After equilibration of the protein-ligand complexes, τRAMD simulations were performed, resulting in 100 independent trajectories with unbinding events for every TLR8-ligand complex simulated. Next, data analysis of the simulations was performed to identify the unbinding pathways and associated probabilities. Two possible unbinding pathways were identified by visual inspection of trajectories, and they were named as internal path, which is directed towards the tollinterleukin-1 receptor (TIR) domain and the cytoplasmic region, and external path, which is directed away from the TIR domain, depending on the direction the ligand took to exit the uridine binding site (Figure ). The classification of unbinding paths for each trajectory was performed through the calculation of the minimum distance of the ligand to representative residues located next to the exit sites of the internal or external unbinding path considering the final frames of the simulations (Figure ). Remarkably, analysis of the pathway probabilities revealed that the TLR8 agonist 1 only undertook the external path for unbinding, while the cationic antagonists 2 and 3 only employed the internal path for unbinding (Figure ). The neutral antagonists 4-6 took either the external or the internal path for unbinding, with each antagonist exhibiting a different path preference (Figure ). ) with the external and internal unbinding paths represented as spheres, which were obtained through the calculation of the center of mass of the ligand during the simulation . In the internal path, the ligand moves towards the tollinterleukin-1 receptor (TIR) domain, which is in the intracellular part of the receptor and not present in the crystal structures, while in the external path the ligand moves away from the TIR domain. B) Probabilities with which the ligands undergo the external or internal unbinding paths in the τRAMD simulations. For each ligand, 100 unbinding events were obtained. The bars show standard errors obtained from bootstrap analysis.