Active legumain promotes invasion and migration of neuroblastoma by regulating epithelial-mesenchymal transition.

Neuroblastoma (NB) is a commonly occurring malignancy in children. Epithelial-mesenchymal transition (EMT) is an adaptive change in promoting tumor metastasis. As an important factor in regulating tumor metastasis, whether legumain could promote metastasis of NB by EMT is still unexplored. Legumain is the active form of prolegumain, abundant in tumor plasma. So in the current study, different forms of legumain were identified in NB. Second, correlation analysis of N-cadherin and active legumain was identified by western blot analysis. Third, legumain gene amplification or gene knockdown were proceeded to examine the effect of legumain on EMT by scratch and transwell assay; meanwhile, active mature legumain or its asparagine endopeptidase (AEP) inhibitor was also added in. Finally, legumain can be detected differently in NB cells. Changes in legumain could influence NB metastasis by regulating EMT markers (e.g., N-cadherin, vimentin, and slug). Besides, the effect of legumain on EMT by its AEP activity was proved by intervention experiment of AEP gene transfection and gene knockdown experiments or adding recombinant human legumain suspension or specific inhibitor of AEP in NB cells (p < 0.05). These results suggest that legumain can promote invasion and migration of NB by regulating EMT, and EMT of NB is regulated by AEP activity of legumain, which can be inhibited by a specific AEP inhibitor.

New azaphilones, phomopsones A-C with biological activities from an endophytic fungus Phomopsis sp. CGMCC No.5416.

Three undescribed azaphilones, phomopsones A-C (1-3) and two known azaphilones (4-5) were isolated from the culture of endophytic fungus Phomopsis sp. CGMCC No.5416 from the stems of Achyranthes bidentata. Their structures were determined by spectroscopic analysis (HRESIMS, 1D and 2D NMR), and the absolute configurations were determined by CD spectroscopy. Compounds 2 and 3 showed significant inhibitory activities against HIV-1 with against HIV-1 with IC50 values of 7.6 and 0.5 µmol/L, respectively. Compounds 2 and 3 also displayed moderate cytotoxicity with CC50 values of 3.2-303 µmol/L against A549, MDA-MB-231 and PANC-1 cell lines. Moreover, compound 3 can induce the early apoptosis of PANC-1 cancer cells with the apoptosis rate of 28.54%.

The Study of Release Mechanisms for Drug in Cyclodextrin Metal-Organic Frameworks.

γ-Cyclodextrin metal-organic frameworks (γCDMOF) recently emerged as biofriendly, highly porous, and crystalline materials with potential applications in drug delivery. However, little is known about their drug entrapment and release characteristics, which are key parameters in the design of drug carriers. The macroscopic properties of a material are determined by its microstructure. Thus, the characteristics of the constitutive units of the cubic crystalline γCDMOF determine their drug loading and release behaviors. In this study, the release profile of prednisolone (PNS) form γCDMOF was predicted, and the mechanism was analyzed based on the γCDMOF molecular structure. For the first time, experimental, molecular simulation, and mathematical modeling methods were combined to gain insights into the drug distribution in cubic porous crystals of γCDMOF as well as on drug release kinetics. The predicted release profile was in good agreement with the experimental results, showing that the modeling method was reliable. The methodology developed here could provide a reference for further investigations of drug penetration and release in supramolecular systems.

Efficient synthesis of an antiviral drug intermediate using an enhanced short-chain dehydrogenase in an aqueous-organic solvent system.

(2R,3S)-N-tert-Butoxycarbonyl-3-amino-1-chloro-2-hydroxy-4-phenylbutane (1b) is key for the synthesis of the antiviral drug atazanavir. It can be obtained via the stereoselective bioreduction of (3S)-3-(N-Boc-amino)-1-chloro-4-phenyl-butanone (1a) with short-chain dehydrogenase/reductase (SDR). However, the stereoselective bioreduction of this hydrophobic and bulky substrate still remained a challenge because of the steric hindrance effect and low mass transfer rate. In this study, SDR isolated from Novosphingobium aromaticivorans (NaSDR) having low activity to 1a, which was engineered to enhance catalytic efficiency through active pocket iterative saturation mutagenesis (ISM). The obtained mutant (muSDR) (G141V/I195L) had 3.57 times higher kcat than the wild type (WT) towards 1a. Molecular docking analysis revealed considerable differences in the distance between the substrate and catalytic residues in WT and mutant SDR. Moreover, muSDR reduced 15 ketones with excellent enantioselectivity, indicating broad substrate acceptance. After optimization of expression and reaction conditions, the conversion was completed in a scale-up reaction (500 mL) using 50% toluene with 500 mM substrate without additional NADH. These results show that muSDR may be a valuable biocatalyst for future industrial applications.

Legumain correlates with neuroblastoma differentiation and can be used in prodrug design.

Neuroblastoma (NB) is a highly malignant solid tumor in children. The cysteine endopeptidase legumain is expressed in adult solid tumors, but its expression in NB has not been examined. In this study, we assayed legumain expression in two NB cell lines and in microarrays of tumor tissues collected from 46 children with undifferentiated NB, differentiated NB, and ganglioneuroblastoma. Correlation analyses showed that legumain was expressed in all NB cell lines tested and that expression correlated with the degree of tumor differentiation. The efficacy, specificity, and toxicity of EMC-AANL-DOX, a novel doxorubicin-based legumain-activated prodrug, were then evaluated in mouse model of NB. Compared with DOX, EMC-AANL-DOX showed greater inhibition of tumor growth and a lower toxicity at high doses. Neither leukocyte or platelet counts nor renal function or cardiac anatomy differed significantly between the EMC-AANL-DOX and control groups (p > .05), suggesting that the prodrug caused minimal bone marrow depression and did not induce renal or cardiac damage. The good specificity and efficacy of EMC-AANL-DOX and low toxicity recommend its use in the treatment of NB. Correlation analyses of NB differentiation and legumain expression may reveal a novel anti-tumor-related functions of the drug and a new strategy for the treatment of pediatric solid tumors.

Structure-toxicity relationship study of para-halogenated styrene analogues in CYP2E1 transgenic cells.

Styrene is one of the most important industrial intermediates consumed in the world and is mainly used as a monomer for reinforced plastics and rubber. Styrene has been found to be hepatotoxic and pneumotoxic in humans and experimental animals. The toxicity of styrene is suggested to be metabolism-dependent. Styrene-7,8-oxide has been considered as the major metabolite responsible for styrene-induced cytotoxicity. The objective of the study was to investigate the correlation between cytotoxicity of styrene and chemical and biochemical properties of the vinyl group of styrene by development of structure activity relationships (SAR). 4-Fluorostyrene, 4-chlorostyrene and 4-bromostyrene were selected for the SAR study. Cytotoxicity of styrene and the halogenated styrene derivatives with an order of 4-bromostyrene>4-chlorostyrene>4-fluorostyrene≈styrene was observed in CYP2E1 transgenic cells. Similar orders in the efficiency of the metabolism of styrene and the halogenated styrene analogues to their oxides and in the electrophilicity of the corresponding oxides were observed. Additionally, the order of the potency of cellular glutathione depletion and the degree of protein adduction induced by styrene and the halogenated styrenes were consistent with that of their cytotoxicities. The wild-type cells were less susceptible to the toxicity of the corresponding model compounds than CYP2E1 cells. The present study provided insight into the roles of the biochemical and chemical properties of styrene in its cytotoxicity.

Water-soluble phosphate prodrugs of pleuromutilin analogues with potent in vivo antibacterial activity against Gram-positive pathogens.

A phosphate prodrug strategy was investigated to address the problem of poor aqueous solubility of pleuromutilin analogues. Water-soluble phosphate prodrugs 6a, 6b and 6c of pleuromutilin analogues were designed and synthesized. Three compounds all exhibited excellent aqueous solubility (>50mg/mL) at near-neutral pH and sufficient stability in buffer solution. In particular, the phenol pleuromutilin prodrug 6c displayed favourable pharmacokinetic profiles and comparable potency with vancomycin against MSSA and MRSA strains in vivo.

Synthesis and biological evaluation of new N-linked 5-triazolylmethyl oxazolidinones.

A new series of oxazolidinones bearing N-linked 5-triazolylmethyl group have been synthesized and their in vitro antibacterial activities (MIC) were evaluated against a spectrum of resistant and susceptible Gram-positive organisms. Some of the analogues in this series displayed activity superior to linezolid and vancomycin. Furthermore, in vivo efficacies and pharmacokinetic properties of the selected compounds were also disclosed herein; the selected compounds showed reasonable bioavailability as well as in vivo efficacy comparable to that of linezolid.

Synthesis and antibacterial activity of oxazolidinones containing triazolyl group.

A new series of oxazolidinones containing triazolyl group has been synthesized and tested for in vitro antibacterial activity by MIC determination against a panel of resistant and susceptible Gram-positive organisms. Most of the analogs in this series displayed activity superior to linezolid and vancomycin in vitro. Further, in vivo efficacies of the selected oxazolidinones were also disclosed herein.