Abstract
The knowledge of the thermodynamics and kinetics of drug-microtubule interaction is essential to understand the structure/affinity relationship of a given ligand family. When a ligand does not show an appropriate signal change (absorbance or fluorescence) upon binding, the extensive direct characterization of its binding affinities and kinetic rate constants of association and dissociation becomes a complex task. In those cases it is possible to obtain these parameters by competition of the ligand with a reference one of the same binding site that shows such change. Nevertheless, although the experimental setup of the competition measurements is easier, the treatment of the data is complex because simultaneous equilibrium/kinetic equations have to be solved. In this chapter, the taxoid-binding site of the microtubules will be used as an example to describe experimental competition and data analysis methods to determine the binding constants and kinetic rates of association and dissociation of ligands for microtubules.
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References
Bollag, D. M., McQueney, P. A., Zhu, J., et al. (1995) Epothilones, a new class of microtubule-stabilizing agents with a taxol-like mechanism of action. Cancer Res. 55, 2325–2333.
Hamel, E., Sackett, D. L., Vourloumis, D., and Nicolaou, K. C. (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, 5490–5498.
Kowalski, R. J., Giannakakou, P., Gunasekera, S. P., Longley, R. E., Day, B. W., and 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, 613–622.
Verdier-Pinard, P., Wang, Z., Mohanakrishnan, A. K., Cushman, M., and Hamel, E. (1999) A steroid derivative with paclitaxel-like effects on tubulin polymerization. Mol. Pharmacol. 57, 568–575.
Edler, M. C., Buey, R. M., Marcus, A. I., et al. (2005) Cyclostreptin (FR182877), a cytotoxic tubulin-polymerizing agent deficient in hypernucleating tubulin assembly. Biochemistry 44, 11,525–11,538.
Paterson, I., Britton, R., Delgado, O., Meyer, A., and Poullennec, K. G. (2004) Total synthesis and configurational assignment of (-)-dictyostatin, a microtubule-stabilizing macrolide of marine sponge origin. Angew. Chem. Int. Ed. Engl. 43, 4629–4633.
Buey, R. M., DÍaz, J. F., Andreu, J. M., et al. (2004) Interaction of epothilone analogs with the paclitaxel binding site: relationship between binding affinity, microtubule stabilization, and cytotoxicity. Chem. Biol. 11, 225–236.
Díaz, J. F., Menéndez, M., and Andreu, J. M. (1993) Thermodynamic of ligand-induced assembly of tubulin. Biochemistry 32, 10,067–10,077.
Evangelio, J. A., Abal, M., Barasoain, I., et al. (1998) Fluorescent taxoids as probes of the microtubule cytoskeleton. Cell Motil. Cytoskeleton 39, 73–90.
Oosawa, F. and Asakura, S. (1975) Thermodynamics of the Polymerization of Protein. Academic Press, London.
Díaz, J. F., Strobe, R., Engelborghs, Y., Souto, A. A., and Andreu, J. M. (2000) Molecular recognition of taxol by microtubules. Kinetics and thermodynamics of binding of fluorescent taxol derivatives to an exposed site. J. Biol. Chem. 275, 26,265–26,276.
Andreu, J. M. and Barasoain, I. (2001) The interaction of baccatin III with the taxol binding site of microtubules determined by a homogeneous assay with fluorescent taxoid. Biochemistry 40, 11,975–11,984.
Pryor, D. E., O’Brate, A., Bilcer, G., et al. (2002) The microtubule stabilizing agent laulimalide does not bind in the taxoid site, kills cells resistant to paclitaxel and epothilones, and may not require its epoxide moiety for activity. Biochemistry 41, 9109–9115.
Nicolaou, K. C., Ritzén, A., Namoto, K., et al. (2002) Chemical synthesis and biological evaluation of novel epothilone B and trans-12,13-cyclopropyl epothilone B analogues. Tetrahedron 58, 6413–6432.
Díaz, J. F., Barasoain, I., and Andreu, J. M. (2003) Fast kinetics of taxol binding to microtubules. Effects of solution variables and microtubule-associated proteins. J. Biol. Chem. 278, 8407–8419.
Gaitanos, T. N., Buey, R. M., Díaz, J. F., et al. (2004) Peloruside A does not bind to the taxoid site on beta-tubulin and retains its activity in multidrug-resistant cell lines. Cancer Res. 64, 5063–5067.
Rao, S., Krauss, N. E., Heerding, J. M., et al. (1994) 3’-(p-azidobenzamido)taxol photolabels the N-terminal 31 amino acids of beta-tubulin J. Biol. Chem. 269, 3132–3134.
Rao, S., Orr, G. A., Chaudhary, A. G., Kingston, D. G., and Horwitz, S. B. (1995) Characterization of the taxol binding site on the microtubule. 2-(m-Azidobenzoyl)taxol photolabels a peptide (amino acids 217–231) of beta-tubulin. J. Biol. Chem. 270, 20,235–20,238.
Rao, S., He, L., Chatkravarty, S., Ojima, I., Orr, G. A., and Horwitz, S. B. (1999) Characterization of the Taxol binding site on the microtubule. Identification of Arg(282) in beta-tubulin as the site of photoincorporation of a 7-benzophenone analogue of Taxol. J. Biol. Chem. 274, 37,990–37,994.
Löwe, J., Li, H., Downing, K. H., and Nogales, E. (2001) Refined structure of alpha beta-tubulin at 3.5 Å resolution. J. Mol. Biol. 313, 1045–1057.
Nogales, E., Wolf, S. G., and Downing, K. (1998) Structure of the alpha-beta tubulin dimer by electron crystallography Nature 391, 199–203.
Melki, R. and Carlier, M. F. (1993) Thermodynamics of tubulin polymerization into zinc sheets: assembly is not regulated by GTP hydrolysis Biochemistry 32, 3405–3413.
Sosa, H., Dias, D. P., Hoenger, A., et al. (1997) A model for the microtubule-Ncd motor protein complex obtained by cryo-electron microscopy and image analysis Cell 90, 217–224.
Meurer-Grob, P., Kasparian, J. and Wade, R. H. (2001) Microtubule structure at improved resolution Biochemistry 40, 8000–8008.
Nogales, E., Whittaker, M., Milligan, R. A., and Downing, K. H. (1999) High-resolution model of the microtubule Cell 96, 79–88.
Dye, R. B., Fink, S. P., and Williams, R. C. (1993) Taxol-induced flexibility of microtubules and its reversal by MAP-2 and Tau. J. Biol. Chem. 268, 6847–6850.
Díaz, J. F., Valpuesta, J. M., Chac-n, P., Diakun, G., and Andreu, J. M. (1998) Changes in microtubule protofilament number induced by Taxol binding to an easily accessible site. Internal microtubule dynamics. J. Biol. Chem. 273, 33,803–33,810.
Díaz, J. F., Barasoain, I., Souto, A. A., Amat-Gerri, F., and Andreu, J. M. (2005) Macromolecular accessibility of fluorescent taxoids bound at a paclitaxel binding site in the microtubule surface. J. Biol. Chem. 280, 3928–3937.
Weisenberg, R. C., Borisy, G. G., and Taylor, E. W. (1968) The colchicine-binding protein of mammalian brain and its relation to microtubules. Biochemistry 7, 4466–4479.
Lee, J. C., Frigon, R. P., and Timasheff, S. N. (1973) The chemical characterization of calf brain microtubule protein subunits. J. Biol. Chem. 248, 7253–7262.
Andreu, J. M., Gorbunoff, M. J., Lee, J. C., and Timasheff, S. N. (1984) Interaction of tubulin with bifunctional colchicine analogues: an equilibrium study. Biochemistry 23, 1742–1752.
Zimmerle, C. T. and Frieden, C. (1989) Analysis of progress curves by simulations generated by numerical integration. Biochem. J. 258, 381–387.
Lackowicz, J. R. (1999) Principles of Fluorescence Spectroscopy. Kluwer Academic/Plenum Publishers, New York, pp. 291–318.
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DÍaz, J.F., Buey, R.M. (2007). Characterizing Ligand-Microtubule Binding by Competition Methods. In: Zhou, J. (eds) Microtubule Protocols. Methods in Molecular Medicine™, vol 137. Humana Press. https://doi.org/10.1007/978-1-59745-442-1_17
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DOI: https://doi.org/10.1007/978-1-59745-442-1_17
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