Marylin Torrentino-Madamet

Atorvastatin Is 10-Fold More Active In Vitro than Other Statins against Plasmodium falciparum

Statins, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, influence a broad array of pathogenic microorganisms. Lovastatin reduces the intracellular growth of Salmonella enterica serovar Typhimurium in cultured macrophages, while atorvastatin does the same in a mouse model (2). Lovastatin additionally reduces the growth of Candida albicans by inhibiting the sterol pathway (11). Statins interfere severely with the growth of protozoan parasites of the Trypanosomatidae family such as Trypanosoma cruzi and various Leishmania species (8, 12, 13). HMG-CoA reductase has been detected in Trypanosoma and Leishmania (3, 8). The presence of an HMG-CoA homolog was not revealed by BLASTX analysis of the Plasmodium falciparum sequence with other protozoal HMG-CoA protein sequences. However, as reported previously, treatment in vitro of Plasmodium falciparum with 120 or 240 μM mevastatin inhibited parasite growth (4, 9). The susceptibilities to simvastatin, simvastatin sodium salt, pravastatin sodium salt, lovastatin, fluvastatin sodium salt, mevastatin, mevastatin sodium salt (Calbiochem, Merck, Germany), and atorvastatin calcium salt (Molekula, United Kingdom) were assessed in vitro against chloroquine-susceptible P. falciparum strains 3D7 (Africa), D6 (Sierra Leone), and IMT031 (Gabon) and chloroquine-resistant strains W2 (Indochina), Bre1 (Brazil), and FCR3 (The Gambia). Lovastatin and mevastatin were converted to the active form by dissolving the lactone form in 100 μl of 100% ethanol, adding 200 μl of 0.2 M KOH, and then adding 0.2 M HCl for neutralization to pH 7.2 (5). Simvastatin, simvastatin sodium salt, pravastatin sodium salt, lovastatin, fluvastatin sodium salt, mevastatin, mevastatin sodium salt, and atorvastatin calcium salt were dissolved in dimethyl sulfoxide 1% (vol/vol) in RPMI. Twofold serial dilutions, with final concentrations ranging from 1.5 μM to 200 μM, were prepared in dimethyl sulfoxide 1% in RPMI and distributed into Falcon 96-well plates just before use. The isotopic microdrug susceptibility test used was described previously (10). Table ​Table11 presents the 50% inhibitory concentrations (IC50) of the different statins for P. falciparum. Simvastatin, fluvastatin, lovastatin, and atorvastatin, in the salt active forms, are more active than simvastatin, mevastatin, and lovastatin, in the lactone form. Pravastatin and mevastatin sodium or potassium salts are inactive against P. falciparum (>200 μM). The results indicate that susceptibility to the salts of simvastatin, fluvastatin, lovastatin, and mevastatin is not dependent on the status of chloroquine resistance. The results observed with the simvastatin salt were similar to those reported by other authors (5). Atorvastatin salt, in the range of 5 to 12 μM, is 10-fold more active against P. falciparum than the other salts. Atorvastatin IC90s ranged from 14.8 to 39 μM. The activity of atorvastatin is independent of the status of chloroquine resistance (4.8 to 5.8 μM against chloroquine-resistant strains versus 5.3 to 11.8 μM for the susceptible strains). TABLE 1. In vitro activities of statins against chloroquine-susceptible (3D7, D6, and IMT031) and chloroquine-resistant (W2, Bre1, and FCR3) P. falciparum strains The chemical structures of simvastatin, lovastatin, mevastatin, and pravastatin are closely related. Those of fluvastatin and atorvastatin are very different from the others. The structural differences between atorvastatin and the other statins could explain differential activity. However, we cannot rule out the action of calcium in the differential activity of atorvastatin. Multiple daily doses of 2.5 to 80 mg of atorvastatin produced steady-state maximum plasma concentrations of 1.95 to 252 μg liter−1 (in the range of 0.2 to 0.3 μM for the maximum) (1). In L6 cells (rat skeletal muscle cell line), atorvastatin at 100 μM induced death in 27% of the cells (7). Although the atorvastatin IC50 for P. falciparum exceeds these reported plasma concentrations, it may be below toxic concentrations. Parasites treated with mevastatin show depressed biosynthesis of dolichol and isoprenoid pyrophosphate (4). In addition, mevastatin decreases the viability of cells by inhibiting proteasome activity. Atorvastatin is an inhibitor for phosphoglycoprotein, an efflux protein, and may be a substrate for this transporter as well (6). A phosphoglycoprotein in P. falciparum, Pgh1, is implicated in quinoline resistance. In conclusion, the present observation suggests that atorvastatin is a good candidate for further studies on the use of statins in malaria treatment.

Limited polymorphisms in k13 gene in Plasmodium falciparum isolates from Dakar, Senegal in 2012–2013

BackgroundThe emergence of Plasmodium falciparum resistance to artemisinin and its derivatives, manifested as delayed parasite clearance following the treatment, has developed in Southeast Asia. The spread of resistance to artemisinin from Asia to Africa may be catastrophic for malaria control and elimination worldwide. Recently, mutations in the propeller domain of the Kelch 13 (k13) gene (PF3D71343700) were associated with in vitro resistance to artemisinin and with delayed clearance after artemisinin treatment in southern Asia. The aim of the study was to characterize the genetic variability of k13 and to evaluate the molecular resistance to artemisinin for the first time in Senegal.MethodsPlasmodium falciparum isolates were collected from 138 malaria patients in Dakar and its districts during the rainy season of October 2012 to January 2013 at the Hôpital Principal de Dakar. The k13 gene was amplified using nested PCR and sequenced.ResultsA very limited variability within the k13 gene in Senegalese P. falciparum isolates was identified. No polymorphism was detected in the six k13-propeller blades. Only two mutations, T149S (6.3%) and K189T (42.2%), and one (N) or two (NN) asparagine insertion at the codon 142 (4.7 and 6.3%, respectively) were detected in the Plasmodium/Apicomplexa-specific domain. None of the polymorphisms associated with artemisinin resistance in Southeast Asia was detected in the 138 P. falciparum from Dakar.DiscussionThe present data do not suggest widespread artemisinin resistance in Dakar in 2012–2013. Notably, the C580Y, R539T or Y493H substitutions that were associated with in vitro resistance or delayed parasite clearance in Southeast Asia were not observed in Dakar, nor were any of the polymorphisms observed in parasites from Southeast Asia, nor the M476I mutation that was selected in vitro with artemisinin pressure in a African parasite line.

Global response of Plasmodium falciparum to hyperoxia: a combined transcriptomic and proteomic approach.

BackgroundOver its life cycle, the Plasmodium falciparum parasite is exposed to different environmental conditions, particularly to variations in O2 pressure. For example, the parasite circulates in human venous blood at 5% O2 pressure and in arterial blood, particularly in the lungs, at 13% O2 pressure. Moreover, the parasite is exposed to 21% O2 levels in the salivary glands of mosquitoes.MethodsTo study the metabolic adaptation of P. falciparum to different oxygen pressures during the intraerythrocytic cycle, a combined approach using transcriptomic and proteomic techniques was undertaken.ResultsEven though hyperoxia lengthens the parasitic cycle, significant transcriptional changes were detected in hyperoxic conditions in the late-ring stage. Using PS 6.0™ software (Ariadne Genomics) for microarray analysis, this study demonstrate up-expression of genes involved in antioxidant systems and down-expression of genes involved in the digestive vacuole metabolism and the glycolysis in favour of mitochondrial respiration. Proteomic analysis revealed increased levels of heat shock proteins, and decreased levels of glycolytic enzymes. Some of this regulation reflected post-transcriptional modifications during the hyperoxia response.ConclusionsThese results seem to indicate that hyperoxia activates antioxidant defence systems in parasites to preserve the integrity of its cellular structures. Moreover, environmental constraints seem to induce an energetic metabolism adaptation of P. falciparum. This study provides a better understanding of the adaptive capabilities of P. falciparum to environmental changes and may lead to the development of novel therapeutic targets.

Atorvastatin Is a Promising Partner for Antimalarial Drugs in Treatment of Plasmodium falciparum Malaria

ABSTRACT Atorvastatin (AVA) is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor. AVA exposure resulted in the reduced in vitro growth of 22 Plasmodium falciparum strains, with the 50% inhibitory concentrations (IC50s) ranging from 2.5 μM to 10.8 μM. A significant positive correlation was found between the strains’ responses to AVA and mefloquine (r = 0.553; P = 0.008). We found no correlation between the responses to AVA and to chloroquine, quinine, monodesethylamodiaquine, lumefantrine, dihydroartemisinin, atovaquone, or doxycycline. These data could suggest that the mechanism of AVA uptake and/or the mode of action of AVA is different from those for other antimalarial drugs. The IC50s for AVA were unrelated to the occurrence of mutations in the transport protein genes involved in quinoline antimalarial drug resistance, such as the P. falciparum crt, mdr1, mrp, and nhe-1 genes. Therefore, AVA can be ruled out as a substrate for the transport proteins (CRT, Pgh1, and MRP) and is not subject to the pH modification induced by the P. falciparum NHE-1 protein. The absence of in vitro cross-resistance between AVA and chloroquine, quinine, mefloquine, monodesethylamodiaquine, lumefantrine, dihydroartemisinin, atovaquone, and doxycycline argues that these antimalarial drugs could potentially be paired with AVA as a treatment for malaria. In conclusion, the present observations suggest that AVA is a good candidate for further studies on the use of statins in association with drugs known to have activities against the malaria parasite.

Plasmodium falciparum infection-induced changes in erythrocyte membrane proteins

Over the past decade, advances in proteomic and mass spectrometry techniques and the sequencing of the Plasmodium falciparum genome have led to an increasing number of studies regarding the parasite proteome. However, these studies have focused principally on parasite protein expression, neglecting parasite-induced variations in the host proteome. Here, we investigated P. falciparum-induced modifications of the infected red blood cell (iRBC) membrane proteome, taking into account both host and parasite proteome alterations. Furthermore, we also determined if some protein changes were associated with genotypically distinct P. falciparum strains. Comparison of host membrane proteomes between iRBCs and uninfected red blood cells using fluorescence-based proteomic approaches, such as 2D difference gel electrophoresis revealed that more than 100 protein spots were highly up-represented (fold change increase greater than five) following P. falciparum infection for both strains (i.e. RP8 and Institut Pasteur Pregnancy Associated Malaria). The majority of spots identified by mass spectrometry corresponded to Homo sapiens proteins. However, infection-induced changes in host proteins did not appear to affect molecules located at the outer surface of the plasma membrane. The under-representation of parasite proteins could not be attributed to deficient parasite protein expression. Thus, this study describes for the first time that considerable host protein modifications were detected following P. falciparum infection at the erythrocyte membrane level. Further analysis of infection-induced host protein modifications will improve our knowledge of malaria pathogenesis.

Dihydroethanoanthracene Derivatives as In Vitro Malarial Chloroquine Resistance Reversal Agents

ABSTRACT The ability of four 9,10-dihydroethanoanthracene derivatives (BG920, BG932, BG958, and BG996), as well as verapamil and promethazine, to reverse chloroquine resistance was assessed against 24 chloroquine-resistant and 10 chloroquine-susceptible strains of Plasmodium falciparum from different countries. The 9,10-dihydroethanoanthracene derivatives clearly increase chloroquine susceptibility only in chloroquine-resistant isolates.

Polymorphism in Plasmodium falciparum Drug Transporter Proteins and Reversal of In Vitro Chloroquine Resistance by a 9,10-Dihydroethanoanthracene Derivative

ABSTRACT BG958 reverses resistance in chloroquine-resistant isolates from different countries. Five mutations in the Plasmodium falciparum crt (pfcrt) gene resulting in the amino acid changes K76T, M74I, N75E, A220S, and R371I are systematically identified in resistance-reversed Asian, African, and Brazilian parasites which possess the pfcrt (CIET) haplotype. In combination with BG958, the activity of chloroquine is increased in parasites with the N86Y mutation in pfmdr1.

Plasmodium falciparumRecrudescence Two Years after a First Treated Uncomplicated Infection without Return in a Malaria Endemic Area

ABSTRACT We report evidence, confirmed by the lack of travel activity outside of France and genetic diversity analysis using polymorphic microsatellite markers, that Plasmodium falciparum malaria infection effectively treated with an artemisinin-based combination can remain dormant and relapse during pregnancy at least 2 years after treatment.