|Authors:||Tran, Thi Ngoc Tuyen|
|Title:||Evaluation of the phage display protocol for target identification of small molecules|
|Other Titles:||Identification and characterization of mitosis modulators|
|Abstract:||Currently, the phage display is the potential method for target identification of small molecules. Thus, the first objective of this thesis was to establish a cDNA phage display protocol for affinity isolation of target proteins binding to small molecules of interest as an alternative to the affinity-based approach. For that reason we generated a cDNA phage display library using HeLa cells, which are usually used for phenotypic studies and for target identification using affinity chromatography in our group. This approach was applied different molecules including melophlin A, CD 267, tubulexin A. and glutathione (GSH) as control. Preincubation the library with streptavidin-biotin or streptavidin-PEG-biotin or inactive compound before biopanning step was employed to reduce on-specific binding. In parallel, specific elution approaches was employed to elute proteins and identified one known hit of GSH. The binders identified by phage display were not identical with the binders identified using affinity purification in the case of melophlin A, CD 267 and tubulexin A. Even though we could succeed in establishing a protocol with the optimal conditions determined. However, there is unlikely to get the same hits of affinity purification for melophlin A, CD 267 and tubulexin A. A separate study focused on the biological evaluation of hit compounds from phenotypic high content screening that monitors changes in cytoskeleton and DNA. Molecules interfering with microtubule dynamics are among the most successful therapeutics for the treatment of cancer. Despite the availability of many tubulin targeting agents, cells can become resistant towards the drugs. Hence, there is high demand for the identification of new anti-tubulin agents that are able to overcome anti-tubulin drug resistance. Within this work, the aim was to further validate the effects of tubulexin A and podoverin A and characterize their mode of action. Tubulexin A and the natural product podoverin A are potent inducers of a G2/M cell cycle arrest and apoptosis. Tubulexin A is the most active compound identified from tetrahydropyran library. Base on target validation showed that tubulexin A inhibits tubulin polymerization by targeting the vinca alkaloid binding site of tubulin and also bind to the protein CAS. Further validation of the effects of those compounds on tubulin polymerization, the cell cycle, and their binding to CAS was done by biophysical measurements, binding assays, tubulin polymerization assays, immunofluorescence and life time imaging, binding assays, and affinity chromatographic methods. Results showed that CAS binds to tubulexin A independent from tubulin. Additionally, tubulin polymerization is inhibited in a synergistic manner in the presence of both, tubulexin A and CAS in vitro. Based on results of overcome vinblastine-resistance, tubulexin A can become a promising antimitotic drug for cancer treatment with the dual mode of action of the tubulexin A and CAS. For the natural product podoverin A is the most active mitosis modulator in natural product library. This compound is a potential target for further study on natural product as mitosis modulator to use for anticancer drugs, also used as natural product scaffold for synthesis library. The third aim of this thesis is discovery new CAS inhibitors as mitosis modulators. Despite the high relevance of CAS as a target in cancer, there is no small molecule targeting CAS published yet. Therefore, additional focus of this work was to discover further compounds that bind to CAS which might interfere with CAS function and thus might inhibit growth of cancer cells. A reverse chemical genomics-approach was chosen in collaboration with the group of Prof. Osada (RIKEN-Wako-Japan). This strategy is based on a chemical array screening. After chemical array screening of approximately 25,000 compounds, 263 potential hit compounds have been obtained. Further validation the hit compounds was based on phenotypic changes and specific function of the target using immunofluorescence and life time imaging, biophysical methods for the determination of the binding affinity, as well as studies on the interaction of the hit compounds with DNA and tubulin polymerization in vitro. The most interesting compound R89 was further validated on HeLa cells and we found that this compound inhibited cell proliferation. Result showed that R89 has effect on mitosis by formation of multipolar mitotic spindles and caused chromosome congression defects and also induces nuclear accumulation of RANBP1. Thus R89 is an interesting mitosis modulator to study for anticancer drugs.|
|Subject Headings:||Tubulexin A|
|Appears in Collections:||Max-Planck-Institut für molekulare Physiologie|
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