Vibrational spectroscopy coupled with machine learning sheds light on the cellular effects induced by rationally designed TLR4 agonists.

In this work, we present the potential of Fourier transform infrared (FTIR) microspectroscopy to compare on whole cells, in an unbiased and untargeted way, the capacity of bacterial lipopolysaccharide (LPS) and two rationally designed molecules (FP20 and FP20Rha) to activate molecular circuits of innate immunity. These compounds are important drug hits in the development of vaccine adjuvants and tumor immunotherapeutics. The biological assays indicated that FP20Rha was more potent than FP20 in inducing cytokine production in cells and in stimulating IgG antibody production post-vaccination in mice. Accordingly, the overall significant IR spectral changes induced by the treatment with LPS and FP20Rha were similar, lipids and glycans signals being the most diagnostic, while the effect of the less potent molecule FP20 on cells resulted to be closer to control untreated cells. We propose here the use of FTIR spectroscopy supported by artificial intelligence (AI) to achieve a more holistic understanding of the cell response to new drug candidates while screening them in cells.

Novel TLR4-Activating Vaccine Adjuvant Enhances the Production of Enterococcus faecium-binding Antibodies.

Vaccines are one of the greatest achievements of modern medicine. Due to their safer profile, the latest investigations usually focus on subunit vaccines. However, the active component often needs to be coupled with an adjuvant to be effective and properly trigger an immune response. We are developing a new synthetic monosaccharide-based TLR4 agonist, such as glucosamine-derived compounds FP18 and FP20, as a potential vaccine adjuvant. In this study, we present a new FP20 derivative, FP20Hmp, with a hydroxylated ester linked to the glucosamine core. We show that the modification introduced improves the activity of the adjuvant and its solubility. This study presents the synthesis of FP20Hmp, its in vitro characterization, and in vivo activity while coupled with the ovalbumin antigen or in formulation with an enterococcal antigen. We show that FP20Hmp enables increased production of antigen-specific antibodies that bind to the whole bacterium.

Cleaner synthesis of preclinically validated vaccine adjuvants.

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.

Overcoming Challenges in Chemical Glycosylation to Achieve Innovative Vaccine Adjuvants Possessing Enhanced TLR4 Activity.

Lipopolysaccharide (LPS) mimicry leading to toll-like receptor 4 (TLR4) active compounds has been so far based mainly on reproducing the lipid A portion of LPS. Our work led to a series of structurally simplified synthetic TLR4 agonists in preclinical development as vaccine adjuvants called FPs. FPs bind MD2/TLR4 similarly to lipid A, inserting the lipid chains in the MD2 lipophilic cavity. A strategy to improve FPs' target affinity is introducing a monosaccharide unit in C6, mimicking the first sugar of the LPS core. We therefore designed a panel of FP derivatives bearing different monosaccharides in C6. We report here the synthesis and optimization of FPs' C6 glycosylation, which presented unique challenges and limitations. The biological activity of glycosylated FP compounds was preliminarily assessed in vitro in HEK-Blue cells. The new molecules showed a higher potency in stimulating TLR4 activation when compared to the parent molecule while maintaining TLR4 selectivity.

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.

Highly Selective SERCA2a Activators: Preclinical Development of a Congeneric Group of First-in-Class Drug Leads against Heart Failure.

The stimulation of sarcoplasmic reticulum calcium ATPase SERCA2a emerged as a novel therapeutic strategy to efficiently improve overall cardiac function in heart failure (HF) with reduced arrhythmogenic risk. Istaroxime is a clinical-phase IIb compound with a double mechanism of action, Na+/K+ ATPase inhibition and SERCA2a stimulation. Starting from the observation that istaroxime metabolite PST3093 does not inhibit Na+/K+ ATPase while stimulates SERCA2a, we synthesized a series of bioisosteric PST3093 analogues devoid of Na+/K+ ATPase inhibitory activity. Most of them retained SERCA2a stimulatory action with nanomolar potency in cardiac preparations from healthy guinea pigs and streptozotocin (STZ)-treated rats. One compound was further characterized in isolated cardiomyocytes, confirming SERCA2a stimulation and in vivo showing a safety profile and improvement of cardiac performance following acute infusion in STZ rats. We identified a new class of selective SERCA2a activators as first-in-class drug candidates for HF treatment.

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.

Increasing the Chemical Variety of Small-Molecule-Based TLR4 Modulators: An Overview.

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.