Fig. 1.

Schematic summary of the strategy used to evaluate the in vivo toxicity and anti-mycobacterial efficacy of HTS-identified lipophilic compounds using the zebrafish embryo model. Candidate compounds with significant anti-mycobacterial activity were identified in a phenotypic HTS campaign using chemical libraries and selected for further evaluation based on their in vitro activity against Mtb (MIC₉₀), and their selectivity index (SI). Prior to in vivo testing in the zebrafish TB model using the fish pathogen Mm, candidate compounds were tested for their in vitro activity against extracellular Mm using a resazurin assay and against intracellular bacteria in mouse macrophages using a CFU assay. Then, candidate compounds were formulated in PMs, primarily to enhance solubility and to enable intravenous injection in the zebrafish embryo. The in vivo toxicity of PM-formulated compounds was first evaluated by mortality analysis after a single injection of different doses at 8 days post treatment (dpt), and compounds with significant toxicity were excluded from further analysis. PM-formulated compounds showing low mortality were then characterized in more detail by assigning a toxicity score to individual embryos at 4 dpt based on different morphological and physiological indicators of toxicity, as outlined in Materials and Methods. For the evaluation of in vivo efficacy, zebrafish embryos were infected with Mm and treated with different doses of PM-formulated compounds. Therapeutic efficacy was then assessed by bacterial burden (FPC) at 4 dpt and by survival analysis. dpf, days post fertilization.