Enzyme Binding Research Articles

Comparative Transcriptomic Analysis Revealing the Potential Mechanisms of Erythritol-Caused Mortality and Oviposition Inhibition in Drosophila melanogaster.

Erythritol has shown excellent insecticidal performance against a wide range of insect species, but the molecular mechanism by which it causes insect mortality and sterility is not fully understood. The mortality and sterility of Drosophila melanogaster were assessed after feeding with 1M erythritol for 72 h and 96 h, and gene expression profiles were further compared through RNA sequencing. Enrichment analysis of GO and KEGG revealed that expressions of the adipokinetic hormone gene (Akh), amylase gene (Amyrel), α-glucosidase gene (Mal-B1/2, Mal-A1-4, Mal-A7/8), and triglyceride lipase gene (Bmm) were significantly up-regulated, while insulin-like peptide genes (Dilp2, Dilp3 and Dilp5) were dramatically down-regulated. Seventeen genes associated with eggshell assembly, including Dec-1 (down 315-fold), Vm26Ab (down 2014-fold) and Vm34Ca (down 6034-fold), were significantly down-regulated or even showed no expression. However, there were no significant differences in the expression of three diuretic hormone genes (DH44, DH31, CAPA) and eight aquaporin genes (Drip, Big brain, AQP, Eglp1, Eglp2, Eglp3, Eglp4 and Prip) involved in osmolality regulation (all p value > 0.05). We concluded that erythritol, a competitive inhibitor of α-glucosidase, severely reduced substrates and enzyme binding, inhibiting effective carbohydrate hydrolysis in the midgut and eventually causing death due to energy deprivation. It was clear that Drosophila melanogaster did not die from the osmolality of the hemolymph. Our findings elucidate the molecular mechanism underlying the mortality and sterility in Drosophila melanogaster induced by erythritol feeding. It also provides an important theoretical basis for the application of erythritol as an environmentally friendly pesticide.

Gene Cloning, Heterologous Expression, and In Silico Analysis of Chitinase B from Serratia marcescens for Biocontrol of Spodoptera frugiperda Larvae Infesting Maize Crops.

Spodoptera frugiperda, the fall armyworm (FAW), is a highly invasive polyphagous insect pest that is considered a source of severe economic losses to agricultural production. Currently, the majority of chemical insecticides pose tremendous threats to humans and animals besides insect resistance. Thus, there is an urgent need to develop new pest management strategies with more specificity, efficiency, and sustainability. Chitin-degrading enzymes, including chitinases, are promising agents which may contribute to FAW control. Chitinase-producing microorganisms are reported normally in bacteria and fungi. In the present study, Serratia marcescens was successfully isolated and identified from the larvae of Spodoptera frugiperda. The bacterial strain NRC408 displayed the highest chitinase enzyme activity of 250 units per milligram of protein. Subsequently, the chitinase gene was cloned and heterologously expressed in E. coli BL21 (DE3). Recombinant chitinase B was overproduced to 2.5-fold, driven by the T7 expression system. Recombinant chitinase B was evaluated for its efficacy as an insecticidal bioagent against S. frugiperda larvae, which induced significant alteration in subsequent developmental stages and conspicuous malformations. Additionally, our study highlights that in silico analyses of the anticipated protein encoded by the chitinase gene (ChiB) offered improved predictions for enzyme binding and catalytic activity. The effectiveness of (ChiB) against S. frugiperda was evaluated in laboratory and controlled field conditions. The results indicated significant mortality, disturbed development, different induced malformations, and a reduction in larval populations. Thus, the current study consequently recommends chitinase B for the first time to control FAW.

Changes in the structure and enzyme binding of starches during in vitro enzymatic hydrolysis using mammalian mucosal enzyme mixtures

Starches are hydrolyzed into monosaccharides by mucosal α-glucosidases in the human small intestine. However, there are few studies assessing the direct digestion of starch by these enzymes. The objective of this study was to investigate the changes in the structure and enzyme binding of starches during in vitro hydrolysis by mammalian mucosal enzymes. Waxy maize (WMS), normal maize (NMS), high-amylose maize (HAMS), waxy potato (WPS), and normal potato (NPS) starches were examined. The order of the digestion rate was different compared with other studies using a mixture of pancreatic α-amylase and amyloglucosidase. NPS was digested more than other starches. WPS was more digestible than WMS. Hydrolyzed starch from NPS, NMS, WPS, WMS, and HAMS after 24 h was 66.4, 64.2, 61.7, 58.7, and 46.2 %, respectively. Notably, a significant change in the morphology, reduced crystallinity, and a decrease in the melting enthalpy of the three starches (NPS, NMS, and WPS) after 24 h of hydrolysis were confirmed by microscopy, X-ray diffraction, and differential scanning calorimetry, respectively. The bound enzyme fraction of NPS, NMS, and WPS increased as hydrolysis progressed. In contrast, HAMS was most resistant to hydrolysis by mucosal α-glucosidases in terms of digestibility, changes in morphology, crystallinity, and thermal properties.

The Analgesic Mechanism of Radix Wikstroemia indica “Sweat Soaking Method” Processed Product Based on Metabolomics and Network Pharmacology

Background: Miao medicine Radix Wikstroemia indica (RWI) is derived from the root of W. indica (L.) C. A. Mey. which has an analgesic effect, but its mechanism is not clear. Because of the toxicity of RWI, the research group used the “Sweat soaking method” to process RWI in the early stage to reduce its toxicity. Therefore, this study explored the analgesic effect and mechanism of processed RWI through network pharmacology and metabolomics. Purpose: To explore the analgesic effect and therapeutic mechanism of RWI processed by “Sweat soaking method”. Materials and methods: The torsion experiment was carried out with acetic acid. The metabolomic analysis of serum samples was carried out based on 1H-NMR technology, and the intersection targets of RWI and pain diseases were screened by network pharmacology for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Results: RWI has an analgesic effect and is related to metabolites such as 4-pyridoxic acid, l-glutamic acid, and agmatine. It is involved in arginine and proline metabolism, arginine biosynthesis, and alanine, aspartate, and glutamate metabolism. In network pharmacology, there were 404 common targets between RWI and pain diseases, and eight core targets were screened, including SRC, STAT3, and HSP90AA1. GO functional enrichment analysis found that RWI had effects on molecular processes such as protein phosphorylation and response to xenobiotic stimulus, cell composition such as receptor complex and membrane raft, and molecular functions such as enzyme binding. KEGG pathway enrichment analysis obtained 193 pathways. Arginine proline metabolism and nitrogen metabolism are involved in the same pathway as metabolomic analysis. Conclusion: RWI has an analgesic effect, and its therapeutic mechanism mainly involves arginine and proline metabolism.

Characterization of a novel 3-quinuclidinone reductase possessing remarkable thermostability

The 3-quinuclidinone reductase plays an irreplaceable role in the biopreparation of (R)-3-quinuclidinol, an intermediate vital for synthesis of various pharmaceuticals. Thermal robustness is a critical factor for enzymatic synthesis in industrial applications. This study characterized a new 3-quinuclidinone reductase, named SaQR, with significant thermal stability. The SaQR was overexpressed in a GST-fused state, and substrate and cofactor screening were conducted. Additionally, three-dimensional structure prediction using AlphaFold and analysis were performed, along with relevant thermostability tests, and the evaluation of factors influencing enzyme activity. The findings highlight the remarkable thermostability of SaQR, retaining over 90% of its activity after 72 h at 50°C, with an optimal operational temperature of 85°C. SaQR showed typical structural traits of the SDR superfamily, with its cofactor-determining residue being aspartic acid, conferring nicotinamide adenine dinucleotide (NAD(H)) preference. Moreover, K+ and Na+, at a concentration of 400 mM, could significantly enhance the activity, while Mg2+ and Mn2+ only display inhibitory effects within the tested concentration range. The findings of molecular dynamics simulations suggest that high temperatures may disrupt the binding of enzyme to substrate by increasing the flexibility of residues 205–215. In conclusion, this study reports a novel 3-quinuclidinone reductase with remarkable thermostability.

Discovery of Red-Shifting Mutations in Firefly Luciferase Using High-Throughput Biochemistry.

Photinus pyralis luciferase (FLuc) has proven a valuable tool for bioluminescence imaging, but much of the light emitted from the native enzyme is absorbed by endogenous biomolecules. Thus, luciferases displaying red-shifted emission enable higher resolution during deep-tissue imaging. A robust model of how protein structure determines emission color would greatly aid the engineering of red-shifted mutants, but no consensus has been reached to date. In this work, we applied deep mutational scanning to systematically assess 20 functionally important amino acid positions on FLuc for red-shifting mutations, predicting that an unbiased approach would enable novel contributions to this debate. We report dozens of red-shifting mutations as a result, a large majority of which have not been previously identified. Further characterization revealed that mutations N229T and T352M, in particular, bring about unimodal emission with the majority of photons being >600 nm. The red-shifting mutations identified by this high-throughput approach provide strong biochemical evidence for the multiple-emitter mechanism of color determination and point to the importance of a water network in the enzyme binding pocket for altering the emitter ratio. This work provides a broadly applicable mutational data set tying FLuc structure to emission color that contributes to our mechanistic understanding of emission color determination and should facilitate further engineering of improved probes for deep-tissue imaging.

Design, synthesis, and molecular dynamics simulation studies of some novel kojic acid fused 2-amino-3-cyano-4H-pyran derivatives as tyrosinase inhibitors.

A novel series of kojic acid fused 2-amino-3-cyano-4H-pyran derivatives were synthesized via a multicomponent reaction involving kojic acid, benzyloxy benzaldehyde, and malonitrile as tyrosinase inhibitors. Subsequently, the structures of the compounds were characterized using FT-IR, 1H-, and 13C-NMR spectroscopic analyses. The designed compounds fall into three series: (1) 4-benzyloxy-phenyl kojopyran 6a-e, (2) 3-benzyloxy- phenyl kojopyran derivatives 6f-j, and (3) 4-benzyloxy-3-methoxy-phenyl kojopyran derivative 6k-o. The assessment of tyrosinase inhibition activity was conducted using L-Dopa as the substrate. Among synthesized compounds, 2-amino-4-(4-((4-fluorobenzyl)oxy)phenyl)-6-(hydroxymethyl)-8-oxo-4,8-dihydropyrano[3,2-b]pyran-3-carbonitrile (6b) demonstrated the highest antityrosinase activity with a competitive inhibition pattern (IC50 = 7.69 ± 1.99μM) as compared to the control agent kojic acid (IC50 = 23.64 ± 2.56µM). Since compound 6b was synthesized as a racemic mixture, in silico studies were performed for both R and S enantiomers. The R- enantiomer showed critical interactions compared with the S-enantiomer. Specifically, it established hydrogen bonds and hydrophobic interactions with crucial and highly conserved amino acids within the enzyme's binding site in the target protein. Moreover, the molecular dynamics simulations revealed that compound 6b demonstrated significant interactions with essential residues of the binding site, resulting in a stable complex throughout the entire simulation run. The drug-like and ADMET properties predictions showed an acceptable profile for compound 6b. Thus, it can serve as a drug candidate to develop more potent antityrosinase agents due to its low toxicity and its high inhibition activity.

Substrate promiscuity of inositol 1,4,5-trisphosphate kinase driven by structurally-modified ligands and active site plasticity.

D-myo-inositol 1,4,5-trisphosphate (InsP3) is a fundamental second messenger in cellular Ca2+ mobilization. InsP3 3-kinase, a highly specific enzyme binding InsP3 in just one mode, phosphorylates InsP3 specifically at its secondary 3-hydroxyl group to generate a tetrakisphosphate. Using a chemical biology approach with both synthetised and established ligands, combining synthesis, crystallography, computational docking, HPLC and fluorescence polarization binding assays using fluorescently-tagged InsP3, we have surveyed the limits of InsP3 3-kinase ligand specificity and uncovered surprisingly unforeseen biosynthetic capacity. Structurally-modified ligands exploit active site plasticity generating a helix-tilt. These facilitated uncovering of unexpected substrates phosphorylated at a surrogate extended primary hydroxyl at the inositol pseudo 3-position, applicable even to carbohydrate-based substrates. Crystallization experiments designed to allow reactions to proceed in situ facilitated unequivocal characterization of the atypical tetrakisphosphate products. In summary, we define features of InsP3 3-kinase plasticity and substrate tolerance that may be more widely exploitable.

Stretchable Nanofiber-Based Felt as a String Electrode for Potential Use in Wearable Glucose Biosensors.

Nanofiber technology is leading the revolution of wearable technology and provides a unique capability to fabricate smart textiles. With the novel fabrication technique of electrospinning, nanofibers can be fabricated and then manufactured into a durable conductive string for the application of smart textiles. This paper presents an electrospun nanofiber mesh-based (NF-Felt) string electrode with a conducting polymer coating for an electrochemical enzymatic glucose sensor. The surface area of a nanofiber matrix is a key physical property for enhanced glucose oxidase (GOx) enzyme binding for the development of an electrochemical biosensor. A morphological characterization of the NF-Felt string electrode was performed using scanning electron microscopy (SEM) and compared with a commercially available cotton-polyester (Cot-Pol) string coated with the same conducting polymer. The results from stress-strain testing demonstrated high stretchability of the NF-Felt string. Also, the electrochemical characterization results showed that the NF-Felt string electrode was able to detect a glucose concentration in the range between 0.0 mM and 30.0 mM with a sensitivity of 37.4 μA/mM·g and a detection limit of 3.31 mM. Overall, with better electrochemical performance and incredible flexibility, the NF-Felt-based string electrode is potentially more suitable for designing wearable biosensors for the detection of glucose in sweat.

Expansion of the neurodevelopmental phenotype of individuals with EEF1A2 variants and genotype-phenotype study.

Translation elongation factor eEF1A2 constitutes the alpha subunit of the elongation factor-1 complex, responsible for the enzymatic binding of aminoacyl-tRNA to the ribosome. Since 2012, 21 pathogenic missense variants affecting EEF1A2 have been described in 42 individuals with a severe neurodevelopmental phenotype including epileptic encephalopathy and moderate to profound intellectual disability (ID), with neurological regression in some patients. Through international collaborative call, we collected 26 patients with EEF1A2 variants and compared them to the literature. Our cohort shows a significantly milder phenotype. 83% of the patients are walking (vs. 29% in the literature), and 84% of the patients have language skills (vs. 15%). Three of our patients do not have ID. Epilepsy is present in 63% (vs. 93%). Neurological examination shows a less severe phenotype with significantly less hypotonia (58% vs. 96%), and pyramidal signs (24% vs. 68%). Cognitive regression was noted in 4% (vs. 56% in the literature). Among individuals over 10 years, 56% disclosed neurocognitive regression, with a mean age of onset at 2 years. We describe 8 novel missense variants of EEF1A2. Modeling of the different amino-acid sites shows that the variants associated with a severe phenotype, and the majority of those associated with a moderate phenotype, cluster within the switch II region of the protein and thus may affect GTP exchange. In contrast, variants associated with milder phenotypes may impact secondary functions such as actin binding. We report the largest cohort of individuals with EEF1A2 variants thus far, allowing us to expand the phenotype spectrum and reveal genotype-phenotype correlations.

Mechanism of improving the digestibility of coconut globulin by atmospheric cold plasma treatment: The perspective of protein structure

As a natural emulsifier in coconut milk, coconut globulin (CG) is a potential plant-based protein source to replace animal protein. However, due to its poor solubility and poor digestive properties, CG is not sufficiently utilized. The aim of this study was to improve the digestibility of CG by employing atmospheric cold plasma (ACP) technology. The influence of ACP treatment on the digestive properties of coconut globulin was evaluated by measurements of digestibility, degree of hydrolysis, free amino acids (FAAs), and polypeptide distribution after gastrointestinal digestion. These results implied that application of ACP treatment facilitated the digestion of CG, resulting in the hydrolysis of the compound into numerous smaller peptides (<1000 Da) and FAAs. The quartz crystal microbalance with dissipation technique was utilized to monitor the dynamic digestion process of CG. The results observed that ACP-treated CG had increased affinity for pepsin and trypsin, especially at 60 kV (the adsorbed mass was 7.60 μg/cm2). Various analytical techniques, including circular dichroism spectroscopy, Fourier transform infrared spectroscopy, fluorescence spectroscopy, and particle size distribution, demonstrated that ACP treatment facilitated the structural unfold of CG, resulting in increased exposure of enzyme binding sites and hydrolysis into smaller fragments. In addition, in vitro and cell-based antioxidant experiments provided evidence of the significant antioxidant activity of the hydrolysate derived from ACP-treated CG. Hence, the utilization of ACP treatment at 60 kV emerged as the optimal approach for enhancing the digestibility of CG (from 71.65% to 78.65%). This finding provides a theoretical basis for the use of ACP treatment in protein-rich foods, thereby providing insights for expanding the application of ACP treatment in the field of food processing.

Dissecting the Allosteric Fine-Tuning of Enzyme Catalysis.

Fully understanding the mechanism of allosteric regulation in biomolecules requires separating and examining all of the involved factors. In enzyme catalysis, allosteric effector binding shifts the structure and dynamics of the active site, leading to modified energetic (e.g., energy barrier) and dynamical (e.g., diffusion coefficient) factors underlying the catalyzed reaction rate. Such modifications can be subtle and dependent on the type of allosteric effector, representing a fine-tuning of protein function. The microscopic description of allosteric regulation at the level of function-dictating factors has prospective applications in fundamental and pharmaceutical sciences, which is, however, largely missing so far. Here, we characterize the allosteric fine-tuning of enzyme catalysis, using human Pin1 as an example, by performing more than half-millisecond all-atom molecular dynamics simulations. Changes of reaction kinetics and the dictating factors, including the free energy surface along the reaction coordinate and the diffusion coefficient of the reaction dynamics, under various enzyme and allosteric effector binding conditions are examined. Our results suggest equal importance of the energetic and dynamical factors, both of which can be modulated allosterically, and the combined effect determines the final allosteric output. We also reveal the potential dynamic basis for allosteric modulation using an advanced statistical technique to detect function-related conformational dynamics. Methods developed in this work can be applied to other allosteric systems.

Hexahydro-1,2,3-triazine Derivatives: Synthesis, Antimicrobial Evaluation, Antibiofilm Activity and Study of Molecular Docking Against Glucosamine-6-Phosphate

The N,N',N''-trisubstituted hexahydro-1,3,5-triazine derivatives (3a–g) had been created and identified through infrared, nuclear magnetic resonance, and mass spectrometry according to their symmetric basic structure. Three molecules of diverse aromatic amines and three molecules of formaldehyde were assembled in a "1+1+1+1+1+1" condensation reaction to produce hexahydrotriazines. Two Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis) and two Gram-negative (Klebsiella pneumonia, Pseudomonas aeruginosa) bacteria were used to evaluate the antimicrobial activity of the produced compounds. The anti-biofilm activity of 3g against S. aureus was also examined. In this investigation, glucosamine-6-phosphate synthase was employed to investigate the binding affinity of 3g within the enzyme's binding site. The results demonstrated that most of the synthesized hexahydro-1,3,5-triazine compounds have mild antimicrobial effects in comparison with the commonly used drug ampicillin, whereas the compounds 3g are potentially anti-biofilm agents. Molecular docking with the Autodock 4.2 tool was applied to study the binding affinity. It was found to hit (3g) in the active center of glucosamine-6-phosphate synthase as the target enzyme for antimicrobial agents. In silico studies reveal that the discovered hit is a promising glucosamine-6-phosphate inhibitor, as well as that the docking data matched up to the in vitro assay.

Novel Benzotriazole-β-lactam Derivatives as Antimalarial Agents: Design, Synthesis, Biological Evaluation and Molecular Docking Studies.

Many people around the world suffer from malaria, especially in tropical or subtropical regions. While malaria medications have shown success in treating malaria, there is still a problem with resistance to these drugs. Herein, we designed and synthesized some structurally novel benzotriazole-β-lactams using 2-(1H-benzo[d][1,2,3]triazol-1-yl)acetic acid as a key intermediate. To synthesize the target molecules, the ketene-imine cycloaddition reaction was employed. First, The reaction of 1H-benzo[d][1,2,3]triazole with 2-bromoacetic acid in aqueous sodium hydroxide yielded 2-(1H-benzo[d][1,2,3]triazol-1-yl)acetic acid. Then, the treatment of 2-(1H-benzo[d][1,2,3]triazol-1-yl)acetic acid with tosyl chloride, triethyl amine, and Schiff base provided new β-lactams in good to moderate yields.The formation of all cycloadducts was confirmed by elemental analysis, FT-IR, NMR and mass spectral data. Moreover, X-ray crystallography was used to determine the relative stereochemistry of 4a compound. The in vitro antimalarial activity test was conducted for each compound against P. falciparum K1. The IC50 values ranged from 5.56 to 25.65 μM. A cytotoxicity profile of the compounds at 200 μM final concentration revealed suitable selectivity of the compounds for malaria treatment. Furthermore, the docking study was carried out for each compound into the P. falciparum dihydrofolate reductase enzyme (PfDHFR) binding site to analyze their possible binding orientation in the active site.

1H-1,2,3-triazolyl-1,6-naphthyridin-7(6H)-ones as Potential Fluorescent Nucleoside Analogues: Synthesis and Optical Properties.

In this article, we present the synthesis and the optical properties of three original molecules as potential fluorescent ribonucleoside analogues incorporating a 1,6-naphthyridin-7(6H)-one scaffold as a fluorescent nucleobase and a 1,2,3-triazole as a linkage. The nucleosides were prepared via a Cu alkyne-azide cycloaddition (CuAAC) reaction between a ribofuranosyl azide and a 4-ethynylpyridine partner. Construction of substituted 1,6-naphthyridin-7(6H)-ones was achieved through two additional steps. Optical property studies were investigated on nucleoside analogues. Powerful fluorescence properties have been evidenced with a remarkable change of emissivity depending on the polarity of the solvent, making these molecules suitable as a new class of artificial fluorescent nucleosides for investigating enzyme binding sites as well as probing nucleic acids. In addition, we are convinced that such analogues could be of great interest in the search for new antiviral or antitumoral drugs based on nucleosides.

Sulfadiazine Exerts Potential Anticancer Effect in HepG2 and MCF7 Cells by Inhibiting TNFα, IL1b, COX-1, COX-2, 5-LOX Gene Expression: Evidence from In Vitro and Computational Studies.

Drug repurposing is a promising approach that has the potential to revolutionize the drug discovery and development process. By leveraging existing drugs, we can bring new treatments to patients more quickly and affordably. Anti-inflammatory drugs have been shown to target multiple pathways involved in cancer development and progression. This suggests that they may be more effective in treating cancer than drugs that target a single pathway. Cell viability was measured using the MTT assay. The expression of genes related to inflammation (TNFa, IL1b, COX-1, COX-2, and 5-LOX) was measured in HepG2, MCF7, and THLE-2 cells using qPCR. The levels of TNFα, IL1b, COX-1, COX-2, and 5-LOX were also measured in these cells using an ELISA kit. An enzyme binding assay revealed that sulfadiazine expressed weaker inhibitory activity against COX-2 (IC50 = 5.27 μM) in comparison with the COX-2 selective reference inhibitor celecoxib (COX-2 IC50 = 1.94 μM). However, a more balanced inhibitory effect was revealed for sulfadiazine against the COX/LOX pathway with greater affinity towards 5-LOX (IC50 = 19.1 μM) versus COX-1 (IC50 = 18.4 μM) as compared to celecoxib (5-LOX IC50 = 16.7 μM, and COX-1 IC50 = 5.9 μM). MTT assays revealed the IC50 values of 245.69 ± 4.1 µM and 215.68 ± 3.8 µM on HepG2 and MCF7 cell lines, respectively, compared to the standard drug cisplatin (66.92 ± 1.8 µM and 46.83 ± 1.3 µM, respectively). The anti-inflammatory effect of sulfadiazine was also depicted through its effect on the levels of inflammatory markers and inflammation-related genes (TNFα, IL1b, COX-1, COX-2, 5-LOX). Molecular simulation studies revealed key binding interactions that explain the difference in the activity profiles of sulfadiazine compared to celecoxib. The results suggest that sulfadiazine exhibited balanced inhibitory activity against the 5-LOX/COX-1 enzymes compared to the selective COX-2 inhibitor, celecoxib. These findings highlight the potential of sulfadiazine as a potential anticancer agent through balanced inhibitory activity against the COX/LOX pathway and reduction in the expression of inflammatory genes.

Mechanisms predictive of Tibetan Medicine Sophora moorcroftiana alkaloids for treatment of lung cancer based on the network pharmacology and molecular docking

BackgroundLeguminous Sophora moorcroftiana (SM) is a genuine medicinal material in Tibet. Many research results have reveal the Sophora moorcroftiana alkaloids (SMA), as the main active substance, have a wide range of effects, such as antibacterial, antitumor and antiparasitic effects. However, there are few reports on the inhibition of lung cancer (LC) and its inhibitory mechanism, and the pharmacological mechanism of SMA is still unclear, Therefore, exploring its mechanism of action is of great significance.MethodsThe SMA active components were obtained from the literature database. Whereas the corresponding targets were screened from the PubChem and PharmMapper database, UniProt database were conducted the correction and transformation of UniProt ID on the obtained targets. The GeneCards and OMIM databases identified targets associated with LC. Venny tools obtained the intersection targets of SMA and LC. R language and Cytoscape software constructed the visual of SMA - intersection targets – LC disease network. The intersection targets protein-protein interaction (PPI) network were built by the STRING database. The functions and pathways of the common targets of SMA and LC were enriched by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Subsequently, molecular docking And A549 cells vitro experiment were performed to further validate our finding.ResultsWe obtained six kinds of alkaloids in SM, 635 potential targets for these compounds, and 1,303 genes related to LC. SMA and LC intersection targets was 33, including ALB, CCND1, ESR1, NOTCH1 and AR. GO enrichment indicated that biological process of SMA was mainly involved in the positive regulation of transcription and nitric oxide biosynthetic process, and DNA-templated, etc. Biological functions were mainly involved in transcription factor binding and enzyme binding, etc. Cell components were mainly involved in protein complexes, extracellular exosome, cytoplasm and nuclear chromatin, etc., Which may be associated with its anti-LC effects. KEGG enrichment analysis showed that main pathways involved in the anti-LC effects of SMA, including pathway in cancer, non small-cell lung cancer, p53, PI3K-Akt and FOXO signaling pathways. Molecular docking analyses revealed that the six active compounds had a good binding activity with the main therapeutic targets 2W96, 2CCH and 1O96. Experiments in vitro proved that SMA inhibited the proliferation of LC A549 cells.ConclusionsResults of the present study, we have successfully revealed the SMA compounds had a multi-target and multi-channel regulatory mechanism in treatment LC, These findings provided a solid theoretical reference of SMA in the clinical treatment of LC.

Identification of miRNA, lncRNA and circRNA associated with gastric cancer metabolism through sequencing and bioinformatics analysis

Gastric cancer remains a highly prevalent malignancy worldwide with its molecular features poorly understood. To gain full insight into its genetic landscape, we performed whole-transcriptome sequencing on human tumors and adjacent non-tumors to predict the function of microRNA, long coding RNA, circular RNA, and mRNA, as well as estimate their correlation with gastric cancer characteristics through construction of ceRNA, WGCNA and PPI network. Functional enrichment analysis annotated nucleic acid binding, enzyme activity and binding related to differentially expressed miRNAs (dif-miRNAs); energy binding and enzyme binding related to dif-lncRNAs; protein binding and enzyme activity related to dif-circRNAs; protein digestion and absorption related to dif-mRNAs. The expression of key miR-135a-5p, lncRNAs-MSTRG.48856.1, ENST00000569981, MSTRG.22826.1, ENST00000564492, circRNAs-CCSER2, FNDC3B, CORO1C, FAM214A were validated by real-time PCR. The ceRNA network filtered 14 miRNAs, 30 lncRNAs, and 6 mRNA in the lncRNA-ceRNA axis and 8 miRNAs, 9 circRNAs, and 3 mRNA in the circRNA-ceRNA axis. Genes involved in ceRNA were annotated to be closely related to tumor material synthesis and metabolism. The WGCNA network filtered gene clusters related to TNM traits and extracted the hub genes CLDN10, CD177, newGene_35523, newGene_51201, CEACAM7, and newGene_46634. These genes were associated with cell proliferation, metabolism, and enzyme activity regulation. The PPI network analyzed the stable interaction relationships of the hub genes. Our research provides a valuable resource for understanding the molecular mechanisms of gastric cancer from the perspective of tumor metabolism.

Structure-based development of 3,5-dihydroxybenzoyl-hydrazineylidene as tyrosinase inhibitor; in vitro and in silico study

A series of new analogs of 3,5-dihydroxybenzoyl-hydrazineylidene conjugated to different methoxyphenyl triazole (11a-n) synthesized using click reaction. The structures of all synthesized compounds were characterized by FTIR, 1H, 13C-NMR spectroscopy, and CHO analysis. The tyrosinase inhibitory potential of the synthesized compounds was studied. The newly synthesized scaffolds were found to illustrate the variable degree of the inhibitory profile, and the most potent analog of this series was that one bearing 4-methoxyphenyl moiety, and exhibited an IC50 value of 55.39 ± 4.93 µM. The kinetic study of the most potent derivative reveals a competitive mode of inhibition. Next, molecular docking studies were performed to understand the potent inhibitor's binding mode within the enzyme's binding site. Molecular dynamics simulations were accomplished to further investigate the orientation and binding interaction over time and the stability of the 11m-tyrosinase complex.

Multi-omics analysis reveals GAPDH posttranscriptional regulation of IFN-γ and PHGDH as a metabolic checkpoint of microglia polarization

A “switch” in the metabolic pattern of microglia is considered to be required to meet the metabolic demands of cell survival and functions. However, how metabolic switches regulate microglial function remains controversial. We found here that exposure to amyloid-β triggers microglial inflammation accompanied by increasing GAPDH levels. The increase of GAPDH, a glycolysis enzyme, leads to the reduced release of interferon-γ (IFN-γ) from inflammatory microglia. Such alternation is translational and is regulated by the binding of glycolysis enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to IFN-γ mRNA. GAPDH, by engaging/disengaging glycolysis and through influencing IFN-γ expression, regulates microglia functions, including phagocytosis and cytokine production. Phosphoglycerate dehydrogenase (PHGDH), screened from different state microglia by metabolomics combined with METARECON analysis, is a metabolic enzyme adjacent downstream of GAPDH and synthesizes serine on the collateral pathway derived from glycolysis. Polarization of microglial with PHGDH as a metabolic checkpoint can be bidirectionally regulated by adding IL-4 or giving PHGDH inhibitors. Therefore, regulation of metabolic enzymes not only reprograms metabolic patterns, but also manipulates microglia functions. Further study should be performed to explore the mechanism of metabolic checkpoints in human microglia or more in vivo animal experiments, and may expand to the effects of various metabolic substrates or enzyme, such as lipids and amino acids, on the functions of microglia.