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Mutations in the P-type cation-transporter ATPase 4, PfATP4, mediate resistance to both aminopyrazole and spiroindolone antimalarials.

Flannery EL, McNamara CW, Kim SW, Kato TS, Li F, Teng CH, Gagaring K, Manary MJ, Barboa R, Meister S, Kuhen K, Vinetz JM, Chatterjee AK, Winzeler EA - ACS Chem. Biol. (2014)

Bottom Line: To investigate their unknown mechanism of action and thus identify their target, we cultured parasites in the presence of a representative member of the aminopyrazole series, GNF-Pf4492, to select for resistance.Whole genome sequencing of three resistant lines showed that each had acquired independent mutations in a P-type cation-transporter ATPase, PfATP4 (PF3D7_1211900), a protein implicated as the novel Plasmodium spp. target of another, structurally unrelated, class of antimalarials called the spiroindolones and characterized as an important sodium transporter of the cell.Our data demonstrate that PfATP4 plays a critical role in cellular processes, can be inhibited by two distinct antimalarial pharmacophores, and supports the recent observations that PfATP4 is a critical antimalarial target.

View Article: PubMed Central - PubMed

Affiliation: Division of Pharmacology and Drug Discovery, Department of Pediatrics, ‡Division of Infectious Diseases, Department of Medicine, University of California San Diego School of Medicine , La Jolla, California 92093, United States.

ABSTRACT
Aminopyrazoles are a new class of antimalarial compounds identified in a cellular antiparasitic screen with potent activity against Plasmodium falciparum asexual and sexual stage parasites. To investigate their unknown mechanism of action and thus identify their target, we cultured parasites in the presence of a representative member of the aminopyrazole series, GNF-Pf4492, to select for resistance. Whole genome sequencing of three resistant lines showed that each had acquired independent mutations in a P-type cation-transporter ATPase, PfATP4 (PF3D7_1211900), a protein implicated as the novel Plasmodium spp. target of another, structurally unrelated, class of antimalarials called the spiroindolones and characterized as an important sodium transporter of the cell. Similarly to the spiroindolones, GNF-Pf4492 blocks parasite transmission to mosquitoes and disrupts intracellular sodium homeostasis. Our data demonstrate that PfATP4 plays a critical role in cellular processes, can be inhibited by two distinct antimalarial pharmacophores, and supports the recent observations that PfATP4 is a critical antimalarial target.

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Phenotypic characterizations of parasitestreated with aminopyrazolesor spiroindolones. (a) The lifecycle stage of action of GNF-Pf4492and KAF246 were determined by addition of either 10× IC50 of GNF-Pf4492 or KAF246 and observation over a 60 h period. (b,c) Standard membrane feeding with P. falciparum todetermine inhibitor action on transmission. Ten to forty mosquitoesper feeding were dissected. (b) Geometric mean number of oocysts countedper feeding. Bars represent median. Squares, GNF-Pf4492 treated; triangles,KAF246 treated. (c) The percent of mosquitoes that had one or moreoocysts per the total number of mosquitoes dissected.
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fig4: Phenotypic characterizations of parasitestreated with aminopyrazolesor spiroindolones. (a) The lifecycle stage of action of GNF-Pf4492and KAF246 were determined by addition of either 10× IC50 of GNF-Pf4492 or KAF246 and observation over a 60 h period. (b,c) Standard membrane feeding with P. falciparum todetermine inhibitor action on transmission. Ten to forty mosquitoesper feeding were dissected. (b) Geometric mean number of oocysts countedper feeding. Bars represent median. Squares, GNF-Pf4492 treated; triangles,KAF246 treated. (c) The percent of mosquitoes that had one or moreoocysts per the total number of mosquitoes dissected.

Mentions: Giventhat mutated PfATP4 mediates aminopyrazoleresistance, we hypothesized that the aminopyrazoles and spiroindolonesshare a mechanism of action. Therefore, we sought to determine whetherparasites treated with inhibitors from either class produced the samephenotype. Synchronized cultures were treated with 10× IC50 GNF-Pf4492 or KAF246 to observe when the compounds act duringthe parasite life cycle. Neither culture treated with inhibitor advancedpast the early trophozoite stage, arresting in the ring or early trophozoitestages (Figure 4a). Furthermore, similar tothe spiroindolones, GNF-Pf4492 diminished protein synthesis activityin the trophozoite stage of the Dd2 parent at 10× and 100×IC50 concentrations as measured by incorporation of radiolabeledmethionine and cysteine (SI Figure S5a).Conversely, protein synthesis progressed unabated in the KAE609R-1clone after addition of either 100× KAF246 or GNF-Pf4492 (SI Figure S5b).


Mutations in the P-type cation-transporter ATPase 4, PfATP4, mediate resistance to both aminopyrazole and spiroindolone antimalarials.

Flannery EL, McNamara CW, Kim SW, Kato TS, Li F, Teng CH, Gagaring K, Manary MJ, Barboa R, Meister S, Kuhen K, Vinetz JM, Chatterjee AK, Winzeler EA - ACS Chem. Biol. (2014)

Phenotypic characterizations of parasitestreated with aminopyrazolesor spiroindolones. (a) The lifecycle stage of action of GNF-Pf4492and KAF246 were determined by addition of either 10× IC50 of GNF-Pf4492 or KAF246 and observation over a 60 h period. (b,c) Standard membrane feeding with P. falciparum todetermine inhibitor action on transmission. Ten to forty mosquitoesper feeding were dissected. (b) Geometric mean number of oocysts countedper feeding. Bars represent median. Squares, GNF-Pf4492 treated; triangles,KAF246 treated. (c) The percent of mosquitoes that had one or moreoocysts per the total number of mosquitoes dissected.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4340351&req=5

fig4: Phenotypic characterizations of parasitestreated with aminopyrazolesor spiroindolones. (a) The lifecycle stage of action of GNF-Pf4492and KAF246 were determined by addition of either 10× IC50 of GNF-Pf4492 or KAF246 and observation over a 60 h period. (b,c) Standard membrane feeding with P. falciparum todetermine inhibitor action on transmission. Ten to forty mosquitoesper feeding were dissected. (b) Geometric mean number of oocysts countedper feeding. Bars represent median. Squares, GNF-Pf4492 treated; triangles,KAF246 treated. (c) The percent of mosquitoes that had one or moreoocysts per the total number of mosquitoes dissected.
Mentions: Giventhat mutated PfATP4 mediates aminopyrazoleresistance, we hypothesized that the aminopyrazoles and spiroindolonesshare a mechanism of action. Therefore, we sought to determine whetherparasites treated with inhibitors from either class produced the samephenotype. Synchronized cultures were treated with 10× IC50 GNF-Pf4492 or KAF246 to observe when the compounds act duringthe parasite life cycle. Neither culture treated with inhibitor advancedpast the early trophozoite stage, arresting in the ring or early trophozoitestages (Figure 4a). Furthermore, similar tothe spiroindolones, GNF-Pf4492 diminished protein synthesis activityin the trophozoite stage of the Dd2 parent at 10× and 100×IC50 concentrations as measured by incorporation of radiolabeledmethionine and cysteine (SI Figure S5a).Conversely, protein synthesis progressed unabated in the KAE609R-1clone after addition of either 100× KAF246 or GNF-Pf4492 (SI Figure S5b).

Bottom Line: To investigate their unknown mechanism of action and thus identify their target, we cultured parasites in the presence of a representative member of the aminopyrazole series, GNF-Pf4492, to select for resistance.Whole genome sequencing of three resistant lines showed that each had acquired independent mutations in a P-type cation-transporter ATPase, PfATP4 (PF3D7_1211900), a protein implicated as the novel Plasmodium spp. target of another, structurally unrelated, class of antimalarials called the spiroindolones and characterized as an important sodium transporter of the cell.Our data demonstrate that PfATP4 plays a critical role in cellular processes, can be inhibited by two distinct antimalarial pharmacophores, and supports the recent observations that PfATP4 is a critical antimalarial target.

View Article: PubMed Central - PubMed

Affiliation: Division of Pharmacology and Drug Discovery, Department of Pediatrics, ‡Division of Infectious Diseases, Department of Medicine, University of California San Diego School of Medicine , La Jolla, California 92093, United States.

ABSTRACT
Aminopyrazoles are a new class of antimalarial compounds identified in a cellular antiparasitic screen with potent activity against Plasmodium falciparum asexual and sexual stage parasites. To investigate their unknown mechanism of action and thus identify their target, we cultured parasites in the presence of a representative member of the aminopyrazole series, GNF-Pf4492, to select for resistance. Whole genome sequencing of three resistant lines showed that each had acquired independent mutations in a P-type cation-transporter ATPase, PfATP4 (PF3D7_1211900), a protein implicated as the novel Plasmodium spp. target of another, structurally unrelated, class of antimalarials called the spiroindolones and characterized as an important sodium transporter of the cell. Similarly to the spiroindolones, GNF-Pf4492 blocks parasite transmission to mosquitoes and disrupts intracellular sodium homeostasis. Our data demonstrate that PfATP4 plays a critical role in cellular processes, can be inhibited by two distinct antimalarial pharmacophores, and supports the recent observations that PfATP4 is a critical antimalarial target.

Show MeSH
Related in: MedlinePlus