The Marine Natural Product Library containing 103 compounds was obtained from the Natural Product Library (Cat #L1400; Selleck, Shanghai, China) by searching the Dictionary of Marine Natural Products in the ChemNetBase website (https://dmnp.chemnetbase.com/faces/chemical/ChemicalSearch.xhtml) and applied for preliminary screens against T. gondii. Estradiol benzoate (Cat #S4110; Selleck) and octyl gallate (Cat #S9338; Selleck) were also obtained from Selleck. These compounds were dissolved with dimethyl sulfoxide (DMSO) into a 10 mM solution and stored at −20°C. Pyrimethamine (Cat # 46706, Sigma-Aldrich, Shanghai, China) was used as a reference drug. All information of compounds is listed in Supplementary Table S1.

T. gondii tachyzoites of the RH-2F strain, expressing luminescence-based β-galactosidase (β-Gal) (Seeber and Boothroyd, 1996), were maintained in monolayers of human foreskin fibroblasts (HFFs, ATCC SCRC-1041) cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Invitrogen, Shanghai, China) made up of 10% (v/v) fetal bovine serum (FBS; Gibco, Invitrogen, Australia) and a cocktail of 1% (v/v) penicillin–streptomycin–glutamine (Cat # 10378016, Gibco) at 37°C and 5% CO₂.

To determine inhibition of parasite growth, β-Gal activity assay was performed in the preliminary screening as described previously (Liu et al., 2020). In brief, each compound was diluted fresh by using DMEM without phenol red to a final concentration of 10 μM and added to confluent HFFs in a 96-well half-area plate. Then mechanically released RH-2F tachyzoites were added at a multiplicity of infection (MOI) of 0.2 (parasite/host cell ratio). HFFs treated with 0.1% DMSO were used as the negative control, and 10 μM pyrimethamine was used as the positive control. After incubation for 72 h, 1 mM chlorophenol red-β-d-galactopyranoside (CPRG; Cat # 220588, Sigma-Aldrich) was added to the medium, and the absorbance was monitored at 560 nm. The number of parasites was calculated according to the standard curves made at the same time. Afterward, those compounds that displayed over 70% parasite inhibition ratio were further chosen to assess their cytotoxicity by using a CellTiter 96R AQueous One Solution Cell Proliferation assay system (Promega Corp, United States) according to manufacturer’s procedure. Compounds exhibiting low cytotoxicity (≥80% cell viability) were evaluated for inhibition efficacy in detail.

Either estradiol benzoate or octyl gallate was added to the first column of HFFs (∼500 cells/well) in a 96-well half-area plate at a final concentration of 50 μM and then diluted serially across the plate by twofold, leaving the final column drug free. As a result, the exact concentration of each well was 50, 25, 12.5, 6.25, 3.125, 1.563, 0.781, 0.391, 0.195, and 0 μM, respectively. Fresh extracellular tachyzoites were added to each well at an MOI of 0.2 and incubated at 37°C and 5% CO₂ for 72 h. The absorbance was detected at 560 nm 24 h after addition of CPRG. Meanwhile, HFF cells (1 × 10⁵ cells/well) treated with different concentrations of each compound were cultured in a 96-well plate at 37°C and 5% CO₂ for 72 h, and then their cytotoxicity was assessed as described above. The exact concentration of the estradiol benzoate was 500, 250, 125, 62.5, 31.25, 15.625, 7.813, 3.906, 1.953, and 0 μM. The exact concentration of the octyl gallate was 100, 50, 25, 12.5, 6.25, 3.125, 1.563, 0.781, 0.391, and 0 μM. The assay was performed in triplicate and repeated three times. The 50% toxicity dose to host cell (TD₅₀) and 50% inhibitory concentration to the parasite (IC₅₀) were calculated, and the therapeutic index (TI) of each compound was defined as TD₅₀/IC₅₀.

The plaque assay was performed as described previously (Liu et al., 2020). Monolayers of HFF in 24-well plates containing 5 μM of either estradiol benzoate or octyl gallate in triplicate were infected with freshly released 100 tachyzoites. DMSO-treated parasites were used as negative control, and the 10 mM pyrimethamine-treated group was used as positive control. Seven days after incubation, the wells were fixed with methanol and stained with 1.5% crystal violet. The plaque area of each group was measured with ImageJ software.

The invasion assay was performed as described previously (Wilson et al., 2020) with minor modifications. RH-2F parasites were inoculated in HFFs and pretreated with 5 μM of each compound for 8 h. Then the freshly released tachyzoites (5 × 10⁶) were added to a new 24-well plate of HFFs with coverslips and each compound. Plates were kept on ice for 15 min and subsequently put at 38°C for 30 min to allow the parasites to invade host cells. Infected cells fixed with 4% paraformaldehyde were stained with mouse anti-T. gondii SAG1 (1:5,000, Cat # ab8313; Abcam, United States) for 1 h followed by antimouse Alexa Fluor 488 (1:1000, Invitrogen, Shanghai, China) for 1 h. Next, cells were permeabilized with 0.25% Triton-X100 and blocked with 3% BSA. An incubation step for 1 h with mouse anti-SAG1 was done followed by staining with anti-mouse Alexa Fluor 594 (1:1,000, Invitrogen, Shanghai, China) for 1 h. Parasite and host cell nuclei were then stained with DAPI (4′,6-diamidino-2-phenylindole) for 5 min. Intracellular parasites were shown as green⁻/red⁺, and the extracellular parasites were shown as green⁺/red⁺ (yellow). The proportion of the number of intracellular parasites to the total number of tachyzoites was termed as the invasion ratio. Ten fields were randomly selected under fluorescent microscope for counting, and three repetitions were performed.

The intracellular proliferation assay was performed as described previously (Liu et al., 2020). Monolayers of HFF in 24-well plates were infected with fresh tachyzoites at MOI = 4. After 3 h, the medium was replaced with fresh culture medium containing 5 μM of each compound. Then 24 h later, the cells were fixed, permeabilized, and stained with mouse anti-SAG1 for 1 h followed by anti-mouse Alexa Fluor 594 for 1 h. At least 100 parasitophorous vacuoles were randomly selected, and the number of parasites in each vacuole was counted.

The egress assay was performed as described previously (Wilson et al., 2020) with minor modifications. Monolayers of HFF in 24-well plates were infected with 1 × 10⁴ tachyzoites followed by incubation at 37°C and 5% CO₂ in a medium containing 5 μM of each compound for 24 h. Prior to egress assays, infected wells were washed three times with prewarmed PBS. After the final wash, the cells were treated with 3 μM A23187. The treated plates were allowed to incubate at 5% CO₂ and 37°C for 5 min before addition of 100% methanol and immediate placement on ice for 10 min. Subsequently, IFA assay was carried out as described in the invasion assay, and the proportion of the number of egressed parasites to the total number of tachyzoites was termed as the egress ratio. Ten fields were randomly selected under fluorescent microscope for counting, and two repetitions were performed.

Flow cytometric analyses was performed as described previously (Liu et al., 2020). Monolayers of HFF were infected with tachyzoites at MOI = 1. After 12 h, synchronization of cell cycle was performed with 80 μM pyrrolidine dithiocarbamate (PDTC, Cat #S1809; Beyotime) at 37°C and 5% CO₂ for 8 h. Then the medium was replaced with culture medium in the presence of 5 μM of each compound. After 8 h, the extracellular parasites were removed by cold PBS washing, and the intracellular tachyzoites were isolated from host cells through a 27-gauge needle and purified with a 3-μm filter. After centrifugation at 4°C for 10 min at 1,000 × g, parasites were fixed with 70% ethanol at −20°C for 24 h followed by cold 2% FBS washing. Parasites were stained with propidium iodide (PI; Cat # PH0530, Phygene) at 37°C for 30 min in the dark and then were filtered with a 5-μm pore filter. Harvested parasites were detected on FACSCanto™II flow cytometer (Beckman Coulter, CA, United States), and the results were analyzed using FlowJo 7.6.1 software (FlowJo LLC, Ashland, OR, United States). At least 50,000 events were collected per sample.

GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, CA, United States) was used to analyze all the data. Data were plotted and are presented as the mean of three replicates ± standard deviation (SD). The TD₅₀ and IC₅₀ values were plotted through a nonlinear regression analysis (curve fit). Phenotypic assay data were analyzed with two-tailed Student’s t-test or one-way ANOVA with Tukey’s post-hoc test. A value of p < 0.05 was considered statistically significant.

We screened 103 marine natural products to identify novel drugs that were anti-T. gondii (Supplementary Table S1). Our selection criteria aimed to identify compounds that caused parasite growth inhibition (at least 70% inhibition ratio) and had low cytotoxicity toward the host cell (at least 80% cell viability ratio). Two compounds, estradiol benzoate (parasite inhibition ratio: 73.0 ± 0.9%; host viability ratio: 136.4 ± 2.5%) and octyl gallate (parasite inhibition ratio: 80.4 ± 1.0%; host viability ratio: 98.6 ± 8.8%), matched our criteria (Figure 1).

To further evaluate the efficiency of estradiol benzoate and octyl gallate on anti-T. gondii activity, the growth inhibition of the parasite by estradiol benzoate and octyl gallate (Figure 2A) was tested against the RH-2F strain expressing β-gal, allowing colorimetric quantitation of T. gondii. The results showed that estradiol benzoate and octyl gallate inhibit parasites at IC₅₀ values of 4.41 ± 0.94 and 5.66 ± 0.35 μM (estradiol benzoate vs. octyl gallate: p = 0.095), respectively (Figure 2B). To analyze the cytotoxicity of each compound on HFF host cells in vitro, we examined cell proliferation using an MTT assay. The TD₅₀ values of estradiol benzoate and octyl gallate were found to be 34.11 ± 2.86 and 26.4 ± 0.98 μM (estradiol benzoate vs. octyl gallate: p = 0.012), respectively (Figures 2C,D). According to the results above, the in vitro TI was calculated to be 7.7 for estradiol benzoate and 4.7 for octyl gallate.

Next, we performed a plaque assay to validate the effect of each compound on parasite and host cell. Seven days after incubation, we observed that the monolayer of HFF was intact, further indicating the low cytotoxicity of the two compounds. Importantly, the parasites treated with estradiol benzoate (0.11 ± 0.04) and octyl gallate (0.71 ± 0.21) displayed approximately 93% and 55% reductions in the plaque area compared with the vehicle control (1.56 ± 0.71), respectively (Figure 3). Also, we noticed that estradiol benzoate inhibition in forming of plaque is comparable with pyrimethamine (0.2 ± 0.07, 87% reduction in plaque area). The results suggest that the two compounds indeed inhibit the replication of tachyzoites.

To unravel the mode of the anti-T. gondii action of the two compounds, we sought to determine whether any step of the lytic cycle of the parasite was impaired. We found that both estradiol benzoate and octyl gallate did not robustly suppress the ability of parasites to invade (estradiol benzoate: 0.52 ± 0.01; octyl gallate: 0.54 ± 0.02; vehicle control: 0.54 ± 0.01) and egress (estradiol benzoate: 0.77 ± 0.01; octyl gallate: 0.81 ± 0.06; vehicle control: 0.76 ± 0.03) (Figure 4). However, both compounds displayed potent ability to inhibit tachyzoite intracellular growth, as shown by the accumulation of vacuoles with mostly one, two, or four tachyzoites (Figures 5A,B). By contrast, the parasites treated with DMSO were mainly composed of vacuoles containing eight tachyzoites (DMSO: 0.51 ± 0.10; estradiol benzoate: 0 ± 0; octyl gallate: 0.18 ± 0.04).

To clarify their potential mode of anti-T. gondii action, we sought to investigate the effect of the two compounds on parasite cell cycle progression by using flow cytometry. We noticed that estradiol benzoate (0.91 ± 0.08), rather than octyl gallate (0.65 ± 0.02), markedly resulted in a G1 accumulation peak compared with the mock treatment (0.64 ± 0.04) (Figures 5C, D), demonstrating that estradiol benzoate deferred or halted the cell cycle of tachyzoites by stumbling progression from G1 to S phase.