Elucidating the active δ-opioid receptor crystal structure with peptide and small-molecule agonists

Abstract

Selective activation of the -opioid receptor (DOP) has great potential for the treatment of chronic pain, benefitting from ancillary anxiolytic and antidepressant-like effects. Moreover, DOP agonists show reduced adverse effects as compared to -opioid receptor (MOP) agonists that are in the spotlight of the current "opioid crisis." Here, we report the first crystal structures of the DOP in an activated state, in complex with two relevant and structurally diverse agonists: the potent opioid agonist peptide KGCHM07 and the small-molecule agonist DPI-287 at 2.8 and 3.3 Å resolution, respectively. Our study identifies key determinants for agonist recognition, receptor activation, and DOP selectivity, revealing crucial differences between both agonist scaffolds. Our findings provide the first investigation into atomic-scale agonist binding at the DOP, supported by site-directed mutagenesis and pharmacological characterization. These structures will underpin the future structure-based development of DOP agonists for an improved pain treatment with fewer adverse effects. RESULTS Activation-related changes in the DOPBoth agonist-bound structures are in an activated state. Unless otherwise indicated, we will use the higher-resolution BRIL-DOP-KGCHM07 structure for comparison with previously published inactive-state antagonist-bound DOP structures [Protein Data Bank (PDB) 4N6H and 4RWD] (16, 17) and with active-state structures of the MOP (PDB 5C1M and 6DDF) (18,20) and KOP (PDB 6B73) (19). First, the agonist-bound DOP structures display large outward movements of the intracellular parts of helices V (4.5 Å) and VI (9.4 to 11.2 Å), and a 3.9 Å inward movement of helix VII ( Fig. 2A), which is a common feature of the active conformational states of GPCRs (21). The shift of helix VII at the level of residue N314 7.49 [superscripts according to the Ballesteros and Weinstein numbering (22)] (Fig. 3A), which leads to a collapse of the allosteric sodium-binding pocket in active-state GPCR structures (23), is even more pronounced in the determined DOP structures as compared to the active MOP and KOP (Fig. 3B and fig. S1). However, this greater shift of N314 7.49 in the DOP might be affected by three mutations in the sodium-binding pocket (N90 2.45 S, D95 2.50 G, N131 3.35 S) that were introduced during construct design. The activation-related outward movement of helix VI at the level of residue F270 6.44 is greater in the agonist-bound DOP than in the MOP and KOP. On the contrary, the very tips of helix VI (at position 6.28 as a reference) are more tilted by 4 to 6 Å in the active-state MOP and KOP ( fig. S1), likely due to the bound G protein or nanobody, respectively, pushing helix VI tips further outward (24). Elucidating the active -opioid receptor crystal structure with peptide and smallmolecule agonists. Sci. Adv. 5, eaax9115 (2019).

N-Phenethyl Substitution in 14-Methoxy-N-methylmorphinan-6-ones Turns Selective µ Opioid Receptor Ligands into Dual µ/δ Opioid Receptor Agonists

“…The recent crystal structure of the δOR (PDB ID: 6PT3) 45 with the co-crystallized agonist DPI-287 supports our proposed binding mode of 4a at the δOR, since DPI-287 also has a phenyl ring which is filling the beforementioned lipophilic subpocket (Fig. 6).…”

“…The structure of the human µOR was remodeled based on the crystal structure of the murine µOR (PDB ID: 5C1M) 38 by using the mutation tool of MOE (Molecular Operating Environment), 2014.09; Chemical Computing Group Inc.) with subsequent sidechain optimization. We used the active crystal structures of the δOR (PDB ID: 6PT3) 45 and κOR (PDB ID: 6B73) 46 for docking experiments. All receptor-ligand docking experiments were performed with the CCDCs software GOLD version 5.1 56 .…”

Biased Opioid Ligands

“…Disrupting this interaction network through mutagenesis transform classical DOR antagonists such as naltrindole into potent β-arrestin2 -biased agonists, possibly opening up new routes towards biased signaling [ 93 ]. Active-like state DOR structures in complex with a peptide (2.8 Å resolution) and a small-molecule agonist (3.3 Å resolution) obtained in 2019 revealed polar networks around the conserved D128 3.32 residue with rearrangements in the agonist-bound binding pocket upon DOR activation [ 94 ]. This residue is crucial for receptor activation as opioid agonists that contain a basic nitrogen interacting with D 3.32 extend deeper into the binding pocket compared to structurally similar antagonists.…”

“…They also found changes in the nonconserved ECL3 during activation, which makes R291 ECL3 available for binding pocket interactions, notably upon the binding of endogenous peptides. Unlike peptides, DOP-selective small molecules address the nonconserved extracellular ends of helices VI and VII with their N , N -diethylbenzamide moiety, which leads to their selectivity over MOP and KOP due to steric clashes in the same region of the latter receptors [ 94 ].…”

Design, Synthesis and Functional Analysis of Cyclic Opioid Peptides with Dmt-Tic Pharmacophore

“…Structural requirements of binding to the MOR and DOR have been studied extensively in the past decades [ 25 , 26 , 27 , 28 ]. The recently solved crystallographic structures of these receptors in the presence of agonists or antagonists of various molecular scaffolds presented a great leap towards the understanding of the basic principles of opioid activity [ 29 , 30 , 31 , 32 , 33 , 34 ]. A common feature of receptor–ligand interactions is the presence of an anchoring salt bridge between Asp 3.32 of the receptors and the protonated N -terminal primary amine of opioid peptides or the basic nitrogen atom of opioid alkaloids.…”