Tubulin inhibitors： pharmacophore modeling,virtua screening and molecular docking

Aim： To construct a quantitative pharmacophore model of tubulin inhibitors and to discovery new leads with potent antitumor activities. Methods： Ligand-based pharmacophore modeling was used to identify the chemical features responsible for inhibiting tubulin polymerization. A set of 26 training compounds was used to generate hypothetical pharmacophores using the HypoGen algorithm. The structures were further validated using the test set, Fischer randomization method, leave-one-out method and a decoy set, and the best model was chosen to screen the Specs database. Hit compounds were subjected to molecular docking study using a Molecular Operating Environment （MOE） software and to biological evaluation in vitro. Results： Hypol was demonstrated to be the best pharmacophore model that exhibited the highest correlation coefficient （0.9582）, largest cost difference （70.905） and lowest RMSD value （0.6977）. Hypol consisted of one hydrogen-bond acceptor, a hydrogen-bond donor, a hydrophobic feature, a ring aromatic feature and three excluded volumes. Hypol was validated with four different methods and had a goodness-of-hit score of 0.81. When Hypol was used in virtual screening of the Specs database, 952 drug-like compounds were revealed. After docking into the colchicine-binding site of tubulin, 5 drug-like compounds with the required interaction with the critical amino acid residues and the binding free energies 〈-4 kcal/mol were selected as representative leads. Compounds 1 and 3 exhibited inhibitory activity against MCF-7 human breast cancer cells in vitro. Conclusion： Hypol is a quantitative pharmacophore model for tubulin inhibitors, which not only provides a better understanding of their interaction with tubulin, but also assists in discovering new potential leads with antitumor activities.     

Molecular modeling,quantum polarized ligand docking and structure-based 3D-QSAR analysis of the imidazole series as dual AT~ and ETA receptor antagonists

Aim： Both endothelin ETA receptor antagonists and angiotensin AT1 receptor antagonists lower blood pressure in hypertensive patients. A dual AT1 and ETA receptor antagonist may be more efficacious antihypertensive drug. In this study we identified the mode and mechanism of binding of imidazole series of compounds as dual AT1 and ETA receptor antagonists. Methods： Molecular modeling approach combining quantum-polarized ligand docking （QPLD）, MM/GBSA free-energy calculation and 3D-QSAR analysis was used to evaluate 24 compounds as dual AT1 and ETA receptor antagonists and to reveal their binding modes and structural basis of the inhibitory activity. Pharmacophore-based virtual screening and docking studies were performed to identify more potent dual antagonists. Results： 3D-QSAR models of the imidazole compounds were developed from the conformer generated by QPLD, and the resulting models showed a good correlation between the predicted and experimental activity. The visualization of the 3D-QSAR model in the context of the compounds under study revealed the details of the structure-activity relationship： substitution of methoxymethyl and cyclooctanone might increase the activity against AT~ receptor, while substitution of cyclohexone and trimethylpyrrolidinone was important for the activity against ETA receptor; addition of a trimethylpyrrolidinone to compound 9 significantly reduced its activity against AT~ receptor but significantly increased its activity against ETA receptor, which was likely due to the larger size and higher intensities of the H-bond donor and acceptor regions in the active site of ETA receptor. Pharmacophore-based virtual screening followed by subsequent Glide SP, XP, QPLD and MM/GBSA calculation identified 5 potential lead compounds that might act as dual AT1 and ETA receptor antagonists. Conclusion： This study may provide some insights into the development of novel potent dual ETA and AT1 receptor antagonists. As a result, five compounds are found to be the best dual antagonists against AT1R and ETA receptors.     

A thienopyridine,CB-20, exerts diuretic activity by inhibiting urea transporters

Urea transporters(UTs)are transmembrane proteins selectively permeable to urea and play an important role in urine concentration.UT-knockout mice exhibit the urea-selective urine-concentrating defect,without affecting electrolyte balance,suggesting that UT-B inhibitors have the potential to be developed as novel diuretics.In this study,we characterized a novel compound 5-ethyl-2-methyl-3-amino-6-methylthieno[2,3-b]pyridine-2,5-dicarboxylate(CB-20)with UT inhibitory activity as novel diuretics with excellent pharmacological properties.This compound was discovered based on high-throughput virtual screening combined with the erythrocyte osmotic lysis assay.Selectivity of UT inhibitors was assayed using transwell chambers.Diuretic activity of the compound was examined in rats and mice using metabolic cages.Pharmacokinetic parameters were detected in rats using LC-MS/MS.Molecular docking was employed to predict the potential binding modes for the CB-20 with human UT-B.This compound dose-dependently inhibited UT-facilitated urea transport with IC50 values at low micromolar levels.It exhibited nearly equal inhibitory activity on both UT-A1 and UT-B.After subcutaneous administration of CB-20,the animals showed polyuria,without electrolyte imbalance and abnormal metabolism.CB-20 possessed a good absorption and rapid clearance in rat plasma.Administration of CB-20 for 5 days did not cause significant morphological abnormality in kidney or liver tissues of rats.Molecular docking showed that CB-20 was positioned near several residues in human UT-B,including Leu364,Val367,and so on.This study provides proof of evidence for the prominent diuretic activity of CB-20 by specifically inhibiting UTs.CB-20 or thienopyridine analogs may be developed as novel diuretics.     

CB-Dock: a web server for cavity detection-guided protein–ligand blind docking

As the number of elucidated protein structures is rapidly increasing,the growing data call for methods to efficiently exploit the structural information for biological and pharmaceutical purposes.Given the three-dimensional(3D)structure of a protein and a ligand,predicting their binding sites and affinity are a key task for computer-aided drug discovery.To address this task,a variety of docking tools have been developed.Most of them focus on docking in the preset binding sites given by users.To automatically predict binding modes without information about binding sites,we developed a user-friendly blind docking web server,named CB-Dock,which predicts binding sites of a given protein and calculates the centers and sizes with a novel curvature-based cavity detection approach,and performs docking with a popular docking program,Autodock Vina.This method was carefully optimized and achieved^70% success rate for the top-ranking poses whose root mean square deviation(RMSD)were within 2Åfrom the X-ray pose,which outperformed the state-of-the-art blind docking tools in our benchmark tests.CB-Dock offers an interactive 3D visualization of results,and is freely available at http://cao.labshare.cn/cb-dock/.     

Benzbromarone, an old uricosuric drug, inhibits human fatty acid binding protein 4 in vitro and lowers the blood glucose level in db/db mice

Aim： Fatty acid-binding protein 4 （FABP4） plays an important role in maintaining glucose and lipid homeostasis. The aim of this study was to find new inhibitors of FABP4 for the treatment of type 2 diabetes. Methods： Human FABP4 protein was expressed, and its inhibitors were detected in 1,8-ANS displacement assay. The effect of the inhibitor on lipolysis activity was examined in mouse 3T3-L1 preadipocytes. The db/db mice were used to evaluate the anti-diabetic activity of the inhibitor. Molecular docking and site-directed mutagenesis studies were carried out to explore the binding mode between the inhibitor and FABP4. Results： From 232 compounds tested, benzbromarone （BBR）, an old uricosuric drug, was discovered to be the best inhibitor of FABP4 with an IC~o value of 14.8 pmol/L. Furthermore, BBR （25 pmol/L） significantly inhibited forskolin-stimulated lipolysis in 3T3-L1 cells. Oral administration of BBR （25 or 50 mg/kg, for 4 weeks） dose-dependently reduced the blood glucose level and improved glucose tolerance and insulin resistance in db/db mice. Molecular docking revealed that the residues Ser55, Asp76, and Arg126 of FABP4 formed important interactions with BBR, which was confirmed by site-directed mutagenesis studies. Conclusion： BBR is an inhibitor of FABP4 and a potential drug candidate for the treatment of type 2 diabetes and atherosclerosis.     

Molecular docking and 3D-QSAR studies of 2-substituted 1-indanone derivatives as acetylcholinesterase inhibitors

Aim： To explore the binding mode of 2-substituted 1-indanone derivatives with acetylcholinesterase （ACHE） and provide hints for the future design of new de- rivatives with higher potency and specificity. Methods： The GOLD-docking con- formations of the compounds in the active site of the enzyme were used in subse- quent studies. The highly reliable and predictive three-dimensional quantitative structure-activity relationship （3D-QSAR） models were achieved by comparative molecular field analysis （CoMFA） and comparative molecular similarity analysis （CoMSIA） methods. The predictive capabilities of the models were validated by an external test set. Moreover, the stabilities of the 3D-QSAR models were veri- fied by the leave-4-out cross-validation method. Results： The CoMFA and CoMSIA models were constructed successfully with a good cross-validated coef- ficient （q2） and a non-cross-validated coefficient （r2）. The q2 and r2 obtained from the leave- 1-out cross validation method were 0.784 and 0.974 in the CoMFA model and 0.736 and 0.947 in the CoMSIA model, respectively. The coefficient isocontour maps obtained from these models were compatible with the geometrical and physi- cochemical properties of ACHE. Conclusion： The contour map demonstrated that the binding affinity could be enhanced when the small protonated nitrogen moi- ety was replaced by a more hydrophobic and bulky group with a highly partial positive charge. The present study provides a better understanding of the inter- action between the inhibitors and ACHE, which is helpful for the discovery of new compounds with more potency and selective activity.     

Energetic factors determining the binding of type I inhibitors to c-Met kinase： experimental studies and quantum mechanical calculations

Aim： To decipher the molecular interactions between c-Met and its type I inhibitors and to facilitate the design of novel c-Met inhibitors. Methods： Based on the prototype model inhibitor 1, four ligands with subtle differences in the fused aromatic rings were synthesized. Quantum chemistry was employed to calculate the binding free energy for each ligand. Symmetry-adapted perturbation theory （SAPT） was used to decompose the binding energy into several fundamental forces to elucidate the determinant factors. Results： Binding free energies calculated from quantum chemistry were correlated well with experimental data. SAPT calculations showed that the predominant driving force for binding was derived from a sandwich n-n interaction with Tyr-1230. Arg-1208 was the differentiating factor, interacting with the 6-position of the fused aromatic ring system through the backbone carbonyl with a force pattern similar to hydrogen bonding. Therefore, a hydrogen atom must be attached at the 6-position, and changing the carbon atom to nitrogen caused unfavorable electrostatic interactions. Conclusion： The theoretical studies have elucidated the determinant factors involved in the binding of type I inhibitors to c-Met.     

Study of effect of oxygen／glucose-deprived culture on the brain-pancreas relative protein in PC12 cells and the mechanism

AIM: To study the effect of oxygen/glucose-deprived (OGD)culture on the expression of a novel protein, brain-pancreas relative protein (BPRP), and the possible regulating mechanism in vitro. BPRP was a key protein found in our previous study of cerebral ischemia. METHODS: PC12 cells was selected and exposed to the Eagle‘s solution containing 1 mmol/L Na2S2O4 for     

Discovery of potent β-secretase （bace-1） inhibitors by the synthesis of isophthalamide-containing hybrids

Aim： The aim of this study was to design and synthesize a series of high activity compounds against aspartyl protease β-secretase （BACE-1） bearing hydroxyethylene （HE） framework. Methods： First, we designed the small library based on our previous work and rational analysis. Subsequently, thirteen compounds were selected and synthesized using skilled solid phase synthetic methods to explore the relationship between structure and activity. We then used molecular modeling to explain the possible binding mode. Results： Thirteen new compounds （6-18） have been designed, synthesized and bioassayed. Their structures were determined by nuclear magnetic resonance （NMR） spectra, low-and high-resolution mass spectra and optical rotation. Most compounds have shown moderate to excellent activities, and compound 10, which contains fewer amino acids and amide bonds than GRL-7234, was about 5-fold more potent than the control compound 4 discovered by Merck. The molecular modeling results have indicated the possible binding mode and explained the difference between compounds 10 and 16, providing direction for further study. Conclusion： This study yielded several high activity compounds bearing fewer amino acids and amide bonds than previous compounds, providing insight into the further development of potent BACE-1 inhibitors for the treatment of Alzheimer＇s disease.     

Discovery of potent inhibitors for phosphodiesterase 5 by virtual screening and pharmacophore analysis

Aim： To explore the potent inhibitor from one of the Traditional Chinese medicine （TCM）, Epimedium sagittatum. Methods： We predicted the potent compound, ESO3b, de novo evolution from the four Epimedium sagittatum components were verified by molecular docking, pharmacophore analysis, and analysis of quantitative structure-activity relationship （QSAR） model, which was constructed by multiple linear regression. Results： ESO3b was chosen to undergo drug modification via de novo evolution. By analyzing the pharmacophore features, we found that the hydrophobic core in the binding site and the hydrogen bond generated at Asn663 played key roles in designing PDE5 inhibitors. ESO3b generated 49 diversities （Evo01-49）. Evo48 had high activity in prediction. Although the value of prediction was overestimated, Evo48 was suggested as the potent lead. Conclusion： In this study, we showed that the hydrophobic core in the binding site and hydrogen bond production on Asn663 played key roles to design PDE5 inhibitors. From several require validation analysis, Evo48 was suggested to be a potent inhibitor.     

Novel 16-substituted bifunctional derivatives of huperzine B： multifunctional cholinesterase inhibitors

Aim： To design novel bifunctional derivatives of huperzine B （HupB） based on the concept of dual binding site of acetylcholinesterase （ACHE） and evaluate their pharmacological activities for seeking new drug candidates against Alzheimer＇s disease （AD）. Methods： Novel 16-substituted bifunctional derivatives of HupB were synthesized through chemical reactions. The inhibitory activities of the derivatives toward AChE and butyrylcholinesterase （BuChE） were determined in vitro by modified EIIman＇s method. Cell viability was quantified by the reduction of MTT. Results： A new preparative method was developed for the generation of 16-substituted derivatives of HupB, and pharmacological trials indicated that the derivatives were multifunctional cholinesterase inhibitors targeting both AChE and BuChE. Among the derivatives tested, 9c, 9e, 9f, and 9i were 480 to 1360 times more potent as AChE inhibitors and 370 to 1560 times more potent as BuChE inhibitors than the parent HupB. Further preliminary pharmacological trials of derivatives 9c and 9i were performed, including examining the mechanism of AChE inhibition, the substrate kinetics of the enzyme inhibition, and protection against hydrogen peroxide （H2O2）-induced cytotoxicity in PC12 cells. Conclusion： Preliminary pharmacological evaluation indicated that 16-substituted derivatives of HupB, particularly 9c and 9i, would be potentially valuable new drug candidates for AD therapy, and further exploration is needed to evaluate their pharmacological and clini- cal efficacies.     

Small envelope protein E of SARS：cloning,expression, purification, CD determination, and bioinformatics analysis

AIM:To obtain the pure sample of SARS small envelope E protein (SARS E protein), study its properties and analyze its possible functions. METHODS: The plasmid of SARS E protein was constructed by the polymerase chain reaction (PCR), and the protein was expressed in the E coli strain. The secondary structure feature of the protein was determined by circular dichroism (CD) technique. The possible functions of this protein were annotated by bioinformatics methods, and its possible three-dimensional model was constructed by molecular modeling. RESULTS: The pure sample of SARS E protein was obtained. The secondary structure feature derived from CD determination is similar to that from the secondary structure prediction. Bioinformatics analysis indicated that the key residues of SARS E protein were much conserved compared to the E proteins of other coronaviruses. In particular, the primary amino acid sequence of SARS E protien is much more similar to that of murine hepatitis virus(MHV) and other mammal coronaviruses. The transmembrane (TM) segment of the SARS E protein is relatively more conserved in the whole protein than other regions. CONCLUSION: The success of expressing the SARS E protein is a good starting point for investigating the structure and functions of this protein and SARS coronavirus itself as well. The SARS E protein may fold in water solution in a similar way as it in membrane-water mixed environment. It is possible that β-sheet I of the SARS E protein interacts with the membrane surface via hydrogen bonding, this β-sheet may uncoil to a random structure in water solution.     

Addictive evaluation of cholic acid-verticinone ester,a potential cough therapeutic agent with agonist action of opioid receptor

Aim： The purpose of this work was to search for potential drugs with potent antitussive and expectorant activities as well as a low toxicity, but without addictive properties. Cholic acid-verticinone ester （CA-Ver） was synthesized based on the clearly elucidated antitussive and expectorant activities of verticinone in bulbs of Fritillaria and different bile acids in Snake Bile. In our previous study, CA-Ver showed a much more potent activity than codeine phosphate. This study was carried out to investigate the central antitussive mechanism and the addictive evaluation of CA-Ver. Methods： Testing on a capsaicin-induced cough model of mice pretreated with naloxone, a non-selective opioid receptor antagonist, was performed for the observation of CA-Ver＇s central antitussive mechanism. We then took naloxone-induced withdrawal tests of mice for the judgment of CA-Ver＇s addiction. Lastly, we determined the opioid dependence of CA-Ver in the guinea pig ileum. Results： The test on the capsaicin-induced cough model showed that naloxone could block the antitussive effect of CA-Ver, suggesting the antitussive mechanism of CA-Ver was related to the central opioid receptors. The naloxone-urged withdrawal tests of the mice showed that CA-Ver was not addictive, and the test of the opioid dependence in the guinea pig ileum showed that CA-Ver had no withdrawal response. Conclusion： These findings suggested that CA-Ver deserved attention for its potent antitussive effects related to the central opioid receptors, but without addiction, and had a good development perspective.     

Development of a quantitative, cell-based, high-content screening assay for epidermal growth factor receptor modulators

Aim： To develop a robust, cell-based, high-content screening （HCS） assay based on receptor internalization for the identification of novel modulators of the epidermal growth factor receptor （EGFR）. Methods： Agonist-induced receptor internalization is part of the signaling cascade of EGFR. Fluorescent-tagged epidermal growth factor （EGF） was used to visualize the internalized receptor- ligand complex. The fluorescent intracellular spots were detected and measured with an ArrayScan HCS reader. Compounds that can competitively bind to EGFR or interfere with EGFR internalization process would result in a reduced number and intensity of intracellular fluorescent spots. This assay was validated, optimized, and applied to a large-scale screening of a library containing 48 000 synthetic compounds. Results： The competition between fluorescent EGF and unlabeled EGF reveals the IC50 of unlabeled EGF is approximately 0.2 nmol/L, which is comparable with other published reports. Thirteen compounds with a relatively high degree of interference with EGFR internalization were identified. One of the compounds was proven to be agonist of the EGFR since it induced phosphorylation of the receptor and extracellular signal-regulated protein kinase （ERK）. Conclusion： This automated, objective, and easy-to-use assay provided abundant information, quantitative results, and demonstrated the potential use of HCS methods in searching membrane receptor modulators.     

Gabapentin effectively alleviates pain sensitization and regulates CACNA2D1 expression in postherpetic neuralgia rats北大核心CSCD

Aim To identify the molecular target of gabapentin in the treatment of postherpetic neuralgia(PHN). Methods The molecular target of gabapentin for PHN was analyzed by network pharmacology and molecular docking and confirmed by coprecipitation test. Rats were randomly divided into control group, model group, model+50 mg·kg-1 gabapentin group, model+100 mg·kg-1 gabapentin group, and model+200 mg·kg-1 gabapentin group, with nine rats in each group. The pain-related behaviors of the rats were measured at different time points. The mRNA and protein expressions of CACNA2D1, Bax, and Bcl-2 in rat spinal cord were determined by immunofluorescence, Western blot, and qPCR. Results CACNA2D1 was the target gene of gabapentin that determined via network pharmacology, molecular docking, and co-precipitation tests. After modeling, mechanical pain threshold and thermal pain threshold significantly decreased, and the number of apoptotic GABA cells significantly increased. However, after intraperitoneal injection of 50, 100, and 200 mg·kg-1 gabapentin, mechanical pain threshold and thermal pain threshold significantly increased(P<0.05), and the number of apoptotic GABA cells significantly decreased(P<0.01). Immunofluorescence and Western blot results showed that compared with the model group, with the increase of gabapentin concentration, the positive expression rate of Bax significantly decreased, and the positive expression rate of Bcl-2 and CACNA2D1 significantly increased. The mRNA expression levels of Bax, Bcl-2 and CACNA2D1 detected by qPCR were consistent with the results of immunofluorescence and Western blot. Conclusions Gabapentin up-regulates the expression of target protein CACNA2D1, inhibits the proapoptotic protein Bax, and promotes the expression of apoptotic inhibitor Bcl-2. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

Derivatives of （phenylsulfonamido-methyl）nicotine and （phenylsulfonamido-methyl）thiazole as novel 11β-hydroxysteroid dehydrogenase type 1 inhibitors： synthesis and biological activities in vitro

Aim： To design and synthese a novel class of 11β-hydroxysteroid dehydrogenase type 1 （11β-HSD1） inhibitors, featuring the （phenylsul- fonamido-methyl）pyridine and （phenylsulfonamido-methyl）thiazole framework. Methods： Our initial lead 4-（phenylsulfonamido-methyl）benzamides were modified. Inhibition of human and mouse 11β-HSD1 enzymatic activities by the new compounds was determined by a scintillation proximity assay （SPA） using microsomes containing 11β- HSDI. Results： Sixteen new compounds （6a-6h, 7a-7h） were designed, synthesized and bioassayed. In dose-response studies, several compounds showed strong inhibitory activities with IC50 values at nanomolar or low nanomolar concentrations. Structure-activity relationships are also discussed with respect to molecular docking results. Conclusion： This study provides two promising new templates for 11β-HSD1 inhibitors.     

Establishment and characteristic analysis of fibroblast-like synoviocytes in rats with adjuvant arthritis北大核心CSCD

Aims To establish the methods of primary culture of fibroblast-like synoviocytes in rats with adjuvant arthritis (AA-FLS) and analyze the feature and to investigate the possibility of AA-FLS as the model for the RA in vitro. Methods The synovial cells obtained from the SD rats were immunized by the Mtb and identified by morphology and immunocytochemistry. The viability of AA-FLS was assessed by Cell Counting Kit-8. ELISA was applied to detect TNF-α and IL-lβ in cell media. Apoptosis was measured by Hochest 33258. The expressions of mitochondrial apoptosis-re-lated molecules, including Bcl-2, Bax, Pro-caspase-3 and Cleave-caspase-3 were determined by Western Blot. Result In isolated primary synovial cells, more than 95% of AA-FLSs were fusiform. Immunocyto-chemistry result showed a positive expression of vimen-tin and a negative expression of CD68 in AA-FLS. Cell proliferation of AA-FLS was higher than FLS and cell apoptosis of AA-FLS was curbed. Western blot data demonstrated that the protein expressions of Bcl-2, Bax were regulated and the expression of caspase-3 was activated in AA-FLS. Conclusions AA-FLS is biologically characterized by high level proliferation activity and inflammatory cytokines and apoptosis suppression. AA-FLS can be used as the model for the RA in vitro.     

Protective effects of the novel adenosine derivative WS0701 in a mouse model of posttraumatic stress disorder

Aim： To investigate the effects of the novel N6-substituted adenosine derivative {（2R,3S,4R,5R）-3,4-dihydroxy-5-[6-[（4-hydroxy-3-methoxybenzyl）amino]-9H-purin-9-yl]tetrahydrofuran-2-yl} methyl decanoate （WS0701） on stress-induced excessive fear, anxiety, and cognitive deficits in a mouse model of posttraumatic stress disorder （PTSD）.
Methods： Molecular modeling approach combining quantum-polarized ligand docking （QPLD）, MM/GBSA free-energy calculation and 3D-QSAR analysis was used to evaluate 24 compounds as dual AT1 and ETA receptor antagonists and to reveal their binding modes and structural basis of the inhibitory activity. Pharmacophore-based virtual screening and docking studies were performed to identify more potent dual antagonists.

Results： WS0701 administration significantly alleviated fear, anxious behaviors and memory deficits in the mouse model of PTSD. Furthermore, WS0701 administration significantly reduced the stress-induced apoptosis of hippocampal neurons, and increased the Bcl-2/Bax ratio in the hippocampus. The positive control drug paroxetine exerted similar effects on PTSD-like behaviors and hippocampal neuron apoptosis in the mouse model of PTSD, which were comparable to those caused by the high dose of WS0701.

Conclusion： WS0701 effectively mitigates stress-induced PTSD-like behaviors in mice, partly via inhibition of neuronal apoptosis in the hippocampus.     

Composite glycidyl methacrylated dextran （Dex-GMA）/gelatin nanoparticles for localized protein delivery

Aim： Localized delivery of growth factors has significant potential as a future therapeutic strategy in tissue engineering and regenerative medicine. A nanoparticle vehicle was created and evaluated in this study with the intent to deliver growth factors for periodontal regeneration.
Methods： Novel composite nanoparticles based on glycidyl methacrylate derivatized dextrans （Dex-GMA） and gelatin were fabricated by a facile method without using any organic solvents. The configurations of the resultant nanoparticles were evaluated by transmission electron microscopy, scanning electron microscopy, and atomic force microscope. Their surfaces were characterized by zeta-potential measurements, after which their properties including swelling, degradation, drug release, and cytotoxicity were also investigated using in vitro models. Results： The particle size of Dex-GMA/gelatin nanoparticles （DG-NPs） ranged from 20 to 100 nm and showed a mono-dis- perse size distribution （mean diameter 53.7 nm） and a strongly negative surface zeta potential （-20 mV）. The DG-NPs were characterized by good swelling and degradation properties in media including dextranase. The in vitro drug release studies showed that the efficient bone morphogenetic protein （BMP） release from DG-NPs was maintained for more than 12 d under degradation conditions, where more than 90% of the loaded BMP was released. No any relevant cell damage caused by DG-NPs was found in the cytotoxicity tests for a period of 24 h.
Conclusion： These combined results demonstrate that DG-NPs fulfill the basic prerequisites for growth factor delivery. With further in vivo studies, those nanoparticles may offer a promising vehicle for the delivery of active drugs to the perio- dontium.     

Oligomannurarate sulfate inhibits CXCL12/SDF-1- mediated proliferation and invasion of human tumor cells in vitro

Aim： JG6 is a novel marine-derived oligosaccharide that has shown to inhibit angiogenesis and tumor metastasis. In this study, we sought to identify the potential target responsible for the anti-cancer activity of JG6. Methods： Human liver cancer cell line Bel-7402 and human cervical cancer cell line HeLa were examined. CXCL12-stimulated cell proliferation and migration were determined using a CCK-8 kit and a transwell assay, respectively. Western blotting was performed to examine the changes in CXCL12/CXCR4 axis. Molecular docking and surface plasmon resonance （SPR） were performed to characterize the possible interaction between JG6 and the CXCL12/CXCR4 axis. Results： Treatment with CXCL12 potently stimulated the proliferation and migration in both Bel-7402 and HeLa cells. Co-treatment of the cells with JG6 （10, 50 and 100 pp=VmL） dose-dependently impeded the CXCL12-stimulated cell proliferation and migration. Furthermore, CXCL12 rapidly induced phosphorylation of AKT, ERK, FAK and Paxillin in Bel-7402 and HeLa cells, whereas pretreatment with JG6 dose-dependently inhibited the CXCL12-induced phosphorylation of these proteins. The SPR assay showed that JG6 bound to CXCL12 with a high affinity. In molecular docking study, JG6 appeared to interact with CXCL12 via multiple polar interactions, including 6 ionic bonds and 7 hydrogen bonds. Conclusion： Inhibition of the CXCL12/CXCR4 axis by JG6 may account for its anticancer activity.