Computational Studies on AC Analog-Tubulin Interaction. To glean additional insight into these differences in activity, we turned to computational modelling of the library in tubulin. Binding constants were calculated using the alchemical Relative Binding Free Energy method (RBFE) with the AToM-OpenMM software version v3.5.0 (), and MTC (3) was used as a reference. MTC and the other compounds were aligned to the crystal structure of colchicine bound to tubulin (PDB 5ITZ). Hamiltonian replica exchange alchemical molecular dynamics calculations were performed with 22 replicas for 20 ns per replica. 100 ns molecular dynamics simulations for the dihedral angle analysis were conducted for each compound bound to tubulin and free in solution. From this data, we were able to extract a series of poses, through which we could profile AC ring dihedral angles bound and unbound (ie, Figure ), and obtain computational binding constants (Figure ).