Impact of Inducible Nitric Oxide Synthase Activation on Endothelial Behavior under Magnesium Deficiency.

Endothelial dysfunction is a crucial event in the early pathogenesis of cardiovascular diseases and is linked to magnesium (Mg) deficiency. Indeed, in endothelial cells, low Mg levels promote the acquisition of a pro-inflammatory and pro-atherogenic phenotype. This paper investigates the mechanisms by which Mg deficiency promotes oxidative stress and affects endothelial behavior in human umbilical vascular endothelial cells (HUVECs). Our data show that low Mg levels trigger oxidative stress initially by increasing NAPDH oxidase activity and then by upregulating the pro-oxidant thioredoxin-interacting protein TXNIP. The overproduction of reactive oxygen species (ROS) activates NF-κB, leading to its increased binding to the inducible nitric oxide synthase (iNOS) promoter, with the consequent increase in iNOS expression. The increased levels of nitric oxide (NO) generated by upregulated iNOS contribute to disrupting endothelial cell function by inhibiting growth and increasing permeability. In conclusion, we provide evidence that multiple mechanisms contribute to generate a pro-oxidant state under low-Mg conditions, ultimately affecting endothelial physiology. These data add support to the notion that adequate Mg levels play a significant role in preserving cardiovascular health and may suggest new approaches to prevent or manage cardiovascular diseases.

Chios Mastic Gum: Chemical Profile and Pharmacological Properties in Inflammatory Bowel Disease: From the Past to the Future.

Chios mastic gum, the product of the tree Pistacia lentiscus var. Chia, has been used for more than 2500 years in traditional Greek medicine for treating several diseases, thanks to the anti-inflammatory and antioxidant properties of its components. Despite the long-time use of mastic in gastroenterology and in particular in chronic-inflammation-associated diseases, to date, the literature lacks reviews regarding this topic. The aim of the present work is to summarize available data on the effects of P. lentiscus on inflammatory bowel disease. A comprehensive review of this topic could drive researchers to conduct future studies aimed at deeply investigating P. lentiscus effects and hypothesizing a mechanism of action. The present review, indeed, schematizes the possible bioactive components of mastic gum. Particular care is given to P. lentiscus var. Chia medicaments' and supplements' chemical compositions and their pharmacological action in inflammatory bowel disease.

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.

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.

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.

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.

Lipid peroxidation inhibition study: A promising case of 1,3-di([1,1'-biphenyl]-3-yl)urea.

In the present study eighteen inhibitors of the hydrolytic enzymes of the endocannabinoid system were investigated for antioxidant activity using lipid peroxidation (LP) method. Among the assayed compounds ten belong to carbamates with phenyl [1,1'-biphenyl]-3-ylcarbamate (6), reported for the first time, and eight are retro-amide derivatives of palmitamine. Interestingly, results indicated that most of the tested compounds have good antioxidant properties. In particular, 1,3-di([1,1'-biphenyl]-3-yl)urea (3) shows IC50 = 26 ± 6 µM comparable to ones obtained for standard antioxidants trolox and quercetin (IC50 = 22 ± 6 µM and 23 ± 6 µM, respectively). Compound 3 was investigated further by means of DFT calculations, to clarify a possible mechanism of the antioxidant action. In order to estimate the capability of 3 to act as radical scavenger the structure was optimized at B3LYP/6-311++G** level and the respective bond dissociation enthalpies were calculated. The calculations in non-polar medium predicted as favorable mechanism a donation of a hydrogen atom to the free radical and formation of N-centered radical, while in polar solvents the mechanism of free radical scavenging by SPLET dominates over HAT H-abstraction. The possible radical scavenging mechanisms of another compound with potent antioxidant properties (IC50 = 53 ± 12 µM), the retro-amide derivative of palmitamine (compound 18), was estimated computationally based on the reaction enthalpies of a model compound (structural analogue to 18). The computations indicated that the most favorable mechanisms are hydrogen atom transfer from the hydroxyl group in meta-position of the benzamide fragment in nonpolar medium, and proton transfer from the hydroxyl group in ortho-position of the benzamide fragment in polar medium.

Simple Synthesis of 17-ß-O-hemisuccinate of Stanozolol for Immunoanalytical Methods.

The use of doping in sports is a global problem that affects athletes around the world. Among the different methods developed to detect doping agents in biological samples, there are antibody-based methods that need an appropriate hapten design. Steroids with a hydroxyl group can be converted to the corresponding hemisuccinates. A novel approach to the synthesis of 17ß-O-hemisuccinate of the common doping agent stanozolol is described here. Acylation of stanozolol with methyl 4-chloro-4-oxobutyrate/4-dimethylaminopyridine, followed by mild alkaline hydrolysis with methanolic sodium hydroxide at room temperature, gave the simultaneous protection and deprotection of pyrazole-nitrogen atoms. The proposed new synthetic method allows the desired hemisuccinate derivative to be obtained in only two steps, and with a good total yield starting from stanozolol.

Urine Endocannabinoids as Novel Non-Invasive Biomarkers for Bladder Cancer at Early Stage.

Due to the involvement of the endocannabinoid system (ECS) in cancer onset and progression and the less studied connection between ECS and bladder cancer, here an evaluation of the ECS modifications associated with bladder cancer is reported. Urine samples were collected from healthy volunteers and patients with bladder cancer at different grades. Endocannabinoids (ECs) and N-acylethanolamides (NAEs) were quantified by HPLC-MS/MS and results normalized for creatinine content. An increase in the urine concentrations of four ECs and NAEs analyzed was observed with a statistically significant increase in the arachidonoylethanolamide (AEA) and stearoylethanoamide (SEA) associated with bladder cancer. Receiver operating characteristic curves built with AEA and SEA data allowed the selection of 160 pg/mL for SEA (area under the curve (AUC) = 0.91, Selectivity (SE) 94%, Specificity (SP) 45%) and 8 pg/mL for AEA (AUC = 0.85, SE 94%, SP 61%) as the best cut-off values. Moreover, data from bladder cancer samples at different grades were derived from The Cancer Genome Atlas, and the expressions of thirteen different components of the "endocannabinoidome" were analyzed. Statistical analysis highlights significant variations in the expression of three enzymes involved in EC and NAE turnover in bladder cancer.

Unambiguous Characterization of p-Cresyl Sulfate, a Protein-Bound Uremic Toxin, as Biomarker of Heart and Kidney Disease.

p-Cresyl sulfate is one of the bound uremic toxins whose level increases in the sera of patients with the severity of chronic kidney disease and is therefore used as a standard for clinical investigations. Our first attempts to obtain p-cresyl sulfate led exclusively to the product of sulfonation of the aromatic ring instead of sulfation on the OH moiety. Nevertheless, this initial discouraging result allowed us to handle both p-cresyl sulfate and 2-hydroxy-5-methylbenzenesulfonic acid obtained by different synthetic pathways. Interestingly, the comparison between the two isomers pointed out that the two molecules show the same fragmentation pattern and are indistinguishable by mass spectrometry. They cannot be separated on several commercially available columns. The only difference between the two compounds is a 10-fold higher ionization yield under negative ion electrospray ionization. NMR spectral studies definitely confirmed the different molecular structures. We present here an unambiguous biomimetic synthetic route for p-cresyl sulfate and the spectroscopic characterization of both the compounds by nuclear magnetic resonance and mass spectrometry.

Set-Up and Validation of a High Throughput Screening Method for Human Monoacylglycerol Lipase (MAGL) Based on a New Red Fluorescent Probe.

Monoacylglycerol lipase (MAGL) is a serine hydrolase that has a key regulatory role in controlling the levels of 2-arachidonoylglycerol (2-AG), the main signaling molecule in the endocannabinoid system. Identification of selective modulators of MAGL enables both to provide new tools for investigating pathophysiological roles of 2-AG, and to discover new lead compounds for drug design. The development of sensitive and reliable methods is crucial to evaluate this modulatory activity. In the current study, we report readily synthesized long-wavelength putative fluorogenic substrates with different acylic side chains to find a new probe for MAGL activity. 7-Hydroxyresorufinyl octanoate proved to be the best substrate thanks to the highest rate of hydrolysis and the best Km and Vmax values. In addition, in silico evaluation of substrates interaction with the active site of MAGL confirms octanoate resorufine derivative as the molecule of choice. The well-known MAGL inhibitors URB602 and methyl arachidonylfluorophosphonate (MAFP) were used for the assay validation. The assay was highly reproducible with an overall average Z' value of 0.86. The fast, sensitive and accurate method described in this study is suitable for low-cost high-throughput screening (HTS) of MAGL modulators and is a powerful new tool for studying MAGL activity.

N6-isopentenyladenosine a new potential anti-angiogenic compound that targets human microvascular endothelial cells in vitro.

N6-isopentenyladenosine is an anti-proliferative and pro-apoptotic atypical nucleoside for normal and tumor cells. Considering the role of angiogenesis in various diseases, we investigated the cytotoxic effect of N6-isopentenyladenosine on human microvascular endothelial cells, protagonists in angiogenesis. Our results show that N6-isopentenyladenosine induced a significant reduction of cell viability, upregulated p21 and promoted caspase-3 cleavage in a dose dependent manner leading to apoptotic cell death as detected by FACS analysis. To understand structure-function relationship of N6-isopentenyladenosine, we investigated the effect of some N6-isopentenyladenosine analogs. Our results suggest that N6-isopentenyladenosine and some of its derivatives are potentially novel angiostatic agents and might be associated with other anti-angiogenic compounds for a better outcome.

Design, synthesis, molecular modelling and in vitro cytotoxicity analysis of novel carbamate derivatives as inhibitors of Monoacylglycerol lipase.

Monoacylglycerol lipase (MAGL) has an essential role in the catabolic pathway of the endocannabinoid 2-arachidonoylglycerol, which makes it a potential target for highly specific inhibitors for the treatment of a number of diseases. We designed and synthesized a series of carbamate analogues of URB602. We evaluated their inhibitory activity toward human MAGL in vitro both in cell culture and lysates. The target compounds exhibited moderate to excellent inhibitory activity against MAGL. The most promising compound 2b showed good inhibitory activity with IC50 value of 4.5 ±â€¯0.70 µM reducing MAGL activity to 82% of controls at 10 µM compared to 66% for the parent compound URB602. Interestingly, compounds 2b and 2c induce cell death through the inhibition of MAGL. Molecular modelling approaches and docking studies, used to investigate inhibitory profiles, indicated that trifluoromethyl substitutions of the aryl group and the benzene ring present at the oxygen side of the carbamate molecule had a significant impact on the activity.

Synthesis and characterization of a new fluorogenic substrate for monoacylglycerol lipase and application to inhibition studies.

Human monoacylglycerol lipase (MAGL), a soluble serine hydrolase that belongs to the α/ß hydrolase fold superfamily, regulates 2-arachidonoyl glycerol level in the endocannabinoid system, which is implicated in a number of severe diseases, and therefore, inhibition of MAGL activity is crucial in the treatment of these diseases. We have synthesized a red fluorogenic substrate, 7-hydroxyresorufinyl-arachidonate (7-HRA), for a new MAGL assay. This assay is simple, sensitive, and reliable and useful for identifying compounds that modulate MAGL activity. In addition, the assay emits red fluorescence, which can significantly reduce interference due to compound fluorescence and dust or lint, all of which fluoresce in the blue wavelength. MAGL catalyzes the hydrolysis of 7-HRA to generate arachidonic acid and a highly red fluorescent resorufin, excitation at 571 nm and emission at 588 nm, with a Km of 0.87 µM and Vmax of 26 nmol min(-1) mg protein(-1). The known MAGL inhibitors URB602, methyl arachidonyl fluorophosphonate, and JZL184 were used to validate the test assay. The assay was highly reproducible with an overall average Z' value of 0.80. This new red fluorogenic substrate and the resulting enzyme assay could be used in high-throughput screening to identify and develop new potential MAGL inhibitors.

N6-isopentenyladenosine and its analogue N6-benzyladenosine induce cell cycle arrest and apoptosis in bladder carcinoma T24 cells.

Cytokinins are phytohormones critically involved in the regulation of plant growth and development. They also affect the proliferation and differentiation of animal cells, thus representing new tools to treat diseases that involve dysfunctional cell growth and/or differentiation. Recently, by performing structure-function studies on human cells, we found that only N6-isopentenyladenosine and its benzyl analogue N6-benzyladenosine suppress the clonogenic activity and the growth of different neoplastic cells. We here broaden our studies on bladder carcinoma T24 cells, because, due to the high recurrence rate of bladder cancer, new active molecules are sought to contrast the growth of this tumor. Early events induced by N6-isopentenyladenosine and N6-benzyladenosine are the alteration of T24 cell morphology and the disorganization of the actin cytoskeleton. After 24 h N6-isopentenyladenosine and N6-benzyladenosine inhibit growth by arresting the cells in the G0/G1 phase of the cell cycle. We also show that the two compounds induce apoptosis, an event linked to the activation of caspase 3. Since DNA damage is a prime factor resulting in cell cycle arrest and apoptosis, it is noteworthy that we do not detect any genotoxic effect upon treatment of T24 cells with N6-isopentenyladenosine and N6- benzyladenosine. Because the disruption of actin filaments leads to G1 arrest and is also implicated in apoptosis, we hypothesize that cytoskeletal rearrangement might be responsible for triggering the antiproliferative and proapotpotic effects of N6-isopentenyladenosine and N6- benzyladenosine in T24 cells.

(1)H, (13)C and (15)N NMR assignments for N- and O-acylethanolamines, important family of naturally occurring bioactive lipid mediators.

The complete (1)H, (13)C and (15)N NMR signal assignments of some N- and O-acylethanolamines, important family of naturally occurring bioactive lipid mediators, were achieved using one-dimensional and two-dimensional experiments (gs-HMQC and gs-HMBC).

Optimized synthesis and characterization of N-acylethanolamines and O-acylethanolamines, important family of lipid-signalling molecules.

The endocannabinoid anandamide (N-arachidonoylethanolamine, AEA), a physiologically occurring bioactive compound on CB1 and CB2 receptors, has multiple physiological functions. Since the discovery of AEA additional non-cannabinoid endogenous compounds such as N-palmitoylethanolamine (PEA), and N-oleoylethanolamine (OEA) have been identified from mammalian tissues. Virodhamine (O-arachidonoylethanolamine, VA) is the only identified new member of the endocannabinoid family that is characterised by an ester linkage between acylic acid and ethanolamine instead of the amide linkage found in AEA and others non-cannabinoid N-acylethanolamines. It has been reported, as a cautionary note for lipid analyses, that VA can be produced nonenzymatically from AEA (and vice versa) as consequence of O,N-acyl migrations. O,N-acyl migrations are well documented in synthetic organic chemistry literature, but are not well described or recognized with regard to methods in lipid isolation or lipid enzyme studies. We here report an economical and effective protocol for large scale synthesis and characterization of some N- and O-acylethanolamines that could be useful as reference standards in order to investigate their possible formation in biological membranes, with potentially interesting biological properties.

Effects of cytokinins, cytokinin ribosides and their analogs on the viability of normal and neoplastic human cells.

We examined the effects of some cytokinins and cytokinin ribosides including a series of adenosine analogs differently substituted in the N(6) position, along with some hypoxanthine derivatives on the viability of normal and neoplastic human cells. Cytokinins such as trans-zeatin, isopentenyladenine and benzyladenine do not show any effect, while cytokinin ribosides such as trans-zeatin riboside, isopentenyladenosine, and benzylaminopurine riboside impair the viability of normal and neoplastic cells, apart from colon carcinoma LoVo cells.

1H, 13C and 15N NMR spectral assignments of adenosine derivatives with different amino substituents at C6-position.

The complete (1)H, (13)C and (15)N NMR signals assignment of adenosine derivatives differently substituted at C(6)-position was achieved using one- and two-dimensional experiments (gs-COSY, gs-NOESY, gs-HSQC and gs-HMBC).

N6-Alkyladenosines: Synthesis and evaluation of in vitro anticancer activity.

A series of adenosine analogues differently substituted in N6-position were synthesized to continue our studies on the relationships between structure and biological activity of iPA. The structures of the compounds were confirmed by standard studies of ¹H NMR, MS and elemental analysis. These molecules were then evaluated for their anti-proliferative activity on bladder cancer cells. We found that some of these compounds possess anti-proliferative activity but have no effect on cell invasion and metalloprotease activity.