A G358S mutation in the Plasmodium falciparum Na+ pump PfATP4 confers clinically-relevant resistance to cipargamin.

Diverse compounds target the Plasmodium falciparum Na+ pump PfATP4, with cipargamin and (+)-SJ733 the most clinically-advanced. In a recent clinical trial for cipargamin, recrudescent parasites emerged, with most having a G358S mutation in PfATP4. Here, we show that PfATP4G358S parasites can withstand micromolar concentrations of cipargamin and (+)-SJ733, while remaining susceptible to antimalarials that do not target PfATP4. The G358S mutation in PfATP4, and the equivalent mutation in Toxoplasma gondii ATP4, decrease the sensitivity of ATP4 to inhibition by cipargamin and (+)-SJ733, thereby protecting parasites from disruption of Na+ regulation. The G358S mutation reduces the affinity of PfATP4 for Na+ and is associated with an increase in the parasite's resting cytosolic [Na+]. However, no defect in parasite growth or transmissibility is observed. Our findings suggest that PfATP4 inhibitors in clinical development should be tested against PfATP4G358S parasites, and that their combination with unrelated antimalarials may mitigate against resistance development.

An Open Drug Discovery Competition: Experimental Validation of Predictive Models in a Series of Novel Antimalarials.

The Open Source Malaria (OSM) consortium is developing compounds that kill the human malaria parasite, Plasmodium falciparum, by targeting PfATP4, an essential ion pump on the parasite surface. The structure of PfATP4 has not been determined. Here, we describe a public competition created to develop a predictive model for the identification of PfATP4 inhibitors, thereby reducing project costs associated with the synthesis of inactive compounds. Competition participants could see all entries as they were submitted. In the final round, featuring private sector entrants specializing in machine learning methods, the best-performing models were used to predict novel inhibitors, of which several were synthesized and evaluated against the parasite. Half possessed biological activity, with one featuring a motif that the human chemists familiar with this series would have dismissed as "ill-advised". Since all data and participant interactions remain in the public domain, this research project "lives" and may be improved by others.

Substrate-mediated regulation of the arginine transporter of Toxoplasma gondii.

Intracellular parasites, such as the apicomplexan Toxoplasma gondii, are adept at scavenging nutrients from their host. However, there is little understanding of how parasites sense and respond to the changing nutrient environments they encounter during an infection. TgApiAT1, a member of the apicomplexan ApiAT family of amino acid transporters, is the major uptake route for the essential amino acid L-arginine (Arg) in T. gondii. Here, we show that the abundance of TgApiAT1, and hence the rate of uptake of Arg, is regulated by the availability of Arg in the parasite's external environment, increasing in response to decreased [Arg]. Using a luciferase-based 'biosensor' strain of T. gondii, we demonstrate that the expression of TgApiAT1 varies between different organs within the host, indicating that parasites are able to modulate TgApiAT1-dependent uptake of Arg as they encounter different nutrient environments in vivo. Finally, we show that Arg-dependent regulation of TgApiAT1 expression is post-transcriptional, mediated by an upstream open reading frame (uORF) in the TgApiAT1 transcript, and we provide evidence that the peptide encoded by this uORF is critical for mediating regulation. Together, our data reveal the mechanism by which an apicomplexan parasite responds to changes in the availability of a key nutrient.

Coordinated action of multiple transporters in the acquisition of essential cationic amino acids by the intracellular parasite Toxoplasma gondii.

Intracellular parasites of the phylum Apicomplexa are dependent on the scavenging of essential amino acids from their hosts. We previously identified a large family of apicomplexan-specific plasma membrane-localized amino acid transporters, the ApiATs, and showed that the Toxoplasma gondii transporter TgApiAT1 functions in the selective uptake of arginine. TgApiAT1 is essential for parasite virulence, but dispensable for parasite growth in medium containing high concentrations of arginine, indicating the presence of at least one other arginine transporter. Here we identify TgApiAT6-1 as the second arginine transporter. Using a combination of parasite assays and heterologous characterisation of TgApiAT6-1 in Xenopus laevis oocytes, we demonstrate that TgApiAT6-1 is a general cationic amino acid transporter that mediates both the high-affinity uptake of lysine and the low-affinity uptake of arginine. TgApiAT6-1 is the primary lysine transporter in the disease-causing tachyzoite stage of T. gondii and is essential for parasite proliferation. We demonstrate that the uptake of cationic amino acids by TgApiAT6-1 is 'trans-stimulated' by cationic and neutral amino acids and is likely promoted by an inwardly negative membrane potential. These findings demonstrate that T. gondii has evolved overlapping transport mechanisms for the uptake of essential cationic amino acids, and we draw together our findings into a comprehensive model that highlights the finely-tuned, regulated processes that mediate cationic amino acid scavenging by these intracellular parasites.

Identifying the major lactate transporter of Toxoplasma gondii tachyzoites.

Toxoplasma gondii and Plasmodium falciparum parasites both extrude L-lactate, a byproduct of glycolysis. The P. falciparum Formate Nitrite Transporter, PfFNT, mediates L-lactate transport across the plasma membrane of P. falciparum parasites and has been validated as a drug target. The T. gondii genome encodes three FNTs that have been shown to transport L-lactate, and which are proposed to be the targets of several inhibitors of T. gondii proliferation. Here, we show that each of the TgFNTs localize to the T. gondii plasma membrane and are capable of transporting L-lactate across it, with TgFNT1 making the primary contribution to L-lactate transport during the disease-causing lytic cycle of the parasite. We use the Xenopus oocyte expression system to provide direct measurements of L-lactate transport via TgFNT1. We undertake a genetic analysis of the importance of the tgfnt genes for parasite proliferation, and demonstrate that all three tgfnt genes can be disrupted individually and together without affecting the lytic cycle under in vitro culture conditions. Together, our experiments identify the major lactate transporter in the disease causing stage of T. gondii, and reveal that this transporter is not required for parasite proliferation, indicating that TgFNTs are unlikely to be targets for anti-Toxoplasma drugs.

Measuring Solute Transport in Toxoplasma gondii Parasites.

The uptake of host-derived nutrients is key to the growth and survival of Toxoplasma gondii parasites. Nutrients are acquired via solute transporters that localize to the plasma membrane of the parasites. In this chapter, we describe methodology by which the uptake of solutes via plasma membrane transporters may be monitored and characterized. These assays, used here to investigate the uptake of amino acids into parasites, have broad applicability in measuring the uptake of a diverse range of solutes.

A 4-cyano-3-methylisoquinoline inhibitor of Plasmodium falciparum growth targets the sodium efflux pump PfATP4.

We developed a novel series of antimalarial compounds based on a 4-cyano-3-methylisoquinoline. Our lead compound MB14 achieved modest inhibition of the growth in vitro of the human malaria parasite, Plasmodium falciparum. To identify its biological target we selected for parasites resistant to MB14. Genome sequencing revealed that all resistant parasites bore a single point S374R mutation in the sodium (Na+) efflux transporter PfATP4. There are many compounds known to inhibit PfATP4 and some are under preclinical development. MB14 was shown to inhibit Na+ dependent ATPase activity in parasite membranes, consistent with the compound targeting PfATP4 directly. PfATP4 inhibitors cause swelling and lysis of infected erythrocytes, attributed to the accumulation of Na+ inside the intracellular parasites and the resultant parasite swelling. We show here that inhibitor-induced lysis of infected erythrocytes is dependent upon the parasite protein RhopH2, a component of the new permeability pathways that are induced by the parasite in the erythrocyte membrane. These pathways mediate the influx of Na+ into the infected erythrocyte and their suppression via RhopH2 knockdown limits the accumulation of Na+ within the parasite hence protecting the infected erythrocyte from lysis. This study reveals a role for the parasite-induced new permeability pathways in the mechanism of action of PfATP4 inhibitors.

The tyrosine transporter of Toxoplasma gondii is a member of the newly defined apicomplexan amino acid transporter (ApiAT) family.

Apicomplexan parasites are auxotrophic for a range of amino acids which must be salvaged from their host cells, either through direct uptake or degradation of host proteins. Here, we describe a family of plasma membrane-localized amino acid transporters, termed the Apicomplexan Amino acid Transporters (ApiATs), that are ubiquitous in apicomplexan parasites. Functional characterization of the ApiATs of Toxoplasma gondii indicate that several of these transporters are important for intracellular growth of the tachyzoite stage of the parasite, which is responsible for acute infections. We demonstrate that the ApiAT protein TgApiAT5-3 is an exchanger for aromatic and large neutral amino acids, with particular importance for L-tyrosine scavenging and amino acid homeostasis, and that TgApiAT5-3 is critical for parasite virulence. Our data indicate that T. gondii expresses additional proteins involved in the uptake of aromatic amino acids, and we present a model for the uptake and homeostasis of these amino acids. Our findings identify a family of amino acid transporters in apicomplexans, and highlight the importance of amino acid scavenging for the biology of this important phylum of intracellular parasites.

Characterization of the ATP4 ion pump in Toxoplasma gondii.

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 Ca2+ 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 Ca2+ 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.

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

The antimalarial activity of chemically diverse compounds, including the clinical candidate cipargamin, has been linked to the ATPase PfATP4 in the malaria-causing parasite Plasmodium falciparum 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 P. falciparum 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 P. falciparum 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 [Ca2+]. With an apparent Km for ATP of 0.2 mm and an apparent Km 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 Km for Na+ is slightly elevated. Our study provides new insights into the biochemical properties and chemical sensitivity of an important new antimalarial drug target.

Diverse antimalarials from whole-cell phenotypic screens disrupt malaria parasite ion and volume homeostasis.

Four hundred structurally diverse drug-like compounds comprising the Medicines for Malaria Venture's 'Pathogen Box' were screened for their effect on a range of physiological parameters in asexual blood-stage malaria (Plasmodium falciparum) parasites. Eleven of these compounds were found to perturb parasite Na+, pH and volume in a manner consistent with inhibition of the putative Na+ efflux P-type ATPase PfATP4. All eleven compounds fell within the subset of 125 compounds included in the Pathogen Box on the basis of their having been identified as potent inhibitors of the growth of asexual blood-stage P. falciparum parasites. All eleven compounds inhibited the Na+-dependent ATPase activity of parasite membranes and showed reduced efficacy against parasites carrying mutations in PfATP4. This study increases the number of chemically diverse structures known to show a 'PfATP4-associated' phenotype, and adds to emerging evidence that a high proportion (7-9%) of the structurally diverse antimalarial compounds identified in whole cell phenotypic screens share the same mechanism of action, exerting their antimalarial effect via an interaction with PfATP4.

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

For an increasing number of antimalarial agents identified in high-throughput phenotypic screens, there is evidence that they target PfATP4, a putative Na+ efflux transporter on the plasma membrane of the human malaria parasite Plasmodium falciparum For several such "PfATP4-associated" compounds, it has been noted that their addition to parasitized erythrocytes results in cell swelling. Here we show that six structurally diverse PfATP4-associated compounds, including the clinical candidate KAE609 (cipargamin), induce swelling of both isolated blood-stage parasites and intact parasitized erythrocytes. The swelling of isolated parasites is dependent on the presence of Na+ in the external environment and may be attributed to the osmotic consequences of Na+ uptake. The swelling of the parasitized erythrocyte results in an increase in its osmotic fragility. Countering cell swelling by increasing the osmolarity of the extracellular medium reduces the antiplasmodial efficacy of PfATP4-associated compounds, consistent with cell swelling playing a role in the antimalarial activity of this class of compounds.

The NMR 'split peak effect' in cell suspensions: Historical perspective, explanation and applications.

The physicochemical environment inside cells is distinctly different from that immediately outside. The selective exchange of ions, water and other molecules across the cell membrane, mediated by integral, membrane-embedded proteins is a hallmark of living systems. There are various methodologies available to measure the selectivity and rates (kinetics) of such exchange processes, including several that take advantage of the non-invasive nature of NMR spectroscopy. A number of solutes, including particular inorganic ions, show distinctive NMR behaviour, in which separate resonances arise from the intra- and extracellular solute populations, without the addition of shift reagents, differences in pH, or selective binding partners. This 'split peak effect/phenomenon', discovered in 1984, has become a valuable tool, used in many NMR studies of cellular behaviour and function. The explanation for the phenomenon, based on the differential hydrogen bonding of the reporter solutes to water, and the various ways in which this phenomenon has been used to investigate aspects of cellular biochemistry and physiology, are the topics of this review.

Cationic amino acid transporters play key roles in the survival and transmission of apicomplexan parasites.

Apicomplexans are obligate intracellular parasites that scavenge essential nutrients from their hosts via transporter proteins on their plasma membrane. The identities of the transporters that mediate amino acid uptake into apicomplexans are unknown. Here we demonstrate that members of an apicomplexan-specific protein family-the Novel Putative Transporters (NPTs)-play key roles in the uptake of cationic amino acids. We show that an NPT from Toxoplasma gondii (TgNPT1) is a selective arginine transporter that is essential for parasite survival and virulence. We also demonstrate that a homologue of TgNPT1 from the malaria parasite Plasmodium berghei (PbNPT1), shown previously to be essential for the sexual gametocyte stage of the parasite, is a cationic amino acid transporter. This reveals a role for cationic amino acid scavenging in gametocyte biology. Our study demonstrates a critical role for amino acid transporters in the survival, virulence and life cycle progression of these parasites.

The Malaria Parasite's Lactate Transporter PfFNT Is the Target of Antiplasmodial Compounds Identified in Whole Cell Phenotypic Screens.

In this study the 'Malaria Box' chemical library comprising 400 compounds with antiplasmodial activity was screened for compounds that perturb the internal pH of the malaria parasite, Plasmodium falciparum. Fifteen compounds induced an acidification of the parasite cytosol. Two of these did so by inhibiting the parasite's formate nitrite transporter (PfFNT), which mediates the H+-coupled efflux from the parasite of lactate generated by glycolysis. Both compounds were shown to inhibit lactate transport across the parasite plasma membrane, and the transport of lactate by PfFNT expressed in Xenopus laevis oocytes. PfFNT inhibition caused accumulation of lactate in parasitised erythrocytes, and swelling of both the parasite and parasitised erythrocyte. Long-term exposure of parasites to one of the inhibitors gave rise to resistant parasites with a mutant form of PfFNT that showed reduced inhibitor sensitivity. This study provides the first evidence that PfFNT is a druggable antimalarial target.

Biochemical and Structural Characterization of Selective Allosteric Inhibitors of the Plasmodium falciparum Drug Target, Prolyl-tRNA-synthetase.

Plasmodium falciparum (Pf) prolyl-tRNA synthetase (ProRS) is one of the few chemical-genetically validated drug targets for malaria, yet highly selective inhibitors have not been described. In this paper, approximately 40,000 compounds were screened to identify compounds that selectively inhibit PfProRS enzyme activity versus Homo sapiens (Hs) ProRS. X-ray crystallography structures were solved for apo, as well as substrate- and inhibitor-bound forms of PfProRS. We identified two new inhibitors of PfProRS that bind outside the active site. These two allosteric inhibitors showed >100 times specificity for PfProRS compared to HsProRS, demonstrating this class of compounds could overcome the toxicity related to HsProRS inhibition by halofuginone and its analogues. Initial medicinal chemistry was performed on one of the two compounds, guided by the cocrystallography of the compound with PfProRS, and the results can instruct future medicinal chemistry work to optimize these promising new leads for drug development against malaria.

Open Source Drug Discovery: Highly Potent Antimalarial Compounds Derived from the Tres Cantos Arylpyrroles.

The development of new antimalarial compounds remains a pivotal part of the strategy for malaria elimination. Recent large-scale phenotypic screens have provided a wealth of potential starting points for hit-to-lead campaigns. One such public set is explored, employing an open source research mechanism in which all data and ideas were shared in real time, anyone was able to participate, and patents were not sought. One chemical subseries was found to exhibit oral activity but contained a labile ester that could not be replaced without loss of activity, and the original hit exhibited remarkable sensitivity to minor structural change. A second subseries displayed high potency, including activity within gametocyte and liver stage assays, but at the cost of low solubility. As an open source research project, unexplored avenues are clearly identified and may be explored further by the community; new findings may be cumulatively added to the present work.

Human dihydrofolate reductase influences the sensitivity of the malaria parasite Plasmodium falciparum to ketotifen - A cautionary tale in screening transgenic parasites.

Ketotifen has recently been reported to inhibit the growth of both asexual and sexual malaria parasites. A parasite transporter, PfgABCG2, has been implicated in its mechanism of action. Human dihydrofolate reductase (hDHFR) is the most commonly used selectable marker to create transgenic Plasmodium falciparum cell lines. Growth assays using transgenic P. falciparum parasites with different selectable markers revealed that the presence of hDHFR rather than the absence of PfgABCG2 is responsible for a shift in the parasite's sensitivity to ketotifen. Employing a range of in vitro assays and liquid chromatography-mass spectrometry we show that ketotifen influences hDHFR activity, but it is not metabolised by the enzyme. Our data also highlights potential pitfalls when functionally characterising transgenic parasites.

A high-sensitivity HPLC assay for measuring intracellular Na(+) and K(+) and its application to Plasmodium falciparum infected erythrocytes.

The measurement of intracellular ion concentrations, and the screening of chemical agents to identify molecules targeting ion transport, has traditionally involved low-throughput techniques. Here we present a novel HPLC method that allows the rapid, high-sensitivity measurement of cell Na(+) and K(+) content, demonstrating its utility by monitoring the ionic changes induced in the intracellular malaria parasite by the new spiroindolone antimalarial KAE609.

Sequestration and metabolism of host cell arginine by the intraerythrocytic malaria parasite Plasmodium falciparum.

Human erythrocytes have an active nitric oxide synthase, which converts arginine into citrulline and nitric oxide (NO). NO serves several important functions, including the maintenance of normal erythrocyte deformability, thereby ensuring efficient passage of the red blood cell through narrow microcapillaries. Here, we show that following invasion by the malaria parasite Plasmodium falciparum the arginine pool in the host erythrocyte compartment is sequestered and metabolized by the parasite. Arginine from the extracellular medium enters the infected cell via endogenous host cell transporters and is taken up by the intracellular parasite by a high-affinity cationic amino acid transporter at the parasite surface. Within the parasite arginine is metabolized into citrulline and ornithine. The uptake and metabolism of arginine by the parasite deprive the erythrocyte of the substrate required for NO production and may contribute to the decreased deformability of infected erythrocytes.