Endosomal Toll-like receptors (TLR3/7/8/9) are highly analogous sensors for various viral or bacterial RNA/DNA molecular patterns. Nonetheless, few small-molecules can selectively modulate these TLRs. In this manuscript, we identified the first human TLR8-specific small-molecule antagonists via a novel inhibition mechanism. Crystal structures of two distinct TLR8-ligand complexes validated a unique binding site on the protein-protein interface of the TLR8 homodimer. Upon binding to this new site, the small-molecule ligands stabilize the preformed TLR8 dimer in its resting state, preventing activation. As a proof of concept of their therapeutic potential, we have demonstrated that these drug-like inhibitors are able to suppress TLR8-mediated proinflammatory signaling in various cell lines, human primary cells, and patient specimens. These results not only suggest a novel strategy for TLR inhibitor design, but also shed critical mechanistic insight into these clinically important immune receptors.

Rational design of drug-like small-molecule ligands based on structural information of proteins remains a significant challenge in chemical biology. In particular, designs targeting protein-protein interfaces have met little success given the dynamic nature of the protein surfaces. Herein, we utilized the structure of a small-molecule ligand in complex with Toll-like receptor 8 (TLR8) as a model system due to TLR8's clinical relevance. Overactivation of TLR8 has been suggested to play a prominent role in the pathogenesis of various autoimmune diseases; however, there are still few small-molecule antagonists available, and our rational designs led to the discovery of six exceptionally potent compounds with ∼picomolar IC values. Two X-ray crystallographic structures validated the contacts within the binding pocket. A variety of biological evaluations in cultured cell lines, human peripheral blood mononuclear cells, and splenocytes from human TLR8-transgenic mice further demonstrated these TLR8 inhibitors' high efficacy, suggesting strong therapeutic potential against autoimmune disorders.

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Background Tumor targeting small molecular inhibitors are the most popular treatments for many malignant diseases, including cancer. However, the lower clinical response and drug resistance still limit their clinical efficacies. HGFK1, the first kringle domain of hepatocyte growth factor, has been defined as a potent anti-angiogenic factor. Here, we aimed to develop and identify novel nanoparticles—PH1/pHGFK1 as potential therapeutic agents for the treatment of renal cell carcinoma (RCC). Methods We produced a novel cationic polymer—PH1 and investigated the anti-tumor activity of PH1/pHGFK1 nanoparticle alone and its combination therapy with sorafenib in RCC cell line xenografted mice model. Then, we figured out its molecular mechanisms in human RCC cell lines in vitro. Results We firstly demonstrated that intravenous injection of PH1/pHGFK1 nanoparticles significantly inhibited tumor growth and prolonged the survival time of tumor-bearing mice, as well as synergistically enhanced anti-tumor activities of sorafenib. Furthermore, we elucidated that recombinant HGFK1 improved sorafenib-induced cell apoptosis and arrested cell cycle. In addition, HGFK1 could also decrease sorafenib-induced autophagy and stemness via blockading NF-κB signaling pathway in RCC both in vitro and in vivo. Conclusions HGFK1 could inhibit tumor growth, synergistically enhance anti-tumor activities of sorafenib and reverse its drug resistance evolution in RCC. Our results provide rational basis for clinical application of sorafenib and HGFK1 combination therapy in RCC patients. Electronic supplementary material The online version of this article (10.1186/s13046-019-1348-z) contains supplementary material, which is available to authorized users.

Small-molecule modulators of TLR8 have drawn much interests as it plays pivotal roles in the innate immune response to single-stranded RNAs (ssRNAs) derived from viruses. However, their clinical uses are limited because they can invoke an uncontrolled, global inflammatory response. The efforts described herein culminate in the fortuitous discovery of a tetrasubstituted imidazole CU-CPD107 which inhibits R848-induced TLR8 signaling. In stark contrast, CU-CPD107 shows unexpected synergistic agonist activities in the presence of ssRNA, while CU-CPD107 alone is unable to influence TLR8 signaling. CU-CPD107’s unique, dichotomous behavior sheds light on a way to approach TLR agonists. CU-CPD107 offers the opportunity to avoid the undesired, global inflammation side effects that have rendered imidazoquinolines clinically irrelevant, providing an insight for the development of antiviral drugs.

Rational designs of small-molecule inhibitors targeting protein−protein interfaces have met little success. Herein, we have designed a series of triazole derivatives with a novel scaffold to specifically intervene with the interaction of TLR8 homomerization. In multiple assays, TH1027 was identified as a highly potent and specific inhibitor of TLR8. A successful solution of the X-ray crystal structure of TLR8 in complex with TH1027 provided an in-depth mechanistic insight into its binding mode, validating that TH1027 was located between two TLR8 monomers and recognized as an unconventional pocket, thereby preventing TLR8 from activation. Further biological evaluations showed that TH1027 dose-dependently suppressed the TLR8-mediated inflammatory responses in both human monocyte cell lines, peripheral blood mononuclear cells, and rheumatoid arthritis patient specimens, suggesting a strong therapeutic potential against autoimmune diseases.

Summary Toll-like receptor 8 (TLR8) is a pattern recognition receptor that senses RNA degradation products and initiates immune responses. TLR8 overactivation is associated with autoimmune diseases. Herein, we describe the evaluation and validation of TLR8 antagonists in HEK-Blue cells via secreted embryonic alkaline phosphatase (SEAP) assay, WST assay, ITC and immunoblotting. These assays can facilitate the development of TLR8 antagonists; this protocol can also be adapted to analyze agonists and antagonists for other TLRs. For complete details on the use and execution of this protocol, please refer to Hu et al. (2018) .

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