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Publications 2019

Application of an in Vitro Blood−Brain Barrier Model in the Selection of Experimental Drug Candidates for the Treatment of Huntington’s Disease

By Di Marco, Annalise; Gonzalez Paz, Odalys; Fini, Ivan; Vignone, Domenico; Cellucci, Antonella; Battista, Maria Rosaria; Auciello, Giulio; Orsatti, Laura; Zini, Matteo; Monteagudo, Edith; et al
From Molecular Pharmaceutics (2019), 16(5), 2069-2082. Language: English, Database: CAPLUS, DOI:10.1021/acs.molpharmaceut.9b00042

Application of an in Vitro Blood-Brain Barrier Model in the Selection of Experimental Drug Candidates for the Treatment of Huntington's DiseaseHuntington’s disease (HD) is a neurodegenerative disease caused by a polyglutamine expansion in the Huntington (HTT) protein. For drug candidates targeting HD, the ability to cross the blood brain barrier (BBB) and reach the site of action in the central nervous system (CNS) is crucial for achieving pharmacol. activity. To assess permeability of selected compds. across the BBB, we utilized a two-dimensional model composed of primary porcine brain endothelial cells and rat astrocytes. Our objective was to use this in vitro model to rank and prioritize compds. for in vivo pharmacokinetic and brain penetration studies. The model was first characterized using a set of validation markers chosen based on their functional importance at the BBB. It was shown to fulfill the major BBB characteristics, including functional tight junctions, high transendothelial elec. resistance (TEER), expression and activity of influx and efflux transporters. The in vitro permeability of fifty-four, structurally diverse, known compds. was detd. and shown to have a good correlation with the in situ brain perfusion data in rodents. We used this model to investigate BBB permeability of a series of new HD compds. from different chem. classes and we found good correlation with in vivo brain permeation, demonstrating the usefulness of the in vitro model for optimizing CNS drug properties and for guiding the selection of lead compds. in a drug discovery setting.

Identification of Isoform 2 Acid-Sensing Ion Channel Inhibitors as Tool Compounds for Target Validation Studies in CNS

By Bencheva Leda Ivanova; De Matteo Marilenia; Ferrante Luca; Ferrara Marco; Prandi Adolfo; Randazzo Pietro; Ronzoni Silvano; Sinisi Roberta; Seneci Pierfausto; Di Fabio Romano; et al
From ACS medicinal chemistry letters (2019), 10(4), 627-632, Language: English, Database: MEDLINE

Acid-sensing ion channels (ASICs) are a family of ion channels permeable to cations and largely responsible for the onset of acid-evoked ion currents both in neurons and in different types of cancer cells, thus representing a potential target for drug discovery. Owing to the limited attention ASIC2 has received so far, an exploratory program was initiated to identify ASIC2 inhibitors using diminazene, a known pan-ASIC inhibitor, as a chemical starting point for structural elaboration. The performed exploration enabled the identification of a novel series of ASIC2 inhibitors. In particular, compound 2u is a brain penetrant ASIC2 inhibitor endowed with an optimal pharmacokinetic profile. This compound may represent a useful tool to validate in animal models in vivo the role of ASIC2 in different neurodegenerative central nervous system pathologies.

Developmental abnormalities in cortical GABAergic system in mice lacking mGlu3 metabotropic glutamate receptors.

By Imbriglio Tiziana; Verhaeghe Remy; Pascarelli Maria Teresa; Chece Giuseppina; Del Percio Claudio; Fucile Sergio; Babiloni Claudio; Battaglia Giuseppe; Limatola Cristina; Nicoletti Ferdinando; et al
From FASEB journal : official publication of the Federation of American Societies for Experimental Biology (2019), 33(12), 14204-14220, Language: English, Database: MEDLINE

Polymorphic variants of the gene encoding for metabotropic glutamate receptor 3 (mGlu3) are linked to schizophrenia. Because abnormalities of cortical GABAergic interneurons lie at the core of the pathophysiology of schizophrenia, we examined whether mGlu3 receptors influence the developmental trajectory of cortical GABAergic transmission in the postnatal life. mGlu3(-/-) mice showed robust changes in the expression of interneuron-related genes in the prefrontal cortex (PFC), including large reductions in the expression of parvalbumin (PV) and the GluN1 subunit of NMDA receptors. The number of cortical cells enwrapped by perineuronal nets was increased in mGlu3(-/-) mice, suggesting that mGlu3 receptors shape the temporal window of plasticity of PV(+) interneurons. Electrophysiological measurements of GABAA receptor-mediated responses revealed a more depolarized reversal potential of GABA currents in the somata of PFC pyramidal neurons in mGlu3(-/-) mice at postnatal d 9 associated with a reduced expression of the K(+)/Cl(-) symporter. Finally, adult mGlu3(-/-) mice showed lower power in electroencephalographic rhythms at 1-45 Hz in quiet wakefulness as compared with their wild-type counterparts. These findings suggest that mGlu3 receptors have a strong impact on the development of cortical GABAergic transmission and cortical neural synchronization mechanisms corroborating the concept that genetic variants of mGlu3 receptors may predispose to psychiatric disorders.-Imbriglio, T., Verhaeghe, R., Martinello, K., Pascarelli, M. T., Chece, G., Bucci, D., Notartomaso, S., Quattromani, M., Mascio, G., Scalabri, F., Simeone, A., Maccari, S., Del Percio, C., Wieloch, T., Fucile, S., Babiloni, C., Battaglia, G., Limatola, C., Nicoletti, F., Cannella, M. Developmental abnormalities in cortical GABAergic system in mice lacking mGlu3 metabotropic glutamate receptors.

Multiple Synthetic Routes to the Mini-Protein Omomyc and Coiled-coil Domain Truncations.

Brown ZZ, Mapelli C, Farasat I, Shoultz AV, Orvieto F, Santoprete A, Bianchi E, Johnson SA, McCracken AB, Chen K, Zhu X, Demma MJ, Lacey BM, Canada KA, Garbaccio RM, O’Neil J, Walji A.
J Org Chem. 2019 Oct 29.

Omomyc Reveals New Mechanisms To Inhibit the MYC Oncogene.

By Demma Mark J; O’Neil Jennifer; Mapelli Claudio; Walji Abbas; Sun Angie; Zelina John; Hall Brian; Ray Kallol; Bodea Smaranda; Ruprecht Benjamin; et al
From Molecular and cellular biology (2019), 39(22), , Language: English, Database: MEDLINE

The MYC oncogene is upregulated in human cancers by translocation, amplification, and mutation of cellular pathways that regulate Myc. Myc/Max heterodimers bind to E box sequences in the promoter regions of genes and activate transcription. The MYC inhibitor Omomyc can reduce the ability of MYC to bind specific box sequences in promoters of MYC target genes by binding directly to E box sequences as demonstrated by chromatin immunoprecipitation (CHIP). Here, we demonstrate by both a proximity ligation assay (PLA) and double chromatin immunoprecipitation (ReCHIP) that Omomyc preferentially binds to Max, not Myc, to mediate inhibition of MYC-mediated transcription by replacing MYC/MAX heterodimers with Omomyc/MAX heterodimers. The formation of Myc/Max and Omomyc/Max heterodimers occurs cotranslationally; Myc, Max, and Omomyc can interact with ribosomes and Max RNA under conditions in which ribosomes are intact. Taken together, our data suggest that the mechanism of action of Omomyc is to bind DNA as either a homodimer or a heterodimer with Max that is formed cotranslationally, revealing a novel mechanism to inhibit the MYC oncogene. We find that in vivo, Omomyc distributes quickly to kidneys and liver and has a short effective half-life in plasma, which could limit its use in vivo.

Aspergillus olivimuriae sp. nov., a halotolerant species isolated from olive brine.

By Crognale Silvia; Pesciaroli Lorena; Felli Martina; Petruccioli Maurizio; D’Annibale Alessandro; Bresciani Alberto; Peterson Stephen W
From International journal of systematic and evolutionary microbiology (2019), 69(9), 2899-2906, Language: English, Database: MEDLINE

A facultative halo-tolerant Aspergillus strain was isolated from olive brine waste, the effluent from the debittering process of table olives. Phenotypic and molecular characteristics showed clearly that the isolate represents a novel species. Based on the source of isolation, the new species has been named Aspergillus olivimuriae. It was found tolerant to high concentrations of NaCl (15 %) or sucrose (60 %) and it exhibits substantial growth under these conditions. Although the new species grew profusely at 37 °C, no growth was observed at 40 °C, conidia en masse were avellaneous on all media. The description of the new species Aspergillus olivimuriae brings the total species of Aspergillus sect. Flavipedes to 15. The type strain of A. olivimuriae sp. nov. is NRRL 66783 (CCF 6208), its whole genome has been deposited as PRJNA498048.

Phospho-S129 Alpha-Synuclein Is Present in Human Plasma but Not in Cerebrospinal Fluid as Determined by an Ultrasensitive Immunoassay.

By Cariulo Cristina; Martufi Paola; Verani Margherita; Azzollini Lucia; Caricasole Andrea; Petricca Lara; Bruni Giordana; Weiss Andreas; Deguire Sean M; Lashuel Hilal A; et al
From Frontiers in neuroscience (2019), 13889, Language: English, Database: MEDLINE

Accumulation and aggregation of misfolded alpha-synuclein is believed to be a cause of Parkinson’s disease (PD). Phosphorylation of alpha-synuclein at S129 is known to be associated with the pathological misfolding process, but efforts to investigate the relevance of this post-translational modification for pathology have been frustrated by difficulties in detecting and quantifying it in relevant samples. We report novel, ultrasensitive immunoassays based on single-molecule counting technology, useful for detecting alpha-synuclein and its S129 phosphorylated form in clinical samples in the low pg/ml range. Using human CSF and plasma samples, we find levels of alpha-synuclein comparable to those previously reported. However, while alpha-synuclein phosphorylated on S129 could easily be detected in human plasma, where its detection is extremely sensitive to protein phosphatases, its levels in CSF were undetectable, with a possible influence of a matrix effect. In plasma samples from a small test cohort comprising 5 PD individuals and five age-matched control individuals we find that pS129 alpha-synuclein levels are increased in PD plasma samples, in line with previous reports. We conclude that pS129 alpha-synuclein is not detectable in CSF and recommend the addition of phosphatase inhibitors to plasma samples at the time of collection. Moreover, the findings obtained on the small cohort of clinical plasma samples point to plasma pS129 alpha-synuclein levels as a candidate diagnostic biomarker in PD.

Fish-derived antimicrobial peptides: Activity of a chionodracine mutant against bacterial models and human bacterial pathogens.

By Buonocore Francesco; Picchietti Simona; Porcelli Fernando; Della Pelle Giulia; Poerio Elia; Fausto Anna Maria; Scapigliati Giuseppe; Olivieri Cristina; Bugli Francesca; Menchinelli Giulia; et al
From Developmental and comparative immunology (2019), 969-17, Language: English, Database: MEDLINE

The increasing resistance to conventional antibiotics is an urgent problem that can be addressed by the discovery of new antimicrobial drugs such as antimicrobial peptides (AMPs). AMPs are components of innate immune system of eukaryotes and are not prone to the conventional mechanisms that are responsible of drug resistance. Fish are an important source of AMPs and, recently, we have isolated and characterized a new 22 amino acid residues peptide, the chionodracine (Cnd), from the Antarctic icefish Chionodraco hamatus. In this paper we focused on a new Cnd-derived mutant peptide, namely Cnd-m3a, designed to improve the selectivity against prokaryotic cells and the antimicrobial activity against human pathogens of the initial Cnd template. Cnd-m3a was used for immunization of rabbits, which gave rise to a polyclonal antibody able to detect the peptide. The interaction kinetic of Cnd-m3a with the Antarctic bacterium Psychrobacter sp. (TAD1) was imaged using a transmission electron microscopy (TEM) immunogold method. Initially the peptide was associated with the plasma membrane, but after 180 min of incubation, it was found in the cytoplasm interacting with a DNA target inside the bacterial cells. Using fluorescent probes we showed that the newly designed mutant can create pores in the outer membrane of the bacteria E. coli and Psychrobacter sp. (TAD1), confirming the results of TEM analysis. Moreover, in vitro assays demonstrated that Cnd-m3a is able to bind lipid vesicles of different compositions with a preference toward negatively charged ones, which mimics the prokaryotic cell. The Cnd-m3a peptide showed quite low hemolytic activity and weak cytotoxic effect against human primary and tumor cell lines, but high antimicrobial activity against selected Gram – human pathogens. These results highlighted the high potential of the Cnd-m3a peptide as a starting point for developing a new human therapeutic agent.

Salivary Metabolome and Soccer Match: Challenges for Understanding Exercise induced Changes.

By Pitti Erica; Petrella Greta; Cicero Daniel Oscar; Di Marino Sara; Summa Vincenzo; Perrone Marco; Bernardini Andrea; D’Ottavio Stefano
From Metabolites (2019), 9(7), , Language: English, Database: MEDLINE

Saliva samples of seventeen soccer players were analyzed by nuclear magnetic resonance before and after an official match. Two different ways of normalizing data are discussed, using total proteins and total metabolite concentrations. Changes in markers related to energy, hydration status, amino acids and other compounds were found. The limits and advantages of using saliva to define the systemic responses to exercise are examined, both in terms of data normalization and interpretation, and the time that the effect lasts in this biofluid, which is shorter to that commonly observed in blood. The heterogeneous nature and different timing of the exercise developed by players also plays an important role in the metabolic changes that can be measured. Our work focuses mainly on three different aspects: The effect that time sampling has on the observed effect, the type of normalization that is necessary to perform in order to cope with changes in water content, and the metabolic response that can be observed using saliva.

Development and characterization of cellular biosensors for HTS of erythroid differentiation inducers targeting the transcriptional activity of γ-globin and β-globin gene promoters.

By Breveglieri Giulia; Finotti Alessia; Cosenza Lucia Carmela; Zuccato Cristina; Borgatti Monica; Gambari Roberto; Breveglieri Giulia; Cosenza Lucia Carmela; Borgatti Monica; Salvatori Francesca; et al
From Analytical and bioanalytical chemistry (2019), 411(29), 7669-7680, Language: English, Database: MEDLINE

There is a general agreement that pharmacologically mediated stimulation of human γ-globin gene expression and increase of production of fetal hemoglobin (HbF) is a potential therapeutic approach in the experimental therapy of β-thalassemia and sickle cell anemia. Here, we report the development and characterization of cellular biosensors carrying enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP) genes under the control of the human γ-globin and β-globin gene promoters, respectively; these dual-reporter cell lines are suitable to identify the induction ability of screened compounds on the transcription in erythroid cells of γ-globin and β-globin genes by FACS with efficiency and reproducibility. Our experimental system allows to identify (a) HbF inducers stimulating to different extent the activity of the γ-globin gene promoter and (b) molecules that stimulate also the activity of the β-globin gene promoter. A good correlation does exist between the results obtained by using the EGFP/RFP clones and experiments performed on erythroid precursor cells from β-thalassemic patients, confirming that this experimental system can be employed for high-throughput screening (HTS) analysis. Finally, we have demonstrated that this dual-reporter cell line can be used for HTS in 384-well plate, in order to identify novel HbF inducers for the therapy of β-thalassemia and sickle cell anemia.

The Outcomes of Decorated Prolines in the Discovery of Antimicrobial Peptides from Temporin-L.

By Buommino Elisabetta; Carotenuto Alfonso; Antignano Ignazio; Bellavita Rosa; Merlino Francesco; Novellino Ettore; Brancaccio Diego; Grieco Paolo; Casciaro Bruno; Loffredo Maria Rosa; et al
From ChemMedChem (2019), 14(13), 1283-1290, Language: English, Database: MEDLINE

Previously, we identified a potent antimicrobial analogue of temporin L (TL), [Pro(3) ]TL, in which glutamine at position 3 was substituted with proline. In this study, a series of analogues in which position 3 is substituted with non-natural proline derivatives, was investigated for correlations between the conformational properties of the compounds and their antibacterial, cytotoxic, and hemolytic activities. Non-natural proline analogues with substituents at position 4 of the pyrrolidine ring were considered. Structure-activity relationship (SAR) studies of these analogues were performed by means of antimicrobial and cytotoxicity assays along with circular dichroism (CD) and NMR spectroscopic analyses for selected compounds. The most promising peptides were additionally evaluated for their activity against some representative veterinary microbial strains to compare with those from human strains. We identified novel analogues with interesting properties that make them attractive lead compounds.

Collagen Prolyl Hydroxylation-Dependent Metabolic Perturbation Governs Epigenetic Remodeling and Mesenchymal Transition in Pluripotent and Cancer Cells.

By D’Aniello Cristina; Cermola Federica; Migliaccio Agnese; Casalino Laura; De Cesare Dario; Patriarca Eduardo J; Minchiotti Gabriella; D’Aniello Cristina; Cermola Federica; Gagliardi Miriam; et al
From Cancer research (2019), 79(13), 3235-3250, Language: English, Database: MEDLINE

Collagen Prolyl Hydroxylation-Dependent Metabolic Perturbation Governs Epigenetic Remodeling and Mesenchymal Transition in Pluripotent and Cancer CellsCollagen prolyl hydroxylation (CPH), which is catalyzed by prolyl 4-hydroxylase (P4H), is the most prevalent posttranslational modification in humans and requires vitamin C (VitC). Here, we demonstrate that CPH acts as an epigenetic modulator of cell plasticity. Increased CPH induced global DNA/histone methylation in pluripotent stem and tumor cells and promoted cell state transition (CST). Interfering with CPH by either genetic ablation of P4H subunit alpha-2 (P4HA2) or pharmacologic treatment reverted epigenetic changes and antagonized CST. Mechanistically, we suggest that CPH modifies the epigenetic landscape by reducing VitC for DNA and histone demethylases. Repurposed drugs targeting CPH-mediated metabolic perturbation, such as the antiasthmatic budesonide, blocked metastatic dissemination of breast cancer cells in vivo by preventing mesenchymal transition. Our study provides mechanistic insights into how metabolic cues and epigenetic factors integrate to control CST and paves the way for the development of novel antimetastatic strategies. SIGNIFICANCE: A phenotype-based high-throughput screening reveals unforeseen metabolic control of cell plasticity and identifies budesonide as a drug candidate for metastatic cancer.

A novel member of Prame family, Gm12794c, counteracts retinoic acid differentiation through the methyltransferase activity of PRC2.

Napolitano G, Tagliaferri D, Fusco S, Cirillo C, De Martino I, Addeo M, Mazzone P, Russo NA, Natale F, Cardoso MC, De Luca L, Lamorte D, La Rocca F, De Felice M, Falco G-
Cell Death Differ. 2019 Jun 11.

Octreotide Conjugates for Tumor Targeting and Imaging.

By Figueras Eduard; Martins Ana; Borbely Adina; Frese Marcel; Sewald Norbert; Martins Ana; Gallinari Paola; Steinkuhler Christian; Le Joncour Vadim; Laakkonen Pirjo; et al
From Pharmaceutics (2019), 11(5), , Language: English, Database: MEDLINE

Tumor targeting has emerged as an advantageous approach to improving the efficacy and safety of cytotoxic agents or radiolabeled ligands that do not preferentially accumulate in the tumor tissue. The somatostatin receptors (SSTRs) belong to the G-protein-coupled receptor superfamily and they are overexpressed in many neuroendocrine tumors (NETs). SSTRs can be efficiently targeted with octreotide, a cyclic octapeptide that is derived from native somatostatin. The conjugation of cargoes to octreotide represents an attractive approach for effective tumor targeting. In this study, we conjugated octreotide to cryptophycin, which is a highly cytotoxic depsipeptide, through the protease cleavable Val-Cit dipeptide linker using two different self-immolative moieties. The biological activity was investigated in vitro and the self-immolative part largely influenced the stability of the conjugates. Replacement of cryptophycin by the infrared cyanine dye Cy5.5 was exploited to elucidate the tumor targeting properties of the conjugates in vitro and in vivo. The compound efficiently and selectively internalized in cells overexpressing SSTR2 and accumulated in xenografts for a prolonged time. Our results on the in vivo properties indicate that octreotide may serve as an efficient delivery vehicle for tumor targeting.

Inhibiting pathologically active ADAM10 rescues synaptic and cognitive decline in Huntington’s disease.

By Vezzoli Elena; Caron Ilaria; Besusso Dario; Conforti Paola; Battaglia Elisa; Rivetti di Val Cervo Pia; Cattaneo Elena; Zuccato Chiara; Vezzoli Elena; Caron Ilaria; et al
From The Journal of clinical investigation (2019), 129(6), 2390-2403, Language: English, Database: MEDLINE

A disintegrine and metalloproteinase 10 (ADAM10) is implicated in synaptic function through its interaction with postsynaptic receptors and adhesion molecules. Here, we report that levels of active ADAM10 are increased in Huntington’s disease (HD) mouse cortices and striata and in human postmortem caudate. We show that, in the presence of polyglutamine-expanded (polyQ-expanded) huntingtin (HTT), ADAM10 accumulates at the postsynaptic densities (PSDs) and causes excessive cleavage of the synaptic protein N-cadherin (N-CAD). This aberrant phenotype is also detected in neurons from HD patients where it can be reverted by selective silencing of mutant HTT. Consistently, ex vivo delivery of an ADAM10 synthetic inhibitor reduces N-CAD proteolysis and corrects electrophysiological alterations in striatal medium-sized spiny neurons (MSNs) of 2 HD mouse models. Moreover, we show that heterozygous conditional deletion of ADAM10 or delivery of a competitive TAT-Pro-ADAM10709-729 peptide in R6/2 mice prevents N-CAD proteolysis and ameliorates cognitive deficits in the mice. Reduction in synapse loss was also found in R6/2 mice conditionally deleted for ADAM10. Taken together, these results point to a detrimental role of hyperactive ADAM10 at the HD synapse and provide preclinical evidence of the therapeutic potential of ADAM10 inhibition in HD.

Design, Synthesis, and Biological Evaluation of New 1-(Aryl-1 H-pyrrolyl)(phenyl)methyl-1 H-imidazole Derivatives as Antiprotozoal Agent.

By Saccoliti Francesco; Madia Valentina Noemi; Tudino Valeria; De Leo Alessandro; Pescatori Luca; Messore Antonella; De Vita Daniela; Scipione Luigi; Costi Roberta; Di Santo Roberto; et al
From Journal of medicinal chemistry (2019), 62(3), 1330-1347, Language: English, Database: MEDLINE

We have designed and synthesized a series of new imidazole-based compounds structurally related to an antiprotozoal agent with nanomolar activity which we identified recently. The new analogues possess micromolar activities against Trypanosoma brucei rhodesiense and Leishmania donovani and nanomolar potency against Plasmodium falciparum. Most of the analogues displayed IC50 within the low nanomolar range against Trypanosoma cruzi, with very high selectivity toward the parasite. Discussion of structure-activity relationships and in vitro biological data for the new compounds are provided against a number of different protozoa. The mechanism of action for the most potent derivatives (5i, 6a-c, and 8b) was assessed by a target-based assay using recombinant T. cruzi CYP51. Bioavailability and efficacy of selected hits were assessed in a T. cruzi mouse model, where 6a and 6b reduced parasitemia in animals >99% following intraperitoneal administration of 25 mg/kg/day dose for 4 consecutive days.

Synthesis and Biological Evaluation of RGD⁻Cryptophycin Conjugates for Targeted Drug Delivery.

By Borbely Adina; Figueras Eduard; Martins Ana; Kemker Isabell; Frese Marcel; Sewald Norbert; Martins Ana; Gallinari Paola; Steinkuhler Christian; Esposito Simone; et al
From Pharmaceutics (2019), 11(4), , Language: English, Database: MEDLINE

Cryptophycins are potent tubulin polymerization inhibitors with picomolar antiproliferative potency in vitro and activity against multidrug-resistant (MDR) cancer cells. Because of neurotoxic side effects and limited efficacy in vivo, cryptophycin-52 failed as a clinical candidate in cancer treatment. However, this class of compounds has emerged as attractive payloads for tumor-targeting applications. In this study, cryptophycin was conjugated to the cyclopeptide c(RGDfK), targeting integrin αvβ3, across the protease-cleavable Val-Cit linker and two different self-immolative spacers. Plasma metabolic stability studies in vitro showed that our selected payload displays an improved stability compared to the parent compound, while the stability of the conjugates is strongly influenced by the self-immolative moiety. Cathepsin B cleavage assays revealed that modifications in the linker lead to different drug release profiles. Antiproliferative effects of Arg-Gly-Asp (RGD)-cryptophycin conjugates were evaluated on M21 and M21-L human melanoma cell lines. The low nanomolar in vitro activity of the novel conjugates was associated with inferior selectivity for cell lines with different integrin αvβ3 expression levels. To elucidate the drug delivery process, cryptophycin was replaced by an infrared dye and the obtained conjugates were studied by confocal microscopy.

A novel method using nuclear magnetic resonance for plasma proteinbinding assessment in drug discovery programs.

By Gallo, Mariana; Matteucci, Sara; Alaimo, Nadine; Pitti, Erica; Orsale, Maria V.; Summa, Vincenzo; Cicero, Daniel O.; Monteagudo, Edith
From Journal of Pharmaceutical and Biomedical Analysis (2019), 167, 21-29. Language: English, Database: CAPLUS, DOI:10.1016/j.jpba.2019.01.049

A new methodol. based on NMR was developed to det. plasma protein binding (PPB) of drug candidates in drug discovery programs. A strong correlation was found between the attenuation of NMR signals of diverse drugs in the presence of different plasma concns. and their fraction bound (fb) reported in the literature. Based on these results, a protocol for a rapid calcn. of fb of small mols. was established. The advantage of using plasma instead of purified recombinant proteins and the possibility of pool anal. to increase throughput were also evaluated. This novel methodol. proved to be very versatile, cost-effective, fast and suitable for automation. As a plus, it contemporarily provides a quality check and soly. of the compd.

Bromoindirubin-3′-oxime intercepts GSK3 signaling to promote and enhance skeletal muscle differentiation affecting miR-206 expression in mice

By Ragozzino, Elvira; Brancaccio, Mariarita; Di Costanzo, Antonella; Scalabri, Francesco; Andolfi, Gennaro; Wanderlingh, Luca G.; Patriarca, Eduardo J.; Minchiotti, Gabriella; Altamura, Sergio; Varrone, Francesca; et al
From Scientific Reports (2019), 9(1), 18091. Language: English, Database: CAPLUS, DOI:10.1038/s41598-019-54574-4

To identify new and alternative compds. with a functional role in skeletal muscle myogenesis, we screened a library of pharmacol. active compds. and selected the small mol. 6-bromoindirubin-3′-oxime as an inhibitor of myoblast proliferation. Using C2C12 cells, we examd. BIO’s effect during myoblast proliferation and differentiation showing that BIO treatment promotes transition from proliferation to myogenic differentiation through the arrest of cell cycle. Here, we show that BIO able promote myogenic differentiation in damaged myotubes in-vitro by enriching population of newly formed skeletal muscle myotubes. Moreover, in-vivo expts. in CTX-damaged TA muscle confirmed the pro-differentiation capability of BIO as shown by the increasing of the percentage of myofibers with centralized nuclei as well as by the increasing of myofibers no. Addnl., we have identified a strong correlation of miR-206 with BIO treatment both in-vitro and in-vivo: the enhanced expression of miR-206 was obsd. in-vitro in BIO-treated proliferating myoblasts, miR-206 restored expression was obsd. in a forced miR-206 silencing conditions antagomiR-mediated upon BIO treatment, and in-vivo in CTX-injured muscles miR-206 enhanced expression was obsd. upon BIO treatment. Taken together, our results highlight the capacity of BIO to act as a pos. modulator of skeletal muscle differentiation in-vitro and in-vivo opening up a new perspective for novel therapeutic targets to correct skeletal muscle defects.

An Ig Transmembrane Domain Motif Improves the Function of TCRs Transduced in Human T Cells: Implications for Immunotherapy

By D’Apice Luciana; Cipria Deborah; Sartorius Rossella; Coscia Maria R; Oreste Umberto; Marzullo Vincenzo M; De Berardinis Piergiuseppe; Cuccaro Fausta; Varriale Sonia; Salerno Massimiliano; et al
From Journal of immunotherapy (Hagerstown, Md. : 1997) (2019), 42(4), 97-109, Language: English, Database: MEDLINE

Adoptive transfer of T lymphocytes (ACT) engineered with T-cell receptors (TCRs) of known antitumor specificity is an effective therapeutic strategy. However, a major constraint of ACT is the unpredictable interference of the endogenous TCR α and β chains in pairing of the transduced TCR. This effect reduces the efficacy of the genetically modified primary T cells and carries the risk of generating novel TCR reactivities with unintended functional consequences. Here, we show a powerful approach to overcome these limitations. We engineered TCR α and β chains with mutations encompassing a conserved motif (FXXXFXXS) required to stabilize the pairing of immunoglobulin heavy chain transmembrane domains. Molecular modeling supported the preferential pairing of mutated TCR and impaired pairing between mutated and wild-type TCRs. Expression of the mutated TCR was similar to wild type and conferred the expected specificity. Fluorescence resonance energy transfer analysis in mouse splenocytes transduced with mutated or wild-type TCRs showed a higher proximity of the former over the latter. Importantly, we show that mutated TCRs effectively outcompete endogenous TCRs and improve in vitro antitumor cytotoxicity when expressed in ex vivo isolated human T cells. This approach should contribute to improving current protocols of anticancer immunetherapy protocols.

Frataxin Structure and Function

By Castro Ignacio Hugo; Pignataro Maria Florencia; Sewell Karl Ellioth; Herrera Maria Georgina; Noguera Martin Ezequiel; Dain Liliana; Nadra Alejandro Daniel; Santos Javier; Castro Ignacio Hugo; Pignataro Maria Florencia; et al
From Sub-cellular biochemistry (2019), 93393-438, Language: English, Database: MEDLINE

Mammalian frataxin is a small mitochondrial protein involved in iron sulfur cluster assembly. Frataxin deficiency causes the neurodegenerative disease Friedreich’s Ataxia. Valuable knowledge has been gained on the structural dynamics of frataxin, metal-ion-protein interactions, as well as on the effect of mutations on protein conformation, stability and internal motions. Additionally, laborious studies concerning the enzymatic reactions involved have allowed for understanding the capability of frataxin to modulate Fe-S cluster assembly function. Remarkably, frataxin biological function depends on its interaction with some proteins to form a supercomplex, among them NFS1 desulfurase and ISCU, the scaffolding protein. By combining multiple experimental tools including high resolution techniques like NMR and X-ray, but also SAXS, crosslinking and mass-spectrometry, it was possible to build a reliable model of the structure of the desulfurase supercomplex NFS1/ACP-ISD11/ISCU/frataxin. In this chapter, we explore these issues showing how the scientific view concerning frataxin structure-function relationships has evolved over the last years.

Improved selective class I HDAC and novel selective HDAC3 inhibitors: Beyond hydroxamic acids and benzamides

By Bresciani, Alberto; Ontoria, Jesus M.; Biancofiore, Ilaria; Cellucci, Antonella; Ciammaichella, Alina; Di Marco, Annalise; Ferrigno, Federica; Francone, Alessandra; Malancona, Savina; Monteagudo, Edith; et al
From ACS Medicinal Chemistry Letters (2019), 10(4), 481-486. Language: English, Database: CAPLUS, DOI:10.1021/acsmedchemlett.8b00517

Improved selective class I HDAC and novel selective HDAC3 inhibitors: Beyond hydroxamic acids and benzamidesThe application of class I HDAC inhibitors as cancer therapies is well established, but more recently their development for nononcol. indications has increased. We report here on the generation of improved class I selective human HDAC inhibitors based on an ethylketone zinc binding group (ZBG) in place of the hydroxamic acid that features the majority of HDAC inhibitors. We also describe a novel set of HDAC3 isoform selective inhibitors that show stronger potency and selectivity than the most commonly used HDAC3 selective tool compd. RGFP966. These compds. are again based on an alternative ZBG with respect to the ortho-anilide that is featured in HDAC3 selective compds. reported to date.

Sulphide as a leaving group: highly stereoselective bromination of alkyl phenyl sulphides

By Canestrari Daniele; Cioffi Caterina; Biancofiore Ilaria; Lancianesi Stefano; Ghisu Lorenza; Adamo Mauro F A; Ibrahim Hasim; Biancofiore Ilaria; Ruether Manuel; O’Brien John
From Chemical science (2019), 10(39), 9042-9050, Language: English, Database: MEDLINE

A conceptionally novel nucleophilic substitution approach to synthetically important alkyl bromides is presented. Using molecular bromine (Br2), readily available secondary benzyl and tertiary alkyl phenyl sulphides are converted into the corresponding bromides under exceptionally mild, acid- and base-free reaction conditions. This simple transformation allows the isolation of elimination sensitive benzylic β-bromo carbonyl and nitrile compounds in mostly high yields and purities. Remarkably, protic functionalities such as acids and alcohols are tolerated. Enantioenriched benzylic β-sulphido esters, readily prepared by asymmetric sulpha-Michael addition, produce the corresponding inverted bromides with high stereoselectivities, approaching complete enantiospecificity at -40 °C. Significantly, the reported benzylic β-bromo esters can be stored without racemisation for prolonged periods at -20 °C. Their synthetic potential was demonstrated by the one-pot preparation of γ-azido alcohol (S)-5 in 90% ee. NMR studies revealed an initial formation of a sulphide bromine adduct, which in turn is in equilibrium with a postulated dibromosulphurane intermediate that undergoes C-Br bond formation.

Synthesis and Evaluation of Bifunctional Aminothiazoles as Antiretrovirals Targeting the HIV-1 Nucleocapsid Protein

By Mori Mattia; Dasso Lang Maria Chiara; Palombi Nastasja; Friggeri Laura; Botta Maurizio; Saladini Francesco; Giannini Alessia; Zazzi Maurizio; Kovalenko Lesia; Mely Yves; et al
From ACS medicinal chemistry letters (2019), 10(4), 463-468, Language: English, Database: MEDLINE

Small molecule inhibitors of the HIV-1 nucleocapsid protein (NC) are considered as promising agents in the treatment of HIV/AIDS. In an effort to exploit the privileged 2-amino-4-phenylthiazole moiety in NC inhibition, here we conceived, synthesized, and tested in vitro 18 NC inhibitors (NCIs) bearing a double functionalization. In these NCIs, one part of the molecule is deputed to interact noncovalently with the NC hydrophobic pocket, while the second portion is designed to interact with the N-terminal domain of NC. This binding hypothesis was verified by molecular dynamics simulations, while the linkage between these two pharmacophores was found to enhance antiretroviral activity both on the wild-type virus and on HIV-1 strains with resistance to currently licensed drugs. The two most interesting compounds 6 and 13 showed no cytotoxicity, thus becoming valuable leads for further investigations.

Whole-Genome Phage Display Libraries: A Powerful Tool for Antigen Discovery

By Beghetto, Elisa; Gargano, Nicola
From Methods in Molecular Biology (New York, NY, United States) (2019), 2024(Immunoproteomics), 181-198. Language: English, Database: CAPLUS, DOI:10.1007/978-1-4939-9597-4_11

In the last two decades, phage display technol. has been used for investigating complex biol. processes and isolating mols. of practical value in several applications. Bacteriophage lambda, representing a classical cloning and expression system, has also been exploited for generating display libraries of small peptides and protein domains. More recently, large cDNA and whole-genome lambda display libraries of human pathogens have been generated for the discovery of new antigens for biomedical applications. Here, we describe the construction of a whole-genome library of a common pathogen- Streptococcus pneumoniae -and the use of this library for the mol. dissection of the human B-cell response against bacterial infection and colonization.

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