RESUMEN
Diverse compounds target the Plasmodium falciparum Na+ pump PfATP4, with cipargamin and (+)-SJ733 the most clinically-advanced. In a recent clinical trial for cipargamin, recrudescent parasites emerged, with most having a G358S mutation in PfATP4. Here, we show that PfATP4G358S parasites can withstand micromolar concentrations of cipargamin and (+)-SJ733, while remaining susceptible to antimalarials that do not target PfATP4. The G358S mutation in PfATP4, and the equivalent mutation in Toxoplasma gondii ATP4, decrease the sensitivity of ATP4 to inhibition by cipargamin and (+)-SJ733, thereby protecting parasites from disruption of Na+ regulation. The G358S mutation reduces the affinity of PfATP4 for Na+ and is associated with an increase in the parasite's resting cytosolic [Na+]. However, no defect in parasite growth or transmissibility is observed. Our findings suggest that PfATP4 inhibitors in clinical development should be tested against PfATP4G358S parasites, and that their combination with unrelated antimalarials may mitigate against resistance development.
Asunto(s)
Antimaláricos , Malaria Falciparum , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , ATPasas Transportadoras de Calcio , Eritrocitos/parasitología , Humanos , Indoles , Iones , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Mutación , Plasmodium falciparum , Sodio , Compuestos de EspiroRESUMEN
The antimalarial activity of chemically diverse compounds, including the clinical candidate cipargamin, has been linked to the ATPase PfATP4 in the malaria-causing parasite Plasmodium falciparum The characterization of PfATP4 has been hampered by the inability thus far to achieve its functional expression in a heterologous system. Here, we optimized a membrane ATPase assay to probe the function of PfATP4 and its chemical sensitivity. We found that cipargamin inhibited the Na+-dependent ATPase activity present in P. falciparum membranes from WT parasites and that its potency was reduced in cipargamin-resistant PfATP4-mutant parasites. The cipargamin-sensitive fraction of membrane ATPase activity was inhibited by all 28 of the compounds in the "Malaria Box" shown previously to disrupt ion regulation in P. falciparum in a cipargamin-like manner. This is consistent with PfATP4 being the direct target of these compounds. Characterization of the cipargamin-sensitive ATPase activity yielded data consistent with PfATP4 being a Na+ transporter that is sensitive to physiologically relevant perturbations of pH, but not of [K+] or [Ca2+]. With an apparent Km for ATP of 0.2 mm and an apparent Km for Na+ of 16-17 mm, the protein is predicted to operate at below its half-maximal rate under normal physiological conditions, allowing the rate of Na+ efflux to increase in response to an increase in cytosolic [Na+]. In membranes from a cipargamin-resistant PfATP4-mutant line, the apparent Km for Na+ is slightly elevated. Our study provides new insights into the biochemical properties and chemical sensitivity of an important new antimalarial drug target.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Antimaláricos/farmacología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Proteínas de Transporte de Catión/antagonistas & inhibidores , Eritrocitos/enzimología , Malaria Falciparum/enzimología , Plasmodium falciparum/enzimología , Sodio/metabolismo , Adenosina Trifosfatasas/genética , Animales , ATPasas Transportadoras de Calcio/genética , Proteínas de Transporte de Catión/genética , Eritrocitos/efectos de los fármacos , Eritrocitos/parasitología , Homeostasis , Humanos , Transporte Iónico , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/genéticaRESUMEN
Four hundred structurally diverse drug-like compounds comprising the Medicines for Malaria Venture's 'Pathogen Box' were screened for their effect on a range of physiological parameters in asexual blood-stage malaria (Plasmodium falciparum) parasites. Eleven of these compounds were found to perturb parasite Na+, pH and volume in a manner consistent with inhibition of the putative Na+ efflux P-type ATPase PfATP4. All eleven compounds fell within the subset of 125 compounds included in the Pathogen Box on the basis of their having been identified as potent inhibitors of the growth of asexual blood-stage P. falciparum parasites. All eleven compounds inhibited the Na+-dependent ATPase activity of parasite membranes and showed reduced efficacy against parasites carrying mutations in PfATP4. This study increases the number of chemically diverse structures known to show a 'PfATP4-associated' phenotype, and adds to emerging evidence that a high proportion (7-9%) of the structurally diverse antimalarial compounds identified in whole cell phenotypic screens share the same mechanism of action, exerting their antimalarial effect via an interaction with PfATP4.