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).

The continuous spread of SARS-CoV-2 calls for more direct-acting antiviral agents to combat the highly infectious variants. The main protease (M pro ) is an promising target for anti-SARS-CoV-2 drug design. Here, we report the discovery of potent non-covalent non-peptide M pro inhibitors featuring a 1,2,4-trisubstituted piperazine scaffold. We systematically modified the non-covalent hit MCULE-5948770040 by structure-based rational design combined with multi-site binding and privileged structure assembly strategies. The optimized compound GC-14 inhibits M pro with high potency (IC 50 = 0.40 μM) and displays excellent antiviral activity (EC 50 = 1.1 μM), being more potent than Remdesivir. Notably, GC-14 exhibits low cytotoxicity (CC 50 > 100 μM) and excellent target selectivity for SARS-CoV-2 M pro (IC 50 > 50 μM for cathepsins B, F, K, L, and caspase 3). X-ray co-crystal structures prove that the inhibitors occupy multiple subpockets by critical non-covalent interactions. These studies may provide a basis for developing a more efficient and safer therapy for COVID-19.

The G protein-coupled adenosine A 2A receptor (A 2A AR) is an important new (potential) drug target in immuno-oncology, and for neurodegenerative diseases. Preladenant and its derivatives belong to the most potent A 2A AR antagonists displaying exceptional selectivity. While crystal structures of the human A 2A AR have been solved, mostly using the A 2A -StaR2 protein that bears 9 point mutations, co-crystallization with Preladenant derivatives has so far been elusive. We developed a new A 2A AR construct harboring a single point mutation (S91 3.39 K) which renders it extremely thermostable. This allowed the co-crystallization of two novel Preladenant derivatives, the polyethylene glycolconjugated (PEGylated) PSB-2113, and the fluorophore-labeled PSB-2115. The obtained crystal structures (2.25 Å and 2.6 Å resolution) provide explanations for the high potency and selectivity of Preladenant derivatives. They represent the first crystal structures of a GPCR in complex with PEG-and fluorophore-conjugated ligands. The applied strategy is predicted to be applicable to further class A GPCRs.

The Gs protein-coupled adenosine A2A receptor (A2AAR) represents an emerging drug target for cancer immunotherapy. The clinical candidate Etrumadenant was developed as an A2AAR antagonist with ancillary blockade of the A2BAR subtype. It constitutes a unique chemotype featuring a poly-substituted 2-amino-4-phenyl-6-triazolylpyrimidine core structure. Herein, we report two crystal structures of the A2AAR in complex with Etrumadenant, obtained with differently thermostabilized A2AAR constructs. This led to the discovery of an unprecedented interaction, a hydrogen bond of T883.36 with the cyano group of Etrumadenant. T883.36 is mutated in most A2AAR constructs used for crystallization, which has prevented the discovery of its interactions. In-vitro characterization of Etrumadenant indicated low selectivity versus the A1AR subtype, which can be rationalized by the structural data. These results will facilitate the future design of AR antagonists with desired selectivity. Moreover, they highlight the advantages of the employed A2AAR crystallization construct that is devoid of ligand binding site mutations.