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Bitter taste receptors (TAS2Rs), a subfamily of G-protein coupled receptors (GPCRs) expressed orally and extraorally, elicit signaling in response to a large set of tastants. Among 25 functional TAS2Rs encoded in the human genome, TAS2R14 is the most promiscuous, and responds to hundreds of chemically diverse ligands. Here we present the cryo-electron microscopy (cryo-EM) structure of the human TAS2R14 in complex with its signaling partner gustducin, and bound to flufenamic acid (FFA), a clinically approved nonsteroidal anti-inflammatory drug. The structure reveals an unusual binding mode, where two copies of FFA are bound at distinct pockets: one at the canonical receptor site within the trans-membrane bundle, and the other in the intracellular facet, bridging the receptor with gustducin. Together with a pocket-specific BRET-based ligand binding assay, these results illuminate bitter taste signaling and provide tools for a site-targeted compound design., Competing Interests: Competing interests The authors declare no competing interests, (© 2024. The Author(s).)

Bitter taste receptors, particularly TAS2R14, play central roles in discerning a wide array of bitter substances, ranging from dietary components to pharmaceutical agents 1,2 . TAS2R14 is also widely expressed in extragustatory tissues, suggesting its extra roles in diverse physiological processes and potential therapeutic applications 3 . Here we present cryogenic electron microscopy structures of TAS2R14 in complex with aristolochic acid, flufenamic acid and compound 28.1, coupling with different G-protein subtypes. Uniquely, a cholesterol molecule is observed occupying what is typically an orthosteric site in class A G-protein-coupled receptors. The three potent agonists bind, individually, to the intracellular pockets, suggesting a distinct activation mechanism for this receptor. Comprehensive structural analysis, combined with mutagenesis and molecular dynamic simulation studies, elucidate the broad-spectrum ligand recognition and activation of the receptor by means of intricate multiple ligand-binding sites. Our study also uncovers the specific coupling modes of TAS2R14 with gustducin and G i1 proteins. These findings should be instrumental in advancing knowledge of bitter taste perception and its broader implications in sensory biology and drug discovery., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

An asymmetric bi-nuclear copper(II) complex with both cytotoxic and immunogenic activity towards breast cancer stem cells (CSCs) is reported. The bi-nuclear copper(II) complex comprises of two copper(II) centres bound to flufenamic acid and 3,4,7,8-tetramethyl-1,10-phenanthroline. The bi-nuclear copper(II) complex exhibits sub-micromolar potency towards breast CSCs grown in monolayers and three-dimensional cultures. Remarkably, the bi-nuclear copper(II) complex is up to 25-fold more potent toward breast CSC mammospheres than salinomycin (a gold standard anti-breast CSC agent) and cisplatin (a clinically administered metallodrug). Mechanistic studies showed that the bi-nuclear copper(II) complex readily enters breast CSCs, elevates intracellular reactive oxygen species levels, induces apoptosis, and promotes damage-associated molecular pattern release. The latter triggers phagocytosis of breast CSCs by macrophages. As far as we are aware, this is the first report of a bi-nuclear copper(II) complex to induce engulfment of breast CSCs by immune cells.

The cytotoxic and immunogenic-activating properties of a cobalt(III)-cyclam complex bearing the non-steroidal anti-inflammatory drug, flufenamic acid is reported within the context of anti-cancer stem cell (CSC) drug discovery. The cobalt(III)-cyclam complex 1 displays sub-micromolar potency towards breast CSCs grown in monolayers, 24-fold and 31-fold greater than salinomycin (an established anti-breast CSC agent) and cisplatin (an anticancer metallopharmaceutical), respectively. Strikingly, the cobalt(III)-cyclam complex 1 is 69-fold and 50-fold more potent than salinomycin and cisplatin towards three-dimensionally cultured breast CSC mammospheres. Mechanistic studies reveal that 1 induces DNA damage, inhibits cyclooxygenase-2 expression, and prompts caspase-dependent apoptosis. Breast CSCs treated with 1 exhibit damage-associated molecular patterns characteristic of immunogenic cell death and are phagocytosed by macrophages. As far as we are aware, 1 is the first cobalt complex of any oxidation state or geometry to display both cytotoxic and immunogenic-activating effects on breast CSCs., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Background: Epilepsy is one of the most common neurologic diseases, and around 30% of all epilepsies, particularly the temporal lobe epilepsy (TLE), are highly refractory to current pharmacological treatments. Abnormal synchronic neuronal activity, brain glucose metabolism alterations, neurodegeneration and neuroinflammation are features of epilepsy. Further, neuroinflammation has been shown to contribute to dysregulation of neuronal excitability and the progression of epileptogenesis. Flufenamic acid (FLU), a non-steroidal anti-inflammatory drug, is also characterized by its wide properties as a dose-dependent ion channel modulator. In this context, in vitro studies have shown that it abolishes seizure-like events in neocortical slices stimulated with a gamma-aminobutyric acid A (GABAA) receptor blocker. However, little is known about its effects in animal models. Thus, our goal was to assess the efficacy and safety of a relatively high dose of FLU in the lithium-pilocarpine rat model of status epilepticus (SE). This animal model reproduces many behavioral and neurobiological features of TLE such as short-term brain hypometabolism, severe hippocampal neurodegeneration and inflammation reflected by a marked reactive astrogliosis., Methods: FLU (100 mg/kg, i.p.) was administered to adult male rats, 150 min before SE induced by pilocarpine. Three days after the SE, brain glucose metabolism was assessed by 2-deoxy-2-[18F]-fluoro-D-glucose ([18F]FDG) positron emission tomography (PET). Markers of hippocampal integrity, neurodegeneration and reactive astrogliosis were also evaluated., Results: FLU neither prevented the occurrence of the SE nor affected brain glucose hypometabolism as assessed by [18F]FDG PET. Regarding the neurohistochemical studies, FLU neither prevented neuronal damage nor hippocampal reactive astrogliosis. On the contrary, FLU increased the mortality rate and negatively affected body weight in the rats that survived the SE., Conclusions: Our results do not support an acute anticonvulsant effect of a single dose of FLU. Besides, FLU did not show short-term neuroprotective or anti-inflammatory effects in the rat lithium-pilocarpine model of SE. Moreover, at the dose administered, FLU resulted in deleterious effects., Competing Interests: Pablo Bascuñana Almarcha is serving as one of the Guest editors of this journal. We declare that Pablo Bascuñana Almarcha had no involvement in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Gernot Riedel. The other authors declare no conflict of interest., (© 2023 The Author(s). Published by IMR Press.)