Synthesis and biological evaluation of 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]-4-ethynylimidazole. A novel and highly potent anti-inflammatory and cytoprotective agent

To explore more potent N-acylimidazole analogues of CDDO than CDDO-Im, which is one of the most potent compounds in several widely used bioassays related to protection against inflammation and carcinogenesis; we have synthesized and evaluated five new N-acyl(acetylenic) imidazole analogues. Among them, 4-ethynylimidazole 4 is nearly equivalent to CDDO-Im in potency in these bioassays. Remarkably, the solid form of 4 is more stable than that of CDDO-Im. These findings suggest that 4 is a very promising anti-inflammatory and cytoprotective agent and its further preclinical evaluation is warranted. (C) 2011 Elsevier Ltd. All rights reserved.</p

RTA 408, A Novel Synthetic Triterpenoid with Broad Anticancer and Anti-Inflammatory Activity

<div><p>Semi-synthetic triterpenoids are antioxidant inflammation modulator (AIM) compounds that inhibit tumor cell growth and metastasis. Compounds in the AIM class bind to Keap1 and attenuate Nrf2 degradation. In the nucleus, Nrf2 increases antioxidant gene expression and reduces pro-inflammatory gene expression. By increasing Nrf2 activity, AIMs reduce reactive oxygen species and inflammation in the tumor microenvironment, which reverses tumor-mediated immune evasion and inhibits tumor growth and metastasis. AIMs also directly inhibit tumor cell growth by modulating oncogenic signaling pathways, such as IKKβ/NF-κB. Here, we characterized the in vitro antioxidant, anti-inflammatory, and anticancer activities of RTA 408, a novel AIM that is currently being evaluated in patients with advanced malignancies. At low concentrations (≤ 25 nM), RTA 408 activated Nrf2 and suppressed nitric oxide and pro-inflammatory cytokine levels in interferon-γ-stimulated RAW 264.7 macrophage cells. At higher concentrations, RTA 408 inhibited tumor cell growth (GI₅₀ = 260 ± 74 nM) and increased caspase activity in tumor cell lines, but not in normal primary human cells. Consistent with the direct effect of AIMs on IKKβ, RTA 408 inhibited NF-κB signaling and decreased cyclin D1 levels at the same concentrations that inhibited cell growth and induced apoptosis. RTA 408 also increased CDKN1A (p21) levels and JNK phosphorylation. The in vitro activity profile of RTA 408 is similar to that of bardoxolone methyl, which was well-tolerated by patients at doses that demonstrated target engagement. Taken together, these data support clinical evaluation of RTA 408 for cancer treatment.</p></div

RTA 408 Inhibits NF-κB and Activates JNK.

<p><b><i>A</i></b>, HeLa/NF-κB-Luc or A549/NF-κB-Luc cells were treated with RTA 408 for one hour and then treated with 10 ng/mL TNFα. Five hours later, luminescence was measured to assess NF-κB activity. Data are presented as percent activity relative to cells treated with TNFα alone. Data points for HeLa and A549 are the mean and SD of three and four experiments, respectively. <b><i>B</i></b>, HeLa cells were pre-treated with RTA 408 or bardoxolone methyl for 6 hours at the indicated concentrations followed by a five-minute treatment with TNFα. Protein levels of phospho-IκBα and total IκBα were evaluated by western blot. Actin was used as loading control. Data are representative of four experiments. <b><i>C</i></b>, Cells were treated with RTA 408 for 24 hours and protein levels of cyclin D1, CDKN1A (p21), and total and phospho-JNK were evaluated by western blot. Actin served as a loading control. Data are representative of two experiments.</p

RTA 408 Inhibits Proliferation and Colony Formation in Wild-Type and <i>Keap1</i>-/- MEFs.

<p><b><i>A</i></b>, Growth of WT and <i>Keap1</i>^-/- MEFs treated with RTA 408. MEFs were treated with RTA 408 and cells were counted at 24-hour intervals. Statistical significance was determined by repeated measures one-way ANOVA and Dunnett’s multiple comparison test. *, <i>P</i> < 0.05; **, P < 0.01 compared to vehicle-treated cells at the same time point. <b><i>B</i></b>, Colony formation by WT and <i>Keap1</i>^-/- MEFs treated with RTA 408. Statistical significance was determined by repeated measures one-way ANOVA and Dunnett’s multiple comparison test. *, <i>P</i> < 0.05; **, P < 0.01 compared to vehicle-treated cells. <b><i>C</i></b>, Nrf2 target gene expression in WT and <i>Keap1</i>^-/- MEFs treated with RTA 408 for 18 hours. mRNA levels of <i>Nqo1</i> and <i>Gclm</i> were measured by qRT-PCR. Values are presented as fold-induction relative to vehicle-treated WT MEFs. <b><i>D</i></b>, RTA 408 increases expression of Nrf2 target genes in human tumor cell lines. MDA-MB-231, HCT 116, and G361 cells were treated with the indicated concentrations of RTA 408 for 18 hours. mRNA levels of <i>NQO1</i>, <i>HMOX1</i>, <i>GCLM</i>, and <i>GCLC</i>, were measured by qRT-PCR. Data are presented as fold-induction relative to vehicle-treated cells for each cell line. Statistical significance was determined by repeated measures one-way ANOVA and Dunnett’s multiple comparison test.*, <i>P</i> < 0.05; **, <i>P</i> <0.01; ***, <i>P</i> < 0.001 compared to vehicle-treated cells. <b><i>E</i></b>, MDA-MB-231, HCT 116, and G361 cells were treated with RTA 408 for 48 hours and cell viability was determined using the SRB assay. Data are presented as percent survival relative to survival in vehicle-treated cells. In all panels, data points are the mean of three independent experiments and error bars are SD.</p

RTA 408 Inhibits Growth and Induces Apoptosis in Human Tumor Cell Lines.

<p><b><i>A</i></b>, Cells were treated with RTA 408 for 72 hours and viability was assessed using the SRB assay. Values are presented as percent growth in RTA 408-treated cells relative to growth in vehicle-treated cells. <b><i>B</i></b>, Human tumor cell lines were treated with RTA 408 for 24 hours and cleavage of the DEVD-AFC peptide was measured as a surrogate for caspase activity. Data are presented as caspase activity in RTA 408-treated cells relative to activity in vehicle-treated cells. Data points are the mean and SD of three experiments. Statistical significance was determined by repeated measures one-way ANOVA and Dunnett’s multiple comparison test. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01 compared to vehicle-treated cells. <b><i>C</i></b>, Cells were treated with RTA 408 for 24 hours and protein levels of caspase-3 and caspase-9 were evaluated by Western blot. Actin served as a loading control. Data are representative of two experiments. <b><i>D</i></b>, Human tumor cell lines and normal primary human cells were treated with RTA 408 for 24 hours and cleavage of the DEVD-AFC peptide was measured as a surrogate for caspase activity. Data are presented as caspase activity in RTA 408-treated cells relative to activity in vehicle-treated cells. Data points are the mean and SD of three experiments.</p

Human Tumor Cell Line Growth Inhibition Following RTA 408 Treatment for 72 hours.

<p>^a Values are mean ± standard deviation of three independent experiments.</p><p>Human Tumor Cell Line Growth Inhibition Following RTA 408 Treatment for 72 hours.</p

RalB GTPase-Mediated Activation of the IκB Family Kinase TBK1 Couples Innate Immune Signaling to Tumor Cell Survival

SummaryThe monomeric RalGTPases, RalA and RalB are recognized as components of a regulatory framework supporting tumorigenic transformation. Specifically, RalB is required to suppress apoptotic checkpoint activation, the mechanistic basis of which is unknown. Reported effector proteins of RalB include the Sec5 component of the exocyst, an octameric protein complex implicated in tethering of vesicles to membranes. Surprisingly, we find that the RalB/Sec5 effector complex directly recruits and activates the atypical IκB kinase family member TBK1. In cancer cells, constitutive engagement of this pathway, via chronic RalB activation, restricts initiation of apoptotic programs typically engaged in the context of oncogenic stress. Although dispensable for survival in a nontumorigenic context, this pathway helps mount an innate immune response to virus exposure. These observations define the mechanistic contribution of RalGTPases to cancer cell survival and reveal the RalB/Sec5 effector complex as a component of TBK1-dependent innate immune signaling