Sterol 14α-Demethylase Ligand-Binding Pocket-Mediated Acquired and Intrinsic Azole Resistance in Fungal Pathogens

Abstract

The fungal cytochrome P450 enzyme sterol 14α-demethylase (SDM) is a key enzyme in the ergosterol biosynthesis pathway. The binding of azoles to the active site of SDM results in a depletion of ergosterol, the accumulation of toxic intermediates and growth inhibition. The prevalence of azole-resistant strains and fungi is increasing in both agriculture and medicine. This can lead to major yield loss during food production and therapeutic failure in medical settings. Diverse mechanisms are responsible for azole resistance. They include amino acid (AA) substitutions in SDM and overexpression of SDM and/or efflux pumps. This review considers AA affecting the ligand-binding pocket of SDMs with a primary focus on substitutions that affect interactions between the active site and the substrate and inhibitory ligands. Some of these interactions are particularly important for the binding of short-tailed azoles (e.g., voriconazole). We highlight the occurrence throughout the fungal kingdom of some key AA substitutions. Elucidation of the role of these AAs and their substitutions may assist drug design in overcoming some common forms of innate and acquired azole resistance.

Keywords: amino acid substitution; antifungal resistance; azole agrochemicals; azole drugs; cross-kingdom pathogens; cytochrome P450; ergosterol biosynthesis; heme-containing active site; imidazole; lanosterol 14α-demethylase; pan-fungal kingdom mutation; point mutations; promotor region; prophylaxis; sterol 14α-demethylase; substrate entry channel; tandem repeat; therapy; treatment; triazole; water-mediated hydrogen bond network.

Figure 1

Structural view of the ligand-binding pocket of S. cerevisiae SDM (sterol 14-alpha demethylase) with the azole inhibitor short-tailed VCZ (voriconazole) and the long-tailed ITC (itraconazole) overlaid. The triazole of each drug is the 5th ligand of the heme iron (large red ball) and C470 is the 6th ligand. AA (amino acid) residues important for azole binding and/or azole resistance discussed in this review are shown with their carbons in light green. A list of this AAs is given in Supplementary Table S3. Relevant structural features, including the heme porphyrin, are given in green. Water-mediated hydrogen bond networks important for the binding of VCZ in the active site and ITC in the substrate entry channel are shown as yellow dashes.

Figure 2

Structural view of the ligand-binding pocket of the human CYP51 D231A H314 mutant catalytic domain (Protein Data Bank ID: 6UEZ) interacting via hydrogen bonds (yellow dashes) with the hydroxyl of lanosterol (carbons in yellow). The heme is given in blue with the iron as a large red ball. Amino acid residues involved in the hydrogen bonding directly or as part of a water-mediated network are indicated. A list of all key AA residues for the main structures of the SDM are given in Supplementary Table S3.