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)

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA016707-15
Application #
3164467
Study Section
Biophysics and Biophysical Chemistry A Study Section (BBCA)
Project Start
1975-02-01
Project End
1990-06-30
Budget Start
1989-07-01
Budget End
1990-06-30
Support Year
15
Fiscal Year
1989
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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