Discovery of Dermorphin-Based Affinity Labels with Subnanomolar Affinity for Mu Opioid Receptors

Abstract

A series of potent electrophilic affinity labels (IC 50 = 0.1-5 nM) containing either a bromoacetamide or isothiocyanate based on the mu opioid receptor (MOR) selective peptide dermorphin were prepared. All four analogs exhibited wash resistant inhibition of [ 3 H]DAMGO binding at subnanomolar to nanomolar concentrations, suggesting that these analogs bind covalently to MOR. To our knowledge these peptides are the highest affinity peptide-based affinity labels for MOR reported to date.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, constipation and physical dependence. 1 Therefore there is an ongoing need to develop novel analgesics with fewer side effects. Understanding receptor-ligand interactions at the molecular level can facilitate the design of novel opioid ligands. Since the cloning of the three major opioid receptors, MOR, delta opioid receptors (DOR) and kappa opioid receptors (KOR), in the 1990s and determination of their sequences, 2, 3 there have been considerable advancements in understanding opioid receptor-ligand interactions. These studies have utilized chimeric receptors (such as MOR/KOR chimeras, etc.) and site-directed mutagenesis. 4 Although these approaches have provided considerable information regarding receptor-ligand interactions, interpreting the results can be complicated by changes in the secondary or tertiary structures of the proteins. 4 Also while these approaches provide information about which residues in the receptor may interact with the ligand, they often do not provide information about what portions of the ligand are involved in these interactions. Affinity labels, which are ligands that interact with their target in a non-equilibrium manner, 5 can provide detailed information about specific receptor-ligand interactions, 6, 7 and the information obtained from affinity labels can compliment results obtained from molecular biology and computational methods. The interaction of affinity labels with the receptor occurs in a two-step manner. 5 In the first step, the ligand binds reversibly to its receptor. In the second step, which can further increase the selectivity of the ligand for its receptor, the ligand binds irreversibly, provided an appropriate nucleophile in the receptor is in close proximity to the reactive group in the ligand. Affinity labels can be either photoaffinity or electrophilic affinity labels. The electrophilic affinity label naltrexamine derivative β-funaltrexamine (β-FNA), a well studied affinity label for MOR, was the first affinity label (and one of only two affinity labels 8 ) for opioid receptors whose covalent attachment point (Lys 233 in MOR) has been successfully determined. 7Although a number of non-peptide affinity labels for opioid receptors have been reported in the literature, 1, 5 until recently peptide-based affinity labels have been mostly limited to photoaffinity labels. 5 A ...

Solution-phase-peptide synthesis via the group-assisted purification (GAP) chemistry without using chromatography and recrystallization

“…The new GAP strategy is presented by the synthesis biphalin peptides that belong to the opioid pain killer series. [33][34][35][36][37] The original biphalin is a highly potent analgesic candidate but shows low selectivity between d and m receptors. [35][36][37][38][39] Its structural modifications are anticipated to generate new pain killers of high potency and selectivity so as to reduce unwanted side effects.…”

Electrophilic, Activation-Free Fluorogenic Reagent for Labeling Bioactive Amines

“…a Determined using fluorescein in basic EtOH as standard (QY: 0.92). [17] In view of the high extinction coefficient and quantum yield of the BODIPY dipolar acid fluoride 2b, we decided to examine its application as a labeling reagent for natamycin, a naturallyoccurring antifungal agent with high affinity and selectivity for fungal cells. Natamycin blocks fungal growth by binding specifically to ergosterol without permeabilizing the membrane where it inhibits vacuole fusion at the priming phase and interferes with membrane protein functions.…”

“…[7] In the context of small molecule labeling, most electrophilic tagging protocols rely on a variety of reactions, including conjugate additions and SNAR, as well as the conventional formation of amides from carboxylic acids and amines with participation of a variety of coupling reagents. [9][10][11][12][13][14][15][16][17][18][19][20] These conjugation reactions proceed with high yields but render side products (e.g. NHS in the case of succinidimyl esters) and, to some extent, are sensitive to small amounts of water.…”

A multi-functional peptide as an HIV-1 entry inhibitor based on self-concentration, recognition, and covalent attachment

“…28,29 After the deprotection, the resin was washed by DCM 5 times then DMF 5 times. To obtain the isothiocyanate derivative, the resin was treated with thiocarbonyldiimidazole (TCD) (4 equiv) in a minimum amount of DMF (3 mL) for 4 h. 30 To obtain the isothiocyanate modified peptides, the cleavage cocktail (following the general procedure) was applied for 0.5 h to detach the peptide from the resin. The purified dry peptide was stored at −20 °C.…”