Taxol, a complex diterpene from Taxus brevifolia (Pacific Yew), has shown excellent potential as an anti-tumor chemotherapeutic agent, especially against ovarian cancer. It acts by binding to microtubules imparting hyperstability to them. Although some studies on the interaction of taxol and some of its derivatives with tubulin and microtubules have been done, nothing is known about its binding site or domain in tubulin. Such information is useful for the further development of active taxol derivatives. The overall aim of this proposal is to synthesize photoaffinity analogues of taxol and to use them to identify peptides in the taxol binding site. The work is divided into three parts. We plan to synthesize phenylazido, nitrophenylazido, and trifluoroethyl benzoyl diazirine derivatives of taxol. These functionalities will be placed at different positions of the baccatin III and phenylisoserine side chain moieties. Not all proposed compounds will be synthesized if we find useful ones early in the work. The taxol derivatives will be compared to taxol in regard to their activities in the microtubule assembly reaction in vitro and on B16 melanoma cells in culture. The binding of the active compounds to microtubules and tubulin will also be measured. The analogues with good activities and binding will be used to label tubulin. This will require the synthesis of radiolabeled analogues. Various digestion and separation procedures together with peptide sequencing and mass spectrometry will be used to identify peptides containing covalently bound analogue.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA055141-01A2
Application #
3199598
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Project Start
1993-01-01
Project End
1995-12-31
Budget Start
1993-01-01
Budget End
1993-12-31
Support Year
1
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Kansas Lawrence
Department
Type
Schools of Arts and Sciences
DUNS #
072933393
City
Lawrence
State
KS
Country
United States
Zip Code
66045
Bode, Claudia J; Gupta, Mohan L; Suprenant, Kathy A et al. (2003) The two alpha-tubulin isotypes in budding yeast have opposing effects on microtubule dynamics in vitro. EMBO Rep 4:94-9
Bode, Claudia J; Gupta Jr, Mohan L; Reiff, Emily A et al. (2002) Epothilone and paclitaxel: unexpected differences in promoting the assembly and stabilization of yeast microtubules. Biochemistry 41:3870-4
Gupta Jr, Mohan L; Bode, Claudia J; Thrower, Douglas A et al. (2002) beta-Tubulin C354 mutations that severely decrease microtubule dynamics do not prevent nuclear migration in yeast. Mol Biol Cell 13:2919-32
Gupta Jr, M L; Bode, C J; Dougherty, C A et al. (2001) Mutagenesis of beta-tubulin cysteine residues in Saccharomyces cerevisiae: mutation of cysteine 354 results in cold-stable microtubules. Cell Motil Cytoskeleton 49:67-77
Panda, D; Chakrabarti, G; Hudson, J et al. (2000) Suppression of microtubule dynamic instability and treadmilling by deuterium oxide. Biochemistry 39:5075-81
Chakrabarti, G; Mejillano, M R; Park, Y H et al. (2000) Nucleoside triphosphate specificity of tubulin. Biochemistry 39:10269-74
Chakrabarti, G; Kim, S; Gupta Jr, M L et al. (1999) Stabilization of tubulin by deuterium oxide. Biochemistry 38:3067-72
Sengupta, S; Boge, T C; Liu, Y et al. (1997) Probing the environment of tubulin-bound paclitaxel using fluorescent paclitaxel analogues. Biochemistry 36:5179-84
Gupta, M L; Toso, R J; Farrell, K W et al. (1995) Commercial [3H]glutamate contains a contaminant that labels tubulin covalently. Anal Biochem 230:350-3
Sengupta, S; Boge, T C; Georg, G I et al. (1995) Interaction of a fluorescent paclitaxel analogue with tubulin. Biochemistry 34:11889-94

Showing the most recent 10 out of 11 publications