Fig. 3: Idelalisib effectively inhibited PfPI3K activity, resulting in a decrease in PI3P levels increased sensitivity to ATRs and reduced ARTs resistance.

The idelalisib at the IC₅₀ value decreased the DHA IC₅₀ value in 3D7-PfKelch13^WT (A) and 3D7-PfKelch13^C580Y (B). The IC₅₀ values were determined by standard 72-h susceptibility assays via the measurement of SYBR Green I fluorescence. The IC₅₀ values were calculated by GraphPad Prism 9.0 software (Analyse-Nonlinear regression curve fit-[Inhibitor] vs. normalized response—variable slope). The definitions of the maximum and minimum response values were 100 and 0%, respectively for the nonlinear regression curve fit. For PfPI3K activity (C) and PI3P level detection (D), tightly synchronized ring-stage parasite cultures were exposed to 0.1% DMSO (control), DHA at the IC₅₀, idelalisib at the IC₅₀ or the combination of DHA and idelalisib (each at the corresponding IC₅₀) for 24 h until the parasites reached the late-trophozoite stage. Briefly, for PfPI3K activity (C), the kinase assay of PfPI3K bound to the immunoprecipitated beads was measured using a Class III PI3-kinase kit from Echelon Biosciences (K-3000) following the manufacturer’s instructions. For PI3P level detection (E), tightly synchronized ring-stage parasite cultures were exposed to 0.1% DMSO (control) or different concentrations of idelalisib for 24 h until the parasites reached the late-trophozoite stage. Briefly for PI3P level detection (D, E), lipids were extracted from the parasite pellet using methanol, chloroform and hydrochloric acid. The PI3P level was assessed (in triplicate) using an Echelon PI3P mass ELISA Kit (K-3300) following the manufacturer’s instructions. In C–E, one-way ANOVA and LSD-T post hoc test comparisons between treatment groups were employed. The means (s.d.) are based on three replicates (the triplicate data points are shown). In F, tightly synchronized early ring parasites (0–3 h post-invasion, 2% hematocrit and 1% parasitemia) were exposed to 700 nM DHA combined with or without 5 μM idelalisib (0.1% DMSO as a control) for 6 h, washed thrice with Albumax-free RPMI and returned to culture for 66 h. Parasite survival rates (%RSAs) were determined by comparing the number of viable parasites between the drug-treated and untreated controls and are expressed as percentages. Two-tailed unpaired Student’s t-test was employed. The means (s.d.) are based on three replicates (the triplicate data points are shown). *p < 0.05, **p < 0.01, ***p ≤ 0.001.