The aims of the proposed research are to arrive at a detailed understanding of the mechanism on the molecular (and submolecular) level of how certain anti-mitotic drugs bind to tubulin and induce it to undergo polymerization reactions to particular structures in each case. The drugs to be studied are colchicine, vinblastine and taxol, as well as their analogs and derivatives, and smaller molecules which correspond to parts of the complex drug structures. Specifically, the linkages between drug binding and self-association or conformational changes of tubulin will be characterized by rigorous thermodynamic and kinetic approaches. The linkage between the interactions of different parts of the same drug molecule with tubulin will be examined by using small molecules, such as vindoline and catharantine in the case of vinblastine. The mutual effects of the binding of the individual drugs on each other will be followed by thermodynamic and conformational techniques to characterize the cooperativity (positive, as in colchicine-vinblastine, or negative as in colchincine-taxol) which exists in their bindings. The relative locations of the drug binding sites on the tubulin molecule and their shifts induced by the binding of other drugs will be determined by measuring distances between the drugs and a reference point on tubulin, such as the exchangeable site GTP, as well as between drug molecules and parts of these molecules. These distances will be measured by nmr, epr and fluorescence energy transfer methods. For this purpose, appropriate fluorinated, spin-labelled and fluorescent marker groups will be introduced into the structures of the drugs and their analogs. The methods used will be those of macromolecular physical biochemistry, including sedimentation, spectrofluorimetry, densimetry, quantitative gel chromatography, light scattering, electrophoresis, Fourier transform infra-red spectroscopy, circular dichroism, nmr and epr, as well as chemical modification and synthesis. This research is aimed at an understanding of the manner in which certain anti-cancer drugs perform their function with the hope of identifying structural features which will be useful in the design of new such drugs. (L)
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