Combined drug action of 2-phenylimidazo[2,1-b]benzothiazole derivatives on cancer cells according to their oncogenic molecular signatures

The development of targeted molecular therapies has provided remarkable advances into the treatment of human cancers. However, in most tumors the selective pressure triggered by anticancer agents encourages cancer cells to acquire resistance mechanisms. The generation of new rationally designed targeting agents acting on the oncogenic path(s) at multiple levels is a promising approach for molecular therapies. 2-phenylimidazo[2,1-b]benzothiazole derivatives have been highlighted for their properties of targeting oncogenic Met receptor tyrosine kinase (RTK) signaling. In this study, we evaluated the mechanism of action of one of the most active imidazo[2,1-b]benzothiazol-2-ylphenyl moiety-based agents, Triflorcas, on a panel of cancer cells with distinct features. We show that Triflorcas impairs in vitro and in vivo tumorigenesis of cancer cells carrying Met mutations. Moreover, Triflorcas hampers survival and anchorage-independent growth of cancer cells characterized by 'RTK swapping' by interfering with PDGFRβ phosphorylation. A restrained effect of Triflorcas on metabolic genes correlates with the absence of major side effects in vivo. Mechanistically, in addition to targeting Met, Triflorcas alters phosphorylation levels of the PI3K-Akt pathway, mediating oncogenic dependency to Met, in addition to Retinoblastoma and nucleophosmin/B23, resulting in altered cell cycle progression and mitotic failure. Our findings show how the unusual binding plasticity of the Met active site towards structurally different inhibitors can be exploited to generate drugs able to target Met oncogenic dependency at distinct levels. Moreover, the disease-oriented NCI Anticancer Drug Screen revealed that Triflorcas elicits a unique profile of growth inhibitory-responses on cancer cell lines, indicating a novel mechanism of drug action. The anti-tumor activity elicited by 2-phenylimidazo[2,1-b]benzothiazole derivatives through combined inhibition of distinct effectors in cancer cells reveal them to be promising anticancer agents for further investigation

Combined drug action of 2-phenylimidazo[2,1-b]benzothiazole derivatives on cancer cells according to their oncogenic molecular signatures

International audienceThe development of targeted molecular therapies has provided remarkable advances into the treatment of human cancers. However, in most tumors the selective pressure triggered by anticancer agents encourages cancer cells to acquire resistance mechanisms. The generation of new rationally designed targeting agents acting on the oncogenic path(s) at multiple levels is a promising approach for molecular therapies. 2-phenylimidazo[2,1-b]benzothiazole derivatives have been highlighted for their properties of targeting oncogenic Met receptor tyrosine kinase (RTK) signaling. In this study, we evaluated the mechanism of action of one of the most active imidazo[2,1-b]benzothiazol-2-ylphenyl moiety-based agents, Triflorcas, on a panel of cancer cells with distinct features. We show that Triflorcas impairs in vitro and in vivo tumorigenesis of cancer cells carrying Met mutations. Moreover, Triflorcas hampers survival and anchorage-independent growth of cancer cells characterized by "RTK swapping" by interfering with PDGFRβ phosphorylation. A restrained effect of Triflorcas on metabolic genes correlates with the absence of major side effects in vivo. Mechanistically, in addition to targeting Met, Triflorcas alters phosphorylation levels of the PI3K-Akt pathway, mediating oncogenic dependency to Met, in addition to Retinoblastoma and nucleophosmin/B23, resulting in altered cell cycle progression and mitotic failure. Our findings show how the unusual binding plasticity of the Met active site towards structurally different inhibitors can be exploited to generate drugs able to target Met oncogenic dependency at distinct levels. Moreover, the disease-oriented NCI Anticancer Drug Screen revealed that Triflorcas elicits a unique profile of growth inhibitory-responses on cancer cell lines, indicating a novel mechanism of drug action. The anti-tumor activity elicited by 2-phenylimidazo[2,1-b]benzothiazole derivatives through combined inhibition of distinct effectors in cancer cells reveal them to be promising anticancer agents for further investigation

Epithelial vanin-1 controls inflammation-driven carcinogenesis in the colitis-associated colon cancer model.

International audienceBACKGROUND:: Vanin-1 is an epithelial pantetheinase that provides cysteamine to tissue and regulates response to stress. Vanin-1 is expressed by enterocytes, and its absence limits intestinal epithelial cell production of proinflammatory signals. A link between chronic active inflammation and cancer is illustrated in patients with ulcerative colitis, who have an augmented risk of developing colorectal cancer. Indeed, sustained inflammation provides advantageous growth conditions to tumors. We examined whether epithelial cells affect tumorigenesis through vanin-1-dependent modulation of colonic inflammation. METHODS:: To vanin-1(-/-) mice, we applied the colitis-associated cancer (CAC) protocol, which combines injection of azoxymethane (AOM) with repeated administrations of dextran sodium sulfate (DSS). We numbered tumors and quantified macrophage infiltration and molecular markers of cell death and proliferation. We also tested DSS-induced colitis. We scored survival, tissue damages, proinflammatory cytokine production, and tissue regeneration. Finally, we explored activation pathways by biochemical analysis on purified colonic epithelial cells (CECs) and in situ immunofluorescence. RESULTS:: Vanin-1(-/-) mice displayed a drastically reduced incidence of colorectal cancer in the CAC protocol and manifested mild clinical signs of DSS-induced colitis. The early impact of vanin-1 deficiency on tumor induction was directly correlated to the amount of inflammation and subsequent epithelial proliferation rather than cell death rate; all this was linked to the modulation of NF-kappaB pathway activation in CECs. CONCLUSIONS:: These results emphasize the importance of the intestinal epithelium in the control of mucosal inflammation acting as a cofactor in carcinogenesis. This might lead to novel anti-inflammatory strategies useful in cancer therapy. Inflamm Bowel Dis 2009

Combined drug action of 2-phenylimidazo[2,1-b]benzothiazole derivatives on cancer cells according to their oncogenic molecular signatures

The development of targeted molecular therapies has provided remarkable advances into the treatment of human cancers. However, in most tumors the selective pressure triggered by anticancer agents encourages cancer cells to acquire resistance mechanisms. The generation of new rationally designed targeting agents acting on the oncogenic path(s) at multiple levels is a promising approach for molecular therapies. 2-phenylimidazo[2,1-b]benzothiazole derivatives have been highlighted for their properties of targeting oncogenic Met receptor tyrosine kinase (RTK) signaling. In this study, we evaluated the mechanism of action of one of the most active imidazo[2,1-b]benzothiazol-2-ylphenyl moiety-based agents, Triflorcas, on a panel of cancer cells with distinct features. We show that Triflorcas impairs in vitro and in vivo tumorigenesis of cancer cells carrying Met mutations. Moreover, Triflorcas hampers survival and anchorage-independent growth of cancer cells characterized by 'RTK swapping' by interfering with PDGFRβ phosphorylation. A restrained effect of Triflorcas on metabolic genes correlates with the absence of major side effects in vivo. Mechanistically, in addition to targeting Met, Triflorcas alters phosphorylation levels of the PI3K-Akt pathway, mediating oncogenic dependency to Met, in addition to Retinoblastoma and nucleophosmin/B23, resulting in altered cell cycle progression and mitotic failure. Our findings show how the unusual binding plasticity of the Met active site towards structurally different inhibitors can be exploited to generate drugs able to target Met oncogenic dependency at distinct levels. Moreover, the disease-oriented NCI Anticancer Drug Screen revealed that Triflorcas elicits a unique profile of growth inhibitory-responses on cancer cell lines, indicating a novel mechanism of drug action. The anti-tumor activity elicited by 2-phenylimidazo[2,1-b]benzothiazole derivatives through combined inhibition of distinct effectors in cancer cells reveal them to be promising anticancer agents for further investigation

Identification of new aminoacid amides containing the imidazo[2,1-b]benzothiazol-2-ylphenyl moiety as inhibitors of tumorigenesis by oncogenic Met signaling.

18The Met receptor tyrosine kinase is a promising target in anticancer therapies for its role during tumor evolution and resistance to treatment. It is characterized by an unusual structural plasticity as its active site accepts different inhibitor binding modes. Such feature can be exploited to identify distinct agents targeting tumor dependence and/or resistance by oncogenic Met. Here we report the identification of bioactive agents, featuring a new 4-(imidazo[2,1-b]benzothiazol-2-yl)phenyl moiety, targeting cancer cells dependent on oncogenic Met. One of these compounds (7c; Triflorcas) impairs survival, anchorage-independent growth, and in vivo tumorigenesis, without showing side effects. Our medicinal chemistry strategy was based on an in-house Met-focused library of aminoacid-amide derivatives enriched through structure-based computer modeling, taking into account the Met multiple-binding-mode feature. Altogether, our findings show how a rational structure-based drug design approach coupled to cell-based drug evaluation strategies can be applied in medicinal chemistry to identify new agents targeting a given oncogenic-dependency setting. Copyright © 2011 Elsevier Masson SAS. All rights reserved.reservedmixedFurlan, A.; Colombo, F.; Kover, A.; Issaly, N.; Tintori, Cristina; Angeli, Lucilla; Leroux, V.; Letard, S.; Amat, M.; Asses, Y.; Maigret, B.; Dubreuil, P.; Botta, Maurizio; Dono, R.; Bosch, J.; Piccolo, O.; Passarella, D.; Maina, F.Furlan, A.; Colombo, F.; Kover, A.; Issaly, N.; Tintori, Cristina; Angeli, Lucilla; Leroux, V.; Letard, S.; Amat, M.; Asses, Y.; Maigret, B.; Dubreuil, P.; Botta, Maurizio; Dono, R.; Bosch, J.; Piccolo, O.; Passarella, D.; Maina, F

Triflorcas perturbs cell cycle progression, leading to mitotic failure.

<p>(A) Graph showing the ratio of percentage of GTL-16 cells in G0/G1 phase over the percentage of cells in S+G2/M phase. Cells were treated with Triflorcas (TFC; 3 µM), or vehicle (cntr) for 48 hours, then stained with propidium iodide. Values are expressed as means ± s.e.m. ***P<0.001; Student-<i>t</i> test (n = 3). Percentages of cells in G0/G1, S, and G2/M phases are reported in the table. (B) GTL-16 cells were grown in the presence of Triflorcas or vehicle and nuclei were visualized using DAPI staining. Arrows show example of cells with multiple nuclei (40X magnification). (C) Quantification of cells with mitotic failure expressed as percentage of the total number of cells analyzed. Triflorcas (TFC; 3 or 10 µM), but not SU11274 (SU; 1 µM), crizotinib (crizo; 1 µM), or PHA665752 (PHA; 1 µM) led to a significant increase in the number of cells with mitotic failure. Values are expressed as means ± s.e.m. ***P<0.001; Student-<i>t</i> test.</p

Triflorcas alters the phosphorylation status of cell cycle-related proteins.

<p>(A) The phosphorylation status of several cell cycle proteins was analyzed by applying the phospho-array KPSS 10.1 (Kinexus Bioinformatics). GTL-16 cells were treated with vehicle (control) or Triflorcas (TFC; 3 µM) for 72 hours (left and right panels, respectively). Red circles highlight constituents of the Akt/mTOR/S6 pathway. Green and blue circles surround nucleophosmin/B23 and Rb phospho-epitopes, respectively. (B) The graph shows the ratio of phosphorylation levels of the indicated proteins in cells untreated versus those exposed to Triflorcas. (C) Phosphorylation levels of nucleophosmin/B23 at Ser₄ and Rb at S₇₈₀ were increased and decreased, respectively, in GTL-16 cells exposed to Triflorcas (TFC; 3 µM for 24 hours).</p

Triflorcas elicits a selective gene expression profile on stress and toxicity pathways.

<p>(A) The expression profile of 84 genes related to cell stress and toxicity was analyzed in GTL-16 cells. Cells were treated with either Triflorcas (black columns; 3 µM) or SU11274 (grey columns; 1 µM) for 24 hours, and gene expression was compared to that of untreated cells. Genes were grouped in clusters corresponding to oxidative/metabolic stress, heat shock, proliferation/carcinogenesis, growth arrest/senescence, inflammation, and apoptosis/necrosis signaling. Only statistically significant changes in gene expression are indicated (<i>P</i><0.05). Triflorcas altered the expression of only 14 genes compared to the alteration of 39 genes induced by SU11274 treatment. Notably, the expression of <i>cyp1A1</i> was increased 611-fold in the presence of Triflorcas. (B) Western blot analysis showing the up-regulation of CYP1A1 protein levels in cells exposed to Triflorcas (3 or 10 µM). Acacetin (ACA; 10 µM) treatment prevented CYP1A1 up-regulation by Triflorcas (TFC). (C) CYP1A1 up-regulation by Triflorcas also occurred in ErbB1-addicted cancer BT474 cells. Gefitinib (Gef; 10 µM) and SU11274 (SU; 2 µM) were used as controls.</p

Small molecule kinase interaction map for Triflorcas.

<p>Compound was screened against a KINOME<i>scan</i> (<a href="http://www.kinomescan.com" target="_blank">http://www.kinomescan.com</a>) panel of 98 kinases. (A) The table indicates the kinases for which Triflorcas reduced more than 30% the binding constant. The ligand binding for each kinase (% of control condition) is indicated. (B) TREEspot image of the 98 kinases screened with the position of Triflorcas targets: Abl wild-type, Abl E255K and Abl T315I mutant forms, IKK-beta, JAK2, MKNK1, and ZAP70. The complete dataset is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046738#pone.0046738.s008" target="_blank">Table S2</a>. TK: tyrosine kinase; TKL: tyrosine kinase like; STE: STE kinase; CK1: cell kinase1; AGC: PKA, PKC, PKG kinases; CAMK: calcium calmodulin-regulated kinase; CMGC: CDK, MAP, GSK, CDK-like kinase.</p