3.3 Results
4.1.2 The binding mode of epothilone
Extensive studies using NMR spectroscopy were done on the binding modes of drug molecules binding to the taxane-binding site ofβ-tubulin. The bound conformation of discodermolide [Sanchez-Pedregal et al., 2006], dictyostatin [Canales et al., 2008], docetaxel [Canales et al., 2011] and tubulysin [Kubicek et al., 2010] were derived using trNOE and INPHARMA data. The natural product epothilone A (epoA, Fig. 4.4) is also in the group of anticancer agents that target the taxane-binding site of β-tubulin. Epothilone showed activity in cancer cells that were resistant against Taxol and the epothilone derivative ixabepilone was released in the United States under the trading name Ixempra. An electron crystallography (EC) derived binding mode of epothilone to Zn-stabilized tubulin sheets is known [Nettles et al., 2004].
There is a severe disagreement between the bound conformation in solu-tion found by the NMR methods trNOE and CCR [Carlomagno et al., 2003]
and the EC-derived structure. An NMR model of the binding mode, based
Figure 4.3: Binding mode of epothilone A to tubulin heterodimers as determined by NMR spectroscopy (left) and to Zn-stabilized tubulin sheets as determined by electron crystallography (right).
on molecular modeling with HADDOCK and scoring by INPHARMA was published [Reese et al., 2007] and shows a very different binding mode of epoA to β-tubulin compared to the EC-derived structure. An investigation by solid state NMR of epothilone B (patupilone) bound to microtubules re-vealed details about the pharmacophore. Still, it was not possible to decide if the EC or the NMR structure is the correct binding mode [Kumar et al., 2010].
Figure 4.4: Constitution and configuration of epothilone A.
The binding site of cyclostreptin was discovered to be in the pore site, which is situated between the two β units of two tubulin dimers. Follow-ing this observation, some authors ( [Canales et al., 2008], [Magnani et al., 2009], [Canales et al., 2011]) proposed that the pore site is also the binding site in solution of paclitaxel-like antimitotic drugs that bind the taxane site inside the microtubule. The logic behind this was, that STD data showed differences in the binding mode between the tubulin and the microtubule samples. Additionally it was observed that the binding affinities are not as strong as expected in the tubulin samples. Therefore these drugs would bind at the pore site between dimers that are formed in solution.
Conse-4.1. INTRODUCTION 107
Figure 4.5: Constitution and configuration of the baccatin III core, derived from paclitaxel.
quently the drugs only bind to the taxane site, if a microtubule is either in the growing phase or already present. An exception could be the Zn-stabilized sheets, that were used to determine the crystal structures of pa-clitaxel and epothilone and which show that the drugs bind to the taxane site. As there are INPHARMA signals between the taxane binding drugs like baccatin, epothilone and discodermolide; epothilone would also be affected.
Indeed, there were also INPHARMA peaks observed between epothilone and tubulysin. Tubulysin is a peptide with a structure very similar to soblidotin, a dolastatin derivative. Dolastatin is known from X-ray crystallography to bind close to the vinblastine and cyclostreptin (pore) binding site [Cormier et al., 2008]. Therefore it had to be concluded that the possibility of another binding site on tubulin in solution is probable [Kubicek et al., 2010].
On the other hand, experimental data obtained by H/D exchange (HDX) followed by mass spectrometry measurements give another picture. Based on these data, the taxane binding site is stabilized and therefore protected against deuteration, if the taxane binding site drugs are in solution [Khrapunovich-Baine et al., 2009]. Furthermore three new binding modes of epothilone A are proposed, which are supported by the H/D data [Khrapunovich-Baine et al., 2011]. These are shown in Fig. 4.6 and are closer to the NMR-, than to the EC-derived structure.
At the final stage of this thesis, a new crystal structure of epothilone bound to tubulin was published [Prota et al., 2013]. The structure is deposited in the PDB under the entry code 4I50 and shows a complex between αβ tubu-lin, the stathmin-like protein RB3, tubulin tyrosine ligase and epoA. The structure is shown later in the text in Fig. 4.19 and epoA is contrary
orien-tated at the binding site to all above mentioned structures. This new crystal structure shows epothilone bound to tubulin with four hydrogen bonds and fixes the M-loop of tubulin as a structured alpha-helix. The hydrogen bonds are induced by OH3, OH7, O1 and the N of the thiazole ring of epoA and are in good agreement with SAR data [Nicolaou et al., 1997, Regueiro-Ren et al., 2002, Buey et al., 2004]. The crystal structure also explains well, why an addition of a methyl group to C12 results in a much stronger binding affinity, as in this structure there is a cavity to fill by the methyl group.
Figure 4.6: Binding mode proposals of epothilone A (shown in magenta) to tubulin, supported by HDX data [Khrapunovich-Baine et al., 2011].
To further investigate the tubulin/epoA system, new STD data were recorded and applied in the following section as a selection criterion to choose between the ambiguous binding modes. As the INPHARMA method depends on two different molecules that target competitively the same binding site, baccatin III (bacIII) was used complementary to epothilone A. BacIII is the core element of paclitaxel (Fig. 4.5, lacking the rest that bears two phenyl rings. The logic behind this is, that bacIII is much better soluble than pa-clitaxel. Interestingly, INPHARMA found for bacIII another binding mode than it would have been expected from the EC structure of paclitaxel. Thus, for bacIII there were also new STD data obtained.