Fig 4. Chemical interrogation of Toxoplasma’s de novo heme production by oxadiazon and its derivatives reduced the intracellular growth of the parasites.

a, Chemical structures of oxadiazon and two derivatives. b, Efficacy determination of oxadiazon and its derivatives in the inhibition of WT Toxoplasma growth using a luciferase-based growth assay. The Δppo::NLuc, ΔppoPPO::NLuc, and WT::NLuc-pTub-TgPPO strains were included for evaluating the target specificity of the inhibitors to TgPPO. Pyrimethamine, an antibiotic targeting folic acid metabolism that is irrelevant to heme biosynthesis, was also included for assessing the specificity of these PPO inhibitors. Data shown in the table represent mean ± SEM of n = 3 biological replicates with 3 technical replicates each. The standard errors for individual IC₅₀s listed in the table were calculated from the IC₅₀s derived from 3 independent biological replicates for each inhibitor. The IC₅₀ values were obtained by curve fitting using the function of “normalized response” vs. “[inhibitor]”, under the “dose-response-inhibition” regression program embedded in GraphPad Prism software (8^th version). c, Heme levels were reduced in the parasites upon treatment with oxadiazon and its derivatives. Data shown here represent mean ± SEM of n = 5 biological replicates. d, Toxoplasma was incapable of taking up extracellular heme to support its intracellular growth. Data represent mean ± SD of n = 3 biological replicates. Statistical significance for the assays described in this figure was determined by two-tailed unpaired Student’s t-test. *, p<0.05; **, p<0.01; ***, p<0.001; n.s., not significant.