthesized as described in [57,58]. 5-(Aziridin-1-yl)-2,4-dinitrobenzamide (CB-1954, Figure 1), synthesized as described in [59], was a generous gift of Dr. Vanda Miškinienė (Institute of Biochemistry, Vilnius, Lithuania). Vinylquinoline-substituted nitrofurans IIIa–h (Figure 1) were synthesized as described in [60,61]. All synthesized compounds were previously verified by determining their melting point, as well as their 1H-NMR, UV, and IR spectra [8,19,25]. The purity of compounds, determined using a high-performance liquid chromatography system equipped with a mass spectrometer (LCMS-2020, Shimadzu, Kyoto, Japan), was >98%. Cytochrome c, NADPH, GSSG, glucose-6-phosphate, glucose-6-phospate dehydrogenase, superoxide dismutase, and other compounds were obtained from Sigma-Aldrich (St. Louis, MO, USA) and used as received.

4.2. Methods

4.2.1. Enzyme Kinetic Studies

All kinetic experiments were carried out spectrophotometrically using a PerkinElmer Lambda 25 UV–VIS spectrophotometer (PerkinElmer, Waltham, MA, USA) in 0.1 M K-phosphate buffer (pH 7.0) containing 1 mM EDTA at 25 °C. The steady-state parameters of reactions, the catalytic constants (kcat(app.)), and the bimolecular rate constants (or catalytic efficiency constants, kcat/Km) of the oxidants at fixed concentrations of NADPH correspond to the reciprocal intercepts and slopes of Lineweaver–Burk plots, [E]/v vs. 1/[oxidant], where v is the reaction rate, and [E] is the enzyme concentration. kcat represents the number of molecules of NADPH oxidized by a single active center of the enzyme per second. The rates of PfFNR- and PfGR-catalyzed NADPH oxidation in the presence of nitroaromatic compounds or GSSG were determined using the value Δε340 = 6.2 mM−1·cm−1. The rates were corrected for the intrinsic NADPH-oxidase activity of enzymes, which were equal to 0.12 s−1 and 0.07 s−1 for PfFNR and PfGR, respectively. In separate experiments, in which 50 µM cytochrome c were included in the reaction mixture, its nitroaromatic-mediated reduction was measured using the value Δε550 = 20 mM−1·cm−1. The kinetic parameters were obtained by the fitting of kinetic data to the parabolic expression using SigmaPlot 2000 version 11.0 (https://systatsoftware.com). The rates of reduction of nitroaromatic compounds (50 μM) by PfFNR in the absence of external oxygen supply were monitored in the presence of an NADPH-regeneration system (50 μM NADPH, 10 mM glucose-6-phosphate, and 50 U/mL yeast glucose-6-phosphate dehydrogenase) at the specific λmax of absorbance of compounds. In these cases, a sealed spectrophotometer cell was completely filled by the solution containing nitroaromatic compound and NADPH-regeneration system, and the reaction was initiated by the injection of PfFNR. In parallel, the rate of oxygen consumption was monitored under identical conditions using a Digital Model 10 Clark electrode (Rank Brothers Ltd., Bottisham, UK). In reversible inhibition studies of PfGR, reaction rates were determined either at fixed NADPH concentration (100 μM) and varied GSSG concentrations (1.0–0.13 mM), or at fixed GSSG concentration (1.0 mM) and varied NADPH concentrations (8–50 μM), and either in the absence or presence of the inhibitor at 4–6 different concentrations. Using tetryl as inhibitor, the reaction rates were corrected for PfGR- catalyzed NADPH oxidation by tetryl, which was typically less than 0.4% of total reaction rate. Since CB-1954 possessed significant absorbance at 340 nm, the reaction rate of PfGR was monitored according to GSH-mediated reduction of 5.5‘-dithiobis-(2-nitrobenzoic acid) (1.0 mM) using the value ∆ε412 = 27.2 mM−1·cm−1. The inhibition constants (Ki) were obtained from the Cleland plots, that is, the dependence of 1/kcat on the inhibitor concentration ([I]).

4.2.2. Antiplasmodial In Vitro Activity Studies

The chloroquine-resistant P. falciparum strain FcB1 from Colombia, which is deposited in the Protist collection of Museum National d’Histoire Naturelle, Paris, France, was kindly provided by Dr. H.D. Heidrich (Max-Planck Institut für Biochemie, Martinsried bei München, Germany). P. falciparum FcB1 strain was maintained in continuous culture of human erythrocytes according to [62]. In vitro antiplasmodial activity was determined using a modification of the semiautomatic microdilution technique [63]. Stock solutions of test compounds in DMSO were serially diluted with culture medium and added to asynchronous parasite cultures (1% parasite infected cells and 1% final hematocrit) for 24 h, at 37 °C, prior to the addition of 1.825 MBq of [3H]-hypoxanthine (0.37–1.11 TBq/mmol), for 24 h. The growth inhibition for each compound concentration was determined according to the radioactivity incorporation into the treated culture as compared with that in the control culture. The experiments were repeated in triplicate.

4.2.3. Statistical Analysis and Calculations

The octanol/water distribution coefficients at pH 7.0 (log D) of compounds were calculated using LogD Predictor (https://chemaxon.com). The multiparameter regression analysis was performed using Statistica (version 4.3, StatSoft, Toronto, ON, Canada).

Supplementary Materials

The following are available online. Figure S1: Dependence of activity of nitroaromatic compounds against P. falciparum FcB1(IC50) on the values of their single-electron reduction midpoint potential (E17), Figure S2: Dependence of activity of nitroaromatic compounds against P. falciparum FcB1(IC50) on the values of their log D, Figure S3: Dependence of activity of nitroaromatic compounds against P. falciparum FcB1(IC50) on the values of their inhibition constant (Ki) of P. falciparum glutathione reductase.

Author Contributions

A.M. and M.L. performed kinetic experiments, E.D.-C. and A.A. purified enzymes, P.G. performed antiplasmodial activity studies, J.Š. synthesized compounds, N.Č. designed and supervised the experiments and wrote the manuscript.

Funding

This work was supported by the European Social Fund (Measure No. 09.33-LMT-K-712, Grant No. DOTSUT-34/09.3.3.-LMT-K712-01-0058/LSS-600000-58) (A.M., M.L., J.Š., N.Č.), and by a grant from the Laboratoire d’Excellence ParaFrap (LabEx ParaFrap ANR-11-LABX-0024) and the ANR-PRC program (grant under the PlasmoPrim project) (E.D.-C.). J.Š. and E.D.C. (MC members) thank the COST Action CM1307, entitled “Targeted Chemotherapy towards Diseases caused by Endoparasites” for covering the exchanges and the costs to publish the Special Issue of Molecules “COST Action CM1307—Proceedings in Medicinal and Natural Product Chemistry”.

Acknowledgments

We thank Justinas Radvila for excellent technical assistance.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Abbreviations

ArNO2Nitroaromatic compound
E17Single-electron reduction midpoint potential of nitroaromatic compound (redox potential of ArNO2/ArNO2−. couple) at pH 7.0
HGRHuman erythrocyte glutathione reductase
IC50Compound concentration causing 50% parasite growth inhibition
kcatEnzyme catalytic constant
kcat/KmEnzyme bimolecular rate constant (catalytic efficiency)
KiEnzyme inhibition constant
log DOctanol/water distribution coefficient at pH 7.0
PfFNRP. falciparum ferredoxin:NADP+ oxidoreductase
PfGRP. falciparum glutathione reductase
ROSReactive oxygen species
TNT2,4,6-Trinitrotoluene
TRTrypanothione reductase

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Sample Availability: Samples of the compounds 3,15–22 (Table 1) are available from the authors.
Molecules 24 04509 g001 550
Figure 1. Formulae of nontrivial nitroaromatic compounds studied in this work: I, tetryl; II, CB-1954; and III, vinylquinoline-substituted nitrofurans.
Figure 1. Formulae of nontrivial nitroaromatic compounds studied in this work: I, tetryl; II, CB-1954; and III, vinylquinoline-substituted nitrofurans.
Molecules 24 04509 g001
Molecules 24 04509 g002 550
Figure 2. Relationship between the reactivity of nitroaromatic compounds in PfFNR-catalyzed reactions (log kcat/Km) and their single-electron reduction midpoint potentials (E17). The numbers of compounds correspond to those in Table 1.
Figure 2. Relationship between the reactivity of nitroaromatic compounds in PfFNR-catalyzed reactions (log kcat/Km) and their single-electron reduction midpoint potentials (E17). The numbers of compounds correspond to those in Table 1.
Molecules 24 04509 g002
Molecules 24 04509 g003 550
Figure 3. (A) Time course of oxygen consumption and (B) spectral changes during reduction of tetryl (trace 1), 1,4-dinitrobenzene (trace 2), nitrofurantoin (trace 3), and N-methylpicramide (trace 4) by 50 nM PfFNR and NADPH-regeneration system under the absence of external oxygen supply. Compound concentration, 50 µM, absorbance monitored at 420 nm (tetryl), 340 nm (1,4-dinitrobenzene), 420 nm (nitrofurantoin), and 343 nm (N-methylpicramide). The arrows indicate the time of introduction of PfFNR.
Figure 3. (A) Time course of oxygen consumption and (B) spectral changes during reduction of tetryl (trace 1), 1,4-dinitrobenzene (trace 2), nitrofurantoin (trace 3), and N-methylpicramide (trace 4) by 50 nM PfFNR and NADPH-regeneration system under the absence of external oxygen supply. Compound concentration, 50 µM, absorbance monitored at 420 nm (tetryl), 340 nm (1,4-dinitrobenzene), 420 nm (nitrofurantoin), and 343 nm (N-methylpicramide). The arrows indicate the time of introduction of PfFNR.
Molecules 24 04509 g003
Molecules 24 04509 g004 550
Figure 4. (A) Inhibition of P. falciparum glutathione reductase (PfGR) by nitrofuran IIIa and by TNT (B) at fixed NADPH concentration, 100 µM, and varied concentrations of GSSG. (A) Concentrations of nitrofuran IIIa: 0.0 µM (line 1), 6.7 µM (line 2), 10.0 µM (line 3), 15.0 µM (line 4), 22.2 µM (line 5), and 50 µM (line 6). (B) Concentrations of TNT: 0.0 µM (line 1), 13.2 µM (line 2), 19.7 µM (line 3), 29.6 µM (line 4), 44.4 µM (line 5), and 100 µM (line 6).
Figure 4. (A) Inhibition of P. falciparum glutathione reductase (PfGR) by nitrofuran IIIa and by TNT (B) at fixed NADPH concentration, 100 µM, and varied concentrations of GSSG. (A) Concentrations of nitrofuran IIIa: 0.0 µM (line 1), 6.7 µM (line 2), 10.0 µM (line 3), 15.0 µM (line 4), 22.2 µM (line 5), and 50 µM (line 6). (B) Concentrations of TNT: 0.0 µM (line 1), 13.2 µM (line 2), 19.7 µM (line 3), 29.6 µM (line 4), 44.4 µM (line 5), and 100 µM (line 6).
Molecules 24 04509 g004
Molecules 24 04509 g005 550
Figure 5. Inhibition of PfGR by nitrofuran IIIa at fixed GSSG concentration, 1.0 mM, and varied concentrations of NADPH. Concentrations of inhibitor: 0.0 µM (line 1), 10.0 µM (line 2), 20.0 µM (line 3), 30.0 µM (line 4), 45.0 µM (line 5), and 66.7 µM (line 6).
Figure 5. Inhibition of PfGR by nitrofuran IIIa at fixed GSSG concentration, 1.0 mM, and varied concentrations of NADPH. Concentrations of inhibitor: 0.0 µM (line 1), 10.0 µM (line 2), 20.0 µM (line 3), 30.0 µM (line 4), 45.0 µM (line 5), and 66.7 µM (line 6).
Molecules 24 04509 g005
Table
Table 1. Single-electron reduction midpoint potentials (E17) of nitroaromatic compounds, their concentrations for 50% Plasmodium falciparum growth inhibition (IC50), their calculated octanol/water distribution coefficients at pH 7.0 (log D), and their apparent bimolecular reduction rate constants by Plasmodium falciparum ferredoxin:NADP+ oxidoreductase (PfFNR) (kcat/Km).
Table 1. Single-electron reduction midpoint potentials (E17) of nitroaromatic compounds, their concentrations for 50% Plasmodium falciparum growth inhibition (IC50), their calculated octanol/water distribution coefficients at pH 7.0 (log D), and their apparent bimolecular reduction rate constants by Plasmodium falciparum ferredoxin:NADP+ oxidoreductase (PfFNR) (kcat/Km).
No.CompoundE17 (V) [35]IC50 (µM) [19]log Dkcat/Km (M−1·s−1)
1Nitrobenzene−0.485473 ± 1131.915.5 ± 0.8 × 101
24-Nitrobenzoic acid−0.425360 ± 16; 450 ± 70.7 b−1.664.5 ± 0.6 × 102
3CB-1954−0.38048.5 ± 5.0 b0.642.8 ± 0.3 × 103
44-Nitroacetophenone−0.355172 ± 8.01.473.3 ± 0.3 × 103
53,5-Dinitrobenzoic acid−0.350390 ± 17−1.793.9 ± 0.3 × 103
61,3-Dinitrobenzene−0.34550.5 ± 2.41.852.7 ± 0.3 × 103
74-Nitrobenzaldehyde−0.32579 ± 281.634.0 ± 0.3 × 103
83,5-Dinitrobenzamide−0.31130.3 ± 3.1; 26.5 ± 6.4 b0.74.9 ± 0.4 × 103
91,2-Dinitrobenzene−0.28711.7 ± 1.11.851.1 ± 0.2 × 104
10Nitrofurantoin−0.25512.9 ± 1.3−0.256.8 ± 0.7 × 104
11Nifuroxime−0.25514.7 ± 0.8−0.343.3 ± 0.4 × 104
121,4-Dinitrobenzene−0.2550.26 ± 0.031.859.3 ± 0.8 × 104
132,4,6-Trinitrotoluene−0.2539.4 ± 7.8 b2.311.3 ± 0.1 × 104
14N-Methylpicramide−0.2477.3 ± 1.1 b1.926.8 ± 0.5 × 104
15Nitrofuran IIIa−0.225 a17.1 ± 1.50.274.8 ± 0.5 × 104
16Nitrofuran IIIb−0.225 a4.5 ± 0.32.641.6 ± 0.2 × 105
17Nitrofuran IIIc−0.225 a7.4 ± 0.32.87n.d.
18Nitrofuran IIId−0.225 a7.4 ± 0.33.23n.d.
19Nitrofuran IIIe−0.225 a9.2 ± 0.32.24n.d.
20Nitrofuran IIIf−0.225 a11.1 ± 0.42.62n.d.
21Nitrofuran IIIg−0.225 a6.4 ± 0.42.458.3 ± 0.7 × 104
22Nitrofuran IIIh−0.225 a4.3 ± 0.32.62n.d.
23Tetryl−0.1914.1 ± 0.8 b1.382.0 ± 0.3 × 105
a The E17 values are taken from [19], b The values of IC50 determined in this work. n.d., not determined.
Table
Table 2. Inhibition constants of nitroaromatic compounds acting on P. falciparum and human erythrocyte glutathione reductases (HGRs), calculated under constant concentration of NADPH (100 μM) and varied concentration of GSSG.
Table 2. Inhibition constants of nitroaromatic compounds acting on P. falciparum and human erythrocyte glutathione reductases (HGRs), calculated under constant concentration of NADPH (100 μM) and varied concentration of GSSG.
No.CompoundKi (μM)
PfGR aHGR b
1Nitrobenzene≥6000≥2000
24-Nitrobenzoic acid1200 ± 180800
3CB-1954350 ± 40≥1000
44-Nitroacetophenone70 ± 11400
53,5-Dinitrobenzoic acid220 ± 29350
61,3-Dinitrobenzene40 ± 5.0320; 350 ± 30a
74-Nitrobenzaldehyde25 ± 4.0290
83,5-Dinitrobenzamide75 ± 9.0≥1000
91,2-Dinitrobenzene30 ± 4.0≥1000
10Nitrofurantoin9.0 ± 1.0200
11Nifuroxime32 ± 5.0200
121,4-Dinitrobenzene0.85 ± 0.1371
132,4,6-Trinitrotoluene8.0 ± 2.06.0 c; 5.2 ± 0.6 a
14N-Methylpicramide5.9 ± 0.610 c
15Nitrofuran IIIa9.0 ± 1.03.0; 3.5 ± 0.2 a
16Nitrofuran IIIb25 ± 3.02.5
17Nitrofuran IIIc115 ± 1725
18Nitrofuran IIId50 ± 6.0≥300
19Nitrofuran IIIe5.0 ± 1.02.5
20Nitrofuran IIIf75 ± 1042.5
21Nitrofuran IIIg35 ± 5.025
22Nitrofuran IIIh100 ± 1245
23Tetryl2.3 ± 0.514 c
aKi determined in present work, b taken from [19], c taken from [42].
Table
Table 3. Kinetic characterization of nitro- and quinone reductase reactions of Plasmodium falciparum flavoenzymes.
Table 3. Kinetic characterization of nitro- and quinone reductase reactions of Plasmodium falciparum flavoenzymes.
EnzymeOxidants
Nitrofurans, NitrobenzenesMenadione
E17 = −0.25–−0.19 V
PfFNRkcat > 20 s−1, kcat/Km = 4.8 × 104 – 1.6×105 M−1·s−1, this workkcat = 14 s−1, kcat/Km = 1.0 ×106 M−1·s−1 [30]
PfGRkcat = 0.06–5.9 s−1, kcat/Km = 7.6 × 103 – 110 M−1·s−1, this workkcat = 0.16 s−1, kcat/Km = 2.0 × 103 M−1·s−1 [54]
P. falciparum thioredoxin reductase kcat = 31 s−1, kcat/Km = 1.6 × 105 M−1·s−1 [54]
P. falciparum type II NADH dehydrogenase kcat = 0.1 s−1 [55]

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Marozienė, A.; Lesanavičius, M.; Davioud-Charvet, E.; Aliverti, A.; Grellier, P.; Šarlauskas, J.; Čėnas, N. Antiplasmodial Activity of Nitroaromatic Compounds: Correlation with Their Reduction Potential and Inhibitory Action on Plasmodium falciparum Glutathione Reductase. Molecules 2019, 24, 4509. https://doi.org/10.3390/molecules24244509

AMA Style

Marozienė A, Lesanavičius M, Davioud-Charvet E, Aliverti A, Grellier P, Šarlauskas J, Čėnas N. Antiplasmodial Activity of Nitroaromatic Compounds: Correlation with Their Reduction Potential and Inhibitory Action on Plasmodium falciparum Glutathione Reductase. Molecules. 2019; 24(24):4509. https://doi.org/10.3390/molecules24244509

Chicago/Turabian Style

Marozienė, Audronė, Mindaugas Lesanavičius, Elisabeth Davioud-Charvet, Alessandro Aliverti, Philippe Grellier, Jonas Šarlauskas, and Narimantas Čėnas. 2019. "Antiplasmodial Activity of Nitroaromatic Compounds: Correlation with Their Reduction Potential and Inhibitory Action on Plasmodium falciparum Glutathione Reductase" Molecules 24, no. 24: 4509. https://doi.org/10.3390/molecules24244509

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