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dc.contributor.advisorMonk, Brian
dc.contributor.advisorTyndall, Joel
dc.contributor.advisorKeniya, Mikhail
dc.contributor.authorSagatova, Alia
dc.date.available2016-07-13T21:18:06Z
dc.date.copyright2016
dc.identifier.citationSagatova, A. (2016). Investigating resistance mutations in the drug target of triazole drugs (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/6697en
dc.identifier.urihttp://hdl.handle.net/10523/6697
dc.description.abstractFungal infections affect a broad spectrum of the population, including premature babies, the elderly and individuals with a range of disease- or medically-induced comorbidities. Fungal pathogens such as Candida albicans and Aspergillus fumigatus cause a variety of conditions, from minor infections to life threatening disease. Some fungi, including C. albicans and Candida glabrata can be commensal living in harmony with the superficial microflora, but these organisms can behave as opportunistic pathogens when individuals become immunodeficient due to comorbidities or become immunocompromised due to AIDS or through medical intervention. Fungal infections have become recognised in recent years as a growing health burden that currently causes around 1.5 million deaths per year worldwide. The azole antifungal drugs (imidazoles and triazoles) are used widely to treat fungal infections and as antifungal prophylaxis. The azole drugs target the fungal enzyme lanosterol 14α-demethylase (Erg11p, CYP51). This monospanning bitopic membrane protein belongs to the CYP51 class in the cytochrome P450 superfamily of enzymes and is involved in the rate-limiting step of ergosterol biosynthesis. Ergosterol is the fungal equivalent of cholesterol and is required for fungal cell growth. Fungal pathogens have evolved several mechanisms of resistance that diminish the action of the azole drugs. The emergence of resistant fungal strains due to mutations in CYP51 can limit therapeutic options and make treatment of fungal infections increasingly problematic. The need for better drugs that overcome resistance is becoming increasingly urgent. The present project builds on the research of Monk et al. who successfully crystallised and obtained the first high-resolution X-ray structures of a fungal CYP51. The aim of this project is to investigate the effect of CYP51 mutations on enzyme structure and function, including different types of triazole drug, by using Saccharomyces cerevisiae Erg11p as a model for the homologous enzymes in pathogenic fungi. A S. cerevisiae overexpression system was used to hyper-express the wild type and the mutant ScErg11p enzymes in order to obtain sufficient quantities of protein for structure-function studies. The C. albicans CYP51 mutations Y132F/H, K143R, G464S and the double mutation Y132F G464S (Y140F/H, K151R, G464S and Y140F G464S 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-terminal hexahistidine-tagged version of S. cerevisiae Erg11p (ScErg11p6×His). In addition, the innate resistance of A. fumigatus CYP51A to fluconazole (FLC) was investigated using the S. cerevisiae Erg11p T322I mutant. Microdilution assays were used to determine triazole susceptibilities of these strains. ScErg11p6×His mutant and wild type enzymes were purified from crude membranes by solubilisation with the detergent n-decyl-β-D-maltoside followed by affinity and size exclusion chromatography. Spectral analysis of the purified protein was used to determine dissociation constants for triazole drugs. Purified preparations of the enzyme were also 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 with triazole drugs and without added ligand, as well as for the wild type enzyme in complex with FLC. Microdilution assays revealed that strains overexpressing ScErg11p6×His Y140F/H or Y140F G464S had reduced susceptibility to the short-tailed triazoles FLC and voriconazole but not the long-tailed triazole itraconazole. Strains overexpressing ScErg11p6×His G464S, T322I and K151R mutants had triazole susceptibility patterns similar to the wild type enzyme overexpressing strain but the G73E/R/W mutants showed increased susceptibility to all triazoles tested. Binding studies revealed that the triazole binding was tight for all the mutant enzymes. The high-resolution (2.05 Å) structure of wild type ScErg11p6×His in complex with FLC revealed a water-mediated hydrogen bonding network between residue Y140 and the hydroxyl group of the drug. The crystal structures of the ScErg11p6×His Y140F/H mutants showed that these mutations disrupted the key water-mediated hydrogen-bonding network seen in the wild type enzyme complex. The disruption of these interactions is proposed to weaken the interactions between the drug and the mutant enzyme leading to resistance. These observations explain reduced susceptibility to FLC and voriconazole and the retention of susceptibility to itraconazole of these mutants. The X-ray crystal structures of the ScErg11p6×His G73E/W mutants in complex with itraconazole showed that the drug bound in different conformations compared to the wild type enzyme structure. The piperazine ring of the itraconazole molecule acts as a hinge, which can adopt different conformations. The crystal structures indicated potential π-anion interactions between the tail of the itraconazole and the E73 residue and π stacking interactions between W73 and the tail of itraconazole. The bending of the drug molecule was found to accommodate each mutation. The conformation of itraconazole bound to the G73W mutant had not been seen previously. These extra interactions between the drug and the site of the mutation in part explain the increased susceptibility of G73E/W mutant strains to itraconazole. The structure of ScErg11p6×His G464S revealed that the mutated residue had replaced polar interactions between a water molecule and the propionate group of the heme. No obvious tilting of the heme was observed in this mutant. The ScErg11p6×His T322I and G73W mutant structures without added ligand revealed some density in the active site and some movement of the carbonyl group of helix I residue G314, previously seen in the wild type ScErg11p6×His structure in complex with lanosterol. Residue G314 may be involved in catalysis by potentially stabilising oxygen bound heme iron intermediates. In summary, this work provides insight into the molecular interactions between the triazole drugs and the CYP51 enzyme. The high-resolution structure of the wild type enzyme in complex with FLC has allowed us to identify the potential basis for resistance of the Y140F/H mutants, which we confirmed by recreating those mutations in our S. cerevisiae system. In addition, other mutations reproduced in our system reveal that despite a relatively high sequence similarity amongst fungal CYP51s, our model does not adequately reflect the effect of the same mutations in pathogenic fungi. This knowledge will aid in the structure-directed design of next generation azole-based antifungal drugs that can be used to overcome antifungal resistance.
dc.format.mimetypeapplication/pdf
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectCytochrome
dc.subjectP450
dc.subjectSaccharomyces cerevisiae
dc.subjectlanosterol 14 alpha demethylase
dc.subjectErg11p
dc.subjectERG11
dc.subjectcrystallography
dc.subjectresistance mutations
dc.titleInvestigating resistance mutations in the drug target of triazole drugs
dc.typeThesis
dc.date.updated2016-07-13T05:03:50Z
dc.language.rfc3066en
thesis.degree.disciplineSir John Walsh Research Institute
thesis.degree.nameDoctor of Philosophy
thesis.degree.grantorUniversity of Otago
thesis.degree.levelDoctoral
otago.openaccessOpen
otago.evidence.presentYes
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