Small-molecule inhibitors of the TLR3/dsRNA complex

The protein-RNA interface has been regarded as "undruggable" despite its importance in many biological processes. The toll-like receptor 3 (TLR3)/double-stranded RNA (dsRNA) complex provides an exciting target for a number of infectious diseases and cancers. We describe the development of a series of small-molecule probes that were shown to be competitive inhibitors of dsRNA binding to TLR3 with high affinity and specificity. In a multitude of assays, compound 4a was profiled as a potent antagonist to TLR3 signaling and also repressed the expression of downstream signaling pathways mediated by the TLR3/dsRNA complex, including TNF-α and IL-1β.     

A Redox-responsive Prodrug Nanogel of TLR7/8 Agonist for Improved Cancer Immunotherapy

The existence of tumor immunosuppressive microenvironment(TIME) is the major determinant for the poor efficacy of current tumor immunotherapy. Tumor-associated macrophages(TAMs) tend to become tumor-promoting M2-like phenotype and hinder immune response in solid tumors. Repolarization of TAMs from M2 to anti-tumor M1 phenotype is robust for remodeling the TIME. Herein, we developed a redoxresponsive nanogel as the delivery system of Toll-like receptor 7 and 8(TLR7/8) agonist(R848) prodrug for po...     

Rosmanol and Carnosol Synergistically Alleviate Rheumatoid Arthritis through Inhibiting TLR4/NF-κB/MAPK Pathway

Callicarpalongissima has been used as a Yao folk medicine to treat arthritis for years in China, although its active anti-arthritic moieties have not been clarified so far. In this study, two natural phenolic diterpenoids with anti-rheumatoid arthritis (RA) effects, rosmanol and carnosol, isolated from the medicinal plant were reported on for the first time. In type II collagen-induced arthritis DBA/1 mice, both rosmanol (40 mg/kg/d) and carnosol (40 mg/kg/d) alone alleviated the RA symptoms, such as swelling, redness, and synovitis; decreased the arthritis index score; and downregulated the serum pro-inflammatory cytokine levels of interleukin 6 (IL-6), monocyte chemotactic protein 1 (MCP-1), and tumor necrosis factor α (TNF-α). Additionally, they blocked the activation of the Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB)/c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways. Of particular interest was that when they were used in combination (20 mg/kg/d each), the anti-RA effect and inhibitory activity on the TLR4/NF-κB/MAPK pathway were significantly enhanced. The results demonstrated that rosmanol and carnosol synergistically alleviated RA by inhibiting inflammation through regulating the TLR4/NF-κB/MAPK pathway, meaning they have the potential to be developed into novel, safe natural combinations for the treatment of RA.     

Recognition of lipopolysaccharide pattern by TLR4 complexes

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria. Minute amounts of LPS released from infecting pathogens can initiate potent innate immune responses that prime the immune system against further infection. However, when the LPS response is not properly controlled it can lead to fatal septic shock syndrome. The common structural pattern of LPS in diverse bacterial species is recognized by a cascade of LPS receptors and accessory proteins, LPS binding protein (LBP), CD14 and the Toll-like receptor4 (TLR4)–MD-2 complex. The structures of these proteins account for how our immune system differentiates LPS molecules from structurally similar host molecules. They also provide insights useful for discovery of anti-sepsis drugs. In this review, we summarize these structures and describe the structural basis of LPS recognition by LPS receptors and accessory proteins.     

Synthesis of serine-based glycolipids as potential TLR4 activators

A new series of monosaccharide-based glycolipids devoid of phosphate groups and with two lipid chains were rationally designed by varying the lipid chain lengths and saccharide structure of a α-GalCer-derived lead compound (CCL-34) that is a potent TLR4 agonist. The NF-κB activity of a 60-membered galactosyl serine-based synthetic library containing compounds with various lipid chain lengths was measured in a HEK293 cell line that stably expressed human TLR4, MD2, and CD14 (293-hTLR4/MD2-CD14). The results showed that the optimal carbon chain lengths for the lipid amine and fatty acid to activate TLR4 were 10-11 and 12, respectively. Evaluation of a 20-membered synthetic glycosyl serine-based lipid library containing compounds with various saccharide moieties and fixed lipid chain lengths revealed that the galactose moiety in CCL-34 could be replaced by glucose without loss of activity (CCL-34-S3 and CCL-34-S16). Changing the orientation of the anomeric glycosidic bond of CCL-34 resulted in a complete loss of activity (β-CCL34). Surprisingly, a change in configuration of the anomeric glycosidic bond in a glucosyl glycolipid is tolerable (CCL-34-S14). Another noteworthy observation is that the activity of a l-fucosyl derived glycolipid (CCL-34-S13) was comparable to that of CCL-34. In sum, this study determines the structural features that are crucial for an optimal TLR4-stimulating activity. It also provides several molecules with immunostimulating potential.     

Recent advances with TLR2-targeting lipopeptide-based vaccines

The next generation of vaccines are being rationally designed according to rules that govern the way in which antigen is recognised by and stimulates the immune system. Amongst the first cells that encounter potentially dangerous agents such as viruses and bacteria are cells of the innate immune system, such as dendritic cells, that are widely distributed throughout the body including the skin. These cells patrol most tissues and have on their surface an array of receptors that have evolved to recognise many of the surface features of pathogens including the lipids and carbohydrates of structural lipoproteins, glycolipids and glycoproteins. Once encountered, recognised and engaged by a particular receptor on the dendritic cell, pathogenic material may then be transported inside the cell and processed for presentation to cells of the adaptive immune system. The result of this concert of events is a specific cellular or antibody response to particular epitopes of the invading pathogen. If then ways can be found to specifically target dendritic cells, through their specific receptors, then the efficacy and potency of vaccines could well be greatly improved. This review covers some of the approaches that we and others are pursuing in order to achieve this result.     

Caragana rosea Turcz Methanol Extract Inhibits Lipopolysaccharide-Induced Inflammatory Responses by Suppressing the TLR4/NF-κB/IRF3 Signaling Pathways

Caragana rosea Turcz, which belongs to the Leguminosae family, is a small shrub found in Northern and Eastern China that is known to possess anti-inflammatory properties and is used to treat fever, asthma, and cough. However, the underlying molecular mechanisms of its anti-inflammatory effects are unknown. Therefore, we used lipopolysaccharide (LPS) in RAW264.7 macrophages to investigate the molecular mechanisms that underlie the anti-inflammatory activities of a methanol extract of Caragana rosea (Cr-ME). We showed that Cr-ME reduced the production of nitric oxide (NO) and mRNA levels of iNOS, TNF-α, and IL-6 in a concentration-dependent manner. We also found that Cr-ME blocked MyD88- and TBK1-induced NF-κB and IRF3 promoter activity, suggesting that it affects multiple targets. Moreover, Cr-ME reduced the phosphorylation levels of IκBα, IKKα/β and IRF3 in a time-dependent manner and regulated the upstream NF-κB proteins Syk and Src, and the IRF3 protein TBK1. Upon overexpression of Src and TBK1, Cr-ME stimulation attenuated the phosphorylation of the NF-κB subunits p50 and p65 and IRF3 signaling. Together, our results suggest that the anti-inflammatory activity of Cr-ME occurs by inhibiting the NF-κB and IRF3 signaling pathways.     

Athrotaxis alkaloids. Part II. Alkaloids of a. selaginoides and a. laxifolia

The taxodeaceous plant A. Selaginoides contains six known homoerythrina-type alkaloids and selaginoidine, which has a furan ring replacing the benzene ring of the latter type. Selaginoidine and five of the six above-mentioned alkaloids also occur in A. laxifolia.     

Exploring the interaction between 3-D structure of TLR 9 and prostaglandin analogues

Toll-like receptor 9 (TLR9) is a class of pattern recognition receptors (PRRs) approved to have an essential role with the development of autoimmune illnesses such as psoriasis and arthritis. TRL9 is predominantly expressed on dendritic cells (DCs) and macrophages, and it mediates antigen presentation to T cells. In this regard, interfering with this interaction by inhibiting TLR9 could be an effective immunotherapy strategy for certain diseases. Based on previous research (Degraaf et al.2014, Farrugia et al., 2017) indicating that prostaglandins play a significant role in regulating or reducing the expression of TLRs, their function against intracellular TLRs such as TLR9 may involve direct inhibition of these receptors. The 3D structure of the human-TLR9 is modelled and described molecularly and then prepared to be docked by prostaglandin analogues, followed by molecular dynamic (MD) simulation, MM/PBSA analysis, and PCA analysis. The study uncovered significant direct interactions between TLR9 and prostaglandin analogues, specifically with the FDA-approved Bimatoprost (BI), which demonstrates the highest binding affinity (calculated as an estimated affinity) and an interesting MM/PBSA score and should be the primary focus of future research into the treatment of autoimmune diseases. In addition, the modelled structure of human-TLR9 and its binding site described in this study could serve as a useful starting point for the development of additional inhibitors.     

Recent progress of fully synthetic carbohydrate-based vaccine using TLR agonist as build-in adjuvant

This review highlights recent advances in developing full synthetic carbohydrate antigen based vaccines, with an emphasis on the structure-activity relationships that provide a primary basis for future vaccine design and immunotherapy developing.     

Circular RNA circRHOT1 contributes to platelet-derived growth factor BB-stimulated proliferation and migration of airway smooth muscle cells by targeting Tip60/TLR4

BackgroundAsthma serves as a chronic inflammatory respiratory diseases disorder. It mainly occurs airway inflammation and remodeling. Circular RNA (circRNA) is mainly the control of cancer disease and asthma. In specific work, we were interested in the function of circRHOT1 in the advances in asthma.MethodsEffects of platelet-derived growth factor BB (PDGF-BB) on airway smooth muscle cells (ASMCs) and remolded simulation cells were assessed. The expressions of circRHOT1, TLR4, and Tip60 were detected by qRT-PCR and the increase and decrease of cell number were analyzed by cell count-8 (CCK-8), Transwell or flow cytometry. At the same time, the levels of PCNA, IGF1R protein were determined by western blot approach. The correlation of circRHOT1, TLR4, and Tip60 was analyzed by qRT-PCR, western blotting, ChIP, and RNA pulldown.ResultsCircRHOT1 was significantly elevated in the serum collected from asthmatic patients. PDGF-BB had a positive effect on ASMCs with enhanced levels of circRHOT1, TLR4, and Tip60. Depletion of circRHOT1 avoided wider impact and migration but induced cell apoptosis. CircRHOT1 contributed PDGF-BB had a direct impact on the proliferation and migration of ASMCs by regulating TLR4. Mechanically, circRHOT1 could cooperate with acetyltransferase Tip60 in ASMCs. CircRHOT1 silencing was easy to have an adverse effect on the enrichment of Tip60 on TLR4 promoter in ASMCs. The depletion of circRHOT1 or Tip60 reduced h3k27ac and RNA polymerase II on the TLR4 promoter. Consistently, the inhibition of circRHOT1 or Tip60 reduced TLR4 expression in ASMCs. The deletion of circRHOT1 could reduce the expression level of TLR4, but the overexpression of Tip60 was easy to have a positive effect on the down-regulation of ASMCs. In addition, Tip60 could inhibit ASMCs proliferation and migration caused by PDGF-BB stimulation. CircRHOT1 could use PDGF-BB to stimulate ASMCs proliferation and migration when Tip60 regulation was implemented.ConclusionsWe can think that based on the related effects of Tip60/TLR4, circular RNA circRHOT1 with platelet-derived growth factor BB can stimulate airway smooth muscle cells to proliferate or migrate, and circRHOT1 is an important target for the treatment of asthma.     

Detection of flagellin by interaction with human recombinant TLR5 immobilized in liposomes

Digestive diseases caused by flagellated bacteria are a huge public health problem worldwide and rapid detection methods are needed for contaminated environments. In this study, we propose a method to detect patterns associated with pathogens based on the properties of the innate immune system. Specifically, we use Toll-like receptor 5 (TLR5), a transmembrane protein that specifically recognizes flagellin (the structural protein of bacterial flagella). TLR5, which was obtained by recombinant production in insect cells, was immobilized into liposomes to form TLR5-proteoliposomes. Through surface plasmon resonance (SPR) and competition flow cytometry assays, the sensitivity of proteoliposomes to recognize Escherichia coli and Salmonella typhimurium flagellin was evaluated. In addition, we compared the results obtained by immobilizing anti-flagellin antibodies into liposomes. The results of the flagellin-affinity tests, expressed as an SPR kinetic rate constant ratio in the equilibrium equation K _D?=?k _d/k _a, showed values of 13.8?×?10^?9 and 7.73?×?10^?9?M for the TLR5-proteoliposomes and anti-flagellin antibodies, respectively, against S. typhimurium. The anti-flagellin affinity results for E. coli showed K _D of 84.1?×?10^?8?M for SPR assays and K _D of 3.5?×?10^?8?M for competitive flow cytometry, which was used as a detection system without the immobilization of proteoliposomes. This research demonstrates the practical possibility of using proteoliposomes as recognition elements in the generation of systems for the rapid detection of flagellated bacteria, which could help avoid consumption of contaminated food by humans and thereby prevent intestinal infections.     

Polysaccharides ofEremurus. X. Characteristics of the polysaccharides ofEremurus lactiflorus andE. luteus

Water-soluble polysaccharides have been isolated from two species ofEremurus — E. lactiflorus andE. luteus — with yields of 13.5% and 20.5%, respectively. They contained mainly glucose and mannose in ratios of 1:5 and 1:3.1. The polysaccharides ofE. lactiflorus were separated from a column of DEAE-cellulose. The yield of neutral fraction was 10.3%. Gel filtration of the polysaccharides on Sephadex G-200 showed their polydispersity. Homogeneous fractions were obtained by fractional precipitation with ethanol. They have been characterized with respect to monosaccharide composition, molecular weight, and IR spectra.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 604–608, September–October, 1979.