For the assay, compounds were prepared in 10 mM DMSO and diluted in HMI-9 to a 40 µM solution (0.4% DMSO). The assay solution was further used to perform serial dilutions (1:2) in a 96-well plate. Mid-log bloodstream forms (100 µL) were added in complete HMI-9 medium at a final cell density of 1×10 4 /mL in a well volume of 200 µL after compound addition, leading to a maximum DMSO concentration of 0.2%. Following incubation for 72 h at 37°C and 5% CO2, 20 µL of 0.5 mM resazurin solution were added and plates were further incubated for 4 h under similar conditions. Fluorescence was then measured using a Synergy 2 multi-mode reader (BioTek) at 540 and 620 nm excitation and emission wavelength, respectively. DesThe efficacy of compounds against L. infantum intracellular amastigotes was determined according to Sereno et al. with slight modifications described in detail in the SI. Liability assays. The hERG cardiotoxicity assay was performed using the Invitrogen Predictor hERG fluorescence polarization (FP) assay. A membrane fraction containing hERG (Predictor hERG membrane) was used together with a red fluorescent high-affinity ligand of the hERG channel (Predictor hERG Tracer Red). Displacement of the latter from hERG by binding of the test compound can be determined in a FP-based format. Cytochrome P450 (CYP450) assays against isoforms 1A2, 2C9, 2C19, 2D6 and 3A4