An NMR Toolkit to Probe Amyloid Oligomer Inhibition in Neurodegenerative Diseases: From Ligand Screening to Dissecting Binding Topology and Mechanisms of Action.

The aggregation of amyloid peptides and proteins into toxic oligomers is a hallmark of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. Inhibition of amyloid oligomers formation and interactions with biological counterparts, as well as the triggering of non-toxic amorphous aggregates, are strategies towards preventive interventions against these pathologies. NMR spectroscopy addresses the need for structural characterization of amyloid proteins and their aggregates, their binding to inhibitors, and rapid screening of compound libraries for ligand identification. Here we briefly discuss the solution experiments constituting the NMR spectroscopist's toolkit and provide examples of their application.

Cell-Free and In Vivo Characterization of the Inhibitory Activity of Lavado Cocoa Flavanols on the Amyloid Protein Ataxin-3: Toward New Approaches against Spinocerebellar Ataxia Type 3.

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder characterized by ataxia and other neurological manifestations, with a poor prognosis and a lack of effective therapies. The amyloid aggregation of the ataxin-3 protein is a hallmark of SCA3 and one of the main biochemical events prompting its onset, making it a prominent target for the development of preventive and therapeutic interventions. Here, we tested the efficacy of an aqueous Lavado cocoa extract and its polyphenolic components against ataxin-3 aggregation and neurotoxicity. The combination of biochemical assays and atomic force microscopy morphological analysis provided clear evidence of cocoa flavanols' ability to hinder ATX3 amyloid aggregation through direct physical interaction, as assessed by NMR spectroscopy. The chemical identity of the flavanols was investigated by ultraperformance liquid chromatography-high-resolution mass spectrometry. The use of the preclinical model Caenorhabditis elegans allowed us to demonstrate cocoa flavanols' ability to ameliorate ataxic phenotypes in vivo. To the best of our knowledge, Lavado cocoa is the first natural source whose extract is able to directly interfere with ATX3 aggregation, leading to the formation of off-pathway species.

Adjuvant Effect of Cinnamon Polyphenolic Components in Colorectal Cancer Cell Lines.

Colorectal cancer (CRC) is the second-leading cause of cancer death, with a worldwide incidence rate constantly increasing; thus, new strategies for its prevention or treatment are needed. Here, we describe the adjuvant effect of the polyphenol-enriched fractions of cinnamon, from cinnamon bark and buds, when co-administered with a potent anticancer drug, cetuximab, used for CRC therapy. The co-administration significantly reduces the cetuximab dose required for the antiproliferative activity against colorectal cancer cell line E705, which is sensitive to EGFR-targeted therapy. The anticancer activity of these cinnamon-derived fractions, whose major components (as assessed by UPLC-HRMS analysis) are procyanidins and other flavonoids, strictly correlates with their ability to induce apoptosis in cancer cell lines through ERK activation and the mitochondrial membrane potential impairment. Due to the severe side effects of cetuximab administration, our results suggest the use of nutraceuticals based on the polyphenolic fractions of cinnamon extracts as adjuvants in the therapy of CRC.

Antarctic Soil Metabolomics: A Pilot Study.

In Antarctica, ice-free areas can be found along the coast, on mountain peaks, and in the McMurdo Dry Valleys, where microorganisms well-adapted to harsh conditions can survive and reproduce. Metabolic analyses can shed light on the survival mechanisms of Antarctic soil communities from both coastal sites, under different plant coverage stages, and inner sites where slow-growing or dormant microorganisms, low water availability, salt accumulation, and a limited number of primary producers make metabolomic profiling difficult. Here, we report, for the first time, an efficient protocol for the extraction and the metabolic profiling of Antarctic soils based on the combination of NMR spectroscopy and mass spectrometry (MS). This approach was set up on samples harvested along different localities of Victoria Land, in continental Antarctica, devoid of or covered by differently developed biological crusts. NMR allowed for the identification of thirty metabolites (mainly sugars, amino acids, and organic acids) and the quantification of just over twenty of them. UPLC-MS analysis identified more than twenty other metabolites, in particular flavonoids, medium- and long-chain fatty acids, benzoic acid derivatives, anthracenes, and quinones. Our results highlighted the complementarity of the two analytical techniques. Moreover, we demonstrated that their combined use represents the "gold standard" for the qualitative and quantitative analysis of little-explored samples, such as those collected from Antarctic soils.

Multivalent calix[4]arene-based mannosylated dendrons as new FimH ligands and inhibitors.

We report the synthesis and biological characterization of a novel class of multivalent glycoconjugates as hit compounds for the design of new antiadhesive therapies against urogenital tract infections (UTIs) caused by uropathogenic E. coli strains (UPEC). The first step of UTIs is the molecular recognition of high mannose N-glycan expressed on the surface of urothelial cells by the bacterial lectin FimH, allowing the pathogen adhesion required for mammalian cell invasion. The inhibition of FimH-mediated interactions is thus a validated strategy for the treatment of UTIs. To this purpose, we designed and synthesized d-mannose multivalent dendrons supported on a calixarene core introducing a significant structural change from a previously described family of dendrimers bearing the same dendrons units on a flexible pentaerythritol scaffold core. The new molecular architecture increased the inhibitory potency against FimH-mediated adhesion processes by about 16 times, as assessed by yeast agglutination assay. Moreover, the direct molecular interaction of the new compounds with FimH protein was assessed by on-cell NMR experiments acquired in the presence of UPEC cells.

Cheese-whey permeate improves the fitness of Escherichia coli cells during recombinant protein production.

BACKGROUND: Escherichia coli cells are the most frequently used hosts in recombinant protein production processes and mainly require molecules such as IPTG or pure lactose as inducers of heterologous expression. A possible way to reduce the production costs is to replace traditional inducers with waste materials such as cheese whey permeate (CWP). CWP is a secondary by-product generated from the production of the valuable whey proteins, which are obtained from ultrafiltration of cheese whey, a main by-product of the dairy industry, which is rich in lactose. RESULTS: The effects of CWP collected from an Italian plant were compared with those of traditional inducers on the production of two model proteins (i.e., green fluorescent protein and the toxic Q55 variant of ataxin-3), in E. coli BL21 (DE3) cells. It was found that the high lactose content of CWP (165 g/L) and the antioxidant properties of its micronutrients (vitamins, cofactors and osmolytes) sustain production yields similar to those obtained with traditional inducers, accompanied by the improvement of cell fitness. CONCLUSIONS: CWP has proven to be an effective and low-cost alternative inducer to produce recombinant proteins. Its use thus combines the advantage of exploiting a waste product with that of reducing the production costs of recombinant proteins.

Conformationally Restricted ß-Sheet Breaker Peptides Incorporating Cyclic α-Methylisoserine Sulfamidates.

Peptides containing variations of the ß-amyloid hydrophobic core and five-membered sulfamidates derived from ß-amino acid α-methylisoserine have been synthesized and fully characterized in the gas phase, solid state and in aqueous solution by a combination of experimental and computational techniques. The cyclic sulfamidate group effectively locks the secondary structure at the N-terminus of such hybrid peptides imposing a conformational restriction and stabilizing non-extended structures. This conformational bias, which is maintained in the gas phase, solid state and aqueous solution, is shown to be resistant to structure templating through assays of in vitro ß-amyloid aggregation, acting as ß-sheet breaker peptides with moderate activity.

Alzheimer's Disease Prevention through Natural Compounds: Cell-Free, In Vitro, and In Vivo Dissection of Hop (Humulus lupulus L.) Multitarget Activity.

The relevant social and economic costs associated with aging and neurodegenerative diseases, particularly Alzheimer's disease (AD), entail considerable efforts to develop effective preventive and therapeutic strategies. The search for natural compounds, whose intake through diet can help prevent the main biochemical mechanisms responsible for AD onset, led us to screen hops, one of the main ingredients of beer. To explore the chemical variability of hops, we characterized four hop varieties, i.e., Cascade, Saaz, Tettnang, and Summit. We investigated the potential multitarget hop activity, in particular its ability to hinder Aß1-42 peptide aggregation and cytotoxicity, its antioxidant properties, and its ability to enhance autophagy, promoting the clearance of misfolded and aggregated proteins in a human neuroblastoma SH-SY5Y cell line. Moreover, we provided evidence of in vivo hop efficacy using the transgenic CL2006Caenorhabditis elegans strain expressing the Aß3-42 peptide. By combining cell-free and in vitro assays with nuclear magnetic resonance (NMR) and MS-based metabolomics, NMR molecular recognition studies, and atomic force microscopy, we identified feruloyl and p-coumaroylquinic acids flavan-3-ol glycosides and procyanidins as the main anti-Aß components of hop.

NMR-Driven Identification of Cinnamon Bud and Bark Components With Anti-Aß Activity.

The anti-Alzheimer disease (AD) activity reported for an aqueous cinnamon bark extract prompted us to investigate and compare the anti-amyloidogenic properties of cinnamon extracts obtained from both bark and bud, the latter being a very little explored matrix. We prepared the extracts with different procedures (alcoholic, hydroalcoholic, or aqueous extractions). An efficient protocol for the rapid analysis of NMR spectra of cinnamon bud and bark extracts was set up, enabling the automatic identification and quantification of metabolites. Moreover, we exploited preparative reverse-phase (RP) chromatography to prepare fractions enriched in polyphenols, further characterized by UPLC-HR-MS. Then, we combined NMR-based molecular recognition studies, atomic force microscopy, and in vitro biochemical and cellular assays to investigate the anti-amyloidogenic activity of our extracts. Both bud and bark extracts showed a potent anti-amyloidogenic activity. Flavanols, particularly procyanidins, and cinnamaldehydes, are the chemical components of cinnamon hindering Aß peptide on-pathway aggregation and toxicity in a human neuroblastoma SH-SY5Y cell line. Together with the previously reported ability to hinder tau aggregation and filament formation, these data indicate cinnamon polyphenols as natural products possessing multitarget anti-AD activity. Since cinnamon is a spice increasingly present in the human diet, our results support its use to prepare nutraceuticals useful in preventing AD through an active contrast to the biochemical processes that underlie the onset of this disease. Moreover, the structures of cinnamon components responsible for cinnamon anti-AD activities represent molecular templates for designing and synthesizing new anti-amyloidogenic drugs.

Green and Roasted Coffee Extracts Inhibit Interferon-ß Release in LPS-Stimulated Human Macrophages.

The anti-inflammatory activity of coffee extracts is widely recognized and supported by experimental evidence, in both in vitro and in vivo settings, mainly murine models. Here, we investigated the immunomodulatory properties of coffee extracts from green (GCE) and medium-roasted (RCE) Coffea canephora beans in human macrophages. The biological effect of GCE and RCE was characterized in LPS-stimulated THP-1-derived human macrophages (TDM) as a model of inflammation. Results showed decreased amounts of TNF-α, IL-6 and IL-1ß and a strong dose-dependent inhibition of interferon-ß (IFN-ß) release. Molecular mechanism of IFN-ß inhibition was further investigated by immunofluorescence confocal microscopy analysis that showed a diminished nuclear translocation of p-IRF-3, the main transcription factor responsible for IFN-ß synthesis. The inhibition of IFN-ß release by RCE and GCE was also confirmed in human primary CD14+ monocytes-derived macrophages (MDM). The main component of coffee extracts, 5-O-caffeoylquinic acid (5-CQA) also inhibited IFN-ß production, through a mechanism occurring downstream to TLR4. Inhibition of IFN-ß release by coffee extracts parallels with the activity of their main phytochemical component, 5-CQA, thus suggesting that this compound is the main responsible for the immunomodulatory effect observed. The application of 5-CQA and coffee derived-phytoextracts to target interferonopathies and inflammation-related diseases could open new pharmacological and nutritional perspectives.

Cross-Linking Effects Dictate the Preference of Galectins to Bind LacNAc-Decorated HPMA Copolymers.

The interaction of multi-LacNAc (Galß1-4GlcNAc)-containing N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers with human galectin-1 (Gal-1) and the carbohydrate recognition domain (CRD) of human galectin-3 (Gal-3) was analyzed using NMR methods in addition to cryo-electron-microscopy and dynamic light scattering (DLS) experiments. The interaction with individual LacNAc-containing components of the polymer was studied for comparison purposes. For Gal-3 CRD, the NMR data suggest a canonical interaction of the individual small-molecule bi- and trivalent ligands with the lectin binding site and better affinity for the trivalent arrangement due to statistical effects. For the glycopolymers, the interaction was stronger, although no evidence for forming a large supramolecule was obtained. In contrast, for Gal-1, the results indicate the formation of large cross-linked supramolecules in the presence of multivalent LacNAc entities for both the individual building blocks and the polymers. Interestingly, the bivalent and trivalent presentation of LacNAc in the polymer did not produce such an increase, indicating that the multivalency provided by the polymer is sufficient for triggering an efficient binding between the glycopolymer and Gal-1. This hypothesis was further demonstrated by electron microscopy and DLS methods.

On-cell saturation transfer difference NMR for the identification of FimH ligands and inhibitors.

We describe the development of an on-cell NMR method for the rapid screening of FimH ligands and the structural identification of ligand binding epitopes. FimH is a mannose-binding bacterial adhesin expressed at the apical end of type 1 pili of uropathogenic bacterial strains and responsible for their d-mannose sensitive adhesion to host mammalian epithelial cells. Because of these properties, FimH is a key virulence factor and an attractive therapeutic target for urinary tract infection. We prepared synthetic d-mannose decorated dendrimers, we tested their ability to prevent the FimH-mediated yeast agglutination, and thus we used the compounds showing the best inhibitory activity as models of FimH multivalent ligands to set up our NMR methodology. Our experimental protocol, based on on-cell STD NMR techniques, is a suitable tool for the screening and the epitope mapping of FimH ligands aimed at the development of new antiadhesive and diagnostic tools against urinary tract infection pathogens. Notably, the study is carried out in a physiological environment, i.e. at the surface of living pathogen cells expressing FimH.

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

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

Targeting GRP receptor: Design, synthesis and preliminary biological characterization of new non-peptide antagonists of bombesin.

We report the rational design, synthesis, and in vitro preliminary evaluation of a new small library of non-peptide ligands of Gastrin Releasing Peptide Receptor (GRP-R), able to antagonize its natural ligand bombesin (BN) in the nanomolar range of concentration. GRP-R is a transmembrane G-protein coupled receptor promoting the stimulation of cancer cell proliferation. Being overexpressed on the surface of different human cancer cell lines, GRP-R is ideal for the selective delivery to tumor cells of both anticancer drug and diagnostic devices. What makes very challenging the design of non-peptide BN analogues is that the 3D structure of the GRP-R is not available, which is the case for many membrane-bound receptors. Thus, the design of GRP-R ligands has to be based on the structure of its natural ligands, BN and GRP. We recently mapped the BN binding epitope by NMR and here we exploited the same spectroscopy, combined with MD, to define BN conformation in proximity of biological membranes, where the interaction with GRP-R takes place. The gained structural information was used to identify a rigid C-galactosidic scaffold able to support pharmacophore groups mimicking the BN key residues' side chains in a suitable manner for binding to GRP-R. Our BN antagonists represent hit compounds for the rational design and synthesis of new ligands and modulators of GRP-R. The further optimization of the pharmacophore groups will allow to increase the biological activity. Due to their favorable chemical properties and stability, they could be employed for the active receptor-mediated targeting of GRP-R positive tumors.

NMR-based Lavado cocoa chemical characterization and comparison with fermented cocoa varieties: Insights on cocoa's anti-amyloidogenic activity.

The metabolic profile of Lavado cocoa was characterized for the first time by NMR spectroscopy, then compared with the profiles of fermented and processed varieties, Natural and commercial cocoa. The significant difference in the contents of theobromine and flavanols prompted us to examine the cocoa varieties to seek correlations between these metabolite concentrations and the anti-amyloidogenic activity reported for cocoa in the literature. We combined NMR spectroscopy, preparative reversed-phase (RP) chromatography, atomic force microscopy, in vitro biochemical and cell assays, to investigate and compare the anti-amyloidogenic properties of extracts and fractions enriched in different metabolite classes. Lavado variety was the most active and the catechins and theobromine were the chemical components of cocoa hindering Aß peptide on-pathway aggregation and toxicity in a human neuroblastoma SH-SY5Y cell line.

Synthesis, Molecular Modeling and Biological Evaluation of Metabolically Stable Analogues of the Endogenous Fatty Acid Amide Palmitoylethanolamide.

Palmitoylethanolamide (PEA) belongs to the class of N-acylethanolamine and is an endogenous lipid potentially useful in a wide range of therapeutic areas; products containing PEA are licensed for use in humans as a nutraceutical, a food supplement, or food for medical purposes for its analgesic and anti-inflammatory properties demonstrating efficacy and tolerability. However, the exogenously administered PEA is rapidly inactivated; in this process, fatty acid amide hydrolase (FAAH) plays a key role both in hepatic metabolism and in intracellular degradation. So, the aim of the present study was the design and synthesis of PEA analogues that are more resistant to FAAH-mediated hydrolysis. A small library of PEA analogues was designed and tested by molecular docking and density functional theory calculations to find the more stable analogue. The computational investigation identified RePEA as the best candidate in terms of both synthetic accessibility and metabolic stability to FAAH-mediated hydrolysis. The selected compound was synthesized and assayed ex vivo to monitor FAAH-mediated hydrolysis and to confirm its anti-inflammatory properties. 1H-NMR spectroscopy performed on membrane samples containing FAAH in integral membrane protein demonstrated that RePEA is not processed by FAAH, in contrast with PEA. Moreover, RePEA retains PEA's ability to inhibit LPS-induced cytokine release in both murine N9 microglial cells and human PMA-THP-1 cells.

Structural Basis of Inhibition of the Pioneer Transcription Factor NF-Y by Suramin.

NF-Y is a transcription factor (TF) comprising three subunits (NF-YA, NF-YB, NF-YC) that binds with high specificity to the CCAAT sequence, a widespread regulatory element in gene promoters of prosurvival, cell-cycle-promoting, and metabolic genes. Tumor cells undergo "metabolic rewiring" through overexpression of genes involved in such pathways, many of which are under NF-Y control. In addition, NF-YA appears to be overexpressed in many tumor types. Thus, limiting NF-Y activity may represent a desirable anti-cancer strategy, which is an ongoing field of research. With virtual-screening docking simulations on a library of pharmacologically active compounds, we identified suramin as a potential NF-Y inhibitor. We focused on suramin given its high water-solubility that is an important factor for in vitro testing, since NF-Y is sensitive to DMSO. By electrophoretic mobility shift assays (EMSA), isothermal titration calorimetry (ITC), STD NMR, X-ray crystallography, and molecular dynamics (MD) simulations, we showed that suramin binds to the histone fold domains (HFDs) of NF-Y, preventing DNA-binding. Our analyses, provide atomic-level detail on the interaction between suramin and NF-Y and reveal a region of the protein, nearby the suramin-binding site and poorly conserved in other HFD-containing TFs, that may represent a promising starting point for rational design of more specific and potent inhibitors with potential therapeutic applications.

Correction to: Different phytotoxic effect of Lolium multiflorum Lam. leaves against Echinochloa oryzoides (Ard.) Fritsch and Oryza sativa L.

In the title, it should be Oryza instead of Oriza.

Effectiveness of Vigna unguiculata seed extracts in preventing colorectal cancer.

Colorectal cancer (CRC) is one of the most common types of cancer, especially in Western countries, and its incidence rate is increasing every year. In this study, for the first time Vigna unguiculata L. Walp. (cowpea) water boiled seed extracts were found to reduce the viability of different colorectal cancer (CRC) cell lines, such as E705, DiFi and SW480 and the proliferation of Caco-2 line too, without affecting CCD841 healthy cell line. Furthermore, the extracts showed the ability to reduce the level of Epidermal Growth Factor Receptor (EGFR) phosphorylation in E705, DiFi and SW480 cell lines and to lower the EC50 of a CRC common drug, cetuximab, on E705 and DiFi lines from 161.7 ng mL-1 to 0.06 ng mL-1 and from 49.5 ng mL-1 to 0.2 ng mL-1 respectively. The extract was characterized in its protein and metabolite profiles by tandem mass spectrometry and 1H-NMR analyses. A Bowman-Birk protease inhibitor was identified within the protein fraction and was supposed to be the main active component. These findings confirm the importance of a legume-based diet to prevent the outbreak of many CRC and to reduce the amount of drug administered during a therapeutic cycle.