BIOder treatment alters expression of apoptotic genes to promote survival of U87MGs.

<p><b>A</b>) U87MGs were treated with 1 µM BIO, 1 µM BIOder, or DMSO and infected with VEEV TC-83. RNA was harvested from infected cells 24 hours post infection and analyzed by RT-PCR for expression of the indicated anti- and pro-apoptotic genes. <b>B</b>) Band intensities corresponding to triplicate samples were quantified and represented as fold change in gene expression over the DMSO control, with the DMSO control being set as a fold change of 1.0.</p

Small molecule inhibitors of GSK-3β impede VEEV replication.

<p><b>A</b>) U87MG astrocytes were treated with BIO (10, 5, 2.5, or 1.25 µM) and cell viability assayed 24 hours later by CellTiter Glo Assay (Promega). DMSO treated cells are displayed at 100% viability and all treatments compared to those values. <b>B</b>) U87MG astrocytes were pretreated for 2 hours with DMSO, or BIO (5, 2.5, or 1.25 µM), infected with VEEV TC-83 at MOI 0.1, and post-treated with compounds. Twenty-four hours post infection viral supernatants were collected and assayed for viral replication by plaque assays. <b>C</b>) U87MG astrocytes were treated with DMSO, SB 415286 (50, 10, or 2 µM), or SB 216763 (50, 10, or 2 µM) and cell viability assayed 24 hours later by CellTiter Glo Assay (Promega). DMSO treated cells are displayed at 100% viability and all treatments compared to those values. <b>D</b>) U87MG astrocytes were pretreated for 2 hours with DMSO, SB 415286 (50, or 10 µM), or SB 216763 (50 or 10 µM), infected with VEEV TC-83 at MOI 0.1, and post-treated with compounds. Twenty-four hours post infection viral supernatants were collected and assayed for viral replication by plaque assays. <b>E</b>) U87MG astrocytes were transfected with negative control siRNA (siNEG) or GSK-3β siRNA (siGSK3) at 50 nM. Forty-eight hours post-transfection, cells were infected with VEEV TC-83 at MOI 0.1. Viral supernatants were collected 24 hours post-infection and assayed for viral replication by plaque assays. Western blot results are displayed below the graph, indicating that GSK-3β expression was decreased following siRNA treatment.</p

Pro- and anti-apoptotic primer sequences used for RT-PCR.

<p>Pro- and anti-apoptotic primer sequences used for RT-PCR.</p

BIOder inhibits VEEV cell death <i>in vivo</i>.

<p><b>A</b>) Female C3H/HeN mice were treated subcutaneously with either DMSO or various concentration of BIOder (10 mg/kg, 20 mg/kg, 40 mg/kg) every day for 5 days. Mice were weighed daily and the average % of the mouse weight is plotted in panel A. <b>B</b>) <b>and </b><b>C</b>) Female C3H/HeN mice were infected intranasally with 5×LD50 (2×10⁷ pfu) of VEEV TC-83. Groups of 10 mice were treated SQ with vehicle, BIO (50 mg/kg) or BIOder (20 mg/kg) on days −1, 1, 3, and 5 and were monitored for survival for 14 days. Kaplan-Meier curves for survival between DMSO and BIO (panel B). Kaplan-Meier curves for survival between DMSO and BIOder (panel C). Significance was determined using Mantel-Cox Log-rank test. P-value of 0.057 between control and BIOder. This experiment was performed one time.</p

Modulation of GSK-3β Activity in Venezuelan Equine Encephalitis Virus Infection

<div><p>Alphaviruses, including Venezuelan Equine Encephalitis Virus (VEEV), cause disease in both equine and humans that exhibit overt encephalitis in a significant percentage of cases. Features of the host immune response and tissue-specific responses may contribute to fatal outcomes as well as the development of encephalitis. It has previously been shown that VEEV infection of mice induces transcription of pro-inflammatory cytokines genes (e.g., IFN-γ, IL-6, IL-12, iNOS and TNF-α) within 6 h. GSK-3β is a host protein that is known to modulate pro-inflammatory gene expression and has been a therapeutic target in neurodegenerative disorders such as Alzheimer's. Hence inhibition of GSK-3β in the context of encephalitic viral infections has been useful in a neuroprotective capacity. Small molecule GSK-3β inhibitors and GSK-3β siRNA experiments indicated that GSK-3β was important for VEEV replication. Thirty-eight second generation BIO derivatives were tested and BIOder was found to be the most potent inhibitor, with an IC₅₀ of ∼0.5 µM and a CC₅₀ of >100 µM. BIOder was a more potent inhibitor of GSK-3β than BIO, as demonstrated through <em>in vitro</em> kinase assays from uninfected and infected cells. Size exclusion chromatography experiments demonstrated that GSK-3β is found in three distinct complexes in VEEV infected cells, whereas GSK-3β is only present in one complex in uninfected cells. Cells treated with BIOder demonstrated an increase in the anti-apoptotic gene, survivin, and a decrease in the pro-apoptotic gene, BID, suggesting that modulation of pro- and anti-apoptotic genes contributes to the protective effect of BIOder treatment. Finally, BIOder partially protected mice from VEEV induced mortality. Our studies demonstrate the utility of GSK-3β inhibitors for modulating VEEV infection.</p> </div

Characterization of BIOder.

<p><b>A</b>) Structure of BIO: 6-bromoindirubin-3′-oxime and BIOder (#6): 6-bromo-5-methyl-1H-indole-2,3-dione 3. <b>B</b>) U87MG astrocytes were pretreated for 2 hours with DMSO or BIOder (10, 1.0, and 0.1 µM), infected with VEEV TC-83 at MOI 0.1, and post-treated. Twenty-four hours post-infection, supernatants were collected and analyzed by plaque assay to determine the amount of infectious virus released. <b>C</b>) U87MG astrocytes were pretreated for 2 hours with DMSO or BIOder (10, 1.0, and 0.1 µM), infected with VEEV TC-83 at MOI 0.1, and post-treated. Seventy hours post infection, CPE was measured by MTT assay. Mock infected cells are displayed at 100% viability. <b>D</b>) Uninfected U87MG astrocytes were treated with BIOder (10, 1.0, and 0.1 µM), and cell viability determined by MTT assay. Untreated (UT) cells are displayed at 100% viability.</p

Presence of novel GSK-3β complexes in VEEV infected cells.

<p><b>A</b>) Samples from both infected and uninfected cells were loaded on a sizing column and separated in presence of a 500 mM salt buffer. No detergents were used during the chromatography steps. Each fraction (over a range of 60 fractions) contained 500 µL and only 250 µL samples were precipitated, resuspend in low volume of TNE₅₀ and 0.1% NP-40 (10 µL) and ran on a gel for western blotting. Samples were western blotted for presence of GSK-3β and actin. <b>B</b>) GSK-3β IPs from fractions 31, 41, 51 (100 µL each) were mixed with GSK-3β Ab (5 µg) overnight and washed next day first with RIPA buffer (1×) and then TNE₅₀ and 0.1% NP-40 (2×) followed by kinase buffer (3×) prior to addition of substrate and ³²P-ATP. Samples were ran on a gel, stained, destained (over 4 hrs), dried and then exposed to phosphoImager cassette. Control immunoprecipitation from these fraction (50 uL each) were mixed and used for IP/Kinase using IgG (5 µg). <b>C</b>) IP/kinase reactions were treated with either BIO or BIOder (0.1 uM or 1 µM) <i>in vitro</i> during the kinase reaction. Samples were ran on a gel and exposed to phosphoImager cassette.</p

IKKβ inhibitors decrease TC-83 replication in rat AP7 neuronal cells.

<p>A) Neurons were pre-treated with DMSO or with IKK inhibitors (1 µM) for 2 hours and 24 hours later cell viability was measured using the Cell-Titer-Glo Luminescent Cell Viability Assay. B) Neurons were pretreated with IKK inhibitors (1 µM), BAY-11-7082, BAY-11-7085 and IKK2-IV for 2 hours. Following the pretreatment, the conditioned media (media containing inhibitor) was removed and the cells infected at MOI: 1 for 1 hour. The viral inoculum was removed and the conditioned media replaced. Supernatants were collected 24 hours post-infection, and infectious viral titers were determined by plaque assay. C) Neurons were pretreated with IKK inhibitors (1 µM) for 2 hours and infected with TC-83 for 1 hour. Conditioned media was replaced after removal of the viral inoculum. Cell viability assay was performed 48 hours later using the Cell-Titer-Glo Luminescent Cell Viability Assay. The red line is representative of the base line for luminescence units, such that luminescence units above this are indicative of increased cell viability. The graphs are representative of 2 independent experiments. Error bars for the independent experiments were calculated and are represented thusly. *** p≤0.005, ** p≤0.01 and * p≤0.05.</p

IKKβ inhibitors are effective in decreasing viral load of wild type VEEV.

<p>A) U87MG cells (A) or neuronal rat AP7 cells (B) were pretreated with 1 µM IKK inhibitors, BAY-11-7082, BAY-11-7085 and IKK2-IV for 2 hours. The cells were infected with the wild type strain of VEEV (TrD) at a MOI: 0.1 (A) or MOI: 1 (B) for 1 hour. The conditioned media (media containing inhibitor) was removed prior to the viral infections and replaced after the viral inoculum was removed. The cells were incubated for an additional 24 hours. The supernatants were collected from all samples and viral titers were determined by plaque assay. The graphs are representative of 2 independent experiments. Error bars (Standard deviations) for 3 replicates within the 2 independent experiments were calculated and are represented thusly. ** p≤0.01.</p

Macromolecular reorganization of the IKKβ complex in TC-83 infected cells.

<p>U87MG cells were infected with TC-83 or UV-TC-83 (MOI: 5) or the cells were treated with Poly I∶C (10 µg/mL) or Imiquimod (2 µg/mL). Cells were lysed 24 hours post-infection and post treatment and protein extracts were quantified. Two milligrams of total protein was subjected to chromatography using a Superose 6 HR 10/30 size-exclusion chromatography column and an AKTA purifier system. Every 5th fraction was acetone precipitated using 4 volumes of ice-cold 100% acetone and incubated for 15 minutes on ice. Lysates were centrifuged at 4°C for 10 minutes at 12,000 rpm, supernatants were removed, and the pellets were allowed to dry for a few minutes at room temperature. The pellets were resuspended in Laemmli buffer and analyzed by immunoblotting using IKKα (A), IKKβ (B), IKKγ (C) and β-actin (D) antibodies. Every fifth fraction ranging from 2.2 mDa to 100 kDa is represented on the western blot. Smaller IKKβ complexes (highlighted in the red box) in the higher fractions suggests a rearrangement of the IKKβ complex in TC-83 infected cells.</p