The computational modeling experiments were conducted using the Schrödinger Small-Molecule Drug Discovery Suite (2018-4) as previously described.7 (link) Briefly, the crystal structure 5EZY was downloaded from PDB and optimized using the Protein Preparation Wizard. The optimized protein structure was simplified by only retaining chain B comprising the taccalonolide AJ-β-tubulin complex. All water molecules were removed except for that forming H-bonds between β-tubulin Thr223 and the C-26 carbonyl group of taccalonolide AJ. The ligand structures were optimized using the Ligand Preparation Wizard (LigPrep). Further covalent docking experiments were performed using CovDock with Asp226 selected as the reactive residue. The docking box was centered on the coordinates X 3.2 / Y −63.5 / Z 22.6 in the length of 20 Å. The top 10 low-energy poses were generated and retained for each docking experiment. The lowest-energy pose showing the correct spatial arrangement of the taccalonolide core structure was selected for analysis of the ligand-protein binding modes.