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Title: Assessment of Aspergillus kinases as targets for antifungal drug discovery
Author: Al-Furaiji, Narjes
ISNI:       0000 0004 9354 1702
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2020
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Fungi cause a wide range of infections with a significant increment in annual burden, and a total estimate of 1.5 million deaths per year, with over a billion being at risk was reported recently. Fungal treatments are limited to certain classes of drugs. However, undesirable side effects, drug–drug interactions and the toxicity associated with some classes and the emergence of resistance with others has limited their use; thus, new antifungal drugs are needed. Protein kinases constitute the second most common group of drug targets after G-protein-coupled receptors, since they regulate most aspects of cell life by phosphorylation. In this project, we aimed to explore the druggability of kinases in the human pathogen A. fumigatus, in an attempt to address the scarcity of known antifungal agents through the validation of completely novel drug targets that can overcome the problems associated with current antifungal drugs, particularly provoking the increasing emergence of antifungal drug resistance. Through a process of identification of protein kinases, 175 kinases were annotated in A. fumigatus A1163, identifying 15 filamentous fungal specific kinases, and other highly conserved kinases that share less than 50% similarity with humans, and they represent prospective antifungal targets. A high-throughput gene knockout strategy enabled the disruption of 115 PK genes, with 39 genes being identified as indispensable for viability; nine out of the total share < 40% sequence similarity with human PKs, while two are Ffks. These findings heighten the potential of developing successful selective kinase inhibitors and maximises the potential therapeutic efficacy while potentially minimising human effects. We developed a competitive fitness study in this project, utilising a PK knockout library of 65 mutants. This study enabled identification of two itraconazole-resistant strains (Δssn3 and Δfus3) and one sensitive strain (Δyak1). Resistance was associated with upregulation of cyp51A, cyp51B and cdr1B by 1.8-, 4.8- and 1.95-fold, respectively, in Δssn3 compared to the wild type, which provide some insight into molecular mechanism of resistance. Ssn3 is highly conserved among Aspergillus species with >90% sequence similarity, while it shares < 50% sequence similarity with human, which maximise its potential as an antifungal drug target. Virulence studies identified three PK mutants that shown reduced fitness: ΔPSK1 in the larval model, ΔRim15 in the larval model, and Δkin4 in the macrophage cell line and larvae models. Interestingly, their orthologs appeared to be contributing to virulence. These findings serve to underscore the importance of these PK genes and their requirement for pathogenicity and to be a potential antifungal target. The current results provide greater insight into the role of PKs in A. fumigatus 1163 and heighten their potential as novel antifungal drugs.
Supervisor: Bromley, Michael ; Bowyer, Paul Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: antifungal ; drug discovery ; Protein kinase