This collaborative research proposal has as its primary aim the development of an improved version of the important anticancer drug paclitaxel, using information derived from a knowledge of the nature of its binding to tubulin and microtubules. The interaction of paclitaxel and tubulin is believed to be crucial to the anticancer activity of the drug, and thus a knowledge of the details of this interaction will facilitate the design and development of improved analogs. The approach adopted will be to prepare paclitaxel analogs at Virginia Polytechnic Institute and State University, which are labeled with one of three different biological probes; fluorescent probes, spin-labeled probes, and stable isotope labeled probes will all be used. The fluorescent probes will be used at the State University of New York at Binghamton to elucidate the nature of the paclitaxel-tubulin interaction through fluorescence spectroscopy, leading to information about the dielectric nature of the fluorophore environment, the fluorophore's accessibility to solvent, and the size, shape, and associative behavior of the tubulin macromolecule. The spin-labeled probes will be used at the University of Toronto to define the conformation and orientation of the analogs at the tubulin binding site. The stable isotope labeled analogs will be analyzed in solid state NMR experiments at Washington University using sophisticated new phasing experiments such as double-REDOR NMR to obtain information on key internuclear distances of paclitaxel bound to microtubules. The information derived from all three approaches will be combined into one model for the conformation of paclitaxel in the binding site on polymerized tubulin, and this model will then be used as the basis of synthetic efforts designed to prepare analogs which are conformationally restricted so as to mimic the active site conformation. Such analogs are expected to show improved bioactivity as compared with paclitaxel itself.
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