New Glucosamine-Based TLR4 Agonists: Design, Synthesis, Mechanism of Action, and In Vivo Activity as Vaccine Adjuvants.

We disclose here a panel of small-molecule TLR4 agonists (the FP20 series) whose structure is derived from previously developed TLR4 ligands (FP18 series). The new molecules have increased chemical stability and a shorter, more efficient, and scalable synthesis. The FP20 series showed selective activity as TLR4 agonists with a potency similar to FP18. Interestingly, despite the chemical similarity with the FP18 series, FP20 showed a different mechanism of action and immunofluorescence microscopy showed no NF-κB nor p-IRF-3 nuclear translocation but rather MAPK and NLRP3-dependent inflammasome activation. The computational studies related a 3D shape of FP20 series with agonist binding properties inside the MD-2 pocket. FP20 displayed a CMC value lower than 5 µM in water, and small unilamellar vesicle (SUV) formation was observed in the biological activity concentration range. FP20 showed no toxicity in mouse vaccination experiments with OVA antigen and induced IgG production, thus indicating a promising adjuvant activity.

Green and Roasted Coffee Extracts Inhibit Interferon-ß Release in LPS-Stimulated Human Macrophages.

The anti-inflammatory activity of coffee extracts is widely recognized and supported by experimental evidence, in both in vitro and in vivo settings, mainly murine models. Here, we investigated the immunomodulatory properties of coffee extracts from green (GCE) and medium-roasted (RCE) Coffea canephora beans in human macrophages. The biological effect of GCE and RCE was characterized in LPS-stimulated THP-1-derived human macrophages (TDM) as a model of inflammation. Results showed decreased amounts of TNF-α, IL-6 and IL-1ß and a strong dose-dependent inhibition of interferon-ß (IFN-ß) release. Molecular mechanism of IFN-ß inhibition was further investigated by immunofluorescence confocal microscopy analysis that showed a diminished nuclear translocation of p-IRF-3, the main transcription factor responsible for IFN-ß synthesis. The inhibition of IFN-ß release by RCE and GCE was also confirmed in human primary CD14+ monocytes-derived macrophages (MDM). The main component of coffee extracts, 5-O-caffeoylquinic acid (5-CQA) also inhibited IFN-ß production, through a mechanism occurring downstream to TLR4. Inhibition of IFN-ß release by coffee extracts parallels with the activity of their main phytochemical component, 5-CQA, thus suggesting that this compound is the main responsible for the immunomodulatory effect observed. The application of 5-CQA and coffee derived-phytoextracts to target interferonopathies and inflammation-related diseases could open new pharmacological and nutritional perspectives.

Synthetic Glycolipids as Molecular Vaccine Adjuvants: Mechanism of Action in Human Cells and In Vivo Activity.

Modern adjuvants for vaccine formulations are immunostimulating agents whose action is based on the activation of pattern recognition receptors (PRRs) by well-defined ligands to boost innate and adaptive immune responses. Monophosphoryl lipid A (MPLA), a detoxified analogue of lipid A, is a clinically approved adjuvant that stimulates toll-like receptor 4 (TLR4). The synthesis of MPLA poses manufacturing and quality assessment challenges. Bridging this gap, we report here the development and preclinical testing of chemically simplified TLR4 agonists that could sustainably be produced in high purity and on a large scale. Underpinned by computational and biological experiments, we show that synthetic monosaccharide-based molecules (FP compounds) bind to the TLR4/MD-2 dimer with submicromolar affinities stabilizing the active receptor conformation. This results in the activation of MyD88- and TRIF-dependent TLR4 signaling and the NLRP3 inflammasome. FP compounds lack in vivo toxicity and exhibit adjuvant activity by stimulating antibody responses with a potency comparable to MPLA.