Research Article: Targeting the innate immune receptor TLR8 using small-molecule agents

Date Published: July 01, 2020

Publisher: International Union of Crystallography

Author(s): Kentaro Sakaniwa, Toshiyuki Shimizu.


The innate immune receptor TLR8 can be positively or negatively regulated by small chemical ligands. Structural views of agonist-bound and antagonist-bound forms have revealed the mechanisms underlying agonism and antagonism.

Partial Text

Toll-like receptors (TLRs) are a family of single transmembrane receptors that recognize molecular patterns from microbes or viruses and activate the innate immune system (Fig. 1 ▸a). Some receptors recognize pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) to rapidly respond to a wide range of invading agents. These receptors are called pattern-recognition receptors (PRRs) and consist of TLRs, Rig-I-like receptors (RLRs), NOD-like receptors (NLRs) and C-type lectin receptors (CLRs) (Takeuchi & Akira, 2010 ▸). TLRs are type I membrane receptors that are located on cell surfaces or in endosomes. Ten TLRs have been identified in humans, including TLR1, TLR2, TLR4, TLR5 and TLR6, which are located on cell surfaces and mainly recognize the components of bacteria; others, including TLR3, TLR7, TLR8 and TLR9, are located in endosomes and recognize pathogen-derived nucleic acids (Kawai & Akira, 2010 ▸). Currently, no consensus has been reached regarding the function of TLR10, despite several proposed hypotheses.

Among the TLRs, TLR3, TLR7, TLR8 and TLR9 are localized in endosomes and recognize patterns of nucleic acids (Zhang et al., 2017 ▸). It has been reported that a chaperone protein, UNC93B1, regulates the stability and/or transportation of these TLRs. Furthermore, TLR researchers have investigated whether UNC93B1 is related to the regulation of TLR functions (Majer, Liu, Kreuk et al., 2019 ▸; Majer, Liu, Woo et al., 2019 ▸). As TLR7, TLR8 and TLR9 share some common features in sequence homology, function and structure, they constitute the TLR7 subfamily and harbor 26 LRRs, thus being the longest members of the TLR family, sense single-stranded nucleic acids and have a characteristic insertion between LRR14 and LRR15 called the Z-loop (Tanji et al., 2013 ▸; Ohto et al., 2015 ▸; Zhang et al., 2016 ▸; Fig. 1 ▸b). The Z-loop is critically important in their functions as it has been reported that cleavage of the Z-loop and reorganization of the TLR is required for activation, as shown by structural analyses and biochemical experiments (Tanji et al., 2016 ▸). TLR7 and TLR8 resemble each other particularly closely in the TLR family. They have a common function in sensing ssRNA and common ligands, such as the antiviral imidazoquinoline resiquimod R848 (Jurk et al., 2002 ▸). Furthermore, they play a principal role in the response to viral infections mediated by the MyD88-dependent pathway, which activates NF-κB and MAPK and induces the production of inflammatory cytokines such as tumor necrosis factor α (TNF-α), IL-6 and IL-12.

In 2013, the crystal structure of the TLR8 ectodomain was reported for the first time in the TLR7 subfamily. Dimeric structures of TLR8 with or without agonistic synthetic imidazoquinoline compounds (R848, CL097 and CL075) were reported (Figs. 2 ▸a and 2 ▸b; Tanji et al., 2013 ▸). These results demonstrated that TLR8 forms an unliganded dimer state, unlike other TLRs localized on cell surfaces, and is converted into an activated dimer state triggered by ligand binding.

In addition to the structures of activated forms of TLR8, TLR8–antagonist complex structures were reported in 2018 (Zhang, Hu et al., 2018 ▸). This study reported CU-CPT compounds as the first human TLR8-specific antagonists, along with structural information in order to understand the TLR8 inhibitory mechanism and accelerate the development of TLR8-targeted medicines with inhibitory effects.

The antagonist-bound site is spatially close to the agonist-bound site, but the binding mechanism differs considerably. A comparison of these structures enabled us to understand the regulatory mechanism of TLR8. As shown in Figs. 2 ▸(d), 2 ▸(e) and 2(f), LRR8, LRR11–13 and LRR15*–18* play key roles in ligand recognition. In the unliganded state, TLR8 forms an inactivated dimer structure in which LRR11–13 encounter LRR15*–16* and LRR8 confronts LRR17*–18*, creating an unoccupied pocket at the interface of the two protomers (Fig. 2 ▸e). The antagonists bind to the pocket in this arrangement (Figs. 2 ▸e, 2 ▸f, 2 ▸h and 2 ▸i), mainly interacting with LRR11–13 and LRR15*, even though LRR8 and LRR17*–18* are partially involved in ligand recognition. In contrast, the agonists primarily interact with LRR11–13 and LRR17*–18* (Figs. 2 ▸d and 2 ▸g). One protomer, TLR8*, is pulled up in the direction from the C-terminus to the N-terminus of the counterpart protomer by the agonist, repositioning LRR17*–18* in front of LRR11–13, which allows rotation of the overall structure and makes the C-terminal regions closer.

TLRs play a vital role in the innate immune system, and they have become notable targets for the development of therapies in certain diseases. Currently, many clinical trials investigating TLR ligands are in progress, and a few TLR agonists have been approved (Smith et al., 2018 ▸).

TLRs are crucial receptors for innate immunity. Previously, structural information on the nucleic acid-sensing TLRs had been limited to TLR3, but the structure of TLR8 was resolved in 2013. Currently, structures are available for all members of the TLR7 subfamily. TLR8 structures showed some characteristic features that are conserved in the TLR7 subfamily, which differs drastically from other subfamilies of TLRs. In addition, ligand-complexed structures of TLRs provide hints to understanding the mechanisms of ligand recognition and signal transduction. Structural information regarding agonist-bound and antagonist-bound TLR8 will accelerate the development of novel therapeutic approaches targeting these TLRs. In particular, the structural information on antagonists may potentially be a paradigm-shifting discovery, even though TLR inhibitor/agonist design has been an active research field, with almost all previous efforts focused on the recognition of activated forms of TLRs.




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