Ligand-tubulin model

Fig. 6 Competition between 3H-paclitaxel and 14C-docetaxel for binding to microtubules. 11.3 pM tubulin was assembled at 37°C in PEDTA, ImM GDP, ImM GTP, 8mM MgCl2, pH6.7 by the addition of paclitaxel and docetaxel at a total concentration of 20 pM, at different molar ratios of paclitaxel to docetaxel. The total concentration of microtubules was 11.0 0.10 pM the concentration of tubulin in supernatants (not polymerized tubulin) varied between ca. 0.4 (in paclitaxel excess) and 0.2 pM (in docetaxel excess). Open circles, 3H-paclitaxel bound per polymerized tubulin dimer solid circles, 14C-docetaxel bound squares, total ligand (paclitaxel plus docetaxel) bound per polymerized tubulin dimer. The solid lines are the best fit to the data, employing a simple competition model of the two ligands for the same site, taken from [35]...

The study of the tubulin-bound conformation of paclitaxel has resulted in a number of protein-ligand models, partially or fully based on the electron diffraction structure of aP-tubulin in paclitaxel-stabilized Zn2+-induced sheets [5, 12], Obviously, the nature of the paclitaxel binding site and the paclitaxel conformation in the binding site have key implications for the design of new MSA. A deep knowledge of the bioactive conformation would also help to explain how compounds as structurally diverse as the epothilones [48], discodermolide [49], and eleutherobin [50] have very similar mechanisms of action. 

It can be expected that the near future will provide tubulin-ligand structures with sufficient accuracy to define precisely the conformation and binding mode for these compounds and thereby validate or reject the current set of models. For the time being, the methodologies schematically reviewed here may provide additional data for understanding the action of MSAs and hopefully to design new potent analogues. 

Fig. 7 The location on tubulin of residues that modulate the sensitivity to MT-destabilizing agents and the location of exogenous inhibitor and nucleotide sites on P tubulin. The a subunit is in semitransparent pink together with a composite P-subunit color-coded as in Fig. 3a with ball-and-stick models of bound taxol (orange), colchicine (yellow) and GDP (magenta). Ball-and-stick models of vinblastine (cyan) are drawn on the two partial vinca sites on a and on P tubulin. The sulfur atom of Cys P12 is highlighted as a yellow sphere. The sites of nine amino acid substitutions [49] that both confer resistance to vinblastine and colchicine and stabilize MTs are depicted as red (on a tubulin) or green (on P tubulin) spheres. Two residues of the P H10 helix whose mutations enhance the sensitivity to colchicine site ligands and destabilize MTs [71] are also shown as blue spheres...

Colchicine site ligands are structurally simpler than taxol or vinblastine site compounds the extent of the colchicine site is also smaller. This is confirmed with the observation that podophyllotoxin interacts with the same P tubulin residues as colchicine [15]. Modehng studies also suggest a pocket of restricted size for other colchicine site ligands [40] while some extensions of the site have been postulated in an approach combining modeling and virtual screening [61]. Biochemical evaluations have confirmed some of the hits as new tubulin inhibitors but no structural data are available yet that confirm whether they actually make use of an additional cavity near the colchicine site. 

The identification of the binding site location within tubulin, together with the clarification of the binding mode for some representative destabilizing compounds, opened new perspectives to modeling studies in this field, making the application possible not only of ligand-based, but also structure-based approaches. 

All ligand-based molecular modeling studies focusing on agents able to bind the colchicine site were performed before the determination of the X-ray structure of the tubulin-colchicine complex (see below). Computational tools in this field were mainly used to derive three-dimensional QS ARs (through comparative molecular field analysis (CoMFA) and other original approaches), but CSI also constituted useful... 

The binding models of the 15 ligands were used to derive a 7-point pharmacophore connecting the different structural classes of CSI, based on consistent structural features and recurring tubulin-ligand interactions. As shown in Fig. 3a, the common... 

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