First in Class Dual Non-ATP-Competitive Glycogen Synthase Kinase 3ß/Histone Deacetylase Inhibitors as a Potential Therapeutic to Treat Alzheimer's Disease.

Despite recent FDA approvals, Alzheimer's disease (AD) still represents an unmet medical need. Among the different available therapeutic approaches, the development of multitarget molecules represents one of the most widely pursued. In this work, we present a second generation of dual ligands directed toward highly networked targets that are deeply involved in the development of the disease, namely, Histone Deacetylases (HDACs) and Glycogen Synthase Kinase 3ß (GSK-3ß). The synthesized compounds are highly potent GSK-3ß, HDAC2, and HDAC6 inhibitors with IC50 values in the nanomolar range of concentrations. Among them, compound 4 inhibits histone H3 and tubulin acetylation at 0.1 µM concentration, blocks hyperphosphorylation of tau protein, and shows interesting immunomodulatory and neuroprotective properties. These features, together with its ability to cross the blood-brain barrier and its favorable physical-chemical properties, make compound 4 a promising hit for the development of innovative disease-modifying agents.

Phenolic and Antioxidant Characterization of Fruit By-Products for Their Nutraceuticals and Dietary Supplements Valorization under a Circular Bio-Economy Approach.

Agri-food by-products, obtained as waste from the food industry, negatively impact the global economy and the environment. In order to valorize waste materials from fruit juices and tomato sauces as upcycled materials rich in health-promoting compounds, they were characterized in terms of polyphenolic and protein content. The results obtained were compared with those collected for their final products. The recovery of polyphenols was performed via ultrasound-assisted extraction (UAE). A high-performance liquid chromatography-diode array detector (HPLC-DAD) method was developed and validated to depict the quali-quantitative polyphenolic profile of both the by-products and the final products. The antioxidant capacity of the resulting extracts was characterized by UV-Vis spectrophotometric assays in terms of total phenolic content (TPC) and total antioxidant status (TAS). Moreover, the protein content was assessed with the Kjeldahl method too. The results highlighted a significant quantity of polyphenols remaining in peach, apricot, and apple by-products, which were able to exert an antioxidant activity (in the range of 4.95 ± 5.69 × 10-1 to 7.06 ± 7.96 × 10-1 mmol Trolox 100 g-1 of dry weight (DW) sample). Conversely, the tomato by-products were highly rich in proteins (11.0 ± 2.00 to 14.4 ± 2.60 g of proteins 100 g-1 DW). The results proved that all by-products may potentially be sustainable ingredients with nutritional and functional value in a circular bio-economy prospect.

Bovine Serum Amine Oxidase and Polyamine Analogues: Chemical Synthesis and Biological Evaluation Integrated with Molecular Docking and 3-D QSAR Studies.

Natural polyamines (PAs) are key players in cellular homeostasis by regulating cell growth and proliferation. Several observations highlight that PAs are also implicated in pathways regulating cell death. Indeed, the PA accumulation cytotoxic effect, maximized with the use of bovine serum amine oxidase (BSAO) enzyme, represents a valuable strategy against tumor progression. In the present study, along with the design, synthesis, and biological evaluation of a series of new spermine (Spm) analogues (1-23), a mixed structure-based (SB) and ligand-based (LB) protocol was applied. Binding modes of BSAO-PA modeled complexes led to clarify electrostatic and steric features likely affecting the BSAO-PA biochemical kinetics. LB and SB three-dimensional quantitative structure-activity relationship (Py-CoMFA and Py-ComBinE) models were developed by means of the 3d-qsar.com portal, and their analysis represents a strong basis for future design and synthesis of PA BSAO substrates for potential application in oxidative stress-induced chemotherapy.

Integrated analytical approaches for the characterization of Spirulina and Chlorella microalgae.

Microalgae are well-known for their content of bioactive molecules such as pigments, proteins, fatty acids, polysaccharides, vitamins and antioxidants, all of which are of great interest in the preparation of a wide range of products such as food, cosmetics and nutraceuticals. The purpose of this project was the analytical characterization of commercial dry microalgal biomass: four samples of Chlorella and five of Spirulina were analysed in order to highlight their content in terms of micro/macro nutrients. The research was oriented towards the development and validation of accurate, fast and reproducible methods for the nutritional assessment of algal biomasses, aiming to provide a guiding methodology. The lipid profiles of algal matrixes were analysed for the content of saturated, unsaturated and polyunsaturated fatty acids. The process was divided into two phases: firstly, the extraction and determination of the total lipids and pigment content; secondly, the trans-esterification of the extracted lipid-pigment portion in order to analyse fatty acid methyl esters (FAMEs) with a GC-MS method. A fingerprinting of MUFAs and PUFAs was obtained regarding microalgae species. The determination of total carotenoids and chlorophylls content in the lipid extracts was evaluated through a fast UV-Vis spectrophotometric analysis, which was validated by a new HPLC-DAD analysis. Furthermore, the total antioxidant activity of each lipid extract was determined along with the determination of the microalgae protein content. Then, with the aid of the principal component analysis (PCA) plots, the two microalgae were clustered in terms of their micro/macro nutrients, for differentiating their properties. Spirulina, resulting to have a greater antioxidant activity, supposedly due to a higher content in pigments and higher protein concentration, could be suggested for an appropriate diet for sporting people. Chlorella, instead, showed a more balanced profile of PUFAs and MUFAs and its use could be suggested for cosmetics and vegan diets. This paper puts forward an overall analytical approach, sustained by a multivariate analysis, for emphasising content differences and activity of two different microalgae strains, in order to underline specific claims for each class, addressed to defined final users.

Glycogen Synthase Kinase 3ß: A New Gold Rush in Anti-Alzheimer's Disease Multitarget Drug Discovery?

Alzheimer's disease (AD), like other multifactorial diseases, is the result of a systemic breakdown of different physiological networks. As result, several lines of evidence suggest that it could be more efficiently tackled by molecules directed toward different dysregulated biochemical targets or pathways. In this context, the selection of targets to which the new molecules will be directed is crucial. For years, the design of such multitarget-directed ligands (MTDLs) has been based on the selection of main targets involved in the "cholinergic" and the "ß-amyloid" hypothesis. Recently, there have been some reports on MTDLs targeting the glycogen synthase kinase 3ß (GSK-3ß) enzyme, due to its appealing properties. Indeed, this enzyme is involved in tau hyperphosphorylation, controls a multitude of CNS-specific signaling pathways, and establishes strict connections with several factors implicated in AD pathogenesis. In the present Miniperspective, we will discuss the reasons behind the development of GSK-3ß-directed MTDLs and highlight some of the recent efforts to obtain these new classes of MTDLs as potential disease-modifying agents.

Targeting topoisomerase II with trypthantrin derivatives: Discovery of 7-((2-(dimethylamino)ethyl)amino)indolo[2,1-b]quinazoline-6,12-dione as an antiproliferative agent and to treat cancer.

Drugs targeting human topoisomerase II (topoII) are used in clinical practice since decades. Nevertheless, there is an urgent need for new and safer topoII inhibitors due to the emergence of secondary malignancies and the appearance of resistance mechanisms upon treatment with topoII-targeted anticancer drugs. In the present investigation, we report the discovery of a new topoII inhibitor, whose design was based on the structure of the natural product trypthantrin, a natural alkaloid containing a basic indoloquinazoline moiety. This new topoII inhibitor, here numbered compound 5, is found to inhibit topoII with an IC50 of 26.6 ± 4.7 µM. Notably, compound 5 is more potent than the template compound trypthantrin, and even than the widely used topoII-targeted clinical drug etoposide. In addition, compound 5 also exhibits high water solubility, and a promising antiproliferative activity on different tumor cell lines such as acute leukemia, colon, and breast cancer. In light of these results, compound 5 represents a promising lead for developing new topoII inhibitors as anti-cancer therapeutic agents.

Correction to "Discovery of the First-in-Class GSK-3ß/HDAC Dual Inhibitor as Disease-Modifying Agent To Combat Alzheimer's Disease".

[This corrects the article DOI: 10.1021/acsmedchemlett.8b00507.].

Discovery of the First-in-Class GSK-3ß/HDAC Dual Inhibitor as Disease-Modifying Agent To Combat Alzheimer's Disease.

Several evidence pointed out the role of epigenetics in Alzheimer's disease (AD) revealing strictly relationships between epigenetic and "classical" AD targets. Based on the reported connection among histone deacetylases (HDACs) and glycogen synthase kinase 3ß (GSK-3ß), herein we present the discovery and the biochemical characterization of the first-in-class hit compound able to exert promising anti-AD effects by modulating the targeted proteins in the low micromolar range of concentration. Compound 11 induces an increase in histone acetylation and a reduction of tau phosphorylation. It is nontoxic and protective against H2O2 and 6-OHDA stimuli in SH-SY5Y and in CGN cell lines, respectively. Moreover, it promotes neurogenesis and displays immunomodulatory effects. Compound 11 shows no lethality in a wt-zebrafish model (<100 µM) and high water solubility.

Novel polyamine-based Histone deacetylases-Lysine demethylase 1 dual binding inhibitors.

Epigenetic modulators Histone deacetylases (HDACs) and Lysine demethylase (LSD1) are validated targets for anticancer therapy. Both HDAC1/2 and LSD1 are found in association with the repressor protein CoREST in a transcriptional co-repressor complex, which is responsible for gene silencing. Combined modulation of both targets results in a synergistic antiproliferative activity. In the present investigation, we report about the design and synthesis of a series of polyamine-based HDACs-LSD1 dual binding inhibitors obtained by coupling Vorinostat and Tranylcypromine. Compound 4 emerged as the most promising of the synthesized series, showing good inhibitory activity towards HDAC1 and LSD1 either in vitro and in cell-based assay (Ki = 42.52 ±â€¯8.94 nM and IC50 = 3.85 µM, respectively). Furthermore, at 70.0 µM compound 4 induced a more pronounced cytotoxic effect than Vorinostat (68.6% vs 56.6% of dead cells) in MCF7 cancer cell line.

Application of an ESI-QTOF method for the detailed characterization of GSK-3ß inhibitors.

The crucial role of Glycogen Synthase Kinase 3 (GSK-3ß) as a pivotal player in Alzheimer's Disease (AD) has recently inspired significant attempts to design and synthesize potent kinase inhibitors. In fact GSK-3ß is considered the main kinase which catalyzes the microtubule-associated protein tau hyper-phosphorylation and the neurofibrillary tangles (NFT) in vitro and in vivo, The first classes of GSK-3ß inhibitors were classified as ATP-competitive and, therefore, they lack of an efficient degree of selectivity over other kinases. In light of this consideration, many efforts are devoted to characterize new non ATP-competitive GSK-3ß inhibitors, endowed with high selectivity. In parallel, there is an urgent need to develop new analytical methodologies for the hit selection (highthroughput screening) and ligand binding characterization in terms of potency, affinity and mechanism of action. The new methodology for GSK-3ß enzymatic activity determination can be adopted as a realistic alternative to the currently used radioactive, luminescence and fluorescence detection methods, each showing limitations in terms of safety and interferences. Herein, we propose an alternative and selective electrospray ionization quadrupole time-of-flight (ESI-QTOF) method, based on the direct quantification of phosphorylated substrate muscle glycogen synthase GSM, a peptide resembling the high affinity sequence of natural substrate muscle glycogen synthase 1, for the detailed characterization of GSK-3ß inhibitors. The method was validated in terms of accuracy and reproducibility of GSM signal intensity with a relative standard deviation RSD% value of 3.55%; Limit of Detection (LOD): 0.006µM; Lower Limit of Quantification (LLOQ): 0.02µM; linearity r2 0.9951. The kinetic constants (KM and vmax) of the GSK-3ß catalyzed kinase reaction and the inhibitory potency of known ligands (IC50), were determined. All the obtained results were in agreement with those reported in literature or obtained in house by the standard reference luminometric approach. The proposed method was applied to the elucidation of well known inhibitors mechanism of action by the construction of a Lineweaver-Burk plot and the Ki determination. Furthermore, the potency, affinity and mechanism of action of a new non ATP-competitive compound were established. We demonstrated the ESI-QTOF method to be more feasible than the classic kinase assays since it avoids drawbacks inherently connected with radioisotope labeling or the indirect detection of kinase activity, so far. It is also scalable to the screening of large library collections and suitable for pharmaceutical industries purposes.

Tacrine-based Multifunctional Agents in Alzheimer's Disease: An Old Story in Continuous Development§.

BACKGROUND: The design of multifunctional agents represents one of the most active research field in medicinal chemistry. In particular, tacrine, a well known Acetylcholinesterase inhibitor, is one of the most used starting point to develop multifunctional ligands and hundreds of papers report about these new agents. This is the third review of a series concerning tacrinebased multifunctional ligands; in particular, in the present, we will summarize and discuss the most intriguing examples of tacrine-based multifunctional agents published since 2013 until 2016. METHODS: We analyzed the bibliographic databases for peer-reviewed publications concerning tacrine-based multifunctional agents possessing biological actions that go beyond the simple "cholinergic" blockage. These papers have been subdivided according to their biological activities. Since this is the third review of a series, we took into considerations only the papers appeared since 2013 until 2016. RESULTS: In this review, we have analyzed more than 33 papers. All the reported compounds retain good inhibitory activity towards acetyl- and butyryl-cholinesterase. The other biological activities concern mostly inhibition of a) ß-amyloid aggregation, b) ß-secretase, c) monoamino oxidases, modulation of τ and ROS and metal chelation. CONCLUSION: The analysis of the current literature reported in this review confirm the importance of tacrine as scaffold to develop multifunctional agents potentially usefull to contrast Alzheimer's disease. Furthermore, the compounds herein reported showed very intriguing biological activities that could be used as starting point to develop new compounds even more interesting and, hopefully, clinically usefull to contrast Alzheimer's Disease.

Naphthalene diimide-polyamine hybrids as antiproliferative agents: Focus on the architecture of the polyamine chains.

Naphthalene diimides (NDIs) have been widely used as scaffold to design DNA-directed agents able to target peculiar DNA secondary arrangements endowed with relevant biochemical roles. Recently, we have reported disubstituted linear- and macrocyclic-NDIs that bind telomeric and non-telomeric G-quadruplex with high degree of affinity and selectivity. Herein, the synthesis, biological evaluation and molecular modelling studies of a series of asymmetrically substituted NDIs are reported. Among these, compound 9 emerges as the most interesting of the series being able to bind telomeric G-quadruplex (ΔTm = 29 °C at 2.5 µM), to inhibit the activity of DNA processing enzymes, such as topoisomerase II and TAQ-polymerase, and to exert antiproliferative effects in the NCI panel of cancer cell lines with GI50 values in the micro-to nanomolar concentration range (i.e. SR cell line, GI50 = 76 nM). Molecular mechanisms of cell death have been investigated and molecular modelling studies have been performed in order to shed light on the antiproliferative and DNA-recognition processes.

Combined inhibition of the EGFR/AKT pathways by a novel conjugate of quinazoline with isothiocyanate.

Epidermal growth factor receptor inhibitors (EGFR-TKIs) represent a class of compounds widely used in anticancer therapy. An increasing number of studies reports on combination therapies in which the block of the EGFR-TK activity is associated with inhibition of its downstream pathways, as PI3K-Akt. Sulforaphane targets the PI3K-Akt pathway whose dysregulation is implicated in many functions of cancer cells. According to these considerations, a series of multitarget molecules have been designed by combining key structural features derived from an EGFR-TKI, PD168393, and the isothiocyanate sulforaphane. Among the obtained molecules 1-6, compound 6 emerges as a promising lead compound able to exert antiproliferative and proapoptotic effects in A431 epithelial cancer cell line by covalently binding to EGFR-TK, and reducing the phosphorylation of Akt without affecting the total Akt levels.

Natural polyamines and synthetic analogs modify the growth and the morphology of Pyrus communis pollen tubes affecting ROS levels and causing cell death.

Polyamines (PAs) are small molecules necessary for pollen maturation and tube growth. Their role is often controversial, since they may act as pro-survival factors as well as factors promoting Programmed Cell Death (PCD). The aim of the present work was to evaluate the effect of exogenous PAs on the apical growth of pear (Pyrus communis) pollen tube and to understand if PAs and reactive oxygen species (ROS) are interconnected in the process of tip-growth. In the present study besides natural PAs, also aryl-substituted spermine and methoctramine (Met 6-8-6) analogs were tested. Among the natural PAs, Spm showed strongest effects on tube growth. Spm entered through the pollen tube tip, then diffused in the sub-apical region that underwent drastic morphological changes, showing enlarged tip. Analogs were mostly less efficient than natural PAs but BD23, an asymmetric synthetic PAs bearing a pyridine ring, showed similar effects. These effects were related to the ability of PAs to cause the decrease of ROS level in the apical zone, leading to cell death, counteracted by the caspase-3 inhibitor Ac-DEVD-CHO (DEVD). In conclusions, ROS are essential for pollen germination and a strict correlation between ROS regulation and PA concentration is reported. Moreover, an imbalance between ROS and PAs can be detrimental thereby driving pollen toward cell death.

Tumor vascular targeted liposomal-bortezomib minimizes side effects and increases therapeutic activity in human neuroblastoma.

Neuroblastoma is a childhood cancer with poor long-term prognosis in advanced stages. A major aim in neuroblastoma therapy is to develop targeted drug delivery systems to ameliorate drug therapeutic index and efficacy. In this study, a novel bortezomib (BTZ) liposomal formulation was set-up and characterized. Since BTZ is freely permeable across the lipidic bilayer, an amino-lactose (LM) was synthesized as complexing agent to entrap BTZ inside the internal aqueous compartment of stealth liposomes. High encapsulation efficiency was achieved by a loading method based on the formation of boronic esters between the boronic acid moiety of BTZ and the hydroxyl groups of LM. Next, NGR peptides were linked to the liposome surface as a targeting-ligand for the tumor endothelial cell marker, aminopeptidase N. Liposomes were characterized for size, Z-potential, polydispersity index, drug content, and release. Lyophilization in the presence of cryoprotectants (trehalose, sucrose) was also examined in terms of particle size changes and drug leakage. BTZ was successfully loaded into non-targeted (SL[LM-BTZ]) and targeted (NGR-SL[LM-BTZ]) liposomes with an entrapment efficiency of about 68% and 57%, respectively. These nanoparticles were suitable for intravenous administration, presenting an average diameter of 170nm and narrow polydispersity. Therefore, orthotopic NB-bearing mice were treated with 1.0 or 1.5mg/kg of BTZ, either in free form or encapsulated into liposomes. BTZ loaded liposomes showed a significant reduction of drug systemic adverse effects with respect to free drug, even at the highest dose tested. Moreover, mice treated with 1.5mg/kg of NGR-SL[LM-BTZ] lived statistically longer than untreated mice (P=0.0018) and SL[LM-BTZ]-treated mice (P=0.0256). Our results demonstrate that the novel vascular targeted BTZ formulation is endowed with high therapeutic index and low toxicity, providing a new tool for future applications in neuroblastoma clinical studies.

Macrocyclic naphthalene diimides as G-quadruplex binders.

The synthesis, biological and molecular modeling evaluation of a series of macrocyclic naphthalene diimides is reported. The present investigation expands on the study of structure-activity relationships of prototype compound 2 by constraining the molecule into a macrocyclic structure with the aim of improving its G-quadruplex binding activity and selectivity. The new derivatives, compounds 4-7 carry spermidine- and spermine-like linkers while in compound 8 the inner basic nitrogen atoms of spermine have been replaced with oxygen atoms. The design strategy has led to potent compounds stabilizing both human telomeric (F21T) and c-KIT2 quadruplex sequences, and high selectivity for quadruplex in comparison to duplex DNA. Antiproliferative effects of the new derivatives 4-8 have been evaluated in a panel of cancer cell lines and all the tested compounds showed activity in the low micromolar or sub-micromolar range of concentrations. In order to rationalize the molecular basis of the DNA G-quadruplex versus duplex recognition preference, docking and molecular dynamics studies have been performed. The computational results support the observation that the main driving force in the recognition is due to electrostatic factors.

Study of the cytotoxic effects of the new synthetic Isothiocyanate CM9 and its fullerene derivative on human T-leukemia cells.

One important strategy to develop effective anticancer agents is based on natural products. Many active phytochemicals are in human clinical trials and have been used for a long time, alone and in association with conventional anticancer drugs, for the treatment of various types of cancers. A great number of in vitro, in vivo and clinical reports document the multi-target anticancer activities of isothiocyanates and of compounds characterized by a naphthalenetetracarboxylic diimide scaffold. In order to search for new anticancer agents with a better pharmaco-toxicological profile, we investigated hybrid compounds obtained by inserting isothiocyanate group(s) on a naphthalenetetracarboxylic diimide scaffold. Moreover, since water-soluble fullerene derivatives can cross cell membranes thus favoring the delivery of anticancer therapeutics, we explored the cytostatic and cytotoxic activity of hybrid compounds conjugated with fullerene. We studied their cytostatic and cytotoxic effects on a human T-lymphoblastoid cell line by using different flow cytometric assays. In order to better understand their pharmaco-toxicological potential, we also analyzed their genotoxicity. Our global results show that the synthesized compounds reduced significantly the viability of leukemia cells. However, the conjugation with a non-toxic vector did not increase their anticancer potential. This opens an interesting research pattern for certain fullerene properties.

Preparation, characterization and in vitro evaluation of sterically stabilized liposome containing a naphthalenediimide derivative as anticancer agent.

The aim of this study was to incorporate a new naphthalenediimide derivative (AN169) with a promising anticancer activity into pegylated liposomes to an extent that allows its in vitro and in vivo testing without use of toxic solvent. AN169-loaded liposomes were prepared using the thin-film hydration method and characterized for size, polydispersity index, drug content and drug release. We examined their lyophilization ability in the presence of cryoprotectants (trehalose, sucrose and lysine) and the long-term stability of the lyophilized products stored at 4 °C for 3 and 6 months by particle size changes and drug leakage. AN169 was successfully loaded into liposomes with an entrapment efficiency of 87.3 ± 2.5%. The hydrodynamic diameter of these liposomes after sonication was ∼ 145 nm with a high degree of monodispersity. Trehalose was found to be superior to the other lyoprotectants. In particular, trehalose 1:10 lipid:cryoprotectant molar ratio may provide stable lyophilized liposomes with the conservation of physicochemical properties upon freeze-drying and long-term storage conditions. We also assessed their in vitro antitumor activity in human cancer cell lines (HTLA-230 neuroblastoma, Mel 3.0 melanoma, OVCAR-3 ovarian carcinoma and SV620 prostate cancer cells). However, only after 72 h incubation, loaded liposomes showed almost the same IC50 as free AN169. In conclusion, we developed a stable lyophilized liposomal formulation for intravenous administration of AN169 as anticancer drug, with the advantage of avoiding the use of potentially toxic solubilizing agents for future in vivo experiments.

Isothiocyanate synthetic analogs: biological activities, structure-activity relationships and synthetic strategies.

Sulforaphane is a natural product that is constantly under biological investigation for its unique biological properties. This naturally occurring isothiocyanate (ITC) and its analogs are the main components of cruciferous vegetables, such as cauliflower, watercress, broccoli, cabbage, Brussels sprouts, widely used as chemopreventive agents. Due to their interesting biological profiles, natural ITCs have been exploited as starting point to develop new synthetic analogs. The present mini-review briefly highlights the most important biological actions of selected new synthetic ITCs focusing on their structure-activity relationships and related synthetic strategies.

Exploring the effects of isothiocyanates on chemotherapeutic drugs.

INTRODUCTION: Chemoprevention has emerged as a promising strategy to reduce the risk and to control cancer. In this context, isothiocyanates (ITCs), found in abundance in the form of glucosinolates in cruciferous vegetables, have gained increasing consideration for their chemopreventive activity. ITCs exert their effects mainly by inducing carcinogen metabolism or by inhibiting tumor cell proliferation. AREAS COVERED: In recent years, novel combination treatments, by coupling chemopreventive agents and typical chemotherapeutics, have been exploited to increase the antitumor activities. The aim of this article is to examine the foremost studies carried out, so far, on the effects of dietary and synthetic ITCs on different signaling pathways involved in the pharmacokinetics and pharmacodynamics of chemotherapeutic agents, in order to enhance their effectiveness. EXPERT OPINION: Undoubtedly, the beneficial anticarcinogenic potential of ITCs, both singly and in combination, has emerged in in vitro and in vivo studies. However, only a few clinical trials have been carried out so far with ITCs, which try to better define both the pharmacokinetic and pharmacodynamic impacts in humans. More toxicological evaluations after long-term administration of ITCs in different species are required for the clinical development of ITCs as anticarcinogenic agents.