Structural insight into the unique binding properties of pyridylethanol(phenylethyl)amine inhibitor in human CYP51

Sterol 14α-demethylase (CYP51) is the main drug target for the treatment of fungal infections. The discovery of new efficient fungal CYP51 inhibitors requires an understanding of the structural requirements for selectivity for the fungal over the human ortholog. In this study, a binding mode of the pyridylethanol(phenylethyl)amine type CYP51 inhibitor to the human ortholog was determined at the atomic level. We isolated and purified a full-length human CYP51. The inhibitor-specific binding and its conformational and dynamic properties were evaluated using UV-visible and NMR spectroscopy. Considering the experimental data in docking calculations and molecular dynamics simulations, the location of the inhibitor moieties and their interactions with the enzyme active site were determined. The inhibitor binds to the enzyme in two diastereomeric forms, which have a common location of aromatic ring moieties, while the less bulky propyl chain can adapt to various hydrophobic regions of the enzyme active site. The halogenated phenyl ring binds in the substrate access channel making numerous contacts with the hydrophobic side chains, and its interactions with the unconserved residues are especially informative. The results reveal the unique binding properties of the investigated inhibitor in comparison to the azoles and provide novel directions for the design of selective fungal inhibitors.