The Design, Synthesis and Evaluation of Peptide Ligands to Study Opioid Receptors

Abstract

The δ opioid receptor (DOR) is involved in the modulation of μ opioid receptor (MOR) agonist mediated side effects such as the development of tolerance, and MOR and DOR may interact to form heterodimers capable of unique pharmacological signaling. Understanding the molecular interactions involved in ligand recognition by opioid receptors will aid in the design of novel therapeutics that target the opioid receptors with fewer side effects. The aim of this research was to develop and use opioid peptide ligands to examine ligand-receptor interactions at the molecular level. We designed a multifunctional peptide-based affinity label derivative of the high affinity DOR selective peptide antagonist TIPP (Tyr-Tic-Phe-Phe, Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) to irreversibly interact with DOR. The multifunctional peptide also contains a fluorescent group and multiple tags to aid in the detection and isolation of labeled DOR. The initial solid phase peptide synthesis of the multifunctional peptide generated multiple side products and required extensive evaluation and optimization to increase the efficiency of peptide synthesis. A model peptide that contained only the affinity label and a fluorescent tag (at 65 nM) demonstrated 95% wash resistant inhibition of binding to DOR suggesting that this peptide is an affinity label for DOR. We also report the synthesis and initial binding analysis of a heterobivalent peptide-based affinity label targeting the proposed MOR-DOR heterodimer. This peptide demonstrated nanomolar affinity to both DOR and MOR in radioligand competition assays, and at 96 nM exhibited 94% wash resistant inhibition of binding to DOR, suggesting that the heterobivalent peptide is an affinity label for DOR. Thus, we successfully prepared two series of novel opioid peptide ligands which appear to be affinity labels for DOR. One series of peptides contains multiple functional groups to aid in DOR isolation, detection and analysis. The second peptide consists of two pharmacophores incorporated into a single entity to study proposed MOR-DOR heterodimers. These peptides will be useful pharmacological tools to study opioid receptors.