In silico comparative proteomics and structural analysis of Theileria species reveals conserved druggable targets for anti-theilerial therapy

Abstract Theileriosis, a tick-borne disease caused by apicomplexan parasites of the genus Theileria, poses a major constraint to livestock productivity worldwide. Existing chemotherapeutics are increasingly limited by host toxicity and emerging resistance, underscoring the need for parasite-specific interventions. In this study, we integrated comparative proteomics, subtractive genomics, and structure-based modeling to systematically identify conserved and novel druggable targets across four clinically important Theileria species (T. parva, T. annulata, T. orientalis Fish Creek, and T. orientalis Goon Nure). Sequence clustering and orthologous analysis yielded 15,439 unique proteins, from which 2,753 were identified as evolutionarily conserved across all species. Host-homology filtering against the Bos taurus proteome excluded 1,688 shared sequences, and integration with essential gene datasets revealed 69 parasite-specific proteins crucial for survival. The top three enzymes, ubiquitin carboxyl-terminal hydrolase (UCH), phosphatidylinositol 3/4-kinase (PI3/PI4K), and DNA polymerase A, were prioritized based on evolutionary conservation, functional essentiality, and predicted druggability. Three-dimensional structural models of these targets were refined and validated for stereochemical accuracy and energetic reliability. Virtual screening of small-molecule libraries identified potent lead candidates (CIDs 163724724, 22409369, and 91149393) exhibiting strong binding affinities within the catalytic sites of the respective proteins. Molecular dynamics simulations using GROMACS over 100-ns further confirmed stable protein-ligand interactions, reflected by minimal RMSD fluctuations (~ 1.3–2.5Å), consistent compactness, and persistent hydrogen bonding. These findings demonstrate that the complexes are energetically stable and maintain their conformations, supporting the suitability of the identified targets and their lead inhibitors. Overall, this integrative approach offers a validated framework for developing anti-Theileria drugs and emphasizes the importance of evolutionarily conserved, host non-homologous proteins as promising initial targets for rational therapeutic development against Theileria infections.