(4-(3-chlorophenyl)piperazin-1-yl)(2-ethoxyphenyl)methanone and Inflammation

We recently identified a series of mitotically acting piperazine-based compounds that potently increase the sensitivity of colon cancer cells to apoptotic ligands. Here we describe a structure-activity relationship study on this compound class and identify a highly active derivative ((4-(3-chlorophenyl)piperazin-1-yl)(2-ethoxyphenyl)methanone), referred to as AK301, the activity of which is governed by the positioning of functional groups on the phenyl and benzoyl rings. AK301 induced mitotic arrest in HT29 human colon cancer cells with an ED50 of ≈115 nm. Although AK301 inhibited growth of normal lung fibroblast cells, mitotic arrest was more pronounced in the colon cancer cells (50% versus 10%). Cells arrested by AK301 showed the formation of multiple microtubule organizing centers with Aurora kinase A and γ-tubulin. Employing in vitro and in vivo assays, tubulin polymerization was found to be slowed (but not abolished) by AK301. In silico molecular docking suggests that AK301 binds to the colchicine-binding domain on β-tubulin, but in a novel orientation. Cells arrested by AK301 expressed elevated levels of TNFR1 on their surface and more readily activated caspases-8, -9, and -3 in the presence of TNF. Relative to other microtubule destabilizers, AK301 was the most active TNF-sensitizing agent and also stimulated Fas- and TRAIL-induced apoptosis. In summary, we report a new class of mitosis-targeting agents that effectively sensitizes cancer cells to apoptotic ligands. These compounds should help illuminate the role of microtubules in regulating apoptotic ligand sensitivity and may ultimately be useful for developing agents that augment the anti-cancer activities of the immune response.

Topics: Apoptosis; Binding Sites; Caspase 8; Caspase 9; Colonic Neoplasms; Fibroblasts; HCT116 Cells; HT29 Cells; Humans; Inflammation; Ligands; Microtubules; Mitosis; Models, Molecular; Piperazines; Signal Transduction; Tumor Necrosis Factor-alpha

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature