The opioid crisis is a severe public health issue in the U.S., with opioid-related deaths increasing significantly from 1999 to 2020. While opioids are crucial for pain management, their addictive nature presents a significant challenge. Opioids exert their effects by binding to opioid receptors, with the µ subtype being primarily responsible for analgesia. This crisis has immense financial consequences, with estimates reaching trillions of dollars annually. Efforts to combat the crisis include developing opioid prescribing guidelines, implementing government-funded programs, and conducting extensive research. A key area of research is developing new drugs for pain management and opioid use disorder (OUD) treatment. This requires a deeper understanding of the opioid receptor's mechanism of action, particularly the µ-opioid receptor (MOR). Advancements in X-ray crystallography and cryo-EM have enabled the resolution of several MOR structures, offering new perspectives on drug design and development. This review focuses on recently solved MOR structures, analyzing the binding site and ligand-receptor interactions, and discussing the allosteric modulation of the MOR. The aim is to highlight the structural details of MOR interactions to inform the development of safer and more effective opioid analgesics.

The existence of opioid receptors was hypothesized in the 1950s based on the structural activity relationship of opioids. The discovery was experimentally demonstrated in 1973. Pharmacological experiments revealed multiple receptor subtypes (µ, δ, κ, σ, ε). Later, four distinct cDNAs were isolated, correlating to µ-(MOR), δ-(DOR), and κ-opioid receptors (KOR), and a fourth receptor binding nociceptin/orphanin FQ (NOR). Opioid receptors are found in pain-modulating pathways and throughout the central nervous system, playing a role in reward and emotion. Endogenous opioid peptides bind to these receptors with varying specificity. Opioid receptors, as G protein-coupled receptors (GPCRs), activate intracellular signaling pathways upon agonist binding. These pathways involve G protein-dependent signaling, β-arrestin-dependent signaling, and their complex-dependent signaling, leading to diverse effects such as cAMP reduction, Ca2+ response decrease, and GIRK channel activation. Functional selectivity, or ligand bias, allows ligands to preferentially activate or inhibit specific pathways. This has led to extensive study of biased agonism and partial agonism. The structure-activity relationship between opioid receptors and their ligands is complex, and despite extensive studies, developing compounds that overcome the adverse effects of existing opioids remains a challenge. The availability of 3D protein structures now provides valuable insights for drug design.

This review compiles and analyzes data from previously published research focusing on the three-dimensional structures of the µ-opioid receptor (MOR). The authors examine the existing structures available in the Protein Data Bank (PDB), focusing on the binding interactions of various ligands, including agonists, partial agonists, and antagonists. The analysis involves a comparative study of atomic-level details of the binding site in these structures. The methodology includes structural alignment of different MOR complexes, identification of conserved and unique binding interactions, and assessment of solvent-accessible surface area and volume of the binding pocket. Molecular dynamics (MD) simulations are also reviewed to provide insights into dynamic interactions and water molecule roles in MOR activation. The authors analyzed the composition of surrounding residues within 5 Å of ligands in all structures and categorized residues by ligand type. Solvent-accessible surface area (SASA) and volume (SAV) were measured for each structure using CASTp 3.0. The review also includes an assessment of the allosteric modulation of the MOR, reviewing various known modulators (cations, lipids, and small molecules) and their proposed binding sites, often informed by in silico studies like docking and MD simulations. Overall, the methodology is based on a comprehensive review and synthesis of existing structural and computational data.

Several high-resolution structures of MOR have been solved since 2012, many using cryo-EM. These include structures bound to antagonists (β-FNA, alvimopan), full agonists (DAMGO, morphine, fentanyl, lofentanil, mitragynine pseudoindoxyl), and partial agonists (PZM21, FH210, oliceridine, SR17018). Analysis reveals that agonists and antagonists bind to the same orthosteric site, but with subtle differences. Conserved interactions include a water-mediated hydrogen bond and an ionic interaction. Fentanyl shows additional interactions compared to morphine, possibly explaining its higher potency. Partial agonists retain the key polar interactions of full agonists but also show high complementarity to a lipophilic vestibule, possibly contributing to their partial agonism and G protein bias. Analysis of residues within 4Å of the ligands showed some residues are shared by all ligand types, some by agonist/antagonist or agonist/partial agonist, while others are unique to each type. Full agonist-bound structures had the smallest solvent-accessible volume compared to antagonist-bound or partial agonist-bound structures. MD simulations support cryo-EM findings and offer additional details about the interactions and the role of water molecules in ligand binding and receptor activation. Water molecules play a significant role in mediating hydrogen bonding interactions. The agonist-bound system shows more intrinsic water molecules and more frequent exchange with extracellular water compared to antagonist-bound systems. Allosteric modulators, including monovalent and divalent cations (Na+, Mg2+), lipids (cholesterol), and small molecules (BMS series), significantly affect MOR activity. Na+ acts as a NAM, stabilizing the inactive conformation, while Mg2+ acts as a PAM, promoting activation. Cholesterol was found co-crystallized with MOR in both active and inactive states. Small molecule PAMs show potential for enhancing the efficacy of existing opioids. Several allosteric binding sites have been predicted in silico.

The findings highlight the importance of considering both the static and dynamic aspects of ligand-receptor interactions. While the orthosteric binding site is crucial, subtle variations in interactions and the surrounding environment profoundly influence the ligand's effect (agonist, partial agonist, or antagonist). The identification of a lipophilic vestibule interacting with partial agonists opens avenues for designing biased ligands that preferentially activate certain signaling pathways. The differing solvent-accessible volumes between agonist, antagonist, and partial agonist bound structures point to an induced fit mechanism where the binding pocket adapts to the ligand. The role of allosteric modulators in influencing MOR conformation and activity adds complexity, providing potential avenues for developing novel therapeutics. By understanding these interactions, researchers can develop safer and more effective analgesics. The in silico predictions of allosteric binding sites could lead to discovering novel, safer analgesics.

This review demonstrates that high-resolution structures of the MOR, combined with computational modeling, significantly advance our understanding of ligand binding and receptor activation. Distinctive binding features of agonists, partial agonists, and antagonists are identified, providing essential information for structure-based drug design. The role of allosteric modulators emphasizes the complexity of MOR function and its modulation. Future research should focus on exploring novel allosteric modulators and structurally divergent compounds to discover safer and more effective opioid analgesics.

The review primarily relies on existing published data, limiting the direct contribution of novel experimental findings. The interpretation of some interactions depends on computational modeling and may not fully capture the complexity of the system. The lack of comprehensive data on the binding site for some allosteric modulators limits the conclusions that can be drawn about their precise mechanism of action. Future studies may refine some of the observations made in this review.