Neuronal Nitric Oxide Synthase and Post-Translational Modifications in the Development of Central Nervous System Diseases: Implications and Regulation.

In the Central Nervous System (CNS), Nitric Oxide (NO) is mainly biosynthesized by neuronal Nitric Oxide Synthase (nNOS). The dysregulated activation of nNOS in neurons is critical in the development of different conditions affecting the CNS. The excessive production of NO by nNOS is responsible for a number of proteins' post-translational modifications (PTMs), which can lead to aberrant biochemical pathways, impairing CNS functions. In this review, we briefly revise the main implications of dysregulated nNOS in the progression of the most prevalent CNS neurodegenerative disorders, i.e., Alzheimer's disease (AD) and Parkinson's disease, as well as in the development of neuronal disorders. Moreover, a specific focus on compounds able to modulate nNOS activity as promising therapeutics to tackle different neuronal diseases is presented.

Anti-Inflammatory, Antioxidant, and WAT/BAT-Conversion Stimulation Induced by Novel PPAR Ligands: Results from Ex Vivo and In Vitro Studies.

Activation of peroxisome proliferator-activated receptors (PPARs) not only regulates multiple metabolic pathways, but mediates various biological effects related to inflammation and oxidative stress. We investigated the effects of four new PPAR ligands containing a fibrate scaffold-the PPAR agonists (1a (αEC50 1.0 µM) and 1b (γEC50 0.012 µM)) and antagonists (2a (αIC50 6.5 µM) and 2b (αIC50 0.98 µM, with a weak antagonist activity on γ isoform))-on proinflammatory and oxidative stress biomarkers. The PPAR ligands 1a-b and 2a-b (0.1-10 µM) were tested on isolated liver specimens treated with lipopolysaccharide (LPS), and the levels of lactate dehydrogenase (LDH), prostaglandin (PG) E2, and 8-iso-PGF2α were measured. The effects of these compounds on the gene expression of the adipose tissue markers of browning, PPARα, and PPARγ, in white adipocytes, were evaluated as well. We found a significant reduction in LPS-induced LDH, PGE2, and 8-iso-PGF2α levels after 1a treatment. On the other hand, 1b decreased LPS-induced LDH activity. Compared to the control, 1a stimulated uncoupling protein 1 (UCP1), PR-(PRD1-BF1-RIZ1 homologous) domain containing 16 (PRDM16), deiodinase type II (DIO2), and PPARα and PPARγ gene expression, in 3T3-L1 cells. Similarly, 1b increased UCP1, DIO2, and PPARγ gene expression. 2a-b caused a reduction in the gene expression of UCP1, PRDM16, and DIO2 when tested at 10 µM. In addition, 2a-b significantly decreased PPARα gene expression. A significant reduction in PPARγ gene expression was also found after 2b treatment. The novel PPARα agonist 1a might be a promising lead compound and represents a valuable pharmacological tool for further assessment. The PPARγ agonist 1b could play a minor role in the regulation of inflammatory pathways.

Activators of Nrf2 to Counteract Neurodegenerative Diseases.

Neurodegenerative diseases are incurable and debilitating conditions that result in progressive degeneration and loss of nerve cells. Oxidative stress has been proposed as one factor that plays a potential role in the pathogenesis of neurodegenerative disorders since neuron cells are particularly vulnerable to oxidative damage. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is strictly related to anti-inflammatory and antioxidative cell response; therefore, its activation and the consequent enhancement of the related cellular pathways have been proposed as a potential therapeutic approach. Several Nrf2 activators with different mechanisms and diverse structures have been reported, but those applied for neurodisorders are still limited. However, in the very last few years, interesting progress has been made, particularly in enhancing the blood-brain barrier penetration, to make Nrf2 activators effective drugs, and in designing Nrf2-based multitarget-directed ligands to affect multiple pathways involved in the pathology of neurodegenerative diseases. The present review gives an overview of the most representative findings in this research area.

Emerging Effects of Resveratrol Derivatives in Cells Involved in Oral Wound Healing: A Preliminary Study.

Recently, there has been an increasing interest in finding new approaches to manage oral wound healing. Although resveratrol (RSV) exhibited many biological properties, such as antioxidant and anti-inflammatory activities, its use as a drug is limited by unfavorable bioavailability. This study aimed to investigate a series of RSV derivatives (1a-j) with better pharmacokinetic profiles. At first, their cytocompatibility at different concentrations was tested on gingival fibroblasts (HGFs). Among them, derivatives 1d and 1h significantly increased cell viability compared to the reference compound RSV. Thus, 1d and 1h were investigated for cytotoxicity, proliferation, and gene expression in HGFs, endothelial cells (HUVECs), and oral osteoblasts (HOBs), which are the main cells involved in oral wound healing. For HUVECs and HGFs, the morphology was also evaluated, while for HOBs ALP and mineralization were observed. The results showed that both 1d and 1h did not exert negative effects on cell viability, and at a lower concentration (5 µM) both even significantly enhanced the proliferative rate, compared to RSV. The morphology observations pointed out that the density of HUVECs and HGFs was promoted by 1d and 1h (5 µM) and mineralization was promoted in HOBs. Moreover, 1d and 1h (5 µM) induced a higher eNOS mRNA level in HUVECs, higher COL1 mRNA in HGFs, and higher OCN in HOBs, compared to RSV. The appreciable physicochemical properties and good enzymatic and chemical stability of 1d and 1h, along with their promising biological properties, provide the scientific basis for further studies leading to the development of RSV-based agents useful in oral tissue repair.

Resveratrol Derivative Exhibits Marked Antiproliferative Actions, Affecting Stemness in Pancreatic Cancer Cells.

Pancreatic cancer (PC) is one of the deadliest malignancies, with an increasing incidence and limited response to current therapeutic options. Therefore, more effective and low-toxic agents are needed to improve PC patients' outcomes. Resveratrol (RSV) is a natural polyphenol with multiple biological properties, including anticancer effects. In this study, we explored the antiproliferative activities of newly synthetized RSV analogues in a panel of PC cell lines and evaluated the physicochemical properties of the most active compound. This derivative exhibited marked antiproliferative effects in PC cells through mechanisms involving DNA damage, apoptosis induction, and interference in cell cycle progression, as assessed using flow cytometry and immunoblot analysis of cell cycle proteins, PARP cleavage, and H2AX phosphorylation. Notably, the compound induced a consistent reduction in the PC cell subpopulation with a CD133+EpCAM+ stem-like phenotype, paralleled by dramatic effects on cell clonogenicity. Moreover, the RSV derivative had negligible toxicity against normal HFF-1 cells and, thus, good selectivity index values toward PC cell lines. Remarkably, its higher lipophilicity and stability in human plasma, as compared to RSV, might ensure a better permeation along the gastrointestinal tract. Our results provide insights into the mechanisms of action contributing to the antiproliferative activity of a synthetic RSV analogue, supporting its potential value in the search for effective and safe agents in PC treatment.

Phenylsulfonimide PPARα Antagonists Enhance Nrf2 Activation and Promote Oxidative Stress-Induced Apoptosis/Pyroptosis in MCF7 Breast Cancer Cells.

The NF-E2-related factor 2 transcription factor (Nrf2) orchestrates the basal and stress-inducible activation of a vast array of antioxidant genes. A high amount of reactive oxygen species (ROS) promotes carcinogenesis in cells with defective redox-sensitive signaling factors such as Nrf2. In breast cancer (BC), emerging evidence indicates that increased Nrf2 activity enhances cell metastatic potential. An interconnection between peroxisome proliferator-activated receptors (PPARs) and Nrf2 pathways in cancer has been shown. In this light, newly synthesized PPARα antagonists, namely IB42, IB44, and IB66, were tested in the BC cell line MCF7 in parallel with GW6471 as the reference compound. Our results show that the most promising compound of this phenylsulfonimide series (IB66) is able to decrease MCF7 proliferation by blocking cells at the G2/M checkpoint. The underlying mechanism has been investigated, disclosing a caspase 3/Akt-dependent apoptotic/pyroptotic pathway induced by the increased generation of oxidative stress. Moreover, the involvement of Nrf2 and COX2 in IB66-treated MCF7 cell response has been highlighted. The reported data lay the groundwork for the development of alternative targeted therapy involving the Nrf2/PPARα molecular axis, able to overcome BC cell chemoresistance and cause better clinical outcomes, promoting other forms of programmed cell death, such as pyroptosis.

New amidine-benzenesulfonamides as iNOS inhibitors for the therapy of the triple negative breast cancer.

Triple negative breast cancer (TNBC) is a specific breast cancer subtype, and poor prognosis is associated to this tumour when it is in the metastatic form. The overexpression of the inducible Nitric Oxide Synthase (iNOS) is considered a predictor of poor outcome in TNBC patients, and this enzyme is reported as a valuable molecular target to compromise TNBC progression. In this work, new amidines containing a benzenesulfonamide group were designed and synthesized as selective iNOS inhibitors. An in vitro biological evaluation was performed to assess compounds activity against both the inducible and constitutive NOSs. The most interesting compounds 1b and 2b were evaluated on MDA-MB-231 cells as antiproliferative agents, and 1b capability to counteract cell migration was also studied. Finally, an in-depth docking study was performed to shed light on the observed potency and selectivity of action of the most promising compounds.

The Inhibition of the Inducible Nitric Oxide Synthase Enhances the DPSC Mineralization under LPS-Induced Inflammation.

Nitric oxide (NO) is a key messenger in physiological and pathological processes in mammals. An excessive NO production is associated with pathological conditions underlying the inflammation response as a trigger. Among others, dental pulp inflammation results from the invasion of dentin by pathogenic bacteria. Vital functions of pulp mesenchymal stem cells (DPSCs, dental pulp stem cells), such as mineralization, might be affected by the inducible NOS (iNOS) upregulation. In this context, the iNOS selective inhibition can be considered an innovative therapeutic strategy to counteract inflammation and to promote the regeneration of the dentin-pulp complex. The present work aims at evaluating two acetamidines structurally related to the selective iNOS inhibitor 1400W, namely CM544 and FAB1020, in a model of LPS-stimulated primary DPSCs. Our data reveal that CM544 and even more FAB1020 are promising anti-inflammatory compounds, decreasing IL-6 secretion by enhancing CD73 expression-levels, a protein involved in innate immunity processes and thus confirming an immunomodulatory role of DPSCs. In parallel, cell mineralization potential is retained in the presence of compounds as well as VEGF secretion, and thus their angiogenetic potential. Data presented lay the ground for further investigation on the anti-inflammatory potential of acetamidines selectively targeting iNOS in a clinical context.

Preface to Nitric Oxide Modulators in Health and Disease I.

Nitric oxide (NO) is a small free radical molecule biosynthesized by nitric oxide synthases (NOS), a family of oxidoreductases responsible for the conversion of the natural substrate L-arginine into L-citrulline and NO [...].

Benzothiazole Derivatives Endowed with Antiproliferative Activity in Paraganglioma and Pancreatic Cancer Cells: Structure-Activity Relationship Studies and Target Prediction Analysis.

The antiproliferative effects played by benzothiazoles in different cancers have aroused the interest for these molecules as promising antitumor agents. In this work, a library of phenylacetamide derivatives containing the benzothiazole nucleus was synthesized and compounds were tested for their antiproliferative activity in paraganglioma and pancreatic cancer cell lines. The novel synthesized compounds induced a marked viability reduction at low micromolar concentrations both in paraganglioma and pancreatic cancer cells. Derivative 4l showed a greater antiproliferative effect and higher selectivity index against cancer cells, as compared to other compounds. Notably, combinations of derivative 4l with gemcitabine at low concentrations induced enhanced and synergistic effects on pancreatic cancer cell viability, thus supporting the relevance of compound 4l in the perspective of clinical translation. A target prediction analysis was also carried out on 4l by using multiple computational tools, identifying cannabinoid receptors and sentrin-specific proteases as putative targets contributing to the observed antiproliferative activity.

HDAC Inhibitors for the Therapy of Triple Negative Breast Cancer.

Triple negative breast cancer (TNBC) is an urgent as well as huge medical challenge, which is associated with poor prognosis and responsiveness to chemotherapies. Since epigenetic changes are highly implicated in TNBC tumorigenesis and development, inhibitors of histone deacetylases (HDACIs) could represent a promising therapeutic strategy. Although clinical trials involving single HDACIs showed disappointing results against TNBC, recent studies emphasize the high potential impact of HDACIs in controlling TNBC. In addition, encouraging results stem from new compounds designed to obtain isoform selectivity and/or polypharmacological HDAC approach. The present review provides a discussion of the HDACIs pharmacophoric models and of the structural modifications, leading to compounds with a potent activity against TNBC progression.

Resveratrol-based compounds and neurodegeneration: Recent insight in multitarget therapy.

Neurodegenerative diseases (NDs) are characterized by gradual and progressive loss of selectively vulnerable populations of neurons, including death of neurons in different regions, leading to nervous system dysfunction. However, pharmacological treatments are only symptomatic, because the exact causes of the disease are not yet known. For this reason, in recent years, the research has been focused on the discovery of new molecules able to target neuropathological pathways involved in NDs. A great deal of attention has been paid to natural polyphenols due to their many biological effects and resveratrol has attracted special interest since its ability to interact simultaneously with the multiple targets implicated in NDs. Moreover, the structural simplicity of the stilbene core, the broad spectrum of possible modifications, and the improved synthetic strategies, made resveratrol an attractive chemical starting point for the search of new entities with extended therapeutic uses in NDs. In this review, a systematic update of the resveratrol-based compounds, and Structure-Activity Relationship analysis were provided as promising drug candidates for the treatment of NDs.

Olea europea L. Leaves and Hibiscus sabdariffa L. Petals Extracts: Herbal Mix from Cardiovascular Network Target to Gut Motility Dysfunction Application.

It is well known that diet and nutrition play a critical role in the etiopathogenesis of many disorders. On the other hand, nutrients or bioactive compounds can specifically target and control various aspects of the mechanism underlying the pathology itself, and, in this context, diseases related to intestinal motility disorders stand out. The Herbal Mix (HM) consisting of Olea europea L. leaf (OEE) and Hibiscus sabdariffa L. (HSE) extracts (13:2) has been proven to be a promising nutraceutical option for many diseases, but its potential in inflammatory-driven gastrointestinal disorders is still unexplored. In this study, HM effects on guinea-pig ileum and colon contractility (induced or spontaneous) and on human iNOS activity, as well as on human colorectal adenocarcinoma Caco-2 cells, were studied. Results showed that the HM can control the ileum and colon contractility without blocking the progression of the food bolus, can selectively inhibit iNOS and possesses a strong pro-apoptotic activity towards Caco-2 cells. In conclusion, the present results suggest that, in some diseases, such as those related to motility disorders, an appropriate nutritional approach can be accompanied by a correct use of nutraceuticals that could help not only in ameliorating the symptoms but also in preventing more severe, cancer-related conditions.

PPAR Ligands Induce Antiviral Effects Targeting Perturbed Lipid Metabolism during SARS-CoV-2, HCV, and HCMV Infection.

The manipulation of host metabolisms by viral infections has been demonstrated by several studies, with a marked influence on the synthesis and utilization of glucose, nucleotides, fatty acids, and amino acids. The ability of virus to perturb the metabolic status of the infected organism is directly linked to the outcome of the viral infection. A great deal of research in recent years has been focusing on these metabolic aspects, pointing at modifications induced by virus, and suggesting novel strategies to counteract the perturbed host metabolism. In this review, our attention is turned on PPARs, nuclear receptors controlling multiple metabolic actions, and on the effects played by PPAR ligands during viral infections. The role of PPAR agonists and antagonists during SARS-CoV-2, HCV, and HCMV infections will be analyzed.

Design, Synthesis and Biological Evaluation of Aromatase Inhibitors Based on Sulfonates and Sulfonamides of Resveratrol.

A library of sulfonate and sulfonamide derivatives of Resveratrol was synthesized and tested for its aromatase inhibitory potential. Interestingly, sulfonate derivatives were found to be more active than sulfonamide bioisosteres with IC50 values in the low micromolar range. The sulfonate analogues 1b-c and 1j exhibited good in vitro antiproliferative activity on the MCF7 cell line, evidenced by MTT and LDH release assays. Structure-activity relationships suggested that electronic and lipophilic properties could have a different role in promoting the biological response for sulfonates and sulfonamides, respectively. Docking studies disclosed the main interactions at a molecular level of detail behind the observed inhibition of the more active compounds whose chemical stability has been evaluated with nano-liquid chromatography. Finally, 1b-c and 1j were highlighted as sulfonates to be further developed as novel and original aromatase inhibitors.

Choline Chloride-Based DES as Solvents/Catalysts/Chemical Donors in Pharmaceutical Synthesis.

DES are mixtures of two or more compounds, able to form liquids upon mixing, with lower freezing points when compared to the individual constituents (eutectic mixtures). This attitude is due to the specific hydrogen-bond interactions network between the components of the mixture. A notable characteristic of DES is the possibility to develop tailor-made mixtures by changing the components ratios or a limited water dilution, for special applications, making them attractive for pharmaceutical purposes. In this review, we focused our attention on application of ChCl-based DES in the synthesis of pharmaceutical compounds. In this context, these eutectic mixtures can be used as solvents, solvents/catalysts, or as chemical donors and we explored some representative examples in recent literature of such applications.

Synthesis, structure-activity relationships and molecular docking studies of phenyldiazenyl sulfonamides as aromatase inhibitors.

The exploration of innovative aromatase inhibitors represents an important approach for the identification of new therapeutic treatments of breast cancer. In this respect, a series of phenyldiazenyl sulfonamides was designed, synthesized and tested. Compounds 3b, 3f and 5f showed an aromatase inhibition in the micromolar range and were evaluated in vitro on the human breast cancer cell line MCF7 by MTT assay, cytotoxicity assay (LDH release), cell cycle analysis and apoptosis, revealing a dose-dependent inhibition profile. In particular, 3f displayed the best reduction in terms of metabolic activity and an anti-proliferative effect on MCF7 cells, being blocked in the G1/S phase checkpoint. Moreover, computational studies were carried out to better understand at a molecular level of detail the rationale behind the effective binding to the active site of aromatase of the more active inhibitor 3f. The obtained results allow to consider this compound as an interesting lead for the development of a new class of non-steroidal aromatase inhibitors.

Selective Inhibitors of the Inducible Nitric Oxide Synthase as Modulators of Cell Responses in LPS-Stimulated Human Monocytes.

Inducible nitric oxide synthase (iNOS) is a crucial enzyme involved in monocyte cell response towards inflammation, and it is responsible for the production of sustained amounts of nitric oxide. This free radical molecule is involved in the defense against pathogens; nevertheless, its continuous and dysregulated production contributes to the development of several pathological conditions, including inflammatory and autoimmune diseases. In the present study, we investigated the effects of two new iNOS inhibitors, i.e., 4-(ethanimidoylamino)-N-(4-fluorophenyl)benzamide hydrobromide (FAB1020) and N-{3-[(ethanimidoylamino)methyl]benzyl}-l-prolinamidedihydrochloride (CM554), on human LPS-stimulated monocytes, using the 1400 W compound as a comparison. Our results show that CM544 and FAB1020 are selective and decrease cytotoxicity, IL-6 secretion and LPS-stimulated monocyte migration. Furthermore, the modulation of iNOS, nitrotyrosine and Nrf2 were analyzed at the protein level. Based on the collected preliminary results, the promising therapeutic value of the investigated compounds emerges, as they appear able to modulate the pro-inflammatory LPS-stimulated response in the low micromolar range in human monocytes.

New azolyl-derivatives as multitargeting agents against breast cancer and fungal infections: synthesis, biological evaluation and docking study.

Nonsteroidal aromatase inhibitors (NSAIs) are well-established drugs for the therapy of breast cancer. However, they display some serious side effects, and their efficacy can be compromised by the development of chemoresistance. Previously, we have reported different indazole-based carbamates and piperidine-sulphonamides as potent aromatase inhibitors. Starting from the most promising compounds, here we have synthesised new indazole and triazole derivatives and evaluated their biological activity as potential dual agents, targeting both the aromatase and the inducible nitric oxide synthase, being this last dysregulated in breast cancer. Furthermore, selected compounds were evaluated as antiproliferative and cytotoxic agents in the MCF-7 cell line. Moreover, considering the therapeutic diversity of azole-based compounds, all the synthesized compounds were also evaluated as antifungals on different Candida strains. A docking study, as well as molecular dynamics simulation, were carried out to shed light on the binding mode of the most interesting compound into the different target enzymes catalytic sites.

Design, synthesis and biological evaluation of imidazole and triazole-based carbamates as novel aromatase inhibitors.

In the search for novel aromatase inhibitors, a series of triazole and imidazole-based carbamate derivatives were designed and synthesized. Final compounds were thus evaluated against human aromatase by in vitro kinetic experiments in a fluorimetric assay in comparison with letrozole. The effect of most active derivatives 13a and 15c was then evaluated in vitro on the human breast cancer cell line MCF7 by MTT assay, cytotoxicity assay (LDH release) and cell cycle analysis, revealing a dose-dependent inhibition profile of cell viability and low micromolar IC50 values. In addition, docking simulations were also carried out to elucidate at a molecular level of detail the binding modes adopted to target human aromatase.