Screening for Anti-Amoebic Compounds

The phenotypic drug susceptibility assays were performed as previously described using CellTiter-Glo v2.0 (CTG) (Promega, Madison, WI)^44,49,63. The ReFrame collection of 12,000 compounds assembled at Calibr–Scripps were prepared in 384-well (Corning, White Flat bottom 3570) plate format at screening concentrations of 5μM (30nL of 10mM stock concentration/well in dimethylsulfoxide (DMSO)) using the Labcyte Echo 555 for acoustic compound dispensing. The first round was to screen in a single-point assay with 3,000 Naegleria/well, 600 Acanthamoeba/well or 4,000 Balamuthia/well in a total volume of 60μl. Prior to initiating drug screening, a maximum of 2% total hits from the library were predetermined for follow up from Calibr-Scripps for all screening projects. Drug spotted plates from Calibr-Scripps were left, at room temperature, to thaw overnight and parasites were plated using the Biomek NX^p automated liquid handler (Beckman Coulter). Hits were defined as compounds that produced > -40% (for Naegleria) or > -50% (for Acanthamoeba and Balamuthia) of normalized growth inhibition compared to the negative growth control (0.5% DMSO) and positive controls that produced ~100% inhibition of amoebae cell populations. Posaconazole, azithromycin and DB2385A all at 1μM were used for controls for Naegleria and Acanthamoeba; artovastatin, fluvastatin and simvastatin all at 1μM were used as controls for Balamuthia. The second round was to verify the initial active hits from round I, hits were identified and serially diluted in duplicate from 5μM to 2nM in a 1:3, 8 point dilution series in 384-well plates with the same number of amoebae for Naegleria, Acanthamoeba and Balamuthia with the exact same controls and concentrations for each respective amoebae to generate the concentration for half-maximal activity derived from the hill equation model (qAC₅₀). Dose-response curve fitting was analysed with Genedata Analyser software using the Smart Fit function. All assayed plates were incubated at the genus optimum growth temperature described above and tested for a period of 72 hours.