Taxanes binding

Docetaxel, another taxane, binds to tubulin to promote microtubule assembly. The pharmacokinetics of docetaxel are best described by a three-compartment model, with an a half-life of 0.08 hours, a 3 half-life of 1.6 to 1.8 hours, and a terminal half-life of 65 to 73 hours.14 Docetaxel has activity in the treatment of breast, non-small cell lung, prostate, bladder, esophageal, stomach, ovary, and head and neck cancers. Dexamethasone, 8 mg twice daily for 3 days starting the day before treatment, is used to prevent the fluid retention syndrome associated with docetaxel and possible hypersensitivity reactions. The fluid... 

Although taxanes bind to p-tubulin promoting microtubule polymerization and stabilization of the spindle complex, they serve to cause a sustained mitotic block at the metaphase/anaphase boundary. This block will occur at a lower concentration than that which is required to increase the microtubule mass (10). However, it is not completely clear how this interaction with microtubules translates into cell death. Morphologic features and the characteristic DNA fragmentation patterns seen in the setting of apoptosis have been documented in tumor cells after therapy with taxanes (10). These observations are accompanied by the phosphorylation of Bcl-2, an anti-apoptotic protein, changing the cellular balance between Bax and Bcl-2 to a status that favors apoptosis (11). 

On the other hand, from a thermodynamical point of view, a common mechanism for assembly induction can be proposed. All microtubule stabilizing agents bind tightly to the assembled form, while they do not bind with a measurable affinity to the dimeric tubulin [33], indicating that the taxane binding pocket is either not formed or not-completely formed in non-microtubular tubulin. 

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 kinetics of taxane binding to microtubule were subsequently determined taxanes and epothilones [10, 22, 23, 44] bind to microtubules extremely fast (Fig. 8, Table 1). 

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. 

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 ... 

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

Trastuzumab, a monoclonal antibody that binds to HER2, produces response rates of 15% to 20% when used as a single agent and increases response rates, time to progression, and OS when combined with chemotherapy. It has been studied in doublet (taxane-trastuzumab vinorelbine-trastuzumab) and triplet (trastuzumab-taxane-platinum) combinations but the optimum regimen is unknown. 

Ng SS, Figg WD, Sparreboom A. Taxane-mediated antiangiogenesis in vitro influence of formulation vehicles and binding proteins. Cancer Res 2004 64 821. 

Although not a taxane, ixabepilone is a novel microtubule inhibitor that was recently approved for metastatic breast cancer in combination with the oral fluoropyrimidine capecitabine or as monotherapy. It is a semisynthetic analog of epothilone B, and is active in the M phase of the cell cycle. This agent binds directly to 6-tubulin subunits on microtubules, leading to inhibition of normal microtubule dynamics. Of note, this agent continues to have activity in drug-resistant tumors that overexpress P-glycoprotein or tubulin mutations. The main adverse effects include myelosuppression, hypersensitivity reactions, and neurotoxicity in the form of peripheral sensory neuropathy. 

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

The binding of taxanes has been well characterized [10, 22] and shows a series of consecutive reactions involving a first fast bimolecular step (k+1 and k, ), a second slow monomolecular step (k+2, k 2) and a third step which is the structural change involving the change in the number of microtubule protofilaments. It can be proved numerically that the first bimolecular fast step of binding is diffusion controlled, thus indicating that taxanes can not directly bind to the lumenal site [22],... 

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. 

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