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

Abstract

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.

Synthesis, Biological Activity and Molecular Docking of Chimeric Peptides Targeting Opioid and NOP Receptors

“…Protein–ligand complexes from selected docked poses were minimized in explicit TIP3P water molecules and equilibrated by molecular dynamics. More details can be found in the literature [ 52 ] and in the experimental section. The overlay of KW-495 and KW-496 into KOP is illustrated in Figure S6 , and shows that the two peptide conjugates occupy the same cavity of the receptor, albeit with distinct orientations.…”

Intracellular pocket conformations determine signaling through the μ opioid receptor

“…Molecular dynamics simulations (MDS) reveal additional 7TMR conformations. Distinct intracellular pocket conformations have been observed in simulations of μOR complexes with a wide variety of ligands 21,[26][27][28][29] . We hypothesized that signaling efficacy is linearly proportional to the equilibrium population of these intracellular receptor conformations.…”

“…We hypothesized that signaling efficacy is linearly proportional to the equilibrium population of these intracellular receptor conformations. To test this hypothesis, we first performed simulations of μOR complexed with 11 ligands from a variety of chemical series, comprising the most comprehensive set of agonists in a single study to date (previous studies 21,[26][27][28][29] included up to 5 complexes). We tested this hypothesis by developing a machine learning model that categorizes configurations from MDS into conformations.…”

Differential Behavior of Conformational Dynamics in Active and Inactive States of Cannabinoid Receptor 1

“…Complimentary experimental and computational techniques such as nuclear magnetic resonance (NMR) spectroscopy, 11,12 hydrogen-deuterium exchange mass spectrometry (HDX-MS), and molecular dynamics (MD) simulations, [13][14][15][16][17] provide dynamic views of receptor flexibility and the structural ensemble. [18][19][20] To date, MD simulations have been instrumental in characterizing GPCR dynamics [21][22][23] and enhancing interpretations of crystallographic data. 24,25 All-atom unbiased MD simulations spanning microsecond to millisecond timescales can comprehensively sample conformational transitions.…”