Molecules

Autophagic clearance of hyperphosphorylated tau is impaired in tauopathies, leading to the progressive accumulation of toxic tau species. Fungal metabolites provide a rich yet largely untapped source of neuroactive molecules with therapeutic potential. Here, we investigated metabolites from Lions Mane (Hericium erinaceus), Magic Mushrooms (Psilocybe spp.), and Ergot fungi (Claviceps spp.) using a computational drug-discovery workflow. We characterised their structural diversity, predicted blood-brain barrier permeability and toxicological properties, and integrated network pharmacology with protein-protein interaction and functional enrichment analyses to identify autophagy-related targets. Peroxisome proliferator-activated receptor gamma (PPARG), glycogen synthase kinase-3 beta (GSK3B), and casein kinase 2 alpha 1 (CSNK2A1) emerged as the three most promising candidates, given their complementary roles linking autophagy and tau pathology and their attractiveness as targets for multi-target drug discovery. Their interactions with fungal metabolites were evaluated by molecular docking, Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) rescoring, molecular dynamics simulations, complemented by machine-learning quantitative structure-activity relationship (QSAR) modelling as an additional, ligand-based line of evidence. Molecular dynamics and MM/GBSA analyses confirmed stable, target-specific binding for Corallocin A and Erinacerin M (PPARG), Chaetopyranin and Ergocryptine (GSK3B), and Hericioic Acid D and Isohericerin (CSNK2A1), alongside Emodin, a reference compound with previously reported activity against all three targets. QSAR predictions were informative primarily for the PPARG candidates, which fell within the models applicability domain; predictions for the remaining candidates fell outside their respective models applicability domains and were therefore not interpretable as evidence for or against their prioritisation. Reanalysis of an independent hippocampal proteomic dataset from Alzheimers disease patients showed CSNK2A1 protein levels to be significantly altered in the CA3 subfield, providing an additional, correlative line of support for this target; PPARG and GSK3B showed no significant changes in protein abundance, which does not preclude their functional involvement given their extensive post-translational regulation. Overall, these findings identify fungal metabolites as promising multi-target autophagy modulator candidates and provide a systematic computational strategy for prioritising them for experimental validation in tauopathies.

Kinema is a traditional fermented soybean food indigenous to the eastern Himalayan regions of Nepal and India. The fermentation process is primarily mediated by the bacterium Bacillus subtilis, which produces several bioactive compounds and enzymes with potential therapeutic applications. Considering the growing burden of cardiovascular diseases and the need for effective fibrinolytic agents for thrombolytic therapy, this study aimed to extract, partially purify, and evaluate the thrombolytic potential of kinemakinase derived from kinema prepared from white soybeans. Partial purification of the enzyme was achieved using ammonium sulfate precipitation. Thrombolytic activity was assessed in vitro using human blood clots, where three enzyme dilutions demonstrated clot lysis ranging from 66% to 68%, indicating considerable fibrinolytic potential. In silico analyses were also performed to investigate the structural and functional characteristics of the enzyme. The tertiary structure obtained from UniProt was modeled using the Robetta server and refined with GalaxyRefine. Docking with fibrin using ClusPro 2.0 and molecular dynamics simulations using iMODS confirmed favorable interaction and structural stability, while disulfide engineering enhanced protein stability. The findings suggest that kinema-derived kinemakinase may serve as a promising alternative thrombolytic agent, warranting further biochemical characterization and dosage optimization.

Hepatitis C virus (HCV) infection remains a major global health challenge despite the success of direct-acting antivirals (DAAs), which are limited by high cost, restricted accessibility, and the emergence of resistant strains. Natural products, particularly phytochemicals, represent a promising reservoir of antiviral agents with diverse mechanisms of action and favorable safety profiles. In this study, we combined wet-lab experimentation with computational approaches to identify plant-derived molecules capable of inhibiting HCV entry. Guided by ethnobotanical evidence, methanolic leaf extracts of Psidium guajava L., Plumeria alba L., Syzygium cumini L., and Tamarindus indica L. were prepared and evaluated for cytotoxicity in Huh7 hepatoma cells. Entry inhibition was assessed using EGFP-labelled HCV pseudoparticles (HCVpp) by qRT-PCR and confocal microscopy. Among the tested plants, Tamarindus indica extract significantly reduced KGFP expression (p < 0.05), confirmed by {Delta}{Delta}Cq analysis and impaired membrane fusion events, while Psidium guajava and Plumeria alba impaired intracellular trafficking without blocking initial attachment. Syzygium cumini showed no inhibitory effect under the tested conditions. Complementary in silico analyses included homology modelling, molecular docking, ADME/toxicity profiling, and molecular dynamics simulations of HCV E2-ligand complexes. Literature mining identified 39 candidate compounds, among which lupeol exhibited stable binding interactions with HCV E2 and favorable pharmacokinetic properties. Critically, in vitro binding assays confirmed that lupeol disrupted the E2-CD81 interaction, reducing bound E2-EGFP to 6% compared to controls. This was supported by HCV-pseudoparticle entry assays confirming inhibition of entry. Together, these findings establish Tamarindus indica and lupeol as potent HCV entry inhibitors.

Background: Subtype-selectivity predictions are scored against measured selectivity and judged against an assumed noise ceiling. We asked what an 2-adrenergic benchmark rewards and which controls change its interpretation. Research design and methods: On a frozen benchmark of 586 paired 2A/2C compounds we evaluated Glide SP docking, CNN rescoring, ligand-only fingerprint models, receptor descriptors and pose contacts, with dopamine D3/D2 as comparator, applying five controls: a measured ceiling, a cluster-identity null, a nonselective reference, a same-receptor floor and a trivial-descriptor baseline. Results: Five descriptors from SMILES reached Spearman 0.645, 72% of the measured ceiling, against 0.071 for Glide SP and 0.188 for CNN rescoring; receptor properties and pose contacts reduced to size under control, while a non-size signal of 0.258 survived. Measured rather than propagated noise raised that ceiling from 0.704 to 0.897; cluster identity alone reached R2 0.499 on D3/D2 and none on 2; a nonselective reference received +1.43 to +4.79 kcal/mol where zero is expected; and a same-receptor floor reached 1.77-fold against 1.88-fold across subtypes. Conclusions: Such benchmarks reward molecular size first; a method must exceed 0.645 before its score indicates structural reasoning. The controls are inexpensive; conclusions rest on two receptor pairs, a three-pair floor and static structures.

Although therapeutic advancements have dramatically improved outcomes for cancer patients in recent decades, advanced cancer remains a significant challenge. There is still an urgent need for new and more effective strategies that minimize toxicity and improve survival outcomes in cancer patients. Natural products from microorganisms are a rich and promising source of bioactive compounds with substantial therapeutic potential in cancer treatment. Recently, B. proteolyticus strain OSUB18, isolated from switchgrass, has been demonstrated to promote plant growth and regulate reactive oxygen species (ROS) production. Here, we investigated the anticancer potential of this plant-associated microbe and demonstrated that its crude extracts exhibited broad-spectrum cytostatic activity across diverse solid tumor cell lines. Importantly, OSUB18 extracts displayed limited cytotoxicity toward normal human mammary epithelial cells and mouse fibroblasts. This potentially selective cytostatic activity was retained within a water soluble, heat-labile, and protease-resistant fraction. The extracts primarily suppressed cell proliferation, accompanied by increased lipid peroxidation, with preliminary evidence suggesting the potential involvement of ferroptosis. These findings suggest that the bioactive component may be a selective cytostatic agent with multifaceted mechanisms of action.

Computational safety evaluation of phytochemical libraries commonly relies on models trained on experimentally tested compounds and applied to much larger and chemically diverse collections. An important but often overlooked question is whether predicted compounds are adequately represented within the experimental chemical space underlying these models. Here, we examined this question for the Indian Medicinal Plants, Phytochemistry and Therapeutics (IMPPAT) database using experimentally characterized datasets for three anti-target endpoints: hERG blockade, cytochrome P450 (CYP) inhibition, and pregnane X receptor (PXR) induction. After structure standardization, 14,956 unique IMPPAT compounds were compared with 8,877 hERG, 4,905 CYP, and 4,138 PXR reference compounds using consensus nearest-neighbor similarity based on Morgan and atom-pair fingerprints. At a Tanimoto similarity threshold of 0.80, only 0.92%, 0.30%, and 0.46% of IMPPAT compounds had close measured analogs in the hERG, CYP, and PXR datasets, respectively. Relative to the internal nearest-neighbor distributions of the reference datasets, a typical IMPPAT compound lay near the 1st percentile, indicating substantial chemical-space separation. Compounds with close experimental analogs also showed distinct molecular characteristics, with descriptor-based logistic regression yielding cross-validated AUC values of 0.77-0.80. Plant-level enrichment was markedly reduced after excluding widely distributed metabolites and was further restricted when the nearest measured analog was required to have pIC50 or pEC50 [&ge;] 5. The remaining signals were concentrated in a limited number of taxa, including Rauvolfia, Sinopodophyllum/Dysosma, Cinchona, and Catharanthus roseus. Overall, most IMPPAT phytochemicals lack close structural representation among currently measured anti-target compounds. Computational safety predictions for these molecules should therefore be interpreted as extrapolations and accompanied by explicit measures of structural support. Absence of a close experimental analog should be regarded as a gap in available evidence rather than evidence of safety.

Here we report genotoxicity and 90-day oral toxicity evaluations of a high-purity, fermentation-derived mogroside preparation rich in mogroside V, the principal sweetener of monk fruit (Siraitia grosvenorii). The test article ([&ge;]95% total mogrosides, 70.3% mogroside V) is produced by a modified Escherichia coli from glucose, offering a higher-purity alternative to traditional monk fruit extracts. The test article was non-mutagenic in a bacterial reverse mutation test (OECD TG 471) and non-clastogenic in an in vitro human lymphocyte micronucleus test (OECD TG 487), up to the maximum recommended concentration. In the 90-day study (OECD TG 408), the test article was given by daily oral gavage at 0, 500, 1000, and 2000 mg/kg body weight/day to Sprague-Dawley rats. There were no deaths and no test article-related effects on clinical signs, ophthalmology, functional observational battery, body weight, food consumption, clinical pathology, thyroid hormones, oestrous cyclicity, or sperm parameters. Minor liver-weight increases lacking any clinical chemistry or histopathological correlates were determined to be non-adverse. No effects were seen on testis weight, sperm endpoints, or spermatogenesis. All other statistically significant differences were minor and considered incidental. The no-observed-adverse-effect level (NOAEL) was 2000 mg/kg body weight/day, the highest dose tested, supporting its safety as a food ingredient.

Ethnopharmacological relevance: Australian Indigenous medicinal plants represent a valuable yet underexplored source of bioactive compounds with potential therapeutic relevance. The Iningai community of Central Queensland has traditionally used native plants to manage conditions associated with inflammation, pain, infection, and general illness. Scientific evaluation of these plants may provide evidence for their customary applications and identify bioactivities relevant to anticancer biodiscovery. Aim of the study: This study evaluated leaf and stem extracts of seven medicinal plants-Pittosporum angustifolium, Alphitonia excelsa, Calytrix microcoma, Geijera parviflora, Melaleuca uncinata, Gossypium australe, and Eucalyptus similis-traditionally used by the Iningai community, focusing on three biological processes relevant to cancer: oxidative stress, inflammation, and cellular proliferation. Materials and methods: Antioxidant activity was assessed using DPPH radical-scavenging and ferric reducing antioxidant power (FRAP) assays. Anti-inflammatory activity was evaluated in lipopolysaccharide (LPS)-stimulated THP-1 macrophage-like cells by profiling IFN-, TNF-, IL-6, IL-12, IL-18, and IL-23. Antiproliferative activity was assessed using MTT-based viability assays in human and murine liver cancer cell lines (Huh7, Hep3B, Hep-55.1c, and A52). Results: The extracts exhibited distinct biological activity profiles. G. parviflora stem and C. microcoma leaf extracts showed the strongest antioxidant activities, whereas P. angustifolium stem exhibited the weakest radical-scavenging capacity. Cytokine responses were extract-specific, with E. similis leaf extract demonstrating broad and pronounced suppression of multiple LPS-induced pro-inflammatory cytokines. Several extracts produced concentration-dependent reductions in liver cancer cell viability, with P. angustifolium stem exhibiting the most consistent and potent antiproliferative activity across the cell lines tested. Notably, strong antioxidant or anti-inflammatory activity did not necessarily correspond with antiproliferative activity. Conclusion: Australian Indigenous medicinal plant extracts demonstrated distinct antioxidant, immunomodulatory, and antiproliferative activities rather than uniform bioactivity across experimental systems. The divergent activities of G. parviflora, C. microcoma, E. similis, and P. angustifolium highlight the importance of integrated biological screening and support the value of Indigenous knowledge-guided biodiscovery. These plants represent promising sources for further investigation of selective bioactive compounds with potential relevance to anticancer drug discovery.

Vanadium (V) is a potential insulinomimetic that can modulate carbohydrate metabolism, but its biological effects are sensitive to chemical form, concentration, and sex. Chelation of vanadium with organic ligands, in particular citrate, allows to increase its bioavailability and optimize pharmacokinetic properties. The aim of the study was to evaluate tissue-, dose-, and sex-dependent changes in physiological parameters and activity of the key glycolytic enzyme - lactate dehydrogenase (LDH) - under the influence of vanadium citrate. The study was conducted on 6-week-old Wistar rats of both sexes. The animals received vanadium citrate orally for 36-38 days at doses of 3, 12.5, and 50 g VCit/kg body weight. LDH activity in skeletal muscle, liver, kidney, and pancreas was investigated. No pronounced toxic effect on physiological parameters was detected: body weight dynamics corresponded to age norms, no behavioral changes were observed. LDH activity demonstrated pronounced sexual dimorphism and depended on the dose received. It was established that the optimal dose, which provides a modulating effect without signs of metabolic stress, for females is 12.5 g VCit/kg, while for males - 3 g VCit/kg. The most significant changes in LDH activity were recorded in the pancreas at a dose of 50 g V/kg, where the indicators decreased from 0.81 to 0.31 mol/(min x mg protein) in females and from 1.02 to 0.28 mol/(min x mg protein) in males. The effect of vanadium citrate on carbohydrate metabolism, as well as its dose-, tissue- and sex-specific nature, is likely determined by a dual action: the insulin-like effect of vanadium (redirecting pyruvate to oxidation) and the allosteric inhibition of glycolysis by the citrate ligand (substrate limitation for LDH). The obtained results emphasize the importance of considering sex and dose in the research and development of metabolically active compounds.

Several potentially potent anticancer drugs have been identified by in vitro evaluation, such as Andrographolide. These compounds show strong anticancer activity in vitro, but struggle to reach effective concentrations in the bloodstream when taken orally because they dissolve poorly in water or break down rapidly in the body. Bioenhancers, which are compounds that have potential to improve drug stability in the body, offer an alternative solution to overcome this limitation. Naringin and Quercetin have been identified as candidate bioenhancers, and have been hypothesized to potentially slow rapid first pass metabolism of poorly bioavailable drugs. Our work focuses on testing Naringin and Quercetin because they are flavonoids with therapeutic potential, due to their anti-inflammatory and antioxidant properties. Data from the hepatic microsomal assays performed on Naringin and Quercetin suggest moderate to proficient periods of stability in the body, with Naringin having 91.86% remaining, while Quercetin had 74.84% remaining. When administered alongside Andrographolide, a drug known to rapidly degrade in the body, Naringin raised its metabolic stability from 38.93% to 80.77% and on the other hand, Quercetin raised Andrographolide metabolic stability from 38.93% to 86.70%. In addition, plasma protein binding assays show the percentage of compounds available at the target site where Naringin was observed to be 49.32% bound and Quercetin found to be 50.14% bound, implying 50.68% of Naringin, and 49.86% of Quercetin available at the target site, respectively. This preliminary study explores whether Quercetin and Naringin could act as bioenhancers by remaining stable and available in plasma and by slowing the metabolism of poorly bioavailable drugs such as Andrographolide.

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

Kaposi's sarcoma (KS) is an angioproliferative tumor associated with infection by human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV). Among the viral genes involved in KS pathogenesis, the viral G protein-coupled receptor (vGPCR) plays a key role in oncogenic transformation and angiogenesis, and its sustained activity contributes to tumor development and maintenance. Quercetin (QUE), a naturally occurring flavonoid with well-recognized anticancer properties, has been shown to reduce cell viability and proliferation and to induce apoptosis in vGPCR-transformed endothelial cells. This study further investigated the mechanisms underlying the antineoplastic effects of QUE in vGPCR cells, an in vitro model of Kaposi's sarcoma. Initially, QUE promoted the activation of MAPK signaling pathways in vGPCR cells, increasing the phosphorylation levels of ERK1/2, JNK1/2, and p38, without affecting AKT phosphorylation. In parallel, QUE increased intracellular oxidant levels, followed by lipid peroxidation and DNA damage, supporting a link between oxidative imbalance and genotoxicity. However, increased total glutathione content, and unaltered Gpx4 mRNA expression, suggest the induction of oxidative stress and compensatory antioxidant responses. Furthermore, increased intracellular oxidant levels were associated with MAPK signaling activation, particularly JNK1/2, whereas ERK1/2 and p38 activation may reflect a compensatory response that is insufficient to overcome QUE-induced cytotoxicity. Taken together, these findings indicate that QUE exerts antiproliferative effects in vGPCR cells through a mechanism involving oxidative stress, MAPK activation, and DNA damage, supporting its potential relevance in the study of therapeutic strategies for virus-associated malignancies.

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.

Objective: One of the strongest genetic associations with coronary artery disease (CAD) risk maps to the metalloproteinase 'a disintegrin and metalloproteinase with thrombospondin motifs 7' (ADAMTS-7) locus. The protein was shown to promote plaque formation and instability. We aimed to generate and evaluate an antibody-based strategy targeting ADAMTS-7 therapeutically to reduce atherosclerotic plaque formation. Approach and Results: A truncated form of human ADAMTS-7 was produced in Nicotiana benthamiana and used as antigen for antibody generation by hybridoma technology. Eight monoclonal antibodies (mAbs) were screened, among which ADAMTS-7-mAb32 (mAb32) demonstrated the highest affinity, as confirmed by surface plasmon resonance analyses and immunoblotting against full-length ADAMTS-7. In vitro, mAb32 inhibited interactions of ADAMTS-7 with its substrates TIMP-1 and SVEP1 in a dose- and time-dependent manner, as assessed by time-resolved Forster resonance energy transfer assays. To assess therapeutic efficacy in vivo, Apoe-/- mice were fed a Western diet for ten weeks and treated with weekly injections of mAb32 or control IgG over the last six weeks. En face aortic Oil Red O staining revealed significantly reduced plaque area in the treatment group, without changes in plasma cholesterol levels or body weight. No evidence of liver or kidney toxicity was observed. Conclusion: Monoclonal antibody-based inhibition of ADAMTS-7 reduced atherosclerotic burden in vivo without affecting lipid metabolism, supporting ADAMTS-7 as a viable therapeutic target in CAD. Further development of mAb32 may provide a cholesterol-independent treatment strategy for atherosclerosis.

Imipramine is known as an older generation tricyclic antidepressant drug. It has been identified in prior studies that imipramine blocks Apolipoprotein E4 (ApoE4)-induced amyloid-{beta}(A{beta}) aggregation and is associated with an improved AD diagnosis [Johnson et al. Alzheimers Research Therapy, 2022, 14, 88]. Using NMR methods such as 1H-1H NOESY and Saturation Transfer Difference Spectroscopy, we demonstrate the binding of A{beta} monomers to imipramine when the full-length A{beta} (1-42) sequence is considered. The more abundant but less toxic form, A{beta} (1-40) does not show interaction with imipramine.

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

Deep learning structure predictors, most prominently AlphaFold2 (the field-standard tool benchmarked against throughout this study), have substantially expanded access to protein structural information, yet characteristically return a single static conformation per target. This is an incomplete representation of the binding-competent state for the many pharmacologically relevant targets whose recognition geometry is intrinsically dependent on receptor flexibility, including cryptic-pocket, induced-fit, and water-mediated binding mechanisms. We present a category-stratified, statistically powered benchmark comparing pose prediction from receptor conformational ensembles against AlphaFold2, used as a matched static-structure baseline, across 29 protein-ligand systems spanning cryptic-pocket, induced-fit, water-mediated, and autoimmune-indication target classes. Considering the most accurate pose available from each methods full candidate output, ensemble-derived poses achieved lower RMSD to the experimental structure than AlphaFold on 21 of 29 targets (72.4%), with a mean RMSD of 3.39 [A] versus 5.60 [A]: a clear, statistically decisive advantage (paired Wilcoxon signed-rank test, W = 93.0, p = 0.0060). Rather than being diffuse, this advantage was concentrated precisely where mechanistic theory predicts it should be: in induced-fit and water-mediated categories, the classes in which static-structure prediction is expected to be least representative of the bound state: a result that constitutes direct, quantitative confirmation of the ensemble hypothesis, not merely a favorable average. Independent assessment against a field-standard physical-validity framework confirmed that this accuracy gain was achieved without any trade-off in chemical or geometric realism. We further quantify, rather than assume, the extent to which this advantage is recoverable by fully autonomous pose selection, using a proprietary ensemble-aware scoring model with no access to the correct answer, and report a substantial, discriminative signal (cross-validated mean AUC 0.92) with a partial, and clearly characterized, recovery under the strictest accuracy criteria (mean AUPR 0.36), which we identify as the principal, now precisely quantified, determinant of near-term translational progress. Under this same fully autonomous, ground-truth-blind setting, AlphaFolds own top-ranked poses currently match or modestly exceed Mavchen-1s autonomously selected poses on strict success-rate criteria (e.g., 17.2% vs. 20.7% at the combined RMSD-and-validity threshold), a result we report without qualification as the clearest current benchmark for near-term development. Together, these results provide compelling, statistically rigorous evidence that conformational ensemble sampling is a mechanistically grounded and substantial source of improved pose accuracy relative to static-structure prediction, and establish a quantitative benchmark against which continued methodological development can be measured and demonstrably improved upon.

Carbohydrates are essential molecules involved in a wide range of biological processes. While their structural representation in the Protein Data Bank (PDB) has been hindered due to their highly diverse and flexible nature, advancements on X-ray crystallography and CryoEM are accelerating the obtaining of their experimental structures, which in turn supports improved analyses of their structural properties, such as the role of their non-chair conformations on biological processes. In this context, the current work provides a comprehensive and up to date overview of carbohydrate structures within the PDB through classification, quantification, and characterization of monosaccharides and glycan chains, incorporating Cremer-Pople puckering coordinates to evaluate pyranose conformations. Our findings show that, even with a steep increase in CryoEM structures including more complex oligosaccharides, carbohydrate data remains concentrate dinsmallglycanchains, with a about 4% of non-chair conformation sintopresolution structures. Free energy landscapes for the pyranose ring in the presence and absence of nine different binding proteins, including both enzymes and non-enzymes, reinforce the preferred stability of chairs and the rarity of conformational induction upon binding.

The muscle-relaxing effects of botulinum neurotoxin (BoNT) serotypes A and B are widely utilized in clinical and aesthetic medicine. While the mouse bioassay remains the gold standard for evaluating the potency of pharmaceutical BoNT preparations, a non-animal alternative would be preferable to comply with European Directive 2010/63/EU. The Binding and Cleavage (BINACLE) assay, which measures BoNT activity based on the receptor-binding and proteolytic properties of the toxin, represents a promising alternative. Previous studies showed that this method allows reliable and sensitive activity determination. Here, we describe a collaborative study involving ten international laboratories, which aimed to characterize the applicability of the BINACLE assay for the potency testing of pharmaceutical BoNT products. Our results demonstrate that the BINACLE assay enables reproducible relative potency determinations for BoNT/A, the most pharmaceutically relevant serotype, with variability within acceptable limits. Reproducible relative potency calculations were also demonstrated for BoNT/B, while the data highlighted opportunities for refinement of the BoNT/B BINACLE protocol to further enhance the robustness of the method. Overall, this work confirms the utility of the BINACLE assay as a reliable and sensitive method for determining BoNT product potency, thereby substantiating its applicability as an alternative to the mouse bioassay.

Marine macroalgae host endophytic microbial communities that serve as valuable reservoirs for drug discovery. In this study, the endophytic actinomycete Streptomyces sp. UL09 was isolated from the internal thallus tissues of the green macroalga Ulva rigida C. Agardh. Fermentation of strain UL09 yielded the nitrobenzoyl sesquiterpenoids insulicolide B (1.63 mg/L) and insulicolide C (1.79 mg/L). The planar structures of compounds 1 and 2 were assigned as insulicolides B and C, respectively, via spectroscopic analysis and comparison with literature data. In vitro bioassays revealed moderate activity against Gram-positive bacteria (MIC 128-512 g/mL), including MRSA Sp3 (MIC 128-256 g/mL). These compounds exhibited cytotoxic activity against human cancer cell lines (HeLa, HepG2, and MDA-MB-231) with IC50 values ranging from 34.35 to 142.64 g/mL. Both compounds demonstrated lower cytotoxicity toward non-malignant Vero cells. Molecular docking simulations predicted that both metabolites target the catalytic ATP-binding cleft of human Polo-like kinase 1 (Plk1), with insulicolide B displaying a favorable docking score ({Delta}G = -9.273 kcal/mol). Furthermore, pkCSM predicted high intestinal absorption and no alerts for the assessed toxicity endpoints. These findings establish Streptomyces sp. UL09 as a viable alternative biological source of insulicolides B and C, highlighting their potential as leads for oncology applications.