Structural characterization of TLRs with novel agonists

Abstract Small molecule TLR4 and TLR2 agonists have been reported, but no structural data reveals their activation mechanism and detailed interactions with the TLRs. We have developed two small molecule agonists, Neoseptin-3 and Diprovocim, that activate TLR4/MD-2 and TLR1/TLR2 complexes, respectively, with exquisitely specific structure activity relationships. These two molecules bear no structural similarity to the natural ligands, lipopolysaccharide (LPS) and tri-acylated lipopeptide (Pam3CSK4). The crystal structures of Neoseptin-3 in complex with mouse TLR4/MD-2 and Diprovocim in complex with human TLR2 provide the first glimpse of how these TLRs bind to unconventional agonists, revealing unique and unexpected binding modes. Neoseptin-3 binds as an asymmetrical dimer within the hydrophobic pocket of MD-2, and induces an active receptor complex (a dimer of TLR4/MD-2) similar to that induced by lipid A. However, Neoseptin-3 and lipid A form different molecular contacts with TLR4/MD-2 to achieve receptor activation. Diprovocim forms a symmetrical dimer and interacts with the same hydrophobic pocket of TLR2 as Pam3CSK4, inducing homodimerization of TLR2 that has a different conformation than the active TLR1/TLR2 heterodimer. Diprovocim binds to TLR2 through an extensive intermolecular hydrogen bonding network that is not observed in the Pam3CSK4/TLR2/TLR1 structure. These two structures are now guiding us in optimization of TLR4/MD-2 and TLR1/TLR2 agonists and antagonists for clinical applications.