Báo cáo khoa học: The bI/bIII-tubulin isoforms and their complexes with antimitotic agents pptx

The bI/bIII-tubulin isoforms and their complexes with antimitotic agents Docking and molecular dynamics studies Matteo Magnani 1 , Francesco Ortuso 2 , Simonetta Soro 3 , Stefano Alcaro 2 , Anna Tramontano 3 and Maurizio Botta 1 1 Dipartimento Farmaco Chimico Tecnologico, Universita ` degli Studi di Siena, Italy 2 Dipartimento di Scienze Farmacobiologiche ‘Complesso Nin’ Barbieri’ Universita ` degli Studi di Catanzaro ‘Magna Graecia’, Roccelletta di Borgia (CZ), Italy 3 Dipartimento di Scienze Biochimiche ‘A. Rossi Fanelli’, Universita ` degli Studi ‘La Sapienza’, Rome, Italy Microtubules are filamentous dynamic polymers com- posed of a ⁄ b-tubulin heterodimers involved in a diverse range of cellular functions including motility, morphogenesis, intracellular trafficking of macromole- cules and organelles, and mitosis and meiosis [1,2]. The role played by microtubules in the cell division process makes them attractive targets for anticancer therapy [3], a perspective that has been explored by using tubulin-binding agents [4]. These compounds are able to disrupt microtubule dynamics and can act either as microtubule destabilizers (such as vinca alka- loids) or as microtubule stabilizers (such as taxanes). Among the latter, paclitaxel (Fig. 1 left) has been dem- onstrated to be effective for the treatment of ovarian, breast, and nonsmall cell lung carcinomas [5]. These molecules have the drawback of being scarcely select- ive, but an even more significant problem that limits their usage in the treatment of malignancies is the emergence of resistance. There are essentially two routes to resistance [4]: (i) expression of the P-glyco- protein [6], which is able to pump the antitumoral compounds out of the tumor cell; and (ii) emergence of structural modification of the microtubules them- selves, both via mutations and modifications of their isotype composition, in particular that of their b-sub- unit [7–9]. In humans, seven isoforms of b-tubulin, displaying different patterns of tissue expression, have been identified [10,11]. In particular, bIis Keywords docking; epothilone A; IDN5390; paclitaxel; tubulin Correspondence M. Botta, Dipartimento Farmaco Chimico Tecnologico, Universita ` degli Studi di Siena, Via Alcide de Gasperi, 2, I-53100 Siena, Italy Fax: +39 577 234333 Tel: +39 577 234306 E-mail: botta@unisi.it (Received 7 April 2006, revised 16 May 2006, accepted 23 May 2006) doi:10.1111/j.1742-4658.2006.05340.x Both microtubule destabilizer and stabilizer agents are important molecules in anticancer therapy. In particular, paclitaxel has been demonstrated to be effective for the treatment of ovarian, breast, and nonsmall cell lung carci- nomas. It has been shown that emergence of resistance against this agent correlates with an increase in the relative abundance of tubulin isoform bIII and that the more recently discovered IDN5390 can be effectively used once resistance has emerged. In this paper, we analyze the binding modes of these antimitotic agents to type I and III isoforms of b-tubulin by com- putational methods. Our results are able to provide a molecular explan- ation of the experimental data. Using the same protocol, we could also show that no preference for any of the two isoforms can be detected for epothilone A, a potentially very interesting drug for which no data about the emergence of resistance is currently available. Our analysis provides structural insights about the recognition mode and the stabilization mech- anism of these antimitotic agents and provides useful suggestions for the design of more potent and selective antimitotic agents. Abbreviation PDB, Protein Data Bank. FEBS Journal 273 (2006) 3301–3310 ª 2006 The Authors Journal compilation ª 2006 FEBS 3301 constitutively expressed and represents, in general, the most abundant isotype, whereas bIII expression is restricted to neuronal tissues and testis. There are sev- eral studies reporting that an increase in the relative abundance of isoform III destabilizes the microtubules [12,13] and clear indications that it correlates with paclitaxel resistance, both in vitro and in vivo [14–18]. Recent studies have shown that a seco-taxan (IDN5390, Fig. 1 middle) [19], although less potent than paclitaxel, is active on tumor cells overexpressing isoform III and therefore could be used in cases where resistance to paclitaxel has emerged [20]. In this study, we use a com- bination of molecular modeling, docking and molecular dynamics techniques to investigate the molecular basis of paclitaxel resistance and IDN5390 sensitivity, through the analysis of the complexes between these two ligands and the isotypes I and III of the human b-tubu- lin. We also investigated the complexes involving the bI and bIII isoforms with epothilone A (Fig. 1 right). Epo- thilones are microtubule stabilizing agents, sharing a common mechanism of action with taxanes [21]. To the best of our knowledge, no data is available about the activity of this class of compounds on different isoforms of tubulin, even though these molecules are gaining more and more attention in antitumoral therapy [22]. Results Analysis of tubulin crystallographic models and docking with the three ligands The structures of ligands used in this study are repor- ted in Fig. 1; with respect to paclitaxel and IDN5390, epothilone A is characterized by a less complex molecular structure. There is some confusion in the lit- erature and in databases about the nomenclature of the various tubulin isoforms. The bI and bIII genes have been recently re-sequenced [17], and we noticed that the protein annotated as tubulin bII chain (Code: TBB2_HUMAN, P07437) in SwissProt corresponds to the sequence of the tubulin bI chain. We also checked that the confusion did not reflect population polymor- phisms: no single nucleotide polymorphism is reported in the human genome in positions that are different between bI and bIII. The problem has now been brought to the attention of the database curators. There are several structural determinations of the tubulin dimer from different sources, some of which have been obtained by binding tubulin to a zinc sheet in order to obtain a bidimensional crystal, some others by fitting the zinc sheet structures in electron microscopy data, some by X-ray diffraction of crystals containing a tubulin dimer in complex with small ligands and ⁄ or other proteins, and some by modeling. No structure determination is available for the human proteins and therefore we needed to build comparative models for the human proteins. In particular, we concentrated on isoforms I and III of the human tubulin b subunit, as this subunit hosts the common binding site for our molecules of interest [23,24]. The sequence identity between tubulin from different sources and their human counterpart is very high (between 89 and 94%), nevertheless it is very difficult to assess which of the available structural determina- tions better reflects the conformation of the protein in physiological conditions and is therefore better suitable to be used as template in the model-building proce- dure. In order to select the appropriate template, we analyzed all tubulin-related entries in the Protein Data Bank (PDB) and performed an all-against-all compar- ison of the complete structures and of the most rele- vant parts of the structures (dimer interface and binding site). The differences in the overall structure are rather high in terms of rmsd (up to 4 A ˚ and more, see supplementary material, Table S1). More limited is the structural variation of the set of residues involved in the interaction with paclitaxel and epothilone A, which are relevant for our purposes (see supplementary material, Table S2). The variability seems to be mostly correlated with the nature of the ligand and, conse- Fig. 1. Chemical structures of paclitaxel (1), IDN5390 (2) and epothilone A (3). Docking of antimitotic agents to tubulin isoforms M. Magnani et al. 3302 FEBS Journal 273 (2006) 3301–3310 ª 2006 The Authors Journal compilation ª 2006 FEBS quently, the best choice seemed to be to select as tem- plates the structures bound to the ligands that we planned to study and we used 1JFF as template to build our comparative model, named hTUB. We used a standard comparative modeling procedure, replacing the residues of the template with those of the target according to the sequence alignment obtained using clustalw [25] with standard parameters. The replaced sidechains were positioned in their most commonly observed conformation [26] and the model was opti- mized using 100 cycles of steepest descent energy mini- mization using discover [27] and the CVFF force field. For the initial positioning of the paclitaxel moi- ety in the complex, we used the orientation found in the 1JFF structure, which contains a dimer of tubulin complexed with paclitaxel [23], to position the ligand in hTUB. In the case of IDN5390, the crystallographic structure of its complex with b-tubulin is not available. Consequently, the ligand was docked into the same binding site of paclitaxel (and epothilone A), assuming that these two very similar molecules act in a similar fashion. As a result of docking (for details, see Experi- mental procedures), IDN5390 was located within the binding site in a conformation which closely resembles that of paclitaxel, with the macrocyclic moiety and the lateral chains occupying the same regions of the pocket (Fig. 2A,B). For simulations involving epothilone, we took advantage of the availability of the 1TVK struc- ture, which contains the structure of a tubulin dimer complexed with epothilone A [24]. 1TVK was superim- posed to hTUB and the ligand positioned in the con- text of the model structure in the same relative orientation as observed in 1TVK (Fig. 2C). Thus, a common tubulin structure (hTUB) was used for all three ligands under analysis (Fig. 3), in an attempt to limit the biases that could derive from using different starting protein structures. The procedures described above were followed to obtain the starting structures of the six complexes of paclitaxel, IDN5390 and epo- thilone A with both bI and bIII isotypes of tubulin. Molecular dynamics and thermodynamics calculations The starting complexes, built as described above, were analyzed by means of molecular dynamics. Such analy- sis involved, at first, paclitaxel and IDN5390, for which data about the activity towards microtubules with different composition in terms of b-tubulin iso- types are reported, and was subsequently extended to epothilone A, for which to date no data is available. Human tubulin bI and bIII isoforms complexed with paclitaxel (referred to as P1 and P3, respectively), and A B C Fig. 2. Location of ligands within the hTUB binding site (solvent- accessible surface representation) in the starting complexes (A) paclitaxel, (B) IDN5390, and (C) epothilone A. Nonpolar hydrogen atoms of the ligands are not shown. M. Magnani et al. Docking of antimitotic agents to tubulin isoforms FEBS Journal 273 (2006) 3301–3310 ª 2006 The Authors Journal compilation ª 2006 FEBS 3303 with IDN5390 (referred to as I1 and I3, respectively), were first energy minimized and then subjected to backbone-constrained molecular dynamics. In the course of each simulation, 100 ligand–protein com- plexes were sampled at regular time intervals and were fully minimized. A conformational clustering analysis was applied to the resulting structures. Representative structures for the whole set of collected and optimized frames were selected by performing a Boltzmann ana- lysis, in order to take into account both the energy and the number of structures within each cluster. The reduced number of selected structures was used to investigate the molecular basis of the difference in the calculated binding energies of both ligands for the bI to bIII-tubulin isoform. Finally, in all simulations, the average drug–protein binding energies (DG-, DH- and DS-values) were computed according to the MOLINE methodology reported by some of us [28]. The results are reported in Table 1. The data predict a higher affinity of paclitaxel for the bI isoform than for the bIII isoform, and an opposite behavior of IDN5390. This is in good agreement with experimental data, as we are able to correctly reproduce the differences in sensitivity to paclitaxel and IDN5390 observed for microtubules with different isotype composition. Figure 4 shows the region around the ligand for both the P1 and P3 complexes, in one of the representative sampled structures (for other representative structures, quite similar considerations can be made). The binding of paclitaxel to bI-tubulin (Fig. 4A) involves both hydrogen bonds and multiple hydrophobic contacts; most interactions are in agreement with the crystallo- graphic structure of the complex (taken as starting structure) and have already been described [22,29,30] (supplementary material, Table S3). In particular, the C2 phenyl ring is involved in hydrophobic interactions with Leu217, His229 and Leu230, while the C4 acetate makes hydrophobic contacts with Phe272, Pro274 and Leu371. Two hydrogen bonds are established between the oxygen of the oxetane ring and the NH backbone of Thr276, and between the C2¢ hydroxyl group and the NH backbone of Gly370. However, as expected, Fig. 3. Ribbon representation of the main secondary structure ele- ments characterizing the b-tubulin binding site for the ligands under analysis: paclitaxel (green), IDN5390 (magenta), epothilone A (orange). Table 1. Free energy, enthalpy and entropy for the drug-protein complexes computed at 300 K. P1 and P3 refer to paclitaxel bound to human b-tubulin isoforms I and III, respectively. I1 and I3 to IDN5390 bound to human b-tubulin isoforms I and III. Complex DG (kcalÆmol )1 ) DH (kcalÆmol )1 ) DS (calÆmol )1 ) P1 )64.47 )64.16 1.01 P3 )54.67 )54.64 0.11 I1 )55.89 )55.49 1.32 I3 )67.52 )67.12 1.33 A B Fig. 4. Paclitaxel in complex with (A) bI-tubulin (P1), and (B) bIII- tubulin (P3). For sake of clarity, nonpolar hydrogen atoms are omit- ted and hydrogen bond interactions are represented by black dashed lines. Docking of antimitotic agents to tubulin isoforms M. Magnani et al. 3304 FEBS Journal 273 (2006) 3301–3310 ª 2006 The Authors Journal compilation ª 2006 FEBS the molecular dynamics simulation is also accompan- ied by structural variations of the initial complex. Especially the flexible M-loop experiences a slight rear- rangement that results in additional interactions with the ligand. As shown in Fig. 4A, in the P1 complex the C7 hydroxyl group of paclitaxel forms two hydro- gen bonds with the main chain carbonyl group and amino group of Ser277 and Gln282, respectively. A hydrogen bond interaction is also established between the C10 acetate and the lateral chain of Arg284. Such hydrogen bonds are stable during the course of the whole simulation, being present in >95% of the sampled structures. Finally, in P1, paclitaxel is also engaged in hydrogen bond interactions with Glu27. Differently from the interactions described above, the latter hydrogen bonds are less stable: in fact, due to the high mobility of the lateral chain of Glu27 they are observed in most of the sampled structures only after minimization. In the complex with bIII-tubulin (P3, Fig. 4B), the ligand is characterized by a very sim- ilar binding conformation compared with P1, while the structure of the M-loop is somewhat different in the two complexes, owing to the replacement of Ser277 in isoform bI with Ala277 in isoform bIII. This results in a different and less effective interaction with the C7– C10 moiety of the ligand. In comparison with P1, Arg278 is directed towards the inside of the binding pocket, establishing two hydrogen bonds with carbonyl at C9, only one of which is consistently observed in the course of the molecular dynamics simulations. Interestingly, in P1 Ser277 forms a hydrogen bond with Ser280, thus directing its carbonyl group towards the C7 position of the paclitaxel ring and forming a hydrogen bond with its OH. The substitution Ser277- Ala, present in the bIII isoform, does not allow this interaction to take place. Similarly, the different rear- rangement of the M-loop in bIII prevents the C10 acetate from interacting with Arg284. In P1, Glu27 is hydrogen bonded to the ligand, while in P3 this inter- action is absent. In this isoform, Glu27 interacts with Arg320, which, in turn, forms a hydrogen bond with Ser241. Such an interaction network cannot take place in P1, where Ser241 is replaced by a cysteine, which does not interact with Arg320. The models of the complexes of IDN5390 with the bI and bIII-tubulin isoforms are shown in Fig. 5. The bound conformation of the ligand is quite similar in the I1 and I3 complexes, in part resembling that of paclitaxel. In fact, similarly to paclitaxel, hydrophobic interactions are established between the C2 phenyl ring of IDN5390 and Leu217, His229 and Leu230, as well as between the C4 acetate and Phe272, Pro274 and Leu371. IDN5390 too is engaged in two hydrogen bonds involving the oxetane ring and the C2¢ hydroxyl group with Thr276 and Gly370, respectively. Further- more, the hydroxyl group in the C2¢ position of paclit- axel forms a second hydrogen bond with Glu27 of bI isotype, while the equivalent atom of IDN5390 is involved in a second hydrogen bond with the sidechain of Asp26 in both isoforms. However, a pattern of interactions different from those observed for paclit- axel is established with the M-loop, whose structural rearrangement is, also in this case, different in the bI and bIII isotypes. In both complexes, the C1 and the C9 hydroxyl groups interact with His229 and Gln282, respectively, through hydrogen bond interactions. Nev- ertheless, the different rearrangement of the M-loop in the two isoforms (due to the replacement of Ser277 in I1 with Ala277 in I3) results in some important differ- ences in the binding of IDN5390. Remarkably, only in the bIII isoform does the conformation of the M-loop allow the lateral chain of Arg278 to move towards the ligand and to favorably interact with it through hydro- gen bonds with the C1 hydroxyl group and the C2 A B Fig. 5. IDN5390 in complex with (A) bI-tubulin (I1), and (B) bIII- tubulin (I3). For sake of clarity, nonpolar hydrogen atoms are omit- ted and hydrogen bond interactions are represented by black dashed lines. M. Magnani et al. Docking of antimitotic agents to tubulin isoforms FEBS Journal 273 (2006) 3301–3310 ª 2006 The Authors Journal compilation ª 2006 FEBS 3305 benzoyl chain (Fig. 5B). Moreover, such location of the sidechain of Arg278 also results in a better ‘entrap- ment’ of the ligand within the binding site compared to isoform bI, as shown in Fig. 6. As mentioned above, in the P3 complex, Arg278 also points towards the inside of the pocket, but it does so to a much lower extent than in the I3 complex. In fact, the differ- ent structure of the macrocycle in paclitaxel with respect to IDN5390 induces a different rearrangement of the M-loop, which does not allow Arg278 to inter- act with the ligand as closely as in the IDN5390 bound structure and therefore the paclitaxel in bIII isotype is less well packed within the protein structure. Taken together, our analysis of the P and I com- plexes reveals that for both paclitaxel or IDN5390 the different calculated binding energies can be mainly ascribed to the replacement of Ser277 of the bI iso- form with Ala277 in bIII. Such a residue is located within the M-loop (which constitutes an important part of the binding pocket, as shown in Fig. 3). The importance of the role of this residue in ligand binding and in the tubulin structure has been pointed out recently [11]. According to our findings, the role played by residue 277 is crucial not only because Ser277 is directly involved in the binding of paclitaxel with the bI isotype, but also because its replacement with Ala277 in bIII induces a conformational rear- rangement of the M-loop which, in turn, results in dif- ferent interactions of the ligands with other residues in the M-loop. In particular, paclitaxel interacts through hydrogen bonds with Ser277, Gln282 and Arg284 in the case of the bI isoform and only with Arg278 in the case of bIII. Similarly, even though IDN5390 interacts with Gln282 in both complexes, it is hydrogen bonded to Arg278 only in the I3 complex. As mentioned above, our results are in accordance with the known pharmacological effects of paclitaxel and of IDN5390 and are able to provide a rational structural basis for them. This prompted us to investigate the mode of binding of the much less well characterized epothilone A. No data about the effect of different isotype com- position of tubulin on the activity of this molecule have been reported so far, therefore we used our pro- cedure to investigate the interactions of epithilone A with the bI- and bIII-tubulin isoforms (indicated as E1 and E3, respectively). The average calculated binding energies of complexes sampled during molecular dynamic simulations and subsequently optimized are shown in Table 2. Our data suggest that epothilone A does not preferentially bind to one of the two iso- forms. The molecular details of the predicted interac- tions are shown in Fig. 7. The position of the ligand in the E1 and E3 structures is not as similar as in the case of paclitaxel and IDN5390 and, especially in E3, substantially differs from the starting complex. In E1, the epothilone is located between the M-loop and helix H7, interacting with them essentially through: (i) hydrogen bonds involving C3 and C7 hydroxyl groups and lateral chains of Arg278 and Gln282, respectively; and (ii) p–p interactions between the thiazole ring and His229. In E3, the ligand is farther away from the M-loop and shifted towards helix H1 and the S9–S10 loop with respect to the E1 complex, and therefore is Table 2. Free energy, enthalpy and entropy of the drug-protein complexes computed at 300 K between epothilone A and isoforms I (E1) and III (E3) of human b-tubulin. Complex DG (kcalÆmol )1 ) DH (kcalÆmol )1 ) DS (calÆmol )1 ) E1 )28.01 )27.99 0.08 E3 )31.00 )30.36 2.13 A B Fig. 6. Solvent accessible surfaces of the bI (A) and bIII (B) iso- forms in complex with IDN5390. Docking of antimitotic agents to tubulin isoforms M. Magnani et al. 3306 FEBS Journal 273 (2006) 3301–3310 ª 2006 The Authors Journal compilation ª 2006 FEBS able to interact with Gly370 (even if this hydrogen bond is not consistently observed during throughout the whole simulation). However, epothilone is still within the van der Waals distance with His229 and the carbonyl in C5 is engaged in a hydrogen bond with Arg278. Also in this case, the structure of the M-loop differs in the two complexes and is stabilized by a dif- ferent pattern of hydrogen bonds involving its residues (in particular, residues 277–280). Similarly to the case of the P3 and I3 complexes, in E3 the rearrangement of the loop directs the sidechain of Arg278 towards the binding pocket, allowing Arg278 to maintain hydrogen bond interactions with the ligand, notwith- standing the shift in the ligand position with respect to the E1 complex. Due to the described differences between the two binding modes, the interactions invol- ving the ligand in E1 and E3 are quite difficult to com- pare. Nevertheless, the analysis of the two complexes suggests that there should be no significant difference in binding energies of epothilone for the two isoforms. These observations, together with the data reported in Table 2, suggest that epothilone A is able to interact with similar affinities with both the bI and bIII iso- forms of tubulin. As a consequence, according to our analysis, it should be useful in cases where resistance mediated by overexpression of the bIII-tubulin isotype arises. Discussion A combination of molecular modeling and molecular dynamics techniques has been applied to investigate the binding modes of three microtubule stabilizing agents, namely paclitaxel, IDN5390 and epothilone A, with isotypes I and III of human b-tubulin. Increased expression of bIII isoform in cancer cells has been cor- related with paclitaxel resistance in several studies, whereas recent findings revealed that the activity of IDN5390 is not affected by bIII-tubulin levels. To our knowledge, no data about the activity of epothilones on tumors characterized by different b-tubulin isotype composition have been reported so far. Six complexes of the three ligands under analysis with the human bI and bIII-tubulin were first built and subjected to molecular dynamics. The average binding energies for structures sampled during the simulations were calcula- ted after energy optimization. Our data rationalize the experimental observations, suggesting a higher affinity of paclitaxel for the bI than for the bIII isoform and an opposite behavior for IDN5390. Interestingly, the calculated binding energies of complexes involving epothilone A are very similar for the bI and bIII iso- forms. Although docking simulation results have to be taken with caution, especially when based on modeled structures, our results suggest an equally effective interaction of this molecule with microtubules with dif- ferent isoform composition. Representative structures of complexes sampled during the course of molecular dynamic simulations were subsequently analyzed, with the aim of detecting specific interactions responsible for the differences in the calculated binding energies of paclitaxel and IDN5390 with bIorbIII isotypes. Such analysis highlighted the crucial role played by the dif- ferent residue present in the 277 position in the two isoforms (serine in bI and alanine in bIII) in determin- ing the different binding affinities of paclitaxel and IDN5390 to the two distinct isoforms. In short, such substitution is responsible for a different rearrange- ment of the M-loop, whose final outcome is a more favorable interaction of paclitaxel and IDN5390 with the bI and bIII isotypes, respectively. Our study sup- ports the hypothesis that the molecular basis of the different activities of paclitaxel and IDN5390 against microtubules expressing variable levels of bIII isoform A B Fig. 7. Epothilone A in complex with (A) bI-tubulin (E1), and (B) bIII- tubulin (E3). For sake of clarity, nonpolar hydrogen atoms are omit- ted and hydrogen bond interactions are represented by dashed lines. M. Magnani et al. Docking of antimitotic agents to tubulin isoforms FEBS Journal 273 (2006) 3301–3310 ª 2006 The Authors Journal compilation ª 2006 FEBS 3307 lie in the different ligand binding mode of the two molecules to the bI and bIII isotypes. The same analy- sis, when applied to epothilone A, predicts that its binding should not be affected by the isoptype compo- sition of tubulin, suggesting that this molecule can have a broader efficacy than paclitaxel and IDN5390 and perhaps be less prone to inducing resistance in tumor cells. Experimental procedures Comparative modeling Human tubulin isoform sequences were downloaded from the Swiss-Prot database (http://www.expasy.org). The identifiers of human bI- and bIII-tubulins are P07437 (TBB2_human) and Q13509 (TBB3-human), respectively. The multiple sequence alignments were obtained using clustalw [25]. The comparative modeling protocol consisted of import- ing the main-chain coordinates of the conserved regions from the template and positioning the replaced sidechains in their most commonly observed conformation [26]. The model was optimized using 100 cycles of steepest descent energy minimization using discover [27]. The PDB identifiers of the three-dimensional structures used in this work are as follows: 1FFX, 1IA0, 1JFF, 1SA0, 1TUB and 1TVK. Molecular dynamics Each complex was subjected to 2000 ps of molecular dynamic simulations with a time step of 1.5 fs. The calcu- lations were performed using macromodel version 7.2 [31] with the AMBER* united atom force field [32]. Sol- vent effects were taken into account by means of the implicit GB ⁄ SA water model [33]. A force constant of 23.9 kcalÆmolÆA ˚ )1 was applied to the protein backbone, while sidechains and ligands were left free. One hundred frames were sampled at regular time intervals for each drug–protein complex and subjected to 5000 steps of the Polak-Ribiere Conjugate Gradient energy minimization algorithm with the same force field and parameters as above. During these optimizations all constrains were removed allowing full relaxation of the system internal degrees of freedom. In order to select the most representa- tive binding modes, a clustering analysis of the optimized conformational ensemble was performed. In details, con- formations with an internal energy difference lower than 1 kcalÆmol )1 were duplicated if their RMS deviation, after superposition of the whole coordinate set, was lower than 0.25 A ˚ . Binding energies and Boltzmann analysis were car- ried out using the thermodynamic module of the moline program [28]. Docking experiments Docking simulations of IDN5390 in the paclitaxel (and epothilone A) binding site of b-tubulin were performed using autodock 3.0.5 software [34]. Both the modeled protein (hTUB) and the ligand (IDN5390, after building and minimization with macromodel version 7.2 [31]) were imported in autodock. Kollman’s united-atoms partial charges and solvent parameters were added to the protein, while Gasteiger atomic charges were calculated for the lig- and. Next, a grid including the binding site of interest was defined and several atom probes (corresponding to the atom types of the ligand) were placed at the grid nodes, in order to calculate the interaction energies between the probe and the protein. Grid maps were generated for each atom probe. The Lamarckian genetic algorithm [34] was employed to explore the orientation ⁄ conformation space of the ligand within the binding pocket. In Lamarckian genetic algorithm docking, the number of individuals within the population and the number of runs were both set to 200. A maximum number of 2 · 10 6 energy evaluations and 50 000 generations was allowed, while all the remaining parameters were kept to their default values. Finally, the conformation with the lowest estimated free energy of binding (and belonging to a well populated cluster) was selected. The reliability of the docking protocol was first assessed by simulating the known binding of paclitaxel. 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J Comput Chem 19, 1639– 1662. M. Magnani et al. Docking of antimitotic agents to tubulin isoforms FEBS Journal 273 (2006) 3301–3310 ª 2006 The Authors Journal compilation ª 2006 FEBS 3309 Supplementary material The following supplementary material is available online: Table S1. RMSd values (A ˚ ) after optimal superposition of the backbone atoms of known tubulin structures. Table S2. RMSd values (A ˚ ) after optimal superposition of the backbone atoms of ligand binding residues. These are defined as the residues within 4 A ˚ of any atom of either paclitaxel in the 1JFF structure or epo- thilone in 1TVK. They are: Glu2 2, Val23, Asp26, Glu27, Leu217, Gly225, Asp226, His229, Leu230, Ala233, Ser236, Phe272, Pro274-Arg278, Arg284, Pro360, Arg369-Leu371 (1JFF numbering). The num- ber of superimposed atoms is 176 per pair with the exception of the superpositions involving 1SA0 due to missing residues in this latter structure. Table S3. Interactions between paclitaxel and tubulin observed in the 1JFF entry as reported by Ligplot (http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/ pdbsum/). This material is avalilable as part of the online arti- cle from http://www.blackwell-synergy.com. Docking of antimitotic agents to tubulin isoforms M. Magnani et al. 3310 FEBS Journal 273 (2006) 3301–3310 ª 2006 The Authors Journal compilation ª 2006 FEBS . between these two ligands and the isotypes I and III of the human b-tubu- lin. We also investigated the complexes involving the bI and bIII isoforms with. interactions with the ligand, notwith- standing the shift in the ligand position with respect to the E1 complex. Due to the described differences between the two