The aims of the proposed research are to gain a detailed understanding on the molecular (and submolecular) level of the thermodynamic mechanism by which certain anti-mitotic drugs interact with tubulin, modulate the ability of this protein to self-assemble into microtubules and other structures, affect its conformational states and, in turn, are affected in their interactions by thermodynamic linkages with variations in tubulin conformational and liganded states. Emphasis will be on colchicine and a series of its newly- synthesized and to-be-synthesized analogs, which bind reversibly to tubulin and which have been designed to resolve the thermodynamic linkages into contributions from various structural features of the drug molecules. Specifically, the features with control """"""""stoichiometric"""""""" vs """"""""substoichiometric"""""""" microtubule inhibition, the generation of GTPase activity and the self-assembly into non-microtubule polymers with microtubule forming thermodynamics will be defined. The control of tubulin conformation (microtubule-forming or ring-forming) by the nature of the nucleotide occupying the E-site will be probed by the use of GTP, GDP, the nonhydrolyzable GTP analog GMPPCP, gamma-F-GTP which binds in the E-site but inhibits microtubule formation competitively, as well as metal fluorides (e.g. Alf4-) which bind strongly in phosphate binding site. The effect of these nucleotides on the standard free energies of microtubule growth (or growth of the tubulin-colchicine complex), ring growth and ring closure will be determined and the linkage free energies available from the binding of these ligands will be deduced. The thermodynamic linkage between colchicine and nucleotide binding will be probed in similar manner by the use of the same series of nucleotides and reversibly binding analogs of colchicine that induce or do not induce the tubulin-colchicine polymerization. The methods used will be those of macromolecular physical biochemistry. These will include sedimentation velocity, light scattering, spectrofluorimetry, quantitative column and batch gel chromatography, difference spectroscopy and densimetry. Drug analogs identified in the physico-chemical studies as having particularly favorable properties (e.g. strong binding, rapid reversibility) will be examined for their ability to reversibility perturb biological function. The rational understanding, based on rigorous thermodynamic foundations, of how certain anti-cancer drugs and their analogs perform their biochemical functions should lead to the identification of structures which will be effective as drugs, but do not have the high toxicity of colchicine.
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