Abstract
<jats:p>This study presents a quantitative in silico docking and spatial profiling analysis of the colchicine-binding site on β-tubulin (PDB: 4O2B) to demonstrate how native posttranslational modifications (PTMs) structurally modulate ligand accessibility and binding free energy (ΔG). High-resolution structural alignments and automated grid-based molecular docking (AutoDock Vina and CB-Dock2) were conducted across native, pointmutated (βV238A, βA250V, βC241A), and O-GlcNAc-modified β-tubulin conformations to evaluate pocket cavity volumes, surface accessibility, and pairwise interaction distances (Å), alongside key control analogs including Combretastatin A-4 and Podophyllotoxin. Baseline docking established a high-affinity interaction between colchicine and Zone A/Cavity 1 (ΔG = −9.7 kcal/mol in AutoDock Vina; −8.7 kcal/mol in CB-Dock2), whereas point mutations such as βA250V disrupted hydrophobic packing, reducing affinity to −6.8 kcal/mol. Crucially, covalent attachment of O-GlcNAc at the βThr238 residue induced severe steric occlusion within the binding funnel, obstructing the entry of colchicine’s trimethoxyphenyl ring and driving a substantial thermodynamic penalty that functionally shields the pocket. Ultimately, these computational findings provide a concrete structural mechanism showing that O-GlcNAcylation can act as a non-mutational, dynamic molecular shield at the colchicine-binding interface, establishing a novel framework for understanding endogenous microtubule regulation and guiding the development of PTMevading antimitotic agents.</jats:p>