Structural Insights and Characterization of a Novel Transmembrane Binding Site for Potential Allosteric Modulation of the Mu‐opioid Receptor

Abstract

Objective Opioids such as morphine, codeine, and oxycodone represent the gold standard for the treatment of pain. These drugs elicit their analgesic action by activating the mu-opioid receptor (mu-OR), a class A G protein-coupled receptor (GPCR). These agonists suffer from serious side effects, including respiratory depression, constipation, tolerance, and drug abuse. Recent advances in the structural biology of GPCRs revealed novel transmembrane lipid-facing binding pockets for allosteric modulators of the beta-2-adrenergic and cannabinoid (CB1) receptors. Allosteric ligands offer distinct advantages such as greater receptor subtype selectivity and the potential to “finetune” endogenous agonism while preserving the temporal and spatial characteristics of the signaling. Most importantly, allosteric modulators can offer unique safety by providing a “ceiling level” for their effects. Our central hypothesis is that a druggable transmembrane allosteric site exists for mu-OR, and high-affinity small-molecule ligands that stabilize the receptor in an active conformation can be identified. In this study, we identified such a novel transmembrane allosteric binding site on mu-OR and several high-affinity positive allosteric modulators through a comprehensive multidisciplinary (computational and experimental) approach. Our results provide the structural and mechanistic basis for the allosteric modulation of the receptor through this site and offer insights into the key binding site residues and membrane interactions that are critical for ligand affinity and receptor stabilization in the active state. Methods We employed a comprehensive computational approach that includes a sequence- and structure-based alignment of several experimental structures of GPCRs bound to their allosteric modulators to identify and characterize lipid-facing transmembrane binding sites. Structure-based pharmacophore models were developed, refined, and used for virtual screening of commercially available compound libraries to identify potential ligands using MOE software. Molecular docking, molecular dynamics simulations, and funnel metadynamics were used to assess the relative binding energies, using GROMACS and PLUMED software. Top ligands were evaluated by multiple in vitro assays for the positive allosteric modulation of mu-OR. Results A lipid-facing transmembrane binding site distinct from the orthosteric site was identified, and several high-affinity small-molecule allosteric modulators with drug-like properties were obtained by a combined computational and experimental approach. A variety of activation signature distances monitored were able to distinguish an active receptor from an inactive. The molecules will be further validated by in vivo methods for potential analgesic actions. Conclusion Our results illustrate a potential allosteric binding site on the transmembrane helices of the µ-opioid receptor that requires the active participation of membrane lipids for stable binding of ligands. The obtained ligands may translate into a safe and potential therapeutic alternative for opioids in the treatment of pain.