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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA016707-17
Application #
3164468
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1975-02-01
Project End
1994-06-30
Budget Start
1991-07-01
Budget End
1992-06-30
Support Year
17
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Brandeis University
Department
Type
Schools of Arts and Sciences
DUNS #
616845814
City
Waltham
State
MA
Country
United States
Zip Code
02454
Perez-Ramirez, B; Gorbunoff, M J; Timasheff, S N (1998) Linkages in tubulin-colchicine functions: the role of the ring C (C') oxygens and ring B in the controls. Biochemistry 37:1646-61
Andreu, J M; Perez-Ramirez, B; Gorbunoff, M J et al. (1998) Role of the colchicine ring A and its methoxy groups in the binding to tubulin and microtubule inhibition. Biochemistry 37:8356-68
Xie, G; Timasheff, S N (1997) Mechanism of the stabilization of ribonuclease A by sorbitol: preferential hydration is greater for the denatured then for the native protein. Protein Sci 6:211-21
Xie, G; Timasheff, S N (1997) Temperature dependence of the preferential interactions of ribonuclease A in aqueous co-solvent systems: thermodynamic analysis. Protein Sci 6:222-32
Lin, T Y; Timasheff, S N (1996) On the role of surface tension in the stabilization of globular proteins. Protein Sci 5:372-81
Perez-Ramirez, B; Andreu, J M; Gorbunoff, M J et al. (1996) Stoichiometric and substoichiometric inhibition of tubulin self-assembly by colchicine analogues. Biochemistry 35:3277-85
Dumortier, C; Gorbunoff, M J; Andreu, J M et al. (1996) Alterations of rings B and C of colchicine are cumulative in overall binding to tubulin but modify each kinetic step. Biochemistry 35:15900-6
Rossi, M; Gorbunoff, M J; Caruso, F et al. (1996) Structural analysis of the substoichiometric and stoichiometric microtuble-inhibiting biphenyl analogues of colchicine. Biochemistry 35:3286-9
Dumortier, C; Gorbunoff, M J; Andreu, J M et al. (1996) Different kinetic pathways of the binding of two biphenyl analogues of colchicine to tubulin. Biochemistry 35:4387-95
Timasheff, S N (1995) Solvent stabilization of protein structure. Methods Mol Biol 40:253-69

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