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.