A focused small-molecule screen identifies 14 compounds with distinct effects on Toxoplasma gondii

Abstract

Toxoplasma gondii is a globally ubiquitous pathogen that can cause severe disease in immunocompromised humans and the developing fetus. Given the proven role of Toxoplasma-secreted kinases in the interaction of Toxoplasma with its host cell, identification of novel kinase inhibitors could precipitate the development of new anti-Toxoplasma drugs and define new pathways important for parasite survival. We selected a small (n = 527) but diverse set of putative kinase inhibitors and screened them for effects on the growth of Toxoplasma in vitro. We identified and validated 14 noncytotoxic compounds, all of which had 50% effective concentrations in the nanomolar to micromolar range. We further characterized eight of these compounds, four inhibitors and four enhancers, by determining their effects on parasite motility, invasion, and the likely cellular target (parasite or host cell). Only two compounds had an effect on parasite motility and invasion. All the inhibitors appeared to target the parasite, and interestingly, two of the enhancers appeared to rather target the host cell, suggesting modulation of host cell pathways beneficial for parasite growth. For the four inhibitors, we also tested their efficacy in a mouse model, where one compound proved potent. Overall, these 14 compounds represent a new and diverse set of small molecules that are likely targeting distinct parasite and host cell pathways. Future work will aim to characterize their molecular targets in both the host and parasite.

Fig 1

Percent luciferase signal compared to that for the vehicle-treated control for 527 compounds tested in the initial growth screen, sorted from the most effective inhibitors to the most effective enhancers. Small molecules that were not obviously toxic to host cells and that either inhibited parasite growth by >90% or increased parasite growth by >200% were categorized as inhibitors and enhancers, respectively, and were selected for further assays. Thirty compounds satisfied these criteria.

Fig 2

Parasite growth against compound treatment. Thirty compounds from the primary screen were reassayed at 10 μM to identify false positives. Fourteen (identified with asterisks) out of the 30 small molecules showed levels of inhibition or enhancement reproducible from the initial screen. These compounds were named C1 to C14 (see Table 1, Table S1 in the supplemental material, and Fig. 3 for structures).

Fig 3

Structures of the 14 small molecules identified in this study. Inhibitors are compounds C1 to C7, and enhancers are compounds C8 to C14.

Fig 4

(A) Effects of compounds on parasite motility. C1 and C5 completely blocked parasite motility, while C3 and C12 slightly enhanced it. V, vehicle control. (B) Effects of four inhibitors and four enhancers on parasite invasion. Cytochalasin D (Cyt-D) was used as a positive control for invasion inhibition. Effects on invasion were assessed by comparing the mean number of invading or uninvading parasites per field of view to that for the vehicle control. Cytochalasin D and C1 significantly decreased the number of invading parasites, while C1 also decreased the number of uninvading (i.e., attached) parasites, although this difference was not significant (P = 0.08). (C) Effect of pretreatment of extracellular parasites for 16 h on parasite growth after washing out the compound. Compounds C1, C2, C3, and C5 significantly inhibited parasite growth after pretreatment, while C8 significantly enhanced parasite growth. On the basis of the invasion assay results, it is likely that very few, if any, parasites attached to the monolayer after C1 treatment. (D) Effects of host cell pretreatment on parasite growth. Host cells were pretreated with the compounds or vehicle for 24 h and extensively washed prior to parasite invasion and 3 days of growth. Assays where the compound was present for the entire 3-day incubation period were also run in parallel. C8 and C11 resulted in significantly enhanced parasite growth after pretreatment that was comparable to that observed when the compound was present for the entire 3-day growth period. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared to the vehicle-treated control for all comparisons).

Fig 5

The ability of parasites to recover from treatment with C1, C2 C3, and C5 was assessed by exposing intracellular parasites for 12 h, followed by compound removal for an additional 12 h. Parasites were harvested both after the 12-h exposure and after the 12-h recovery period. Data are expressed as the fold increase in luminescent signal during the 12-h recovery period. C3-treated parasites fully recovered from the treatment, while C1-treated parasites did not. C2- and C5-treated parasites were found to only moderately recover. ***, P < 0.0001 compared to control.

Fig 6

Parasite ultrastructure after exposure to four inhibitory compounds. (A to C) Parasites were treated with either vehicle (A), C3 (B), or C5 (C) for 40 h. (B) C3-treated parasites have a phenotype most similar to the phenotype of parasites treated with the vehicle control, with the exception of growth arrest over the 40-h incubation period. (C) C5-treated parasites have empty (*) vacuoles compared to parasites treated with vehicle. (D to F) In contrast to treatment with C1 and C3, treatment with C2 (E) and C1 (F) showed dramatic effects on parasite morphology compared to treatment with vehicle (D) over a 24-h period. C2-treated parasites have empty vacuoles within them (*) and a ruffled PVM and parasite plasma membrane (white arrowheads), while C1-treated parasites have reduced electron density both in the nucleus (n) and in the cytoplasm (c). Magnifications, ×12,000 (A), ×20,000 (B and D to F), and ×25,000 (C).

Fig 7

(A) Effect of C2 treatment in vivo after infection with 10,000 PB3-10 parasites. p/sec/cm^∧2/sr, photons per second per cm² per steradian. (B and C) C2 treatment significantly reduced the parasite growth by day 4 (D4; P < 0.001) and had a significant effect on mouse survival, although all mice eventually became morbid.

Fig 8

(A) Effect of C2 treatment in vivo after infection with 10,000 5A10 parasites. (B and C) C2 treatment again significantly reduced the parasite burden over the course of the experiment (P < 0.001 on day 7) and slightly prolonged mouse survival, although this difference was not significant.