Probing the Antiplasmodial Properties of Plakortinic Acids C and D: An Uncommon Pair of Marine Peroxide-Polyketides Isolated from a Two-Sponge Association of Plakortis symbiotica and Xetospongia deweerdtae Collected near Puerto Rico.

Plakortinic acids C (1) and D (2), an unseparable pair of endoperoxide polyketides isolated and purified from the symbiotic association of Caribbean Sea sponges Plakortis symbiotica-Xestospongia deweerdtae, underwent in vitro evaluation for antiplasmodial activity against the malaria parasite Plasmodium berghei using a drug luminescence assay. Initial screening at 10 µM revealed 50% in vitro parasite growth inhibition. The title compounds displayed antiplasmodial activity with an EC50 of 5.3 µM toward P. berghei parasites. The lytic activity against erythrocytes was assessed through an erythrocyte cell lysis assay, which showed non-lytic activity at lower concentrations ranging from 1.95 to 3.91 µM. The antiplasmodial activity and the absence of hemolytic activity support the potential of plakortinic acids C (1) and D (2) as promising lead compounds. Moreover, drug-likeness (ADMET) properties assessed through the pkCSM server predicted high intestinal absorption, hepatic metabolism, and volume of distribution, indicating favorable pharmacokinetic profiles for oral administration. These findings suggest the potential suitability of these metabolites for further investigations of antiplasmodial activity in multiple parasitic stages in the mosquito and Plasmodium falciparum. Notably, this study represents the first report of a marine natural product exhibiting the unique 7,8-dioxatricyclo[4.2.2.02,5]dec-9-ene motif being evaluated against malaria.

Novel hydrazone compounds with broad-spectrum antiplasmodial activity and synergistic interactions with antimalarial drugs.

The development of novel antiplasmodial compounds with broad-spectrum activity against different stages of Plasmodium parasites is crucial to prevent malaria disease and parasite transmission. This study evaluated the antiplasmodial activity of seven novel hydrazone compounds (referred to as CB compounds: CB-27, CB-41, CB-50, CB-53, CB-58, CB-59, and CB-61) against multiple stages of Plasmodium parasites. All CB compounds inhibited blood stage proliferation of drug-resistant or sensitive strains of Plasmodium falciparum in the low micromolar to nanomolar range. Interestingly, CB-41 exhibited prophylactic activity against hypnozoites and liver schizonts in Plasmodium cynomolgi, a primate model for Plasmodium vivax. Four CB compounds (CB-27, CB-41, CB-53, and CB-61) inhibited P. falciparum oocyst formation in mosquitoes, and five CB compounds (CB-27, CB-41, CB-53, CB-58, and CB-61) hindered the in vitro development of Plasmodium berghei ookinetes. The CB compounds did not inhibit the activation of P. berghei female and male gametocytes in vitro. Isobologram assays demonstrated synergistic interactions between CB-61 and the FDA-approved antimalarial drugs, clindamycin and halofantrine. Testing of six CB compounds showed no inhibition of Plasmodium glutathione S-transferase as a putative target and no cytotoxicity in HepG2 liver cells. CB compounds are promising candidates for further development as antimalarial drugs against multidrug-resistant parasites, which could also prevent malaria transmission.

Antimalarial Drug Combination Predictions Using the Machine Learning Synergy Predictor (MLSyPred©) tool.

PURPOSE: Antimalarial drug resistance is a global public health problem that leads to treatment failure. Synergistic drug combinations can improve treatment outcomes and delay the development of drug resistance. Here, we describe the implementation of a freely available computational tool, Machine Learning Synergy Predictor (MLSyPred©), to predict potential synergy in antimalarial drug combinations. METHODS: The MLSyPred© synergy prediction method extracts molecular fingerprints from the drugs' biochemical structures to use as features and also cleans and prepares the raw data. Five machine learning algorithms (Logistic Regression, Random Forest, Support vector machine, Ada Boost, and Gradient Boost) were implemented to build prediction models. Implementation and application of the MLSyPred© tool were tested using datasets from 1540 combinations of 79 drugs and compounds biologically evaluated in pairs for three strains of Plasmodium falciparum (3D7, HB3, and Dd2). RESULTS: The best prediction models were obtained using Logistic Regression for antimalarials with the strains Dd2 and HB3 (0.81 and 0.70 AUC, respectively) and Random Forest for antimalarials with 3D7 (0.69 AUC). The MLSyPred© tool yielded 45% precision for synergistically predicted antimalarial drug combinations that were annotated and biologically validated, thus confirming the functionality and applicability of the tool. CONCLUSION:  The MLSyPred© tool is freely available and represents a promising strategy for discovering potential synergistic drug combinations for further development as novel antimalarial therapies.

Structure-Based Screening of Plasmodium berghei Glutathione S-Transferase Identifies CB-27 as a Novel Antiplasmodial Compound.

Plasmodium falciparum parasites are increasingly drug-resistant, requiring the search for novel antimalarials with distinct modes of action. Enzymes in the glutathione pathway, including glutathione S-transferase (GST), show promise as novel antimalarial targets. This study aims to better understand the biological function of Plasmodium GST, assess its potential as a drug target, and identify novel antiplasmodial compounds using the rodent model P. berghei. By using reverse genetics, we provided evidence that GST is essential for survival of P. berghei intra-erythrocytic stages and is a valid target for drug development. A structural model of the P. berghei glutathione S-transferase (PbGST) protein was generated and used in a structure-based screening of 900,000 compounds from the ChemBridge Hit2Lead library. Forty compounds were identified as potential inhibitors and analyzed in parasite in vitro drug susceptibility assays. One compound, CB-27, exhibited antiplasmodial activity with an EC50 of 0.5 µM toward P. berghei and 0.9 µM toward P. falciparum multidrug-resistant Dd2 clone B2 parasites. Moreover, CB-27 showed a concentration-dependent inhibition of the PbGST enzyme without inhibiting the human ortholog. A shape similarity screening using CB-27 as query resulted in the identification of 24 novel chemical scaffolds, with six of them showing antiplasmodial activity ranging from EC50 of 0.6-4.9 µM. Pharmacokinetic and toxicity predictions suggest that the lead compounds have drug-likeness properties. The antiplasmodial potency, the absence of hemolytic activity, and the predicted drug-likeness properties position these compounds for lead optimization and further development as antimalarials.

Association of Plasmodium falciparum with Human Endothelial Cells in vitro.

Endothelial abnormalities play a critical role in the pathogenesis of malaria caused by the human pathogen, Plasmodium falciparum. In serious infections and especially in cerebral malaria, red blood cells infected with the parasite are sequestered in small venules in various organs, resulting in endothelial activation and vascular occlusion, which are believed to be largely responsible for the morbidity and mortality caused by this infection, especially in children. We demonstrate that after incubation with infected red blood cells (iRBCs), cultured human umbilical vein endothelial cells (HUVECs) contain parasite protein, genomic DNA, and RNA, as well as intracellular vacuoles with apparent parasite-derived material, but not engulfed or adherent iRBCs. The association of this material with the HUVECs is observed over 96 hours after removal of iRBCs. This phenomenon may occur in endothelial cells in vivo by the process of trogocytosis, in which transfer of material between cells depends on direct cell contact. This process may contribute to the endothelial activation and disruption involved in the pathogenesis of cerebral malaria.

Marvels and Shadows: Science and Education at the University of Puerto Rico School of Tropical Medicine under the Auspices of Columbia University: An Introduction.

This essay introduces a series of five historical articles on the scientific and educational contributions of the University of Puerto Rico School of Tropical Medicine (STM), under the auspices of Columbia University (1926-1949), to the fields of tropical medicine and public health. The articles will appear in several consecutive issues, and will address various themes as follows: 1) historical antecedents of the STM, particularly institutional precedents; 2) the educational legacy of the STM; 3) a history of the STM scientific journal ("The Puerto Rico Journal of Public Health and Tropical Medicine"); 4) the scientific practices and representations that prevailed at the institution; and, 5) a brief sociocultural history of malaria in Puerto Rico, mainly from the perspective of the STM's scientific and public health activities. The authors have systematically and comprehensively studied a wide variety of documents from different sources based on multiple archives in Puerto Rico, the United States and England. The authors treat the fluid meanings of the examined historical encounters from a research perspective that privilege complex reciprocal interactions, multiple adaptations and elaborate sociocultural constructs present in a collaborative exemplar of the modernity of medical science in a neocolonial tropical context.

Glutathione-deficient Plasmodium berghei parasites exhibit growth delay and nuclear DNA damage.

Plasmodium parasites are exposed to endogenous and exogenous oxidative stress during their complex life cycle. To minimize oxidative damage, the parasites use glutathione (GSH) and thioredoxin (Trx) as primary antioxidants. We previously showed that disruption of the Plasmodium berghei gamma-glutamylcysteine synthetase (pbggcs-ko) or the glutathione reductase (pbgr-ko) genes resulted in a significant reduction of GSH in intraerythrocytic stages, and a defect in growth in the pbggcs-ko parasites. In this report, time course experiments of parasite intraerythrocytic development and morphological studies showed a growth delay during the ring to schizont progression. Morphological analysis shows a significant reduction in size (diameter) of trophozoites and schizonts with increased number of cytoplasmic vacuoles in the pbggcs-ko parasites in comparison to the wild type (WT). Furthermore, the pbggcs-ko mutants exhibited an impaired response to oxidative stress and increased levels of nuclear DNA (nDNA) damage. Reduced GSH levels did not result in mitochondrial DNA (mtDNA) damage or protein carbonylations in neither pbggcs-ko nor pbgr-ko parasites. In addition, the pbggcs-ko mutant parasites showed an increase in mRNA expression of genes involved in oxidative stress detoxification and DNA synthesis, suggesting a potential compensatory mechanism to allow for parasite proliferation. These results reveal that low GSH levels affect parasite development through the impairment of oxidative stress reduction systems and damage to the nDNA. Our studies provide new insights into the role of the GSH antioxidant system in the intraerythrocytic development of Plasmodium parasites, with potential translation into novel pharmacological interventions.

Multidrug ATP-binding cassette transporters are essential for hepatic development of Plasmodium sporozoites.

Multidrug resistance-associated proteins (MRPs) belong to the C-family of ATP-binding cassette (ABC) transport proteins and are known to transport a variety of physiologically important compounds and to be involved in the extrusion of pharmaceuticals. Rodent malaria parasites encode a single ABC transporter subfamily C protein, whereas human parasites encode two: MRP1 and MRP2. Although associated with drug resistance, their biological function and substrates remain unknown. To elucidate the role of MRP throughout the parasite life cycle, Plasmodium berghei and Plasmodium falciparum mutants lacking MRP expression were generated. P. berghei mutants lacking expression of the single MRP as well as P. falciparum mutants lacking MRP1, MRP2 or both proteins have similar blood stage growth kinetics and drug-sensitivity profiles as wild type parasites. We show that MRP1-deficient parasites readily invade primary human hepatocytes and develop into mature liver stages. In contrast, both P. falciparum MRP2-deficient parasites and P. berghei mutants lacking MRP protein expression abort in mid to late liver stage development, failing to produce mature liver stages. The combined P. berghei and P. falciparum data are the first demonstration of a critical role of an ABC transporter during Plasmodium liver stage development.

Implications of Glutathione Levels in the Plasmodium berghei Response to Chloroquine and Artemisinin.

Malaria is one of the most devastating parasitic diseases worldwide. Plasmodium drug resistance remains a major challenge to malaria control and has led to the re-emergence of the disease. Chloroquine (CQ) and artemisinin (ART) are thought to exert their anti-malarial activity inducing cytotoxicity in the parasite by blocking heme degradation (for CQ) and increasing oxidative stress. Besides the contribution of the CQ resistance transporter (PfCRT) and the multidrug resistant gene (pfmdr), CQ resistance has also been associated with increased parasite glutathione (GSH) levels. ART resistance was recently shown to be associated with mutations in the K13-propeller protein. To analyze the role of GSH levels in CQ and ART resistance, we generated transgenic Plasmodium berghei parasites either deficient in or overexpressing the gamma-glutamylcysteine synthetase gene (pbggcs) encoding the rate-limiting enzyme in GSH biosynthesis. These lines produce either lower (pbggcs-ko) or higher (pbggcs-oe) levels of GSH than wild type parasites. In addition, GSH levels were determined in P. berghei parasites resistant to CQ and mefloquine (MQ). Increased GSH levels were detected in both, CQ and MQ resistant parasites, when compared to the parental sensitive clone. Sensitivity to CQ and ART remained unaltered in both pgggcs-ko and pbggcs-oe parasites when tested in a 4 days drug suppressive assay. However, recrudescence assays after the parasites have been exposed to a sub-lethal dose of ART showed that parasites with low levels of GSH are more sensitive to ART treatment. These results suggest that GSH levels influence Plasmodium berghei response to ART treatment.

Diversity-oriented synthesis-facilitated medicinal chemistry: toward the development of novel antimalarial agents.

Here, we describe medicinal chemistry that was accelerated by a diversity-oriented synthesis (DOS) pathway, and in vivo studies of our previously reported macrocyclic antimalarial agent that derived from the synthetic pathway. Structure-activity relationships that focused on both appendage and skeletal features yielded a nanomolar inhibitor of P. falciparum asexual blood-stage growth with improved solubility and microsomal stability and reduced hERG binding. The build/couple/pair (B/C/P) synthetic strategy, used in the preparation of the original screening library, facilitated medicinal chemistry optimization of the antimalarial lead.

Novel S-adenosyl-L-methionine decarboxylase inhibitors as potent antiproliferative agents against intraerythrocytic Plasmodium falciparum parasites.

S-adenosyl-l-methionine decarboxylase (AdoMetDC) in the polyamine biosynthesis pathway has been identified as a suitable drug target in Plasmodium falciparum parasites, which causes the most lethal form of malaria. Derivatives of an irreversible inhibitor of this enzyme, 5'-{[(Z)-4-amino-2-butenyl]methylamino}-5'-deoxyadenosine (MDL73811), have been developed with improved pharmacokinetic profiles and activity against related parasites, Trypanosoma brucei. Here, these derivatives were assayed for inhibition of AdoMetDC from P. falciparum parasites and the methylated derivative, 8-methyl-5'-{[(Z)-4-aminobut-2-enyl]methylamino}-5'-deoxyadenosine (Genz-644131) was shown to be the most active. The in vitro efficacy of Genz-644131 was markedly increased by nanoencapsulation in immunoliposomes, which specifically targeted intraerythrocytic P. falciparum parasites.

Identification and validation of tetracyclic benzothiazepines as Plasmodium falciparum cytochrome bc1 inhibitors.

Here we report the discovery of tetracyclic benzothiazepines (BTZs) as highly potent and selective antimalarials along with the identification of the Plasmodium falciparum cytochrome bc(1) complex as the primary functional target of this novel compound class. Investigation of the structure activity relationship within this previously unexplored chemical scaffold has yielded inhibitors with low nanomolar activity. A combined approach employing genetically modified parasites, biochemical profiling, and resistance selection validated inhibition of cytochrome bc(1) activity, an essential component of the parasite respiratory chain and target of the widely used antimalarial drug atovaquone, as the mode of action of this novel compound class. Resistance to atovaquone is eroding the efficacy of this widely used antimalarial drug. Intriguingly, BTZ-based inhibitors retain activity against atovaquone resistant parasites, suggesting this chemical class may provide an alternative to atovaquone in combination therapy.

Aminoindoles, a novel scaffold with potent activity against Plasmodium falciparum.

This study characterizes aminoindole molecules that are analogs of Genz-644442. Genz-644442 was identified as a hit in a screen of ~70,000 compounds in the Broad Institute's small-molecule library and the ICCB-L compound collection at Harvard Medical School. Genz-644442 is a potent inhibitor of Plasmodium falciparum in vitro (50% inhibitory concentrations [IC50s], 200 to 285 nM) and inhibits P. berghei in vivo with an efficacy of > 99% in an adapted version of Peters' 4-day suppressive test (W. Peters, Ann. Trop. Med. Parasitol. 69:155-171, 1975). Genz-644442 became the focus of medicinal chemistry optimization; 321 analogs were synthesized and were tested for in vitro potency against P. falciparum and for in vitro absorption, distribution, metabolism, and excretion (ADME) properties. This yielded compounds with IC50s of approximately 30 nM. The lead compound, Genz-668764, has been characterized in more detail. It is a single enantiomer with IC50s of 28 to 65 nM against P. falciparum in vitro. In the 4-day P. berghei model, when it was dosed at 100 mg/kg of body weight/day, no parasites were detected on day 4 postinfection. However, parasites recrudesced by day 9. Dosing at 200 mg/kg/day twice a day resulted in cures of 3/5 animals. The compound had comparable activity against P. falciparum blood stages in a human-engrafted NOD-scid mouse model. Genz-668764 had a terminal half-life of 2.8 h and plasma trough levels of 41 ng/ml when it was dosed twice a day orally at 55 mg/kg/day. Seven-day rat safety studies showed a no-observable-adverse-effect level (NOAEL) at 200 mg/kg/day; the compound was not mutagenic in Ames tests, did not inhibit the hERG channel, and did not have potent activity against a broad panel of receptors and enzymes. Employing allometric scaling and using in vitro ADME data, the predicted human minimum efficacious dose of Genz-668764 in a 3-day once-daily dosing regimen was 421 mg/day/70 kg, which would maintain plasma trough levels above the IC90 against P. falciparum for at least 96 h after the last dose. The predicted human therapeutic index was approximately 3, on the basis of the exposure in rats at the NOAEL. We were unable to select for parasites with >2-fold decreased sensitivity to the parent compound, Genz-644442, over 270 days of in vitro culture under drug pressure. These characteristics make Genz-668764 a good candidate for preclinical development.

Novel inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase with anti-malarial activity in the mouse model.

Plasmodium falciparum, the causative agent of the most deadly form of human malaria, is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in the pyrimidine biosynthetic pathway and represents a potential target for anti-malarial therapy. A high throughput screen and subsequent medicinal chemistry program identified a series of N-alkyl-5-(1H-benzimidazol-1-yl)thiophene-2-carboxamides with low nanomolar in vitro potency against DHODH from P. falciparum, P. vivax, and P. berghei. The compounds were selective for the parasite enzymes over human DHODH, and x-ray structural data on the analog Genz-667348, demonstrated that species selectivity could be attributed to amino acid differences in the inhibitor-binding site. Compounds from this series demonstrated in vitro potency against the 3D7 and Dd2 strains of P. falciparum, good tolerability and oral exposure in the mouse, and ED(50) values in the 4-day murine P. berghei efficacy model of 13-21 mg/kg/day with oral twice-daily dosing. In particular, treatment with Genz-667348 at 100 mg/kg/day resulted in sterile cure. Two recent analogs of Genz-667348 are currently undergoing pilot toxicity testing to determine suitability as clinical development candidates.

A concise silylamine approach to 2-amino-3-hydroxy-indoles with potent in vivo antimalaria activity.

The development of a concise strategy to access 2-amino-3-hydroxy-indoles, which are disclosed as novel antimalarials with potent in vivo activity, is reported. Starting from isatins the target compounds are synthesized in 2 steps and in good yields via oxoindole intermediates by employing tert-butyldimethylsilyl amine (TBDMSNH(2)) as previously unexplored ammonia equivalent.

Glutathione reductase-null malaria parasites have normal blood stage growth but arrest during development in the mosquito.

Malaria parasites contain a complete glutathione (GSH) redox system, and several enzymes of this system are considered potential targets for antimalarial drugs. Through generation of a gamma-glutamylcysteine synthetase (gamma-GCS)-null mutant of the rodent parasite Plasmodium berghei, we previously showed that de novo GSH synthesis is not critical for blood stage multiplication but is essential for oocyst development. In this study, phenotype analyses of mutant parasites lacking expression of glutathione reductase (GR) confirmed that GSH metabolism is critical for the mosquito oocyst stage. Similar to what was found for gamma-GCS, GR is not essential for blood stage growth. GR-null parasites showed the same sensitivity to methylene blue and eosin B as wild type parasites, demonstrating that these compounds target molecules other than GR in Plasmodium. Attempts to generate parasites lacking both GR and gamma-GCS by simultaneous disruption of gr and gamma-gcs were unsuccessful. This demonstrates that the maintenance of total GSH levels required for blood stage survival is dependent on either de novo GSH synthesis or glutathione disulfide (GSSG) reduction by Plasmodium GR. Our studies provide new insights into the role of the GSH system in malaria parasites with implications for the development of drugs targeting GSH metabolism.

The glutathione biosynthetic pathway of Plasmodium is essential for mosquito transmission.

Infection of red blood cells (RBC) subjects the malaria parasite to oxidative stress. Therefore, efficient antioxidant and redox systems are required to prevent damage by reactive oxygen species. Plasmodium spp. have thioredoxin and glutathione (GSH) systems that are thought to play a major role as antioxidants during blood stage infection. In this report, we analyzed a critical component of the GSH biosynthesis pathway using reverse genetics. Plasmodium berghei parasites lacking expression of gamma-glutamylcysteine synthetase (gamma-GCS), the rate limiting enzyme in de novo synthesis of GSH, were generated through targeted gene disruption thus demonstrating, quite unexpectedly, that gamma-GCS is not essential for blood stage development. Despite a significant reduction in GSH levels, blood stage forms of pbggcs(-) parasites showed only a defect in growth as compared to wild type. In contrast, a dramatic effect on development of the parasites in the mosquito was observed. Infection of mosquitoes with pbggcs(-) parasites resulted in reduced numbers of stunted oocysts that did not produce sporozoites. These results have important implications for the design of drugs aiming at interfering with the GSH redox-system in blood stages and demonstrate that de novo synthesis of GSH is pivotal for development of Plasmodium in the mosquito.

Identification and bioinformatic characterization of a multidrug resistance associated protein (ABCC) gene in Plasmodium berghei.

BACKGROUND: The ATP-binding cassette (ABC) superfamily is one of the largest evolutionarily conserved families of proteins. ABC proteins play key roles in cellular detoxification of endobiotics and xenobiotics. Overexpression of certain ABC proteins, among them the multidrug resistance associated protein (MRP), contributes to drug resistance in organisms ranging from human neoplastic cells to parasitic protozoa. In the present study, the Plasmodium berghei mrp gene (pbmrp) was partially characterized and the predicted protein was classified using bioinformatics in order to explore its putative involvement in drug resistance. METHODS: The pbmrp gene from the P. berghei drug sensitive, N clone, was sequenced using a PCR strategy. Classification and domain organization of pbMRP were determined with bioinformatics. The Plasmodium spp. MRPs were aligned and analysed to study their conserved motifs and organization. Gene copy number and organization were determined via Southern blot analysis in both N clone and the chloroquine selected line, RC. Chromosomal Southern blots and RNase protection assays were employed to determine the chromosomal location and expression levels of pbmrp in blood stages. RESULTS: The pbmrp gene is a single copy, intronless gene with a predicted open reading frame spanning 5820 nucleotides. Bioinformatic analyses show that this protein has distinctive features characteristic of the ABCC sub-family. Multiple sequence alignments reveal a high degree of conservation in the nucleotide binding and transmembrane domains within the MRPs from the Plasmodium spp. analysed. Expression of pbmrp was detected in asexual blood stages. Gene organization, copy number and mRNA expression was similar in both lines studied. A chromosomal translocation was observed in the chloroquine selected RC line, from chromosome 13/14 to chromosome 8, when compared to the drug sensitive N clone. CONCLUSION: In this study, the pbmrp gene was sequenced and classified as a member of the ABCC sub-family. Multiple sequence alignments reveal that this gene is homologous to the Plasmodium y. yoelii and Plasmodium knowlesi mrp, and the Plasmodium vivax and Plasmodium falciparum mrp2 genes. There were no differences in gene organization, copy number, or mRNA expression between N clone and the RC line, but a chromosomal translocation of pbmrp from chromosome 13/14 to chromosome 8 was detected in RC.

Plasmodium yoelii: identification and partial characterization of an MDR1 gene in an artemisinin-resistant line.

The molecular mechanisms by which the malarial parasite has managed to develop resistance to many antimalarial drugs remain to be completely elucidated. Mutations in the pfmdr1 gene of Plasmodium falciparum, as well as an increase in pfmdr1 copy number, have been associated with resistance to the quinoline-containing antimalarial drugs. We investigated the mechanisms of drug resistance in Plasmodium using a collection of P. yoelii lines with different drug resistance profiles. The mdr1 gene of P. yoelii (pymdr1) was identified and characterized. A 2- to 3-fold increase in the pymdr1 gene copy number was observed in the P. yoelii ART line (artemisinin resistant) when compared with the NS parental line. The pymdr1 gene was mapped to a chromosome of 2.1 Mb in all lines analyzed. Reverse transcriptase-polymerase chain reaction and Western blot experiments confirmed the expression of the gene at the RNA and protein levels.

Plasmodium berghei: analysis of the gamma-glutamylcysteine synthetase gene in drug-resistant lines.

The rapid emergence of multidrug-resistant Plasmodium falciparum is a worldwide concern. Despite the magnitude of the problem, the mechanisms involved in this phenomenon are not well understood. One current proposal suggests that toxic heme molecules are degraded by glutathione (GSH), and that anti-malarial drugs, such as chloroquine (CQ), inhibit this degradation, thus implicating GSH in drug resistance. Furthermore, in some strains of Plasmodium berghei and P. falciparum, chloroquine resistance is accompanied by an increase in glutathione levels and increased activity in GSH-related enzymes. We are investigating the relationship between the gamma-glutamylcysteine synthetase (ggcs) gene, the rate-limiting enzyme in de novo synthesis of GSH, and drug resistance in P. berghei at the molecular level. In this report, we have demonstrated an increase in pbggcs mRNA levels associated with CQ and mefloquine (MFQ) resistance. In addition, the pbggcs gene locus structure was shown to be similar and localized to chromosome 8 in four parasite lines of P. berghei with different drug resistance profiles. This work suggests a link between increased GSH levels and drug resistance in Plasmodium.