This study investigates the potential effects of essential oils (EOs) in enhancing the efficacy of clindamycin against Methicillin-resistant Staphylococcus aureus (MRSA) using in vitro and computer simulations. The research seeks to identify essential oils that exhibit synergistic activity with clindamycin and determine their potential key active components.

Essential oils commonly used in traditional medicine were tested for their antimicrobial activity against MRSA. The minimum inhibitory concentration (MIC) was determined using in vitro microdilution assays. A synergistic test with clindamycin was performed, and molecular docking studies evaluated the interaction between a key compound (trans-cinnamaldehyde) and MRSA protein.

EOs from Cinnamomum verum, Rosmarinus officinalis, Salvia officinalis, and Thymus vulgaris demonstrated significant inhibitory and synergistic activities against MRSA, standard strain, and human clinical isolates. Gas Chromatography/Mass Spectroscopy identified trans-cinnamaldehyde, eucalyptol, and thymol as prominent antibacterial compounds. Molecular docking studies confirmed trans-cinnamaldehyde's strong binding to MRSA's AgrA protein, elucidating its enhanced efficacy.

The study underscores the potential of plant-based therapies to augment the effectiveness of conventional antibiotics like clindamycin in combating MRSA and addressing antibiotic resistance by integrating traditional plant remedies with modern medical approaches.

In light of searching for new breast cancer therapies, DNA-targeted small molecules were rationally designed to simultaneously bind DNA and inhibit human dihydrofolate reductase (hDHFR). Fourteen new arylidene-hydrazinyl-1,3-thiazoles (5-18) were synthesised and their dual DNA groove binding potential and in vitro hDHFR inhibition were performed. Two compounds, 5 and 11, proved their dual efficacy. Molecular docking and molecular dynamics simulations were performed for those active derivatives to explore their mode of binding and stability of interactions inside DHFR active site. Anti-breast cancer activity was assessed for 5 and 11 on MCF-7 cells using MTX as reference. IC50 measurements revealed that both compounds were more potent and selective than MTX. Cytotoxicity was examined against normal skin fibroblasts to examine safety and selectivity Moreover, mechanistic studies including apoptosis induction and wound healing were performed. Further in silico ADMET assessment was conducted to determine their eligibility as drug leads suitable for future optimisation and development.

The current study aims to identify the key pathways and potential therapeutic targets for pulmonary arterial hypertension (PAH) and to further evaluate the anti-PAH effects of isorhamnetin.

The dataset of gene expression profiling for PAH (GSE113439) was downloaded from the gene expression omnibus (GEO) database. Isorhamnetin target genes were extracted from the comparative toxicogenomics database (CTD). Various bioinformatics methods were employed to identify the core pathways associated with PAH and potential intervention targets. Molecular docking was conducted between the interacting target and the candidate compound, isorhamnetin.

One thousand nine hundred sixty-two upregulated genes and 642 downregulated genes were identified. Molecular complex detection analyses revealed that the significant biological processes associated with upregulated genes included DNA damage response, mitotic cell cycle, and chromosome organization. In contrast, the signifi ant biological processes related to downregulated genes encompassed cellular response to growth factor stimulus, response to growth factor, and blood vessel development. Immune infilt ation analysis indicated that PAH is associated with signifi ant changes in the distribution of immune cells and differential expression of immune checkpoints. Furthermore, 58 isorhamnetin targets were extracted from the CTD, and we identified 1 interacting gene, NFE2L2, among the differentially expressed genes (DEGs), DEGs related to ferroptosis, and isorhamnetin targets. Isorhamnetin demonstrated strong affinities with vascular endothelial growth factor (VEGF) receptors and transcription factors (ATM and ZNF24) associated with VEGFs, as well as the ferroptosis protein NFE2L2.

Pulmonary arterial hypertension is characterized by a series of abnormalities in downstream molecular signaling pathways, including DNA damage, immune dysregulation, VEGF signaling deficienc , and the ferroptosis process. These may represent the core pathophysiological mechanisms of PAH. Ferroptosis-related genes, such as NFE2L2 and TF (ATM, ZNF24) associated with VEGFs, are potential therapeutic targets that contribute to the mechanisms mentioned above. Isorhamnetin is a promising candidate compound for the treatment of PAH.

Ginger (Zingiber officinale) is widely recognised for its functional benefits, primarily attributed to its diverse phytochemicals. However, its proteome remains largely unexplored. This study hypothesised that isolated peptides may exhibit different bioactivities or more targeted mechanisms of action and could be investigated at a molecular level. Proteins were enzymatically hydrolysed under five conditions, and peptides were identified using LC-MS/MS. In silico screening suggested antioxidant, ACE-inhibitory, and antibacterial properties, further assessed through molecular docking and in vitro validation. 41 potentially bioactive peptides were identified. In vitro assays confirmed these properties for selected peptides, P1 (GSPVWIIPEPT), P2 (FASYPVKK), P3 (GPEKIFYDGPYL), and P4 (IAISPSYPIK). Notably, P4 exhibited potent mixed-type ACE-inhibition and bacteriostatic effects. Molecular docking provided mechanistic insights into these interactions. These findings highlight ginger as a promising source of bioactive peptides while underscoring the need to complement AI tools with in vitro and in vivo validations due to observed discrepancies.

Nanomaterials (NMs) and the exploration of their comprehensive uses is an emerging research area of interest. They have improved physicochemical and biological properties and diverse functionality owing to their unique shape and size and therefore they are being explored for their enormous uses, particularly as medicinal and therapeutic agents. Nanoparticles (NPs) including metal and metal oxide-based NPs have received substantial consideration because of their biological applications. Computer-aided drug design (CADD) involving different strategies like homology modelling, molecular docking, virtual screening (VS), quantitative structure-activity relationship (QSAR) etc. and virtual screening hold significant importance in CADD used for lead identification and target identification. Despite holding importance, there are very few computational studies undertaken so far to explore their binding to the target proteins and macromolecules. Although the structural properties of nanomaterials are well documented, it is worthwhile to know how they interact with the target proteins making it a pragmatic issue for comprehension. This review discusses some important computational strategies like molecular docking and simulation, Nano-QSAR, quantum chemical calculations based on Density functional Theory (DFT) and computational nanotoxicology. Nano-QSAR modelling, based on semiempirical calculations and computational simulation can be useful for biomedical applications, whereas the DFT calculations make it possible to know about the behaviour of the material by calculations based on quantum mechanics, without the requirement of higher-order material properties. Other than the beneficial interactions, it is also important to know the hazardous consequences of engineered nanostructures and NPs can penetrate more deeply into the human body, and computational nanotoxicology has emerged as a potential strategy to predict the delirious effects of NMs. Although computational tools are helpful, yet more studies like in vitro assays are still required to get the complete picture, which is essential in the development of potent and safe drug entities.

Adverse Outcome Pathways (AOPs) in toxicology describe the sequence of key events from chemical exposure to adverse outcomes, facilitating the development of predictive models. The EU ONTOX project uses this framework to predict liver, developmental brain, and kidney toxicity without animal testing. Focusing on Molecular Initiating Events (MIEs), more concretely on the interaction of chemicals with key proteins, we have developed an automated workflow for docking small molecules onto over 20 pre-processed protein structures, implemented in the online tool DockTox. This tool generates conformers of small molecules, performs docking on MIE-associated proteins, and provides binding energy, interacting residues, and interaction maps. Additionally, it compares the interactions to a reference list of known ligands, producing an interaction fraction as an additional similarity measure. Evaluation of the docking workflow's predictive performance on Peroxisome Proliferator-Activated Receptor α (PPARα) showed that interaction fraction values are more informative than binding energy alone for distinguishing binders from non-binders. This unique feature enhances the understanding of target protein interactions. DockTox supports the virtual screening of small molecules targeting MIE-associated proteins, offering insights into binding energies and interaction profiles. It is a valuable tool for anticipating adverse outcomes from chemical exposure in a tiered risk assessment approach.

Nitazenes are a class of novel synthetic opioids with exceptionally high potency. Currently, an experimental structure of μOR-opioid receptor (μOR) in complex with a nitazene is lacking. Here we used a suite of computational tools, including consensus docking, conventional molecular dynamics (MD) and metadynamics simulations, to investigate the μOR binding modes of nitro-containing meto-, eto-, proto-, buto-, and isotonitazenes and nitro-less analogs, metodes-, etodes-, and protodesnitazenes. Docking generated three binding modes, whereby the nitro-substituted or unsubstituted benzimidazole group extends into SP1 (subpocket 1 between transmembrane helix or TM 2 and 3), SP2 (subpocket 2 between TM1, TM2, and TM7) or SP3 (subpocket 3 between TM5 and TM6). Simulations suggest that etonitazene and likely also other nitazenes favor the SP2-binding mode. Comparison to the experimental structures of μOR in complex with BU72, fentanyl, and mitragynine pseudoindoxyl (MP) allows us to propose a putative model for μOR-ligand recognition in which ligand can access hydrophobic SP1 or hydrophilic SP2, mediated by the conformational change of Gln1242.60. Interestingly, in addition to water-mediated hydrogen bonds, the nitro group in nitazenes forms a π-hole interaction with the conserved Tyr751.39. Our computational analysis provides new insights into the mechanism of μOR-opioid recognition, paving the way for investigations of the structure-activity relationships of nitazenes.

Peripheral nerve injury enhances mitochondrial translocator protein (TSPO) expression in the spinal cord and dorsal root ganglia (DRG), which is associated with neuroinflammation and mitochondrial dysfunction contributing to chronic pain development. Here, we investigate the effect of TSPO ligand Etifoxine, on the development of chronic pain and motor dysfunction following sciatic nerve injury. Mechanical and thermal sensitivity, as well as motor function, were measured in rats before and after sciatic nerve crush (SNC). Rats were treated with the Etifoxine (50 mg/kg, twice daily) for one week. At the end of the experiment, RT-PCR and immunohistochemistry (IHC) were performed to assess mitochondrial stress and neuroinflammation. Additionally, high-resolution respirometry (O2k) was used to evaluate mitochondrial function in the spinal cord following mitochondrial permeability transition pore (mPTP) induction by Ca2+. Etifoxine treatment post-SNC alleviated mechanical and thermal hypersensitivity, as well as motor dysfunction in rats. In addition, Etifoxine treatment modulates neuroinflammation and mitochondrial stress. Specifically, we found a significant reduction in microglia presence and the transcription of pro-inflammatory cytokines (TNFα, IL-6, IL-1β) in the DRG and spinal cord of the SNC/etifoxine-treated group. Furthermore, Etifoxine treatment prevent the decline in mitochondrial respiration, including non-phosphorylation, ATP-linked respiration, and maximal respiration, after mPTP induction by Ca2+. Our findings suggest that TSPO-ligand Etifoxine protects against motor dysfunction and the development of chronic pain by reducing neuroinflammation and apoptosis in the DRG and spinal cord. Importantly, the beneficial effects of TSPO-ligands are reflected in the restoration of the mitochondrial function under challenging conditions.

Recently, there has been a growing approach that approved drugs have been tested for additional purposes. Efavirenz is a non-nucleoside reverse transcriptase inhibitor used to treat human immunodeficiency virus infection. In addition, it has selective cytotoxic effects against cancer cells. This study constructed an electrochemical dsDNA nanobiosensor to monitor Efavirenz-dsDNA interaction based on the amine-functionalized multi-walled carbon nanotubes. The experimental conditions of the nanobiosensor, such as dropping the volume of nanomaterial suspension, activation of the nanosensor, and dsDNA concentration, were optimized. The peak currents of dsDNA bases were enhanced, and the peak potentials of Efavirenz have shifted to the less positive potential thanks to the modified sensor with amine-functionalized multi-walled carbon nanotubes. The interaction mechanism was also evaluated in incubated solutions. Docking calculations showed that Efavirenz is active in the large cleft regions of DNA that suggest minor groove binding. The effect of efavirenz on the expression profile of particular stress and possible DNA genotoxicity was studied via examining gene polymorphisms in hepatic cells. These findings align with previously released research that shows Efavirenz-treated hepatic cells to have altered mitochondrial function and elevated ROS levels.

Modified cyclodextrins (CDs) are cyclic oligosaccharides with many applications in drug delivery, catalysis, and as active pharmaceutical ingredients. In general, they exist as distributions of structurally diverse molecules rather than single-isomer compounds. Their performance depends on the number of glucopyranose units (GPUs), and the type, number, and position of chemical substitutions in their hydroxyl groups. Effectively targeting individual species within these distributions is essential for optimizing CDs for specific applications. Computational techniques can generate large datasets to AI-driven structural optimization, but the absence of a standardized nomenclature system for modified CDs presents a major barrier to progress in this direction. This lack of consensus limits effective communication, data sharing, automation, and collaboration. To address this, a clear and extensible nomenclature for modified CDs is proposed. In this framework, GPUs are treated like amino-acid residues, with unsubstituted GPUs as reference building-blocks and substituted ones considered as mutations. This approach precisely defines substitution types and patterns, resolves cyclic permutation ambiguities, and offers versatility for both simple and complex modifications, including chiral center alterations and covalently linked CD oligomers. By introducing this standardized nomenclature, we aim to enhance molecular design, improve reproducibility, and streamline both experimental and computational research in the CD field.

Tuberculosis is one of the oldest diseases that still affects millions of people worldwide and remains a significant public health challenge. The N-acetyltransferase 2 (NAT2) enzyme metabolizes isoniazid (INH), a primary antibiotic in tuberculosis treatment. The single nucleotide polymorphisms (SNPs) of NAT2 affect the metabolism and function of isoniazid. The rs1801280 (T341C) and rs1208 (G803A) variants of NAT2 are associated with INH drug responses. Individuals with the slow-metabolizing rs1801280 variant of the NAT2 enzyme are at a higher risk of INH-induced liver damage and require lower doses or longer treatment regimens. At the same time, individuals with the fast-metabolizing rs1208 variant are at risk of treatment failure due to rapid drug metabolism. Genotyping of the NAT2 variants can help clinicians personalize tuberculosis treatment, optimize drug doses, and thus minimize adverse effects. Under this study, an allele-specific PCR (ASPCR) method was developed for genotyping the NAT2 variants, and the results were validated through targeted sequencing. The allele frequencies at the rs1801280 locus were 0.60 for the T allele and 0.40 for the C. For rs1208, the participants' allele frequencies were 0.27 for the G allele and 0.73 for the A allele. This ASPCR method is quick, affordable, and could be used in routine genotyping to personalize the treatment for tuberculosis patients, leading to more effective and safer treatments. We also used molecular docking to study how the rs1801280 and rs1208 variants affect the interaction between the NAT2 enzyme and drugs. A slight change was visible in the flexibility of the amino acid residues. However, those amino acids were not involved in the ligand binding mechanism.

The C-C chemokine receptor type 5 is a G protein-coupled receptor expressed on various immune cells, playing a crucial role in inflammation and chemotaxis. Beyond its physiological functions, C-C chemokine receptor type 5 is implicated in numerous diseases, including cardiovascular, central nervous system, immune system, and infectious diseases, as well as in the progression of cancer. The therapeutic potential of C-C chemokine receptor type 5 inhibition has been demonstrated by antagonists targeting the extracellular domain, notably maraviroc, a Food and Drug Administration-approved Human Immunodeficiency Virus entry inhibitor. However, challenges such as suboptimal pharmacokinetics and efficacy necessitate new antagonists with unique modes of action. Recent advancements in G protein-coupled receptor structural characterization have identified a novel intracellular allosteric binding site in chemokine receptors. This study introduces a series of disubstituted [1,2,5]oxadiazolo-[3,4-b]pyrazines targeting the intracellular allosteric binding pocket of C-C chemokine receptor type 5. Among these, compound 3ad emerged as a promising C-C chemokine receptor type 5-selective allosteric antagonist with a half-maximal inhibitory concentration of 1.09 μM and an almost 30-fold selectivity over C-C chemokine receptor type 2. Molecular dynamics simulations and a competition assay with a Gαq11 mimetic were used to confirm the intracellular binding mode of these compounds. This novel class of C-C chemokine receptor type 5-selective intracellular antagonists offers a foundation for developing molecular tools and therapeutic agents, potentially overcoming the limitations of current extracellular C-C chemokine receptor type 5 antagonists.

Herein, a highly efficient and recyclable biocatalyst was developed using stabilized enzyme aggregates on amino-functionalized magnetic biochar for removing persistent organic pollutants from water. The biochar derived from biomass featured abundant hydroxyl functional groups, after functionalization with amino functional groups and magnetic nanoparticles, it was employed for laccase immobilization via enzyme electrostatic adsorption, precipitation and cross-linking in a favorable orientation. This immobilized enzyme aggregates exhibited enhanced pH tolerance, thermal and storage stability than free enzyme. Complete removal of 20 mg/L bisphenol A was achieved within 60 min via C-C bond cleavage and hydroxylation. Notably, the removal efficiency remained at approximately 90 % even after six cycles. Furthermore, this biocatalyst was also successfully applied to efficiently remove other various persistent organic pollutants and demonstrated applicability in real environmental water samples. This study highlights the substantial potential of enzyme-based biocatalysts, presenting a sustainable and efficient approach for water purification and biomass resource recovery.

Hypoxic pulmonary hypertension (HPH) is characterized by sustained elevation of pulmonary arterial pressure and vascular remodeling. The present study is to investigate the efficacy of acacetin on HPH and its potential molecular mechanism. C57/BL6 mice were exposed to hypobaric hypoxia for six weeks. At 4th week of hypoxia exposure, mice were administrated with the water-soluble prodrug of acacetin (5, 10, 20 mg/kg) or equivalent normal saline for another two weeks. The haemodynamic and pathohistological assessment were performed. Primary pulmonary artery smooth muscle cells (PASMCs) were cultured to examine the anti-proliferation efficacy of acacetin (0.3-3 μM). The activity and expression of sirtuin1 (SIRT1) acetylation and distribution of high-mobility group box 1 (HMGB1) were determined in lungs and/or cultured PASMCs with or without RNA interference of SIRT1. Macromolecular docking and molecular dynamics simulation were done to explore the potential binding between acacetin and SIRT1. Results showed that acacetin prodrug significantly reversed the increased pulmonary pressure and vascular remodeling in HPH mice, which is associated with inhibiting the reduction in SIRT1 and the increase in HMGB1, and inhibiting the nucleocytoplasmic translocation of HMGB1. In cultured PASMCs, acacetin inhibited the hyper-proliferation induced by hypoxia, reversed the SIRT1 reduction and inhibited the nucleocytoplasmic translocation of HMGB1 and HMGB1 increase. Silencing SIRT1 abolished all the beneficial effects of acacetin. These results demonstrate that acacetin is very effective in reversing HPH by inhibiting PASMC hyper-proliferation via regulating SIRT1-HMGB1 signaling, suggesting that acacetin is likely a promising drug candidate for treating patients with HPH.

The molecular interactions between Chikungunya virus (CHIKV) non-structural protein 3 (nsP3) and the host GTPase Activating SH3 Domain Binding Protein 1 (G3BP1) are critical for CHIKV replication. The C-terminus hypervariable domain (HVD) of nsP3 protein binds to the nuclear transport factor 2 (NTF2)-like domain of G3BP1 through two tandem FGDF motifs, aiding in the disruption of stress granule (SG) formation. Given G3BP1's role in the antiviral response, it presents an attractive target for antiviral drug development. In this study, seven potential small molecules targeting the FGDF motif binding pocket of G3BP1 were identified using a structure-based virtual screening approach. The binding modes of these molecules were further investigated through molecular docking and simulations. Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC) experiments confirmed their binding to purified G3BP1 with micromolar (μM) affinity. The antiviral efficacy of these molecules was assessed using in vitro cell culture-based assays, revealing that L-7, WIN, SB2, NAL, DHD, GSK, and FLU effectively inhibited CHIKV replication with EC50 values of 1.99, 0.40, 5.38, 1.52, 7.39, 3.66, and 0.61 μM, respectively. Additionally, CHIKV-infected cells treated with these compounds exhibited fewer virus-induced SGs compared to untreated controls without affecting SG formation under oxidative stress conditions. These findings indicate that identified inhibitors successfully block G3BP1-nsP3 interactions and suppress CHIKV replication. This is one of the first reports of small antiviral molecules targeting G3BP1, a host protein essential for stress granule formation in the antiviral cellular response and CHIKV replication.

Melanoma, an aggressive and highly metastatic form of skin cancer, remains challenging to treat due to its resistance to conventional therapies and frequent mutations in the BRAF signaling pathway. In this study, we report the design and synthesis of a novel series of thirteen quinazolinone derivatives, featuring a phenyl thiazole moiety linked via a triazole acetamide spacer. These compounds were developed as potential dual inhibitors of BRAFV600E and EGFR, which should offer a promising therapeutic strategy for melanoma treatment. The antiproliferative activity of these compounds was evaluated against the NCI-60 cell line panel, with six compounds advancing to a five-dose screening. Three compounds, 7k, 7l, and 7m, exhibited broad-spectrum anticancer activity, with mean growth inhibition (GI%) exceeding 100 %. Compound 7l demonstrated exceptional efficacy against melanoma subpanels (GI% = 152 %) and potent dual kinase inhibition, with IC50 values of 0.048 μM against B-RAFV600E and 0.037 μM against EGFR. In vitro studies of compound 7l revealed significant cytotoxicity against MALME-3 M (IC50 = 3.16 μM) and LOX-IMVI (IC50 = 2.50 μM) melanoma cell lines, with minimal toxicity towards normal Vero cells. Cell cycle analysis showed G1-phase arrest and disrupted DNA synthesis in melanoma cells, while apoptosis assays demonstrated a dramatic increase in early apoptotic cells from 7.28 % to 40.69 %. Compound 7l modulated key apoptotic markers, increasing the BAX/Bcl-2 ratio by 14.42-fold and elevating caspase 3 and 9 levels, indicating its potential to overcome drug resistance and enhance therapeutic efficacy in melanoma treatment.

Newcastle disease virus (NDV) classified in the Avian avulavirus 1 [genus Orthoavulavirus, subfamily Avulavirinae, family Paramyxoviridae] constitutes a serious financial risk to the global poultry market. Available vaccines do not show good results in catering to the virus. Currently there is no FDA-approved drug to treat the disease. Nucleoprotein (NP) is a structural protein playing that constitutes a serious financial risk to the global poultry market.a valuable role in the virus replication process and encapsidation. This study is an effort to screen phytochemicals, from the plant family Moringaceae, as potential inhibitors of the N protein. ADMET (adsorption, distribution, metabolism, excretion and toxicity) analysis was performed to screen potential phytochemicals with drug likeliness. Molecular Docking was performed for the binding affinities. Gas Chromatography-Mass Spectrometry (GC-MS) and Density Function Theory (DFT) were performed to evaluate the phytochemicals bioavailability and reactivity, respectively. The stability of protein-ligand complexes was examined by 50 ns MD simulations and MM/PBSA values were calculated. Out of 128 phytochemicals, 22 phytochemicals were selected following ADMET screening. Based on the binding energies and the number of H bonding the following 10 phytochemicals were suggested as potential inhibitors to N protein of NDV - cis-11,14-eicosadienoic acid methyl ester, aurantiamide acetate, α-tocopherol, 4,8,12,16-tetramethylheptadecan-4-olide, 3,7,11,15-tetramethyl-2-hexadecen-1-ol, β-amyrin, β-sitosterol-3-O-β-d-galactopyranoside, α-amyrin, pterygospermin and sitogluside. Furthermore, DFT results showed that the 4 pytochemicals - Cis-11,14-eicosadienoic acid methyl ester, aurantiamide acetate, α-tocopherol, and 3,7,11,15-tetramethyl-2-hexadecen-1-ol were most reactive and thus could be used as potential inhibitors of NDV N protein. Further studies are required to validate the selected four phytochemicals as drug candidates against NDV.

This study introduces a novel joint technique combining "parallel peptide synthesis" and "de novo sequencing", which facilitates the high-throughput screening and structure validation of biological peptides derived from food and traditional Chinese medicine (TCM). Sea cucumber (Stichopus japonicus) was used as a model organism, undergoing simulated gastrointestinal digestion followed by de novo sequencing to predict potential peptides. Subsequently, cost-effective filter pipette tips were innovatively employed as parallel reaction vessels, enabling efficient peptide synthesis through the microfluidic flow of amino acid solutions over the loaded resin. After high-throughput biological activity screening, peptide YPGQLT was identified as the most potent antioxidant and acetylcholinesterase (AChE) inhibitor. It was then subjected to a molecular docking study to further explore its potential ligand-receptor interactions. This synergistic effect highlights peptide YPGQLT as a promising candidate for Alzheimer's disease (AD) treatment, thereby enhancing the potential biomedical application of sea cucumber. Notably, de novo predicted sequences can be further verified by comparing their characteristics, such as retention time and MS/MS spectrum, with those from the standard reference synthesized using this parallel synthesis protocol.

Peptide metabolites are emerging biomolecules with numerous possibilities in biomaterial-based regenerative medicine due to their inherent bioactivities. These small, naturally occurring compounds are intermediates or byproducts of larger proteins and peptides, and they can have profound effects, such as antiviral therapeutics, proangiogenic agents, and regenerative medicinal applications. This study is among the first to focus on using thymosin β4 protein-derived metabolites to pioneer novel applications for peptide metabolites in biomaterials. This study found that the novel peptide metabolite acetyl-thymosin β4 (amino acid 1-17) (Ac-Tβ1-17) exhibited significant protease inhibition activity against SARS-CoV-2, surpassing its precursor protein. Additionally, Ac-Tβ1-17 demonstrated beneficial effects, such as cell proliferation, wound healing, and scavenging of reactive oxygen species (ROS) in human umbilical vein endothelial cells (HUVEC). Integrating Ac-Tβ1-17 into a peptide-based scaffold facilitated cell growth and angiogenesis inside the scaffold and through gradual release into the surrounding environment. The Ac-Tβ1-17 peptide treatment induced significant biochemical responses in HUVEC, increasing Akt, ERK, PI3K, MEK, and Bcl-2 gene expression and proangiogenic proteins. Ac-Tβ1-17 peptide treatment showed similar results in ex vivo by enhancing mouse fetal metatarsal growth and angiogenesis. These findings highlight the potential of natural protein metabolites to generate biologically active peptides, offering a novel strategy for enhancing biomaterial compatibility. This approach holds promise for developing therapeutic biomaterials using peptide metabolites, presenting exciting prospects for future research and applications.

Sepsis is a life-threatening disease caused by the dysregulation of the immune response. It is important to identify influential genes modulating the immune response in sepsis. In this study, we used P-NET, a biologically informed explainable artificial intelligence model, to evaluate the gene importance for sepsis. About 688 important genes were identified, and these genes were enriched in pathways involved in inflammation and immune regulation, such as the PI3K-Akt signalling pathway, necroptosis and the NF-κB signalling pathway. We further selected differentially expressed genes both at bulk and single-cell levels and found TIMP1, GSTO1 and MYL6 exhibited significant different expressions in multiple cell types. Moreover, the expression levels of these 3 genes were correlated with the abundance of important immune cells, such as M-MDSC cells. Further analysis demonstrated that these three genes were highly expressed in sepsis patients with worse outcomes, such as severe, non-survived and shock sepsis patients. Using a drug repositioning strategy, we found navitoclax, curcumin and rotenone could down-regulate and bind to these genes. In conclusion, TIMP1, GSTO1 and MYL6 may serve as promising biomarkers and targets for sepsis treatment.

Esophageal cancer is one of the most migratory, invasive, and lethal malignancies and has a poor prognosis, highlighting the urgent need to develop more effective drugs for its treatment. Given that PSMD14 and HDAC play an important role in the treatment of esophageal cancer, thiolutin is used as a lead compound to design and synthesize a series of dual-target PSMD14/HDAC small molecule inhibitors, aiming to discover more effective anti-esophageal cancer drugs. Through the in vitro screening of PSMD14/HDAC enzyme inhibitory activities of a series of thiolutin derivatives, it was found that compound 8b, with a linker length of 8 and a Zn2+-chelating group of 1,2-phenylenediamine, exhibited the most balanced inhibitory activity against PSMD14/HDAC.The impact of 8b on PSMD14/HDAC at the cellular level was evaluated, and its drug-like properties were further assessed in vivo. Compound 8b demonstrates balanced dual-target activity (PSMD14 IC50 = 238.7 ± 27 nM, HDAC1 IC50 = 141.2 ± 10.3 nM) and excellent cytotoxicity against esophageal cancer cells (IC50 = 30-250 nM), effectively reversing epithelial-mesenchymal transition in cancer cells. Moreover, 8b exhibited excellent pharmacokinetic characteristics. More importantly, in a nude mouse xenograft model with subcutaneous transplantation of KYSE 30 cells, compound 8b (0.8 mg/kg, BID, PO, TGI = 81 %; 0.8 mg/kg, Q3D, SC, TGI = 77 %) significantly inhibited tumor growth, outperforming single-agent or combination treatments, thereby highlighting the therapeutic advantages of dual-target inhibition. These findings highlight the potential of dual-target PSMD14/HDAC inhibitors as a promising strategy for developing anti-esophageal cancer drugs.

Bone cement implantation syndrome is a critical complication of orthopaedic surgery, characterised by hypotension and hypoxemia. This syndrome is hypothesised to result from obstruction caused by fat droplets and the biochemical release of histamine caused by bone cement components. This study aimed to elucidate the histamine release mechanism, focusing on Mas-related G protein-coupled receptor X2 expressed on mast cells, which is hypothesised to be activated by bone cement components. Using a mast cell-deficient mouse femur fracture model, we examined bone cement's effect on serum histamine. Rat basophil-like cells expressing Mas-related G protein-coupled receptor X2 were exposed to monomethyl methacrylate, a bone cement component, to assess degranulation via β-hexosaminidase release. Our findings demonstrated that histamine levels significantly increased in wild-type mice post-cement application, from 27.7 ± 11.1 to 35.3 ± 12.9 ng/mL (p = 0.016). Furthermore, Mas-related G protein-coupled receptor X2 expressing cells showed a marked increase in β-hexosaminidase release upon monomethyl methacrylate stimulation (p = 4.30 × 10-5). These results support the hypothesis that activating Mas-related G protein-coupled receptor X2 by monomethyl methacrylate contributes to bone cement implantation syndrome via histamine release. Bone cement implantation syndrome can manifest as a condition involving either peripheral vascular embolism, the release of chemical mediators, or a combination of both. Our research elucidates the role of chemical mediators, particularly histamine-induced vasodilation, in the pathophysiology of bone cement implantation syndrome, providing valuable insights that pave the way for targeted interventions to mitigate this severe complication during orthopaedic surgery.

The molecular and genetic mechanisms underlying vascular calcification remain unclear. This study aimed to determine the differences in calcification marker-related gene expression in macrophages.

The expression profiling datasets GSE104140 and GSE235995 were analysed to identify differentially expressed genes (DEGs) between fibroatheroma with calcification and diffuse intimal thickening. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, Weighted Gene Co-expression Network Analysis (WGCNA), and Gene Set Enrichment Analysis (GSEA) were performed to assess functional characteristics. Hub genes were identified through a protein-protein interaction (PPI) network and machine learning approaches. Single-cell RNA sequencing data (GSE159677) validated the expression of calcification-related genes in macrophages, while Mendelian randomization analysis explored their potential causal relationship with coronary calcification. Further validation was conducted using enzyme-linked immunosorbent assay (ELISA) on coronary calcification samples and immunohistochemistry in ApoE-/- mice. Intravascular ultrasound was performed to assess coronary calcification severity.

Two key biomarkers, ITGAX and MYD88, were identified as diagnostic indicators of cardiovascular calcification. Both biomarkers were significantly upregulated in calcified samples and were strongly associated with immune processes. Single-cell RNA sequencing confirmed their high expression in multiple immune cell types. Additionally, molecular docking analysis revealed that retinoic acid interacted with both biomarkers, suggesting potential therapeutic relevance. Immunohistochemical and ELISA analyses further validated their elevated expression in calcified samples. These findings provide novel insights into the molecular mechanisms of vascular calcification and highlight potential diagnostic and therapeutic targets.

In the present study, an efficient method for the expression and purification of recombinant HIV Rev protein with a C-terminal hexahistidine tag was proposed. Noteworthy, this method circumvents the precipitation of the protein into inclusion bodies and their subsequent aggregation during purification. It does not necessitate denaturing isolation conditions, in contrast to currently widely used protocols. As a result, protocols for HIV Rev isolation have been developed allowing the production of non-aggregated Rev protein in a good yield, high purity, and free of bacterial RNA impurities. This high-purity result became possible due to high salt extraction buffer usage. Complementary [α-32P]-labeled Rev response element (RRE) RNA has been synthesized and an inhibitor test system was developed based on Rev-RRE complex formation. We were able to reveal a novel class of potential Rev-RRE inhibitors based on dimeric benzimidazole derivatives and used those results to validate the testing system. The proposed protocols for screening and structure-activity relationship for new inhibitors of Rev binding to viral RNA broaden the scope of potential candidates for anti-HIV drug development.

Olfaction biosensors are playing crucial roles in detecting volatile organic compounds (VOCs) in various domains, while the response pattern of biosensors to different alcohols and the underlying reasons for the differences in response remain unclear. Herein, this study presents a sensitive electrochemical olfactory biosensor utilizing Drosophila odorant-binding protein (LUSH) as a sensing material for the detection of 11 alcohols with different molecular structures (alkyl chain lengths, hydroxyl group numbers, and cyclic alcohols) and phenol. The electrodes covalently immobilized with the LUSH proteins were characterized by atomic force microscopy (AFM), electrochemical impedance spectroscopy (EIS), and cyclic voltammetry (CV), and their ability to detect alcohols was investigated through EIS. Results showed that the LUSH modified biosensor exhibited ultrasensitive detection of multiple alcohols (detection limits: 10-100 fM), with linear ranges from 10-14 to 10-7 M and coefficients of determination (R2) of 0.948-0.992. In addition, the biosensor demonstrated high selectivity toward interfering compounds (selectivity coefficients <0.22), excellent reproducibility (relative standard deviation, RSD: 1.2%, n = 4 for parallel sensors), and good stability (response decreased by 10.2% on the 10th day). Notably, the sensitivity of the biosensor to alcohols showed alkyl chain-length dependence of n-alcohols and was influenced by the number of hydroxyl groups and the cyclic structure. More importantly, molecular docking revealed the binding modes, binding energies, and key amino acids involved in the LUSH-alcohol interaction and explained the response discrepancies.

Synergistic effect is one of the main properties of umami substances, as a new natural umami agent, umami peptide synergy has not been systematically explored. Presently, conventional methods relying on human sensory evaluation and intelligent instrument analysis pose challenges due to their time-consuming and lack of high throughput. This research provides a detailed molecular-level understanding of multiple umami peptides interact with T1R1-VFT simultaneous, revealing that multiple umami peptides promotes stronger binding affinity and more effective receptor activation (from -7.3 kcal mol-1 to -11.19 kcal mol-1). The kinetic simulations demonstrated a significant reduction in the average fluctuation of protein amino acid residues during the binding process. Moreover, the hydrophobic regions on the protein surface were diminished following binding, and the resultant complex structure was more tightly packed, these phenomena may collectively represent the manifestation of synergistic effects. To validate the simulation results, biolayer interferometry sensing strategies were developed to measure the interaction process, indicating that umami peptides and T1R1-VFT could association and dissociation in solution without significant interactions with other proteins. When multiple umami peptides interacted with T1R1-VFT, the kinetic equilibrium constant decreased and affinity increased (from 1.2 e-6 M to 8.3 e-7 M), showing significant synergistic effect. Furthermore, the practical application ability of this sensing strategy was verified in a complex matrix with multiple real samples. Overall, this comprehensive study combined micro-molecular simulation and biological experiment verification, offering a deeper understanding of umami peptide synergy and paving the way for innovative approaches in flavor science and food product development.

The design of new antimicrobial agents is an important challenge due to the growing resistance of microorganisms to existing antibiotics. In recent years, the trend towards the development of compounds and materials with (bio)degradable properties has emerged. In this work, we propose and develop a method for the synthesis of new peptidomimetics, i.e., water-soluble macrocyclic quaternary ammonium salts containing L-tyrosine fragments based on p-tert-butylthiacalix[4]arene in various stereoisomeric forms (cone, partial cone, and 1,3-alternate). These compounds have low cytotoxicity (IC50 = 80-267 μM) and high antibacterial activity (MIC = 0.5-15.6 μM) against Gram-positive bacterial strains including methicillin-resistant Staphylococcus aureus (MRSA). The obtained peptidomimetics can bind α-chymotrypsin with the formation of supramolecular systems and their subsequent degradation. Our results demonstrate the first example of multi-action thiacalixarene derivatives with antibacterial activity, protein binding ability and degradation induced by binding to α-chymotrypsin. The obtained results open the possibility of creating multi-action peptidomimetic systems with antimicrobial and biodegradable effect.

The relationship between prenatal exposure to per- and polyfluoroalkyl substances (PFASs), a well-known endocrine disruptor, and thyroid hormones (THs) levels remains unclear. Therefore, this study aimed to investigate this relationship in a birth cohort during the second trimester.

This prospective study included 562 pregnant women in the Wuxi Birth Cohort from 2019 to 2021 and quantified the serum concentrations of 23 PFASs and 5 THs. Multiple statistical models were used to assess the associations between individual or combined PFASs concentrations and THs, while molecular docking simulated the interactions between PFASs and four thyroid-related proteins.

The median concentration of ∑23PFASs was 71.91 ng/mL, with perfluorovaleric acid (PFPeA) (18.13 ng/mL) emerging as the predominant PFAS. Most PFASs were negatively associated with maternal free thyroxine (FT4) and thyroid-stimulating hormone (TSH) levels, whereas perfluorobutane sulfonate (PFBS) was positively correlated with TSH levels. A similar trend was observed in the weighted quantile sum (WQS) model, in which combined PFASs exposure was inversely associated with the FT4 and TSH levels. Molecular docking results showed that compared with TH natural ligand thyroxine (T4), perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), and 6:2 chlorinated polyfluorinated ether sulfonate (6:2 Cl- PFESA) exhibited relatively high binding affinity with thyroid-related proteins (-6.6 to -9.8 kcal/mol vs. T4: -5.6 to -8.6 kcal/mol). Furthermore, PFASs with medium chain lengths and sulfonic acid groups exhibited enhanced protein-binding properties.

PFASs exposure may affect THs homeostasis during pregnancy. Moreover, different types and concentrations of PFASs have different effects on THs in the maternal serum.

Rheumatoid arthritis (RA) is an inflammation-mediated autoimmune disease. Tinosporine (TIN), derived from Tinospora sinensis (Lour.) Merr. has significant anti-inflammatory and immunosuppressive effects. However, the anti-RA effect and mechanism of TIN have not been fully elucidated.

This study elucidates the mechanism of TIN in alleviating collagen-induced arthritis (CIA) in rats based on the gut microbiome-metabolomic-immunity axis.

We established CIA rat model to evaluate the efficacy of TIN. Based on 16S rRNA sequencing analysis, fecal metabolomics profiling and the concentrations of short-chain fatty acids determination to investigate the effect of TIN on gut microbiota composition and metabolome changes. Histopathology showed that TIN protected the intestinal barrier, then use ELISA and flow cytometry to analyze the mechanism of TIN, and qRT-PCR and WB were employed for verification.

TIN ameliorated joint damage and inflammation in CIA rats, histopathological observation confirmed that TIN had protective effect on intestinal barrier. 16S rRNA sequencing analysis and fecal metabolomics profiling confirmed that TIN intervention regulates the composition of gut microbiome and promote the propagation of probiotics, the abundance changes of 12 serum metabolites in the CIA group were reversed by TIN intervention. After intervention with TIN, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and caproic acid rise significantly, but acetic acid cannot be reversed. Then ELISA and flow cytometry confirmed that TIN intervention could regulate the level of inflammatory factors, maintain the integrity of the intestinal barrier and restoring the imbalance of Th1/Th2 and Th17/Treg ratios in the colon of CIA rats.

TIN inhibited the inflammatory response of CIA rats by regulating the gut microbiome-metabolome-immunity axis, reversed the abnormalities of intestinal flora and differential metabolites, and maintained the integrity of the intestinal barrier.

Percutaneous neoadjuvant therapy has proven effective in diminishing tumor size and the surgical intervention area, which couldeffectively mitigate the risk of tumor recurrence and enhance immunotherapy efficacy. Lenalidomide, an approved medication orally used to treat myeloma, was loaded into nanosuspensions-based hydrogels (Len-NBHs) for transdermal administration as a percutaneous neoadjuvant therapy. This study was designed to investigate the inhibitory effect and mechanism of Len-NBHs on melanoma. Network pharmacology and transcriptomic analyses identified key targets and signaling pathways. The effects of lenalidomide on melanoma were further verified through Western blotting, immunohistochemistry, immunofluorescence, and quantitative real-time polymerase chain reaction，using both in vitro cell experiments and in vivo melanoma mouse models. Lenalidomide could induce melanoma cells apoptosis, disrupt cell cycle progression, impede cell migration and invasion, and modify tumor microenvironment (TME). Mechanistically, lenalidomide reversed the abnormal activation of the PI3K-AKT signaling pathway and the overexpression of CD93, while also recruiting CD8+ T cells, CD4+ T cells, and dendritic cells to infiltrate the tumor site. Transdermal administration of Len-NBHs represents a promising adjuvant therapy for the treatment of malignant melanoma. Preoperative administration of Len-NBHs can inhibit the outward spread of melanoma, reduce tumor size, thereby decreasing the surgical excision area and improving patient survival rates and prognosis.

Focal adhesion kinase (FAK) is a critical drug target implicated in various disease pathways, including hematological malignancies and breast cancer. Therefore, identifying FAK inhibitors with novel scaffolds could offer new opportunities for developing effective therapeutic compounds. Herein, we disclosed the discovery of a new backbone inhibitor of FAK using an "internal" database, employing a structure-based high-transparency permeability virtual screening (HTVS) and a DeepDock algorithm based on geometric deep learning. Subsequently, molecular docking was conducted at different precisions to identify 10 compounds for further evaluation of biological activity. Ultimately, compound 4, a pyrimidin-4-amine derivative, demonstrated inhibitory activity against FAK and breast cancer cells, further supporting its potential as a FAK inhibitor. Moreover, molecular dynamics simulations were carried out to gain more detailed insights into the binding mechanism between compound 4 and FAK to guide subsequent structural optimization.

The compilation of ligand and structure-based molecular modeling methods has become an important practice in virtual screening applied to drug discovery. This systematic review addresses and ranks various virtual screening strategies to drive the selection of the optimal method for studies that have as their starting point a multi-ligand investigation and investigation based on the protein structure of a therapeutic target. This study shows examples of applications and an evaluation based on the objective and problematic of a series of virtual screening studies present in the ScienceDirect® database. The results showed that the molecular docking technique is widely used in scientific production, indicating that approaches that use protein structure as a starting point are the most promising strategy for drug discovery that relies on virtual screening-based research.

The Plasmodium falciparum Protein Kinase 6 (PfPK6) is a serine/threonine protein kinase categorized under the CMGC group, displaying both cyclin-dependent kinases (CDKs) and mitogen-activated protein kinases (MAPKs) activity. Previous research has indicated that PfPK6 is expressed during the trophozoite and schizont stages of the Plasmodium falciparum asexual blood stage. Unlike typical cyclin-dependent kinases, PfPK6 demonstrates kinase activity independent of cyclin, making it a promising target for drug identification. In this study, we utilized a computational approach to identify a novel PfPK6 inhibitor through virtual screening of small inhibitor compounds from diverse datasets, employing a structure-based approach and a Deep Learning (DL) model. The most promising inhibitor molecule, TCMDC-132409 from the Tres Cantos Antimalarial Set, exhibited a binding affinity of -13.553 kcal/mol against PfPK6. Additionally, a 200ns molecular dynamics simulation study confirmed the stability of the binding mode, indicating the potential of TCMDC-132409 as an antiplasmodial inhibitor for further investigation.

Theileriosis, caused by protozoan parasites of genus Theileria, primarily affects both domestic and wild ruminants. It can lead to significant economic losses in livestock farming due to decreased productivity and high mortality rates in susceptible animals, while treatment measures are not cost-effective. Since most of mechanisms of this disease remain unknown, this study investigates the differences in the mode of pathogenesis between transforming and non-transforming groups through an in silico comparative proteomics approach to recognize the key players involved in host cell transformation. Although the major biological processes and molecular functions are almost conserved between the two groups, PEST-motif containing secretory proteins of SfiI, SVSP, and Tash-AT gene families were identified as important candidates with the potential to transform infected host cells. Several members of PEST-motif containing proteins possess signal peptides, nuclear localization signals, and trans-membrane helices, further supporting their potential to transform host cells. Additionally, structural analysis helped in the identification of a parasitic protein (SfiIp) from SfiI family as a plausible drug target. Virtual screening revealed FDA-approved drugs (i.e. atogepant and rimegepant) as promising compounds, showing the highest affinity for SfiIp during molecular docking. Further studies, including molecular dynamics simulation, principal component analysis, and free energy landscape, suggested that these drug molecules exhibit the stable interaction with protein. Therefore, our research could facilitate the identification and targeting of novel drug candidates that may be further implemented to recognize effective therapeutics against Theileria infections.

Diet habits and nutrition quality significantly impact health and disease. Here is delve into the intricate relationship between diet habits, nutrition quality, and their direct impact on health and homeostasis. Focusing on (-)-Epicatechin, a natural flavanol found in various foods like green tea and cocoa, known for its positive effects on cardiovascular health and diabetes prevention. The investigation encompasses the absorption, metabolism, and distribution of (-)-Epicatechin in the human body, revealing a diverse array of metabolites in the circulatory system. Notably, (-)-Epicatechin demonstrates an ability to activate nitric oxide synthase (eNOS) through the G protein-coupled estrogen receptor (GPER). While the precise role of GPER and its interaction with classical estrogen receptors (ERs) remains under scrutiny, the study employs computational methods, including density functional theory, molecular docking, and molecular dynamics simulations, to assess the physicochemical properties and binding affinities of key (-)-Epicatechin metabolites with GPER. DFT analysis revealed distinct physicochemical properties among metabolites, influencing their reactivity and stability. Rigid and flexible molecular docking demonstrated varying binding affinities, with some metabolites surpassing (-)-Epicatechin. Molecular dynamics simulations highlighted potential binding pose variations, while MMGBSA analysis provided insights into the energetics of GPER-metabolite interactions. The outcomes elucidate distinct interactions, providing insights into potential molecular mechanisms underlying the effects of (-)-Epicatechin across varied biological contexts.

As the world's only commercially available paclitaxel liposome, Lipusu (Lip) has been clinically used in chemotherapy for >20 years, but the design concept of Lip remains largely unchanged since its initial development. Based on the study of Acetyl-CoA-carboxylase 1 (ACC1) in nasopharyngeal carcinoma (NPC), we proposed the concept of next-generation liposomes (NGL) utilizing lipid demand balance. In this study, we evaluated the feasibility of ACC1 and integrin αVβ3 as NPC targets, and designed 10 conjugates of 5-tetradecyloxy-2-furoic acid (TOFA) and c(RGDfK) that can bind to Lip. Considering the results of chemical parameter prediction, molecular docking, molecular dynamics simulation (MD) and other aspects, we finally selected and synthesized the compound F, and successfully constructed F-Lip by simple incubation method. Compared with Lip, F-Lip showed stronger toxicity in both HONE-1 cells and corresponding tumor-bearing mice. In conclusion, by regulating the balance of lipid demand, the toxicity of Lip can be improved so as to achieve the goal of inhibiting the proliferation of NPC. This study provides a new model for the future design and development of Lip.

NUDIX hydrolase 5 (NUDT5) is an enzyme involved in the hydrolysis of nucleoside diphosphates linked to other moieties, such as ADP-ribose. This cofactor is vital in redox reactions and is essential for the activity of sirtuins and poly(ADP-ribose) polymerases, which are involved in DNA repair and genomic stability. It has been shown that NUDT5 activity can also influence NAD+ homeostasis, thereby affecting cancer cell metabolism and survival. In this regard, the discovery of NUDT5 inhibitors has emerged as a potential therapeutic approach in cancer treatment. In this study, we conducted a high-throughput virtual screening of marine bacterial compounds against the NUDT5 enzyme and four molecules were selected based on their docking scores. These compounds established strong interactions within the NUDT5 active site, with molecular analysis highlighting the key role of Trp28A and Trp46B residues. Molecular dynamics simulations over 200 ns indicated a stable behavior, in association with root mean square deviation values always below 3 Å, suggesting conformational stability. Free energy landscape analysis further supported their potential as NUDT5 inhibitors, offering avenues for novel therapeutic strategies against NUDT5-associated breast cancer.

Demyelination is a common feature of numerous neurological disorders including multiple sclerosis and leukodystrophies. Although myelin can be regenerated spontaneously following injury, this process is often inadequate, potentially resulting in neurodegeneration and exacerbating neurological dysfunction. Several drugs aimed at promoting the differentiation of oligodendrocyte precursor cells (OPCs) have yielded unsatisfactory clinical effects. A recent study has shifted the strategy of pro-OPC differentiation towards enhancing myelinogenesis. In this study we identified the pro-myelinating drug using a zebrafish model. Five traditional Chinese medicine monomers including gastrodin, paeoniflorin, puerarin, salidroside and scutellarin were assessed by bath-application in Tg (MBP:eGFP-CAAX) transgenic line at 1-5 dpf. Among the 5 monomers, only gastrodin exhibited significant pro-myelination activity. We showed that gastrodin (10 µM) enhanced myelin sheath formation and oligodendrocyte (OL) maturation without affecting the number of OLs. Gastrodin markedly increased the phosphorylation levels of PI3K, AKT, and mTOR in primary cultured OLs via direct interaction with PI3K. Co-treatment with the PI3K inhibitor LY294002 (5 µM) mitigated gastrodin-induced OL maturation. Furthermore, injection of gastrodin (100 mg·kg-1·d-1, i.p.) effectively facilitated remyelination in a lysophosphatidylcholine-induced demyelinating mouse model and alleviated demyelination in the experimental autoimmune encephalomyelitis mice. These results identify gastrodin as a promising therapeutic agent for demyelinating diseases and highlight the potential of the zebrafish model for screening pro-myelinogenic pharmacotherapy.