Antimalarial screening via large‐scale purification of Plasmodium falciparum Ca²⁺‐ATPase 6 and in vitro studies

Abstract

The most severe form of human malaria is caused by the parasite Plasmodium falciparum. Despite the current need, there is no effective vaccine and parasites are becoming resistant to most of the antimalarials available. Therefore, there is an urgent need to discover new drugs from targets that have not yet suffered from drug pressure with the aim of overcoming the problem of new emerging resistance. Membrane transporters, such as P. falciparum Ca2+‐ATPase 6 (PfATP6), the P. falciparum sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase (SERCA), have been proposed as potentially good antimalarial targets. The present investigation focuses on: (a) the large‐scale purification of PfATP6 for maintenance of its enzymatic activity; (b) screening for PfATP6 inhibitors from a compound library; and (c) the selection of the best inhibitors for further tests on P. falciparum growth in vitro. We managed to heterologously express in yeast and purify an active form of PfATP6 as previously described, although in larger amounts. In addition to some classical SERCA inhibitors, a chemical library of 1680 molecules was screened. From these, we selected a pool of the 20 most potent inhibitors of PfATP6, presenting half maximal inhibitory concentration values in the range 1–9 μm. From these, eight were chosen for evaluation of their effect on P. falciparum growth in vitro, and the best compound presented a half maximal inhibitory concentration of ~ 2 μm. We verified the absence of an inhibitory effect of most of the compounds on mammalian SERCA1a, representing a potential advantage in terms of human toxicity. The present study describes a multidisciplinary approach allowing the selection of promising PfATP6‐specific inhibitors with good antimalarial activity.

Overexpression of Membrane Proteins in Saccharomyces cerevisiae for Structural and Functional Studies: A Focus on the Rabbit Ca2+-ATPase Serca1a and on the Yeast Lipid “Flippase” Complex Drs2p/Cdc50p

“…After overexpression, the purification yield of PfATP6 is similar to that of the rabbit Serca1a, and although the two proteins share a rather high degree of sequence similarity they exhibit significant differences with respect to their sensitivity to known SERCA inhibitors, e.g., thapsigargin and cyclopiazonic acid (Cardi et al 2010b;Arnou et al 2011). At variance with studies from others, our work demonstrated that PfATP6 is probably not the target of the antimalarial drug artemisinin; recently, our purified recombinant PfATP6 was used for screening from a compound library inhibitors that might potentially be used as new antimalarial drugs (David-Bosne et al 2013).…”

SC83288 is a clinical development candidate for the treatment of severe malaria

“…In comparison, cyclopiazonic acid (CPA; 10 μM), an established inhibitor of PfATP6 (ref. 37 ), induced a strong Ca 2+ responses in the parasite's cytoplasm ( Supplementary Fig. 12 ).…”

Identification of Antifungal H + -ATPase Inhibitors with Effect on Plasma Membrane Potential

“…Given that ebselen has previously been reported to inhibit PfATP6 and the H + ,K + -ATPase (13, 14), this compound is likely an unspecific P-type ATPase inhibitor.…”

“…Ebselen is known to inhibit the mammalian H + ,K + -ATPase (13) and the Ca 2+ -ATPase ATP6 of Plasmodium falciparum (PfATP6), in addition to Pma1 (14). Due to the reactivity of ebselen with protein thiols, it is believed to target several enzymes and modify a range of biological activities (15).…”