The overall objectives of this research proposal are the further development of a multidisciplinary program for studying the biochemical mechanism of action of, and the molecular basis of resistance to, antitumor drugs. The ultimate goal is to use the knowledge acquired in these studies to: 1) develop taxol analogs that will maintain or improve the therapeutic activity of the drug and be amenable to production in concentrations required for clinical studies and 2) design therapeutic protocols to avoid and circumvent drug resistance. The specific objectives of this research proposal are to: 1) understand the molecular mechanisms by which cells become resistant to taxol. Taxol-resistant cells isolated in the laboratory will be analyzed for the presence of P-glycoprotein and altered forms of tubulin. Cell lines grown in the presence of taxol and cis-platinum will be isolated and analyzed for drug resistance. 2) prepare antibodies to taxol that can be used for a) visualizing the drug in normal and tumor cells by immunofluorescence and on the microtubule by immunoelectron microscopy utilizing gold spheres, b) developing an radioimmune assay (RIA) for the quantitation of taxol in body fluids and tissues, and in crude extracts of plants, and c) domain mapping of the taxol binding site in the microtubule. 3) determine the drug binding site(s) on the microtubule by using photoaffinity labeled radioactive taxol analogs that will covalently bind to the microtubule. Isolation and analysis of a peptide(s) containing the radiolabeled taxol analog should provide the amino acid sequence with which taxol interacts. This information, taken together with the known structural features of taxol that are required for binding, plus what is known about the tertiary structure of tubulin, should produce, at least a partial definition of the binding site for taxol on the microtubule. 4) further define the structure-activity profile of taxol with specific emphasis on the A-ring side chain. Such information will provide insight into the chemical features of taxol that are important in its interaction with microtubules and allow the design of new therapeutic analogues.
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