Biochemical characterization and chemical inhibition of PfATP4-associated Na+-ATPase activity in Plasmodium falciparum membranes

Abstract

The antimalarial activity of chemically diverse compounds, including the clinical candidate cipargamin, has been linked to the ATPase PfATP4 in the malaria-causing parasite 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 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 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 [Ca]. With an apparent for ATP of 0.2 mm and an apparent 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 for Na is slightly elevated. Our study provides new insights into the biochemical properties and chemical sensitivity of an important new antimalarial drug target.

<p>Recent Progress in the Development of New Antimalarial Drugs with Novel Targets</p>

“…Several compounds like MMV007839 and MMV000972, kill asexual blood-stage P. falciparum parasites via inhibition of the lactate: H+ transporter. 67…”

“…Several compounds including, cipargamin, which is in Phase 2, (+)-SJ733, which is in Phase1 and KAE609, which is in phase 2 target PfATP4 as their Mechanism of action. 67,69…”

Characterization of the ATP4 ion pump in Toxoplasma gondii

“…Furthermore, addition to isolated P. falciparum parasites of either the spiroindolone NITD246 (10) or the pyrazoleamide PA21A050 (15) (for which resistance is mediated by mutation of PfATP4 (7, 15)) resulted in an increase in [Na ϩ ] cyt and pH cyt but no change in [Ca 2ϩ ] cyt (10,15). Consistent with this, Ca 2ϩ did not stimulate the cipargamin-sensitive ATPase activity measured in P. falciparum membrane preparations (17). Our observations that cipargamin had no significant effect on [Ca 2ϩ ] cyt in T. gondii and that TgATP4 knockdown parasites were no less effective than TgATP4-expressing parasites in regulating their [Ca 2ϩ ] cyt in the face of increasing external [Ca 2ϩ ] provide further evidence against a role for ATP4 in parasite Ca 2ϩ regulation.…”

“…Second, spiroindolone-resistant parasites with mutations in PfATP4 are less sensitive to the disruption of [Na ϩ ] cyt by spiroindolones and also have a higher resting [Na ϩ ] cyt , consistent with resistanceconferring mutations in PfATP4 causing some impairment of its Na ϩ transport function (10,13). Third, spiroindolones inhibit Na ϩ -dependent ATPase activity in parasite membrane preparations (10,16,17).…”

The Functioning of Na+-ATPases from Protozoan Parasites: Are These Pumps Targets for Antiparasitic Drugs?

“…These mechanisms together promote a remarkable rapid clearance of parasites [ 48 ]. The Na + -dependent ATPase activity of membrane fractions of Plasmodium is also inhibited by cipargamin, an effect that is less pronounced in cells with a single mutation in PfATP4, demonstrating a direct effect of cipargamin on Na + -ATPase activity [ 49 ]. Spiroindolones cause swelling of malaria parasites in a Na + -dependent way, which may be due to an increase in intracellular Na + .…”