title: 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 Ca(2+)-ATPase 6 (PfATP6), the P. falciparum sarcoplasmic/endoplasmic reticulum Ca(2+)-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.
authors.affiliation: UMR 8221 CNRS, Université Paris-Sud and CEA, Gif-sur-Yvette, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: David-Bosne
authors.forename: Isabelle
authors.initials: I
authors.lastname: Florent
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Lund-Winther
authors.forename: John B
authors.initials: JB
authors.lastname: Hansen
authors.forename: Morten
authors.initials: M
authors.lastname: Buch-Pedersen
authors.forename: Paul
authors.initials: P
authors.lastname: Machillot
authors.forename: Marc
authors.initials: M
authors.lastname: le Maire
authors.forename: Christine
authors.initials: C
authors.lastname: Jaxel
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D015153:Blotting, Western; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D016778:Malaria, Falciparum / Q000188:drug therapy* / Q000201:enzymology; D010963:Plasmodium falciparum / Q000187:drug effects*; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D054852:Small Molecule Libraries
chemical_list: C513500:ATP6 protein, Plasmodium falciparum; D000962:Antimalarials; D054852:Small Molecule Libraries; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases
keywords: Ca2+-ATPase activity; PfATP6; Saccharomyces cerevisiae; antiplasmodial activity; recombinant protein