Cancer Chemotherapy and Pharmacology

, Volume 53, Issue 5, pp 397–403 | Cite as

A quantitative evaluation of the effects of inhibitors of tubulin assembly on polymerization induced by discodermolide, epothilone B, and paclitaxel

  • Donnette A. Dabydeen
  • Gordon J. Florence
  • Ian Paterson
  • Ernest Hamel
Original Article



To determine whether inhibitors of microtubule assembly inhibit polymerization induced by discodermolide and epothilone B, as well as paclitaxel, and to quantitatively measure such effects.


Inhibition was quantitated by measuring polymer formation either by turbidimetry or by centrifugation, and the amount of inhibitor required to inhibit 50% relative to an appropriate control reaction was determined.


The inhibitory drugs evaluated were four colchicine site agents (combretastatin A-4, podophyllotoxin, nocodazole, and N-acetylcolchinol-O-methyl ether), maytansine, which competitively inhibits the binding of Catharanthus alkaloids to tubulin, halichondrin B and phomopsin A, which noncompetitively inhibit the binding of Catharanthus alkaloids to tubulin, and the depsipeptide dolastatin 15. While relative inhibitory effects were highly variable, a few broad generalizations can be made. First, assembly reactions that were either enhanced or dependent upon all three stimulatory drugs were subject to inhibition by all inhibitors. Second, the more readily the tubulin assembled, the greater the concentration of inhibitor required to inhibit polymerization. Drug IC50 values were generally lowest with no stimulatory drug and highest when discodermolide was present; IC50 values were higher as reaction temperature increased; and IC50 values were higher as the tubulin concentration increased. Third, inhibition of assembly by inhibitors of Catharanthus alkaloid binding to tubulin changed much less as a function of changes in reaction conditions than inhibition by inhibitors of colchicine binding.


Since there was no apparent quantitative predictability of combined drug interactions with tubulin, any combination of interest must be studied in detail.


Discodermolide Epothilone B Paclitaxel Tubulin assembly 



Microtubule-associated proteins




  1. 1.
    Bai R, Pettit GR, Hamel E (1990) Binding of dolastatin 10 to tubulin at a distinct site for peptide antimitotic agents near the exchangeable nucleotide and vinca alkaloid sites. J Biol Chem 265:17141PubMedGoogle Scholar
  2. 2.
    Bai R, Paull KD, Herald CL, Malspeis L, Pettit GR, Hamel E (1991) Halichondrin B and homohalichondrin B, marine natural products binding in the vinca domain of tubulin: discovery of tubulin-based mechanism of action by analysis of differential cytotoxicity data. J Biol Chem 266:15882PubMedGoogle Scholar
  3. 3.
    Bai R, Friedman SJ, Pettit GR, Hamel E (1992) Dolastatin 15, a potent antimitotic depsipeptide derived from Dolabella auricularia: interaction with tubulin and effects on cellular microtubules. Biochem Pharmacol 43:2637CrossRefPubMedGoogle Scholar
  4. 4.
    Bollag DM, McQueney PA, Zhu J, Hensens O, Koupal L, Liesch J, Goetz M, Lazarides E, Woods CM (1995) Epothilones, a new class of microtubule-stabilizing agents with a taxol-like mechanism of action. Cancer Res 55:2325PubMedGoogle Scholar
  5. 5.
    Cruz-Monserrate Z, Mullaney JT, Harran PG, Pettit GR, Hamel E (2003) Dolastatin 15 binds in the vinca domain of tubulin as demonstrated by Hummel-Dreyer chromatography. Eur J Biochem 270:3822CrossRefPubMedGoogle Scholar
  6. 6.
    Grover S, Hamel E (1994) The magnesium-GTP interaction in microtubule assembly. Eur J Biochem 222:163PubMedGoogle Scholar
  7. 7.
    Hamel E, Lin CM (1984) Separation of active tubulin and microtubule-associated proteins by ultracentrifugation and isolation of a component causing the formation of microtubule bundles. Biochemistry 23:4173PubMedGoogle Scholar
  8. 8.
    Hamel E, Sackett DL, Vourloumis D, Nicolaou KC (1999) The coral-derived natural products eleutherobin and sarcodictyins A and B: effects on the assembly of purified tubulin with and without microtubule-associated proteins and binding at the polymer taxoid site. Biochemistry 38:5490CrossRefPubMedGoogle Scholar
  9. 9.
    Hoebeke J, Van Nijen G, De Brabander M (1976) Interaction of oncodazole (R 17934), a new antitumoral drug, with rat brain tubulin. Biochem Biophys Res Commun 69:319PubMedGoogle Scholar
  10. 10.
    Iorio MA (1984) Contraction of the tropolonic ring of colchicine by hydrogen peroxide oxidation. Heterocycles 22:2207Google Scholar
  11. 11.
    Jordan MA (2002) Mechanism of action of antitumor drugs that interact with microtubules and tubulin. Curr Med Chem Anti-Canc Agents 2:1Google Scholar
  12. 12.
    Kang G-J, Getahun Z, Muzaffar A, Brossi A, Hamel E (1990) N-acetylcolchinol O-methyl ether and thiocolchicine, potent analogs of colchicine modified in the C ring: evaluation of the mechanistic basis for their enhanced biological properties. J Biol Chem 265:10255PubMedGoogle Scholar
  13. 13.
    Kowalski RJ, Giannakakou P, Gunasekera SP, Longley RE, Day BW, Hamel E (1997) 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. Mol Pharmacol 52:613PubMedGoogle Scholar
  14. 14.
    Kowalski RJ, Giannakakou P, Hamel E (1997) Activities of the microtubule-stabilizing agents epothilones A and B with purified tubulin and in cells resistant to paclitaxel (Taxol). J Biol Chem 272:2534CrossRefPubMedGoogle Scholar
  15. 15.
    Kumar N (1981) Taxol-induced polymerization of purified tubulin: mechanism of action. J Biol Chem 256:10435PubMedGoogle Scholar
  16. 16.
    Lin CM, Ho HH, Pettit GR, Hamel E (1989) Antimitotic natural products combretastatin A-4 and combretastatin A-2: studies on the mechanism of their inhibition of the binding of colchicine to tubulin. Biochemistry 28:6984PubMedGoogle Scholar
  17. 17.
    Long BH, Carboni JM, Wasserman AJ, Cornell LA, Casazza AM, Jensen PR, Lindel T, Fenical W, Fairchild CR (1998) Eleutherobin, a novel cytotoxic agent that induces tubulin polymerization, is similar to paclitaxel (Taxol). Cancer Res 58:1111PubMedGoogle Scholar
  18. 18.
    Mandelbaum-Shavit F, Wolpert-DeFilippes MK, Johns DG (1976) Binding of maytansine to rat brain tubulin. Biochem Biophys Res Commun 72:47PubMedGoogle Scholar
  19. 19.
    Manfredi JJ, Horwitz SB (1984) Taxol: an antimitotic agent with a new mechanism of action. Pharmacol Ther 25:83PubMedGoogle Scholar
  20. 20.
    Nogales E, Wolf SG, Downing KH (1998) Structure of the αβ tubulin dimer by electron crystallography. Nature 391:199PubMedGoogle Scholar
  21. 21.
    Paterson I, Florence GJ, Gerlach K, Scott JP, Sereinig N (2001) A practical synthesis of (+)-discodermolide and analogues: fragment union by complex aldol reactions. J Am Chem Soc 123:9535CrossRefPubMedGoogle Scholar
  22. 22.
    Schiff PB, Fant J, Horwitz SB (1979) Promotion of microtubule assembly in vitro by taxol. Nature 277:665PubMedGoogle Scholar
  23. 23.
    Takoudju M, Wright M, Chenu J, Guéritte-Voegelein F, Guénard D (1988) Absence of 7-acetyl taxol binding to unassembled brain tubulin. FEBS Lett 227:96CrossRefPubMedGoogle Scholar
  24. 24.
    Ter Haar E, Kowalski RJ, Hamel E, Lin CM, Longley RE, Gunasekera SP, Rosenkranz HS, Day BW (1996) Discodermolide, a cytotoxic marine agent that stabilizes microtubules more potently than taxol. Biochemistry 35:243Google Scholar
  25. 25.
    Tonsing EM, Steyn PS, Osborn M, Weber K (1984) Phomopsin A, the causative agent of lupinosis, interacts with microtubules in vivo and in vitro. Eur J Cell Biol 35:156Google Scholar
  26. 26.
    Wegner A, Engel J (1975) Kinetics of the cooperative association of actin to actin filaments. Biophys Chem 3:215CrossRefPubMedGoogle Scholar
  27. 27.
    Wilson L (1970) Properties of colchicine binding protein from chick embryo brain. Interactions with vinca alkaloids and podophyllotoxin. Biochemistry 9:4999PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Donnette A. Dabydeen
    • 1
  • Gordon J. Florence
    • 2
  • Ian Paterson
    • 2
  • Ernest Hamel
    • 1
    • 3
  1. 1.Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at FrederickNational Institutes of HealthFrederickUSA
  2. 2.Department of ChemistryUniversity of CambridgeCambridgeUK
  3. 3.Building 469, Room 104National Cancer Institute at FrederickFrederickUSA

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