Screening and identification of halofuginone direct binding target proteins based on thermal proteomics profiling

Anticoccidial drugs remain the primary strategy for controlling avian coccidiosis caused by Eimeria parasites. However, widespread drug resistance and limited understanding of the molecular mechanisms underlying the action of existing anticoccidials severely impede the development of novel agents. In this study, simplified thermal proteome profiling coupled with precipitate-supported thermal proteome profiling was employed to globally identify halofuginone-binding proteins in Eimeria tenella. Sixteen candidate interacting proteins were prioritized through integrated bioinformatics analyses and molecular docking simulations. Three targets-U6L2Z8, U6L2C0, and U6L6M2-were recombinantly produced and purified, and their direct, dose-dependent binding to halofuginone was rigorously confirmed using thermal shift assays, differential scanning fluorimetry, and surface plasmon resonance, yielding dissociation constants in the micromolar range. RT-qPCR analysis showed that halofuginone induces significant changes in the mRNA expression profiles of target genes in Eimeria tenella. These transcriptional alterations provide a plausible explanation that HAL interferes with mitochondrial function and activates phosphorylation-related compensatory signaling pathways via transcriptional perturbation of these target genes, thereby inhibiting the growth and development of parasites. Collectively, this work identifies three novel, experimentally validated direct targets of halofuginone, significantly advancing the mechanistic understanding of its anticoccidial activity and establishing a robust foundation for target-informed rational design of next-generation anticoccidial drugs. SIGNIFICANCE: Anticoccidial agents serve as the primary measure to control Eimeria coccidiosis in modern poultry farming. The widespread drug resistance and poorly understood action mechanisms of currently commercialized anticoccidials have severely hindered the discovery of novel drug candidates. This study, using the anticoccidial drug halofuginone as an example, based on thermal proteomics analysis, for the first time constructs an in vitro screening and identification model for anticoccidial drug target proteins. By integrating structural thermal proteome profiling with molecular docking, in vitro recombinant protein synthesis, thermal proteome profiling - western blotting, differential scanning fluorimetry and surface plasmon resonance, this study successfully screened and validated U6L2Z8, U6L2C0 and U6L6M2 as novel direct binding targets of halofuginone in Eimeria tenella. These direct target-binding identifications fill a critical knowledge gap regarding the anticoccidial mechanism of halofuginone. Meanwhile, our research findings provide innovative strategies for the rational pharmacophore design targeting parasite-specific proteins and the development of novel anticoccidial drugs.