Antiamoebic Activity of Lonafarnib

Lonafarnib (Sigma) was tested for its activity against European KUL; US Davis, CAMP, TY; and Australian CDC:V1005 strains of N. fowleri. Lonafarnib was dissolved as 20 mM stock in DMSO. Starting at 50 μM, the compound was serially diluted with DMSO to eight concentrations. 0.5 μL from each concentration was transferred in triplicate into a flat white bottom 96 well microtiter plate. 0.5% DMSO was used as a vehicle control and 50 μM of amphotericin B as a positive control. Density of 10,000 N. fowleri trophozoites per 99.5 μL was seeded across the plate. In between each transfer, the cells were evenly resuspended throughout the medium. After 48 h of incubation at 37 °C, the plates were taken out and cooled to room temperature. CellTiter-Glo^® Luminescent Cell Viability Assay (Promega Corporation, Madison, WI, USA) was utilized to measure generation of ATP-bioluminescence [31 (link)]. The assay solution contains luciferase, which in the presence of cellular ATP and oxygen, catalyzes the transformation of luciferin into oxyluciferin, yielding PPi, AMP, and light. The luminescence was quantified using EnVision Multilabel plate reader (PerkinElmer, Inc. Waltham, MA, USA). Microsoft Excel and GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA) softwares were used for statistical analysis of experiments and determination of EC₅₀ of Lonafarnib.