Drugs that target dynamic microtubules: a new molecular perspective

Abstract

Microtubules have long been considered an ideal target for anticancer drugs because of the essential role they play in mitosis, forming the dynamic spindle apparatus. As such, there is a wide variety of compounds currently in clinical use and in development that act as antimitotic agents by altering microtubule dynamics. Although these diverse molecules are known to affect microtubule dynamics upon binding to one of the three established drug domains (taxane, vinca alkaloid, or colchicine site), the exact mechanism by which each drug works is still an area of intense speculation and research. In this study, we review the effects of microtubule-binding chemotherapeutic agents from a new perspective, considering how their mode of binding induces conformational changes and alters biological function relative to the molecular vectors of microtubule assembly or disassembly. These "biological vectors" can thus be used as a spatiotemporal context to describe molecular mechanisms by which microtubule-targeting drugs work.

Figure 1

A depicts a cervical cancer HeLa cell with individual microtubule filaments stained with FITC-labeled anti-α-tubulin antibody and nucleus stained red using the DNA binding dye, DAPI. B. Microtubules show a typical arrangement of 13 protofilaments (top view in C). Packaging of protofilaments adjacent to each other in a hollow cylinder forms the interacting surface of microtubule polymer. D shows an atomic-resolution model of a 13 protofilament microtubule built using coordinates of the refined electron crystallographic structure of αβ tubulin dimer at 3.5 Å, which has been fit within the 8-Å resolution electron microscopy data of Li and Downing.E shows the apical side view of this model, with the three-start seam seen at the bottom of the figure. One straight protofilament is shown in detail, with the nucleotides (GDP or GTP of each tubulin monomer) highlighted as space-filling molecular models. A straight axis of growth connecting the nucleotides of the protofilament is in green to show the vector of growth of the microtubule in the plus direction. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 2

Confocal immunofluorescence microscopy using HeLa cells showing two interphase cells and a mitotic cell (lower right). Microtubules are stained in green, the chromatin material (DNA) in blue and the arrow denotes the red “centrosome” stained using antibody against γ-tubulin.The centrosome is also referred to as the MTOC responsible for nucleating microtubular arrays. Scale bar = 10 μM. MTOC, microtubule organizing center. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 3

A shows the treadmilling of a microtubule, whereby tubulin dimers are added to the positive end of the microtubule while peeling off the negative end. B shows the end view of atomic-resolution microtubule model (built as described earlier). Proto-filaments peel off of the microtubule or thogonally to the microtubule surface, showing kinks of 12 and 18° at the intra and interdimer interface of tubulin, respectively, forming bending protofilament.C shows the same figure from a side apical perspective. A green axis is shown connecting the nucleotides of tubulin subunits in the direction of straight protofilament growth while the cyan vector shows the direction of the protofilament peel. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 4

Exquisite spatiotemporal regulation of microtubule dynamicity dictates cell division, a process by which a parent cell divides into two daughters. Panels show confocal immunomicrographs of HeLa cells with microtubules (green) and DNA (red) displaying hallmarks of a typical cell division process, in particular, interphase (A) showing distinct filamentous microtubular arrays; metaphase (B) showing a characteristic bipolar mitotic apparatus (featuring mitotic and astral microtubules) with all chromosomes (red) perfectly aligned at the metaphase plate; anaphase (C) visualizing the push-pull of spindle microtubules that govern accurate and precise portioning of the genomic material into two daughter cells; telophase (D) where the chromosomes have arrived at the poles of their respective spindles. Nuclear envelope reforms before the chromosomes decondense and the spindle fibers begin to disassemble; initiation of mid-body formation (E) to accomplish the cytokinetic process of splitting the daughter cells apart by formation of a cleavage furrow that pinches the two cells apart; cytokinetic abscission (F) showing the deposition of membrane between the daughter cells and sealing of the cytoplasmic bridge between them to complete their separation. The mid-body is usually inherited by one of the progeny cells. Finally, each daughter cell receives an identical complement of chromosomes. The microtubular arrays appear to be spreading again into interphase arrays and the chromosomes have completely decondensed. Scale bar = 10 μM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 5

A shows the structure of RB3-stathmin-like domain (orange helix) complexed with two tubulin subunits as solved by Dorleans et al. superimposed onto the microtubule structure for reference. Orange vector connects sugar ring of the nucleotides in the tubulin subunits within the complex. Green and cyan vectors showing alignment of nucleotides in a straight and peeling protofilament, respectively, are included for reference. B shows the same structure from the axial perspective of the growing end of the microtubule. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 6

Confocal micrographs showing dual color staining of microtubules (green) and DNA (red) for control untreated, paclitaxel-treated, and vinblastine-treated MCF-7 cells. Paclitaxel facilitates tubulin assembly resulting in highly resistant tubulin polymers with shorter and highly polymerized microtubules. Vinblastine binds free tubulin heterodimers, resulting in the formation of paracrystals, spirals, and tubules. Scale bar = 10 μM. [Color figure can be viewed inthe online issue, which is available at wileyonlinelibrary.com.]

Figure 7

Major taxane domain-binding drugs: Paclitaxel, Epothilone, and Discodermolide.

Figure 8

Lumenal view of paclitaxel (green spacefill) bound to a straight protofilament of a microtubule as solved by Lowe et al. Note that paclitaxel is bound completely in the β-subunit of the dimer. The green vector shows the direction of growth of a straight protofilament, to which the tubulin–paclitaxel vector is aligned. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 9

A shows the lumenal view of taxol bound to a straight protofilament of microtubule as solved by Lowe et al. Taxol supports the growth of microtubules by stabilizing the straight structure of protofilaments as illustrated by the green vector connecting the nucleotides of tubulin subunits along a straight axis. B shows the position of taxol from the perspective of the microtubule lumen. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 10

Major vinca domain-binding drugs: Vinblastine, Dolastatin10, and Cryptophycin.

Figure 11

Depolymerizing drug, vinblastine, is shown as space-filling model in blue, bound to the interdimer interface of the peeling protofilament as determined by Gigant et al. As in previous figures, the green axis shows direction of growth of a straight protofilament while the cyan axis connects nucleotides of a peeling protofilament. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 12

Depolymerizing drug-binding positions on a peeling protofilament. Purple cylinder shows position of several colchicine-domain binding drug binders (colchicine, ABT751, and T138067) as determined by Dorleans et al. at the intradimer interface of a peeling protofilament. Superimposed on this structure, Vinblastine is shown as space-filling model in blue, bound to the interdimer interface of the peeling protofilament as determined by Gigant et al. As in previous figures, green axis shows direction of growth of a straight protofilament while cyan axis connects nucleotides of a peeling protofilament. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 13

Structure of colchicine showing the characteristic three hexameric rings (A, B, C).

Figure 14

Depolymerizing drug, Colchicine is shown as a space-filling model (magenta, highlighted with arrow) at the intradimer interface at the start of the peeling protofilament as determined by Ravelli et al. As in previous figures, the green axis shows direction of growth of a straight protofilament while the cyan axis connects nucleotides of a peeling protofilament. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 15

Major colchicine-domain binding drugs: 2-Methoxyestradiol, Combretastatin A4, and Chalcone.

Figure 16

Molecular structure of noscapine.

Figure 17

Apical side view (A) and axial view (B) of biological vectors of three phases of tubulin polymerization shown in context of microtubule superstructure. All axes are made by connecting the nucleotides of sequential tubulin subunits to one another. Green represents straight protofilaments, which are integrated into microtubule structure and supported by the polymerizing drug taxol (shown as green space-filling molecule model).^, Cyan axis shows directionality of peeling protofilament, a microtubule depolymerizing action supported by the colchicine-domain drugs (binding domain shown as purple cylinder)^, and the vinca-domain drugs (represented by the space-filling model of vinblastine shown in blue). The orange vector showing the directionality of an RB3/stathmin-like protein and two tubulin complexes are shown superimposed on growing end of microtubule as a reference, as this structure has been used to determine the binding domain of several depolymerizing drugs.^,, [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]