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    Characterization and Exploitation of Divergent Substrate Binding Within HspA5 for the Development of Selective Inhibitors

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    Author
    Ambrose, Andrew
    Issue Date
    2020
    Keywords
    Assay
    Cancer
    Evolution
    Grp78
    Hsp70
    Inhibitor
    Advisor
    Chapman, Eli
    
    Metadata
    Show full item record
    Publisher
    The University of Arizona.
    Rights
    Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction, presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
    Embargo
    Release after 04/07/2021
    Abstract
    Hsp70s are among the most highly conserved proteins in all of biology. These molecular machines function through cofactor interaction and ATP binding and hydrolysis. Through iterative binding and release of exposed hydrophobic residues on client proteins, Hsp70s are able to prevent intermolecular aggregation and promote folding progression toward the native sate. The human proteome contains 8 canonical Hsp70 proteins that vary in tissue specificity, inducibility, and organellular localization. Because Hsp70s are relatively promiscuous with regard to client selection, Hsp70s play a significant role in folding a large proportion of the proteome. Hsp70s are implicated in numerous neurodegenerative diseases and cancer because of their ability to regulate protein homeostasis within cells. In recent years, many groups have attempted to develop selective inhibitors of Hsp70 isoforms to better understand the role of individual isoforms in biology. Through rational design, forced localization, and serendipity, inhibitors have been discovered, however discovery of a molecule that interacts with a single isoform remains elusive. Herein lies a report validating the substrate binding domain of Hsp70 as a targetable site for the development of selective inhibitors. Using a fluorescence polarization (FP) peptide binding assay, a pilot screen revealed hexachlorophene as a selective inhibitor of HspA5. While this molecule is not a lead, it serves as a proof a concept that small molecules can be selective towards Hsp70 isoforms through interaction with the substrate binding domain. From here, an expansion of the assay and scale up of the screen revealed 4.8 as a potent (IC50 = 162 nM) and selective HspA5 inhibitor (20-fold for HspA5 vs HspA9 and at least 300-fold for all other Hsp70s). Through the development of the assay used to discover these compounds it was discovered that Hsp70 isoforms appear to interact differently with substrate peptides. From here, the peptide binding pattern of each canonical human Hsp70 isoform was characterized which led to the development of an algorithm for predicting client interactions with specific Hsp70s. These results highlight the functional variance that exists between Hsp70s in their interaction with peptide substrates, and the possibility for exploiting these differences in inhibitor development.
    Type
    text
    Electronic Dissertation
    Degree Name
    Ph.D.
    Degree Level
    doctoral
    Degree Program
    Graduate College
    Pharmacology & Toxicology
    Degree Grantor
    University of Arizona
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