Raccor, Brianne S.
(2009)
Mechanism and Kinetics of Microtubule Perturbing Agents.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Microtubules are cellular cytoskeletal components that play an integral part in many cell functions. Compounds that bind to microtubules and alter their dynamics are highly sought as a result of the clinical success of paclitaxel and docetaxel. A series of analogues of the microtubule stabilizing dictyostatin were examined to probe biological and biochemical structure-activity relationships. The results were consistent with previous reports showing that 16-normethyldictyostatin and 15Z,16-normethyldictyostatin lose potency in pacitaxel-resistant cell lines that have a Phe270-to-Val mutation in the taxoid binding site of â-tubulin. 6-epi-Dictyostatin and 7-epi-dictyostatin were potent analogues of dictyostatin, and 6-epi-dictyostatin was chosen for milligram scale for pre-clinical studies. The thalidomide analogue 5HPP-33 was identified as an easily synthesized small microtubule perturbing agent, and experiments with isolated tubulin were performed to determine its mechanism of action. Tubulin polymerization was used to determine the effect of 5HPP-33 on normal microtubule formation. In experiments utilizing microtubule associated proteins (MAPs) to induce polymer formation, 5HPP-33 inhibited tubulin polymerization, but under a different set of conditions appeared to form and stabilize microtubules. The polymer was imaged using electron microscopy, which showed that 5HPP-33 caused the formation of spirals and rings. Due to 5HPP-33 failing to compete with known radiolabeled microtubule perturbing agents for their respective binding sites, a tritiated version of 5HPP-33 was synthesized. The binding experiments performed showed that [3H]5HPP-33 had a slight affinity for isolated MAPs, and this was the reason for the discrepancy between the tubulin polymerization experiments. A binding site for 5HPP-33 could not be determined, making it a possible novel microtubule perturbing agent. (-)-Pironetin is a microtubule inhibitor that appears to form a covalent linkage to the tubulin heterodimer. Although immunoflourescent images showed (-)-pironetin to work in the same manner as vinblastine, cellular and biochemical experiments proved that (-)-pironetin is mechanistically different from vinblastine. The tubulysins are known microtubule destabilizers and bind to the vinca domain on â-tubulin. Three analogues of the tubulysins were synthesized and their effects on cell growth and microtubule perturbation experiments were determined. WZY-111-63C (N14-desacetoxytubulysin H) was found to be 50 times more cytotoxic than paclitaxel and vincristine.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
29 January 2009 |
| Date Type: |
Completion |
| Defense Date: |
17 September 2008 |
| Approval Date: |
29 January 2009 |
| Submission Date: |
9 October 2008 |
| Access Restriction: |
5 year -- Restrict access to University of Pittsburgh for a period of 5 years. |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Chemistry |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
chemotherapeutics; kinetics; microtubules |
| Other ID: |
http://etd.library.pitt.edu/ETD/available/etd-10092008-160103/, etd-10092008-160103 |
| Date Deposited: |
10 Nov 2011 20:02 |
| Last Modified: |
15 Nov 2016 13:50 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/9449 |
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