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

AbstractThe 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+. Whether, and if so how, such chemically distinct compounds interact with PfATP4, and how such interactions lead to parasite death, is not yet clear. The fact that multiple different chemical classes have converged upon PfATP4 highlights its significance as a potential target for new generation antimalarial agents. A spiroindolone (KAE609, now known as cipargamin) has progressed through Phase I and IIa clinical trials with favourable results. In this review we consider the physiological role of PfATP4, summarise the current repertoire of antimalarial compounds for which PfATP4 is implicated in their mechanism of action, and provide an outlook on translation from target identification in the laboratory to patient treatment in the field

Na+ regulation in the intraerythrocytic malaria parasite

The maintenance of a low intracellular [Na⁺] ([Na⁺]i) is a crucial aspect of cellular physiology. In mammalian cells this is achieved through the extrusion of Na⁺ via the well-characterised Na⁺/K⁺-ATPase. Approximately 12 hr after invasion of the human erythrocyte by the malaria parasite there is a profound increase in the permeability of the erythrocyte membrane to a wide range of solutes, including Na⁺. Na⁺ enters the infected erythrocyte via parasite-induced 'New Permeability Pathways' and there is, as a result, an increase in [Na⁺] in the erythrocyte compartment, with [Na⁺]i eventually reaching levels similar to those in the extra erythrocytic plasma (~130 mM). The parasite itself maintains a low [Na⁺]i. The resulting large inwardly-directed electrochemical Na⁺ gradient across the parasite plasma membrane energises the accumulation within the parasite of at least one essential nutrient (inorganic phosphate). The aim of this thesis was to characterise the mechanisms involved in Na⁺ regulation in the mature asexual 'trophozoite' stage of the human malaria parasite Plasmodium falciparum. The Na⁺-sensitive, fluorescent dye Sodium-binding BenzoFuran Isophthalate (SBFI) was used to measure [Na⁺]i in parasites functionally isolated from their host cells by saponin-permeabilisation of the host erythrocyte membrane. Under physiologically relevant conditions the resting [Na⁺]i in isolated trophozoites was estimated to be ~11 mM. Maintenance of [Na⁺]i was sensitive to the P-type ATPase inhibitor orthovanadate, consistent with Na⁺ extrusion being via a P-type Na⁺-ATPase, similar to the ENA (exitus natru; exit of sodium)-type ATPases that operate in some other protozoa, fungi and lower plants. ENA ATPases have been predicted to antiport H⁺ and the data obtained here are consistent with this being true of the P. falciparum Na⁺ extrusion system. The P. falciparum genome encodes a number of putative P-type ATPases; one of these, PfATP4, was found to share significant sequence similarities to ENA ATPases of other protozoa. A recent study showed that mutations in PfATP4 confer resistance to a newly-described class of antimalarials, the spiroindolones. The effect of the spiroindolones on ion regulation was therefore investigated. Several spiroindolones were shown to cause a profound disruption of [Na⁺]i regulation. In parasites with mutant PfATP4 there was both an impairment of Na⁺ regulation and a decrease in the spiroindolone-sensitivity of Na⁺ regulation. These results are consistent with PfATP4 being a Na⁺-ATPase and the target of the spiroindolones. The physiological role of another putative Na⁺ transporter, the Na⁺/H⁺-exchanger PfNHE was also investigated, as previous studies on its contribution to regulation of [Na⁺]i and intracellular pH (pHi) have been controversial. On the basis of a bioinformatics analysis it was predicted that the protein functions as an amiloride-insensitive, plasma membrane Na⁺-extruder, like its closely related plant homologues. However physiological studies revealed no significant role for such an NHE in either pHi or [Na⁺]i regulation in the P. falciparum trophozoite. This study constitutes a significant advance in our understanding of fundamental aspects of the cell physiology of the intraerythrocytic parasite, as well as shedding light on the mode of action of what promises to be an important new class of antimalarials, the spiroindolones

(+)-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 i

Violacein-Induced Chaperone System Collapse Underlies Multistage Antiplasmodial Activity

Antimalarial drugs with novel modes of action and wide therapeutic potential are needed to pave the way for malaria eradication. Violacein is a natural compound known for its biological activity against cancer cells and several pathogens, including the malaria parasite, Plasmodium falciparum (Pf). Herein, using chemical genomic profiling (CGP), we found that violacein affects protein homeostasis. Mechanistically, violacein binds Pf chaperones, PfHsp90 and PfHsp70-1, compromising the latter's ATPase and chaperone activities. Additionally, violacein-treated parasites exhibited increased protein unfolding and proteasomal degradation. The uncoupling of the parasite stress response reflects the multistage growth inhibitory effect promoted by violacein. Despite evidence of proteotoxic stress, violacein did not inhibit global protein synthesis via UPR activation - a process that is highly dependent on chaperones, in agreement with the notion of a violacein-induced proteostasis collapse. Our data highlight the importance of a functioning chaperone-proteasome system for parasite development and differentiation. Thus, a violacein-like small molecule might provide a good scaffold for development of a novel probe for examining the molecular chaperone network and/or antiplasmodial drug design.publishersversionpublishe

Multistage and transmission-blocking targeted antimalarials discovered from the open-source MMV Pandemic Response Box

Chemical matter is needed to target the divergent biology associated with the different life cycle stages of Plasmodium. Here, we report the parallel de novo screening of the Medicines for Malaria Venture (MMV) Pandemic Response Box against Plasmodium asexual and liver stage parasites, stage IV/V gametocytes, gametes, oocysts and as endectocides. Unique chemotypes were identified with both multistage activity or stage-specific activity, including structurally diverse gametocyte-targeted compounds with potent transmission-blocking activity, such as the JmjC inhibitor ML324 and the antitubercular clinical candidate SQ109. Mechanistic investigations prove that ML324 prevents histone demethylation, resulting in aberrant gene expression and death in gametocytes. Moreover, the selection of parasites resistant to SQ109 implicates the druggable V-type H+-ATPase for the reduced sensitivity. Our data therefore provides an expansive dataset of compounds that could be redirected for antimalarial development and also point towards proteins that can be targeted in multiple parasite life cycle stages.Supplementary Data 1: Data of the supra-hexagonal plot in Figure 2ASupplementary Data 2: Complete dataset of all MMV PRB compounds’ activity on Plasmodium life cycle stagesSupplementary Data 3: Full SMFA dataset to support Figure 5CSupplementary Data 4: Transcriptome analysis of MMV1580488 (ML324) treated parasites to support Figure 6C.The Medicines for Malaria Venture and South African Technology Innovation Agency (TIA). This project was in part supported by the South African Medical Research Council with funds received from the South African Department of Science and Innovation, in partnership with the Medicines for Malaria Venture; and the DST/NRF South African Research Chairs Initiative Grant; and CSIR Parliamentary Grant funding as well as the Bill and Melinda Gates Foundation and the Australian NHMRC (APP1072217).http://www.nature.com/ncommshj2021BiochemistryGeneticsMicrobiology and Plant PathologyUP Centre for Sustainable Malaria Control (UP CSMC

Protein export into malaria parasite-infected erythrocytes: Mechanisms and functional consequences

Phylum Apicomplexa comprises a large group of obligate intracellular parasites of high medical and veterinary importance. These organisms succeed intracellularly by effecting remarkable changes in a broad range of diverse host cells. The transformation of the host erythrocyte is particularly striking in the case of the malaria parasite Plasmodium falciparum. P. falciparum exports hundreds of proteins that mediate a complex cellular renovation marked by changes in the permeability, rigidity, and cytoadherence properties of the host erythrocyte. The past decade has seen enormous progress in understanding the identity and function of these exported effectors, as well as the mechanisms by which they are trafficked into the host cell. Here we review these advances, place them in the context of host manipulation by related apicomplexans, and propose key directions for future research