Modulation of CD14 and TLR4⋅MD‐2 Activities by a Synthetic Lipid A Mimetic

Abstract

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.

TLR4 antagonist FP7 inhibits LPS-induced cytokine production and glycolytic reprogramming in dendritic cells, and protects mice from lethal influenza infection

“…For DCs, FP7 IC 50 was 0.22 μM for IL-6, 0.44 μM for IL-8, and 0.32 μM for MIP-1β secretion. These results correlate with the previous data obtained with HEK-Blue™-hTLR4 cells40. Monocyte and DC viability measurements showed that FP7 was not toxic at the maximal concentration of 10 μM (Supplementary Fig.…”

“…Previous results have shown that FP7 antagonized LPS signalling in HEK-Blue™-hTLR4 cells stably co-expressing TLR4, MD-2, and CD14 with an IC 50 of 0.46 μM for 10 ng/ml of LPS40. To evaluate the IC 50 of FP7 on primary cells, monocytes and DCs were pre-treated with increasing doses of FP7 for 15 minutes before stimulation with 10 ng/ml of LPS.…”

“…The selectivity of FP7 for TLR4 had not been investigated previously40. TLR2 forms a heterodimer with TLR1 to bind triacylated lipopeptides45 and with TLR6 to recognize diacylated lipopeptides46.…”

“…In an effort to characterize a synthetic small-molecule drug candidate that can modulate innate immunity, we developed the potent TLR4 antagonist FP740 with the chemical structure of a diacylated, di-phosphorylated monosaccharide (Fig. 1c).…”

Curcumin Analog L48H37 Prevents Lipopolysaccharide-Induced TLR4 Signaling Pathway Activation and Sepsis via Targeting MD2

“…Subsequent TLR4 activation leads to the recruitment of myeloid differentiation primary-response gene 88 (MyD88), activating downstream nuclear factor (NF)-kB and mitogen-activated protein kinase (MAPK) pathways. It is well known that LPS from Gram-negative bacteria is a potent stimulant of the immune response (Rossol et al, 2011), and the activation of the NF-kB and MAPK pathways induces the upregulation and increased expression of proinflammatory genes, contributing to multiple organ dysfunction and systemic inflammatory response syndrome (Park et al, 2012;Cighetti et al, 2014).…”