搜索结果：Nature structural & molecular biology

Obesity represents one of the most critical global public health challenges. Pancreatic lipase (PL) serves as a key therapeutic target for obesity control, whereas clinical synthetic PL inhibitors are greatly restricted by adverse reactions. Traditional Chinese medicines (TCMs) have a long-standing history in regulating lipid metabolism and ameliorating obesity-related disorders, and are characterized by remarkable structural diversity, low toxicity, and mild side effects, thus representing a promising source for developing safe and efficient PL inhibitors. In this work, an integrated strategy combining in silico screening and in vitro validation was employed to identify potential PL inhibitors from TCM components, including molecular docking, molecular dynamics simulation, MM/PBSA binding free energy computation, and in vitro enzymatic assay. Six compounds with docking scores ranging from -9.9 to -9.0 kcal/mol were selected for further investigation. Molecular dynamics simulations verified the favorable structural stability of the corresponding ligand-PL complexes, and MM/PBSA calculations demonstrated negative binding free energies from -21.24 ± 0.39 to -12.03 ± 0.40 kcal/mol. In vitro experiments indicated that three compounds (Hydroxygenkwanin, Atractylenolide I, and Peiminine) showed effective PL inhibitory activity, with IC50 values of 0.128 ± 0.009, 0.584 ± 0.031, and 0.748 ± 0.042 mM, respectively. These values are comparable to quercetin (0.231 ± 0.034 mM) but significantly higher than orlistat (0.481 ± 0.023 μM), which is attributed to their non-covalent binding pattern. Collectively, this study validated the reliability of the integrated in silico and in vitro screening strategy, identified three effective pancreatic lipase inhibitors derived from TCMs, established a robust paradigm for the discovery of natural PL inhibitors, and laid a solid foundation for subsequent research on natural anti-obesity agents.

Over the last two decades, we have routinely published phylogenies describing the evolution of organisms and viruses using protein structure information. The structure-based trees offer several advantages over traditional approaches, including improved resolution of basal branches of the trees, a more realistic representation of evolutionary events such as gene duplication, loss, gain, and horizontal gene transfer, better handling of fast-evolving (micro)-organisms and organisms with parasitic tendencies (viruses), and reduced susceptibility to artifacts arising from sequence alignment and reconstruction in complex genomic datasets. Here, we present a generic protocol for phylogenomic analysis of molecular structure, which is timely considering the recent revolution in AI-driven models of protein structure prediction. While the protocol is illustrated with viral protein structures, the procedure is generic in nature and can be easily adapted for other structures (e.g., RNA) and molecular characteristics (e.g., molecular functions, pathways), thereby enriching the phylogenetic toolkit available to molecular biologists.

Triple Negative Breast Cancer (TNBC) is one of the most aggressive subtypes of breast cancer (BC), which is associated with a very poor prognosis. It is a broad category of tumors with a variety of biological, clinical, and morphological characteristics. FOXM1 is a pivotal transcription factor that modulates proliferation-associated genes through complex protein-DNA and protein-protein interactions, making it a highly attractive target in cancer therapy. However, existing small-molecule inhibitors often suffer from limited specificity and efficacy. In this study, we designed, synthesized, and evaluated novel series of 2-aminothiazole derivatives (C1-C15) as potential FOXM1 inhibitors. Molecular docking and molecular dynamics (MD) simulations were employed to investigate the binding interactions of these compounds with the FOXM1 DNA-binding domain (FOXM1-DBD). Structural analysis highlighted the importance of crucial residues, including Asn283, His287, and Arg286, in mediating inhibitory activity. Among the synthesized compounds, C11 exhibited remarkable structural alignment and interaction patterns with FOXM1-DBD, comparable to the reference inhibitor FDI-6. In vitro studies using TNBC cell lines (MDA-MB-231, BT-549, and BT-20) demonstrated that compound C11 significantly outperformed FDI-6 in potency. Western blot analysis revealed that C11 effectively suppressed FOXM1 transcriptional activity at concentrations of 10 µM in BT-549 cells and 20 µM in MDA-MB-231 cells. These findings underscore the potential of C11 as a potent FOXM1 inhibitor and highlight its promise for further development in TNBC therapy.

Single-molecule tracking in living cells measures protein diffusivity but requires sparse imaging, limiting high-density mapping. Here we introduce single-molecule localization and diffusivity microscopy (SMLDM), a deep learning-based approach that accurately estimates single-molecule movement tracks and diffusion coefficients directly from single-frame snapshots, eliminating the need for trajectory linking. Implemented as mobility photoactivated localization microscopy (MPALM) with bright photoactivatable fluorophores and U-Net-based single-molecule segmentation, this method achieves a 50- to 300-fold increase in data density compared to conventional tracking-based approaches, generating high-density, spatially super-resolved maps of molecular diffusivity and organization in living human cells. We applied MPALM to diverse dynamic cellular processes, uncovering nucleosome clustering into low-mobility chromatin domains, pathway-biased μ-opioid receptor dynamic clustering, focal adhesion movement and nonuniform molecular diffusivity and microcondensate organization during early droplet coalescence. SMLDM provides a powerful tool for resolving biomolecular organization and dynamics at single-molecule resolution in live cells.

Hierarchical chirality, arising from the coupling of chiral surfaces, units, assemblies, and overall architectures, represents a structural paradigm with profound implications for enantioselective catalysis, molecular recognition, and chiral photonics. Unraveling and regulating its formation across multiple length scales is crucial for understanding structure-property relationships and guiding the design of chiral nanomaterials. Here, we present a strategy for constructing step-rich Pt@Au nanostructures with multilevel chirality. Using octahedral Pt nanoframes as templates and cysteine as a chiral inducer, rotated and oriented Au triangular pyramids with high-Miller-index chiral facets are generated, leading to the formation of fishbone-like, pinwheel-like, and tilted-step surface features that collectively constitute a hierarchical chiral nanostructure. Mechanistic studies identify cysteine and Pt nanoframes as indispensable for directing step formation and hierarchical growth, thereby defining the overall morphology and chiroplasmonic responses. Leveraging the abundant chiral surface area and the strong chiral electromagnetic nearfields generated under plasmonic excitation, we fabricate monolayer-modified electrodes based on the Pt@Au nanostructures that enabled plasmon-enhanced enantioselective electrochemical recognition of penicillamine enantiomers and determination of their enantiomeric purity. This strategy clarifies the origin of multilevel chirality in step-rich architectures and establishes a structural engineering approach for designing chiral nanomaterials with enhanced near-field properties for enantioselective recognition.

Monkeypox virus (MPXV) is emerging as a global public health concern due to its nature of spread. There are limited treatment options, as the sole drug for treatment is lacking, highlighting the need for new therapeutic options. The use of computer-aided drugs discovery such as molecular docking, molecular dynamic (MD) simulations and post-simulation analysis are important tools in identifying potential compounds that can target specific proteins of the virus, such as DNA polymerase to stop virus replication. This study employed molecular docking and molecular simulation with the aim to identify potential inhibitors for MPXV treatment from the ZINC Database. Molecular docking was performed using PyRx 0.8 version after virtual screening of the ZINC database using the Tranches tool; then, toxicity prediction of the selected compounds was performed using the ProTox-3.0 web server. Molecular dynamics simulation was conducted using GROMACS version 4.5 to evaluate the structural stability and dynamic behavior of the protein-ligand complex for the best interacting compound. Furthermore, post-simulation analysis was conducted using standard GROMACS utilities for visualizing time-dependent properties from MD simulations. A total of 16 compounds were shortlisted based on their molecular docking scores and interaction profiles with the monkeypox virus DNA polymerase (PDB ID: 8HG1). The leading compound, ZINC000019418450, demonstrated strong binding affinity (-7.4 kcal/mol). According to post-simulation analysis, all top compounds formed between one and five hydrogen bonds and up to eleven hydrophobic contacts with residues within the active site, thus providing strong geometric and energetic evidence for binding stability. Notably, our identification of ZINC000104288636 as a Class 6 compound with an LD50 of 23,000 mg/kg adds translational value by highlighting candidates with low predicted acute toxicity. Overall, this study lays a solid foundation for the rational design of next-generation monkeypox antiviral therapeutics. Future work is needed for experimental validation of prioritized compounds to assess their biochemical efficacy and pharmacological potential.

Pneumocystis pneumonia (PcP) caused by Pneumocystis jirovecii is one of the most common and serious opportunistic infections in immunocompromised patients. People at risk for developing Pneumocystis pneumonia include human immunodeficiency virus positive individuals, cancer patients, people receiving immunosuppressive therapy, organ transplant recipients and people with compromised immune systems. Despite effective treatment and prophylaxis, mortality is reported to be between 15-40%, even in people with immunodeficiency other than acquired immunodeficiency syndrome. Although the number of immunosuppressed patients is increasing, diagnostic and epidemiological studies for this agent are still insufficient. This study aimed to investigate the prevalance and epidemiological characteristics of P.jirovecii isolated from various patient groups. Among the 469 bronchoalveolar lavage fluids or sputum samples sent to the medical microbiology laboratory from various units, 114 samples were selected for indirect fluorescent antibody (IFA) testing and molecular studies based on an examination of the patients' files to determine underlying diseases, use of immunosuppressive drugs, corticosteroid treatments received and the presence of patchy or nodular ground-glass opacity on computed tomography scans and the presence of a cyst-like structure on May-Grunwald Giemsa staining. The presence of P.jirovecii was investigated by IFA and polymerase chain reaction methods and genotyping was performed by sequencing of mtLSU rRNA and internal transcribed spacer (ITS) region. The obtained ITS gene region DNA sequences were aligned and TCS network analysis was performed in the aligned region. As a result, positivity was found in eight samples out of 469 samples by using mtLSU rRNA genotyping, Genotype 1 (n= 3; 37.5%) was found to be the most common genotype in our samples. Genotype 2 (n= 2, 25%), genotype 4 (n= 2, 25%) and genotype 3 were the most frequently detected other genotypes, respectively. When the relationships between the sequences were examined, it was observed that our samples were generally related to the samples originating from Iran. In the present study, the genotyping analysis of the ITS region, constructed using the consensus sequence employed in the study by Lee et al., revealed that the most common genotype was Eh (n=3; 42.85%), followed by Bg (n= 2; 28.57%), Bi (n= 1; 14.28%) and Eg (n=1; 14.28%). Our study is the first genotyping study conducted in our country using the ITS gene region. Different epidemiological findings were found in P.jirovecii genotype frequencies in studies conducted in different geographies. This suggests that genetic variations in P.jirovecii have a geographical component and this may affect the distribution of P.jirovecii strains among humans. According to TCS network analysis, our samples are generally associated with samples originating from Iran. None of our samples are found alongside samples originating from India. However, in certain areas of the analysis, New World and Old-World samples exist together. It can be assumed that globalization and thus the increase in human movement over time has led to the spread of different genotypes to different geographical regions and the formation of genotypic mosaics in certain geographical regions. Man-made destruction of nature and the consequent intertwining of urban and rural boundaries, as well as global warming and climate change have undeniably contributed to these movements.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder characterized by cholinergic dysfunction, amyloid-β aggregation, mitochondrial stress, and aberrant kinase activity. Carotenoids, naturally occurring pigments with antioxidant and neuroprotective properties, have emerged as promising candidates for AD intervention. In this study, we performed a systematic stepwise computational screening of a large carotenoid library (n = 1191) to identify multitarget candidates against AD-related proteins. The workflow consisted of predefined ADMET filtering (oral absorption > 90%, Caco-2 > 0.9, logBB > -1, and absence of major CYP inhibition and toxicity alerts), reducing the dataset to 61 compounds, followed by multi-target molecular docking against AChE, BChE, BACE-1, MAO-B, and GSK3-β. Compounds were ranked using an aggregated mean docking score across all five targets, and the top-performing candidate was subjected to detailed mechanistic analyses. Hopkinsiaxanthin emerged as the highest-ranked multitarget carotenoid and was further evaluated using frontier molecular orbital (FMO) analysis, pharmacophore modeling, 100 ns molecular dynamics (MD) simulations, MM/PBSA binding free energy calculations, and per-residue decomposition. Docking predicted favorable estimated binding affinities toward all targets. MD simulations confirmed stable receptor-ligand complexes with low RMSD values (0.278-0.285 nm). MM/PBSA analysis indicated favorable binding free energies, particularly for GSK3-β (-22.73 kcal/mol) and AChE (-21.50 kcal/mol). Per-residue decomposition identified key hotspot residues driving stabilization. Overall, this structured computational framework identifies Hopkinsiaxanthin as a promising multitarget scaffold and supports its prioritization for experimental validation in AD models.

Restoring the tumor-suppressor function of p53 by inhibiting its negative regulator, MDM2, represents a significant therapeutic avenue for cancers that maintain wild-type p53. This research aimed to identify new MDM2 inhibitors through a phylogenetically guided strategy that involved the construction of a focused virtual library of metabolites derived from the Penicillium genus. A comprehensive computational framework was developed, employing machine learning-based quantitative structure-activity relationship (ML-QSAR) modeling, ensemble molecular docking, network pharmacology, molecular dynamics (MD) simulations, and ADMET profiling. The gradient boosting ML-QSAR model achieved a test set R2 of 0.80 and was externally validated against 39 known MDM2 inhibitors (R2 = 0.82, RMSE = 0.80 pIC50 units), confirming its predictive reliability. Ensemble docking studies against 13 conformations of MDM2 highlighted three leading candidates (CNP0147553.1, CNP0154476.3, and CNP0154476.4) demonstrating binding affinities comparable to the known control inhibitor Nutlin-3a, with docking scores validated against experimental binding data. Further investigations through 500 ns MD simulations provided insights into the stability of the CNP0147553.1-MDM2 complex, which maintained a mean ligand RMSD of 0.039 nm and a complex RMSD of 0.176 nm, alongside a favorable binding free energy of -25.82 kcal/mol. Key residue analysis revealed that CNP0147553.1 achieved pronounced stabilization of critical binding pocket residues, including an 81.6% reduction in flexibility of HIS96. Network pharmacology analysis revealed a polypharmacology potential, indicating that the hub genes related to the identified compounds predominantly converged on the PI3K-AKT-mTOR and RAS-RAF-MAPK signaling pathways. ADMET profiling suggested promising pharmacokinetic and safety profiles for the lead candidates, establishing the basis for future experimental validation.

A ∼ 20 kDa heteroglucan composed of glucose (Glc) and glucuronic acid (GlcA) was isolated from Hypsizygus marmoreus. Preliminary analysis through methylation analysis and nuclear magnetic resonance (NMR) showed the backbone consisted of β-1,6-linked d-glucopyranosyl (Glcp) residues, with branches at O-3 positions containing t-β-D-Glcp residues. The linkages of GlcA was carried out using partial acid and glycosidase hydrolysis. HILIC-FLR/ESI-MSn, methylation and NMR of the partial acid and glycosidase hydrolysis products revealed GlcpA was in the forms of t-β-D-GlcpA and β-1,4-D-GlcpA attached to the C-3 position of part of the Glc in the main chain. Furthermore, the β-glucan exhibited structural heterogeneity, comprising three distinct domains: (a) unsubstituted β-1,6-glucan; (b) β-1,6-heteroglucan substituted with GlcA (1-2 sugar residues); and (c) β-1,6-heteroglucan substituted with Glc (single sugar residue). The proposed heteropolysaccharide structure provides critical insights into the structural diversity of mushroom-derived β-1,6-glucans, thereby facilitating future investigations into their structure-activity relationships.

Marine-derived bioactive peptides have attracted increasing attention as value-added functional ingredients. In this study, peptides (<3 kDa) were prepared from yellowfin tuna processing by-products and further fractionated by Sephadex G-25 gel filtration. The major fraction (TBP-MF) exhibited markedly improved compositional homogeneity compared with the unfractionated hydrolysate (TBP), providing a well-defined peptide system for subsequent characterization and biological evaluation. Physicochemical analyses demonstrated that TBP-MF possessed enhanced thermal stability and a more ordered secondary structure, characterized by pronounced &#x3b2;-sheet enrichment, as revealed by TGA/DSC, FTIR, and circular dichroism analyses. Morphological and colloidal characterization further showed that TBP-MF formed relatively uniform lamellar and fibrous assemblies with a narrower particle size distribution and reduced electrostatic stabilization, indicating a higher tendency toward ordered self-association. Peptidomic profiling combined with in silico analysis revealed that TBP-MF was enriched in short peptides with relatively higher PeptideRanker scores and a functional motif distribution containing relatively more neuro-related annotations, although angiotensin-converting enzyme (ACE)- and dipeptidyl peptidase IV (DPP-IV)-related motifs remained predominant in both groups. In differentiated PC12 cells, TBP-MF exhibited excellent cytocompatibility and induced a stable, concentration-dependent increase in the Cell Counting Kit-8 (CCK-8) readout (OD450), indicating enhanced cellular metabolic activity and/or increased cell number. In addition, TBP-MF significantly increased intracellular levels of key neurochemical factors associated with sleep-related regulation, including tetrahydrobiopterin (BH4), serotonin (5-HT), and &#x3b3;-aminobutyric acid (GABA). Overall, this study highlights yellowfin tuna by-products as a promising marine resource for bioactive peptides and suggests that fractionation-driven structural refinement is associated with neuro-related biological activity in differentiated PC12 cells. These findings support the potential application of marine by-product-derived peptides as functional ingredients in health-related fields.

Hepatocellular carcinoma (HCC) is an aggressive malignancy with limited treatment options. While natural products offer a vast chemical space for drug discovery, the literature at the oncology-phytochemistry interface remains fragmented. This study provides a high-resolution bibliometric curation to map research trends and pharmacological hotspots in HCC therapy over the last decade. A systematic search was conducted via Scopus for studies published between 2015 and 2025. Following manual curation of 1477 eligible articles, VOSviewer and the Bibliometrix R-package were utilized for analysis. A distinctive feature was the integration of taxonomic validation and manual text mining to ensure high accuracy of botanical and phytochemical data. Our findings reveal an exponential growth in scientific production, led by China, the USA, and India. The editorial landscape spans over 400 journals and 90 publishers, such as Elsevier, Frontiers, MDPI and Wiley. Phytomedicine and Journal of Ethnopharmacology were the most prolific journals. We identified over 700 distinct phytochemicals, notably quercetin, curcumin, and resveratrol, and approximately 600 plant species. Thematic clustering demonstrated that these compounds modulate critical cellular events, including apoptosis, oxidative stress, cell-cycle checkpoints, epithelial-mesenquimal transition, and distinct but complementary signaling pathway, such as Bax-Bcl-2, PI3K/Akt, mTOR, VEGF, SIRT1/NRF2, MAPK/ERK, CHOP and GRP78, and also different classes of miRNAs. Nanotechnology-based delivery systems emerged as a major trend to overcome bioavailability challenges. This bibliometric curation provides a mapping of the field, identifying critical thematic transitions and current knowledge gaps. The systematic cataloging of authors, institutions, publishers, journals, and phytochemicals offers a foundation for prioritizing molecules in future trials, providing objective insights for drug discovery and potential novel targeted therapies for HCC.

The papain-like protease (PLpro) of SARS-CoV-2 plays fundamental roles in its replication, and its mechanistic inhibition can impede the virus's replication and infection. Most Plpro inhibitors identified thus far are chemically synthesized and subject to numerous restrictions regarding stability and adverse side effects. Nevertheless, the inhibitors of those compounds can be replaced with natural, selective PLpro inhibitors that are highly stable and have minimal adverse effects. Since ancient times, extracts of Boesenbergia rotunda (L.) Mansf. have been recognized for their antiviral and other properties. Consequently, the objective of the investigation was to investigate the inhibitory activity of B. rotunda extract compounds against the virus, with the intention of inhibiting PLpro's signaling function in its replicative pathway, as a result, preventing viral infections. Molecular docking was initially suggested to evaluate the level of binding affinity among 57 natural compounds identified from B. rotunda to the desired protein. The results of this computational analysis have additionally been compared against molnupiravir, which has been addressed experimentally for its interacting efficiency towards the PLpro receptor protein of SARS-CoV-2 lately. This comparison indicates that the proposed dietary compounds have a significantly noticeable interaction efficiency regarding binding efficiency and other energetic contributions. Furthermore, the structure of PLpro was significantly influenced by compounds in MD-simulation experiments that were validated through some standard analyses, such as RMSF (root mean square fluctuation), RMSD (root mean square deviation), solvent accessible surface area, radius of gyration, MolSA, and PSA. The most promising three phytochemicals that could be established as an antiviral curative option against SARS-CoV-2 infection have been identified through computational approaches: rubranine, boesenbergin B, and panduratin A. The results of our computational investigation indicate that our proposed medications require clinical experimentation; consequently, they may be a superior treatment against SARS-CoV-2 viral infection.

Dengue virus (DENV) infection imposes a major global health burden, with around 100 million symptomatic cases and about 40 000 deaths each year. Despite decades of effort, no direct-acting antivirals are licensed and current vaccines show serotype-dependent and baseline serostatus-dependent efficacy, raising concerns about antibody-dependent enhancement. Early phase therapeutic trials therefore rely mainly on quantitative viraemia as a virological endpoint. However, DENV RNA in blood becomes undetectable soon after presentation, and may be an imperfect surrogate for infectious virus owing to defective interfering particles and continued replication in tissues. The secreted non-structural protein 1 (NS1) correlates with viral replication and disease severity in many studies and remains detectable after virological clearance, making it an attractive complementary biomarker. Here, we review mechanistic and clinical evidence linking NS1 to viral burden, vascular leakage and thrombocytopenia, and summarize data from human challenge studies, natural infection cohorts and pharmacometric modelling. We highlight the extended analytical window and growing availability of quantitative NS1 assays and propose practical analysis frameworks for incorporating NS1 into early phase dengue trials alongside viral RNA. We also discuss limitations, including serotype, immune status and assay-dependent effects and outline priorities for standardization of NS1-based endpoints. This article is part of the Theo Murphy meeting issue 'Evaluating anti-infective drugs'.

The formation of RIP-homotypic interaction motif (RHIM)-based heteromeric amyloid assemblies between effector proteins such as receptor-interacting protein kinases 1, Z-DNA Binding Protein 1, or TRIF and the kinase RIPK3 serves as regulating signals for the necroptosis process, a key element of innate immune defense. Murine cytomegalovirus expresses the M45-encoded viral inhibitor of RIP activation which inhibits necroptosis in a RHIM-dependent manner. A pivotal question is how viral M45 forms heteroamyloids with RIPK3 to effectively create an inhibitory assembly. We report a high-resolution structure of the M45:RIPK3 complex where M45 and RIPK3 alternately stack in an amyloid-state structure. Mutagenesis of the residues flanking the IQIG tetrad in M45 results in specific impacts on coassembly with RIPK3, indicating an extended interface in the heteromeric fibrils. Other key interactions support the formation of stable viral:host fibrils. The M45:RIPK3 heteroamyloid is likely to act as an antinecroptotic signal by competing with formation of other pronecroptotic species and introducing a barrier to RIPK3 autophosphorylation.

Skin ageing emerges from the interplay of intrinsic biological decline, environmental exposures and disease-related remodelling, yet the pathway-level epigenetic programs that integrate these processes remain largely unresolved. Here, we develop a skin-specific PathwayAge clock that aggregates DNA methylation into biologically interpretable gene ontology pathways, enabling both precise quantification of epigenetic age and mechanistic dissection of cutaneous ageing. The model demonstrates high accuracy and strong generalisability across multiple independent cohorts, providing a robust foundation for mapping pathway-level ageing biology in human skin. Comprehensive analyses reveal four core functional modules underlying skin epigenetic ageing, encompassing cellular process, stress and immune responses, developmental process, and signal and regulation. These programs show substantial concordance with systemic ageing pathways in blood, indicating shared epigenetic architecture across tissues. Sun-exposed skin exhibits pronounced age acceleration driven by oxidative stress, inflammatory activation and metabolic perturbation, defining the molecular signature of photoageing. In disease settings, actinic keratosis and squamous cell carcinoma display marked ageing acceleration with proliferative and extracellular matrix dysregulation; melanoma progression involves coordinated remodelling of stress, immune, structural and metabolic ageing programs; and psoriasis demonstrates inflammation- and differentiation-driven premature ageing. Together, these findings position the skin-specific PathwayAge clock as a mechanistic and quantitative framework for decoding intrinsic ageing, photoageing and disease-associated remodelling. This work advances a unified understanding of cutaneous ageing biology and provides a foundation for precision assessment and targeted intervention in skin ageing.

Thioester chemistry is exploited in Nature by many CoA-dependent enzymes. However, the covalent nature of CoA attachment largely prevents the use of these enzymes in many applications. Replacing the CoA moiety with simpler, truncated fragments, such as its pantetheine (PAN) moiety, is also hampered by the lack of understanding of the function of the CoA moiety in enzymatic conversions. Herein, we describe the utilization of the enzyme (2E)-enoyl-CoA hydratase (ECH) using PAN thioesters and an activator, 3',5'-ADP (PAP). ECH catalyzes the hydration of the carbon-carbon double bond of (2E)-enoyl-CoA substrates in the &#x3b2;-oxidation lipid-degrading pathway. The hydration reaction is very challenging to carry out by traditional chemical synthesis, as no selective catalysts are available. Structural enzymology of ECH and its complexes with (3S)-hydroxyacyl-CoA products show that hydrogen bonds between the adenine 6-amino group of the ADP moiety of CoA and loop-2 induce a small structural change in this active site loop, tightening the NN distance between the hydrogen bond donors of the oxyanion hole from 5.2 &#xc5; (unliganded) to 4.0 &#xc5; and forming a competent oxyanion hole at the catalytic site. A structurally similar and catalytically competent oxyanion hole is observed in the complex with (3S)-hydroxyhexanoyl PAN and the activator 3',5'-ADP, both bound at the active site. The use of 3',5'-ADP as the activator enables the synthetic use of ECH for the hydration of a wide range of (2E)-enoyl-PAN substrates with different steric demands and functionalities. The products, 3-hydroxyacyl-PAN thioesters, were obtained in good isolated yields and excellent stereoselectivities (typically >99:<1 3S:3R). Even for acyl chains that contain reactive groups such as bromide or methyl ester functionalities at C7, no side products resulting from potentially competing cyclization could be detected in the enzymatic hydration protocol.

Neuropathic pain remains a major unmet clinical challenge. Growing evidence identifies sigma receptors (&#x3c3;Rs) as pivotal intracellular modulators of maladaptive stress signaling, positioning them as promising non-opioid targets for chronic pain management. Notably, despite the pleiotropic nature of &#x3c3;Rs in regulating diverse cellular pathways-which might theoretically suggest a high risk of off-target effects-current selective antagonists have demonstrated remarkable safety and tolerability profiles. Sigma-1 and sigma-2 receptors (&#x3c3;1R and &#x3c3;2R) are molecularly and functionally distinct proteins that regulate neuronal excitability, proteostasis, and neuroimmune communication, all mechanisms that characterize neuronal excitability and cellular stress adaptation. &#x3c3;1R acts as a ligand-operated molecular chaperone at the mitochondria-associated endoplasmic reticulum membrane. Extensive preclinical data demonstrate that &#x3c3;1R antagonism attenuates peripheral and central sensitization, suppresses neuroinflammation, and restores opioid analgesic efficacy. These findings are supported by the advanced clinical candidate E-52862, which has shown efficacy and a favorable safety profile in neuropathic pain conditions. &#x3c3;2R, identified as transmembrane protein 97 (&#x3c3;2R/TMEM97), functions as a regulator of cholesterol trafficking, lysosomal integrity, and integrated stress response (ISR). &#x3c3;2R modulation alleviates neuropathic pain by restoring proteostatic balance and reducing ISR-driven neuronal vulnerability rather than directly suppressing excitability. Emerging &#x3c3;2R ligands such as FEM-1689, UKH-1114, and CM-398 provide compelling proof-of-concept for durable, disease-modifying analgesia. A structured literature search was conducted using PubMed, Scopus, and Web of Science to identify studies published within the last decade describing &#x3c3;1R and &#x3c3;2R/TMEM97 biology, ligand development, and their preclinical or clinical evaluation in neuropathic pain. Reference lists were manually screened to ensure comprehensive coverage. This review synthesizes pharmacology, ligand development, and translational evidence supporting &#x3c3;Rs as next-generation targets for neuropathic pain therapy, highlighting convergent roles of &#x3c3;1R and &#x3c3;2R in pain chronification and outlining future directions for structure-guided therapeutic strategies.

At present, the characteristics of key enzyme genes in the upstream pathway for triterpenoid saponin biosynthesis in P. grandiflorum, as well as their expression patterns over the growth duration, have not been systematically analyzed. This study, at the whole-genome level, conducts the first bioinformatics and expression analyses of the SS and SE gene families in P. grandiflorum. Four PgSS and seven PgSE genes were identified and distributed across six chromosomes. Members within the same subfamily exhibited highly conserved sequences and structures, while distinct structural divergence was observed between different subfamilies. Phylogenetic analysis showed that PgSS and PgSE genes were closely related to those of dicotyledons such as Panax ginseng and Polygala tenuifolia, suggesting high evolutionary conservation. Promoter analysis revealed abundant light- and hormone-responsive elements and MYB/MYC binding sites, indicating regulation by multiple signals. Protein secondary structures were dominated by the Alpha helix and were structurally stable. Quantitative real-time polymerase chain reaction (qPCR) demonstrated that expression levels of PgSS and PgSE in one-year-old Platycodonis Radix were significantly higher than in perennial Platycodonis Radix, especially for the PgSE family. This study characterized the basic biological features and growth-stage-dependent expression patterns of the SS and SE gene families in P. grandiflorum. The results identify key candidate genes and molecular targets for regulating triterpenoid saponin biosynthesis, and provide data supporting quality improvement and active metabolite research in this medicinal plant.