Fig. 2: In vitro individual antimalarial activity and synergistic antimalarial effects of five PI3K inhibitors with DHA against P. falciparum 3D7.

The in vitro individual antimalarial activity was determined by standard 72-h susceptibility assays by the measurement of SYBR Green I fluorescence. Synchronized 2- to 4 h post-invasion ring-stage parasites were exposed to a serial dilution of idelalisib (A, ⅰ), umbralisib (A, ⅱ), taselisib (A, iii), AZD6482 (A, ⅳ) and copanlisib (A, ⅴ). The means (s.d.) were based on four replicates. 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. The antimalarial activity of the combination of DHA and PI3K inhibitors was determined by two coculture methods: (1) The synchronized 2- to 4 h post-invasion ring-stage parasites were cocultured with DHA at the IC₅₀ and gradient concentration (from 1/4 × IC₅₀ to 4 × IC₅₀) of idelalisib (B, ⅰ), umbralisib (B, ⅱ), taselisib (B, ⅲ), AZD6482 (B, ⅳ) and copanlisib (B, ⅴ); 2) The synchronized 2- to 4 h post-invasion ring-stage parasites were cocultured with gradient concentrations of DHA (from 1/4 × IC₅₀ to 4 × IC₅₀) and with or without idelalisib (C, ⅰ), umbralisib (C, ⅱ), taselisib (C, ⅲ), AZD6482 (C, ⅳ) or copanlisib (C, ⅴ) at their IC₅₀ values for 24 h. After treatment with the two compounds, the culture medium was replaced, and the parasite cultures were further incubated for another 48 h. The parasitemia rates (survival rates) were monitored by counting infected erythrocytes in Giemsa-stained thin blood smears using light microscopy. In B, the relative survival rate of no drug exposure was defined as 100% (first column), and the values of the other columns (parasitemia) were compared with the mean value of the first column. In C, the relative survival rate of no drug exposure was defined as 100% (first column), and the value of the other column (parasitemia) were compared with the mean value of the first column. The means (s.d.) are based on three replicates (the triplicate data points are shown). One-way ANOVA and LSD-T post hoc test comparisons between treatment groups were conducted. *p < 0.05, **p < 0.01, ***p ≤ 0.001.