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Toll-Like Receptor 4 (TLR4) is one of the receptors of innate immunity. It is activated by Pathogen- and Damage-Associated Molecular Patterns (PAMPs and DAMPs) and triggers pro-inflammatory responses that belong to the repertoire of innate immune responses, consequently protecting against infectious challenges and boosting adaptive immunity. Mild TLR4 stimulation by non-toxic molecules resembling its natural agonist (lipid A) provided efficient vaccine adjuvants. The non-toxic TLR4 agonist monophosphoryl lipid A (MPLA) has been approved for clinical use. This suggests the development of other TLR4 agonists as adjuvants or drugs for cancer immunotherapy. TLR4 excessive activation by a Gram-negative bacteria lipopolysaccharide (LPS) leads to sepsis, while TLR4 stimulation by DAMPs is a common mechanism in several inflammatory and autoimmune diseases. TLR4 inhibition by small molecules and antibodies could therefore provide access to innovative therapeutics targeting sepsis as well as acute and chronic inflammations. The potential use of TLR4 antagonists as anti-inflammatory drugs with unique selectivity and a new mechanism of action compared to corticosteroids or other non-steroid anti-inflammatory drugs fueled the search for compounds of natural or synthetic origin able to block or inhibit TLR4 activation and signaling. The wide spectrum of clinical settings to which TLR4 inhibitors can be applied include autoimmune diseases (rheumatoid arthritis, inflammatory bowel diseases), vascular inflammation, neuroinflammations, and neurodegenerative diseases. The last advances (from 2017) in TLR4 activation or inhibition by small molecules (molecular weight <2 kDa) are reviewed here. Studies on pre-clinical validation of new chemical entities (drug hits) on cellular or animal models as well as new clinical studies on previously developed TLR4 modulators are reported. Innovative TLR4 modulators discovered by computer-assisted drug design and an artificial intelligence approach are described. Some "old" TLR4 agonists or antagonists such as MPLA or Eritoran are under study for repositioning in different pharmacological contexts. The mechanism of action of the molecules and the level of TLR4 involvement in their biological activity are critically discussed. [ABSTRACT FROM AUTHOR]

We developed synthetic glycophospholipids based on a glucosamine core (FP compounds) with potent and selective activity in stimulating Toll-Like Receptor 4 (TLR4) as agonists. These compounds have activity and toxicity profiles similar to the clinically approved adjuvant monophosphoryl lipid A (MPLA), included in several vaccine formulations, and are now in the preclinical phase of development as vaccine adjuvants in collaboration with Croda International PLC. FP compound synthesis is shorter and less expensive than MPLA preparation but presents challenges due to the use of toxic solvents and hazardous intermediates. In this paper we describe the optimization of FP compound synthesis. The use of regio- and chemoselective reactions allowed us to reduce the number of synthesis steps and improve process scalability, overall yield, safety, and Process Mass Intensity (PMI), thus paving the way to the industrial scale-up of the process., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Romerio and Peri.)

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.

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.