Synthesis and In Vitro Anti-Influenza Virus Evaluation of Novel Sialic Acid (C-5 and C-9)-Pentacyclic Triterpene Derivatives.

The emergence of drug resistant variants of the influenza virus has led to a great need to identify novel and effective antiviral agents. In our previous study, a series of sialic acid (C-2 and C-4)-pentacyclic triterpene conjugates have been synthesized, and a five-fold more potent antiviral activity was observed when sialic acid was conjugated with pentacyclic triterpene via C-4 than C-2. It was here that we further reported the synthesis and anti-influenza activity of novel sialic acid (C-5 and C-9)-pentacyclic triterpene conjugates. Their structures were confirmed by ESI-HRMS, ¹H-NMR, and 13C-NMR spectroscopic analyses. Two conjugates (26 and 42) showed strong cytotoxicity to MDCK cells in the CellTiter-Glo assay at a concentration of 100 µM. However, they showed no significant cytotoxicity to HL-60, Hela, and A549 cell lines in MTT assay under the concentration of 10 µM (except compound 42 showed weak cytotoxicity to HL-60 cell line (10 µM, ~53%)). Compounds 20, 28, 36, and 44 displayed weak potency to influenza A/WSN/33 (H1N1) virus (100 µM, ~20-30%), and no significant anti-influenza activity was found for the other conjugates. The data suggested that both the C-5 acetylamide and C-9 hydroxy of sialic acid were important for its binding with hemagglutinin during viral entry into host cells, while C-4 and C-2 hydroxy were not critical for the binding process and could be replaced with hydrophobic moieties. The research presented herein had significant implications for the design of novel antiviral inhibitors based on a sialic acid scaffold.

Discovery of novel heteroarylmethylcarbamodithioates as potent anticancer agents: Synthesis, structure-activity relationship analysis and biological evaluation.

A series of new analogs based on the structure of lead compound 10 were designed, synthesized and evaluated for their in vitro anti-cancer activities against four selected human cancer cell lines (HL-60, Bel-7402, SK-BR-3 and MDA-MB-468). Several synthesized compounds exhibited improved anti-cancer activities comparing with lead compound 10. Among them, 1,3,4-oxadiazole analogs 17o showed highest bioactivity with IC50 values of 1.23, 0.58 and 4.29 µM against Bel-7402, SK-BR-3 and MDA-MB-468 cells, respectively. It is noteworthy that 17o has potent anti-proliferation activity toward a panel of cancer cells with relatively less cytotoxicity to nonmalignant cells. The further mechanistic study showed that it induced apoptosis and cell cycle arrest through disrupting spindle assembly in mitotic progression, indicating these synthesized dithiocarbamates represented a novel series of anti-cancer compounds targeting mitosis.

Anti-H1N1 virus, cytotoxic and Nrf2 activation activities of chemical constituents from Scutellaria baicalensis.

ETHNOPHARMACOLOGICAL RELEVANCE: Huang-Qin, derived from the roots of Scutellaria baicalensis Georgi, is a popular Chinese herbal medicine mainly used to treat influenza and cancer. This study aims to elucidate the anti-influenza, anti-cancer and anti-oxidation effective components of S. baicalensis. MATERIALS AND METHODS: Various column chromatography techniques and semi-preparative HPLC were used to isolate Scutellaria compounds, and their structures were identified by HRESIMS and NMR spectroscopic analysis. The pure compounds were evaluated for anti-influenza activities against A/WSN/33 (H1N1) virus in MDCK cells, cytotoxic activities against HepG2, SW480 and MCF7 human cancer cells by MTS assay, and antioxidant activities by Nrf2 luciferase reporter assay. In addition, the contents of 12 major compounds in 27 batches of S. baicalensis were simultaneously determined by a fully validated UPLC/UV method. RESULTS: A total of thirty compounds (1-30), including four new ones (3, 7, 11 and 23), were isolated from S. baicalensis. Baicalin (15), baicalein (26), wogonin (27), chrysin (28) and oroxylin A (30) showed potent anti-H1N1 activities, with IC50 values of 7.4, 7.5, 2.1, 7.7 and 12.8 µM, respectively, which were remarkably more potent than the positive drug Osv-P (oseltamivir phosphate, IC50 45.6 µM). Most free flavones (26-28 and 30) showed significant cytotoxic activities at 10 µM (up to 61.2% inhibition rate). Furthermore, 30 could activate Nrf2 transcription by 3.8-fold of the control at 10 µM. UPLC analysis indicated the 12 major compounds (including the bioactive ones) accounted for 195.93 ± 43.9 mg g(-)(1) of the herbal materials. CONCLUSION: This study demonstrated that free flavones showed potent anti-influenza, anti-cancer and anti-oxidative activities. They are important effective components of S. baicalensis, and can be used as chemical markers for quality control of this herbal medicine.

Intestinal Absorption of Ergostane and Lanostane Triterpenoids from Antrodia cinnamomea Using Caco-2 Cell Monolayer Model.

Antrodia cinnamomea is a precious medicinal mushroom. It exhibits promising therapeutic effects on cancer, intoxication, hypertension, hepatitis, and inflammation. Its major bioactive constituents are ergostane and lanostane triterpenoids. In this study, we used intestinal Caco-2 cell monolayer model to reveal the intestinal absorption property of 14 representative triterpenoids from A. cinnamomea. The bidirectional transport through the monolayer at different time points was monitored by a fully validated LC/MS/MS method. In the case of pure compounds, ergostanes 5 (25R-antcin H), 6 (25S-antcin H) and 10 (25R-antcin B) could readily pass through the Caco-2 cell layer, whereas lanostanes 13 (dehydroeburicoic acid) and 14 (eburicoic acid) could hardly pass through. When the cells were treated with A. cinnamomea extract, antcins A, B, C, H and K (1-6 and 9-11) were absorbed via passive transcellular diffusion, and showed high P AB and P BA values (> 2.5 × 10(-5) cm/s). Meanwhile, the lanostanes dehydrosulphurenic acid (8), 15α-acetyldehydrosulphurenic acid (12), 13 and 14 exhibited poor permeability. Transport features of these compounds were consistent with their pharmacokinetic behaviors in rats. This study could also be helpful in predicting the intestinal absorption of A. cinnamomea in human.

IC-4, a new irreversible EGFR inhibitor, exhibits prominent anti-tumor and anti-angiogenesis activities.

Accumulating evidence suggested that the irreversible tyrosine kinase inhibitors (TKIs) have potential to override the acquired resistance to target-based therapies. Herein, we reported IC-4 as a novel irreversible TKI for epidermal growth factor receptor (EGFR). IC-4 potentially suppressed proliferation, induced apoptosis and a G2/M cell cycle arrest in breast cancer cells, correlating with inhibition of EGF-induced EGFR activation, but independent of DNA damage. In addition, IC-4 exhibited anti-angiogenetic activities both in vitro and in vivo. It suppressed cell viability and proliferation induced by various growth factors in human umbilical vein endothelial cells (HUVECs). IC-4 also inhibited HUVECs migration and tube formation. In transgenic zebrafish embryo model, IC-4 was shown to suppress formation of intersegmental vessel and development of subintestinal vessels. Taken together, these results demonstrated that IC-4 is a new irreversible EGFR-TKI, exhibiting potent anti-breast cancer and anti-angiogenetic effects.

Elucidation of the pharmacophore of echinocystic acid, a new lead for blocking HCV entry.

To elucidate the pharmacophore of echinocystic acid (EA), an oleanane-type triterpene displaying substantial inhibitory activity on HCV entry, two microbial strains, Rhizopus chinensis CICC 3043 and Alternaria alternata AS 3.4578, were utilized to modify the chemical structure of EA. Eight new metabolites with regio- and stereo-selective introduction of hydroxyl and lactone groups at various inert carbon positions were obtained. The anti-HCV entry activity of the metabolites 2-13, along with their parental compound EA and other analogs 14-15, were evaluated. Most of the metabolites showed no improvement but detrimental effect on potency except compound 5 and 6, which showed similar and even a litter higher anti-HCV entry activity than that of EA. The results demonstrated that ring A, B, C and the left side of ring E of EA are highly conserved, while ring D and the right side of ring E of EA are flexible. Introduction of a hydroxyl group at C-16 enhanced the triterpene potency. Further analysis indicated that the hemolytic effect of EA disappeared upon such modifications.

Conjugation of cyclodextrin with fullerene as a new class of HCV entry inhibitors.

An α-cyclodextrin-[60]fullerene conjugate with a flexible linker at the secondary face of α-cyclodextrin has been prepared, which displays significant water solubility and, more importantly, acts as a new class of HCV entry inhibitor with IC(50) at 0.17 µM level.

Differential gene expression profiles of DNA repair genes in esophageal cancer cells after X-ray irradiation.

BACKGROUND AND OBJECTIVE: Various factors affect the radioresistance of tumor cells, with unknown molecular mechanism(s). Many genes have been found to associate with the radioresistance of tumor cells, however, the precise mechanism of these genes have not been elucidated. This paper was to analyze the differential expressions of DNA repair genes in esophageal carcinoma cells at different time after X-ray irradiation, and to investigate the role of these DNA repair genes in radiation resistance. METHODS: Esophageal cancer parental cells Seg-1 were treated with continuous 2 Gy of fractionated irradiation until the total dose reached 60 Gy to establish the radioresistant cell line Seg-1R. Total RNA was extracted from each cell line at 0, 8, and 24 h after irradiation. Illumine Human-6 V3 microarray was used to identify differentially expressed genes between parental and radioresistant cells. Ten genes involved in DNA repair were obtained and their expressions at different time points after irradiation were analyzed by Gene Ontology analysis. RESULTS: Ten DNA repair associated genes were found to be differentially expressed. Three of these genes, SLK, HMGB1, and PMS1, were not only differentially expressed between parental and radioresistant cell lines, but also expressed differently at different time points after irradiation in the same cell line. CONCLUSIONS: PMS1 may be an important factor involved in the mechanism of radioresistance of esophageal carcinoma cells.

Identification of differentially expressed genes related to radioresistance of human esophageal cancer cells.

BACKGROUND AND OBJECTIVE: Radioresistant cells in esophageal cancer is one of the important reasons for the local failure of radiotherapy. In recent years, some researchers used gene chip technology to screen the differentially expressed genes between parental and radioresistant human esophageal cancer cells. But there were some problems in these studies, for example comparing cells at only one time interval, and genetic background not matching. In this study, we selected 3 different pairs of parental and radioresistant human esophageal cancer cells, and compared the gene expression profiles by cDNA microarray at 3 time intervals to identify and analyze the differentially expressed genes between parental and radioresistant human esophageal cancer cells. METHODS: We compared the gene expression profiles between parental cells (TE13, Seg-1, Kyse170) and radioresistant cells (TE13R, Seg-1R, Kyse170R) before, and at 8 h and 24 h after irradiation with a cDNA microarray consisting of 48 000 genes (Human Genome). We identified differentially expressed genes by Pathway and GO analyses, and verified the differentially expressed genes LEF1 and CTNNB1 by RT-PCR. RESULTS: A total of 460, 451, and 397 differentially expressed genes were found before, and at 8 h and 24 h after irradiation. After Pathway and GO analyses, 14 differentially expressed genes, participating in cell growth, apoptosis, cell cycle regulation, gene repair and signal transmission, were selected to further research. LEF1 and CTNNB1 were verified by RT-PCR, and the results were consistent with those of cDNA microarray. CONCLUSIONS: The WNT signal pathway may be an important pathway participating in the formation of radioresistance of esophageal cancer cells. LEF1 and CTNNB1 may be the important genes causing the esophageal cancer cell radioresistance.

A model supported by GIS for locating and quantifying PM2.5 emission originated from residential wood burning.

A research method was presented for spatially quantifying and allocating the potential activity of a fine particle matter emission (PM2.5), which originated from residential wood burning (RWB) in this study. Demographic, hypsographic, climatic and topographic data were compiled and processed within a geographic information system (GIS), and as independent variables put into a linear regression model for describing spatial distribution of the potential activity of residential wood burning as primary heating source. In order to improve the estimation, the classifications of urban, suburban and rural were redefined to meet the specifications of this application. Also, several definitions of forest accessibility were tested for estimation. The results suggested that the potential activity of RWB was mostly determined by elevation of a location, forest accessibility, urban/non-urban position, climatic conditions and several demographic variables. The linear regression model could explain approximately 86% of the variation of surveyed potential activity of RWB. The analysis results were validated by employing survey data collected mainly from a WebGIS based phone interview over the study area in central California. Based on lots free public GIS data, the model provided an easy and ideal tool for geographic researchers, environmental planners and administrators to understand where and how much PM2.5 emission from RWB was contributed to air quality. With this knowledge they could identify regions of concern, and better plan mitigation strategies to improve air quality. Furthermore, it allows for future adjustment on some parameters as the spatial analysis method is implemented in the different regions or various eco-social models.

Existence of efficient divalent metal ion-catalyzed and inefficient divalent metal ion-independent channels in reactions catalyzed by a hammerhead ribozyme.

The hammerhead ribozyme is generally accepted as a well characterized metalloenzyme. However, the precise nature of the interactions of the RNA with metal ions remains to be fully defined. Examination of metal ion-catalyzed hammerhead reactions at limited concentrations of metal ions is useful for evaluation of the role of metal ions, as demonstrated in this study. At concentrations of Mn2+ ions from 0.3 to 3 mM, addition of the ribozyme to the reaction mixture under single-turnover conditions enhances the reaction with the product reaching a fixed maximum level. Further addition of the ribozyme inhibits the reaction, demonstrating that a certain number of divalent metal ions is required for proper folding and also for catalysis. At extremely high concentrations, monovalent ions, such as Na+ ions, can also serve as cofactors in hammerhead ribozyme-catalyzed reactions. However, the catalytic efficiency of monovalent ions is extremely low and, thus, high concentrations are required. Furthermore, addition of monovalent ions to divalent metal ion-catalyzed hammerhead reactions inhibits the divalent metal ion-catalyzed reactions, suggesting that the more desirable divalent metal ion-ribozyme complexes are converted to less desirable monovalent metal ion-ribozyme complexes via removal of divalent metal ions, which serve as a structural support in the ribozyme complex. Even though two channels appear to exist, namely an efficient divalent metal ion-catalyzed channel and an inefficient monovalent metal ion-catalyzed channel, it is clear that, under physiological conditions, hammerhead ribozymes are metalloenzymes that act via the significantly more efficient divalent metal ion-dependent channel. Moreover, the observed kinetic data are consistent with Lilley's and DeRose's two-phase folding model that was based on ground state structure analyses.

Comparison of metal-ion-dependent cleavages of RNA by a DNA enzyme and a hammerhead ribozyme.

Joyce's DNA enzyme catalyzes cleavage of RNAs with almost the same efficiency as the hammerhead ribozyme. The cleavage activity of the DNA enzyme was pH dependent, and the logarithm of the cleavage rate increased linearly with pH from pH 6 to pH 9 with a slope of approximately unity. The existence of an apparent solvent isotope effect, with cleavage of RNA by the DNA enzyme in H(2)O being 4.3 times faster than cleavage in D(2)O, was in accord with the interpretation that, at a given pH, the concentration of the active species (deprotonated species) is 4.3 times higher in H(2)O than the concentration in D(2)O. This leads to the intrinsic isotope effect of unity, demonstrating that no proton transfer occurs in the transition state in reactions catalyzed by the DNA enzyme. Addition of La(3+) ions to the Mg(2+)-background reaction mixture inhibited the DNA enzyme-catalyzed reactions, suggesting the replacement of catalytically and/or structurally important Mg(2+) ions by La(3+) ions. Similar kinetic features of DNA enzyme mediated cleavage of RNA and of hammerhead ribozyme-mediated cleavage suggest that a very similar catalytic mechanism is used by the two types of enzyme, despite their different compositions.