# Structural and functional characterization of TgGSK3, a druggable kinase in Toxoplasma gondii

## TL;DR

Researchers found that the drug LY2090314 effectively inhibits two harmful parasites and revealed how it works by studying the structure of a key parasite protein.

## Contribution

The first crystal structure of LY2090314 bound to TgGSK3 is reported, validating it as a druggable target for parasitic infections.

## Key findings

- LY2090314 is a potent inhibitor of Toxoplasma gondii and Cryptosporidium growth in mammalian cells.
- TgGSK3 kinase is identified as the primary molecular target of LY2090314 through target deconvolution strategies.
- The X-ray crystal structure of LY2090314 bound to TgGSK3 reveals a type I ATP-competitive interaction mode.

## Abstract

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 clinical efficacy is often limited 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 medication to treat diarrhea in adults and children older than 1 year of age. 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 identifies 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 elucidate the parasiticidal mechanism of LY2090314 and demonstrate 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. Furthermore, interactome analysis uncovers functional connections between TgGSK3 and key cytoskeletal and signaling regulators, providing insights into compound’s effects. Collectively, these findings validate TgGSK3 as a promising therapeutic target for toxoplasmosis and offer mechanistic insights into apicomplexan GSK3 biology.

Researchers identify the cancer drug LY2090314 as a potent inhibitor of Toxoplasma gondii and Cryptosporidium and report the first crystal structure of the parasite kinase TgGSK3 bound to it, validating TgGSK3 as a therapeutic target.