Triazole resistance mediated by mutations of a conserved active site tyrosine in fungal lanosterol 14α-demethylase

Abstract

Emergence of fungal strains showing resistance to triazole drugs can make treatment of fungal disease problematic. Triazole resistance can arise due to single mutations in the drug target lanosterol 14α-demethylase (Erg11p/CYP51). We have determined how commonly occurring single site mutations in pathogenic fungi affect triazole binding using Saccharomyces cerevisiae Erg11p (ScErg11p) as a target surrogate. The mutations Y140F/H were introduced into full-length hexahistidine-tagged ScErg11p. Phenotypes and high-resolution X-ray crystal structures were determined for the mutant enzymes complexed with short-tailed (fluconazole and voriconazole) or long-tailed (itraconazole and posaconazole) triazoles and wild type enzyme complexed with voriconazole. The mutations disrupted a water-mediated hydrogen bond network involved in binding of short-tailed triazoles, which contain a tertiary hydroxyl not present in long-tailed triazoles. This appears to be the mechanism by which resistance to these short chain azoles occurs. Understanding how these mutations affect drug affinity will aid the design of azoles that overcome resistance.

Synthesis, Molecular Docking Studies, and Antifungal Activity Evaluation of New Benzimidazole-Triazoles as Potential Lanosterol 14α-Demethylase Inhibitors

“…The affinity of azole antifungals to the lanosterol 14a-demethylase is determined not only by the coordination binding of the nitrogen of azole ring to the heme iron in the active side (N-4 of triazole and N-3 of imidazole) but also by the affinity of N-l substituent for the apoprotein part of the enzyme. The remaining part of the azole antifungal fits in the similar way like lanosterol in the hydrophobic groove of lanosterol 14--demethylase [13][14][15][16][17].…”

Structural and Functional Elucidation of Yeast Lanosterol 14α-Demethylase in Complex with Agrochemical Antifungals

“…Like the dioxolane moiety in ITC [20], the 4-methyl-1,3-dioxolane ring of DFC occupies the position where the key water 743 is found in the presence of FLC, R - and S -DPZ. The residue M509 projects into the binding site adjacent to H381 when compared to other structures, indicating a degree of flexibility around this position only previously seen in the ScErg11p6×His-VRC structure (PDB ID:5HS1) [22]. The elasticity at this position is generally dependent on the size of the inhibitor, with larger medium and long chain azoles occupying the space.…”

“…The central quaternary carbons of the enantiomers are approximately 1 Å apart, with R -TBZ projecting deeper into the active site cavity which easily accommodates the smaller tert -butyl substituent (Fig 3C). The key water 743 identified in the wild type FLC structure [21, 22] is not seen in either TBZ structure. This may be due to the lower resolution of this structure but could easily be accommodated.…”

“…We have determined the X-ray crystal structures of hexahistidine-tagged Saccharomyces cerevisiae lanosterol 14α-demethylase in complex with its substrate lanosterol, the pseudosubstrate estriol and the triazole drugs itraconazole (ITC), posaconazole (PCZ), fluconazole (FLC) and voriconazole (VCZ) [20–22]. These structures provided the first complete crystallographic analysis for a bitopic monospanning membrane protein, the first crystal structure for a fungal lanosterol 14α-demethylase and the first crystal structure to visualise the transmembrane domain of cytochrome 450, including its interaction with the enzyme’s catalytic domain.…”

Synthesis, structural and spectroscopic features, and investigation of bioactive nature of a novel organic-inorganic hybrid material 1H-1,2,4-triazole-4-ium trioxonitrate

“…The target of the triazole antifungals is the cytochrome P450 (CYP)-dependent 14-alpha-demethylase (CYP51). The crystal structures of various mutants of the CYP51 with fluconazole, itraconazole, posaconazole, and voriconazole has been reported in the literature [34]. Depending on the structure of the triazole compound, the binding affinities and interaction profile shows significant variations resulting in different antifungal activities and side effects [35].…”