Biological activities of BAY61-3606 derivatives.

<p>(<b>a</b>) GI50 values for BAY61-3606 derivatives performed in HCT-116 (red) or HKe-3 (blue) cells. Derivative 6 was chosen for further study for its increased potency and specificity for K-RAS mutant cells. (<b>b</b>) Cell viability quantified by Syto60 after 72 hours exposure to BAY derivative 6 in HCT-116 (red) or HKe-3 (blue) cells. Relative cell viability was normalized to DMSO vehicle treated control for each cell line. Error bars represent SEM for 3 independent experiments. HCT-116 cells were relatively sensitive to BAY derivative 6.</p

Identification and attempted validation of BAY61-3606 targets.

<p>(<b>a</b>) TREEspot image representing the inhibitory activity of BAY61-3606. Kinases that are inhibited for ATP binding by BAY61-3606 are indicated by red dots on the phylogenetic tree of kinases. The size of the red dot corresponds to the amount of inhibition by 1 μM BAY61-3606. The identity of the individual kinases can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041343#pone.0041343.s008" target="_blank">Table S1</a>. (<b>b</b>) Cell viability quantified by Syto60 after shRNA-mediated knockdown of the potential BAY61-3606 targets in DLD-1 (red) or DKs-8 (blue) cell lines. Relative cell viability is normalized to the parental cell lines infected with vector only. Error bars represent SEM for 2 independent experiments.</p

Inhibition of SYK is not responsible for the BAY61-3606 effect on cell viability in colorectal cancer cells.

<p>(<b>a</b>) Knockdown of SYK protein in DLD-1 cells via shRNA, as shown by Western Blotting. The Ramos (R) cell line, a hematopoetic cell line with a high expression of SYK, was used as a positive control. (<b>b</b>) Cell viability of DLD-1 cells (red), and its K-RAS wild-type (DKs-8 – blue) and SYK knockdown (red shaded) derivatives after 72 hours of treatment with 1 <b>μ</b>M of BAY61-3606 or R406, a distinct SYK inhibitor. Relative cell viability was normalized to DMSO vehicle treated control for each cell line. Error bars represent SEM for 3 independent experiments. DLD-1 cells and its K-RAS wild-type derivative did not exhibit sensitivity to R406, while knocking down SYK in DLD-1 cells only minimally affected its sensitivity to BAY61-3606.</p

BAY61-3606 affects viability in cells expressing mutant K-RAS or B-RAF through a MAPK-independent pathway.

<p>(<b>a</b>) Cell viability quantified by Syto60 after 72 hours of AZ-628, CI-1040 or BAY61-3606 treatment in HCT-116 (K-RAS^G13D/+, red) or HKe-3 (K-RAS^−/+, blue) cell lines. Relative cell viability was normalized to DMSO vehicle treated control for each cell line. Error bars represent SEM for 3 independent experiments. Cells expressing mutant K-RAS were relatively sensitive to AZ-628 and BAY61-3606, but not CI-1040. (<b>b</b>) Cell cycle profiles, as determined by propidium iodide staining, of colorectal cancer cells with mutant B-RAF (HT-29) treated with CI-1040 or mutant K-RAS (DLD-1) treated with BAY61-3606. While inhibition of MEK induces G1 arrest in HT-29 cells, as evidenced by the loss of the 4N peak, BAY61-3606 did not appear to alter the profile of DLD-1 cells. (<b>c</b>) Cell viability of colorectal cancer cells expressing mutant B-RAF (V600E) after 72 hours of treatment with BAY61-3606 and/or CI-1040. Cells expressing mutant B-RAF (HT-29 – solid outline and RKO – dotted outline) were sensitive to both BAY61-3606 and CI-1040 and these two inhibitors cooperated to produce an enhanced response. (<b>d</b>) Phospho-MEK1 (Ser217/Ser221) and phospho-ERK1/2 (Thr202/Tyr204, Thr185/Tyr187) levels in K-RAS wild-type and mutant cells after 45 minute exposure to 1 µM AZ-628, 1 µM BAY61-3606, or vehicle control. Signals were measured using Bio-Plex assays. Relative signal was normalized to a master control lysate. AZ-628 treatment reduced the level of phospho-MEK and phospho-ERK, but BAY61-3606 did not.</p

Activity of BAY61-3606 against selected kinases.

*<p>% inhibition of ATP binding as assessed by KINOMEscan™ (Ambit Biosciences).</p>**<p><i>In vitro</i> IC50 were determined by SelectScreen® (Invitrogen).</p

MAP4K2 is a target for BAY61-3606 that modulates the response of wild-type cells to AZ-628.

<p>(<b>a</b>) Cell viability quantified by Syto60 after 72 hours of combinatorial treatment with varying concentrations of BAY61-3606 and 1 µM AZ-628 in HCT-116 (red line) or HKe-3 (blue lines) cells. Relative cell viability was normalized to DMSO vehicle treated control for each cell line. Error bars represent SEM for 3 independent experiments. The two inhibitors cooperate in wild-type cells, but not in cells expressing mutant K-RAS. (<b>b</b>) Cell viability after 72 hours of combinatorial treatment with varying concentrations of BAY derivative 6 and 1 µM AZ-628 in HCT-116 and HKe-3 cells. BAY derivative 6 does not confer additional sensitivity to AZ-628 upon HKe-3 cells. (<b>c</b>) Cell viability quantified by Syto60 after 72 hours of AZ-628 treatment in HCT-116 or HKe-3 cell lines with MAP4K2 knockdown. Loss of MAP4K2 does not affect AZ-628 response in cells expressing mutant K-RAS, but enhances the effect of AZ-628 in cells expressing wild-type K-RAS. (<b>d</b>) Cell viability after 72 hours of combinatorial treatment with 1 µM BAY61-3606 and 1 µM AZ-628 (shaded) or 1 µM AZ-628 alone (clear) in HKe-3 cells with MAP4K2 knockdown. Relative cell viability was normalized to 1 µM AZ-628 treated samples. In parental HKe-3 cells, BAY61-3606 confers sensitivity to AZ-628. Upon loss of MAP4K2, BAY61-3606 no longer sensitizes.</p

Identification of IKKα as a target of BAY61-3606.

<p>(A) Inhibition of AD169 replication by BAY compounds. HFF cells were infected with AD169 at an MOI of 1 then treated with 1μM of the drug indicated in the figure or the equivalent volume of DMSO. Viral supernatants were harvested at 96 h.p.i. and viral titre (p.f.u./ml) was determined. (B and C) In vitro kinase assays in the presence of BAY61-3606. The ability of 10 μM BAY61-3606 (Fig 3B) or a range of BAY61-3606 concentrations (Fig 3C) to inhibit the kinase activity of each of the indicated kinase proteins was assayed. Each data point in each figure represents the percentage kinase activity in the presence of drug compared to DMSO treated controls. Each data point shows the mean value of two experiments. (D-G) Analysis of HFF treated with siRNA. HFF cells were treated with either Crtl or IKKα siRNA. After 72 hours incubation with siRNA cell lysates were prepared for western blotting (Fig 3D) or infected with 1x10⁵ p.f.u. of HCMV to analyze virus replication by virus titration (Fig 3E). The data in Fig 3E is represented as p.f.u./ml at 96 h.p.i. and shows the mean and standard deviation of 3 experiments. Also, cell lysates from siRNA treated cells infected AD169 were prepared for western blotting 24 h.p.i. (Fig 3F). In Fig 3G, samples from lanes 1 and 5 of Fig 3E were diluted in a 2-fold series (lanes 2–4 and 6–8, respectively). The siRNA used in each case is indicated in each panel. In Fig 3D, 3F and 3G proteins recognized by the antibodies used in each experiment are indicated to the right of each figure and the positions of molecular weight markers (kDa) are indicated to the left of each figure. Band intensities are expressed in arbitrary units above or below each panel.</p

Viral inhibition and cytotoxicity assays using BAY61-3606.

Viral inhibition and cytotoxicity assays using BAY61-3606.</p

MAP4K2 modulates NFκβ signaling.

<p>(<b>a</b>) Time course of phospho-Iκβα (Ser32/Ser36) activation after 1 µM AZ-628 treatment in HCT-116 (red lines) or HKe-3 (blue line) cells with MAP4K2 knock down, as measured via Bio-Plex. Relative signal was normalized to a master control lysate. Error bars represent SEM for 3 independent experiments. NFκβ signaling was enhanced in HKe-3 cells after exposure to AZ-628 and this was dependent upon MAP4K2. (<b>b</b>) Cell viability quantified by Syto60 after 72 hours of combinatorial treatment with IKK inhibitor VII and 1 µM AZ-628. Relative cell viability was normalized to DMSO vehicle treated control for each cell line. Like BAY61-3606, IKK inhibitor VII enhanced the effect of AZ-628 specifically in K-RAS wild-type cells. (<b>c</b>) Cell viability after 72 hours of combinatorial treatment of 1 µM IKK inhibitor VII and 1 µM AZ-628 (shaded) or 1 µM AZ-628 alone (clear) in HKe3 cells with MAP4K2 knockdown. Relative cell viability was normalized to 1 µM AZ-628 treated samples. Loss of MAP4K2 abrogated the ability of IKK inhibitor VII to sensitize HKe-3 cells to AZ-628.</p

Investigation of histone H3 modifications in BAY61-3606 and siRNA treated cells.

<p>(A, C and D) Western blotting of HFF cells treated with BAY61-3606. HFF cells were uninfected or infected with AD169 at an MOI of 1, then treated with either 1μM BAY61-3606 or the equivalent volume of DMSO. Cell lysates were prepared for western blotting at the time points (hours post infection (h.p.i.)) indicated above the figure. Uninfected cells harvested at the time of infection are shown as 0 h.p.i.. (B) Western blotting of HFF cells treated with siRNA. HFF cells were treated with either Crtl or IKKα siRNA. After 72 hours incubation cells infected with 1x10⁵ p.f.u. of AD169 and then prepared for western blotting at 72 h.p.i.. The siRNA used is indicated above the figure. In each figure proteins recognized by the antibodies used in each experiment are indicated to the right of each figure. The positions of molecular weight markers (kDa) are indicated to the left of each figure.</p