In vivo selection of Plasmodium falciparum parasites carrying the chloroquine-susceptible pfcrt K76 allele after treatment with artemether-lumefantrine in Africa

Background: Artemether-lumefantrine (AL) is a major and highly effective artemisinin-based combination therapy that is becoming increasingly important as a new first-line therapy against Plasmodium falciparum malaria. However, recrudescences occurring after AL treatment have been reported. Identification of drug-specific parasite determinants that contribute to treatment failures will provide important tools for the detection and surveillance of AL resistance. Methods: The findings from a 42-day follow-up efficacy trial in Tanzania that compared AL with sulfadoxinepyrimethamine (SP) were analyzed to identify candidate markers for lumefantrine tolerance/resistance in the chloroquine resistance transporter gene (pfcrt) and multidrug resistance gene 1 (pfmdr1). The findings were corroborated in vitro with genetically modified isogenic P. falciparum parasite lines. Results: Treatment with AL selected for the chloroquine-susceptible pfcrt K76 allele (P \u3c .0001) and, to a lesser extent, the pfmdr1 N86 (P = .048) allele among recurrent infections. These genotypes were not selected during SP treatment. No pfmdr1 gene amplifications were observed. Isogenic pfcrt-modified parasite lines demonstrated a 2-fold increase in susceptibility to lumefantrine, which was directly attributable to the K76T mutation. Conclusions: Our findings suggest that the pfcrt K76T mutation is a drug-specific contributor to enhanced P. falciparum susceptibility to lumefantrine in vivo and in vitro, and they highlight the benefit of using AL in areas affected by chloroquine-resistant P. falciparum malaria. © 2009 by the Infectious Diseases Society of America. All rights reserved

Stage-dependent localization of a novel gene product of the malaria parasite, Plasmodium falciparum

A novel Plasmodium falciparum gene, MB2, was identified by screening a sporozoite cDNA library with the serum of a human volunteer protected experimentally by the bites of P. falciparum-infected and irradiated mosquitoes. The single-exon, single-copy MB2 gene is predicted to encode a protein with an Mr of 187,000. The MB2 protein has an amino-terminal basic domain, a central acidic domain, and a carboxyl-terminal domain with similarity to the GTP-binding domain of the prokaryotic translation initiation factor 2. MB2 is expressed in sporozoites, the liver, and blood-stage parasites and gametocytes. The MB2 protein is distributed as a ~120-kDa moiety on the surface of sporozoites and is imported into the nucleus of blood-stage parasites as a ~66-kDa species. Proteolytic processing is favored as the mechanism regulating the distinct subcellular localization of the MB2 protein. This differential localization provides multiple opportunities to exploit the MB2 gene product as a vaccine or therapeutic target

Plasmodium falciparum K13 Mutations Differentially Impact Ozonide Susceptibility and Parasite Fitness In Vitro

The emergence and spread in Southeast Asia of Plasmodium falciparum resistance to artemisinin (ART) derivatives, the cornerstone of first-line artemisinin-based combination therapies (ACTs), underscore the urgent need to identify suitable replacement drugs. Discovery and development efforts have identified a series of ozonides with attractive chemical and pharmacological properties that are being touted as suitable replacements. Partial resistance to ART, defined as delayed parasite clearance in malaria patients treated with an ART derivative or an ACT, has been associated with mutations in the P. falciparum K13 gene. In light of reports showing that ART derivatives and ozonides share similar modes of action, we have investigated whether parasites expressing mutant K13 are cross-resistant to the ozonides OZ439 (artefenomel) and OZ227 (arterolane). This work used a panel of culture-adapted clinical isolates from Cambodia that were genetically edited to express variant forms of K13. Phenotypic analyses employed ring-stage survival assays (ring-stage survival assay from 0 to 3 h [RSA0–3h]), whose results have earlier been shown to correlate with parasite clearance rates in patients. Our results document cross-resistance between OZ277 and dihydroartemisinin (DHA), a semisynthetic derivative of ART, in parasites carrying the K13 mutations C580Y, R539T, and I543T. For OZ439, we observed cross-resistance only for parasites that carried the rare K13 I543T mutation, with no evidence of cross-resistance afforded by the prevalent C580Y mutation. Mixed-culture competition experiments with isogenic lines carrying modified K13 revealed variable growth deficits depending on the K13 mutation and parasite strain and provide a rationale for the broad dissemination of the fitness-neutral K13 C580Y mutation throughout strains currently circulating in Southeast Asia

Select pyrimidinones inhibit the propagation of the malarial parasite, Plasmodium falciparum

Plasmodium falciparum, the Apicomplexan parasite that is responsible for the most lethal forms of human malaria, is exposed to radically different environments and stress factors during its complex lifecycle. In any organism, Hsp70 chaperones are typically associated with tolerance to stress. We therefore reasoned that inhibition of P. falciparum Hsp70 chaperones would adversely affect parasite homeostasis. To test this hypothesis, we measured whether pyrimidinone-amides, a new class of Hsp70 modulators, could inhibit the replication of the pathogenic P. falciparum stages in human red blood cells. Nine compounds with IC50 values from 30 nM to 1.6 μM were identified. Each compound also altered the ATPase activity of purified P. falciparum Hsp70 in single-turnover assays, although higher concentrations of agents were required than was necessary to inhibit P. falciparum replication. Varying effects of these compounds on Hsp70s from other organisms were also observed. Together, our data indicate that pyrimidinone-amides constitute a novel class of anti-malarial agents. © 2009 Elsevier Ltd. All rights reserved

Degrees of chloroquine resistance in Plasmodium - Is the redox system involved?

Chloroquine (CQ) was once a very effective antimalarial drug that, at its peak, was consumed in the hundreds of millions of doses per year. The drug acts against the Plasmodium parasite during the asexual intra-erythrocytic phase of its lifecycle. Unfortunately, clinical resistance to this drug is now widespread. Questions remain about precisely how CQ kills malaria parasites, and by what means some CQ-resistant (CQR) parasites can withstand much higher concentrations of the drug than others that also fall in the CQR category. In this review we investigate the evidence for and against the proposal that CQ kills parasites by generating oxidative stress. Further, we examine a long-held idea that the glutathione system of malaria parasites plays a role in CQ resistance. We conclude that there is strong evidence that glutathione levels modulate CQ response in the rodent malaria species Plasmodium berghei, but that a role for redox in contributing to the degree of CQ resistance in species infectious to humans has not been firmly established.Adele M. Lehane, Christopher A. McDevitt, Kiaran Kirk, David A. Fidoc

The Mu subunit of Plasmodium falciparum clathrin-associated adaptor protein 2 modulates in vitro parasite response to artemisinin and quinine.

The emergence of drug-resistant parasites is a serious threat faced by malaria control programs. Understanding the genetic basis of resistance is critical to the success of treatment and intervention strategies. A novel locus associated with antimalarial resistance, ap2-mu (encoding the mu chain of the adaptor protein 2 [AP2] complex), was recently identified in studies on the rodent malaria parasite Plasmodium chabaudi (pcap2-mu). Furthermore, analysis in Kenyan malaria patients of polymorphisms in the Plasmodium falciparum ap2-mu homologue, pfap2-mu, found evidence that differences in the amino acid encoded by codon 160 are associated with enhanced parasite survival in vivo following combination treatments which included artemisinin derivatives. Here, we characterize the role of pfap2-mu in mediating the in vitro antimalarial drug response of P. falciparum by generating transgenic parasites constitutively expressing codon 160 encoding either the wild-type Ser (Ser160) or the Asn mutant (160Asn) form of pfap2-mu. Transgenic parasites carrying the pfap2-mu 160Asn allele were significantly less sensitive to dihydroartemisinin using a standard 48-h in vitro test, providing direct evidence of an altered parasite response to artemisinin. Our data also provide evidence that pfap2-mu variants can modulate parasite sensitivity to quinine. No evidence was found that pfap2-mu variants contribute to the slow-clearance phenotype exhibited by P. falciparum in Cambodian patients treated with artesunate monotherapy. These findings provide compelling evidence that pfap2-mu can modulate P. falciparum responses to multiple drugs. We propose that this gene should be evaluated further as a potential molecular marker of antimalarial resistance

Recent highlights in antimalarial drug resistance and chemotherapy research

This review summarizes recent investigations into antimalarial drug resistance and chemotherapy, including reports of some of the many exciting talks and posters on this topic that were presented at the third Molecular Approaches to Malaria meeting held in Lorne, Australia, in February 2008 (MAM 2008). After surveying this area of research, we focus on two important questions: what is the molecular contribution of pfcrt to chloroquine resistance, and what is the mechanism of action of artemisinin? We conclude with thoughts about the current state of antimalarial chemotherapy and priorities moving forward

Preclinical antimalarial combination studies: the case of M5717, a P. falciparum elongation factor 2 inhibitor and pyronaridine, a hemozoin formation inhibitor

Antimalarial drug resistance in the; Plasmodium falciparum; parasite poses a constant challenge for drug development. To mitigate this risk, new antimalarial medicines should be developed as fixed-dose combinations. Assessing the pharmacodynamic interactions of potential antimalarial drug combination partners during early phases of development is essential in developing the targeted parasitological and clinical profile of the final drug product. Here, we have studied the combination of M5717, a; P. falciparum; translation elongation factor 2 inhibitor, and pyronaridine, an inhibitor of hemozoin formation. Our test cascade consisted of; in vitro; isobolograms as well as; in vivo; studies in the; P. falciparum; severe combined immunodeficient (SCID) mouse model. We also analyzed pharmacokinetic and pharmacodynamic parameters, including genomic sequencing of recrudescent parasites. We observed no pharmacokinetic interactions with the combination of M5717 and pyronaridine. M5717 did not negatively impact the rate of kill of the faster-acting pyronaridine, and the latter was able to suppress the selection of M5717-resistant mutants, as well as significantly delay the recrudescence of parasites both with suboptimal and optimal dosing regimens

Pan-active imidazolopiperazine antimalarials target the Plasmodium falciparum intracellular secretory pathway.

A promising new compound class for treating human malaria is the imidazolopiperazines (IZP) class. IZP compounds KAF156 (Ganaplacide) and GNF179 are effective against Plasmodium symptomatic asexual blood-stage infections, and are able to prevent transmission and block infection in animal models. But despite the identification of resistance mechanisms in P. falciparum, the mode of action of IZPs remains unknown. To investigate, we here combine in vitro evolution and genome analysis in Saccharomyces cerevisiae with molecular, metabolomic, and chemogenomic methods in P. falciparum. Our findings reveal that IZP-resistant S. cerevisiae clones carry mutations in genes involved in Endoplasmic Reticulum (ER)-based lipid homeostasis and autophagy. In Plasmodium, IZPs inhibit protein trafficking, block the establishment of new permeation pathways, and cause ER expansion. Our data highlight a mechanism for blocking parasite development that is distinct from those of standard compounds used to treat malaria, and demonstrate the potential of IZPs for studying ER-dependent protein processing

PNAS plus: plasmodium falciparum responds to amino acid starvation by entering into a hibernatory state

The human malaria parasite Plasmodium falciparum is auxotrophic for most amino acids. Its amino acid needs are met largely through the degradation of host erythrocyte hemoglobin; however the parasite must acquire isoleucine exogenously, because this amino acid is not present in adult human hemoglobin. We report that when isoleucine is withdrawn from the culture medium of intraerythrocytic P. falciparum, the parasite slows its metabolism and progresses through its developmental cycle at a reduced rate. Isoleucine-starved parasites remain viable for 72 h and resume rapid growth upon resupplementation. Protein degradation during starvation is important for maintenance of this hibernatory state. Microarray analysis of starved parasites revealed a 60% decrease in the rate of progression through the normal transcriptional program but no other apparent stress response. Plasmodium parasites do not possess a TOR nutrient-sensing pathway and have only a rudimentary amino acid starvation-sensing eukaryotic initiation factor 2α (eIF2α) stress response. Isoleucine deprivation results in GCN2-mediated phosphorylation of eIF2α, but kinase-knockout clones still are able to hibernate and recover, indicating that this pathway does not directly promote survival during isoleucine starvation. We conclude that P. falciparum, in the absence of canonical eukaryotic nutrient stress-response pathways, can cope with an inconsistent bloodstream amino acid supply by hibernating and waiting for more nutrient to be provided