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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.)

Transcriptional enhanced associate domain (TEAD) is a family of transcription factors that plays a significant role during embryonic developmental processes, and its dysregulation is responsible for tumour progression. TEAD is considered as druggable targets in various diseases, namely cancer, cardiovascular diseases and neurodegenerative disorders. Previous structural studies revealed the importance of the central hydrophobic pocket of TEAD as a potential target for small-molecule inhibitors and demonstrated flufenamic acid (FLU) (a COX-2 enzyme inhibitor) to bind and inhibit TEAD2 functions. However, to date, no drug candidates that bind specifically to TEAD2 with high selectivity and efficacy have been developed or proposed. Within this framework, we present here a case study where we have identified potential TEAD2 inhibitor candidates by integrating multiple computational approaches. Among the candidates, the top two ranked compounds ZINC95969481 (LG1) which is a fused pyrazole derivative and ZINC05203789 (LG2), a fluorene derivative resulted in much favourable binding energy scores than the reference ligand, FLU. The drug likeliness of the best compounds was also evaluated in silico to ensure the bioavailability of these compounds particularly LG1 as compared to FLU thus providing a strong rationale for their development as leads against TEAD. Molecular dynamics simulations results highlighted the role of key residues contributing to favourable interactions in TEAD2-LG1 complex with much favourable interaction and binding free energy values with respect to the reference compound. Altogether, this study provides a starting platform to be more exploited by future experimental research towards the development of inhibitors against TEAD, a persuasive strategy for therapeutic intervention in cancer treatment., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Cosmetic hair removal procedures are everyday routines in our society. However, it is unclear if such routines lead to increased uptake of applied substances such as drugs or formulation compounds, potentially resulting in skin irritation or sensitization. The aim of this study was to elucidate the effect of common depilation and epilation methods on skin penetration of two surfactants and four model drugs of different physicochemical properties using the porcine ear model. It should be elucidated whether the substances' skin penetration behavior would be affected by hair removal procedures and if potential effects would be related to their polarity. Confocal Raman spectroscopy revealed no effect of hair removal on total penetration depths of SDS and sulfathiazole. Significantly higher relative penetrated amounts within 0-6 µm of stratum corneum depth were found for SDS after dry shaving, depilatory cream and waxing and for sulfathiazole after all depilation methods and partly after epilation. ATR-FTIR spectroscopy revealed no effect of hair removal on the penetration depth of lecithin LPC80, but higher relative amounts at the skin surface after wet shaving and electric epilation. Diffusion cell experiments using a lecithin-based microemulsion as carrier system for fluconazole, fludrocortisone acetate and flufenamic acid showed higher cumulative amounts, higher drug fluxes and shorter lag times for the more lipophilic drugs for some of the methods, but only shorter lag times in some cases for fluconazole. In summary, the observed effects appeared to depend on drug polarity and experimental setup., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Copper(II) complexes containing non-steroidal anti-inflammatory drugs (NSAIDs) have been the subject of many research papers and reviews. Here we report the synthesis, spectroscopic study and biological activity of novel mixed copper(II) complexes with NSAIDs: tolfenamic (tolf), mefenamic (mef) and flufenamic (fluf) acids and phenanthroline (phen): [Cu(tolf-O,O') 2 (phen)] (1), [Cu(mef-O,O') 2 (phen)] (2), [Cu(fluf-O,O') 2 (phen)] (3). Complexes were characterized by X-ray analysis and EPR spectroscopy. Complexes 1-3 are monomeric, six-coordinate and crystallize in a monoclinic space group. Interaction of Cu(II) complexes with DNA was studied by means of absorption titrations, viscosity measurements and gel electrophoresis. The relative ability of the complexes to cleave DNA even in the absence of hydrogen peroxide is in the order 3 > 2 > 1. Application of the reactive oxygen species (ROS) scavengers, L-histidine, DMSO and SOD confirmed that singlet oxygen, hydroxyl radicals (Fenton reaction) and superoxide radical were formed, respectively. Thus, in addition to mechanism of intercalation, redox-cycling mechanism which in turn lead to the formation of ROS contribute to DNA damage. Cu(II) complexes exhibit excellent SOD-mimetic activity in the order 3~1 > 2. The fluorescence spectroscopy revealed that albumin may act as a targeted drug delivery vehicle for Cu(II) complexes (K~10 6 ). The anticancer activities of complexes 1-3 were investigated using an MTS assay (reduction of the tetrazolium compound) against three cancer cell lines (HT-29 human colon adenocarcinoma, HeLa and T-47D breast cancer cells) and mesenchymal stromal cells (MSC). The most promising compound, from the viewpoint of its NSAID biological activity is 3, due to the presence of the three fluorine atoms participating in the formation of weak hydrogen-bonds at the DNA surface., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Little is known about the course of the plasma concentration and the bioavailability of non-steroidal anti-inflammatory drugs (NSAIDs) contained in dermal patches. We compared an etofenamate prototype patch (patent EP 1833471) and a commercially available diclofenac epolamine patch regarding the bioavailability of the active ingredients relative to respective i.m. applications and regarding their plasma concentration-time course. Twenty-four healthy human volunteers were treated using a parallel group design (n = 12 per group) with a single dermal patch (removed after 12 hr) followed (after a latency of 48 hr) by eight consecutive dermal patches every 12 hr to reach steady-state conditions. The patches were generally well tolerated, but one volunteer treated with etofenamate developed an allergic contact dermatitis. After the first patch, C max was 0.81 ± 0.11 (mean ± S.E.M.) ng/mL (reached 12 hr after patch removal) for diclofenac and 31.3 ± 3.8 ng/mL for flufenamic acid (reached at patch removal), the main metabolite of etofenamate. Etofenamate was not detectable. After repetitive dosing, trough plasma concentrations after the eighth dose were 1.72 ± 0.32 ng/mL for diclofenac and 48.7 ± 6.6 ng/mL for flufenamic acid. Bioavailabilities (single dose) relative to i.m. applications were 0.22 ± 0.04% for diclofenac and 1.15 ± 0.06% for flufenamic acid. In conclusion, the relative bioavailability (compared to the respective i.m. application) of both drugs is low. The maximal plasma concentrations after topical administration of these drugs are well below the IC 50 values for COX-1 and COX-2, explaining the absence of dose-dependent toxicities., (© 2017 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Nuclear factor kappa B (NF-κB)-mediated inflammatory responses play crucial roles in the pathogenesis of diarrhea caused by the Vibrio cholerae El Tor variant (EL), which is a major bacterial strain causing recent cholera outbreaks. Flufenamic acid (FFA) has previously been demonstrated to be a potent activator of AMP-activated protein kinase (AMPK), which is a negative regulator of NF-κB signaling. This study aimed to investigate the anti-diarrheal efficacy of FFA in a mouse model of EL infection and to investigate the mechanisms by which FFA activates AMPK in intestinal epithelial cells (IEC). In a mouse closed loop model of EL infection, FFA treatment (20mg/kg) significantly abrogated EL-induced intestinal fluid secretion and barrier disruption. In addition, FFA suppressed NF-κB nuclear translocation and expression of proinflammatory mediators and promoted AMPK phosphorylation in the EL-infected mouse intestine. In T84 cells, FFA induced AMPK activation. Furthermore, FFA promoted tight junction assembly and prevented interferon gamma (IFN-γ)-induced barrier disruption in an AMPK-dependent manner. Biochemical and molecular docking analyses indicated that FFA activates AMPK via a direct stimulation of calcium/calmodulin-dependent protein kinase kinase beta (CaMKKβ) activity. Collectively, our data indicate that FFA represents a class of existing drugs that may be of potential utility in the treatment of cholera caused by EL infection via AMPK-mediated suppression of NF-κB signaling in IEC., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Skin penetration amounts of a highly lipophilic drug, ufenamate, prepared in four oily vehicles, including white petrolatum (WP), liquid paraffin (LP), isopropyl myristate (IPM), and isocetyl stearate (ICS), were compared. Ufenamate was mixed in each vehicle at 5% and applied at a rate of 2 mg/cm 2 to intact, stripped, and delipidized Yucatan micropig skin. The amounts of ufenamate and IPM in the stratum corneum (SC), epidermis, and dermis were determined. The skin penetration amounts of ufenamate from liquid oils were significantly higher than those from WP; the amounts of ufenamate were in the order WP

Background: Salmonella typhimurium is the main cause of gastrointestinal illness in humans, and treatment options are decreasing because drug-resistant strains have emerged., Objective: The objective of this study was to use computational drug repurposing to identify a novel candidate with an effective mechanism of action to circumvent the drug resistance., Methods: We used the Mantra 2.0 database to initially screen drug candidates that share similar gene expression profiles to those of quinolones. Data were further reduced using pharmacophore mapping theory. Finally, we employed molecular-simulation studies to calculate the binding affinity of the screened candidates with DNA gyrase, alongside an analysis of side effects., Results: A total of 16 drug candidates from the Mantra 2.0 database were screened. The pharmacophoric features of the screened candidates were examined and nalidixic acid features compared using the PharamGist program. A total of 11 compounds with the highest pharmacophore score were considered for binding energy calculation. Finally, we analysed the side effects of the eight drug candidates that showed significant binding affinity in the simulation study., Conclusion: Overall, flufenamic acid and sulconazole may be potential drug candidates that could be studied in vitro to assess their resistance profile against Salmonella enterica Typhimurium.

For some time, in-silico models to address substance transport into and through the skin are gaining more and more importance in different fields of science and industry. In particular, the mathematical prediction of in-vivo skin absorption is of great interest to overcome ethical and economical issues. The presented work outlines a strategy to address this problem and in particular, investigates in-vitro and in-vivo skin penetration experiments of the model compound flufenamic acid solved in an ointment by means of a mathematical model. Experimental stratum corneum concentration-depth profiles (SC-CDP) for various time intervals using two different in-vitro systems (Franz diffusion cell, Saarbruecken penetration model) were examined and simulated with the help of a highly optimized three compartment numerical diffusion model and compared to the findings of SC-CDPs of the in-vivo scenario. Fitted model input parameters (diffusion coefficient and partition coefficient with respect to the stratum corneum) for the in-vitro infinite dose case could be used to predict the in-use conditions in-vitro. Despite apparent differences in calculated partition coefficients between in-vivo and in-vitro studies, prediction of in-vivo scenarios from input parameters calculated from the in-vitro case yielded reasonable results., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: In the last 50 years, the use of medical implants has increased dramatically. Failure of implanted devices and biomaterials is a significant source of morbidity and increasing healthcare expenditures. An important cause of implant failure is the host inflammatory response. Recent evidence implicates extracellular ATP as an important inflammatory signaling molecule. A major pathway for release of cytoplasmic ATP into the extracellular space is through connexin hemichannels, which are the unpaired constituents of gap junction intercellular channels. Blockade of hemichannels of the connexin 43 (Cx43) isoform has been shown to reduce inflammation and improve healing. We have developed a Cx43 mimetic peptide (JM2) that targets the microtubule-binding domain of Cx43. The following report investigates the role of the Cx43 microtubule-binding domain in extracellular ATP release by Cx43 hemichannels and how this impacts early inflammatory events of the foreign body reaction., Methods: In vitro Cx43 hemichannel-mediated ATP release by cultured human microvascular endothelial cells subjected to hypocalcemic and normocalcemic conditions was measured after application of JM2 and the known hemichannel blocker, flufenamic acid. A submuscular silicone implant model was used to investigate in vivo ATP signaling during the early foreign body response. Implants were coated with control pluronic vehicle or pluronic carrying JM2, ATP, JM2+ATP, or known hemichannel blockers and harvested at 24 h for analysis., Results: JM2 significantly inhibited connexin hemichannel-mediated ATP release from cultured endothelial cells. Importantly, the early inflammatory response to submuscular silicone implants was inhibited by JM2. The reduction in inflammation by JM2 was reversed by the addition of exogenous ATP to the pluronic vehicle., Conclusions: These data indicate that ATP released through Cx43 hemichannels into the vasculature is an important signal driving the early inflammatory response to implanted devices. A vital aspect of this work is that it demonstrates that targeted molecular therapeutics, such as JM2, provide the capacity to regulate inflammation in a clinically relevant system.

Although microemulsions are one of the most promising dermal carrier systems, their clinical use is limited due to their skin irritation potential. Therefore, microemulsions based on naturally derived monoacyl phosphatidylcholine (MAPL) were developed. The influence of the water, oil and surfactant content on dermal delivery of flufenamic acid was systematically investigated for the first time. A water-rich microemulsion led to significantly higher in vitro skin penetration of flufenamic acid compared to other microemulsions. The superiority of the water-rich microemulsion over a marketed flufenamic acid containing formulation was additionally confirmed. Differences in drug delivery could be explained by alterations of the microemulsions after application. Evaporation of isopropanol led to crystal-like structures of MAPL on the skin surface from the surfactant- or oleic acid-rich microemulsions. In contrast, the formation of this additional barrier was hindered in case of the water-rich microemulsion. The skin penetration of MAPL was additionally analyzed by combined ATR-FTIR and tape stripping experiments, where MAPL itself penetrated only into the initial layers of the stratum corneum, independent of the microemulsion composition. Since a surfactant must penetrate the skin to cause irritation, MAPL can be presumed as a skin-friendly emulsifier with the ability to stabilize pharmaceutically acceptable microemulsions., (Copyright © 2014 Elsevier B.V. All rights reserved.)