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The supramolecular structure in peptides’ prolonged-released gel formulations is the most critical parameter for the determination of the pharmaceutical profile of the drug. Here, we report our investigation on lanreotide Autogel as a case study. For the first time, we describe the use of the pulsed field gradient (PFG) diffusion-ordered spectroscopy (DOSY) magic-angle spinning NMR to characterize the supramolecular self-assembly and molecular mobility of different samples of lanreotide Autogel formulations prepared according to different formulation protocols. The diffusion coefficient was used to calculate the hydrodynamic radii of supramolecular assemblies and build relative molecular models. DOSY data were integrated with NMR imaging (MRI) measurements and atomic force microscopy (AFM) imaging.

Amyotrophic lateral sclerosis (ALS) is a multifactorial neurodegenerative pathology of the upper or lower motor neuron. Evaluation of ALS progression is based on clinical outcomes considering the impairment of body sites. ALS has been extensively investigated in the pathogenetic mechanisms and the clinical profile; however, no molecular biomarkers are used as diagnostic criteria to establish the ALS pathological staging. Using the source-reconstructed magnetoencephalography (MEG) approach, we demonstrated that global brain hyperconnectivity is associated with early and advanced clinical ALS stages. Using nuclear magnetic resonance (1H-NMR) and high resolution mass spectrometry (HRMS) spectroscopy, here we studied the metabolomic profile of ALS patients’ sera characterized by different stages of disease progression—namely early and advanced. Multivariate statistical analysis of the data integrated with the network analysis indicates that metabolites related to energy deficit, abnormal concentrations of neurotoxic metabolites and metabolites related to neurotransmitter production are pathognomonic of ALS in the advanced stage. Furthermore, analysis of the lipidomic profile indicates that advanced ALS patients report significant alteration of phosphocholine (PCs), lysophosphatidylcholine (LPCs), and sphingomyelin (SMs) metabolism, consistent with the exigency of lipid remodeling to repair advanced neuronal degeneration and inflammation.

Salivary gland tumors are relatively uncommon neoplasms that represent less than 5% of head and neck tumors, and about 90% are in the parotid gland. The wide variety of histologies and tumor characteristics makes diagnosis and treatment challenging. In the present study, Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used to discriminate the pathological regions of patient-derived biopsies of parotid neoplasms by metabolomic and lipidomic profiles. Fresh frozen parotid tissues were analyzed by MALDI time-of-flight (TOF) MSI, both in positive and negative ionization modes, and additional MALDI-Fourier-transform ion cyclotron resonance (FT-ICR) MSI was carried out for metabolite annotation. MALDI-TOF-MSI spatial segmentation maps with different molecular signatures were compared with the histologic annotation. To maximize the information related to specific alterations between the pathological and healthy tissues, unsupervised (principal component analysis, PCA) and supervised (partial least squares-discriminant analysis, PLS-DA) multivariate analyses were performed presenting a 95.00% accuracy in cross-validation. Glycerophospholipids significantly increased in tumor tissues, while sphingomyelins and triacylglycerols, key players in the signaling pathway and energy production, were sensibly reduced. In addition, a significant increase of amino acids and nucleotide intermediates, consistent with the bioenergetics request of tumor cells, was observed. These results underline the potential of MALDI-MSI as a complementary diagnostic tool to improve the specificity of diagnosis and monitoring of pharmacological therapies.

A variety of cyclen and hexacyclen derivatives decorated with (S)-1-phenylethyl side chains or (S)-pyrrolidine units have been prepared via a reductive approach from the corresponding cyclic peptoids containing N-(S)-(1-phenylethyl)glycine and L-proline residues. Spectroscopic and DFT studies on their Na+ complexes show that point chirality and ring size play a crucial role in controlling the structural dynamism of 1,2-diaminoethylene units and pendant arms. The detection of highly symmetric C4- and C3-symmetric metalated species demonstrates that a full understanding of the relationship between the structure and conformational properties of peraza-macrocyclic metal complexes is possible.

Intrathecal delivery of Nusinersen–an antisense oligonucleotide that promotes survival motor neuron (SMN) protein induction–is an approved therapy for spinal muscular atrophy (SMA). Here, we employed nuclear magnetic resonance (NMR) spectroscopy to longitudinally characterize the unknown metabolic effects of Nusinersen in the cerebrospinal fluid (CSF) of SMA patients across disease severity. Modulation of amino acid metabolism is a common denominator of biochemical changes induced by Nusinersen, with distinct downstream metabolic effects according to disease severity. In severe SMA1 patients, Nusinersen stimulates energy-related glucose metabolism. In intermediate SMA2 patients, Nusinersen effects are also related to energy homeostasis but involve ketone body and fatty acid biosynthesis. In milder SMA3 patients, Nusinersen mainly modulates amino acid metabolism. Moreover, Nusinersen modifies the CSF metabolome of a more severe clinical group towards the profile of untreated SMA patients with milder disease. These findings reveal disease severity-specific neurometabolic signatures of Nusinersen treatment, suggesting a selective modulation of peripheral organ metabolism by this CNS-directed therapy in severe SMA patients.

Alzheimer’s disease is a neurodegenerative disease characterized by the formation of amyloid plaques constituted prevalently by amyloid peptides. Due to the well-known challenges related to the study in solution of these peptides, several membrane-mimicking systems such as micelle constituted by detergent—i.e., DPC and SDS—have been deeply investigated. Additionally, the strategy of studying short fragments instead of the full-length peptide turned out to be advantageous in exploring the structural properties of the different moieties in Aβ in order to reproduce its pathologic effects. Several studies reveal that among Aβ fragments, Aβ(25−35) is the shortest fragment able to reproduce the aggregation process. To enrich the structural data currently available, in the present work we decided to evaluate the conformational changes adopted by Aβ(25−35) in SDS combining CD and NMR spectroscopies at different times. From the solved structures, it emerges that Aβ(25−35) passes from an unordered conformation at the time of the constitution of the system to a more ordered and energetically favorable secondary structure at day 7, which is kept for 2 weeks. These preliminary data suggest that a relatively long time affects the kinetic in the aggregation process of Aβ(25−35) in a micellar system, favoring the stabilization and the formation of a soluble helix conformation.

Almonds are the tasty seeds of Prunus dulcis plants globally appreciated for the pleasant palatability and remarkable nutritional value, therefore it is very spread as snack and as basic ingredient of the confectionery products. The HR-MAS-NMR is a simple spectroscopy able to directly and quickly explore the chemical composition of powdered seed samples dispersed in D2O. 1H spectra witness the remarkable presence of triglyceride fatty esters together with sucrose; other minor water soluble metabolites are also detectable. This very rough approach is effectively providing chemical profiles featuring almond samples. In this analysis we were able to statistically distinguish the “Avola” almonds from other marketed products submitted to the same analysis. This is just a first investigation based on the main compounds but it might pave the way toward the quantitative evaluation of many other compounds in the almond therefore implementing the HR-MAS-NMR knowledge of these precious seeds.

Alzheimer’s disease (AD) is a neurodegenerative pathology characterized by the presence of neurofibrillary tangles and amyloid plaques, the latter mainly composed of Aβ(1–40) and Aβ(1–42) peptides. The control of the Aβ aggregation process as a therapeutic strategy for AD has prompted the interest to investigate the conformation of the Aβ peptides, taking advantage of computational and experimental techniques. Mixtures composed of systematically different proportions of HFIP and water have been used to monitor, by NMR, the conformational transition of the Aβ(1–42) from soluble α-helical structure to β-sheet aggregates. In the previous studies, 50/50 HFIP/water proportion emerged as the solution condition where the first evident Aβ(1–42) conformational changes occur. In the hypothesis that this solvent reproduces the best condition to catch transitional helical-β-sheet Aβ(1–42) conformations, in this study, we report an extensive NMR conformational analysis of Aβ(1–42) in 50/50 HFIP/water v/v. Aβ(1–42) structure was solved by us, giving evidence that the evolution of Aβ(1–42) peptide from helical to the β-sheet may follow unexpected routes. Molecular dynamics simulations confirm that the structural model we calculated represents a starting condition for amyloid fibrils formation.

Plant-based systems continue to play a pivotal role in healthcare, and their use has been extensively documented. Asphodelus L. is a genus comprising various herbaceous species, known by the trivial name Asphodelus. These plants have been known since antiquity for both food and therapeutic uses, especially for treating several diseases associated with inflammatory and infectious skin disorders. Phytochemical studies revealed the presence of different constituents, mainly anthraquinones, triterpenoids, phenolic acids, and flavonoids. Although extensive literature has been published on these constituents, a paucity of information has been reported regarding the carbohydrate composition, such as fructans and fructan-like derivatives. The extraction of water-soluble neutral polysaccharides is commonly performed using water extraction, at times assisted by microwaves and ultrasounds. Herein, we reported the investigation of the alkaline extraction of root-tubers of Asphodelus ramosus L., analyzing the water-soluble polysaccharides obtained by precipitation from the alkaline extract and its subsequent purification by chromatography. A polysaccharide was isolated by alkaline extraction, the HPTLC study to determine its composition showed fructose as the main monosaccharide. FT-IR analysis showed the presence of an inulin-type structure, and NMR analyses allowed us to conclude that A. ramosus roots contain polysaccharide with an inulin-type fructooligosaccharide with a degree of polymerization of 7–8.

Alzheimer's disease (AD), is the most common neurodegenerative disorder of the aging population resulting in progressive cognitive and functional decline. Accumulation of amyloid plaques around neuronal cells is considered a critical pathogenetic event and, in most cases, a hallmark of the pathology. In the attempt to identify anti-AD drug candidates, hundreds of molecules targeting Aβ peptides have been screened. Peptide molecules have been widely explored, appreciating chemical stability, biocompatibility, and low production cost. More recently, many anti-Aβ(1-42) monoclonal antibodies have been developed, given the excellent potential of immunotherapy for treating or preventing AD. Antibodies are versatile ligands that bind a large variety of molecules with high affinity and specificity; however, their extensive therapeutic application is complex and requires huge economic investments. Novel approaches to identify alternative antibody formats are considered with great interest. In this context, taking advantage of the favorable peptide properties and the availability of Aβ-antibodies structural data, we followed an innovative research approach to identify short peptide sequences on the model of the binding sites of Aβ(1-42)/antibodies. WAibH and SYSTPGK were designed as mimics of solanezumab and aducanumab, respectively. Circular dichroism and nuclear magnetic resonance analysis reveal that the antibody-derived peptides interact with Aβ(1-42) in the soluble monomeric form. Moreover, AFM microscopy imaging shows that WAibH and SYSTPGK are capable of controlling the Aβ(1-42) aggregation. The strategy to identify WAibH and SYSTPGK is innovative and can be widely applied for new anti-Aβ antibody mimicking peptides.

d-Amino acids were believed to occur only in bacteria and invertebrates. Today, it is well known that d-amino acids are also present in mammalian tissues in a considerable amount. In particular, high levels of free d-serine (d-Ser) and d-aspartate (d-Asp) are found in the brain. While the functions of d-Ser are well known, many questions remain unanswered regarding the role of d-Asp in the central nervous system. d-Asp is very abundant at the embryonic stage, while it strongly decreases after birth because of the expression of d-aspartate oxidase (Ddo) enzyme, which catalyzes the oxidation of this d-amino acid into oxaloacetate, ammonium, and hydrogen peroxide. Pharmacologically, d-Asp acts as an endogenous agonist of N-methyl d-aspartate and mGlu5 receptors, which are known to control fundamental brain processes, including brain development, synaptic plasticity, and cognition. In this work, we studied a recently generated knockin mouse model (R26ddo/ddo), which was designed to express DDO beginning at the zygotic stage. This strategy enables d-Asp to be almost eliminated in both prenatal and postnatal lives. To understand which biochemical pathways are affected by depletion of d-Asp, in this study, we carried out a metabolomic and lipidomic study of ddo knockin brains at different stages of embryonic and postnatal development, combining nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS) techniques. Our study shows that d-Asp deficiency in the brain influences amino acid pathways such as threonine, glycine, alanine, valine, and glutamate. Interestingly, d-Asp is also correlated with metabolites involved in brain development and functions such as choline, creatine, phosphocholine (PCho), glycerophosphocholine (GPCho), sphingolipids, and glycerophospholipids, as well as metabolites involved in brain energy metabolism, such as GPCho, glucose, and lactate.

SARS-CoV-2 is a virus belonging to the betacoronavirus family, causing fatal respiratory disease in humans, which became pandemic in 2020. Italy is one of the most affected countries by COVID-19, particularly in the northern regions. Several studies consider COVID-19 a zoonotic disease and, since Italy is the repository of a high biodiversity, SARS-CoV-2 infection in animals can be considered as a reservoir of the virus or favor the spreading between animals and humans. In this work, we analyzed the amino acid sequences of ACE2 protein of the most common domestic and wild animals present in Italy. Among the latter, we focused on ACE2 of the Chiroptera species present in Italy to identify the primary reservoir in this region. First, we reproduced in silico the Chiroptera ACE2/viral spike (S) protein interactions on the human ACE2/SARS-CoV-2 S complex model and identified the critical residues for the binding. In silico molecular docking of ACE2 belonging to Chiroptera vs SARS-CoV-2 S protein pointed to Rhinolophus ferrumequinum as a bat living in Italy, that may be a potential primary reservoir of the virus. On the other hand, a sequence similarity search on ACE2 of domestic and wild animals living in Italy pointed to domestic (horses, cats, cattle and sheep) and wild (European rabbits and grizzly bears) animal species as potential SARS-CoV-2 secondary reservoirs. Molecular docking of ACE2 belonging to these species vs S protein of Bat coronavirus (Bt-CoV/Rp3/2004) suggests that the primary reservoir Rhinolophus ferrumequinum may infect the secondary reservoirs, domestic and worldwide animals living in Italy, determining a specific risk of SARS-CoV-2 infection., Bioinformatics; Virology; SARS-CoV-2; COVID-19; Italian biodiversity; molecular docking and dynamics; sequence alignment; host range prediction.

Gp36 is the virus envelope glycoproteins catalyzing the fusion of the feline immunodeficiency virus with the host cells. The peptide C8 is a tryptophan-rich peptide corresponding to the fragment 770W-I777 of gp36 exerting antiviral activity by binding the membrane cell and inhibiting the virus entry. Several factors, including the membrane surface charge, regulate the binding of C8 to the lipid membrane. Based on the evidence that imperceptible variation of membrane charge may induce a dramatic effect in several critical biological events, in the present work we investigate the effect induced by systematic variation of charge in phospholipid bilayers on the aptitude of C8 to interact with lipid membranes, the tendency of C8 to assume specific conformational states and the re-organization of the lipid bilayer upon the interaction with C8. Accordingly, employing a bottom-up multiscale protocol, including CD, NMR, ESR spectroscopy, atomistic molecular dynamics simulations, and confocal microscopy, we studied C8 in six membrane models composed of different ratios of zwitterionic/negatively charged phospholipids. Our data show that charge content modulates C8-membrane binding with significant effects on the peptide conformations. C8 in micelle solution or in SUV formed by DPC or DOPC zwitterionic phospholipids assumes regular β-turn structures that are progressively destabilized as the concentration of negatively charged SDS or DOPG phospholipids exceed 40%. Interaction of C8 with zwitterionic membrane surface is mediated by Trp1 and Trp4 that are deepened in the membrane, forming H-bonds and cation-π interactions with the DOPC polar heads. Additional stabilizing salt bridge interactions involve Glu2 and Asp3. MD and ESR data show that the C8-membrane affinity increases as the concentration of zwitterionic phospholipid increases. In the lipid membrane characterized by an excess of zwitterionic phospholipids, C8 is adsorbed at the membrane interface, inducing a stiffening of the outer region of the DOPC bilayer. However, the bound of C8 significantly perturbs the whole organization of lipid bilayer resulting in membrane remodeling. These events, measurable as a variation of the bilayer thickness, are the onset mechanism of the membrane fusion and vesicle tubulation observed in confocal microscopy by imaging zwitterionic MLVs in the presence of C8 peptide.

Hypercholesterolaemia is considered an important cause of atherosclerotic cardiovascular disease. In a previous investigation, we demonstrated that cultured hepatoma cells treated with hypercholesterolaemic sera compared with cells treated with normocholesterolaemic sera show overexpression of mRNAs related to mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGCS2). In the present work, using an NMR metabolomic analysis, we demonstrate that the hypercholesterolaemic blood sera previously used to treat cultured hepatoma cells are characterized by a metabolomic profile that is significantly different from the normocholesterolaemic sera. Acetate, acetone, 2-hydroxybutyrate, cysteine, valine, and glutamine are the metabolites distinguishing the two groups. Abnormalities in the concentrations of these metabolites reflect alterations in energy-related pathways, such as pantothenate and CoA biosynthesis, pyruvate, glycolysis/gluconeogenesis, the citrate cycle, and ketone bodies. Regarding ketone bodies, the pathway is regulated by HMGCS2; therefore, serum samples previously found to be able to increase HMGCS2 mRNA levels in cultured cells also contain higher amounts of the metabolites of its encoded enzyme protein product.

Cancers affecting the salivary glands have been an increasing incidence. Salivary gland cancer is not detected until it reaches an advanced stage, which would generally result in a poor prognosis and survival rate. Therefore, early detection as well as the screening of high risk populations with precancerous lesions remains an unmet medical need. In the present work, we present a NMR-based metabolomic study of the saliva of patients suffering from salivary gland tumours. Analysis of data was done using a combined approach based on PRICONA quantitative analysis and statistical multivariate analysis. Interestingly, both the analytical methods indicate that individuals affected by parotid tumour have a characteristic metabolomic profile characterized by abnormalities in the concentration of several aminoacids. Among these the most significant are those relative to Alanine and Leucine suggestive of an alteration in the metabolic pathways of glycogenic aminoacids and ketone bodies. Our data, describing the preliminary metabolomics fingerprint of parotid tumour, are consistent with the recent view that oncogenic signalling corresponds to alteration in the metabolism of nutrient pull (Vander Heiden et al., 2009), rather than to a single metabolite.

N6-Isopentenyladenosine (i6A) is a naturally occurring modified nucleoside displaying in vitro and in vivo antiproliferative and pro-apoptotic properties. In our previous studies, including an in silico inverse virtual screening, NMR experiments and in vitro enzymatic assays, we demonstrated that i6A targeted farnesyl pyrophosphate synthase (FPPS), a key enzyme involved in the mevalonate (MVA) pathway and prenylation of downstream proteins, which are aberrant in several cancers. Following our interest in the anticancer effects of FPPS inhibition, we developed a panel of i6A derivatives bearing bulky aromatic moieties in the N6 position of adenosine. With the aim of clarifying molecular action of N6-benzyladenosine analogs on the FPPS enzyme inhibition and cellular toxicity and proliferation, herein we report the evaluation of the N6-benzyladenosine derivatives' (compounds 2a-m) effects on cell viability and proliferation on HCT116, DLD-1 (human) and MC38 (murine) colorectal cancer cells (CRC). We found that compounds 2, 2a and 2c showed a persistent antiproliferative effect on human CRC lines and compound 2f exerted a significant effect in impairing the prenylation of RAS and Rap-1A proteins, confirming that the antitumor activity of 2f was related to the ability to inhibit FPPS activity. ispartof: MOLECULES vol:26 issue:23 ispartof: location:Switzerland status: published

The role of β-amyloid (Aβ) in the pathogenesis of Alzheimer's disease (AD) is still considered crucial. The state of Aβ aggregation is critical in promoting neuronal loss and neuronal function impairment. Recently, we demonstrated that Acetylcholine (ACh) is neuroprotective against the toxic effects of Aβ in the cholinergic LAN-2 cells. In biophysical experiments, ACh promotes the soluble Aβ peptide conformation rather than the aggregation-prone β-sheet conformation. In order to better understand the biological role of ACh in AD, we studied the effect of Aβ on the phosphorylation of the cytosolic phospholipase A2 (cPLA2) in the TB neuroectodermal cell line, which differentiates toward a neuronal phenotype when cultured in the presence of retinoic acid (RA). We chose the phosphorylated form of cPLA2 (Ser505, Phospho-cPLA2) as a biomarker to test the influence of ACh on the effects of Aβ in both undifferentiated and RA-differentiated TB cells. Our results show that TB cells are responsive to Aβ. Moreover, in undifferentiated cells 1 h treatment with Aβ induces a 2.5-fold increase of the Phospho-cPLA2 level compared to the control after 24 h in vitro, while no significant difference is observed between Aβ-treated and non-treated cells after 4 and 7 days in vitro. The RA-differentiated cells are not sensitive to Aβ. In TB cell line ACh is able to blunt the effects of Aβ. The ability of ACh to protect non-cholinergic cells against Aβ reinforces the hypothesis that, in addition to its role in cholinergic transmission, ACh could also act as a neuroprotective agent.

Farnesyl pyrophosphate synthase (FPPS) is a crucial enzyme for the synthesis of isoprenoids and the key target of nitrogen-containing bisphosphonates (N-BPs). N-BPs are potent and selective FPPS inhibitors that are used in the treatment of bone-related diseases, but have poor pharmacokinetic properties. Given the key role played by FPPS in many cancer-related pathways and the pharmacokinetic limits of N-BPs, hundreds of molecules have been screened to identify new FPPS inhibitors characterized by improved drug-like properties that are useful for broader therapeutic applications in solid, non-skeletal tumours. We have previously shown that N6-isopentenyladenosine (i6A) and its related compound N6-benzyladenosine (2) exert anti-glioma activity by interfering with the mevalonate pathway and inhibiting FPPS. Here, we report the design and synthesis of a panel of N6-benzyladenosine derivatives (compounds 2a-m) incorporating different chemical moieties on the benzyl ring. Compounds 2a-m show in vitro antiproliferative activity in U87MG glioma cells and, analogous to the bisphosphonate FPPS inhibitors, exhibit immunogenic properties in ex vivo γδ T cells from stimulated peripheral blood mononuclear cells (PBMCs). Using saturation transfer difference (STD) and quantitative 1H nuclear magnetic resonance (NMR) experiments, we found that 2f, the N6-benzyladenosine analogue that includes a tertbutyl moiety in the para position of the benzyl ring, is endowed with increased FPPS binding and inhibition compared to the parent compounds i6A and 2. N6-benzyladenosine derivatives, characterized by structural features that are significantly different from those of N-BPs, have been confirmed to be promising chemical scaffolds for the development of non N-BP FPPS inhibitors, exerting combined cytotoxic and immunostimulatory activities.

Psoriasis is an inflammatory disease of the epidermis based on an immunological mechanism involving Langerhans cells and T lymphocytes that produce pro-inflammatory cytokines. Genetic factors, environmental factors, and improper nutrition are considered triggers of the disease. Numerous studies have reported that in a high number of patients, psoriasis is associated with obesity. Excess adipose tissue, typical of obesity, causes a systemic inflammatory status coming from the inflammatory active adipose tissue; therefore, weight reduction is a strategy to fight this pro-inflammatory state. This study aimed to evaluate how a nutritional regimen based on a ketogenic diet influenced the clinical parameters, metabolic profile, and inflammatory state of psoriasis patients. To this end, 30 psoriasis patients were subjected to a ketogenic nutritional regimen and monitored for 4 weeks by evaluating the clinical data, biochemical and clinical parameters, NMR metabolomic profile, and IL-2, IL-1β, TNF-α, IFN-γ, and IL-4 concentrations before and after the nutritional regimen. Our data show that a low-calorie ketogenic diet can be considered a successful strategy and therapeutic option to gain an improvement in psoriasis-related dysmetabolism, with significant correction of the full metabolic and inflammatory status.

Aim: The present study aims at investigating the possible correlation between peripheral markers of inflammation and brain networks. Introduction: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease dominated by progressive motor impairment. Among the complex mechanisms contributing to the pathogenesis of the disease, neuroinflammation, which is associated with altered circulating cytokine levels, is suggested to play a prominent role. Methods: Based on magnetoencephalography data, we estimated topological properties of the brain networks in ALS patients and healthy controls. Subsequently, the blood levels of a subset of cytokines were assayed. Finally, we modeled the brain topological features in the function of the cytokine levels. Results: Significant differences were found in the levels of the cytokines interleukin (IL)-4, IL-1β, and interferon-gamma (IFN-γ) between patients and controls. In particular, IL-4 and IL-1β levels increased in ALS patients, while the IFN-γ level was higher in healthy controls. We also detected modifications in brain global topological parameters in terms of hyperconnectedness. Despite both blood cytokines and brain topology being altered in ALS patients, such changes do not appear to be in a direct relationship. Conclusion: Our results would be in line with the idea that topological changes relate to neurodegenerative processes. However, the absence of correlation between blood cytokines and topological parameters of brain networks does not preclude that inflammatory processes contribute to the alterations of the brain networks. Impact statement The progression of amyotrophic lateral sclerosis entails both neurodegenerative and inflammatory processes. Furthermore, disease progression induces global modifications of the brain networks, with advanced stages showing a more compact, hyperconnected network topology. The pathophysiological processes underlying topological changes are unknown. In this article, we hypothesized that the global inflammatory profile would relate to the topological alterations. Our results showed that this is not the case, as modeling the topological properties as a function of the inflammatory state did not yield good predictions. Hence, our results suggest that topological changes might directly relate to neurodegenerative processes instead.

Feline immunodeficiency virus (FIV) is a naturally occurring Lentivirus causing acquired immunodeficiency syndrome in felines. It is considered a useful non-primate model to study HIV infection, and to test anti-HIV vaccine. Similarly to HIV, FIV enters cells via a mechanism involving a surface glycoprotein named gp36. C8 is a short synthetic peptide corresponding to the residues 770WEDWVGWI777 of gp36 membrane proximal external region (MPER). It elicits antiviral activity by inhibiting the fusion of the FIV and host cell membrane. C8 is endowed with evident membrane binding property, inducing alteration of the phospholipid bilayer and membrane fusion. The presence and the position of tryptophan residues in C8 are important for antiviral activity: the C8 derivative C6a, obtained by truncating the N-terminal 770WE771 residues, exhibits conserved antiviral activity, while the C8 derivative C6b, derived from the truncation of the C-terminal 776WI777, is nearly inactive. To elucidate the structural factors that induce the different activity profiles of C6a and C6b, in spite of their similarity, we investigated the structural behaviour of the two peptides in membrane mimicking environments. Conformational data on the short peptides C6a and C6b, matched to those of their parent peptide C8, allow describing a pharmacophore model of antiviral fusion inhibitors. This includes the essential structural motifs to design new simplified molecules overcoming the pharmacokinetic and high cost limitations affecting the antiviral entry inhibitors that currently are in therapy.

N6-isopentenyladenosine (i6A), a modified nucleoside belonging to the cytokinin family, has shown in humans many biological actions, including antitumoral effects through the modulation of the farnesyl pyrophosphate synthase (FPPS) activity. To investigate the relationship between i6A and FPPS, we undertook an inverse virtual screening computational target searching, testing i6A on a large panel of 3D protein structures involved in cancer processes. Experimentally, we performed an NMR investigation of i6A in the presence of FPPS protein. Both inverse virtual screening and saturation transfer difference (STD) NMR outcomes provided evidence of the structural interaction between i6A and FPPS, pointing to i6A as a valuable lead compound in the search of new ligands endowed with antitumoral potential and targeting FPPS protein.

C8, a short peptide characterized by three regularly spaced Trp residues, belongs to the membrane-proximal external functional domains of the feline immunodeficiency virus coat protein gp36. It elicits antiviral activity as a result of blocking cell entry and exhibits membranotropic and fusogenic activities. Membrane-proximal external functional domains of virus coat proteins are potential targets in the development of new anti-HIV drugs that overcome the limitations of the current anti-retroviral therapy. In the present work, we studied the conformation of C8 and its interaction with micellar surfaces using circular dichroism, nuclear magnetic resonance and fluorescence spectroscopy. The experimental data were integrated by molecular dynamics simulations in a micelle–water system. Our data provide insight into the environmental conditions related to the presence of the fusogenic peptide C8 on zwitterionic or negatively charged membranes. The membrane charge modulates the conformational features of C8. A zwitterionic membrane surface induces C8 to assume canonical secondary structures, with hydrophobic interactions between the Trp residues and the phospholipid chains of the micelles. A negatively charged membrane surface favors disordered C8 conformations and unspecific superficial interactions, resulting in membrane destabilization.

A key component to success in structure-based drug design is reliable information on protein-ligand interactions. Recent development in NMR techniques has accelerated this process by overcoming some of the limitations of X-ray crystallography and computational protein-ligand docking. In this work we present a new scoring protocol based on NMR-derived interligand INPHARMA NOEs to guide the selection of computationally generated docking modes. We demonstrate the performance in a range of scenarios, encompassing traditionally difficult cases such as docking to homology models and ligand dependent domain rearrangements. Ambiguities associated with sparse experimental information are lifted by searching a consensus solution based on simultaneously fitting multiple ligand pairs. This study provides a previously unexplored integration between molecular modeling and experimental data, in which interligand NOEs represent the key element in the rescoring algorithm. The presented protocol should be widely applicable for protein-ligand docking also in a different context from drug design and highlights the important role of NMR-based approaches to describe intermolecular ligand-receptor interactions.

Phosphodiesterases (PDE) hydrolyze both cyclic AMP and GMP (cAMP/cGMP) and are responsible for the regulation of their levels in a multitude of cellular functions. PDE10A is expressed in the brain and is a validated target for both schizophrenia and Huntington disease. Here, we address the identification of novel chemical scaffolds that may bind PDE10A via structure-based drug design. For this task, we use INPHARMA, an NMR-based method that measures protein-mediated interligand NOEs between pairs of weakly, competitively binding ligands. INPHARMA is applied to a combination of four chemically diverse PDE10A binding fragments, with the aim of merging their pharmacophoric features into a larger, tighter binding molecule. All four ligands bind the PDE10A cAMP binding domain with affinity in the micromolar range. The application of INPHARMA to identify the correct docking poses of these ligands is challenging due to the nature of the binding pocket and the high content of water-mediated intermolecular contacts. Nevertheless, ensemble docking in the presence of conserved water molecules generates docking poses that are in agreement with all sets of INPHARMA data. These poses are used to build a pharmacophore model with which we search the ZINC database.

The recently introduced Cu(I)-catalyzed azide-alkyne 1,3-dipolar Huisgen cycloaddition as a prototypic "click chemistry reaction" presented an opportunity for introducing the 1,4-disubstituted-[1,2,3]triazolyl moiety as a new isostere for peptide bonds in the backbone. Previous study in our lab focused on the synthesis of a model i-to-i+4 side chain-to-side chain 1,4- and 4,1-disubstituted-[1,2,3]triazolyl-bridged cyclo-nonapeptide I (Scheme 1) as analogues of its structurally related helical i-to-i+4 lactam-bridged cyclo-nonapeptide [Lys¹³ (&¹), Asp¹⁷ (&²)]parathyroid hormone related peptide (PTHrP)(11-19)NH₂ (1) a truncated version of the α-helical and potent parathyroid hormone receptor 1 agonist [Lys¹³ (&¹), Asp¹⁷ (&²)]PTHrP(1-34)NH₂, (2,3) N(α) -Ac-Lys-Gly-Lys(&¹)-Ser-Ile-Gln-Asp(&²)-Leu-Arg-NH₂]. Systematic [1,2,3]triazolyl-containing bridge structure-conformation relationship studies in hexafluoroacetone/water mixture included incorporation of bridges varied in size and position and orientation of the triazolyl ring within the bridge. These studies revealed that the size of methylene bridge flanking triazolyl moiety is critical to reproduce in the heterodetic cyclo-nonapeptides. The conformational features of the analogues cyclo-nonapeptide in which Lys¹³ and Asp¹⁷ are bridged by the isosteric lactam. Here, we extend our conformational analysis to dimethyl sulfoxide/water mixture in an effort to characterize inherent conformational properties of the heterodetic cyclopeptides that are solvent independent. Our present study shows that the physicochemical properties of the structure-supporting solvent cannot override the effect of the size of methylene bridge to form helical mimetic structures. Moreover, we prove that [1,2,3]triazolyl ring is not a simple bioisostere of lactam bond, but it affects the secondary structure of the peptide, in relation to its positioning orientation.

Hemopressin, a bioactive nonapeptide derived from the α1 chain of hemoglobin, was recently shown to possess selective antagonist activity at the cannabinoid CB(1) receptor [Heimann, A. S., et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 20588-20593]. CB(1) receptor antagonists have been extensively studied for their possible therapeutic use in the treatment of obesity, drug abuse, and heroin addiction. In particular, many compounds acting as CB(1) receptor antagonists have been synthesized and subjected to experiments as possible anti-obesity drugs, but their therapeutic application is still complicated by important side effects. Using circular dichroism and nuclear magnetic resonance spectroscopy, this work reports the conformational analysis of hemopressin and its truncated, biologically active fragment hemopressin(1-6). The binding modes of both hemopressin and hemopressin(1-6) are investigated by molecular docking calculations. Our conformational data indicate that regular turn structures in the central portion of hemopressin and hemopressin(1-6) are critical for an effective interaction with the receptor. The results of molecular docking calculations, indicating similarities and differences in comparison to the most accepted CB(1) pharmacophore model, suggest the possibility of new chemical scaffolds for the design of new CB(1) antagonist lead compounds.

Aggregation of β-amyloid peptides into senile plaques has been identified as one of the hallmarks of Alzheimer’s disease. An attractive therapeutic strategy for Alzheimer’s disease is the inhibition of the soluble β-amyloid aggregation using synthetic β-sheet breaker peptides that are capable of binding Aβ but are unable to become part of a β-sheet structure. As the early stages of the Aβ aggregation process are supposed to occur close to the neuronal membrane, it is strategic to define the β-sheet breaker peptide positioning with respect to lipid bilayers. In this work, we have focused on the interaction between the β-sheet breaker peptide acetyl-LPFFD-amide, iAβ5p, and lipid membranes, studied by ESR spectroscopy, using either peptides alternatively labeled at the C- and at the N-terminus or phospholipids spin-labeled in different positions of the acyl chain. Our results show that iAβ5p interacts directly with membranes formed by the zwitterionic phospholipid dioleoyl phosphatidylcholine and this interaction is modulated by inclusion of cholesterol in the lipid bilayer formulation, in terms of both peptide partition coefficient and the solubilization site. In particular, cholesterol decreases the peptide partition coefficient between the membrane and the aqueous medium. Moreover, in the absence of cholesterol, iAβ5p is located between the outer part of the hydrophobic core and the external hydrophilic layer of the membrane, while in the presence of cholesterol it penetrates more deeply into the lipid bilayer. Copyright c � 2010 European Peptide Society and John Wiley & Sons, Ltd.

Deposition of senile plaques composed of fibrillar aggregates of Abeta-amyloid peptide is a characteristic hallmark of Alzheimer's disease. A widely employed approach in the study of anti-Alzheimer agents involves the identification of substances able to prevent amyloid aggregation, or to disaggregate the amyloid fibrils through a direct structural interaction with the soluble or aggregated forms of the peptide. Here, we report the synthesis of a set of 1,3-dihydro-3,6-disubstituted-imidazo[1,5-c]thiazole-5,7-dione derivatives supporting different alkyl, aryl and alkylamine side chains. The ability of these compounds to interact with the Abeta(25-35) peptide was evaluated using circular dichroism, nuclear magnetic resonance and thioflavin fluorescence spectroscopy. A molecular model for Abeta(25-35)-ligand interactions was calculated by molecular docking procedures. Our data show that the ability of the synthesized compounds to modify the structural behaviour of Abeta(25-35) varies as a function of the overall structural features of the ligands rather contributions from specific individual substituents.

Background: For long time Alzheimer's disease has been attributed to a cholinergic deficit. More recently, it has been considered dependent on the accumulation of the amyloid beta peptide (Aβ), which promotes neuronal loss and impairs neuronal function. Results/methodology: In the present study, using biophysical and biochemical experiments we tested the hypothesis that in addition to its role as a neurotransmitter, acetylcholine may exert its action as an anti-Alzheimer agent through a direct interaction with Aβ. Conclusion: Our data provide evidence that acetylcholine favors the soluble peptide conformation and exerts a neuroprotective effect against the neuroinflammatory and toxic effects of Aβ. The present paper paves the way toward the development of new polyfunctional anti-Alzheimer therapeutics capable of intervening on both the cholinergic transmission and the Aβ aggregation.

The effect of the 665-683 fragment of the HIV fusion glycoprotein 41, corresponding to the MPER domain of the protein and named gp41MPER, on the microscopic structure and mesoscopic arrangement of palmitoyl oleoyl phosphatidylcholine (POPC) and POPC/sphingomyelin (SM)/cholesterol (CHOL) lipid bilayers is analyzed. The microscopic structuring of the bilayers has been studied by Electron Spin Resonance (ESR) spectroscopy, using glycerophosphocholines spin-labelled in different positions along the acyl chain. Transitions of the bilayer liquid crystalline state have been also monitored by Differential Scanning Calorimetry (DSC). Changes of the bilayers morphology have been studied by determining the dimension of the liposomes through Dynamic Light Scattering (DLS) measurements. The results converge in showing that the sample preparation procedure, the bilayer composition and the peptide/lipid ratio critically tune the lipid response to the peptide/membrane interaction. When gp41MPER is added to preformed liposomes, it positions at the bilayer interface and the lipid perturbation is limited to the more external segments. In contrast, if the peptide is mixed with the lipids during the liposome preparation, it assumes a trans-membrane topology. This happens at all peptide/lipid ratios for fluid POPC bilayers, while in the case of rigid POPC/SM/CHOL membranes a minimum ratio has to be reached, thus suggesting peptide self-aggregation to occur. Peptide insertion results in a dramatic increase of the lipid ordering and bilayer stiffening, which reflect in significant changes in liposome average dimension and distribution. The biological implications of these findings are discussed.

A broad biophysical analysis was performed to investigate the molecular basis of the neuroprotective action of Curcuma longa extracts in Alzheimer’s disease. By combining circular dichroism and electron paramagnetic resonance experiments with molecular modeling calculations, the minor components of Curcuma longa extracts, such as demethoxycurcumin (2, DMC), bisdemethoxycurcumin (3, BDMC) and cyclocurcumin (4, CYC), were analyzed in a membrane environment mimicking the phospholipid bilayer. Our study provides the first evidence on the relative role of single curcuminoids interacting with Aβ-peptide. When the CYC and curcumin metabolite tetrahydrocurcumin (5, THC) were inserted into an anionic lipid solution, a significant modification of the Aβ CD curves was detected. These data were implemented by EPR experiments, demonstrating that CYC reaches the inner part of the bilayer, while the other curcuminoids are localized close to the membrane interface. Computational studies provided a model for the curcuminoid-Aβ interaction, highlighting the importance of a constrained “semi-folded” conformation to interact with Aβ analogously to the pattern observed in α-helical coiled-coil peptide structures. This combined approach led to a better understanding of the intriguing in vitro and in vivo activity of curcuminoids as anti-Alzheimer agents, paving a new path for the rational design of optimized druggable analogues.

Antiphospholipid syndrome (APS) is a rheumatic inflammatory chronic autoimmune disease inducing hypercoagulable state associated with vascular thrombosis and pregnancy loss in women. Cardiac, cerebral and vascular strokes in these patients are responsible for reduction in life expectancy. Timely diagnosis and accurate monitoring of disease are decisive to improve the accuracy of therapy. In the present work, we present a NMR-based metabolomic study of blood sera of APS patients. Our data show that individuals suffering APS have a characteristic metabolomic profile with abnormalities associated to the metabolism of methyl group donors, ketone bodies and amino acids. We have identified for the first time the metabolomic fingerprint characterizing APS disease having potential application to improve APS timely diagnosis and appropriate therapeutic approaches.

Side chain-to-side chain cyclizations represent a strategy to select a family of bioactive conformations by reducing the entropy and enhancing the stabilization of functional ligand-induced receptor conformations. This structural manipulation contributes to increased target specificity, enhanced biological potency, improved pharmacokinetic properties, increased functional potency, and lowered metabolic susceptibility. The Cu(I)-catalyzed azide-alkyne 1,3-dipolar Huisgen's cycloaddition, the prototypic click reaction, presents a promising opportunity to develop a new paradigm for an orthogonal bioorganic and intramolecular side chain-to-side chain cyclization. In fact, the proteolytic stable 1,4- or 4,1-disubstituted [1,2,3]triazolyl moiety is isosteric with the peptide bond and can function as a surrogate of the classical side chain-to-side chain lactam forming bridge. Herein we report the design, synthesis, conformational analysis, and functional biological activity of a series of i-to-i+5 1,4- and 4,1-disubstituted [1,2,3]triazole-bridged cyclopeptides derived from MT-II, the homodetic Asp(5) to Lys(10) side chain-to-side chain bridged heptapeptide, an extensively studied agonist of melanocortin receptors.

Viral fusion glycoproteins present a membrane-proximal external region (MPER) which is usually rich in aromatic residues and exhibits a marked tendency to stably reside at the membrane interfaces, leading, through unknown mechanisms, to a destabilization of the bilayer structure. This step has been proposed to be fundamental for the fusion process between target membrane and viral envelope. In the present work, we investigate the interaction between an octapeptide (C8) deriving from the MPER domain of gp36 of feline immunodeficiency virus and POPC bilayers by combining experimental results obtained by neutron reflectivity, electron spin resonance, circular dichroism, and fluorescence spectroscopy with molecular dynamics simulations. Our data indicate that C8 binds to the lipid bilayer adsorbing onto the membrane surface without deep penetration. As a consequence of this interaction, the bilayer thickness decreases. The association of the peptide with the lipid membrane is driven by hydrogen bonds as well as hydrophobic interactions that the Trp side chains form with the lipid headgroups. Upon peptide-bilayer interaction, C8 forms transient secondary structures ranging from 3(10) helices to turn conformations, while acyl chains of the peptide-exposed POPC molecules assume a more ordered packing. At the same time, lipid headgroups' hydration increases. The asymmetric lipid bilayer perturbation is proposed to play a fundamental role in favoring the membrane fusion process.

Many drugs are available for the treatment of systemic or superficial mycoses, but only a limited number of them are effective antifungal drugs, devoid of toxic and undesirable side effects. Furthermore, resis- tance development and fungistatic rather than fungicidal activities represent limitations of current antifungal therapy. Therefore an urgent need for a new generation of antifungal agents remains. We recently synthesised a set of linear and cyclic peptides characterized by sequences typical of membrane-active antimicrobial peptides (AMP). AMT2, cyclo-AMT2, AMT3 and cyclo-AMT3 (Scheme 1) were tested against different yeast species and exhibited general antifungal activity, with a specificity against Cryptococcus neoformans. To evaluate the role of the membrane cell in the mechanism of antifungal activity, we investigated the conformational behaviour of AMT2, cyclo-AMT2, AMT3 and cyclo-AMT3 in different bio-membrane mimicking systems using a combined approach based on spectroscopy and microscopy techniques. Our data highlight the behaviour of the peptides to interact with the bilayer surface, excluding their ability to destabilize or permeabilize the fungal cell wall. Microbial membrane, indeed, may be an important platform for specific interactions of peptides with specific targets involved in the cell wall synthesis.

Alzheimer’s disease is characterized by the deposition of aggregates of the b-amyloid peptide (Ab) in the brain. A potential therapeutic strategy for Alzheimer’s disease is the use of synthetic b-sheet breaker pep- tides, which are capable of binding Ab but unable to become part of a b-sheet structure, thus inhibiting the peptide aggregation. Many studies suggest that membranes play a key role in the Ab aggregation; consequently, it is strategic to investigate the interplay between b-sheet breaker peptides and Ab in the presence of lipid bilayers. In this work, we focused on the effect of the b-sheet breaker peptide acetyl-LPFFD-amide, iAb5p, on the interaction of the Ab(25–35) fragment with lipid membranes, studied by Electron Spin Resonance spectroscopy, using spin-labeled membrane components (either phospholip- ids or cholesterol). The ESR results show that iAb5p influences the Ab(25–35) interaction with the bilayer through a cholesterol-mediated mechanism: iAb5p withholds cholesterol in the inner hydrophobic core of the bilayer, making the interfacial region more fluid and capable to accommodate Ab(25–35). As a con- sequence, iAb5p prevents the Ab(25–35) release from the lipid membrane, which is the first step of the b-amyloid aggregation process.

Many drugs are available for the treatment of systemic or superficial mycoses, but only a limited number of them are effective antifungal drugs, devoid of toxic and undesirable side effects. Furthermore, resistance development and fungistatic rather than fungicidal activities represent limitations of current antifungal therapy. Therefore there remains an urgent need for a new generation of antifungal agents. According to a polypharmacological approach, the present work concerns the synthesis and antifungal activity of a set of peptides designed to simultaneously target the fungal cell surface and lanosterol demethylase, a key enzyme involved in ergosterol synthesis. Our peptides include amino acid sequences characteristic of membrane-active antimicrobial peptides (AMP), and due to the presence of His residues, they carry the imidazole ring characteristic of azole compounds. The peptides synthesized by us, were tested against different yeast species, and displayed general antifungal activity, with a therapeutically promising antifungal specificity against Cryptococcus neoformans.

Abeta (16-35) is the hydrophobic central core of beta-amyloid peptide, the main component of plaques found in the brain tissue of Alzheimer's disease patients. Depending on the conditions present, beta-amyloid peptides undergo a conformational transition from random coil or alpha-helical monomers, to highly toxic beta-sheet oligomers and aggregate fibrils. The behavior of beta-amyloid peptide at plasma membrane level has been extensively investigated, and membrane charge has been proved to be a key factor modulating its conformational properties. In the present work we probed the conformational behavior of Abeta (16-35) in response to negative charge modifications of the micelle surface. CD and NMR conformational analyses were performed in negatively charged pure SDS micelles and in zwitterionic DPC micelles "doped" with small amounts of SDS. To analyze the tendency of Abeta (16-35) to interact with these micellar systems, we performed EPR experiments on three spin-labeled analogues of Abeta (16-35), bearing the methyl 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl) methanethiolsulfonate spin label at the N-terminus, in the middle of the sequence and at the C-terminus, respectively. Our conformational data show that, by varying the negative charge of the membrane, Abeta (16-35) undergoes a conformational transition from a soluble helical-kink-helical structure, to a U-turn shaped conformation that resembles protofibril models.

Background: Synthesis, computational study and biological evaluation of peptidomimetic analogues of FR235222 (3), a natural immunosuppressant and HDAC inhibitor, have been reported. These new compounds, bearing α-hydroxyketone moiety, as more stable zinc binding group (ZBG), were evaluated in vitro as HDAC inhibitors against the human HDACs isoforms 1-9 and in cellular antiproliferative assays on U937 human leukemia cell line. The 1,4-benzodiazepin-2,5-dione (BDZ), capping group and the natural ZBG, (S,R)-2-amino-9-hydroxy-8-oxodecanoic acid (Ahoda), were evaluated in order to probe HDAC inhibition and/or paralogue selectivity. Some of the new derivatives showed an interesting activity against a number of HDAC isozymes. The observed activity profile was rationalized by a computational assisted SAR study, in order to understand how the BDZ classes interact with the enzyme into the catalytic pocket. Despite its poor solubility, compound 17b showed significant antiproliferative profile and HDAC inhibition activity. Result: In order to assess how the solubility issue could have affected the biological outcome, bioassay conditions were reproduced and quantification of precipitated particulate material was evaluated by turbidimetric and NMR studies together with physicochemical descriptors prediction. Thus, BDZ 17b has been chosen to be promising lead compounds for further optimization, in order to elucidate molecule- enzyme surface recognition.