Taxane-binding site

The modification of the microtubule structure should come from the perturbation of the interprotofilament contacts, which allows the accommodation of extra protofilaments in the microtubule lattice. The experimental fact that taxane binding modifies the interprotofilament contacts rapidly lead to the conclusion that the taxane binding site in microtubules was located in the interprotofilament space [18, 19]. 

The first structural location of the taxane binding site [42] placed it in the interprotofilament space, thus supporting the biochemical results. However, this changed when the first high resolution 3D structure of the paclitaxel-tubulin complex was solved by electron-crystallography of a two-dimensional zinc-induced tubulin polymer [5]. The fitting of this structure into a three-dimensional reconstruction of microtubules from cryoelectron microscopy allowed a pseudo atomic resolution model of microtubules [43] in which the paclitaxel binding site was placed inside the lumen of the microtubules hidden from the outer solvent. 

The route of taxanes was finally unveiled with the use of a covalent ligand of the taxane binding site [21], Cyclostreptin, abacterial natural product [46,47] with weak, but irreversible tubulin assembly activity and strong apparent binding affinity for the paclit-axel site, covalently labels both a residue placed in the lumenal site of paclitaxel Asn228 and a residue previously proposed to be in the external binding site Thr220 (Fig. 3a). 

Several natural compounds of diverse structure, shown in Fig. (12), affecting the taxane-binding site on tubulin are currently developed in clinical trials, namely (+)-Discodermolide, Epothilone B, BMS-247550 (Azaepothilone B, NSC-710428) and Eleutherobin. 

Taxane-binding sites of -tubulin (view from the interior of the microtubule). 

Since the lumenal site of microtubules was well supported by the structural data and also by the fact that mutations in the lumenal site confer resistance to taxanes [45], an alternative mechanism with binding to an initial exposed binding site located in pore type I of the microtubule wall and later transportation of the ligand to the lumenal site was proposed in [22] (Fig. 9). 

Specificity of binding is supported by the observation of protein-mediated interligand NOEs [75, 76, 112] between pairs of ligands known to compete for the PTX-binding site (epothilones, taxanes, discodermolide, and baccatins) but not between ligands known to bind to different sites in MT, such as EpoA/vinblastine [76],... 

The framework for this discussion will be formed around three important classes of MT stabilizers shown in Scheme 1. Given the chemical similarity between epothilones and laulimalide, it is particularly surprising that, while taxanes and epothilones compete for the same binding site, laulimalide has been shown to be non-competitive with either. In fact, laulimalide may bind simultaneously with taxanes and produce a synergistic effect. This chapter provides details of experiments and analyses we have done to formulate and test hypotheses about these binding mechanisms. 

The combination of structural simplicity of epothilones with respect to paclitaxel, together with their very interesting activity profile appealed different research teams, interested to overcome limitations of taxanes (poor solubility, multi drug-resistance - MDR [61-63]). Investigations on epothilones were focused on the interaction between the ligands and the paclitaxel binding site (as well as the possible similar portions between epothilones and taxanes) in order to find common pharmacopores to be used in activity improvement and design of novel molecules. 

Other binding sites have been postulated according to tubulin interfering agents with a binding behaviour distinct to that of taxanes, vinca alkaloids and colchinoids. 

Anticancer taxanes initially were isolated from the bark of the Pacific yew Taxus brevifolia) but are now produced semisynthetically from an inactive natural precursor found in the leaves of the European yew (Taxus baccata) a renewable resource. Taxanes bind to polymerized (elongated) (3-tubulin at a specific receptor site located within the tubular lumen. At standard therapeutic doses (which should lead to intracellular concentrations of 1-20 pM), taxane-tubulin binding renders the microtubules resistant to depolymerization and prone to polymerization (69). This promotes the elongation phase of microtubule dynamic instability at the expense of the shortening phase, and it inhibits the disassembly of the tubule into the mitotic spindle. In turn, this interrupts the normal process of cell division. At these concentrations, extensive polymerization causes the formation of large and ... 

Maccari L, Manetti F, Corelli F, et al. 3D QSAR studies for the (3-tubulin binding site of microtubule-stabilizing anticancer agents (MSAAs). A pseudoreceptor model for taxanes based on the experimental structure of tubulin. II Farmaco 2003 58 659-668. 

Soulere, L. (2009) Toward docking-based virtual screening for discovering antitubulin agents by targeting taxane and colchicine binding sites. ChemMedChem, 4, 161-163. 

L. Soulere, Toward Docking-Based Virtual Screening for Discovering Anti-tubulin Agents by Targeting Taxane and Colchicine Binding Sites , Che-MedChem, 2009, 4, 161. 

Table 1 Kinetic rates of taxane site ligands binding to microtubules (37°C)...

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