Next Article in Journal
Evaluating the Effects of Sugar Shift® Symbiotic on Microbiome Composition and LPS Regulation: A Double-Blind, Placebo-Controlled Study
Previous Article in Journal
The Role of Inflammation in the Pathogenesis of Viral Respiratory Infections
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Microorganisms
Volume 12
Issue 12
10.3390/microorganisms12122524
microorganisms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Evaluation of In Vitro Inhibition of β-Hematin Formation: A Step Towards a Comprehensive Understanding of the Mechanism of Action of New Arylamino Alcohols

by
Céline Damiani
1,2,*,
Floriane Soler
2,
Yohann Le Govic
1,2,
Anne Totet
1,2,
Guillaume Bentzinger
1,
Anne Bouchut
1,
Romain Mustière
1,
Patrice Agnamey
1,
Alexandra Dassonville-Klimpt
1 and
Pascal Sonnet
1
1
Agents Infectieux, Résistance et Chimiothérapie (AGIR), UR 4294, Université de Picardie Jules Verne, 1 rue des Louvels, 80037 Amiens, France
2
Laboratoire de Parasitologie et Mycologie, Centre de Biologie Humaine, CHU Amiens-Picardie, 1 Rond-Point du Pr Cabrol, 80054 Amiens, France
*
Author to whom correspondence should be addressed.
Microorganisms 2024, 12(12), 2524; https://doi.org/10.3390/microorganisms12122524
Submission received: 29 October 2024 / Revised: 21 November 2024 / Accepted: 4 December 2024 / Published: 7 December 2024
(This article belongs to the Section Antimicrobial Agents and Resistance)
Browse Figures
Versions Notes

Abstract

:
Currently, artemisinin-based combination therapy is recommended as first-line treatment of uncomplicated falciparum malaria. Arylamino alcohols (AAAs) such as mefloquine (MQ) are the preferred partner drugs due to their longer half-life, reliable absorption and strong antimalarial activity. However, the mode of action of MQ remains poorly understood and its neurotoxicity limits its use. Furthermore, the emergence of drug-resistant parasites requires development of new antimalarial drugs. The aim of this study was to evaluate the β-hematin inhibition capacity of three pairs of enantiopure AAAs 1–3 (a/S and b/R) derived from MQ or enpiroline (ENP), a pyridine-based MQ analog with strong antimalarial activity. Inhibition of β-hematin—the synthetic counterpart of hemozoin formation—was determined for each compound. Antimalarial activity against W2 and 3D7 Plasmodium falciparum strains as well as percentages of inhibition of β-hematin formation were compared to those of reference molecules, i.e., chloroquine (CQ), MQ and ENP. Furthermore, a cytotoxicity study on the human-derived hepatocarcinoma cell line HepG2 was performed. With high antimalarial activity, stronger ability to inhibit β-hematin formation and low cytotoxicity, AAAs 1a-b and 2a are the most promising. These findings provide a better understanding of their potential mechanisms of action and may pave the way toward developing new lead compounds.

1. Introduction

Malaria remains one of the most significant infectious diseases in the world. In 2022, there were a total of 249 million new cases of malaria worldwide and 608,000 deaths, 76% of which were children under five [1]. Plasmodium falciparum (Pf) is the causative agent of the most dangerous form of the disease in humans. Nutrients and ions essential for the parasite’s growth and development are located in the host’s red blood cells (RBC). During intraerythrocytic growth, trophozoite stages of Pf can digest up to 80% of the RBC’s hemoglobin content to source amino acids required for protein production [2]. Hemoglobin is then degraded by proteases within the parasite’s acidic digestive vacuole (DV) into two entities, namely heme and globin. Globin is cleaved by enzymes into amino acids, essential for parasite metabolic functions and growth. Heme is initially oxidized into hematin, a toxic ferriprotoporphyrin IX (Fe(III)PPIX) whose accumulation can lead to Plasmodium death through reactive oxygen species (ROS) production. To avoid toxicity, Plasmodium parasites neutralize hematin into inert and insoluble hemozoin (Hz) crystals. Hz, whose synthetic counterpart is called β-hematin, represents an attractive target for common antimalarial drugs since aminoquinolines like chloroquine (CQ) and arylamino alcohols (AAAs) such as mefloquine (MQ, Figure 1) interfere with the crystallization process (Scheme 1) [3,4]. According to different studies [5,6,7], CQ and MQ are able to inhibit in vitro hemozoin formation by up to 90% and from 6% to 70%, respectively. The inhibition of hemozoin formation is the main mechanism of action of CQ. MQ has several targets within the parasites (including disruption of protein synthesis, impairment of lipid-binding proteins function and the induction of apoptosis) and is also able to prevent hemozoin formation even though it is not its main mechanism of action [8,9,10,11,12]. The mechanism of action of enpiroline (ENP, Figure 1) is not understood; notably, there is no data on the percentage of inhibition of β-hematin formation.
Nowadays, due to the parasite’s resistance to antimalarial drugs, and especially against artemisinin and its derivatives, agents displaying innovative chemistry have to be developed. Owing to their schizonticidal activity and good pharmacokinetic properties such as a long half-life, AAAs are interesting partners for artemisinin-based combination therapies (ACTs) [14]. Mainly used in combination with artesunate, MQ is one of the WHO Model List of Essential Medicines and has been commercially available since the 1970s as a racemic mixture of erythro enantiomers. However, MQ exhibits neurotoxic effects by disrupting calcium ion (Ca2+) homeostasis, resulting in a massive release of Ca2+ and ROS production [15,16]. Furthermore, it has been shown that (−)-RS-MQ enantiomer accumulates strongly in the central nervous system [17,18]. High affinity for brain adenosine receptors suggests its implication in neurological events associated with MQ usage [19]. In contrast, better antimalarial efficiency and lower neurotoxicity have been reported for (+)-SR-MQ enantiomer. To avoid adverse effects as well as to increase MQ antiplasmodial activity, the synthesis of quinoline analogs with an open-chain has been proposed [20].
In this context, we developed enantiopure analogs with an open-chain [6,21,22]. Thus, novel enantiopure MQ analogs (Series 1, Figure 2) with a linear alkyl chain (C4-C7, n = 2–5) have been synthesized in five steps with good yields and excellent enantiomeric excesses. Their antimalarial activities ranged from 12.7 to 254.0 nM against the MQ-reduced susceptibility strain Pf3D7, and from 7.0 to 142.0 nM against the CQ-resistant strain PfW2. In addition, (S)-enantiomers were 2–15 times more active than their (R)-counterpart, depending on the side chain length [6]. Then, focusing on the role of the aromatic ring, we dissociated the quinoline core to 6-phenylpyridines of Series 2 (Figure 2), considered as ENP analogs. The newly synthesized pyridines were more active than their quinoline counterparts (IC50 (Pf3D7) = 17.7–56.7 nM and IC50 (PfW2) = 3.5–10.0 nM), but clearly showed fewer differences between (S)- and (R)- enantiomers (1.1–3.1 times). From these 4-aminoalcohol pyridine derivatives, the alkyl chain position was modified to afford the corresponding 3-aminoalcohol pyridines (Series 3, Figure 2). The resulting compounds showed weaker antimalarial activity than compounds of Series 1 and 2, with IC50 values ranging from 97.0 to 1379.7 nM against Pf3D7 and from 24.4 to 522.8 nM against PfW2. However, eudysmic ratios were surprisingly higher than those of the other two Series (1.2–20 times), constantly in favor of (S)-enantiomer.
These previous works confirmed the interest in developing new enantiopure AAAs as MQ analogs (marketed drug), and ENP analogs (non-marketed drug) to fight against resistant strains of Pf. However, efforts should be made to better understand their mode of action, which could explain the differences in antimalarial activity observed in these three Series of AAAs (a/S and b/R). The aim of the present study was to evaluate the influence of the heterocyclic ring on the inhibition of hemozoin formation, which is assumed to be one of the main mechanisms of action of these compounds (Scheme 1). For this purpose, AAAs 1a-b, 2a-b and 3a-b were selected, because the structure of their alkyl amino alcohol chain (shown in blue in Figure 2) confers potent antimalarial activities. The corresponding results would enable us to establish new structure–activity relationships (SAR) considering β-hematin (synthetic Hz analog) as target. The β-hematin inhibition assay was performed to evaluate the corresponding inhibition percentages of compounds 1a-b, 2a-b and 3a-b and the three reference molecules CQ, MQ and ENP. To complete these data, a cytotoxicity study on the human-derived hepatocarcinoma cell line HepG2 was also carried out on the most active compounds to consider their selectivity index (SI).

2. Materials and Methods

2.1. Arylamino Alcohols Derivatives 1a-b, 2a-b and 3a-b

The enantioselective synthesis of AAA derivatives 1a-b, 2a-b and 3a-b was carried out according to the procedures previously described [23].
IC50s, corresponding to in vitro antimalarial activity against PfW2 and Pf3D7, was previously determined for compounds 2a-b, 3a-b and reference molecules using the SYBR Green I fluorescence-based method. As compounds 1a-b were previously screened using the hypoxanthine-tritied method [6], they were here re-assayed with the SYBR Green I fluorescence-based method described elsewhere [24,25] for comparison with the other two Series (2 and 3) and reference molecules. When possible, eudysmic ratios were calculated. The SYBR green I fluorescence-based method is detailed below.
Compounds were dissolved in DMSO and then diluted in sterile water in order to obtain a range of concentrations from 40 nM to 40 mM for the first screening against culture-adapted Plasmodium falciparum reference strains 3D7 and W2. The former strain is susceptible to CQ but displays a decreased susceptibility to MQ; the latter is considered resistant to CQ. These two strains were obtained from the collection of the National Museum of Natural History (Paris, France). The parasites were cultivated in RPMI medium (Sigma-Aldrich, Saint Quentin Fallavier, France) supplemented with 0.5% Albumax I (Life Technologies Corporation, Paisley, UK), hypoxanthine (Sigma-Aldrich, Saint Quentin Fallavier, France) and gentamicin (Sigma-Aldrich, Saint Quentin Fallavier, France) with human erythrocytes and were incubated at 37 °C with 5% CO2 [26]. The P. falciparum drug susceptibility test was carried out in 96-well flat-bottomed sterile plates in a final volume of 250 μL. After a 48 h incubation period with the drugs, quantities of DNA in treated and control cultures of parasites in human erythrocytes were quantified using the SYBR Green I (Sigma-Aldrich, Saint Quentin Fallavier, France) fluorescence-based method [27,28]. Briefly, after incubation, the plates were frozen at −20 °C until use. The plates were then thawed for 2 h at room temperature, and 100 μL of each homogenized culture was transferred to a well of a 96-well flat-bottomed sterile black plate (Sigma-Aldrich, Saint Quentin Fallavier, France) that contained 100 μL of the SYBR Green I lysis buffer (2xSYBR Green, 20 mM Tris base pH 7.5, 5 mM EDTA, 0.008% w/v saponin, 0.08% w/v Triton X-100). Negative controls treated with solvent (typically DMSO or H2O) and positive controls (CQ and MQ) were added to each set of experiments. Plates were incubated for 1 h at room temperature and then read on a fluorescence plate reader (Tecan Trading AG, Männedorf, Switzerland) using excitation and emission wavelengths of 485 and 535 nm, respectively. The concentrations at which the screening drug or antimalarial can inhibit 50% of parasitic growth (IC50s) were calculated from a sigmoid inhibition model Emax with an estimate of IC50s by non-linear regression (IC Estimator version 1.2) and were reported as means calculated from three independent experiments [29].

2.2. Inhibition of β-Hematin Formation

Inhibition of β-hematin formation of 1a-b, 2a-b, 3a-b and reference molecules was assessed using the assay previously described in [5,30]. Stock solutions (20 mM) of CQ-phosphate were prepared in water while MQ and AAA derivatives were solubilized in MeOH/DMSO (4/1, v/v). Inhibition experiments were carried out over a concentration range of 0–2 mM (final concentration of the drug in wells). Briefly, 100 µL of a fresh 6.5 mM solution of hemin (Sigma-Aldrich, SaintQuentin Fallavier, France) dissolved in 0.2 M NaOH was mixed with 100 µL of 3 M sodium acetate, 25 µL of 17.4 M acetic acid and 25 µL of the tested drug or controls (water and MeOH/DMSO). Magnetic stirring of the solution was used to avoid sedimentation. After 24 h incubation at 37 °C under shaking, the supernatant resulting from centrifugation for 15 min at 3300× g was discarded. The pellet was washed twice with 200 µL DMSO. After a final wash with water, the pellet was dissolved in 200 µL with 0.1 M NaOH. After a further 1:30 dilution, absorption at 405 nm was read using a microplate photometer (Multiskan® EX, Thermo Scientific, Waltham, MA, USA). CQ, MQ, ENP (considered as positive controls), water and MeOH/DMSO (considered as negative controls) were included in each experiment. Results were expressed as percentages of inhibition of β-hematin formation at 2 mM in comparison to the negative control result, calculated by the following formula [5]:
% inhibition = 100 × (1 − (OD molecule)/(OD control)).
Results were given as mean ± standard deviation of values obtained from three independent experiments and, when achievable, IC50 values were determined graphically.
Comparisons of percentages of inhibition of β-hematin formation were made using the Mann–Whitney signed rank test using Graphpad Prism 5.0 software. Statistical significance was defined as a value of p ≤ 0.05.

2.3. In Vitro Toxicity

The cytotoxicity of the most active compounds and reference molecules was evaluated with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay adapted from [31]. Briefly, in 96-well plates, 100 µL of a cell culture adjusted to 25,000 cells/mL was dispensed and the plates were incubated at 37 °C in a humidified 5% CO2 atmosphere for 24 h to allow the cells to adhere to the bottom of the wells. After microscopic verification of the adherent cells, the culture medium was removed and replaced with 100 μL of medium with the compounds at various concentrations dissolved in DMSO (final concentration less than 0.5% v/v), and the plates were incubated for 72 h at 37 °C. Controls were DMSO in medium (0.5% v/v) without compound as blank. Then, the supernatant from each well was removed prior to the addition of 100 µL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) solution (0.5 mg/mL in culture medium) per well. After 2 h of incubation, the supernatant from each well was removed and 100 µL of DMSO was added to dissolve the resulting blue formazan crystals accumulated at the bottom of the wells. The plates were incubated for 5 min at 37 °C in a humidified 5% CO2 atmosphere. Then, the wells were homogenized by shaking the plates for 10 min with a microplate shaker. The absorbance was read using the Tecan’s Sunrise™ absorbance microplate reader at 570 nm with 630 nm as the reference wavelength. The 50% cytotoxic concentration (CC50) values were determined by nonlinear regression analysis processed on dose–response curves, using the Quest Graph™ IC50 Calculator1. Results were given as mean ± standard deviation of values obtained from three independent experiments. Selectivity index (SI) was calculated and defined as the ratio between CC50 values on HepG2 and IC50.

3. Results

Results of in vitro antimalarial activity against PfW2 and Pf3D7 strains, inhibition of β-hematin formation and in vitro cytotoxicity results against HepG2 for 1–3 and reference molecules (CQ, MQ, ENP) are reported in Table 1 and Table 2.
ENP exhibited better antimalarial activity, whatever the Pf strain, compared with CQ and MQ (IC50 (PfW2) = 11.5 nM vs. 198.8 nM and 31.8 nM, respectively; IC50 (Pf3D7) = 21.6 nM vs. 75.9 nM and 79.7 nM, respectively). In addition, 1a-b were the more active compounds. In particular, they were more active than MQ on both PfW2 and Pf3D7 (IC50 (Pf3D7) = 15.1 nM and 15.1 nM vs. 79.7 nM, and IC50 (Pf3W2) = 5 nM and 7.3 nM vs. 31.8 nM, respectively).
Considering the results of inhibition of β-hematin formation expressed in IC50 (mM), CQ was significantly more efficient in inhibiting β-hematin formation than all tested compounds except 1a-b. By contrast, ENP and 3a-b exhibited the highest IC50 > 2 mM. No statistically significant difference was observed between enantiomers 1a and 1b (1.17 vs. 1.31 mM, p > 0.05). However, 1a-b displayed a significantly higher inhibition of β-hematin formation than its parental drug MQ (p = 0.0004 and 0.015, respectively).
Likewise, 2a-b showed significantly higher inhibition of β-hematin formation compared to their parental drug ENP (p = 0.00016 and 0.013, respectively). Moreover, 2a-b presented significantly higher inhibition than 3a-b (IC50 = 1.45 and 1.77 vs. >2 mM, p < 0.05), but no significant difference was observed between enantiomers 2a and 2b (IC50 = 1.45 vs. 1.77 mM, p > 0.05) or between 3a and 3b (48.3% vs. 40.6% at 2 mM, p > 0.05).
Concerning in vitro cytotoxicity results against HepG2, 2a-b were as cytotoxic as their respective parental drug ENP (CC50 at 4 and 4 vs. 4.4 µM, respectively), but their selectivity index (SI) was 87 and 70, respectively. The most active compounds, 1a-b, were also among the less cytotoxic drugs and they were three to four times less cytotoxic than MQ (25.6 and 37.8 vs. 8.6 µM, respectively). Compound 1b had the best SI, estimated at 2503.

4. Discussion

Regarding β-hematin as target, CQ is considered as the most potent molecule for inhibiting its formation. Indeed, CQ is known to inhibit in vivo hemozoin formation, defining its main action mode. In our study, its percentage of inhibition reached 86.9%, which is consistent with a previous report [4]. In contrast, the modes of action of MQ are still poorly understood, but it is believed to play a lesser role in inhibition of hemozoin formation [9,10,32,33,34]. This is consistent with our results, since IC50 of β-hematin formation for MQ was significantly higher than that of CQ (1.79 vs. 1.13 mM, p = 0.0004). Furthermore, the present study provides the first data concerning the percentage of inhibition of β-hematin formation for ENP (20.3%). Thus, ENP exhibited a significantly weaker β-hematin inhibition capacity but better antimalarial activity, whatever the Pf strain, compared with CQ and MQ. These results suggest that the slight structural difference that exists between MQ and ENP, i.e., the dissociation of the quinoline ring of MQ into 6-phenylpyridine in ENP, has a significant impact on the mode of action of this class of antimalarial drugs.
Compounds 1a-b can be considered as open-MQ analogs as they possess a quinoline core with an open-butyl chain in place of the piperidine ring at the 4-position. However, these enantiomers are stronger for inhibited β-hematin formation than MQ, with IC50 at 1.17 and 1.31 mM, respectively, vs. 1.79 mM. In addition, compounds 1a-b were more active than MQ on both PfW2 and Pf3D7, with SI 15–23 times higher than that of their respective parental drug. Thus, the opening of the piperidine ring present in MQ could have an impact on both antimalarial activity and, to a lesser extent, the mode of action of these AAAs.
Compounds 2a-b and 3a-b have the same open butyl chain as 1a-b, but the quinoline core of 1a-b was dissociated into 6-phenylpyridine. Compounds 2a-b and 3a-b share the same heterocycle but differ from each other by the branched chain located either at the 4- or at the 3-position of the pyridine core. Inhibition of β-hematin formation for 2a-b was significantly stronger than that of ENP. These results suggest that compound 2 may have a different mechanism of action than ENP. Interestingly, the antimalarial activity of 2a-b was close to that of ENP against the PfW2 strain (but not against Pf3D7) while 3a-b were the least active compounds. Indeed, compared with ENP, the antimalarial activity of 3a-b was 11 to 45 times weaker against PfW2 and 22 to 63 times weaker against Pf3D7. Remarkably, the enantiomer with a (S)-absolute configuration 3a was found to be significantly more active than its (R)-counterpart 3b, as shown by eudysmic ratios of 4 and 2.9 against PfW2 and Pf3D7, respectively. This difference in activity does not seem to be correlated with the inhibition of β-hematin because the percentages of inhibition of 3a and 3b, at 2 mM, were similar. These results suggest another target or a differential mode of transport for these enantiomers in the DV. Furthermore, antimalarial activity against both Pf strains and inhibition of β-hematin formation were stronger for compounds with an open-butyl chain located at the 4-position of the pyridine ring (2a-b) than for those branched at the 3-position (3a-b). Thus, SAR study on the open-butyl chain of compounds 2–3 indicates that the position of the alkyl chain has a greater impact on the antimalarial activity and the inhibition of β-hematin formation than the dissociation of the quinoline core. Altogether, these results show that the opening of the piperidine ring led to the formation of compounds 2a-b, which were more active than and as toxic as ENP. AAAs 2a-b seem to have different mechanisms of action from ENP and possess a stronger capacity to inhibit β-hematin formation. The AAAs 1–3, along with the references MQ and ENP, are currently being evaluated across strains with different resistance profiles (PfDd2, PfF32, PfC580Y) to gain deeper insights into their mode of action and antimalarial spectrum.

5. Conclusions

Regarding hematin crystallization as an antiplasmodial target of interest, this work brought new SAR in the AAAs family. When comparing the piperidine compounds (MQ and ENP), the dissociation of the quinoline ring seems to be discriminant for in vitro β-hematin inhibition, suggesting a different mode of action for these two references. Contrastingly, regarding the 4-substituted open-butyl chain derivatives 1a-b and 2a-b, β-hematin formation is inhibited in the same way by quinoline and 6-phenylpyridine-based compounds. Moreover, when considering the 6-phenylpyridines 2a-b and 3a-b, the position of the alkyl chain appears important for both antimalarial activity and inhibition of β-hematin formation. Overall, in our study, the 4-substituted open-butyl chain compounds were the best inhibitors of β-hematin formation regardless of the heterocyclic core. Thus, 1a-b and 2a were the most active compounds on the two strains. In particular, they were more active than their parental drugs on the resistant strain PfW2. Moreover, regarding their cytotoxicity profiles, 1a-b are safer than MQ and 2a is as toxic as ENP. In contrast to the piperidine ring opening, dissociation of the quinoline core is less discriminating for the inhibition of β-hematin formation. The data obtained in this study regarding the heme detoxification pathway represent a significant step towards a comprehensive understanding of the mechanism of action of the AAA derivatives. We are now considering further deciphering of this mechanism by assessing the transcriptomic and metabolomic responses of the parasite when cultured with these compounds. To fully characterize the mechanism of action, we also plan to identify mutations induced during ramping selection assays on the PfW2 strain through a whole-genome sequencing approach.

Author Contributions

In vitro tests: F.S., G.B., P.A., R.M. and A.B.; original draft preparation: F.S.; writing and revising the manuscript: C.D., with support from A.D.-K.; scheme and figures: A.D.-K.; proofreading: P.S., Y.L.G. and A.T. All authors have read and agreed to the published version of the manuscript.

Funding

This project was financed in part by DGA (Direction Générale de l’Armement, Ministère de la Défense, France), ANR Astrid (project ANR-12-STR-003), Région Hauts-de-France and the SATT Nord.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors thank Philippe Grellier (Muséum National d’Histoire Naturelle) who graciously provided P. falciparum strains.

Conflicts of Interest

The authors declare no conflicts 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.

References

  1. World Health Organization. 2023. Available online: https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2023 (accessed on 28 October 2024).
  2. Egan, T.J. Haemozoin formation. Mol. Biochem. Parasitol. 2008, 157, 127–136. [Google Scholar] [CrossRef] [PubMed]
  3. de Villiers, K.A.; Egan, T.J. Heme Detoxification in the Malaria Parasite: A Target for Antimalarial Drug Development. Acc. Chem. Res. 2021, 54, 2649–2659. [Google Scholar] [CrossRef] [PubMed]
  4. Wiser, M.F. The Digestive Vacuole of the Malaria Parasite: A Specialized Lysosome. Pathogens 2024, 13, 182. [Google Scholar] [CrossRef]
  5. Baelmans, R.; Deharo, E.; Munoz, V.; Sauvain, M.; Ginsburg, H. Experimental Conditions for Testing the Inhibitory Activity of Chloroquine on the Formation of beta-Hematin. Exp. Parasitol. 2000, 96, 243–248. [Google Scholar] [CrossRef]
  6. Mullié, C.; Jonet, A.; Desgrouas, C.; Taudon, N.; Sonnet, P. Differences in anti-malarial activity of 4-aminoalcohol quinoline enantiomers and investigation of the presumed underlying mechanism of action. Malar. J. 2012, 11, 65. [Google Scholar] [CrossRef]
  7. Herraiz, T.; Guillén, H.; González-Peña, D.; Arán, V.J. Antimalarial Quinoline Drugs Inhibit β-Hematin and Increase Free Hemin Catalyzing Peroxidative Reactions and Inhibition of Cysteine Proteases. Sci. Rep. 2019, 9, 15398. [Google Scholar] [CrossRef]
  8. Combrinck, J.M.; Mabotha, T.E.; Ncokazi, K.K.; Ambele, M.A.; Taylor, D.; Smith, P.J.; Hoppe, H.C.; Egan, T.J. Insights into the role of heme in the mechanism of action of antimalarials. ACS Chem. Biol. 2013, 8, 133e137. [Google Scholar] [CrossRef]
  9. Wong, W.; Bai, X.C.; Sleebs, B.E.; Triglia, T.; Brown, A.; Thompson, J.K.; Jackson, K.E.; Hanssen, E.; Marapana, D.S.; Fernandez, I.S.; et al. The antimalarial Mefloquine targets the Plasmodium falciparum 80S ribosome to inhibit protein synthesis. Nat. Microbiol. 2017, 2, 17031. [Google Scholar] [CrossRef]
  10. Kumar, A.; Ghosh, D.K.; Ali, J.; Ranjan, A. Characterization of Lipid Binding Properties of Plasmodium falciparum Acyl-Coenzyme A Binding Proteins and Their Competitive Inhibition by Mefloquine. ACS Chem. Biol. 2019, 14, 901–915. [Google Scholar] [CrossRef]
  11. Kumar, A.; Ghosh, D.K.; Ranjan, A. Differential Stabilities of Mefloquine-Bound Human and Plasmodium Falciparum Acyl-CoA-Binding Proteins. ACS Omega. 2021, 6, 1883–1893. [Google Scholar] [CrossRef]
  12. Gunjan, S.; Singh, S.K.; Sharma, T.; Dwivedi, H.; Chauhan, B.S.; Imran Siddiqi, M.; Tripathi, R. Mefloquine Induces ROS Mediated Programmed Cell Death in Malaria Parasite: Plasmodium. Apoptosis 2016, 21, 955–964. [Google Scholar] [CrossRef] [PubMed]
  13. Karle, J.M.; Olmeda, R.; Gerena, L.; Milhous, W.K. Plasmodium falciparum: Role of Absolute Stereochemistry in the Antimalarial Activity of Synthetic Amino Alcohol Antimalarial Agents. Exp. Parasitol. 1993, 76, 345–351. [Google Scholar] [CrossRef] [PubMed]
  14. World Health Organization. Guidelines for the Treatment of Malaria, 3rd ed.; World Health Organization: Geneva, Switzerland, 2015; Available online: https://www.afro.who.int/sites/default/files/2017-06/9789241549127_eng.pdf (accessed on 28 October 2024).
  15. Hood, J.E.; Jenkins, J.W.; Milatovic, D.; Rongzhu, L.; Aschner, M. Mefloquine induces oxidative stress and neurodegeneration in primary rat cortical neurons. Neurotoxicology 2010, 31, 518–523. [Google Scholar] [CrossRef] [PubMed]
  16. Görlach, A.; Bertram, K.; Hudecova, S.; Krizanova, O. Calcium and ROS: A mutual interplay. Redox Biol. 2015, 6, 260–271. [Google Scholar] [CrossRef] [PubMed]
  17. Baudry, S.; Pham, Y.T.; Baune, B.; Vidrequin, S.; Crevoisier, C.; Gimenez, F.; Farinotti, R. Stereoselective Passage of Mefloquine Through the Blood-Brain Barrier in the Rat. J. Pharm. Pharmacol. 1997, 49, 1086–1090. [Google Scholar] [CrossRef]
  18. Barraud de Lagerie, S.; Comets, E.; Gautrand, C.; Fernandez, C.; Auchere, D.; Singlas, E.; Mentre, F.; Gimenez, F. Cerebral uptake of mefloquine enantiomers with and without the P-gp inhibitor elacridar (GF1210918) in mice. Br. J. Pharmacol 2004, 141, 1214–1222. [Google Scholar] [CrossRef]
  19. Fletcher, A.; Shepherd, R. Use of (+)-Mefloquine for the Treatment of Malaria, WO1998039003A1. 6 March 1998. Available online: https://patentimages.storage.googleapis.com/4c/8a/4d/c470c01e488865/WO1998039003A1.pdf (accessed on 28 October 2024).
  20. Dow, G.S.; Heady, T.N.; Bhattacharjee, A.K.; Caridha, D.; Gerena, L.; Gettayacamin, M.; Lanteri, C.A.; Obaldia, N., 3rd; Roncal, N.; Shearer, T.; et al. Utility of Alkylaminoquinolinyl Methanols as New Antimalarial Drugs. Antimicrob. Agents Chemother. 2006, 50, 4132–4143. [Google Scholar] [CrossRef]
  21. Mullié, C.; Taudon, N.; Degrouas, C.; Jonet, A.; Pascual, A.; Agnamey, P.; Sonnet, P. Enantiomerically pure amino-alcohol quinolines: In vitro anti-malarial activity in combination with dihydroartemisinin, cytotoxicity and in vivo efficacy in a Plasmodium berghei mouse model. Malar. J. 2014, 13, 407. [Google Scholar] [CrossRef]
  22. Bentzinger G, Sonnet P, Dassonville-Klimpt A, Mullié Demailly C, Agnamey P. Novel aminopyridinemethanol compounds and their use. WO2019086614A1. 5 September 2019. Available online: https://patentimages.storage.googleapis.com/c3/41/16/d407e74fa01cb4/WO2019086614A1.pdf (accessed on 28 October 2024).
  23. Jonet, A.; Dassonville-Klimpt, A.; Da Nascimento, S.; Leger, J.M.; Guillon, J.; Sonnet, P. First enantioselective synthesis of 4-aminoalcohol quinoline derivatives through a regioselective SN2 epoxide opening mechanism. Tetrahedron Asymmetry 2011, 22, 138–148. [Google Scholar] [CrossRef]
  24. Desjardins, R.E.; Canfield, C.J.; Haynes, J.D.; Chulay, J.D. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob. Agents Chemother. 1979, 16, 710–718. [Google Scholar] [CrossRef]
  25. Bennett, T.N.; Paguio, M.; Gligorijevic, B.; Seudie, C.; Kosa, A.D.; Davidson, E.; Roepe, P.D. Novel, Rapid, and Inexpensive Cell-Based Quantification of Antimalarial Drug Efficacy. Antimicrob. Agents Chemother. 2004, 48, 1807–1810. [Google Scholar] [CrossRef] [PubMed]
  26. Bacon, D.J.; Latour, C.; Lucas, C.; Colina, O.; Ringwald, P.; Picot, S. Comparison of a SYBR Green I-Based Assay with a Histidine- Rich Protein II Enzyme-Linked Immunosorbent Assay for In Vitro Antimalarial Drug Efficacy Testing and Application to Clinical Isolates. Antimicrob. Agents Chemother. 2007, 51, 1172–1178. [Google Scholar] [CrossRef] [PubMed]
  27. Kaddouri, H.; Nakache, S.; Houzé, S.; Mentré, F.; Le Bras, J. Assessment of the Drug Susceptibility of Plasmodium falciparum Clinical Isolates from Africa by Using a Plasmodium Lactate Dehydrogenase Immunodetection Assay and an Inhibitory Maximum Effect Model for Precise Measurement of the 50-Percent Inhibitory Concentration. Antimicrob. Agents Chemother. 2006, 50, 3343–3349. [Google Scholar] [PubMed]
  28. Dassonville-Klimpt, A.; Schneider, J.; Damiani, C.; Tisnerat, C.; Cohen, A.; Azas, N.; Marchivie, M.; Guillon, J.; Mullié, C.; Agnamey, P.; et al. Design, synthesis, and characterization of novel aminoalcohol quinolines with strong in vitro antimalarial activity. Eur. J. Med. Chem. 2022, 228, 113981. [Google Scholar] [CrossRef]
  29. Albenque-Rubio, S.; Guillon, J.; Agnamey, P.; Damiani, C.; Savrimoutou, S.; Mustière, R.; Pinaud, N.; Moreau, S.; Mergny, J.-L.; Ronga, L.; et al. Design, Synthesis, and In Vitro Antimalarial Evaluation of New 1,3,5-Tris[(4-(Substituted-Aminomethyl) Phenoxy)Methyl]Benzenes. Drugs Drug Candidates 2024, 3, 615–637. [Google Scholar] [CrossRef]
  30. Egan, T.J.; Ross, D.C.; Adams, P.A. Quinoline anti-malarial drugs inhibit spontaneous formation of beta-haematin (malaria pigment). FEBS Lett. 1994, 352, 54–57. [Google Scholar] [CrossRef]
  31. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55–63. [Google Scholar] [CrossRef]
  32. Price, R.N.; Uhlemann, A.C.; Brockman, A.; McGready, R.; Ashley, E.; Phaipun, L.; Patel, R.; Laing, K.; Looareesuwan, S.; White, N.J.; et al. Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number. Lancet 2004, 364, 438–447. [Google Scholar] [CrossRef]
  33. Sidhu, A.B.; Uhlemann, A.C.; Valderramos, S.G.; Valderramos, J.C.; Krishna, S.; Fidock, D.A. Decreasing pfmdr1 copy number in Plasmodium falciparum malaria heightens susceptibility to mefloquine, lumefantrine, halofantrine, quinine, and artemisinin. J Infect. Dis. 2006, 194, 528–535. [Google Scholar] [CrossRef]
  34. Sanchez, C.P.; Dave, A.; Stein, W.D.; Lanzer, M. Transporters as mediators of drug resistance in Plasmodium falciparum. Int. J. Parasitol. 2010, 40, 1109–1118. [Google Scholar] [CrossRef]
Microorganisms 12 02524 g001
Figure 1. Structures of mefloquine (MQ) and enpiroline (ENP) enantiomers [13]. * stereogenic center.
Figure 1. Structures of mefloquine (MQ) and enpiroline (ENP) enantiomers [13]. * stereogenic center.
Microorganisms 12 02524 g001
Microorganisms 12 02524 sch001
Scheme 1. Schematic representation of the hemoglobin degradation process in Plasmodium falciparum and of the potential target of new AAAs.
Scheme 1. Schematic representation of the hemoglobin degradation process in Plasmodium falciparum and of the potential target of new AAAs.
Microorganisms 12 02524 sch001
Microorganisms 12 02524 g002
Figure 2. Structure of the three Series 1, 2 and 3 previously synthesized and structure of (S)-enantiomers 1a, 2a and 3a and their (R)-counterparts 1b, 2b and 3b selected for this study [6,22]. * stereogenic center.
Figure 2. Structure of the three Series 1, 2 and 3 previously synthesized and structure of (S)-enantiomers 1a, 2a and 3a and their (R)-counterparts 1b, 2b and 3b selected for this study [6,22]. * stereogenic center.
Microorganisms 12 02524 g002
Table 1. In vitro antimalarial activity against Plasmodium falciparum W2 and 3D7 strains and eudysmic ratios for six arylamino alcohols (1a-b, 2a-b, 3a-b) and three reference molecules (chloroquine, mefloquine, enpiroline).
Table 1. In vitro antimalarial activity against Plasmodium falciparum W2 and 3D7 strains and eudysmic ratios for six arylamino alcohols (1a-b, 2a-b, 3a-b) and three reference molecules (chloroquine, mefloquine, enpiroline).
CompoundConfig.IC50 (nM) a,bEudysmic Ratio c
PfW2 dPf3D7 ePfW2Pf3D7
Chloroquine198.8 ± 27.075.9 ± 3.0NANA
Mefloquine 31.8 ± 1.079.7 ± 8.5NANA
Enpiroline 11.5 ± 2.3 21.6 ± 1.4 NANA
11a5.0 ± 0.315.1 ± 3.31.46 (S)1
1b7.3 ± 0.915.1 ± 3.4
22a9.0 ± 0.4 45.8 ± 2.3 1.1 (S)1.2 (S)
2b10.0 ± 0.7 56.7 ± 4.8
33a131.7 ± 10.3 479.0 ± 38.2 4.0 (S)2.9 (S)
3b522.8 ± 95.8 1379.7 ± 992.8
a 50% inhibitory concentration (IC50) corresponds to the mean ± standard deviation from three independent experiments. b IC50s (nM) against PfW2 and Pf3D7 of compounds 1–3 was determined using the SYBR Green I fluorescence-based method. c The eudysmic ratio was calculated using the following formula: IC50 eutomer/IC50 distomer. The eutomer configuration is between parentheses. d Resistant to CQ and sensitive to MQ. e Susceptible to CQ but displays a decreased susceptibility to MQ. NA: Not Applicable.
Table 2. Inhibition of β-hematin formation and in vitro cytotoxicity results against HepG2 for six arylamino alcohols (1a-b, 2a-b, 3a-b) and three reference molecules (chloroquine, mefloquine, enpiroline).
Table 2. Inhibition of β-hematin formation and in vitro cytotoxicity results against HepG2 for six arylamino alcohols (1a-b, 2a-b, 3a-b) and three reference molecules (chloroquine, mefloquine, enpiroline).
Inhibition of β-Hematin FormationCytotoxicity Results Against HepG2
CompoundConfig.Inhibition (%) a
at 2 mM		IC50
(mM) a		CC50 (µM)
on HepG2 a		Selectivity Index b
HepG2/3D7
Chloroquine86.9 ± 4.81.13 ± 0.2024.1 ± 0.8317
Mefloquine 61.3 ± 8.51.79 ± 0.098.6 ± 2.6108
Enpiroline 20.3 ± ND c>24.4 ± 0.6204
11a81.8 ± 8.41.17 ± 0.3225.6 ± 2.71695
1b71.3 ± 10.31.31 ± 0.5437.8 ± 1.92503
22a73.5 ± 18.01.45 ± 0.304.0 ± 0.987
2b54.0 ± 22.71.77 ± 0.224.0 ± 0.770
33a48.3 ± ND c>211.6 ± 0.724
3b40.6 ± 28.3>2>100>72
a Mean ± standard deviation from three independent experiments. b Selectivity index was defined as the ratio between the CC50 value on the HepG2 cell line and the IC50 value against the Pf3D7 strain. c Results obtained in simplicate. ND: Not Determined.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Damiani, C.; Soler, F.; Le Govic, Y.; Totet, A.; Bentzinger, G.; Bouchut, A.; Mustière, R.; Agnamey, P.; Dassonville-Klimpt, A.; Sonnet, P. Evaluation of In Vitro Inhibition of β-Hematin Formation: A Step Towards a Comprehensive Understanding of the Mechanism of Action of New Arylamino Alcohols. Microorganisms 2024, 12, 2524. https://doi.org/10.3390/microorganisms12122524

AMA Style

Damiani C, Soler F, Le Govic Y, Totet A, Bentzinger G, Bouchut A, Mustière R, Agnamey P, Dassonville-Klimpt A, Sonnet P. Evaluation of In Vitro Inhibition of β-Hematin Formation: A Step Towards a Comprehensive Understanding of the Mechanism of Action of New Arylamino Alcohols. Microorganisms. 2024; 12(12):2524. https://doi.org/10.3390/microorganisms12122524

Chicago/Turabian Style

Damiani, Céline, Floriane Soler, Yohann Le Govic, Anne Totet, Guillaume Bentzinger, Anne Bouchut, Romain Mustière, Patrice Agnamey, Alexandra Dassonville-Klimpt, and Pascal Sonnet. 2024. "Evaluation of In Vitro Inhibition of β-Hematin Formation: A Step Towards a Comprehensive Understanding of the Mechanism of Action of New Arylamino Alcohols" Microorganisms 12, no. 12: 2524. https://doi.org/10.3390/microorganisms12122524

APA Style

Damiani, C., Soler, F., Le Govic, Y., Totet, A., Bentzinger, G., Bouchut, A., Mustière, R., Agnamey, P., Dassonville-Klimpt, A., & Sonnet, P. (2024). Evaluation of In Vitro Inhibition of β-Hematin Formation: A Step Towards a Comprehensive Understanding of the Mechanism of Action of New Arylamino Alcohols. Microorganisms, 12(12), 2524. https://doi.org/10.3390/microorganisms12122524

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop