Structural and Functional Characterization of TgGSK3, a Druggable Kinase in Toxoplasma gondii

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 performe 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. Methods: RNA profiles were generated from HFF cells infected by RH wild-type or L2090314-resistant T. gondii strains. Total RNAs were extracted and purified using TRIzol (Invitrogen, Carlsbad, CA, USA) and RNeasy Plus Mini Kit (Qiagen). RNA-sequencing was performed by Novogene (Munich, Germany).RNA quantity, integrity, and purity were determined using the Agilent 5400 Fragment Analyzer System (Agilent Technologies, Palo Alto, California, USA). The integrity values or RIN ranged from 9.1 to 10 for all samples, which was considered sufficient. Messenger RNAs (mRNA) were purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers. For directional libraries, the second strand cDNA was synthesized using dUTP, instead of dTTP. The directional library was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced on Illumina platforms, according to effective library concentration and data amount. The samples were sequenced using the Illumina NovaSeq platform, employing both non-stranded (for variant calling analysis) and strand-specific sequencing (for gene expression analysis) with 2 x 150 bp reads and generated ~40 million paired-end reads for each sample.