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Publications 2015

High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting.

By Ullman Christopher; Mathonet Pascale; Oleksy Arkadiusz; Diamandakis Agata; Tomei Licia; Demartis Anna; Nardi Chiara; Sambucini Sonia; Missineo Antonino; Alt Karen; et al
From PloS one (2015), 10(8), e0135278, Language: English, Database: MEDLINE

Abdurins are a novel antibody-like scaffold derived from the engineering of a single isolated CH2 domain of human IgG. Previous studies established the prolonged serum half-life of Abdurins, the result of a retained FcRn binding motif. Here we present data on the construction of large, diverse, phage-display and cell-free DNA display libraries and the isolation of high affinity binders to the cancer target, membrane-bound ephrin receptor tyrosine kinase class A2 (EphA2). Antigen binding regions were created by designing combinatorial libraries into the structural loops and Abdurins were selected using phage display methods. Initial binders were reformatted into new maturation libraries and low nanomolar binders were isolated using cell-free DNA display, CIS display. Further characterization confirmed binding of the Abdurins to both human and murine EphA2 proteins and exclusively to cell lines that expressed EphA2, followed by rapid internalization. Two different EphA2 binders were labeled with 64Cu, using a bifunctional MeCOSar chelator, and administered to mice bearing tumors from transplanted human prostate cancer cells, followed by PET/CT imaging. The anti-EphA2 Abdurins localized in the tumors as early as 4 hours after injection and continued to accumulate up to 48 hours when the imaging was completed. These data demonstrate the ability to isolate high affinity binders from the engineered Abdurin scaffold, which retain a long serum half-life, and specifically target tumors in a xenograft model.

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Detection of huntingtin exon 1 phosphorylation by Phos-Tag SDS-PAGE: Predominant phosphorylation on threonine 3 and regulation by IKKß.

By Bustamante, Maria Blaire; Ansaloni, Annalisa; Pedersen, Jeppe Falsig; Azzollini, Lucia; Cariulo, Cristina; Wang, Zhe-Ming; Petricca, Lara; Verani, Margherita; Puglisi, Francesca; Park, Hyunsun; et al
From Biochemical and Biophysical Research Communications (2015), 463(4), 1317-1322. Language: English, Database: CAPLUS, DOI:10.1016/j.bbrc.2015.06.116

Expansion of a CAG triplet repeat within the first exon of the HUNTINGTIN gene encoding for a polyglutamine tract is the cause of a progressive neurodegenerative disorder known as Huntington’s disease. N-terminal fragments of mutant huntingtin have a strong propensity to form oligomers and aggregates that have been linked to the Huntington’s disease pathol. by different mechanisms, including gain of toxic functions. The biol. and biophys. properties of the polyglutamine expansion within these huntingtin fragments are influenced by neighboring domains, in particular by the first 17 amino acids of huntingtin (N17), which precede the polyglutamine expansion. It has been suggested that N17 phosphorylation modulate mutant huntingtin aggregation and toxicity, but the study of its functional and pathol. relevance requires the capacity to detect this modification in biol. samples in a simple, robust way, that ideally provides information on the abundance of a phosphorylated species relative to the total pool of the protein of interest. Using a modified SDS-PAGE protocol (Phos-Tag) followed by Western blotting with specific anti-HUNTINGTIN antibodies, we efficiently resolved huntingtin fragments expressed in cellular contexts based on the presence of phosphorylated residues, we defined threonine 3 as the major site of huntingtin N17 phosphorylation and, finally, we identified IKK-beta as a kinase capable of phosphorylating threonine 3 in N-terminal hungtingtin fragments.

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A chemogenomic screening identifies CK2 as a target for pro-senescence therapy in PTEN-deficient tumours.

By Kalathur Madhuri; Chen Jingjing; Revandkar Ajinkya; Alimonti Andrea; Toso Alberto; Guccini Ilaria; Alajati Abdullah; Sarti Manuela; Pinton Sandra; Brambilla Lara; et al
From Nature communications (2015), 67227, Language: English, Database: MEDLINE

Enhancement of cellular senescence in tumours triggers a stable cell growth arrest and activation of an antitumour immune response that can be exploited for cancer therapy. Currently, there are only a limited number of targeted therapies that act by increasing senescence in cancers, but the majority of them are not selective and also target healthy cells. Here we developed a chemogenomic screening to identify compounds that enhance senescence in PTEN-deficient cells without affecting normal cells. By using this approach, we identified casein kinase 2 (CK2) as a pro-senescent target. Mechanistically, we show that Pten loss increases CK2 levels by activating STAT3. CK2 upregulation in Pten null tumours affects the stability of Pml, an essential regulator of senescence. However, CK2 inhibition stabilizes Pml levels enhancing senescence in Pten null tumours. Taken together, our screening strategy has identified a novel STAT3-CK2-PML network that can be targeted for pro-senescence therapy for cancer.

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A single-run liquid chromatography mass spectrometry method to quantify neuroactive kynurenine pathway metabolites in rat plasma.

By Orsatti, Laura; Speziale, Roberto; Orsale, Maria Vittoria; Caretti, Fulvia; Veneziano, Maria; Zini, Matteo; Monteagudo, Edith; Lyons, Kathryn; Beconi, Maria; Chan, Kelvin; et al
From Journal of Pharmaceutical and Biomedical Analysis (2015), 107, 426-431. Language: English, Database: CAPLUS, DOI:10.1016/j.jpba.2015.01.030

Neuroactive metabolites in the kynurenine pathway of tryptophan catabolism are assocd. with neurodegenerative disorders. Tryptophan is transported across the blood-brain barrier and converted via the kynurenine pathway to N-formyl-L-kynurenine, which is further degraded to L-kynurenine. This metabolite can then generate a group of metabolites called kynurenines, most of which have neuroactive properties.The assocn. of tryptophan catabolic pathway alterations with various central nervous system (CNS) pathologies has raised interest in anal. methods to accurately quantify kynurenines in body fluids. We here describe a rapid and sensitive reverse-phase HPLC-MS/MS method to quantify L-kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxy-L-kynurenine (3HK) and anthranilic acid (AA) in rat plasma. Our goal was to quantify these metabolites in a single run; given their different physico-chem. properties, major efforts were devoted to develop a chromatog. suitable for all metabolites that involves plasma protein pptn. with acetonitrile followed by chromatog. sepn. by C18 RP chromatog., detected by electrospray mass spectrometry. Quantitation range was 0.098-100 ng/mL for 3HK, 9.8-20,000 ng/mL for KYN, 0.49-1000 ng/mL for KYNA and AA. The method was linear (r > 0.9963) and validation parameters were within acceptance range (calibration stds. and QC accuracy within ±30%).

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Development and validation of a luminescence-based, medium-throughput assay for drug screening in Schistosoma mansoni.

By Lalli Cristiana; Guidi Alessandra; Ruberti Giovina; Gennari Nadia; Altamura Sergio; Bresciani Alberto
From PLoS neglected tropical diseases (2015), 9(1), e0003484, Language: English, Database: MEDLINE

Schistosomiasis, one of the world’s greatest neglected tropical diseases, is responsible for over 280,000 human deaths per annum. Praziquantel, developed in the 1970s, has high efficacy, excellent tolerability, few and transient side effects, simple administration procedures and competitive cost and it is currently the only recommended drug for treatment of human schistosomiasis. The use of a single drug to treat a population of over 200 million infected people appears particularly alarming when considering the threat of drug resistance. Quantitative, objective and validated methods for the screening of compound collections are needed for the discovery of novel anti-schistosomal drugs. METHODOLOGY/PRINCIPAL FINDINGS: The present work describes the development and validation of a luminescence-based, medium-throughput assay for the detection of schistosomula viability through quantitation of ATP, a good indicator of metabolically active cells in culture. This validated method is demonstrated to be fast, highly reliable, sensitive and automation-friendly. The optimized assay was used for the screening of a small compound library on S. mansoni schistosomula, showing that the proposed method is suitable for a medium-throughput semi-automated screening. Interestingly, the pilot screening identified hits previously reported to have some anti-parasitic activity, further supporting the validity of this assay for anthelminthic drug discovery. CONCLUSIONS: The developed and validated schistosomula viability luminescence-based assay was shown to be successful and suitable for the identification of novel compounds potentially exploitable in future schistosomiasis therapies.

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Polyglutamine- and temperature-dependent conformational rigidity in mutant huntingtin revealed by immunoassays and circular dichroism spectroscopy.

Fodale V, Kegulian NC, Verani M, Cariulo C, Azzollini L, Petricca L, Daldin M, Boggio R, Padova A, Kuhn R, Pacifici R, Macdonald D, Schoenfeld RC, Park H, Isas JM, Langen R, Weiss A, Caricasole A. (2014, 2015)
PLoS One. 2014 Dec 2;9(12):e112262. Erratum in: PLoS One. 2015;10(6):e0129986.

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Peptide nucleic acids targeting ß-globin mRNAs selectively inhibit hemoglobin production in murine erythroleukemia cells.

By Montagner Giulia; Gemmo Chiara; Fabbri Enrica; Bianchi Nicoletta; Finotti Alessia; Breveglieri Giulia; Salvatori Francesca; Borgatti Monica; Lampronti Ilaria; Gambari Roberto; et al
From International journal of molecular medicine (2015), 35(1), 51-8, Language: English, Database: MEDLINE

In the treatment of hemoglobinopathies, amending altered hemoglobins and/or globins produced in excess is an important part of therapeutic strategies and the selective inhibition of globin production may be clinically beneficial. Therefore the development of drug-based methods for the selective inhibition of globin accumulation is required. In this study, we employed peptide nucleic acids (PNAs) to alter globin gene expression. The main conclusion of the present study was that PNAs designed to target adult murine β-globin mRNA inhibit hemoglobin accumulation and erythroid differentiation of murine erythroleukemia (MEL) cells with high efficiency and fair selectivity. No major effects were observed on cell proliferation. Our study supports the concept that PNAs may be used to target mRNAs that, similar to globin mRNAs, are expressed at very high levels in differentiating erythroid cells. Our data suggest that PNAs inhibit the excess production of globins involved in the pathophysiology of hemoglobinopathies.

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