Toll-like receptor (TLR) family proteins belong to PRRs. They play a crucial role in the initiation of the hosts’ immune responses, linking innate immunity and adaptive immunity. TLRs recruit a series of adaptor proteins, which trigger the pro-inflammatory signaling cascades. Therefore, they result in the activation of NF-κB, and upregulation of inflammatory cytokines and chemokines. This TLR response is crucial in helping eliminate the pathogen and establishing long-lasting adaptive responses but also can cause various autoimmune diseases and inflammatory disorders. Endosomal TLR3/7/8/9 are highly analogous sensors for various viral or bacterial RNA/DNA molecular patterns. In this study, CU-CPT9b is a specific TLR8 antagonist with an IC50 of 0.7 nM.
Crystal structures of two distinct TLR8-ligand complexes validated a unique binding site on the protein-protein interface of the TLR8 homodimer. CU-CPT9b strongly binds to this new site with a Kd of 21 nM. Thus, it stabilizes the preformed TLR8 dimer in its resting state, preventing activation. In detail, CU-CPT9b binds to the inactive TLR8 dimer and utilizes hydrogen bonds with G351 and V520*. They are conserved among TLR8/antagonist structures.
Additionally, CU-CPT9b forms water-mediated contacts with S516* and Q519*. It suggests that the enhanced potency of CU-CPT9b derives from the new interactions with these polar residues. The orientation of Y567* also changes to facilitate van der Waals interactions with CU-CPT9b.
In summary, CU-CPT9b not only stabilizes the preformed TLR8 dimer but also prevents further conformational changes that are necessary for TLR8 activation. CU-CPT9b is a chemical probe to understand the biological relevance of TLR8 in different pathogenesis processes and presents significant therapeutic development potential.