Identification of Small-Molecule Inhibitors of Babesia bovis by High-Throughput SYBR Green I Screening

Background: Bovine babesiosis, caused by Babesia bovis, remains a major constraint to cattle production in endemic regions, while the current control relies on only a few drugs with concerns over resistance and residues. Methods: This study screened 9600 chemically diverse compounds from the Drug Discovery Initiative, University of Tokyo, against B. bovis using a 96-well SYBR Green I high-throughput assay at 1 µM. Primary hits were subjected to dose–response testing to determine IC50 values, followed by Madin–Darby bovine kidney cell cytotoxicity assays for selectivity index calculation. Combination effects with diminazene aceturate were assessed using combination indices, whereby three compounds were selected for in silico ADME, drug-likeness and target prediction analyses. DDI-1101229 was further assessed by exploratory docking against BbHT1 and BbCDPK4. Results and Conclusions: Eight compounds showed ≥50% inhibition in the primary screen. DDI-1101229, DDI-8231037 and DDI-15106103 showed sub-micromolar activity (IC50 = 0.27–0.68 µM), selectivity indices > 14.7 and strong synergy with diminazene aceturate (CI < 0.4). Overall, this study identified selective, chemically diverse non-diamidine and non-imidazoline small-molecule inhibitors of B. bovis, highlighting alternative chemotypes with antibabesial activity.