Evaluation of Compounds Against N. fowleri Trophozoites

The compounds were screened and reassayed for EC₅₀ determination against N. fowleri trophozoites using a final 8-point concentration range. 2.5 μL of 5 mM stock compounds in 100% DMSO were diluted with 17.5 μL sterile water to yield 625 μM working concentration of compounds. A three-fold serial dilution was then performed yielding a concentration range 625 μM-0.25 μM. From this dilution plate, 4 μL were transferred into the 96-well screen plates followed by addition of 96 μL of trophozoites (10,000 amebas per well) to yield a final 8-point concentration range spanning 25–0.01 μM in final 0.5% DMSO (Debnath et al., 2012 (link)). Miltefosine was tested at a concentration range of 200 μM-1.56 μM. The assays were performed in triplicate and assay plates were incubated for 48 h at 37°C. At the end of incubation, the assay plates were equilibrated to room temperature for 30 min, 50 μL of CellTiter-Glo Luminescent Cell Viability Assay (Promega) were added in each well of the 96-well plates. The plates were then placed on an orbital shaker at room temperature for 10 min to induce cell lysis. After lysis, the plates were again equilibrated at room temperature for 10 min to stabilize luminescent signal. The resulting ATP bioluminescence of the trophozoites was measured at room temperature using an EnVision Multilabel Reader (PerkinElmer, Waltham, MA). Negative controls in the screen plates contained 0.5% DMSO and positive controls contained 50 μM amphotericin B (Sigma-Aldrich).
In parallel, to determine the effect of ebselen, BAY 11-7082 and BAY 11-7085 on the growth of N. fowleri, 10⁴ amebae were incubated at a concentration range of 50–0.39 μM of ebselen, BAY 11-7082 and BAY 11-7085 for 48 h at 37°C. Control trophozoites were incubated with 0.5% DMSO. Cell numbers were calculated by hemocytometer at the end of incubation. The percentage of viable trophozoites due to treatment at different concentrations of compound was determined by the standard trypan blue exclusion method. Cells stained blue were considered non-viable.