Tailoring a lead-like compound targeting multiple G-quadruplex structures.
Publication Date: 27/11/2018, on European journal of medicinal chemistry
by Amato J, Platella C, Iachettini S, Zizza P, Musumeci D, Cosconati S, Pagano A, Novellino E, Biroccio A, Randazzo A, Pagano B, Montesarchio D
A focused library of analogs of a lead-like G-quadruplex (G4) targeting compound (4), sharing a furobenzoxazine naphthoquinone core and differing for the pendant groups on the N-atom of the oxazine ring, has been here analyzed with the aim of developing more potent and selective ligands. These molecules have been tested vs. topologically different G4s by the G4-CPG assay, an affinity chromatography-based method for screening putative G4 ligands. The obtained results showed that all these compounds were able to bind several G4 structures, both telomeric and extra-telomeric, thus behaving as multi-target ligands, and two of them fully discriminated G4 vs. duplex DNA. Biological assays proved that almost all the compounds produced effective DNA damage, showing marked antiproliferative effects on tumor cells in the low μM range. Combined analysis of the G4-CPG binding assays and biological data led us to focus on compound S4-5, proved to be less cytotoxic than the parent compound 4 on normal cells. An in-depth biophysical characterization of the binding of S4-5 to different G4s showed that the here identified ligand has higher affinity for the G4s and higher ability to discriminate G4 vs. duplex DNA than 4. Molecular docking studies, in agreement with the NMR data, suggest that S4-5 interacts with the accessible grooves of the target G4 structures, giving clues for its increased G4 vs. duplex selectivity.
Clogging the Ubiquitin-Proteasome Machinery with Marine Natural Products: Last Decade Update.
Publication Date: 26/11/2018, on Marine drugs
by Della Sala G, Agriesti F, Mazzoccoli C, Tataranni T, Costantino V, Piccoli C
The ubiquitin-proteasome pathway (UPP) is the central protein degradation system in eukaryotic cells, playing a key role in homeostasis maintenance, through proteolysis of regulatory and misfolded (potentially harmful) proteins. As cancer cells produce proteins inducing cell proliferation and inhibiting cell death pathways, UPP inhibition has been exploited as an anticancer strategy to shift the balance between protein synthesis and degradation towards cell death. Over the last few years, marine invertebrates and microorganisms have shown to be an unexhaustive factory of secondary metabolites targeting the UPP. These chemically intriguing compounds can inspire clinical development of novel antitumor drugs to cope with the incessant outbreak of side effects and resistance mechanisms induced by currently approved proteasome inhibitors (e.g., bortezomib). In this review, we report about (a) the role of the UPP in anticancer therapy, (b) chemical and biological properties of UPP inhibitors from marine sources discovered in the last decade, (c) high-throughput screening techniques for mining natural UPP inhibitors in organic extracts. Moreover, we will tell about the fascinating story of salinosporamide A, the first marine natural product to access clinical trials as a proteasome inhibitor for cancer treatment.
Neuro-Immune Hemostasis: Homeostasis and Diseases in the Central Nervous System.
Publication Date: 26/11/2018, on Frontiers in cellular neuroscience
by De Luca C, Colangelo AM, Alberghina L, Papa M
Coagulation and the immune system interact in several physiological and pathological conditions, including tissue repair, host defense, and homeostatic maintenance. This network plays a key role in diseases of the central nervous system (CNS) by involving several cells (CNS resident cells, platelets, endothelium, and leukocytes) and molecular pathways (protease activity, complement factors, platelet granule content). Endothelial damage prompts platelet activation and the coagulation cascade as the first physiological step to support the rescue of damaged tissues, a flawed rescuing system ultimately producing neuroinflammation. Leukocytes, platelets, and endothelial cells are sensitive to the damage and indeed can release or respond to chemokines and cytokines (platelet factor 4, CXCL4, TNF, interleukins), and growth factors (including platelet-derived growth factor, vascular endothelial growth factor, and brain-derived neurotrophic factor) with platelet activation, change in capillary permeability, migration or differentiation of leukocytes. Thrombin, plasmin, activated complement factors and matrix metalloproteinase-1 (MMP-1), furthermore, activate intracellular transduction through complement or protease-activated receptors. Impairment of the neuro-immune hemostasis network induces acute or chronic CNS pathologies related to the neurovascular unit, either directly or by the systemic activation of its main steps. Neurons, glial cells (astrocytes and microglia) and the extracellular matrix play a crucial function in a "tetrapartite" synaptic model. Taking into account the neurovascular unit, in this review we thoroughly analyzed the influence of neuro-immune hemostasis on these five elements acting as a functional unit ("pentapartite" synapse) in the adaptive and maladaptive plasticity and discuss the relevance of these events in inflammatory, cerebrovascular, Alzheimer, neoplastic and psychiatric diseases. Finally, based on the solid reviewed data, we hypothesize a model of neuro-immune hemostatic network based on protein-protein interactions. In addition, we propose that, to better understand and favor the maintenance of adaptive plasticity, it would be useful to construct predictive molecular models, able to enlighten the regulating logic of the complex molecular network, which belongs to different cellular domains. A modeling approach would help to define how nodes of the network interact with basic cellular functions, such as mitochondrial metabolism, autophagy or apoptosis. It is expected that dynamic systems biology models might help to elucidate the fine structure of molecular events generated by blood coagulation and neuro-immune responses in several CNS diseases, thereby opening the way to more effective treatments.
Genome-wide mapping of 8-oxo-7,8-dihydro-2'-deoxyguanosine reveals accumulation of oxidatively-generated damage at DNA replication origins within transcribed long genes of mammalian cells.
Publication Date: 20/11/2018, on Nucleic acids research
by Amente S, Di Palo G, Scala G, Castrignanò T, Gorini F, Cocozza S, Moresano A, Pucci P, Ma B, Stepanov I, Lania L, Pelicci PG, Dellino GI, Majello B
8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is one of the major DNA modifications and a potent pre-mutagenic lesion prone to mispair with 2'-deoxyadenosine (dA). Several thousand residues of 8-oxodG are constitutively generated in the genome of mammalian cells, but their genomic distribution has not yet been fully characterized. Here, by using OxiDIP-Seq, a highly sensitive methodology that uses immuno-precipitation with efficient anti-8-oxodG antibodies combined with high-throughput sequencing, we report the genome-wide distribution of 8-oxodG in human non-tumorigenic epithelial breast cells (MCF10A), and mouse embryonic fibroblasts (MEFs). OxiDIP-Seq revealed sites of 8-oxodG accumulation overlapping with γH2AX ChIP-Seq signals within the gene body of transcribed long genes, particularly at the DNA replication origins contained therein. We propose that the presence of persistent single-stranded DNA, as a consequence of transcription-replication clashes at these sites, determines local vulnerability to DNA oxidation and/or its slow repair. This oxidatively-generated damage, likely in combination with other kinds of lesion, might contribute to the formation of DNA double strand breaks and activation of DNA damage response.
A Novel 12q13.2-q13.3 Microdeletion Syndrome With Combined Features of Diamond Blackfan Anemia, Pierre Robin Sequence and Klippel Feil Deformity.
Publication Date: 19/11/2018, on Frontiers in genetics
by Roberti D, Conforti R, Giugliano T, Brogna B, Tartaglione I, Casale M, Piluso G, Perrotta S
Diamond-Blackfan anemia (DBA) is a rare congenital erythroid aplasia with a highly heterogeneous genetic background; it usually occurs in infancy. Approximately 30-40% of patients have other associated congenital anomalies; in particular, facial anomalies, such as cleft palate, are part of about 10% of the DBA clinical presentations. Pierre Robin sequence (PRS) is a heterogeneous condition, defined by the presence of the triad of glossoptosis, micrognathia and cleft palate; it occurs in 1/8500 to 1/14,000 births. Klippel Feil (KF) syndrome is a complex of both osseous and visceral anomalies, characterized mainly by congenital development defects of the cervical spine. We describe the case of a 22-years-old woman affected by DBA, carrying a deletion about 500 Kb-long at 12q13.2-q13.3 that included and, at least, others 25 flanking genes. The patient showed craniofacial anomalies due to PRS and suffered for KF deformities (type II). Computed Tomography study of cranio-cervical junction (CCJ) drew out severe bone malformations and congenital anomalies as atlanto-occipital assimilation (AOA), arcuate foramen and occipito-condylar hyperplasia. Foramen magnum was severely reduced. Atlanto-axial instability (AAI) was linked to atlanto-occipital assimilation, congenital vertebral fusion and occipito-condyle bone hyperplasia. Basilar invagination and platybasia were ruled out on CT and Magnetic Resonance Imaging (MRI) studies. Furthermore, the temporal Bone CT study showed anomalies of external auditory canals, absent mastoid pneumatization, chronic middle ear otitis and abnormal course of the facial nerve bones canal. The described phenotype might be related to the peculiar deletion affecting the patient, highlighting that genes involved in the in the breakdown of extracellular matrix (), in cell cycle regulation (, vesicular trafficking (, in ribonucleoprotein complexes formation () and muscles function ( and ) could be potentially related to bone-developmental disorders. Moreover, it points out that multiple associated ribosomal deficits might play a role in DBA-related phenotypes, considering the simultaneous deletion of three of them in the index case ( and , and it confirms the association among functional disruption and severe myopia. This report highlights the need for a careful genetic evaluation and a detailed phenotype-genotype correlation in each complex malformative syndrome.
Suggestive association between OPRM1 and impulse control disorders in Parkinson's disease.
Publication Date: 16/11/2018, on Movement disorders : official journal of the Movement Disorder Society
by Cormier-Dequaire F, Bekadar S, Anheim M, Lebbah S, Pelissolo A, Krack P, Lacomblez L, Lhommée E, Castrioto A, Azulay JP, Defebvre L, Kreisler A, Durif F, Marques-Raquel A, Brefel-Courbon C, Grabli D, Roze E, Llorca PM, Ory-Magne F, Benatru I, Ansquer S, Maltête D, Tir M, Krystkowiak P, Tranchant C, Lagha-Boukbiza O, Lebrun-Vignes B, Mangone G, Vidailhet M, Charbonnier-Beaupel F, Rascol O, Lesage S, Brice A, Tezenas du Montcel S, Corvol JC,
Impulse control disorders are frequently associated with dopaminergic therapy in Parkinson's disease. Genetic studies have suggested a high heritability of impulse control disorders in the general population and in PD. The aim of this study was to identify candidate gene variants associated with impulse control disorders and related behaviors in PD.
Disordered Peptides Looking for Their Native Environment: Structural Basis of CB1 Endocannabinoid Receptor Binding to Pepcans.
Publication Date: 16/11/2018, on Frontiers in molecular biosciences
by Emendato A, Guerrini R, Marzola E, Wienk H, Boelens R, Leone S, Picone D
Endocannabinoid peptides, or "pepcans," are endogenous ligands of the CB1 cannabinoid receptor. Depending on their length, they display diverse activity: For instance, the nona-peptide Pepcan-9, also known as hemopressin, is a powerful inhibitor of CB1, whereas the longer variant Pepcan-12, which extends by only three amino acid residues at the N-terminus, acts on both CB1 and CB2 as an allosteric modulator, although with diverse effects. Despite active research on their pharmacological applications, very little is known about structure-activity relationships of pepcans. Different structures have been proposed for the nona-peptide, which has also been reported to form fibrillar aggregates. This might have affected the outcome and reproducibility of bioactivity studies. In an attempt of elucidating the determinants of both biological activity and aggregation propensity of Pepcan-9 and Pepcan-12, we have performed their structure characterization in solvent systems characterized by different polarity and pH. We have found that, while disordered in aqueous environment, both peptides display helical structure in less polar environment, mimicking the proteic receptor milieu. In the case of Pepcan-9, this structure is fully consistent with the observed modulation of the CB1. For Pepcan-12, whose allosteric binding site is still unknown, the presented structure is compatible with the binding at one of the previously proposed allosteric sites on CB1. These findings open the way to structure-driven design of selective peptide modulators of CB1.
Left inferior parietal and posterior temporal cortices mediate the effect of action observation on semantic processing of objects: evidence from rTMS.
Publication Date: 08/11/2018, on Psychological research
by De Bellis F, Magliacano A, Sagliano L, Conson M, Grossi D, Trojano L
Previous studies showed that motor information related to tool use (i.e., functional actions) could affect processing of objects semantic properties, whereas motor information related to grasping or moving tool (i.e., structural actions) cannot. However, little is known about the neural correlates mediating such interaction between motor and semantic information. Here, healthy participants performed a semantic judgment task requiring identification of semantic relations among objects, after observing a functional, a structural or a pointing action prime. In a within-subject design, during prime presentation the participants underwent repetitive transcranial magnetic stimulation (rTMS) over the left supramarginal gyrus (SMG), the left posterior middle temporal gyrus (pMTG) or received sham stimulation. Results showed that in the sham condition observing functional actions (vs. structural and pointing actions) favoured processing of semantic relations based on function similarity (i.e., taxonomic relations), but not of relations based on co-occurrence within an event schema (i.e., thematic relations). Moreover, stimulation of both left SMG and pMTG abolished the effect of functional action primes worsening subsequent judgment about taxonomic relations, and this effect was greater after pMTG stimulation. rTMS did not affect processing of thematic semantic relations. We suggest that action observation triggers activation of functional motor information within left inferior parietal cortex, and that integration between functional motor and conceptual information in left temporal cortex could impact high-level semantic processing of tools.
Controlled Pore Glass-based oligonucleotide affinity support: towards High Throughput Screening methods for the identification of conformation-selective G-quadruplex ligands.
Publication Date: 07/11/2018, on Analytica chimica acta
by Platella C, Musumeci D, Arciello A, Doria F, Freccero M, Randazzo A, Amato J, Pagano B, Montesarchio D
Target selectivity is one of the main challenges in the search for small molecules able to act as effective and non-toxic anticancer and/or antiviral drugs. To achieve this goal, handy, rapid and reliable High Throughput Screening methodologies are needed. We here describe a novel functionalization for the solid phase synthesis of oligonucleotides on Controlled Pore Glass, including a flexible hexaethylene glycol spacer linking the first nucleoside through the nucleobase via a covalent bond stable to the final deprotection step. This allowed us preparing fully deprotected oligonucleotides still covalently attached to their supports. In detail, on this support we performed both the on-line synthesis of different secondary structure-forming oligonucleotides and the affinity chromatography-based screenings of conformation-selective G-quadruplex ligands. By using a fluorescent core-extended naphthalene diimide with different emitting response upon binding to sequences folding into G-quadruplexes of different topologies, we have been able to discriminate not only G-quadruplex vs. duplex DNA structures, but also different G-quadruplex conformations on the glass beads by confocal microscopy.
Bergamot Polyphenols Boost Therapeutic Effects of the Diet on Non-Alcoholic Steatohepatitis (NASH) Induced by "Junk Food": Evidence for Anti-Inflammatory Activity.
Publication Date: 01/11/2018, on Nutrients
by Parafati M, Lascala A, La Russa D, Mignogna C, Trimboli F, Morittu VM, Riillo C, Macirella R, Mollace V, Brunelli E, Janda E
Wrong alimentary behaviors and so-called "junk food" are a driving force for the rising incidence of non-alcoholic fatty liver disease (NAFLD) among children and adults. The "junk food" toxicity can be studied in "cafeteria" (CAF) diet animal model. Young rats exposed to CAF diet become obese and rapidly develop NAFLD. We have previously showed that bergamot () flavonoids, in the form of bergamot polyphenol fraction (BPF), effectively prevent CAF diet-induced NAFLD in rats. Here, we addressed if BPF can accelerate therapeutic effects of weight loss induced by a normocaloric standard chow (SC) diet. 21 rats fed with CAF diet for 16 weeks to induce NAFLD with inflammatory features (NASH) were divided into three groups. Two groups were switched to SC diet supplemented or not with BPF (CAF/SC±BPF), while one group continued with CAF diet (CAF/CAF) for 10 weeks. BPF had no effect on SC diet-induced weight loss, but it accelerated hepatic lipid droplets clearance and reduced blood triglycerides. Accordingly, BPF improved insulin sensitivity, but had little effect on leptin levels. Interestingly, the inflammatory parameters were still elevated in CAF/SC livers compared to CAF/CAF group after 10 weeks of dietary intervention, despite over 90% hepatic fat reduction. In contrast, BPF supplementation decreased hepatic inflammation by reducing interleukin 6 () mRNA expression and increasing anti-inflammatory , which correlated with fewer Kupffer cells and lower inflammatory foci score in CAF/SC+BPF livers compared to CAF/SC group. These data indicate that BPF mediates a specific anti-inflammatory activity in livers recovering from NASH, while it boosts lipid-lowering and anti-diabetic effects of the dietary intervention.
Selegiline in Patients With Disorder of Consciousness: An Open Pilot Study.
Publication Date: 01/11/2018, on The Canadian journal of neurological sciences. Le journal canadien des sciences neurologiques
by Masotta O, Trojano L, Loreto V, Moretta P, Estraneo A
This open study investigated the clinical effects of 10-week selegiline administration in six patients in vegetative state and in four patients in a minimally conscious state, at least 6 months after onset. Clinical outcome was assessed by Coma Recovery Scale-Revised once a week during selegiline administration and 1 month later. Three patients stopped treatment because of possible side effects. After treatment and at 1 month of follow-up, four patients showed improvements in clinical diagnosis, and three patients showed an increase in arousal level only. Selegiline might represent a relatively safe option to enhance arousal and promote recovery in brain-injured patients with disorders of consciousness.
"Pure apathy" and cognitive dysfunctions in Parkinson's disease: A meta-analytic study.
Publication Date: 01/11/2018, on Neuroscience and biobehavioral reviews
by D'Iorio A, Maggi G, Vitale C, Trojano L, Santangelo G
Parkinson's Disease (PD) is characterized by motor and non-motor symptoms such as cognitive deficit and behavioural disturbances. Apathy seems to be related to cognitive impairment, but some studies failed to confirm the relationship due to different methodological procedures across studies. A meta-analysis on 8 studies was performed to explore the cognitive correlates of apathy without depression and dementia (pure apathy). Global cognitive function, memory, executive functions, processing speed/attention/working memory, visuospatial abilities and language were the outcomes. The effect size of the relationship between "pure apathy" and reduced global cognitive functioning, executive functions, processing speed/attention/working memory, visuospatial functions, long-term verbal memory was moderate, whereas apathy was strongly associated with inhibition dysfunctioning. Our results revealed a strong association between "pure apathy" and cognitive dysfunctions, particularly deficit of memory and executive functions related to altered prefronto-subcortical circuitries.
Publication Date: 01/11/2018, on Stem cells (Dayton, Ohio)
by Squillaro T, Finicelli M, Peluso G, Galderisi U
Validation of the Nine Hole Peg Test as a measure of dexterity in myotonic dystrophy type 1.
Publication Date: 01/11/2018, on Neuromuscular disorders : NMD
by Cutellè C, Rastelli E, Gibellini M, Greco G, Frezza E, Botta A, Terracciano C, Massa R
We aimed to validate the Nine Hole Peg Test as a measure of dexterity in myotonic dystrophy type 1 (DM1). Fifty patients with adult-onset, genetically confirmed DM1 were evaluated by Nine Hole Peg Test and re-evaluated at one week. Myotonia was not a limiting factor. The first test was compared with that performed by normal subjects (n = 28). Contextually, patients underwent handgrip and three-finger pinch assessments by handheld dynamometer. The Nine Hole Peg Test showed high intra-rater and inter-rater reliability in DM1 [ICC 0.86/0.83 for dominant and 0.90/0.88 for non-dominant hand, respectively]. Inverse correlation with handgrip and pinch strength values (r = -0.4; p < 0.01) and direct correlation with Muscular Impairment Rating Scale (r = 0.4; p < 0.01) were found for both DH and NDH. The test was able to differentiate severe DM1 patients, stratified by extent of muscle impairment, from mildly affected and normal controls, with a sensitivity of 97% and 95% for dominant hand and non-dominant hand, respectively (p < 0.0001). In conclusion, we showed that the Nine Hole Peg Test is a reliable, valid and sensitive test of dexterity in DM1, and that it can be considered as a candidate outcome measure to monitor natural history of disease and, possibly, therapeutic response in clinical trials.
The extreme hyper-reactivity of Cys94 in lysozyme avoids its amorphous aggregation.
Publication Date: 30/10/2018, on Scientific reports
by Bocedi A, Cattani G, Martelli C, Cozzolino F, Castagnola M, Pucci P, Ricci G
Many proteins provided with disulfide bridges in the native state undergo amorphous irreversible aggregation when these bonds are not formed. Here we show that egg lysozyme displays a clever strategy to prevent this deleterious aggregation during the nascent phase when disulfides are still absent. In fact, when the reduced protein assembles into a molten globule state, its cysteines acquire strong hyper-reactivity towards natural disulfides. The most reactive residue, Cys94, reacts with oxidized glutathione (GSSG) 3000 times faster than an unperturbed protein cysteine. A low pK of its sulfhydryl group (6.6/7.1) and a productive complex with GSSG (K = 0.3 mM), causes a fast glutathionylation of this residue (t = 3 s) and a complete inhibition of the protein aggregation. Other six cysteines display 70 times higher reactivity toward GSSG. The discovery of extreme hyper-reactivity in cysteines only devoted to structural roles opens new research fields for Alzheimer's and Parkinson diseases.