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    INVESTIGATION OF PROTEIN AND SMALL-MOLECULE SELF-ASSOCIATION IN THE CONTEXT OF C-MYC/MAX INHIBITION

    Cover for INVESTIGATION OF PROTEIN AND SMALL-MOLECULE SELF-ASSOCIATION IN THE CONTEXT OF C-MYC/MAX INHIBITION
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    View/Open: Gerhart_georgetown_0076D_13488.pdf (39.MB) Bookview

    Creator
    Gerhart, Kaitlyn
    Advisor
    Metallo, Steven J
    Abstract
    The intrinsically disordered c-Myc oncoprotein is deregulated in ~70% of human cancers and is considered a promising but challenging drug target. To regulate transcription, c-Myc undergoes coupled folding and binding with another intrinsically disordered protein, Max, forming a basic-helix-loop-helix leucine zipper (bHLHZip) motif that interacts with the E-box DNA consensus sequence. In the early c-Myc literature, homo-oligomerization of c-Myc was observed but dismissed as only occurring at high (>20 µM) concentrations. Although it has been demonstrated in a mouse model that interfering with the c-Myc/Max interaction can lead to complete tumor regression, a clinically viable small-molecule inhibitor of the c-Myc/Max interaction remains elusive.
     
    In this work, it is found that the c-Myc bHLHZip domain dramatically quenches the fluorescent probe Alexa488 upon conjugation. Quenching occurs irrespective of c-Myc labeling position and it is determined through specific cleavage of the bHLHZip that the residues responsible for quenching are located at the N-terminus. It is shown that the quenching occurs intramolecularly and the c-Myc bHLHZip forms a disordered, compact state in solution. Unexpectedly, quenching by the bHLHZip was concentration dependent, with relief occurring at higher c-Myc concentrations, providing evidence for an intermolecular interaction between c-Myc molecules. This association begins at ~100 nM, which is much lower than the previously identified concentration for c-Myc homo-oligomerization. The formation of higher order c-Myc bHLHZip oligomers displays a pH and salt dependence.
     
    In addition to describing c-Myc self-association, the c-Myc-Max inhibitor 10074-A4 is investigated. This small-molecule was previously shown to interact enantiospecifically with the bHLHZip of c-Myc. By synthesizing the individual enantiomers of 10074-A4, the enantiospecific interaction with c-Myc is further characterized. The small-molecule undergoes formation of a chiral self-assembly which may have interfered with previous affinity measurements. The affinities of R-10074-A4 and S-10074-A4 for Myc353-437 are tenfold stronger than previously reported (Kd=1.9±0.2 µM and 2.6±0.5 µM respectively). Synthesis of 10074 A4 derivatives an initial structure activity relationship for 10074-A4/c-Myc binding is reported. Although this system is prone to challenges stemming from the self-association of both small-molecule inhibitors and the c-Myc protein, these studies elaborate upon an example of an IDP/small-molecule interaction with single-digit micromolar affinity.
     
    Description
    Ph.D.
    Permanent Link
    http://hdl.handle.net/10822/1042819
    Date Published
    2016
    Subject
    10074-A4; Alexa 488 quenching; c-Myc; fuzzy complex; intrinsically disordered proteins; small-molecule inhibitor; Biophysics; Biochemistry; Biophysics; Biochemistry;
    Type
    thesis
    Embargo Lift Date
    2019-01-06
    Publisher
    Georgetown University
    Extent
    340 leaves
    Collections
    • Graduate Theses and Dissertations - Chemistry
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    Georgetown University Seal
    ©2009 - 2022 Georgetown University Library
    37th & O Streets NW
    Washington DC 20057-1174
    202.687.7385
    digitalscholarship@georgetown.edu
    Accessibility