Abstract
Aspergillus fumigatus is the opportunistic fungal pathogen that predominantly affects the immunocompromised population and causes 600,000 deaths/year. The cytochrome P450 51 (CYP51) inhibitor voriconazole is currently the drug of choice, yet the treatment efficiency remains low, calling for rational development of more efficient agents. A. fumigatus has two CYP51 genes, CYP51A and CYP51B, which share 59% amino acid sequence identity. CYP51B is expressed constitutively, whereas gene CYP51A is reported to be inducible. We expressed, purified, and characterized A. fumigatus CYP51B, including determination of its substrate preferences, catalytic parameters, inhibition, and x-ray structure in complexes with voriconazole and the experimental inhibitor (R)-N-(1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide (VNI). The enzyme demethylated its natural substrate eburicol and the plant CYP51 substrate obtusifoliol at steady-state rates of 17 and 16 min(-1), respectively, but did not metabolize lanosterol, and the topical antifungal drug miconazole was the strongest inhibitor that we identified. The x-ray crystal structures displayed high overall similarity of A. fumigatus CYP51B to CYP51 orthologs from other biological kingdoms but revealed phylum-specific differences relevant to enzyme catalysis and inhibition. The complex with voriconazole provides an explanation for the potency of this relatively small molecule, whereas the complex with VNI outlines a direction for further enhancement of the efficiency of this new inhibitory scaffold to treat humans afflicted with filamentous fungal infections.
Highlights
The fungus Aspergillus fumigatus causes human diseases that are treated with cytochrome P450 51 (CYP51) azole inhibitors
We report the expression and purification of A. fumigatus CYP51B and its substrate preferences, catalytic parameters, inhibition, and x-ray structures in complexes with voriconazole and (R)-N-(1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide (VNI), an experimental inhibitor of protozoan CYP51 enzymes
Titration with lanosterol (the major natu- lated lanosterol analog C4-norlanosterol did not strate in T. brucei and Leishmania [30]) did not induce any spectral response, whereas the response to the plant CYP51 substrate obtusifoliol (ϳ14% low to high spin transition in the heme iron) was quite similar to the response to eburicol, with the apparent Kd being 1.7 versus 1.3 M (Fig. 5C), respectively, implying that in order to be productively bound to A. fumigatus CYP51B, the sterol substrates must contain a methylene group at the C24 position and/or not a C24ϭC25 double bond
Summary
The fungus Aspergillus fumigatus causes human diseases that are treated with CYP51 azole inhibitors. Azole-, pyridine-, or pyrimidine-based compounds block ergosterol biosynthesis and cause accumulation of the 14␣-methylated precursors [16, 17] These drugs act as competitive CYP51 inhibitors that occupy the P450 active site, preventing substrate binding and oxidation. Human, and other vertebrate genomes contain only one CYP51 gene; A. fumigatus and some other filamentous ascomycetes [25] have two CYP51 paralogs (CYP51A and CYP51B; Fig. 2), each present as a single copy [26] (chromosomes 4 and 7, respectively) Both genes had been suggested to be active in A. fumigatus, it was later reported that the gene CYP51B encodes the enzyme primarily responsible for sterol 14␣-demethylation. We report the expression and purification of A. fumigatus CYP51B and its substrate preferences, catalytic parameters, inhibition, and x-ray structures in complexes with voriconazole and (R)-N-(1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide (VNI), an experimental inhibitor of protozoan CYP51 enzymes. The x-ray structures confirm high three-dimensional similarity as the molecular basis for the CYP51 catalytic conservation across phyla, but some structural features appear to be fungus-specific and potentially useful in antifungal drug development involving rational structure-based design of novel drugs
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