British Journal of Pharmacology

Background and Purpose The antiepileptic drug ethosuximide (ETX) suppresses epileptiform activity in a mouse model of GNB1 syndrome, caused by mutations in Gβ1 protein, likely through the inhibition of G‐protein gated K⁺ (GIRK) channels. Here, we investigated the mechanism of ETX inhibition (block) of different GIRKs. Experimental Approach We studied ETX inhibition of GIRK channels expressed in Xenopus oocytes with or without their physiological activator, the G protein subunit dimer Gβγ. ETX binding site and mode of action were analysed using molecular dynamic (MD) simulations and kinetic modelling, and the predictions were tested by mutagenesis and functional testing. Key Results We show that ETX is a subunit‐selective, allosteric blocker of GIRKs. The potency of ETX block is increased by Gβγ, in parallel with channel activation. MD simulations and mutagenesis locate the ETX binding site in GIRK2 to a region associated with phosphatidylinositol‐4,5‐bisphosphate (PIP2) regulation, and suggest that ETX acts by closing the helix bundle crossing (HBC) gate and altering channel's interaction with PIP2. The apparent affinity of ETX block is highly sensitive to changes in channel gating caused by mutations in Gβ1 or GIRK subunits. Conclusion and Implications ETX block of GIRKs is allosteric, subunit‐specific, and enhanced by Gβγ through an intricate network of allosteric interactions within the channel molecule. Our findings pose GIRK as a potential therapeutic target for ETX and ETX as a potent allosteric GIRK blocker and a tool for probing gating‐related conformational changes in GIRK.

Background and purpose Autophagy–lysosomal pathway dysfunction leads to postoperative cognitive dysfunction (POCD). Dexmedetomidine (Dex) improves POCD, and we probed the effects of Dex on autophagy–lysosomal pathway dysfunction in a POCD model. Experimental approach A POCD mouse model was established and intraperitoneally injected with Dex. Cognitive function was evaluated by Morris water maze/open field test/novel object recognition assay. Levels of neurotransmitters/inflammatory cytokines in hippocampus, and NLRP3/ASC/Cleaved Caspase‐1 proteins were determined by ELISA/Western blot. NLRP3 inflammasome‐mediated microglial activation/astrocyte A1 differentiation in the hippocampal CA1 region were assessed by immunofluorescence assay. BV‐2 cells were treated with lipopolysaccharide (LPS) and Dex and/or the NLRP3 inflammasome activator Nigericin, and transfected with si‐TFEB for co‐culture with primary reactive astrocytes (RAs) to verify the function of Dex in vitro. Key Results Dex alleviated cognitive dysfunction in POCD mice and repressed NLRP3 inflammasome‐mediated microglial activation and astrocyte A1 differentiation. NLRP3 inflammasome activation partially reversed the protective effect of Dex on the POCD condition. In vitro experiments verified the inhibitory properties of Dex on microglial activation and astrocyte A1 differentiation. Dex induces TFEB nuclear translocation, microglial autophagy and lysosomal biogenesis. By activating the autophagy–lysosome pathway, Dex regulated NLRP3 inflammasome‐mediated microglial activation, inhibited astrocyte A1 differentiation and alleviated POCD in vivo. Conclusion and implications Dex regulates NLRP3 inflammasome‐mediated hippocampal microglial activation by promoting TFEB nuclear translocation and activating the autophagy–lysosome pathway and inhibits astrocyte A1 differentiation, thereby alleviating POCD.

Background and purpose Pituitary adenylate cyclase activating polypeptide (PACAP) is a human migraine trigger that is being targeted for migraine. The δ‐opioid receptor (δ‐receptor) is a novel target for the treatment of migraine, but its mechanism remains unclear. The goals of this study were to develop a mouse PACAP‐headache model using clinically significant doses of PACAP; determine the effects of δ‐receptor activation in this model; and investigate the co‐expression of δ‐receptors, PACAP and PACAP‐PAC1 receptor. Experimental approach Cephalic allodynia to low doses of acute and chronic PACAP were tested. A triptan (sumatriptan) and a CGRP receptor antagonist (olcegepant) were tested in this model. The δ‐receptor agonist SNC80 was tested in PACAP and CGRP‐induced headache models. Expression of PACAP, PAC1, CRLR and δ‐receptors was determined using in situ hybridisation. Key results Low doses of PACAP produced dose‐dependent acute and chronic cephalic allodynia, blocked by sumatriptan but not by olcegepant. The PAC1 antagonist (M65) did not inhibit CGRP‐induced allodynia. There was moderate co‐expression of PAC1 and CRLR transcripts in migraine‐related regions. SNC80 blocked PACAP‐ and CGRP‐induced allodynia. There was low co‐expression of PACAP and δ‐receptors in brain regions measured. However, there was high co‐expression of PAC1 and δ‐receptors in somatosensory cortex, hippocampus and trigeminal nucleus caudalis. Conclusion and implications We developed a translationally significant model of PACAP‐induced headache, which was mechanistically distinct from CGRP. Activation of δ‐receptors blocked PACAP‐ and CGRP‐induced allodynia, and δ‐receptors were highly co‐expressed with the PACAP‐ergic system. Future studies will examine the functional relationship between δ‐receptors and PAC1.

Background and Purpose Tumour hypoxia frequently presents a major challenge in the treatment of neuroblastoma (NBL). The neuroblastoma cells produce carbonic anhydrase IX (CA IX), an enzyme crucial for the survival of cancer cells in low‐oxygen environments. Experimental Approach We designed and synthesised a novel high‐affinity inhibitor of CA IX. The highest to‐date. The affinities were determined for all human catalytically active CA isozymes showing significant selectivity for CA IX over other isozymes. The inhibitor effect on neuroblastoma cancer cell growth was determined in vitro and in vivo via a mice xenograft model. Key Results The novel designed inhibitor effectively mitigated the acidification induced by CA IX and reduced spheroid growth under hypoxic conditions in the SK‐N‐AS cell line. It also diminished the secretion of pro‐tumour chemokines IL‐8 (CXCL2) and CCL2. When we combined this novel CA IX inhibitor with a compound that inhibits the chemokine receptor CCR2 protein activity, we observed a reduction in mouse tumour growth. The combined treatment also prompted tumours to exhibit adaptive resistance by producing higher levels of vascular endothelial growth factor receptors (VEGFR) and other compensatory signals. Conclusions and Implications This research underscores the pivotal role of CA IX in cancer and the potential of a novel CA IX inhibitor‐based combination intervention therapy for neuroblastoma treatment.

Background and Purpose Irritable bowel syndrome (IBS) is a common condition that is challenging to treat, and novel drugs are needed for this condition. Previously, a chronic vicarious social defeat stress (cVSDS) mouse model exhibits IBS‐like symptoms. Also agonists of the opioid δ‐receptor exert anti‐stress effects in rodents with minimal adverse effects. Here, we evaluated the effects of δ‐receptor agonists on the IBS‐like symptoms in cVSDS mice. Experimental Approach cVSDS mice (male C57BL/6J mice) were prepared following a 10‐day exposure to witness of social defeat stress. Subsequently, intestinal peristaltic motility and abdominal hyperalgesia were evaluated using the charcoal meal test (CMT) and capsaicin‐induced hyperalgesia test (CHT), respectively. Extracellular glutamate levels were measured using in vivo brain microdialysis. The drug was singly administrated 30 min before testing. Key Results In cVSDS mice, systemic (10 mg kg⁻¹) and intracerebroventricular (30 nmol) administration of a δ‐receptor agonist regulated intestinal peristalsis in the CMT and relieved abdominal pain in the CHT. Effects of systemic administration were blocked by intracerebroventricular injection of a δ‐receptor inhibitor. Local infusion of the δ‐receptor agonist (0.6 nmol) into the insular cortex improved cVSDS‐induced intestinal hypermotility. The in vivo brain microdialysis study showed that re‐exposure to VSDS elevated the extracellular glutamate levels in the IC, which was restored by the δ‐receptor agonist. Conclusions and Implications We propose that agonists of opioid δ‐receptors are potential drugs for the radical treatment of IBS because they can ameliorate IBS‐like symptoms via the CNS, specifically the insular cortex.

Background and Purpose Alzheimer's disease (AD) is a widespread neurodegenerative condition characterized by amyloid‐beta (Aβ) plaques and tau protein aggregates, leading to significant cognitive decline. Existing treatments primarily offer symptomatic relief, underscoring the urgent need for novel therapies that address multiple AD pathways. This study evaluates the efficacy of DK02, a hydroxyl chalcone derivative, in a scopolamine‐induced dementia model in zebrafish, hypothesizing that it targets several neurodegenerative mechanisms simultaneously. Experimental Approach We employed a blend of experiments, including in silico docking, in vitro enzyme inhibition assays and in vivo zebrafish models, to assess therapeutic effects of DK02. Methods included molecular docking to forecast interaction sites, acetylcholinesterase (AChE) inhibition testing, and various behavioural and histopathological analyses to gauge DK02's cognitive and neuroprotective impacts. Key Results DK02 emerged as a potent AChE inhibitor via virtual screening, and significantly enhanced cognitive functions in zebrafish, by improving memory retention and reducing anxiety‐like behaviours. DK02 also displayed strong antioxidant properties, reducing oxidative stress‐induced neuronal damage. Histopathological analysis confirmed its neuroprotective effects by showing decreased amyloid plaque burden and mitigated structural brain damage. Conclusion and Implications DK02 shows promise as a multi‐target‐directed ligand for AD, offering a new therapeutic path by simultaneously addressing cholinergic, oxidative and amyloid pathways. Its potential to enhance cognitive functions and curtail neurodegeneration suggests advantages over current symptomatic treatments. Further research into DK02 mechanisms and long‐term impacts is essential for its development in AD therapy.

Background and Purpose Glucagon‐like peptide‐1 receptor agonists (GLP‐1RAs) exert cardiovascular benefits in diabetic patients, but the underlying mechanisms remain incompletely understood. Semaglutide, a novel long‐acting GLP‐1RA, has shown a reduced risk of cardiovascular events. Based on these results, we investigated the therapeutic potential of semaglutide in diabetic cardiomyopathy and sought to elucidate the underlying mechanisms. Experimental Approach Mice with diabetes induced by high‐fat diet/streptozotocin were treated with semaglutide. The mechanisms underlying the cardioprotective effects of semaglutide were analysed using animal and cell experiments. Key Results In diabetic mice, semaglutide alleviated metabolic disorders, ameliorated myocardial fibrosis, improved cardiac function, antagonized oxidative stress and suppressed cardiomyocyte apoptosis. More significantly, semaglutide attenuated cardiac inflammation through restoring Raf kinase inhibitor protein (RKIP) expression and inhibiting downstream TANK‐binding kinase 1 (TBK1)‐NF‐κB pathway. Meanwhile, decreased RKIP expression and activated TBK1‐NF‐κB signalling pathway were also found in tissues from human diabetic hearts. RKIP deficiency exacerbated cardiac inflammation and offset the cardioprotective effect of semaglutide in diabetic mice. Moreover, semaglutide also restored the expression level of Sirtuin 3(Sirt3), which served as a modulator against cardiac inflammation by regulating RKIP‐dependent pathway. In diabetic mice, RKIP deficiency abolished the cardioprotective benefits conferred by the Sirt3 activator honokiol. We also found that cAMP/PKA signalling, rather than glucose lowering, contributed to the anti‐inflammatory effect of semaglutide through Sirt3‐dependent RKIP pathway. Conclusions and Implications Semaglutide exerted cardioprotective effects against diabetic heart failure by alleviating cardiac inflammation through Sirt3‐dependent RKIP signalling pathway.

The past decade has seen a huge increase in clinical research with psychedelic drugs and 3,4‐methylenedioxymethamphetamine (MDMA), which have revealed great potential for treating mental health conditions. Given this progress in research, as well as the current unmet clinical need of millions of patients, in 2023, the Australian Therapeutic Goods Administration (TGA) approved the use of psilocybin for treatment‐resistant depression and MDMA for PTSD to take effect from 1 July 2023. The campaign for TGA approval was led by a coalition comprising the Australian charity Mind Medicine Australia with support from Professor David Nutt, Drug Science, Professor Arthur Christopolous, Professor Chris Langmead (both from Monash University) and from large numbers of clinical, academic and patient groups. Under the rescheduling, current prescribing rights are limited to psychiatrists who have become authorised prescribers under the TGA's Authorised Prescriber Scheme, and psilocybin can only be used for treatment resistant depression and MDMA can only be used for PTSD. This paper reviews the background for this decision, its implications for approvals in other jurisdictions, as well as for the development pathways for other psychedelic drugs.

Background and Purpose Stimulator of interferon response cGAMP interactor 1 (STING), a central hub protein of cyclic GMP‐AMP synthase (cGAS)–STING signalling pathway, has a crucial role in regulating type I interferons (IFNs) production and response. Recent studies indicate that excessive activation of STING is strongly associated with autoimmune diseases, including systemic lupus erythematosus (SLE). Searching immunomodulators that negatively regulate STING might greatly contribute to the suppression of autoimmunity. Experimental Approach The peripheral blood mononuclear cells (PBMCs) of SLE patients, Hela cells, L929 cells and bone marrow‐derived macrophages (BMDMs) from mice were used as in vitro models. While, Trex1 KO mouse autoimmune disease model was used as in vivo model. After treatment with cordycepin, a nucleoside from Cordyceps mushrooms, type I IFNs production and response were determined by western blotting, real‐time polymerase chain reaction (PCR), dual‐luciferase assay, enzyme‐linked immunosorbent assay (ELISA), haematoxylin–eosin staining and RNA‐seq. Key Results Cordycepin inhibited type I IFNs production and response in human and murine systems following cGAS–STING signalling activation. Importantly, cordycepin markedly attenuates the autoinflammatory and autoimmune responses in Trex1 KO BMDMs and Trex1 KO mice. Furthermore, cordycepin effectively suppressed the production of type I IFNs and interferon‐stimulated genes (ISGs) in the PBMCs of SLE patients. Mechanistically, cordycepin promoted STING degradation via autophagy pathway upon DNA stimulation. Conclusion and Implications This study shows that cordycepin promotes STING autophagic degradation to alleviate autoimmunity upon DNA stimulation. Cordycepin might be a potential therapeutic candidate for alleviating aberrant type I IFNs in autoimmune and autoinflammatory diseases.

Background and Purpose Orexins have important biological effects on the central and peripheral nervous systems. Their primary ability is to regulate the sleep–wake cycle. Orexins and their antagonists, via OX1 receptor have been shown to have proapoptotic and antitumor effects on various digestive cancers cell models. We investigated, (1) the ability of orexin‐A and its antagonists to regulate OX1 receptor expression at the cell surface and (2), how OX1 antagonists induced proapoptotic effect in cancer cells models. Experimental Approach The OX1 receptor internalisation is determined by imaging flow cytometry in colon cancer cell models and the OX1 receptor coupling to G proteins via bioluminescence resonance energy transfer and molecular dynamic simulation. Key Results Orexin‐A induced rapid receptor internalisation within 15 min via β‐arrestin 2 recruitment, whereas antagonists had no effect. Furthermore, Gq is critical for receptor internalisation and signalling pathways, and no other G proteins appear to be recruited. Surprisingly, antagonists induced recruitment and conformational changes in Gq protein. Simulated molecular dynamics of agonists/orexin receptor/Gq complexes show that antagonists exhibits a similar binding mode, stable at the binding site and show conformational changes of ECL2, similar to that of the agonists. Conclusion and Implications OX1 receptor activation induced orexin/β‐arrestin‐dependent internalisation, which was independent of the apoptotic pathway induced by orexins and antagonists. In addition, antagonists activate the Gq protein, suggesting its putative partial dissociation. These results suggest that the development of OX1 receptor targeting molecules, including orexin antagonists with antitumor properties, may pave the way for innovative cancer therapies.

Background and Purpose Ulcerative colitis (UC) is an idiopathic inflammatory bowel disease, and the range of current clinical treatments is not ideal. We previously found that polysaccharide of Atractylodes macrocephala Koidz (PAMK) is beneficial in DSS‐induced colitis, and we aimed to investigate the underlying mechanisms in this study. Experimental Approach PAMK was used to treat DSS‐induced colitis in mice, 16S rRNA sequencing analysis was used to detect changes in the intestinal microbiota, targeted metabolomics analysis was used to determine the content of tryptophan‐metabolizing bacteria, and western blotting was used to determine aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR) levels. Furthermore, antibiotic‐mediated depletion of gut microbiota and faecal microbiota transplantation were performed to assess the role of the gut microbiota in PAMK alleviation of colitis. Key Results PAMK treatment relieved intestinal microbiota dysbiosis in mice with colitis, contributed to the proliferation of tryptophan‐metabolizing bacteria, and increased the levels of tryptophan metabolites, resulting in a significant increase in the nuclear translocation of PXR and expression of PXR and its target genes, but not AhR. The gut microbiota is important in PAMK treatment of colitis, including in the alleviation of symptoms, inhibition of inflammation, maintenance of the integrity of the intestinal barrier, and the regulation of the Th17/Treg cell balance. Conclusion and Implications Based on our findings, we elucidate a novel mechanism by which PAMK alleviates DSS‐induced colitis and thus provides evidence to support the potential development of PAMK as a new clinical drug against UC.

Background and Purpose Sarcoplasmic reticulum Ca²⁺‐ATPase (SERCA2a) is impaired in heart failure. Phosphodiesterases (PDEs) are implicated in the modulation of local cAMP signals and protein kinase A (PKA) activity essential for cardiac function. We characterise PDE isoforms that underlie decreased activities of SERCA2a and reduced cardiac contractile function in diabetic cardiomyopathy. Experimental Approach Wild type mice were fed with either normal chow or a high‐fat diet (HFD). Cardiomyocytes were isolated for excitation–contraction coupling (ECC), fluorescence resonant energy transfer PKA biosensor and proximity ligation assays. Key Results The upregulated PDE4D3 and PDE4D9 isoforms in HFD cardiomyocytes specifically bound to SERCA2a but not ryanodine receptor 2 (RyR2) on the sarcoplasmic reticulum (SR). The increased association of PDE4D isoforms with SERCA2a in HFD cardiomyocytes led to reduced local PKA activities and phosphorylation of phospholamban (PLB) but minimally effected the PKA activities and phosphorylation of RyR2. These changes correlate with slower calcium decay tau in the SR and attenuation of ECC in HFD cardiomyocytes. Selective inhibition of PDE4D3 or PDE4D9 restored PKA activities and phosphorylation of PLB at the SERCA2a complex, recovered calcium decay tau, and increased ECC in HFD cardiomyocytes. Therapies with PDE4 inhibitor roflumilast, PDE4D inhibitor BPN14770 or genetical deletion of PDE4D restored PKA phosphorylation of PLB and cardiac contractile function. Conclusion and Implications The current study identifies upregulation of specific PDE4D isoforms that selectively inhibit SERCA2a function in HFD‐induced cardiomyopathy, indicating that this remodelling can be targeted to restore cardiac contractility in diabetic cardiomyopathy.

Background and Purpose Alterations in tryptophan‐kynurenine (TRP‐KYN) pathway are implicated in major depressive disorder (MDD). α7 nicotinic acetylcholine (α7nACh) receptor regulates the hypothalamic–pituitary–adrenal (HPA) axis. We have shown that deficiency of kynurenine 3‐monooxygenase (KMO) induces depression‐like behaviour via kynurenic acid (KYNA; α7nACh antagonist). In this study, we investigated the involvement of the TRP‐KYN pathway in stress‐induced behavioural changes and the regulation of the HPA axis. Experimental Approach Mice were exposed to chronic unpredictable mild stress (CUMS) and subjected to behavioural tests. We measured TRP‐KYN metabolites and the expression of their enzymes in the hippocampus. KMO heterozygous mice were used to investigate stress vulnerability. We also evaluated the effect of nicotine (s.c.) on CUMS‐induced behavioural changes and an increase in serum corticosterone (CORT) concentration. Key Results CUMS decreased social interaction time but increased immobility time under tail suspension associated with increased serum corticosterone concentration. CUMS increased KYNA levels via KMO suppression with microglial decline in the hippocampus. Kmo+/− mice were vulnerable to stress: they exhibited social impairment and increased serum corticosterone concentration even after short‐term CUMS. Nicotine attenuated CUMS‐induced behavioural changes and increased serum corticosterone concentration by inhibiting the increase in corticotropin‐releasing hormone. Methyllycaconitine (α7nACh antagonist) inhibited the attenuating effect of nicotine. Conclusions and Implications CUMS‐induced behavioural changes and the HPA axis dysregulation could be induced by the increased levels of KYNA via KMO suppression. KYNA plays an important role in the pathophysiology of MDD as an α7nACh antagonist. Therefore, α7nACh receptor is an attractive therapeutic target for MDD.

Background and Purpose As a highly heterogeneous cancer, hepatocellular carcinoma (HCC) shows different response rates to the multi‐kinase inhibitor lenvatinib. Thus, it is important to explore genetic biomarkers for precision lenvatinib therapy in HCC. Experimental Approach The effect and mechanism of AXIN1 mutation on HCC were revealed by cell proliferation assay, long‐term clone formation assay, sphere formation assay and small molecule inhibitor library screening. A new therapeutic strategy targeting HCC with AXIN1 mutation was evaluated in humanized models (patient‐derived xenograft [PDX] and patient‐derived organoid [PDO]). Key Results Based on The Cancer Genome Atlas (TCGA) data, we screened 6 most frequently lost tumour suppressor genes in HCC (TP53, ARID1A, AXIN1, CDKN2A, ARID2 and PTEN) and identified AXIN1 as the most crucial gene for lenvatinib sensitivity. Further study showed that AXIN1‐knockout HCC cells had a more malignant phenotype and lower sensitivity to lenvatinib in vitro and in vivo. Mechanistically, the WNT pathway and its target gene c‐Myc were activated when AXIN1 was missing, and the expression of tumour suppressor p15 was inhibited by transcription co‐repressors c‐Myc and Miz‐1, resulting in the exacerbation of the resistant phenotype. Screening of a library of epigenetic‐related enzyme inhibitors showed that a KDM5B inhibitor up‐regulated p15 expression, leading to increased sensitivity to lenvatinib in vitro and in vivo. Conclusion and Implications AXIN1‐deficient patients have a lower response to lenvatinib, which may be associated with suppression of p15 mediated by WNT pathway activation. KDM5B inhibitors can restore p15 levels, resulting in efficient killing of resistant cells in HCC.

Background and Purpose Pharmacological intervention of thrombosis is challenging, requiring a fined tune balance between beneficial antithrombotic effect versus risk of major bleeding complications. In this investigation, we elucidated the antithrombotic capacity of the novel 90‐mer RNA aptamer Apta‐1 and its underlying mechanism of action. Experimental Approach We utilized three independent in vivo animal models to establish antithrombotic activity and bleeding risk of Apta‐1. Several cellular and molecular techniques were utilized to extensively characterize the effects of Apta‐1 on primary and secondary haemostasis. Key Results Apta‐1 significantly reduced thrombus weight in ferric chloride‐induced carotid artery thrombosis. A consistent reduction in thrombus weight was also observed in arteriovenous shunt thrombosis in rats, whereas tail bleeding time was unaffected. Cellular and molecular analyses revealed that Apta‐1 interacted with thrombin, resulting in significant inhibition of protease‐activated receptor (PAR) signalling in platelets. On the other hand, Apta‐1 shortened both thrombin generation and thrombin‐induced clotting times. Conclusions and Implications Apta‐1 targets the heparin‐binding motif exosite II on thrombin leading to significant suppression of platelet PAR1 and PAR4 signalling. Intriguingly, Apta‐1 produces substantial antithrombotic activity without anticoagulant or general antiplatelet properties. In fact, we found that Apta‐1 accelerates the formation of blood clots and thus supports haemostasis without exhibiting typical anticoagulant properties. We suggest that Apta‐1 may be a promising future drug candidate for treatment of thrombosis in diseases/conditions where there are significant risks of serious bleeding complications.

Background and Purpose Slow‐acting biogenic amines, such as dopamine, are known to modulate fast neurotransmitters e.g. glutamate. In the striatum, dopamine (DA) interacts with glutamate, influencing neural excitability and promoting synaptic plasticity. The exact mechanism of such interaction is not fully understood. This study investigates, in detail, how dopamine overactivity in dopamine transporter knockout (DAT−/−) rats, alters the homeostasis of the striatal glutamate synapse from a molecular, behavioural and functional point of view. Experimental Approach The expression, localisation, retention and electrophysiological properties of N‐methyl‐D‐aspartate (NMDA) receptors as well as dendritic spine density and morphology were investigated in the striatum of DAT−/− rats, at baseline and after treatment with the non‐competitive NMDA receptor antagonist memantine (30 mg kg⁻¹). Key Results Dopamine overactivity dramatically reorganises the striatal glutamate synapse, redistributing NMDA receptors in the synapse as typified by reduced synaptic availability and reduced expression of NMDA scaffolding proteins, as well as by increased GluN2B‐containing NMDA receptors in the extra synapse. Such changes are accompanied by reduced spine density, suggesting dopamine‐induced structural rearrangements. These results converge into a compromised plasticity, as shown by the impaired ability to promote long‐term depression (LTD) in the striatum of DAT−/−rats. Notably, memantine counteracts hyperlocomotion, reverses spine alterations and abolishes the extrasynaptic movements of NMDA receptors in the striatum of DAT−/− rats, thus restoring functional LTD. Conclusion and Implications A hyperdopaminergic condition seems to alter striatal homeostasis by increasing extrasynaptic NMDA receptors. These findings may be relevant to manipulate disorders characterised by elevated dopaminergic activity.

Background and Purpose Gene regulation is frequently altered in diseases in unique and patient‐specific ways. Hence, personalised strategies have been proposed to infer patient‐specific gene‐regulatory networks. However, existing methods do not scale well because they often require recomputing the entire network per sample. Moreover, they do not account for clinically important confounding factors such as age, sex or treatment history. Finally, a user‐friendly implementation for the analysis and interpretation of such networks is missing. Experimental Approach We present DysRegNet, a method for inferring patient‐specific regulatory alterations (dysregulations) from bulk gene expression profiles. We compared DysRegNet to the well‐known SSN method, considering patient clustering, promoter methylation, mutations and cancer‐stage data. Key Results We demonstrate that both SSN and DysRegNet produce interpretable and biologically meaningful networks across various cancer types. In contrast to SSN, DysRegNet can scale to arbitrary sample numbers and highlights the importance of confounders in network inference, revealing an age‐specific bias in gene regulation in breast cancer. DysRegNet is available as a Python package (https://github.com/biomedbigdata/DysRegNet_package), and analysis results for 11 TCGA cancer types are available through an interactive web interface (https://exbio.wzw.tum.de/dysregnet). Conclusion and Implications DysRegNet introduces a novel bioinformatics tool enabling confounder‐aware and patient‐specific network analysis to unravel regulatory alteration in complex diseases.

Background and Purpose Our previous study reported that fructose intake increased systemic blood pressure and reduced nitric oxide (NO) in the nucleus tractus solitarius (NTS) due to oxidative stress and neuroinflammation. However, it remains unclear how reactive oxygen species (ROS) reduce NO and how this process impacts neuroinflammation in the NTS. This study aimed at investigating the effect of ROS on acetylation of high mobility group box 1 protein (HMGB1) in the NTS of fructose‐induced hypertensive rats. Experimental Approach Male Wistar‐Kyoto (WKY) rats were fed with 10% fructose water to elevate blood pressure. Thereafter, CLI‐095 and glycyrrhizic acid (GA) treatments were delivered for up to 2 weeks (1 mg·12 μL⁻¹·day⁻¹, by intracerebroventricular injection) to reduce the negative effects of toll‐like receptor 4 (TLR4) and HMGB1 activation. Key Results Two weeks of CLI‐095 and GA treatment reduced systemic blood pressure and significantly preserved neuronal and endothelial nitric oxide synthase (nNOS and eNOS) availability against the inflammatory insults of fructose consumption. Both CLI‐095 and GA halted the interaction of acetylated HMGB1 and TLR4. Two weeks of CLI‐095 and GA treatment markedly reduced NTS inflammation (pro‐inflammatory cytokines and microglial activation) and lowered serum norepinephrine levels. Conclusion and Implications Our data reveal novel pharmacological properties for CLI‐095 and GA, which improved blood pressure and inflammatory conditions by decreasing the interaction of acetylated HMGB1 with TLR4. These findings challenge the commonly accepted dogma that essential hypertension is specifically mediated by neuroinflammation due to acetylated HMGB1 coupling to TLR4.

Background and Purpose The current therapy cannot meet the needs of glioblastoma (GBM). V‐domain immunoglobulin suppressor of T‐cell activation (VISTA) is significantly up‐regulated in GBM patients; however, its therapeutic potential in GBM is still unclear. Experimental Approach Flow cytometry was used to detect the expression of VISTA and the co‐expression pattern of VISTA and programmed death receptor 1 (PD‐1) on brain infiltrating lymphocytes of GBM mice. Monoclonal antibody therapy was used to evaluate the therapeutic effect of α‐VISTA monotherapy and α‐VISTA combined with α‐PD‐1 on GBM mice. Transcriptome analysis, flow cytometry, and immunofluorescence were used to detect changes of immune microenvironment in mouse brain tumours. Immunofluorescence and TCGA data analysis were used to further validate the combined treatment strategy on patient data. Key Results Compared with normal mice, the frequency of VISTA expression and co‐expression of VISTA and PD‐1 on tumour‐infiltrating lymphocytes (TILs) in tumour‐bearing mice was increased. Anti‐VISTA monotherapy significantly up‐regulated multiple immune stimulation‐related pathways and moderately prolonged mouse survival time. Blocking the immune checkpoint VISTA and PD‐1 significantly prolonged the survival time of mice and cured about 80% of the mice; CD8⁺ T cells played an important role in this process. In addition, we found that the expression of VISTA and PD‐1 was significantly up‐regulated in GBM patients by immunofluorescence, and patients with high expression of VISTA and PD‐1 were associated with poor overall survival. This combination of blocking the immune checkpoint VISTA and PD‐1 may achieve clinical transformation in GBM.

Hypertension is a major contributor to cardiovascular disease and its associated morbidity and mortality. The low efficacy observed with some anti‐hypertensive therapies has been attributed partly to inter‐individual genetic variability. This paper reviews the major findings regarding these genetic variabilities that modulate responses to anti‐hypertensive therapies such as angiotensin converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), diuretics, calcium channel blockers (CCBs) and β‐adrenoceptor blockers. The importance of studying these genetic polymorphisms stems from the goal to optimise anti‐hypertensive therapy for each individual patient, aiming for the highest efficacy and lowest risk of adverse effects. It is important to recognise that environmental and epigenetic factors can contribute to the observed variations in drug responses. Owing to the multigenic and multifactorial nature of drug responses, further research is crucial for translating these findings into clinical practice and the establishment of reliable recommendations.

Background and Purpose Paracetamol has been found to alleviate inflammatory pain by modulating KV7 channels. Its metabolite N‐acetyl‐4‐benzoquinoneimine (NAPQI) increases currents through these channels via a stretch of three cysteine residues in the channel S2–S3 linker. Through this effect, the excitability of neurons in the pain pathway is dampened. Inflammatory mediators, in turn, enhance the excitability of sensory neurons by inhibiting KV7 channels. Here, a specific interaction between NAPQI and the so‐called inflammatory soup was investigated. Experimental Approach Currents through KV7 channels were measured in sensory neurons and after heterologous expression in tsA201 cells. In addition, changes in cytosolic Ca²⁺ and in the distribution of PIP2 (PI(4,5)P2) between membrane and cytosol were determined by fluorescence microscopy. Key Results NAPQI abolished Ca²⁺‐mediated inhibitory effects of an ‘inflammatory soup’ containing ADP, ATP, bradykinin, histamine, 5‐hydroxytryptamine, prostaglandin E2, substance P and a PAR2 agonist on KV7 channel currents in sensory neurons. Moreover, the increase of KV7.2 channel currents by quenching of cytosolic Ca²⁺ as well as the current decrease by depletion of membrane PIP2 was impaired by NAPQI. These effects were lost in mutant channels lacking the three cysteines in the S2–S3 linker. Conclusion and Implication NAPQI targets the three‐cysteine motif in the S2–S3 linker of KV7.2 channels to counteract the signalling cascades employed by inflammatory mediators that inhibit these channels. In sensory neurons, this abolishes the closure of KV7 channels by the inflammatory soup. This mechanism is likely involved in the alleviation of inflammatory pain by paracetamol.

Background and Purpose Abdominal pain is a leading cause of morbidity for people living with gastrointestinal disease. Whereas the transient receptor potential vanilloid 4 (TRPV4) ion channel has been implicated in the pathogenesis of abdominal pain, the relative paucity of TRPV4 expression in colon‐projecting sensory neurons suggests that non‐neuronal cells may contribute to TRPV4‐mediated nociceptor stimulation. Experimental Approach Changes in murine colonic afferent activity were examined using ex vivo electrophysiology in tissues with the gut mucosa present or removed. ATP and glutamate release were measured by bioluminescence assays from human colon organoid cultures and mouse colon. Dorsal root ganglion sensory neuron activity was evaluated by Ca²⁺ imaging when cultured alone or co‐cultured with colonic mucosa. Key Results Bath application of TRPV4 agonist GSK1016790A elicited a robust increase in murine colonic afferent activity, which was abolished by removing the gut mucosa. GSK1016790A promoted ATP and glutamate release from human colon organoid cultures and mouse colon. Inhibition of ATP degradation in mouse colon enhanced the afferent response to GSK1016790A. Pretreatment with purinoceptor or glutamate receptor antagonists attenuated and abolished the response to GSK1016790A when given alone or in combination, respectively. Sensory neurons co‐cultured with colonic mucosal cells produced a marked increase in intracellular Ca²⁺ to GSK1016790A compared with neurons cultured alone. Conclusion and Implications Our data indicate that mucosal release of ATP and glutamate is responsible for the stimulation of colonic afferents following TRPV4 activation. These findings highlight an opportunity to target the gut mucosa for the development of new visceral analgesics.

Background and Purpose The limited effectiveness of current pharmacological treatments for alcohol use disorder (AUD) highlights the need for novel therapies. These may involve the glucagon‐like peptide‐1 receptor or the amylin receptor, as treatment with agonists targeting either of these receptors lowers alcohol intake. The complexity of the mechanisms underlying AUD indicates that combining agents could enhance treatment efficacy. While a combination of amylin receptor and GLP‐1 receptor agonists reduced food intake and body weight synergistic‐like, its influence on alcohol intake is unknown. Experimental Approach Effects of a range of dose‐combinations of GLP‐1 receptor (dulaglutide) and amylin receptor (salmon calcitonin; sCT) agonists on alcohol intake were explored in male and female rats. We used dose combinations that either lowered alcohol intake as monotherapy (0.1 mg·kg⁻¹ + 5 μg·kg⁻¹), or that did not affect alcohol consumption per se (0.075 mg·kg⁻¹ + 2 μg·kg⁻¹). Key Results Acute administration of dulaglutide and sCT (0.1 mg·kg⁻¹ + 5 μg·kg⁻¹) reduced alcohol intake in males, but not in females. When higher doses were evaluated in female rats, a decrease in alcohol intake was observed. Furthermore, the low dose combination (0.075 mg·kg⁻¹ + 2 μg·kg⁻¹) decreased, in in a synergistic‐like manner, alcohol intake and prevented abstinence‐induced drinking without affecting kaolin intake in males. However, tolerance developed during sub‐chronic treatment. Conclusion and Implications Collectively, these findings show that the combination of dulaglutide and sCT decreased, in in a synergistic‐like manner, alcohol consumption in male rats. Contrarily, higher doses are required for females.

Background and Purpose Cardiac glycosides (CGs), traditionally prescribed for heart failure and arrhythmias, show anticancer potential. However, their mechanisms for preferential inhibition of tumour tissue and constituent malignant cells are not fully elucidated. This study aims to elucidate the therapeutic benefits of CGs in targeting specific tumours and dissect their multi‐targeting mechanisms that confer their cytotoxicity against malignant cells. Experimental Approach We designed an integrated workflow to identify therapeutic CGs with high toxicity to certain cancers, investigating their multi‐target effects, assessing their toxicity to malignant cells and analysing the prognostic relevance of CGs' target genes. The computational findings were confirmed through gene knockdown, cell viability assays, reactive oxygen species (ROS) measurements and so forth. Key Results CGs modulate multiple genes crucial for ion homeostasis, oxidative stress and apoptosis, with a particularly strong inhibitory effects on uveal melanoma (UVM). Notably, digitoxin suppresses UVM cell proliferation and induces ROS levels by simultaneously targeting STAT3 and KLF5. Single‐cell transcriptomic analysis revealed that malignant cells are likely more vulnerable to CGs due to their higher expression of CG target genes compared with surrounding cells in the UVM microenvironment. Conclusions and Implications Given UVM's limited options, our study highlights the potential of digitoxin as a promising novel therapeutic agent for this aggressive and rare ocular cancer. Our comprehensive approach is effective in identifying the potent, cancer‐specific therapeutic agents from herbal plants.

Mechanopharmacology is an emerging interdisciplinary field that investigates drug action using biomechanically appropriate in vitro systems to the relevant (patho)physiology. This review outlines emerging technologies and techniques which aim to bridge the gap between mechanical cues influencing cellular biology and conventional pharmacology. We delve into the impact of mechanopharmacology on drug development in cancers and fibrotic diseases. Mechanical cues such as stretch, stiffness, circadian rhythms, fluid flow, intercellular signalling cascades and cytoskeletal structures can modulate drug interactions with molecular targets with implications for drug discovery and development. Models incorporating mechanopharmacological cues to investigate pharmacokinetics, pharmacodynamics and therapeutic outcomes are outlined. Furthermore, this review discusses innovations in the use of biomaterials and microfluidics to further enable the emulation of the mechanical microenvironment. We advocate for the application of mechanopharmacological considerations to improve the physiological relevance of methods used in the drug discovery pipeline.