¹H NMR spectra showing NH of indole moiety and aromatic protons of 1 in the free state (R = Htel/[1] = 0) and at different R. High and low R values must be related to the free and bound state of 1 in solution respectively.

<p>¹H NMR spectra showing NH of indole moiety and aromatic protons of 1 in the free state (R = Htel/[1] = 0) and at different R. High and low R values must be related to the free and bound state of 1 in solution respectively.</p

Results obtained by the competitive dialysis assays.

<p>The amount of ligand bound to each DNA structure is shown as a bar graph.</p

Pharmacological Characterization of μ-Opioid Receptor Agonists with Biased G Protein or β-Arrestin Signaling, and Computational Study of Conformational Changes during Receptor Activation

In recent years, G protein vs. β-arrestin biased agonism at opioid receptors has been proposed as an opportunity to produce antinociception with reduced adverse effects. However, at present this approach is highly debated, a reason why more information about biased ligands is required. While the practical relevance of bias in the case of µ-opioid receptors (MOP) still needs to be validated, it remains important to understand the basis of this bias of MOP (and other GPCRs). Recently, we reported two cyclopeptides with high affinity for MOP, the G protein biased Dmt-c[d-Lys-Phe-pCF3-Phe-Asp]NH2 (F-81), and the β-arrestin 2 biased Dmt-c[d-Lys-Phe-Asp]NH2 (C-33), as determined by calcium mobilization assay and bioluminescence resonance energy transfer-based assay. The biased character of F-81 and C-33 has been further analyzed in the [35S]GTPγS binding assay in human MOP-expressing cells, and the PathHunter enzyme complementation assay, used to measure β-arrestin 2 recruitment. To investigate the structural features of peptide-MOP complexes, we performed conformational analysis by NMR spectroscopy, molecular docking, and molecular dynamics simulation. These studies predicted that the two ligands form alternative complexes with MOP, engaging specific ligand–receptor contacts. This would induce different displays of the cytosolic side of the seven-helices bundle, in particular by stabilizing different angulations of helix 6, that could favor intracellular coupling to either G protein or β-arrestin

Structure of 1 and 2.

<p>Structure of 1 and 2.</p

Imino protons regions of the NMR spectra of Htel (a) Htel/1(b) and Htel/2 (c) at different temperatures.

<p>Imino protons regions of the NMR spectra of Htel (a) Htel/1(b) and Htel/2 (c) at different temperatures.</p

¹H NMR spectra (11.5–10.2 ppm and 9.0–7.0 ppm), acquired at T = 25°C in H₂O, containing 25 mM KH₂PO₄, KCl 150 mM and EDTA 1 mM (pH 6.7), showing resonance of imino protons G4, G5 and G6 as well as the aromatic protons at different R = [1]/Htel_.

<p>¹H NMR spectra (11.5–10.2 ppm and 9.0–7.0 ppm), acquired at T = 25°C in H₂O, containing 25 mM KH₂PO₄, KCl 150 mM and EDTA 1 mM (pH 6.7), showing resonance of imino protons G4, G5 and G6 as well as the aromatic protons at different R = [1]/Htel_.</p

¹H chemical shift assignments for Htel (δ) in the presence of 1 and 2^a.

a<p>Measured in ppm at 25°C, <i>R</i> = [drug]/[DNA] = 3.0.</p

¹H NMR spectra (11.5–10.2 ppm and 9.0–7.0 ppm) showing resonance of imino protons G4, G5 and G6 as well as the aromatic protons at different R = [2]/Htel.

<p>¹H NMR spectra (11.5–10.2 ppm and 9.0–7.0 ppm) showing resonance of imino protons G4, G5 and G6 as well as the aromatic protons at different R = [2]/Htel.</p

Time dependence of hydrogen bond distances observed between G-quadruplex and ligand 1 (A and B) and 2 (C and D).

<p>A)1. Hb and N1A10 (cyan) 2. Hb and N3A10 (green) 3. Hf and OPA10 (blue) 4. O_D and HN2G25 (red). B) 1. Hi and OaA17 (blue) 2. Hi and OPA17 (red). C) 1. Hb and N1A10 (blue) 2. Nc and HN6A10 (red) 3. Hf and OPA10 (green) D) 1. Hb and N2G11 (blue) 2. Hb and N7A17 (red) 3. Od and HN2G25 (green).</p

Lateral (A) and upper (B) views of the best docked conformations for 1 and 2.

<p><b>1</b> is shown in green stick and <b>2</b> in yellow stick. In DNA, the base pairs are shown using the ladder representation, with the backbones displayed as arrows.</p