Proteomics analysis of antimalarial targets of Garcinia mangostana Linn

Objective: To investigate possible protein targets for antimalarial activity of Garcinia mangostana Linn. (G. mangostana) (pericarp) in 3D7 Plasmodium falciparum clone using 2-dimensional electrophoresis and liquid chromatography mass-spectrometry (LC/MS/MS). Methods: 3D7 Plasmodium falciparum was exposed to the crude ethanolic extract of G.mangostana Linn. (pericarp) at the concentrations of 12μg/mL (IC50 level: concentration that inhibits parasite growth by 50%) and 30 μg/mL (IC90 level: concentration that inhibits parasite growth by 90%) for 12 h. Parasite proteins were separated by 2-dimensional electrophoresis and identified by LC/MS/MS.Results:At the IC50 concentration, about 82% of the expressed parasite proteins were matched with the control (non-exposed), while at the IC90 concentration, only 15% matched proteins were found. The selected protein spots from parasite exposed to the plant extract at the concentration of 12 μg/mL were identified as enzymes that play role in glycolysis pathway, i.e., phosphoglycerate mutase putative, L-lactate dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase, and fructose-bisphosphate aldolase/phosphoglycerate kinase. The proteosome was found in parasite exposed to 30 μg/mL of the extract.Conclusions:Results suggest that proteins involved in the glycolysis pathway may be the targets for antimalarial activity of G. mangostana Linn. (pericarp).

Polymorphic patterns of pfcrt and pfmdr1 in Plasmodium falciparum isolates along the Thai-Myanmar border

Objective: To investigate the distribution and patterns of pfcrt and pfmdr1 polymorphisms in Plasmodium falciparum (P. falciparum) isolates collected from the malaria endemic area of Thailand along Thai-Myanmar border. Methods: Dried blood spot samples were collected from 172 falciparum malaria patients prior received treatment. The samples were extracted using chelex to obtain parasite DNA. PCR-RFLP was employed to detect pfcrt mutation at codons 76, 220, 271, 326, 356 and 371, and the pfmdr1 mutation at codon 86. Pfmdr1 gene copy number was determined by SYBR Green I real-time PCR. Results:Mutant alleles of pfcrt and wild type allele of pfmdr1 were found in almost all samples. Pfmdr1 gene copy number in isolates collected from all areas ranged from 1.0 to 5.0 copies and proportion of isolates carrying>1 gene copies was 38.1%. The distribution and patterns of pfcrt and pfmdr1 mutations were similar in P. falciparum isolates from all areas. However, significant differences in both number of pfmdr1 copies and prevalence of isolates carrying>1 gene copies were observed among isolates collected from different areas. The median pfmdr1 copy number in P. falciparum collected from Kanchanaburi and Mae Hongson were 2.5 and 2.0, respectively and more than half of the isolates carried>1 gene copies.

Comparison of protein patterns between Plasmodium falciparum mutant clone T9/94-M1-1(b3) induced by pyrimethamine and the original parent clone T9/94

Objective: To compare the protein patterns from the extracts of the mutant clone T9/94-M1-1(b3) induced by pyrimethamine, and the original parent clone T9/94 following separation of parasite extracts by two-dimensional electrophoresis (2-DE). Methods: Proteins were solubilized and separated according to their charges and sizes. The separated protein spots were then detected by silver staining and analyzed for protein density by the powerful image analysis software. Results:Differentially expressed protein patterns (up- or down-regulation) were separated from the extracts from the two clones. A total of 223 and 134 protein spots were detected from the extracts of T9/94 and T9/94-M1-1(b3) clones, respectively. Marked reduction in density of protein expression was observed with the extract from the mutant (resistant) clone compared with the parent (sensitive) clone. A total of 25 protein spots showed at least two-fold difference in density, some of which exhibited as high as ten-fold difference. Conclusions: These proteins may be the molecular targets of resistance of Plasmodium falciparum to pyrimethamine. Further study to identify the chemical structures of these proteins by mass spectrometry is required.

Assessment of in vitro antimalarial interactions between dihydroartemisinin and fosmidomycin.

Malaria remains one of the leading causes of morbidity and mortality in the tropics with an annual estimate of 500 million clinical cases and 2 million deaths. The treatment and control of malaria is becoming increasingly difficult due to Plasmodium falciparum resistance to commonly used antimalarials. Combination therapy is currently the strategy for combating multi-drug resistant falciparum malaria, through exploiting pharmacodynamic synergistic effects and delaying the emergence of drug resistance. The combination of artemisinin derivatives with fosmidomycin, which have different modes of action, appears to be one of the most promising combinations. The objective of the present study was to investigate the antimalarial interactions between dihydroartemisinin and fosmidomycin in vitro, against chloroquine-resistant (K1) and chloroquine-sensitive (G112) P. falciparum strains. Concentration-response analysis was performed based on an in vitro schizont maturation inhibition test. The fixed concentration ratios of dihydroartemisinin: fosmidomycin used were 0:5,000, 2:4,500, 6:3,500, 10:2,500, 14:1,500, 18:500 and 20:0 nM. The highest final concentrations of dihydroartemisinin and fosmidomycin were 20 and 5,000 nM, respectively. Results showed IC50 (drug concentration which produced 50% schizont maturation inhibition) medians (range) for dihydroartemisinin against K1 and G112 strains to be 1.6 (1.2-2.0) and 2.5 (2.4-2.6) nM, respectively. The IC50 medians (range) for fosmidomycin against K1 and G112 strains were 1,347 (1,068-1,625) and 786 (737-834) nM, respectively. An isobologram revealed an increasing trend for the fraction IC50 (FIC), which indicates marked antagonism of this drug combination against both chloroquine resistant and chloroquine sensitive strains.

Comparison of two in vitro sensitivity tests for Plasmodium falciparum.

The main purpose of the study was to compare the in vitro sensitivity results obtained from the two widely-used in vitro systems: (1) standard WHO micro-technique based on schizont maturation inhibition using fresh isolates (M-I), and (2) micro-technique based on incorporation of [3H]-hypoxanthine using culture-adapted isolates (M-II). The study was conducted during 1998 and 2002. A total of 473 Plasmodium falciparum isolates were collected from five highly malaria endemic areas of Thailand, ie, Mae Sot district, Tak (north-western), Kanchanaburi (western), Ranong (south-western), Ratchaburi (south-western) and Chantaburi (eastern) Provinces. The antimalarials tested were: mefloquine, quinine, chloroquine, artemisinin and dihydroartemisinin. The sensitivity results for mefloquine obtained from the two methods were significantly different from each other. The IC50 values for M-II was less than M-I. The median (95%C.I.) IC50 value for mefloquine using the M-II method was significantly lower [696.47 (393.11-1,233.2) nM] than for M-I [3,955.4 (1,035.61-5,108.9) nM]. The in vitro sensitivity results for quinine were significantly different from each other. The median (95% C.I.) IC50 value for M-II [161 (42-351) nM] was 2.5-fold that of M-I [66 (24-450) nM].