Computational Identification and Inhibitor Design Targeting Sterol 14α-Demethylase (CYP51) of Balamuthia mandrillaris, a Neglected Causative Agent of Fatal Granulomatous Amoebic Encephalitis

Balamuthia mandrillaris is a free-living amoeba responsible for granulomatous amoebic encephalitis (GAE), a rare but almost universally fatal central nervous system infection with a case fatality rate exceeding 90%. No effective treatment exists. Recent experimental work has established sterol 14α-demethylase (CYP51) as a validated drug target in the closely related amoeba Acanthamoeba castellanii (Hargrove et al., 2024); however, no structural or inhibitor data exist for Balamuthia CYP51. Here we report the first computational structural characterization of B. mandrillaris CYP51 using AlphaFold2 structure prediction, binding pocket analysis, and molecular docking. We identify the active site architecture, characterize 75 pocket-lining residues, and discover four residues unique to Balamuthia CYP51 relative to human CYP51 that represent selectivity opportunities: LYS112, ASP222, PRO370-371, and THR311. Two candidate small-molecule inhibitors are proposed and evaluated for binding affinity and drug-like properties. Candidate V1 achieves a predicted binding affinity of −9.2 kcal/mol against the heme-containing Balamuthia CYP51 model, exceeding the predicted affinity of fluconazole against its target. Candidate V2, a simplified scaffold, demonstrates predicted blood-brain barrier permeability and favorable leadlikeness. This work establishes the first structural framework for rational drug design targeting Balamuthia CYP51 and provides actionable hypotheses for experimental validation.