PfATP4 and the biochemical signature of PfATP4-associated compounds

Abstract

In 2016 more than 200 million cases of malaria were reported and nearly 500 000 people died from the disease. Although there are antimalarial drugs available, the most virulent species of the disease-causing parasite, Plasmodium falciparum, has evolved some level of resistance to many of them. A global effort to accelerate antimalarial drug discovery has led to the finding that one particular parasite protein, the P-type ATPase PfATP4, appears to be the target of a number of potent novel antimalarial compounds. Two of these ‘PfATP4-associated compounds’ have entered the clinical pipeline; one of these is the spiroindolone KAE609 (also known as cipargamin).

PfATP4 is located on the plasma membrane of the parasite. Although an early study yielded evidence consistent with PfATP4 being a Ca2+ transporter, more recent studies have provided evidence that PfATP4 extrudes Na+ ions from the parasite while importing H+ ions, allowing the parasite to maintain a large inward Na+ concentration gradient. When parasites are exposed to PfATP4-associated compounds, there is an increase in the Na+ concentration and the pH inside the parasite.

In the work reported in this Thesis I have investigated the nature and function of PfATP4, extended the characterisation of the effects of PfATP4-associated compounds on mature asexual-stage P. falciparum parasites, and identified a number of new compounds of this class.

A phylogenetic analysis revealed that PfATP4 belongs to a unique subgroup of Type II P-type ATPases, specific to apicomplexan parasites and their closest relatives.

Previous studies have reported that PfATP4-associated compounds cause parasite swelling. The effect of KAE609 on the volume of isolated parasites and parasitised erythrocytes was characterised using a Coulter Multisizer. KAE609 caused isolated parasites to swell in a Na+-dependent manner. KAE609 also caused intact infected erythrocytes to swell and thereby increase in osmotic fragility. Another six PfATP4-associated compounds were also shown to induce parasite swelling. Protecting parasitised erythrocytes from excessive swelling by growing them in a hyperosmotic medium increased the concentration of KAE609 that was required to kill the parasite, consistent with cell swelling playing a role in the mechanism of action of KAE609.

The ‘biochemical signature’ of PfATP4-associated compounds was used to identify novel PfATP4-associated compounds from a set of 400 diverse drug-like compounds. Experiments investigating the effects of these compounds on parasite Na+, pH and volume, as well as cross-resistance studies using KAE609-resistant parasites, provided evidence that eleven compounds act in a manner consistent with inhibition of PfATP4. These compounds add to the chemical diversity of known PfATP4-associated compounds.

PfATP4 has previously been proposed to be a Ca2+ transporter. The role of Ca2+ in the mechanism of action of PfATP4-associated compounds was explored using a physiological approach. The results support the prevailing view that PfATP4-associated compounds disrupt Na+ homeostasis directly, and are inconsistent with a role for Ca2+ in this phenomenon.

This study furthers our understanding of the effects of PfATP4-associated compounds on parasite physiology and, in extending our knowledge of the characteristic biochemical signature of these compounds, provides a basis for identifying further such compounds. Show more