The nucleolus as a therapeutic target for A fumigatus

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DESCRIPTION (provided by the applicant): Aspergillus fumigatus is an opportunistic fungal pathogen that causes severe and often fatal infections in patients with depressed immunity. Although the incidence of invasive aspergillosis is continuing to rise, reflecting the increased population of immunosuppressed patients, there are only a few antifungal agents available for treatment and their efficacy is severely hampered by problems with toxicity and drug resistance. Since most patients will die from aspergillosis despite having received treatment, there is an urgent need for the development of new drugs to treat this disease. In this grant we are proposing that nucleolar proteins are important considerations for novel therapeutic targets because of their potent effects on protein synthesis. Our preliminary data has identified a conserved fungal-specific nucleolar protein that is essential for growth in S. cerevisiae, and we are proposing to test the hypothesis that the A. fumigatus ortholog, AfcgrA, defines a pathway of nucleolar function that would be an effective new target for therapy. The goal of Aim I is to use AfcgrA-deficient mutants of A. fumigatus to model the therapeutic efficacy of a putative nucleolus-targeting drug. The AfcgrA gene will be disrupted by gene targeting approaches and the extent to which the mutant alleles impair the rate of disease progression or pattern of tissue dissemination will be compared in two in vivo models of aspergillosis. The goal of Aim II is to acquire information on AfcgrA interactions that will facilitate future application of structure-based drug design to the identification of AfcgrA inhibitors. We will use the yeast two-hybrid system to identify the molecular partners of AfcgrA, and gain insight into the structural requirements for these interactions by determining the domain organization of AfcgrA with circular dichroism spectroscopy. The goal of Aim III is to determine the regions of AfcgrA that specify nucleolar localization. We propose to define these sequences, anticipating that an understanding of the mechanism used to localize AfcgrA could be exploited in the design of selective inhibitors of this transport process.