An Acid-loading Chloride Transport Pathway in the Intraerythrocytic Malaria Parasite, Plasmodium falciparum

Henry, Roselani I.; Cobbold, Simon A.; Khan, Asif; Hayward, Rhys; Lehane, Adele M.; Bray, Patrick G.; Howitt, Susan; Biagini, Giancarlo A.; Saliba, Kevin J.; Kirk, Kiaran

doi:10.1074/jbc.m110.120980

Cited by 10 publications

(16 citation statements)

References 27 publications

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“…The nonspecific anion transport inhibitor DIDS prevented the alkalinization from occurring (Figure D, orange trace), as reported previously, and consistent with the Cl – transporter(s) being inhibited. This compound has also been shown to inhibit (radioactive) 36 Cl – transport across the parasite plasma membrane . The PfFNT inhibitor MMV007839 also decreased the rate of alkalinization in these conditions (Figure D, purple trace); this was unexpected and therefore explored further below.…”

Section: Resultssupporting

confidence: 90%

“…47 There is physiological evidence for an acid-loading Cl − transporter on the parasite plasma membrane that plays a role in pH regulation (aiding parasite recovery from an alkalinization event) and allows the parasite to maintain a cytosolic Cl − concentration higher than what would be expected if Cl − were at electrochemical equilibrium. 48 The molecular identity of the acid-loading Cl − transporter is not known, and it is possible that more than one transporter is involved.…”

Section: Introductionmentioning

confidence: 99%

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A pH Fingerprint Assay to Identify Inhibitors of Multiple Validated and Potential Antimalarial Drug Targets

Lindblom,

Zhang,

Lehane

2024

ACS Infect. Dis.

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New drugs with novel modes of action are needed to safeguard malaria treatment. In recent years, millions of compounds have been tested for their ability to inhibit the growth of asexual blood-stage Plasmodium falciparum parasites, resulting in the identification of thousands of compounds with antiplasmodial activity. Determining the mechanisms of action of antiplasmodial compounds informs their further development, but remains challenging. A relatively high proportion of compounds identified as killing asexual blood-stage parasites show evidence of targeting the parasite's plasma membrane Na + -extruding, H +importing pump, PfATP4. Inhibitors of PfATP4 give rise to characteristic changes in the parasite's internal [Na + ] and pH. Here, we designed a "pH fingerprint" assay that robustly identifies PfATP4 inhibitors while simultaneously allowing the detection of (and discrimination between) inhibitors of the lactate:H + transporter PfFNT, which is a validated antimalarial drug target, and the V-type H + ATPase, which was suggested as a possible target of the clinical candidate ZY19489. In our pH fingerprint assays and subsequent secondary assays, ZY19489 did not show evidence for the inhibition of pH regulation by the V-type H + ATPase, suggesting that it has a different mode of action in the parasite. The pH fingerprint assay also has the potential to identify protonophores, inhibitors of the acid-loading Cl − transporter(s) (for which the molecular identity(ies) remain elusive), and compounds that act through inhibition of either the glucose transporter PfHT or glycolysis. The pH fingerprint assay therefore provides an efficient starting point to match a proportion of antiplasmodial compounds with their mechanisms of action.

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Section: Resultssupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

A pH Fingerprint Assay to Identify Inhibitors of Multiple Validated and Potential Antimalarial Drug Targets

Lindblom,

Zhang,

Lehane

2024

ACS Infect. Dis.

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“…A chloride concentration inside the parasite of 48 mM [59] is adequate for DPAP1 activation. We envision DPAP1 acting on a wide range of sequence-diverse oligopeptides that are generated by the action of endopeptidases on globin polypeptides.…”

Section: Discussionmentioning

confidence: 99%

Biochemical characterization of Plasmodium falciparum dipeptidyl aminopeptidase 1

Wang

Krai

Deu

et al. 2011

Molecular and Biochemical Parasitology

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Dipeptidyl aminopeptidase 1 (DPAP1) is an essential food vacuole enzyme with a putative role in hemoglobin catabolism by the erythrocytic malaria parasite. Here, the biochemical properties of DPAP1 have been investigated and compared to those of the human ortholog cathepsin C. To facilitate the characterization of DPAP1, we have developed a method for the production of purified recombinant DPAP1 with properties closely resembling those of the native enzyme. Like cathepsin C, DPAP1 is a chloride-activated enzyme that is most efficient in catalyzing amide bond hydrolysis at acidic pH values. The monomeric quaternary structure of DPAP1 differs from the homotetrameric structure of cathepsin C, which suggests that tetramerization is required for a cathepsin C-specific function. The S1 and S2 subsite preferences of DPAP1 and cathepsin C were profiled with a positional scanning synthetic combinatorial library. The S1 preferences bore close similarity to those of other C1-family cysteine peptidases. The S2 subsites of both DPAP1 and cathepsin C accepted aliphatic hydrophobic residues, proline, and some polar residues, yielding a distinct specificity profile. DPAP1 efficiently catalyzed the hydrolysis of several fluorogenic dipeptide substrates; surprisingly, however, a potential substrate with a P2-phenylalanine residue was instead a competitive inhibitor. Together, our biochemical data suggest that DPAP1 accelerates the production of amino acids from hemoglobin by bridging the gap between the endopeptidase and aminopeptidase activities of the food vacuole. Two reversible cathepsin C inhibitors potently inhibited both recombinant and native DPAP1, thereby validating the use of recombinant DPAP1 for future inhibitor discovery and characterization.

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“…The observation that the uptake of a high (10 mM) concentration of putrescine to an isolated parasite suspension had no significant effect on cytosolic pH (contrasting with the substantial alkalinization seen on addition of an equivalent concentration of NH 4 + ) argues against there being a rapid diffusion of unprotonated putrescine across the membrane bilayer, both at pH 7.1 and pH 8.1. However, it should be noted that the parasite does have pH regulatory systems that allow it to counter a cytosolic alkalinization (Henry et al, 2010). The possibility therefore cannot be excluded that a fraction of the polyamines do enter via diffusion, much more slowly than NH 3 /NH 4 + , and that the parasite effectively counters (and therefore masks) the potential pH changes.…”

Section: Discussionmentioning

confidence: 99%

Polyamine uptake by the intraerythrocytic malaria parasite, Plasmodium falciparum

Niemand

Louw

Birkholtz

et al. 2012

International Journal for Parasitology

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Polyamines and the enzymes involved in their biosynthesis are present at high levels in rapidly proliferating cells, including cancer cells and protozoan parasites. Inhibition of polyamine biosynthesis in asexual blood-stage malaria parasites causes cytostatic arrest of parasite development under in vitro conditions, but does not cure infections in vivo. This may be due to replenishment of the parasite's intracellular polyamine pool via salvage of exogenous polyamines from the host. However the mechanism(s) of polyamine uptake by the intra-erythrocytic parasite are not well understood. In this study, the uptake of the polyamines putrescine and spermidine into Plasmodium falciparum parasites functionally isolated from their host erythrocyte were investigated using radioisotope flux techniques. Both putrescine and spermidine were taken up into isolated parasites via a temperature-dependent process that showed cross-competition between different polyamines. There was also some inhibition of polyamine uptake by basic amino acids. Inhibition of polyamine biosynthesis led to an increase in the total amount of putrescine and spermidine taken up from the extracellular medium. The uptake of putrescine and spermidine by isolated parasites was independent of extracellular Na + but increased with increasing external pH. Uptake also showed a marked dependence on the parasite's membrane potential, decreasing with membrane depolarization and increasing with membrane hyperpolarization. The data are consistent with polyamines being taken up into the parasite via an electrogenic uptake process, energized by the parasite's inwardly negative membrane potential.

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An Acid-loading Chloride Transport Pathway in the Intraerythrocytic Malaria Parasite, Plasmodium falciparum

Abstract: The intraerythrocytic malaria parasite exerts tight control over its ionic composition. In this study,

Cited by 10 publications

References 27 publications

A pH Fingerprint Assay to Identify Inhibitors of Multiple Validated and Potential Antimalarial Drug Targets

A pH Fingerprint Assay to Identify Inhibitors of Multiple Validated and Potential Antimalarial Drug Targets

Biochemical characterization of Plasmodium falciparum dipeptidyl aminopeptidase 1

Polyamine uptake by the intraerythrocytic malaria parasite, Plasmodium falciparum

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