Quinoline Antimalarials Research Articles

Leveraging the Aggregated Protein Dye YAT2150 for Malaria Chemotherapy.

Background/Objectives: YAT2150 is a first-in-class antiplasmodial compound that has been recently proposed as a new interesting drug for malaria therapy. Methods/Results: The fluorescence of YAT2150 rapidly increases upon its entry into Plasmodium, a property that can be of use for the design of highly sensitive diagnostic approaches. YAT2150 blocks the in vitro development of the ookinete stage of Plasmodium and, when added to an infected blood meal, inhibits oocyst formation in the mosquito. Thus, the compound could possibly contribute to future transmission-blocking antimalarial strategies. Cell influx/efflux studies in Caco-2 cells suggest that YAT2150 is internalized by endocytosis and also through the OATP2B1 transporter, whereas its main export route would be via OSTα. YAT2150 has an overall favorable drug metabolism and pharmacokinetics profile, and its moderate cytotoxicity can be significantly reduced upon encapsulation in immunoliposomes, which leads to a dramatic increase in the drug selectivity index to values close to 1000. Although YAT2150 binds amyloid-forming peptides, its in vitro fluorescence emission is stronger upon association with peptides that form amorphous aggregates, suggesting that regions enriched in unstructured proteins are the preferential binding sites of the drug inside Plasmodium cells. The reduction of protein aggregation in the parasite after YAT2150 treatment, which has been suggested to be directly related to the drug's mode of action, is also observed following treatment with quinoline antimalarials like chloroquine and primaquine. Conclusions: Altogether, the data presented here indicate that YAT2150 can represent the spearhead of a new family of compounds for malaria diagnosis and therapy due to its presumed novel mode of action based on the interaction with functional protein aggregates in the pathogen.

Distribution patterns of molecular markers of antimalarial drug resistance in Plasmodium falciparum isolates on the Thai-Myanmar border during the periods of 1993–1998 and 2002–2008

BackgroundPolymorphisms of Plasmodium falciparum chloroquine resistance transporter (pfcrt), Plasmodium falciparum multi-drug resistance 1 (pfmdr1) and Plasmodium falciparum kelch 13-propeller (pfk13) genes are accepted as valid molecular markers of quinoline antimalarials and artemisinins. This study investigated the distribution patterns of these genes in P. falciparum isolates from the areas along the Thai-Myanmar border during the two different periods of antimalarial usage in Thailand.ResultsPolymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were used to detect pfcrt mutations at codons 76, 220, 271, 326, 356, and 371 as well as pfmdr1 mutation at codon 86. The prevalence of pfcrt mutations was markedly high (96.4–99.7%) in samples collected during both periods. The proportions of mutant genotypes (number of mutant/total isolate) at codons 76, 220, 271, 326, 356 and 371 in the isolates collected during 1993–1998 (period 1) compared with 2002–2008 (period 2) were 97.9% (137/140) vs. 97.1% (401/413), 97.9% (140/143) vs. 98.8% (171/173), 97.2% (139/143) vs. 97.1% (333/343), 98.6% (140/142) vs. 99.7% (385/386), 96.4% (134/139) vs. 98.2% (378/385) and 97.8% (136/139) vs. 98.9% (375/379), respectively. Most isolates carried pfmdr1 wild-type at codon 86, with a significant difference in proportions genotypes (number of wild type/total sample) in samples collected during period 1 [92.9% (130/140)] compared with period 2 [96.9% (379/391)]. Investigation of pfmdr1 copy number was performed by real-time PCR. The proportions of isolates carried 1, 2, 3 and 4 or more than 4 copies of pfmdr1 (number of isolates carried correspondent copy number/total isolate) were significantly different between the two sample collecting periods (65.7% (90/137) vs. 87.8% (390/444), 18.2% (25/137) vs. 6.3%(28/444), 5.1% (7/137) vs. 1.4% (6/444) and 11.0% (15/137) vs. 4.5% (20/444), for period 1 vs. period 2, respectively). No pfk13 mutation was detected by nested PCR and nucleotide sequencing in all samples with successful analysis (n = 68).ConclusionsThe persistence of pfcrt mutations and pfmdr1 wild-types at codon 86, along with gene amplification in P. falciparum, contributes to the continued resistance of chloroquine and mefloquine in P. falciparum isolates in the study area. Regular surveillance of antimalarial drug resistance in P. falciparum, incorporating relevant molecular markers and treatment efficacy assessments, should be conducted.

Antimalarial Agents Derived from Metal-Amodiaquine Complexes with Activity in Multiple Stages of the Plasmodium Life Cycle†.

Quinolines are excellent ligands for metal coordination and are deployed as drugs for malaria treatment. There is a growing body of evidence indicating that metal complexes can be conjugated with antimalarial quinolines to be used as chemical tools to overcome the disadvantages of quinolines, improving their bioactive speciation, cellular distribution, and subsequently broadening the spectrum of activity to multiple stages of the complex Plasmodium life cycle. In this study, four novel complexes of ruthenium(II) and gold(I) containing amodiaquine (AQ) were synthesized and a careful chemical characterization revealed the precise coordination site of AQ to the metals. Their speciation in solution was investigated, demonstrating the stability of the quinoline-metal bond. Ru(II)- and Au(I)-AQ complexes were demonstrated to be potent and efficacious in inhibiting parasite growth in multiple stages of the Plasmodium life cycle assayed in vitro and in vivo. These properties could be attributed to the ability of the metal-AQ complexes to reproduce the suppression of heme detoxification induced by AQ, while also inhibiting other processes in the parasite life cycle, which can be attributed to the action of the metallic species. These findings indicate that metal coordination is a potential chemical tool for drug design and discovery in malaria.

PfCRT mutations conferring piperaquine resistance in falciparum malaria shape the kinetics of quinoline drug binding and transport.

The chloroquine resistance transporter (PfCRT) confers resistance to a wide range of quinoline and quinoline-like antimalarial drugs in Plasmodium falciparum, with local drug histories driving its evolution and, hence, the drug transport specificities. For example, the change in prescription practice from chloroquine (CQ) to piperaquine (PPQ) in Southeast Asia has resulted in PfCRT variants that carry an additional mutation, leading to PPQ resistance and, concomitantly, to CQ re-sensitization. How this additional amino acid substitution guides such opposing changes in drug susceptibility is largely unclear. Here, we show by detailed kinetic analyses that both the CQ- and the PPQ-resistance conferring PfCRT variants can bind and transport both drugs. Surprisingly, the kinetic profiles revealed subtle yet significant differences, defining a threshold for in vivo CQ and PPQ resistance. Competition kinetics, together with docking and molecular dynamics simulations, show that the PfCRT variant from the Southeast Asian P. falciparum strain Dd2 can accept simultaneously both CQ and PPQ at distinct but allosterically interacting sites. Furthermore, combining existing mutations associated with PPQ resistance created a PfCRT isoform with unprecedented non-Michaelis-Menten kinetics and superior transport efficiency for both CQ and PPQ. Our study provides additional insights into the organization of the substrate binding cavity of PfCRT and, in addition, reveals perspectives for PfCRT variants with equal transport efficiencies for both PPQ and CQ.

Development and Validation of an Ion-Pair HPLC-UV Method for the Quantitation of Quinoline and Indoloquinoline Alkaloids in Herbal and Pharmaceutical Antimalarial Formulations

Quinine- and cryptolepine-based antimalarials serve as valuable alternatives to artemisinin-based combination therapies (ACTs) in Ghana. Their use, however, is associated with adulteration and substandard quality challenges. An HPLC method targeting quinoline and indoloquinoline antimalarial alkaloids was developed, validated, and applied to evaluate herbal and pharmaceutical antimalarial formulations (HPAFs) and starting materials (APIs). The separation/quantitation of the alkaloids (including quinine, quinidine, cinchonine, cinchonidine, dihydroquinine, dihydroquinidine, and cryptolepine) was achieved on a Zorbax SB-CN column (250 mm × 4.6 mm, 5 μm), with an isocratic elution system of methanol: trifluoroacetic acid (0.1%, v/v) (15 : 85, v/v) at 1.5 mL/min and 223 nm. Method validation was according to ICH Q2(R1) guidelines. It was then used to assess the quality of APIs (n = 3) and HPAFs (n = 44) including quinine-based pharmaceutical antimalarial formulations (QBPAFs) (n = 23) and herbal antimalarial products (HAMPs). The method was found to be specific, selective, accurate, precise, and robust toward the alkaloids with linearity achieved within specified concentration ranges (r2 > 0.995 for all analytes). Analyte stability ranged between 6 and 12 hours. All the APIs contained quinine <99.0%–101.0%, with dihydroquinine and cinchonidine at levels compliant with the established acceptance criteria. The QBPAFs had quinine content ranging between 50.2% and 151.2%, with 43.5% (n = 10/23) of them complying with the acceptance criteria. The related alkaloids observed in the QBPAFs included quinidine (56.5%, n = 13/23), dihydroquinine (100%, n = 23/23), dihydroquinidine (21.7%, n = 5/23), cinchonine (17.4%, n = 4/23), and cinchonidine (95.7%, n = 22/23). For the HAMPs, 81.0% (n = 17/21) were adulterated with quinine (0.59 ± 0.04 mg/10 mL–86.03 ± 0.02 mg/10 mL). Cryptolepine was identified in 19% (n = 4/21) of the HAMPs with concentration ranging between 43.99 ± 0.43 μg/mL and 747.86 ± 0.34 μg/mL. In conclusion, the application of the ion-pair HPLC method targeting quinoline and indoloquinoline antimalarials has demonstrated the presence of quality and poor-quality HPAFs on the Ghanaian market.

A Review of Modifications of Quinoline Antimalarials: Mefloquine and (hydroxy)Chloroquine.

Late-stage modification of drug molecules is a fast method to introduce diversity into the already biologically active scaffold. A notable number of analogs of mefloquine, chloroquine, and hydroxychloroquine have been synthesized, starting from the readily available active pharmaceutical ingredient (API). In the current review, all the modifications sites and reactivity types are summarized and provide insight into the chemistry of these molecules. The approaches include the introduction of simple groups and functionalities. Coupling to other drugs, polymers, or carriers afforded hybrid compounds or conjugates with either easily hydrolyzable or more chemically inert bonds. The utility of some of the compounds was tested in antiprotozoal, antibacterial, and antiproliferative assays, as well as in enantiodifferentiation experiments.

Prevalence of Mutations in the pfcoronin Gene and Association with Ex Vivo Susceptibility to Common Quinoline Drugs against Plasmodium falciparum.

Background: Artemisinin-based combination therapy (ACT) was recommended to treat uncomplicated falciparum malaria. Unlike the situation in Asia where resistance to ACT has been reported, artemisinin resistance has not yet emerged in Africa. However, some rare failures with ACT or patients continuing to be parasitaemic on day 3 after ACT treatment have been reported in Africa or in travellers returning from Africa. Three mutations (G50E, R100K, and E107V) in the pfcoronin gene could be responsible for artemisinin resistance in Africa. Methods: The aims of this study were first to determine the prevalence of mutations in the pfcoronin gene in African P. falciparum isolates by Sanger sequencing, by targeting the 874 samples collected from patients hospitalised in France after returning from endemic areas in Africa between 2018 and 2019, and secondly to evaluate their association with in vitro reduced susceptibility to standard quinoline antimalarial drugs, including chloroquine, quinine, mefloquine, desethylamodiaquine, lumefantrine, piperaquine, and pyronaridine. Results: The three mutations in the pfcoronin gene (50E, 100K, and 107V) were not detected in the 874 P. falciparum isolates. Current data show that another polymorphism (P76S) is present in many countries of West Africa (mean prevalence of 20.7%) and Central Africa (11.9%) and, rarely, in East Africa (4.2%). This mutation does not appear to be predictive of in vitro reduced susceptibility to quinolines, including artemisinin derivative partners in ACT such as amodiaquine, lumefantrine, piperaquine, pyronaridine, and mefloquine. Another mutation (V62M) was identified at low prevalence (overall prevalence of 1%). Conclusions: The 76S mutation is present in many African countries with a prevalence above 10%. It is reassuring that this mutation does not confer in vitro resistance to ACT partners.

Quinoline Antimalarials Increase the Antibacterial Activity of Ampicillin.

Bacterial and malaria co-infections are common in malaria endemic countries and thus necessitate co-administration of antibiotics and antimalarials. There have long been anecdotal clinical reports of interactions between penicillins and antimalarial agents, but the nature and mechanisms of these interactions remain to be investigated. In this study, we employed antimicrobial interaction testing methods to study the effect of two antimalarials on the antibacterial activity of ampicillin in vitro. Paper strip diffusion, a modified disc diffusion and checkerboard methods were used to determine the nature of interactions between ampicillin and quinoline antimalarials, chloroquine and quinine, against Gram-positive and Gram-negative bacteria. The impact of antimalarials and ampicillin-antimalarial drug combinations on cell integrity of test bacteria were determined by measuring potassium release. The tested antimalarials did not show substantial antibacterial activity but quinine was bactericidal at high concentrations. Chloroquine and quinine increased ampicillin activity, with increasing concentrations extending the antibacterial’s inhibition zones by 2.7-4.4 mm and from 1.1 to over 60 mm, respectively. Observed interactions were largely additive with Fractional Inhibitory Concentration Indices of >0.5-1 for all ampicillin-antimalarial combinations. Quinine and, to a lesser extent, chloroquine increase the activity of ampicillin and potentially other β-lactams, which has implications for combined clinical use.

Repurposing Quinoline and Artemisinin Antimalarials as Therapeutics for SARS-CoV-2: Rationale and Implications.

The coronavirus disease-2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected more than 116 million individuals globally and resulted in over 2.5 million deaths since the first report in December 2019. For most of this time, healthcare professionals have had few tools at their disposal. In December 2020, several vaccines that were shown to be highly effective have been granted emergency use authorization (EUA). Despite these remarkable breakthroughs, challenges include vaccine roll-out and implementation, in addition to deeply entrenched antivaccination viewpoints. While vaccines will prevent disease occurrence, infected individuals still need treatment options, and repurposing drugs circumvents the lengthy and costly process of drug development. SARS-CoV-2, like many other enveloped viruses, require the action of host proteases for entry. In addition, this novel virus employs a unique method of cell exit of deacidified lysosomes and exocytosis. Thus, inhibitors of lysosomes or other players in this pathway are good candidates to target SARS-CoV-2. Chemical compounds in the quinoline class are known to be lysomotropic and perturb pH levels. A large number of quinolines are FDA-approved for treatment of inflammatory diseases and antimalarials. Artemisinins are another class of drugs that have been demonstrated to be safe for use in humans and are widely utilized as antimalarials. In this Review, we discuss the use of antimalarial drugs in the class of quinolines and artemisinins, which have been shown to be effective against SARS-CoV-2 in vitro and in vivo, and provide a rationale in employing quinolines as treatment of SARS-CoV-2 in clinical settings.

Randomized Controlled Trial of the Electrocardiographic Effects of Four Antimalarials for Pregnant Women with Uncomplicated Malaria on the Thailand-Myanmar Border.

Quinoline antimalarials cause drug-induced electrocardiographic QT prolongation, a potential risk factor for torsade de pointes. The effects of currently used antimalarials on the electrocardiogram (ECG) were assessed in pregnant women with malaria. Pregnant women with microscopy-confirmed parasitemia of any malaria species were enrolled in an open-label randomized controlled trial on the Thailand-Myanmar border from 2010 to 2016. Patients were randomized to the standard regimen of dihydroartemisinin-piperaquine (DP) or artesunate-mefloquine (ASMQ) or an extended regimen of artemether-lumefantrine (AL+). Recurrent Plasmodium vivax infections were treated with chloroquine. Standard 12-lead electrocardiograms were assessed on day 0, 4 to 6 h following the last dose, and day 7. QT was corrected for the heart rate by a linear mixed-effects model-derived population-based correction formula (QTcP = QT/RR0.381). A total of 86 AL+, 82 ASMQ, 88 DP, and 21 chloroquine-treated episodes were included. No patients had an uncorrected QT interval nor QTcP of >480 ms at any time. QTcP corresponding to peak drug concentration was longer in the DP group (adjusted predicted mean difference, 17.84 ms; 95% confidence interval [CI], 11.58 to 24.10; P < 0.001) and chloroquine group (18.31 ms; 95% CI, 8.78 to 27.84; P < 0.001) than in the AL+ group, but not different in the ASMQ group (2.45 ms; 95% CI, -4.20 to 9.10; P = 0.47) by the multivariable linear mixed-effects model. There was no difference between DP and chloroquine (P = 0.91). QTc prolongation resulted mainly from widening of the JT interval. In pregnant women, none of the antimalarial drug treatments exceeded conventional thresholds for an increased risk of torsade de pointes.

The in-vivo Mode of Action of Quinoline Antimalarial Drugs Unveiled by X-ray Microscopy

The <i>in-vivo</i> Mode of Action of Quinoline Antimalarial Drugs Unveiled by X-ray Microscopy

Multi-omic Characterization of the Mode of Action of a Potent New Antimalarial Compound, JPC-3210, Against Plasmodium falciparum

The increasing incidence of antimalarial drug resistance to the first-line artemisinin combination therapies underpins an urgent need for new antimalarial drugs, ideally with a novel mode of action. The recently developed 2-aminomethylphenol, JPC-3210, (MMV 892646) is an erythrocytic schizonticide with potent in vitro antimalarial activity against multidrug-resistant Plasmodium falciparum lines, low cytotoxicity, potent in vivo efficacy against murine malaria, and favorable preclinical pharmacokinetics including a lengthy plasma elimination half-life. To investigate the impact of JPC-3210 on biochemical pathways within P. falciparum-infected red blood cells, we have applied a "multi-omics" workflow based on high resolution orbitrap mass spectrometry combined with biochemical approaches. Metabolomics, peptidomics and hemoglobin fractionation analyses revealed a perturbation in hemoglobin metabolism following JPC-3210 exposure. The metabolomics data demonstrated a specific depletion of short hemoglobin-derived peptides, peptidomics analysis revealed a depletion of longer hemoglobin-derived peptides, and the hemoglobin fractionation assay demonstrated decreases in hemoglobin, heme and hemozoin levels. To further elucidate the mechanism responsible for inhibition of hemoglobin metabolism, we used in vitro β-hematin polymerization assays and showed JPC-3210 to be an intermediate inhibitor of β-hematin polymerization, about 10-fold less potent then the quinoline antimalarials, such as chloroquine and mefloquine. Further, quantitative proteomics analysis showed that JPC-3210 treatment results in a distinct proteomic signature compared with other known antimalarials. While JPC-3210 clustered closely with mefloquine in the metabolomics and proteomics analyses, a key differentiating signature for JPC-3210 was the significant enrichment of parasite proteins involved in regulation of translation. These studies revealed that the mode of action for JPC-3210 involves inhibition of the hemoglobin digestion pathway and elevation of regulators of protein translation. Importantly, JPC-3210 demonstrated rapid parasite killing kinetics compared with other quinolones, suggesting that JPC-3210 warrants further investigation as a potentially long acting partner drug for malaria treatment.

Mode of action of quinoline antimalarial drugs in red blood cells infected by Plasmodium falciparum revealed in vivo

The most widely used antimalarial drugs belong to the quinoline family. Their mode of action has not been characterized at the molecular level in vivo. We report the in vivo mode of action of a bromo analog of the drug chloroquine in rapidly frozen Plasmodium falciparum-infected red blood cells. The Plasmodium parasite digests hemoglobin, liberating the heme as a byproduct, toxic to the parasite. It is detoxified by crystallization into inert hemozoin within the parasitic digestive vacuole. By mapping such infected red blood cells with nondestructive X-ray microscopy, we observe that bromoquine caps hemozoin crystals. The measured crystal surface coverage is sufficient to inhibit further hemozoin crystal growth, thereby sabotaging heme detoxification. Moreover, we find that bromoquine accumulates in the digestive vacuole, reaching submillimolar concentration, 1,000-fold more than that of the drug in the culture medium. Such a dramatic increase in bromoquine concentration enhances the drug's efficiency in depriving heme from docking onto the hemozoin crystal surface. Based on direct observation of bromoquine distribution in the digestive vacuole and at its membrane surface, we deduce that the excess bromoquine forms a complex with the remaining heme deprived from crystallization. This complex is driven toward the digestive vacuole membrane, increasing the chances of membrane puncture and spillage of heme into the interior of the parasite.

Antimalarial Quinoline Drugs Inhibit \u03b2-Hematin and Increase Free Hemin Catalyzing Peroxidative Reactions and Inhibition of Cysteine Proteases

Malaria caused by Plasmodium affects millions people worldwide. Plasmodium consumes hemoglobin during its intraerythrocytic stage leaving toxic heme. Parasite detoxifies free heme through formation of hemozoin (β-hematin) pigment. Proteolysis of hemoglobin and formation of hemozoin are two main targets for antimalarial drugs. Quinoline antimarial drugs and analogs (β-carbolines or nitroindazoles) were studied as inhibitors of β-hematin formation. The most potent inhibitors were quinacrine, chloroquine, and amodiaquine followed by quinidine, mefloquine and quinine whereas 8-hydroxyquinoline and β-carbolines had no effect. Compounds that inhibited β-hematin increased free hemin that promoted peroxidative reactions as determined with TMB and ABTS substrates. Hemin-catalyzed peroxidative reactions were potentiated in presence of proteins (i.e. globin or BSA) while antioxidants and peroxidase inhibitors decreased peroxidation. Free hemin increased by chloroquine action promoted oxidative reactions resulting in inhibition of proteolysis by three cysteine proteases: papain, ficin and cathepsin B. Glutathione reversed inhibition of proteolysis. These results show that active quinolines inhibit hemozoin and increase free hemin which in presence of H2O2 that abounds in parasite digestive vacuole catalyzes peroxidative reactions and inhibition of cysteine proteases. This work suggests a link between the action of quinoline drugs with biochemical processes of peroxidation and inhibition of proteolysis.

The arrhythmogenic cardiotoxicity of the quinoline and structurally related antimalarial drugs: a systematic review

BackgroundSeveral quinoline and structurally related antimalarial drugs are associated with cardiovascular side effects, particularly hypotension and electrocardiographic QT interval prolongation. A prolonged QT interval is a sensitive but not specific risk marker for the development of Torsade de Pointes—a potentially lethal polymorphic ventricular tachyarrhythmia. The increasing use of quinoline and structurally related antimalarials in mass treatments to eliminate malaria rapidly highlights the need to review their cardiovascular safety profiles.MethodsThe primary objective of this systematic review was to describe the documented clinical and electrocardiographic cardiovascular side effects of quinine, mefloquine, lumefantrine, piperaquine, halofantrine, chloroquine, sulfadoxine-pyrimethamine, amodiaquine, and primaquine. Trials in healthy subjects or patients with Plasmodium falciparum or P. vivax infection were included if at least two ECGs were conducted during the trial. All trial designs were included except case reports and pooled analyses. Secondary outcomes were the methods adopted by trials for measuring and reporting the QT interval.ResultsData from trials published between 1982 and July 2016 were included. A total of 177 trials met the inclusion criteria. 35,448 participants received quinoline antimalarials in these trials, of which 18,436 participants underwent ECG evaluation. Subjects with co-medication use or comorbidities including cardiovascular disease were excluded from the majority of trials. Dihydroartemisinin-piperaquine was the drug most studied (5083 participants). Despite enormous use over the past 60 years, only 1076, 452, and 150 patients had ECG recordings reported in studies of chloroquine, amodiaquine, and primaquine respectively. Transiently high concentrations of quinine, quinidine, and chloroquine following parenteral administration have all been associated with hypotension, but there were no documented reports of death or syncope attributable to a cardiovascular cause, nor of electrocardiographic recordings of ventricular arrhythmia in these trials. The large volume of missing outcome information and the heterogeneity of ECG interval reporting and measurement methodology did not allow pooled quantitative analysis of QT interval changes.ConclusionsNo serious cardiac adverse effects were recorded in malaria clinical trials of 35,548 participants who received quinoline and structurally related antimalarials with close follow-up including 18,436 individuals who underwent ECG evaluation. While these findings provide further evidence of the rarity of serious cardiovascular events after treatment with these drugs, they also underscore the need for continued strengthening of pharmacovigilance systems for robust detection of rare drug adverse events in real-world populations. A standardised approach to measurement and reporting of ECG data in malaria trials is also needed.Trial registrationPROSPERO CRD42016036678

Heterologous Expression of a Novel Drug Transporter from the Malaria Parasite Alters Resistance to Quinoline Antimalarials

Antimalarial drug resistance hampers effective malaria treatment. Critical SNPs in a particular, putative amino acid transporter were recently linked to chloroquine (CQ) resistance in malaria parasites. Here, we show that this conserved protein (PF3D7_0629500 in Plasmodium falciparum; AAT1 in P. chabaudi) is a structural homologue of the yeast amino acid transporter Tat2p, which is known to mediate quinine uptake and toxicity. Heterologous expression of PF3D7_0629500 in yeast produced CQ hypersensitivity, coincident with increased CQ uptake. PF3D7_0629500-expressing cultures were also sensitized to related antimalarials; amodiaquine, mefloquine and particularly quinine. Drug sensitivity was reversed by introducing a SNP linked to CQ resistance in the parasite. Like Tat2p, PF3D7_0629500-dependent quinine hypersensitivity was suppressible with tryptophan, consistent with a common transport mechanism. A four-fold increase in quinine uptake by PF3D7_0629500 expressing cells was abolished by the resistance SNP. The parasite protein localised primarily to the yeast plasma membrane. Its expression varied between cells and this heterogeneity was used to show that high-expressing cell subpopulations were the most drug sensitive. The results reveal that the PF3D7_0629500 protein can determine the level of sensitivity to several major quinine-related antimalarials through an amino acid-inhibitable drug transport function. The potential clinical relevance is discussed.

Iron is a substrate of the Plasmodium falciparum chloroquine resistance transporter PfCRT in Xenopus oocytes

The chloroquine resistance transporter of the human malaria parasite Plasmodium falciparum, PfCRT, is an important determinant of resistance to several quinoline and quinoline-like antimalarial drugs. PfCRT also plays an essential role in the physiology of the parasite during development inside erythrocytes. However, the function of this transporter besides its role in drug resistance is still unclear. Using electrophysiological and flux experiments conducted on PfCRT-expressing Xenopus laevis oocytes, we show here that both wild-type PfCRT and a PfCRT variant associated with chloroquine resistance transport both ferrous and ferric iron, albeit with different kinetics. In particular, we found that the ability to transport ferrous iron is reduced by the specific polymorphisms acquired by the PfCRT variant as a result of chloroquine selection. We further show that iron and chloroquine transport via PfCRT is electrogenic. If these findings in the Xenopus model extend to P. falciparum in vivo, our data suggest that PfCRT might play a role in iron homeostasis, which is essential for the parasite's development in erythrocytes.

The Effects of Quinoline and Non-Quinoline Inhibitors on the Kinetics of Lipid-Mediated β-Hematin Crystallization.

The throughput of a biomimetic lipid-mediated assay used to investigate the effects of inhibitors on the kinetics of β-hematin formation has been optimized through the use of 24-well microplates. The rate constant for β-hematin formation mediated by monopalmitoyl-rac-glycerol was reduced from 0.17 ± 0.04 min-1 previously measured in Falcon tubes to 0.019 ± 0.002 min-1 in the optimized assay. While this necessitated longer incubation times, transferring aliquots from multiple 24-well plates to a single 96-well plate for final absorbance measurements actually improved the overall turnaround time per inhibitor. This assay has been applied to investigate the effects of four clinically relevant antimalarial drugs (chloroquine, amodiaquine, quinidine, and quinine) as well as several short-chain 4-aminoquinoline derivatives and non-quinoline (benzamide) compounds on the kinetics of β-hematin formation. The adsorption strength of these inhibitors to crystalline β-hematin (Kads) was quantified using a theoretical kinetic model that is based on the Avrami equation and the Langmuir isotherm. Statistically significant linear correlations between lipid-mediated β-hematin inhibitory activity and Kads values for quinoline (r2 = 0.76, P-value = 0.0046) and non-quinoline compounds (r2 = 0.99, P-stat = 0.0006), as well as between parasite inhibitory activity (D10) and Kads values for quinoline antimalarial drugs and short-chain chloroquine derivatives (r2 = 0.64, P-value = 0.0098), provide a strong indication that drug action involves adsorption to the surface of β-hematin crystals. Independent support in this regard is provided by experiments that spectrophotometrically monitor the direct adsorption of antimalarial drugs to preformed β-hematin.

Antimalarials inhibit hematin crystallization by unique drug–surface site interactions

In malaria pathophysiology, divergent hypotheses on the inhibition of hematin crystallization posit that drugs act either by the sequestration of soluble hematin or their interaction with crystal surfaces. We use physiologically relevant, time-resolved in situ surface observations and show that quinoline antimalarials inhibit β-hematin crystal surfaces by three distinct modes of action: step pinning, kink blocking, and step bunch induction. Detailed experimental evidence of kink blocking validates classical theory and demonstrates that this mechanism is not the most effective inhibition pathway. Quinolines also form various complexes with soluble hematin, but complexation is insufficient to suppress heme detoxification and is a poor indicator of drug specificity. Collectively, our findings reveal the significance of drug-crystal interactions and open avenues for rationally designing antimalarial compounds.

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

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.