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Pyrazoleamide compounds are potent antimalarials that target Na+ homeostasis in intraerythrocytic Plasmodium falciparum.

Vaidya AB, Morrisey JM, Zhang Z, Das S, Daly TM, Otto TD, Spillman NJ, Wyvratt M, Siegl P, Marfurt J, Wirjanata G, Sebayang BF, Price RN, Chatterjee A, Nagle A, Stasiak M, Charman SA, Angulo-Barturen I, Ferrer S, Belén Jiménez-Díaz M, Martínez MS, Gamo FJ, Avery VM, Ruecker A, Delves M, Kirk K, Berriman M, Kortagere S, Burrows J, Fan E, Bergman LW - Nat Commun (2014)

Bottom Line: Investigations involving pyrazoleamide-resistant parasites, whole-genome sequencing and gene transfers reveal that mutations in two proteins, a calcium-dependent protein kinase (PfCDPK5) and a P-type cation-ATPase (PfATP4), are necessary to impart full resistance to these compounds.A pyrazoleamide compound causes a rapid disruption of Na(+) regulation in blood-stage Plasmodium falciparum parasites.Similar effect on Na(+) homeostasis was recently reported for spiroindolones, which are antimalarials of a chemical class quite distinct from pyrazoleamides.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Center for Molecular Parasitology, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, Pennsylvania 190129, USA.

ABSTRACT
The quest for new antimalarial drugs, especially those with novel modes of action, is essential in the face of emerging drug-resistant parasites. Here we describe a new chemical class of molecules, pyrazoleamides, with potent activity against human malaria parasites and showing remarkably rapid parasite clearance in an in vivo model. Investigations involving pyrazoleamide-resistant parasites, whole-genome sequencing and gene transfers reveal that mutations in two proteins, a calcium-dependent protein kinase (PfCDPK5) and a P-type cation-ATPase (PfATP4), are necessary to impart full resistance to these compounds. A pyrazoleamide compound causes a rapid disruption of Na(+) regulation in blood-stage Plasmodium falciparum parasites. Similar effect on Na(+) homeostasis was recently reported for spiroindolones, which are antimalarials of a chemical class quite distinct from pyrazoleamides. Our results reveal that disruption of Na(+) homeostasis in malaria parasites is a promising mode of antimalarial action mediated by at least two distinct chemical classes.

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Rapid influx of Na+ in P. falciparum trophozoites following exposure to PA21A050.Intracellular concentrations of Na+, Ca2+ and pH in saponin-isolated parasites loaded with appropriate fluorescent probes were determined by ratiometric methods as described in ref. 10. In a,c,d, the pyrazoleamide was added at the time point indicated by the closed triangle. The traces and influx rates shown are, in each case, representative of those obtained from at least three independent cell preparations. In control experiments it was shown that there was no direct effect of the pyrazoleamide on the fluorescence signals of any of the three fluorescent ion indicators used (not shown). (a) Concentration-dependent increase in [Na+]i in response to PA21A050. (b) Initial Na+ influx rate (±s.e.m.; calculated as described in ref. 10) plotted as function of [21A050]. The estimated IC50 for this effect was 0.08 nM. (c) PA21A050 at 1 nM caused a rapid rise in pHi from 7.3 to 7.45, consistent with a lifting of the ‘acid-load’ proposed to be imposed by the action of the (H+ counter-transporting) Na+ ATPase1033. (d) PA21A050, at 1 nM, had no significant effect on [Ca2+]i in parasites suspended in an EGTA-buffered medium containing 1 μM free Ca2+. Addition of the endoplasmic reticulum Ca2+ pump inhibitor cyclopiazonic acid (unfilled triangle) resulted in a transient spike in [Ca2+]i.
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f4: Rapid influx of Na+ in P. falciparum trophozoites following exposure to PA21A050.Intracellular concentrations of Na+, Ca2+ and pH in saponin-isolated parasites loaded with appropriate fluorescent probes were determined by ratiometric methods as described in ref. 10. In a,c,d, the pyrazoleamide was added at the time point indicated by the closed triangle. The traces and influx rates shown are, in each case, representative of those obtained from at least three independent cell preparations. In control experiments it was shown that there was no direct effect of the pyrazoleamide on the fluorescence signals of any of the three fluorescent ion indicators used (not shown). (a) Concentration-dependent increase in [Na+]i in response to PA21A050. (b) Initial Na+ influx rate (±s.e.m.; calculated as described in ref. 10) plotted as function of [21A050]. The estimated IC50 for this effect was 0.08 nM. (c) PA21A050 at 1 nM caused a rapid rise in pHi from 7.3 to 7.45, consistent with a lifting of the ‘acid-load’ proposed to be imposed by the action of the (H+ counter-transporting) Na+ ATPase1033. (d) PA21A050, at 1 nM, had no significant effect on [Ca2+]i in parasites suspended in an EGTA-buffered medium containing 1 μM free Ca2+. Addition of the endoplasmic reticulum Ca2+ pump inhibitor cyclopiazonic acid (unfilled triangle) resulted in a transient spike in [Ca2+]i.

Mentions: As highlighted in a recent study10, PfATP4 bears a close resemblance to the closely related ENA (exitus natrum) Na+ ATPases that extrude Na+ ions from the cells of fungi and lower plants23. In the same study it was shown that antimalarial spiroindolones at therapeutic doses rapidly disrupt Na+ homeostasis in blood-stage P. falciparum parasites, and that spiroindolone-resistant parasites with mutations in PfATP4 (ref. 9) had reduced sensitivity to disruption of Na+ homeostasis by a spiroindolone10. As shown in Fig. 4a, the pyrazoleamide PA21A050 treatment of saponin-isolated parasites led to rapid increase in intracellular Na+ concentration ([Na+]i), doing so in a dose-dependent manner. Remarkably, a maximum initial Na+ influx rate of ~0.07 mM s−1 was reached with the addition of just 1 nM PA21A050 (Fig. 4b). The increase in [Na]i was accompanied by an increase in intracellular pH (pHi, Fig. 4c), whereas there was no significant change in intracellular Ca2+ ([Ca2+]i) on addition of PA21A050 (Fig. 4d). These results are similar to those reported for the spiroindolones10 except that PA21A050 was more potent in its activity in this assay. Antimalarials such as artesunate and chloroquine have no effect on [Na+]i in this assay10.


Pyrazoleamide compounds are potent antimalarials that target Na+ homeostasis in intraerythrocytic Plasmodium falciparum.

Vaidya AB, Morrisey JM, Zhang Z, Das S, Daly TM, Otto TD, Spillman NJ, Wyvratt M, Siegl P, Marfurt J, Wirjanata G, Sebayang BF, Price RN, Chatterjee A, Nagle A, Stasiak M, Charman SA, Angulo-Barturen I, Ferrer S, Belén Jiménez-Díaz M, Martínez MS, Gamo FJ, Avery VM, Ruecker A, Delves M, Kirk K, Berriman M, Kortagere S, Burrows J, Fan E, Bergman LW - Nat Commun (2014)

Rapid influx of Na+ in P. falciparum trophozoites following exposure to PA21A050.Intracellular concentrations of Na+, Ca2+ and pH in saponin-isolated parasites loaded with appropriate fluorescent probes were determined by ratiometric methods as described in ref. 10. In a,c,d, the pyrazoleamide was added at the time point indicated by the closed triangle. The traces and influx rates shown are, in each case, representative of those obtained from at least three independent cell preparations. In control experiments it was shown that there was no direct effect of the pyrazoleamide on the fluorescence signals of any of the three fluorescent ion indicators used (not shown). (a) Concentration-dependent increase in [Na+]i in response to PA21A050. (b) Initial Na+ influx rate (±s.e.m.; calculated as described in ref. 10) plotted as function of [21A050]. The estimated IC50 for this effect was 0.08 nM. (c) PA21A050 at 1 nM caused a rapid rise in pHi from 7.3 to 7.45, consistent with a lifting of the ‘acid-load’ proposed to be imposed by the action of the (H+ counter-transporting) Na+ ATPase1033. (d) PA21A050, at 1 nM, had no significant effect on [Ca2+]i in parasites suspended in an EGTA-buffered medium containing 1 μM free Ca2+. Addition of the endoplasmic reticulum Ca2+ pump inhibitor cyclopiazonic acid (unfilled triangle) resulted in a transient spike in [Ca2+]i.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263321&req=5

f4: Rapid influx of Na+ in P. falciparum trophozoites following exposure to PA21A050.Intracellular concentrations of Na+, Ca2+ and pH in saponin-isolated parasites loaded with appropriate fluorescent probes were determined by ratiometric methods as described in ref. 10. In a,c,d, the pyrazoleamide was added at the time point indicated by the closed triangle. The traces and influx rates shown are, in each case, representative of those obtained from at least three independent cell preparations. In control experiments it was shown that there was no direct effect of the pyrazoleamide on the fluorescence signals of any of the three fluorescent ion indicators used (not shown). (a) Concentration-dependent increase in [Na+]i in response to PA21A050. (b) Initial Na+ influx rate (±s.e.m.; calculated as described in ref. 10) plotted as function of [21A050]. The estimated IC50 for this effect was 0.08 nM. (c) PA21A050 at 1 nM caused a rapid rise in pHi from 7.3 to 7.45, consistent with a lifting of the ‘acid-load’ proposed to be imposed by the action of the (H+ counter-transporting) Na+ ATPase1033. (d) PA21A050, at 1 nM, had no significant effect on [Ca2+]i in parasites suspended in an EGTA-buffered medium containing 1 μM free Ca2+. Addition of the endoplasmic reticulum Ca2+ pump inhibitor cyclopiazonic acid (unfilled triangle) resulted in a transient spike in [Ca2+]i.
Mentions: As highlighted in a recent study10, PfATP4 bears a close resemblance to the closely related ENA (exitus natrum) Na+ ATPases that extrude Na+ ions from the cells of fungi and lower plants23. In the same study it was shown that antimalarial spiroindolones at therapeutic doses rapidly disrupt Na+ homeostasis in blood-stage P. falciparum parasites, and that spiroindolone-resistant parasites with mutations in PfATP4 (ref. 9) had reduced sensitivity to disruption of Na+ homeostasis by a spiroindolone10. As shown in Fig. 4a, the pyrazoleamide PA21A050 treatment of saponin-isolated parasites led to rapid increase in intracellular Na+ concentration ([Na+]i), doing so in a dose-dependent manner. Remarkably, a maximum initial Na+ influx rate of ~0.07 mM s−1 was reached with the addition of just 1 nM PA21A050 (Fig. 4b). The increase in [Na]i was accompanied by an increase in intracellular pH (pHi, Fig. 4c), whereas there was no significant change in intracellular Ca2+ ([Ca2+]i) on addition of PA21A050 (Fig. 4d). These results are similar to those reported for the spiroindolones10 except that PA21A050 was more potent in its activity in this assay. Antimalarials such as artesunate and chloroquine have no effect on [Na+]i in this assay10.

Bottom Line: Investigations involving pyrazoleamide-resistant parasites, whole-genome sequencing and gene transfers reveal that mutations in two proteins, a calcium-dependent protein kinase (PfCDPK5) and a P-type cation-ATPase (PfATP4), are necessary to impart full resistance to these compounds.A pyrazoleamide compound causes a rapid disruption of Na(+) regulation in blood-stage Plasmodium falciparum parasites.Similar effect on Na(+) homeostasis was recently reported for spiroindolones, which are antimalarials of a chemical class quite distinct from pyrazoleamides.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Center for Molecular Parasitology, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, Pennsylvania 190129, USA.

ABSTRACT
The quest for new antimalarial drugs, especially those with novel modes of action, is essential in the face of emerging drug-resistant parasites. Here we describe a new chemical class of molecules, pyrazoleamides, with potent activity against human malaria parasites and showing remarkably rapid parasite clearance in an in vivo model. Investigations involving pyrazoleamide-resistant parasites, whole-genome sequencing and gene transfers reveal that mutations in two proteins, a calcium-dependent protein kinase (PfCDPK5) and a P-type cation-ATPase (PfATP4), are necessary to impart full resistance to these compounds. A pyrazoleamide compound causes a rapid disruption of Na(+) regulation in blood-stage Plasmodium falciparum parasites. Similar effect on Na(+) homeostasis was recently reported for spiroindolones, which are antimalarials of a chemical class quite distinct from pyrazoleamides. Our results reveal that disruption of Na(+) homeostasis in malaria parasites is a promising mode of antimalarial action mediated by at least two distinct chemical classes.

Show MeSH
Related in: MedlinePlus