In vitro C. parvum growth inhibition assay

The primary screening of compounds for C. parvum was performed as follows: HCT-8 cells were seeded in 96-well plates at a density of 2 × 10⁴ cells/well and allowed to grow overnight. Before infecting cells, Cryptosporidium oocysts were excysted to sporozoites. 87 compounds were tested against C. parvum at a single concentration of 1 μM. Each compound was tested once. Infected HCT-8 cells and uninfected HCT-8 cells were used as positive and negative controls, respectively, in medium containing 0.1% DMSO. Nitazoxanide was used as a standard comparative drug. In vitro inhibition assays were carried out as previously described [22, 23]. Briefly, oocysts were suspended in 1 ml of 0.1% sodium hypochlorite in PBS, incubated for 5 min at 4°C, centrifuged for 10 min at 3000 rpm, and washed three times in PBS. The oocysts were then suspended in excystation solution consisting of 0.75% sodium taurocholate and 0.25% trypsin dissolved in 0.1 M phosphate buffer (with 93.4 mM K₂HPO₄ and 6.5 mM KH₂PO₄; pH 8.0) and incubated at 37°C for 1 h. The oocyst/sporozoite suspensions were then rinsed in 1 x PBS (with 137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, and 2 mM KH₂PO₄; pH 7.5) once more before being filtered through a 5-μm pore-size PVDF filter (Millipore, Burlington, MA, USA) with 5 ml syringe to remove the oocyst wall. HCT-8 cells were seeded in 96-well plates (Thermo Fisher Scientific Inc., MA, USA) and incubated overnight. The cells were then infected with C. parvum sporozoites (4 ×10⁴ sporozoites/well) containing in RPMI-1640 medium for 3 h. After 3h inoculation, the non-infected parasite or debris were washed with new RPMI-1640 media. At this point, in RPMI-1640 medium containing 1 μM library compounds were added. Infected cells were incubated for an additional 45 h. Following incubation, the infected cells were fixed for 10 minutes in ice-cold 100% methanol at room temperature. Plates were then carefully rinsed 3 times with PBS. The parasite infected cells were then blocked with 1% bovine serum albumin (BSA) in PBS for 30 minutes and then stained with Sporo-Glo (an anti-Cryptosporidium polyclonal antibody) (Waterborne Environmental, Inc., VA, USA) protected from light for 1 h at room temperature. Finally, cells were washed twice with 1 x PBS-T (1 x PBS supplemented with 0.01% Tween). A Keyence BZ-II 9000 was used to capture the images (KEYENCE, Osaka, Japan). The plates were imaged with a HS all-in-one fluorescence microscope using a 20 x objective. The image output was imported into Microsoft excel for data organization and analyses. All C. parvum parasite in the well were counted. Compounds that showed more than 60% inhibition of C. parvum growth relative to the DMSO control were selected as hit compounds.

For a second investigation, four selective hit compounds were used to determine the half-maximum inhibitory concentration (EC₅₀). The parasites were treated with various doses of compounds (ranging from 0.008–1 μM). HCT-8 cells (2 × 10⁴ cells/well) were grown for 24 h before being infected with 4 × 10⁴ C. parvum sporozoites in 96-well plates. The sporozoites that did not invade were washed with RPMI-1640 3 h after inoculation and treated with the four hit compounds from the original screening at 0.008–1 μM, then incubated for another 45 h. Sporo-Glo was used for C. parvum staining as described above. Fluorescence microscopy with a Keyence BZ-II 9000 imager was used to count all Cryptosporidium in the wells (KEYENCE, Osaka, Japan). EC₅₀ vales were determined by analyzing dose-response curves made by GraphPad Prism (GraphPad Software, CA, USA).