Analysis of the intramolecular 1,7-lactone of N-acetylneuraminic acid using HPLC-MS: relationship between detection and stability.

A subclass of the sialic acid family consists of intramolecular lactones that may function as key indicators of physiological and pathological states. However, the existence of these compounds in free form is highly improbable, since they are unlikely to exist in an aqueous solution due to their lability. Current analytical method used to detect them in biological fluids has not recognized their reactivity in solution and is prone to misidentification. However, recent advances in synthetic methods for 1,7-lactones have allowed the preparation of these sialic acid derivatives as authentic reference standards. We report here the development of a new HPLC-MS method for the simultaneous detection of the 1,7-lactone of N-acetylneuraminic acid, its γ-lactone derivative, and N-acetylneuraminic acid that overcomes the limitations of the previous analytical procedure for their identification.

Small-Molecules as Chemiluminescent Probes to Detect Lipase Activity.

The set-up of highly sensitive detection tools to evaluate lipase activity remains a central goal in different fields. In this context, we proposed new chemiluminescent 1,2-dioxetane luminophores, sharing an octanoyl triggerable group, to monitor lipase activity. We herein report the synthesis and both the evaluation of their luminescence emission profile and their enzyme-substrate specificity, generated by three different commercial lipases (Candida cylindracea, Pseudomonas fluorescens, and Mucor miehei) and one esterase (porcine liver esterase, PLE, as a literature control). Remarkably, the present study confirmed the applicability of these 1,2-dioxetane luminophores as (i) highly efficient, broad-range, chemiluminescent probes for the detection and the enzymatic activity evaluation of lipases and as (ii) promising candidates for the future development of both flash- and glow-type luminescence assays.

Neu3 Sialidase Activates the RISK Cardioprotective Signaling Pathway during Ischemia and Reperfusion Injury (IRI).

Coronary reperfusion strategies are life-saving approaches to restore blood flow to cardiac tissue after acute myocardial infarction (AMI). However, the sudden restoration of normal blood flow leads to ischemia and reperfusion injury (IRI), which results in cardiomyoblast death, irreversible tissue degeneration, and heart failure. The molecular mechanism of IRI is not fully understood, and there are no effective cardioprotective strategies to prevent it. In this study, we show that activation of sialidase-3, a glycohydrolytic enzyme that cleaves sialic acid residues from glycoconjugates, is cardioprotective by triggering RISK pro-survival signaling pathways. We found that overexpression of Neu3 significantly increased cardiomyoblast resistance to IRI through activation of HIF-1α and Akt/Erk signaling pathways. This raises the possibility of using Sialidase-3 activation as a cardioprotective reperfusion strategy after myocardial infarction.

Human Sarcopenic Myoblasts Can Be Rescued by Pharmacological Reactivation of HIF-1α.

Sarcopenia, an age-related decline in muscle mass and strength, is associated with metabolic disease and increased risk of cardiovascular morbidity and mortality. It is associated with decreased tissue vascularization and muscle atrophy. In this work, we investigated the role of the hypoxia inducible factor HIF-1α in sarcopenia. To this end, we obtained skeletal muscle biopsies from elderly sarcopenic patients and compared them with those from young individuals. We found a decrease in the expression of HIF-1α and its target genes in sarcopenia, as well as of PAX7, the major stem cell marker of satellite cells, whereas the atrophy marker MURF1 was increased. We also isolated satellite cells from muscle biopsies and cultured them in vitro. We found that a pharmacological activation of HIF-1α and its target genes caused a reduction in skeletal muscle atrophy and activation of PAX7 gene expression. In conclusion, in this work we found that HIF-1α plays a role in sarcopenia and is involved in satellite cell homeostasis. These results support further studies to test whether pharmacological reactivation of HIF-1α could prevent and counteract sarcopenia.

2-Arachidonoylglycerol Synthesis: Facile and Handy Enzymatic Method That Allows to Avoid Isomerization.

A simple and practical synthesis of 2-arachidonoyl glycerol (2-AG), an endogenous agonist for cannabinoid receptors, based on a two-step enzymatic process and a chemical coupling, was achieved with a good yield and negligible amount of the isomerization product 1-AG. Commercial preparation of immobilized lipase from Mucor miehei (MML) was selected as the most suitable enzyme to catalyze the efficient protection of glycerol using vinyl benzoate as an acyl transfer reagent in tetrahydrofuran. The same enzyme was used to remove the protective groups in positions 1 and 3. Owing to the mild neutral conditions and easy suitability of the method, 2-AG was obtained without any isomerization to the more stable 1-AG and air oxidation of acid chain. The synthetic method proposed here allows us to easily obtain 2-AG from the protected precursor in a one-step reaction without purification requirement.

The antithetic role of ceramide and sphingosine-1-phosphate in cardiac dysfunction.

Cardiovascular diseases (CVDs) are the leading cause of death globally and the number of cardiovascular patients, which is estimated to be over 30 million in 2018, represent a challenging issue for the healthcare systems worldwide. Therefore, the identification of novel molecular targets to develop new treatments is an ongoing challenge for the scientific community. In this context, sphingolipids (SLs) have been progressively recognized as potent bioactive compounds that play crucial roles in the modulation of several key biological processes, such as proliferation, differentiation, and apoptosis. Furthermore, SLs involvement in cardiac physiology and pathophysiology attracted much attention, since these molecules could be crucial in the development of CVDs. Among SLs, ceramide and sphingosine-1-phosphate (S1P) represent the most studied bioactive lipid mediators, which are characterized by opposing activities in the regulation of the fate of cardiac cells. In particular, maintaining the balance of the so-called ceramide/S1P rheostat emerged as an important novel therapeutical target to counteract CVDs. Thus, this review aims at critically summarizing the current knowledge about the antithetic roles of ceramide and S1P in cardiomyocytes dysfunctions, highlighting how the modulation of their metabolism through specific molecules, such as myriocin and FTY720, could represent a novel and interesting therapeutic approach to improve the management of CVDs.

Role of sialidase Neu3 and ganglioside GM3 in cardiac fibroblasts activation.

Cardiac fibrosis is a key physiological response to cardiac tissue injury to protect the heart from wall rupture. However, its progression increases heart stiffness, eventually causing a decrease in heart contractility. Unfortunately, to date, no efficient antifibrotic therapies are available to the clinic. This is primarily due to the complexity of the process, which involves several cell types and signaling pathways. For instance, the transforming growth factor beta (TGF-ß) signaling pathway has been recognized to be vital for myofibroblasts activation and fibrosis progression. In this context, complex sphingolipids, such as ganglioside GM3, have been shown to be directly involved in TGF-ß receptor 1 (TGF-R1) activation. In this work, we report that an induced up-regulation of sialidase Neu3, a glycohydrolytic enzyme involved in ganglioside cell homeostasis, can significantly reduce cardiac fibrosis in primary cultures of human cardiac fibroblasts by inhibiting the TGF-ß signaling pathway, ultimately decreasing collagen I deposition. These results support the notion that modulating ganglioside GM3 cell content could represent a novel therapeutic approach for cardiac fibrosis, warranting for further investigations.

A New Ultrasensitive Bioluminescence-Based Method for Assaying Monoacylglycerol Lipase.

A novel bioluminescent Monoacylglycerol lipase (MAGL) substrate 6-O-arachidonoylluciferin, a D-luciferin derivative, was synthesized, physico-chemically characterized, and used as highly sensitive substrate for MAGL in an assay developed for this purpose. We present here a new method based on the enzymatic cleavage of arachidonic acid with luciferin release using human Monoacylglycerol lipase (hMAGL) followed by its reaction with a chimeric luciferase, PLG2, to produce bioluminescence. Enzymatic cleavage of the new substrate by MAGL was demonstrated, and kinetic constants Km and Vmax were determined. 6-O-arachidonoylluciferin has proved to be a highly sensitive substrate for MAGL. The bioluminescence assay (LOD 90 pM, LOQ 300 pM) is much more sensitive and should suffer fewer biological interferences in cells lysate applications than typical fluorometric methods. The assay was validated for the identification and characterization of MAGL modulators using the well-known MAGL inhibitor JZL184. The use of PLG2 displaying distinct bioluminescence color and kinetics may offer a highly desirable opportunity to extend the range of applications to cell-based assays.

Clarifying the Use of Benzylidene Protecting Group for D-(+)-Ribono-1,4-Lactone, an Essential Building Block in the Synthesis of C-Nucleosides.

In the last two years, nucleosides analogues, a class of well-established bioactive compounds, have been the subject of renewed interest from the scientific community thanks to their antiviral activity. The COVID-19 global pandemic, indeed, spread light on the antiviral drug Remdesivir, an adenine C-nucleoside analogue. This new attention of the medical community on Remdesivir prompts the medicinal chemists to investigate once again C-nucleosides. One of the essential building blocks to synthetize these compounds is the D-(+)-ribono-1,4-lactone, but some mechanistic aspects linked to the use of different carbohydrate protecting groups remain unclear. Here, we present our investigations on the use of benzylidene as a ribonolactone protecting group useful in the synthesis of C-purine nucleosides analogues. A detailed 1D and 2D NMR structural study of the obtained compounds under different reaction conditions is presented. In addition, a molecular modeling study at the B3LYP/6-31G* level of theory with the SM8 solvation model for CHCl3 and DMSO to support the obtained results is used. This study allows for clarifying mechanistic aspects as the side reactions and structural rearrangements liked to the use of the benzylidene protecting group.

Relationship between saliva and plasma rufinamide concentrations in patients with epilepsy.

The assay of saliva samples provides a valuable alternative to the use of blood samples for therapeutic drug monitoring (TDM), at least for certain categories of patients. To determine the feasibility of using saliva sampling for the TDM of rufinamide, we compared rufinamide concentrations in paired samples of saliva and plasma collected from 26 patients with epilepsy at steady state. Within-patient relationships between plasma rufinamide concentrations and dose, and the influence of comedication were also investigated. Assay results in the two tested fluids showed a good correlation (r2  = .78, P < .0001), but concentrations in saliva were moderately lower than those in plasma (mean saliva to plasma ratio = 0.7 ± 0.2). In eight patients evaluated at three different dose levels, plasma rufinamide concentrations increased linearly with increasing dose. Patients receiving valproic acid comedication had higher dose-normalized plasma rufinamide levels than patients comedicated with drugs devoid of strong enzyme-inducing or enzyme-inhibiting activity. Overall, these findings indicate that use of saliva represents a feasible option for the application of TDM in patients treated with rufinamide. Because rufinamide concentrations are lower in saliva than in plasma, a correction factor is needed if measurements made in saliva are used as a surrogate for plasma concentrations.

2ß-3,4-Unsaturated sialic acid derivatives: Synthesis optimization, and biological evaluation as Newcastle disease virus hemagglutinin-neuraminidase inhibitors.

The optimization of the synthetic protocol to obtain the 3,4-unsaturated sialic acid derivatives, through the fine-tuning of both the Ferrier glycosylation conditions and the subsequent hydrolysis work-up, is herein reported. The accomplishment of the desired ß-anomers and some selected α-ones, in pure form, led us to evaluate their specific inhibitory activity towards NDV-HN and human sialidase NEU3. Importantly, the resulting data allowed the identification, for the first time, of three active 3,4-unsaturated sialic acid analogs, showing IC50 values against NDV-HN in the micromolar range.

Intramolecular Lactones of Sialic Acids.

The so-called "sialo-chemical-biology" has become an attractive research area, as an increasing number of natural products containing a sialic acid moiety have been shown to play important roles in biological, pathological, and immunological processes. The intramolecular lactones of sialic acids are a subclass from this crucial family that could have central functions in the discrimination of physiological and pathological conditions. In this review, we report an in-depth analysis of the synthetic achievements in the preparation of the intramolecular lactones of sialic acids (1,4-, 1,7- and γ-lactones), in their free and/or protected form. In particular, recent advances in the synthesis of the 1,7-lactones have allowed the preparation of key sialic acid derivatives. These compounds could be used as authentic reference standards for their correct determination in biological samples, thus overcoming some of the limitations of the previous analytical procedures.

Lactonization Method To Assign the Anomeric Configuration of the 3,4-Unsaturated Congeners of N-Acetylneuraminic Acid.

Assigning the correct configuration at C2 in sialosides is a standing problem because of the absence of an anomeric hydrogen. All different empirical rules that have been proposed over the years lack general applicability. In particular, the correct configuration of several 3,4-unsaturated derivatives of N-acetylneuraminic acid (Neu5Ac), which have been recently shown to be novel sialidase/neuraminidase inhibitors, could only be tentatively assigned by similarity with the reported 3,4-unsaturated 2O-methyl sialosides. In this work, we overcome this problem as we devised a rapid synthetic method to unequivocally resolve the anomeric configuration of the 3,4-unsaturated Neu5Ac derivatives through the synthesis of the corresponding unreported unsaturated 1,7-lactones. Moreover, we discovered a diagnostic 13C nuclear magnetic resonance signal that allows the formulation of a new empirical rule for the direct assignment of the C2 stereochemistry of these molecules, even when only one of the two C2 epimers is available.

Synthesis and biological evaluation of several dephosphonated analogues of CMP-Neu5Ac as inhibitors of GM3-synthase.

Previous studies demonstrated that reducing the GM3 content in myoblasts increased the cell resistance to hypoxic stress, suggesting that a pharmacological inhibition of the GM3 synthesis could be instrumental for the development of new treatments for ischemic diseases. Herein, the synthesis of several dephosphonated CMP-Neu5Ac congeners and their anti-GM3-synthase activity is reported. Biological activity testes revealed that some inhibitors almost completely blocked the GM3-synthase activity in vitro and reduced the GM3 content in living embryonic kidney 293A cells, eventually activating the epidermal growth factor receptor (EGFR) signaling cascade.

Elucidation of several neglected reactions in the GC-MS identification of sialic acids as heptafluorobutyrates calls for an urgent reassessment of previous claims.

The current analytical protocol used for the GC-MS determination of free or 1,7-lactonized natural sialic acids (Sias), as heptafluorobutyrates, overlooks several transformations. Using authentic reference standards and by combining GC-MS and NMR analyses, flaws in the analytical protocol were pinpointed and elucidated, thus establishing the scope and limitations of the method. It was demonstrated that (a) Sias 1,7-lactones, even if present in biological samples, decompose under the acidic hydrolysis conditions used for their release; (b) Sias 1,7-lactones are unpredicted artifacts, accidentally generated from their parent acids; (c) the N-acetyl group is quantitatively exchanged with that of the derivatizing perfluorinated anhydride; (d) the partial or complete failure of the Sias esterification-step with diazomethane leads to the incorrect quantification and structure attribution of all free Sias. While these findings prompt an urgent correction and improvement of the current analytical protocol, they could be instrumental for a critical revision of many incorrect claims reported in the literature.

Chemical structure, biosynthesis and synthesis of free and glycosylated pyridinolines formed by cross-link of bone and synovium collagen.

This review focuses on the chemical structure, biosynthesis and synthesis of free and glycosylated pyridinolines (Pyds), fluorescent collagen cross-links, with a pyridinium salt structure. Pyds derive from the degradation of bone collagen and have attracted attention for their use as biochemical markers of bone resorption and to assess fracture risk prediction in persons suffering from osteoporosis, bone cancer and other bone or collagen diseases. We consider and critically discuss all reported syntheses of free and glycosylated Pyds evidencing an unrevised chemistry, original and of general utility, analysis of which allows us to also support a previously suggested non-enzymatic formation of Pyds in collagen better rationalizing and justifying the chemical events.

Cenobamate: A Review of its Pharmacological Properties, Clinical Efficacy and Tolerability Profile in the Treatment of Epilepsy.

Cenobamate is a novel antiseizure medication (ASM) commercially available in Europe and in the U.S. for the treatment of focal seizures in adults. The mechanisms responsible for its antiseizure activity include enhancement of the inactivated state of voltage-gated sodium channels with blockade of the persistent sodium current and positive allosteric modulation of GABAA receptors at a non-benzodiazepine binding site. Cenobamate has a high oral bioavailability that is not influenced by food intake. The terminal half-life is 50-60 hours, allowing for once-daily dosing. Cenobamate is a CYP2C19 inhibitor and an inducer of CYP3A4 and CYP2B6, and consequently, it can cause a number of drug-drug interactions. Efficacy and safety have been evaluated in two randomized, double-blind, placebo-controlled adjunctive therapy trials in adults with focal seizures. In both trials, cenobamate decreased significantly the frequency of focal seizures, with relatively high seizure freedom rates. Adverse events most commonly reported in double-blind trials included dizziness, somnolence, headache, fatigue, and diplopia. The occurrence of three cases of drug reaction with eosinophilia and systemic symptoms (DRESS) during early clinical development led to the conduction of a Phase 3 open-label long-term safety study in a total of 1339 patients. In this study, no serious idiosyncratic adverse reactions were observed using a start-low and go-slow approach. Further studies are required to determine whether the clinical activity profile of cenobamate extends to protection against other seizure types and to evaluate its efficacy and safety profile in special patient groups such as infants, children, the elderly, and patients with comorbid conditions.

Design, Synthesis, and Antiviral Evaluation of Sialic Acid Derivatives as Inhibitors of Newcastle Disease Virus Hemagglutinin-Neuraminidase: A Translational Study on Human Parainfluenza Viruses.

Global infections with viruses belonging to the Paramyxoviridae, such as Newcastle disease virus (NDV) or human parainfluenza viruses (hPIVs), pose a serious threat to animal and human health. NDV-HN and hPIVs-HN (HN hemagglutinin-neuraminidase) share a high degree of similarity in catalytic site structures; therefore, the development of an efficient experimental NDV host model (chicken) may be informative for evaluating the efficacy of hPIVs-HN inhibitors. As part of the broad research in pursuit of this goal and as an extension of our published work on antiviral drug development, we report here the biological results obtained with some newly synthesized C4- and C5-substituted 2,3-unsaturated sialic acid derivatives against NDV. All developed compounds showed high neuraminidase inhibitory activity (IC50 0.03-13 µM). Four molecules (9, 10, 23, 24) confirmed their high in vitro inhibitory activity, which caused a significant reduction of NDV infection in Vero cells, accompanied by very low toxicity.

Therapeutic Salivary Monitoring of Perampanel in Patients with Epilepsy Using a Volumetric Absorptive Microsampling Technique.

The objective of this study was to validate a novel assay using the volumetric absorptive microsampling (VAMS) technique combined with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for the determination of the antiseizure medication perampanel in saliva and its clinical applicability in patients with epilepsy. VAMS tips were loaded with 30 µL of saliva and dried for 60 min. Analytes were extracted with methanol. The supernatant was evaporated under a gentle stream of nitrogen and reconstituted with 60 µL of methanol. Separation and quantification were achieved on a monolithic column connected to a mass spectrometer. Calibration curves were linear between 0.5 and 300 ng/mL. Intra- and inter-day accuracy was within 85.6-103.2% and intra-day and inter-day precision did not exceed 12.1%. Perampanel was stable in samples collected by VAMS and stored under different storage conditions. The VAMS-LC-MS/MS method was validated according to internationally accepted criteria and tested in patients with epilepsy who were receiving a combination of perampanel and other antiseizure medications. The method showed adequate bioanalytical performances, holding great potential as an alternative strategy to support domiciliary TDM in patients with epilepsy treated with perampanel according to the simplicity of sample collection.

Melatonin versus Sleep Deprivation for Sleep Induction in Nap Electroencephalography: Protocol for a Prospective Randomized Crossover Trial in Children and Young Adults with Epilepsy.

Electroencephalography (EEG) continues to be a pivotal investigation in children with epilepsy, providing diagnostic evidence and supporting syndromic classification. In the pediatric population, electroencephalographic recordings are frequently performed during sleep, since this procedure reduces the number of artifacts and activates epileptiform abnormalities. To date, no shared guidelines are available for sleep induction in EEG. Among the interventions used in the clinical setting, melatonin and sleep deprivation represent the most used methods. The main purpose of this study is to test the non-inferiority of 3-5 mg melatonin versus sleep deprivation in achieving sleep in nap electroencephalography in children and young adult patients with epilepsy. To test non-inferiority, a randomized crossover trial is proposed where 30 patients will be randomized to receive 3-5 mg melatonin or sleep deprivation. Each enrolled subject will perform EEG recordings during sleep in the early afternoon for a total of 60 EEGs. In the melatonin group, the study drug will be administered a single oral dose 30 min prior to the EEG recording. In the sleep deprivation group, parents will be required to subject the child to sleep deprivation the night before registration. Urinary and salivary concentrations of melatonin and of its main metabolite 6-hydroxymelatonin will be determined by using a validated LC-MS method. The present protocol aims to offer a standardized protocol for sleep induction to be applied to EEG recordings in those of pediatric age. In addition, melatonin metabolism and elimination will be characterized and its potential interference in interictal abnormalities will be assessed.