Investigating resistance mutations in the drug target of triazole drugs
Fungal infections affect a broad spectrum of the population, including prematurebabies, the elderly and individuals with a range of disease- or medically-induced comorbidities.Fungal pathogens such as Candida albicans and Aspergillus fumigatuscause a variety of conditions, from minor infections to life threatening disease. Somefungi, including C. albicans and Candida glabrata can be commensal living inharmony with the superficial microflora, but these organisms can behave asopportunistic pathogens when individuals become immunodeficient due to comorbiditiesor become immunocompromised due to AIDS or through medicalintervention. Fungal infections have become recognised in recent years as a growinghealth burden that currently causes around 1.5 million deaths per year worldwide.The azole antifungal drugs (imidazoles and triazoles) are used widely to treat fungalinfections and as antifungal prophylaxis. The azole drugs target the fungal enzymelanosterol 14α-demethylase (Erg11p, CYP51). This monospanning bitopic membraneprotein belongs to the CYP51 class in the cytochrome P450 superfamily of enzymesand is involved in the rate-limiting step of ergosterol biosynthesis. Ergosterol is thefungal equivalent of cholesterol and is required for fungal cell growth. Fungalpathogens have evolved several mechanisms of resistance that diminish the action ofthe azole drugs. The emergence of resistant fungal strains due to mutations in CYP51can limit therapeutic options and make treatment of fungal infections increasinglyproblematic. The need for better drugs that overcome resistance is becomingincreasingly urgent. The present project builds on the research of Monk et al. whosuccessfully crystallised and obtained the first high-resolution X-ray structures of afungal CYP51. The aim of this project is to investigate the effect of CYP51 mutationson enzyme structure and function, including different types of triazole drug, by usingSaccharomyces cerevisiae Erg11p as a model for the homologous enzymes inpathogenic fungi.A S. cerevisiae overexpression system was used to hyper-express the wild type andthe mutant ScErg11p enzymes in order to obtain sufficient quantities of protein forstructure-function studies. The C. albicans CYP51 mutations Y132F/H, K143R,G464S and the double mutation Y132F G464S (Y140F/H, K151R, G464S and Y140FG464S S. cerevisiae numbering), as well as the CYP51A G54E/R/W mutations of A.fumigatus (G73E/R/W S. cerevisiae numbering) have been reproduced in a C-terminalhexahistidine-tagged version of S. cerevisiae Erg11p (ScErg11p6×His). In addition,the innate resistance of A. fumigatus CYP51A to fluconazole (FLC) was investigatedusing the S. cerevisiae Erg11p T322I mutant. Microdilution assays were used todetermine triazole susceptibilities of these strains. ScErg11p6×His mutant and wildtype enzymes were purified from crude membranes by solubilisation with thedetergent n-decyl-β-D-maltoside followed by affinity and size exclusionchromatography. Spectral analysis of the purified protein was used to determinedissociation constants for triazole drugs. Purified preparations of the enzyme werealso used to obtain crystals for X-ray crystallographic analysis. High-resolution (1.98– 2.35 Å) X-ray crystal structures were obtained for mutant enzymes in complex withtriazole drugs and without added ligand, as well as for the wild type enzyme incomplex with FLC.Microdilution assays revealed that strains overexpressing ScErg11p6×His Y140F/Hor Y140F G464S had reduced susceptibility to the short-tailed triazoles FLC andvoriconazole but not the long-tailed triazole itraconazole. Strains overexpressingScErg11p6×His G464S, T322I and K151R mutants had triazole susceptibility patternssimilar to the wild type enzyme overexpressing strain but the G73E/R/W mutantsshowed increased susceptibility to all triazoles tested. Binding studies revealed thatthe triazole binding was tight for all the mutant enzymes.The high-resolution (2.05 Å) structure of wild type ScErg11p6×His in complex withFLC revealed a water-mediated hydrogen bonding network between residue Y140 andthe hydroxyl group of the drug. The crystal structures of the ScErg11p6×HisY140F/H mutants showed that these mutations disrupted the key water-mediatedhydrogen-bonding network seen in the wild type enzyme complex. The disruption ofthese interactions is proposed to weaken the interactions between the drug and themutant enzyme leading to resistance. These observations explain reducedsusceptibility to FLC and voriconazole and the retention of susceptibility toitraconazole of these mutants.The X-ray crystal structures of the ScErg11p6×His G73E/W mutants in complex withitraconazole showed that the drug bound in different conformations compared to thewild type enzyme structure. The piperazine ring of the itraconazole molecule acts as ahinge, which can adopt different conformations. The crystal structures indicatedpotential π-anion interactions between the tail of the itraconazole and the E73 residueand π stacking interactions between W73 and the tail of itraconazole. The bending ofthe drug molecule was found to accommodate each mutation. The conformation ofitraconazole bound to the G73W mutant had not been seen previously. These extrainteractions between the drug and the site of the mutation in part explain the increasedsusceptibility of G73E/W mutant strains to itraconazole.The structure of ScErg11p6×His G464S revealed that the mutated residue hadreplaced polar interactions between a water molecule and the propionate group of theheme. No obvious tilting of the heme was observed in this mutant. TheScErg11p6×His T322I and G73W mutant structures without added ligand revealedsome density in the active site and some movement of the carbonyl group of helix Iresidue G314, previously seen in the wild type ScErg11p6×His structure in complexwith lanosterol. Residue G314 may be involved in catalysis by potentially stabilisingoxygen bound heme iron intermediates.In summary, this work provides insight into the molecular interactions between thetriazole drugs and the CYP51 enzyme. The high-resolution structure of the wild typeenzyme in complex with FLC has allowed us to identify the potential basis forresistance of the Y140F/H mutants, which we confirmed by recreating thosemutations in our S. cerevisiae system. In addition, other mutations reproduced in oursystem reveal that despite a relatively high sequence similarity amongst fungalCYP51s, our model does not adequately reflect the effect of the same mutations inpathogenic fungi. This knowledge will aid in the structure-directed design of next generationazole-based antifungal drugs that can be used to overcome antifungalresistance.
Advisor: Monk, Brian; Tyndall, Joel; Keniya, Mikhail
Degree Name: Doctor of Philosophy
Degree Discipline: Sir John Walsh Research Institute
Publisher: University of Otago
Keywords: Cytochrome; P450; Saccharomyces cerevisiae; lanosterol 14 alpha demethylase; Erg11p; ERG11; crystallography; resistance mutations
Research Type: Thesis