A Plant Extract of Ribes nigrum folium Possesses Anti-Influenza Virus Activity In Vitro and In Vivo by Preventing Virus Entry to Host Cells

Infections with influenza A viruses (IAV) are still amongst the major causes of highly contagious severe respiratory diseases not only bearing a devastating effect to human health, but also significantly impact the economy. Besides vaccination that represents the best option to protect from IAV infections, only two classes of anti-influenza drugs, inhibitors of the M2 ion channel and the neuraminidase, often causing resistant IAV variants have been approved. That is why the need for effective and amply available antivirals against IAV is of high priority. Here we introduce LADANIA067 from the leaves of the wild black currant (Ribes nigrum folium) as a potent compound against IAV infections in vitro and in vivo. LADANIA067 treatment resulted in a reduction of progeny virus titers in cell cultures infected with prototype avian and human influenza virus strains of different subtypes. At the effective dose of 100 µg/ml the extract did not exhibit apparent harming effects on cell viability, metabolism or proliferation. Further, viruses showed no tendency to develop resistance to LADANIA067 when compared to amantadine that resulted in the generation of resistant variants after only a few passages. On a molecular basis the protective effect of LADANIA067 appears to be mainly due to interference with virus internalisation. In the mouse infection model LADANIA067 treatment reduces progeny virus titers in the lung upon intranasal application. In conclusion, an extract from the leaves of the wild black currant might be a promising source for the development of new antiviral compounds to fight IAV infections

Vemurafenib Limits Influenza A Virus Propagation by Targeting Multiple Signaling Pathways

Influenza A viruses (IAV) can cause severe global pandemic outbreaks. The currently licensed antiviral drugs are not very effective and prone to viral resistance. Thus, novel effective and broadly active drugs are urgently needed. We have identified the cellular Raf/MEK/ERK signaling cascade as crucial for IAV replication and suitable target for an antiviral intervention. Since this signaling cascade is aberrantly activated in many human cancers, several clinically approved inhibitors of Raf and MEK are now available. Here we explored the anti-IAV action of the licensed B-RafV600E inhibitor Vemurafenib. Treatment of B-RafWT cells with Vemurafenib induced a hyperactivation of the Raf/MEK/ERK cascade rather than inhibiting its activation upon IAV infection. Despite this hyperactivation, which has also been confirmed by others, Vemurafenib still strongly limited IAV-induced activation of other signaling cascades especially of p38 and JNK mitogen-activated protein kinase (MAPK) pathways. Most interestingly, Vemurafenib inhibited virus-induced apoptosis via impaired expression of apoptosis inducing cytokines and led to hampered viral protein translation most likely due to the decreased activation of p38 and JNK MAPK. These multiple actions resulted in a profound and broadly active inhibition of viral replication, up to a titer reduction of three orders of a magnitude. Thus, while Vemurafenib did not act similar to MEK inhibitors, it displays strong antiviral properties via a distinct and multi-target mode of action. <br

Vemurafenib Limits Influenza A Virus Propagation by Targeting Multiple Signaling Pathways

Influenza A viruses (IAV) can cause severe global pandemic outbreaks. The currently licensed antiviral drugs are not very effective and prone to viral resistance. Thus, novel effective and broadly active drugs are urgently needed. We have identified the cellular Raf/MEK/ERK signaling cascade as crucial for IAV replication and suitable target for an antiviral intervention. Since this signaling cascade is aberrantly activated in many human cancers, several clinically approved inhibitors of Raf and MEK are now available. Here we explored the anti-IAV action of the licensed B-RafV600E inhibitor Vemurafenib. Treatment of B-RafWT cells with Vemurafenib induced a hyperactivation of the Raf/MEK/ERK cascade rather than inhibiting its activation upon IAV infection. Despite this hyperactivation, which has also been confirmed by others, Vemurafenib still strongly limited IAV-induced activation of other signaling cascades especially of p38 and JNK mitogen-activated protein kinase (MAPK) pathways. Most interestingly, Vemurafenib inhibited virus-induced apoptosis via impaired expression of apoptosis-inducing cytokines and led to hampered viral protein expression most likely due to the decreased activation of p38 and JNK MAPK. These multiple actions resulted in a profound and broadly active inhibition of viral replication, up to a titer reduction of three orders of a magnitude. Thus, while Vemurafenib did not act similar to MEK inhibitors, it displays strong antiviral properties via a distinct and multi-target mode of action

A plant extract of Ribes nigrum folium possesses anti-influenza virus activity in vitro and in vivo by preventing virus entry to host cells.

Infections with influenza A viruses (IAV) are still amongst the major causes of highly contagious severe respiratory diseases not only bearing a devastating effect to human health, but also significantly impact the economy. Besides vaccination that represents the best option to protect from IAV infections, only two classes of anti-influenza drugs, inhibitors of the M2 ion channel and the neuraminidase, often causing resistant IAV variants have been approved. That is why the need for effective and amply available antivirals against IAV is of high priority. Here we introduce LADANIA067 from the leaves of the wild black currant (Ribes nigrum folium) as a potent compound against IAV infections in vitro and in vivo. LADANIA067 treatment resulted in a reduction of progeny virus titers in cell cultures infected with prototype avian and human influenza virus strains of different subtypes. At the effective dose of 100 µg/ml the extract did not exhibit apparent harming effects on cell viability, metabolism or proliferation. Further, viruses showed no tendency to develop resistance to LADANIA067 when compared to amantadine that resulted in the generation of resistant variants after only a few passages. On a molecular basis the protective effect of LADANIA067 appears to be mainly due to interference with virus internalisation. In the mouse infection model LADANIA067 treatment reduces progeny virus titers in the lung upon intranasal application. In conclusion, an extract from the leaves of the wild black currant might be a promising source for the development of new antiviral compounds to fight IAV infections

Regrowing the adult brain: NF-kappaB controls functional circuit formation and tissue homeostasis in the dentate gyrus

Imielski Y, Schwamborn JC, Lüningschrör P, et al. Regrowing the adult brain: NF-kappaB controls functional circuit formation and tissue homeostasis in the dentate gyrus. PLoS ONE. 2012;7(2): e30838.Cognitive decline during aging is correlated with a continuous loss of cells within the brain and especially within the hippocampus, which could be regenerated by adult neurogenesis. Here we show that genetic ablation of NF-kappaB resulted in severe defects in the neurogenic region (dentate gyrus) of the hippocampus. Despite increased stem cell proliferation, axogenesis, synaptogenesis and neuroprotection were hampered, leading to disruption of the mossy fiber pathway and to atrophy of the dentate gyrus during aging. Here, NF-kappaB controls the transcription of FOXO1 and PKA, regulating axogenesis. Structural defects culminated in behavioral impairments in pattern separation. Re-activation of NF-kappaB resulted in integration of newborn neurons, finally to regeneration of the dentate gyrus, accompanied by a complete recovery of structural and behavioral defects. These data identify NF-kappaB as a crucial regulator of dentate gyrus tissue homeostasis suggesting NF-kappaB to be a therapeutic target for treating cognitive and mood disorders

LADANIA067 inhibits IAV replication at early times of infection.

<p>(A) A549 cells were left untreated or treated with 100 µg/ml of LADANIA067, which was added at the indicated times during infection with FPV (H7N7) (MOI = 1). (B-D) A549 cells and/or virus were left untreated or treated with 100 µg/ml of LADANIA067 pre-infection or post-infection (p.i.). For infection FPV (H7N7) (MOI = 0.1), SO-IAV (H1N1pan) (MOI = 1) and PR8 (H1N1) (MOI = 5) have been used. Supernatants were assayed for progeny virus particles 8 h p.i. Data represent means ±SD of 4 biological samples (A) or 6 biological samples (B–D). Statistical significance was assessed by students t-test (*p<0.05, **p<0.01, ***p<0.001).</p

LADANIA067 is not toxic or proapoptotic in A549 cells.

<p>(A) A549 cells were left untreated or treated with LADANIA067 with the amounts and times as indicated. The impact of LADANIA067 on cell morphology was visualized by an Axiovert 2000 ApoTome microscope (100-fold magnification) and monitored by photography. (B) A549 cells were left untreated or treated with 50 µg/ml, 100 µg/ml or 200 µg/ml LADANIA067 or 1 µM Staurosporine for the indicated time periods. Cell proliferation/viability was estimated via colorimetric assay that followed incubation of cells with MTT. The proliferating cells are depicted, whereby the untreated controls were arbitrarily set as 100%. Data represent ±SD of twelve biological samples. (C) A549 cell were transfected with a constitutive active CMV promoter luciferase plasmid. Eight hours post transfection cells were left untreated or treated with the indicated amounts of LADANIA067 or 10 µg/ml Cycloheximide respectively for 18 hours. Data represent ±SD of nine biological samples and are given as the ratio of luciferase activity relative to the untreated control. (D) A549 cells were incubated in the presence and absence of 50 µg/ml, 100 µg/ml or 200 µg/ml LADANIA for 24 h, 48 h and 72 h. A549 cells that were treated with Staurosporine (1 µg/ml) in addition to EDTA (5 mM) for 4 hours served as control. Percentage of viable cells was measured by staining of dead cells with propidium iodide (PI) as described in <i>Materials and Methods.</i> Data represent ±SD of nine biological samples. Statistical significance was assessed by students t-test (*p<0.05, **p<0.01, ***p<0.001) (B, C, D). (E) A549 cells were left untreated or were treated with 1 µM Staurosporine for 8 h or 50 µg/ml or 100 µg/ml LADANIA067 for 24 h or 48 h. Upon stimulation cell lysates were prepared and subjected to Western blot, and caspase mediated PARP cleavage was determined as a measure for apoptosis induction. ERK2 expression was monitored as loading control.</p

LADANIA067 impairs IAV replication.

<p>A549 cells were left untreated or treated with the indicated amounts of LADANIA067 before and during infection with the influenza virus strains A/FPV/Bratislava/79 (FPV) (H7N7) (8 h: MOI = 0.1; 24 h: MOI = 0.01) (A), A/NRW/173/09, an pandemic swine-origin-IAV (SO-IAV) (H1N1pan) (8 h: MOI = 1.5; 24 h: MOI = 0.5) (B) or A/Puerto Rico/8/34 (PR8) (H1N1) (MOI = 0.01) (C). In (C) cells were left untreated or treated with the indicated amounts (left panel) or 50 µg/ml of LADANIA067 before and during infection. (A–C) The virus was pretreated with LADANIA067 as well. Supernatants were assayed for progeny virus particles at the times indicated. Data represent means ±SD of 4 biological samples. Statistical significance was assessed by students t-test (*p<0.05, **p<0.01, ***p<0.001).</p

LADANIA067 inhibits viral internalisation.

<p>(A) A549 cells were left untreated or treated with 100 µg/ml LADANIA067 for 1 h at 37°C. Biotinylated <i>Sambucus nigra</i> agglutinin (SNA) (1 µg/ml) was added and cells were incubated for 1 h at 4°C with A549 cells. Subsequently, cells were incubated with Cy3-conjugated streptavidin to detect lectins for fluorescence microscopy using an Axiovert 2000 ApoTome microscope with AxioCam digital camera and AxioVision software (Zeiss) (20-fold magnification) (B) LADANIA067 was diluted as indicated. Influenza A/FPV/Bratislava/79 was diluted in PBS (4×10⁵ pfu/well) and 50 µl was added per well of a 96-well plate. After preincubation of 45 min, chicken erythrocytes (1∶20 in PBS) were mixed with the solution. In samples where viruses were preincubated with LADANIA067 or CYSTUS052, up to a certain dilution the virus particles are no longer capable of agglutinating erythrocytes, indicating an interaction of LADANIA067 or CYSTUS052 with the viral HA. (C) A549 cells (upper panel) or MDCK cells (lower panel) were left untreated or treated with 200 µg/ml LADANIA067 30 min before infection. Also the influenza virus A/Puerto Rico/8/34 was left untreated or was incubated with the same amount of LADANIA067 at 4°C for 30 min. Subsequently cells were left untreated or infected (MOI = 10) with virus for 30 min at 4°C till a temperature shift to 37°C for further 15 min. Upon stimulation cells were washed twice with PBS and cell lysates were prepared and subjected to Western blot. Viral M1 protein was determined as a measure for attached and internalized virus particles. A549 cells were left untreated or treated with 100 µg/ml LADANIA067 for 4 h prior to stimulation with (D) 5 ng/ml TNFα for 20 min or (E) 30 ng/ml EGF for 10 min. (F) Influenza virus A/Puerto Rico/8/34 was left untreated or was incubated with the indicated amounts of LADANIA067 supplemented with medium for 30 min at 4°C. Subsequently, cells were washed with PBS and incubated for 60 min at 37°C without virus or with virus which was left untreated or was pretreated with LADANIA067. Upon stimulation cell lysates were prepared and subjected to Western blot. (D) Expression of IκBα, (E, F) phosphorylation of EGFR and ERK2 were detected and (D, E, F) EGFR or ERK2 expression was monitored as loading control.</p