BAY 11-7082 Is a Broad-Spectrum Inhibitor with Anti-Inflammatory Activity against Multiple Targets

BAY 11-7082 (BAY) is an inhibitor of κB kinase (IKK) that has pharmacological activities that include anticancer, neuroprotective, and anti-inflammatory effects. In this study, BAY-pharmacological target pathways were further characterized to determine how this compound simultaneously suppresses various responses. Primary and cancerous (RAW264.7 cells) macrophages were activated by lipopolysaccharide, a ligand of toll-like receptor 4. As reported previously, BAY strongly suppressed the production of nitric oxide, prostaglandin E2, and tumor necrosis factor-α and reduced the translocation of p65, major subunit of nuclear factor-κB, and its upstream signaling events such as phosphorylation of IκBα, IKK, and Akt. In addition, BAY also suppressed the translocation and activation of activator protein-1, interferon regulatory factor-3, and signal transducer and activator of transcription-1 by inhibiting the phosphorylation or activation of extracellular signal-related kinase, p38, TANK-binding protein, and Janus kinase-2. These data strongly suggest that BAY is an inhibitor with multiple targets and could serve as a lead compound in developing strong anti-inflammatory drugs with multiple targets in inflammatory responses.

BAY11-7082 (BAY, Figure 1(a)) is a representative IKK inhibitor. Although IKK/NF-κB is important biochemical factor, the pharmacological activities of BAY such as inhibition of inflammatory cytokines [11], induction of heme oxygenase-1 [12], suppression of ICAM-1 expression [13], reduction of ATPase activity of NLRP3 inflammasome [14], and increase in neutrophil apoptosis [15] strongly indicate that it is not a selective inhibitor of only IKK. Therefore, in this study, we explored its inhibitory potency for inflammatory signals leading to the production of NO, PGE 2 , and TNF-α under LPS-activated conditions, using primary and cancerous (RAW264.7 cells) macrophages.    [19,20]. At 1 or 3 h before culture termination, 10 μL MTT solution (10 mg/mL in phosphate-buffered saline, pH 7.4) was added to each well, and the cells were continuously cultured until termination of the experiment. Incubation was halted by the addition of 15% sodium dodecyl sulfate (SDS) into each well, solubilizing the formazan [21]. Absorbance at 570 nm (OD 570-630 ) was measured using a SpectraMax 250 microplate reader.

Preparation of Cell Lysates and Nuclear Fraction, and
Immunoblotting. RAW264.7 cells (5 × 10 6 cells/mL) were washed three times in cold PBS with 1 mM sodium orthovanadate and lysed by a sonicator in lysis buffer (20 mM tris-HCl, pH 7.4, 2 mM EDTA, 2 mM ethyleneglycotetraacetic acid, 50 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 1% Triton X-100, 10% glycerol, 10 μg/mL aprotinin, 10 μg/mL pepstatin, 1 mM benzimide, and 2 mM PMSF) for 30 min with rotation at 4 • C. Lysates were clarified by centrifugation at 16,000 xg for 10 min at 4 • C and stored at −20 • C until needed. Nuclear lysates were prepared in a three-step procedure [22]. After treatment, cells were collected with a rubber policeman, washed with PBS, and lysed in 500 μL lysis buffer containing 50 mM KCl, 0.5% Nonidet P-40, 25 mM HEPES (pH 7.8), 1 mM phenylmethylsulfonyl fluoride, 10 μg/mL leupeptin, 20 μg/mL aprotinin, and 100 μM 1,4dithiothreitol (DTT) on ice for 4 min. Cell lysates were centrifuged at 19,326 xg for 1 min in a microcentrifuge. In the second step, the nuclear fraction pellet was washed once in washing buffer (lysis buffer without Nonidet P-40). In the final step, nuclei were treated with an extraction buffer of lysis buffer with 500 mM KCl and 10% glycerol. The nuclei/ extraction buffer mixture was frozen at −80 • C, thawed on ice and centrifuged at 19,326 xg for 5 min. The supernatant was collected as a nuclear extract. Soluble cell lysates were immunoblotted and protein levels determined as previously reported [23].

Results and Discussion
As previously reported, BAY showed strong inhibition of NF-κB activation. BAY blocked the production of NO, PGE 2 , and TNF-α, well-known inflammatory responses generated by activated NF-κB [29], in LPS-treated RAW264.7 cells (Figures 1(b) and 1(d)) and peritoneal macrophages (Figure 1(c)). It suppressed the translocation of NF-κB subunit (p65) in a time-dependent manner (Figure 2(a) left panel) and blocked the phosphorylation of p50 and p65 in whole cell lysates (Figure 2(b)), without altering cell viability up to 20 μM (Figure 2(a) right panel), indicating that it could specifically block the activation and translocation pathway of NF-κB as reported previously [30].  (TRIF, MyD88, and TBK1), as assessed by reporter gene assay. Furthermore, IκBα phosphorylation [31], a representative upstream pathway for NF-κB translocation, was also strongly suppressed in LPS-treated RAW264.7 cells (Figure 2(g) left panel) and peritoneal macrophages (Figure 2(g) right panel), as reported previously [32], due to its direct inhibition of IKK [33].
Unexpectedly, however, the pharmacological activity of BAY seemed not to be limited to IKK inhibition. It clearly suppressed IKK phosphorylation (Figure 3(a) left panel), mediated by Akt in inflammatory signaling. Furthermore, it blocked the phosphorylation of Akt in both LPS-treated RAW264.7 cells (Figure 3(a) right panel) and peritoneal macrophages (Figure 3(a) right panel), indicating that the target of BAY in inflammatory signaling was upstream of IKK and Akt. Since Src and Syk are representative upstream kinases that activate PI3K, PDK1, and Akt by cascade phosphorylation [34], we evaluated whether these enzymes were suppressed by BAY. As shown in Figures 3(b) and  3(c), BAY did not suppress the phosphorylation of p85, a regulatory subunit of PI3K [35], or its upstream kinases Syk and Src, but diminished p-IKK levels at 2 min. This suggested that the target of BAY in the NF-κB inhibitory pathway was not IKK, but a protein activated upstream of IKK and downstream of Src and Syk. Currently, we do not know which enzyme directly contributes to inhibition by BAY. One of three PI3K isoforms or PDK1 could be the actual target that is regulated by BAY. We plan to conduct future experiments to determine this.
In addition to NF-κB, AP-1 is a representative transcription factor that is activated in inflammatory responses [36]. Therefore, we examined whether BAY blocked the activation of AP-1 in LPS-treated RAW264.7 cells, by measuring the nuclear translocated level of AP-1 components, c-Jun and c-Fos, and using a reporter gene (luciferase) assay with DNA construct containing an AP-1-binding promoter region. As seen in Figures 4(a)-4(d), BAY suppressed the promoterbinding activity of AP-1 induced by PMA and TBK1, but not TRIF and MyD88, as assessed by luciferase activity. Similarly, BAY also strongly suppressed nuclear translocation of c-Fos and c-Jun (Figure 4(e)), indicating that it could modulate the activation pathway required for the translocation of AP-1. Indeed, BAY reduced important upstream events for AP-1 translocation. Thus, it diminished the phosphorylation levels of ERK at 5 min and p38 at 15 min although it also increased the phosphorylation of ERK at 15 to 30 min (Figure 4(f)). Therefore, our data suggested that BAY had a negative effect on AP-1 activation pathway, managed by PKC and TBK1, via indirectly suppressing MAPK (ERK and p38) activation pathway. The fact that U0126 (U0), an ERK inhibitor, and SB203580 (SB), a p38 inhibitor, blocked the production of PGE 2 and TNF-α (data not shown), but not NO (Figure 4(g)), implies that BAY-mediated inhibition of ERK and p38 phosphorylation did not affect its NO inhibitory action.
Recent work on inflammatory signaling demonstrated a critical role for IRF-3 in releasing type I interferons such as IFN-α and IFN-β [37], and additional inflammatory responses by these cytokines via activation of the JAK2/STAT-1 pathway [38]. The activation of the JAK2/STAT-1 pathway is important in the expression of iNOS and COX-2 and other proinflammatory cytokines such as IL-1β and TNF-α [39]. Blockade of the IRF-3 pathway with BX795, a TBK1 inhibitor [40], blocked the expression of iNOS and COX-2 and suppressed the production of NO and PGE 2 ( Figure 5(a)). AG490, an inhibitor of JAK2/STAT-1 pathway also elicited strong anti-inflammatory responses ( Figure 5(b)). Therefore, we examined whether these pathways were also targeted by BAY. BAY strongly suppressed the translocation of IRF3 into the nucleus (Figure 5(c)) and its phosphorylation in the cytosol (Figure 5(d)). This indicated that the IRF-3 regulatory pathway was also directly modulated by BAY. In agreement, BAY diminished the upregulation of luciferase activity induced by TBK1 ( Figure 5(e)), suggesting that the TBK1-mediated inflammatory pathway was a BAY target. Suppression of the JAK2/STAT-1 pathway by BAY was determined by measuring the nuclear translocation of phospho-STAT-1 and its upstream kinase. This also strongly indicated involvement in BAY-mediated inhibition. BAY strongly suppressed the phosphorylation of STAT-1 at 120 min in the nucleus ( Figure 5(f)) and JAK-2 at 0.25 and 2 min in whole cell lysates ( Figure 5(g)), suggesting that the JAK-2/STAT-1-mediated inflammatory responses were also targeted by BAY.
The mechanism of broad-spectrum pharmacological activity of BAY in various inflammatory signaling pathways is not clear. The main factor of this could be derived by its structural properties. Several compounds (e.g., celecoxib, phenylpropanoid derivatives) with a methylphenyl group in their backbone display anti-inflammatory activity by inhibiting various enzyme targets [41,42]. Recently, we have also found that 8-(tosylamino)quinoline (8-TQ), with a similar structural backbone, strongly suppresses various inflammatory signaling cascades (Jung et al., submitted). Therefore, our data suggested that a structural feature of BAY contributed to its multiple pharmacological activities. Since a compound, 3-(4-(tert-octyl)phenoxy)propane-1,2diol, with multiple inhibitory targets such as Syk, IKK, and p38, was found to display higher in vivo efficacy [43], it seems to be worth to further develop BAY derivatives. In view of this, we are currently collaborating with several Chemists to synthesize and develop pharmacologically stronger BAY derivatives.
JAK-2/STAT-1. The suppressive activity of BAY was linked to the suppression of NO, TNF-α, and PGE 2 release. Although the direct target of BAY needs to be identified, our data strongly implied that BAY inhibited the TLR4-activated signaling cascade and the subsequent inflammatory response by targeting multiple signaling enzymes and transcription factors. Considering that inflammatory responses occurred through multiple signaling pathways, and simultaneous inhibition of these pathways contributes to maximum therapeutic potential, the chemical optimization of BAY could be helpful in developing strong BAY-derived anti-inflammatory drugs with multiple targets.