Cell Swelling Induced by the Antimalarial KAE609 (Cipargamin) and Other PfATP4-Associated Antimalarials

Dennis, Adelaide S. M.; Lehane, Adele M.; Ridgway, Melanie C.; Holleran, John; Kirk, Kiaran

doi:10.1128/aac.00087-18

Cited by 36 publications

(41 citation statements)

References 47 publications

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“…There is functional evidence that PfATP4 serves as the parasite's primary Na ϩ -efflux pump. On addition of cipargamin or related antimalarial spiroindolones to trophozoite-stage parasites, there is an increase in the parasite's Na ϩ content (9,12) and [Na ϩ ] cyt (13)(14)(15). The data are consistent with the spiroindolones inhibiting Na ϩ extrusion via PfATP4, resulting in a net uptake of Na ϩ as the ion moves into the parasite, down its electrochemical gradient.…”

supporting

confidence: 61%

Biochemical characterization and chemical inhibition of PfATP4-associated Na+-ATPase activity in Plasmodium falciparum membranes

Rosling

Ridgway

Summers

et al. 2018

Journal of Biological Chemistry

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The antimalarial activity of chemically diverse compounds, including the clinical candidate cipargamin, has been linked to the ATPase PfATP4 in the malaria-causing parasite The characterization of PfATP4 has been hampered by the inability thus far to achieve its functional expression in a heterologous system. Here, we optimized a membrane ATPase assay to probe the function of PfATP4 and its chemical sensitivity. We found that cipargamin inhibited the Na-dependent ATPase activity present in membranes from WT parasites and that its potency was reduced in cipargamin-resistant PfATP4-mutant parasites. The cipargamin-sensitive fraction of membrane ATPase activity was inhibited by all 28 of the compounds in the "Malaria Box" shown previously to disrupt ion regulation in in a cipargamin-like manner. This is consistent with PfATP4 being the direct target of these compounds. Characterization of the cipargamin-sensitive ATPase activity yielded data consistent with PfATP4 being a Na transporter that is sensitive to physiologically relevant perturbations of pH, but not of [K] or [Ca]. With an apparent for ATP of 0.2 mm and an apparent for Na of 16-17 mm, the protein is predicted to operate at below its half-maximal rate under normal physiological conditions, allowing the rate of Na efflux to increase in response to an increase in cytosolic [Na]. In membranes from a cipargamin-resistant PfATP4-mutant line, the apparent for Na is slightly elevated. Our study provides new insights into the biochemical properties and chemical sensitivity of an important new antimalarial drug target.

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supporting

confidence: 61%

Biochemical characterization and chemical inhibition of PfATP4-associated Na+-ATPase activity in Plasmodium falciparum membranes

Rosling

Ridgway

Summers

et al. 2018

Journal of Biological Chemistry

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“…The plant-like vacuole has been associated with parasite tolerance to salt stress (36), and its apparent increase in size in extracellular parasites lacking TgATP4 may represent a parasite response to the presence of high [Na ϩ ] cyt . Our observation that extracellular T. gondii parasites swell upon depletion of TgATP4 is in line with previous studies reporting that P. falciparum parasites swell on exposure to compounds believed to inhibit PfATP4 (9,13,15,16). The observation that the pH cyt of TgATP4-knockdown parasites was not significantly different from that of TgATP4-expressing parasites indicates that although, when present, TgATP4 imposes a significant acid load on the parasite, it does not play a major role in maintaining the normal resting pH cyt .…”

Section: Characterization Of Atp4 In Toxoplasma Gondiisupporting

confidence: 93%

“…The disruption of ion regulation by the PfATP4-associated antimalarials triggers a variety of detrimental events in the parasite (9,20). It is not known whether ATP4 homologues in other apicomplexan parasites are similarly vulnerable.…”

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confidence: 99%

Characterization of the ATP4 ion pump in Toxoplasma gondii

Lehane

Dennis

Bray

et al. 2019

Journal of Biological Chemistry

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Edited by Roger J. Colbran The Plasmodium falciparum ATPase PfATP4 is the target of a diverse range of antimalarial compounds, including the clinical drug candidate cipargamin. PfATP4 was originally annotated as a Ca 2؉ transporter, but recent evidence suggests that it is a Na ؉ efflux pump, extruding Na ؉ in exchange for H ؉. Here we demonstrate that ATP4 proteins belong to a clade of P-type ATPases that are restricted to apicomplexans and their closest relatives. We employed a variety of genetic and physiological approaches to investigate the ATP4 protein of the apicomplexan Toxoplasma gondii, TgATP4. We show that TgATP4 is a plasma membrane protein. Knockdown of TgATP4 had no effect on resting pH or Ca 2؉ but rendered parasites unable to regulate their cytosolic Na ؉ concentration ([Na ؉ ] cyt). PfATP4 inhibitors caused an increase in [Na ؉ ] cyt and a cytosolic alkalinization in WT but not TgATP4 knockdown parasites. Parasites in which TgATP4 was knocked down or disrupted exhibited a growth defect, attributable to reduced viability of extracellular parasites. Parasites in which TgATP4 had been disrupted showed reduced virulence in mice. These results provide evidence for ATP4 proteins playing a key conserved role in Na ؉ regulation in apicomplexan parasites.

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“…Inhibition of PfATP4 results in an increased [Na + ] inside the parasite cytoplasm (9). As a consequence, multiple events such as swelling of the parasite (8,10), changes in the lipid composition of the parasite plasma membrane (PPM) and premature schizogony (11), and eryptosis (7) seem to contribute to parasite demise. Interestingly, 7-9% of the collection of antimalarial compounds included in the Malaria and the Pathogen Boxes distributed by Medicines for Malaria Venture (MMV) appear to inhibit PfATP4 as judged by their ability to cause Na + influx into the parasite and disruption of lipid homeostasis within the PPM (12)(13)(14).…”

Section: Introductionmentioning

confidence: 99%

WITHDRAWN: Oligomerization of the antimalarial drug targetPfATP4 is essential for parasite survival

Ramanathan

Morrisey

Daly

et al. 2019

Preprint

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Plasmodium falciparum P-type ATPase (PfATP4) is a Na + efflux pump crucial for maintaining low [Na + ] i in malaria parasites during their intraerythrocytic development cycle. In recent years, multiple studies have shown PfATP4 to be the target of a large number of chemical scaffolds, including current candidate antimalarials KAE609 and SJ733. Here we show that PfATP4 exists as a large complex. Immunopurification and proteomic studies revealed the complex to be homooligomeric in nature. The complex appears to be assembled cotranslationally. Phylogenetic analysis suggests that ATP4 from apicomplexans and chromerids form a distinct class of P-type ATPases having fewer transmembrane helices compared to their orthologues. We hypothesized that reduction of transmembrane helices in PfATP4 might necessitate oligomerization to maintain its function. We further suspected potential involvement of π-π interactions between aromatic amino acids within the terminal transmembrane helix of each monomer to be critical for oligomerization. To test this hypothesis, we mutated three aromatic amino acids in the last transmembrane helix of PfATP4. Wildtype and the mutated PfATP4 genes were introduced at an ectopic locus in a P. falciparum line, in which endogenous PfATP4 was conditionally expressed. Whereas the wildtype copy of PfATP4 expressed from the ectopic locus was able to form the oligomeric complex, the mutant PfATP4 failed to do so. Strikingly, unlike the wildtype, the mutant PfATP4 failed to functionally complement the knockdown of the endogenous gene, leading to parasite demise.These results strongly suggest that co-translational oligomerization of PfATP4 is essential for its function and for parasite survival. Significance StatementPlasmodium falciparum ATP4 (PfATP4) is a Na + efflux pump and is the target of at least two antimalarial drug candidates (KAE609, SJ733) currently in clinical trials. With a rapid parasite clearance rate (t 99 =12h) in initial clinical studies, PfATP4-active drugs present the prospect of taking us a step closer to 3 malaria elimination in a world that is currently threatened by artemisinin resistance. In this study, we have established that PfATP4 exists as a homooligomeric complex, which is assembled co-translationally through interactions facilitated by aromatic amino acids in its last transmembrane helix. Crucially, its existence as a complex is essential for its function. This exposes a vulnerability in the target that could be potentially exploited for drug design.

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Cell Swelling Induced by the Antimalarial KAE609 (Cipargamin) and Other PfATP4-Associated Antimalarials

Cited by 36 publications

References 47 publications

Biochemical characterization and chemical inhibition of PfATP4-associated Na+-ATPase activity in Plasmodium falciparum membranes

Biochemical characterization and chemical inhibition of PfATP4-associated Na+-ATPase activity in Plasmodium falciparum membranes

Characterization of the ATP4 ion pump in Toxoplasma gondii

WITHDRAWN: Oligomerization of the antimalarial drug targetPfATP4 is essential for parasite survival

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