The mechanism of poly-galloyl-glucoses preventing Influenza A virus entry into host cells.

Hemagglutinin (HA) is essential for Influenza A virus infection, but its diversity of subtypes presents an obstacle to developing broad-spectrum HA inhibitors. In this study, we investigated the molecular mechanisms by which poly-galloyl glucose (pGG) analogs inhibit influenza hemagglutinin (HA) in vitro and in silico. We found that (1) star-shaped pGG analogs exhibit HA-inhibition activity by interacting with the conserved structural elements of the receptor binding domain (RBD); (2) HA inhibition depends on the number of galloyl substituents in a pGG analog; the best number is four; and when PGG binds with two HA trimers at their conserved receptor binding domains (loop 130, loop 220, and 190-α-helix), PGG acts as a molecular glue by aggregating viral particles so as to prevent viral entry into host cells (this was revealed via an in silico simulation on the binding of penta-galloyl-glucose (PGG) with HA). pGGs are also effective on a broad-spectrum influenza A subtypes (including H1, H3, H5, H7); this suggests that pGG analogs can be applied to most influenza A subtypes as a prophylactic against influenza viral infections.

Anti-hepatitis B virus effects of dehydrocheilanthifoline from Corydalis saxicola.

In this report, the anti-hepatitis B virus (HBV) activity of dehydrocheilanthifoline (DHCH), a quaternary ammonium alkaloid isolated from the traditional Chinese medicine Corydalis saxicola Bunting (Papaveraceae), was determined in vitro. Following six days of treatment, DHCH efficiently suppressed the secretions of HBsAg and HBeAg in HepG2.2.15 cell cultures, with a half-maximal inhibitory concentration (IC(50)) of 15.84 and 17.12 µM, and with a therapeutic index (TI) of 7.32 and 6.77, respectively. Further studies revealed that DHCH reduced the levels of extracellular DNA, intracellular DNA and covalently closed circular DNA (cccDNA) of HBV in a dose-dependent and time-dependent manner, with IC(50) values of 15.08, 7.62 and 8.25 µM, respectively after six days of treatment. In contrast, the level of viral pre-genomic RNA (pgRNA) increased 6.13-fold after treatment with DHCH. Together, it was demonstrated for the first time that DHCH could significantly inhibit the replication of HBV, which warrants further studies on the antiviral mechanisms of DHCH, and suggests that it may be a promising candidate in the therapy of HBV infection.

The selective Hsp90 inhibitor BJ-B11 exhibits potent antitumor activity via induction of cell cycle arrest, apoptosis and autophagy in Eca-109 human esophageal squamous carcinoma cells.

BJ-B11 is a selective heat shock protein 90 (Hsp90) inhibitor that has been reported to possess significant antitumor activity in multiple types of cancer cells; however, the mechanism of action needs to be further clarified. We investigated, for the first time, the antitumor activity and the molecular mechanism underlying growth inhibition in Eca-109 cells. The results revealed that BJ-B11 inhibited the proliferation of Eca-109 cells in a time- and concentration-dependent manner, with 50% inhibitory concentration (IC(50)) values of 0.31±0.01 µM after 48-h incubation. BJ-B11 induced concentration-dependent G2/M cell cycle arrest and apoptosis. The cleavage of caspase 3 and PARP signals detected might originate from mitochondrial dysfunction, which was supported by the results of reactive oxygen species (ROS) production, cytochrome c release and the mitochondrial membrane potential (MMP) reduction. The general caspase inhibitor Z-VAD-fmk did not completely abolish BJ-B11-induced cell death. Furthermore, inhibition of the Akt/mTOR/p70S6K signaling pathway might be involved in the process of BJ-B11-induced autophagy, which was characterized by the production of autophagic vacuoles and upregulation of LC3-II protein in a time- and concentration-dependent manner. Meanwhile, the general autophagy inhibitor 3-MA decreased the apoptotic ratio. Furthermore, BJ-B11 induced the polymerization of cytoskeleton ß-tubulin and F-actin. Taken together, our results suggest that the growth inhibition of Eca-109 cells induced by BJ-B11 may result from the induction of G2/M cell cycle arrest, apoptosis and autophagy.

Anti-herpes simplex virus efficacies of 2-aminobenzamide derivatives as novel HSP90 inhibitors.

After the widespread use of the acyclic purine nucleoside analogues for therapy of herpes simplex virus (HSV) infection since the 1980s, new antiviral strategies are urgently needed to counter the emergence of drug-resistant clinical isolates. In this report, we define the anti-HSV efficacies of three optimized 2-aminobenzamide derivatives in vitro and in vivo. The synthetic analogues SNX-25a, SNX-2112 and SNX-7081, which selectively bind to the N-terminal ATP pocket of heat shock protein 90 (HSP90), exhibited significant anti-HSV-1 and HSV-2 activities at non-cytotoxic concentrations in Vero cells, with EC(50) values close to that of acyclovir (ACV). The in vivo antiviral potentials were then confirmed using a herpes simplex keratitis (HSK) rabbit model, where eye gels containing 0.1% or 0.025% SNX-25a displayed the highest efficacies against HSV-1 infection, which were better than that obtained with 0.1% ACV. SNX-2112 and SNX-7081 gels were also effective against HSV-1 with different magnitude of activities. Our results for the first time confirmed the anti-HSV efficacies of these 2-aminobenzamide derivatives and suggest that with alternative mechanisms of action these novel HSP90 inhibitors, especially SNX-25a, could be potent as new anti-HSV clinical trial candidates.

Cloning, soluble expression, rapid purification and characterization of human Cofilin1.

Cofilin1 is an actin-binding protein that plays a critical role in the regulation of actin cytoskeleton and consequently affects various physiological processes. In this study, the human Cofilin1 cDNA was cloned into the expression vector pET-28a(+) with a 6 × His tag and expressed as soluble protein in Escherichia coli BL21(DE3). Approximately 78 mg of Cofilin1, which showed high activity as determined by native PAGE, could be purified from each liter of LB medium by His-tag affinity chromatography and gel filtration. Further, high-titer IgG against Cofilin1 was positively detected after immunization in rabbits and the polyclonal antibodies were purified and identified. Together, this report provides the first protocol to efficiently obtain human Cofilin1 with high biological activity and immunogenicity using E. coli BL21 (DE3) expression system.

[In vitro study on the anti-HSV-1 and HBV activities of extracts from the fruit of Eucalyptus maidenii].

OBJECTIVE: To investigate the antiviral activities of three kinds of extracts from the fruit of Eucalyptus maidenii against herpes simplex virus typel and Hepatitis B Virus. METHODS: Cytotoxicity of extracts on Vero cell lines were estimated using MTT method and anti-HSV-1 activity was observed and determined with CPE and plaque reduction assay. The inhibitory effects of extracts on HBsAg and HBeAg secretion in HepG2.2.15 cell culture were detected using ELISA. RESULTS: Aqueous extract (pl8-E3) had conspicuous anti-HSV-1 activity, the IC50 was 126.77 microg/mL,but the EtOAc extracts( pl8-E1 )and MeOH extracts (pl8-E2) showed little anti-HSV-1 activity. None of these extracts had significant inhibitory eflect on HBsAg and HBeAg secretion in HepG2.2.15 cell culture. CONCLUSION: Aqueous extract(p18-E3) from the fruit of Eucalyptus maidenii has conspicuous anti-HSV-1 activity. It could inactivate virus directly,and inhibit virus attachment,but had no influence on virus penetration. The mechanism that p18-E3 inactivates virus might involve in viral envelope alteration.

SNX-2112, a novel Hsp90 inhibitor, induces G2/M cell cycle arrest and apoptosis in MCF-7 cells.

SNX-2112 is a heat shock protein 90 (Hsp90) inhibitor with anticancer properties currently in clinical trials. This study investigated the effects of SNX-2112 on inhibition of cell growth, the cell cycle, and apoptosis in MCF-7 human breast cancer cells, in addition to the various molecular mechanisms. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometric analysis suggest that SNX-2112 inhibits cell growth in a time- and dose-dependent manner more potently than 17-(allylamino)-17-demethoxygeldanmycin (17-AAG), a traditional Hsp90 inhibitor, probably as a result of cell-cycle arrest at the G2/M phase and the induction of apoptosis. Downregulation of Bcl-2 and Bcl-xL, upregulation of Bax, cleavage of caspase-9 and poly (ADP-ribose) polymerase (PARP), and degradation of the breast cancer-related Hsp90 client proteins human epidermal growth factor receptor-2 (HER2), Akt, Raf-1, and nuclear factor kappa-B kinase (IKK) were observed in SNX-2112 treated cells by Western blot assay. These findings suggest that the molecular mechanisms of cell-growth inhibition by SNX-2112 involve activation of the mitochondrial apoptotic pathway and the degradation of breast cancer-related proteins.

BJ-B11, a novel Hsp90 inhibitor, induces apoptosis in human chronic myeloid leukemia K562 cells through the mitochondria-dependent pathway.

In the past few years heat shock protein 90 (Hsp90) inhibitors have been reported to possess significant antitumor activity. We investigated, for the first time, the antitumor activity of a novel Hsp90 inhibitor 2-(4-acetyloxycyclohexylamino)-4-(3, 6, 6-trimethyl-4-oxo-4, 5, 6, 7-tetrahydro-1H-indazol-1-yl)-benzamide (BJ-B11) and the molecular mechanism underlying the apoptosis it induces in human chronic myeloid leukemia K562 cells. The results revealed that BJ-B11 triggered growth inhibition in K562 cells and other malignant cell lines in vitro with only minor toxicity in a normal human cell line. BJ-B11 inhibited the proliferation of K562 cells in a concentration- and time-dependent manner, with IC(50) values of 1.1 ± 0.2 µM and 0.4 ± 0.1 µM after 48 and 72 h incubations respectively. This most likely results from cell cycle arrest at the G(0)/G(1) phase and the induction of apoptosis. In addition, BJ-B11 degraded the Hsp90 client proteins Bcr-Abl and Akt, induced activation of caspase-9 and caspase-3, and subsequent cleavage of PARP. The caspase signals may originate from mitochondrial dysfunction, which is supported by the finding of cytochrome c release. In addition, inactivation of the Akt signaling pathway may be involved in the process of BJ-B11-induced apoptosis. Taken together, our data provide a putative molecular mechanism for the anticancer effect of BJ-B11 on K562 cells, and suggest a potential application for BJ-B11 in chronic myeloid leukemia therapy.

Antiviral activity and possible mechanisms of action of pentagalloylglucose (PGG) against influenza A virus.

Influenza A virus (IAV) infection is a major public health threat leading to significant morbidity and mortality. The emergence of drug-resistant virus strains highlights the urgent need to develop novel antiviral drugs with alternative modes of action. Pentagalloylglucose (PGG), a naturally occurring polyphenolic compound, possesses a broad spectrum of biological activities. In this study, we found that PGG has anti-influenza-virus activity, and investigated its possible mechanism(s) of action in vitro. Both pre-incubation of virus prior to infection and post-exposure of infected cells with PGG significantly inhibited virus yields. Influenza-virus-induced hemagglutination of chicken red blood cells was inhibited by PGG treatment, suggesting that PGG can inhibit IAV infection by interacting with the viral hemagglutinin. PGG did not affect viral protein synthesis or nuclear transport of viral nucleoprotein (NP) but greatly reduced plasma membrane accumulation of NP protein at the late stage of the replication cycle. Furthermore, PGG significantly reduced virus budding and progeny virus release from infected cells. This study revealed for the first time that PGG can inhibit IAV replication with a dual mode of action and offers new insights into its underlying mechanisms of antiviral action.

Synthesis and in vitro anti-HSV-1 activity of a novel Hsp90 inhibitor BJ-B11.

In this study, a novel Hsp90 inhibitor BJ-B11, was synthesized and evaluated for in vitro antiviral activity against several viruses. Possible anti-HSV-1 mechanisms were also investigated. BJ-B11 displayed no antiviral activity against coxsackievirus B(3) (CVB(3)), human respiratory syncytial virus (RSV) and influenza virus (H1N1), but exhibited potent anti-HSV-1 and HSV-2 activity with EC(50) values of 0.42±0.18 µM and 0.60±0.21 µM, respectively. Additionally, the inhibitory effects of BJ-B11 against HSV-1 were likely to be introduced at early stage of infection. Our results indicate that BJ-B11 with alternative mechanisms of action is potent as an anti-HSV clinical trial candidate.

Effect of siRNAs on HSV-1 plaque formation and relative expression levels of RR mRNA.

RNA interference (RNAi) is a process by which introduced small interfering RNA (siRNA) can cause the specific degradation of mRNA with identical sequences. The human herpes simplex virus type 1 (HSV-1) RR is composed of two distinct homodimeric subunits encoded by UL39 and UL40, respectively. In this study, we applied siRNAs targeting the UL39 and UL40 genes of HSV-1. We showed that synthetic siRNA silenced effectively and specifically UL39 and UL40 mRNA expression and inhibited HSV-1 replication. Our work offers new possibilities for RNAi as a genetic tool for inhibition of HSV-1 replication.

Pentagalloylglucose downregulates cofilin1 and inhibits HSV-1 infection.

To investigate the anti-herpesvirus mechanism of pentagalloylglucose (PGG), we compared the proteomic changes between herpes simplex virus type 1 (HSV-1) infected MRC-5 cells with or without PGG-treatment, and between non-infected MRC-5 cells with or without PGG-treatment by 2-DE and MS-based analysis. Differentially expressed cellular proteins were mainly involved with actin cytoskeleton regulation. Significantly, PGG can down-regulate cofilin1, a key regulator of actin cytoskeleton dynamics. PGG can inhibit HSV-1-induced rearrangements of actin cytoskeleton which is important for infectivity. Furthermore, cofilin1 knockdown by siRNA also inhibited the HSV-1-induced actin-skeleton rearrangements. Both PGG-treatment and cofilin1 knockdown can reduce HSV-1 DNA, mRNA, protein synthesis and virus yields. Altogether, the results suggested that down-regulating cofilin1 plays a role in PGG inhibiting HSV-1 infection. PGG may be a promising anti-herpesvirus agent for drug development.

Effects of 1,2,4,6-tetra-O-galloyl-ß-D-glucose from P. emblica on HBsAg and HBeAg secretion in HepG2.2.15 cell culture.

A polyphenolic compound, 1,2,4,6-tetra-O-galloyl-ß-D-glucose (1246TGG), was isolated from the traditional Chinese medicine Phyllanthus emblica L. (Euphorbiaceae) and assayed for its potential as an anti-hepatitis B virus (HBV) agent. The cytotoxicity of 1246TGG on HepG2.2.15 as well as HepG2 cells was determined by observing cytopathic effects, and the effects of 1246TGG on secretion of HBsAg and HBeAg in HepG2.2.15 cells were assayed by enzyme immunoassay. Results indicates that treatment with 1246TGG (6.25 µg/mL, 3.13 µg/mL), reduced both HBsAg and HBeAg levels in culture supernatant, yet the inhibitory effects tend to decline with the assay time. This study provides a basis for further investigation of the anti-HBV activity and possible mechanism of action of 1246TGG.