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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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GNF-Pf4492-R lines show cross-resistance to the malariabox inhibitorsMMV666124 and MMV020660. Chemical structure of compounds known tohave blood-stage antimalarial activity (a) MMV666124 and (b) MMV020660.(c) Log2 fold change in IC50 for GNF-Pf4492Rlines compared to the Dd2EF1 parent. Bars represent meanlog2(IC50 fold change) from a minimum of 3 experimentsconducted in duplicate. Error bars = SEM; Significance values weredetermined using one-way ANOVA followed by Dunnett’s multiplecomparison post-test to test for a difference in mean log2(IC50) between each strain and the Dd2EF1 parent;*p < 0.05, **p < 0.01.
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fig3: GNF-Pf4492-R lines show cross-resistance to the malariabox inhibitorsMMV666124 and MMV020660. Chemical structure of compounds known tohave blood-stage antimalarial activity (a) MMV666124 and (b) MMV020660.(c) Log2 fold change in IC50 for GNF-Pf4492Rlines compared to the Dd2EF1 parent. Bars represent meanlog2(IC50 fold change) from a minimum of 3 experimentsconducted in duplicate. Error bars = SEM; Significance values weredetermined using one-way ANOVA followed by Dunnett’s multiplecomparison post-test to test for a difference in mean log2(IC50) between each strain and the Dd2EF1 parent;*p < 0.05, **p < 0.01.

Mentions: To determine if any other publicly available compoundswould haveactivity against PfATP4, we screened several inhibitors from the “malariabox,” a set of compounds previously identified in cellularscreens with activity against blood-stage P. falciparum, for cross-resistance with the GNF-Pf4492R lines.32 Two of these compounds, MMV666124 (Figure 3a) and MMV020660 (Figure 3b), showedcross-resistance with the GNF-Pf4492R lines (Figure 3c). As with the phenotype observed with the spiroindolones,GNF-Pf4492R-1 did not exhibit cross-resistance with MMV020660 butrather was more sensitive than the Dd2EF1 parent. Interestingly,the structure of MMV020660 shows some similarity to GNF-Pf4492 (Figure 3b).


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)

GNF-Pf4492-R lines show cross-resistance to the malariabox inhibitorsMMV666124 and MMV020660. Chemical structure of compounds known tohave blood-stage antimalarial activity (a) MMV666124 and (b) MMV020660.(c) Log2 fold change in IC50 for GNF-Pf4492Rlines compared to the Dd2EF1 parent. Bars represent meanlog2(IC50 fold change) from a minimum of 3 experimentsconducted in duplicate. Error bars = SEM; Significance values weredetermined using one-way ANOVA followed by Dunnett’s multiplecomparison post-test to test for a difference in mean log2(IC50) between each strain and the Dd2EF1 parent;*p < 0.05, **p < 0.01.
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Related In: Results  -  Collection

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

fig3: GNF-Pf4492-R lines show cross-resistance to the malariabox inhibitorsMMV666124 and MMV020660. Chemical structure of compounds known tohave blood-stage antimalarial activity (a) MMV666124 and (b) MMV020660.(c) Log2 fold change in IC50 for GNF-Pf4492Rlines compared to the Dd2EF1 parent. Bars represent meanlog2(IC50 fold change) from a minimum of 3 experimentsconducted in duplicate. Error bars = SEM; Significance values weredetermined using one-way ANOVA followed by Dunnett’s multiplecomparison post-test to test for a difference in mean log2(IC50) between each strain and the Dd2EF1 parent;*p < 0.05, **p < 0.01.
Mentions: To determine if any other publicly available compoundswould haveactivity against PfATP4, we screened several inhibitors from the “malariabox,” a set of compounds previously identified in cellularscreens with activity against blood-stage P. falciparum, for cross-resistance with the GNF-Pf4492R lines.32 Two of these compounds, MMV666124 (Figure 3a) and MMV020660 (Figure 3b), showedcross-resistance with the GNF-Pf4492R lines (Figure 3c). As with the phenotype observed with the spiroindolones,GNF-Pf4492R-1 did not exhibit cross-resistance with MMV020660 butrather was more sensitive than the Dd2EF1 parent. Interestingly,the structure of MMV020660 shows some similarity to GNF-Pf4492 (Figure 3b).

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