Mechanisms of potential novel antimalarials and proteomics of plasmodium falciparum resistance

Introduction: Plasmodium falciparum is the dominant cause of severe malaria in humans, with the highest number of global deaths occurring in Africa and Southeast Asia. Even though artemisinin-derivative combination therapies are readily available in Africa there are numerous reports of poor quality products which may lead to the development of drug resistance in Plasmodium parasites. Other aspects such as counterfeit medicines with sub-therapeutic levels of active ingredients may also be a contributing factor. The exact mechanism of P. falciparum drug resistance is still poorly understood, but numerous proteins linked to multi-drug resistance have been identified. The aim of this study was to ascertain whether clofazimine can act as a chemotherapeutic sensitiser as reported, and to determine its ability to alter the expression of chloroquine resistance gene products in vitro. Materials and Methods: In this study the 50% inhibitory concentrations of (IC50) humic and fulvic acid, the 3-hydroxy-3-methyl-glutaryl-Coenzyme A (HMG-CoA) reductase inhibitor mevinolin (lovastatin) and a riminophenazine antibiotic clofazimine were established using SYBR® green dye and fluorometric assays using chloroquine sensitive (3D7), chloroquine sensitive- pyrimethamine resistant (HB3) and chloroquine resistant (W2) parasites which demonstrate mefloquine, antifolate and chloroquine resistance respectively. Eflornithine (DFMO) is a polyamine synthesis inhibitor that was used in sensitivity assays for comparison. The selective toxicity of each of these agents was determined by Sulforhodamine B (SRB) colorimetric assays in hepatocarcinoma cells (HepG2). Flow cytometry techniques were employed to establish any alterations in life-cycle progression. Comparative proteomics of possible resistance proteins expressed on the membrane of the parasite food vacuole was conducted on W2, 3D7 and clofazimine-treated W2 strains of P. falciparum. Food vacuoles isolated by MidiMACS magnetic purification were separated using one dimensional gel electrophoresis, followed by in-gel trypsinisation, sample clean-up, fractionation by nano-liquid chromatography and mass spectrometric analysis by Matrix-Assisted Laser Desorption/Ionisation (MALDI) and Time of Flight (TOF) assays. Proteins were further identified in silico through the use of proteomic databases and homology comparison software. Results: Mevinolin showed poor antiplasmodial efficacy (IC50 of 1.19 x105 ± 1.02 nM), in comparison to artemisinin and chloroquine (32.61 ± 1.03 nM and 8.36 ± 1.03 nM, respectively) in 3D7 strains. Clofazimine showed greater antiplasmodial efficacy than DFMO in W2 strains (IC50 of 272.00 ± 1.04 and 3.39 x105 ± 1.06 nM, respectively). W2 strains were confirmed to be less susceptible to chloroquine (p<0.001). All test compounds showed decreased toxicity for model mammalian cells (p<0.001), except for mevinolin. Clofazimine (constant dose 375 nM) sensitised W2 strains to the actions of chloroquine (p