A drug repositioning strategy identifies a compound inhibiting GSK3 activity in Toxoplasma gondii

The phylum Apicomplexa encompasses many of the world"s pre-eminent protozoan pathogens, including Toxoplasma gondii causing toxoplasmosis, a highly prevalent foodborne parasitic disease. Despite its widespread impact, existing therapies exhibit limited effectiveness, highlighting the urgent need for potent drugs targeting both T. gondii and related pathogens within the phylum. Here, compound 209 (C209) was identified in a phenotypic screen using a repurposed drug library aiming to uncover novel antiparasitic compounds and their corresponding targets. Previously assessed in clinical trials for cancer therapy, C209 demonstrated significant parasiticidal activity by disrupting the intracellular development of tachyzoites in the nanomolar range. Using a forward genetic approach, we identified the essential cell cycle-associated kinase TgGSK3 as the primary target of C209, identifying three single nucleotides substitutions conferring resistance to the drug. Through high-resolution crystallization and structure determination of the GSK3/C209 complex, we elucidated the compound's unique binding mode which displays a remarkable fold of its own and we could rationalize the interfering effect of the three amino acid mutations within the compound binding cavity. Biochemical analysis confirmed this inhibition, revealing a 2- to 18-fold loss of compound binding affinity in mutated proteins and a 2- to 5-fold decrease in kinase activity compared to the native protein. The conservation of GSK3 in Cryptosporidium has triggered interest in its potential as a new target offering an avenue for the development of broader anti-parasitic drugs.