Raw diffraction data from a crystal of Toxoplasma gondii GSK3b bound to LY2090314

Toxoplasma gondii and Cryptosporidium species are apicomplexan parasites of significant medical and veterinary importance. Although current therapeutic options for toxoplasmosis and cryptosporidiosis demonstrate notable efficacy, their utility is often compromised by suboptimal efficacy and frequent adverse effects. Moreover, therapeutic alternatives remain limited or nonexistent, particularly for cryptosporidiosis, for which nitazoxanide is currently the only approved treatment. To identify alternative therapeutic options for addressing these health challenges, we performed a phenotypic screening of an FDA-approved drug repurposing library against Toxoplasma. This screening led to the identification of LY2090314 as a potent inhibitor of T. gondii and Cryptosporidium growth in mammalian cells. Through a target deconvolution strategy combining forward genetics, transcriptome sequencing, and computational mutation analysis, we elucidated the parasiticidal mechanism of LY2090314, demonstrating that TgGSK3 kinase is its primary molecular target. We also report the first X-ray crystal structure of LY2090314 bound to TgGSK3, resolved at 2.1 Å, which reveals an interaction mode characteristic of type I ATP-competitive inhibitors. Collectively, our findings highlight a novel drug-target pair, providing a promising foundation for the development of new therapeutics against apicomplexan parasites.