Frontiers in Chemistry

Human RNase 2 (eosinophil-derived neurotoxin, EDN) is a major eosinophil granule protein of the vertebrate-specific RNase A superfamily and is involved in antiviral response and inflammation. Identifying ligand-binding pockets in EDN is thus relevant to structure-based drug design. In our laboratory we identified by protein crystallography a conserved site at the protein surface binding to carboxylic anion molecules (malonate, tartrate and citrate). Searching for potential biomolecules rich in anion groups and considering previous report of EDN binding to glycosaminoglycans, we explored the protein binding to saccharides. Next, EDN crystals were soaked with mono- and disaccharides, and the 3D structures of ten complexes were solved by X-ray crystallography at atomic resolution. We identified protein binding pockets to glucose, fucose, mannose, sucrose, galactose, trehalose, N-acetyl-D-glucosamine, N-acetylmuramic acid, and the sialic acid N-acetylneuraminic acid. A main site for glucose, fucose, and galactose was located adjacent to the spotted carboxylic anion site. Secondarily, N-acetylneuraminic acid, N-acetylmuramic acid, sucrose, galactose, and mannose shared another protein surface region. Overall, the saccharides clustered into seven defined sites, outlining a conserved recognition pattern, which was further analysed by molecular modelling. Interestingly, within the RNase A family, we find amphibian RNases that were initially isolated as carbohydrate binding proteins and named as leczymes, combining enzymatic and lectin properties. The present data is the first systematic structural characterization of a mammalian sugar-binding RNase within the family. The results highlight unique EDN residues that mediate its sugar specific interactions, of particular interest for a better understanding of the protein physiological role. HighlightsO_LIstructure of RNase 2 in complex with mono and disaccharides at atomic resolution C_LIO_LIidentification of RNase 2 unique sugar binding sites C_LIO_LIcharacterization of a mammalian RNase A family enzyme with lectin properties C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/713198v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1d805f7org.highwire.dtl.DTLVardef@16fcc49org.highwire.dtl.DTLVardef@ccfd92org.highwire.dtl.DTLVardef@1b8f1e_HPS_FORMAT_FIGEXP M_FIG C_FIG

A cassane diterpene, 6{beta}-cinnamoyl-7-hydroxyvouacapen-5-ol (6{beta}CHV), isolated from Caesalpinia pulcherrima, has emerged as a promising anticancer drug lead with reported Wnt/{beta}-catenin pathway inhibitory activity and in vivo safety. The present study reports the in vivo pharmacokinetics and tissue distribution of 6{beta}CHV in Wistar rats following a single oral dose of 200 mg/kg. A reproducible RP-HPLC-UV method was developed and validated for quantifying 6{beta}CHV in rat plasma and tissues. Chromatographic separation was achieved using a gradient elution of methanol and water. The method was subsequently applied to investigate the pharmacokinetics and tissue distribution of 6{beta}CHV. Plasma pharmacokinetic analysis revealed delayed and moderate absorption, with a Tmax of 4 h and a Cmax of 1314.12 ng/mL. Following absorption, 6{beta}CHV is distributed widely across peripheral tissues, including the liver, heart, lungs, spleen, and kidneys, as well as pharmacological sanctuary sites such as the brain and testes. The highest concentrations were observed in the stomach, small intestine, and liver, with detectable levels persisting up to 24 h, reflecting extensive tissue partitioning and retention. Overall, these findings demonstrate that oral administration of 6{beta}CHV is feasible. However, the delayed absorption suggests that further optimization of formulation or alternative administration routes may enhance systemic exposure. This study provides the first comprehensive pharmacokinetic and tissue distribution profile of 6{beta}CHV, supporting its continued preclinical development as a potential anticancer therapeutic. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/715187v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@4ae86forg.highwire.dtl.DTLVardef@1e1e51aorg.highwire.dtl.DTLVardef@1881c43org.highwire.dtl.DTLVardef@f7789f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Inositols are a family of cyclic sugar alcohols comprising nine stereoisomers. Myo-inositol is the most abundant isomer found in humans and has been studied most extensively. It plays an important role in osmoregulation and is incorporated into membrane-anchored phosphatidylinositols. Scyllo-inositol is the second most abundant inositol isomer in the human brain and aberrant concentrations are associated with various diseases; however, its biological functions remain poorly understood. Here, the development and application of [13C6]scyllo-inositol as an isotopic tracer to study its metabolism is reported. A concise and robust synthetic route was established to obtain [13C6]scyllo-inositol from [13C6]myo-inositol in good yield. The uptake of [13C6]scyllo-inositol and responses of endogenous inositol isomers were measured in multiple cell lines by HILIC-MS/MS, showcasing the advantages of isotopic tracing. [13C6]scyllo-inositol proved to be a versatile isotopic tracer, when coupled with MS-based lipidomics and 2D NMR experiments. These experiments provide evidence that scyllo-inositol is incorporated into phosphatidylinositols in different cell lines. The results suggest a previously underappreciated role of scyllo-inositol in mammalian cells. The utilization of [13C6]scyllo-inositol will help to elucidate the role of scyllo-inositol metabolism in healthy and diseased states. SignificanceScyllo-inositol is a cyclic sugar alcohol found predominantly in the human brain. Changes in its concentration are associated with different diseases, and scyllo-inositol has been investigated as a potential drug against Alzheimers disease in clinical trials. However, its metabolic fate in mammalian cells is not well understood. We report here a synthetic strategy to obtain [13C6]scyllo-inositol and demonstrate, through isotopic tracing, its incorporation into phosphatidylinositols in different human-derived cell lines. This new stable isotopic tracer enables the investigation of the biological role of scyllo-inositol in mammals and beyond. HighlightsO_LIConcise synthesis of [13C6]scyllo-inositol C_LIO_LI[13C6]scyllo-inositol uptake and response of endogenous inositol isomers studied in multiple cell lines C_LIO_LIUse of [13C6]scyllo-inositol as an isotopic tracer in metabolomics and lipidomics experiments C_LIO_LIEvidence for scyllo-inositol incorporation into phosphatidylinositol in mammalian cells C_LI

Age-accompanied chronic, low-grade systemic inflammation (inflammaging) drives the onset and progression of neurodegenerative disorders like Parkinsons disease (PD). Currently, no disease-modifying therapies are available for PD. Exposure to environmental toxicants, including paraquat (PQ), rotenone, and neurotoxic metals, increases disease risk. Conversely, sustained consumption of dietary soft electrophiles, such as flavonoids, carotenoids, vitamin E vitamers, and essential fatty acids, has been associated with increased lifespan and delayed age-related neurological decline. Omega-3 and select omega-6 fatty acids also serve as precursors of lipid-derived specialized pro-resolving mediators (SPMs), which exert potent anti-inflammatory and inflammation-resolving activities. Here, we report the development of a robust analytical method to quantify pro-resolving oxylipins in a PQ-induced Drosophila melanogaster model of PD, enabling investigation of how dietary phytochemicals modulate anti-inflammatory and pro-resolving lipid metabolism in vivo. We hypothesized that plant-derived soft electrophiles promote active resolution of neuroinflammation by enhancing the production of pro-resolving oxylipins derived from essential fatty acids, and that their neuroprotective effects are linked to their soft electrophilic properties. Our results demonstrate that specific lipophilic plant-derived soft electrophiles significantly upregulate pro-resolving oxylipins in Drosophila heads following PQ exposure. We identify a subset of flavones and structurally related phytochemicals that selectively enhance SPM biosynthesis and show that this response involves the NF-{kappa}B orthologue relish. Additionally, feeding modality and sex-specific dimorphisms were found to influence oxylipin production. Collectively, these findings indicate that structurally related dietary soft electrophiles enhance endogenous pro-resolving lipid pathways, promote resolution of toxin-induced neuroinflammation, and have potential preventive and therapeutic relevance for neuroinflammation-associated neurodegenerative diseases. HighlightsO_LIQuantification of pro-resolving lipids in a Drosophila Parkinsons model. C_LIO_LISpecific structural features of phytochemicals contribute to in vivo bioactivity. C_LIO_LILipophilic soft electrophiles show therapeutic potential against neuroinflammation. C_LIO_LIFeeding modality and sexual dimorphism also regulate oxylipin production. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/714080v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@2088cforg.highwire.dtl.DTLVardef@1f5d026org.highwire.dtl.DTLVardef@134aa44org.highwire.dtl.DTLVardef@965e28_HPS_FORMAT_FIGEXP M_FIG C_FIG

Prasugrel is a prodrug, widely used in antiplatelet strategy for secondary prevention after acute coronary syndrome. The metabolism of prasugrel leads to the formation of the Prasugrel Active Metabolite (PAM), an irreversible P2Y12 receptor antagonist. Its mode of binding has not yet been fully established, although it is known that it binds covalently to P2Y12 by forming a disulfide bridge with cysteines and its sulfur moiety. PAM is a molecule with two chiral centers, resulting in four stereoisomers which appear to be stereoselective upon binding. A combination of different molecular modeling methods, such as molecular dynamics, ensemble docking, and Density Functional Theory (DFT), were used to rationalize these differences in antagonism observed in vitro and to elucidate the mode of binding of PAM to P2Y12. PAM is found to bind to the closed P2Y12 conformation in a preferential way. Although the four stereoisomers have comparable affinity, the location of the RS stereoisomer makes the formation of a disulfide bond with cysteines more favorable, particularly with cysteine 175. Compared to the RR stereoisomer, the RS stereoisomer interacts less deeply with the P2Y12 receptor, interacting in particular with the second and third extracellular loops, explaining the competition observed with cangrelor and an intermediate metabolite of prasugrel. Furthermore, DFT calculations have shown that the formation of a disulfide bridge is energetically more favorable with the RS stereoisomer than with the RR stereoisomer. The physical interactions and chemical reaction between the RS stereoisomer and the P2Y12 receptor are key factors in explaining the stereoselective binding of PAM to P2Y12.

The genetically authenticated Finnish hop genotype LUKE 2541 obtained from wild was evaluated for antibacterial, anti-inflammatory, and anticancer activities. Water extracts from hop cones inhibited the Gram-positive bacteria Staphylococcus aureus and Bacillus cereus, with MIC values of 0.094- 0.188mg/mL, whereas Gram-negative strains showed limited sensitivity. In LPS-primed THP-1 cells, both IPA and IPA-Control extracts reduced reactive oxygen species formation in a dose-dependent manner, exhibiting similar IC50 values (50.41{micro}g/mL and 35.41{micro}g/mL). This hop genotype also displayed clear tissue- and solvent-dependent antiproliferative effects in human cancer cell lines. Bioactivity was strongly enriched in hop cones and predominantly associated with non-polar extracts, particularly hexane and dichloromethane fractions, which produced marked, dose-dependent reductions in cell viability. In contrast, aqueous and methanolic extracts were largely inactive, underscoring the critical importance of extraction chemistry and tissue selection. Sensitivity varied among cancer cell lines, with colorectal cells generally more responsive and leukemia cells less affected, highlighting cell-specific susceptibility. Further research is needed to elucidate underlying mechanisms, determine selectivity toward non-malignant cells, and identify the active compounds responsible for all in all investigated effects.

High-resolution binding site mapping is important for in-depth activity assessment of new therapeutics including AI-designed antibodies. However, complex protein targets such as glycosylated antigens are challenging for many methods including crystallography. PD1 is a highly glycosylated antigen, and with the traditional HDX-MS method, only 51% sequence coverage could be obtained with multiple epitope residues undetected for Pembrolizumab. By implementing glyco-peptide detection, subzero temperature LC-MS and electron based MSMS fragmentation, the new HDX FineMapping methodology enabled 100% sequence coverage and complete epitope characterization for the Pembrolizumab-PD1 system, with amino acid level resolution. Furthermore, HDX FineMapping detects binding epitopes directly in solution, without any mutation or modification to either the antigen or the antibody. The amino acid level resolution combined with low cost, minimal sample consumption, fast turnaround time, and no need of mutant library or crystallization makes it a competitive methodology for binding mode validation of AI-designed therapeutics.

Neuroactive steroids modulate GABAA and NMDA receptors allosterically, typically requiring specific structural features for their activity. In this study, we characterize YX84, a novel neuroactive steroid bearing a 3{beta} sulfate and p-trifluoroacetylbenzyl alcohol attached in an ether linkage to a hydroxyl group at steroid carbon 17. This compound and similar analogues exhibit an atypical pharmacological profile, with three distinct actions at GABAA receptors. First, YX84 is a full agonist, with EC50 near 1 {micro}M and comparable efficacy to GABA at GABAA receptors in native hippocampal neurons. It presents as a full agonist relative to GABA at 4/{delta} subunit-containing receptors. Second, YX84 acts as a slow-onset, potent positive allosteric modulator (PAM) of GABAA receptors at concentrations below those that gate a response. Finally, YX84 exhibits rapid desensitizing and/or blocking kinetics; voltage dependence is consistent with a contribution of channel block. Structure- activity relationship analyses reveal that both functional groups are essential for gating activity, while classical requirements such as carbon 3 hydroxyl stereoselectivity and carbon 5 reduction are dispensable. YX84 also modestly inhibits NMDA receptor currents, suggesting weak negative allosteric modulation. Behavioral assays show that intraperitoneal administration of YX84 (30 mg/kg) does not impair sensorimotor function, unlike allopregnanolone. These findings identify YX84 as a structurally distinct neuroactive steroid with dual receptor activity and favorable behavioral tolerability, offering a promising scaffold for therapeutic development targeting excitatory/inhibitory imbalance in neuropsychiatric disorders if pharmacokinetic considerations can be overcome.

Lung cancer remains the leading cause of cancer-related deaths worldwide, with cisplatin as the primary chemotherapy despite its limitations. Organotin(IV) dithiocarbamates have emerged as promising anticancer agents due to their potent cytotoxicity and stability. This study reports the successful synthesis of four novel organotin(IV) dithiocarbamates: dimethyltin(IV) N-methyl-N-benzyldithiocarbamate (DioSn-1), diphenyltin(IV) N-methyl-N-benzyldithiocarbamate (DioSn-2), triphenyltin(IV) N-methyl-N-benzyldithiocarbamate (TriSn-3), and triphenyltin(IV) N-ethyl-N-benzyldithiocarbamate (TriSn-4). Their cytotoxicity against A549 lung carcinoma cells was evaluated via MTT assay, while Annexin V-FITC/PI staining determined the mode of cell death. DioSn-2, TriSn-3, and TriSn-4 exhibited potent cytotoxicity (IC: 0.52-1.86 M), whereas DioSn-1 was inactive (IC > 50 M). Apoptotic features such as cell shrinkage and membrane blebbing were observed, with apoptosis rates ranging from 58% to 91%. DioSn-2 was the most selective (SI = 6.45) and induced early DNA damage within 30 minutes, followed by mitochondrial depolarization and excessive ROS generation. Caspase-9 activation exceeded caspase-8, confirming intrinsic apoptosis. NAC treatment reduced apoptosis by 52%, highlighting oxidative stress as a key cytotoxic mechanism. These findings suggest DioSn-2 as a promising alternative to cisplatin for lung cancer therapy.

Globally, about 264 million individuals across all age groups are impacted by depression, a prevalent central nervous system (CNS) condition. Chronic and enduring depression might result in significant health consequences. Numerous pharmaceutical antidepressants exist for the management of mild to severe depression, largely functioning by modifying neurotransmitter levels in the brain. Nevertheless, these drugs frequently induce a variety of side effects, such as insomnia, constipation, exhaustion, drowsiness, and anxiety. Saffron (Crocus sativus L.) is widely acknowledged as a natural antidepressant with little adverse effects. This study investigated the potential antidepressant mechanisms of saffrons principal bioactive compounds safranal, crocin, and picrocrocin via molecular docking against critical target proteins associated with depression, namely the dopamine transporter (DAT), serotonin transporter (SERT), and monoamine oxidase B (MAO-B). Molecular docking was conducted with AutoDock 4.2 to assess the binding affinity and interaction energy of these drugs with the target proteins. Furthermore, Discovery Studio facilitated the viewing and study of both interacting and non-interacting residues at the docking sites, juxtaposing these interactions with those of established inhibitors in crystal structures. The permeability of the blood-brain barrier (BBB), pharmacokinetic characteristics, and toxicity profiles of saffron components were evaluated using SWISS ADME, DataWarrior, and Osiris Molecular Property Explorer. Among the evaluated elements, safranal had the greatest potential as a competitive inhibitor of the dopamine transporter, according to its notable blood-brain barrier permeability, robust binding affinity, and analogous interaction residues in comparison to nortriptyline, a recognized inhibitor. Our findings indicate that safranal may be a viable natural alternative to traditional antidepressants, with minimized adverse effects.

Natural products, especially polyphenol-rich medicinal plants, are increasingly investigated as multitarget therapeutics in both human and veterinary medicine for angiogenic regenerative properties and for inflammation based-diseases. Recent developments in natural product formulation, notably microencapsulation, have been shown to improve the stability, bioavailability, and controlled release of bioactive compounds. The integration of complementary in vitro and in vivo models is critical for evaluating both efficacy and translational potential. In this context, the present study assessed the phytochemical composition and biological activity of a microencapsulated Ecuadorian Vaccinium floribundum extract (VFM), using a combination of in vitro and in vivo approaches. VFM biochemical characterization identified 15 compounds, including flavonoids, procyanidins, dihydrochalcones, and phenolic acids, with chlorogenic acid and quercetin as the most abundant metabolites. Anthocyanins ideain and petunidin were also detected, confirming a rich bioactive profile. Primary porcine thoracic aortic endothelial cells (pAECs) were treated with VFM to assess cell viability and angiogenic potential and challenged with bacterial lipopolysaccharide (LPS) in the presence or absence of the extract. Anti-inflammatory effects were further evaluated in vivo using a carrageenan-induced mouse paw edema model. VFM enhanced endothelial cell viability, promoted capillary-like network and modulated early angiogenic signaling pathways. It mitigated LPS-induced endothelial dysfunction by reducing pro-inflammatory cytokines and oxidative stress markers. In vivo, paw edema assays confirmed its anti-inflammatory efficacy, with microencapsulation likely sustaining bioactive release. These findings support the traditional use of Vaccinium floribundum and highlight its potential for developing nutraceutical formulations targeting vascular and inflammatory disorders.

Lacritin is an abundantly expressed glycoprotein in tear fluid and plays key roles in immune response, tear secretion, and bacterial killing. These biological functions are tightly regulated through several biochemical mechanisms including multimerization, proteolysis, and alternative splicing, especially within its C-terminal domain. Given its critical role at the ocular surface, lacritin is currently under investigation as a diagnostic biomarker and therapeutic candidate for dry eye disease (DED). However, despite over three decades since its initial discovery, the functional significance of the O-glycans that comprise more than 50% of its molecular weight remain largely unknown. To address this gap, we leveraged mass spectrometry (MS)-based glycoproteomics and molecular dynamics (MD) to explore the structural role of site-specific O-glycans on C-terminal lacritin. In doing do, we identified distinct glycosylation profiles between monomeric and multimeric lacritin, particularly at glycosites located near crosslinking residues (Lys101 and Lys104) that modulate multimer formation. Building on our glycoproteomics data, we performed MD simulations on monomer and multimer glycoforms and revealed that O-glycans participate in intra-glycan-protein interactions, thereby affecting the conformational flexibility of lacritin and the spatial arrangement of Lys101 and Lys104. Finally, we quantified the solvent-accessible surface area (SASA) of Lys101 and Lys104, highlighting that proximal O-glycosylation is predicted to affect the propensity of these residues to participate in crosslinking. Taken together, these findings underscore a central role for lacritin O-glycans in affecting structural topology with implications for its downstream biological activity.

Our current understanding of carbohydrate-binding module (CBM) function is limited by the fact that most CBM research has focused on single-binding-site modules. CBM family 92 (CBM92) is a recently characterized family of predominantly trivalent proteins that bind {beta}-1,3- and {beta}-1,6-glucans with high specificity. CpCBM92A from Chitinophaga pinensis stands out as the first trivalent member of the family to be structurally determined. Multivalent CBM families are rare, and the way in which the three binding sites cooperate in ligand recognition remains unclear. Here, we use NMR spectroscopy to demonstrate how each of the proteins binding sites plays distinct roles in ligand binding. One binding site, referred to as the {beta} site, can be identified as the primary attachment point because of its higher affinity for all tested ligands, consistent with previous biochemical data suggesting it is the strongest binding site on CpCBM92A. The other two binding sites, referred to as and {gamma}, preferentially bind longer segments of {beta}-1,3- and {beta}-1,6-glucan chains, respectively. We further show that the glycosidic bond position and anomeric configuration of the binding glucosyl unit strongly affects protein affinity due to a preferred ligand pose in the binding sites. Our results provide insight into how the trivalent architecture of CBM92 might enable cross-linking of scleroglucan chains, which may guide the development of new applications for CBMs in biotechnology.

This study presents a multidisciplinary approach to evaluate the structure formation and digestion of lupin protein crosslinked with transglutaminase (TG). TG was applied at 0-10 U/g protein, and structural development was assessed by oscillatory rheology (G, G"), while SDS-PAGE and o-phthaldialdehyde (OPA) assays were used to evaluate protein participation and the reduction of free {varepsilon}-amino groups, respectively. Proteomics was further employed to characterise molecular features associated with crosslinking behaviour. Lupin protein showed a clear dose-dependent increase in gel strength during incubation, with G values reaching 214 {+/-} 43.9 Pa at 10 U/g TG, compared to 7.2 {+/-} 0.6 Pa in the untreated control. Across all conditions, G remained higher than G" throughout frequency sweeps, and low tan {delta} values confirmed the formation of elastic networks driven by covalent crosslinks. SDS-PAGE and OPA results consistently demonstrated efficient crosslink formation, which increased with both incubation time and TG dosage, with SDS-PAGE indicating involvement of specific protein fractions. Proteomic analysis revealed disordered structural domains in the protein are preferred regions to form crosslinks. Furthermore, TG treatment was found to slow the digestibility of the crosslinked lupin protein. Overall, this work demonstrates how integrating proteomic insights with functional measurements can guide the selection and optimisation of plant proteins for enzymatic structuring. The approach offers a rational pathway to enhance the functionality of alternative protein sources such as lupin, supporting the development of sustainable food systems, including applications in meat and dairy analogues.

Accurate, simultaneous, and efficient quantification of chemically diverse phytohormone species is a critical task towards understanding the complex system of phytohormone signaling pathways. Quantification of phytohormones with the commonly used technique liquid chromatography coupled to tandem mass spectrometry is susceptible to the influence of non-phytohormone components present in the sample, a phenomenon referred to as matrix effect. To reduce matrix effect, some phytohormone quantification methods include additional steps of cleanup of crude extracts. However, to what extent additional purification steps provide increased accuracy compared to simpler, less laborious methods is seldomly evaluated. We evaluated three previously described phytohormone extraction methods, two of which include solid-phase extraction and one that does not, in their ability to minimize matrix effect and generate accurate estimates of phytohormone species spanning six classifications, from fruit and leaf tissue of Solanum lycopersicum cv. Micro-Tom (tomato). Our results show that, while the methods that included solid phase extraction occasionally outperformed each other regarding matrix effect and/or recovery efficiency for broad range of phytohormones, they rarely outperformed the simpler single-phase extraction method. Short AbstractAccurate, simultaneous quantification of chemically diverse phytohormones by LC-MS/MS is frequently confounded by matrix effects, leading to the incorporation of additional purification steps. We systematically compared three published extraction protocols with or without solid-phase extraction in tomato tissues across six hormone classes. Solid-phase methods occasionally improved matrix suppression or recovery, but did not consistently outperform the single-phase approach, questioning the added value of extra cleanup steps, particularly when high-throughput is desired, as in the case of systems biology interrogations.

Synaptic vesicle glycoproteins 2 (SV2) are integral membrane proteins essential for neurotransmitter release and are implicated in neurological disorders including epilepsy and Parkinsons disease. In the attempt to develop a ligand selective for SV2C, and in collaboration with UCB, UCB-F was identified as a potential candidate. However, the affinity of UCB-F to SV2C was found to be temperature dependent, decreasing by about 10-fold from +4 to 37 degrees. UCB1A was subsequently identified as SV2C ligand displaying in vitro a 100-fold selectivity for SV2C compared with SV2A. In this study we investigated whether the binding of UCB-1A to SV2A and SV2C was affected by the temperature. A combination of experimental binding assay data and molecular dynamics (MD) simulations were used. The binding studies revealed that UCB1A affinity for SV2A decreased significantly at 37 {degrees}C compared with 4 {degrees}C, whereas binding to SV2C remained largely unchanged. MD simulations reproduced these observations, namely that ligand RMSD values at 310 K showed that UCB1A binding fluctuated markedly in the SV2A complex, with many trajectories exceeding the 3.0 [A] stability cutoff, whereas UCB1A remained relatively well-anchored in SV2C under the same conditions. Structural analysis showed that, while UCB1A adopts a conserved binding pose across all isoforms stabilized by {pi}- {pi} stacking and a hydrogen bond with Asp, SV2C possesses a unique stabilizing feature. In SV2C, Tyr298 is less exposed to the solvent and engages in a persistent hydrogen bond with Asparagine, a structural feature that reinforces pocket stability and limits temperature-induced destabilization. This interaction is absent in SV2A, consistent with its greater temperature sensitivity. Together, these findings provide a mechanistic explanation for the experimentally observed temperature independence of UCB1A binding to SV2C. More broadly, the results highlight the importance of incorporating physiologically relevant temperatures into SV2 ligand evaluation and demonstrate how combining experiments with simulations can uncover isoform-specific mechanisms of ligand recognition and stability.

Cannabinoid receptors are therapeutically promising GPCRs that are also interesting test systems for structure-based methods, which have targeted them previously. Here we used the CB2 receptor as a template to explore several topical questions in library docking. Whereas an earlier campaign against the CB1 receptor led to potent but relatively non-selective ligands, here we found that targeting interactions with polar, orthosteric site residues led to subtype-selective ligands. Docking hit rate and especially hit affinity improved in moving from a 7 million to a 2.6 billion molecule library. Similar to earlier studies, docking against active and inactive states of the receptor did not reliably bias toward the discovery of agonists or inverse agonists. Cryo-EM structures of two of the new agonists, each in a different chemotype, superposed well on the docking predictions. Correspondingly, structure-based optimization led to 10- to 140-fold improvements within three different series, also consistent with well-behaved ligand families. Hit rates with a fully enumerated 2.6 billion molecule library resembled those of an implied 11 billion molecule library from a building-block method, consistent with the latters ability to explore this space, though higher affinities were discovered from the fully enumerated set. Overall, eight diverse families of ligands, with potencies <100 nM and mostly unrelated to previously known ligands were found. Implications for future studies are considered.

Tox21 assays compile extensive chemical bioactivity data across diverse biological targets, making them widely utilized resources for in silico model development. Nuclear receptor-specific assays within this dataset are particularly valuable for screening potential endocrine disrupting chemicals. This study presents a comprehensive benchmarking of diverse machine learning (ML), deep learning (DL), and transformer-based architectures with varied chemical feature representations across nuclear receptor assays. First, 43 datasets associated with 18 nuclear receptors within Tox21 assays were systematically curated from ToxCast invitrodb v4.3. Upon testing across these datasets, model performance was found to be dependent on the degree of class imbalance. Tree-based ML models such as random forest (RF) and extreme gradient boosting (XGBoost) trained on descriptors, or combination of descriptors and fingerprints, consistently outperformed in datasets with higher proportions of active chemicals (>10%), while DL models showed greater robustness for those with moderate proportions (5-10%). Further analysis revealed that approximately 40% of misclassified active chemicals occupied structurally isolated regions of the chemical space, suggesting absence of close structural analogues in the training set potentially contributed to their misclassification. External validation using in vitro and in vivo androgen and estrogen receptor bioactivity data showed generally good concordance. Finally, a systematic literature review revealed that the models in this study span wider range of architectures, feature representations, and assay endpoints, and are broadly comparable to or better than existing work. Overall, insights from this study can inform the development of more reliable in silico tools supporting new approach methodologies for nuclear receptor bioactivity predictions.

MAVISp (Multi-layered Assessment of VarIants by Structure for proteins) is a structure-based framework for facilitating mechanistic interpretation of missense variants, with protein stability as one of its core analytical layers. When software tools are updated, a key consideration for database curation is whether the new version can be adopted without compromising compatibility with existing entries. This study evaluated the effect of replacing FoldX5 with FoldX5.1 on the results of the MAVISp stability workflow. We compared predicted changes in folding free energy for 539,809 shared variants across 119 proteins. We found high overall agreement with a mean Pearson correlation of 0.933 and a mean Cohen coefficient of 0.814. Most proteins showed strong concordance, whereas only three (NUPR1, TSC1, and TMEM127) showed poor agreement. The number of disagreements was higher at sites with low AlphaFold2 confidence for NUPR1 and TSC1. These outliers did not display systematic inter-version bias, as mean shifts in folding free energies between versions were minimal. Collectively, these findings support adopting FoldX5.1 for future MAVISp data collection. We will include a transition period, during which existing entries retain FoldX5 annotations until their scheduled annual update, while new or updated entries are processed with FoldX5.1. To facilitate this transition, the FoldX software version has been added as a new metadata annotation in the MAVISp database.

IntroductionRetinoids are bioactive vitamin A derivatives that regulate cellular differentiation and gene expression, yet their reliable quantification remains challenging due to low abundance, structural isomerism, and sensitivity to ionization conditions while handling. ObjectivesIn this study, we performed a systematic optimization of liquid chromatography-mass spectrometry (LC-MS)-based detection of retinoids across tissues and biofluids. MethodsChromatographic separation, adduct formation, ionization parameters, fragmentation behavior, and extraction procedures were evaluated in an integrated workflow. ResultsChromatographic conditions influenced not only retention time but also the ionic species detected, affecting precursor selection for MS{superscript 2} analysis. Retinoids exhibited compound-dependent responses to electrospray ionization and collision energy, requiring tailored acquisition parameters. Extraction experiments demonstrated differential recovery among retinoid classes and revealed matrix-dependent behavior, indicating that protocols used for tissues cannot be directly transferred to low-abundance biofluids. Using optimized conditions, retinoids were detected in mouse cerebrospinal fluid (CSF) at concentrations approaching the analytical detection limit, where MS{superscript 2} confirmation was necessary for reliable identification. ConclusionTogether, our results provide a framework for reproducible retinoid profiling across biological matrices and enables comparative studies of retinoid biology in low-volume and low-abundance biofluids.