T-Taxol

Walle UK, Walle T. Taxol transport by human intestinal epithelial Caco-2 cells. Drug Metab Dispos 1998 26(4) 343-346. 

Computational studies have been of paramount importance in order to integrate the results of the experimental studies indicated above with the electron crystallographic data known. According to Snyder and co-workers, a satisfactory and experimentally verifiable model of the tubulin-binding site and of the conformation of paclitaxel has been obtained by computational methods on the first electron crystallographic model. In this context, a new paclitaxel conformer, T-Taxol (Fig. 10c), was proposed [59, 81, 82],... 

Further refinement of the electron crystallographic structure of tubulin-paclitaxel at 3.5A resolution delivered a similar result. Nevertheless, the T-Taxol model has not been completely accepted as the actual bioactive conformation [78], It is evident that the low 3.5-3.7A resolution of the complex limits the precision of the resulting model. In addition, the Zn2+-stabilized tubulin preparation employed in the electron crystallographic study involves antiparallel protofilaments organized in sheets, which strongly differ from genuine microtubules. Consequently, concern has been expressed that the sheets may not be representative of cellular microtubules and that sheet-bound paclitaxel geometry may differ from its bioactive form in microtubules. 

Fig. 8 Conformations of PTX in solution (C blue, O red, N green). Left nonpolar conformation. Middle T-taxol conformation. Right polar conformation. The drawings are representative structures of each class and do not correspond exactly to any physically determined structure. Coordinates were generated by manual manipulation with Kryomol (http //galileo.usc.es/ armando/ software) of the PTX coordinates in the PDB entry 1JFF...

Two independent studies based on the refinement of the EC structure of tubulin/taxol concluded that T-taxol is the conformation that best fits the EC density [25, 83], despite the fact that the two intemuclear distances of T-taxol (9.1 A and 9.9 A, respectively) are in slight disagreement with those derived by REDOR NMR. 

Another conformation different from T-taxol was proposed based on molecular dynamics simulations and computational docking restrained by the two REDOR distances [85], The main difference between this conformer (named REDOR-taxol or PTX-NY) and T-taxol is the conformation of the C13 side chain, that places the 2 -OH in a different orientation. However, subsequent studies demonstrated that PTX-NY is not consistent with the EC density [86, 87],... 

If all these facts are taken into consideration, the T-taxol conformation can be regarded as consistent with the two 13C-19F REDOR distances. Other conformations apart from T-taxol that also conform with the REDOR distances, such as the polar PTX or PTX-NY, can be rejected on the basis of their poorer fitting to the EC density (Table 1). 

Polar model3 Nonpolar model15 PTX-NY model04 1JFF" T-taxol model44 REDOR distance... 

More recently, additional 2H "I J REDOR measurements on new PTX derivatives bound to MT provided three new interatomic distances that helped to define more precisely the bound conformation of PTX [87], The two PTX analogues labeled with fluorine and deuterium were designed to determine the three key distances based on the measurement of 2II— "I distances, as indicated in Fig. 9 (compounds 4 and 5). These interatomic distances rule out the polar and nonpolar conformers and further support the T-taxol conformation (Tablel). The polar conformation is rejected on the basis of distance IV, as the 4.5 A is much shorter than the > 8 A determined by REDOR. The nonpolar conformation can be ruled out on the basis of distances I and II, that are too short in the nonpolar conformer compared to the experimental ones (1.8 and 3.6 A short). Besides, bridged taxanes resembling the nonpolar conformation displayed no activity in the tubulin assembly assay [88], PTX-NY closely matches the REDOR distances. Nevertheless, it is discarded as the bioactive form of PTX due to its poor fit to the EC density [87],... 

In summary, the T-taxol conformation is the only one that is compatible with the EC density and with the intemuclear distances measured by REDOR ss-NMR. The conformation of T-taxol has been used as a template to design novel bridged taxanes. The fact that some of them possess improved bioactivity [89, 90] provides indirect additional evidence that T-taxol represents indeed the bioactive conformation. 

Fig. 8 T -Taxol within the MAID-optimized complex, yellow. Crystallographically refined tubulin model ljff in white. Primary optimization occurred with the internal geometry of the C-3 benzamido side chain...

Scheme 2 Newman projections for PTX conformations in terms of the improper torsion angles 0-C2-C3 -N(Bz) and 0-C2-C3 -C(Ph) (< )1, < )2). The front atom is C-2, the unseen back atom, C-3. a The nonpolar conformation as derived by NMR in CDC13. b The polar conformation as observed in the solid state, c T-Taxol conformation bound to 3 tubulin from EC...

Fig. 9 The hydrophobic binding pocket for T -Taxol on beta tubulin. Color on the surface model ranges from most lipophilic (brown) to most hydrophilic (blue). The ribbon view of beta tubulin is colored by secondary structure with red - alpha helices and blue - beta sheets...

Fig. 14.1 T-Taxol(PTX, blue) in P-tubulin in which the PTX-NY orange) conformer is superimposed on matching atoms in the baccatin core (reproduced from [9], with the permission of the American Chemical Society)...

I. Ojima who suggested the PTX-NY conformer, in which the C-13 side chain is proposed to adopt a different conformation and an alternative hydrogen-bonding pattern in the tubulin binding site [8]. The two conformers were compared to show that only T-Taxol fits the PTX-derived electron crystallographic density [9]. 

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