Leu-enkephalin analogues, in which the amide bonds were sequentially and systematically replaced either by ester or N-methyl amide bonds, were prepared using classical organic chemistry as well as solid phase peptide synthesis (SPPS). The peptidomimetics were characterized using competition binding, ERK1/2 phosphorylation, receptor internalization, and contractility assays to evaluate their pharmacological profile over the delta opioid receptor (DOPr). The lipophilicity (LogD7.4) and plasma stability of the active analogues were also measured. Our results revealed that the last amide bond can be successfully replaced by either an ester or an N-methyl amide bond without significantly decreasing the biological activity of the corresponding analogues when compared to Leu-enkephalin. The peptidomimetics with an N-methyl amide function between residues Phe and Leu were found to be more lipophilic and more stable than Leu-enkephalin. Findings from the present study further revealed that the hydrogen-bond donor properties of the fourth amide of Leu-enkephalin are not important for its biological activity on DOPr. Our results show that the systematic replacement of amide bonds by isosteric functions represents an efficient way to design and synthesize novel peptide analogues with enhanced stability. Our findings further suggest that such a strategy can also be useful to study the biological roles of amide bonds.
In this study, we used a combination of traditional signaling investigation approaches, bioluminescence resonance energy transfer (BRET) biosensors, and the label-free approach surface plasmon resonance (SPR) spectroscopy to monitor the signaling cascades of the m-opioid receptor (MOP). In human embryonic kidney cells stably expressing a Flag-tagged version of human MOP, we compared the signals triggered by the noninternalizing and internalizing MOP agonists morphine and DAMGO (Tyr-DAla-Gly-N-methyl-Phe-Gly-ol), respectively. We studied three major and well described components of MOP signaling: receptor internalization, G protein coupling, and activation of extracellular signal-regulated kinase ERK1/ERK2. Our results show that morphine and DAMGO display different profiles of receptor internalization and a similar ability to trigger the phosphorylation of ERK1/ERK2. Our SPR analyses revealed that morphine and DAMGO evoke similar SPR signatures and that Ga i , cAMP-dependent pathways, and ERK1/ERK2 have key roles in morphine-and DAMGO-mediated signaling. Most interestingly, we found that the so-called MOP neutral antagonists CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH 2 ), naloxone, and naltrexone behave like partial agonists. Even more intriguing, BRET experiments indicate that CTAP (D-Phe-CysTyr-D-Trp-Arg-Thr-Pen-Thr-NH 2 ) induces similar conformational changes as naltrexone at the Ga i -bg interface, whereas it appears as an inverse agonist based on its SPR response thus indicating distinct signaling mechanisms for the two ligands. Taken together, our results support the usefulness of labelfree methods such as SPR to study whole-cell responses and signaling cascades triggered by G protein-coupled receptors and complement the conventional approaches by revealing cellular responses that would have been otherwise undetectable.
In this review, the loading efficacies of helper and Cationic Lipids Cholesterol
(CHOL), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), Dioctadecyl Dimethyl-
Ammonium Bromide (DDAB) and Dioleoyl Phosphatidylethanolamine (DOPE) with milk β-
lactoglobulin, α-casein and β-casein were compared in aqueous solution at physiological conditions.
Structural analysis showed that lipids bind milk proteins via hydrophilic, hydrophobic
and H-bonding contacts with DOTAP and DDAB forming more stable protein conjugates.
Loading efficacy was 30-50% and enhanced with cationic lipids. Lipid conjugation altered
protein conformation, causing a partial protein structural destabilization. Milk proteins are
capable of transporting lipids in vitro.
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