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    Design, Synthesis and Evaluation of Peptide-Based Affinity Labels for Mu Opioid Receptors

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    Issue Date
    2009-07-17
    Author
    Sinha, Bhaswati
    Publisher
    University of Kansas
    Format
    215 pages
    Type
    Dissertation
    Degree Level
    Ph.D.
    Discipline
    Medicinal Chemistry
    Rights
    This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
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    Abstract
    Narcotic analgesics produce pain relief generally through activation of &mu opioid receptors (MOR), but the use of these analgesics is limited by their side effects, namely respiratory depression, tolerance, physical dependence and constipation. Understanding receptor-ligand interactions at the molecular level could facilitate the design of novel opioid ligands potentially with less deleterious side effects. This task is challenging since there is no crystal structure available for opioid receptors. With the aim of understanding MOR-ligand interactions, we designed novel MOR selective peptide ligands containing a reactive affinity label group. Affinity labels that interact with the receptor in a non-equilibrium manner can provide information about specific receptor-ligand interactions. We selected two MOR selective peptides: dermorphin, an endogenous ligand present in South American frog skin, and the synthetic enkephalin analog DAMGO ([D-Ala2,NMePhe4,glyol]enkephalin), for developing electrophilic affinity label derivatives. We substituted D-Orn or D-Lys in position 2 (in place of D-Ala) in both dermorphin and DAMGO, and attached a bromoacetamide or an isothiocyanate group as the electrophilic functionality to the side chain amines of the D-amino acids. For the dermorphin derivatives, we successfully identified several affinity labels with high MOR affinity (IC50 = 0.1-5 nM) and high selectivity for MOR that exhibit wash-resistant inhibition of binding to these receptors. Among these, [D-Lys(=C=S)2]dermorphin was further modified to include a purification tag (d-desthiobiotin) and a fluorescent tag (Oregon Green or 5-carboxyrhodamine B). This multifunctional affinity label peptide was synthesized successfully using an Fmoc-solid phase synthetic strategy. Initial fluorescent microscopy studies suggest irreversible labeling of MOR expressed on SH-SY5Y cells by this multifunctional peptide, thus demonstrating the utility of the fluorescent tag. For the DAMGO series of analogs, the bromoacetamide derivatives exhibited subnanomolar binding affinity (IC50 = 0.45 nM) to MOR. However, the isothiocyanate derivatives resulted in the formation of an unexpected cyclic O-alkyl thiocarbamate side product. This side reaction was successfully overcome by replacing the glyol in DAMGO by the glycylamide, yielding affinity label derivatives that exhibited subnanomolar affinity (IC 50 = 0.3-0.8 nM) and wash-resistant inhibition of MOR binding. These high affinity peptide-based affinity labels will be useful pharmacological tools to study MOR.
    URI
    http://hdl.handle.net/1808/5581
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    • Dissertations [4321]
    • Medicinal Chemistry Dissertations and Theses [81]

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    785-864-8983
    KU Libraries
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    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
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    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

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