Toll-like receptors (TLRs) form the first barrier of the innate immune system. TLR8 is an important target to treat autoimmune diseases since its ligand-induced degree of activation regulates immune response and associated hyperinflammation. Molecular dynamics (MD) simulations have been used to investigate interactions of TLRs with ligands, but the mechanism of ligand unbinding remains elusive. We therefore applied τ-random acceleration molecular dynamics (τRAMD) simulations to characterize the unbinding paths of one TLR8 agonist and five TLR8 antagonists. Data analysis of the simulations led to the discovery of two possible unbinding pathways: the internal pathway, directed toward the Toll-interleukin-1 receptor (TIR) domain, and the external pathway, pointing away from the TIR domain. Remarkably, some ligands showed clear path preferences: the TLR8 agonist exited through the external unbinding pathway only, while the cationic antagonists exited through the internal pathway only. The neutral antagonists used both pathways. The mechanistic insights obtained can assist in the design of improved TLR modulators.
