Title:
Crystallographic studies of TLR8 sensing single stranded RNA in innate immune system

Toshiyuki Shimizu
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan

Abstract:
Toll-like receptors (TLRs) are a family of pattern-recognition receptors that recognize microbial components and initiate subsequent immune responses. Ten members of the human TLR family (TLR1 to TLR10) have been identified to date. The extracellular domains have leucine rich repeats (LRRs) and are responsible for binding so-called “pathogen-associated molecular patterns”. TLR7 and TLR8 recognize ssRNA and initiate innate immune responses. Moreover, several small molecule compounds have been identified as TLR7 and TLR8 activators. We determined the crystal structures of unliganded and ligand-induced activated human TLR8 dimers [1]. Upon ligand stimulation, the TLR8 dimer was reorganized such that the two C-termini were brought into proximity. Ligand binding induces reorganization of the TLR8 dimer, which enables downstream signaling processes.
To elucidate how TLR8 recognizes its natural ligand (ssRNA), as well as how the receptor can be activated by ssRNA that is structurally and chemically so different from the chemical ligands, we performed crystallographic studies of TLR8 in complex with ssRNAs. TLR8 recognizes, at distinct sites, uridine and small oligonucleotides derived from degradation of ssRNA. Uridine bound the site on the dimerization interface where small chemical ligands are recognized, whereas short oligonucleotides bound a newly identified site.
A C2-butyl furo[2,3-c]quinoline was reported with purely TLR8 agonistic activity. This compound was successfully co-crystallized with the human TLR8 ectodomain. Focused structure-based ligand design studies led to the identification of 3-pentyl-quinoline-2-amine as a novel, structurally simple, and highly potent human TLR8-specific agonist [2].

References:
[1] Tanji, H, Ohto, U, Shibata, T, Miyake, M, and Shimizu, T (2013), “Structural reorganization of the Toll-like receptor 8 dimer induced by agonistic ligands”, Science 339, pp1426-1429
[2] Kokatla HP, Sil D, Tanji H, Ohto U, Malladi SS, Fox LM, Shimizu T, David SA. (2014), “Structure-Based Design of Novel Human Toll-like Receptor 8 Agonists”, ChemMedChem. 9, 719-723