Rapid identification of chemical compositions from three species of Siegesbeckiae Herba by ultra-performance liquid chromatography-electrospray ionization-quadrupole time of flight-mass spectrometry in combination with deoxyribonucleic acid barcoding.

Siegesbeckiae Herba, a traditional Chinese medicine, originates from Siegesbeckia orientalis, S. glabrescens, and S. pubescens in the Pharmacopoeia of the People's Republic of China. However, accurate identification of decoction pieces from the three plants remains a challenge. In this study, 26 batches of Siegesbeckiae Herba were identified by deoxyribonucleic acid barcoding, and their chemical compositions were determined using ultra-performance liquid chromatography-electrospray ionization-quadrupole time of flight-mass spectrometry. The results showed that the internal transcribed spacer 2 and internal transcribed spacer 1-5.8 S- internal transcribed spacer 2 sequences could distinguish three species. In total, 48 compounds were identified including 12 marker compounds screened for three species using the partial least square discriminant analysis. Among these, two diterpenoids 16-O-malonylkirenol and 15-O-malonylkirenol, and a novel diterpenoid 15,16-di-O-malonylkirenol were isolated and identified. A convenient method for the identification of Siegesbeckiae Herba was established using kirenol and 16-O-acetlydarutoside as control standards by thin-layer chromatography. Unexpectedly, none of the batches of S. orientalis contained kirenol, which did not meet the quality standards of Siegesbeckiae Herba, suggesting that the rationality of kirenol as a quality marker for S. orientalis should be further investigated. The results of this study will contribute to the quality control of Siegesbeckiae Herba.

Leuconostoc performance in soy-based fermentations - Survival, acidification, sugar metabolism, and flavor comparisons.

Leuconostoc spp. is often regarded as the flavor producer, responsible for the production of acetoin and diacetyl in dairy cheese. In this study, we investigate seven plant-derived Leuconostoc strains, covering four species, in their potential as a lyophilized starter culture for flavor production in fermented soy-based cheese alternatives. We show that the process of lyophilization of Leuconostoc can be feasible using a soy-based lyoprotectant, with survivability up to 63% during long term storage. Furthermore, the storage in this media improves the subsequent growth in a soy-based substrate in a strain specific manner. The utilization of individual raffinose family oligosaccharides was strain dependent, with Leuconostoc pseudomesenteroides NFICC99 being the best consumer. Furthermore, we show that all investigated strains were able to produce a range of volatile flavor compounds found in dairy cheese products, as well as remove certain dairy off-flavors from the soy-based substrate like hexanal and 2-pentylfuran. Also here, NFICC99 was strain producing most cheese-related volatile flavor compounds, followed by Leuconostoc mesenteroides NFICC319. These findings provide initial insights into the development of Leuconostoc as a potential starter culture for plant-based dairy alternatives, as well as a promising approach for generation of stable, lyophilized cultures.

Evaluation of the fermentation potential of lactic acid bacteria isolated from herbs, fruits and vegetables as starter cultures in nut-based milk alternatives.

Fermentation of plant-based milk alternatives (PBMAs), including nut-based products, has the potential to generate new foods with improved sensorial properties. In this study, we screened 593 lactic acid bacteria (LAB) isolates from herbs, fruits and vegetables for their ability to acidify an almond-based milk alternative. The majority of the strongest acidifying plant-based isolates were identified as Lactococcus lactis, which were found to lower the pH of almond milk faster than dairy yoghurt cultures. Whole genome sequencing (WGS) of 18 plant-based Lc. lactis isolates revealed the presence of sucrose utilisation genes (sacR, sacA, sacB and sacK) in the strongly acidifying strains (n = 17), which were absent in one non-acidifying strain. To confirm the importance of Lc. lactis sucrose metabolism in efficient acidification of nut-based milk alternatives, we obtained spontaneous mutants defective in sucrose utilisation and confirmed their mutations by WGS. One mutant containing a sucrose-6-phosphate hydrolase gene (sacA) frameshift mutation was unable to efficiently acidify almond, cashew and macadamia nut milk alternatives. Plant-based Lc. lactis isolates were heterogeneous in their possession of the nisin gene operon near the sucrose gene cluster. The results of this work show that sucrose-utilising plant-based Lc. lactis have potential as starter cultures for nut-based milk alternatives.

Comparison of Chemical Compositions and Antioxidant Activities for the Immature Fruits of Citrus changshan-huyou Y.B. Chang and Citrus aurantium L.

Quzhou Aurantii Fructus (QAF), the dried immature fruit of Citrus changshan-huyou Y.B. Chang, is similar to Aurantii Fructus (AF), the dried immature fruit of Citrus aurantium L. or its cultivars, in terms of composition, pharmacological action, and appearance. However, potential chemical markers to distinguish QAF from AF remain unknown owing to the lack of a comprehensive systematic chemical comparison aligned with discriminant analysis. To achieve a better understanding of the differences in their composition, this study aimed to identify the basic chemical compounds in QAF (n = 42) and AF (n = 8) using ultra-performance liquid chromatography coupled with electron spray ionization and quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) and gas chromatography coupled with mass spectrometry (GC-MS). Principal component analysis (PCA), orthogonal partial least squares-discriminant analysis (OPLS-DA), and hierarchical clustering analysis (HCA) were used to further analyze, screen, and verify potential chemical markers; the antioxidant capacity was assayed in vitro. A total of 108 compounds were found in QAF and AF, including 25 flavonoids, 8 limonoids, 2 coumarins, and 73 volatile components. The chemometric analysis indicated that the main components in QAF and AF were very similar. Trace differential components, including 9 flavonoids, 2 coumarins, 5 limonoids, and 26 volatile compounds, were screened as potential chemical markers to distinguish between QAF and AF. Additionally, the antioxidant capacity of QAF was found to be greater than that of AF. This research provides insights into the quality control and clinical application of QAF.

Broadband stealth devices based on encoded metamaterials.

Based on the generalized Snell's law, the relationship between the phase gradient of the metasurface and the incident frequency is demonstrated, and the principle of the achromatic metasurface is developed. By adjusting the phase gradient and linear dispersion simultaneously, the function of achromatic aberration is realized, and the influence of chromatic aberration on the metasurface is reduced. We propose a metasurface stealth device with achromatic multilayer frame metasurfaces with beam deflection, steering, and collection functions so that the incident electromagnetic beam is transmitted around the stealth object without scattering. In the range of 0.45-0.9 THz, the stealth function can be achieved. We have shown that the achromatic principle, design method, and stealth structure provide a guide for achieving transmissive cloaking.

Toosendanin inhibits colorectal cancer cell growth through the Hedgehog pathway by targeting Shh.

Colorectal cancer (CRC) is one of the most common gastrointestinal cancers worldwide. This complex and often fatal disease has a high mortality rate. The Hedgehog (Hh) signaling pathway is crucial in CRC. Many studies have indicated that Shh is overexpressed in cancer stem cells (CSCs), and shh overexpression is positively correlated with CRC tumorigenesis. New drugs that kill CRC cells through the Hh pathway are needed. Toosendanin (TSN), a natural triterpenoid saponin extracted from the bark or fruit of Melia toosendan Sieb. et Zucc, can inhibit various tumors. Here, we investigated the effects of TSN in CRC and explored the possible targets and mechanisms. Shh-Light Ⅱ cells were treated with TSN and tested by dual luciferase reporter assays to determine the relationship with the Hh pathway. Cell Counting Kit-8 (CCK-8) assays were used to test the inhibitory effects of TSN on CRC cells. The expression of Hh components after TSN treatment was detected using western blots and quantitative reverse transcription polymerase chain reaction. Cellular thermal shift assays confirmed the targets of TSN. The same effects of TSN on xenograft tumor growth were investigated in vivo. The average weight, volume of the finally resected tumor, and the expression of Shh in the TSN-treated groups were significantly lower than those of the control group. This result strongly suggested that TSN administration inhibited CRC growth in vivo. Our research preliminarily demonstrated that the target of TSN was Shh and that TSN inhibits CRC cell growth by inhibiting the Hh pathway, identifying a new anticancer molecular mechanism of TSN in CRC.

[Diterpenoids as PPARγ agonists from Siegesbeckia pubescens and their anti-inflammatory effects in vitro].

This study aims to investigate the PPARγ agonists isolated from the aqueous extract of Siegesbeckia pubescens( SPA) and their anti-inflammatory activities in vitro. The 293 T cells transfected transiently with PPARγ recombinant plasmid were used as a screening model to guide the isolation of PPARγ activitating components,and then PPARγ activities were measured by double luciferase reporter gene assay. The chemical structures were identified by chromatography or spectroscopic techniques. Furthermore,a UC inflammatory model in vitro was established on HT-29 cells by stimulating with TNF-α． The mRNA levels and secretion of proinflammatory cytokines on HT-29 cells,such as IL-1ß,TNF-α,IL-8,were detected by RT-PCR and ELISA. The results showed that five diterpenoids were obtained from the fraction D_(50) with the strongest PPARγ activity among others in SPA,and determined as kirenol( 1),darutigenol( 2),enantiomeric-2-ketone-15,16,19-three hydroxypinomane-8( 14)-ene-19-O-ß-D-glucoside( 3),darutoside( 4),enantiomeric-2-ß,15,16,19-four hydroxypinomane-8( 14)-ene-19-O-ß-D-glucoside( 5),respectively. All the compounds exhibited active effects on PPARγ in a concentration-dependent manner( P<0. 01). In addition,compound 1 significantly inhibited the expression of IL-1ß mRNA and secretion of IL-8 on HT-29 cells inflammation model( P<0. 001); both compounds 2 and 3 effectively inhibited the expression of IL-1ß,TNF-α,IL-8 mRNA and secretion of IL-8( P<0. 01 or P<0. 001),although at different extent; compound 4 significantly inhibited the expression of IL-1ß and TNF-α mRNA( P<0. 01 or P<0. 001),while compound 5 inhibited the expression of IL-1ß mRNA obviously( P<0. 001). In conclusion,the diterpenoids 1-5 isolated from S. pubescens have the PPARγ activation activities and potential effects of anti-UC in vitro.

ASD v3.0: unraveling allosteric regulation with structural mechanisms and biological networks.

Allosteric regulation, the most direct and efficient way of regulating protein function, is induced by the binding of a ligand at one site that is topographically distinct from an orthosteric site. Allosteric Database (ASD, available online at http://mdl.shsmu.edu.cn/ASD) has been developed to provide comprehensive information featuring allosteric regulation. With increasing data, fundamental questions pertaining to allostery are currently receiving more attention from the mechanism of allosteric changes in an individual protein to the entire effect of the changes in the interconnected network in the cell. Thus, the following novel features were added to this updated version: (i) structural mechanisms of more than 1600 allosteric actions were elucidated by a comparison of site structures before and after the binding of an modulator; (ii) 261 allosteric networks were identified to unveil how the allosteric action in a single protein would propagate to affect downstream proteins; (iii) two of the largest human allosteromes, protein kinases and GPCRs, were thoroughly constructed; and (iv) web interface and data organization were completely redesigned for efficient access. In addition, allosteric data have largely expanded in this update. These updates are useful for facilitating the investigation of allosteric mechanisms, dynamic networks and drug discoveries.

Improved Method for the Identification and Validation of Allosteric Sites.

Allosteric regulation induced by modulators binding to different, often distant, allosteric sites allows for exquisite control of protein functional activity. The structural diversity of allosteric sites endows allosteric modulators with high selectivity and low toxicity. Targeting allosteric sites, a novel tactic in drug discovery, has garnered much attention in the scientific community, and the identification of allosteric sites has become an important component of the development of allosteric drugs. Here we present AllositePro, a method which predicts allosteric sites in proteins by combining pocket features with perturbation analysis. The performance of AllositePro is superior to that of the other currently available methods. Using AllositePro, we predicted a novel allosteric site in cyclin-dependent kinase 2 (CDK2) and validated it by site-directed mutagenesis assay. Thus, the AllositePro method provides an effective way to identify allosteric sites and could be a useful strategy for allosteric drug discovery.

A novel allosteric site in casein kinase 2α discovered using combining bioinformatics and biochemistry methods.

Casein kinase 2 (CK2) is a highly pleiotropic serine-threonine kinase, which catalyzed phosphorylation of more than 300 proteins that are implicated in regulation of many cellular functions, such as signal transduction, transcriptional control, apoptosis and the cell cycle. On the other hand, CK2 is abnormally elevated in a variety of tumors, and is considered as a promising therapeutic target. The currently available ATP-competitive CK2 inhibitors, however, lack selectivity, which has impeded their development in cancer therapy. Because allosteric inhibitors can avoid the shortcomings of conventional kinase inhibitors, this study was aimed to discover a new allosteric site in CK2α and to investigate the effects of mutations in this site on the activity of CK2α. Using Allosite based on protein dynamics and structural alignment, we predicted a new allosteric site that was partly located in the αC helix of CK2α. Five residues exposed on the surface of this site were mutated to validate the prediction. Kinetic analyses were performed using a luminescent ADP detection assay by varying the concentrations of a peptide substrate, and the results showed that the mutations I78C and I78W decreased CK2α activity, whereas V31R, K75E, I82C and P109C increased CK2α activity. Potential allosteric pathways were identified using the Monte Carlo path generation approach, and the results of these predicted allosteric pathways were consistent with the mutation analysis. Multiple sequence alignments of CK2α with the other kinases in the family were conducted using the ClustalX method, which revealed the diversity of the residues in the site. In conclusion, we identified a new allosteric site in CK2α that can be altered to modulate the activity of the kinase. Because of the high diversity of the residues in the site, the site can be targeted using rational drug design of specific CK2α inhibitors for biological relevance.

ASBench: benchmarking sets for allosteric discovery.

Allostery allows for the fine-tuning of protein function. Targeting allosteric sites is gaining increasing recognition as a novel strategy in drug design. The key challenge in the discovery of allosteric sites has strongly motivated the development of computational methods and thus high-quality, publicly accessible standard data have become indispensable. Here, we report benchmarking data for experimentally determined allosteric sites through a complex process, including a 'Core set' with 235 unique allosteric sites and a 'Core-Diversity set' with 147 structurally diverse allosteric sites. These benchmarking sets can be exploited to develop efficient computational methods to predict unknown allosteric sites in proteins and reveal unique allosteric ligand-protein interactions to guide allosteric drug design.

The structural basis of ATP as an allosteric modulator.

Adenosine-5'-triphosphate (ATP) is generally regarded as a substrate for energy currency and protein modification. Recent findings uncovered the allosteric function of ATP in cellular signal transduction but little is understood about this critical behavior of ATP. Through extensive analysis of ATP in solution and proteins, we found that the free ATP can exist in the compact and extended conformations in solution, and the two different conformational characteristics may be responsible for ATP to exert distinct biological functions: ATP molecules adopt both compact and extended conformations in the allosteric binding sites but conserve extended conformations in the substrate binding sites. Nudged elastic band simulations unveiled the distinct dynamic processes of ATP binding to the corresponding allosteric and substrate binding sites of uridine monophosphate kinase, and suggested that in solution ATP preferentially binds to the substrate binding sites of proteins. When the ATP molecules occupy the allosteric binding sites, the allosteric trigger from ATP to fuel allosteric communication between allosteric and functional sites is stemmed mainly from the triphosphate part of ATP, with a small number from the adenine part of ATP. Taken together, our results provide overall understanding of ATP allosteric functions responsible for regulation in biological systems.

Novel inhibitor against Malassezia globosa LIP1 (SMG1), a potential anti-dandruff target.

Compelling evidence have demonstrated the role of lipase activity in the pathogenicity of Malassezia globosa toward dandruff and seborrheic dermatitis (D/SD). As a representative secreted lipase from M. globosa CBS 7966, Malassezia globosa LIP1 (SMG1) is considered a potential anti-dandruff target. In this study, homology modeling, docking-based virtual screening and in vitro lipase-based assay were integrated to identify the first hit compound against SMG1, with an IC50 of 20 µM against synthetic lipase substrate, and of 0.19 µM when using natural lipase substrate. Evaluation of similar compounds, along with docking, offered information on the binding patterns of the hit compound. This work is expected to serve as a starting point for the rational design of more potent inhibitors against SMG1.

Allosite: a method for predicting allosteric sites.

MOTIVATION: The use of allosteric modulators as preferred therapeutic agents against classic orthosteric ligands has colossal advantages, including higher specificity, fewer side effects and lower toxicity. Therefore, the computational prediction of allosteric sites in proteins is receiving increased attention in the field of drug discovery. Allosite is a newly developed automatic tool for the prediction of allosteric sites in proteins of interest and is now available through a web server. AVAILABILITY: The Allosite server and tutorials are freely available at http://mdl.shsmu.edu.cn/AST CONTACT: jian.zhang@sjtu.edu.cn SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

CBC-1 as a Cynanbungeigenin C derivative inhibits the growth of colorectal cancer through targeting Hedgehog pathway component GLI 1.

Colorectal cancer (CRC) is one of the most common gastrointestinal cancers that results in death in worldwide. The Hedgehog (HH) signalling pathway regulates the initiation and progression of CRC. Inhibiting the HH pathway has been presented as a potential treatment strategy in recent years. Cynanbungeigenin C (CBC) is a new type of C21 steroid that has been previously reported for the treatment of medulloblastoma. However, its further investigation was limited by its poor water solubility. In this study, six new CBC derivatives were synthesized through the structural modification of CBC, and four of them showed better water solubility than CBC. Moreover, their antiproliferative activities on CRC were evaluated. It was found that CBC-1 presented the best inhibitory effect on three types of CRC cell lines, and this effect was superior to that of CBC. Mechanistically, CBC-1 inhibited the proliferation of CRC cells through regulation of mRNA and proteins of the HH pathway according to qRT-PCR and Western blotting analysis. Furthermore, Cellular Thermal Shift Assay results indicated that CBC-1 regulated this signalling pathway by targeting glioma­associated oncogene (GLI 1).In addition, cell apoptosis was induced increasingly by transfection with GLI 1 siRNA or treatment with CBC-1 to downregulate GLI 1. Last, the in vivo results demonstrated that CBC-1 significantly reduced tumour size and downregulated GLI 1 in CRC. Therefore, this study suggests that CBC-1, a new GLI 1 inhibitor derived from natural products, may be developed as a potential antitumour candidate for CRC treatment.

How calcium inhibits the magnesium-dependent kinase gsk3ß: a molecular simulation study.

Glycogen synthase kinase 3ß (GSK3ß) is a ubiquitous serine/threonine kinase that plays a pivotal role in many biological processes. GSK3ß catalyzes the transfer of γ-phosphate of ATP to the unique substrate Ser/Thr residues with the assistance of two natural activating cofactors Mg(2+). Interestingly, the biological observation reveals that a non-native Ca(2+) ion can inhibit the GSK3ß catalytic activity. Here, the inhibitory mechanism of GSK3ß by the displacement of native Mg(2+) at site 1 by Ca(2+) was investigated by means of 80 ns comparative molecular dynamics (MD) simulations of the GSK3ß···Mg(2+)-2/ATP/Mg(2+) -1 and GSK3ß···Mg(2+)-2/ATP/Ca(2+)-1 systems. MD simulation results revealed that using the AMBER point charge model force field for Mg(2+) was more appropriate in the reproduction of the active site architectural characteristics of GSK3ß than using the magnesium-cationic dummy atom model force field. Compared with the native Mg(2+) bound system, the misalignment of the critical triphosphate moiety of ATP, the erroneous coordination environments around the Mg(2+) ion at site 2, and the rupture of the key hydrogen bond between the invariant Lys85 and the ATP O(ß2) atom in the Ca(2+) substituted system were observed in the MD simulation due to the Ca(2+) ion in active site in order to achieve its preferred sevenfold coordination geometry, which adequately abolish the enzymatic activity. The obtained results are valuable in understanding the possible mechanism by why Ca(2+) inhibits the GSK3ß activity and also provide insights into the mechanism of Ca(2+) inhibition in other structurally related protein kinases.

Effects of inoculating feruloyl esterase-producing Lactiplantibacillus plantarum A1 on ensiling characteristics, in vitro ruminal fermentation and microbiota of alfalfa silage.

BACKGROUND: Ferulic acid esterase (FAE)-secreting Lactiplantibacillus plantarum A1 (Lp A1) is a promising silage inoculant due to the FAE's ability to alter the plant cell wall structure during ensiling, an action that is expected to improve forage digestibility. However, little is known regarding the impacts of Lp A1 on rumen microbiota. Our research assessed the influences of Lp A1 in comparison to a widely adopted commercial inoculant Lp MTD/1 on alfalfa's ensilage, in vitro rumen incubation and microbiota. RESULTS: Samples of fresh and ensiled alfalfa treated with (either Lp A1 or Lp MTD/1) or without additives (as control; CON) and ensiled for 30, 60 and 90 d were used for fermentation quality, in vitro digestibility and batch culture study. Inoculants treated silage had lower (P < 0.001) pH, acetic acid concentration and dry matter (DM) loss, but higher (P = 0.001) lactic acid concentration than the CON during ensiling. Compared to the CON and Lp MTD/1, silage treated with Lp A1 had lower (P < 0.001) aNDF, ADF, ADL, hemicellulose, and cellulose contents and higher (P < 0.001) free ferulic acid concentration. Compared silage treated with Lp MTD/1, silage treated with Lp A1 had significantly (P < 0.01) improved ruminal gas production and digestibility, which were equivalent to those of fresh alfalfa. Real-time PCR analysis indicated that Lp A1 inoculation improved the relative abundances of rumen's total bacteria, fungi, Ruminococcus albus and Ruminococcus flavefaciens, while the relative abundance of methanogens was reduced by Lp MTD/1 compared with CON. Principal component analysis of rumen bacterial 16S rRNA gene amplicons showed a clear distinction between CON and inoculated treatments without noticeable distinction between Lp A1 and Lp MTD/1 treatments. Comparison analysis revealed differences in the relative abundance of some bacteria in different taxa between Lp A1 and Lp MTD/1 treatments. Silage treated with Lp A1 exhibited improved rumen fermentation characteristics due to the inoculant effects on the rumen microbial populations and bacterial community. CONCLUSIONS: Our findings suggest that silage inoculation of the FAE-producing Lp A1 could be effective in improving silage quality and digestibility, and modulating the rumen fermentation to improve feed utilization.

Utilization of plant derived lactic acid bacteria for efficient bioconversion of brewers' spent grain into acetoin.

Brewers' spent grain (BSG) is a major side-stream from the beer industry, with an annual estimated production of 39 million tons worldwide. Due to its high nutritional value, high abundance and low price, it has been proposed as an ingredient in human food. Here we investigated the ability of different lactic acid bacteria to produce the flavor molecule acetoin in liquid BSG extract, in order to broaden the possibilities of utilization of BSG in human food. All the investigated lactic acid bacteria (LAB) covering the Leuconostoc, Lactobacillus and Lactoccocus species were able to convert the fermentable sugars in liquid BSG into acetoin. Production levels varied significantly between the different LAB species, with Leuconostoc pseudomesenteroides species reaching the highest titers of acetoin with only acetate as the main byproduct, while also being the fastest consumer of the fermentable sugars present in liquid BSG. Surprisingly, the currently best investigated LAB for acetoin production, L. lactis, was unable to consume the maltose fraction of liquid BSG and was therefore deemed unfit for full conversion of the sugars in BSG into acetoin. The production of acetoin in Leu. pseudomesenteroides was pH dependent as previously observed in other LAB, and the conversion of BSG into acetoin was scalable from shake flasks to 1 L bioreactors. While all investigated LAB species produced acetoin under aerobic conditions, Leu. pseudomesenteroides was found to be an efficient and scalable organism for bioconversion of liquid BSG into a safe acetoin rich food additive.

Insights into the role of magnesium triad in myo-inositol monophosphatase: metal mechanism, substrate binding, and lithium therapy.

myo-Inositol monophosphatase (IMPase) plays a pivotal role in the intracellular phosphatidylinositol cell signaling pathway. It has attracted considerable attention as a putative therapeutic target for lithium therapy in the treatment of bipolar disorder. A trio of activated cofactor Mg²âº ions is required for inositol monophosphate hydrolysis by IMPase. In the present study, computational studies, including two-layered ONIOM-based quantum mechanics/mechanical mechanics (QM/MM) calculations, molecular modeling, and molecular dynamics (MD) simulations, were performed to ascertain the role of the Mg²âº triad in the IMPase active site. The QM/MM calculations show that the structural identity of the nucleophilic water molecule W1 shared by Mg²âº-1 and Mg²âº-3, activated by Thr95/Asp47 dyad, is a hydroxide ion. Moreover, Mg²âº-3 needs to be conjugated with Mg²âº-1 in the binding site to create the activated nucleophilic hydroxide ion in accordance with the three-metal ion catalytic mechanism. The MD simulation of the IMPase-substrate-Mg²âº complex shows that the three Mg²âº ions promote substrate binding and help fix the phosphate moiety of the substrate for nucleophilic attack by the hydroxide ion. When Mg²âº-2 is displaced with Li⁺, the MD simulations of the postreaction complex indicate that the conformation of the catalytic loop (residues 33 to 44) is disrupted and water molecule W2 does not coordinate with Li⁺. This disruption traps the inorganic phosphate and inositolate in the active site, which lead to IMPase inhibition. By contrast, in the native Mg²âº system, the W2 ligated by Mg²âº-2 and Asp200 aids in protonation of the leaving inositolate moiety.

Identifying Active Compounds and Mechanisms of Citrus changshan-Huyou Y. B. Chang against URTIs-Associated Inflammation by Network Pharmacology in Combination with Molecular Docking.

Purpose: The ripe fruits of Citrus changshan-huyou, known as Quzhou Fructus Aurantii (QFA), have been commonly used for respiratory diseases. The purpose of this study was to investigate their active compounds and demonstrate their mechanism in the treatment of upper respiratory tract infections (URTIs) through network pharmacology and molecular docking. Methods: The prominent compounds of QFA were acquired from TCMSP database. Their targets were retrieved from SwissTargetPrediction database, and target genes associated with URTIs were collected from DisGeNET and GeneCards databases. The target protein-protein interaction (PPI) network was constructed by using STRING database and Cytoscape. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were enriched. Visual compound-target-pathway network was established with Cytoscape. The effects of compounds were verified on the inhibitory activities against phosphoinositide 3-kinases (PI3Ks). Finally, the molecular docking was carried out to confirm the binding affinity of the bioactive compounds and target proteins. Results: Five important active compounds, naringenin (NAR), tangeretin (TAN), luteolin (LUT), hesperetin (HES), and auraptene (AUR), were obtained. The enrichment analysis demonstrated that the pathways associated with inflammation mainly contained PI3K/Akt signalling pathway, TNF signalling pathway, and so on. The most important targets covering inflammation-related proteins might be PI3Ks. In vitro assays and molecular docking exhibited that TAN, LUT, and AUR acted as PI3Kγ inhibitors. Conclusion: The results revealed that QFA could treat URTIs through a multi-compound, multi-target, multi-pathway network, in which TAN, LUT, and AUR acted as PI3Kγ inhibitors, probably contributing to a crucial role in treatment of URTIs.