Structural basis of μ-opioid receptor targeting by a nanobody antagonist

Nature Communications volume  15, Article number: 8687 (2024 ) Cite this article

The μ-opioid receptor (μOR), a prototypical G protein-coupled receptor (GPCR), is the target of opioid analgesics such as morphine and fentanyl. Due to the severe side effects of current opioid drugs, there is considerable interest in developing novel modulators of μOR function. Most GPCR ligands today are small molecules, however biologics, including antibodies and nanobodies, represent alternative therapeutics with clear advantages such as affinity and target selectivity. Here, we describe the nanobody NbE, which selectively binds to the μOR and acts as an antagonist. We functionally characterize NbE as an extracellular and genetically encoded μOR ligand and uncover the molecular basis for μOR antagonism by determining the cryo-EM structure of the NbE-μOR complex. NbE displays a unique ligand binding mode and achieves μOR selectivity by interactions with the orthosteric pocket and extracellular receptor loops. Based on a β-hairpin loop formed by NbE that deeply protrudes into the μOR, we design linear and cyclic peptide analogs that recapitulate NbE’s antagonism. The work illustrates the potential of nanobodies to uniquely engage with GPCRs and describes lower molecular weight μOR ligands that can serve as a basis for therapeutic developments.

G protein-coupled receptors (GPCRs) represent key therapeutic targets due to their central roles in cellular signaling and control over a plethora of physiological processes. Developing new ligands that bind a given GPCR with high selectivity remains a significant challenge in drug discovery1,2,3. Small molecule ligands have historically dominated the landscape of GPCR-targeted drugs, but recently biologics, including antibodies and nanobodies (Nbs), have emerged as an alternative class of ligands that offer distinct advantages and hold promise for therapeutic developments4,5. Nbs are single-domain antibody fragments derived from heavy chain-only antibodies, which naturally occur in camelids and cartilaginous fish, and are characterized by small size, high antigen binding affinity, and binding loops that can access deep cavities on target proteins6. Nbs can show enhanced selectivity over small molecules due to their ability to interact with unique and extended epitope surfaces. Over the last decade, Nbs that bind GPCRs on their intracellular side have served as innovative research tools to uncover GPCR signal transduction mechanisms7,8. For example, Nbs were used as crystallization chaperones or as fiducial markers in high-resolution structural studies9,10,11,12. Conformation-selective Nbs were also repurposed into biosensors to report GPCR activity in living cells13,14. Only recently, several Nbs that bind GPCRs as extracellular ligands and thereby modulate receptor function have been described15,16,17,18,19,20. Generating knowledge on GPCR-targeting Nbs is key to unlocking their potential as both versatile research tools and therapeutic compounds.

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