API

Event: 1580

Key Event Title

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Binding of microtubule stabilizing agents (MSA) to microtubules

Short name

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Binding of MSAs to microtubules

Biological Context

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Level of Biological Organization
Molecular

Cell term

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Organ term

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Key Event Components

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Process Object Action

Key Event Overview


AOPs Including This Key Event

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AOP Name Role of event in AOP
Microtubule interacting drugs lead to peripheral neuropathy MolecularInitiatingEvent

Stressors

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Taxonomic Applicability

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Life Stages

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Sex Applicability

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Key Event Description

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MSAs bind to polymerized tubulin. [1, 2] In several studies, MSAs were shown to inhibit cell proliferation and possess antineoplastic activity. [3-9] The taxane pocket found on the β-subunit of tubulin dimers was identified as the binding site for taxol and other MSAs like the above mentioned epothilones. [6-8, 10-20] Binding of MSAs is reversible. [6-8, 13-15]


How It Is Measured or Detected

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- Electron microscopy of tannin-embedded tubulin crystals stabilized with taxol. Projection density maps were created from the electron microscopy data and further processed to difference maps of tubulin to visualize the binding site of taxol in polymerized tubulin [12, 16]

- Direct photolabeling of tubulin with radiolabelled [ 3 H]taxol [13], and [ 3 H]taxoid-derivatives to identify binding sites of taxol in tubulin [10, 11, 14, 15, 18]

- Mapping studies: Tubulin is photolabelled with [ 3 H]taxoid-derivatives. To identify the photoincorporation site, the labelled complex is then digested by formic acid or CNBr in combination with either clostripain or trypsin. The obtained peptide fragments are analysed via reverse phase HPLC and the radioactive peak is sequenced. [14, 15, 18]

- X-ray crystallography [17]

- NMR studies [20] and solid-state rotational echo double-resonance (REDOR) NMR [19] to determine the orientation of microtubule-bound taxol

- Fluorescence energy transfer (FRET) spectroscopy to determine the conformation of microtubule-bound taxol [19]


Domain of Applicability

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Evidence for Perturbation by Stressor


Overview for Molecular Initiating Event

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References

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1. Manfredi, J.J., J. Parness, and S.B. Horwitz, Taxol binds to cellular microtubules. The Journal of Cell Biology, 1982. 94(3): p. 688-696. Confidential

2. Parness, J. and S. Horwitz, Taxol binds to polymerized tubulin in vitro. The Journal of Cell Biology, 1981. 91(2): p. 479-487.

3. Riondel, J., et al., Therapeutic response to taxol of six human tumors xenografted into nude mice. Cancer Chemotherapy and Pharmacology, 1986. 17(2): p. 137-142.

4. Wani, M.C., et al., Plant antitumor agents. VI. Isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. Journal of the American Chemical Society, 1971. 93(9): p. 2325-2327.

5. Schiff, P.B., J. Fant, and S.B. Horwitz, Promotion of microtubule assembly in vitro by taxol. Nature, 1979. 277(5698): p. 665-667.

6. Bollag, D.M., et al., Epothilones, a New Class of Microtubule-stabilizing Agents with a Taxol-like Mechanism of Action. Cancer Research, 1995. 55(11): p. 2325-2333.

7. Hung, D.T., J. Chen, and S.L. Schreiber, (+)-Discodermolide binds to microtubules in stoichiometric ratio to tubulin dimers, blocks taxol binding and results in mitotic arrest. Chemistry & Biology, 1996. 3(4): p. 287-293.

8. Kowalski, R.J., et al., The Microtubule-Stabilizing Agent Discodermolide Competitively Inhibits the Binding of Paclitaxel (Taxol) to Tubulin Polymers, Enhances Tubulin Nucleation Reactions More Potently than Paclitaxel, and Inhibits the Growth of Paclitaxel-Resistant Cells. Molecular Pharmacology, 1997. 52(4): p. 613-622.

9. ter Haar, E., et al., Discodermolide, A Cytotoxic Marine Agent That Stabilizes Microtubules More Potently Than Taxol. Biochemistry, 1996. 35(1): p. 243-250.

10. Combeau, C., et al., Predominant Labeling of. beta.-over. alpha.-Tubulin from Porcine Brain by a Photoactivatable Taxoid Derivative. Biochemistry, 1994. 33(21): p. 6676-6683. Confidential

11. Dasgupta, D., et al., Synthesis of a photoaffinity taxol analog and its use in labeling tubulin. Journal of medicinal chemistry, 1994. 37(18): p. 2976-2980.

12. Nogales, E., S.G. Wolf, and K.H. Downing, Structure of the αβ tubulin dimer by electron crystallography. Nature, 1998. 391: p. 199.

13. Rao, S., S.B. Horwitz, and I. Ringel, Direct Photoaffinity Labeling of Tubulin With Taxol. JNCI: Journal of the National Cancer Institute, 1992. 84(10): p. 785-788.

14. Rao, S., et al., 3'-(p-azidobenzamido)taxol photolabels the N-terminal 31 amino acids of beta-tubulin. Journal of Biological Chemistry, 1994. 269(5): p. 3132-3134.

15. Rao, S., et al., Characterization of the Taxol Binding Site on the Microtubule 2-(m-AZIDOBENZOYL) TAXOL PHOTOLABELS A PEPTIDE (AMINO ACIDS 217-231) of β-TUBULIN. Journal of Biological Chemistry, 1995. 270(35): p. 20235-20238.

16. Nogales, E., et al., Structure of tubulin at 6.5 Å and location of the taxol-binding site. Nature, 1995. 375(6530): p. 424-427.

17. Prota, A.E., et al., Molecular mechanism of action of microtubule-stabilizing anticancer agents. Science, 2013. 339(6119): p. 587-590.

18. Rao, S., et al., Characterization of the Taxol Binding Site on the Microtubule: IDENTIFICATION OF Arg282 IN β-TUBULIN AS THE SITE OF PHOTOINCORPORATION OF A 7-BENZOPHENONE ANALOGUE OF TAXOL. Journal of Biological Chemistry, 1999. 274(53): p. 37990-37994.

19. Li, Y., et al., Conformation of Microtubule-Bound Paclitaxel Determined by Fluorescence Spectroscopy and REDOR NMR. Biochemistry, 2000. 39(2): p. 281-291.

20. Snyder, J.P., et al., The binding conformation of Taxol in β-tubulin: A model based on electron crystallographic density. Proceedings of the National Academy of Sciences, 2001. 98(9): p. 5312-5316.