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Design, Synthesis and Evaluation of Peptide-Based Affinity Labels for Mu Opioid Receptors
Sinha, Bhaswati
Sinha, Bhaswati
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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.
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Date
2009-07-17
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University of Kansas
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Keywords
Pharmaceutical chemistry, Affinity labels, Damgo, Dermorphin, Multifunctional, Opioids, Peptides