CU-CPT-9b (TLR8-specific antagonist 1)

Discovery of Novel Small Molecule Dual Inhibitors Targeting Toll-Like Receptors 7 and 8

Endosomal toll-like receptors (TLRs) 7 and 8 recognize viral single-stranded RNAs, a class of imidazoquinoline compounds, 8-oxo-adenosines, 8-aminobenzodiazepines, pyrimidines, and guanosine analogues. Substantial evidence is present linking chronic inflammation mediated specifically by TLR7 to the progression of autoimmunity. We identified a new TLR7/8 dual inhibitor (1) and a TLR8-specific inhibitor (2) based on our previous screen targeting TLR8. Compound 1, bearing a benzanilide scaffold, was found to inhibit TLR7 and TLR8 at low micromolar concentrations. We envisioned making modifications on the benzanilide scaffold of 1 resulting in a class of highly specific TLR7 inhibitors. Our efforts led to the discovery of a new TLR8 inhibitor (CU-115) and identification of a TLR7/8 dual inhibitor (CU-72), bearing a distinct diphenyl ether skeleton, with potential for TLR7 selectivity optimization. Given the role of TLR8 in autoimmunity, we also optimized the potency of 2 and developed a new TLR8 inhibitor bearing a 1,3,4-oxadiazole motif.

Small-molecule inhibition of TLR8 through stabilization of its resting state

Endosomal Toll-like receptors (TLR3, TLR7, TLR8, and TLR9) are highly analogous sensors for various viral or bacterial RNA and 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.

Identification of a Human Toll-Like Receptor (TLR) 8-Specific Agonist and a Functional Pan-TLR Inhibitor in 2-Aminoimidazoles

Activation of human toll-like receptor-8 (TLR8), expressed in myeloid dendritic cells, monocytes, and monocyte-derived dendritic cells, evokes a distinct cytokine profile which favors the development of Type 1 helper T cells. Part-structures of the 2-aminobenzimidazole scaffold were examined with a view to identifying structural requisites corresponding to the smallest possible fragment of the benzimidazole core that would allow for retention of TLR8-agonistic activity. TLR8-specific agonistic activity was retained in 1-pentyl-4-phenyl-1H-imidazol-2-amine. The crystal structure of this compound bound to the TLR8 ectodomain displayed binding interactions that are common to other TLR8 agonists. This compound showed markedly attenuated proinflammatory properties in ex vivo human blood models. SAR studies revealed that 4-(2-(benzyloxy)phenyl)-1-pentyl-1H-imidazol-2-amine inhibited TLR signaling in a variety of TLR reporter cell lines, as well as in pharmacologically relevant human blood model systems. A kinase screen of this compound showed relative specificity for calmodulin kinases.

Endosomal recognition of Lactococcus lactis G121 and its RNA by dendritic cells is key to its allergy-protective effects

Background: Bacterial cowshed isolates are allergy protective in mice; however, the underlying mechanisms are largely unknown. We examined the ability of Lactococcus lactis G121 to prevent allergic inflammatory reactions.
Objective: We sought to identify the ligands and pattern recognition receptors through which L lactis G121 confers allergy protection.
Methods: L lactis G121-induced cytokine release and surface expression of costimulatory molecules by untreated or inhibitor-treated (bafilomycin and cytochalasin D) human monocyte-derived dendritic cells (moDCs), bone marrow-derived mouse dendritic cells (BMDCs), and moDC/naive CD4⁺ T-cell cocultures were analyzed by using ELISA and flow cytometry. The pathology of ovalbumin-induced acute allergic airway inflammation after adoptive transfer of BMDCs was examined by means of microscopy.
Results: L lactis G121-treated murine BMDCs and human moDCs released T_H1-polarizing cytokines and induced T_H1 T cells. Inhibiting phagocytosis and endosomal acidification in BMDCs or moDCs impaired the release of T_H1-polarizing cytokines, costimulatory molecule expression, and T-cell activation on L lactis G121 challenge. In vivo allergy protection mediated by L lactis G121 was dependent on endosomal acidification in dendritic cells (DCs). Toll-like receptor (Tlr) 13^-/- BMDCs showed a weak response to L lactis G121 and were unresponsive to its RNA. The T_H1-polarizing activity of L lactis G121-treated human DCs was blocked by TLR8-specific inhibitors, mediated by L lactis G121 RNA, and synergistically enhanced by activation of nucleotide-binding oligomerization domain-containing protein (NOD) 2.
Conclusion: Bacterial RNA is the main driver of L lactis G121-mediated protection against experimentally induced allergy and requires both bacterial uptake by DCs and endosomal acidification. In mice L lactis G121 RNA signals through TLR13; however, the most likely intracellular receptor in human subjects is TLR8.

Icariside II induces cell cycle arrest and differentiation via TLR8/MyD88/p38 pathway in acute myeloid leukemia cells

Acute myeloid leukemia (AML) is a devastating hematological malignancy, characterized by differentiation arrest and unscheduled proliferation of immature cells of the myeloid lineage. Inducing AML cell differentiation has emerged as a promising therapeutic strategy for the therapy of AML. Icariside II, an active component of Herba Epimedii, has been well defined to promote osteogenic differentiation. However, the differentiation-inducing effect of Icariside II on AML cells has not been explored. In this study, we investigated the differentiation-inducing effect and underlying mechanism of Icariside II in AML HL-60 and THP-1 cell lines. Icariside II induced G1 phase cell cycle arrest by down-regulating Cyclin-dependent kinases (CDK2, CDK4 and CDK6) and up-regulating Cyclin-dependent kinase inhibitor (p21 and p27). Importantly, Icariside II could induce differentiation of AML cells, accompanied by the up-regulation of Toll-like receptor 8 (TLR8), myeloid differentiation factor 88 (MyD88) and phosphorylated p38. Further study indicated the cell cycle arrest and differentiation induced by Icariside II could be abrogated by TLR8-specific inhibitor CU-CPT9a. Collectively, these findings firstly demonstrate Icariside II induces cell cycle arrest and differentiation of AML cells via activation of TLR8/MyD88/p38 pathway, suggesting Icariside II could be developed into a novel differentiation-inducing agent for AML.