High-Throughput Repurposing Screen Reveals Compounds with Activity against Toxoplasma gondii Bradyzoites

Abstract

Toxoplasma gondii causes widespread chronic infections that are not cured by current treatments due to the inability to affect semidormant bradyzoite stages within tissue cysts. To identify compounds to eliminate chronic infection, we developed an HTS using a recently characterized strain of T. gondii that undergoes efficient conversion to bradyzoites in vitro. Stage-specific expression of luciferase was used to selectively monitor the growth inhibition of bradyzoites by the Library of Pharmacological Active Compounds, consisting of 1280 drug-like compounds. We identified 44 compounds with >50% inhibitory effects against bradyzoites, including new highly potent compounds, several of which have precedent for antimicrobial activity. Subsequent characterization of the compound sanguinarine sulfate revealed potent and rapid killing against in vitro-produced bradyzoites and bradyzoites harvested from chronically infected mice, including potent activity against intact cysts. These findings provide a platform for expanded screening and identify promising compounds for further preclinical development against T. gondii bradyzoites that are responsible for chronic infection.

Keywords: chronic infection; high throughput screening; toxoplasmosis.

Figure 1

Development of a High Throughput Screening assay for growth inhibition of Tg68 tachyzoites. (A) A tachyzoite-specific firefly luciferase (Fluc) reporter strain of Tg68 was generated using the pTUB1 promoter. (B) Confluent HFF cells in 384-well plates were infected with Tg68 tachyzoites, compounds were added immediately in a dilution series, and luciferase activity was measured on day 3. (C) Replicates of 12 wells from six independent plates were analyzed for luciferase activity. Fluc expression increased more than 1,000-fold and was found significantly higher at day 3 (72 h) compared to day 0 (4 h postinfection). Data accumulated from 12 wells across 6 plates. Mann–Whitney test, P < 0.02. (D) EC₅₀ values were determined for Tg68-pTub1:Fluc and ME49-Fluc treated with serial dilutions of BRD7929 or Atovaquone. All EC₅₀ values are presented as the mean of three biological replicates (n = 3).

Figure 2

Development of a High Throughput Screening assay for the growth inhibition of Tg68 bradyzoites. (A) A bradyzoite-specific Nanoluc luciferase (nLuc) reporter strain of Tg68 was generated using the pBAG1 promoter. Parasites were grown in alkaline medium (pH 8.2), CO₂ free, or in the absence of glucose supplemented with glutamine, which stimulates in vitro development of bradyzoites. (B) Confluent HFFs in 384-well plates were infected with Tg68 tachyzoites for two h, washed, and then cultured either in D10 under normal conditions (Tz) or switched to alkaline or glutamine conditions to induce bradyzoites. The cultures were then maintained under CO₂-free conditions for 10 days with media changes on days 3 and 6, with a compound treatment beginning at day 6 and readout at day 10. (C) Luciferase signals from tachyzoites (Tz) harvested at day 0 (4 h postinfection) or bradyzoites induced for different times (day 0 (4 h) to day 10) by culture in alkaline or glutamine media. Comparisons between sequential time points using the Mann–Whitney test, ****P < 0.0001. (D) Determination of EC₅₀ values for BRD7929 and atovaquone treatment of in vitro induced bradyzoites culture in alkaline or glutamine media. All EC₅₀ values are presented as the mean of four biological replicates (n = 4).

Figure 3

Summary of LOPAC screening for the growth inhibition of T. gondii. (A) Venn diagram showing the number of compounds with ≥50% growth inhibition at 10 μM in each of three growth assays. Tz = tachyzoite growth assay, Alk = alkaline induced bradyzoite growth assay, Gln = glutamine induced bradyzoite growth assay. Red circled numbers indicate the selection criteria for Primary Hits. (B). Summary of LOPAC screen and prioritization of Hits for follow up. Of 44 Primary Hits, 36 were available for dilution series to determine EC₅₀ values. Top 9 Hits were available for further biological testing.

Figure 4

Testing of compounds for activity against ex vivo bradyzoites. (A) Schematic for the isolation of bradyzoites from chronically infected mice and testing in vitro during continuous treatment or after 4 h treatment and washout. (B) Testing of sanguinarine and reference compounds in continuous treatment vs 4 h treatment and washout. Values are determined from plaque counts after 10–14 days of outgrowth and are normalized to the DMSO control for each condition. (C) Schematic for 4 or 24 h treatment of intact cysts followed by in vitro plaque formation in media with or without compounds. (D) Testing of sanguinarine and reference compounds on intact cysts for 4 h treatment. (E) Testing of sanguinarine and reference compounds on intact cyst for 24 h treatment. Values are determined from plaque counts after 10–14 days of outgrowth and are normalized to the DMSO control for each condition. Compounds were used at 3XEC₉₀ based on the sensitivity of Tg68Fluc tachyzoites (Table S3). Data from 3 to 6 biological replicates, bar graph represents the average of percentage of number of plaques in the treatment group normalized to DMSO control.