Pipettierroboter
Final Report Abstract
We are interested in the process of chromosome segregation in higher eukaryotes. Specifically, our research focuses on the function and regulation of motor proteins involved in spindle assembly and chromosome congression, the regulation of cell cycle progression by ubiquitin-ligases and the coordination of karyokinesis (mitosis) with cytokinesis. For these studies we combine cell biological approaches in in human tissue culture cells and Xenopus egg extract with biochemical in vitro analyses. In addition to classical cell biological approaches, we use small molecules to modulate protein activity on a fast time scale. For these studies we have to identify specific small molecule inhibitors. By screening a library comprising 10.000 drug-like compounds, we succeeded in identifying an inhibitor for the mitotic kinesin Kif18A. Kif18A is a unique kinesin in that integrates both plus-end directed motility and microtubule depolymerization activity. Cells depleted of Kif18A have elongated spindles, hyperstable microtubules and severe defects in chromosome congression. The identified inhibitor, BTB-1, inhibits Kif18A in an ATP- competitive but microtubule-uncompetitive manner. Of the tested mitotic kinesins, BTB-1 specifically inhibits Kif18A. In cells, BTB-1 induces a mitotic delay which is consistent with the essential function of Kif18A in mitosis. Future studies are required to characterize the mode of action of this inhibitor in cells. Further screens identified small molecule inhibitors for DNA polymerases, specific inhibitors for the mitotic kinesin Eg5, and small molecule binders of the human telomeric repeat sequence. In addition to small molecule screens, we performed RNAi-based screens. One screened designed to identify genes which when depleted rescue spindle bipolarity in Eg5-inhibited revealed that changes in the dynamic behavior of microtubules suppresses the monopolar spindle phenotype in Eg5-inhibited cells. Eg5 is a homotetrameric kinesin that uses its plus-end directed motility to push spindle poles apart and, thus, is essential for spindle bipolarization in mammalian cells. In the absence of Eg5 activity, subtle stabilization of microtubules is sufficient to assemble bipolar spindles via a pathway that depends on the activity of Kif15, a kinesin which under normal conditions is dispensable for the establishment of spindle bipolarity. Given that alterations in microtubule dynamics are a common mechanism responsible for the resistance of tumor cells to microtubule poisons, e.g. vinblastine, our insights suggest that cells resistant to microtubule drugs might also be insensitive to drug-induced inhibition of Eg5. This observation might be important for the evaluation of the efficacy of Eg5 inhibitors which are currently tested in clinical phases. In summary, the identified small molecule inhibitors will provide novel insights into the process of mitosis not amenable by classical cell biological approaches and, therefore, will be key for a better understanding of the mechanism underlying chromosome segregation in mammalian cells.
Publications
- (2009). BTB-1: The first small molecule inhibitor of the mitotic motor protein Kif18A. Angew. Chem. Int. Ed., 48, 9072-9076
Catarinella M, Grüner, T, Strittmatter, T, Marx, A, and Mayer, TU
(See online at https://doi.org/10.1002/anie.200904510) - (2010). Monastrol analogs: a synthesis of pyrazolopyridine, benzopyranopyrazolopyridine, and oxygen-bridged azolopyrimidine derivatives and their biological screening. Bioorg Med Chem Lett. 20, 4073-4076
Svetlik, J., Veizerová, L., Mayer, T.U., Catarinella, M.
(See online at https://dx.doi.org/10.1016/j.bmcl.2010.05.085) - (2011). A Versatile Framework for the Analysis of High-Throughput Screening Data. 8th International Workshop on Computational Systems Biology (WCSB), 57-60
Hamecher, J., Rieß, T., Bertini, E., Kozak, K., Kastl, J., Mayer, T.U., Merhof, D.
- (2011). Identification of Novel Quadruplex Ligands from Small Molecule Libraries by FRET-Based High-Throughput Screening. Chembiochem, 12, 1422-1426
Benz, A., Singh, V., Mayer, T.U., Hartig, J.S.
(See online at https://doi.org/10.1002/cbic.201100094) - (2011). Small Molecule Inhibitors of Human DNA Polymerase lambda. ACS Chem Biol, 6, 314-319
Strittmatter, T., Bareth, B., Immel, T.A., Huhn, T., Mayer, T.U., Marx, A.
(See online at https://doi.org/10.1021/cb100382m) - HiTSEE: A Visualization Tool for Hit Selection and Analysis in High-Throughput Screening Experiments. 1st IEEE Symposium on Biological Data Visualization (IEEE BioVis), pp. 95-102, 2011
Enrico Bertini, Hendrik Strobelt, Joachim Braun, Oliver Deussen, Ulrich Groth, Thomas U. Mayer, Dorit Merhof
(See online at https://doi.org/10.1109/BioVis.2011.6094053)
