Identification of New Anti-Trypanosoma brucei Leads: An Integrated CRK12-Guided Virtual Screen With Experimental Validation.

Human African trypanosomiasis (HAT), caused by Trypanosoma brucei, remains a neglected tropical disease with a critical shortage of therapeutic options, underscoring the need for new chemotypes. Cell division cycle-2-related kinase 12 (CRK12) has emerged as a genetically essential and chemically validated target in kinetoplastids, yet experimentally supported CRK12-targeting chemotypes against T. brucei remain limited, and no experimental CRK12 structure is available. Here, we developed an integrated computational-experimental screening workflow to accelerate CRK12-guided hit discovery, combining ligand-based prescreening, structure-based virtual screening, molecular dynamics (MD) refinement, and experimental validation. An 8.6-million-compound library was prescreened using MACCS fingerprint screening and complex-based pharmacophore screening, followed by staged docking and MD simulations to prioritize candidates for experimental testing. Notably, four of ten purchased compounds showed strong growth inhibition against T. brucei at 10 μM, with IC50 values of 6.09, 1.47, 0.81, and 1.33 μM, demonstrating a promising hit rate for this data-limited target. Binding-mode analysis revealed a conserved hinge-anchoring interaction pattern in the ATP-binding pocket, providing a structural rationale for follow-up analogue design. Overall, this study identifies new chemical starting points for anti-T. brucei drug discovery and demonstrates the utility of an AlphaFold2-enabled CRK12-guided screening strategy for targets with limited structural and ligand data.