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    Targeting Melanocortin and Cholecystokinin Receptors via Multivalent Molecules Bearing Peptide Ligands

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    Author
    Nakath Gamlath Ralalage, Dayan Elshan
    Issue Date
    2014
    Keywords
    Cholecystokinin
    HEK293
    Melanocortin
    Multivalent
    Time resolved fluorescence
    Binding assay
    Chemistry
    Advisor
    Mash, Eugene A Jr.
    
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    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 or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
    Abstract
    Peptide receptor overexpression in diseased cells and tissues, including carcinomas provides an opportunity to develop therapeutics and imaging agents that selectively bind to such cells and tissues. This dissertation presents tools and processes that can be utilized to target melanocortin and cholecystokinin receptors through multivalent binding. In Chapter 2, improved synthesis and purification methods are described for the production of Eu-chelated probes that serve to evaluate the binding efficacy of multivalent molecules through competition binding assays. Specifically, a xylenol orange-based assay for quantification of unchelated metal ions was used to determine unbound metal ion contamination and the success of metal ion removal. The use of Empore™ chelating disks was determined to be the method of choice for the selective removal of unchelated Eu ions from several Eu-diethylenetriaminepentaacetic acid chelate-peptide conjugates. Applying new synthesis and purification strategies, the TRF probe Eu-DTPA-PEGO-CCK4 targeted to cholecystokinin receptors was synthesized (Chapter 2) and validated via saturation and competition binding assays (Chapter 4) using a HEK293 cell line overexpressing the human cholecystokinin 2 receptor. In Chapter 3, short and efficient syntheses of multivalent molecules targeted to melanocortin receptors based on three commercially available trigonal core scaffolds, phloroglucinol, tripropargylamine, and 1,4,7-triazacyclononane, are described. These constructs were designed to further test the 24 ±5 Å inter-ligand distance suggested in recent literature for multivalent binding to melanocortin receptors. The bioactivities of these compounds were evaluated using a competitive binding assay that employed HEK293 cells engineered to overexpress the human melanocortin 4 receptor. In the course of conducting these bioassays, novel in vitro binding assay protocols were established, which led to high repeatability and robustness of the bioassays compared to previous methods. The divalent molecules exhibited 10- to 30-fold higher levels of inhibition when compared to the corresponding monovalent molecules, consistent with divalent binding. The trivalent molecules were only statistically (~2-fold) better than the divalent molecules, still consistent with divalent binding but inconsistent with trivalent binding. Possible reasons for these behaviors and planned refinements of the multivalent constructs targeting melanocortin receptors based on these scaffolds are discussed in Chapters 3 and 6.
    Type
    text
    Electronic Dissertation
    Degree Name
    Ph.D.
    Degree Level
    doctoral
    Degree Program
    Graduate College
    Chemistry
    Degree Grantor
    University of Arizona
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