In vivo and in vitro antimalarial properties of azithromycin-chloroquine combinations that include the resistance reversal agent amlodipine.

Evidence of emerging Plasmodium falciparum resistance to artemisinin-based combination therapies, documented in western Cambodia, underscores the continuing need to identify new antimalarial combinations. Given recent reports of the resurgence of chloroquine-sensitive P. falciparum parasites in Malawi, after the enforced and prolonged withdrawal of this drug, and indications of a possible synergistic interaction with the macrolide azithromycin, we sought to further characterize chloroquine-azithromycin combinations for their in vitro and in vivo antimalarial properties.

Use of high-density tiling microarrays to identify mutations globally and elucidate mechanisms of drug resistance in Plasmodium falciparum.

Background: The identification of genetic changes that confer drug resistance or other phenotypic changes in pathogens can help optimize treatment strategies, support the development of new therapeutic agents, and provide information about the likely function of genes. Elucidating mechanisms of phenotypic drug resistance can also assist in identifying the mode of action of uncharacterized but potent antimalarial compounds identified in high-throughput chemical screening campaigns against Plasmodium falciparum.

Results: Here we show that tiling microarrays can detect de novo a large proportion of the genetic changes that differentiate one genome from another.

The fatty acid biosynthesis enzyme FabI plays a key role in the development of liver-stage malarial parasites.

The fatty acid synthesis type II pathway has received considerable interest as a candidate therapeutic target in Plasmodium falciparum asexual blood-stage infections. This apicoplast-resident pathway, distinct from the mammalian type I process, includes FabI. Here, we report synthetic chemistry and transfection studies concluding that Plasmodium FabI is not the target of the antimalarial activity of triclosan, an inhibitor of bacterial FabI.

Gene encoding a deubiquitinating enzyme is mutated in artesunate- and chloroquine-resistant rodent malaria parasites.

Artemisinin- and artesunate-resistant Plasmodium chabaudi mutants, AS-ART and AS-ATN, were previously selected from chloroquine-resistant clones AS-30CQ and AS-15CQ respectively. Now, a genetic cross between AS-ART and the artemisinin-sensitive clone AJ has been analysed by Linkage Group Selection. A genetic linkage group on chromosome 2 was selected under artemisinin treatment.

In vitro efficacy, resistance selection, and structural modeling studies implicate the malarial parasite apicoplast as the target of azithromycin.

Azithromycin (AZ), a broad-spectrum antibacterial macrolide that inhibits protein synthesis, also manifests reasonable efficacy as an antimalarial. Its mode of action against malarial parasites, however, has remained undefined. Our in vitro investigations with the human malarial parasite Plasmodium falciparum document a remarkable increase in AZ potency when exposure is prolonged from one to two generations of intraerythrocytic growth, with AZ producing 50% inhibition of parasite growth at concentrations in the mid to low nanomolar range.

Decreasing pfmdr1 copy number in plasmodium falciparum malaria heightens susceptibility to mefloquine, lumefantrine, halofantrine, quinine, and artemisinin.

The global dissemination of drug-resistant Plasmodium falciparum is spurring intense efforts to implement artemisinin (ART)-based combination therapies for malaria, including mefloquine (MFQ)-artesunate and lumefantrine (LUM)-artemether. Clinical studies have identified an association between an increased risk of MFQ, MFQ-artesunate, and LUM-artemether treatment failures and pfmdr1 gene amplification. To directly address the contribution that pfmdr1 copy number makes to drug resistance, we genetically disrupted 1 of the 2 pfmdr1 copies in the drug-resistant FCB line, which resulted in reduced pfmdr1 mRNA and protein expression.

Genetic linkage of pfmdr1 with food vacuolar solute import in Plasmodium falciparum.

The P-glycoprotein homolog of the human malaria parasite Plasmodium falciparum (Pgh-1) has been implicated in decreased susceptibility to several antimalarial drugs, including quinine, mefloquine and artemisinin. Pgh-1 mainly resides within the parasite's food vacuolar membrane. Here, we describe a surrogate assay for Pgh-1 function based on the subcellular distribution of Fluo-4 acetoxymethylester and its free fluorochrome.

pfmdr1 mutations contribute to quinine resistance and enhance mefloquine and artemisinin sensitivity in Plasmodium falciparum.

The emergence and spread of multidrug resistant Plasmodium falciparum has severely limited the therapeutic options for the treatment of malaria. With ever-increasing failure rates associated with chloroquine or sulphadoxine-pyrimethamine treatment, attention has turned to the few alternatives, which include quinine and mefloquine. Here, we have investigated the role of pfmdr1 3' coding region point mutations in antimalarial drug susceptibility by allelic exchange in the GC03 and 3BA6 parasite lines.

A critical role for PfCRT K76T in Plasmodium falciparum verapamil-reversible chloroquine resistance.

Chloroquine resistance (CQR) in Plasmodium falciparum is associated with mutations in the digestive vacuole transmembrane protein PfCRT. However, the contribution of individual pfcrt mutations has not been clarified and other genes have been postulated to play a substantial role. Using allelic exchange, we show that removal of the single PfCRT amino-acid change K76T from resistant strains leads to wild-type levels of CQ susceptibility, increased binding of CQ to its target ferriprotoporphyrin IX in the digestive vacuole and loss of verapamil reversibility of CQ and quinine resistance.

Evidence for a central role for PfCRT in conferring Plasmodium falciparum resistance to diverse antimalarial agents.

Chloroquine resistance in Plasmodium falciparum is primarily conferred by mutations in pfcrt. Parasites resistant to chloroquine can display hypersensitivity to other antimalarials; however, the patterns of crossresistance are complex, and the genetic basis has remained elusive. We show that stepwise selection for resistance to amantadine or halofantrine produced previously unknown pfcrt mutations (including S163R), which were associated with a loss of verapamil-reversible chloroquine resistance.

Drug resistance-associated pfCRT mutations confer decreased Plasmodium falciparum digestive vacuolar pH.

Elucidating the altered physiology of various chloroquine resistant (CQR) strains of Plasmodium falciparum is essential for understanding the molecular basis of CQR. In this study, we have devised several new methods for analyzing digestive vacuolar (DV) pH for individual intraerythrocytic parasites under continuous perfusion. These use controlled illumination power and novel data acquisition software, and are based on either acridine orange (AO) emission spectra or ratiometric 5-(and 6-)carboxy-2',7'-dimethyl-3'-hydroxy-6'-N-ethylaminospiro [isobenzofuran-1(3H),9'-(9H)xanthen]-3-one (DM NERF) excitation.

Chloroquine resistance modulated in vitro by expression levels of the Plasmodium falciparum chloroquine resistance transporter.

Plasmodium falciparum malaria is increasingly difficult to treat and control due to the emergence of parasite resistance to the major antimalarials, notably chloroquine. Recent work has shown that the chloroquine resistance phenotype can be conferred by multiple amino acid mutations in the parasite digestive vacuole transmembrane protein PfCRT. Here, we have addressed whether chloroquine resistance can also be affected by changes in expression levels of this protein.

Chloroquine resistance in Plasmodium falciparum malaria parasites conferred by pfcrt mutations.

Plasmodium falciparum chloroquine resistance is a major cause of worldwide increases in malaria mortality and morbidity. Recent laboratory and clinical studies have associated chloroquine resistance with point mutations in the gene pfcrt. However, direct proof of a causal relationship has remained elusive and most models have posited a multigenic basis of resistance.

Structural elucidation of the specificity of the antibacterial agent triclosan for malarial enoyl acyl carrier protein reductase.

The human malaria parasite Plasmodium falciparum synthesizes fatty acids using a type II pathway that is absent in humans. The final step in fatty acid elongation is catalyzed by enoyl acyl carrier protein reductase, a validated antimicrobial drug target. Here, we report the cloning and expression of the P.