Biochemical Pharmacology

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.

3:(-)-trans-Cannabidiol ((-)-CBD) is a principal phytocannabinoid from Cannabis sativa. (-)-CBD has complex pharmacology but is a relatively weak inhibitor of CB1 and CB2 receptor signalling. Cannabidiol has two chiral centres and thus four stereoisomers. (+)-trans-CBD ((+)-CBD) has a higher affinity than (-)-CBD at CB1 and CB2, but its pharmacodynamic effects at these receptors are incompletely described. We examined the activity of (+)-CBD at human CB1 and CB2 receptors using a fluorescence-based assay of membrane potential in AtT20 cells stably expressing CB1 or CB2 receptors. (+)-CBD produced a rapid, concentration-dependent hyperpolarization in CB2-expressing cells (pEC50 6.63 {+/-} 0.08) with a maximal effect [~]90% of the response to CP55940. The CB2 response was blocked by pertussis toxin pretreatment and competitively inhibited by the CB2 antagonist AM630 (Schild slope 1.1 {+/-} 0.1). (+)-CBD was a low-efficacy, low-potency CB1 agonist and inhibited somatostatin-receptor effects at high concentrations (10-30 {micro}M). It had no effect on the membrane potential of AtT20 wild-type cells. In silico modelling of ligand interactions with CB2 indicated that (+)-CBD but not (-)-CBD formed an H-bond with Ser285, a residue crucial for agonist activation of CB2. Our data suggests (+)-CBD acted as a CB2 agonist via the orthosteric binding site on the receptor. Synthetic CBD, including (+)-CBD, has previously been administered in clinical trials, presumably without consideration of its potential CB2 agonist activity. Given the relative safety of (-)-CBD in people, (+)-CBD may be a useful drug to explore CB2-sensitive disease states, should it prove similarly safe.

The platelet P2Y1 receptor (P2Y1R) is necessary for inflammation, signalling via Rho-GTPase pathways to elicit functions that are distinct from aggregation (PLC-dependent canonical signalling pathway). Whether these distinct platelet inflammatory functions can be selectively suppressed to preserve hemostasis through the rational design of P2Y1R antagonists has not been explored. In silico molecular docking analysis examined biased nucleotide interactions within the P2Y1R binding pocket. The identified possible key amino acid residues guided rational design to synthesize compounds for pathway selective inhibition, evolving from nucleotide to non-nucleotide structures. The nucleotide analogue KMR-82-13 was predicted to engage distinct regions of the binding pocket and selectively inhibited platelet chemotaxis while preserving aggregation. These findings informed the design of a non-nucleotide compound KSN-159-27, aiming to retain key KMR-82-13-like interactions while improving drug-like properties. Docking and molecular dynamics simulation supported a stable but dynamic binding mode for KSN-159-27 within the P2Y1R pocket, consistent with pathway-selective inhibition. KSN-159-27 displayed characteristics of a pathway selective inverse agonist at P2Y1R towards G12/13-mediated pathways, but not those associated by Gq activation in P2Y1R-transfected HEK293T cells. KSN-159-27 showed functionally selective inhibition for platelet P2Y1R-mediated functions. In vivo, KSN-159-27 suppressed inflammatory cell recruitment, whilst preserving bleeding time and ADP-induced thromboembolic responses, in contrast to the neutral P2Y1R antagonist MRS2500. This first demonstration for the rational design of a pathway selective inverse agonist at platelet P2Y1Rs has significant implications for novel therapeutic strategies developed to safely target platelet activation during inflammation, in contrast to current anti-platelet drugs used in the prevention of thrombosis. Key PointsO_LIBiased inverse platelet P2Y1R agonists selectively supress inflammation whilst preserving hemostasis and the ability of platelets to aggregate. C_LIO_LIBiased inverse agonism selectively inhibited P2Y1R G12/13 (Rho-GTPAse functions) but not Gq activities (PLC functions). C_LI

Small-molecule agonists of class B1 G-protein-coupled receptors (GPCRs) remain rare because these receptors typically require large extracellular peptide ligands for activation. PCO371 is a notable exception: an intracellular agonist, originally developed for osteoporosis treatment, that activates parathyroid hormone 1 receptor (PTH1R) from the cytoplasmic face of the receptor. In this study, we compared the functional, pharmacological and structural properties of PCO371 with the canonical extracellular peptide PTH1-34 at the PTH1R to define the mechanism underlying PCO371s unusual signalling profile. Functionally, PCO371 exhibited markedly lower functional affinity and a strong dependence on receptor reserve, achieving full agonism only at high receptor expression, whereas PTH1-34 maintained robust signalling under receptor depletion. Across Gs, Gi3, Gq(R183Q), cAMP, and {beta}-arrestin-2 pathways, operational model analysis showed that PCO371 is non-biased, engaging the same transducers as PTH1-34 but with [~]1000-fold lower potency. Our findings establish PCO371 as a non-biased but globally less potent agonist, compared to PTH1-34, whose signalling efficacy depends on receptor reserve and G-protein engagement. PCO371 binding is {beta}-arrestin-compatible but only drives measurable {beta}-arrestin-2 recruitment when PTH1R is highly expressed. Overall, these insights define the mechanistic basis of intracellular agonism at a class B1 GPCR and provide a framework for designing next-generation small-molecule modulators that exploit this emerging pharmacological space. HIGHLIGHTSO_LIPCO371 requires high PTH1R expression to achieve full agonism. C_LIO_LIPCO371 activates Gas, Gai and Gaq families but is significantly less potent than PTH1-34. Operational modelling shows no detectable signalling bias for PCO371; reduced signalling is global rather than pathway selective. C_LIO_LIPCO371-bound PTH1R structure is compatible with {beta}-arrestin engagement, where PCO371 elicits a measurable {beta}-arrestin-2 response only under high receptor expression. C_LIO_LIMolecular dynamic simulations reveal that PCO371 becomes stably bound only within a preassembled PTH1R-G-protein complex. C_LIO_LIPCO371 stabilises a distinct rearrangement in PTH1R:Gs/Gq/Gi3 TM6 and the TM1/TM7 bundle and forms G-protein subtype specific 5 helix interactions. C_LIO_LIEstablish a mechanistic basis for intracellular agonism, informing future design of therapeutically relevant modulators. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/728112v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@13cb194org.highwire.dtl.DTLVardef@1ae82bforg.highwire.dtl.DTLVardef@a00d4org.highwire.dtl.DTLVardef@bc657f_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG

Temozolomide is the gold standard chemotherapeutic agent used in the treatment of glioblastoma multiforme. Yet its pharmacological use has been linked to the emergence of depressive- and/or anxiety-like behaviors, probably through the inhibition of hippocampal neurogenesis. Since prior studies reporting these negative effects were based on prolonged treatment paradigms (i.e., from 2 weeks to up to 6 months), and given the few reports that have included female rodents in their studies, our approach aimed at further characterizing the behavioral effects induced by temozolomide (25 mg/kg, 1 or 2 cycles, 5 days/cycle) in a mixed-sex cohort of adult rats. To do so, rats were scored across time through specific behavioral tests that capture diverse manifestations of affective-like responses (forced-swim, open field, novelty-suppressed feeding and sucrose preference) or cognitive performance (Barnes maze). At the neurochemical level, we ascertained the effects of 2 cycles of temozolomide on hippocampal neurogenesis (neural progenitors with NeuroD) and other potential neuroplasticity targets (i.e., FADD, BDNF). The main results showed that temozolomide induced unexpected antidepressant-like responses in a treatment-duration manner while decreased hippocampal FADD, a neuroplastic marker previously associated with the acute and repeated actions of most antidepressants. These results break the prior dogma linking increased hippocampal neurogenesis with antidepressant-like efficacy, and suggest that other mechanisms of action, such as the one described through the neuroplastic molecule FADD, might be responsible for the antidepressant-like actions of temozolomide, even in the presence of impaired neurogenesis. Our results, in conjunction with the prior data, suggested cycle- and/or length-dependent treatment effects in terms of temozolomides antidepressant- vs. depressant-like profile, while proposing a novel biomarker of its treatment response.

BackgroundEpidemiological studies consistently report inverse associations between caffeinated coffee consumption and dementia risk. However, the molecular mechanisms linking coffee-derived phytochemicals to neuroprotection remain only partially understood. ObjectiveTo evaluate, through integrated in silico pharmacology, the relative contribution of adenosine receptor modulation versus direct amyloidogenic enzyme and kinase inhibition in mediating the putative neuroprotective effects of major coffee constituents. MethodsMolecular docking analyses were conducted for caffeine, paraxanthine, chlorogenic acid, trigonelline, cafestol, and kahweol against adenosine A2A and A1 receptors (A2AR, A1R), {beta}-secretase 1 (BACE1), glycogen synthase kinase-3{beta} (GSK-3{beta}), and NLRP3 inflammasome components. Docking was performed using the CB-Dock2 platform. Binding affinities, interaction patterns, and ligand efficiency metrics were assessed. Blood-brain barrier permeability and ADMET properties were predicted using pkCSM. ResultsCaffeine and paraxanthine demonstrated structurally coherent binding within the orthosteric pockets of A2AR and A1R, supported by favorable predicted blood-brain barrier penetration and high unbound fractions. Ligand efficiency analysis identified adenosine receptors as the most pharmacologically plausible targets for small xanthine derivatives. Although larger phytochemicals exhibited stronger absolute docking scores at BACE1, GSK-3{beta}, and NLRP3, predicted pharmacokinetic constraints suggest a small biological effect due to a limited central exposure. ConclusionsThese findings support an adenosine receptor-centered mechanism as the dominant molecular axis linking caffeinated coffee consumption to reduced dementia risk, favoring neuroinflammatory and signaling modulation over direct enzymatic inhibition. Experimental validation is warranted to confirm translational relevance. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/723029v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1a02629org.highwire.dtl.DTLVardef@129890dorg.highwire.dtl.DTLVardef@1e4c05corg.highwire.dtl.DTLVardef@110ec7a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Migraine headaches affect over one billion people internationally and can be defined as episodes of acute severe pain wrapping around the head and are normally accompanied by nausea, blurry vision, and sensitivity to light and sound. While triggers that cause migraines may vary among patients, evidence shows they are involved with the trigeminovascular system (a network of blood vessels in the brain in conjunction with the trigeminal nerve). Activation of the trigeminal neurons triggers the release of vasoactive neuropeptides, such as calcitonin gene-related peptide (CGRP), leading to neurogenic inflammation and vasodilation of cranial blood vessels. The development of 5-HT1B serotonin agonist drugs, commonly known as triptans, have been an effective measure of migraine relief. The drugs created in this research were found to have improved docking scores within the 5-HT1B binding site compared to that of naratriptan. The two drugs proposed in this paper would need to undergo further investigation to determine the feasibility of laboratory synthesis and clinical trials.

Malignant brain tumors are among the most aggressive and difficult to treat human cancers. Glioblastomas (World Health Organization grade IV gliomas) are particularly lethal and refractory to treatment. Few drugs exist that are even somewhat effective. Our investigation of the physiologic role of fatty acid (FA) activating enzymes (acyl-CoA synthetase; ACS) identified an ACS that was widely expressed in gliomas but not in normal glial cells. Depletion of this enzyme, ACSVL3 (very long-chain ACS3), by knockdown or knockout decreased the malignant behavior of several glioma cell models including U87MG and Mayo-22 cells both in culture and when grown as xenografts. Hypothesizing that ACSVL3 is a potential therapeutic target in glioma, we conducted a search for inhibitors of this enzyme and found that CB5 (grassofermata) was a promising candidate. Treating U87MG glioma cells with CB5 slowed growth in monolayer culture; the growth rate was similar to that seen in cells in which ACSVL3 was either knocked down or knocked out. CB5 inhibited growth in a dose-dependent manner over a narrow range, and concentrations above 10 M were toxic. Treatment at the lower dose of 3 M inhibited growth of U87MG cells but was reversible, suggesting that this dose was not toxic. CB5- treated U87MG cells exhibited an altered morphology with a larger size and longer projections. In contrast, normal human fibroblasts treated with 10 M CB5, a concentration that was toxic to U87MG cells, showed no effect on either growth rate or morphology. Treating U87MG cells with 3 M CB5 induced differentiation as shown by increased expression of the astrocyte-specific marker glial fibrillary acidic protein (GFAP). In contrast, GFAP levels remained low in ACSVL3 knockdown cells. CB5- treated U87MG cells were less invasive, and thus less malignant, than either untreated cells or ACSVL3 knockout cells when assessed by a scratch wound healing assay. Acute treatment of U87MG cells with 3 M CB5 decreased the ability of these cells to degrade FA of differing chain lengths from 16-24 carbons by {beta}-oxidation, suggesting that decreased ACS enzyme activity contributes at least in part to the drugs mechanism of action. NOD/SCID mice receiving up to 32 mg/kg/day CB5 by intraperitoneal injection showed no obvious side effects, suggesting that the drug was well-tolerated. Xenografts induced by subcutaneous injection of U87MG cells in the flanks of NOD/SCID mice were allowed to grow for 8 days after which half of the mice were treated with 2 mg/kg/day CB5. After 7 days of treatment, xenograft growth slowed in the treated mice and by 12 days tumor size had begun to decrease, suggesting therapeutic efficacy. When a similar study was done using xenografts induced by subcutaneous injection of Mayo-22 cells, which are maintained as subcutaneous tumors in mice rather than in cell culture, the effect of CB5 on tumor growth or weight at sacrifice was not statistically significant. The results of these studies suggest that CB5 may have therapeutic value in malignant glioma. Additional studies using other glioma models and other drugs chemically related to CB5 seem warranted.

Glioblastoma remains a highly aggressive and therapy-resistant brain tumor, with limited benefit from the current standard-of-care regimen combining surgery, radiotherapy, and temozolomide. Overcoming chemoresistance therefore represents a critical unmet clinical need. Here, we investigate the anticancer potential of Succisa pratensis and its ability to enhance TMZ efficacy in GBM models. Treatment with S. pratensis markedly reduced cell proliferation and migration while significantly increasing sensitivity to TMZ. Integrated multi-omics analyses revealed extensive metabolic rewiring, characterized by suppression of central carbon metabolism and activation of stress-adaptive pathways. Mechanistically, we identify the Pregnane X Receptor, a key regulator of drug metabolism and chemoresistance, as a central node affected by treatment. Although S. pratensis increased PXR expression, this was not accompanied by induction of canonical downstream targets, including MDR1 and ALDH1A1, indicating a functional impairment of PXR transcriptional activity. Consistently, pharmacological inhibition of PXR using the antagonist SPA70 further potentiated the cytotoxic effects of S. pratensis and TMZ. Docking analyses suggest that specific secondary metabolites, including apigenin-derived compounds, may interact with the PXR ligand-binding domain, providing a potential molecular basis for this effect. Collectively, our findings indicate that S. pratensis enhances TMZ efficacy by inducing metabolic vulnerability and functionally impairing PXR signaling. These results highlight the therapeutic potential of plant-derived metabolites as adjuvant strategies to overcome chemoresistance in glioblastoma. Article HighlightsO_LISuccisa pratensis enhances temozolomide efficacy in glioblastoma by reducing proliferation, migration, and clonogenic growth. C_LIO_LIIntegrated proteomic and metabolomic analyses reveal extensive metabolic rewiring, with suppression of central carbon metabolism and induction of stress-adaptive pathways. C_LIO_LIPregnane X Receptor (PXR), a key regulator of chemoresistance, is functionally impaired despite increased expression, resulting in reduced activation of drug-resistance genes. C_LIO_LIPharmacological inhibition of PXR further potentiates the antitumor effects of Succisa pratensis and temozolomide, promoting apoptotic cell death. C_LIO_LIApigenin-derived metabolites show high affinity for the PXR ligand-binding domain and emerge as promising candidates to overcome temozolomide resistance in glioblastoma. C_LI

The three opioid receptors, mu (mu-OR), delta (delta-OR), and kappa (kappa-OR), are key G-protein-coupled receptors (GPCRs) involved in mediating pain relief. Yet, there has been a lack of understanding regarding how changes in the extracellular pH associated with inflammation affect their signal transduction pathways. This study demonstrates that all opioid receptors act as proton-gated coincidence detectors. A humanized yeast-based platform called DCyFIR, which isolates the pH environment outside the cell from potential confounding factors inside the cell, was employed to examine the activation of opioid receptors over a range of physiologically relevant to pathologically low pH environments. As such, the results show that agonist efficacy decreases with decreasing pH. This indicates that the receptor itself contains one or more protonatable sidechains that act as an intrinsic "pH sensor". To determine the structural basis of this pH-sensing mechanism, structure-based pHinder calculations were used to identify potential pH sensing residues. Subsequent variant profiling identified the protonation of one conserved aspartic acid residue, D149 (also known as D3.32), located in a region near the binding site of opioid ligands, which, when exposed to acidic pH conditions, resulted in the disruption of the salt bridge necessary for effective docking of opioid ligands with the receptor. Consequently, the receptor lost all ability to be activated by agonists. Further characterization using a variety of functional assays on more than twenty different mu-OR alternative splicing variants indicated that all isoforms that possessed a continuous 7-transmembrane domain retained this proton-sensing capability. These studies collectively provide insight into the structural mechanisms underlying pH-dependent opioid receptor activation and lay the foundation for the design of new generations of selective analgesics that activate opioid receptors only in acidic inflammatory tissue.

Large-conductance calcium-activated potassium (BKCa) channels are ubiquitously expressed in mammalian cells and regulate electrical activity, intracellular calcium signaling, and cell survival. Although BKCa dysfunction has been linked to multiple diseases, the number of selective channel modulators is limited. In this study, we characterize dibenzoylmethane (DBM), a plant-derived compound isolated from Hottonia palustris, as a novel inhibitor of BKCa channel activity in both plasma membrane and mitochondrial BKCa. Electrophysiological recordings revealed that DBM lowers the open probability of BKCa channels in a concentration-dependent fashion and markedly reduces mean open time, leading to a pronounced flickering behavior - hallmarks of pore-targeted blockade. Competition experiments demonstrated that DBM antagonizes the effect of paxilline, a high-affinity pore-binding inhibitor, suggesting overlapping binding sites. Molecular dynamics simulations further supported this hypothesis, showing that several DBM molecules can block the pore by employing {pi}-{pi} interactions with each other and pore residues. On top of the pore, the carbonyl groups of DBM block the nearest potassium ion in the selectivity filter. The presence of DBM induces the removal of water molecules from the pore. To assess the structural requirements for activity, we tested three DBM analogs: phenyl-1,3-butanedione (PBD), trans-chalcone (T-Ch), and (E)-1,3-diphenylprop-2-en-1-ol (DPE). T-Ch and DPE inhibited BKCa channels with comparable efficacy to DBM, whereas PBD was significantly less potent. These results indicate that diphenyl substitution and structural rigidity are critical determinants of inhibitory activity. Our findings position DBM and its analogs as promising chemical scaffolds for the development of selective BKCa channel modulators with potential pharmacological applications.

The KCa2.2 and KCa3.1 channels are fundamental regulator of cellular K+ concentration, and promising target to treat diseases such as spinocerebellar ataxia and cancer. To fully exploit their therapeutic potential, and to continue studying their pathophysiological role, it is crucial to develop selective modulators for each of these two channels. Here we present a computational study to identify the molecular determinants behind the selectivity of two recently reported KCa2.2 modulators. We leveraged a protocol combining in silico mutagenesis, molecular dynamics simulations, and protein-ligand docking to analyse the pockets targeted by these ligands. We identified a Ser353/Pro245 substitution to be the main driver of the distinct pocket shapes in KCa2.2 and KCa3.1 channels, ultimately defining modulator selectivity. This approach provides novel insights into the structural differences of this binding site across potassium channel subtypes, shedding light on the selectivity determinants of modulators targeting this pocket.

Cyclooxygenase-1 (COX-1) is classically regarded as a constitutive enzyme that produces eicosanoids with housekeeping functions, but recent evidence indicates that it may also be involved in the acute phase of severe systemic inflammation. There is evidence indicating that COX-1 is selectively activated in the spleen via post-translational mechanisms early the course of LPS-induced systemic inflammation. However, the mechanistic link between COX-1 and the spleen has not yet been demonstrated in direct experiments. The present study was conducted to fill this gap. The effects of the COX-1 inhibitor SC-560 on the LPS-induced severity triad (hypotension, hypothermia and acidosis) were evaluated in rats subjected to splenectomy or in sham-operated controls. In the sham-operated group, SC-560 significantly attenuated the severity triad independently of changes in plasma cytokines (TNF and IL-1{beta}). In the splenectomized rats, SC-560 completely lost its ability to attenuate the hypotension and the acidosis induced by LPS. The effect of SC-560 on LPS-induced hypothermia was also impaired by splenectomy, though not completely. We then conducted a lipidomic screening to identify which COX-1-derived eicosanoids might be responsible for mediating the severity triad. Based on spleen-blood correlations, the screening identified PGE2 and PGD2 as putative candidates. In conclusion, the present study provides direct evidence for a mechanistic link between the spleen and COX-1 in the mediation of severity in systemic inflammation, and identifies PGE2 and PGD2 as putative candidates involved.

BackgroundThe discontinuation of Fasiglifam (TAK-875), a GPR40/FFAR1 full agonist, during Phase 3 clinical trials due to hepatotoxicity led to widespread abandonment of GPR40 as a viable therapeutic target for type 2 diabetes mellitus (T2DM). However, mechanistic evidence suggests that Fasiglifams hepatotoxicity arises from mitochondrial liability driven by high lipophilicity (aLogP = 5.31), rather than from on-target GPR40 signaling. We hypothesized that target-level failure was incorrectly inferred from compound-level safety concerns, and that superior candidates exist within publicly available databases. MethodsWe queried ChEMBL Release 36 (28 GB SQLite, 74 tables) for all compounds with documented GPR40/FFAR1 activity (UniProt: O14842). Compounds were filtered by EC50 [&le;] 10 nM in nM units with standard relation "=". Drug-likeness was assessed using Lipinskis Rule of Five (Ro5), aLogP, molecular weight (MW), hydrogen bond donors/acceptors (HBD/HBA), and polar surface area (PSA). A parallel analysis of Therapeutic Target Database (TTD v10.1.01, 4,298 targets) provided clinical context. A real-world evidence (RWE) patient stratification framework was constructed using EMR data from tens of millions of patients with >10 years of longitudinal follow-up. ResultsOf 2,637 GPR40-active compounds in ChEMBL 36, 526 (19.9%) demonstrated EC50 < 100 nM and 102 (3.9%) demonstrated EC50 < 10 nM. Eight compounds met stringent drug-likeness criteria (Ro5 violations = 0, aLogP < 5.0, EC50 [&le;] 1 nM). The lead compound (CHEMBL4859651) exhibited EC50 = 0.04 nM (8.75-fold more potent than Fasiglifam), MW = 297 Da (43% lower), and aLogP = 4.30 (19% lower), with zero Ro5 violations. Mean MW of the eight candidates was 317 {+/-} 28 Da versus 524 Da for Fasiglifam. A parallel GCK analysis identified a protein-protein interaction target (CHEMBL3885579, GCK-GKRP interface) harboring 40 exclusive compounds as an orthogonal strategy for partial GCK activation. ConclusionsSystematic cheminformatic analysis reveals that compounds with substantially superior activity and drug-likeness profiles relative to Fasiglifam exist within ChEMBL 36. Fasiglifams hepatotoxicity is attributable to compound-specific physicochemical properties, not GPR40-mediated toxicity. RWE patient stratification may further mitigate hepatotoxicity risk for next-generation GPR40 agonists. These findings argue for systematic reappraisal of GPR40 as a viable therapeutic target for T2DM.

Metabolic dysfunction-associated steatotic liver disease (MASLD) and Clostridioides difficile (C. difficile) infection (CDI) are clinically associated, yet there is limited effective treatment for both diseases. Bile salt analogs (BSAs) have demonstrated potential in treating either MASLD or CDI. We screened a library of BSAs (n=112) previously synthesized as potential inhibitors of C. difficile spore germination, for their therapeutic potential in reducing intracellular accumulation of fatty acids in HepG2 cells as candidates for prevention and treatment of both MASLD and CDI. The screening was based on an in vitro model established by incubating HepG2 cells with free fatty acids, with obeticholic acid (OCA), a known BSA with anti-MASLD activity as a control. Gene and protein expressions were quantified to validate the treatment effect. We found that compounds C13, C24, C25, C74, C98, and C101 demonstrated significant effectiveness in both preventing the intracellular accumulation of lipids and removing pre-loaded cellular lipids. Gene expression analysis showed that C24, C25, and C74 produced a similar pattern characterized by a robust induction of FGF21 expression, while C13, C98, and C101 produced a transcription pattern that mirrors the effect of OCA. Structurally, while C13, C24, and C25 do not display drug-like properties, C74, C98, and C101 are drug-like and share a similar structure. Interestingly, C101 is a potent inhibitor of C. difficile spore germination. OCA shows a weak anti-gemination effect. Our study identified lead compound candidates for the development of novel therapeutics capable of treating both MASLD and CDI. Significance statementThe clinical association between MASLD and CDI remains an unmet need for dual acting therapeutic strategies. Given the reported potential of BSA, we screened 112 previously synthesized as potential inhibitors of C. difficile spore germination, for their therapeutic potential in reducing intracellular accumulation of fatty acids in HepG2 cells. Our study identified compounds that effectively reduce intracellular lipid accumulation and inhibit C. difficile spore germination. These results nominate lead candidates for developing dual-acting therapeutics targeting both MASLD and CDI.

Background and PurposeEpilepsy is a neurological condition characterized by recurring seizures and neuronal hyperexcitability. Cell-based high-throughput screening applications have been essential for drug development and discovering novel biological processes. However, cell-based screens do not provide information on how drug-targeted pathways are integrated into a whole animal. Our objective was to develop and evaluate a screening application using zebrafish larvae to identify signalling mechanisms that modulate neural activity. Experimental ApproachWe developed an in vivo automated high-content screening assay using zebrafish larvae expressing the calcium sensor CaMPARI (calcium-modulated photoactivatable ratiometric integrator) in neurons. This assay can quantify neural activity of multiple individual larvae per well in a 96-well format. We quantified neural activity in 8725 individual larvae, in response to 1292 different drugs to identify molecules that protect against convulsant-induced neuronal hyperexcitability. Key ResultsThe assay was effective at identifying drugs that target diverse neurotransmitter signalling systems. While some commonly used anti-convulsants (e.g. phenytoin, carbamazepine, valproic acid) had poor activity in the assay, Kv7 potassium channel activators were consistently effective (ICA-069673, ICA-27243, ICA-110381, retigabine, and ML213). Many compounds approved for treatment of other conditions, including amitriptyline (depression), cyclobenzaprine (muscle spasm), clomipramine (obsessive-compulsive disorder) and ganaxolone (seizures), also strongly suppressed excitability in the assay. Conclusion and ImplicationsNeuronal CaMPARI expression in zebrafish larvae is a powerful tool for plate-based compound library screening to identify drugs that suppress hyperexcitability in vivo. Bullet Point SummaryO_ST_ABSWhat is already knownC_ST_ABSO_LICaMPARI is an integrative Ca2+ sensor that can be used to identify active neurons. C_LIO_LIKv7 activators (retigabine, ML213, and ICA-069673) are effective at reducing convulsant-induced (4-AP) neuronal hyperexcitability. C_LI What this study addsO_LIAn automated in vivo high-content drug screening assay to quantify neural activity. C_LIO_LIA series of drug targets that influence convulsant-induced hyperexcitability. C_LI Clinical significanceO_LIOur new tool will help identify novel compounds and signalling mechanisms that could be pursued as therapeutic targets for diseases involving electrical hyperexcitability. C_LI

Background & Aims: Activation of apoptosis signal-regulating kinase 1 (ASK1), a ubiquitous redox-sensitive kinase, results in inflammation, apoptosis, and fibrosis, key common pathways in human liver disease. SRT-015 is a novel, small molecule inhibitor of ASK1. This study evaluated the in vitro efficacy of SRT-015, compared it to other ASK1 inhibitors, and determined the in vivo efficacy of SRT-015 across multiple acute and chronic liver disease models. Methods: In vitro studies determined the kinase potency and selectivity of SRT-015, and cellular studies were used to demonstrate direct mechanisms of action. The cardiac hERG channel inhibition was assessed and PK determined in rodents and nonhuman primates. In vivo studies evaluated SRT-015 efficacy in rodent models of drug-induced hepatotoxicity (acetaminophen (APAP) overdose), alcohol-associated liver disease (ALD), metabolic-disease associated steatohepatitis (MASH) and cholestatic disease (bile duct ligation, BDL). Results: SRT-015, was demonstrated a selective ASK1 kinase, and SRT-015 treatment directly inhibited fibrosis, apoptosis and inflammation in activated human fibroblasts, hepatocytes and PBMCs, respectively without safety signals or hERG inhibition. Other ASK1 inhibitors had safety concerns or limited functional activity. Liver and kidney selective PK were observed for SRT-015 in all species evaluated. In vivo, SRT-015 treatment was efficacious in the acute mouse APAP overdose and ALD model significantly (P<0.05) decreasing serum ALT. Using a therapeutic diet-induced obesity (DIO)-MASH model with biopsy-verified fibrosis, SRT-015 treatment significantly (P<0.05) inhibited DIO-induced liver enzymes, hepatomegaly, fibrosis, inflammation, and apoptosis independent of body weight loss whereas treatment with selonsertib was ineffective. In a rat cholestatic model, SRT-015 treatment significantly (P<0.05) decreased fibrosis and stellate cell activation. Conclusions: These findings support SRT-015 as a potential therapeutic for human liver diseases of any etiology.

BackgroundTriple-negative breast cancer (TNBC) is an aggressive subtype lacking well-defined molecular targets, leaving chemotherapy as the primary treatment despite drug resistance, systemic toxicity, and high recurrence rates. Therefore, the development of effective and less toxic therapeutic agents is essential. This study investigated the anti-cancer potential of gloriosine, a bioactive alkaloid with antiproliferative activity and low toxicity toward normal breast cells. MethodsPotential targets of gloriosine were predicted using SwissTargetPrediction, TargetNet, and PharmMapper, and overlapping genes related to TNBC and glutamine metabolism were selected. Protein-protein interaction networks, Gene Ontology, and KEGG pathway enrichment analyses were performed. Molecular docking evaluated binding affinity, followed by in vitro validation using cell viability, colony formation, and wound healing assays. ROS levels were measured by DCFDA and GSH assays, and ferroptosis was assessed by Western blot and FerroOrange staining in MDA{square}MB{square}231 cells. ResultsA total of 100 potential targets were identified, with 60 overlapping with TNBC and glutamine metabolism-related genes. Key targets included SRC, EGFR, mTOR, and HSP90AA1. Enrichment analyses indicated involvement in cancer progression, metabolic regulation, and resistance pathways, including central carbon metabolism, EGFR inhibitor resistance, and ErbB signaling. Gloriosine showed strong binding affinity toward hub targets. Experimental studies confirmed concentration-dependent inhibition of cell proliferation and migration. Mechanistically, gloriosine suppressed glutamine metabolism via GLS1 downregulation and induced ferroptosis, evidenced by increased ROS, glutathione depletion, GPX4 downregulation, and elevated intracellular iron levels. ConclusionsGloriosine exerts significant anti-cancer effects in TNBC through multi-target modulation and induction of ferroptosis, highlighting its potential as a promising therapeutic candidate. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/725321v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@ce0ebcorg.highwire.dtl.DTLVardef@29603borg.highwire.dtl.DTLVardef@6d0025org.highwire.dtl.DTLVardef@249700_HPS_FORMAT_FIGEXP M_FIG C_FIG Flow chart of the network pharmacological and in vitro study of gloriosine

The hippocampus is essential for memory consolidation, a process mediated by high-frequency oscillations known as ripples during non-rapid eye movement (NREM) sleep. Ramelteon, a selective MT1/MT2 receptor agonist, has been reported to possess cognitive-enhancing properties; however, its impact on the fine-scale dynamics of hippocampal ripples remains unclear. We performed chronic local field potential recordings from the dorsal hippocampus and prefrontal cortex in mice. Following the intraperitoneal administration of either vehicle or ramelteon, we evaluated sleep architecture and characterized ripple properties, including occurrence rate, amplitude, instantaneous frequency, and duration during NREM sleep. Ramelteon administration significantly increased NREM sleep occupancy. Notably, we found that ramelteon significantly enhanced both the occurrence rate and amplitude of hippocampal ripples compared to the control group. While a slight increase in intra-ripple frequency was observed, other structural features, such as ripple duration and asymmetry index, remained unaffected. Our findings demonstrate that ramelteon facilitates hippocampal ripple dynamics by increasing their occurrence and synchrony during NREM sleep. Given the critical role of ripples in memory consolidation, these neurophysiological changes may underlie the procognitive effects of ramelteon. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=55 SRC="FIGDIR/small/723673v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@c798c7org.highwire.dtl.DTLVardef@1ff616eorg.highwire.dtl.DTLVardef@1557dc8org.highwire.dtl.DTLVardef@1b4e89e_HPS_FORMAT_FIGEXP M_FIG C_FIG

White, beige and brown adipocytes store energy as lipids, secrete hormones and produce heat, playing an important role in the regulation of energy balance through not completely defined mechanisms. We investigate herein the impact of the almost complete absence of mature adipocytes (severe lipoatrophy) in the determination of energy balance (energy intake and expenditure) and homeothermy in mice. For this, mice with severe lipoatrophy induced by adipocyte deletion of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) (PPAR{gamma} flox adiponectin-Cre) and littermate controls (PPAR{gamma} flox) were evaluated for energy balance, thermoneutral zone, core body temperature, locomotor activity, and gene expression profiles at different ambient temperatures. Severely lipoatrophic mice are heavier, hypermetabolic and hyperphagic and feature a widened thermoneutral zone, lower ambulatory activity, and metabolic inflexibility at both 23 and 17{degrees}C, along with unstable thermal behavior characterized by hyperthermia at 30{degrees}C, normothermia at 23{degrees}C, and bouts of hypothermia at 17{degrees}C. Noteworthy, lipoatrophic mice hypermetabolism at 30{degrees}C is not due to thyroid hormones, impaired insulation or increased body and lean masses and is not altered by pharmacological blockade of either {beta}-adrenergic receptor signaling with propranolol or skeletal muscle sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA) and sarcolipin (SLN)-mediated calcium cycling with dantrolene, but is partially attenuated by pharmacological inhibition of acetyl-CoA carboxylase (ACC) and de novo lipogenesis with ND-630. In conclusion, severe lipoatrophy causes hypermetabolism and hyperthermia at 30{degrees}C partly through the activation of liver de novo fatty acid synthesis.