Mechanistic and Structural Insights on Difluoromethyl-1,3,4-oxadiazole Inhibitors of HDAC6.

Histone deacetylase 6 (HDAC6) is increasingly recognized for its potential in targeted disease therapy. This study delves into the mechanistic and structural nuances of HDAC6 inhibition by difluoromethyl-1,3,4-oxadiazole (DFMO) derivatives, a class of non-hydroxamic inhibitors with remarkable selectivity and potency. Employing a combination of nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) kinetic experiments, comprehensive enzymatic characterizations, and X-ray crystallography, we dissect the intricate details of the DFMO-HDAC6 interaction dynamics. More specifically, we find that the chemical structure of a DMFO and the binding mode of its difluoroacetylhydrazide derivative are crucial in determining the predominant hydrolysis mechanism. Our findings provide additional insights into two different mechanisms of DFMO hydrolysis, thus contributing to a better understanding of the HDAC6 inhibition by oxadiazoles in disease modulation and therapeutic intervention.

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.

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.

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.

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.

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.

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 study of Bombesin binding to GRP receptor.

We report the NMR characterization of the molecular interaction between Gastrin Releasing Peptide Receptor (GRP-R) and its natural ligand bombesin (BN). GRP-R is a transmembrane G-protein coupled receptor promoting the stimulation of cancer cell proliferation; in addition, being overexpressed on the surface of different human cancer cell lines, it is ideal for the development of new strategies for the selective targeted delivery of anticancer drugs and diagnostic devices to tumor cells. However, the design of new GRP-R binders requires structural information on receptor interaction with its natural ligands. The experimental protocol presented herein, based on on-cell STD NMR techniques, is a powerful tool for the screening and the epitope mapping of GRP-R ligands aimed at the development of new anticancer and diagnostic tools. Notably, the study can be carried out in a physiological environment, at the surface of tumoral cells overespressing GRP-R. Moreover, to the best of our knowledge, this is the first example of an NMR experiment able to detect and investigate the structural determinants of BN/GRP-R interaction.

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.

Epigallocatechin-3-gallate and related phenol compounds redirect the amyloidogenic aggregation pathway of ataxin-3 towards non-toxic aggregates and prevent toxicity in neural cells and Caenorhabditis elegans animal model.

The protein ataxin-3 (ATX3) triggers an amyloid-related neurodegenerative disease when its polyglutamine stretch is expanded beyond a critical threshold. We formerly demonstrated that the polyphenol epigallocatechin-3-gallate (EGCG) could redirect amyloid aggregation of a full-length, expanded ATX3 (ATX3-Q55) towards non-toxic, soluble, SDS-resistant aggregates. Here, we have characterized other related phenol compounds, although smaller in size, i.e. (-)-epigallocatechin gallate (EGC), and gallic acid (GA). We analysed the aggregation pattern of ATX3-Q55 and of the N-terminal globular Josephin domain (JD) by assessing the time course of the soluble protein, as well its structural features by FTIR and AFM, in the presence and the absence of the mentioned compounds. All of them redirected the aggregation pattern towards soluble, SDS-resistant aggregates. They also prevented the appearance of ordered side-chain hydrogen bonding in ATX3-Q55, which is the hallmark of polyQ-related amyloids. Molecular docking analyses on the JD highlighted three interacting regions, including the central, aggregation-prone one. All three compounds bound to each of them, although with different patterns. This might account for their capability to prevent amyloidogenesis. Saturation transfer difference NMR experiments also confirmed EGCG and EGC binding to monomeric JD. ATX3-Q55 pre-incubation with any of the three compounds prevented its calcium-influx-mediated cytotoxicity towards neural cells. Finally, all the phenols significantly reduced toxicity in a transgenic Caenorhabditis elegans strain expressing an expanded ATX3. Overall, our results show that the three polyphenols act in a substantially similar manner. GA, however, might be more suitable for antiamyloid treatments due to its simpler structure and higher chemical stability.

Targeting Bacterial Biofilm: A New LecA Multivalent Ligand with Inhibitory Activity.

Biofilm formation by bacterial pathogens is a hallmark of chronic infections and is associated to increased antibiotic tolerance that makes pathogens difficult to eradicate with conventional antibiotic therapies. Infections caused by Pseudomonas aeruginosa are of great concern, especially for immunocompromised and cystic fibrosis patients. P. aeruginosa lectins LecA and LecB are virulence factors and play a key role in establishing biofilm; therefore, inhibition of the function of these proteins has potential in dismantling the bacterium from the protective biofilm environment and in restoring the activity of antibiotics. Here, we report the NMR characterization of the binding of a galactose-based dendrimer (Gal18) to LecA. Moreover, we demonstrate the activity of the Gal18 molecule in inhibiting P. aeruginosa biofilm formation in vitro.

bioNMR-based identification of natural anti-Aß compounds in Peucedanum ostruthium.

The growing interest in medicinal plants for the identification of new bioactive compounds and the formulation of new nutraceuticals and drugs prompted us to develop a powerful experimental approach allowing the detailed metabolic profiling of complex plant extracts, the identification of ligands of macromolecular targets of biomedical relevance and a preliminary characterization of their biological activity. To this end, we selected Peucedanum ostruthium, a plant traditionally employed in Austria and Italy for its several potential therapeutic applications, as case study. We combined the use of NMR and UPLC-HR-MS for the identification of the metabolites present in its leaves and rhizome extracts. Due to the significant content of polyphenols, particularly chlorogenic acids, recently identified as anti-amyloidogenic compounds, polyphenols-enriched fractions were prepared and tested for their ability to prevent Aß1-42 peptide aggregation and neurotoxicity in a neuronal human cell line. STD-NMR experiments allowed the detailed identification of Aß oligomers' ligands responsible for the anti-amyloidogenic activity. These data provide experimental protocols and structural information suitable for the development of innovative molecular tools for prevention, therapy and diagnosis of Alzheimer's disease.

Protein Kinase A Activation Promotes Cancer Cell Resistance to Glucose Starvation and Anoikis.

Cancer cells often rely on glycolysis to obtain energy and support anabolic growth. Several studies showed that glycolytic cells are susceptible to cell death when subjected to low glucose availability or to lack of glucose. However, some cancer cells, including glycolytic ones, can efficiently acquire higher tolerance to glucose depletion, leading to their survival and aggressiveness. Although increased resistance to glucose starvation has been shown to be a consequence of signaling pathways and compensatory metabolic routes activation, the full repertoire of the underlying molecular alterations remain elusive. Using omics and computational analyses, we found that cyclic adenosine monophosphate-Protein Kinase A (cAMP-PKA) axis activation is fundamental for cancer cell resistance to glucose starvation and anoikis. Notably, here we show that such a PKA-dependent survival is mediated by parallel activation of autophagy and glutamine utilization that in concert concur to attenuate the endoplasmic reticulum (ER) stress and to sustain cell anabolism. Indeed, the inhibition of PKA-mediated autophagy or glutamine metabolism increased the level of cell death, suggesting that the induction of autophagy and metabolic rewiring by PKA is important for cancer cellular survival under glucose starvation. Importantly, both processes actively participate to cancer cell survival mediated by suspension-activated PKA as well. In addition we identify also a PKA/Src mechanism capable to protect cancer cells from anoikis. Our results reveal for the first time the role of the versatile PKA in cancer cells survival under chronic glucose starvation and anoikis and may be a novel potential target for cancer treatment.

1H NMR To Evaluate the Metabolome of Bronchoalveolar Lavage Fluid (BALf) in Bronchiolitis Obliterans Syndrome (BOS): Toward the Development of a New Approach for Biomarker Identification.

This report describes the application of NMR spectroscopy to the profiling of metabolites in bronchoalveolar lavage fluid (BALf) of lung transplant recipients without bronchiolitis obliterans syndrome (BOS) (stable, S, n = 10), and with BOS at different degrees of severity (BOS 0p, n = 10; BOS I, n = 10). Through the fine-tuning of a number of parameters concerning both sample preparation/processing and variations of spectra acquisition modes, an efficient and reproducible protocol was designed for the screening of metabolites in a pulmonary fluid that should reflect the status of airway inflammation/injury. Exploiting the combination of mono- and bidimensional NMR experiments, 38 polar metabolites, including amino acids, Krebs cycle intermediates, mono- and disaccharides, nucleotides, and phospholipid precursors, were unequivocally identified. To determine which signature could be correlated with the onset of BOS, the metabolites' content of the above recipients was analyzed by multivariate (PCA and OPLS-DA) statistical methods. PCA analysis (almost) totally differentiated S from BOS I, and this discrimination was significantly improved by the application of OPLS-DA, whose model was characterized by excellent fit and prediction values (R2 = 0.99 and Q2 = 0.88). The analysis of S vs BOS 0p and of BOS 0p vs BOS I samples showed a clear discrimination of considered cohorts, although with a poorer efficiency compared to those measured for S vs BOS I patients. The data shown in this work assess the suitability of the NMR approach in monitoring different pathological lung conditions.

1H NMR To Explore the Metabolome of Exhaled Breath Condensate in α1-Antitrypsin Deficient Patients: A Pilot Study.

The metabolomic analysis of exhaled breath condensate (EBC) may provide insights on both the pathology of pulmonary disorders and the response to therapy. This pilot study describes the ability of nuclear magnetic resonance (NMR)-based metabolomics to discriminate α1-antitrypsin deficient (AATD)-patients, who were diagnosed with moderate to severe emphysema, from healthy individuals. Comparative analysis of samples from these two homogeneous cohorts of individuals resulted in the generation of NMR profiles that were different from both a qualitative and a quantitative point-of-view. Among the identified metabolites that separated patients from controls, acetoin, propionate, acetate, and propane-1,2 diol were those presenting the biggest difference. Unambiguous confirmation that the two groups could be completely differentiated on the basis of their metabolite content came from the application of univariate and multivariate statistical analysis (principal component analysis, partial least squares discriminant analysis (PLS-DA), and orthogonal PLS-DA). MetaboAnalyst 3.0 platform, used to define a relationship among metabolites, allowed us to observe that pyruvate metabolism is the most-involved pathway, most of metabolites being originated from pyruvate. These preliminary data suggest that NMR, with its ability to differentiate the metabolic fingerprint of EBC of AATD patients from that of healthy controls, has a potential "clinical applicability" in this area.

Enhanced amino acid utilization sustains growth of cells lacking Snf1/AMPK.

The metabolism of proliferating cells shows common features even in evolutionary distant organisms such as mammals and yeasts, for example the requirement for anabolic processes under tight control of signaling pathways. Analysis of the rewiring of metabolism, which occurs following the dysregulation of signaling pathways, provides new knowledge about the mechanisms underlying cell proliferation. The key energy regulator in yeast Snf1 and its mammalian ortholog AMPK have earlier been shown to have similar functions at glucose limited conditions and here we show that they also have analogies when grown with glucose excess. We show that loss of Snf1 in cells growing in 2% glucose induces an extensive transcriptional reprogramming, enhances glycolytic activity, fatty acid accumulation and reliance on amino acid utilization for growth. Strikingly, we demonstrate that Snf1/AMPK-deficient cells remodel their metabolism fueling mitochondria and show glucose and amino acids addiction, a typical hallmark of cancer cells.

Performance Assessment in Fingerprinting and Multi Component Quantitative NMR Analyses.

An interlaboratory comparison (ILC) was organized with the aim to set up quality control indicators suitable for multicomponent quantitative analysis by nuclear magnetic resonance (NMR) spectroscopy. A total of 36 NMR data sets (corresponding to 1260 NMR spectra) were produced by 30 participants using 34 NMR spectrometers. The calibration line method was chosen for the quantification of a five-component model mixture. Results show that quantitative NMR is a robust quantification tool and that 26 out of 36 data sets resulted in statistically equivalent calibration lines for all considered NMR signals. The performance of each laboratory was assessed by means of a new performance index (named Qp-score) which is related to the difference between the experimental and the consensus values of the slope of the calibration lines. Laboratories endowed with a Qp-score falling within the suitable acceptability range are qualified to produce NMR spectra that can be considered statistically equivalent in terms of relative intensities of the signals. In addition, the specific response of nuclei to the experimental excitation/relaxation conditions was addressed by means of the parameter named NR. NR is related to the difference between the theoretical and the consensus slopes of the calibration lines and is specific for each signal produced by a well-defined set of acquisition parameters.

How Epigallocatechin-3-gallate and Tetracycline Interact with the Josephin Domain of Ataxin-3 and Alter Its Aggregation Mode.

Epigallocatechin-3-gallate (EGCG) and tetracycline are two known inhibitors of amyloid aggregation able to counteract the fibrillation of most of the proteins involved in neurodegenerative diseases. We have recently investigated their effect on ataxin-3 (AT3), the polyglutamine-containing protein responsible for spinocerebellar ataxia type 3. We previously showed that EGCG and tetracycline can contrast the aggregation process and toxicity of expanded AT3, although by different mechanisms. Here, we have performed further experiments by using the sole Josephin domain (JD) to further elucidate the mechanism of action of the two compounds. By protein solubility assays and FTIR spectroscopy we have first observed that EGCG and tetracycline affect the JD aggregation essentially in the same way displayed when acting on the full-length expanded AT3. Then, by saturation transfer difference (STD) NMR experiments, we have shown that EGCG binds both the monomeric and the oligomeric JD form, whereas tetracycline can only interact with the oligomeric one. Surface plasmon resonance (SPR) analysis has confirmed the capability of the sole EGCG to bind monomeric JD, although with a KD value suggestive for a non-specific interaction. Our investigations provide new details on the JD interaction with EGCG and tetracycline, which could explain the different mechanisms by which the two compounds reduce the toxicity of AT3.

Natural compounds against neurodegenerative diseases: molecular characterization of the interaction of catechins from green tea with Aß1-42, PrP106-126, and ataxin-3 oligomers.

By combining NMR spectroscopy, transmission electron microscopy, and circular dichroism we have identified the structural determinants involved in the interaction of green tea catechins with Aß1-42, PrP106-126, and ataxin-3 oligomers. The data allow the elucidation of their mechanism of action, showing that the flavan-3-ol unit of catechins is essential for interaction. At the same time, the gallate moiety, when present, seems to increase the affinity for the target proteins. These results provide important information for the rational design of new compounds with anti-amyloidogenic activity and/or molecular tools for the specific targeting of amyloid aggregates in vivo.