Toll-like receptors (TLRs), including TLR4, play a crucial role in innate immunity activation, and small molecular TLR4 activators (agonists) are in preclinical and clinical phase of development as vaccine adjuvant or tumor immunotherapeuutics. .Recently, we have generated novel glucosamine-derived compounds, FP molecules, that are active as TLR4 agonists. Despite their chemical structure different from LPS, some of this compounds, included compound FP18, mimic the biological activity of LPS and its capacity to activate TLR4 and its downstream pathways.. Differently from FP18, compound FP20 has an immunostimulant activity only partially due to TLR4 agonism. This activity is mainly associated to NLRP3 inflammasome activation. We generated a panel of glycosylated -P20 derivatives (glyco-FP20) bearing different monosaccharides linked to C6 of the glucosamine. The biological activity of glyco-FP20 is related to TLR4 activation, as assessed from preliminary experiments in HEK-Blue cells. We present here a comprehensive study of the mechanism of action of glyco-FP20 derivatives and their effect on TLR4 signalling leading to macrophage M1 polarisation and pyroptosis in THP-1 derived macrophages. Results revealed that similarly to LPS, and differently from FP20, glyco-FP20 were potent TLR4 agonists inducing TLR4/MyD88 signalling pathways leading to M1 macrophage polarisation associated with NF-kB activation/translocation and release of a number of proinflammatory mediators in THP-1-derived macrophages. In particular, compound FP20 Rha activates TLR4/TRIF signalling associated with phosphorylation of STAT1/IRF3 leading to production of IFN- in THP-1 derived macrophages. Furthermore, using specific GSD inhibitor (U73) we demonstrated the ability of FP20 and glyco-FP20 to induce GSD-dependent pyroptosis which
Towards more efficient synthetic immunomodulators: biological characterization and mechanism of action of monosaccharide-derived TLR4 agonists
