Biomedicine & Pharmacotherapy

Successful treatment of primary Clostridioides difficile infection (CDI) while preventing its recurrence remains a major clinical challenge, with recurrent CDI (R-CDI) affecting 20-30% of treated patients. Persistence of C. difficile spores within the intestinal mucosa is thought to drive R-CDI following antibiotic treatment. Here, we show that intracellular C. difficile spores remain protected from taurocholate-triggered germination, and that nystatin, an FDA-approved antifungal that blocks spore entry into host cells, reduces this protected intracellular population and redirects spores toward an extracellular, germinating population accessible to antibiotics. In a murine model of R-CDI, oral co-administration of taurocholate with nystatin and antibiotics during primary CDI reduced R-CDI incidence from 100% to 44%. When administered during an active R-CDI episode, the same formulation reduced a second R-CDI from 100% to 62%. To improve bioavailability, we encapsulated nystatin and vancomycin in chitosan nanoparticles. Oral administration of nanoparticle-encapsulated vancomycin, alone or combined with nanoparticle-encapsulated nystatin, reduced R-CDI colonization to 33% and 44%, respectively, compared with 100% in mice receiving free vancomycin. Together, these findings support a therapeutic strategy that repurposes nystatin and taurocholate, in combination with antibiotics and nanoparticle-based delivery, to target intracellular spore persistence and reduce the incidence of C. difficile recurrence..

Background: Relapsed and refractory osteosarcoma (OS) remains a major therapeutic challenge, with fewer than 20% of patients surviving beyond 3 years. Increasing evidence indicates that MYC amplification/overexpression is associated with inferior survival. Small molecule inhibitors and immunotherapies have limited single-agent efficacy in pediatric solid tumors. Using syngeneic cell lines derived from p53-driven and MYC-hyperactivated genetically engineered mouse models (GEMMs) of OS, we tested cabozantinib, a multi-tyrosine kinase inhibitor with immunomodulatory properties, with TIGIT immune checkpoint blockade and investigated mechanisms underlying therapeutic response and resistance. Methods: In vitro cabozantinib sensitivity was established in GEMM-derived cell lines. Mice bearing tibial tumors were randomized to vehicle control, cabozantinib, anti-TIGIT antibody, or combination therapy, and tumor growth and survival assessed after a 3-week treatment period. Temporal RNA sequencing was performed at early (8-15 days) and late (18-24 days) time points to characterize transcriptomic changes associated with efficacy. Results: MYC-hyperactivated cell lines were more resistant to cabozantinib in vitro than p53-driven lines (mean IC50 5.51 vs 0.65 mciroM, p=0.0016). In p53-driven orthotopic models, combination therapy significantly decreased tumor growth and improved survival compared to solvent and cabozantinib alone, while in MYC-hyperactivated models cabozantinib-containing regimens delayed tumor progression relative to control or anti-TIGIT monotherapy, however the addition of anti-TIGIT did not significantly improve survival over cabozantinib alone. Temporal transcriptomics revealed upregulated anti-tumor immune-response pathways and decreased M2 macrophages only with combination treatment in the p53-driven model. In contrast, combination-treated MYC-hyperactivated models demonstrated increased TNF signaling and elevated Cxcl5 and Ccr2 expression, indicative of increased myeloid cell recruitment, and upregulation of extracellular matrix (ECM) remodeling pathways suggest a therapy-induced stress adapted state that propagates treatment resistance over time. Conclusion: New therapies are needed for patients with relapse or refractory OS. By targeting tumor-intrinsic resistance mechanisms and modulating the tumor microenvironment using cabozantinib and anti-TIGIT therapy, improved tumor control and survival was achieved in p53-driven orthotopic OS models. MYC-hyperactivated models were able to overcome therapeutic pressure and employ myeloid recruitment and ECM remodeling programs to achieve treatment resistance. Targeting of these programs should be considered in future studies investigating therapeutic strategies in relapsed and refractory OS.

Ogerin, a positive regulator of GPR68, reduces infarct volume and attenuates blood-brain barrier (BBB) leakage post transient middle cerebral artery occlusion (tMCAO). These data support the potential use of Ogerin for ischemic protection. For therapeutic interventions, tissue distribution, kinetics, and brain penetration are important characteristics for a compound. For these reasons, we performed liquid chromatography-mass spectrometry (LC-MS) analysis of Ogerin in mice. At 3 hours following intraperitoneal (IP) injection, liver exhibited highest level of Ogerin among all major organs assessed. We further analyzed time-dependent changes in Ogerin concentration in serum following intravenous delivery. Brain Ogerin level was ~140 ng/g at 2 hour and ~0.06ng/g at 24 hours. The brain-to-serum ratio of Ogerin was ~0.6 at 5 hr. To determine the effect of Ogerin on signaling, we collected samples 24 hours after tMCAO with or without Ogerin delivered and performed Western blotting. Consistent with the diminished level of Ogerin in brain at 24 hour, tMCAO increased similar changes in phosphorylation of PKA substrates and PKC substrates in vehicle- and Ogerin-treated groups. The pharmacokinetic data suggests that Ogerin is effective mainly during acute hours after injection. This finding generates important insights on the design of effective protocol for Ogerin delivery to potentiate GPR68 function in vivo.

Spinocerebellar ataxia type 3 (SCA3) is most common subtype of autosomal dominant spinocerebellar ataxia worldwide and the most prevalent CAG expansion disorder in the east Asian population, but disease-modifying therapies remain unavailable. SCA3 is caused by the accumulation of polyglutamine- (polyQ-) expanded mutant ataxin-3 (mAtxn3), while the wild-type ataxin-3 (wtAtxn3) plays an important cellular role. Thus, allele-selective clearance of mAtxn3 without affecting wtAtxn3 is highly desired for SCA3 treatment. To achieve this goal, we tested previously reported compounds that have been shown to selectively degrade the Huntingtons disease causing mutant HTT protein by tethering the expanded polyQ to the autophagosome protein LC3. Here, we demonstrated that two of these compounds, AN2 and GW5074, selectively reduced both soluble and aggregated mAtxn3 with expanded polyQ (84Q), but not wtAtxn3, in both cellular and mouse models of SCA3. Mechanistically, both compounds lowered mAtxn3 by enhancing its autophagic degradation without obvious activation of global autophagy. At the molecular level, both compounds increased mAtxn3-LC3 interaction and colocalization to enhance the allele-selectively autophagic degradation of mAtxn3. Phenotypically, both compounds rescued cell viability and ATP content of SCA3 cellular models. In SCA3 patient pluripotent stem cells (iPSCs) derived cortical neurons, both compounds allele-selectively reduced mAtxn3 and partially rescued neurite shortening. Importantly, in a well-established SCA3 transgenic mouse model (MJD84.2), intracerebroventricular, intraperitoneal, and oral administration allele-selectively reduced mAtxn3 in multiple brain regions and rescued motor function deficits. Oral dosing achieved nanomolar cerebellar exposure and showed no overt liver, kidney, or glial toxicity over 28 days. Analyses of 1303 Chinese patients with SCA3 and ATXN3 constructs of different polyglutamine lengths further supported the compounds polyQ length-dependent activity with relative sparing of normal-length ataxin-3. Together, these findings provide preclinical proof of concept that utilizing orally available small molecule compounds to allele-selectively enhance mAtxn3 autophagic degradation for SCA3 treatment. One-sentence summarySmall-molecule autophagosome-tethering compounds selectively lower mutant ataxin-3 through an LC3- and autophagy-dependent mechanism and improve disease-related phenotypes in preclinical models of SCA3.

Background: Alpha-mannosidosis (AM) is a debilitating lysosomal storage disorder (LSD) caused by insufficient activity of alpha-mannosidase, an enzyme responsible for cleavage of alpha-linked mannose residues from N-glycan-type oligosaccharides during their degradation. As a result, specific oligosaccharides accumulate in tissues and are also detectable in body fluids. AM is a progressive multi-organ disease for which hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT) are therapeutic options, however, treatment results depend considerably on early diagnosis. Therefore, inclusion of AM into newborn screening panels would significantly contribute to improving therapeutic outcome. Methods: A rapid and fully quantitative method for the specific AM oligosaccharide biomarker Man2GlcNAc in dried blood spots (DBS) using ultra-performance liquid chromatography coupled to tandem-mass spectrometry was developed. DBS from 420 controls, 27 AM patients (including five newborn screening samples), and 35 patients with other LSDs were analyzed. Results: A distinct separation in biomarker concentrations was observed between unaffected individuals and untreated AM patients (0.1 - 0.8 vs 3.2 - 11.3 mol Man2GlcNAc/L, p<0.0001). Furthermore, Man2GlcNAc concentrations significantly differed between untreated patients and those on ERT (n = 17, p<0.0001) or after HSCT (n = 15, p<0.0001), and between both treatment groups (p<0.0001). Moreover, we demonstrated that this method is appropriate for monitoring treatment efficacy by analysis of samples from individual patients before and after initiation of therapy. We retrospectively analyzed residual samples of dried blood spots (DBS) from routine newborn screening that had been stored from patients with later-confirmed AM, which resulted in clear separation from the concentrations observed in a newborn screening control cohort (7.7 - 11.3 vs 0.1 - 0.8 mol Man2GlcNAc/L, p=0.0001). Conclusion: We present a simple and rapid method for quantitative analysis of the specific AM oligosaccharide biomarker Man2GlcNAc, which is remarkably stable compared to enzymatic assays. Thus, the method is useful for selective screening in suspected cases as well as for monitoring the response to different forms of treatment. Furthermore, Man2GlcNAc has the potential to serve as a useful biomarker for newborn screening offering the prospect of earlier treatment initiation, improved therapeutic outcome and higher quality of life for AM patients.

Screening of the open-access CRESTdb small-molecule library identified the imidazothiazole carboxamide sCQG200 (1) as an initial hit, with activity against H37Rv and Erdman Mycobacterium tuberculosis strains in cholesterol-containing medium (MIC90= 6.88 uM and 4.48 uM, respectively) and against intracellular Mtb (EC50= 3.80 uM). sCQG200 retained activity against drug-resistant Mtb strains, including clinical MDR/XDR isolates. SAR optimization led to analogs 21 and 24 with substantially improved antimycobacterial potency. Mouse pharmacokinetic studies following oral and intravenous administration showed 62.1% and 29.5% oral bioavailability for 21 and 24, respectively. This structurally differentiated chemotype provides a promising starting point for further optimization as antitubercular agent.

BackgroundSerotonergic signaling plays an important role in the modulation of absence seizures (ASs), and activation of serotonin 2A receptors (5-HT2ARs) with TCB-2 suppresses spontaneous absence seizures (ASs) in Genetic Absence Epilepsy Rats from Strasbourg (GAERS). Lisuride is a pharmacologically complex serotonergic and dopaminergic ligand with low 5-HT2AR signaling efficacy and a receptor profile distinct from that of TCB-2. We therefore investigated its effects on spontaneous ASs and the contribution of 5-HT2ARs to its actions. MethodsThe effects of systemic lisuride (0.1 and 0.5 mg/kg) on spontaneous ASs were examined in freely moving GAERS over 120 min by quantifying time spent in seizure, mean seizure duration and seizure number. The contribution of 5-HT2ARs was investigated by administering lisuride (0.5 mg/kg) following pretreatment with the selective 5-HT2AR antagonist MDL11,939 (0.5 mg/kg). ResultsLisuride produced marked dose- and time-dependent effects on ASs. At 0.1 mg/kg, lisuride elicited modest pro-absence effect. In contrast, 0.5 mg/kg produced a pronounced biphasic response, with a marked reduction in seizure activity during the first 40 min, followed by rebound increase during the second hour. The early suppression involved reductions in both seizure number and duration, whereas the delayed pro-absence effect was predominantly associated with prolonged ASs. MDL11,939 did not prevent the early seizure suppression but blocked the increase in seizure duration while producing a transient increase in seizure number. ConclusionsLisuride is a dose- and time-dependent bidirectional modulator of ASs in GAERS. The persistence of the profound early anti-absence effect during 5-HT2AR blockade indicates that this response does not require 5-HT2AR activation, whereas the modification of the delayed response by MDL11,939 supports a 5-HT2AR-dependent component in the later phase. These findings distinguish lisuride pharmacologically from TCB-2 and suggest that its biphasic effects arise from the dynamic engagement of multiple receptor mechanisms, potentially including dopaminergic signaling.

Objective To systematically evaluate the predictability of drug chemical structures against the complete set of MedDRA Preferred Terms (PTs), construct a comprehensive structure adverse reaction landscape, validate the utility of molecular fingerprint representations in pan-screening tasks, and establish quantitative tools for drug safety pre-screening. Methods Drug adverse event association data were compiled from SIDER 4.1 and its derivative compilation Nsider, encompassing 2,005 approved drugs, of which 1,120 small-molecule drugs with successfully retrieved chemical structures were retained for analysis. For each Preferred Term (PT) meeting the minimum sample-size threshold ([&ge;]25 positive and [&ge;]30 negative drugs; n = 768), XGBoost classifiers were trained using MACCS 166-bit fingerprints, with model performance evaluated via 5-fold stratified cross-validation. A PT was designated as high-confidence if it achieved a validation AUC [&ge;] 0.75, a cross-validation AUC [&ge;] 0.70, and passed Benjamini Hochberg false discovery rate (FDR) correction (q < 0.05). The entire analytical pipeline was replicated using Morgan ECFP4 2048-bit fingerprints to assess the robustness of fingerprint selection. The robustness of significance screening was independently validated by permutation testing (500 random label permutations), and model generalizability was evaluated through Bemis-Murcko scaffold-based stratified split validation. SHAP (SHapley Additive exPlanations) analysis was employed to identify critical structural features, and mechanism-based case studies further demonstrated concordance between model-identified features and established pharmacophores. Results Among the 768 modelable PTs, 71 (9.2%) met the high-confidence significance criteria (FDR q < 0.05), predominantly comprising nervous system disorders (10 PTs) and psychiatric disorders (9 PTs). The toxicities exhibiting the strongest structural predictability included hyperprolactinaemia (AUC = 0.968), dermatitis acneiform (AUC = 0.966), tardive dyskinesia (AUC = 0.952), and breast tenderness (AUC = 0.979). Under a unified XGBoost hyperparameter configuration, the low-dimensional MACCS fingerprint (166-bit) demonstrated near-equivalent performance to the high-dimensional Morgan fingerprint (2048-bit) in pan-screening tasks ({Delta}AUC = -0.012, Cohen's d = 0.16), and achieved comparable performance at a substantially lower dimensionality across 18 of 22 organ systems. SHAP analysis identified key structural fragments corresponding to established pharmacophores (six-membered rings, tertiary amine cores, heteroatomic bonds, and bridged heteroatomic chains), and mechanism-based case studies further demonstrated that the model captured structural patterns concordant with known pharmacophores of D2 antagonists, EGFR inhibitors, and related agents. Bemis-Murcko scaffold-based stratified split validation revealed a median AUC decrease of merely 0.042, with 66% of PTs maintaining an AUC [&ge;] 0.70 under scaffold-split conditions. Immune-mediated and multifactorial endpoints remained structurally opaque under the current data conditions. Conclusion This study constructed a comprehensive structure-adverse reaction predictability landscape encompassing 768 MedDRA PTs; seventy-one PTs achieved high-confidence criteria. The MACCS 166-bit fingerprint achieved near-equivalent performance to the Morgan 2048-bit fingerprint at a markedly lower dimensionality, and its chemically explicit features enable direct translation of model outputs into structural alerts, providing a computational tool for prospective screening of potential adverse reaction profiles for any novel compound. A tiered assessment strategy is proposed: machine learning ensemble models for high-confidence screening, substructure alert rules for high-specificity exclusion, and pharmacovigilance signal detection for complementary coverage of structurally unpredictable endpoints. Bemis-Murcko scaffold-based stratified split validation further demonstrated that the median generalization performance decrease was merely 0.042 AUC, with 66% of PTs maintaining an AUC [&ge;] 0.70 under scaffold-split conditions, corroborating the existence of a genuine chemical basis underlying structure-adverse reaction mappings.

Background: Severe hepatotoxicity has been reported when sotorasib follows immune checkpoint inhibitor (ICI) therapy, and fixed washout intervals have been proposed. We assessed prior ICI exposure and the ICI-to-sotorasib interval as separate risk factors. Materials and methods: We searched PubMed, Europe PMC, Embase, Web of Science, Scopus, CENTRAL, and meeting proceedings for comparative studies of grade 3 or higher hepatotoxicity after KRAS G12C inhibitors. Matched risk ratios (RRs) and unadjusted cohort data were synthesized separately, the latter with an exact conditional odds ratio (OR). Certainty was rated with GRADE. Results: Of 530 unique records screened, seven sources were included, all involving sotorasib. In the only matched analysis (meeting abstract, 386 patients per arm), prior ICI exposure was associated with higher risk (13.2% vs 4.4%, RR 3.00, 95% CI 1.76-5.10), whereas matched comparisons at 6 weeks (RR 1.11, 0.61-2.03) and 12 weeks (RR 1.08, 0.52-2.27) detected no difference but could not exclude important effects. All five unadjusted cohorts showed higher risk with shorter intervals, including 21/74 versus 8/75 patients in the earlier versus later interval quartiles of the CodeBreaK 200 trial, although effect sizes were heterogeneous (exact homogeneity P=0.006). Certainty was very low throughout. Conclusions: Prior ICI exposure was associated with higher risk of severe sotorasib-associated hepatotoxicity, with very low certainty. A shorter interval was associated with higher risk in unadjusted data only, and current evidence does not establish a mandatory washout period. Liver-test monitoring per the prescribing information applies to all patients, whereas additional surveillance after ICI exposure remains an untested precaution.

Alkaline ceramidase 2 (ACER2) catalyzes the hydrolysis of ceramides into sphingosine and free fatty acids, playing crucial roles in regulating various biological processes, including but not limited to cell proliferation, survival, and the DNA damage response. However, a pharmacological tool that regulates its catalytic and signaling functions remains to be developed. In this study, N-[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]dodecane-1-sulfonamide (KPB82) was identified as a potent and selective ACER2 inhibitor. In vitro enzymatic assays showed that KPB82 inhibited ACER2 with an IC50 of 190 nM, while displaying minimal activity against acid and neutral ceramidases (IC50> 100 {micro}M) and no significant inhibition of alkaline ceramidase 1 or alkaline ceramidase 3. Enzymatic kinetic studies revealed that KPB82 is a competitive inhibitor of ACER2. The three-dimensional structure of ACER2 was modeled in the AlphaFold3 server, revealing key Zn2+-coordinating histidine residues and a substrate-binding pocket. Molecular docking studies demonstrated that KPB82 binds ACER2 within this pocket, forming hydrogen bonds with the catalytically important amino residues, Asp93 and His215, and interacting with several hydrophobic residues. Lactate dehydrogenase release assays revealed that KPB82 significantly reduced cytotoxicity in HeLa cells overexpressing ACER2, as well as in HCT116 cells, RT4 cells, and human umbilical vein endothelial cells treated with the DNA-damaging agent doxorubicin. Lipidomic analysis showed that KPB82 inhibited sphingosine production in response to ACER2 overexpression or doxorubicin treatment. Collectively, these findings demonstrate that KPB82 is a potent and selective ACER2 inhibitor capable of suppressing ACER2-dependent biological processes and highlight its potential as a tool for studying ceramide metabolism and ACER2-mediated signaling pathways.

Gastrointestinal infections are a leading cause of death and morbidity worldwide. Antibiotics as current treatments are ineffective or not recommended due to the emergence of multidrug resistant organisms. The initial step of the pathogenesis for most of the enteric infections is the binding of the pathogen to oligosaccharide receptors on the surface of host cells, making these receptors attractive targets for novel antimicrobial strategies that do not rely on conventional antibiotics. Here, we developed an alternative therapeutic intervention based on a novel microbiome-based chemical engineering approach. We synthesized cholesteryl-peptides as flexible chemical scaffold for the presentation of carbohydrate GM1 mimic receptors in probiotic bacteria acting as a decoy for cholera toxin neutralization. The GM1-cholesteryl-peptides were stably embedded into the membranes of both gram positive and gram negative probiotics, Lactobacillus reuteri DSM and Escherichia coli Nissle 1917 respectively. The resulting GM1-coated probiotics demonstrated to quench nearly 100% of cholera toxin in vitro, functioning as decoys by preventing toxin binding to epithelial cells. The present study strongly suggests that our new and versatile biomimetic non-recombinant system could be employed to neutralize the functional effect of infections mediated by oligosaccharide receptors.

Hypertriglyceridemia is commonly managed with lipid-lowering drugs such as statins; however, adverse effects, dose intolerance, and financial barriers may limit their use. Therefore, affordable and potentially safer plant-derived alternatives warrant investigation. This study evaluated the lipid-lowering effects of ethanol extracts of Fucus vesiculosus and Phytolacca berry, administered individually or in combination, in mice fed a butter-enriched diet for 12 weeks. Forty-eight mice were randomly assigned to six groups: normal control (Group 1), butter control (Group 2), statin control (Group 3), Fucus vesiculosus extract (Ext-1; Group 4), Phytolacca berry extract (Ext-2; Group 5), and the combined extracts (Group 6). Body weight, organ-to-body-weight ratios, serum lipid parameters, and histopathological changes were assessed. Group 3 showed the lowest final body weight, while Group 6 demonstrated a comparable body-weight trajectory. Organ-to-body-weight ratios varied among groups, with Group 3 generally showing the highest values and Group 6 comparatively lower ratios for several organs. Group 5 had the lowest total cholesterol concentration, followed by Group 3, whereas Group 6 showed cholesterol levels comparable to the normal control and statin groups. Notably, Group 6 exhibited the lowest serum triglyceride concentration (129.9 mg/dL). Histopathological examination revealed relatively mild hepatic alterations and limited vascular plaque formation in Group 6. The combined extracts demonstrated favorable effects on body-weight gain and serum lipid profiles, particularly triglycerides, with comparatively mild histopathological changes. Further mechanistic, dose-response, toxicity, and long-term studies are warranted before clinical application can be considered.

BackgroundTherapeutics that modulate peripheral and central immune responses are emerging as promising approaches for neurodegenerative disease. However, the biological context in which these interventions are deployed may critically influence therapeutic outcomes. An attenuated Brucella melitensis 16M strain and a similar strain engineered to express the metabolite indole have previously demonstrated therapeutic efficacy in preclinical models of cancer and autoimmunity through immune system modulation. Here, we tested whether repeated administration of these bacterial strains could modify pathological outcomes in a mouse model of tauopathy. MethodsAttenuated Brucella strains were administered to transgenic mice overexpressing mutant tau protein on a human APOE4 knock-in background via intraperitoneal injection every 5 weeks starting at 6 months of age. Mice were analyzed at 9.5 months of age for tau pathology, neurodegeneration, and changes in innate and adaptive immune responses. ResultsBrucella strains distributed predominantly in the periphery following repeated intraperitoneal treatment in transgenic mice, with minimal detection in the brain and no detection in the dural meninges. Repeated administration of either bacterial strain was well tolerated, with no evidence of adverse safety events. Surprisingly, repeated treatment with the engineered Brucella strain exacerbated neurodegenerative pathology, resulting in reduced hippocampal volume and increased phosphorylated tau pathology compared with control- and non-engineered Brucella strain -treated animals. Immunohistochemical analyses revealed increased numbers of hippocampal CD4+ T cells following treatment with either Brucella strain relative to PBS controls. However, only treatment with the engineered Brucella increased microglial reactivity within the hippocampus. ConclusionThese findings demonstrate that peripheral administration of an engineered bacterial therapeutic can differentially influence neuroimmune responses and disease progression. These results underscore the importance of disease context when developing bacteria-based immunomodulatory therapies for neurodegenerative disorders.

CC10137 is a first-in-class peripherally restricted 2A-adrenergic receptor (2AAR) agonist with broad-spectrum analgesic efficacy and a favorable safety profile. Systematic exploration of the chemical space surrounding first-in-class leads is important for defining series boundaries and guiding continued optimization, but conventional analogue-by-analogue medicinal chemistry samples only a small fraction of the accessible structural space. Here, we used a scaffold-seeded enumeration strategy to expand the chemical space surrounding CC10137 from four SAR-informed seed compounds comprising CC10137 and three closely related structural variants. Application of predefined medicinal chemistry transformation rules in StarDrop generated a virtual library of 16,601,163 unique structures. Morgan fingerprint-based principal component analysis indicated that the library occupied a highly multidimensional structural space involving variation in scaffold substitution, peripheral functional groups, and side-chain composition. A retrospective comparison set of 43 compounds independently designed and experimentally characterized in the earlier CC10137 program represented only approximately 0.00026% of the 16.6-million-member library, yet all 43 were recovered as exact structural matches. Three compounds selected directly from the virtual library retained 2AAR binding affinity and agonist potency below 25 nM. Five representative compounds further showed significant anti-allodynic effects in the in vivo spared nerve injury model, with inhibition rates ranging from 39.3% to 55.7%. These findings support scaffold-seeded computational enumeration as a practical strategy for systematic chemical-space mapping around a first-in-class lead and for identifying additional pharmacologically active structural regions for further optimization.

Objective Based on the bioinformatics method of multi-database integration, this study systematically analyzes the expression characteristics, clinical pathological correlation, prognostic value, potential molecular mechanisms, and immune infiltration patterns of hepatocyte growth factor receptor (MET) in gastric cancer. Methods The UALCAN and GEPIA databases were employed to examine the differential expression of MET between gastric cancer and normal gastric mucosal tissues, as well as its associations with clinicopathological features. Kaplan-Meier Plotter was utilized to evaluate the impact of MET expression on overall survival (OS) and progression-free survival (PFS). Protein-protein interaction (PPI) network was constructed via LinkedOmics, followed by Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of co-expressed genes. Four algorithms, TIMER, CIBERSORT, EPIC, and MCPcounter, were used to cross evaluate the correlation between MET expression and immune cell infiltration; Further validate the cell type specific expression of MET using the gastric cancer single-cell sequencing queues (GSE134520, GSE167297) built into the TISCH database. Results MET expression was significantly elevated in gastric cancer tissues compared with normal gastric mucosa (P < .05), and its expression level was significantly correlated with tumor grade and TNM stage. Patients with high MET expression exhibited significantly poorer OS and PFS than those with low MET expression (P < .05). The PPI network revealed that MET could interact with 20 key proteins, including EGFR, ERBB2, HGF, STAT3, and GRB2 etc. GO enrichment analysis suggests that differentially expressed genes are significantly enriched in functions such as the ERBB signaling pathway, cadherin binding, and DNA repair complexes; KEGG enrichment analysis showed that MET related genes were significantly enriched in pathways such as homologous recombination, nuclear cytoplasmic transport, mismatch repair, and oxidative phosphorylation. Immune infiltration analysis showed that the negative association between MET and B cells infiltration has cross algorithm robustness, while the association with neutrophils, CD8+ T cells, CD4+ T cells, and macrophages exhibits algorithmic heterogeneity or insignificance; There is no significant correlation between MET and common immune checkpoint molecules such as PD-1, PD-L1, CTLA4, etc. Single cell validation further confirmed that MET is mainly enriched in malignant epithelial cells and endothelial cells, and is almost not expressed in immune cells. Conclusions Multidimensional bioinformatic analyses demonstrate that elevated MET expression serves as an independent risk factor for unfavorable prognosis in gastric cancer. MET may mediate dual drug resistance in gastric cancer via crosstalk with multiple signaling molecules (including EGFR, ERBB2, HGF, STAT3 and GRB2) and dysregulation of the homologous recombination repair pathway. Results from multiple-algorithm immune infiltration analysis, single-cell dataset analysis and immune checkpoint correlation analysis indicate that MET exerts only modest direct regulatory effects on the gastric cancer immune microenvironment. This exploratory study offers systematic bioinformatic evidence supporting MET as a candidate prognostic biomarker and potential therapeutic target for gastric cancer. Further functional experiments and prospective cohort studies are required to validate its molecular mechanisms and clinical utility.

Chemobrain, characterized by cognitive dysfunction, has emerged as a significant health concern following cancer treatment. Doxorubicin, an FDA-approved chemotherapeutic agent, is widely used to treat various cancers. However, its adverse effects raise concerns regarding toxicity in non-cancerous cell types. Microglia, the resident immune cells of the brain, play a central role in maintaining neural homeostasis. Despite this, the mechanisms by which microglia become dysfunctional following doxorubicin exposure remain poorly understood. Understanding the transcriptomic alterations induced by doxorubicin in microglia could provide critical insights into mechanisms underlying its neurotoxic effects. In this study, the transcriptomic response of human microglia to doxorubicin using mRNA sequencing combined with bioinformatic analysis was investigated. Exposure to doxorubicin significantly altered microglial morphology, promoting a shift toward a process-bearing phenotype in human HMC3 cells. This phenotypic change was accompanied by elevated levels of DNA damage and was associated with the induction of cellular senescence. At the gene regulation level, the Illumina-based transcriptomic analysis provided 25,834 gene expression profiling in which 5,669 were differentially expressed. The future analyses revealed that 3,431 (upregulated) and 2,238 (downregulated) were differentially expressed under the treatment of doxorubicin. Genes associated with the p53 signaling pathway and inflammatory responses were upregulated, whereas genes involved in translation, DNA replication, and cell-cycle progression were downregulated. This transcriptional dysregulation was accompanied by differential expression of key transcription factors. This study provides transcriptomic evidence elucidating the genotoxic response to doxorubicin in microglial cells, offering mechanistic insights into chemobrain and highlighting key molecular pathways as potential targets for therapeutic intervention.

Background: More than 90% of rare diseases lack an approved therapy, partly reflecting economic barriers in developing treatments for small patient populations. A common bottleneck is costly and resource-intensive hit-to-lead prioritization. Congenital contractures of the limbs and face, hypotonia, and developmental delay (CLIFAHDD) is an ultra-rare neurodevelopmental disease often caused by de novo gain-of-function variants in the sodium leak channel NALCN, with no approved treatments. Methods: We developed a cost and resource-efficient, AI-enabled translational drug discovery framework integrating phenotypic high-throughput screening (HTS), structural biology foundation models for hit-to-lead prioritization, and orthogonal experimental validation. We applied this framework for drug repurposing to identify NALCN inhibitors as therapeutic candidates for CLIFAHDD. We screened ~1,200 approved drugs using a fluorescence-based membrane potential assay in doxycycline-inducible HEK293 cells stably expressing the NALCN channelosome. Boltz-2 modeling predicted binding affinity and mode to prioritize hits. Lead repurposing candidates were selected by a multidisciplinary expert review panel. Lead candidates were evaluated using patch-clamp electrophysiology in HEK293 cells expressing wild-type or CLIFAHDD-associated NALCN variants, primary mouse hippocampal neurons, and a C. elegans NALCN gain-of-function model. Results: HTS identified 11 candidate NALCN inhibitors. Boltz-2 prioritized candidates based on predicted binding affinity and predicted binding within the NALCN pore, consistent with a direct pore-blocking mechanism. Aprepitant emerged as the lead candidate after multidisciplinary evaluation based on its predicted mechanism, pharmacologic properties, blood-brain barrier penetration, established safety profile, and regulatory approval in children and adults. Patch-clamp electrophysiology confirmed partial inhibition of NALCN-mediated sodium leak currents in HEK293 cells (IC50;=2.22 M), with comparable potency against Cav3.1 (IC50=1.63 M). In primary mouse hippocampal neurons, aprepitant reduced sodium leak currents, hyperpolarized resting membrane potential, and decreased spontaneous firing. Aprepitant rescued hyperlocomotor and dystonic phenotypes in a C. elegans gain-of-function NALCN model. There was notable concordance between AI-predicted hits, phenotypic screening hits, and experimental validation. Conclusion: These findings identify aprepitant as a lead therapeutic candidate for CLIFAHDD, and establish an AI-enabled translational drug discovery framework integrating phenotypic screening, structural modeling, and experimental validation for NALCN channelopathies. This scalable and resource-conscious strategy may accelerate hit-to-lead prioritization and drug discovery for rare and ultra-rare diseases lacking effective therapies.

The delivery of small-molecule therapeutics across cellular membranes remains a fundamental challenge in drug development, particularly for compounds whose physicochemical properties limit intracellular bioavailability. Inspired by the low toxicity, biodegradability, and synthetic accessibility of triethanolamine-derived lipids, we investigated a series of C18 aminotriester lipids as excipients for the codelivery of anticancer therapeutics. Four structurally related triester lipids bearing saturated, cis-monounsaturated, trans-monounsaturated, and di-unsaturated C18 tails were synthesized through straightforward esterification reactions with triethanolamine. These lipids were evaluated for their ability to enhance the antiproliferative activity of several anticancer agents, including paclitaxel, doxorubicin, amonafide, homoharringtonine, camptothecin, and SN-38. Across multiple drug classes, the aminotriester lipids exhibited minimal intrinsic cytotoxicity and generally produced limited enhancement of drug potency. However, the fully saturated tris-stearate aminotriester lipid selectively enhanced the antiproliferative activity of amonafide in HCT-116 colorectal cancer cells, particularly at lower concentrations. In contrast, unsaturated lipid analogues displayed negligible enhancement effects, and the activity of the tris-stearate lipid was substantially attenuated in A549 lung adenocarcinoma cells, weakly observed in HT-29 colorectal cancer cells, and absent in CT26 murine colorectal cancer cells. Lactate dehydrogenase release assays indicated that these effects were not associated with membrane lysis or nonspecific cytotoxicity. Further, fluorescence microscopy time course experiments suggest improved membrane trafficking of amonafide when codelivered stoichiometrically with fully saturated C18 aminotriester lipids. Collectively, these findings demonstrate that saturated triethanolamine-based aminotriester lipids may function as selective and biocompatible codelivery excipients for certain small-molecule therapeutics. The observed dependence on both lipid saturation state and cellular context suggests that membrane organization and lipid packing may play important roles in mediating these effects. These results establish a foundation for further mechanistic investigations into aminotriester lipid-assisted small-molecule delivery systems.

Mesaconate (MES, an immunosuppressive metabolite) but not itaconate is upregulated in stool of Crohns Disease (CD) patients, albeit below therapeutic levels.. However, colonic MES level is difficult to maintain because of its high absorption rate, limiting its therapeutic potential for CD. Herein, we developed two strategies that elevate MES level and suppress inflammation: EcN-CAD, an engineered probiotic Escherichia coli Nissle 1917 (EcN) that overexpresses cis-aconitate decarboxylase (CAD, produces itaconic acid that is converted to MES); and MES+Zn, a Zinc-MES ionically crosslinked formulation. Mechanistically, MES modulates T cell function by downregulating glycolytic and mitochondrial activity and decrease ROR{gamma}t expression. In preclinical SAMP1/YitFC dexamethasone-withdrawal model and TNFDARE model, MES+Zn decreased gut permeability significantly compared to controls and reduced myeloperoxidase (MPO, an inflammatory marker) 1000-fold compared to controls. EcN-CAD probiotics decreased ROR{gamma}t expression in CD4+ and CD8+ T cells, gut permeability and inflammation in preventive SAMP1/YitFC model, and SAMP1/YitFC dexamethasone-withdrawal model. Overall, MES rewires T cell responses and technologies that enhance MES levels can be effective in gut-related inflammatory diseases.

Glioblastoma and medulloblastoma are characterized by an immunosuppressive tumor microenvironment, in which tumor-associated macrophages may impair T-cell function, in part through the expression of PD-L1 and CSF1R. Single-target therapies have shown inconsistent results in brain tumors, likely due to the complex interplay involving tumor-associated macrophages and immunosuppression. We developed two immunoliposomes functionalized with antibodies against PD-L1 and CSF1R to co-target these receptors on M2-like tumor-associated macrophages. TAM2Ms were generated by polarizing THP-1 monocytes with glioblastoma (A172, U87MG) and medulloblastoma (DAOY, ONS-76) tumor-conditioned medium together with IL-4 and IL-13. In co-culture with activated Jurkat T cells under tumor-conditioned medium-influenced conditions, combined PD-L1 and CSF1R immunoliposomes enhanced T-cell proliferation, reduced apoptosis, and improved migration toward tumor spheroids compared to single-target liposomal treatments or free antibodies. These benefits varied between glioblastoma and medulloblastoma in vitro models, reflecting distinct TAM2M phenotypes and microenvironment contexts. Dual immunoliposome targeting of PD-L1 and CSF1R may offer a promising strategy for reprogramming tumor-associated macrophages in brain tumor immunotherapy and warrants further evaluation in primary human macrophages and in vivo models to clarify efficacy and mechanisms.