The identification of the bacterial endotoxin receptors for innate immunity, most notably TLR4 (Toll-like receptor 4), has sparked great interest in therapeutic manipulation of the innate immune system. In the present mini-review, several natural and synthetic molecules that modulate the TLR4-mediated LPS (lipopolysaccharide) signalling in animals and humans are considered, and their mechanisms of action are discussed. The process of LPS sensing and signal amplification in humans is based on the sequential action of specific receptors situated in the extracellular side of the innate immunity cells, which bind and transfer LPS to TLR4: LBP (LPS-binding protein), CD14, MD-2 (myeloid differentiation protein 2). We classified the compounds active on TLR4 pathway depending on the specific molecular targets (LPS, LBP, CD14, MD-2 or TLR4). Small molecules developed by our group are described that inhibit LPS-stimulated TLR4 activation by selectively targeting the LPS-CD14 interaction. These compounds have an interesting antiseptic shock, anti-inflammatory and anti-neuropathic pain activity in vivo.

Monosaccharide lipid A mimetics composed by a glucosamine core linked to two fatty acid chains and bearing one or two phosphates have been synthesized. While compounds 1 and 2, with one phosphate group, were practically inactive in inhibiting LPS-induced TLR4 signaling and cytokine production in HEK-blue™ cells and murine macrophages, compound 3 with two phosphates was found to be active in efficiently inhibiting TLR4 signal in both cell types. The direct interaction of molecule 3 with MD-2 co-receptor has been investigated by means of NMR and molecular modeling/docking analysis. This compound also interacts directly with CD14 receptor, stimulating its internalization by endocytosis. Experiments on macrophages show that the effect on CD14 reinforces the activity on MD-2.TLR4, because compound 3 activity is higher when CD14 is important for TLR4 signaling i,e, at low LPS concentration. The dual MD-2 and CD14 targeting, accompanied by good solubility in water and lack of toxicity, suggests the use of monosaccharide 3 as a lead compound to develop drugs directed against TLR4-related syndromes.

An increasing number of pathologies have been linked to Toll-like receptor 4 (TLR4) activation and signaling, therefore new hit and lead compounds targeting this receptor activation process are urgently needed. We report on the synthesis and biological properties of glycolipids based on glucose and trehalose scaffolds which potently inhibit TLR4 activation and signaling in vitro and in vivo. Structure-activity relationship studies on these compounds indicate that the presence of fatty ester chains in the molecule is a primary prerequisite for biological activity and point to facial amphiphilicity as a preferred architecture for TLR4 antagonism. The cationic glycolipids here presented can be considered as new lead compounds for the development of drugs targeting TLR4 activation and signaling in infectious, inflammatory, and autoimmune diseases. Interestingly, the biological activity of the best drug candidate was retained after adsorption at the surface of colloidal gold nanoparticles, broadening the options for clinical development.

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Toll-like receptor 4 (TLR4), the receptor of bacterial endotoxins in mammalians, plays a pivotal role in the induction of innate immunity and inflammation. TLR4 activation by bacterial lipopolysaccharide (LPS) is achieved by the coordinate and sequential action of three other proteins, the lipopolysaccharide binding protein (LBP), the cluster differentiation antigen CD14, and the myeloid differentiation protein (MD-2) receptors, that bind LPS and present it in a monomeric form to TLR4 by forming the activated [TLR4·MD-2·LPS]2 complex. Small molecules and nanoparticles active in modulating the TLR4 signal by targeting directly the MD-2·TLR4 complex or by interfering in other points of the TLR4 signaling are presented in this paper. These compounds have great pharmacological interest as vaccine adjuvants, immunotherapeutics, anti-sepsis, and anti-inflammatory agents.

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