Finally, the simulations with the neutral antagonists 4, 5 and 6 were also visually inspected to identify the main motions during unbinding through the internal and external paths. The neutral antagonists 4, 5 and 6 form a hydrogen bond with the backbone of Gly351, identical to the cationic antagonists, but they lack the ionic interaction with Glu427. The lack of this interaction allows the formation of a hydrogen bond between the side chain of Glu427 and the side chain of Tyr567*, which sterically hinders 4 from exiting the binding site via the internal path, while residues Lys350, Gly351 and Val520* sterically hinder 4 from exiting the binding site via the external path (Figure ). During the simulations, Lys350 and Gly351 move away from Val520*, allowing 4 to exit through the external path due to the increased space (Figure ). The external path of neutral antagonists presents small differences in comparison to the path of agonist 1 due to the structural differences between the active and inactive TLR8. While Tyr353 plays an important role in guiding unbinding of 1 via the external path, it is less involved in the external path of neutral antagonists.