Sugar coated armour: exploring UDP galactopyranose mutase (UGM) as a potential antifungal target
Lead Research Organisation:
University of Dundee
Department Name: College of Life Sciences
Abstract
Modern medical treatments including advances in cancer therapy and organ transplantation render patients highly susceptible to infection due to a weakened immune system. Fungal infections are often fatal in these patients. Aspergillus fumigatus is a fungus that produces spores found widely in the environment. In healthy people inhaled spores are harmless and eliminated by the immune system but in certain groups, including those with existing lung problems, they cause disease. The most serious, ‘invasive Aspergillosis‘, leads to life-threatening infections that are notoriously difficult to diagnose and treat.
A shield of sugar-coated armour makes up the cell wall surrounding and protecting the fungus. There are still many gaps in our knowledge of this important structure. These need addressed by scientists and clinicians using novel laboratory based approaches. With assistance from the MRC, I seek to improve our understanding of the structure and function of a protein (enzyme) responsible for producing one of the novel sugars and to devise a way of identifying small molecules to block its function. This work is a crucial first step towards ultimately improving diagnostic tests and treatments for the increasing burden of fungal infections.
A shield of sugar-coated armour makes up the cell wall surrounding and protecting the fungus. There are still many gaps in our knowledge of this important structure. These need addressed by scientists and clinicians using novel laboratory based approaches. With assistance from the MRC, I seek to improve our understanding of the structure and function of a protein (enzyme) responsible for producing one of the novel sugars and to devise a way of identifying small molecules to block its function. This work is a crucial first step towards ultimately improving diagnostic tests and treatments for the increasing burden of fungal infections.
Technical Summary
The fungal cell wall is a dynamic and multi-layered structure almost entirely built from polysaccharides, consisting of a core of cross linked chitin and glucan. Less well characterised is the outermost galactomannan layer of linear mannan branched by short galactofuranose (Galf) chains that are displayed to the host immune system. Crucially, Galf is produced by a single enzyme, UDP-galactopyranose mutase (UGM), that catalyses the reaction UDP-galactopyranose to UDP-Galf.
Understanding the biosynthetic pathways in cell wall synthesis is imperative in the drive for structure-based drug design of the next generation of antifungals. This is particularly pressing given the rise in fungal infections in the immunocompromised with cases of invasive aspergillosis due to Aspergillus fumigatus invariably fatal having limited diagnostic and therapeutic options. There is renewed interest in agents targeting the fungal cell wall following the introduction of the echinocandins and this proposal explores a potential new target.
Using a multidisciplinary approach covering genetics, structural biology and high throughput screening I aim to provide concerted genetic and chemical validation of A. fumigatus UGM (AfUGM). Specific objectives and methodologies include:
AfUGM genetic validation: based on two reported knockout strains, the role of AfUGM catalytic activity will be studied by complementing these strains with catalytically inactive mutant forms followed by phenotypic analyses.
AfUGM molecular mechanism: the AfUGM crystal structure will be determined based on initial crystals/recombinant protein already available in the laboratory, an existing enzyme assay will be optimised and the mechanism/active site probed by site-directed mutagenesis.
AfUGM inhibitors: the enzyme assay will be developed into a high-throughput screening (HTS) format and fragment/drug-derived libraries available in the Drug Discovery Unit screened to evaluate the druggability of AfUGM as a target.
The output will be increased knowledge of AfUGM and the functional implications of impaired Galf synthesis alongside rigorous research training.
Understanding the biosynthetic pathways in cell wall synthesis is imperative in the drive for structure-based drug design of the next generation of antifungals. This is particularly pressing given the rise in fungal infections in the immunocompromised with cases of invasive aspergillosis due to Aspergillus fumigatus invariably fatal having limited diagnostic and therapeutic options. There is renewed interest in agents targeting the fungal cell wall following the introduction of the echinocandins and this proposal explores a potential new target.
Using a multidisciplinary approach covering genetics, structural biology and high throughput screening I aim to provide concerted genetic and chemical validation of A. fumigatus UGM (AfUGM). Specific objectives and methodologies include:
AfUGM genetic validation: based on two reported knockout strains, the role of AfUGM catalytic activity will be studied by complementing these strains with catalytically inactive mutant forms followed by phenotypic analyses.
AfUGM molecular mechanism: the AfUGM crystal structure will be determined based on initial crystals/recombinant protein already available in the laboratory, an existing enzyme assay will be optimised and the mechanism/active site probed by site-directed mutagenesis.
AfUGM inhibitors: the enzyme assay will be developed into a high-throughput screening (HTS) format and fragment/drug-derived libraries available in the Drug Discovery Unit screened to evaluate the druggability of AfUGM as a target.
The output will be increased knowledge of AfUGM and the functional implications of impaired Galf synthesis alongside rigorous research training.