Acta Pharmaceutica Sinica B

Cigarette smoke-induced lung inflammation is a central driver of pulmonary diseases. The limited efficacy of current anti-inflammatory agents underscores the need for structurally novel therapeutics with distinct mechanisms. We recently demonstrated that AB-free kava, a flavokavains A/B-depleted formulation from Piper methysticum containing six major kavalactones, effectively suppresses cigarette smoke-induced lung inflammation in mice. This study aims to identify the bioactive constituent(s) and elucidate underlying mechanisms. These kavalactones revealed a clear structure-activity relationship in suppressing lipopolysaccharide (LPS)-stimulated prostaglandin E2 (PGE2) production in macrophages with desmethoxyyangonin (DMY) as the most potent kavalactone whereas dihydrokavain (DHK, a structurally similar analog) with minimal activity. DMY also effectively reduced LPS-induced interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-) production while DHK was ineffective. Mechanistically, DMY, but not DHK, attenuated COX-2 induction and reduced phosphorylation of cAMP response element-binding protein (CREB). Pharmacological inhibition of protein kinase A (PKA) similarly reduced p-CREB, COX-2 and PGE2, supporting a PKA-dependent CREB/COX-2 signaling in mediating PGE2 suppression while these effects were independent of nuclear factor kappa B (NF-{kappa}B) and activator protein 1 (AP-1) signaling. Similar results were observed for DMY and DHK in attenuating cigarette smoke condensate-induced proinflammatory pathways and PGE2 production. Consistently, DMY demonstrated significant in vivo efficacy in suppressing cigarette smoke-induced lung inflammation while DHK was not effective. Interestingly, dihydromethysticin (DHM) demonstrated the greatest in vivo anti-inflammatory efficacy, although it only exhibited moderate in vitro potency, likely due to its superior bioavailability over DMY. Concordantly, cigarette smoke exposure elevated p-CREB and COX-2 expressions in mouse lungs, which were attenuated by AB-free kava and its bioactive kavalactones with the extent of suppression correlating with their in vivo anti-inflammatory efficacy. DHM effectively suppressed LPS-induced neutrophil accumulation in mouse lungs as well. Collectively, these studies identify bioactive kavalactones in AB-free kava that suppress cigarette smoke- and LPS-induced lung inflammation through the modulation of the PKA/CREB/COX-2 signaling axis, providing a foundation for developing structurally distinct anti-inflammatory agents, particularly targeting smoke-induced inflammation and associated pulmonary diseases.

Excessive osteoclast activity contributes to pathological bone loss in osteoporosis, rheumatoid arthritis, and osteolytic malignancies. The effects of small catechol derivatives on receptor activator of nuclear factor-{kappa}B ligand (RANKL)-induced osteoclastogenesis remain poorly understood. This study investigated the effects of 4-methylcatechol (4-MC) on RANKL-induced NF-{kappa}B activation and osteoclast differentiation. 4-MC reduced RANKL-induced NF-{kappa}B luciferase activity in HEK-293T/RANK cells. 4-MC also suppressed RANKL-induced TRAP activity in RAW264.7 cells in a concentration-dependent manner and reduced the number of TRAP-positive multinucleated osteoclasts, without affecting cell viability. Molecular docking predicted non-covalent binding of 4-MC within the ATP-binding hinge region of IKK{beta} (PDB: 4KIK), forming a close polar contact with Glu97, predicted hydrogen bonds with Cys99, and a hydrophobic contact with Ile165, within the pocket occupied by the co-crystallized inhibitor K252a. Covalent docking predicted that the oxidized quinone form of 4-MC engages Cys179 in the IKK{beta} activation loop. Quantum chemical calculations confirmed a markedly higher electrophilicity index for the oxidized quinone than for the parent catechol, supporting this mechanism. In silico ADMET profiling indicated favorable drug-likeness and safety. These findings identify IKK{beta} as a plausible molecular target of 4-MC through both non-covalent and covalent mechanisms. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/741661v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@35a0d3org.highwire.dtl.DTLVardef@d19458org.highwire.dtl.DTLVardef@1623fadorg.highwire.dtl.DTLVardef@1429e8b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Plant-derived small molecules possess highly diverse physicochemical properties, and the computational design of their protein recognition elements depends not only on the global structural quality of candidate backbones, but also on whether the local binding pocket, ligand-contact pattern, and predefined recognition conformation can be consistently retained after sequence design and structural back-prediction. To explore pocket-design strategies for different types of natural-product small molecules, this study selected capsaicin, (4R)-limonene, and quercetin as model ligands, representing a flexible amphipathic molecule, a compact hydrophobic monoterpene, and a rigid polyphenolic flavonoid scaffold, respectively, and covering the dimensions of pungent sensory flavor, volatile aroma, and flavonoid functional constituents. A ligand- physicochemical-property-guided computational design and multi-stage prioritization framework was established for candidate protein binders. The results showed that candidates with favorable initial global structural scores did not necessarily form reasonable local small-molecule binding pockets, indicating that evaluation of the local ligand environment is essential for candidate prioritization. After screening, 31 partial- pocket candidate backbones for capsaicin, 75 buried hydrophobic-pocket candidate backbones for (4R)-limonene, and 56 pocket-qualified candidate backbones for quercetin were obtained. Further sequence design and structural back-prediction analyses indicated that a subset of candidates could maintain the original pocket geometry and major ligand-contact patterns after sequence realization. Overall, these results suggest that the physicochemical properties of different plant-derived small molecules substantially influence the efficiency of de novo protein pocket formation, with compact hydrophobic ligands being more compatible with buried hydrophobic- pocket strategies, whereas flexible or multipolar ligands require a more refined balance between hydrophobic burial and polar exposure. This study provides a pre- experimental computational prioritization framework for natural-product small- molecule-recognizing proteins and offers candidate resources for subsequent protein expression, in vitro binding validation, active-constituent enrichment, and development of small-molecule biorecognition tools. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/743643v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@8fe6c2org.highwire.dtl.DTLVardef@176cef2org.highwire.dtl.DTLVardef@10c8201org.highwire.dtl.DTLVardef@2b28cf_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

Glyceraldehyde (GA) contributes to the development of various diseases, such as diabetes and Alzheimer's disease via protein glycation and the formation of advanced glycation end products (AGEs); however, effective strategies for neutralizing GA are limited. Carnosine (Car), an imidazole dipeptide (IDP) that is abundant in meat, suppresses protein glycation by scavenging reactive aldehydes. There are only a few reports on the antiglycation activity of Car against GA. For other IDPs, such as anserine, balenine (Bal), and homocarnosine, there are almost no reports on their antiglycation activity. In this study, we demonstrated the antiglycation activity of four types of IDPs and 2-oxocarnosine (2-oxo-Car), an oxidized form of Car, against GA-induced intracellular protein glycation and neuronal cytotoxicity. Car and Bal exhibited significantly higher reactivity with GA compared with other IDPs and 2-oxo-Car. An in silico analysis suggested that the difference in reactivity is dependent upon intramolecular hydrogen bond formation and the conformation of each IDP. Although there were differences in reactivity with GA, LC-MS analysis revealed that all of the IDPs and 2-oxo-Car reacted with two molecules of GA to form adducts containing pyridinium rings. Car and Bal exhibited high reactivity with GA and markedly suppressed GA-induced cytotoxicity in SH-SY5Y cells. Western blot and qPCR analyses revealed that IDPs suppressed GA-induced protein glycation and the upregulation of endoplasmic reticulum and oxidative stress response genes. Our results indicate that IDPs represent a novel preventive approach to AGE-related diseases and provide a foundation for the development of strategies to treat GA-related neurotoxicity.

Obesity-driven metabolic syndrome poses a critical global threat, yet standard therapies like GLP-1 receptor agonists trigger substantial lean mass wasting, with muscle loss accounting for up to 40% of reduced weight. Here we identify a non-canonical metabolic application for dronedarone hydrochloride, an anti-arrhythmic benzofuran derivative. In diet-induced and ob/ob obese mice, short-term dronedarone hydrochloride administration dose-dependently reduces food intake, clears visceral and subcutaneous adiposity, and reverses steatohepatitis. Head-to-head trials show that dronedarone hydrochloride achieves glycemic control and fat clearance non-inferior to semaglutide, tirzepatide, and empagliflozin, but uniquely and completely preserves skeletal muscle mass. Mechanistically, dronedarone hydrochloride operates independently of central hypothalamic appetite-regulating neuropeptides and the peripheral leptin pathway. By decoupling fat reduction from sarcopenia, our findings establish dronedarone hydrochloride as a muscle-sparing therapeutic candidate for metabolic syndrome.

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

Protein Kinase Novel 3 (PKN3) understudied kinase with a diverse array of biological functions that are yet to be fully defined. Here, we report the design and development of a novel advanced functional chemical tool inhibitor for PKN3. A pyridyl imidazole series has been synthesized and evaluated against PKN3 in vitro and in cells. These efforts led to the discovery of 6e (URS03-06), a submicromolar cell active functional inhibitor with a narrow kinome spectrum, to enable the elucidation and interrogation of PKN3 cellular biology.

GLP-1 receptor agonists achieve weight loss but are associated with clinically significant reductions in lean mass. Activin type II receptors (ActRIIA and ActRIIB) mediate signaling of myostatin and activin A, both of which negatively regulate muscle growth, suggesting that dual blockade of these receptors may preserve or increase lean mass while promoting fat loss. In this study, we developed anti-ActRIIA/B antibodies using AI-driven platforms (AlfaDAX) and selected the lead candidate AB130-165 based on in vitro binding, functional blocking, and developability assessments. Compared with a laboratory-prepared bimagrumab analog, AB130-165 exhibited potent dual inhibition of ActRIIA/B signaling, with a 9.5-fold higher functional blocking activity against activin A-induced SMAD signaling and 1054-fold improvements in binding affinity for ActRIIA (KD = 0.204 pM), 10-fold for ActRIIB (KD = 0.243 pM), respectively. In diet-induced obese mice, combination therapy with AB130-165 and semaglutide resulted in a 33.4% body weight reduction, which was superior to semaglutide monotherapy (-24.3%) and the bimagrumab combination group (-25.5%). Moreover, the combination significantly improved body composition, reducing fat mass percentage by 77.8% (vs. 65.0% in the bimagrumab combination group) and increasing the lean-to-body weight ratio to 67.3% (vs. 62.3%), demonstrating superior fat loss with better preservation of lean mass. Collectively, these findings establish AB130-165 as a differentiated anti-ActRII antibody that enables high-quality weight loss, and its combination with semaglutide shows superior efficacy over bimagrumab-based regimens. With favorable developability and potential for long-acting subcutaneous administration, AB130-165 represents a promising next-generation therapeutic candidate for obesity and muscle-sparing weight management.

Targeting viral macrodomains (Mac) has emerged as a promising strategy for antiviral drug development, especially after the outbreak of COVID-19 that claimed millions of lives worldwide. Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Mac1 inhibitors have been reported in the past few years. In the present work, we converted GS-441524 (IC50 of [~]10 M for SARS-CoV-2 Mac1) to KP-S54 (18c), a potent inhibitor of both SARS-CoV-2 Mac1 (IC50: 44 nM) and Middle East respiratory syndrome coronavirus (MERS-CoV) Mac1 (IC50: 91 nM) through an iterative direct-to-biology approach. This approach leverages efficient amide-coupling reaction and the mix-and-read fluorescence polarization (FP) assays where reaction mixtures could be screened directly without purification. Cocrystal structure of a selected derivative (12p) binding to SARS-CoV-2 Mac1 revealed the binding mode, which will guide future drug development against viral macrodomains.

Dysregulated gut homing of leukocytes drives chronic inflammation in Crohns disease (CD). We employed phage display selection campaigns with libraries of stabilized, constrained peptides against endogenous conformation states of the cannabinoid receptor CB2R on human T cells, to discover novel receptor antagonists with potential to inhibit gut homing. Cluster and frequency analysis of 50,000 enriched sequences resulted in expression and functional characterisation of 10 protein candidates using assays of glucose uptake, ERK phosphorylation (pERK) and beta-arrestin recruitment. Each candidate antagonised CB2R activity with recorded IC50 values of between 5-10 nM. Cannabinoid receptor nanodisc binding experiments and SPR confirmed CB2R selectivity. SLKC_09 with an IC50 of 5.4 nM, was studied in a mouse model of chronic ileitis where it significantly inhibited gut homing of CD4+ & CD8+ naive, effector and memory cell types. Our findings highlight an alternative route to therapeutic inhibition of leukocyte trafficking in CD with a biologic inhibitor of CB2R.

Inflammatory bowel disease (IBD) is associated with chronic intestinal inflammation and gut microbial dysbiosis, yet effective microbiome-targeted therapeutics remain limited. Here, we investigated the anti-inflammatory and microbiome-modulating activities of metabolites isolated from Garcinia brassii, an endemic species of the Australian Wet Tropics. Five compounds, including a new natural product named garcitine, were isolated and structurally characterised. In human immune cells, garcinol and garcinia biflavonoid 1 significantly suppressed lipopolysaccharide-induced production of IL-1{beta}, IL-6, and TNF without detectable cytotoxicity, while parvifoliol F selectively inhibited IL-1{beta} release. Therapeutic efficacy was further evaluated in a TNBS-induced murine colitis model, where garcinia biflavonoid 1 and parvifoliol F significantly reduced colonic inflammation and improved histopathological outcomes. 16S rRNA sequencing demonstrated that both compounds restored gut microbial homeostasis by reversing colitis-associated dysbiosis and reducing inflammation-associated microbial signatures. Functional pathway prediction further suggested suppression of pro-inflammatory microbial metabolic pathways following treatment. Together, these findings demonstrate that Garcinia-derived metabolites alleviate experimental colitis through coordinated immunomodulatory and microbiome-reprogramming mechanisms and identify garcinia biflavonoid 1 and parvifoliol F as promising candidates for microbiome-targeted IBD therapeutics.

Cancer immunotherapy has transformed cancer treatment; however, durable responses remain limited by suppressive myeloid populations within the tumor microenvironment. Leukocyte immunoglobulin-like receptor B4 (LILRB4/ILT3) is an emerging myeloid immune checkpoint implicated in immune evasion and resistance to immunotherapy, yet small molecule targeting of ILT3 remains largely unexplored. Here, we report the discovery of small molecule ILT3 modulators identified using a Dianthus-based temperature-related intensity change (TRIC) screening platform. Screening of an 8,961-member Enamine Library identified multiple direct ILT3 binders, with lead compound ICB-7 demonstrating high-affinity binding to recombinant human ILT3 by microscale thermophoresis and robust cellular target engagement in CETSA assays. Molecular docking and molecular dynamics simulations revealed a stable hydrophobic binding pocket within the D2 domain of ILT3. Functionally, ICB-7 disrupted the ILT3-SCG2 interaction and inhibited downstream SHP1, SHP2, and STAT3 signaling. In patient-derived colorectal cancer and acute myeloid leukemia co-culture models, ICB-7 enhanced cytotoxic T-cell activity, and reduced tumor-cell viability. The compound also demonstrated favorable pharmacokinetic and safety properties together with significant anti-tumor efficacy in the CT26 syngeneic colorectal carcinoma model. Collectively, these findings establish ILT3 as a tractable target for small-molecule immunomodulation and support pharmacological targeting of suppressive myeloid checkpoints as a promising cancer immunotherapy strategy.

BackgroundMetabolic dysfunction is characterized by dysregulated lipid metabolism, lipotoxicity, insulin resistance, and chronic low-grade inflammation, contributing to obesity and metabolic dysfunction-associated steatotic liver disease (MASLD). Multi-target therapeutic strategies that restore lipid homeostasis are of growing interest. Patolakaturohiniyadi Kashayam (PKR), a classical Ayurvedic polyherbal formulation, was investigated for its potential to modulate lipid metabolism and ameliorate metabolic dysfunction. MethodsAn integrated approach combining network pharmacology, in vitro, lipidomics, and in vivo studies was employed. Hub gene identification and KEGG pathway enrichment were performed to elucidate molecular targets. Anti-steatotic and anti-adipogenic effects were assessed in hepatocytes and adipocytes, followed by lipidomic profiling. Efficacy was further evaluated in a high-fat high-fructose diet (HFHFD)-induced animal model. ResultsNetwork pharmacology identified key targets including TP53, AKT1, IL6, TNF, and STAT3, enriched in pathways related to lipid metabolism, inflammation, and metabolic regulation. PKR significantly reduced lipid droplet accumulation and intracellular triglyceride levels in vitro. Lipidomics revealed suppression of diacylglycerol-mediated lipotoxicity and restoration of phospholipid balance, characterized by increased lysophospholipids and phosphatidylethanolamines with normalization of phosphatidylcholine species. In vivo, PKR reduced body, liver, and adipose tissue weights, improved serum lipid profiles, and decreased AST and ALT levels. Histological analyses demonstrated reduced lipid accumulation and inflammation, along with preservation of adipose tissue architecture. PKR also improved glucose tolerance and significantly elevated plasma GLP-1 levels. ConclusionPKR exerts potent anti-steatotic and anti-obesogenic effects through coordinated regulation of lipid metabolism, inflammation, and incretin signalling, highlighting its potential as a multi-target therapeutics for metabolic dysfunction.

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.

BackgroundCannabis derivatives have been reported to possess antinociceptive properties. However, oral delivery is limited by poor bioavailability, stability, and reliability of effects. Previously, we reported an analgesic effect of the aqueous complex {Delta}9-tetrahydrocannabinol/2-hydroxypropyl-{beta}-cyclodextrin (THC/HP{beta}CD) after intracerebroventricular administration in male rats. MethodsHere, we investigated the analgesic effects of the THC/HP{beta}CD complex after oral administration (0.3 and 3 mg/kg) by the tail flick test after both acute and chronic administration (15 days) in female rats. Locomotor activity and anxiety-like behavior were also evaluated at the same experimental conditions. Moreover, dopamine and glutamate content in the periaqueductal gray (PAG), a key area for the antinociceptive action of THC, were also measured by HPLC. ResultsAfter acute administration, the antinociceptive effect of the complex was seen at 3 but not 0.3 mg/kg THC, with a maximum effect observed at 30 min (MPE 60%). Similar results were obtained after 15 days of treatment, although partially reduced (max MPE 20%). Reductions in locomotor activity with the dose of 3 mg/kg and a slight biphasic effect of the two doses on anxiety-like behavior were also observed. Finally, neurochemical analyses revealed that the dose of 3 mg/kg significantly increased dopamine and glutamate content in the PAG, an effect no longer present after 15 days of treatment. ConclusionsOur results highlight the antinociceptive efficacy of the THC/HP{beta}CD complex also after oral administration, notably higher than that previously seen with other carriers, although with some degree of tolerance after chronic administration. From a translational point of view, these results are relevant for the development of THC-based oral formulations with analgesic properties for the treatment of pain in humans. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/742765v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@fff791org.highwire.dtl.DTLVardef@d672f4org.highwire.dtl.DTLVardef@1150b3forg.highwire.dtl.DTLVardef@956403_HPS_FORMAT_FIGEXP M_FIG C_FIG

GPR183 is an oxysterol-sensing GPCR predominantly expressed in lymphoid organs and tissues. Activation of the receptor by oxysterol 7,25-OHC leads to Gi protein-mediated signaling as well as {beta}-arrestin2 recruitment. GPR183/oxysterol signaling modulates localization of lymphoid cells, consequently the receptor is associated with several inflammation-associated diseases and is an interesting potential drug target. Previously, we reported the discovery of moderately potent G protein-biased partial agonists for GPR183 from a virtual screening based on the scaffold of the antagonist NIBR189. Herein, we present the detailed structure-activity investigations and optimizations, which led to the identification of full agonists for GPR183 with complete bias for Gi protein signaling and low nanomolar potency, including 63 (TUG-2604) with potency and efficacy similar to 7,25-OHC. Notably, 63 was unable to induce migration of human dendritic cells but inhibited migration induced by 7,25-OHC. This compound will be valuable for further explorations of the signaling-specific function and drug target potential of GPR183.

Formyl peptide receptor 1 (FPR1) is a promising therapeutic target for the treatment of inflammatory and infectious diseases. Although multiple classes of peptides are known to modulate FPR1 activity, comprehensive studies systematically linking N-terminal modifications to binding, mechanism of action and functional outcomes remain limited. In this study, we aimed to rationalise the binding and activity of three peptide series (MLF, FLFLF, and MLFYLA) featuring diverse N-terminal modifications from a structural point of view. A combined in silico and in vitro approach was employed to evaluate the activity of newly designed peptide agonists and to generate mechanistic binding hypotheses. Our findings led to the identification of a transmembrane binding pocket in FPR1, which provides a structural basis for the observed antagonist and partial agonist behaviours and leads to a generalisable strategy for tuning the functional outcome of peptidic ligands.

N-acylethanolamines (NAEs), including the endocannabinoid anandamide, are bioactive fatty acid amides that are normally hydrolyzed by fatty acid amide hydrolase (FAAH) or N-acyl acid amidohydrolase (NAAA). Strikingly, when canonical NAE degradation is blocked, NAE levels do not increase indefinitely but instead reach a plateau. This apparent metabolic ceiling suggests that additional, underexplored pathways contribute to NAE homeostasis. Identifying these pathways is essential to determine whether NAEs are converted into inactive metabolites or products with distinct biological properties. Here, we identify NAE glycosylation as a metabolic pathway that links endocannabinoid-related lipid metabolism to glycosphingolipid turnover. We synthesized glycosylated NAEs and their isotope-encoded standards and developed targeted LC-MS/MS assays to monitor their enzymatic processing and quantify their abundance in mouse and human cells, tissues, and plasma. We show that non-lysosomal glucosylceramidase GBA2 transfers glucose or galactose to anandamide, N-oleoylethanolamine and N-palmitoylethanolamine, and lysosomal glucosylceramidase GCase hydrolyses {beta}-Glycosylated-NAEs ({beta}-Glyco-NAE) back to their parent NAEs. {beta}-Glyco-NAEs occur endogenously in macrophages and neuronal cells, increase when canonical NAE degradation is impaired, and accumulate in human samples with GCase deficiency, including Gaucher disease and GBA1-associated Parkinsons disease. {beta}-Glyco-NAEs do not engage the cannabinoid receptors, TRPV1, or PPAR, and potentiate inflammatory cytokine release, including IL6 and TNF, from microglia. Based on these findings, we pose that GBA2-dependent NAE glycosylation may constitute an overflow lipid-remodeling pathway that connects NAE metabolism to lysosomal dysfunction, inflammation and neurodegeneration.

BackgroundAtherosclerosis is a chronic inflammatory vascular disorder with persistent residual inflammation even after standard lipid-lowering therapy. Mounting evidence from bench to bedside suggests that diabetes and obesity accelerate atherosclerosis development. Tirzepatide (TZP), a dual Glucagon-Like Peptide-1 Receptor/Glucose-Dependent Insulinotropic Polypeptide Receptor (GLP-1R/GIPR) agonist approved for treating diabetes and obesity, has demonstrated proven cardiometabolic efficacy in large cardiovascular outcome trials. However, it remains largely uncertain whether TZP attenuates atherosclerosis independent of its anti-diabetic and anti-obese effects through direct actions on the vasculature. MethodsWe established atherosclerotic mouse models under diabetic, obese, and non-diabetic/non-obese conditions. Analysis of covariance (ANCOVA) and pair-feeding experiments were applied to experimentally decouple weight-dependent metabolic improvement from intrinsic vasculoprotection. Molecular and cell biological assays in human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) were performed to dissect the underlying signaling mechanisms. ResultsTZP markedly reduced aortic plaque burden and inflammation, restrained necrotic core enlargement, and improved plaque stability across all experimental mouse models. Both ANCOVA and pair-feeding experiments confirmed that these atheroprotective effects were independent of food intake and body weight loss. Furthermore, TZP attenuated systemic and vascular inflammation in Tumor Necrosis Factor- (TNF)-treated C57BL/6J mice, and this protection occurred without changes in body weight or blood glucose levels. Mechanistically, TZP directly targeted endothelial cells and activated the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/endothelial nitric oxide synthase (eNOS) pathway, increased eNOS phosphorylation and nitric oxide bioavailability, consequently downregulating the expression of the pro-inflammatory adhesion molecules Vascular Cell Adhesion Molecule-1 (VCAM-1) and Intercellular Adhesion Molecule-1 (ICAM-1). ConclusionsTZP arrests atherosclerosis progression through weight loss-independent anti-inflammatory mechanisms. These findings implicate TZP as a promising therapeutic drug for mitigating residual vascular inflammation in patients with atherosclerotic cardiovascular disease (ASCVD), irrespective of glycemic status or obesity. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LITirzepatide exerts direct anti-atherosclerotic effects in preclinical mouse models of atherosclerosis under diabetic, obese, and non-obese conditions. C_LIO_LITirzepatide directly targets endothelial GLP-1R/GIPR and downstream cAMP/PKA/eNOS signaling pathway to suppress NF-{kappa}B-driven vascular inflammation, thereby uncovering a previously unrecognized vasculoprotective mechanism underlying its cardiovascular benefits C_LI What Are the Clinical Implications?O_LITirzepatide exerts direct vascular protective effects independent of body weight reduction, suggesting that its cardiovascular benefits may extend beyond glycemic control and obesity management. C_LIO_LITirzepatide may represent a promising therapeutic drug for addressing residual vascular inflammation in ASCVD patients, including those without overt diabetes or obesity C_LI