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.

A new advanced cellular model of functional cholinergic-like neurons developed by reprogramming the human SH-SY5Y neuroblastoma cell line.

Modeling human neuronal properties in physiological and pathological conditions is essential to identify novel potential drugs and to explore pathological mechanisms of neurological diseases. For this purpose, we generated a three-dimensional (3D) neuronal culture, by employing the readily available human neuroblastoma SH-SY5Y cell line, and a new differentiation protocol. The entire differentiation process occurred in a matrix and lasted 47 days, with 7 days of pre-differentiation phase and 40 days of differentiation, and allowed the development of a 3D culture in conditions consistent with the physiological environment. Neurons in the culture were electrically active, were able to establish functional networks, and showed features of cholinergic neurons. Hence here we provide an easily accessible, reproducible, and suitable culture method that might empower studies on synaptic function, vesicle trafficking, and metabolism, which sustain neuronal activity and cerebral circuits. Moreover, this novel differentiation protocol could represent a promising cellular tool to study physiological cellular processes, such as migration, differentiation, maturation, and to develop novel therapeutic approaches.

Selective SERCA2a activator as a candidate for chronic heart failure therapy.

BACKGROUND: The sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2a) depression substantially contributes to diastolic dysfunction in heart failure (HF), suggesting that SERCA2a stimulation may be a mechanism-based HF therapy. Istaroxime is a drug endowed with both a SERCA2a stimulatory activity and a Na+/K+ pump inhibitory activity for acute HF treatment. Its main metabolite PST3093 shows a more favorable therapeutic profile as compared to the parent drug, but it is still unsuitable for chronic usage. Novel PST3093 derivatives have been recently developed for oral (chronic) HF treatment; compound 8 was selected among them and here characterized. METHODS: Effects of compound 8 were evaluated in a context of SERCA2a depression, by using streptozotocin-treated rats, a well-known model of diastolic dysfunction. The impact of SERCA2a stimulation by compound 8 was assessed at the cellular level ad in vivo, following i.v. infusion (acute effects) or oral administration (chronic effects). RESULTS: As expected from SERCA2a stimulation, compound 8 induced SR Ca2+ compartmentalization in STZ myocytes. In-vivo echocardiographic analysis during i.v. infusion and after repeated oral administration of compound 8, detected a significant improvement of diastolic function. Moreover, compound 8 did not affect electrical activity of healthy guinea-pig myocytes, in line with the absence of off-target effects. Finally, compound 8 was well tolerated in mice with no evidence of acute toxicity. CONCLUSIONS: The pharmacological evaluation of compound 8 indicates that it may be a safe and selective drug for a mechanism-based treatment of chronic HF by restoring SERCA2a activity.

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.

A small-molecule TLR4 antagonist reduced neuroinflammation in female E4FAD mice.

BACKGROUND: APOE genotype is the greatest genetic risk factor for sporadic Alzheimer's disease (AD). APOE4 increases AD risk up to 12-fold compared to APOE3, an effect that is greater in females. Evidence suggests that one-way APOE could modulate AD risk and progression through neuroinflammation. Indeed, APOE4 is associated with higher glial activation and cytokine levels in AD patients and mice. Therefore, identifying pathways that contribute to APOE4-associated neuroinflammation is an important approach for understanding and treating AD. Human and in vivo evidence suggests that TLR4, one of the key receptors involved in the innate immune system, could be involved in APOE-modulated neuroinflammation. Consistent with that idea, we previously demonstrated that the TLR4 antagonist IAXO-101 can reduce LPS- and Aß-induced cytokine secretion in APOE4 glial cultures. Therefore, the goal of this study was to advance these findings and determine whether IAXO-101 can modulate neuroinflammation, Aß pathology, and behavior in mice that express APOE4. METHODS: We used mice that express five familial AD mutations and human APOE3 (E3FAD) or APOE4 (E4FAD). Female and male E4FAD mice and female E3FAD mice were treated with vehicle or IAXO-101 in two treatment paradigms: prevention from 4 to 6 months of age or reversal from 6 to 7 months of age. Learning and memory were assessed by modified Morris water maze. Aß deposition, fibrillar amyloid deposition, astrogliosis, and microgliosis were assessed by immunohistochemistry. Soluble levels of Aß and apoE, insoluble levels of apoE and Aß, and IL-1ß were measured by ELISA. RESULTS: IAXO-101 treatment resulted in lower Iba-1 coverage, lower number of reactive microglia, and improved memory in female E4FAD mice in both prevention and reversal paradigms. IAXO-101-treated male E4FAD mice also had lower Iba-1 coverage and reactivity in the RVS paradigm, but there was no effect on behavior. There was also no effect of IAXO-101 treatment on neuroinflammation and behavior in female E3FAD mice. CONCLUSION: Our data supports that TLR4 is a potential mechanistic therapeutic target for modulating neuroinflammation and cognition in APOE4 females.

Use of Fluorescent Chemical Probes in the Study of Toll-like Receptors (TLRs) Trafficking.

Fluorescent chemical probes are used nowadays as a chemical resource to study the physiology and pharmacology of several important endogenous receptors. Different fluorescent groups have been coupled with known ligands of these receptors, allowing the visualization of their localization and trafficking. One of the most important molecular players of innate immunity and inflammation are the Toll-Like Receptors (TLRs). These Pattern-Recognition Receptors (PRR) have as natural ligands microbial-derived pathogen-associated molecular patterns (PAMPs) and also endogenous molecules called danger-associated molecular patterns (DAMPs). These ligands activate TLRs to start a response that will determine the host's protection and overall cell survival but can also lead to chronic inflammation and autoimmune syndromes. TLRs action is tightly related to their subcellular localization and trafficking. Understanding this trafficking phenomenon can enlighten critical molecular pathways that might allow to decipher the causes of different diseases. In this chapter, the study of function, localization and trafficking of TLRs through the use of chemical probes will be discussed. Furthermore, an example protocol of the use of fluorescent chemical probes to study TLR4 trafficking using high-content analysis will be described.

Emerging Extracellular Molecular Targets for Innovative Pharmacological Approaches to Resistant Mtb Infection.

Emerging pharmacological strategies that target major virulence factors of antibiotic-resistant Mycobacterium tuberculosis (Mtb) are presented and discussed. This review is divided into three parts corresponding to structures and functions important for Mtb pathogenicity: the cell wall, the lipoarabinomannan, and the secretory proteins. Within the cell wall, we further focus on three biopolymeric sub-components: mycolic acids, arabinogalactan, and peptidoglycan. We present a comprehensive overview of drugs and drug candidates that target cell walls, envelopes, and secretory systems. An understanding at a molecular level of Mtb pathogenesis is provided, and potential future directions in therapeutic strategies are suggested to access new drugs to combat the growing global threat of antibiotic-resistant Mtb infection.

Microbiota-Derived Natural Products Targeting Cancer Stem Cells: Inside the Gut Pharma Factory.

Cancer stem cells (CSCs) have drawn much attention as important tumour-initiating cells that may also be crucial for recurrence after chemotherapy. Although the activity of CSCs in various forms of cancer is complex and yet to be fully elucidated, opportunities for therapies targeting CSCs exist. CSCs are molecularly distinct from bulk tumour cells, so they can be targeted by exploiting their signature molecular pathways. Inhibiting stemness has the potential to reduce the risk posed by CSCs by limiting or eliminating their capacity for tumorigenesis, proliferation, metastasis, and recurrence. Here, we briefly described the role of CSCs in tumour biology, the mechanisms involved in CSC therapy resistance, and the role of the gut microbiota in cancer development and treatment, to then review and discuss the current advances in the discovery of microbiota-derived natural compounds targeting CSCs. Collectively, our overview suggests that dietary intervention, toward the production of those identified microbial metabolites capable of suppressing CSC properties, is a promising approach to support standard chemotherapy.

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.

Istaroxime Metabolite PST3093 Selectively Stimulates SERCA2a and Reverses Disease-Induced Changes in Cardiac Function.

Heart failure (HF) therapeutic toolkit would strongly benefit from the availability of ino-lusitropic agents with a favorable pharmacodynamics and safety profile. Istaroxime is a promising agent, which combines Na+/K+ pump inhibition with sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) stimulation; however, it has a very short half-life and extensive metabolism to a molecule named PST3093. The present work aims to investigate whether PST3093 still retains the pharmacodynamic and pharmacokinetic properties of its parent compound. We studied PST3093 for its effects on SERCA2a and Na+/K+ ATPase activities, Ca2+ dynamics in isolated myocytes, and hemodynamic effects in an in vivo rat model of diabetic [streptozotocin (STZ)-induced] cardiomyopathy. Istaroxime infusion in HF patients led to accumulation of PST3093 in the plasma; clearance was substantially slower for PST3093 than for istaroxime. In cardiac rat preparations, PST3093 did not inhibit the Na+/K+ ATPase activity but retained SERCA2a stimulatory activity. In in vivo echocardiographic assessment, PST3093 improved overall cardiac performance and reversed most STZ-induced abnormalities. PST3093 intravenous toxicity was considerably lower than that of istaroxime, and it failed to significantly interact with 50 off-targets. Overall, PST3093 is a "selective" SERCA2a activator, the prototype of a novel pharmacodynamic category with a potential in the ino-lusitropic approach to HF with prevailing diastolic dysfunction. Its pharmacodynamics are peculiar, and its pharmacokinetics are suitable to prolong the cardiac beneficial effect of istaroxime infusion. SIGNIFICANCE STATEMENT: Heart failure (HF) treatment would benefit from the availability of ino-lusitropic agents with favourable profiles. PST3093 is the main metabolite of istaroxime, a promising agent combining Na+/K+ pump inhibition and sarcoplasmic reticulum Ca2+ ATPase2a (SERCA2a) stimulation. PST3093 shows a longer half-life in human circulation compared to istaroxime, selectively activates SERCA2a, and improves cardiac performance in a model of diabetic cardiomyopathy. Overall, PST3093 as a selective SERCA2a activator can be considered the prototype of a novel pharmacodynamic category for HF treatment.

A Global Picture of Molecular Changes Associated to LPS Treatment in THP-1 Derived Human Macrophages by Fourier Transform Infrared Microspectroscopy.

Macrophages are among the first immune cells involved in the initiation of the inflammatory response to protect the host from pathogens. THP-1 derived macrophages (TDM) are used as a model to study the pro-inflammatory effects of lipopolysaccharide (LPS) exposure. Intact TDM cells were analysed by Fourier transform infrared (FTIR) microspectroscopy, supported by multivariate analysis, to obtain a snapshot of the molecular events sparked by LPS stimulation in macrophage-like cells. This spectroscopic analysis enabled the untargeted identification of the most significant spectral components affected by the treatment, ascribable mainly to lipid, protein, and sulfated sugar bands, thus stressing the fundamental role of these classes of molecules in inflammation and in immune response. Our study, therefore, shows that FTIR microspectroscopy enabled the identification of spectroscopic markers of LPS stimulation and has the potential to become a tool to assess those global biochemical changes related to inflammatory and anti-inflammatory stimuli of synthetic and natural immunomodulators different from LPS.

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.

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.

A Highly Sensitive Luminescent Biosensor for the Microvolumetric Detection of the Pseudomonas aeruginosa Siderophore Pyochelin.

The pyochelin (PCH) siderophore produced by the pathogenic bacterium Pseudomonas aeruginosa is an important virulence factor, acting as a growth promoter during infection. While strong evidence exists for PCH production in vivo, PCH quantification in biological samples is problematic due to analytical complexity, requiring extraction from large volumes and time-consuming purification steps. Here, the construction of a bioluminescent whole cell-based biosensor, which allows rapid, sensitive, and single-step PCH quantification in biological samples, is reported. The biosensor was engineered by fusing the promoter of the PCH biosynthetic gene pchE to the luxCDABE operon, and the resulting construct was inserted into the chromosome of the ΔpvdAΔpchDΔfpvA siderophore-null P. aeruginosa mutant. A bioassay was setup in a 96-well microplate format, enabling the contemporary screening of several samples in a few hours. A linear response was observed for up to 40 nM PCH, with a lower detection limit of 1.64 ± 0.26 nM PCH. Different parameters were considered to calibrate the biosensor, and a detailed step-by-step operation protocol, including troubleshooting specific problems that can arise during sample preparation, was established to achieve rapid, sensitive, and specific PCH quantification in both P. aeruginosa culture supernatants and biological samples. The biosensor was implemented as a screening tool to detect PCH-producing P. aeruginosa strains on a solid medium.

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.

Synthetic glycolipid-based TLR4 antagonists negatively regulate TRIF-dependent TLR4 signalling in human macrophages.

TLRs, including TLR4, play a crucial role in inflammatory-based diseases, and TLR4 has been identified as a therapeutic target for pharmacological intervention. In previous studies, we investigated the potential of FP7, a novel synthetic glycolipid active as a TLR4 antagonist, to inhibit haematopoietic and non-haematopoietic MyD88-dependent TLR4 pro-inflammatory signalling. The main aim of this study was to investigate the action of FP7 and its derivative FP12 on MyD88-independent TLR4 signalling in THP-1 derived macrophages. Western blotting, Ab array and ELISA approaches were used to explore the effect of FP7 and FP12 on TRIF-dependent TLR4 functional activity in response to LPS and other endogenous TLR4 ligands in THP-1 macrophages. A different kinetic in the inhibition of endotoxin-driven TBK1, IRF3 and STAT1 phosphorylation was observed using different LPS chemotypes. Following activation of TLR4 by LPS, data revealed that FP7 and FP12 inhibited TBK1, IRF3 and STAT1 phosphorylation which was associated with down-regulation IFN-ß and IP-10. Specific blockage of the IFN type one receptor showed that these novel molecules inhibited TRIF-dependent TLR4 signalling via IFN-ß pathways. These results add novel information on the mechanism of action of monosaccharide FP derivatives. The inhibition of the TRIF-dependent pathway in human macrophages suggests potential therapeutic uses for these novel TLR4 antagonists in pharmacological interventions on inflammatory diseases.

Electrospinning for drug delivery applications: A review.

Drug delivery devices are promising tools in the pharmaceutical field, as they are able to maximize the therapeutic effects of the delivered drug while minimizing the undesired side effects. In the past years, electrospun nanofibers attracted rising attention due to their unique features, like biocompatibility and broad flexibility. Incorporation of active principles in nanofibrous meshes proved to be an efficient method for in situ delivery of a wide range of drugs, expanding the possibility and applicability of those devices. In this review, the principle of electrospinning and different fields of applications are treated to give an overview of the recent literature, underlining the easy tuning and endless combination of this technique, that in the future could be the new frontier of personalized medicine.

The Multi-Level Mechanism of Action of a Pan-Ras Inhibitor Explains its Antiproliferative Activity on Cetuximab-Resistant Cancer Cells.

Ras oncoproteins play a crucial role in the onset, maintenance, and progression of the most common and deadly human cancers. Despite extensive research efforts, only a few mutant-specific Ras inhibitors have been reported. We show that cmp4-previously identified as a water-soluble Ras inhibitor- targets multiple steps in the activation and downstream signaling of different Ras mutants and isoforms. Binding of this pan-Ras inhibitor to an extended Switch II pocket on HRas and KRas proteins induces a conformational change that down-regulates intrinsic and GEF-mediated nucleotide dissociation and exchange and effector binding. A mathematical model of the Ras activation cycle predicts that the inhibitor severely reduces the proliferation of different Ras-driven cancer cells, effectively cooperating with Cetuximab to reduce proliferation even of Cetuximab-resistant cancer cell lines. Experimental data confirm the model prediction, indicating that the pan-Ras inhibitor is an appropriate candidate for medicinal chemistry efforts tailored at improving its currently unsatisfactory affinity.