Molecular mechanism underlying the TLR4 antagonistic and antiseptic activities of papiliocin, an insect innate immune response molecule

Antimicrobial peptides are innate immune molecules playing essential roles in insects, which lack the adaptive immune system. Insects possess Toll9, the innate pattern-recognition receptor highly similar to the mammalian Toll-like receptor 4 (TLR4), which is involved in recognizing lipopolysaccharide (LPS). TLR4 is an important therapeutic target, as it causes uncontrolled immune response in sepsis; therefore, identification of TLR4-targeting molecules is imperative. Papiliocin, an insect cecropin derived from the larvae of the swallowtail butterfly, possesses potent antibacterial activities against gram-negative bacteria. We investigated the molecular mechanism underlying the TLR4-antagonistic and antiseptic activities of papiliocin. Binding analysis, docking simulation, and flow cytometry showed that papiliocin inhibited LPS-induced TLR4 signaling by directly binding to TLR4/MD-2 and causing rapid dissociation of LPS from the TLR4/MD-2 complex. R13 and R16 in the N-terminal helix, conserved in insect cecropins, were the key binding sites at the TLR4/MD-2 interface, along with the flexible hinge region, which promoted the interaction of the hydrophobic carboxyl-terminal helix with the MD-2 pocket to competitively inhibit the LPS–TLR4/MD-2 interaction. Papiliocin, an antiendotoxin molecule and TLR4 inhibitor, rescued the pathology of Escherichia coli–induced sepsis in mice more effectively and with lower nephrotoxicity compared to polymyxin B. Our results provide insight into the key structural components and mechanism underlying the TLR4-antagonistic activities of papiliocin, which is essential for the innate immune response of the insect against microbial infection. Papiliocin may be useful for developing a multifunctional alternative to polymyxin B for treating gram-negative sepsis.