Comment

Na⁺/H⁺ Antiporter, Chloroquine Uptake and Drug Resistance: Inconsistencies in a Newly Proposed Model

Malaria remains one of the major health concerns, especially in tropical countries. Although drugs such as artemisinin-based combinations are efficient for treating Plasmodium falciparum, the growing threat from multi-drug resistance has become a major challenge. Thus, there is a constant need to identify and validate new combinations to sustain current disease control strategies to overcome the challenge of drug resistance in the malaria parasites. To meet this demand, liquiritigenin (LTG) has been found to positively interact in combination with the existing clinically used drug chloroquine (CQ), which has become unfunctional due to acquired drug resistance.

To evaluate the best interaction between LTG and CQ against CQ- resistant strain of P. falciparum. Furthermore, the in vivo antimalarial efficacy and possible mechanism of action of the best combination was also assessed.

The in vitro anti-plasmodial potential of LTG against CQ- resistant strain K1 of P. falciparum was tested using Giemsa staining method. The behaviour of the combinations was evaluated using the fix ratio method and evaluated the interaction of LTG and CQ by calculating the fractional inhibitory concentration index (FICI). Oral toxicity study was carried out in a mice model. In vivo antimalarial efficacy of LTG alone and in combination with CQ was evaluated using a four-day suppression test in a mouse model. The effect of LTG on CQ accumulation was measured using HPLC and the rate of alkalinization of the digestive vacuole. Cytosolic Ca²⁺ level, mitochondrial membrane potential, caspase-like activity, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and Annexin V Apoptosis assay to assess anti-plasmodial potential. Proteomics analysis was evaluated by LC-MS/MS analysis.

LTG possesses anti-plasmodial activity on its own and it showed to be an adjuvant of CQ. In in vitro studies, LTG showed synergy with CQ only in the ratio (CQ: LTG-1:4) against CQ-resistant strain (K1) of P. falciparum. Interestingly, in vivo studies, LTG in combination with CQ showed higher chemo-suppression and enhanced mean survival time at much lower concentrations compared to individual doses of LTG and CQ against CQ- resistant strain (N67) of Plasmodium yoelli nigeriensis. LTG was found to increase the CQ accumulation into digestive vacuole, reducing the rate of alkalinization, in turn increasing cytosolic Ca²⁺ level, loss of mitochondrial potential, caspase-3 activity, DNA damage and externalization of phosphatidylserine of the membrane (in vitro). These observations indicate the involvement of apoptosis-like death of P. falciparum that might be due to the accumulation of CQ.

LTG showed synergy with CQ in the ratio LTG: CQ, 4:1) in vitro and was able to curtail the IC₅₀ of CQ and LTG. Interestingly, in vivo in combination with CQ, LTG showed higher chemo-suppression as well as enhanced mean survival time at a much lower concentrations of both the partners as compared to an individual dose of CQ and LTG. Thus, synergistic drug combination offers the possibility to enhance CQ efficacy in chemotherapy.

Au cours de leur évolution, les micro-organismes ont su déjouer les pièges qui leur sont tendus par l’environnement et notamment leur hôte (immunité et utilisation de molécules anti-infectieuses). L’émergence et la diffusion de la résistance aux antipaludiques posent un sérieux problème de santé publique. Plasmodium falciparum est maintenant résistant à tous les antipaludiques utilisés même aux derniers commercialisés comme les associations à base d’artémisinine. Les échecs prophylactiques ou thérapeutiques entraînent une ré-émergence du paludisme s’accompagnant d’une augmentation de la transmission, de la morbidité et de la mortalité. La connaissance des mécanismes de résistance permet le développement de nouvelles molécules qui diminueront la résistance, d’identifier les cibles de nouveaux antipaludiques et enfin d’identifier des marqueurs moléculaires pour la surveillance de la résistance aux antipaludiques. La résistance est souvent associée 1) à une altération d’enzymes clé qui sont des cibles d’antipaludiques, 2) à l’altération de l’accumulation de l’antipaludique dans le parasite résultant d’une diminution d’entrée ou d’une augmentation de sortie (efflux) de la molécule, voire aux deux. Des données épidémiologiques, les modes d’action, les mécanismes de résistance et les marqueurs moléculaires de résistance sont présentés pour chaque antipaludique actuellement utilisé.

Throughout the course of evolution, micro-organisms have thwarted traps set by the environment including those designed by man (immunity and use of anti-infectious drugs). The emergence and spread of antimalarial drug resistance poses a severe and increasing public health threat. The Plasmodium falciparum parasite is now resistant to all of the used antimalarial drugs, even to the latest artemisinin-based combination treatments. Failures in prophylaxis or treatments induce the re-emergence of parasite-related morbidity and mortality. Knowledge about resistance mechanisms involved may allow the development of new drugs that minimize or circumvent drug resistance, may allow the identification of new targets for drug development and to identify molecular markers for malaria resistance surveillance. Resistance is often associated with 1) inhibition of alteration of key enzymes that are targets for antimalarial drugs or 2) alteration of drug accumulation into the parasite which results from reduced uptake of the drug, an increased efflux, or a combination of the two processes. Epidemiological data, mode of action, mechanisms of resistance and resistance molecular markers are presented for each antimalarial drugs used at the present time.

The intraerythrocytic malaria parasite, Plasmodium falciparum maintains an intracellular pH (pH_i) of around 7.3. If subjected to an experimentally imposed acidification the parasite extrudes H⁺, thereby undergoing a pH_i recovery. In a recent study, Bennett et al. [Bennett TN, Patel J, Ferdig MT, Roepe PD. P. falciparum Na⁺/H⁺ exchanger activity and quinine resistance. Mol Biochem Parasitol 2007;153:48–58] used the H⁺ ionophore nigericin, in conjunction with an acidic medium, to acidify the parasite cytosol, and then used bovine serum albumin (BSA) to scavenge the nigericin from the parasite membrane. The ensuing Na⁺-dependent pH_i recovery, seen following an increase in the extracellular pH, was attributed to a plasma membrane Na⁺/H⁺ exchanger. This is at odds with previous reports that the primary H⁺ extrusion mechanism in the parasite is a plasma membrane V-type H⁺-ATPase. Here we present evidence that the Na⁺-dependent efflux of H⁺ from parasites acidified using nigericin/BSA is attributable to Na⁺/H⁺ exchange via residual nigericin remaining in the parasite plasma membrane, rather than to endogenous transporter activity.

Live cell fluorescence microscopy has been widely used to study physiological processes in the human malarial parasitePlasmodium falciparum, including pH homeostasis, Ca²⁺ signaling and protein targeting. However, the reproducibility of the data is often poor. Controversial statements exist regarding cytosolic and vacuolar baseline pH, as well as regarding the subcellular localization of some of the fluorochromes used. When trying to reproduce published baseline values, we observed an unexpected light sensitivity of P. falciparum, which manifests itself in the form of a strong cytoplasmic acidification. Even short exposure times with moderate to low light intensities caused the parasite cytosol to acidify. We show that this effect arises from the selective disruption of the parasite's acidic food vacuole, brought about by lipid peroxidation initiated by light-induced generation of hydroxyl radicals. Our data suggest that heme serves as a photosensitizer in this process. Our findings have major implications for the use of live cell microscopy in P. falciparum and add a cautionary note to previous studies where live cell fluorometry has been used to determine physiological parameters in P. falciparum.

Malaria has plagued humans throughout recorded history and results in the death of over 2 million people per year. The protozoan parasite Plasmodium falciparum causes the most severe form of malaria in humans. Chemotherapy has become one of the major control strategies for this parasite; however, the development of drug resistance to virtually all of the currently available drugs is causing a crisis in the use and deployment of these compounds for prophylaxis and treatment of this disease. The genome sequence of P. falciparum is providing the informational base for the use of whole-genome strategies such as bioinformatics, microarrays and genetic mapping. These approaches, together with the availability of a high-resolution genome linkage map consisting of hundreds of microsatellite markers and the advanced technologies of transfection and proteomics, will facilitate an integrated approach to address important biological questions. In this review we will discuss strategies to identify novel genes involved in the molecular mechanisms used by the parasite to circumvent the lethal effect of current chemotherapeutic agents.