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.
Vestibular schwannomas (VS) are benign neoplasms of the VIII cranial nerve; they account for 6–8% of all intracranial tumors and about 80% of tumors of the cerebellopontine angle. Cystic forms of VS may be particularly dangerous, as they demonstrate faster growth and worse postoperative outcomes. In this study, we performed proteomic analysis of cerebrospinal fluid (CSF) samples obtained from patients with solid and cystic forms of VS, as well as cyst fluid from patients with cystic VS. CSF samples from patients with hemifacial spasm were used as controls. Mass spectrometric (MS) analysis resulted in identification of 816 proteins in all samples and 111 proteins were upregulated in samples obtained from VS patients compared to the control group. The upregulated proteins belonged to the groups of components and regulators of the complement system, proteins associated with glucose and lipid metabolism. MS analysis of CSF obtained from patients with cystic and solid forms of VS resulted in identification of two proteomic clusters enriched in proteins associated with the development of the nervous system, cell adhesion, and myelination processes. The data obtained suggest the involvement of these biological processes in the pathogenesis of cystic VS. Significance This study provides a comprehensive proteomic characterization of cerebrospinal fluid (CSF) and cyst fluid from patients with solid and cystic vestibular schwannomas (VS), offering valuable insights into the molecular mechanisms underlying these tumors. Our discovery that complement system components and regulators are consistently upregulated in CSF and cyst fluid from VS patients, along with proteins involved in lipid metabolism, points to a potential mechanism of blood-brain barrier disruption and cyst formation, which is particularly relevant given the more aggressive clinical course of cystic VS. These findings not only contribute to understanding of VS pathogenesis but also suggest candidate protein targets for future diagnostic, prognostic, and therapeutic strategies, particularly for the more challenging cystic form of the disease.
Resistance to tyrosine kinase inhibitors (TKI) remains a major clinical challenge in the treatment of renal cell carcinoma (RCC). In this study, we established renal cancer cell models resistant to the TKI lenvatinib and cabozantinib to investigate the molecular mechanisms underlying acquired resistance. Using a quantitative proteomic approach, we profiled protein alterations associated with the resistant phenotype. A moderate-to-strong linear correlation was observed between the proteomic profiles of drug-treated control cells and matched resistant cells (Pearson r = 0.80 for lenvatinib and r = 0.66 for cabozantinib), suggesting shared alterations associated with the resistant phenotype. Notably, 35 proteins were commonly upregulated, and 40 proteins were commonly downregulated, across both resistant models, highlighting shared adaptive mechanisms driving cross-resistance. Among these, AXL tyrosine kinase receptor and enolase 2 (ENO2) emerged as key resistance-associated druggable targets that are overexpressed in clear cell RCC (ccRCC) tumors and associated with patient poor overall survival. Functional validation confirmed that pharmacological inhibition of AXL and ENO2 effectively reduced cell viability of renal cancer cells. These findings unveil association of AXL and ENO2 with resistant phenotype in RCC cells, and reveal AXL and ENO2 as promising alternative therapeutic targets in renal cancer. Significance Acquired resistance to tyrosine kinase inhibitors (TKIs) remains a major limitation in the treatment of renal cell carcinoma, underscoring the need to better understand the underlying mechanisms. In this study, we established lenvatinib- and cabozantinib-resistant renal cancer models to investigate shared resistance pathways. Quantitative proteomic analysis identified 35 proteins consistently upregulated in resistant cells, pointing to common adaptive mechanisms. Among these, AXL and enolase 2 were also overexpressed in patient tumors and associated with poor prognosis in ccRCC. Functional assays showed that inhibition of AXL and enolase 2 reduces the viability of renal cancer cells. Together, these findings identify proteins associated with the resistant phenotype that remain pharmacologically targetable and highlight potential targets for improving treatment of drug-refractory renal cancer.
