The potent and selective Gq protein inhibitor depsipeptide FR900359 (FR), originally discovered as the product of an uncultivable plant endosymbiont, is synthesized by a complex biosynthetic system comprising two nonribosomal peptide synthetase (NRPS) assembly lines. Here we characterize a cultivable bacterial FR producer, enabling detailed investigations into biosynthesis and attachment of the functionally important FR side chain. We reconstitute side chain assembly by the monomodular NRPS FrsA and the non-heme monooxygenase FrsH, and characterize intermolecular side chain transesterification to the final macrocyclic intermediate FR-Core, mediated by the FrsA thioesterase domain. We harness FrsA substrate promiscuity to generate FR analogs with altered side chains and demonstrate indispensability of the FR side chain for efficient Gq inhibition by comparative bioactivity, toxicity and docking studies. Finally, evolution of FR and side chain biosynthesis is discussed based on bioinformatics analyses. Side chain transesterification boosts potency and target affinity of selective Gq inhibitor natural products.

Frizzleds (ten paralogs: FZD_1-10) belong to the class F of G protein-coupled receptors (GPCRs), which remains poorly understood despite its crucial role in multiple key biological functions including embryonic development, stem cell regulation, and homeostasis in the adult. FZD₇, one of the most studied members of the family, is more specifically involved in the migration of mesendoderm cells during the development and renewal of intestinal stem cells in adults. Moreover, FZD₇ has been highlighted for its involvement in tumor development predominantly in the gastrointestinal tract. This study reports the structure of inactive FZD₇, without any stabilizing mutations, determined by cryo-electron microscopy (cryo-EM) at 1.9 Å resolution. We characterize a fluctuating water pocket in the core of the receptor important for FZD₇ dynamics. Molecular dynamics simulations are used to investigate the temporal distribution of those water molecules and their importance for potential conformational changes in FZD₇. Moreover, we identify lipids interacting with the receptor core and a conserved cholesterol-binding site, which displays a key role in FZD₇ association with a transducer protein, Disheveled (DVL), and initiation of downstream signaling and signalosome formation.

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.

The Wingless/Int-1 (WNT) signaling network is essential to orchestrate central physiological processes such as embryonic development and tissue homeostasis. In the currently held tenet, WNT/β-catenin signaling is initiated by WNT-induced recruitment of Frizzleds (FZDs) and LRP5/6 followed by the formation of a multiprotein signalosome complex. Here, we use bioluminescence resonance energy transfer (BRET) to show that different WNT paralogs dynamically trigger FZD-LRP6 association. While WNT-induced receptor interaction was independent of C-terminal LRP6 phosphorylation, it was allosterically modulated by binding of the phosphoprotein Dishevelled (DVL) to FZD. WNT-16B emerged as a ligand of particular interest, as it efficiently promoted FZD-LRP6 association but, unlike WNT-3A, did not lead to WNT/β-catenin signaling. Transcriptomic analysis further revealed distinct transcriptional fingerprints of WNT-3A and WNT-16B stimulation in HEK293 cells. Additionally, single-molecule tracking demonstrated that, despite increasing FZD₅ and LRP6 confinement, WNT-16B stimulation did not result in formation of higher-order receptor clusters, in contrast to WNT-3A. Our results suggest that FZD-WNT-LRP5/6 complex formation alone is not sufficient for the initiation of WNT/β-catenin signaling. Instead, we propose a two-step model, where initial ligand-induced FZD-LRP6 association must be followed by receptor clustering into higher-order complexes and subsequent phosphorylation of LRP6 for efficient activation of WNT/β-catenin signaling.

Background and Purpose: G proteins are intracellular switches that transduce and amplify extracellular signals from GPCRs. The G_q protein subtypes, which are coupled to PLC activation, can act as oncogenes, and their expression was reported to be up‐regulated in cancer and inflammatory diseases. G_q inhibition may be an efficient therapeutic strategy constituting a new level of intervention. However, diagnostic tools and therapeutic drugs for G_q proteins are lacking.Experimental Approach: We have now developed G_q‐specific, cell‐permeable ³H‐labelled high‐affinity probes based on the macrocyclic depsipeptides FR900359 (FR) and YM‐254890 (YM). The tracers served to specifically label and quantify G_q proteins in their native conformation in cells and tissues with high accuracy.Key Results: FR and YM displayed low nanomolar affinity for Gα_q, Gα₁₁ and Gα₁₄ expressed in CRISPR/Cas9 Gα_q‐knockout cells, but not for Gα₁₅. The two structurally very similar tracers showed strikingly different dissociation kinetics, which is predicted to result in divergent biological effects. Computational studies suggested a “dowel” effect of the pseudoirreversibly binding FR. A high‐throughput binding assay led to the discovery of novel G_q inhibitors, which inhibited G_q signalling in recombinant cells and primary murine brown adipocytes, resulting in enhanced differentiation.Conclusions and Implications: The Gq protein inhibitors YM and FR are pharmacologically different despite similar structures. The new versatile tools and powerful assays will contribute to the advancement of the rising field of G protein research.

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.