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Publications 2018

A liquid chromatography high-resolution mass spectrometry in vitro assay to assess metabolism at the injection site of subcutaneously administered therapeutic peptides.

Esposito S, de Leonibus ML, Ingenito R, Bianchi E, Orsatti L, Monteagudo E. (2018)
J Pharm Biomed Anal 2018 Jul 10; 159;449-458 [Epub ahead of print]

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Development of a Broadly Applicable Assay for Measurement of Glycan-Directed Enzymatic Activity.

By Bresciani, Alberto; Cecchetti, Ottavia; Missineo, Antonino; Pacifici, Pier Giorgio; Tomei, Licia; Rodems, Steven
From SLAS Discovery (2018), 23(9), 941-950. Language: English, Database: CAPLUS, DOI:10.1177/2472555218782625

Glycosylation is a key posttranslational modification that tags protein to membranes, organelles, secretory pathways, and degrdn. Aberrant protein glycosylation is present both in acquired diseases, such as cancer and neurodegeneration, and in congenital disorders of glycosylation (CDGs). Consequently, the ability to interrogate the activity of enzymes that can modify protein glycan moieties is key for drug discovery projects aimed at finding modulators of these enzymes. To date, low-throughput technologies such as SDS-PAGE and mass spectrometry have been used, which are not suitable for compd. screening in drug discovery. In the present work, a broadly applicable time-resolved fluorescence resonance energy transfer (TR-FRET) assay was developed that can det. the activity of endoglycosidase enzymes in high-throughput formats. The assay was validated using PNGaseF and EndoH as tool deglycosylases. Even though the current setup is based on the recognition of glycans that bind Con A (ConA), the assay concept can be adapted to glycans that bind other lectins.

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Early metabolic response to acute myocardial ischaemia in patients undergoing elective coronary angioplasty.

By Di Marino Sara; Lembo Angelo; Summa Vincenzo; Cicero Daniel Oscar; Viceconte Nicola; Tanzilli Gaetano; Truscelli Giovanni; Mangieri Enrico; Gaudio Carlo; Raparelli Valeria; et al
From Open heart (2018), 5(1), e000709, Language: English, Database: MEDLINE

Balloon-induced transient coronary ischaemia represents a model of myocardial ischaemia and reperfusion. We are interested in the very early systemic metabolic response to this event. Methods: Blood samples of patients with stable angina (SA) were collected before and after coronary angioplasty. Serum metabolic profiles were obtained using nuclear magnetic resonance spectroscopy. Univariate and multivariate analyses were used to investigate changes in metabolite concentrations. Results: Thirty-four consecutive patients with SA, undergoing elective coronary angioplasty at Policlinico Umberto I of Rome, were included in this study. Changes in metabolites concentration induced by balloon occlusion in venous and arterial sera were detected. In both serum types, a significant increase in ketone bodies, 2-hydroxybutyrate, glutamine and O-acetylcarnitine concentration is observed, while alanine, lactate, phenylalanine and tyrosine decreased after intervention. Most significant metabolic changes were detected in arterial serum. Conclusions: Our study points out two main global metabolic changes in peripheral blood after balloon-induced coronary ischaemia: ketone bodies increase and lactate decrease. Both could be related to compensation mechanisms finalised to fulfil heart’s needs after short period of myocardial ischaemia and probably after reperfusion.

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Peptidomimetic nitrile inhibitors of malarial protease falcipain-2 with high selectivity against human cathepsins.

By Nizi, Emanuela; Sferrazza, Alessio; Fabbrini, Danilo; Nardi, Valentina; Andreini, Matteo; Graziani, Rita; Gennari, Nadia; Bresciani, Alberto; Paonessa, Giacomo; Harper, Steven
From Bioorganic & Medicinal Chemistry Letters (2018), 28(9), 1540-1544. Language: English, Database: CAPLUS, DOI:10.1016/j.bmcl.2018.03.069

Falcipain-2 (FP2) is an essential enzyme in the lifecycle of malaria parasites such as Plasmodium falciparum, and its inhibition is viewed as an attractive mechanism of action for new anti-malarial agents. Selective inhibition of FP2 with respect to a family of human cysteine proteases (that include cathepsins B, K, L and S) is likely to be required for the development of agents targeting FP2. Here the authors describe a series of P2-modified aminonitrile based inhibitors of FP2 that provide a clear strategy toward addressing selectivity for the P. falciparum and show that it can provide potent FP2 inhibitors with strong selectivity against all four of these human cathepsin isoforms.

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Quantifying autophagy using novel LC3B and p62 TR-FRET assays.

By Bresciani, Alberto; Spiezia, Maria Carolina; Boggio, Roberto; Cariulo, Cristina; Nordheim, Anja; Altobelli, Roberta; Kuhlbrodt, Kirsten; Dominguez, Celia; Munoz-Sanjuan, Ignacio; Wityak, John; et al
From PLoS One (2018), 13(3), e0194423/1-e0194423/18. Language: English, Database: CAPLUS, DOI:10.1371/journal.pone.0194423

Autophagy is a cellular mechanism that can generate energy for cells or clear misfolded or aggregated proteins, and upregulating this process has been proposed as a therapeutic approach for neurodegenerative diseases. Here we describe a novel set of LC3B-II and p62 time-resolved fluorescence resonance energy transfer (TR-FRET) assays that can detect changes in autophagy in the absence of exogenous labels. Lipidated LC3 is a marker of autophagosomes, while p62 is a substrate of autophagy. These assays can be employed in high-throughput screens to identify novel autophagy upregulators, and can measure autophagy changes in cultured cells or tissues after genetic or pharmacol. interventions. We also demonstrate that different cells exhibit varying autophagic responses to pharmacol. interventions. Overall, it is clear that a battery of readouts is required to make conclusions about changes in autophagy.

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Determination of acetyl coenzyme A in human whole blood by ultra-performance liquid chromatography-mass spectrometry

By Speziale, Roberto; Montesano, Camilla; De Leonibus, Maria Lucia; Bonelli, Fabio; Fezzardi, Paola; Beconi, Maria G.; Monteagudo, Edith; Elbaum, Daniel; Orsatti, Laura
From Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences (2018), 1083, 57-62. Language: English, Database: CAPLUS, DOI:10.1016/j.jchromb.2018.02.039

Acetyl CoA is involved in several key metabolic pathways. Its concn. can vary considerably in response to physiol. or pathol. conditions making it a potentially valuable biomarker. However, little information about the measurement and concn. of acetyl CoA in human whole blood is found in the literature. The aim of this study was the development of an accurate method for the detn. of acetyl CoA in human whole blood by LC-MS/MS. The method, involving extn. from whole blood by a rapid protein pptn. procedure was thoroughly validated: limit of quantitation was 3.91 ng mL-1. Accuracy and precision were calcd. at five concns. and were within ± 1 %. The av. endogenous level of acetyl CoA in human whole blood was detd. in 17 healthy individuals to be 220.9 ng mL-1 (ranging from 124.0 to 308.0 ng mL-1). This represents, to the authors’ knowledge, the first report of acetyl CoA levels in human whole blood, and the first practical and reliable method for its detn.

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Fosmetpantotenate (RE-024), a phosphopantothenate replacement therapy for pantothenate kinase-associated neurodegeneration:
Mechanism of action and efficacy in nonclinical models.

By Elbaum Daniel; Beconi Maria G; Quinton Maria S; Vaino Andrew; Monteagudo Edith; Di Marco Annalise; Lyons Kathryn A; Harper Steven
From PloS one (2018), 13(3), e0192028, Language: English, Database: MEDLINE

In cells, phosphorylation of pantothenic acid to generate phosphopantothenic acid by the pantothenate kinase enzymes is the first step in coenzyme A synthesis. Pantothenate kinase 2, the isoform localized in neuronal cell mitochondria, is dysfunctional in patients with pantothenate kinase-associated neurodegeneration. Fosmetpantotenate is a phosphopantothenic acid prodrug in clinical development for treatment of pantothenate kinase-associated neurodegeneration, which aims to replenish phosphopantothenic acid in patients. Fosmetpantotenate restored coenzyme A in short-hairpin RNA pantothenate kinase 2 gene-silenced neuroblastoma cells and was permeable in a blood-brain barrier model. The rate of fosmetpantotenate metabolism in blood is species-dependent. Following up to 700 mg/kg orally, blood exposure to fosmetpantotenate was negligible in rat and mouse, but measurable in monkey. Consistent with the difference in whole blood half-life, fosmetpantotenate dosed orally was found in the brains of the monkey (striatal dialysate) but was absent in mice. Following administration of isotopically labeled-fosmetpantotenate to mice, ~40% of liver coenzyme A (after 500 mg/kg orally) and ~50% of brain coenzyme A (after 125 μg intrastriatally) originated from isotopically labeled-fosmetpantotenate. Additionally, 10-day dosing of isotopically labeled-fosmetpantotenate, 12.5 μg, intracerebroventricularly in mice led to ~30% of brain coenzyme A containing the stable isotopic labels. This work supports the hypothesis that fosmetpantotenate acts to replace reduced phosphopantothenic acid in pantothenate kinase 2-deficient tissues.

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Polypharmacy through Phage Display: Selection of Glucagon and GLP-1 Receptor Co-agonists from a Phage-Displayed Peptide Library.

By Demartis Anna; Tomei Licia; Beghetto Elisa; Di Biasio Valentina; Orvieto Federica; Frattolillo Francesco; Bianchi Elisabetta; Lahm Armin; Pessi Antonello; Carrington Paul E; et al
From Scientific reports (2018), 8(1), 585, Language: English, Database: MEDLINE

A promising emerging area for the treatment of obesity and diabetes is combinatorial hormone therapy, where single-molecule peptides are rationally designed to integrate the complementary actions of multiple endogenous metabolically-related hormones. We describe here a proof-of-concept study on developing unimolecular polypharmacy agents through the use of selection methods based on phage-displayed peptide libraries (PDL). Co-agonists of the glucagon (GCG) and GLP-1 receptors were identified from a PDL sequentially selected on GCGR- and GLP1R-overexpressing cells. After two or three rounds of selection, 7.5% of randomly picked clones were GLP1R/GCGR co-agonists, and a further 1.53% were agonists of a single receptor. The phages were sequenced and 35 corresponding peptides were synthesized. 18 peptides were potent co-agonists, 8 of whom showed EC50 ≤ 30 pM on each receptor, comparable to the best rationally designed co-agonists reported in the literature. Based on literature examples, two sequences were engineered to stabilize against dipeptidyl peptidase IV cleavage and prolong the in vivo half-life: the engineered peptides were comparably potent to the parent peptides on both receptors, highlighting the potential use of phage-derived peptides as therapeutic agents. The strategy described here appears of general value for the discovery of optimized polypharmacology paradigms across several metabolically-related hormones.

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Discovery of 2-(1H-imidazo-2-yl)piperazines as a new class of potent and non-cytotoxic inhibitors of Trypanosoma brucei growth in vitro

By Ferrigno, Federica; Biancofiore, Ilaria; Malancona, Savina; Ponzi, Simona; Paonessa, Giacomo; Graziani, Rita; Bresciani, Alberto; Gennari, Nadia; Di Marco, Annalise; Kaiser, Marcel; et al
From Bioorganic & Medicinal Chemistry Letters (2018), 28(23-24), 3689-3692. Language: English, Database: CAPLUS, DOI:10.1016/j.bmcl.2018.10.028

The identification of a new series of growth inhibitors of Trypanosoma brucei rhodesiense, causative agent of Human African Trypanosomiasis (HAT), is described. A selection of compds. from our inhouse compd. collection was screened in vitro against the parasite leading to the identification of compds. with nanomolar inhibition of T. brucei growth. Preliminary SAR on the hit compd. led to the identification of compd. 34 that shows low nanomolar parasite growth inhibition (T. brucei EC50 5 nM), is not cytotoxic (HeLa CC50 > 25,000 nM) and is selective over other parasites, such as Trypanosoma cruzi and Plasmodium falciparum (T. cruzi EC50 8120 nM, P. falciparum EC50 3624 nM).

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DNA replication and repair kinetics of Alu, LINE-1 and satellite III genomic repetitive elements

By Natale Francesco; Scholl Annina; Rapp Alexander; Yu Wei; Rausch Cathia; Cardoso M Cristina; Natale Francesco; Yu Wei
From Epigenetics & chromatin (2018), 11(1), 61, Language: English, Database: MEDLINE

Preservation of genome integrity by complete, error-free DNA duplication prior to cell division and by correct DNA damage repair is paramount for the development and maintenance of an organism. This holds true not only for protein-encoding genes, but also it applies to repetitive DNA elements, which make up more than half of the human genome. Here, we focused on the replication and repair kinetics of interspersed and tandem repetitive DNA elements. RESULTS: We integrated genomic population level data with a single cell immunofluorescence in situ hybridization approach to simultaneously label replication/repair and repetitive DNA elements. We found that: (1) the euchromatic Alu element was replicated during early S-phase; (2) LINE-1, which is associated with AT-rich genomic regions, was replicated throughout S-phase, with the majority being replicated according to their particular histone marks; (3) satellite III, which constitutes pericentromeric heterochromatin, was replicated exclusively during the mid-to-late S-phase. As for the DNA double-strand break repair process, we observed that Alu elements followed the global genome repair kinetics, while LINE-1 elements repaired at a slower rate. Finally, satellite III repeats were repaired at later time points. CONCLUSIONS: We conclude that the histone modifications in the specific repeat element predominantly determine its replication and repair timing. Thus, Alu elements, which are characterized by euchromatic chromatin features, are repaired and replicated the earliest, followed by LINE-1 elements, including more variegated eu/heterochromatic features and, lastly, satellite tandem repeats, which are homogeneously characterized by heterochromatic features and extend over megabase-long genomic regions. Altogether, this work reemphasizes the need for complementary approaches to achieve an integrated and comprehensive investigation of genomic processes.

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Identification and binding mode of a novel Leishmania Trypanothione reductase inhibitor from high throughput screening

By Turcano, Lorenzo; Torrente, Esther; Missineo, Antonino; Andreini, Matteo; Gramiccia, Marina; Muccio, Trentina Di; Genovese, Ilaria; Fiorillo, Annarita; Harper, Steven; Bresciani, Alberto; et al
From PLoS Neglected Tropical Diseases (2018), 12(11), e0006969. Language: English, Database: CAPLUS, DOI:10.1371/journal.pntd.0006969

Trypanothione reductase (TR) is considered to be one of the best targets to find new drugs against Leishmaniasis. This enzyme is fundamental for parasite survival in the host since it reduces trypanothione, a mol. used by the tryparedoxin/tryparedoxin peroxidase system of Leishmania to neutralize hydrogen peroxide produced by host macrophages during infection. In order to identify new lead compds. against Leishmania we developed and validated a new luminescence-based high-throughput screening (HTS) assay that allowed us to screen a library of 120,000 compds. We identified a novel chem. class of TR inhibitors, able to kill parasites with an IC50 in the low micromolar range. The X-ray crystal structure of TR in complex with a compd. from this class (compd. 3) allowed the identification of its binding site in a pocket at the entrance of the NADPH binding site. Since the binding site of compd. 3 identified by the X-ray structure is unique, and is not present in human homologs such as glutathione reductase (hGR), it represents a new target for drug discovery efforts.

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Identification of novel multi-stage histone deacetylase (HDAC) inhibitors that impair Schistosoma mansoni viability and egg production

By Guidi Alessandra; Saccoccia Fulvio; Gimmelli Roberto; Lalli Cristiana; Papoff Giuliana; Ruberti Giovina; Gennari Nadia; Paonessa Giacomo; Bresciani Alberto; Nizi Emanuela
From Parasites & vectors (2018), 11(1), 668, Language: English, Database: MEDLINE

Novel anti-schistosomal multi-stage drugs are needed because only a single drug, praziquantel, is available for the treatment of schistosomiasis and is poorly effective on larval and juvenile stages of the parasite. Schistosomes have a complex life-cycle and multiple developmental stages in the intermediate and definitive hosts. Acetylation and deacetylation of histones play pivotal roles in chromatin structure and in the regulation of transcription in eukaryotic cells. Histone deacetylase (HDAC) inhibitors modulate acetylation of several other proteins localized both in the nucleus and in the cytoplasm and therefore impact on many signaling networks and biological processes. Histone post-translational modifications may provide parasites with the ability to readily adapt to changes in gene expression required for their development and adaptation to the host environment. The aim of the present study was to screen a HDAC class I inhibitor library in order to identify and characterize novel multi-stage hit compounds. METHODS: We used a high-throughput assay based on the quantitation of ATP in the Schistosoma mansoni larval stage (schistosomula) and screened a library of 1500 class I HDAC inhibitors. Subsequently, a few hits were selected and further characterized by viability assays and phenotypic analyses on adult parasites by carmine red and confocal microscopy. RESULTS: Three compounds (SmI-124, SmI-148 and SmI-558) that had an effect on the viability of both the schistosomula larval stage and the adult worm were identified. Treatment with sub-lethal doses of SmI-148 and SmI-558 also decreased egg production. Moreover, treatment of adult parasites with SmI-148, and to a lesser extent Sm-124, was associated with histone hyperacetylation. Finally, SmI-148 and SmI-558 treatments of worm pairs caused a phenotype characterized by defects in the parasite reproductive system, with peculiar features in the ovary. In addition, SmI-558 induced oocyte- and vitelline cell-engulfment and signs of degeneration in the uterus and/or oviduct. CONCLUSIONS: We report the screening of a small HDAC inhibitor library and the identification of three novel compounds which impair viability of the S. mansoni larval stage and adult pairs. These compounds are useful tools for studying deacetylase activity during parasite development and for interfering with egg production. Characterization of their specificity for selected S. mansoni versus human HDAC could provide insights that can be used in optimization and compound design.

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Structure-Based Identification of HIV-1 Nucleocapsid Protein Inhibitors Active against Wild-Type and Drug-Resistant HIV-1 Strains

By Mori Mattia; Dasso Lang Maria Chiara; Botta Maurizio; Kovalenko Lesia; Pires Manuel; Humbert Nicolas; Real Eleonore; Mely Yves; Kovalenko Lesia; Malancona Savina; et al
From ACS chemical biology (2018), 13(1), 253-266, Language: English, Database: MEDLINE

HIV/AIDS is still one of the leading causes of death worldwide. Current drugs that target the canonical steps of the HIV-1 life cycle are efficient in blocking viral replication but are unable to eradicate HIV-1 from infected patients. Moreover, drug resistance (DR) is often associated with the clinical use of these molecules, thus raising the need for novel drug candidates as well as novel putative drug targets. In this respect, pharmacological inhibition of the highly conserved and multifunctional nucleocapsid protein (NC) of HIV-1 is considered a promising alternative to current drugs, particularly to overcome DR. Here, using a multidisciplinary approach combining in silico screening, fluorescence-based molecular assays, and cellular antiviral assays, we identified nordihydroguaiaretic acid (6), as a novel natural product inhibitor of NC. By using NMR, mass spectrometry, fluorescence spectroscopy, and molecular modeling, 6 was found to act through a dual mechanism of action never highlighted before for NC inhibitors (NCIs). First, the molecule recognizes and binds NC noncovalently, which results in the inhibition of the nucleic acid chaperone properties of NC. In a second step, chemical oxidation of 6 induces a potent chemical inactivation of the protein. Overall, 6 inhibits NC and the replication of wild-type and drug-resistant HIV-1 strains in the low micromolar range with moderate cytotoxicity that makes it a profitable tool compound as well as a good starting point for the development of pharmacologically relevant NCIs.

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