Modes of Action

The continual emergence of drug-resistant Plasmodium strains means that developing new drugs with novel MoAs is important [129]. Below, are summarized some newer MoAs that are currently being pursued and which are exemplified by the compounds described above.

Translational elongation factor 2 (Pf eEF2) inhibitor

The P. falciparum translational elongation factor 2 (Pf eEF2) is one of the essential factors for eukaryotic protein synthesis, catalysing the translocation of tRNA and mRNA [130]. The overall efficacy of drugs that target this elongation factor may be increased due to Pf eEF2 being expressed in multiple stages of the parasite life-cycle [78].

M5717 has been shown to inhibit Pf eEF2 as its MoA, but a binding pocket for M5717 could not be elucidated from the modelling studies [78]. This MoA was found by culturing blood stage parasites with five times the EC₅₀ of M5717, leading to resistance in the 3D7, 7G8 and Dd2 strains. Through whole-genome sequencing of these resistance lines, a common mutation was found which identified Pf eEF2 as the target.

P-type Na⁺–ATPase inhibitor (Pf ATP4) inhibitor

The malaria parasite must maintain a low intracellular Na⁺ concentration to survive, especially in the presence of the high concentration of extracellular sodium ions. In this case, the parasite’s influx of Na⁺ is regulated by using a P-type ATPase transporter (Pf ATP4) that shuttles sodium ions out of the cell. Inhibition of this transporter results in a build up of Na⁺ inside the parasite, ultimately leading to cell death [102].

A number of structurally diverse compounds (including (+)-SJ733 and KAE609) are thought to target Pf ATP4 as their MoA again suggested by the development of resistant mutants and sequencing [124]. The Kirk group have developed a fluorescence assay for this MoA [131], which has been used by the OSM project to implicate Pf ATP4 as a MoA for the Series 4 triazolopyrazines [132]. Of note, however, is that this MoA is suggested for a very broad range of chemotypes [133, 134], and there is no direct evidence for binding of these compounds to Pf ATP4, the structure of which remains unsolved.

V-type H⁺–ATPase inhibitor

At the same time that the parasite is regulating its Na⁺ concentration through the above mechanism, it also imports H⁺ through the same pathway. To regulate this increasing H⁺ concentration and maintain an intracellular pH of ∼ 7.3, the parasite uses a complementary V-type ATPase transporter to efflux H⁺ [135].

MMV253 has been shown to inhibit the V-type H⁺ ATPase as its MoA through mutant selection and whole-genome sequencing [81].

Phosphatidylinositol 4-kinase (Pf PI4K) inhibitor

Phosphatidylinositol 4-kinase (PI4K) is a eukaryotic enzyme that phosphorylates lipids, allowing them to regulate intracellular signalling and trafficking [87]. Inhibition of the ATP-binding pocket of PI4K leads to disruption of the intracellular distribution of PI4-phosphate (PI4P), which in turn results in decreased late-stage parasite development.

By culturing resistant lines and identifying the genomic mutations, two compounds (UCT943 and MMV048) have been found to inhibit the Pf PI4K enzyme [87].

Dihydroorotate dehydrogenase (Pf DHODH) inhibitor

To be able to replicate in the human erythrocyte, the Plasmodium parasite requires pyrimidines, which are essential metabolites in all cells. As the parasite is unable to use endogenous pyrimidines, it must synthesize them de novo. A key step in the biosynthesis of pyrimidines is the oxidation of dihydroorotate to produce orotate, a reaction that is catalysed by the enzyme dihydroorotate dehydrogenase (DHODH). By inhibiting this enzyme, the malaria parasite can no longer obtain the required metabolites to survive, and is killed [136].

The P. falciparum D10 strain (chloroquine sensitive) has been known to be less susceptible to bc₁ complex inhibitors and Pf DHODH inhibitors. Addition of proguanil to this strain is known to restore the sensitivity to bc₁ complex inhibitors but not to Pf DHODH inhibitors. This cell line was found to be resistant to DSM265, regardless of whether proguanil was present or not, which identifies inhibition of Pf DHODH as its MoA [127, 137].

Dihydrofolate reductase (Pf DHFR) inhibitor

Similar to the above case, the malaria parasite also requires folates in order to maintain their high rate of replication. The parasite is able to scavenge folates or synthesize them de novo. Dihydrofolate reductase (DHFR) is an enzyme that catalyses a step required for the recycling of folates, which in turn are used in the synthesis of thymidylate, purines and methionine [138].

Pf DHFR is a known target of a number of well known antifolate anti-malarial drugs including cycloguanil and pyrimethamine. Through examinations of the key interactions in the cocrystal structure of known inhibitors with the enzyme, P218 was designed as a potent inhibitor [98].