Computational analysis of Amsacrine resistance in human topoisomerase II alpha mutants (R487K and E571K) using homology modeling, docking and all-atom molecular dynamics simulation in explicit solvent

Amsacrine is an effective topoisomerase II enzyme inhibitor in acute lymphatic leukemia. Previous experimental studies have successfully identified two important mutations (R487K and E571K) conferring 100 and 25 fold resistance to Amsacrine respectively. Although the reduction of the cleavage ligand-DNA-protein ternary complex has been well thought as the major cause of drug resistance, the detailed energetic, structural and dynamic mechanisms remain to be elusive. In this study, we constructed human topoisomerase II alpha (hTop2α) homology model docked with Amsacrine based on crystal structure of human Top2β in complex with etoposide. This wild type complex was used to build the ternary complex with R487K and E571K mutants. Three 500 ns molecular dynamics simulations were performed on complex systems of wild type and two mutants. The detailed energetic, structural and dynamic analysis were performed on the simulation data. Our binding data indicated a significant impairment of Amsacrine binding energy in the two mutants compared with the wild type. The order of weakening (R487K > E571K) was in agreement with the order of experimental drug resistance fold (R489K > E571K). Our binding energy decomposition further indicated that weakening of the ligand-protein interaction rather than the ligand-DNA interaction was the major contributor of the binding energy difference between R487K and E571K. In addition, key residues contributing to the binding energy (ΔG) or the decrease of the binding energy (ΔΔG) were identified through the energy decomposition analysis. The change in ligand binding pose, dynamics of protein, DNA and ligand upon the mutations were thoroughly analyzed and discussed. Deciphering the molecular basis of drug resistance is crucial to overcome drug resistance using rational drug design.