Binding, Tubulin

The site of action is the tubulin in the cytoplasm. Tubulins represent a large superfamily, and several isotypes are described for both α and β tubulin in mammalian cells [Luduena, 2013]. At least six different isotypes of the α subunit are known, while eight isotypes are known for the β subunit. These subunits share a high degree of homology (90% similarity). In addition to α- and β-tubulin, other tubulin homologues have been identified (γ, δ and ε), but their roles in the life cycle of the cell are uncertain [Bhattacharya and Cabral, 2009]. All available isotypes are incorporated within microtubules, although with different tissue distributions in normal cells [Berrieman et al., 2004]. The currently known microtubule-disrupting agents bind to all isotypes, having only a slight preference for one over another [Miller et al., 2010].

Binding sites on the α/β-tubulin heterodimer: Conventionally, microtubule-interfering agents are categorized into two main groups: (1) microtubule destabilizers, including colchicine and a variety of vinca alkaloids; and (2) microtubule stabilizers, including taxanes and epothilones. Most agents interact with known binding pockets of α/β-tubulin; however, there are compounds that bind to tubulin on undefined sites. Three distinct sites are well characterized in the literature [Marchetti et al., 2016; Botta et al., 2009]: (1) the colchicine-binding domain at the interface between the α- and β-tubulin dimers; (2) the vinca domain surrounding the GTP binding site on β- and α-tubulin; and, (3) the taxane domain located on β-tubulin [Botta et al., 2009].

Colchicine binding domain on tubulin: The colchicine binding domain is a deep pocket located at the α/β interface of tubulin heterodimers. Crystal structures for tubulin and different ligands are available, although their resolution is not high [Lu et al., 2012; Massarotti et al., 2012]. Notwithstanding its deep location, significant conformational changes in the protein are necessary for accommodating the inhibitors. Both the A and C rings of colchicine are necessary for high affinity binding, while the B ring may only function as a linker between the other two. Three methoxy residues are present in the A ring and all of them are involved in the high affinity binding to tubulin. The C ring of colchicine interacts through van der Waals contacts with Valα181, Serα178, and Valβ315. The carbonyl group behaves as a hydrogen bond acceptor, interacting with Val181a. The A ring is buried in a hydrophobic pocket delimited by Lysβ352, Asnβ350, Leuβ378, Alaβ316, Leuβ255, Lysβ254, Alaβ250, and Leuβ242, and the methoxy group at position 3 is involved in a hydrogen bond interaction within the thiol group of Cysβ241 [Marchetti et al., 2016]. Different ligands may compete with colchicine for the same binding site, even in the absence of high structural correspondence [Lu et al., 2012].

There is no OECD guideline for measuring chemical binding to tubulin, however, binding of colchicine to tubulin is one of the most studied chemical interactions with biological materials and methodology for its measurement is well established and standardized [Hamel and Lin, 1981; Verdier-Pinard et al., 1998].

Binding properties to tubulin are generally evaluated in vitro, typically on tubulin extracts derived from brain tissues [Miller and Wilson 2010]. To determine whether a compound can bind to tubulin, a competitive [3H]colchicine tubulin-binding assay is conducted in vitro to measure whether the binding of colchicine is inhibited by the presence of the test agent [Verdier-Pinard et al. 1998]. A reaction mixture containing tubulin, [3H]colchicine and a potential inhibitor is incubated and after the addition of the scintillation fluid, the radioactivity of [3H]colchicine-bound tubulin is measured using a scintillation counter. The reduction of [3H]colchicine-bound tubulin value is inversely proportional to the test agent binding affinity [Hamel and Lin 1981]. A reaction mixture with only tubulin and [3H]colchicine is generally used as an experimental control standard. The inhibition constant (Ki) of colchicine is 5.75 μM [Zavala et al. 1980] and the ability of new chemicals to interfere with colchicine binding to tubulin is benchmarked against this value.

Chemical binding to tubulin has been measured in somatic and germ cells in a variety of species, from rodents in vivo to human cells in culture. Theoretically, chemical binding to tubulin can occur in any cell type in any organism.

Berrieman HK, Lind MJ, Cawkwell L. 2004. Do beta-tubulin mutations have a role in resistance to chemotherapy? Lancet Oncol 5:158-164.

Bhattacharya R, Cabral F. 2009. Molecular basis for class V beta-tubulin effects on microtubule assembly and paclitaxel resistance. J Biol Chem 284:13023-13032.

Botta M, Forli S, Magnani M, Manetti F. 2009. Molecular Modeling Approaches to Study the Binding Mode on Tubulin of Microtubule Destabilizing and Stabilizing Agents. In: Carlomagno T, editor. Tubulin-Binding Agents: Springer Berlin Heidelberg. p 279-328.

Clement MJ, Rathinasamy K, Adjadj E, Toma F, Curmi PA, Panda D. 2008. Benomyl and colchicine synergistically inhibit cell proliferation and mitosis: evidence of distinct binding sites for these agents in tubulin. Biochemistry 47:13016-13025.

Cutts JH, Beer CT, Noble RL. 1960. Biological properties of Vincaleukoblastine, an alkaloid in Vinca rosea Linn, with reference to its antitumor action. Cancer Res 20:1023-1031.

D'Amato RJ, Lin CM, Flynn E, Folkman J, Hamel E. 1994. 2-Methoxyestradiol, an endogenous mammalian metabolite, inhibits tubulin polymerization by interacting at the colchicine site. Proc Natl Acad Sci USA 91:3964-3968.

Desbene S, Giorgi-Renault S. 2002. Drugs that inhibit tubulin polymerization: the particular case of podophyllotoxin and analogues. Curr Med Chem Anticancer Agents 2:71-90.

Engelborghs Y. 1998. General features of the recognition by tubulin of colchicine and related compounds. Eur Biophys J 27:437-445.

Garland DL. 1978. Kinetics and mechanism of colchicine binding to tubulin: evidence for ligand-induced conformational change. Biochemistry 17:4266-4272.

Gigant B, Wang C, Ravelli RB, Roussi F, Steinmetz MO, Curmi PA, Sobel A, Knossow M. 2005. Structural basis for the regulation of tubulin by vinblastine. Nature 435:519-522.

Hamel E, Lin CM. 1981. Stabilization of the colchicine-binding activity of tubulin by organic acids. Biochim Biophys Acta 675:226-231.

Himes RH. 1991. Interactions of the catharanthus (Vinca) alkaloids with tubulin and microtubules. Pharmacol Ther 51:257-267.

Kingston DG. 2009. Tubulin-interactive natural products as anticancer agents. J Nat Prod 72:507-515.

Lambeir A, Engelborghs Y. 1981. A fluorescence stopped flow study of colchicine binding to tubulin. J Biol Chem 256:3279-3282.

Lu Y, Chen J, Xiao M, Li W, Miller DD. 2012. An overview of tubulin inhibitors that interact with the colchicine binding site. Pharm Res 29:2943-2971.

Luduena RF. 2013. A hypothesis on the origin and evolution of tubulin. Int Rev Cell Mol Biol 302:41-185.

Marchetti A, Massarotti A, Yauk CL, Pacchierotti F, Russo A. Submitted. The adverse outcome pathway (AOP) for chemical binding to tubulin in oocytes leading to aneuploid offspring. Environ Mol Mutagen.

Massarotti A, Coluccia A, Silvestri R, Sorba G, Brancale A. 2012. The tubulin colchicine domain: a molecular modeling perspective. ChemMedChem 7:33-42.

Miller HP, Wilson L. 2010. Chapter 1 - Preparation of Microtubule Protein and Purified Tubulin from Bovine Brain by Cycles of Assembly and Disassembly and Phosphocellulose Chromatography. In: Leslie W, John JC, editors. Methods Cell Biol: Academic Press. p 2-15.

Miller LM, Xiao H, Burd B, Horwitz SB, Angeletti RH, Verdier-Pinard P. 2010. Chapter 7 - Methods in Tubulin Proteomics. In: Leslie W, John JC, editors. Methods Cell Biol: Academic Press. p 105-126.

Rai SS, Wolff J. 1996. Localization of the vinblastine-binding site on beta-tubulin. J Biol Chem 271:14707-14711.

Ravelli RB, Gigant B, Curmi PA, Jourdain I, Lachkar S, Sobel A, Knossow M. 2004. Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain. Nature 428:198-202.

Stanton RA, Gernert KM, Nettles JH, Aneja R. 2011. Drugs that target dynamic microtubules: a new molecular perspective. Med Res Rev 31:443-481.

Toso RJ, Jordan MA, Farrell KW, Matsumoto B, Wilson L. 1993. Kinetic stabilization of microtubule dynamic instability in vitro by vinblastine. Biochemistry 32:1285-1293.

Warfield RK, Bouck GB. 1974. Microtubule-macrotubule transitions: intermediates after exposure to the mitotic inhibitor vinblastine. Science 186:1219-1221.

Weisenberg RC, Timasheff SN. 1970. Aggregation of microtubule subunit protein. Effects of divalent cations, colchicine and vinblastine. Biochemistry 9:4110-4116.

Wilson L, Jordan MA, Morse A, Margolis RL. 1982. Interaction of vinblastine with steady-state microtubules in vitro. J Mol Biol 159:125-149.

Xu K, Schwarz PM, Ludueña RF. 2002. Interaction of nocodazole with tubulin isotypes. Drug Dev Res 55:91-96.

Zavala F, Guenard D, Robin JP, Brown E. 1980. Structure--antitubulin activity relationship in steganacin congeners and analogues. Inhibition of tubulin polymerization in vitro by (+/-)-isodeoxypodophyllotoxin. J Med Chem 23:546-549.