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, , by targeting ATP4, an essential ion pump on the parasite surface. The structure of ATP4 has not been determined. Here, we describe a public competition created to develop a predictive model for the identification of ATP4 inhibitors, thereby reducing project costs associated with the synthesis of inactive compounds.

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.

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.

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.

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.

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.

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.

Biochemical characterization and chemical inhibition of PfATP4-associated Na-ATPase activity in 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 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.

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.

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 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 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.

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.

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.

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.

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.

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.

Ion Regulation in the Malaria Parasite.

Some hours after invading the erythrocytes of its human host, the malaria parasite Plasmodium falciparum induces an increase in the permeability of the erythrocyte membrane to monovalent ions. The resulting net influx of Na(+) and net efflux of K(+), down their respective concentration gradients, converts the erythrocyte cytosol from an initially high-K(+), low-Na(+) solution to a high-Na(+), low-K(+) solution. The intraerythrocytic parasite itself exerts tight control over its internal Na(+), K(+), Cl(-), and Ca(2+) concentrations and its intracellular pH through the combined actions of a range of membrane transport proteins.

The malaria parasite cation ATPase PfATP4 and its role in the mechanism of action of a new arsenal of antimalarial drugs.

The intraerythrocytic malaria parasite, Plasmodium falciparum, maintains a low cytosolic Na(+) concentration and the plasma membrane P-type cation translocating ATPase 'PfATP4' has been implicated as playing a key role in this process. PfATP4 has been the subject of significant attention in recent years as mutations in this protein confer resistance to a growing number of new antimalarial compounds, including the spiroindolones, the pyrazoles, the dihydroisoquinolones, and a number of the antimalarial agents in the Medicines for Malaria Venture's 'Malaria Box'. On exposure of parasites to these compounds there is a rapid disruption of cytosolic Na(+).

A lactate and formate transporter in the intraerythrocytic malaria parasite, Plasmodium falciparum.

The intraerythrocytic malaria parasite relies primarily on glycolysis to fuel its rapid growth and reproduction. The major byproduct of this metabolism, lactic acid, is extruded into the external medium. In this study, we show that the human malaria parasite Plasmodium falciparum expresses at its surface a member of the microbial formate-nitrite transporter family (PfFNT), which, when expressed in Xenopus laevis oocytes, transports both formate and lactate.

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

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.