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    Targeting the Histone Acetyl-Transferase, RTT109, for Novel Anti-Fungal Drug Development: A Dissertation

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    Authors
    Lopes da Rosa-Spiegler, Jessica
    Faculty Advisor
    Paul Kaufman, Ph.D.
    Academic Program
    Interdisciplinary Graduate Program
    UMass Chan Affiliations
    Molecular, Cell and Cancer Biology
    Document Type
    Doctoral Dissertation
    Publication Date
    2012-05-03
    Keywords
    Histone Acetyltransferases
    Candida albicans
    Antifungal Agents
    Molecular Targeted Therapy
    Biochemistry, Biophysics, and Structural Biology
    Cells
    Chemical Actions and Uses
    Enzymes and Coenzymes
    Fungi
    Hemic and Immune Systems
    Immunology and Infectious Disease
    Microbiology
    Pathology
    Pharmaceutical Preparations
    Therapeutics
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    Abstract
    Discovery of new antifungal chemo-therapeutics for humans is limited by the large degree of conservation among eukaryotic organisms. In recent years, the histone acetyl-transferase Rtt109 was identified as the sole enzyme responsible for an abundant and important histone modification, histone H3 lysine 56 (H3K56) acetylation. In the absence of Rtt109, the lack of acetylated H3K56 renders yeast cells extremely sensitive to genotoxic agents. Consequently, the ability to sustain genotoxic stress from the host immune system is crucial for pathogens to perpetuate an infection. Because Rtt109 is conserved only within the fungal kingdom, I reasoned that Rtt109 could be a novel drug target. My dissertation first establishes that genome stability provided by Rtt109 and H3K56 acetylation is required for Candida albicans pathogenesis. I demonstrate that mice infected with rtt109 -/- cells experience a significant reduction in organ pathology and mortality rate. I hypothesized that the avirulent phenotype of rtt109 -/- cells is due to their intrinsic hypersensitivity to the genotoxic effects of reactive oxygen species (ROS), which are utilized by phagocytic cells of the immune system to kill pathogens. Indeed, C. albicans rtt109 -/- cells are more efficiently killed by macrophages in vitro than are wild-type cells. However, inhibition of ROS generation in macrophages renders rtt109 -/- and wild-type yeast cells equally resilient to killing. These findings support the concept that ability to resist genotoxic stress conferred by Rtt109 and H3K56 acetylation is a virulence factor for fungal pathogens and establish Rtt109 as an opportune drug- target for novel antifungal therapeutics. Second, I report the discovery of a specific chemical inhibitor of Rtt109 catalysis as the initial step in the development of a novel antifungal agent. We established a collaboration with the Broad Institute (Cambridge, MA) to perform a high-throughput screen of 300,000 compounds. From these, I identified a single chemical, termed KB7, which specifically inhibits Rtt109 catalysis, with no effect on other HAT enzymes tested. KB7 has an IC50 value of approximately 60 nM and displays noncompetitive inhibition regarding both acetyl-coenzyme A and histone substrates. With the genotoxic agent camptothecin, KB7 causes a synergistic decrease in C. albicans growth rate. However, this effect is only observed in an efflux-pump mutant, suggesting that this compound would be more effective if it were better retained intracellularly. Further studies through structure-activity relationship (SAR) modifications will be conducted on KB7 to improve its effective cellular concentration.
    DOI
    10.13028/w35r-7869
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/31973
    Rights
    Copyright is held by the author, with all rights reserved.
    ae974a485f413a2113503eed53cd6c53
    10.13028/w35r-7869
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