Triaging of VLS hits via primary screen and validation through secondary parasite assays. a Venn diagrams of our primary screen using VLS, enzyme activity assay and thermal shift data. 36 compounds showed activity in more than one assay. b Venn diagram of those compounds overlapping in at least two assays in our primary screens, gliding motility assay and liver invasion assay. Compound 24 emerges from five different assays and was the only one successfully visualized through our co-crystallization efforts. c Flowchart depicting the sequence of steps in the lead identification and verification process described here. While initially 315,105 chemical structures were screened using VLS, after each step fewer viable drug candidates remained. At the end of this sequence of computational and biochemical experiments, compound 24 emerged as the most promising lead.