API

Event: 718

Key Event Title

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Binding, Tubulin

Short name

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Binding, Tubulin

Key Event Component

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Process Object Action
tubulin binding tubulin increased

Key Event Overview


AOPs Including This Key Event

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AOP Name Role of event in AOP
Chemical binding to tubulin in oocytes leading to aneuploid offspring MolecularInitiatingEvent

Stressors

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Level of Biological Organization

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Biological Organization
Molecular

Cell term

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Cell term
eukaryotic cell


Organ term

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Taxonomic Applicability

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Term Scientific Term Evidence Link
mouse Mus musculus Strong NCBI
Homo sapiens Homo sapiens Moderate NCBI

Life Stages

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Sex Applicability

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Term Evidence
Female Strong

How This Key Event Works

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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., submitted; 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., submitted]. Different ligands may compete with colchicine for the same binding site, even in the absence of high structural correspondence [Lu et al. 2012].


How It Is Measured or Detected

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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.


Consider the following criteria when describing each method: 1. Is the assay fit for purpose? Yes 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? Yes 3. Is the assay repeatable? Yes 4. Is the assay reproducible? Yes


Evidence Supporting Taxonomic Applicability

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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.


Evidence for Perturbation by Stressor


Overview for Molecular Initiating Event

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Colchicine is a prototypical spindle poison that has been extensively used to investigate binding to tubulin. The kinetics of colchicine binding are well established [Lambeir and Engelborghs 1981; Engelborghs 1998] and can be measured experimentally with high precision [Hamel and Lin 1981]. Colchicine binds non-polymerized α/β dimeric tubulin by a two-step process. The first step is rapid but weak, resulting in the formation of an initial pre-equilibrium complex, which involves a low affinity binding of colchicine that is reversible. This is followed by slow conformational changes in tubulin, which finally lead to the formation of an irreversible final state tubulin–colchicine complex that has high activation energy [Garland 1978]. The conformational change in tubulin heterodimers, followed by the addition of the complex at the ends of microtubules, is responsible for the suppressed polymerization at microtubule ends leading to their depolymerization [Ravelli et al. 2004]. The binding kinetics have been studied at different temperatures. The standard enthalpy change of the first step (ΔH°1 = -33±8 kJ · mol–1) and the activation energy of the second step (ΔH°2 = 100±5 kJ · mol–1) were determined based on the temperature dependence [Lambeir and Engelborghs 1981]. Using eight different analogues to study the binding mechanisms of colchicine, it was demonstrated that the C-ring of colchicine is responsible for the first step of the binding mechanism, while the second step involves the rearrangement of the initial complex to interact with the A-ring [Engelborghs 1998].


Other chemicals that bind to tubulin: Other chemicals are also known to bind to tubulin [Marchetti et al., submitted]. These chemicals can be grouped in two general classes: colchicine domain binders and vinca domain binders.

Known colchicine domain binders:

1. Podophyllotoxin ((5R,5aR,8aR,9R)-5-hydroxy-9-(3,4,5-trimethoxyphenyl)-5a,6,8a,9-tetrahydro-5H-[2]benzofuro[5,6-f][1,3]benzodioxol-8-one, POD) has a trimethoxybenzoic chemical structure similar to colchicine. It inhibits colchicine binding to the colchicine-binding domain of tubulin. However, although colchicine and podophyllotoxin bind in the same pocket on β-tubulin, their binding sites are not completely overlapping [Desbene and Giorgi-Renault 2002].

2. 2-methoxyestradiol ((8R,9S,13S,14S,17S)-2-methoxy-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthrene-3,17-diol, 2ME) binds to the colchicine domain of tubulin [D'Amato et al. 1994].

3. Nocodazole (methyl (5-[2-thienylcarbonyl]-1H-benzimidazol-2-yl, NOC) belongs to the group of benzimidazole derivatives that were patented, as a class, for the treatment of cancer in conjunction with other pharmaceuticals, and has been shown to bind in the colchicine domain [Xu et al. 2002].

4. Benomyl (methyl N-[1-(butylcarbamoyl)benzimidazol-2-yl]carbamate, BEN), another benzimidazole derivative, is the active compound in several agricultural fungicides. The benomyl-binding site is located in the core of β-tubulin at a site distinct from the colchicine domain [Clement et al. 2008].

5. Carbendazim (methyl N-(1H-benzimidazol-2-yl)carbamate, MBC) is a fungicide commonly used in agriculture for the control of a wide range of fungal diseases. Carbendazim is the methylbenzimidazolcarbamate product of the spontaneous hydrolyzation process incurred by benomyl in aqueous solution (it is the major metabolite). Therefore, it is at least partially responsible for the benomyl effects observed in vivo. The affinity of carbendazim for mammalian tubulin is less than that of benomyl, most probably because it lacks the position 1 side chain of benomyl. Like benomyl, carbendazim does not compete with colchicine for binding to tubulin [INSERT REFERENCE].

6. Thiabendazole (4-(1H-benzimidazol-2-yl)-1,3-thiazole, TBZ) is a benzimidazole-derived anthelmintic and an agricultural fungicide, structurally related to NOC, benomyl and MBC.

INSERT WHERE THIABENDAZOLE BINDS HERE.

Vinca domain binders:

The vinca alkaloids, a class of antimitotic compounds derived from the periwinkle plant, Catharanthus roseus [Cutts et al. 1960], bind near the GTP-binding site on the β-subunit of tubulin at a site distinct from the colchicine-binding one [Rai and Wolff 1996]. Vinblastine and vincristine are first-generation vinca alkaloids [Kingston 2009]. At low concentrations, vincas bind to the plus ends of microtubules producing a conformational change of dimers from a straight ‘‘growing’’ vector to a curved ‘‘peeling’’ vector [Toso et al. 1993]. At higher concentrations, the vinca alkaloids have affinity for free tubulin heterodimers, again potentially forming an altered, curved geometry of the dimeric biological vector [Warfield and Bouck 1974]. Although vincas do not share structural similarity with colchicine and bind to a different site on tubulin, they similarly act by destabilizing microtubules [Stanton et al. 2011].

Vinca domain binders include:

1. Vinblastine (dimethyl (2β,3β,4β,5α,12β,19α)-15-[(5S,9S)-5-ethyl-5-hydroxy-9-(methoxycarbonyl)-1,4,5,6,7,8,9,10-octahydro-2H-3,7-methanoazacycloundecino[5,4b]indol-9-yl]-3-hydroxy-16-methoxy-1-methyl-6,7-didehydroaspidospermidine-3,4-dicarboxylate, VBL) is an anticancer drug that is used extensively. The crystal structure of vinblastine bound to tubulin has been determined [Gigant et al. 2005]. In contrast with the binding site for colchicine, which is mostly embedded in β-tubulin subunit, the vinblastine binding site is shared equally between α/β-heterodimer [Marcehtti et al., submitted]. In the β-subunit, vinblastine interacts through van der Waals contacts with residues Serβ174-Aspβ179, Asnβ206-Aspβ211, Pheβ214 and Tyrβ224; while in the α-subunit, Pheα351, Lysα352, Valα353 and Ileα355 delimit the pocket occupied by VBL. Amino acids Proβ222 and Asnα329 are also involved in hydrogen bond interactions with VBL [Marchetti et al., submitted]. Following a mechanism similar to colchicine, VBL binds to tubulin in two consecutive steps: formation of a rapid equilibrium complex followed by a slower rearrangement linked to changes in the structure of the heterodimer. A major effect of VBL is the formation of spiral-like tubulin aggregates [Weisenberg and Timasheff 1970; Himes 1991]. VBL binds to microtubule ends [Wilson et al. 1982] and at low concentrations suppresses the dynamic instability of plus ends [Toso et al. 1993]. When used at much higher concentrations, VBL depolymerizes microtubules, giving rise in particular to protofilament spirals and curls.


VINCRISTINE?



References

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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.