Figure 3

MB14 and its analog MB10 inhibited Na⁺ dependent ATPase activity in P. falciparum membrane preparations consistent with the compounds targeting PfATP4. (A) The effects of various compounds on P. falciparum membrane ATPase activity under high-[Na⁺] (152 mM) and low-[Na⁺] (2 mM) conditions in the presence and absence of cipargamin (50 nM). Dihydroartemisinin (DHA) was tested at a concentration of 50 nM; MB14, MB10 and RK18 were each tested at a concentration of 5 μM. The data were obtained with 3D7 parasites and the bars show the mean (+SEM) from four independent experiments, each performed on different days with different membrane preparations. The symbols show the data from the individual independent experiments. The P_i produced is shown as a percentage of that measured in the 152 mM Na⁺ Control. P_i production in the 152 mM Na⁺ Control varied from 30 to 86 nmol per mg (total) protein per min in the different experiments. For each compound, the (pre-normalised data) were tested for statistical significance compared to the Control (0.4% v/v DMSO or 50 nM cipargamin only) data for the same (high-[Na⁺] or low-[Na⁺]) condition; *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA with ‘experiment’ nominated as a ‘blocking factor’). (B) Potency of MB14 (black circles) and MB10 (red circles) against PfATP4-associated ATPase activity in membranes from 3D7 parasites. The data are the mean (shown + or − SEM) obtained from five independent experiments (with the exception of the highest and the lowest concentrations of MB10, for which data are n = 2 and n = 3, respectively). (C) Potency of MB14 against PfATP4-associated ATPase activity in membranes from B7 parasites expressing PfATP4^S374R (white squares) and matched control parasites expressing wild-type PfATP4 (3D7_2; black circles). The data are the mean (shown + or − SEM) obtained from 5–6 independent experiments (with the exception of the two highest concentrations of MB14 in B7 parasites, for which data are n = 2). In (B and C), the PfATP4-associated ATPase activity was calculated by subtracting the total ATPase activity measured in the presence of 50 nM cipargamin from that measured in the absence of cipargamin. Each experiment was performed on a different day with a different membrane preparation.