Discrimination of Ligands with Different Flexibilities Resulting from the Plasticity of the Binding Site in Tubulin

Abstract

Tubulin, an α,β heterodimer, has four distinct ligand binding sites (for paclitaxel, peloruside/laulimalide, vinca, and colchicine). The site where colchicine binds is a promising drug target for arresting cell division and has been observed to accommodate compounds that are structurally diverse but possess comparable affinity. This investigation, using two such structurally different ligands as probes (one being colchicine itself and another, TN16), aims to provide insight into the origin of this diverse acceptability to provide a better perspective for the design of novel therapeutic molecules. Thermodynamic measurements reveal interesting interplay between entropy and enthalpy. Although both these parameters are favourable for TN16 binding (ΔH < 0, ΔS > 0), but the magnitude of entropy has the determining role for colchicine binding as its enthalpic component is destabilizing (ΔH > 0, ΔS > 0). Molecular dynamics simulation provides atomistic insight into the mechanism, pointing to the inherent flexibility of the binding pocket that can drastically change its shape depending on the ligand that it accepts. Simulation shows that in the complexed states both the ligands have freedom to move within the binding pocket; colchicine can switch its interactions like a "flying trapeze", whereas TN16 rocks like a "swing cradle", both benefiting entropically, although in two different ways. Additionally, the experimental results with respect to the role of solvation entropy correlate well with the computed difference in the hydration: water molecules associated with the ligands are released upon complexation. The complementary role of van der Waals packing versus flexibility controls the entropy-enthalpy modulations. This analysis provides lessons for the design of new ligands that should balance between the "better fit" and "flexibility"', instead of focusing only on the receptor-ligand interactions.

A new antiproliferative noscapine analogue: chemical synthesis and biological evaluation

“…Both combretastatin A4 and compound 8 bind deeper into β-tubulin than colchicine, which supports previous work that found flexible ligands bind more deeply [36]. For each compound, the methoxy-containing A ring is directed into β-tubulin near Cys241, and overlap of these rings is observed for the compounds studied, as previously found for colchicine and combretastatin A4 [35, 37, 38].…”

“…The top-ranked docking poses for colchicine resemble the crystal structure pose, providing confidence in our docking protocols. Small variations exist in the orientation of the acetamide relative to the crystal structure, which has been previously shown to have high mobility in the binding site [36]. The top poses of the other ligands are also similar, which indicates a common binding motif can be established.…”

Biological Characterization of an Improved Pyrrole-Based Colchicine Site Agent Identified through Structure-Based Design

“…Analogous to the vinca domain, the different binding poses prompted the suggestion that the colchicine site be referred to as the colchicine domain, taking into account these various orientations (Dorléans et al, 2009). A continuing challenge in the rational development of new colchicine site agents is the low resolution (∼3.6 Å) of the existing crystal structures of this site, and a further challenge in designing new colchicine site agents with improved binding properties is the inherent flexibility of the colchicine binding pocket as predicted by molecular simulations (Ravelli et al, 2004;Dorléans et al, 2009;Chakraborti et al, 2012) and the fact that more than 56 chemical scaffolds can interact within this pocket.…”

How to Deal with Low-Resolution Target Structures: Using SAR, Ensemble Docking, Hydropathic Analysis, and 3D-QSAR to Definitively Map the αβ-Tubulin Colchicine Site

“…5,8 The ability of the colchicine site to accommodate such structural diversity is due to the inherent flexibility of the site, which has been demonstrated by X-ray crystal structures of the protein complexed with different agents (PDBID: 1SA0, 1SA1, 3HKC, 3HKE, 3HKD, 3N2K and 3N2G) 9–11 and molecular dynamics simulations. 12 …”