Exploring the potential mechanism of WuFuYin against hypertrophic scar using network pharmacology and molecular docking.

BACKGROUND: Hypertrophic scar (HTS) is dermal fibroproliferative disorder, which may cause physiological and psychological problems. Currently, the potential mechanism of WuFuYin (WFY) in the treatment of HTS remained to be elucidated. AIM: To explore the potential mechanism of WFY in treating HTS. METHODS: Active components and corresponding targets were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform. HTS-related genes were obtained from the GeneCards, DisGeNET, and National Center for Biotechnology Information. The function of targets was analyzed by performing Gene Ontology and Kyoto Encyclopaedia of Genes and Genome (KEGG) enrichment analysis. A protein + IBM-protein interaction (PPI) network was developed using STRING database and Cytoscape. To confirm the high affinity between compounds and targets, molecular docking was performed. RESULTS: A total of 65 core genes, which were both related to compounds and HTS, were selected from multiple databases. PPI analysis showed that CKD2, ABCC1, MMP2, MMP9, glycogen synthase kinase 3 beta (GSK3B), PRARG, MMP3, and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma (PIK3CG) were the hub targets and MOL004941, MOL004935, MOL004866, MOL004993, and MOL004989 were the key compounds of WFY against HTS. The results of KEGG enrichment analysis demonstrated that the function of most genes were enriched in the PI3K-Akt pathway. Moreover, by performing molecular docking, we confirmed that GSK3B and 8-prenylated eriodictyol shared the highest affinity. CONCLUSION: The current findings showed that the GSK3B and cyclin dependent kinase 2 were the potential targets and MOL004941, MOL004989, and MOL004993 were the main compounds of WFY in HTS treatment.

Prediction and analysis of components and functions of Ixeris chinensis based on network pharmacology and molecular docking.

Background: It is reported that the Ixeris chinensis has high medicinal value, but there are few reports about its potential molecular mechanism. We used a network pharmacology approach to predict the active ingredients, targets of action and possible interventions in diseases of Ixeris chinensis. Methods: We employed various databases and software to predict the active ingredients, target genes, protein interactions, signaling pathways, network diagrams, and molecular docking of Ixeris chinensis. Simultaneously, we searched multiple Chinese and English databases and conducted meta-analyses of five randomized controlled trials. Results: The analysis results revealed 12 effective components, including apigenin ß-sitosterol, baicalin, baicalein, and luteolin; and selected 40 key targets, including AKT1, TNF, EGFR, ESR1, SRC, among others. GO analysis generated 225 biological processes, 39 cellular components, and 65 molecular functions; KEGG analysis revealed 103 signaling pathways. Molecular docking results indicated that the main active components of Ixeris chinensis can bind well with key targets. Five randomized controlled trials were included. Meta-analysis showed that Ixeris extract can effectively reduce animal blood lipid levels. Conclusion: This study revealed the main active ingredients and key targets of Ixeris chinensis, analyzed the signaling pathways of potential targets, conducted disease prediction, and performed molecular docking prediction, providing a basis for research on the pathways of Ixeris treatment for related diseases and subsequent new drug development.

Identification of immune-related genes and small-molecule drugs in hypertension-induced left ventricular hypertrophy based on machine learning algorithms and molecular docking.

Background: Left ventricular hypertrophy (LVH) is a common consequence of hypertension and can lead to heart failure. The immune response plays an important role in hypertensive LVH; however, there is no comprehensive method to investigate the mechanistic relationships between immune response and hypertensive LVH or to find novel therapeutic targets. This study aimed to screen hub immune-related genes involved in hypertensive LVH as well as to explore immune target-based therapeutic drugs. Materials and methods: RNA-sequencing data from a mouse model generated by angiotensin II infusion were subjected to weighted gene co-expression network analysis (WGCNA) to identify core expression modules. Machine learning algorithms were applied to screen immune-related LVH characteristic genes. Heart structures were evaluated by echocardiography and cardiac magnetic resonance imaging (CMRI). Validation of hub genes was conducted by RT-qPCR and western blot. Using the Connectivity Map database and molecular docking, potential small-molecule drugs were explored. Results: A total of 1215 differentially expressed genes were obtained, most of which were significantly enriched in immunoregulation and collagen synthesis. WGCNA and multiple machine learning strategies uncovered six hub immune-related genes (Ankrd1, Birc5, Nuf2, C1qtnf6, Fcgr3, and Cdca3) that may accurately predict hypertensive LVH diagnosis. Immune analysis revealed that fibroblasts and macrophages were closely correlated with hypertensive LVH, and hub gene expression was significantly associated with these immune cells. A regulatory network of transcription factor-mRNA and a ceRNA network of miRNA-lncRNA was established. Notably, six hub immune-related genes were significantly increased in the hypertensive LVH model, which were positively linked to left ventricle wall thickness. Finally, 12 small-molecule compounds with the potential to reverse the high expression of hub genes were ruled out as potential therapeutic agents for hypertensive LVH. Conclusion: This study identified and validated six hub immune-related genes that may play essential roles in hypertensive LVH, providing new insights into the potential pathogenesis of cardiac remodeling and novel targets for medical interventions.

Pseudomonas aeruginosa Recombinant L-asparaginase: PEGylation with Low Molecular Weight Polyethylene Glycol, Molecular Dynamics Simulation, In vitro and In vivo Serum half-life and Biochemical Characterization.

BACKGROUND: Microbial L-asparaginase (L-ASNase, EC 3.5.1.1) is a pivotal biopharmaceutical drug-protein that catalyzes the hydrolysis of the non-essential amino acid L-asparagine (L-Asn) into L-aspartic acid (L-Asp) and ammonia , resulting in deplenishing the cellular L-Asn pool, which leads to the ultimate death of the L-asparagine synthetase (L-ASNS) deficient cancerous cells. OBJECTIVE: This study aimed to investigate the impact of conjugating low molecular weight polyethylene glycol to recombinant P. aeruginosa L-ASNase by examining the pharmacokinetic properties, affinity towards the substrate, and enzyme stability prior to and following the reaction. METHODS: The recombinant P. aeruginosa L-ASNase was affinity purified and then PEGylated by attaching polyethylene glycol (MW= 330 Da) site-specifically to the protein's N-terminus end. After which, the PEGylated L-ASNase was examined by SDS-PAGE (15%), FTIR, and UV/Vis spectrophotometry and subsequently biochemically characterized. RESULTS: The Km and Vmax values of free P. aeruginosa rL-ASNase were determined to be 0.318 ±1.76 mM and 2915 µmol min-1and following the PEGylation, they were found to be 0.396 ±1.736 mM and 3193 µmol min-1, respectively. Polyethylene glycol (330 Da) has markedly enhanced LASNase thermostability at 37, 45, 50, and 55 °C, as opposed to the free enzyme, which retained 19.5% after 1 h of incubation at 37 °C. The PEGylated L-ASNase was found to be stable upon incubation with human serum for 28 h, in contrast to the sharp decline in the residual bioactivity of the free rL-ASNase after 4 h incubation. Accordingly, an in vivo study was used for validation, and it demonstrated that PEGylated rL-ASNase exhibited longer bioactivity for 24 h, while the free form's activity vanished entirely from the rats' blood sera after 8 h. Molecular dynamics simulation indicated that PEG (330 Da) has affected the hydrodynamic volume of L-ASNase and increased its structural stability. Docking analysis has explored the position of PEG with respect to binding sites and predicted a similar binding affinity to that of the free enzyme. CONCLUSION: For the first time, recombinant L-ASNase was modified by covalently attaching PEG (330 Da). The resultant novel proposed PEGylated rL-ASNase with remarkably increased stability and prolonged in vivo half-life duration, which could be considered an alternative to mitigate the high molecular weight of PEGylation's drawbacks.

Network Pharmacology and Molecular Docking Integrated with Molecular Dynamics Simulations Investigate the Pharmacological Mechanism of Yinchenhao Decoction in the Treatment of Non-alcoholic Fatty Liver Disease.

BACKGROUND: Non-Alcoholic Fatty Liver Disease (NAFLD) has become a significant health and economic burden globally. Yinchenhao decoction (YCHD) is a traditional Chinese medicine formula that has been validated to exert therapeutic effects on NAFLD. OBJECT: The current study aimed to explore the pharmacological mechanisms of YCHD on NAFLD and further identify the potential active compounds acting on the main targets. METHODS: Compounds in YCHD were screened and collected from TCMSP and published studies, and their corresponding targets were obtained from the SWISS and SEA databases. NAFLD-related targets were searched in the GeneCards and DisGeNet databases. The "compound- intersection target" network was constructed to recognize the key compounds. Moreover, a PPI network was constructed to identify potential targets. GO and KEGG analyses were performed to enrich the functional information of the intersection targets. Then, molecular docking was used to identify the most promising compounds and targets. Finally, molecular dynamics (MD) simulations were performed to verify the binding affinity of the most potential compounds with the key targets. RESULTS: A total of 53 compounds and 556 corresponding drug targets were collected. Moreover, 2684 NAFLD-related targets were obtained, and 201 intersection targets were identified. Biological processes, including the apoptotic process, inflammatory response, xenobiotic metabolic process, and regulation of MAP kinase activity, were closely related to the treatment of NAFLD. Metabolic pathways, non-alcoholic fatty liver disease, the MAPK signaling pathway, and the PI3K-Akt signaling pathway were found to be the key pathways. Molecular docking showed that quercetin and isorhamnetin were the potential active compounds, while AKT1, IL1B, and PPARG were the most promising targets. MD simulations further verified that the binding of PPARG-isorhamnetin (-35.96 ± 1.64 kcal/mol) and AKT1-quercetin (-31.47 ± 1.49 kcal/mol) was due to their lowest binding free energy. CONCLUSION: This study demonstrated that YCHD exerts therapeutic effects for the treatment of NAFLD through multiple targets and pathways, providing a theoretical basis for further pharmacological research on the potential mechanisms of YCHD in NAFLD.

In silico Screening and in vitro Cytotoxicity Study of Achyranthes aspera Phytochemicals Against Oral Cancer: A Possible Step towards the Development of Anti-cancer Agents.

BACKGROUND: Oral cancer poses a significant threat to public health worldwide. In addition, because many chemotherapy treatments have negative side effects, natural herbs may be beneficial for oral cancer therapy. Achyranthes aspera (AA), a potential medicinal herb, exerts various pharmacological and biochemical activities. OBJECTIVE: The present study aimed to predict the anti-oral cancer potential of AA using in silico tools and cell death by in vitro testing. METHODS: A total of fourteen bioactive constituents from AA herb were selected using phytochemical databases. The toxicity of AA herb extract was analysed through MTT assay against oral carcinoma A253 cell line. The binding activities of the phytocomponents against serine/ threonine-specific protein kinases isoforms, namely Akt1 (PDB ID: 3qkk) and Akt2 (PDB ID: 2jdo) proteins, were analysed using Discovery Studio 2021 and PyRx docking software. RESULTS: Cell viability data revealed that AA extract decreased the viability and reduced the number of live cells of the oral carcinoma A253 cell line in a dose-dependent manner. The halfmaximal concentration (IC50) value of AA was assessed as 204.74 µg/ml. Based on binding affinity, saponin C (-CDOCKER energy = -77.9862), oleanolic acid (-CDOCKER energy = - 49.4349), spinasterol (-CDOCKER energy = -38.1246), 36,47-dihydroxyhenpentacontan-4-one (-CDOCKER energy = -32.4386), and 20-hydroxyecdysone (-CDOCKER energy = -31.9138) were identified as the best compounds against Akt1, while, compounds saponin C (-CDOCKER energy = -134.412), oleanolic acid (-CDOCKER energy = -90.0846), spinasterol (-CDOCKER energy = -78.3213), 20-hydroxyecdysone (-CDOCKER energy = -80.1049), and ecdysone (- CDOCKER energy = -73.3885) were identified as Akt2 inhibitors. These top compounds fulfilled drug score values, pharmacokinetic and physicochemical characteristics, and druglikeness parameters. CONCLUSION: The present findings reveal that the lead phytomolecules of AA could be effective and developed as a prospective drug against oral cancer.

Luteolin enhances drug chemosensitivity by downregulating the FAK/PI3K/AKT pathway in paclitaxel­resistant esophageal squamous cell carcinoma.

Drug resistance is a key factor underlying the failure of tumor chemotherapy. It enhances the stem­like cell properties of cancer cells, tumor metastasis and relapse. Luteolin is a natural flavonoid with strong anti­tumor effects. However, the mechanism(s) by which luteolin protects against paclitaxel (PTX)­resistant cancer cell remains to be elucidated. The inhibitory effect of luteolin on the proliferation of EC1/PTX and EC1 cells was detected by cell counting kit­8 assay. Colony formation and flow cytometry assays were used to assess clonogenic capacity, cell cycle and apoptosis. Wound healing and Transwell invasion tests were used to investigate the effects of luteolin on the migration and invasion of EC1/PTX cells. Western blotting was used to detect the protein levels of EMT­related proteins and stem cell markers after sphere formation. Parental cells and drug­resistant cells were screened by high­throughput sequencing to detect the differential expression of RNA and differential genes. ELISA and western blotting were used to verify the screened PI3K/Akt signaling pathway, key proteins of which were explored by molecular docking. Hematoxylin and eosin staining and TUNEL staining were used to observe tumor xenografts on morphology and apoptosis in nude mice. The present study found that luteolin inhibited tumor resistance (inhibited proliferation, induced cell cycle arrest and apoptosis and hindered migration invasion, EMT and stem cell spherification) in vitro in PTX­resistant esophageal squamous cell carcinoma (ESCC) cells. In addition, luteolin enhanced drug sensitivity and promoted the apoptosis of drug­resistant ESCC cells in combination with PTX. Mechanistically, luteolin may inhibit the PI3K/AKT signaling pathway by binding to the active sites of focal adhesion kinase (FAK), Src and AKT. Notably, luteolin lowered the tumorigenic potential of PTX­resistant ESCC cells but did not show significant toxicity in vivo. Luteolin enhanced drug chemosensitivity by downregulating the FAK/PI3K/AKT pathway in PTX­resistant ESCC and could be a promising agent for the treatment of PTX­resistant ESCC cancers.

Multireceptor Analysis for Evaluating the Antidiabetic Efficacy of Karanjin: A Computational Approach.

BACKGROUND: Diabetes mellitus, notably type 2, is a rising global health challenge, prompting the need for effective management strategies. Common medications such as metformin, insulin, repaglinide and sitagliptin can induce side effects like gastrointestinal disturbances, hypoglycemia, weight gain and specific organ risks. Plant-derived therapies like Karanjin from Pongamia pinnata present promising alternatives due to their historical use, holistic health benefits and potentially fewer adverse effects. This study employs in silico analysis to explore Karanjin's interactions with diabetes-associated receptors, aiming to unveil its therapeutic potential while addressing the limitations and side effects associated with conventional medications. METHODOLOGY: The research encompassed the selection of proteins from the Protein Data Bank (PDB), followed by structural refinement processes and optimization. Ligands such as Karanjin and standard drugs were retrieved from PubChem, followed by a comprehensive analysis of their ADMET profiling and pharmacokinetic properties. Protein-ligand interactions were evaluated through molecular docking using AutoDockTools 1.5.7, followed by the analysis of structural stability using coarse-grained simulations with CABS Flex 2.0. Molecular dynamics simulations were performed using Desmond 7.2 and the OPLS4 force field to explore how Karanjin interacts with proteins over 100 nanoseconds, focusing on the dynamics and structural stability. RESULTS: Karanjin, a phytochemical from Pongamia pinnata, shows superior drug candidate potential compared to common medications, offering advantages in efficacy and reduced side effects. It adheres to drug-likeness criteria and exhibits optimal ADMET properties, including moderate solubility, high gastrointestinal absorption and blood-brain barrier penetration. Molecular docking revealed Karanjin's highest binding energy against receptor 3L2M (Pig pancreatic alpha-amylase) at -9.1 kcal/mol, indicating strong efficacy potential. Molecular dynamics simulations confirmed stable ligand-protein complexes with minor fluctuations in RMSD and RMSF, suggesting robust interactions with receptors 3L2M. CONCLUSION: Karanjin demonstrates potential in pharmaceutical expansion for treating metabolic disorders such as diabetes, as supported by computational analysis. Prospects for Karanjin in pharmaceutical development include structural modifications for enhanced efficacy and safety. Nanoencapsulation may improve bioavailability and targeted delivery to pancreatic cells, while combination therapies could optimize treatment outcomes in diabetes management. Clinical trials and experimental studies are crucial to validate its potential as a novel therapeutic agent.

Computational Insights into Papaveroline as an In Silico Drug Candidate for Alzheimer's Disease via Fyn Tyrosine Kinase Inhibition.

Alzheimer's disease (AD) poses a significant global health challenge, necessitating the exploration of novel therapeutic strategies. Fyn Tyrosine Kinase has emerged as a key player in AD pathogenesis, making it an attractive target for drug development. This study focuses on investigating the potential of Papaveroline as a drug candidate for AD by targeting Fyn Tyrosine Kinase. The research employed high-throughput virtual screening and QSAR analysis were conducted to identify compounds with optimal drug-like properties, emphasizing adherence to ADMET parameters for further evaluation. Molecular dynamics simulations to analyze the binding interactions between Papaveroline and Staurosporine with Fyn Tyrosine Kinase over a 200-ns period. The study revealed detailed insights into the binding mechanisms and stability of the Papaveroline-Fyn complex, showcasing the compound's potential as an inhibitor of Fyn Tyrosine Kinase. Comparative analysis with natural compounds and a reference compound highlighted Papaveroline's unique characteristics and promising therapeutic implications for AD treatment. Overall, the findings underscore Papaveroline's potential as a valuable drug candidate for targeting Fyn Tyrosine Kinase in AD therapy, offering new avenues for drug discovery in neurodegenerative diseases. This study contributes to advancing our understanding of molecular interactions in AD pathogenesis and paves the way for further research and development in this critical area.

Comprehensive analysis of hub genes associated with cisplatin-resistance in ovarian cancer and screening of therapeutic drugs through bioinformatics and experimental validation.

BACKGROUND: To identify key genes associated with cisplatin resistance in ovarian cancer, a comprehensive analysis was conducted on three datasets from the GEO database and through experimental validation. METHODS: Gene expression profiles were retrieved from the GEO database. DEGs were identified by comparing gene expression profiles between cisplatin-sensitive and resistant ovarian cancer cell lines. The identified genes were further subjected to GO, KEGG, and PPI network analysis. Potential inhibitors of key genes were identified through methods such as LibDock nuclear molecular docking. In vitro assays and RT-qPCR were performed to assess the expression levels of key genes in ovarian cancer cell lines. The sensitivity of cells to chemotherapy and proliferation of key gene knockout cells were evaluated through CCK8 and Clonogenic assays. RESULTS: Results showed that 12 genes influenced the chemosensitivity of the ovarian cancer cell line SKOV3, and 9 genes were associated with the prognosis and survival outcomes of ovarian cancer patients. RT-qPCR results revealed NDRG1, CYBRD1, MT2A, CNIH3, DPYSL3, and CARMIL1 were upregulated, whereas ERBB4, ANK3, B2M, LRRTM4, EYA4, and SLIT2 were downregulated in cisplatin-resistant cell lines. NDRG1, CYBRD1, and DPYSL3 knock-down significantly inhibited the proliferation of cisplatin-resistant cell line SKOV3. Finally, photofrin, a small-molecule compound targeting CYBRD1, was identified. CONCLUSION: This study reveals changes in the expression level of some genes associated with cisplatin-resistant ovarian cancer. In addition, a new small molecule compound was identified for the treatment of cisplatin-resistant ovarian cancer.

Crocin-Phospholipid Complex: Molecular Docking, Molecular Dynamics Simulation, Preparation, Characterization, and Antioxidant Activity.

Background: Crocin is a water-soluble carotenoid compound present in saffron (Crocus sativus L.), known for its wide range of pharmacological activities, including cardioprotective, hepatoprotective, anti-tumorigenic, anti-atherosclerosis, and anti-inflammatory effects. Objectives: The instability of crocin, its low miscibility with oils, and poor bioavailability pose challenges for its pharmaceutical applications. This study aimed to design and prepare a crocin-phospholipid complex (CPC) and assess its physicochemical properties. Methods: The study investigated the formation of the complex and its binding affinity through molecular docking. Molecular dynamics (MD) simulations were conducted to find the optimal molar ratio of crocin to phospholipid for the complex's preparation. The CPC was produced using the solvent evaporation method. Techniques such as X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), field-emission scanning electron microscopy (FE-SEM), nuclear magnetic resonance (NMR), and solubility studies were utilized to characterize and confirm the formation of CPC. Additionally, the in vitro antioxidant activity of crocin and CPC was evaluated. Results: Molecular dynamic simulations explored molar ratios of 1: 1, 1: 1.5, and 1: 2 for crocin to phospholipid. The ratio of 1: 2 was found to be the most stable, exhibiting the highest probability of hydrogen bond formation. Molecular docking, FTIR, and NMR studies indicated hydrogen bond interactions between crocin and phospholipid, confirming CPC's formation. XRD and FE-SEM analyses showed a decrease in crocin's crystallinity within the phospholipid complex. Furthermore, the solubility of crocin in n-octanol was enhanced post-complexation, indicating an increase in crocin's lipophilic nature. Conclusions: Phospholipid complexation emerges as a promising technique for enhancing the physicochemical characteristics of crocin.

Natural products can be potential inhibitors of metalloproteinase II from Bacteroides fragilis to intervene colorectal cancer.

Bacteroides fragilis, a gram negative and obligate anaerobe bacterium, is a member of normal gut microbiota and facilitates many essential roles being performed in human body in normal circumstances specifically in Gastrointestinal or GI tract. Sometimes, due to genetics, epigenetics, and environmental factors, Bacteroides fragilis and their protein(s) start interacting with intestinal epithelium thus damaging the lining leading to colorectal cancers (CRC). To identify these protein(s), we incorporated a novel subtractive proteomics approach in the study. Metalloproteinase II (MPII), a Bacteroides fragilis toxin (bft), was investigated for its virulence and unique pathways to demonstrate its specificity and uniqueness in pathogenicity followed by molecular docking against a set of small drug-like natural molecules to discover potential inhibitors against the toxin. All these identified inhibitor-like molecules were analyzed for their ADMET calculations and detailed physiochemical properties to predict their druggability, GI absorption, blood brain barrier and skin permeation, and others. Resultantly, a total of ten compounds with the least binding energies were obtained and were subjected to protein-compound interaction analysis. Interaction analysis revealed the most common ligand-interacting residues in MPII are His 345, Glu 346, His 339, Gly 310, Tyr 341, Pro 340, Asp 187, Phe 309, Lys 307, Ile 185, Thr 308, and Pro 184. Therefore, top three compounds complexed with MPII having best binding energies were selected in order to analyze their trajectories. RMSD, RMSF, Rg and MMPBSA analysis revealed that all compounds showed good binding and keeping the complex stable and compact throughout the simulation time in addition to all properties and qualities of being a potential inhibitor against MPII.

Evaluation of suitability and biodegradability of the organophosphate insecticides to mitigate insecticide pollution in onion farming.

Organophosphates constitute a major class of pesticides widely employed in agriculture to manage insect pests. Their toxicity is attributed to their ability to inhibit the functioning of acetylcholinesterase (AChE), an essential enzyme for normal nerve transmission. Organophosphates, especially chlorpyrifos, have been a key component of the integrated pest management (IPM) in onions, effectively controlling onion maggot Delia antiqua, a severe pest of onions. However, the growing concerns over the use of this insecticide on human health and the environment compelled the need for an alternative organophosphate and a potential microbial agent for bioremediation to mitigate organophosphate pesticide pollution. In the present study, chloropyrifos along with five other organophosphate insecticides, phosmet, primiphos-methyl, isofenphos, iodofenphos and tribuphos, were screened against the target protein AChE of D. antiqua using molecular modeling and docking techniques. The results revealed that iodofenphos showed the best interaction, while tribuphos had the lowest interaction with the AChE based on comparative binding energy values. Further, protein-protein interaction analysis conducted using the STRING database and Cytoscap software revealed that AChE is linked with a network of 10 different proteins, suggesting that the function of AChE is disrupted through interaction with insecticides, potentially leading to disruption within the network of associated proteins. Additionally, an in silico study was conducted to predict the binding efficiency of two organophosphate degrading enzymes, organophosphohydrolase (OpdA) from Agrobacterium radiobacter and Trichoderma harzianum paraoxonase 1 like (ThPON1-like) protein from Trichoderma harzianum, with the selected insecticides. The analysis revealed their potential to degrade the pesticides, offering a promising alternative before going for cumbersome onsite remediation.

Phytochemical analysis, in-vitro and in-silico study of antiproliferative activity of ethyl acetate fraction of Launaea cornuta (Hochst. ex Oliv. & Hiern) C. Jeffrey against human cervical cancer cell line.

Introduction: Cervical cancer is one of the leading causes of death among women globally due to the limitation of current treatment methods and their associated adverse side effects. Launaea cornuta is used as traditional medicine for the treatment of a variety of diseases including cancer. However, there is no scientific validation on the antiproliferative activity of L. cornuta against cervical cancer. Objective: This study aimed to evaluate the selective antiproliferative, cytotoxic and antimigratory effects of L. cornuta and to explore its therapeutical mechanisms in human cervical cancer cell lines (HeLa-229) through a network analysis approach. Materials and methods: The cytotoxic effect of L. cornuta ethyl acetate fraction on the proliferation of cervical cancer cells was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) bioassay and the antimigratory effect was assessed by wound healing assays. Compounds were analysed using the qualitative colour method and gas chromatography-mass spectroscopy (GC-MS). Subsequently, bioinformatic analyses, including the protein-protein interaction (PPI) network analysis, Gene Ontology (GO), and Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis, were performed to screen for potential anticervical cancer therapeutic target genes of L. cornuta. Molecular docking (MD) was performed to predict and understand the molecular interactions of the ligands against cervical cancer. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to validate the network analysis results. Results: L. cornuta ethyl acetate fraction exhibited a remarkable cytotoxic effect on HeLa-229 proliferation (IC50 of 20.56 ± 2.83 µg/mL) with a selectivity index (SI) of 2.36 with minimal cytotoxicity on non-cancerous cells (Vero-CCL 81 (IC50 of 48.83 ± 23.02). The preliminary screening revealed the presence of glycosides, phenols, saponins, terpenoids, quinones, and tannins. Thirteen compounds were also identified by GC-MS analysis. 124 potential target genes associated with the effect of L. cornuta ethyl acetate fraction on human cervical cancer were obtained, including AKT1, MDM2, CDK2, MCL1 and MTOR were identified among the top hub genes and PI3K/Akt1, Ras/MAPK, FoxO and EGFR signalling pathways were identified as the significantly enriched pathways. Molecular docking results showed that stigmasteryl methyl ether had a good binding affinity against CDK2, ATK1, BCL2, MDM2, and Casp9, with binding energy ranging from -7.0 to -12.6 kcal/mol. Tremulone showed a good binding affinity against TP53 and P21 with -7.0 and -8.0 kcal/mol, respectively. This suggests a stable molecular interaction of the ethyl acetate fraction of L. cornuta compounds with the selected target genes for cervical cancer. Furthermore, RT-qPCR analysis revealed that CDK2, MDM2 and BCL2 were downregulated, and Casp9 and P21 were upregulated in HeLa-229 cells treated with L. cornuta compared to the negative control (DMSO 0.2%). Conclusion: The findings indicate that L. cornuta ethyl acetate fraction phytochemicals modulates various molecular targets and pathways to exhibit selective antiproliferative and cytotoxic effects against HeLa-229 cells. This study lays a foundation for further research to develop innovative clinical anticervical cancer agents.

Exploration of Berberine Against Ulcerative Colitis via TLR4/NF-κB/HIF-1α Pathway by Bioinformatics and Experimental Validation.

Purpose: This study aimed to delineate the molecular processes underlying the therapeutic effects of berberine on UC by employing network pharmacology tactics, molecular docking, and dynamic simulations supported by empirical validations both in vivo and in vitro. Patients and Methods: We systematically screened potential targets and relevant pathways affected by berberine for UC treatment from comprehensive databases, including GeneCards, DisGeNET, and GEO. Molecular docking and simulation protocols were used to assess the interaction stability between berberine and its principal targets. The predictions were validated using both a DSS-induced UC mouse model and a lipopolysaccharide (LPS)-stimulated NCM460 cellular inflammation model. Results: Network pharmacology analysis revealed the regulatory effect of the TLR4/NF-κB/HIF-1α pathway in the ameliorative action of berberine in UC. Docking and simulation studies predicted the high-affinity interactions of berberine with pivotal targets: TLR4, NF-κB, HIF-1α, and the HIF inhibitor KC7F2. Moreover, in vivo analyses demonstrated that berberine attenuates clinical severity, as reflected by decreased disease activity index (DAI) scores, reduced weight loss, and mitigated intestinal inflammation in DSS-challenged mice. These outcomes include suppression of the proinflammatory cytokines IL-6 and TNF-α and downregulation of TLR4/NF-κB/HIF-1α mRNA and protein levels. Correspondingly, in vitro findings indicate that berberine decreases cellular inflammatory injury and suppresses TLR4/NF-κB/HIF-1α signaling, with notable effectiveness similar to that of the HIF-1α inhibitor KC7F2. Conclusion: Through network pharmacology analysis and experimental substantiation, this study confirmed that berberine enhances UC treatment outcomes by inhibiting the TLR4/NF-κB/HIF-1α axis, thereby mitigating inflammatory reactions and improving colonic pathology.

Integrating network pharmacology with experimental validation to investigate the mechanism of Wuwei Zishen formula in improving perimenopausal syndrome.

OBJECTIVES: To investigate the role of the Wuwei Zishen formula (WWZSF) in treating and preventing perimenopausal syndrome (PMS) and to understand its mechanism. METHODS: Network pharmacology and molecular docking was used to predict active compounds, potential targets, and pathways for PMS treatment using WWZSF. Female Sprague-Dawley (SD) rats were induced with D-galactose (D-gal) to establish a PMS model and treated with Kunbao pill (KBP) and WWZSF. Estrus cycles were observed using vaginal smears. Serum sex hormones were measured using the enzyme-linked immunosorbent assay (ELISA). Histological changes in the uterus and ovaries were evaluated using hematoxylin-eosin staining (HE). Western blot was used to assess the protein expression levels of Cleaved Caspase-3, p62, BAX/Bcl-2, p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR in the uterus and ovaries. RESULTS: A total of 70 active compounds and 440 potential targets were screened out. Important targets and pathways, including AKT1, Bcl-2, Caspase-3, mTOR, and the PI3K/AKT/mTOR pathways, and molecular docking verified their high affinities to key WWZSF components. In vivo experiments showed that WWZSF can ameliorate the morphological abnormalities of the uterus and ovaries, increase sex hormone levels and organ index, and restore the estrus cycles in PMS rats. Moreover, the western blot results showed decreased Cleaved Caspase-3 and BAX/Bcl-2 protein levels in the ovarian and uterine tissues after WWZSF therapy. Concurrently, there was an increase in the expression of p62 and the ratios of p-AKT/AKT, p-mTOR/mTOR, and p-PI3K/PI3K. CONCLUSION: The PI3K/AKT/mTOR signaling pathway-mediated apoptosis and autophagy pathways may be how WWZSF efficiently reduces PMS.

In-silico study MM/GBSA binding free energy and molecular dynamics simulation of some designed remdesivir derivatives as the inhibitory potential of SARS-CoV-2 main protease.

Background and purpose: Coronavirus disease (COVID-19) is one of the greatest challenges of the twentieth century. Recently, in silico tools help to predict new inhibitors of SARS-CoV-2. In this study, the new compounds based on the remdesivir structure (12 compounds) were designed. Experimental approach: The main interactions of remdesivir and designed compounds were investigated in the 3CLpro active site. The binding free energy of compounds by the MM-GBSA method was calculated and the best compound (compound 12 with the value of -88.173 kcal/mol) was introduced to the molecular dynamics simulation study. Findings/Results: The simulation results were compared with the results of protein simulation without the presence of an inhibitor and in the presence of remdesivir. Additionally, the RMSD results for the protein backbone showed that compound 12 in the second 50 nanoseconds has less fluctuation than the protein alone and in the presence of remdesivir, which indicates the stability of the compound in the active site of the Mpro protein. Furthermore, protein compactness was investigated in the absence of compounds and the presence of compound 12 and remdesivir. The Rg diagram shows a fluctuation of approximately 0.05 A, which indicates the compressibility of the protein in the presence and absence of compounds. The results of the RMSF plot also show the stability of essential amino acids during protein binding. Conclusion and implications: Supported by the theoretical results, compound 12 could have the potential to inhibit the 3CLpro enzyme, which requires further in vitro studies and enzyme inhibition must also be confirmed at protein levels.

In silico molecular modeling and in vitro biological screening of novel benzimidazole-based piperazine derivatives as potential acetylcholinesterase and butyrylcholinesterase inhibitors.

New series of benzimidazole incorporating piperazine moieties in single molecular framework has been reported. The structures of the synthesized derivatives were assigned by 1H-NMR, 13C-NMR, and HR-MS techniques. The hybrid derivatives were evaluated for their acetylcholinesterase and butyrylcholinesterase inhibition effect. All the synthesized analogs showed good to moderate inhibitory effect ranging from IC50 value 0.20 ± 0.01 µM to 0.50 ± 0.10 µM for acetylcholinesterase and from IC50 value 0.25 ± 0.01 µM to 0.70 ± 0.10 µM for butyrylcholinesterase except one that showed least potency with IC50 value 1.05 ± 0.1 µM and 1.20 ± 0.1 µM. The differences in inhibitory potential of synthesized compounds were due to the nature and position of substitution attached to the main ring. Additionally, molecular docking study was carried out for most active in order to explore the binding interactions established by ligand (active compounds) with the active residues of targeted AChE & BuChE enzyme.

Fluorescence Studies on the Binding Affinity and Determination of Vitamin B12 in the Presence of Fibrinogen.

The binding properties between vitamin B12 (vitB12, cyanocobalamin) and fibrinogen (Fib) were investigated by UV-vis absorption and steady-state/three-dimentional (3D) fluorescence spectra techniques as well as molecular docking. The experimental results showed that the intrinsic fluorescence of Fib quenched by vitB12 with static mechanism to form a non-fluorescent complex. The positive signs of thermodynamic parameters, ΔH (92.18 kJ/mol) and ΔS (433.5 J/molK), indicated that the hydrophobic forces were dominant in the binding mode. The molecular docking data were found to be in agreement with these experimental results and were confirmed by three hydrophobic interactions between the Trp430, Try390 residues of Fib and the vitamin. 3D spectra showed that fibrinogen undergoes a conformation change when it interacts with vitB12. Based on non-radiative energy transfer theory, binding distance was calculated to be 3.94 nm between donor (tryptophan residues of Fib) and acceptor (vitB12). The limit of detection (LOD) of vitB12 was calculated as 2.08 µM in the presence of fibrinogen. The relative standard deviation (RSD) of method was 4.28% for determinations (n = 7) of a vitB12 solution with the concentration of 7.80 µM.

Isolated auto-citrullinated regions of PADI4 associate to the intact protein without altering their disordered conformation.

PADI4 is one of the human isoforms of a group of enzymes intervening in the conversion of arginine to citrulline. It is involved in the development of several types of tumors, as well as other immunological illnesses, such as psoriasis, multiple sclerosis, or rheumatoid arthritis. PADI4 auto-citrullinates in several regions of its sequence, namely in correspondence of residues Arg205, Arg212, Arg218, and Arg383. We wanted to study whether the citrullinated moiety affects the conformation of nearby regions and its binding to intact PADI4. We designed two series of synthetic peptides comprising either the wild-type or the relative citrullinated versions of such regions - i.e., a first series of peptides comprising the first three arginines, and a second series comprising Arg383. We studied their conformational properties in isolation by using fluorescence, far-ultraviolet (UV) circular dichroism (CD), and 2D1H NMR. Furthermore, we characterized the binding of the wild-type and citrullinated peptides in the two series to the intact PADI4, by using isothermal titration calorimetry (ITC), fluorescence, and biolayer interferometry (BLI), as well as by molecular docking simulations. We observed that citrullination did not alter the local conformational propensities of the isolated peptides. Nevertheless, for all the peptides in the two series, citrullination slowed down the kinetic koff rates of the binding reaction to PADI4, probably due to differences in electrostatic effects compared to the presence of arginine. The affinities of PADI4 for unmodified peptides were slightly larger than those of the corresponding citrullinated ones in the two series, but they were all within the same range, indicating that there were no relevant variations in the thermodynamics of binding due to sequence effects. These results highlight details of the self-citrullination of PADI4 and, more generally, of possible auto-catalytic mechanisms taking place in vivo for other citrullinating enzymes or, alternatively, in proteins undergoing citrullination passively.