Crystal structures provide first atomic-level insights into δ-opioid receptor activation by two structurally diverse agonists.

Orthomyxo- and bunyaviruses steal the 5′ cap portion of host RNAs to prime their own transcription in a process called “cap snatching.” We report that RNA modification of the cap portion by host 2′-O-ribose methyltransferase 1 (MTr1) is essential for the initiation of influenza A and B virus replication, but not for other cap-snatching viruses. We identified with in silico compound screening and functional analysis a derivative of a natural product from Streptomyces , called trifluoromethyl-tubercidin (TFMT), that inhibits MTr1 through interaction at its S -adenosyl- l -methionine binding pocket to restrict influenza virus replication. Mechanistically, TFMT impairs the association of host cap RNAs with the viral polymerase basic protein 2 subunit in human lung explants and in vivo in mice. TFMT acts synergistically with approved anti-influenza drugs.

The G protein‐coupled adenosine A_2A receptor (A_2AAR) is an important new (potential) drug target in immuno‐oncology, and for neurodegenerative diseases. Preladenant and its derivatives belong to the most potent A_2AAR antagonists displaying exceptional selectivity. While crystal structures of the human A_2AAR 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_2AAR construct harboring a single point mutation (S91^3.39K) which renders it extremely thermostable. This allowed the co‐crystallization of two novel Preladenant derivatives, the polyethylene glycol‐conjugated (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.

GPR55 is an orphan G protein-coupled receptor (GPCR) and represents a promising drug target for cancer, inflammation, and metabolic diseases. The endogenous activation of lipid GPCRs can be solely mediated by membrane components and different lipids have been proposed as endogenous activators of GPR55, such as cannabinoids and lysophosphatidylinositols. Here, we determine high-resolution cryo-electron microscopy structures of the activated GPR55 in complex with heterotrimeric G₁₃ and two structurally diverse ligands: the putative endogenous agonist 1-palmitoyl-2-lysophosphatidylinositol (LPI) and the synthetic agonist ML184. These results reveal insights into ligand recognition at GPR55, G protein coupling and receptor activation. Notably, an orthosteric binding site opening towards the membrane is observed in both structures, enabling direct interaction of the agonists with membrane lipids. The structural observations are supported by mutagenesis and functional experiments employing G protein dissociation assays. These findings will be of importance for the structure-based development of drugs targeting GPR55.

The G_s protein-coupled adenosine A_2A receptor (A_2AAR) represents an emerging drug target for cancer immunotherapy. The clinical candidate Etrumadenant was developed as an A_2AAR antagonist with ancillary blockade of the A_2BAR 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 A_2AAR in complex with Etrumadenant, obtained with differently thermostabilized A_2AAR constructs. This led to the discovery of an unprecedented interaction, a hydrogen bond of T88^3.36 with the cyano group of Etrumadenant. T88^3.36 is mutated in most A_2AAR 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 A_2AAR crystallization construct that is devoid of ligand binding site mutations.