Fig. 1. Emergence of resistance to DSM267 and DSM265 in P. falciparum in vitro.

(A) Structure of the DHODH inhibitor DSM265 used in in vitro resistance selection experiments [selection 1 (S1)]. (B) Representative dose-response curves for bulk-selected parasite populations. Parasite populations in three independent culture flasks containing human blood were exposed to DSM265 at the EC₉₉ and allowed to recrudesce. Cultures recovered after selection were tested in a dose-response assay to determine the resistance phenotype (flask 1, yellow; flask 2, blue; Flask 3, red). (C) Shown is the dose-response phenotype of clones isolated from in vitro resistance selection experiments using pulsed treatments with DSM265. Bulk resistant populations were cloned in the absence of compound pressure. Clonal parasite lines from flasks 1 and 3 exhibited heterogeneous resistance phenotypes. Each clonal line is labeled on the x axis with a unique identifier that includes the selection number (S), flask number (F), and clone number (C), for example, S1-F1-C1. Individual replicate EC₅₀ values are shown as a scatter plot, with error bars depicting mean and SD. Statistically significant differences in specific clonal phenotypes relative to the wild-type (WT) 3D7 A10 parental parasite line are indicated on the graph. Significance was calculated using a nonparametric one-way ANOVA (Kruskal-Wallis) with post hoc multiple comparisons (Dunn’s test): **P < 0.01, ****P < 0.0001; ns, not significant. (D to F) Increasing continuous drug selection pressure yielded parasites with highly drug-resistant phenotypes. (D) Structure of the DHODH inhibitor DSM267 used in in vitro drug resistance selection experiments (selection 2). (E) Parasites were exposed to two steps of selective pressure (steps 1 and 2). In step 1, parasites were exposed to a single pulse of 25 nM DSM267, leading to ~10-fold resistance in two of three flasks (flask 1, yellow; flask 2, blue; flask 3, green). In step 2, the drug-resistant populations from flasks 2 and 3 were continuously exposed to DSM267 at an increased dose (50 nM) for 7 to 8 weeks. Shown are representative dose-response curves for bulk-selected populations from step 1. (F) Shown are dose-response phenotypes of clones isolated from step 1 and step 2 of selection 2 (S2). Each clonal line is labeled on the x axis with a unique identifier that includes the selection number (S), flask number (F), and clone number (C), for example, S2-F2-C1. Individual replicate EC₅₀ values are shown as a scatter plot, with error bars depicting mean and SD. Statistically significant differences in specific clonal phenotypes relative to the wild-type 3D7 A10 parental line are indicated on the graph. Significance was calculated using a nonparametric one-way ANOVA (Kruskal-Wallis) with post hoc multiple comparisons (Dunn’s test): *P < 0.05, **P < 0.01; ns, not significant.