Towards more efficient synthetic immunomodulators: biological characterization and mechanism of action of monosaccharide-derived TLR4 agonists

Toll-like receptors (TLRs), including TLR4, play a crucial role in innate immunity activation, and small molecular TLR4 activators (agonists) are in the preclinical and clinical phases of development as vaccine adjuvants or tumor immunotherapeutics. Recently, we generated novel glucosamine-derived compounds, FP molecules, that are active as TLR4 agonists. Despite their chemical structure differing from LPS, some of these compounds, including compound FP18, mimicked the biological activity of LPS and its capacity to activate TLR4 and its downstream pathways. In contrast to FP18, compound FP20 showed immunostimulant activity that was only partially due to TLR4 agonism. This activity was mainly associated with NLRP3 inflammasome activation. We generated a panel of glycosylated FP20 derivatives (glyco-FP20) bearing different monosaccharides linked to C6 of the glucosamine. The biological activity of glyco-FP20 was related to TLR4 activation, as assessed from preliminary experiments in HEK-Blue cells. We presented 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 derivatives were potent TLR4 agonists inducing TLR4/MyD88 signalling pathways that led 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 activated TLR4/TRIF signalling, associated with phosphorylation of STAT1/IRF3, leading to the production of IFN-β in THP-1-derived macrophages. Furthermore, using a specific GSD inhibitor (U73), we demonstrated the ability of FP20 and glyco-FP20 to induce GSD-dependent pyroptosis, which was associated with IL-1β/IL-18 and LDH release in THP-1-derived macrophages. These results show that the optimization of FP20 glycosylation can increase the biological potency of the parent molecule and can be used in preclinical development as vaccine adjuvants or macrophage-based cancer immunotherapy.