Fully Flexible Binding of Taxane-Site Ligands to Tubulin via Enhanced Sampling MD Simulations

Microtubules (MTs) are cytoskeleton components involved in a plenty of cellular functions such as transport, motility, and mitosis. Being polymers made up of α/β-tubulin heterodimers, in order to accomplish these functions, they go through large variations in their spatial arrangement switching between polymerization and depolymerization phases. Because of their role in cellular division, interfering with MTs dynamic behavior has been proven to be suitable for anticancer therapy as tubulin-binding agents induce mitotic arrest and cell death by apoptosis. However, how microtubule-stabilizing agents like taxane-site ligands act to promote microtubule assembly and stabilization is still argument of debate. As in the case of tubulin, traditional docking techniques lack the necessary capabilities of treating protein flexibility that are central to certain binding processes. For this reason, the aim of this project is to put in place a protocol for dynamic docking of taxane-site ligands to β-tubulin by means of enhanced sampling MD simulation techniques. Firstly, the behavior of the binding pocket has been investigated with classical MD simulations. It has been observed that the most flexible part of the taxane site is the so-called “M-loop”, the one involved into the lateral associations of tubulin heterodimers and that is supposed to be stabilized by taxane-site ligands. Secondly, the protocol for the dynamic docking has been put in place using the MD-Binding technique developed by BiKi Technologies. It showed to be effective in reproducing the binding mode of epothilone A and discodermolide as in their X-ray crystal structures. Finally, the protocol has been tested against paclitaxel, a drug for which no X-ray crystal structure is currently available. These results showed the potential of such an approach and strengthen the belief that in the future dynamic docking will replace traditional static docking in the drug discovery and development process.