British Journal of Pharmacology

The P2X3 receptor (P2X3R), an ATP-gated non-selective cation channel of the P2X receptor family, is expressed in sensory neurons and involved in nociception. P2X3R inhibition was shown to reduce chronic and neuropathic pain. In a previous screening of 2000 approved drugs, natural products and bioactive substances, various non-steroidal anti-inflammatory drugs (NSAIDs) were found to inhibit P2X3R-mediated currents. To investigate whether the inhibition of P2X receptors contributes to the analgesic effect of NSAIDs, we characterized the potency and selectivity of various NSAIDs at P2X3R and other P2XR subtypes using two-electrode voltage clamp electrophysiology. We identified diclofenac as a hP2X3R and hP2X2/3R antagonist with micromolar potency (with IC50 values of 138.2 {micro}M and 76.7 {micro}M, respectively). A weaker inhibition of hP2X1R, hP2X4R and hP2X7R by diclofenac was determined. Flufenamic acid (FFA) proved to inhibit hP2X3R, rP2X3R and hP2X7R (IC50 values of 221{micro}M, 264.1{micro}M and [~] 900{micro}M, respectively), questioning its widespread use as a nonselective ion channel blocker, when P2XR-mediated currents are under study. Inhibition of the hP2X3R or hP2X2/3R by diclofenac could be overcome by prolonged ATP-application or increasing concentrations of the agonist ,{beta}-meATP, respectively, indicating competition of diclofenac and the agonists. Molecular dynamics simulation showed that diclofenac largely overlaps with ATP bound to the open state of the hP2X3R. Our results strongly support a competitive antagonism through which diclofenac, by interacting with residues of the ATP-binding site, left flipper, and dorsal fin domains inhibits gating of P2X3R by conformational fixation of the left flipper and dorsal fin domains. In summary, we demonstrate the inhibition of the human P2X3 receptor by various NSAIDs. Diclofenac proved to be the most effective antagonist with a strong inhibition of hP2X3R and hP2X2/3R and a weaker inhibition of hP2X1R, hP2X4R and hP2X7R. Considering their involvement in nociception, inhibition of hP2X3R and hP2X2/3R by micromolar concentrations of diclofenac may contribute to the analgesic effect as well as the side effect of taste disturbances of diclofenac and represent an additional mode of action besides the well-known high potency COX inhibition.

The action of the antidepressants imipramine, amitriptyline, and paroxetine on LPA3 receptors was studied in cellulo, using receptor-transfected HEK 293 Flp-In TREx cells, and in silico, through docking simulations. These drugs showed a low affinity for LPA3 receptors with lesser efficacy than LPA (paroxetine {approx} 60% and imipramine and amitriptyline {approx} 30%). When LPA-treated cells (with the agonist present) were challenged with the antidepressants, paroxetine triggered a robust increase in intracellular calcium, whereas imipramine and amitriptyline decreased the calcium concentration below baseline values. For ERK 1/2 phosphorylation, imipramine induced a rapid and potent increase, whereas amitriptyline and paroxetine reduced ERK 1/2 phosphorylation below baseline. Similarly, imipramine produced rapid and robust ERK phosphorylation in LPA-stimulated cells, but amitriptyline decreased ERK 1/2 phosphorylation. Activation with the antidepressants leads to LPA3 internalization; dramatic morphological changes accompany these actions. Docking simulations showed these drugs interact with an LPA3 receptor pocket, denominated Upper Cavity. Although the agonist binding cavity was the same, the amino acids interacting with the various ligands were distinct due to their different chemical structure. The manuscript advances knowledge on the mechanisms of antidepressant effects on LPA3 receptors, which might have potential therapeutic implications.

Cannabigerol (CBG), a non-psychotropic phytocannabinoid, is a precursor for cannabis derivatives, {Delta}9-tetrahydrocannabinol and cannabidiol (CBD). Like CBD, CBG has been suggested as an analgesic. A previous study reported CBG (10 M) blocks voltage-gated sodium (Nav) currents in CNS neurons. However, the manner in which CBG inhibits Nav channels, and whether this effect contributes to CBGs potential analgesic behavior remain unknown. Genetic and functional studies have validated Nav1.7 as an opportune target for analgesic drug development. The efforts to develop therapeutic selective Nav1.7 blockers have been unsuccessful thus far, possibly due to issues in occupancy; drugs have been administered at concentrations many folds above IC50, resulting in loss of isoform-selectivity, and increasing off-target effects. We reasoned that an alternative approach could use compounds possessing 2 important properties: ultra-hydrophobicity and functional selectivity. Hydrophobicity could enhance absorption into neuronal cells especially with local administration. Functional selectivity could reduce likelihood of side-effects. As CBG is ultra-hydrophobic (cLogD=7.04), we sought to determine whether it also possesses functional selectivity against Nav channels that are expressed in dorsal root ganglion (DRG). We found that CBG is a ~10-fold state-dependent Nav inhibitor (KI-KR: ~2-20 M) with an average Hill-slope of ~2. We determined that at lower concentrations, CBG predominantly blocks sodium Gmax and slows recovery from inactivation; however, as concentration is increased, CBG also hyperpolarizes Nav inactivation curves. Our modeling and multielectrode array recordings suggest that CBG attenuates DRG excitability, which is likely linked with Nav inhibition. As most Nav1.7 channels are inactivated at DRG resting membrane potential, they are more likely to be inhibited by lower CBG concentrations, suggesting functional selectivity against Nav1.7 compared to other Navs (via Gmax block).

ATP-sensitive potassium (KATP) channel opener levcromakalim is a potent inducer of vasodilation, headache, and migraine attacks in humans and tactile hypersensitivity in mice. Other migraine-inducing agents such as nitric oxide (NO) donors, CGRP, and PACAP are thought to activate second messengers leading to KATP opening. Yet, how KATP channel opening leads to migraine remains unclear. Here, we investigated the contribution of nitric oxide synthase (NOS) isoforms and downstream signaling cascades in a mouse model of migraine-relevant tactile hypersensitivity induced by repeated administration of levcromakalim. The non-selective NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) effectively prevented levcromakalim-induced hypersensitivity. Gene expression analysis in the dura mater suggested contributions from endothelial NOS (eNOS) and inducible NOS (iNOS). Semi-selective nNOS inhibition with S-methyl-L-Thiocitrulline (SMTC) or genetic deletion of neuronal NOS (nNOS) had minimal effect on hypersensitivity and no effect on vasodilation. In contrast, eNOS-/- mice were partially protected from levcromakalim-induced hypersensitivity and exhibited impaired vascular response, highlighting eNOS as a key mediator. Inhibition of iNOS with S-methylisothiourea (SMT) revealed a possible contribution from iNOS as well. Surprisingly, inhibition of soluble guanylate cyclase (sGC) had no effect, while the peroxynitrite decomposition catalyst FeTPPS partially attenuated hypersensitivity, implicating nitrosative stress--rather than classical NO-sGC-cGMP signaling--as the critical downstream pathway. We propose that levcromakalim induces both coupled and uncoupled eNOS activity, enhanced NO production and generation of reactive nitrogen species, including peroxynitrite. Our findings reveal a pivotal role for eNOS and peroxynitrite in KATP channel-induced migraine-relevant hypersensitivity and support targeting nitrosative stress as a potential therapeutic strategy.

BackgroundThe etiology of essential tremor (ET) remains unclear but may involve abnormal firing of Purkinje cells, which receive excitatory inputs from granule cells in the cerebellum. Since 6 subunit-containing GABAA receptors (6GABAARs) are abundantly expressed in granule cells, we validated a hypothesis that 6GABAAR-selective positive allosteric modulators (PAMs) are promising pharmacological interventions for ET therapy. MethodsEmploying the harmaline-induced ET model in male ICR mice, we evaluated the possible anti-tremor effects of four 6GABAAR-selective PAMs, the pyrazoloquinolinones Compound 6 and LAU-463 and their respective deuterated derivatives. Propranolol, a clinical anti-tremor agent, was employed as positive control. To investigate the involvement of cerebellar 6GABAARs in the antitremor effect of intraperitoneal (i.p.) Compound 6, furosemide, an 6GABAAR antagonist, was intracerebellarly (i.cb.) co-administered with Compound 6. The burrowing activity, an indicator of wellbeing in rodents, was measured concurrently. ResultsHarmaline (10-30 mg/kg, s.c.) induced action tremor in ICR mice dose-dependently and markedly reduced their burrowing activity. Compound 6 (3 and 10 mg/kg, i.p.) significantly attenuated harmaline (20 mg/kg)-induced action tremor and burrowing activity impairment. Propranolol (20 mg/kg, i.p.) diminished tremor but failed to restore the burrowing activity in harmaline-treated mice. Importantly, both anti-tremor and burrowing activity restorative effects of Compound 6 (10 mg/kg, i.p.) was significantly reversed by co-administration of i.cb. furosemide at a dose (10 nmol/0.5 l) having no effect per se. All four 6GABAAR PAMs exhibited a similar therapeutic efficacy. Conclusion6GABAAR-selective PAMs significantly attenuated action tremor and restored physical well-being in a mouse model mimicking ET by acting in the cerebellum. Thus, 6GABAAR-selective PAMs may be potential therapeutic agents for ET.

Chronic pain affects a significant portion of the global population and imposes substantial clinical and socioeconomic burdens. Current treatments mainly rely on opioid analgesics, which carry serious risks of dependence and misuse, underscoring the urgent need for alternative therapeutic strategies. Galanin receptors (GALR1-3) are known to be involved in modulating pain, yet their specific roles remain poorly understood due to the lack of receptor subtype-selective ligands. Recently, spexin has been identified as an endogenous peptide that selectively activates GALR2 and GALR3, offering a new scaffold for developing pharmacological tools targeting these receptor subtypes. In this study, we report the design and characterization of a modified spexin analog, LIT-01-144, engineered through N-terminal functionalization with a fluorocarbon chain to improve metabolic stability while preserving receptor selectivity. In vitro assays showed that LIT-01-144 has high potency at GALR2 and GALR3, with minimal activity at GALR1. Pharmacokinetic studies revealed a significantly longer plasma half-life compared to native spexin and no central nervous system penetration. In mice, intracerebroventricular administration of LIT-01-144 produced strong antinociceptive effects at doses ten times lower than spexin. While systemic administration showed no notable antinociception in naive animals, LIT-01-144 significantly reduced pain responses in a mouse model of persistent inflammatory pain induced by complete Freunds adjuvant (CFA). This antinociceptive activity was specifically mediated through GALR2 and was independent of opioid receptor pathways. In situ hybridization further showed an increase in Galr2-positive neurons in dorsal root ganglia of inflamed mice. Overall, these findings highlight GALR2 as a promising peripheral target for developing non-opioid analgesics and demonstrate the potential of LIT-01-144 as a valuable tool for understanding GALR2-mediated mechanisms of pain modulation.

BackgroundNeuropathic pain is a major source of disability and distress with few pharmacological options for treatment. Opioid drugs can be effective, but high doses are needed, leading to unwanted effects. BMS-986122 is a positive allosteric modulator of the mu opioid receptor that potentiates acute opioid antinociception without increasing opioid-induced constipation, reward, or respiratory depression. Therefore, we asked if BMS-986122 could increase the effects of low-dose opioid analgesics in chronic neuropathic pain. MethodsWe employed the spared nerve injury and tibial neuroma models in rats and assessed the tactile hypersensitivity of the hind paw and site of neuroma, respectively. ResultsAdministration of low doses of (R)-methadone, morphine, or buprenorphine slightly reduced the tactile hypersensitivity of the hind paw the in spared nerve injury model. Pretreatment with BMS-986122 significantly enhanced the reversal of hypersensitivity, reaching the effect of high-dose gabapentin, a standard of care in neuropathic pain. Pretreatment with BMS-986122 similarly increased the anti-allodynic effects of low dose (R)-methadone on neuroma pain. A similar effect of (R)-methadone in the absence of BMS-986122 was only observed at a dose where respiratory distress was seen. ConclusionsThese findings show that allosteric modulators of the mu opioid receptor such as BMS-986122 can enhance opioid activity that could translate to a safe and effective treatment for chronic neuropathic pain.

Chronic pain is a complex, debilitating, and escalating health problem worldwide, impacting one in five adults. Current treatment is compromised by dose-limiting side effects including high abuse liability, loss of ability to function socially and professionally, fatigue, drowsiness, and apathy. PICK1 has emerged as a promising target for the treatment of chronic pain conditions. Here, we develop and characterize a cell-permeable fatty acid conjugated bivalent peptide inhibitor of PICK1 and assess its effects on acute and chronic pain. The myristoylated myr-NPEG4-(HWLKV)2, (mPD5), self-assembles into core-shell micelles that provide favourable pharmacodynamic properties and relieves ongoing and evoked mechanical hypersensitivity, thermal hypersensitivity as well as anxio-depressive symptoms in mouse models of neuropathic and inflammatory pain following subcutaneous administration. No overt no side effects were associated with mPD5 administration, and it has no effect on acute nociception. Finally, neuropathic pain is relieved far into the chronic phase (18 weeks post SNI surgery) and while the effect of a single injection ceases after a few hours, repeated administration provides pain relief lasting up to 20 hours after the last injection.

Hypertension-induced erectile dysfunction is associated with endothelial dysfunction in the corpus cavernosum. Membrane depolarization activates the NLRP3 inflammasome, with downregulation of endothelial Ca2+-activated K+ channels type 2.3 (KCa 2.3) and upregulation of endothelin-1 (ET-1) linked to erectile dysfunction. However, underlying mechanisms remain incompletely understood. We hypothesized that activating KCa 2.2/2.3 channels reverses erectile dysfunction and ET-1-induced NLRP3 activation in hypertensive DOCA/salt mice. Hypertension was induced in mice using a DOCA/salt model, with unilaterally nephrectomized mice as controls. We measured blood pressure, intracavernous pressure (ICP), and corpus cavernosum (CC) contractility, and performed immunoblots for KCa 2.3, caspase-1, and interleukin-1{beta} (IL-1{beta}). DOCA/salt mice showed impaired erectile function and increased IL-1{beta} activity and KCa 2.3 expression. Treatment with the endothelin receptor antagonist bosentan or the KCa 2.2/2.3 channel opener NS13001 reversed these dysfunctions and reduced ET-1-induced NLRP3 activation. NS13001 also restored decreased currents in endothelial cells exposed to ET-1. These findings establish that hypertension-induced erectile dysfunction involves an ET-1/membrane depolarization/NLRP3 inflammasome axis in corpus cavernosum endothelial cells, and that targeting endothelial KCa2.2/2.3 channels represents a promising therapeutic strategy to counteract erectile dysfunction. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/611748v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@19aef56org.highwire.dtl.DTLVardef@d7250forg.highwire.dtl.DTLVardef@c2222borg.highwire.dtl.DTLVardef@1516fe9_HPS_FORMAT_FIGEXP M_FIG Graphic abstract Overview of the KCa2.2/2.3 regulation on the ET-1-induced NLRP3 inflammasome activation in ECs. NLRP3 inflammasome activation in ECs depends on endothelin receptor B. On activation, NLRP3 recruits and forms a complex with ASC as well as procaspase 1. In the final step, the assembled inflammasome platform cleaves pro-caspase-1, and caspase-1 cleaves pro-IL-1 to activate IL-1. NS13001 activates KCa2.2/2.3, which inhibits ET-1-induced NLRP3 activation. Apamin inhibits KCa2.2/2.3 opening. Bosentan directly inhibits ETB receptors in ECs, preventing the NLRP3 inflammasome activation. C_FIG

This study focuses on RU-1205, a new kappa-opioid agonist exhibiting analgesic effect without causing dysphoric or aversive reactions. It is assumed that the absence of dysphoric or aversive effects can be attributed to functional selectivity or it might be due to an additional mechanism of action that involves blocking the p38 mitogen-activated protein kinase (MAPK). The aimof this study was the experimental identification of the mechanisms of action of RU-1205 associated with inhibition of MAPK p38 and functional selectivity at kappa-opioid receptors. Materials and methodsRats weighing 260-280 g were implanted with chronic cortical and deep electrodes. LFP activity was recorded after intracerebroventricular administration of well-studied reference substances: the selective kappa-opioid agonist U-50488 at a dose of 100 g; the MAPK p38 blocker SB203580 at a dose of 1 g; and the investigational compound RU-1205 at 350 g. The weighted phase lag index (WPLI) was calculated. Subsequently, machine learning techniques were employed to reduce dimensionality and extract connectivity features using the principal component analysis method. Finally, signal classification was conducted using models based on Gaussian processes. By applying the patch-clamp technique in the whole-cell configuration, the spike activity of pyramidal neurons in the basolateral amygdala was studied. The neurons were identified by their accommodation properties. After local perfusion of the test compounds, 3 dose-response curves were obtained for: (1) U-50488 at concentrations ranging from 0.001 to 10 M; (2) combinations of U-50488 (0.001-10 M) and RU-1205 (10 M); and (3) combinations of U-50488 (0.01-10 M) and RU-1205 (100 M). ResultsThe developed models were able to classify the compound RU-1205 as a <<non-inhibitor>> of MAPK p38 with a probability of 0.89. The results obtained were confirmed in patch clamp experiments on acute brain slices, where it was demonstrated that U-50488 statistically significantly increases the spike activity of pyramidal neurons in the basolateral amygdala (p <0.05) and RU-1205 interacts with U-50488, suppressing its effect on the spike activity of neurons. ConclusionsThe findings suggest that compound RU-1205 displays properties consistent with a functional kappa opioid receptor agonist and does not have a significant effect on MAPK p38. The study demonstrates the possibility of integrating electrophysiological measurements and advanced data analysis methods for a deep understanding of neuronal mechanisms of drug action and underscores the potential for further research in this area.

Nitrous oxide (N2O; laughing gas) has recently been reported as a putative rapid-acting antidepressant, but little is known about the underlying mechanisms. We performed transcriptomics, in situ hybridization, and electrophysiological studies to examine the potential shared signatures induced by 1 h inhalation of 50% N2O and a single subanesthetic dose of ketamine in the medial prefrontal cortex (mPFC) in adult mice. Both treatments similarly affected the transcription of several negative regulators of mitogen-activated protein kinases (MAPKs), namely, dual specificity phosphatases. The effects were primarily located in the pyramidal cells. Notably, the overall effects of N2O on mRNA expression were much more prominent and widespread compared to ketamine. Ketamine caused an elevation of the spiking frequency of putative pyramidal neurons and increased gamma activity (30-100 Hz) of cortical local field potentials. However, N2O produced no such effects. Spiking amplitudes and spike-to-local field potential phase locking of putative pyramidal neurons and interneurons in this brain area showed no uniform changes across treatments. Thus, this study characterized the electrophysiological and transcriptome-wide changes in mPFC triggered by exposure to N2O and compared them with those caused by the rapid-acting antidepressant ketamine in terms of both the direction of their regulation and localization.

Approximately 50 million Americans suffer from chronic pain, and opioids are commonly prescribed for such individuals. Unfortunately, nearly a quarter of chronic pain patients have reported misusing their prescription. We are investigating the effect of chronic pain on drug-seeking behavior at the neuronal level. Repeated drug-seeking is associated with reactivation of an ensemble of neurons sparsely scattered throughout the dorsomedial prefrontal cortex (dmPFC). Prior research has demonstrated that chronic pain increases intrinsic excitability of dmPFC neurons, which may increase the likelihood of reactivation during drug seeking. We tested the hypothesis that chronic pain would increase oxycodone seeking behavior, and that the pain state would differentially increase intrinsic excitability in dmPFC drug seeking ensemble neurons. TetTag mice self-administered intravenous oxycodone. After 7 days of forced abstinence, a drug seeking session (extinction conditions) was performed and the ensemble was tagged. Mice received spared nerve injury (SNI) to induce chronic pain during the period between a first and second seeking session, and we measured persistence of seeking between the two sessions to determine if the SNI exacerbated seeking. Following the second seeking session we performed electrophysiology on individual neurons within the dmPFC to assess intrinsic excitability of the drug-seeking ensemble and non-ensemble neurons. We found significant sex differences in the effect of SNI on oxycodone seeking and electrophysiology, such that the induction of chronic pain could modulate seeking behavior in mice that have previously self-administered oxycodone prior to injury. HighlightsO_LIOxycodone seeking was higher in females following SNI that came after the 10-day SA timeline. C_LIO_LIAn increase in intrinsic excitability was detected among non-ensemble neurons from female mice that received SNI, and this correlated with an increase in seeking behavior. C_LI

Opioid withdrawal is associated with heightened pain sensitivity, including allodynia. Although opioid-induced allodynia is well-documented in humans and animal models, the relationship between the severity of opioid withdrawal-induced allodynia and individual addiction-like behaviors remains poorly understood. To address this gap, Heterogeneous Stock rats underwent long access (12 hours/day) intravenous oxycodone self-administration, followed by measurement of mechanical sensitivity at six timepoints across three weeks of abstinence. Rats were stratified by an Addiction Index derived from individual differences in the escalation of oxycodone intake, motivation to consume oxycodone, tolerance to oxycodones analgesic effects, and acute withdrawal-induced mechanical pain sensitivity. Here, we show that oxycodone withdrawal induces significant and prolonged allodynia for up to three weeks, with High Addiction Index rats exhibiting greater intensity and longer duration of pain sensitivity than Low Addiction Index rats. Results remained consistent even when excluding allodynia from the Addiction Index, highlighting the robustness of the association between addiction-like severity and protracted allodynia. Linear regression associations revealed that self-administration behaviors, particularly oxycodone intake escalation and motivation to seek oxycodone, predicted subsequent withdrawal-induced allodynia severity. These findings demonstrate that greater addiction-like severity is associated with more intense and prolonged withdrawal-induced pain, supporting mechanical allodynia as a marker of addiction severity. These results motivate future work to define the mechanisms linking addiction severity to protracted opioid withdrawal-induced pain, with the goal of informing targeted clinical interventions for individuals most susceptible to severe abstinence-related allodynia.

Acid-sensing ion channels (ASICs) are members of the DEG/ENaC family that includes the only known peptide-gated ion channels. While ASICs are gated by protons, they have kept sensitivity to peptides and are notably modulated by the molluscan FMRFamide and other related mammalian neuropeptides ending by the RFamide motif. Screening efforts have been made to identify and characterize natural peptides able to modulate ASICs activity, and some peptides from different species are already known to modulate ASIC1a, ASIC1b and ASIC3. We identified here a set of synthetic short amidated hexapeptides, initially designed thirty years ago for their ability to inhibit the Na/Ca exchanger, as potent and selective positive modulators of the ASIC3 acid-induced activity. We focused on two of them, a RFamide peptide (FRCCRFamide) and a CFamide peptide (FR[C][R][C]Famide), demonstrating that they have similar specificity for and effects on ASIC3. The potentiating effects of the two peptides are due to a strong slow-down of the current desensitization, leading to an increase in the amount of current induced by acid pH ([&le;]pH6.6), with apparent affinities ranging from 1 to 5 {micro}M. Surprisingly, the washout kinetic for the FR[C][C]RFamide peptide was much slower than those of FR[C][R][C]Famide and other known RFamide peptides, suggesting potential differences in their mechanisms of action. Computational modeling and structure-function analysis reveal interactions of both peptides with the non-proton binding site of ASIC3 initially identified for the synthetic compound GMQ (2-guanidine-4-methylquinazoline), as already reported before for other RFamide peptides, but our data also suggest possible additional effects of FR[C][C]RFamide involving directly or indirectly the proton binding domain. These findings expand our understanding of peptide modulation of ASIC channels and identify novel pharmacological tools selective among ASICs for investigating ASIC3 function.

Risperidone acts on monoaminergic signaling to alleviate psychosis. At the cellular level, through both direct and indirect effects, the drug induces a specific pattern of gene expression, which differs between the basal ganglia and frontal cortex. These risperidone-regulated changes influence neuronal plasticity and are crucial for both its antipsychotic and extrapyramidal effects. Here, we employed sequencing-based spatial transcriptomics to comprehensively characterize gene expression changes in the male mouse (Mus musculus L.) forebrain after an acute dose of risperidone (0.5 mg/kg, i.p.). The transcriptional patterns were structure-specific, and unsupervised clustering of spatial profiles accurately identified cortical divisions, layers, and basal ganglia subregions. Differential gene expression was subsequently analyzed within each anatomically defined cluster using a customized statistical framework. Risperidone significantly altered the levels of 95 transcripts across 12 brain regions. The largest number of changes was observed in ventral brain areas, including the olfactory tubercle (25 differentially regulated transcripts), the diagonal band nucleus (22), the corpus callosum and commissures (13), and the lateral septal nucleus (9). Notably, 21 of the 95 differentially expressed genes were previously associated with schizophrenia, including Olig2, Smpd3, and Cacna1i. Overall, our results indicate that the strongest effects of risperidone are in medial and ventral brain regions rich in oligodendrocytes and glial cells. Furthermore, the enrichment analysis provides robust evidence of a molecular link between the drugs mechanism of action and genetic factors involved in schizophrenia. HighlightsO_LIUnsupervised clustering accurately identifies transcripts localization in the brain C_LIO_LIAcute risperidone treatment alters spatial transcriptional patterns of 95 genes C_LIO_LIRisperidone-regulated transcripts include 21 genes previously linked to schizophrenia C_LIO_LISpatial gene expression analysis offers novel insight into the drug action mechanism C_LI

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is re-emerging in clinical settings as a candidate for the treatment of specific psychiatric disorders (e.g. post-traumatic stress disorder) in combination with psychotherapy. MDMA is a psychoactive drug, typically regarded as an empathogen or entactogen, which leads to transporter-mediated monoamine release. Despite its therapeutic potential, MDMA can induce dose-, individual-, and context-dependent untoward effects outside safe settings. In this study, we investigated whether three new methylenedioxy bioisosteres of MDMA improve its off-target profile. In vitro methods included radiotracer assays, transporter electrophysiology, bioluminescence resonance energy transfer and fluorescence-based assays, pooled human liver microsome/S9 fraction incubation with isozyme mapping, and liquid chromatography coupled to high-resolution mass spectrometry. In silico methods included molecular docking. Compared with MDMA, all three MDMA bioisosteres (ODMA, TDMA, and SeDMA) showed similar pharmacological activity at human serotonin and dopamine transporters (hSERT and hDAT, respectively) but decreased activity at 5-HT2A/2B/2C receptors. Regarding their hepatic metabolism, they differed from MDMA, with N-demethylation being the only metabolic route shared, and without forming phase II metabolites. Additional screening for their interaction with human organic cation transporters (hOCTs) and plasma membrane transporter (hPMAT) revealed a weaker interaction of the MDMA analogs with hOCT1, hOCT2, and hPMAT. Our findings suggest that these new MDMA analogs might constitute appealing therapeutic alternatives to MDMA, sparing the primary pharmacological activity at hSERT and hDAT, but displaying a reduced activity at 5-HT2A/2B/2C receptors and reduced hepatic metabolism. Whether these MDMA bioisosteres may pose lower risk alternatives to the clinically re-emerging MDMA warrants further studies.

The excitability of afferents involved in nociceptive signaling reflects the interaction of several co-expressed membrane receptors. Current studies have shown that Toll-like receptor-4 (TLR-4) signaling can exacerbate excitation evoked by transient receptor potential vanilloid type 1 (TRPV1) activity, and this interaction plays a key role in driving and sustaining facilitated pain states. The mechanism by which this potentiated TRPV1 activity secondary to TLR-4 agonism occurs in sensory neurons remains unknown, although intracellular kinase activity is a strong candidate. To address this hypothesized linkage, neuronal cell cultures prepared from dorsal root ganglia (DRG) of male wildtype (WT) and Tlr4-/- mice were used to evaluate calcium transients of neurons after capsaicin administration in culture, pre-treated for 30 minutes with the TLR-4 agonist, lipopolysaccharide (LPS). TRPV1 protein expression at the neuron surface in cultured DRG cells with or without LPS treatment was quantified by flow cytometry assay. The roles of protein kinase A (PKA) and C were assessed using selective inhibitors (KT5720 for PKA and Chelerythrine chloride for PKC) applied to WT-DRG neurons or administered in vivo by intraplantar or intrathecal injection, prior to LPS and capsaicin administration. Behavioral effects of in vivo TRPV1 activation were assessed through paw flinch responses evoked by intraplantar capsaicin injection and by hind paw tactile thresholds measured by von Frey filaments. LPS incubation in cultured DRG neurons enhances the intensity of calcium influx following TRPV1 activation in WT but not Tlr4-/ cells. The augmented calcium influx evoked by capsaicin was prevented by the inhibition of PKA but not PKC. Similarly, mice treated with LPS in the hind paw displayed greater nociceptive responding after capsaicin and increased tactile allodynia. The facilitated component was prevented by the local pre-treatment with the PKA inhibitor. Correspondingly, lumbar spinal blockade of PKA resulted in temporary reversal of hyperalgesia induced by intrathecal LPS injection in mice. Together, these results demonstrate the relevance of TLR-4 in modulating the excitability of nociceptor signaling by regulating TRPV1, thereby influencing pain transmission through PKA signaling.

Type 2 ryanodine receptor (RyR2) is a Ca2+ release channel on the endoplasmic/sarcoplasmic reticulum (ER/SR) that plays a central role in the excitation-contraction coupling in the heart. Hyperactivity of RyR2 has been linked to ventricular arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT) and heart failure, where spontaneous Ca2+ release via hyperactivated RyR2 depolarizes diastolic membrane potential to induce triggered activity. In such cases, drugs that suppress RyR2 activity are expected to prevent the arrhythmias, but there is no clinically available RyR2 inhibitors at present. In this study, we searched for RyR2 inhibitors from a well-characterized compound library using a recently developed ER Ca2+-based assay, where the inhibition of RyR2 activity was detected by the increase in ER Ca2+ signals from R-CEPIA1er, a genetically encoded ER Ca2+ indicator, in RyR2-expressing HEK293 cells. By screening 1535 compounds in the library, we identified three compounds (chloroxylenol, methyl orsellinate and riluzole) that greatly increased the ER Ca2+ signal. All of the three compounds suppressed spontaneous Ca2+ oscillations in RyR2-expressing HEK293 cells and correspondingly reduced the Ca2+-dependent [3H]ryanodine binding activity. In cardiomyocytes from RyR2-mutant mice, the three compounds effectively suppressed abnormal Ca2+ waves without substantial effects on the action-potential-induced Ca2+ transients. These results confirm that ER Ca2+-based screening is useful for identifying modulators of ER Ca2+ release channels and suggest that RyR2 inhibitors have potential to be developed as a new category of antiarrhythmic drugs. Significance statementWe successfully identified three compounds having RyR2 inhibitory action from a well-characterized compound library using an ER Ca2+-based assay, and demonstrated that these compounds suppressed arrhythmogenic Ca2+ wave generation without substantially affecting physiological action-potential induced Ca2+ transients in cardiomyocytes. This study will facilitate the development of RyR2 specific inhibitors as a potential new class of drugs for life-threatening arrhythmias induced by hyperactivation of RyR2.

TRPA1 is a non-selective cation channel that plays a crucial role in several pain and inflammatory conditions. Agents reducing membrane cholesterol decrease TRPA1 activation, but it remains unclear how cholesterol-lowering medications affect TRPA1 function. Given that TRPA1 is activated by a wide variety of chemicals, we explored whether statins have acute effects on this channel. We found that five commonly used statins activate human and mouse TRPA1 in a reversible and concentration-dependent manner. The effective concentrations were above the micromolar range, in the order: simvastatin {approx} lovastatin < fluvastatin < atorvastatin < pravastatin. Statin-induced activation was not correlated to changes in membrane order, nor mediated by N-terminal cysteine residues contributing to electrophilic compound agonism. Molecular docking calculations and the functional characterization of single-point mutants revealed two separate putative binding sites, one situated close to the kink of transmembrane segment 5 (TM5) and the other at the interface between TM4 and TM5. The mTRPA1 inhibitor A-967079 largely abrogated the response to the electrophilic agonist allyl isothiocyanate, but had weaker and varied effects across different statins and menthol. Mutation T877L strongly altered the effect of A-967079, also in an agonist-dependent manner, suggesting competitive binding between this antagonist and the non-electrophilic agonists. The identification of two distinct agonist binding sites may help explaining how TRPA1 is able to respond to a large variety of non-electrophilic compounds, while the finding of competitive interactions at one of these sites may help guide the development of agonist-specific antagonists of therapeutic relevance.

The sodium (Na+) leak channel (NALCN) is a member of the four-domain voltage-gated cation channel family that includes the prototypical voltage-gated sodium and calcium channels (NaVs and CaVs, respectively). Unlike NaVs and CaVs, which have four lateral fenestrations that serve as routes for lipophilic compounds to enter the central cavity to modulate channel function, NALCN has bulky residues (W311, L588, M1145 and Y1436) that block these openings. Structural data suggest that oc-cluded lateral fenestrations underlie the pharmacological resistance of NALCN to lipophilic compounds, but functional evidence is lacking. To test this hypothesis, we unplugged the fenestrations of NALCN by substituting the four aforementioned resi-dues with alanine (AAAA) and compared the effects of NaV, CaV and NALCN block-ers on both wild-type (WT) and AAAA channels. Most compounds behaved in a simi-lar manner on both channels, but phenytoin and 2-aminoethoxydiphenyl borate (2-APB) elicited additional, distinct responses on AAAA channels. Further experiments using single alanine mutants revealed that phenytoin and 2-APB enter the inner cav-ity through distinct fenestrations, implying structural specificity to their modes of ac-cess. Using a combination of computational and functional approaches, we identified amino acid residues critical for 2-APB activity, supporting the existence of drug bind-ing site(s) within the pore region. Intrigued by the activity of 2-APB and its ana-logues, we tested additional compounds containing the diphenylmethane/amine moiety on WT channels. We identified compounds from existing clinically used drugs that exhibited diverse activity, thus expanding the pharmacological toolbox for NALCN. While the low potencies of active compounds reiterate the resistance of NALCN to pharmacological targeting, our findings lay the foundation for rational drug design to develop NALCN modulators with refined properties. Significance statementThe sodium leak channel (NALCN) is essential for survival: mutations cause life-threatening developmental disorders in humans. However, no treatment is currently available due to the resistance of NALCN to pharmacological targeting. One likely reason is that the lateral fenestrations, a common route for clinically used drugs to enter and block related ion channels, are occluded in NALCN. Using a combination of computational and functional approaches, we unplugged the fenestrations of NALCN which led us to the first molecularly defined drug binding site within the pore region. Besides that, we also identified additional NALCN modulators from existing clinically used therapeutics, thus expanding the pharmacological toolbox for this leak channel.