New methodologies for studying the biological activity of platinum complexes
| Autor: | Sandman, Karen E. (Karen Elizabeth), 1972 |
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| Jazyk: | angličtina |
| Rok vydání: | 1998 |
| Předmět: | |
| Druh dokumentu: | Diplomová práce |
| Popis: | Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1998. Vita. Includes bibliographical references. A review of existing approaches to platinum drug discovery highlighted the need for new methods to perform mechanism-based, high-throughput screening of platinum complexes. A new solution phase screening method was based on the importance of high mobility group (HMG) domain proteins in the cytotoxicity of cisplatin. Platinum compounds were evaluated based on their ability to form DNA adducts that bind specifically to HMG-domain proteins. A mixture of platinum amino acid complexes was prepared and combined with DNA to form platinum adducts, some of which were recognized by HMG1 in a gel shift assay. In order to identify the platinum complex(es) responsible for this behavior, a sublibrary synthesis and screening approach was employed. After three iterations of sublibrary synthesis and screening, [Pt(lysine)C12] (Kplatin) was identified as an (N,O)-chelated platinum(II) complex with DNA adducts recognized by HMG-domain proteins. Kplatin and analogous (N,O)-chelates were less toxic towards HeLa cells than cisplatin, presumably because of charge considerations. A solid-phase assay was envisioned in which platinum-modified DNA could be covalently linked to a solid support, and the binding of a fluorescently labeled HMGdomain protein measured to predict the cytotoxicity of the platinum complexes. A fluorescent HMG-domain protein was generated by expressing HMG1 as a fusion with the green fluorescent protein (GFPuv). The product HMG1-GFPuv retained the fluorescence and DNA-binding properties of its protein components. To screen platinum complexes for biological activity in human cancer cells, a transcription inhibition assay was developed. A stable HeLa cell line was generated with doxycycline-inducible enhanced green fluorescent protein (EGFP). The effect of platinum complexes and other cytotoxic agents on EGFP expression was assessed. Most cytotoxic agents including trans platinum complexes stimulated EGFP transcription, whereas cis platinum complexes inhibited EGFP expression in a dosedependent fashion. Transient transfection of the HeLa cells with testis-specific HMG enhanced the inhibitory effect of cisplatin. A second reporter gene assay using a fluorescent - lactamase substrate demonstrated the inhibition of gene expression by cisplatin but not by ineffective platinum complexes. by Karen E. Sandman. Ph.D. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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