Adele M. Lehane

PfCRT and its role in antimalarial drug resistance

Plasmodium falciparum resistance to chloroquine, the former gold standard antimalarial drug, is mediated primarily by mutant forms of the chloroquine resistance transporter (PfCRT). These mutations impart upon PfCRT the ability to efflux chloroquine from the intracellular digestive vacuole, the site of drug action. Recent studies reveal that PfCRT variants can also affect parasite fitness, protect immature gametocytes against chloroquine action, and alter P. falciparum susceptibility to current first-line therapies. These results highlight the need to be vigilant in screening for the appearance of novel pfcrt alleles that could contribute to new multi-drug resistance phenotypes.

(+)-SJ733, a clinical candidate for malaria that acts through ATP4 to induce rapid host-mediated clearance of Plasmodium

Significance Useful antimalarial drugs must be rapidly acting, highly efficacious, and have low potential for developing resistance. (+)-SJ733 targets a Plasmodium cation-transporting ATPase, ATP4. (+)-SJ733 cleared parasites in vivo as quickly as artesunate by specifically inducing eryptosis/senescence in infected, treated erythrocytes. Although in vitro selection of pfatp4 mutants with (+)-SJ733 proceeded with moderate frequency, during in vivo selection of pbatp4 mutants, resistance emerged slowly and produced marginally resistant mutants with poor fitness. In addition, (+)-SJ733 met all other criteria for a clinical candidate, including high oral bioavailability, a high safety margin, and transmission blocking activity. These results demonstrate that targeting ATP4 has great potential to deliver useful drugs for malaria eradication. Drug discovery for malaria has been transformed in the last 5 years by the discovery of many new lead compounds identified by phenotypic screening. The process of developing these compounds as drug leads and studying the cellular responses they induce is revealing new targets that regulate key processes in the Plasmodium parasites that cause malaria. We disclose herein that the clinical candidate (+)-SJ733 acts upon one of these targets, ATP4. ATP4 is thought to be a cation-transporting ATPase responsible for maintaining low intracellular Na+ levels in the parasite. Treatment of parasitized erythrocytes with (+)-SJ733 in vitro caused a rapid perturbation of Na+ homeostasis in the parasite. This perturbation was followed by profound physical changes in the infected cells, including increased membrane rigidity and externalization of phosphatidylserine, consistent with eryptosis (erythrocyte suicide) or senescence. These changes are proposed to underpin the rapid (+)-SJ733-induced clearance of parasites seen in vivo. Plasmodium falciparum ATPase 4 (pfatp4) mutations that confer resistance to (+)-SJ733 carry a high fitness cost. The speed with which (+)-SJ733 kills parasites and the high fitness cost associated with resistance-conferring mutations appear to slow and suppress the selection of highly drug-resistant mutants in vivo. Together, our data suggest that inhibitors of PfATP4 have highly attractive features for fast-acting antimalarials to be used in the global eradication campaign.

Common dietary flavonoids inhibit the growth of the intraerythrocytic malaria parasite

BackgroundFlavonoids are abundant plant phenolic compounds. More than 6000 have been identified to date, and some have been shown to possess antiparasitic activity. Here we investigate the effects of a range of common dietary flavonoids on the growth of two strains of the human malaria parasite Plasmodium falciparum.FindingsA chloroquine-sensitive (3D7) and a chloroquine-resistant (7G8) strain of P. falciparum were tested for in vitro susceptibility to a range of individual dietary flavonoids and flavonoid combinations. Parasite susceptibility was measured in 96-well plates over 96 h using a previously described [3H]hypoxanthine incorporation assay. Of the eleven flavonoids tested, eight showed antiplasmodial activity against the 3D7 strain (with IC50 values between 11 and 66 μM), and all showed activity against the 7G8 strain (with IC50 values between 12 and 76 μM). The most active compound against both strains was luteolin, with IC50 values of 11 ± 1 μM and 12 ± 1 μM for 3D7 and 7G8, respectively. Luteolin was found to prevent the progression of parasite growth beyond the young trophozoite stage, and did not affect parasite susceptibility to the antimalarial drugs chloroquine or artemisinin. Combining low concentrations of flavonoids was found to produce an apparent additive antiplasmodial effect.ConclusionCertain common dietary flavonoids inhibit the intraerythrocytic growth of the 3D7 and 7G8 strains of P. falciparum. Flavonoid combinations warrant further investigation as antiplasmodial agents.

Globally prevalent PfMDR1 mutations modulate Plasmodium falciparum susceptibility to artemisinin-based combination therapies

Antimalarial chemotherapy, globally reliant on artemisinin-based combination therapies (ACTs), is threatened by the spread of drug resistance in Plasmodium falciparum parasites. Here we use zinc-finger nucleases to genetically modify the multidrug resistance-1 transporter PfMDR1 at amino acids 86 and 184, and demonstrate that the widely prevalent N86Y mutation augments resistance to the ACT partner drug amodiaquine and the former first-line agent chloroquine. In contrast, N86Y increases parasite susceptibility to the partner drugs lumefantrine and mefloquine, and the active artemisinin metabolite dihydroartemisinin. The PfMDR1 N86 plus Y184F isoform moderately reduces piperaquine potency in strains expressing an Asian/African variant of the chloroquine resistance transporter PfCRT. Mutations in both digestive vacuole-resident transporters are thought to differentially regulate ACT drug interactions with host haem, a product of parasite-mediated haemoglobin degradation. Global mapping of these mutations illustrates where the different ACTs could be selectively deployed to optimize treatment based on regional differences in PfMDR1 haplotypes.

A verapamil-sensitive chloroquine-associated H + leak from the digestive vacuole in chloroquine-resistant malaria parasites

Chloroquine resistance in the malaria parasite Plasmodium falciparum has made malaria increasingly difficult to control. Chloroquine-resistant parasites accumulate less chloroquine than their chloroquine-sensitive counterparts; however, the mechanism underlying this remains unclear. The primary site of accumulation and antimalarial action of chloroquine is the internal acidic digestive vacuole of the parasite, the acidity of which is maintained by inwardly-directed H+ pumps, working against the (outward) leak of H+. In this study we have investigated the leak of H+ from the digestive vacuole of the parasite by monitoring the alkalinisation of the vacuole following inhibition of the H+-pumping V-type ATPase by concanamycin A. The rates of alkalinisation observed in three chloroquine-resistant strains were two- to fourfold higher than those measured in three chloroquine-sensitive strains. On addition of chloroquine there was a dramatic increase in the rate of alkalinisation in the chloroquine-resistant strains, whereas chloroquine caused the rate of alkalinisation to decrease in the chloroquine-sensitive strains. The chloroquine-associated increase in the rate of alkalinisation seen in chloroquine-resistant parasites was inhibited by the chloroquine-resistance reversal agent verapamil. The data are consistent with the hypothesis that in chloroquine-resistant parasites chloroquine effluxes from the digestive vacuole, in association with H+, via a verapamil-sensitive pathway.

Efflux of a range of antimalarial drugs and ‘chloroquine resistance reversers’ from the digestive vacuole in malaria parasites with mutant PfCRT

Chloroquine‐resistant malaria parasites (Plasmodium falciparum) show an increased leak of H+ ions from their internal digestive vacuole in the presence of chloroquine. This phenomenon has been attributed to the transport of chloroquine, together with H+, out of the digestive vacuole (and hence away from its site of action) via a mutant form of the parasites chloroquine resistance transporter (PfCRT). Here, using transfectant parasite lines, we show that a range of other antimalarial drugs, as well as various ‘chloroquine resistance reversers’ induce an increased leak of H+ from the digestive vacuole of parasites expressing mutant PfCRT, consistent with these compounds being substrates for mutant forms, but not the wild‐type form, of PfCRT. For some compounds there were significant differences observed between parasites having the African/Asian Dd2 form of PfCRT and those with the South American 7G8 form of PfCRT, consistent with there being differences in the transport properties of the two mutant proteins. The finding that chloroquine resistance reversers are substrates for mutant PfCRT has implications for the mechanism of action of this class of compound.

Chloroquine Resistance-Conferring Mutations in pfcrt Give Rise to a Chloroquine-Associated H+ Leak from the Malaria Parasite's Digestive Vacuole

ABSTRACT Chloroquine resistance in the malaria parasite Plasmodium falciparum is conferred by mutations in the P. falciparum chloroquine resistance transporter (PfCRT). PfCRT localizes to the membrane of the parasites internal digestive vacuole, an acidic organelle in which chloroquine accumulates to high concentrations and exerts its toxic effect. Mutations in PfCRT are thought to reduce chloroquine accumulation in this organelle. How they do so is the subject of ongoing debate. Recently we have shown that in the presence of chloroquine there is an increased leak of H+ from the digestive vacuole in chloroquine-resistant but not chloroquine-sensitive parasites. Here, using transfectant parasite strains of a single genetic background and differing only in their pfcrt allele, we show that chloroquine resistance-conferring PfCRT mutations are responsible for this chloroquine-associated H+ leak. This is consistent with the hypothesis that the chloroquine resistance-conferring forms of PfCRT mediate the efflux of chloroquine, in association with H+, from the malaria parasites digestive vacuole.

Membrane transport in the malaria parasite and its host erythrocyte.

As it grows and replicates within the erythrocytes of its host the malaria parasite takes up nutrients from the extracellular medium, exports metabolites and maintains a tight control over its internal ionic composition. These functions are achieved via membrane transport proteins, integral membrane proteins that mediate the passage of solutes across the various membranes that separate the biochemical machinery of the parasite from the extracellular environment. Proteins of this type play a key role in antimalarial drug resistance, as well as being candidate drug targets in their own right. This review provides an overview of recent work on the membrane transport biology of the malaria parasite-infected erythrocyte, encompassing both the parasite-induced changes in the membrane transport properties of the host erythrocyte and the cell physiology of the intracellular parasite itself.

Bacteriophage-encoded glucosyltransferase gtrll of Shigella flexneri : membrane topology and identification of critical residues

The Shigella flexneri serotypes differ in the nature of their O-antigens. The addition of glucosyl or O-acetyl groups to the common backbone repeat units gives rise to the different serotypes. GtrII glucosylates rhamnose III of the O-antigen repeat unit, thus converting serotype Y (which has no modifications to the basic O-antigen repeat unit) into serotype 2a, the most prevalent serotype. In the present study, the topology of GtrII has been determined. GtrII has nine transmembrane helices, a re-entrant loop and three large periplasmic regions. Four critical residues (Glu40, Phe414, Cys435 and Lys478) were identified in two of the periplasmic regions. Despite the lack of sequence similarity between GtrII and the Gtrs from other serotypes, three of the critical residues identified are conserved in the remaining Gtrs. This is consistent with some degree of mechanistic conservation in this functionally related group of proteins.

Balancing drug resistance and growth rates via compensatory mutations in the Plasmodium falciparum chloroquine resistance transporter

The widespread use of chloroquine to treat Plasmodium falciparum infections has resulted in the selection and dissemination of variant haplotypes of the primary resistance determinant PfCRT. These haplotypes have encountered drug pressure and within‐host competition with wild‐type drug‐sensitive parasites. To examine these selective forces in vitro, we genetically engineered P. falciparum to express geographically diverse PfCRT haplotypes. Variant alleles from the Philippines (PH1 and PH2, which differ solely by the C72S mutation) both conferred a moderate gain of chloroquine resistance and a reduction in growth rates in vitro. Of the two, PH2 showed higher IC50 values, contrasting with reduced growth. Furthermore, a highly mutated pfcrt allele from Cambodia (Cam734) conferred moderate chloroquine resistance and enhanced growth rates, when tested against wild‐type pfcrt in co‐culture competition assays. These three alleles mediated cross‐resistance to amodiaquine, an antimalarial drug widely used in Africa. Each allele, along with the globally prevalent Dd2 and 7G8 alleles, rendered parasites more susceptible to lumefantrine, the partner drug used in the leading first‐line artemisinin‐based combination therapy. These data reveal ongoing region‐specific evolution of PfCRT that impacts drug susceptibility and relative fitness in settings of mixed infections, and raise important considerations about optimal agents to treat chloroquine‐resistant malaria.