Synthetic lethality and experimental evaluation of halofantrine against L. major promastigotes. Synthetic lethality analysis highlights an interaction between genes in the combination; a simultaneous knockout of genes in the combination causes a perturbation in the functioning of associated reactions in the network, thereby leading to an adverse effect on biomass production. A similar effect on biomass would not occur if the genes in the combination were knocked out individually. Panel A illustrates the concept of a drug acting on multiple targets (that are synthetically lethal) in order to inhibit growth of L. major. The effect of halofantrine evaluated at different concentrations against L. major promastigotes is presented in Panel B. The y-axis indicates alamarBlue fluorescence normalized to the 'No Drug' control. AMP-B refers to Amphotericin B, which is used as a positive control in the assay. In Panel C, a four-parameter log-logistic regression was performed on the concentration response data to compute the IC50 for halofantrine against L. major promastigotes. Panel D provides a setup for the experimental conditions and corresponding reagents used for the ATP bioluminescence assay to determine the effects of halofantrine on ATP levels in L. major. The results of the ATP bioluminescence assay are presented in Panel E. Parasites were incubated with or without halofantrine at 10 μM and in the presence of mitochondrial and/or glycolytic ATP blocks for 2 hours. The absorbance was monitored at 18 hours and the results are shown in Panel F. All error bars indicate standard error. Statistical significance in panels B and E was determined using a one-tailed Student's t-test, while statistical significance in panel F was determined using a two-tailed Student's t-test. As an additional note, the absorbance measurements in panel F between parasites incubated with and without halofantrine are significant for two conditions. However, this significance is attributable to the precision of the microplate reader rather than any meaningful biological implications regarding variations in absorbance. In Panels E and F, the condition where parasites are incubated with both mitochondrial and glycolytic ATP blocks is displayed with a dashed bar as it serves as a reference. In this particular case, halofantrine was not added.