Centro Congressi d’Ateneo “Federico II” – Via Partenope ... · Centro Congressi d’Ateneo “Federico II” – Via Partenope, Napoli June 12-14, 2014 14th Naples Workshop

Centro Congressi d’Ateneo “Federico II” – Via Partenope, Napoli

June 12-14, 2014

14th Naples Workshop on Bioactive Peptides

THE RENAISSANCE ERA OF PEPTIDES IN DRUG DISCOVERY

Organized by

Centro Interuniversitario di Ricerca sui Peptidi Bioattivi (CIRPeB)

Università di Napoli “Federico II” – Dipartimento di Farmacia

Istituto di Biostrutture e Bioimmagini del Consiglio Nazionale delle Ricerche

Istituto di Cristallografia del Consiglio Nazionale delle Ricerche

DFM Scarl

Under the auspicies ofEuropean Peptide Society


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Editors

Prof. Giancarlo [email protected]. Paolo [email protected]. Michele [email protected]

Organizing Secretariat

YES MEETVia S. Nicola, 480067 - Sorrento (NA) - ItalyTel. +39 0818770604Fax + 39 [email protected] [email protected]

Official Photographer

Ludovica [email protected]

Publisher

EDIZIONI ZIINO – Italy www.massmediacomunicazione.com [email protected]


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Honorary Chair

Prof. Carlo Pedone

Co-Chairmen

Prof. Giancarlo MorelliProf. Paolo Grieco

Dr. Michele Saviano

Organizing Committee

G. Morelli University of Naples “Federico II” P. Grieco University of Naples “Federico II” M. Saviano Institute of Crystallography, CNR A. Accardo University of Naples “Federico II” L. De Luca Institute of Biostructures and Bioimaging, CNR M. Ruvo Institute of Biostructures and Bioimaging, CNR D. Tesauro University of Naples “Federico II”

L. Vitagliano Institute of Biostructures and Bioimaging, CNR

Scientific Committee

D. Andreu Universitat Pompeu Fabra - Barcelona, Spain A. Ceci (Satellite Workshop) University of Bari - Bari, Italy P. Grieco University of Naples “Federico II” - Naples Italy F. Hudecz Eötvös L. University - Budapest, Hungary G. Morelli University of Naples “Federico II” - Naples Italy L. Moroder Max Planck Institute - Martinsried, Germany P. Rovero University of Florence - Florence, Italy M. Ruvo Institute of Biostructures and Bioimaging, CNR - Naples, Italy M. Saviano Institute of Crystallography, CNR - Bari, Italy L. Stella University of Rome “Tor Vergata” - Rome Italy C. Toniolo University of Padua - Padua, Italy


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Acknowledgments

The Organizing Committee gratefully acknowledges the support

and assistance of the following Institutions:

Centro Interuniversitario di Ricerca sui Peptidi Bioattivi (CIRPeB)

Università di Napoli “Federico II” – Dipartimento di Farmacia

Istituto di Biostrutture e Bioimmagini del Consiglio Nazionale delle Ricerche

Istituto di Cristallografia del Consiglio Nazionale delle Ricerche

DFM Scarl – Projects: “Farmabionet” and “Bersagli”

Regione Campania Assessorato Università e Ricerca Scientifica

European Peptide Society (EPS)

Ordine dei Farmacisti della Provincia di Napoli


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Under the auspicies ofEuropean peptides Society

With support of DFM SCARLFinancial co-supported projects

included in the POR Campania FESR 2007 - 2013 objective 2.1


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The Organizing Committee gratefully acknowledges the contribution given to the organization of the event by the following companies


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PROGRAM


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THURSDAY, JUNE 12th

09.30 Registration (onwards)

10.30 - 13.00 SATELLITE MEETING “MicroRNA: Potential for Cancer Detection and Diagnosis” Chairmen: M. Saviano & M. Caraglia

10.30 - 11.15 PL1-SM - Roberto Gambari (University of Ferrara, Italy) “Targeting biological functions of disease-associated microRNAs: novel frontiers in miRNA-Therapeutics”

11.15 - 11.50 Coffee Break

11.50 - 12.30 KN1-SM - Isabella Bray (Royal College of Surgeons in Ireland, Dublin, Ireland) “Modulation of chemotherapeutic drug resistance by miRNA”

12.30 - 13.10 KN2- SM Amelia Cimmino (Institute of Genetics and Biophysics CNR, Napoli, Italy) “Fingerprinting of ultra conserved long noncoding RNAs in bladder cancer analysis reveals a network between non-coding RNA and miRNA”

Opening of 14th Naples Workshop on Bioactive Peptides“The Renaissance era of Peptides in Drug Discovery”

14.00 - 14.15 Welcome addresses

Session I A Antimicrobial Peptides Chairmen: P. Rovero & L. Stella

14.15 - 15.00 PL1 - Robert E.W. Hancock (University of British Columbia, Vancouver, BC, Canada) “New therapies for antibiotic resistant infections”

15.00 - 15.35 KN1 - William C. Wimley (University of Tulane, New Orleans, LA, USA) “Discovery of novel membrane-active peptides by synthetic molecular evolution”

15.35 - 15.55 O1 - Burkhard Bechinger (University of Strasbourg, France) “Biophysical investigations of the mechanism of action of antimicrobial peptides and their synergistic interactions”

15.55 - 16.15 O2 - Patricio Carvajal-Rondanelli (Pontificia Universidad Catolica de Valparaiso, Chile) “Antimicrobial effect of proline and alanine scan on short cationic Homopeptides”

16.15 - 16.40 Coffee break

16.40 - 17.00 O3 - Luciano Polonelli (University of Parma, Italy) “Bioactive peptides from the inside of the antibodies”


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17.00 - 17.20 O4 - Marta De Zotti (University of Padua, Padova, Italy) “Peptide cytotoxicity as a function of capping moieties: trichogin GA IV”

17.20 - 17.40 O5 - Andrea Farrotti (University of Rome Tor Vergata, Roma, Italy) “Computational methods to determine peptide orientation in membranes”

17.40 - 18.00 O6 - Bart De Spiegeleer (Ghent University, Ghent, Belgium) “How the exploration of the chemical space of cell-penetrating peptides helps to understand their functionality”

18.00 - 18.20 O7 - Manuel N. Melo (University of Groningen, Netherlands) “There is no such thing as a typical AMP. Maybe”

18.20 - 18.40 O8 - Marina Gobbo (University of Padua, Padova, Italy) “Photosensitizing activity of porphyrin-antimicrobial peptides conjugates toward prokaryotic and eukaryotic cells”

18.40 - 19.00 O9 - Fabian Zehender (NanoTemper Tech. GmbH, Munich, Germany) “Some like it hot: Biomolecule analytics using microScale thermophoresis”

19.30 - 21.00 Welcome reception at Aula Magna Partenope


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FRIDAY, JUNE 13th

Session II Peptides in Nanomedicine for diagnostic and therapeutic applications Chairmen: G. Morelli & D. Andreu

08.45 - 09.30 PL2 - Alberto Bianco (CNRS, Strasbourg, France) “Adamantane-based dendrons for the multipresentation of therapeutic peptides and small drugs”

09.30 - 10.05 KN2 - Hayat Onyuksel (University of Illinois, Chicago, IL, USA) “Safe and Stable VIP in Phopholipid Micelles”

10.05 - 10.25 O10 - Felisa Cilurzo (University “Magna Graecia”, Catanzaro, Italy) “Gene therapy innovation: targeted Peg-protamine as potential nanocarrier for gene transfection”


10.50 - 11.10 O11 - Ferenc Hudecz (Eötvös Loránd University, Budapest, Hungary) “Targeting of daunomycin with oligo/polypeptide bioconjugates: the effect of the partner on functional properties”

11.10 - 11.30 O12 - Mariano Venanzi (University of Rome Tor Vergata, Roma, Italy) “Tuning the aggregation of conformationally constrained oligopeptides”

11.30 - 12.05 KN3 - Nuno Correira Santos (University of Lisbon, Portugal) “Atomic force microscopy and AFM-based force spectroscopy on the study of peptides against dengue virus and bacterial infections”

12.05 - 12.50 PL3 - Rassoul Dinarvand (Tehran University of Medical Sciences, Iran) “Albumin coated PLGA nanoparticles for the ocular delivery of bevacizumab as treatment for retinal and choroidal neovascularization”

12.50 - 14.00 Lunch

Session III Peptides in Immunology Chairmen: M. Ruvo & C. Toniolo

14.00 - 14.45 PL4 - David Andreu (Pompeu Fabra University, Barcelona, Spain) Peptide vaccines: successful despite only modest enthusiasm

14.45 - 15.20 KN4 - Arvind Patel (University of Glasgow, UK) “The structural and functional basis of hepatitis C virus neutralization by a broadly neutralizing antibody” 15.20 - 15.55 KN5 - Anna Maria Papini (University of Florence, Firenze, Italy) “From an N-glucopeptide synthetic probe to an hyper-glucosylated protein antigen: a bacterial infection triggering an antibody mediated form of multiple sclerosis?”


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15.55 - 16.15 O13 - Cedric Rentier (University of Cergy-Pontoise, Cergy-Pontoise, France) “Peptides of the dihydrolipoamide acetyltrnsferase for the detection of autoantibodies in autoimmune diseases”


Session I B Antimicrobial Peptides Chairmen: A. Papini & M. Venanzi

16.40 - 17.00 O14 - Lorenzo Stella (University of Rome Tor Vergata, Roma, Italy) “Behaviour of antimicrobial peptides in phospholipid membranes: insights from combined spectroscopic and simulative studies”

17.00 - 17.20 O15 - Erik Strandberg (Karlsruhe Institute of Technology, Germany) “Pore-forming antimicrobial peptides as ‘molecular rulers’ to measure bacterial membrane thickness”

17.20 - 17.40 O16 - Benoit Odaert (University of Bordeaux, Pessac, France) “Mechanism of action on membrane models of Clausin, a lantibiotic peptide from Bacillus clausii”

17.40 - 18.00 O17 - Alessandro Pini (University of Siena, Italy) “A novel synthetic antimicrobial peptide. A new weapon for multi-drug resistant bacteria?”

18.00 - 18.20 O18 - Antonello Pessi (Peptipharma, Roma, Italy) “Peptide antivirals directly from viral genome information”

18.20 - 19.30 Poster Session 1 - Discussion of posters

Free evening


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SATURDAY, JUNE 14th

Session IV A Peptides in Chemical Biology Chairmen: F. Hudecz & P. Grieco

08.45 - 09.30 PL5 - William Lubell (University of Montreal, Montreal, QC, Canada) “Aza-peptide tools for studying peptide chemical-biology in pursuit of CD36 receptor modulators to treat age-related macular degeneration”

09.30 - 10.05 KN6 - Knud J. Jensen (University of Copenaghen, Denmark) “Cyclic peptides as inhibitors of cancer-related proteases”

10.05 - 10.25 O19 - Michele Caraglia (Second University of Naples, Napoli, Italy) “Urotensin II receptor: its role as prognostic marker and potential therapeutic targets in human epithelial cancers”


10.55 - 11.30 KN7 - Victor J. Hruby (University of Arizona, AZ, USA) “New approaches for drug design: Design of multivalent ligands for treatment and detection of degenerative diseases”

11.30 - 11.50 O20 - Ildiko Szabo (Eötvös L. University, Budapest, Hungary) “GnRH antagonist as potential targeting units - synthesis and in vitro Evaluation”

11.50 - 12.10 O21 - Sara Pellegrino (University of Milan, Milano, Italy) “Modulation of the c-Maf transcription factor: a new perspective for multiple Myeloma”

12.10 - 12.30 022 - Massimo Zollo (CEINGE and University of Naples “Federico II” Napoli, Italy) “Targeting Nm23-H1/h-Prune interaction impairs cell growth, survival and migration in Prostate cancer”

12.30 - 12.50 O23 - Minying Cai (University of Arizona, AZ, USA) “Developing bioavailable melanotropin peptides”

12.50 - 13.10 O24 - Jean-Marie Swiecicki (Ecole Normale Supérieure, Paris, France) “Unravelling the penetration and subcellular distribution of cell penetrating Peptides”

13.10 - 14.20 Break


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Session IV B Peptides in Chemical Biology Chairmen: L. Moroder & S. Galdiero

14.20 - 14.40 O25 - Isabel Alves (University of Bordeaux, Pessac, France) “How do a proapoptotic and a cell penetrating peptide work together to kill cancer cells?”

14.40 - 15.00 O26 - Ivan De Paola (CIRPeB, University of Naples “Federico II” & IBB CNR, Napoli, Italy) “Stapled Peptides for Cullin3-BTB interface targeting”

15.00 - 15.20 O27 - Sandro De Falco (Institute of Genetics and Biophysics, CNR, Napoli, Italy) “Inhibition of pathological angiogenesis antagonizing VEGF Receptor 1”

15.20 - 15.40 O28 - Zoltan Banoczi (Eötvös L. University, Budapest, Hungary) “Cell-penetrating conjugates of calpain inhibitors”

15.40 - 16.00 O29 - Biancamaria Farina (CIRPeB, University of Naples “Federico II” & IBB CNR, Napoli, Italy) “NMR interaction studies of RGDechi-hCit peptide with integrins embedded into cell membranes”

16.00 - 16.20 O30 - Luisa Bracci (University of Siena, Italy) “Targeting sulfated glycans in cancer cells and tissues by branched peptides”

16.20 - 16.40 O31 - Evelien Wynendaele (Ghent University, Ghent, Belgium) “Crosstalk between mammalian cells and the microbiome through quorum sensing peptides, influencing cancer metastasis”


Session V Peptides and Industrial Applications: Successes and Perspectives Chairmen: L. Vitagliano & L. Moroder

17.00 - 17.35 KN8 - Paolo Botti (ArisGen, Switzerland) “Enteral delivery of the peptide MIF-1 using ArisCrown technology”

17.35 - 18.10 KN9 - Immaculada Rentero (Ecole Polytechnique Fédérale de Lausanne, Switzerland) “Phage selection of bicyclic peptides for therapeutic applications”

18.10 - 18.30 Concluding Remarks: G. Morelli, F. Hudecz, L. Moroder

18.30 - 19.45 Poster Session 2 - Discussion of posters

21.00 Gala Dinner at “Transatlantico” Restaurant


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POSTER SESSION


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POSTER SESSION 1

P1-SM Ida Autiero Molecular Dynamics simulations of two PNA-based systems using new parameters implemented in the GROMACS package

P2-SM Raffaella Ummarino Synthesis and characterization of g-thiazole orange mono and bifunctionalized PNA to light up PNA targets

Antimicrobial Peptides (P1-P15)

P1 Novella Incoronato Conformational modifications and antiviral activity of gB from Herpes simplex virus type 1 analyzed by synthetic peptides

P2 Tecla Ciociola Single residue contribution to self-aggregation of a fungicidal immunoglobulin-derived peptide

P3 Fernando Formaggio Cotton fibers functionalized with peptaibiotics

P4 Martina Sperindè Candidacidal properties of peptides encoded by immunoglobulin genes

P5 Lorenzo Stella Trichogin GA IV forms ion channels of well-defined size in planar lipid membranes

P6 Lorenzo Stella How many AMP molecules kill a bacterium? Spectroscopic determination of PMAP-23 binding to E. coli.

P7 Lorenzo Stella Testing the “Sand in the Gearbox” model: Antimicrobial Peptide effects on membrane dynamics

P8 Concetta Avitabile Circular Dichroism and NMR studies to elucidate the mechanism of action of antimicrobial peptides with bacterial cells

P9 Lucia Lombardi Antiviral activity of a peptido-dendrimer

P10 Thelma Pertinhez Dissection of the structural features and fungicidal activity of an antibody-derived peptide

P11 Carla Esposito Dimerization of HBHA adhesion from Mycobacterium tuberculosis, insights into bacterial agglutina-tion

P12 Maria Romano Structure and Function of RNase AS, a Polyadenylate-Specific Exoribonuclease Affecting Mycobac-terial Virulence In Vivo


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P13 Luciano Pirone The identification and characterization of a novel CAMP from Stf76, a Sulfolobus islandicus plasmid-virus pSSVx transcription factor

P14 Marco Cantisani Structure-Activity Relations of Myxinidin, an Antibacterial Peptide Derived from the Epidermal Mu-cus of Hagfish

P15 Francesco Merlino Synthesis and Biological Activity of Temporin L Analogues

Peptides in Nanomedicine for diagnostic and therapeutic applications (P16-P31)

P16 Diego Tesauro EGF analog peptide functionalized micelles for target-selective sorafenib delivery

P17 Adriano Mollica Multi-Target Peptides for chronic pain control: opioid agonists / omega-conotoxin analogues

P18 Roberta Iannitti Target-selective theranostic micelles for Bombesin receptors incorporating Au(III)-dithiocarbamate complex

P19 Nicoletta Depalo RGD Peptide–Conjugated Silica Coated PbS Nanocrystals with Tunable Emission in the Near Infra-red Region for Molecular Targeted Imaging

P20 Nicoletta Depalo Nanocrystalline Semiconducting-Magnetic Heterostructures decorated with cyclic RGD peptide for Integrin Targeting

P21 Bernhard Ay Identifying IgE-Binding Sites of Cor a 8 by SPOT-Synthesis on PU-Modified Cellulose Membranes

P22 Andrea Caporale Investigating the substrate specificity and use of M-TGase to graft peptide synthons onto proteins

P23 Barbara Monaco RGD: SPPS and on-resin functionalization suitable for Nuclear Medicine application

P24 Domenica Musumeci Solid phase synthesis and nucleic acids binding studies of a thymine-functionalized oligolysine

P25 Accardo Antonella Liposomes doubly functionalized with the CCK8 and gH625 peptides for intracellular drug delivery

P26 Annarita Falanga Dendrimer’s functionalized with the membrane-interacting peptide gH625: mechanism of interaction with liposomes

P27 Emiliana Perillo In vitro investigation on cancer cell uptake and toxicity of liposomes functionalized with the membra-notropic peptide gH625


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P28 Nunzia Migliaccio Peptide-based siRNA for the treatment of cancer cells

P29 Linda Piras Development of new fluorescent probes for RNA imaging in cell

P30 Luigi Aloj Novel peptide based CXCR4 antagonists for cancer imaging

P31 Enrico Iaccino Harnessing the potential of idiotypic peptides to design smart drug delivery system

Peptides in Immunology (P32-P39)

P32 Angela Ostuni Aldehyde modification and alum synergize to enhance anti-Tnfα vaccination and mitigates arthritis in rats

P33 Rosita Aitoro Gliadin peptide P31-43: structure and biological effects

P34 Carmen Aiello Small peptide inhibitors of Protein-Protein interactions essential in JAK-STAT pathway

P35 Bianca Carfora Cripto recognition by the Loop-Helix Motif [44-67] of Nodal: an AlaScan Analysis

P36 Giuseppina Focà Comparative binding to VEGF165 of PEGylated Bevacizumab fragments

P37 Alessandro Arcovito Study of the interaction of a salivary proline-rich peptide with SH3 domains from the SRC kinases family

P38 Silvia Scaramuzza PEGylated Trastuzumab Fabs

P39 Luca Sanguigno Characterization of Herceptin antibody fragments in in vitro assays


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POSTER SESSION 2

Peptides in Chemical Biology (P40-P85)

P40 Alfonso Bavoso Spectroscopic investigation of auranofin binding to zinc finger nucleocapsidic domains

P41 Benoit Odaert NMR structural investigation of the membrane proteins involved in the dimerization of the mitochon-drial ATP-synthase

P42 Laure Guilhaudis Investigation of the bioactive conformation of 26RFa, an orexigenic peptide

P43 Lucia Falcigno BMP-2 fragments: synthesis, structural characterization, binding properties and biological activity

P44 Stefania De Luca Lipidated peptides via post-synthetic thioalkylation promoted by molecular sieves

P45 Marta De Zotti Cytotoxicity and interaction with cellular membranes of Trichogin GA IV analogs

P46 Donatella Diana Functional binding surface of a ß-hairpin VEGF receptor targeting peptide determined by NMR in living cells

P47 Izabela Małuch Synthesis of modified peptides containing P1 arginine mimetics

P48 Luisa Calvanese De novo design of Nodal mimetic peptides

P49 Rosario Oliva Thermodynamics of interaction between a small peptide derived from glycoprotein gp36 of Feline Immunodeficiency Virus and model membrane systems

P50 Teresa Łepek Capillary electrophoresis as a tool for analysis of the Conus geographus venom profile: proof-of-concept study

P51 Daniela Barone Dynamic interplay of the N- and the C-terminal domains in KCTD5

P52 Luigi Vitagliano Loop insertions in helices: a novel structural motif

P53 Monika Lewandowska Research of potent inhibitors of furin modified in position P5

P54 Simona Pascarella Mimicking SERPINH1 chaperone function in collagen type I biosynthetic pathway via the TASP ap-proach


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P55 Lucia De Rosa Peptides mimicking a discontinuos VEGF binding epitope

P56 Concetta Di Natale Conformational studies of nucleophosmin C-terminal leukemia-associated regions: new insights from protein dissection approach

P57 Nicole Balasco Local backbone geometry and conformational preferences of amino acids

P58 Fabiola Mascanzoni Structured-based optimization of AIF(370-394), an inhibitor of the AIF/CypA lethal complex

P59 Biancamaria Farina Structural insights on the recognition of AIF by CypA revealed by NMR spectroscopy

P60 Gianluigi Di Sorbo Cis-trans prolyl isomerase activity: new internally quenched fluorogenic substrates for HTS assay

P61 Giovanni Smaldone Defining the minimal interacting region in Cul3-BTB complexes

P62 Alessia Ruggiero Dissecting domain swapping in the Arginine Binding Protein isolated from Thermotoga maritima

P63 Annalia Focà Inhibition of APEH with a click

P64 Carla Marra Site-specific mono-pegylation of glucagon-like peptide 1 and its mutants using prokaryotic microbial transglutaminase

P65 Marilisa Leone Conformational and binding studies of peptides spanning the EphA2 interacting region of the first Sam domain of Odin

P66 Virginia Lorenzo Inhibition of Prep1-p160 complex by peptides targeting the Prep1 binding site on p160

P67 Rosanna Palumbo APEH-mediated downregulation of proteasome by a potential anticancer peptide

P68 Carmen Lammi Characterization of the cholesterol-lowering effects of soy and lupin peptides at HepG2 cell line

P69 Emma Fenude Conformational study of bioactive peptides finalized to design of tailored delivery systems

P70 Veronica Celentano Probing the helical stability in a proangiogenic peptide

P71 Rossella Di Stasi VEGF/VEGF receptor interaction: a structural analysis on living cells


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P72 Anna Russomanno An intein-based strategy for the preparation of isotopically labelled peptides

P73 Laura Zaccaro RGDechi-hCit peptide: new insights into biological behavior on melanoma metastatic cells

P74 Antonia Spensiero Design and synthesis of new Integrase inhibitors

P75 Marian Vincenzi Solution conformational features of intrinsically disordered phosphopeptides: a new class of potential targets in drug discovery

P76 Fabio Selis Use of a novel Transglutaminase Glutamine-containing consensus sequence for the identification of conjugation site of proteins by mass spectroscopy

P77 Maria Carmina Scala 4-(1-Piperidinyl)aspartate formation during the preparation of lactam constrained cyclic peptides

P78 Gilmar Salgado Biophysical characterization of the Helicobacter pylori A1 toxin

P79 Anna Maria D’Ursi Gp41 MPER in membrane mimetics characterized by mixed lipid composition

P80 Ermelinda Vernieri Design, synthesis and efficacy of lactam-constrained GRK2 peptide inhibitors

P81 Martina Buonanno Nuclear transport factors Importin Kapß2 and Exportin CRM1 interact with the tumor enzyme hCA IX

P82 Angela Meccariello Carbonic anhydrases as new targets against the bacterial pathogen Brucella suis

P83 Anna Di Fiore Carbonic anhydrase inhibitors: X-ray crystallographic studies for the binding of molecules containing a sulfamide moiety

P84 Ali Munaim Yousif Synthesis and Biological activity of novel peptides active on urokinase system

P85 Remo Guerrini A novel and facile synthesis of tetra branched derivatives of nociceptin/orphanin FQ

Peptides and Industrial Applications: Successes and Perspectives (P86-P91)

P86 Lucilla Scarnato The BIORICE European project: BIO technology for the recovery of valuable peptides from indus-trial RICE by-products and production of added value ingredients for nutraceuticals, functional foods and cosmetics


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P87 Mario Scrima Molecular Dynamics studies of PMMA polymer film- c02 peptide interfacial phenomena

P88 Federica Donnarumma Design, production and characterization of new protein sweeteners P89 Paolo Grieco SAR Study on P5U and Urantide by replacing Tyr9 with uncoded and constrained amino acids.

P90 Paolo Grieco Conformational Analysis of Urotensin-II Related Peptide in Membrane Mimetic Environment

P91 Giusy Corvino Investigating the oxidative refolding mechanism of Cripto CFC domain


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PLENARY LECTURES


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New therapies for antibiotic resistant infections

R.E.W. (Bob) Hancock

Centre for Microbial Diseases and Immunity Research, University of British Columbia, Vancouver, BC, Canada

Cationic host defence (antimicrobial) peptides are produced by virtually all organisms, ranging from plants and insects to humans, as a major part of their innate defences against infection. We and others have demonstrated that they are a key component of innate immunity and have multiple mechanisms that enable them to deal with infections and inflammation including direct antimicrobial activity, an ability to dissolve bacterial biofilms (the cause of 65% of all human infections) and an ability to favour-ably modulate the innate immune system.While their activity against free swimming (planktonic) bacteria is often weak we have recently dem-onstrated that specific peptides act against biofilms formed by multiple species of bacteria in a manner that is independent of their activity vs. planktonic bacteria. We have now developed novel anti-biofilm peptides that (i) kill multiple species of bacteria in biofilms (MBEC < 1 µg/ml), including P. aerugi-nosa and other major clinically relevant Gram negative bacteria, especially the so-called ESKAPE pathogens, as well as Gram positive MRSA, Listeria and Enterococcus, (ii) work synergistically with antibiotics in multiple species, and (iii) are effective in animal models of biofilm infections. These peptides can prevent biofilm formation and cause dispersal and/or death of bacteria in pre-formed biofilms. Their action is dependent on their ability to trigger the degradation of the nucleotide stress signal (p)ppGpp.The manipulation of natural innate immunity represents a new adjunctive therapeutic strategy against antibiotic-resistant infections. Cationic host defence peptides boost protective innate immunity while suppressing potentially harmful inflammation/sepsis, and work synergistically with conventional thera-py. Using the principle of selective boosting of innate immunity we have developed novel small innate defence regulator (IDR) peptides with no direct antibacterial activity, that are nevertheless able to pro-tect in animal models against many different microbial infections, including antibiotic resistant infec-tions and cerebral malaria, as well as inflammatory diseases, providing a new concept in anti-infective therapy. For example we have demonstrated protection in infection models against the superbug methi-cillin resistant Staph aureus (MRSA), and Vancomycin resistant Enterococcus (VRE) as well as E. coli, P. aeruginosa, MDR tuberculosis, and cerebral malaria. Good activity in models of wound healing, pre-term birth and cystic fibrosis has also been achieved. Systems approaches have helped considerably in understanding the how these agents work and they are currently being developed pre-clinically to treat diseases of animals and man.

PL1


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Adamantane-based dendrons for the multipresentation of therapeutic peptides and small drugs

A. Bianco

CNRS, Institut de Biologie Moléculaire et Cellulaire, Laboratoire d’Immunopathologie et Chimie Thérapeutique, 15, Rue René Descartes, 67084 Strasbourg, France

Dendrimers and dendrons, corresponding to wedge-shaped dendrimer sections, are currently gaining a lot of interest for therapeutic and diagnostic uses.[1] The diversity of functions that can be introduced on these ramified structures has direct impact on biocompatibility, stability and specificity. All these properties make dendrimers and dendrons good candidates for drug or gene delivery and molecular targeting. In this context, we are investigating dendron structures based on adamantane because this rigid molecule with a well defined 3D structure can bring further advantages to other proposed dendron conformations, for example on multivalent ligand/receptor interactions. We have designed a synthesis of the unsymmetrical tetrasubstituted adamantane with one amino and three identical carboxylic acid functions in order to build the dendron only by formation of amide bonds, up to the third generation.[2] In a proof-of-concept study we linked ibuprofen to the first and second generation dendrons, leading to an enhanced anti-inflammatory activity of the drug in vitro in comparison to the drug alone.[3] More recently, we have elucidated the uptake mechanism and the cytotoxicity impact of this type of molecules.[4] We have also used the dendrons as multivalent scaffolds to multimerize an apoptogenic peptide that mimics the natural tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).[5] The trimeric and hexameric forms of the peptide exerted an increased affinity to TRAIL receptor-2 of about 1500- and 20000-fold, respectively, relative to the monomer. Similarly, a peptide (discovered in our laboratory and termed P140) involved in the modulation of the autoimmune disease systemic lupus erythematosus was conjugated to the adamantane-dendron to study the effect of the multipresentation on the biological activity in comparison to the single peptide.[6] In this communication, we will summarize our recent advances on the potential applications of adamantane-based dendritic structures for the delivery of therapeutic peptides and small drugs. Uptake mechanisms and impact on cells will be also discussed.

References1. G. R. Newkome,C. Shreiner Chem. Rev. (2010) 110, 6338.2. G. Lamanna, J. Russier, C. Ménard-Moyon, A. Bianco Chem. Commun. (2011) 47, 8955.3. G. Lamanna, J. Russier, H. Dumortier, A. Bianco Biomaterials (2012) 33, 5610.4. M. Grillaud, J. Russier, A. Bianco J. Am. Chem. Soc. 2014, 136, 810.5. G. Lamanna, C. R. Smulski, N. Chekkat, K. Estieu-Gionnet, G. Guichard, S. Fournel, A. Bianco Chem. Eur. J.

(2013) 19, 1762.6. M. Grillaud, C. Macri, G. Lamanna, O. Chaloin S. Muller, A. Bianco, submitted.

PL2


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PL3

Albumin coated PLGA nanoparticles for the ocular delivery of bevacizumab as treatment for retinal

and choroidal neovascularization

Reyhaneh Varshochian1,2, Ahmad Reza Mahmoudi3, Fatemeh Atyabi1,2, Mohammad Riazi Esfahani5, Rassoul Dinarvand1,2,*

1. Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran2. Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran

1417614411, Iran3. Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran4. Farabi Eye Hospital, Tehran University of Medical Sciences, Qazvin Square, Tehran, Iran

Rapid growth in the application of protein therapeutics specifies the necessity to design novel drug delivery systems. Bevacizumab, a whole antibody against vascular endothelial growth factor (VEGF), has demonstrated promising effects in treatment of retinal and choroidal neovascularization which both are crucial sight threatening conditions. However, the weak point of this treatment is the short half-life of the drug in vitreous which necessitates repetitive intravitreal injections. Accordingly employing controlled release drug delivery systems such as polymeric nanoparticles has been suggested.In the present study bevacizumab loaded poly(lactic-co-glicolic) acid (PLGA) nanoparticles were for-mulated by water-in-oil-in-water emulsion method intended for reducing the number of intravitreal injections and thus its side effects as well. However, protein inactivation and aggregation are the ma-jor drawbacks of this technique; therefore protective ability of various stabilizers was studied during entrapment process. Results revealed that the protein interfacial adsorption is the main destabilizing factor in double emulsion method and incorporation of appropriate concentrations of albumin could reduce bevacizumab instabilities and protect the drugs against entrapment stress. Bevacizumab ex vivo release from albuminated PLGA nanoparticles in rabbit vitreous indicated the ability of nanoparticles in prolonged release of the active antibody. In vivo results revealed that the bevacizumab vitreous concentration following the intravitreal injection of nanoparticles in rabbits maintained above the minimum concentration which completely blocks VEGF for about 8 weeks and 3.3-fold increase was observed in the drug vitreous mean residence time (MRT) in comparison with Avastin injected group (control). Moreover following the intravitreal injection of coumarin-6 loaded albuminated PLGA nanoparticles, fluorescence emissions in posterior tissues were observed for 56 days which confirmed the nanoparticles persistence in ocular tissues during the test period.


29

PL4

Peptide vaccines: successful despite only modest enthusiasm

D. Andreu1, E. Blanco, F. Sobrino2, Ll. Ganges4 and B.G. de la Torre1

1 Department of Experimental & Health Sciences, Universitat Pompeu Fabra, 08003 Barcelona, Spain2 Centro de Investigación en Sanidad Animal (CISA-INIA), Valdeolmos, 28130 Madrid, Spain3 Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Cantoblanco, 28049 Madrid, Spain 4 Centre Recerca en Sanitat Animal (CReSA), Universitat Autònoma de Barcelona, 01893 Bellaterra, Spain

Ever since the firsts accounts some three decades ago[1], interest in peptide vaccines has gone through several surge-and-wane cycles, not unlike those experienced by peptide pharmaceuticals. Reasons for the cautious attitude from pharma companies, health agencies, etc. towards synthetic peptide vaccines include the difficulty in defining relevant B and/or T-cell epitopes protective for a given disease, or the generally low immunogenicity of peptides, requiring enhancement by oligomerization, conjugation or adjuvancy[2]. At a more basic—almost philosophical—level there is mild skepticism that peptide vaccine designers can adequately handle the subtleties of epitope/paratope recognition—i.e., a set of fuzzy, no-clear-boundary binding events—, or that reductionistic attempts to reproduce complex conformational epitopes by simple peptide constructions can ever succeed[3]. Against this somewhat apathetic setting, practitioners in the field are tempted to quote American novelist James Baldwin (“those who say it can’t be done are usually interrupted by others doing it”). Indeed, in recent years, a steady stream of successful reports seems to confirm that peptide vaccines are here to stay, even though the need for renewed efforts to sort out the abovementioned challenges must be fully admitted and addressed. This presentation will review some significant realizations in the peptide vaccine field, then discuss recent work from our group on developing effective, fully protecting vaccine candidates against foot-and-mouth disease (FMD), the most economically disrupting infection of farm animals. Our prototype B4T, integrating in a single molecular entity 4 and 1 copies of B- and T-cell epitopes from FMD virus (FMDV), respectively, elicited high mucosal IgA levels that underscored the solid protection found in pigs[4]. More recently, again on branched BnT (n=2,4) designs, we investigated how B epitope multiplicity and other structural differences—epitope N- or C-terminal attachment to the branching core, and type of connecting linkage—affect the immune response. Somehow unexpectedly, B2T constructs consistently perform better than B4T counterparts, particularly with maleimide connecting units[5,6]. After successful (100% protection) trials with the B2T prototype in swine, steps towards massive field evaluation and commercialization in the PRC are currently under way. A similar approach is being used to develop classical swine fever vaccines.

References1. Shinnick TM, Sutcliffe JG, Green N, Lerner RA, Annu Rev Microbiol, 1983, 37, 425-446.2. Zauner W, Lingnau K, Mattner F, von Gabain A, Buschle M, Biol Chem, 2001, 382, 581-595.3. Van Regenmortel MH, Front Immunol, 2012, 3, 194.4. Cubillos C, de la Torre BG, Jakab A, Clementi G, Borràs E, Bárcena J, Andreu D, Sobrino F, Blanco E, J Virol

2008,82, 7223−7230.5. Monsó M, de la Torre BG, Blanco E, Moreno N, Andreu D, Bioconjugate Chem, 2013, 24, 578-585.6. Blanco E, Cubillos C, Moreno N, Bárcena J, de la Torre BG, Andreu D, Sobrino F, Dev Comp Immunol, 2013,

475960.


30

Aza-peptide tools for studying peptide chemical-biology in pursuit of CD36 receptor modulators to treat age-related macular degeneration

Traore M., Zhang J., Doan N. D., Turcotte S., Garcia-Ramos Y., Pohankova P., Chemtob S., Ong, H. and Lubell W. D.

Université de Montréal, Départements de Chimie et d’Ophtalmologie, et Faculté de Pharmacie, Montréal, Quebec, H3C 3J7, Canada

Employment of semicarbazides as amino acid surrogates in peptides causes electronic interactions that manifest turn conformations [1]. Moreover, the resulting azapeptides have exhibited improve po-tency, selectivity and pharmacodynamics compared to their parent peptides. Developing and employ-ing methods for synthesizing azapeptides, we are pursuing a novel approach to provide treatments of the leading cause of blindness in the elderly, age-related macular degeneration (AMD). Targeting the cluster of differentiation 36 (CD36) receptor, because this multi-functional scavenger receptor is ex-pressed on three main sub-retinal cell types and plays roles in the uptake of cytotoxic oxidized lipids, inflammation and neovascularization characteristic of AMD, we have developed selective CD36 recep-tor ligands by introducing aza-amino acid residues into growth hormone releasing peptide 6 (GHRP-6: His-D-Trp-Ala-Trp-D-Phe-Lys-NH2). For example, [azaTyr4]-GHRP-6 has exhibited relatively tight affinity for the CD36 receptor in a surface plasmon resonance assay, and reduced neovascularization relative to control in a microvascular sprouting assay on mouse choroidal explants [2]. Our presentation will highlight advances in the synthetic methods of azapeptides and structure-activity studies of CD36 ligands towards effective prototypes to treat the pathology of AMD.

References1. Proulx, C.; Sabatino, D.; Hopewell, R.; Jochen, S.; Garcia-Ramos, Y.; Lubell, W. D. Future Medicinal Chemistry

2011, 3, 1139.2. Proulx, C.; Picard, E.; Boeglin, B.; Pohankova, P.; Chemtob, S.; Ong, H.; Lubell, W., D. J. Med. Chem. 2012, 55,

6502–6511.

PL5


31

KEYNOTES


32

KN1

Discovery of Novel Membrane-active Peptides by Synthetic Molecular EvolutionWC. Wimley1

1 Department of Biochemistry, Tulane University School of Medicine, New Orleans LA, 70112

Membrane-active peptides have many potential applications in biotechnology and medicine, including use as antibiotics, antivirals, anticancer drugs, drug delivery vehicles, biosensors and more. However, their full development is inhibited because their mechanism of action can rarely, if ever, be described in the detailed, molecular terms. This lack of explicit sequence-function relationships is a significant roadblock to the rational development of useful sequences. We have begun to circumvent this roadblock using synthetic molecular evolution, or high-throughput screening in combination with iterative, feedback driven peptide library design. Using synthetic molecular evolution to engineer pore-forming peptides, we have identified two sequence families with unique properties that are not found in known, naturally-occurring pore forming peptides. Using synthetic molecular evolution to select for membrane translocation we have discovered spontaneous membrane translocating peptides capable of direct cargo delivery across the plasma membranes of cells. Using synthetic molecular evolution to identify membrane permeabilizing peptides, we have discovered new classes of antimicrobial peptides. Our synthetic molecular evolution–based approach, taken together with the new high–throughput tools we have developed, enables the identification, refinement and optimization of unique membrane active peptides.


33

KN2

Safe and Stable Peptide Delivery using Phospholipid MicellesH. Onyuksel

University of Illinois, Chicago Illinois, USA

Peptide drugs are not stable in blood due to enzymatic degradation, and when administered at high quantities by intravenous (iv) infusion they can cause serious side effects. To overcome these problems we have developed a safe delivery system composed of PEGylated phospholipids, sterically stabilized micelles (SSM), for iv administration of peptide drugs. We have shown that peptide drug can self-assemble into SSM, change its conformation from random coil to alfa-helical, and circulate in blood long hours without being degraded. Half-life of a model neuropeptide, vasoactive intestinal peptide (VIP) was increased from 20 minutes to 9.6hrs [1]. Furthermore, the blood pressure-lowering side effect of VIP was completely eliminated when delivered in SSM [2]. Phospholipid micelles are ~ 15nm in size and cannot extravasate out of the circulation at normal tissues and VIP receptors are not expressed in the endothelial lining of blood vessels, but primarily in the extra vascular space. Therefore VIP can-not reach its receptors at normal tissues and show any side effects. However at pathologic cases such as cancer or inflammation there exist leaky vasculatures where the 15nm peptide nanomedicine can extravasate locally and show its therapeutic effect. My talk will show efficacy data for VIP, glucagon like peptide-1 [3] and pancreatic polypeptide [4] on animal models of rheumatoid arthritis, acute lung injury and diabetes.

References1. V. Sethi, I. Rubinstein, A. Kuzmis, J. Artwohl, H. Kastrissios, and H. Onyuksel “Novel, biocompatible, Dis-

ease Modifying Nanomedicine of VIP for Rheumatoid Arthritis” Molecular Pharmaceutics (2013) 10: 728-738,. http://dx.doi.org/10.1021/mp300539f

2. A. Banerjee, H. Onyuksel “Peptide Delivery using Phospholipid Micelles” WIRE’s Nanomed Nanobiotechnol (2012) 4:562-74.

3. S.B. Lim, I. Rubinstein, R.T. Sadikot, J.E. Artwohl, and H. Onyuksel “A Novel Peptide Nanomedicine Against Acute Lung Injury: GLP- in Phospholipid Micelles” Pharm Res. (2011) 28: 662-672.

4. A. Banerjee, H. Onyuksel, “A Novel Peptide Nanomedicine for Treatment of Pancreatogenic Diabetes” Nanomedicine: Nanotechnology, Biology, and Medicine, (2013) 9: 722-728. http://dx.doi.org/10.1016/j.nano.2012.12.005


34

KN3

Atomic force microscopy and AFM-based force spectroscopy on the development of a peptide against dengue virus infection

Filomena A. Carvalho1, Ivo C. Martins1, André F. Faustino1, Fabiana A. Carneiro2, Iranaia Assunção-Miranda2, Ronaldo Mohana-Borges2, Miguel Castanho1, Fabio Almeida2, Andrea T. Da Poian2, Nuno C. Santos1

1. Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Portugal.2. Universidade Federal do Rio de Janeiro, Brazil.

Dengue virus (DENV) affects millions of people, causing more than 20 thousand deaths annually. No effective treatment is currently available. We studied the interaction between DENV capsid (C) protein and lipid droplets (LD), recently shown to be essential for viral replication. Atomic force microscopy (AFM)-based force spectroscopy measurements were performed with DENV C-functionalized AFM tips, tapping at the surface of the sample until the binding between tip and LD, and measuring afterwards the force necessary for the unbinding at the single-molecule level. AFM studies were complemented with NMR, zeta-potential and cell biology studies. DENV C-LD interaction is dependent on the high intracellular concentrations of potassium. Inhibition of Na+/K+-ATPase in DENV-infected cells resulted in a 50-fold inhibition of virus production. Limited proteolysis of LD surface impaired the interaction. Force measurements in the presence of specific antibodies indicate perilipin 3 (TIP47) as the major DENV C ligand on LD. A peptide designed based on a conserved segment of DENV C intrinsically disordered N-terminal domain (pep14-23) was shown to successfully inhibit DENV C-LD interaction, and the related specific binding of DENV C to very low-density lipoproteins (VLDL). These advances pave the way to drug development approaches, in which inhibitory efficiency may be improved through lead optimization. A similar strategy may be used for other flaviviruses.

References Carvalho et al. (2012) J. Virol., 86, 2096-2108.Martins et al. (2012) Biochem. J., 444, 405-415.Patent WO/2012/159187, PCT/BR2012/000162.Faustino et al. (2014) Nanomedicine, in press.


35

KN4

The Structural and Functional Basis of Hepatitis C Virus Neutralization by a Broadly Neutralizing Antibody

Arvind H. Patel

MRC – University of Glasgow Centre for Virus Research, University of Glasgow, Glasgow G11 5JR, UK

Hepatitis C virus (HCV) infection is a major public health problem. It is a leading cause of liver disease. Over 185 million people world-wide are infected with the virus, and 3 to 4 million more get infected every year. The majority of infected individuals develop chronic hepatitis, which can progress to cirrhosis and hepatocellular carcinoma. In most individuals, the immune system fails to clear the virus and a chronic infection is established. Treatment of HCV infection is becoming very effective, but there is an urgent need for a vaccine that will protect from infection with the virus, but as yet, no such vaccine exists. One of the obstacles to vaccine development is the high genetic diversity of the viral envelope glycoproteins. An effective vaccine needs to focus the immune response on conserved, functionally important regions that are normally poorly immunogenic. HCV, a member of the Flaviviridae family of positive strand RNA viruses, is composed of a nucleocapsid core enveloped by a lipid bilayer in which the two surface glycoproteins, E1 and E2, are anchored. These proteins play an essential role in viral entry into target cell. The entry process is known to involve a number of host cell surface entry factors including CD81, scavenger receptor class B type I and the tight junction proteins Claudin-1 and Occludin. E2 is a major target for neutralizing antibodies (nAbs) and contains hypervariable region 1 (HVR1), which is immunodominant and highly variable in sequence. Consequently, while antibodies to HVR1 can be neutralizing, they tend to be isolate specific and are unable to recognize E2 from other genotypes or isolates. While more broadly neutralizing antibodies exist, the majority of these recognize conformational epitopes on E2 that are non-contiguous and therefore extremely challenging to mimic in a potential vaccine. There is a great deal of interest in nAbs that are directed against conserved, linear epitopes. Our lab has focused on a mouse monoclonal antibody (mAb) AP33 that binds to a highly conserved protective epitope on the HCV E2 and potently neutralises all genotypes of HCV. The AP33 epitope, which spans amino acid residues 412 to 423 of E2, is linear, highly conserved and encompasses a tryptophan residue that plays a critical role in CD81 recognition. To further understand the mechanism by which AP33 neutralizes HCV infection, and to aid the development of a potential epitope vaccine, we recently determined the X-ray crystal structure of the Fab portion of AP33 in complex with its epitope peptide to 1.8Å. Additionally, we have characterized the interaction between AP33 and E2 by cross-competition analyses, alanine-scanning mutagenesis of E2 and by mutagenesis of AP33 at key epitope-binding residues. The structural and functional details of the E2 epitope, which will be presented at this meeting, provide a starting point for the design of an immunogen capable of eliciting AP33-like antibodies.


36

KN5

From N-glucopeptide synthetic probes to an hyper-glucosylated protein antigen: a bacterial infection triggering an antibody mediated form of

multiple sclerosis?Anna Maria Papini1,2,3

1 French-Italian Interdepartmental Laboratory of Peptide & Protein Chemistry & Biology2 University of Florence, Department of Chemistry “Ugo Schiff”, 50019 – Sesto Fiorentino (Italy)3 University of Cergy-Pontoise, PeptLab@UCP-SOSCO EA4505, 95031 – Cergy-Pontoise (France)

Aberrant glycosylations are known to affect in various manner immune responses and to exert marked effects on immune tolerance in humans. In line with these observations, we established in 1999 the ability of the glucosylated analogue of the immunodominant epitope of myelin oligodendrocyte glycoprotein (MOG), [Asn31(N-ßGlc)]hMOG(30-50), to detect autoantibodies in multiple sclerosis (MS) patients, but not in healthy controls, the unglucosylated analogue hMOG(30-50) being inactive. A model type I’ ß-turn structure of an N-ß-D-glucopyranosyl peptide allowed the characterization of a statistically significant population of relapsing-remitting MS patients and autoantibody titre correlates with disease progression. Type I’ ß-turn increased antibody recognition in the solid-phase conditions of the ELISA. Moreover, for an efficient antibody recognition, the epitope shall contain Asn(ß-Glc), but the conventional sequence motif for eukaryotic N-linked glycosylation NX(S/T) is not compulsory.[1]

Prokaryotes are able to glycosylate proteins and over 70 bacterial glycoproteins have been reported, those glycoproteins are surface exposed and play a vital role in bacteria adhesion to host cells or evasion of host immunity. The Haemophilus influenzae HMW1C protein is a glycosyltransferase, which has been shown to transfer glucose residues to Asn sites in HMW1 adhesin, identifying for the first time a new family of bacterial glycosyltransferases.[2] In 2011, Aebi’s group reported that the cytoplasmic N-glucosyltransferase of Actinobacillus pleuropneumoniae was an inverting enzyme, which transferred a glucose moiety from UDP-Glucose onto an asparagine side chain. We will report herein the first insight that the N-glucosylated beta-turn peptide probes detecting autoantibodies in MS are mimicking bacterial proteins N-glucosylated by a specific bacterial N-glucosyltransferase possibly involved in an early anti-N(Glc) antibody response [B. Imperiali & A.M. Papini unpublished results].Our hypothesis of an in vivo aberrant N-glucosylation related to a bacterial infection is supported by the functional transfer of the machinery of A. pleuropneumoniae N-glucosyltransferase into E. coli that enabled in vitro N-glucosylation of heterologous proteins.[3]

References1. (a)F. Lolli et al. P.N.A.S. (2005) 102, 10273; (b)S. Pandey et al. J. Med. Chem. (2012) 55, 10437.2. K.-J. Choi et al. PlosOne (2010) 5(12), e15888 ; (b) S. Grass et al. PlosPathog. (2010) 6(5), e1000919.3. A. Naegeli et al. J. Biol. Chem. (2014) 289, 2170-2179.


37

KN6

Cyclic peptides as inhibitors of cancer-related proteases

Masood Hosseini, Renée Roodbeen, Mingdong Huang, Peter Andreasen, Knud J. Jensen

University of Copenhagen, Department of Chemistry, Denmark

Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small molecule serine protease inhibitors generally suffer from specificity problems, peptide inhibitors, isolated from phage-displayed peptide libraries, have attracted considerable attention. Here we have investigated the mechanism of binding of peptide inhibitors to their serine protease targets. Our model is upain-1 (CSWRGLENHRMC), a disulfide bond-constrained competitive inhibitor of human urokinase-type plasminogen activator with a non-canonical inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we have characterized the upain-1-uPA complex with X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analyzed binding kinetics and thermodynamics by surface plasmon resonance and isothermal titration calorimetry. We found that upain-1 changes both main chain conformation and side-chain orientations as it binds to the protease, in particular its Trp3 residue and the surrounding backbone. Finally, we have rationally designed bicyclic peptide inhibitors of the serine protease urokinase-type plasminogen activator on the basis of the established monocyclic peptide, upain-2. These results provide a uniquely detailed description of the binding of a peptide pro-tease inhibitor to its target and are of general importance in development of peptide inhibitors with high specificity and new inhibitory mechanisms.

References1. Longguang Jiang, Anna S. P. Svane, Hans Peter Sørensen, Jan K. Jensen, Masood Hosseini, Zhuo Chen, Caroline

Weydert, Jakob T. Nielsen, Anni Christensen, Cai Yuan, Knud J. Jensen, Niels C. Nielsen, Anders Malmendal, Mingdong Huang, Peter A. Andreasen, , J. Mol. Biol., 2011, 412 (2), 235-250.

2. Masood Hosseini, Longguang Jiang, Hans Peter Sørensen, Jan K. Jensen, Anni Christensen, Sarah Fogh, Cai Yuan, Lisbeth M. Andersen, Mingdong Huang, Peter A. Andreasen, and Knud J. Jensen, 2011, Mol. Pharm., 2011, 80 (4), 585-597.

3. Zhuo Liu, Tobias Kromann-Hansen, Ida K. Lund, Masood Hosseini, Knud J. Jensen, Gunilla Høyer-Hansen, Peter A. Andreasen, Hans Peter Sørensen, Biochemistry, 2012, 51(39), 7804-7811.

4. Renée Roodbeen, Berit Paaske, Longguang Jiang, Jan K. Jensen, Anni Christensen, Jakob T. Nielsen, Mingdong Huang, Frans A. A. Mulder, Niels Chr. Nielsen, Peter A. Andreasen, and Knud J. Jensen, ChemBioChem, 2013, 2179-2188


38

KN7

New Approaches for Drug Design: Design of Multivalent Ligands for Treatment and Detection

of Degenerative DiseasesVictor J. Hruby

Regents Professor, Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721 USA

Major degenerative diseases such as cancer, prolonged pain, cardiovascular disease, diabetes, etc. re-sult from numerous changes in the expressed genome. Not surprisingly, our efforts to treat them with ligands which interact with a single receptor acceptor/enzyme/etc. do not work well. A new paradigm for drug discovery is needed. During the past two decades we have been exploring the design of multi-valent ligands that contain two or more pharmacaphores that can interact with two or more targets that are involved in a single molecule.In this talk we will discuss the design and synthesis of multivalent peptide and peptidomimetic ligands that act as agonists or antagonists at multiple receptors that are involved in prolonged and neuropathic pain, the most ubiquitous disease in the world, without the development of tolerance, addiction and other toxicities associated with current drugs. We will also discuss hetero- and homo-multivalent li-gands for the detection and treatment of cancer. The rationales for the design and consequences of multivalency for biological activity will be emphasized.Supported in part by grants from the U.S. Public Health Service, National Institute of Health, NIDA and NCI.


39

KN8

Enteral delivery of the peptide MIF-1 using ArisCrown technology

Anthony Padfield1, Sylvie Tchertchian2, Roger Causon1, Doriane Theurillat2, Tim Luker1, Martin Quibell1, Timothy Schulz-Utermoehl1, Fraser Murray1, Andrew Parker1 and Paolo Botti2

1. Exploratory Projects, Shire Pharmaceuticals, Unity Place, Hampshire International Business Park, Basingstoke, Hampshire RG24 8EP, UK.

2. ArisGen SA, 14 chemin des Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland.

Peptide therapeutics represent a large growing market with significant potential to treat diseases that have major unmet medical needs. However their successful development as novel therapeutics continues to be hampered by the very nature of peptide biochemistry and metabolism despite advances in delivery technology[1] and drug design strategies such as PEGylation[2] and lipidation. Oral delivery of therapeutic peptides is significantly restricted by the digestive system which is designed to cleave peptide backbones into single amino acids and prevent passive permeation of highly polar molecules into the bloodstream and thereby negatively impacting peptide bioavailability. In this context, the efficiency of ArisCrown technology was evaluated to orally deliver bioactive peptide L-prolyl-L-leucyl-glycinamide (MIF-1) in male Sprague Dawley rats. MIF-1 is a polar tri peptide exerting multiple effects in the CNS, with therapeutic potential in PD and depression. The pharmacokinetic parameters of MIF-1 peptide upon intraduodenal (ID) administration in proprietary formulations were determined and compared to exposure following ID, intraperitoneal, oral and intravenous dosing in standard vehicles. The ArisCrown formulations led to excellent bioavailability enhancement (F = 25 to 51% compared to <1%) at different MIF-1 concentrations (2, 6, 15 & 20 mg/ml) and doses (1, 3, 10 & 25 mg/kg).

References1. Jain A, et al., (2013) Crit. Rev. Ther. Drug Carrier Syst.,30(4), pp 293-329.2. Simerska P, et al (2011) Med. Res. Rev. 31(4), pp 520-47.


40

KN9

Phage selection of bicyclic peptides for therapeutic applicationsI. Rentero Rebollo1, L. Pollaro1, S. Chen1, C. Heinis1

1 Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland

Bicyclic peptides combine key qualities of antibody therapeutics (high affinity and specificity) and advantages of small molecule drugs (access to chemical synthesis, diffusion into tissue). Bicyclic peptide ligands to a desired target can be generated using an approach based on phage display. In brief, large combinatorial libraries of linear peptides are chemically modified to obtain combinatorial libraries of phage-displayed bicyclic peptides[1]. We were able to generate bicyclic peptide ligands with nanomolar or even picomolar binding affinity to a range of disease targets including plasma kallikrein, urokinase-type plasminogen activator, coagulation factor XII, matrix metalloproteinase 2 and sortase A. Towards the therapeutic application of the peptides, we have extended their circulation time to several days in mice[2] and we are now assessing the therapeutic effect of some of the peptides in vivo. Additionally, we are developing novel peptide macrocyclic formats with even better binding properties.[3,4]

Figure 1. A. Strategy for the selection of bicyclic peptides based on phage display. B. Example of the binding mode of a bicyclic peptide inhibitor (UK18) of the serine protease uPA.

References1. C. Heinis, T. Rutherford, S. Freund, G. Winter. Nature chemical biology (2009), 5, 502 - 5072. A. Angelini, J. Morales-Sanfrutos, P. Diderich, S. Chen, C. Heinis. J. Med. Chem. (2012), 55, 10187-101973. S. Chen, D. Bertoldo, A. Angelini, F. Pojer, C. Heinis. Angew. Chem. (2014), 126, 1628–16324. I. Rentero Rebollo, A. Angelini, C. Heinis. Med. Chem. Comm. (2013), 4, 145-150


41

ORAL PRESENTATIONS


42

O1

Biophysical Investigations of the Mechanism of Action of Antimicrobial Peptides and their Synergistic Interactions

B. Bechinger1, E. S. Salnikov1, E. Glattard1, A. Marquette1 and C. Aisenbrey3

1 Institut de chimie UMR7177, University of Strasbourg/CNRS, F-67070 – Strasbourg

Biophysical investigations, that aim to explain the antimicrobial activities of cationic linear peptides will be presented [1,2]. In particular we aim to understand how some mixtures of these peptides exhibit synergistic activities. Therefore, the structure, topology and dynamics of PGLa and magainin 2 were investigated in oriented phospholipid bilayers using solid-state NMR in the presence or absence of the other peptide and as a function of the membrane lipid composition [2,3]. Furthermore, fluorescence spectroscopy was used to investigate how the peptides interact with each other within the lipid bilayer environment.Whereas, magainin 2 exhibits stable in-planar alignments under all conditions investigated PGLa adopts a number of different membrane topologies with considerable tilt angle variations [2,3] Notably, the hydrophobic thickness modulates the alignment of PGLa [1]. In equimolar mixtures of PGLa and magainin 2 the former adopts transmembrane orientations in DMPC when at the same time magainin 2 remains associated with the surface [1]. In contrast in bilayers, which represent better the natural membrane composition (1-palmitoyl-2-oleoyl-phospholipids), both peptides adopt a surface oriented topology. Therefore, lipid-mediated interactions play a fundamental role in determining the topology of membrane peptides and proteins [3] and thereby potentially also the regulation of their activities. These results have important consequences for the mechanistic models explaining synergistic activities of the peptide mixtures and will be discussed in the context of unpublished data where membrane structure and interactions are correlated with biological activities..[1]

References1. B. Bechinger, J. Pep. Scie, 17, 306-314 (2010) 2. B. Bechinger, J. Resende, J., & C. Aisenbrey, Biophysical Chemistry 153, 115-125 (2011)3. E. Salnikov & B. Bechinger, Biophys J. 100, 1473-1480 (2011)


43

O2

Antimicrobial effect of proline and alanine scan on short cationic homopeptides

Guzman F.1, Albericio F.2, Ojeda C.1, Marshall SH.3, Aróstica M.4, Rojas R.4, Carvajal-Rondanelli P.5

1. Núcleo de Biotecnología Curauma, Pontificia Universidad Católica de Valparaíso (PUCV), Avenida Brasil 2950, 2. Valparaíso, Chile.3. Department of Organic Chemistry, University of Barcelona, Barcelona, Spain.4. Instituto de Biología, PUCV, Valparaíso, Chile.5. Instituto de Química, PUCV, Valparaíso, Chile.6. Escuela de Alimentos, PUCV, Valparaíso, Chile.

In the selection or design of antimicrobial peptides, the key role played by the number of cationic amino acids, and their position in the chain on the inhibitory potency of antimicrobial peptides is not clear. Thus, studying cationic homopeptides as model component we previously demonstrated that Lys homopeptides with an odd number of residues of 9, 11 or 13 were capable to inhibit the growth of bacteria in a broader spectrum and more efficient manner than those with an even number of Lys residues or Arg homopeptides of the same size [1]. To understand more in depth the inhibitory effect of 11- and 13-residue Lys homopeptides we performed an Ala and Pro scanning analysis by Fmoc solid-phase synthesis in order to assess the significance of every lysyl side chains for antibacterial activity. The antimicrobial activity of these analogs was analyzed by the microbroth dilution method against a wide range of bacteria. In general, Lys residues at either the next-to-last C-terminal or N-terminal end of both homopeptides played a crucial role for bacterial inhibition compared to Lys residues located at the center or nearby, as demonstrated by Ala exchange. Pro substitutions along the chain did not appreciably affect the inhibitory efficacy of 11- or 13- Lys homopeptides as Ala substitutions. Ala substitutions at any posi-tion of both homopeptides determined a more pronounced change of the polyproline type II (PPII) structure compared to Pro substitutions, demonstrated by circular dichroism measurement. Indeed, a high propensity of the PPII structure reduced significantly the inhibitory action. We concluded that the antibacterial efficacy effect of short lysine homopeptides is essentially determined by both a denser lysyl charge and a more flexible backbone located at the N- or C- terminal end of the homopeptide.

Key wordsSCAN, homolysine, antibacterial peptides

AcknowledgementWork funded by Grant 1140926 from FONDECYT, Chile

References1. Guzmán F, Marshall S, Ojeda C, Albericio F, Carvajal-Rondanelli P. J Pept Sci 19(12):792. 2013


44

O3

Bioactive peptides from the inside of the antibodiesL. Polonelli1, W. Magliani1, T. Ciociola1, C. Santinoli1, L. Giovati1, M. Sperindè1 and S. Conti1

1 Dept. Biomedical, Biotechnological and Translational Sciences (S.Bi.Bi.T.), Microbiology and Virology Unit, University of Parma, Parma, Italy

Synthetic peptides representative of sequences included in the complementarity determining regions (CDRs) and constant region (Fc) of antibodies (Abs), proved to exert in vitro, ex vivo and/or in vivo antimicrobial, antiviral, antitumor and/or immunomodulatory activities, conceivably mediated by dif-ferent mechanisms of action and regardless of the specificity and isotype of the belonging immuno-globulin (Ig).[1-6] Ab fragments can show intrinsic properties of self-aggregation in ß structures, leading to the formation of hydrogels which can assemble on molecular targets and dissociate spontaneously.[7] While the self-assembled state provides protection against proteases and the slow kinetic of disso-ciation assures a release of the active form over time, the receptor affinity is responsible for targeted delivery. Peptides derived from single amino acid substitution of bioactive CDRs and Fc fragments, adopted as surrogates of natural point mutations, displayed further differential biological activities. Phosphorylated and/or acetylated Ig fragments were identified in the proteomas of the human serum. A selected serum peptide, derived from Ig µ chain C region conserved domain, proved to exert a potent in vitro wide-spectrum fungicidal activity inclusive of strains resistant to conventional antifungal drugs, and a significant therapeutic effect against experimental candidiasis, without showing toxic or geno-toxic effects on mammalian cells. Synthetic peptides representative of the products of the Ig encoding genes J lambda, J kappa, D heavy and J heavy proved to display microbicidal activity in vitro against pathogenic eukaryotic and prokaryotic microorganisms and therapeutic effects against experimental candidiasis. Overall, our data lead to postulate that Abs could represent an unlimited source of new an-tiinfective and antitumor peptides as evolutionary result of the adaptive combination of gene products that ancestrally were devoted to functions of innate immunity.

References1. L. Polonelli, J. Ponton, N. Elguezabal, M.D. Moragues, C. Casoli, E. Pilotti, P. Ronzi, A.S. Dobroff, E.G. Rodrigues,

M.A. Juliano, D.L. Maffei, W. Magliani, S. Conti, L.R. Travassos PLoS ONE (2008), 3, e2371.2. E. Gabrielli, E. Pericolini, E. Cenci, F. Ortelli, W. Magliani, T. Ciociola, F. Bistoni, S. Conti, A. Vecchiarelli, L.

Polonelli PLoS ONE (2009), 4, e8187.3. W. Magliani, S. Conti, R.L. Cunha, L.R. Travassos, L. Polonelli Curr. Med. Chem. (2009), 16, 2305-2323.4. D.C. Arruda, L.C. Santos, F.M. Melo, F.V. Pereira, C.R. Figueiredo, A.L. Matsuo, R.A. Mortara, M.A. Juliano, E.G.

Rodrigues, A.S. Dobroff, L. Polonelli, L.R. Travassos J. Biol. Chem. (2012), 287, 14912-14922.5. L. Polonelli, T. Ciociola, W. Magliani, P.P. Zanello, T. D’Adda, S. Galati, F. De Bernardis, S. Arancia, E. Gabrielli, E.

Pericolini, A. Vecchiarelli, D.C. Arruda, M.R. Pinto, L.R. Travassos, T.A. Pertinhez, A. Spisni, S. Conti PLoS ONE (2012), 7, e34105.

6. E. Gabrielli, E. Pericolini, E. Cenci, C. Monari, W. Magliani, T. Ciociola, S. Conti, R. Gatti, F. Bistoni, L. Polonelli, A. Vecchiarelli PLoS ONE (2012), 7, e43972.

7. T.A. Pertinhez, S. Conti, E. Ferrari, W. Magliani, A. Spisni, L. Polonelli Mol. Pharm. (2009), 6, 1036-1039.


45

O4

Peptide cytotoxicity as a function of capping moieties: trichogin GA IV

M. De Zotti1, R. Tavano2,3, B. Biondi1, C. Peggion1, E. Papini2,3 and F. Formaggio1

1 Institute of Biomolecular Chemistry, CNR, Padova Unit, Department of Chemistry, University of Padova, 35131 - Padova (Italy)

2 Department of Biomedical Sciences, University of Padova, 35121 - Padova (Italy)

Various ascomycetes species secrete non-ribosomally produced peptides, called peptaibiotics, characterized by the unique presence of α,α-dialkylated α-amino acids, a rigid helical conformation, and membrane permeation properties. The antibacterial and cytotoxic activities of the short-chain peptaibiotic trichogin GA IV from the plant saprophytic and human opportunistic mold Trichomonas longibrachiatum were analyzed against Gram– and Gram+ bacteria, and a large panel of immortalized and primary human cells. Unexpectedly, the killing efficacy of trichogin was much stronger on human cells than on bacteria, due to specific mechanisms occurring in nucleated eukaryotic cells but not in erythrocytes. We thus synthesized and fully characterized an array of analogs. We discovered that we can effectively alter the selectivity of trichogin by acting on its N- or C-terminal moiety. The insertion of different capping groups results in a small yet significant modulation of the peptide 3D-structure (from 310- to α-helix) in a membrane mimetic environment (Figure 1). This finding suggests that a different peptide-membrane interaction takes place when the capping groups are modified. We hypothesize that the tuning of the peptide selectivity may originate from the alteration of its efficacy to modify and/or permeate biological membranes.

Figure 1. CD spectra of trichogin GA IV (tric GA IV, nOct-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Lol, with nOct, n-octanoyl; Lol, 1,2-amino alcohol leucinol) and its analogs in a membrane mimetic environment.


46

O5

Computational methods to determine peptide orientation in membranes

A. Farrotti1, G. Bocchinfuso1, A. Palleschi1, N.Rosato2, B. Bechinger3, L. Stella1

1 University of Rome Tor Vergata, Department of Chemical Sciences and Technologies, 00133 – Rome, Italy2 University of Rome Tor Vergata, Department of Experimental Medicine and Surgery, 00133 – Rome, Italy3 Université de Strasbourg, Institut de Chimie , F-67000 – Strasbourg, France

Antimicrobial peptides (AMPs) represent an efficient alternative to classic antibiotics in order to fight drug-resistant bacteria. Molecular dynamics simulations provide atomic details on the interactions between AMPs and membranes, but the limited length of simulations leads to sampling problems. In order to overcome these limitations we used three different approaches: the “minimum-bias”,[1, 2] coarse-graining (CG) and potential of mean force (PMF) methods. We tested these techniques on the artificial AMP LAH4. As demonstrated by solid-state NMR, thanks to the presence of four His residues in its sequence, this peptide is located at membrane surface at acidic pH, while it inserts into the bilayer hydrophobic core at higher pH values.[3]

Figure 1. Final structures of “minimum-bias” (left) and CG (middle) simulations and PMF profiles (right) of LAH4 with neutral (up) and charged (down) His residues.

In both minimum-bias and CG simulations, the peptide was located within the membrane when its His residues were neutral, while His protonation promoted the interaction with phospholipid headgroups. Similarly, PMF profiles exhibited in correspondence of the bilayer center a deep free-energy minimum for LAH4 with neutral His and a maximum for charged His LAH4.

References1. S. Esteban-Martìn, S. Salgado Biophys. J. (2007), 92, 903.2. G. Bocchinfuso et al., J. Pept. Sci. (2009), 15, 550.3. J. Georgescu, V.H. Munhos, B. Bechinger, Biophys. J. (2010), 99, 2507.


47

O6

How the exploration of the chemical space of cell-penetrating peptides helps to understand their functionality

S. Stalmans1, E. Wynendaele1 and B. De Spiegeleer1

1 Drug Quality and Registration (DruQuaR) group, Faculty of Pharmaceutical Sciences, Ghent University, 9000-Ghent.

Cell-penetrating peptides (CPPs) gained in recent years a lot of interest in the applied biomedical research field because of their ability to cross cellular barriers without causing significant lethal membrane damage. They have already been successfully applied to bring cell-impermeable cargoes into the cell interior. However, to date, only limited applications have reached the clinical phase of drug development. Currently, harmonization is needed in the cellular uptake studies of CPPs in order to efficiently draw reliable conclusions on their uptake mechanisms and structural properties. Therefore, we explored the chemical space of representative CPPs using chemo-molecular descriptors, which numerically express their chemical properties. Together with a newly defined cell-penetrating (CP) response for cellular influx, calculated using available quantitative cellular uptake data, the chemo-molecular descriptors were used to clarify which structural characteristics of the CPPs influence to what extent their cell-penetrating properties.[1] Our study indicated that CPPs are a chemically and CP-functionally diverse group of peptides.[1] Moreover, the newly defined CP-response and subgroups of CPPs help to select representative model peptides for further investigating their functionalities like e.g. uptake mechanisms and behaviour at different membrane interfaces, like the blood-brain barrier.[2] Another application of the exploration of the chemical space of the CPPs, is to investigate their chemical and functional relationship with the antimicrobial peptides, which are historically investigated as a separate peptide group.[3]

References1. S. Stalmans et al. Plos One. (2013), 8, e71752.2. S. Stalmans et al. (submitted, 2014)3. S. Stalmans et al. Protein Peptide Lett (2014), 21, 399-406.


48

O7There is no such thing as a typical AMP. Maybe

Manuel N. Melo1, Juanjuan Su, Siewert J. Marrink

1 University of Groningen, Groningen - Netherlands

The class of antimicrobial peptides (AMPs) is broad and diverse. Several sub-classifications have been proposed since its discovery in order to better systematize the understanding—and the prediction—of AMP action. However, exceptions seem to be the rule to any categorization made thus far and overarch-ing patterns of peptide action are faint, at best.One such sought trend is the separation of charged and zwitterionic membrane lipids upon binding of AMPs, and how this could bring about further membrane disruption. Recently, different degrees of peptide-induced lipid segregation have been experimentally observed for a number of AMPs [1]. In our work we use coarse-grained and atomistic molecular dynamics simulations to observe such lipid segre-gation. We replicate the conditions under which segregation was experimentally observed and test the effects of the AMPs Magainin2, BP100, and MSI-103. Indeed, charge-based lipid segregation is visible in our simulations at the molecular level with a trend compatible with the experimental findings. How-ever, other individual features of the peptides stand out for their disparity—namely the propensities to aggregate and to induce membrane tension. This occurs despite the three peptides often being classified together under the “cationic alpha-helical” AMP category. We follow up on those features to the limits of our simulation models with interesting and surprising results.Our observations support one well-preserved rule of AMP action: that there is no such thing as ‘typical’ when it comes to AMPs. Maybe

Different self-aggregation and clustering actions of magainin2 (left) and BP100 (right) on cardiolipin-POPE mixtures (red and green, respectively); peptides are in blue.

References1. Wadhwani, P., et al (2012). Membrane-active peptides and the clustering of anionic lipids. Biophysical journal, 103,

265


49

O8

Photosensitizing activity of porphyrin-antimicrobial peptides conjugates toward prokaryotic and eukaryotic cells

M. Gobbo1, R. Dosselli,2 F. Moret,2 S. Nonell3 and E. Reddi2

1 University of Padova, Department of Chemical Sciences, 35131- Padova, Italy2 University of Padova, Department of Biology, 35131- Padova, Italy3 Universitat Ramon Llull, Institut Químic de Sarrià, 080017 - Barcelona, Spain

Cationic antimicrobial peptides and photodynamic therapy (PDT) are attractive tools to contrast infectious diseases and further development of antibiotic resistance.[1] To enhance the efficacy of PDT against Gram-negative bacteria, the binding of the photosensitizer (PS) to cationic antimicrobial peptides offers the attractive prospect for improving both the water solubility and the localization of the photoactive drug into bacteria. The have synthesized conjugates between neutral or cationic porphyrins and the antimicrobial peptides apidaecin 1b,[2] buforin II and magainin 2 and evaluated their phototoxic activity against bacterial and mammalian cells.

The neutral and hydrophobic porphyrin, which is not photoactive per se against Gram-negative bacteria, when conjugated to antimicrobial peptides reduced the survival of E. coli cells, upon light activation, by 3-5 log at 1.5 µM concentration. PS-peptide conjugates were considerably more potent against methicillin-resistant S. aureus, resulting phototoxic at one-tenth concentration. In protocols requiring repeated washings after sensitizer delivering, peptide conjugation strengthened considerably the interaction of cationic porphyrins with bacterial cells, retaining their photoinactivation activity. On the other hand porphyrin conjugates exhibited strong interaction as well as phototoxicity toward eukaryotic cells (human skin fibroblasts) losing the selectivity against bacteria exhibited by cationic antimicrobial peptides.

References1. T. Dai, Y.Y. Huang, M. R. Hamblin Photodiagn. Photodyn. Ther. (2009), 6, 170.2. R. Dosselli, C. Tampieri, R. Ruiz-Gonzáles, et al. J. Med.Chem. (2012), 56, 1052.


50

O9

Some like it hot: Biomolecule Analytics using MicroScale Thermophoresis

Fabian Zehender1

1 NanoTemper Technologies GmbH, Floessergasse 4, 81369 Muenchen, Germany

The technology is called “Microscale Thermophoresis”, which means that we measure the directed motion of molecules along a local temperature gradient generated with infrared laser radiation[1]. Thermophoresis depends on the size, the charge and the solvation entropy of the molecules in solution. Since one of the parameters changes in virtually every binding event, we can measure protein-peptide/protein, protein-nucleic acid, and protein-ribosome interactions. Technology allows even measurement of the interactions of small molecules (drugs, sugars, ions) with proteins. Measurements require less than 5 µl of sample volume at nanomolar concentrations and takes just 10 minutes. The method is also suited for the measurement in complex biological liquids as serum or cell lysate and can detect aggregates in the sample[2]. Orthogonal methods include Surface Plasmon Resonance and Isothermal Titration Calorimetry.

Figure 1. MST setup and experiments. (A) The Monolith NT.115 from NanoTemper Technologies GmbH. (B) MST is measured in capillaries. (C) Typical signal of a MST experiment, thermophoretic movement of the labelled molecules out of the heated sample volume can be detected. (D) The thermophoretic movement of a fluorescent molecule changes upon binding to a non-fluorescent

ligand. A titration of the ligand yields a binding curve which can be fitted to derive binding constants.

References1. Seidel, S. A., et al. (2013). “Microscale thermophoresis quantifies biomolecular interactions under previously

challenging conditions.” Methods.2. Jerabek-Willemsen, M., et al. (2014). “MicroScale Thermophoresis: Interaction analysis and beyond.” Journal of

Molecular Structure.


51

O10

Gene therapy innovation: targeted Peg-protamine as potential nanocarrier for gene transfection

Felisa Cilurzo1, Donatella Paolino2, Donato Cosco2, Anna Mero3, Massimo Fresta2 and Gianfranco Pasut3

1 Centro di Ricerca Interregionale sulla Sicurezza Alimentare e la Salute, University Magna Graecia of Catanzaro, Department of Health Science, 88100 – Catanzaro2 University Magna Graecia of Catanzaro, Department Health Science, 88100 – Catanzaro3 University of Padua, Department of Drug Science, 35131 – Padua

In this investigation folic acid-based PEG-protamine (Folate-Protamine) was synthesized and proposed as a nanocarrier for gene transfection (PRRRRSSRRPVRSPRVSRRRRRRGGRRRR). Folate-Protamine was developed to take advantage of a possible synergistic effect between folic acid and protamine by combining the destabilizing effect of protamine on cell membranes, thus improving the endosomal escape and facilitating the DNA cytoplasmic entrance, and the targeting features of folate moiety towards cells overexpressing the folate receptor. The advantage of this strategy may be the achievement of a safe and efficacious nanocarrier for the selective delivery of therapeutic DNA to be used for the treatment of genetic disease. It has been hypothesized that the condensation of DNA into a toroidal-like structure is essential for the protection of DNA from enzymatic degradation as well as to facilitate entry of the DNA into the nucleus [1].The conjugate was synthesized in a three-step reaction and every reaction intermediate has been characterized and purified individually in order to assess the yield and purity. The chemical and technological characterization was carried out by RP-HPLC, SDS-PAGE, 1H-NMR. The new compound showed an amphyphilic feature, leading to a self-assembling in the form of a colloidal nanocarrier, which was physico-chemically characterized by dynamic light scattering to determine both the mean size and the zeta potential (table 1), as a function of the nanocarrier/DNA ratio. The plasmid DNA coding for the green fluorescent protein (GFP) was used as a model for these preliminary tests.

Table 1: Table of photon correlation spectroscopy of free folate-PEG-protamine and DNA complexed polymer in different molar ratio

Compound Potenziale Z Size PdI

Fol-PEG-Protamine +16,29±2 83,5±0.5 0,3

Fol-PEG-Protamine/DNA 20/20 -51,4±4,2 -- --

Fol-PEG-Protamine/DNA 40/20 -17,4±2,6 -- --

Fol-PEG-Protamine/DNA 80/20 +8,5±2 137±0.3 0,32

The results regarding to the purity of the compounds and the ability of complexing the plasmid were extremely positive and a further investigation and development is envisaged.

References1. Kabanov AV et al., J. Controll. Rel. (1995).


52

O11

Targeting of daunomycin with oligo/polypeptide bioconjugates: the influence of the partner on functional properties

G. Csik1, G. Schlosser2, E. Orbán2, Z. Bánóczi2, R. Szabó2, Sz. Bősze2 and F. Hudecz2,3

1. Institute of Biophysics and Radiation Biology, Semmelweis Medical University, Budapest2. Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös Loránd University, H-1518 Budapest3. Department of Organic Chemistry, Eötvös Loránd University, H-1518 Budapest, Hungary

The effect of chemotherapy is frequently restricted by dose-limiting toxicities, including side effects (e.g. cardiotoxicity, multidrug resistance [MDR]). One of the novel approaches to destroy target cells is to deliver drug by its covalent peptide conjugate.[1,2] We have developed two groups of conjugates in which drugs (e.g. daunomycin [Dau], vinblastine or ferrocene derivatives are attached to branched chain polymeric polypeptides, (poly[Lys-(DL-Alam-Xi)] (X = Glu, Ser, m ≈ 3, i < 1) with different side chain composition [3] or to Argn oligopeptides (n = 4, 6 or 8) [4] . We found that these peptide conjugates without known “recognition unit” - could significantly influence anti-tumour activity of the drug in vitro even in MDR resistant cells. [5-8] Using these two new classes of Dau-conjugates we have studied the effect of the chemical structure of the conjugates (including the influence of the type of the covalent linkage between the two partners) on the fluorescence properties (spectra, intensity, quantum yield), on the in vitro cytostatic/cytotoxic effect on relevant cell lines (e.g. HL-60, HepG2), on cellular uptake and also on the protein expression profile of the conjugate in comparison with those of the free drug. We found marked influence of the charge properties on fluorescence features of the conjugate. Similarly, cellular uptake mechanism was very much dependent on the size of the peptide even within the same group (e.g. Arg4 vs. Arg8). In contrast, little effect could be documented in relation to the type of covalent linkage and of spacer applied between Argn oligopeptide and Dau. In conclusion, by the rational combination of structural elements there is a room to improve antitumour potency of Dau by attachment of poly/oligopeptide.

AcknowledgementsThese studies were supported by the Hungarian Research Fund (K104385).

References1. F. Hudecz, Gy. Kóczán, J. Reményi. In: Molecular pathomechanisms and new trends in drug research (Eds.: Keri,

Gy. and Toth, I.) (2003) Taylor and Francis Group, London, pp. 553. 2. F. Hudecz, Z. Bánóczi, G. Csík, Med. Res. Rev. (2005), 25, 679.3. R. Szabó, G. Mező, et al. Bioconjugate Chemistry (2008), 19, 1078.4. Z. Bánóczi, B. Peregi, E. Orbán, et al. ARKIVOC, (2008), 140.5. Zs. Miklán, E. Orbán, G. Csík, et al. Biopolymers (2009), 92, 489.6. Zs. Miklán, E. Orbán, Z. Bánóczi et al. J. Peptide Sci. (2011), 17, 805. 7. E. Orbán, M. Manea et al. Bioconjugate Chemistry (2011), 22, 2154.8. Z. Bánóczi, Z., Csik, G. et al. In: Proc. 4th Asia-Pacific International Peptide Symposium/50th Japanese Peptide

Symposium, Osaka, Japan, November 6-8, 2013 (in press)


53

O12

Tuning the aggregation of conformationally constrained oligopeptides

M. Venanzi1, M. Caruso1, G. Bocchinfuso1, E. Gatto,1 A. Palleschi1, C. Aleman2, D. Zanuy2, F. Formaggio3 and C. Toniolo3

1 University of Rome Tor Vergata , Department of Chemical Sciences and Technologies, 00133 – Rome 2 ICB, Padua Unit, CNR, University of Padua, Department of Chemical Sciences, - Padua3 Department of Chemical Engineering, Polytechnic University of Catalunya, E-08028, Barcelona

Peptide aggregation is determined by a complex interplay of dynamical and structural factors, including the secondary structure attained by the peptide chain and its dynamical properties, the interactions between the peptide chains, the solvation of single peptide molecules, the presence of residues giving rise to specific site-to-site interactions (aromatic side-chains, charged groups, sulphur atoms). All these factors govern the formation of peptide aggregates of different morphologies (nanospheres, fibrils, nanotubes).In this contribution we report on two case studies concerning the aggregation propensity of conformationally constrained oligopeptides, i.e. peptides formed almost exclusively by Cα-tetrasubstituted residues. In the former, we discuss the aggregation of homoAib oligopeptides of different length (n=6, 12, 15) in methanol/water solution. Experiments showed that the aggregation propensity increases with increasing the length of the peptide chain. When the peptides are immobilized on mica, the interplay of aromatic-aromatic and interhelix interactions determines the morphology of the peptide aggregates, as revealed by AFM imaging.[1]

In the second system investigated, the aggregation properties of two Ala-based pentapeptides were studied by spectroscopic techniques and Molecular Dynamics simulations. The two peptides, both functionalized at the N-terminus with a pyrenyl group, differ in the insertion of an α-aminoisobutyric acid at position 4. This single substitution is crucial in determining the aggregation propensity and the morphology of mesoscopic aggregates.[2]

The insights that such model studies can provide to the understanding of the early stages of the process leading to the formation of neurotoxic peptide aggregates will be also discussed at the conference.

References1. M. Caruso, E. Placidi, E. Gatto, C. Mazzuca, L. Stella, G. Bocchinfuso, A. Palleschi, F. Formaggio, C. Toniolo, M.

Venanzi J. Phys. Chem. B (2013), 117, 5448-5459.2. M. Caruso, P. Flamigni, E. Gatto, E. Placidi, G. Ballano, F. Formaggio, C. Toniolo, D. Zanuy, C. Aleman, M. Venanzi

Soft Matter (2014), 10, 2508-2519.


54

O13

Peptides of the dihydrolipoamide acetyltrnsferase for the detection of autoantibodies in autoimmune diseases

Cedric Rentier 1,2,3, Giulia Pacini 1,4, Francesca Nuti 1,3, Carlo Selmi 5, Pier-Maria Battezzati 6, Paolo Rovero 1,4 and Anna-Maria Papini 1,2,3

1. French-Italian Laboratory of Peptide and Protein Chemistry & Biology PeptLab www.peptlab.eu2. Laboratoire SOSCO EA4505, University of Cergy-Pontoise, 5 mail Gay-Lussac, Neuville-sur-Oise 95000 Cergy-

Pontoise3. Department of Chemistry “Ugo Schiff”, Via della Lastruccia 3/13, Polo Scientifico e Tecnologico, University of

Florence, I-50019 Sesto Fiorentino, Italy4. Department NeuroFarBa, Section of Pharmaceutical Sciences and Nutraceutics, Via Ugo Schiff 6, University of

Florence, I-50019 Sesto Fiorentino, Italy5. University of Milan, IRCCS-Istituto Clinico Humanitas, via A. Manzoni 56, 20089 Rozzano, Milan, Italy6. Ospedale San Paolo - Department of Health Sciences, University of Milan, via Di Rudinì 8, I-20142 Milano, Italy

Primary Biliary Cirrhosis (PBC) is a cholestatic autoimmune disease affecting the liver, characterized by the progressive destruction of intrahepatic bile ducts [1]. Its triggering mechanism remains unclear, but it is believed to be a combination of genetic and environmental factors (such as bacterial infection or xenobiotics contaminations) [2,3].Evidence from various studies shows that antimitochondrial autoantibodies (AMA) are mainly directed toward lipoylated epitopes of the E2 component of the pyruvate dehydrogenase complex (PDC-E2). In particular, the sequence ETDK*AT has been found in common in inner and outer lipoyl domains of the PDC-E2, although it is unclear if the epitope is linear or conformational-based. [4]

We propose to use peptides including ETDK*AT as synthetic probes to characterize different families of autoantibodies in PBC possibly related to other immune-mediated diseases (i.e., scleroderma and type-1 diabetes) to highlight sequence and/or conformational effects. In particular we investigated the role of several post-translational modifications (i.e., lipoylation and N-glucosylation), as well as of beta-turn structures, through a comparison with modified analogs of the original type I’ beta-turn peptide CSF114 [5]. The synthesis and characterization of such peptides is herein described.

Key wordsautoantibodies, autoimmune disease, post-translational modification, beta-turn

References1. Selmi, C. et al. Lancet, 377, 1600 (2011)2. Selmi, C.; et al. Hepatology, 38, 1250 (2003)3. Leung P.S.C. et al. Trends in Molecular Medicine 18(10), 577 (2012)4. Braun, S. et al., World J. Gastroenterol,. 16(8), 973 (2010)5. Carotenuto, A.; et al., J. Med. Chem., 49, 5072 (2006)


55

O14

Behaviour of antimicrobial peptides in phospholipid membranes: insights from combined spectroscopic and simulative studies

L. Stella1, G. Bocchinfuso1, S. Bobone1, A. Farrotti1, D. Roversi1, Z. Vaezi1, A. Palleschi1, Y. Park2, M. De Zotti3, F. Formaggio3, C. Toniolo3

1 University of Rome Tor Vergata, Department of Chemical Sciences and Technologies, 00133 – Rome, Italy2 Chosun University, Department of Biotechnology, 501-759 – Gwangju, Korea3 CNR, ICB, Padova Unit and University of Padova, Department of Chemistry, 35131 – Padova, Italy.

Antimicrobial peptides (AMPs) have multiple roles, but their first activity is bactericidal, through perturbation of the permeability of microbial membranes. Due to this mechanism of action, AMPs represent promising lead compounds against drug-resistant bacteria.Through the combined application of spectroscopic techniques and molecular dynamics simulations,[1-8] we investigated several aspects of the interaction of different AMPs with membranes, such as selectivity, location in the bilayer, mechanism of pore formation, and effects on lipid dynamics.Overall, these studies provide an atomic level picture of AMP behaviour in lipid membranes, which is the basis for the rational design of new molecules with improved, drug-like properties.

References1. S. Bobone, et al. Biochim. Biophys. Acta (2013), 1828, 1013.2. S. Bobone, et al. J. Pept. Sci. (2013), 19, 758.3. S. Bobone, et al. Biochemistry (2012), 51, 10124.4. G. Bocchinfuso, et al. Cell. Mol. Life Sci. (2011), 68, 2281.5. S. Bobone, et al. J. Pept. Sci. (2011), 17, 335.6. C. Mazzuca, et al. Biophys. J. (2010), 99, 1791.7. B. Orioni, et al. Biochim. Biophys. Acta (2009), 1788, 1523.8. G. Bocchinfuso, et al. J. Pept. Sci. (2009), 15, 550.


56

O15

Pore-forming antimicrobial peptides as “molecular rulers” to measure bacterial membrane thickness

Erik Strandberg1, Ariadna Grau-Campistany2, Parvesh Wadhwani1, Johannes Reichert1, Jochen Bürck1, Francesc Rabanal2, and Anne S. Ulrich1,3

1 Karlsruhe Institute of Technology (KIT), Institute of Biological Interfaces (IBG-2), POB 3640, 76021 Karlsruhe, Germany2 Departament de Química Orgànica, Facultat de Química, Universitat de Barcelona, Barcelona, Spain3 KIT, Institute of Organic Chemistry, Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany

MSI-103 [sequence (KIAGKIA)3-NH2] is a designer-made antimicrobial peptide based on the sequence of PGLa, a host defence peptide from the African frog Xenopus laevis, with high activity against bacteria.[1,2] It forms an amphipathic α-helix upon binding to a lipid bilayer, and has been proposed to kill bacteria by forming membrane pores with peptides in a transmembrane orientation. If this were the case, shorter analogues that are not long enough to span the membrane should not be able to form pores and should be inactive. To test this hypothesis we have synthesized a series of analogues of MSI-103, called KIA peptides, with a length of 14 to 28 amino acids, all of which were shown to be α-helical by circular dichroism spectroscopy. We tested their antimicrobial and haemolytic activities and the ability to induce vesicle leakage, and found that there is a threshold length needed for activity, supporting the pore formation hypothesis. Interestingly, different bacteria had different thresholds, as the shortest peptides are completely inactive, while KIA17 and longer peptides could kill E. coli, even longer peptides could kill also other bacteria like S. aureus, whereas only the longest peptides of at least 24 amino acids were active against E. faecalis. To induce vesicle leakage in different lipid systems with different acyl chain lengths, the necessary peptide lengths were found to fit almost perfectly to the hydrophobic thickness of the membranes used. Assuming that the same correlation holds also for the case of bacterial membranes, the KIA peptide series can thus be used as a molecular ruler to estimate the thickness of different bacterial membranes.Using solid-state 15N-NMR on isotopically labelled peptides, we also investigated the orientation of the different KIA peptides in membranes of different lipid composition. In some lipid systems we observed a systematic dependence of orientation on peptide length, in line with our previous result on MSI-103, where we noted a strong correlation between peptide orientation and lipid spontaneous curvature.[3]

References1. J. Blazyk, R. Wiegand, J. Klein, J. Hammer, R.M. Epand, R.F. Epand, W.L. Maloy, U.P. Kari, J. Biol. Chem. (2001),

276, 27899-27906.2. E. Strandberg, N. Kanithasen, J. Bürck, P. Wadhwani, D. Tiltak, O. Zwernemann, A.S. Ulrich, Biochemistry (2008),

47, 2601-2616.3. E. Strandberg, D. Tiltak, S. Ehni, P. Wadhwani, A.S. Ulrich, Biochim. Biophys. Acta (2012) 1818, 1764-1776.


57

O16

Mechanism of action on membrane modelsof Clausin, a lantibiotic peptide from Bacillus clausii

J. Toupé1, A. Bouhss2, J-M. Bressollier1, B. Desbat1, R. Oda1, J-M. Schmitter1, J. Gallay2, EJ. Dufourc1, MC. Urdaci1 , and B. Odaert1

1. Chimie et Biologie des Membranes et des Nanoobjets (CBMN), UMR 5248, CNRS - Université de Bordeaux – Institut Polytechnique de Bordeaux, Pessac, France. [email protected]

2. Institut de Biochimie et Biophysique Moléculaire et Cellulaire (IBBMC), UMR 8619, Université Paris-Sud 11 CNRS, Orsay, France.

The emergence of “super-bacteria” ultra-resistant to antibiotics urges us to find new drugs for combating the coming-back of infectious diseases. Natural AntiMicrobial Peptides (AMPs) may constitute templates for rational drug design.We study Clausin, a peptide produced by Bacillus Clausii and active against Staphylococcus Aureus. By combining Mass and NMR spectroscopies, we showed that Clausin is a new lantibiotic[1], partly homolog to Nisin which is known to block bacterial cell wall formation through binding to bactoprenol lipids. Fluorescence with dansylated-Lipid II (LII) and ITC with Undecaprenyl PyroPhosphate (UPP) showed sub-nanomolar stoichiometric complex formation with Clausin[2].We elucidated the NMR structure of Clausin in membrane mimicking media. Solid State NMR spectroscopy with bacterial mimicking membrane models showed slight change of the dynamics for the polar head group (31P), but none for the core (2H). Clausin adsorbs by itself to the membrane surface due of its hydrophobic nature.Solid State NMR spectroscopy showed a drastic ordering for the membrane core (2H) with bactoprenol UPP in the presence of clausin, suggesting cluster formation due to peptide insertion. Ellipsometry microscopy imaging allowed us to confirm this hypothesis. Epifluorescence with dansylated-LII allowed us to see fiber formation of the bactoprenol lipis in the presence of Clausin. These results support the view that short lantibiotics block cell division by sequestering bactoprenol lipids[3].

References1. Chatterje C, Van der Donk, WA. (2005). Chem. Rev., 105,633-683.2. Bouhss A, Al-Dabbagh B, Vincent M, Odaert B, Aumont-Nicaire M, Bressollier P, Desmadril M, Mengin-Lecreulx

D, Urdaci MC, Gallay J (2009). Biophys J, 97, 1-8.3. Hasper HE, Kramer NE, Smith JL, Hillman JD, Zachariah C, Kuipers OP, de Kruijf B, Breukink, E. (2006). Science,

313, 1636-37.


58

O17

A novel synthetic antimicrobial peptide. A new weapon for multi-drug-resistant bacteria?

J. Brunetti1, C. Falciani2, L. Lozzi1, G. Roscia1, L. Quercini1, E. Ibba1, L. Bracci1 and A. Pini1

1 University of Siena , Department of Medical Biotechnology, via Aldo Moro 2, 53100 - Siena2 SetLance srl, Toscana Life Sciences, via Fiorentina 1, 53100- Siena

M33 is a synthetic antimicrobial peptide with a strong activity against different multi-resistant Gram-negative bacteria, including MDR clinical isolates of Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumanii and other enterobacteriaceae. It becomes active against Gram positives when synthesized with D-aminoacids. M33 activity is based on the following two-step mechanism: 1- high affinity binding to bacterial LPS or LTA, and 2- disruption of bacterial membranes. No haemolytic activity and no toxicity against eukaryotic cells was observed. The peptide was optimized by a sequence selected by a random library [1-2]. It was characterized for its activity on biofilms [3], modification with Peg [4], procedures for manufacturing, for its in vivo efficacy in animal models of sepsis, lung and skin infections, and for the low toxicity in vivo and low propensity to select bacterial resistance. The latter test produced better outcomes in comparison with antimicrobial peptides already used in the clinic. M33 also showed a strong anti-inflammatory activity neutralizing LPS and reducing the expression of cytokines involved in inflammation. M33 is synthesized in a tetra-branched form, a well-defined molecular structure that confers to the molecule a high resistance to circulating proteases, hence overcoming the problem of peptide short half-life [5]. Presently, it is under study for encapsulation in nano-particles for aerosol delivery

NH

O

NH

NH

O

NH

NH

O

NH

NH O

OH

O

KKIRVRLSA

O

KKIRVRLSA

O

KKIRVRLSA

O

KKIRVRLSA

Figure 1. Structure of tetra-branched peptide M33. Aminoacids of the four peptide sequences are indicated with the one letter code

M33 is a strong candidate for the development of a new antibacterial drug. Currently, it is under preclinical development and it is expected to enter in clinical trials within the next two years.

References1. A. Pini, et al., Antimicrob Agents Chemother. (2005), 49, 26652. A. Pini et al., FASEB J. (2010), 24, 10153. C. Falciani et al., PlosOne. (2012), 7, e462594. C. Falciani et al., AminoAcids. (2014), In press5. L. Bracci et al., J Biol Chem. (2003), 278, 46590


59

O18

Peptide Antivirals Directly From Viral Genome InformationA. Pessi1,2

1 PeptiPharma, 00144 Roma, Italy2 CEINGE, 80145 Napoli, Italy

Over the past decade, the global effort to meet the challenge of emerging viral pathogens has resulted in a greatly enhanced ability to identify and genetically fingerprint causative agents, often with extraordinary speed. However, this ability has not yet been translated into equally rapid development of new therapies, since drug discovery requires information that does not immediately derive from knowledge of the viral genome. A telling example is represented by the 2002-03 SARS epidemic, where identification of the novel SARS coronavirus and sequencing of its genome was accomplished in a few weeks, but in the absence of a specific antiviral the only effective measure to curb the epidemics was quarantine of infected people. Indeed, a 2006 retrospective analysis of the treatments used concluded that it was not possible to determine whether any of them benefited patients during the outbreak, and that some may have actually been harmful [1].We describe a technology that would enable preparation and shelving of specific inhibitors for enveloped viral pathogens in response to genetic diagnostic information, in advance of, or concurrently with an outbreak of viral disease. This platform may form the basis for an efficacious and timely emergency response, immediately following identification of genetic information about potentially dangerous new enveloped viruses. In our strategy, candidate lead molecules come in the form of peptides corresponding to key domains of the viral fusion machinery, which are endowed with improved antiviral and pharmaceutical properties via simple chemical modifications: the attachment of a cholesterol group (“cholesterol-tagging”) and sequence dimerization. We have successfully used this strategy for several enveloped viruses [2-7], yielding proof of concept for this genomic-based emergency antiviral response.

References1. L.J. Stockman, R. Bellamy, P. Garner PLoS Med. (2006) 3, e343.2. P. Ingallinella, E. Bianchi, N.A. Ladwa, Y.J. Wang, R. Hrin, M. Veneziano, F. Bonelli F, T.J. Keta, J.P. Moore, M.D.

Miller, A. Pessi Proc. Natl. Acad. Sci. U.S.A. (2009) 106, 5801.3. M. Porotto, C.C. Yokoyama, L.M. Palermo, B. Mungall, M. Aljofan, R. Cortese, A. Pessi, A. Moscona J. Virol.

(2010) 84, 6760.4. M. Porotto, B. Rockx, C.C. Yokoyama, A. Talekar, I. DeVito, L.M. Palermo, J. Liu, R. Cortese, M. Lu, H. Feldmann,

A. Pessi, A. Moscona PLoS Pathog. (2010) 6, e1001168.5. K.K. Lee, A. Pessi, L. Gui, A. Santoprete, A. Talekar, A. Moscona, M. Porotto J. Biol. Chem. (2011) 286, 42141.6. A. Pessi, A. Langella, E. Capitò, S. Ghezzi, E. Vicenzi, G. Poli, T.J. Ketas, C. Mathieu, R. Cortese, B. Horvat, A.

Moscona, M. Porotto PLoS One (2012) 7, e36833.7. J.C. Welsch, A. Talekar, C. Mathieu, A. Pessi, A. Moscona, B. Horvat, M. Porotto J. Virol. (2013) 87, 13785.


60

O19

Urotensin II receptor: its role as prognostic marker and potential therapeutic targets in human epithelial cancers

M. Caraglia1, S. Zappavigna1, A. Luce1, R. Franco2, A. Federico3, E. Novellino4, P. Grieco4

1 Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, 80138 - Naples, Italy2 Pathology Unit, INT Pascale 80131, Naples Italy3 Department of Clinical and Experimental Medicine and Surgery, Second University of Naples, 80131 - Naples, Italy 4 Department of Pharmacy, University “Federico II” of Naples, 80131 – Naples Italy

Urotensin-II (UT-II) is a potent vasoconstrictor peptide and its receptor (UT) was correlated with human cortico-adrenal carcinoma proliferation [1]. Based upon these data we have evaluated the expression and function of UT on several epithelial cancer cells both in vitro and in vivo. We have assessed UT expression on a tissue micro-array of different normal and tumour tissues of different histogenesis and we have detected higher levels of UT on prostate, colon and bladder cancer if compared with normal counterparts. We have firstly evaluated the correlation between UT expression and prognosis of human prostate adenocarcinoma. UT mRNA and protein were expressed at high levels only in androgen-dependent LNCaP cells. The immunohistochemical expression of UT in vivo in 195 human prostate tissue samples was always present at low intensity in hyperplastic tissues and at high intensity in well-differentiated carcinomas (Gleason 2-3). The UT antagonist Urantide induced a dose-dependent decrease of motility and invasion of LNCaP cells whose characteristic ameboid movement seems to be advantageous for their malignancy. [2]. Similarly also colon cancer cell lines expressed UII protein, and UT protein expression was detected in 5-30% of epithelial cells in 45 normal controls, in 30-48% in 21 adenomatous polyps and in 65-90% in 48 colon adenocarcinomas. UT mRNA expression was increased by threefold in adenomatous polyps and eight-fold in colon cancer, compared with normal colon [3]. Finally, UT discriminated between non muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC) and showed a significant correlation between low UT expression and shorter disease free survival in NMIBC. In all these studies, we evaluated the effects of some superagonist and antagonist recently developed in our lab. Our data suggest that UT is a new molecular prognostic marker and a potential new target in epithelial cancers.

References1. F. Merlino, S. Di Maro, A. Munaim Yousif, M. Caraglia, P. Grieco. Urotensin-II Ligands: An Overview from Peptide

to Nonpeptide Structures. J Amino Acids. 2013;2013:979016.2. Grieco P, Franco R, Bozzuto G, Toccacieli L, Sgambato A, Marra M, Zappavigna S, Migaldi M, Rossi G, Striano

S, Marra L, Gallo L, Cittadini A, Botti G, Novellino E, Molinari A, Budillon A, Caraglia M. J Cell Biochem. 2011 Jan;112(1):341-53.

3. Federico A, Zappavigna S, Romano M, Grieco P, Luce A, Marra M, Gravina AG, Stiuso P, D’Armiento FP, Vitale G, Tuccillo C, Novellino E, Loguercio C, Caraglia M. Eur J Clin Invest. 2014 Mar;44(3):285-94.


61

O20

GnRH antagonist as potential targeting units - synthesis and in vitro evaluation

I. Szabó1 and Sz. Bősze1

1 MTA-ELTE Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös L. University, 1117– Budapest

GnRH-I agonists and antagonists are widely used in the therapy of cancers, endometriosis, as well as reproductive medicine. The antiproliferative effect of GnRH-II analogues have been studied a few years ago. In contrast to GnRH-I analoges, GnRH-II agonists and antagonists induce apoptosis in many different cell types. The different GnRH agonists and antagonists induced effects, as well as antiproliferative effect, are mediated via GnRH-I receptor. In contrast to GnRH-I the main advantageof GnRH-II antagonists is the absence of endocrine side effects. [1-3]

Our aim was to synthesize daunomycin containing [D-Lys6]GnRH antagonist based conjugates and determine their in vitro cellular uptake rate, cytotoxic and apoptotic effects. Synthesis of [D-Lys6]GnRH antagonists was carried out on solid phase using Fmoc/tBu strategy. Daunomycin were attached directly to the GnRH antagonists via oxime bond. In vitro cytotoxic effects of GnRH conjugates were determined by MTT-assay using human colon carcinoma (HT-29) cell culture. Cellular uptake of the drug containing antagonists was studied on HT-29 cells using flow cytometry and fluorescence microscopy. The apoptotic effect was evaluated by flow cytometry applying APC-Annexin-V and PI. Coupling of drug molecule to the GnRH antagonists could increase the in vitro cytotoxic activity of the parent hormone analogs (IC50: 30-50 M). However GnRH antagonist can be taken up more effectively, than the agonist ones, they have less in vitro cytotoxic activity (IC50: 5-15 M), which correlates with their moderate in vitro apoptotic effect (15-35%). According to the cellular uptake profile, GnRH antagonists could be potential candidate to target different intracellular apoptotic pathway elements.

References1. K.Y. Kim, P.C. Leung J. Clin. Endocrinol. Metab. (2004), 89, 3020.2. I.S. Hong, P.C. Leung J. Clin. Endocrinol. Metab. (2008), 93, 3179.3. N. Eicke, C. Gründker Int. J. Oncol. (2006), 29, 1223.


62

O21

Modulation of the c-Maf transcription factor: a new perspective for multiple myeloma

S, Pellegrino1, L. Ronda2, G. Paredi3, R. Piano4, A. Contini1, S. Bettati2,3, A. Mozzarelli3,4 and M.L. Gelmi1

1 DISFARM-Sez. “A.Marchesini”, Università degli Studi di Milano, via Venezian 21, 20133, Milano, Italy 2 Department of Neuroscience, University of Parma, Via Volturno, 39, 43125, Parma, Italy3 SITEIA.PARMA Interdepartmental Center, University of Parma, Parco Area delle Scienze, 181/A, 43124, Parma, Italy4 Department of Pharmacy, University of Parma, Parco Area delle Scienze, 23/A, 43124, Parma, Italy

Multiple myeloma (MM) is a malignant hematological disorder characterized by clonal proliferation of plasma cells in the bone marrow, recently proposed as a model system for the comprehension of tumor biology[1]. Over-expression of Maf basic leucine zipper (LZ) transcription factors, either by specific chromosomal translocations or by other mechanisms, occurs in 50% of MM cases[2]. c-Maf signature, in particular, is associated with a poor prognosis, high proliferation index and low median survival.The deregulation of c-Maf plays an important pathogenetic role, leading to an abnormal expression of a wide set of genes, including CCND2, ITGB7 and ARK5 and others, whose products regulate processes such as cell proliferation, adhesion, invasion and cell-cell communication[3].

In this work, Maf basic LZ has been produced by solid phase peptide synthesis (SPPS)[4] and characterized in terms of secondary structure and dimerization properties. Peptidic c-Maf dimerization inhibitors have been rationally designed and their ability to interact with synthetic LZ was studied using circular dichroism spectroscopy and MALDI TOF techniques. The putative inhibitors interacted selectively with c-Maf LZ domain, affecting the degree of structural organization and destabilizing homodimers. These molecules were able to drive the folding of c-Maf, an intrinsically disordered protein, suggesting an alternative way to interfere with transcription factors, based on a “folding distractor” rather than a “folding disruptor”. Finally, we demonstrated that the combination between protein domains SPPS and secondary and quaternary structure analysis allow to build up a simple experimental platform to test LZ dimerization inhibitors [5].

References1. W. Matsui Nature (2011), 480, S58.2. R. Fonseca et al Leukemia (2009), 23, 2210.3. A. Eychene et al. Nature reviews (2008), 8, 6834. S. Pellegrino et al Amino Acids (2012), 43, 19955. S. Pellegrino, L. Ronda et al BBA: molecular basis of disease submitted

Dimeric Maf in complex with DNA


63

O22

Targeting Nm23-H1/h-Prune interaction impairs cell growth, survival and migration in Prostate cancer

Marianeve Carotenuto1,2, Maria Cristina Chiarolla1,2, Carmela Attanasio1,2, Nadia Aiese1,2, Valentina Damiani1,2, Benedetta Accordi3, Giuseppe Basso3, Ciro Imbimbo4 and Massimo Zollo1,2,.

1 Centro di Ingegneria Genetica e Biotecnologie Avanzate (CEINGE), Naples, Italy2 Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università ‘Federico II’ di Naples, Italy3 Haemato-Oncology Laboratory, Department of Paediatrics, University of Padova, Padua, Italy4 Dipartimento di Neuroscienze e Scienze riproduttive ed odontostomatologiche, Università ‘Federico II’ di Naples, Italy

Nm23-H1 gene is a metastasis suppressor gene active in various cancers, where its overexpression was found associated with reduced cell motility. However, high level of Nm23-H1 expression has been associated with reduction in survival for hematological malignances, and additionally, in neuroblastoma and osteosarcoma. We think this dichotomy of function of Nm23-H1 may be regulated by the acquired function of interacting proteins, creating new protein complexes. We have already demonstrated that h-Prune through its C-terminal region, upon Nm23-H1 phosphorylation on Ser120, Ser122 by Casein kinase I, binds Nm23-H1. Our hypothesis implies that this protein complex in turns regulates cancer progression and its specific impairment could result in new therapeutic strategies in oncology. Here we present the proof of concept using xenografted studies by the prostate cancer human cell lines (PC3).A competitive permeable peptide (CPP) was developed and delivered by AdenoVirus (AdV-CPP) in tumorigenic cell thus showing that is able to impair cell proliferation and motility in several tumorigenic cells representing breast, colorectal, and prostate carcinoma and thus including tumorigenic cell of CNS and PNS origin (medulloblastoma and neuroblastoma, respectively). We found that CPP leads to inhibition of AKT/mTOR and NF-kB signalling pathways thus resulting on enhancing Caspase 3 activation. In a heterotopic delivery of xenografted prostate cancer cell line, generated by injection of PC3-Luc cells pre-infected by AdV-CPP, we saw regression of established tumors once treated by an intra-tumor adenovirus injection. In addition, we demonstrated that AdV-CPP also impairs tumorigenic initiation and metastases formations in vivo by a metastatic lung assay. Altogether these findings prompt us to consider the use of CPP as a new potential clinical application, being a mimetic nm23-H1 peptide, able to impair h-Prune/Nm23-H1 complex formation, and its application in topic treatment of prostate cancer.


64

O23

Developing Bioavailable Melanotropin PeptidesM. Cai1, D. Craik 2,, H. Kessler 3 and V. J. Hruby1

1 Department of Chemistry & Biochemistry, The University of Arizona ,Tucson AZ, 85721, USA2 Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia 3 Center for Integrated Protein Science and Institute for Advanced Study at the Technische Universität München,

Lichtenbergstr. 4, 85747 Garching, Germany

There has been a resurgence of interest in peptide pharmaceuticals recently as they have an advantage of potency, selectivity and less toxicity compared with small-molecule therapeutics. In addition, the diverse applications of peptide are due to their distinctive properties, including ease of synthesis and characterization, introduction of chemical diversity by novel amino acid substitution, and modulation of 3D structure by chemical modification. The application of peptides as drugs stems from their key roles in many target recognition and signal transduction pathways, which makes them an attractive avenue to target diseases. However, the major drawback of many peptides is lack of stability in biological media. In this context, cyclization and N-methylation of peptides have become useful approaches for improving in vivo stability of peptides.[1-3] These two strategies have rendered, in some cases, oral bioavailability, cell permeability, improved potency at the target receptor, selectivity against receptor subtypes and improved stability to enzymes. Several new modalities in constraining peptides also have been developed over recent years and this work highlights some of these developments. Further understanding the rules governing cell permeability, oral absorption and stability for enzymatic degradation of peptides can help peptides to enter the clinical phases for many unmet medical needs.

References1. E. Biron, J. Chatterjee, O, Ovadia, D. Langenegger, J. Brueggen, D. Hoyer, H. Schmid, R. Jelinek, C. Gilon, A.

Hoffman, H. Kessler Angew.Chem Int. Ed. (2008), 47, 25952. V. J. Hruby Nature Reviews Drug Discovery (2002), 1, 8473. K. J. Rosengren, N.L. Daly, M.R. Plan, C. Waine, D. J. Craik J. Biol. Chem. (2003) 278, 8606-16.


65

O24

Unravelling the penetration and subcellular distribution of cell penetrating peptides

J.-M. Swiecicki1, F. Thiebaut1, J. Tailhades1, C. Mansuy1, F. Burlina2, S. Chwetzoff3, G. Trugnan3, G. Chassaing1 and S. Lavielle1

1 Laboratoire des BioMolécules, UMR 7203, UPMC-CNRS-ENS, 24 rue Lhomond, 75005 – Paris2 Laboratoire des BioMolécules, UMR 7203, UPMC-CNRS, 4 place Jussieu, 75005 – Paris3 Laboratoire des BioMolécules, UMR 7203, UPMC-INSERM, 27 rue de Chaligny, 75012 – Paris

Cell penetrating peptides (CPPs) are short cationic peptides able to translocate through membranes. As they can shuttle bioactive cargoes into eukaryotic cells, they have opened new avenues in intracellular drug delivery. Nevertheless their therapeutic use is currently limited because their exact internalization mechanisms and final intracellular localizations remain to be elucidated.[1]

To investigate the mechanism of cellular uptake and the intracellular distribution of CPPs, these peptides are commonly labelled using various fluorophores. Nevertheless, conclusions based on a quantitative mass spectrometry assay and those based on confocal microscopy seem to be contradictory.[2,3,4] This discrepancy might originate from fluorescence self-quenching. We developed a “dilution” protocol in which fluorescent CPPs are incubated with living cells in presence of an excess of their unlabelled analogues. By varying the ratio of labelled over unlabelled CPPs, we ranked subcellular regions depending on their CPP content. These observations helped us to understand the subcellular distribution of CPPs and to elucidate their penetration mechanism. More generally, this study proposes a broadly applicable protocol to study the subcellular distribution of fluorescently labelled small biomolecules.

Figure 1. MA-104 cells incubated 10 min with the CPP nonaarginine conjugated or not to fluorescein. (a) Incubation with 20 µM nonaarginine conjugated to fluorescein. A diffuse intracellular fluorescence is observed. (b) Incubation with 600 nM nonaarginine

conjugated fluorescein and 19.4 µM of free nonaarginine. The fluorescence is localized into endosomes. (Scale bar: 20 µm)

References1. R. Brock Bioconj. Chem. (2014) DOI: 10.1021/bc500017t.2. F. Burlina, S. Sagan, G. Bolbach, G. Chassaing Angew. Chem. Int. Ed. (2005), 44, 4244. 3. C.-Y. Jiao, D. Delaroche, F. Burlina, I. D. Alves, G. Chassaing, S. Sagan J. Biol. Chem. (2009), 284, 33957.4. F. Duchardt, M. Fotin-Mleczek, H. Schwarz, R. Fischer, R. Brock Traffic (2007), 8, 848.

(a) (b)


66

O25

How do a proapoptotic and a cell penetrating peptide work together to kill cancer cells?

Isabel D. Alves1, Manon Carré2, Diane Braguer2, Solange Lavielle3

1 Chimie et Biologie des Membranes et Nanoobjets, CBMN CNRS UMR 5248, Université Bordeaux 1, Allée Geoffroy de Saint Hilaire, 33600 Pessac, France

2 INSERM UMR911 Centre de Recherche en Oncologie biologique et Oncopharmacologie Faculté de Pharmacie, Aix-Marseille Université, 27 Bd Jean Moulin - 13385 Marseille Cedex 05

3 UPMC Univ Paris 6, Laboratoire des BioMolecules, 4 place Jussieu, F-75005 Paris, CNRS UMR 7203, LBM, ENS, LBM, 24 rue Lhomond, F-75005

The peptide KLA (acetyl-(KLAKLAK)2-NH2), which is rather non toxic for eukaryotic cell lines, becomes active when coupled to the cell penetrating peptide, penetratin (Pen, RQIKIWFQNRRMKWKK), by a disulfide bridge. Once inside cells, the disulfide bridge should be reduced and KLA peptide released. Remarkably, we have determined that the conjugate KLA-Pen is cytotoxic, at low micromolar concentrations, against a panel of seven human tumor cell lines of various tissue origins, including cells resistant to conventional chemotherapy agents but not to normal human cell lines. Live microscopy on cells possessing fluorescent labeled mitochondria shows that in tumor cells, KLA-Pen had a strong impact on mitochondria tubular organization instantly resulting in their aggregation, while the unconjugated KLA and Pen peptides had no effect. But, mitochondria in various normal cells were not affected by KLA-Pen.[1]

To understand the mode of action of KLA-Pen in mitochondria and its selectivity towards cancer cells, its interaction with membrane models were studied using DLS, calorimetry, plasmon resonance, CD and ATR-FTIR. Lipid model systems composed of zwitterionic lipids were used as mimics of normal cell membranes and anionic lipids as mimics of tumor cell and mitochondria membrane. A very distinct mode of interaction with the two model systems was observed. Indeed, much stronger interactions and lipid perturbations were observed in contact with anionic lipids. The studies indicated that asides from electrostatic interactions, lipid interactions of KLA and KLA-Pen are highly influenced by membrane fluidity. Moreover, such parameters (electrostatics, membrane fluidity) influence the oligomerization state of the peptide strongly affecting its action mode. KLA-Pen may exert its deleterious action by the formation of pores with an oblique orientation in the membrane and establishment of important hydrophobic interactions. These results suggest that KLA-Pen could be a lead compound for the design of cancer therapeutics.

References1. I.D. Alves, M. Carré, S. Castano, S. Lecomte, R. Marquant, P. Lecorche, F. Burlina, C. Schatz, S. Sagan, G.

Chassaing, D. Braguer, S. Lavielle BBA Biomembranes (2014) in press.


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O26

Stapled Peptides for Cullin3-BTB interface targetingI. de Paola1, L. Pirone2, M. Palmieri3, D. Mazzà4, S. Di Gaetano1, L. Vitagliano1, G. Malgieri3,

L. Di Marcotullio4, E. Pedone1, L. Zaccaro1

1. Institute of Biostructures and Bioimaging - CNR, Naples, Italy2. Institute of Cristallography - CNR, Bari, Italy3. Department of Environmental, Biological and Pharmaceutical Science and Technology, Second University of Naples, Caserta, Italy4. Department of Molecular Medicine, University of Rome La Sapienza, Rome, Italy

Peptides mimicking interacting helices represent a valuable tool for modulating protein-protein interactions. Unfortunately, α-helical regions eradicated from their protein context tend to lose their structure, thus limiting their efficacy. Recently an intriguing approach to stabilize a-helical conformation is achieved through the “stapling” strategy using a hydrocarbon cross-linker (staple)[1]. This approach has been shown improving the performance of peptides thereby increasing their potential as therapeutic agents.In this study the stapling approach has been extended to the interaction of Cullin3 (Cul3) with a family of proteins containing BTB domains. Cul3 is an important component of Cullin3-Ring ubiquitin Ligases which facilitates the transfer of ubiquitin to protein substrates[2]. Cul3 is able to interact with different BTB-containing proteins which potentially bind a variety of different substrate to be ubiquitinated. In this scenario, the development of molecules able to interfere with Cul3/BTB interaction could represent an important step for regulating a variety of different processes. We here targeted the interaction of Cul3 with the BTB containing protein KCTD11, one of the best characterized members of the emerging class of multidomain proteins denoted as KCTD (Potassium Channel Tetramerization Domain containing proteins)[3,4]. The stapling strategy was adopted with the aim to develop a probe useful to explore the important interaction between Cul3 and KCTD family members and to deepen the pathway in which such interaction is involved.

References1. Schafmeister, C. E.; Po, J.; Verdine, G. L. J. Am. Chem. Soc. 2000, 122, 5891–22. Pintard, L.; Willems, A.; Peter, M. EMBO J. 2004, 2, 31681-73. Pirone, L.; Correale, S.; de Paola, I.; Zaccaro L.; De Simone, G.; Vitagliano, L.; Pedone, E.; Di Gaetano S. J Pept

Sci. 2011, 17, 373-376.4. Canettieri, G.; Di Marcotullio, L.; Greco, A.; Coni, S.; Antonucci, L.; Infante, P.; Pietrosanti, L.; De Smaele,

E.; Ferretti, E.; Miele, E.; Pelloni, M.; De Simone, G.; Pedone, EM.; Gallinari, P.; Giorgi, A.; Steinkühler, C.; Vitagliano, L.; Pedone, C.; Schinin, ME.; Screpanti, I.; Gulino, A. Nat Cell Biol. 2010, 12, 132-42


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O27

Inhibition of pathological angiogenesis antagonizing VEGF Receptor 1

Valeria Cicatiello1, Ivana Apicella1, Valeria Tarallo1, Laura Tudisco1, Annamaria Sandomenico2, Roberto Bianco3, Jayakrishna Ambati4, Menotti Ruvo2 and Sandro De Falco1

1 Angiogenesis Lab, Institute of Genetics and Biophysics - CNR, 80131 Naples2 Institute of Biostructures and Bioimaging, - CNR, 80134 Naples3 Dept of Endocrinology and Molecular Oncology, University of Naples “Federico II”, 80131Naples.4 Dept. of Ophthalmology & Visual Sciences, University of Kentucky, 40506 Lexington, KY, USA

The pro-angiogenic members of vascular endothelial growth factor (VEGF) family, VEGF-A, VEGF-B and placental growth factor (PlGF), which accomplish their action interacting with the two receptors VEGFR-1 and VEGFR-2, play a central role in the modulation of pathological angiogenesis. These molecular players are still the preferential targets for anti-angiogenic therapy.Recently, a great attention has been devoted on PlGF/VEGFR-1 axis because it is important not only for the stimulation of endothelial cells but mainly for the modulation of inflammatory response associated to the pathological angiogenesis as well as for vessel stabilization. Indeed, it has been reported that overexpression of PlGF in tumor cells induced an impressive recruitment of inflammatory cells (F4/80 positive cells) and increased significantly the number of vessels surrounded by smooth muscle cells. It is important also consider that due to the strict biochemical and functional relationship between VEGFs and related receptors it appear evident how the ability to interfere with more than one of these factors may represent an advantage in term of therapeutic outcome.We have reported the identification of a new synthetic tetrameric tripeptide[1], named inhibitor of VEGFR-1 (iVR1), which specifically binds VEGFR-1 preventing its activation by VEGF-A, VEGF-B and PlGF, at low micromolar range. This peptide inhibits tumor growth and associated angiogenesis in syngenic and xenograft colon cancer model, with an extent comparable to that of Avastin and higher compared to that of 16D3 anti-PlGF antibody. The iVR1 showed synergic activity with the chemotherapy agent irinotecan. The main effect of the peptide was a potent inhibition of F4/80 positive cells recruitment confirmed by in vitro inhibition of RAW 246.7 macrophages cell line and ex-vivo isolated murine peritoneal macrophage migration induced by VEGF or PlGF. In addition, iVR1 potently prevent lung colonization by tumor cells injected into caudal vein, resulting more effective than Avastin. Finally, it was able to inhibit neoangiogenesis also in the model of laser-induced choroid neovascularization.Our results confirm how the specific block of VEGFR-1 represents a successful strategy to inhibit pathological angiogenesis in pre-clinical models. Further preclinical and combination therapy data are needed to propose further development of iVR1 as a new tool for anti-angiogenic therapy.

References1. Ponticelli S. et al, Journal of Biological Chemistry (2008), 283: 34250-9.


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O28

Cell-penetrating conjugates of calpain inhibitorsZ. Bánóczi1, L. E. Dókus1, Á. Tantos2 and F. Hudecz1,3

1 MTA-ELTE Research Group of Peptide Chemistry, Eötvös L. University, Pázmány P. S. 1/A, 1117 Budapest, Hungary

2 Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academic of Sciences, Magyar tudósok körútja 2, 1117 Budapest, Hungary

3 Department of Organic Chemistry, Eötvös L. University, POB 32, 1518 Budapest 112, Hungary

Calpains are intracellular cysteine proteases and are of considerable interest due to their implication in numerous physiological events. Besides these functions, they could play a key role in some well-studied pathological processes. The overactivation of calpains, which is resulted in by the disorder in Ca2+ homeostasis, increases the degradation of the enzyme substrates and could contribute to the development of the Alzheimer and/or Huntington diseases and also to death of nervous cells caused by traumatic brain injury, spinal cord injury.[1] Thus the inhibition of calpain may be important in blocking of calpain over-activation. This claim requires more selective inhibitors, with proper cell-membrane permeability.Our aim is to develop peptide based calpain inhibitors, using the sequence of substrate (TPLKSPPPSPR) which was identified by us. [2] In the new inhibitor family, the Lys residue after which the calpain cleaves the substrate was replaced with azaglycine [3] or epoxysuccinyl moiety. Different number of amino acids, derived from the reference matrix, [2] at N- and C-terminal was also incorporated. The inhibitory activity of these peptide derivatives was studied on isolated m-calpain and other cysteine proteinases. Derivatives with enzyme inhibitory activity were attached to octaarginine as cell-penetrating peptide. The internalisation and enzyme inhibitory ability of free and conjugated compounds were compared. These cell-penetrating inhibitors may be useful in calpain function studies to refine our knowledge of their role in different cellular processes.

AcknowledgementsThis study was supported by grants: OTKA K-68285, OTKA PD-83923 and GVOP-3.2.1-2004-04-0352/3.0. Bánóczi, Z. acknowledges the support of the MTA Bolyai János Scholarship.

References1. M.E. Saez, et al. Drug Discov. Today. (2006), 11, 917.2. P. Tompa, et al. J. Biol. Chem. (2004), 279, 20775.3. Z. Bánóczi et al. J. Pept. Sci. (2013), 19, 370.


70

O29

NMR interaction studies of RGDechi-hCit peptide with integrins embedded into cell membranes

B. Farina1,2, I. de Paola2, A. Liquori2, A. Del Gatto2, D. Capasso2, S. Di Gaetano2, D. Comegna2, M. Saviano3, L. Zaccaro2 and R. Fattorusso4

1 Interuniversity Centre for Research on Bioactive Peptides (CIRPeB), University of Naples “Federico II”, 80134 – Naples, Italy

2 Institute of Biostructures and Bioimaging, CNR, 80134 – Naples, Italy3 Institute of Crystallography, CNR, 70126 – Bari, Italy4 Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of

Naples, 81100 – Caserta, Italy

Integrins are heterodimeric transmembrane receptors that mediate cell-cell and cell-extracellular matrix adhesion processes and provide the traction for cell mobility and invasion.[1] They are involved in tumour cell proliferation, migration and survival. Among these, αvß3 and αvß5 integrins play a prominent role in progression of various tumour types, such as melanoma[2] They bind as primary recognition sequence the Arg-Gly-Asp (RGD) triad found in many extracellular matrix proteins (i.e., vitronectin) and disintegrins. Residues flanking the RGD sequence of high-affinity ligands modulate the specificity of interaction with integrins. In recent years, both preclinical and clinical studies have demonstrated the effectiveness of various αvß3 and αvß5 integrin antagonists in blocking tumour progression. Moreover, while αvß5 is widely expressed by many malignant tumour cells, αvß3 has a relatively limited cellular distribution compared with that of αvß5.

[3] Recently, we have designed and biologically characterized a novel and selective ligand for αvß3 integrin, named RGDechi-hCit, containing a cyclic RGD motif covalently linked to two echistatin C-terminal moieties.[4-6] In this study, we report NMR Saturation Transfer Difference and transferred NOESY experiments aimed at the direct observation of interactions between RGDechi-hCit and integrins in both intact cells and isolated cell membranes.

References1. R. O. Hynes Cell (1992), 69, 11.2. S. M. Albelda, S. A. Mette, D. E. Elder, R. Stewart, L. Damjanovich, M. Herlyn, C. A. Buck Cancer Res. (1999), 59,

6757.3. R. Pasqualini, J. Bodorova, M. E. Hemler J. Cell Sci. (1993), 105, 101.4. A. Del Gatto, L. Zaccaro, P.Grieco, E. Novellino,A. Zannetti,S. Del Vecchio, F. Iommelli, M. Salvatore, C. Pedone C,

M. Saviano J Med Chem (2006), 49, 3416.5. A. Zannetti, S. Del Vecchio, F. Iommelli, A. Del Gatto, S. De Luca, L. Zaccaro, A. Papaccioli, J. Sommella, M.

Panico, A. Speranza, P. Grieco, E. Novellino, M. Saviano, C. Pedone, M. Salvatore. Clin Cancer Res. (2009), 15, 5224.

6. M. Pisano, I. de Paola, V. Nieddu, I. Sassu, S. Cossu, G. Galleri, A. Del Gatto, M. Budroni, A. Cossu, M. Saviano, G. Palmieri, L. Zaccaro, C. Rozzo Anticancer Res. 2013, 33, 871.


71

O30

Targeting sulfated glycans in cancer cells and tissues by branched peptides

A.Pini, J. Brunetti, C. Falciani, L. Depau, G. Roscia, M. Mazzacuva, L. Lozzi, S. Scali and L Bracci

Department of Medical Biotechnologies, University of Siena, Via Aldo Moro, 53100 Siena

We have shown in previous works that a tetra-branched form of neurotensin, which we have synthe-sized and called NT4, has an extraordinary selectivity, towards different human cancer (i.e. colon, pan-creas and urinary bladder cancer), which is not shared by native neurotensin. We have recently found that the much higher binding of NT4 peptides, in respect to native neurotensin, to cancer cells and human cancer surgical samples is generated by a switch in selectivity towards additional membrane receptors, which are specifically expressed by different human cancers. We demonstrated that the branched structure provides NT4 with ability to bind membrane sulfated glycosaminoglycans (HSPG) as well as different membrane endocytic receptors belonging to the low density lipoprotein receptor (LDLR) family, including LRP1 and LRP6, all of them already known to be potential druggable tumor markers involved in cancer biology. Systematic modification of neurotensin sequence in NT4 peptides, led to identification of a multimeric positively charged motif, which mediates interaction with both HSPG s and endocytic receptors [1].HSPG modulate cell–cell and cell–ECM interactions by interacting with several bioactive molecules including chemokines, cytokines, growth factors, morphogens, adhesion molecules and matrix com-ponents, like collagen, fibronectin, laminin and vitronectin. HSPG are thus essential mediators of cancer progression by modulating epithelial mesenchimal transition (EMT), invasion and metastasis. Since direct binding of HSPG to adhesion molecules, integrins and matrix component, can directly influence cancer cell adhesion and migration, we have analyzed the effect of NT4 branched peptides on adhesion and motility of cancer cells on different supports. We have found that NT4 can inhibit both binding and migration of cancer cells on ECM protein supports. Our results confirm that NT4 peptides are very promising tumor ligands which might ensure a high and broad cancer selectivity. Moreover, by selectively binding sulphated glycans, NT4 peptides can be use-ful tools for uravelling the still unclear role of HSPG in many aspects of cancer progression.

References1. Falciani C, Brunetti J, Lelli B, Ravenni N, Lozzi L, Depau L, Scali S, Bernini A, Pini A, Bracci L. J Med Chem.

2013;56:5009-18.


72

O31

Crosstalk between mammalian cells and the microbiome through quorum sensing peptides, influencing cancer metastasis

E. Wynendaele1, F. Verbeke1, M. D’Hondt1, A. Hendrix2, C. Van De Wiele3, C. Burvenich4, K. Peremans4, O. De Wever2, M. Bracke2 and B. De Spiegeleer1

1 Drug Quality and Registration (DruQuaR) group, Faculty of Pharmaceutical Sciences, Ghent University, 9000 – Ghent

2 Department of Radiation Oncology and Experimental Cancer Research, Faculty of Medicine and Health Sciences, Ghent University Hospital, 9000 – Ghent

3 Department of Radiology and Nuclear Medicine, Faculty of Medicine and Health Sciences, Ghent University Hospital, 9000 – Ghent

4 Department of Medical Imaging, Medicine and Clinical Biology of Small Animals and Comparative Physiology and Biometrics, Faculty of Veterinary Medicine, Ghent University, 9820 – Merelbeke

To date, the precise role of the human microbiome in health and disease remains largely unknown. Research mainly focused on the effect of toxins or DNA-damaging reactive oxygen species (ROS), produced by commensal or pathogenic bacteria, on inflammation or carcinogenesis.[1,2] In this study however, we demonstrate that quorum sensing peptides, secreted by intestinal bacteria, can also influence cancer cell behaviour: Phr0662 (Bacillus sp., ERNNT), EntF-metabolite (Enterococcus faecium, SNLVECVFSLFKKCN) and EDF-derived (Escherichia coli, NWN) peptides initiate tumour cell invasion and migration through epithelial to mesenchymal (EMT)-like transition as well as promote angiogenesis. Transcriptome profiling after peptide treatment of the HCT-8/E11 cells confirmed their tumour-promoting properties by up- or downregulation of different microRNAs (e.g. miR-664a and miR-222) and other genes (e.g. CXorf61 and Histone cluster 1 H4).[3] Our results indicate that the human microbiome, through their quorum sensing peptides, is one of the factors responsible for cancer metastasis.

Figure 1: EMT-like behaviour of HCT-8/E11 cells (left) and angiogenesis (right) after Phr0662 addition, compared to placebo sample.

References1. C.S. Plottel et al. Cell Host Microbe (2011), 10, 324-335.2. R.F. Schwabe et al. Science (2012), 338, 52-53.3. E. Wynendaele et al. (submitted, 2014).


73

POSTER PRESENTATIONS


74

P1

Conformational modifications and antiviral activity of gB fromHerpes simplex virus type 1 analyzed by synthetic peptides

Novella Incoronato 1, Marco Cantisani 2,3, Annarita Falanga 2, Francesca Martora 1, Alfonso De Simone 4, Rita Berisio 5, Maria Teresa Vitiello 1,6, Stefania Galdiero 2,3,5, Massimiliano Galdiero 1,3

1. Department of Experimental Medicine II University of Naples, Napoli, Italy; 2. Department of Pharmacy University of Naples Federico II, Napoli, Italy; 3. Centro Interuniversitario di Ricerca sui Peptidi Bioattivi, University of Naples Federico II, Napoli, Italy; 4. Division of Molecular Biosciences, Imperial College, London, United Kingdom; 5. Istituto di Biostrutture e Bioimmagini, Cnr, Napoli, Italy6. Department of Clinical Pathology and Transfusion Medicine, University Hospital “Ruggi d’Aragona”, Salerno, Italy

Entry of enveloped viruses requires fusion of viral and cellular membranes, driven by conformational changes of viral glycoproteins. Glycoprotein B of Herpes simplex virus belongs to the newly formed Class III of viral fusion proteins. Several studies prove that as other class III fusion proteins gB under-goes a pH dependent switch between the pre and post fusion conformation. The gB coil arm complex consists of residues 500 to 544 in the coil and 670 to 690 in the arm with an arrangement that brings the C-terminal TM region in proximity with the fusion loops. Inhibitors that prevent the fusion function of gB may be developed starting from peptide analogues able to interfere with the membrane fusion event, thus preventing conformational changes that occur as gB refolds to its post-fusion form by en-ergetically trapping gB in a prefusion state. We focused on the long helix spanning residues 500-544 of gB ectodomain and tested a set of peptides of different length derived from this sequence in order to determine the minimal sequence able to elicit inhibition of viral infectivity. Our data make gB peptide analogs attractive candidates for further drug development against HSV and may represent a prototype for other Class III viruses.


75

P2

Single residue contribution to self-aggregation of a fungicidal immunoglobulin-derived peptide

T. Ciociola1, T.A. Pertinhez2, L. Giovati1, M. Sperindè1, W. Magliani1, L. Polonelli1, A. Spisni3 and S. Conti1

1 Dept. Biomedical, Biotechnological and Translational Sciences (S.Bi.Bi.T.), Microbiology and Virology Unit, University of Parma,

2 C.I.M. Laboratory - Technopole Parma, University of Parma3 Dept. Surgical Sciences, University of Parma, Parma, Italy

Peptide fragments related to the complementarity determining regions (CDRs) of antibodies (Abs) may exert differential antimicrobial, antiviral, antitumor and/or immunomodulatory activity in vitro and in vivo, involving different mechanisms of action, regardless of the specificity of the parental Ab.[1, 2] Likewise CDR peptides, fragments of the constant region of Abs (Fc-Pepts) may also display fungicidal and/or immunomodulatory activity in vitro and/or in vivo, regardless of the isotype of the belonging Ab.[3, 4] Alanine substituted derivatives (asd) of Fc-Pepts showed unaltered, increased or decreased candidacidal activity in vitro.[3] As the IgG-derived Fc-Pept N10K (NQVSLTCLVK) showed the ability to spontaneously self-assemble, the aim of this study was to evaluate the critical role of each residue in this process by circular dichroism spectroscopy. Transmission and scanning electron microscopy studies were performed, while potential apoptotic effects against Candida albicans cells were evaluated by flow cytometry. Overall, our results indicate the critical role of some residues in self-aggregation process, other than in candidacidal activity in vitro.

References1. L. Polonelli, J. Ponton, N. Elguezabal, M.D. Moragues, C. Casoli, E. Pilotti, P. Ronzi, A.S. Dobroff, E.G.

Rodrigues, M.A. Juliano, D.L. Maffei, W. Magliani, S. Conti, L.R. Travassos PLoS ONE (2008), 3, e2371.2. E. Gabrielli, E. Pericolini, E. Cenci, F. Ortelli, W. Magliani, T. Ciociola, F. Bistoni, S. Conti, A. Vecchiarelli, L.

Polonelli PLoS ONE (2009), 4, e8187.3. L. Polonelli, T. Ciociola, W. Magliani, P.P. Zanello, T. D’Adda, S. Galati, F. De Bernardis, S. Arancia, E. Gabrielli,

E. Pericolini, A. Vecchiarelli, D.C. Arruda, M.R. Pinto, L.R. Travassos, T.A. Pertinhez, A. Spisni, S. Conti PLoS ONE (2012), 7, e34105.



76

P3

Cotton fibers functionalized with peptaibiotics

A. Orlandin1, G. Hilma2,3, D. Coman3, S. Oancea2, F. Formaggio1 and C. Peggion1

1 ICB, CNR, Padova Unit, Department of Chemistry, University of Padova, 35131 Padova (Italy)2 Publich Health Directorate of Sibiu, 551130 Sibiu (Romania)3 University “Lucian Blaga” of Sibiu, 550012 Sibiu (Romania)

The resistance to commonly used antibiotics by some bacterial strains has increased the risk of contracting infections and calls for materials that prevent proliferation, mutation and transmission of harmful bacteria. We recently started the preparation of fabrics containing covalently-bound, antibacterial compounds. Our aim is to produce safety clothing for health care workers and for immuno-compromised or debilitated people who need to be protected from infections.[1] Garments functionalized with antimicrobial agents on the market today contain salts or silver nanoparticles. However, there is no serious investigation on the possible harmful effects of silver-based products.[2] In addition, the antimicrobial protection is limited in time due to the gradual release of the active ingredient. Therefore, we decided to prepare cotton garments functionalized with peptaibiotics, a class of antibiotic peptides of natural origin extensively investigated by our group.[3,4] In particular, we linked to cotton samples the following peptides:

FA-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-OHFA-Aib-Hyp-Leu-Val-Gln-Leu-OH

FA-Leu-Aib-Leu-Aib-Phe-OH

where FA is a fatty acid chain, Aib is α-aminoisobutyric acid and Hyp is trans-4-hydroxy-L-proline. The three sequences were modeled after the peptaibiotics trichogin GA IV,[4] halovir[5] and peptaibolin,[6] respectively. We exploited a recently proposed method to functionalize with an amine the free hydroxyl groups of cellulose.[7] The peptide was then coupled directly to this amine or grown step-by-step (SPPS-like) by introducing one residue at a time. An appropriate experimental set-up was devised to test the activity of our cotton fibres against Gram-positive and Gram-negative bacterial strains. A moderate protection against Staphylococcus aureus was detected in few samples.

References1. D. Coman, N. Vrînceanu, S. Oancea, D. Vlad. In: Proceedings of the Fourth International Proficiency Testing

Conference, Brasov, Romania, 2013, pp. 263-274.2. X. Chen, H. J. Schluesener, Toxicol. Lett. (2008), 176, 1. 3. C. Peggion, B. Biondi, M. De Zotti, S. Oancea, F. Formaggio, C. Toniolo, J. Pept. Sci. (2013), 19, 246.4. C.Toniolo, M. Crisma, F. Formaggio, C. Peggion, R. F. Epand, R. M. Epand, Cell. Mol. Life Sci. (2001), 58, 1179. 5. D. C. Rowley, S. Kelly, C. A. Kauffman, P. R. Jensen, W. Fenical, Bioorg. Med. Chem. (2003), 11, 4263.6. H. Hulsmann, S. Heinze, M. Ritzau, B. Schlegel, U. Grafe, J. Antibiot. (1998) 11, 1055.7. M. Nakamura, T. Iwasaki, S. Tokino, A. Asaoka, M. Yamakawa, J. Ishibashi. Biomacromolecules (2011), 12, 1540.


77

P4

Candidacidal properties of peptides encoded by immunoglobulin genes

M. Sperindè1, T. Ciociola1, T.A. Pertinhez2, L. Giovati1, C. Santinoli1, S. Conti1, L. Polonelli1, A. Spisni3 and W. Magliani1

1 Dept. Biomedical, Biotechnological and Translational Sciences (S.Bi.Bi.T.), Microbiology and Virology Unit,University of Parma, Parma, Italy2 C.I.M. Laboratory -Technopole Parma, University of Parma, Parma, Italy3 Dept. Surgical Sciences, University of Parma, Parma, Italy

Peptides representing complementarity determining regions (CDRs) or constant region (Fc) fragments of different antibodies (Abs) were previously shown to exert differential antimicrobial, antiviral, an-titumor and/or immunomodulatory activities in vitro, ex vivo and in vivo, regardless of specificity and isotype of the originating Ab.[1-6] Based on these observations, in this work we report that some pep-tides encoded by immunoglobulin (Ig) genes show significant candidacidal properties. Peptides were selected through the analysis of protein sequences encoded by the light (L) chain genes (V, J and C) and the heavy (H) chain genes (V, D, J and C) of Abs on the basis of some physical and chemical charac-teristics, such as length and molecular weight, positive and net charges, isoelectric point, hydrophobic/hydrophilic pattern, presence of cysteines, and the occurrence of interspecies conserved amino acids. In particular, two of the selected peptides (L12P, LGVRRRDQADRP, and L18R, LLVLRSLGPWHP-GHCLLR) showed to exert their fungicidal activity against several Candida strains, even characterized by resistance to conventional antifungal drugs. The chemical-physical characteristics and potential mechanism of action of L12P and L18R on C. albicans cells were investigated by circular dichroism spectroscopy, flow cytometry, confocal microscopy, transmission and scanning electron microscopy. The main purpose of this work is to claim the evolutionary hypothesis that Igs are the result of the as-sociation of genes coding for proteins or peptides characterized by ancestral biological functions of the innate immunity. This study, moreover, allowed the selection of peptides that could be exploited for the production of new antimicrobial drugs.

References

1. L. Polonelli, J. Ponton, N. Elguezabal, M.D. Moragues, C. Casoli, E. Pilotti, P. Ronzi, A.S. Dobroff, E.G. Rodrigues, M.A. Juliano, D.L. Maffei, W. Magliani, S. Conti, L.R. Travassos PLoS ONE (2008), 3, e2371.

2. E. Gabrielli, E. Pericolini, E. Cenci, F. Ortelli, W. Magliani, T. Ciociola, F. Bistoni, S. Conti, A. Vecchiarelli, L. Polonelli PLoS ONE (2009), 4, e8187.

3. A.S. Dobroff, E.G. Rodrigues, M.A. Juliano, D.M. Friaca, E.S. Nakayasu, I.C. Almeida, R.A. Mortara, J.F. Jacysyn, G.P. Amarante-Mendes, W. Magliani, S. Conti, L. Polonelli, L.R. Travassos Transl. Oncol. (2010), 3, 204-217.

4. D.C. Arruda, L.C. Santos, F.M. Melo, F.V. Pereira, C.R. Figueiredo, A.L. Matsuo, R.A. Mortara, M.A. Juliano, E.G. Rodrigues, A.S. Dobroff, L. Polonelli, L.R. Travassos J. Biol. Chem. (2012), 287, 14912-14922.

5. L. Polonelli, T. Ciociola, W. Magliani, P.P. Zanello, T. D’Adda, S. Galati, F. De Bernardis, S. Arancia, E. Gabrielli, E. Pericolini, A. Vecchiarelli, D.C. Arruda, M.R. Pinto, L.R. Travassos, T.A. Pertinhez, A. Spisni, S. Conti PLoS ONE (2012), 7, e34105.



78

P5

Trichogin GA IV forms ion channels of well-defined size in planar lipid membranes

S. Iftemi1, M. De Zotti2, F. Formaggio2, C. Toniolo2, L. Stella3, and T. Luchian1, 4

1 A.I. Cuza University, Department of Physics, 700506 - Iasi, Romania.2 CNR, ICB, Padova Unit, and University of Padova, Department of Chemistry, 35131 – Padova, Italy.3 University of Rome “Tor Vergata”, Department of Chemical Sciences and Technologies, 00133 – Rome, Italy.4 A.I. Cuza University, Department of Interdisciplinary Science, 700506 – Iasi, Romania.

Trichogin GA IV, an antimicrobial peptaibol, exerts its function by augmenting membrane permeability, but the molecular aspects of its pore-forming mechanism are still debated. Several lines of evidence indicate a “barrel-stave” channel structure, similar to that of alamethicin, but the length of a trichogin helix is too short to span a normal bilayer[1]. Herein, we present electrophysiology measurements in planar bilayers showing that trichogin does form channels of a well-defined size (R=4.2·109 W, corresponding at least to a trimeric aggregate) that span the membrane and allow ion diffusion. However, these channels do not exhibit voltage-dependent rectification, unlike those of alamethicin [2].

References1. S. Bobone, Y. Gerelli, M. De Zotti, G. Bocchinfuso, A. Farrotti, B. Orioni, F. Sebastiani, E. Latter, J. Penfold, R.

Senesi, F. Formaggio, A. Palleschi, C. Toniolo, G. Fragneto, L. Stella Biochim. Biophys. Acta (2013), 1828, 1013.2. S. Iftemi, M. De Zotti, F. Formaggio, C. Toniolo, L. Stella, T. Luchian, Chem. Biodivers., in press.


79

P6

How many AMP molecules kill a bacterium?Spectroscopic determination of PMAP-23 binding to E. coli

D.Roversi1, V. Luca2, S. Aureli1, Y. Park3, M. L. Mangoni2 and L.Stella1

1 University of Rome Tor Vergata, Department of Chemical Sciences and Technologies, 00133 - Rome, Italy 2 Sapienza Rome University, Department of Biochemical Sciences “A. Rossi Fanelli”, 00185 - Rome, Italy3 Chosun University, Department of Biotechnology, 501-759 - Gwangju, Korea

Antimicrobial peptides (AMPs) kill bacteria mainly through the permeabilization of the plasma mem-brane. Experiments on these molecules focus on their biophysical characterization in model mem-branes, or on their activity on bacterial cells, but studies demonstrating a correlation between biological activity and behaviour in liposomes are still lacking.One unanswered issue is the minimal amount of bound peptide that is necessary to kill a bacterium. Different attempts to assess this quantity [1,2] reached conclusions differing by almost a factor of 103.Trying to fill the hiatus between biological and biophysical studies, we determined by fluorescence measurements the affinity of a dansyl-labeled analogue of the PMAP-23 AMP [3] for both liposomes and E. coli cells by using the same experimental conditions of the bactericidal assay. Our results show that a high membrane coverage is necessary to induce bacterial death, as predicted by the carpet model hypothesized for PMAP-23.

Figure 1.Peptide fraction bound to bacteria.

References1. W. Wimley; ACS Chem. Biol. (2010), 5, 905.2. N.M. Melo, R.Ferre, M.A.R.B. Castanho; Nat. Rev. Microbiol. (2009), 7, 245.3. B.Orioni, G.Bocchinfuso, J.Y. Kim, A.Palleschi, G.Grande, S.Bobone, Y.Park, J.I. Kim, K.Hahm, L. Stella; Biochim.

Biophys. Acta (2009), 1788, 1523.


80

P7

Testing the “Sand in the Gearbox” model: Antimicrobial Peptide effects on membrane dynamics

Z. Vaezi1, D. Roversi1, D. Branca2, and L. Stella1

1 University of Rome Tor Vergata, Department of Chemical Sciences and Technologies, 00133 – Rome, Italy2 IRBM Science Park Spa, 00040 – Pomezia (RM), Italy.

Antimicrobial peptides (AMPs) are thought to kill bacteria by forming pores in their cell membranes. However, they might also interfere with vital cellular functions taking place in the membrane, by perturbing bilayer dynamics and structure (“sand in a gearbox” model) [1]. We previously observed that the cationic AMP PMAP-23 profoundly affects lipid motions in the membrane [2]. To further test the “sand in the gearbox” model, we extend this study to melittin and magainin, whose mechanism of pore formation is well characterized. Fluorescence anisotropy measurements on liposomes comprising probes localized at different depths in the bilayer, we observed that both peptides perturb membrane fluidity and order. Pyrene excimer-formation experiments showed a peptide-induced reduction in lipid lateral mobility. Finally, Laurdan fluorescence indicated reduced water penetration. All these effects were observed at higher peptide concentrations than that causing vesicle leakage, and are likely due to a tightening of the bilayer driven by the strong electrostatic peptide-lipid interactions.

Figure 1. Steady-state anisotropy of NBD-PE (1%) in PC/PG (2:1) liposomes, in the presence of increasing peptide concentrations.

References1. U. Pag, et al. J. Antimicrob. Chemother. (2008), 61, 341. 2. D. Roversi, et al. J. Pept. Sci. (2012), 18, S64.


81

P8

Circular Dichroism and NMR studies to elucidate the mechanism of action of antimicrobial peptides with bacterial cells

C. Avitabile1, M. Palmieri2, G. Malgieri2, C. Isernia2, R. Ummarino2, L.D. D’Andrea2, R. Fattorusso2 and A. Romanelli3

1 Diagnostica e Farmaceutica Molecolari Scarl, via Mezzocannone 16, Napoli, Italy2 Seconda Università di Napoli, Dipartimento di Scienze e Tecnologie Ambientali Biologiche e Farmaceutiche, via

Vivaldi 433 Istituto di Biostrutture e Bioimmagini, via Mezzocannone 16, Napoli, Italy4 Università di Napoli “Federico II”, Dipartimento di Farmacia, via Mezzocannone 16, Napoli, Italy

The therapeutic relevance of antimicrobial peptides (AMPs) led research efforts towards studies on their mechanism of action and in particular on their interactions with the bacterial membranes. To this aim different biophysical techniques (CD, NMR) and different model systems, such as lipid mixtures or LPS, able to mimic the outer leaflet of bacterial membranes, have been employed. Due to bacterial environment heterogeneity it is not yet clear how closely these results reproduce what really happens when AMPs interact with bacterial cells. Recent studies carried out by solid state NMR and time lapse fluorescence showed that the behavior of the peptides on the membranes depends on the composition of the membranes[1,2]. For these reasons, in order to investigate the mechanism by which antimicrobial peptides kill bacterial cells, we carried out studies of interaction of AMPs with the whole cells. We recently reported secondary structure studies of two antimicrobial peptides, magainin 2 and cecropin A, in the presence of E.coli cells by Circular Dichroism (CD)[3]. In this work we explored the interactions of peptides belonging to the temporin family (TB, TL and the analogue TB_KKG6A) with E.coli cells by CD. NMR studies in the presence of E.coli cells were carried out on the peptide TB_KKG6A. Upon interaction with E.coli cells the peptide adopts a helical conformation, slightly different from that obtained with LPS4, confirming how the structure and the orientation of antimicrobial peptides upon interaction with bacterial membranes is related to the composition of the latter.

References 1. K. J. Hallock, D. K. Lee, A. Ramamoorthy, Biophys J. (2003) 84, 3052 .2. M. L. Gee, M. Burton, A.Grevis-James, M.A. Hossain, S.McArthur, E.A. Palombo, J.D. Wade, A.H.A. Clayton Sci

Rep. (2013), 3, 1557.3. Avitabile, L.D. D’Andrea, A. Romanelli Sci. Rep, (2014) 4 doi:10.1038/srep04293.4. C. Avitabile, F. Netti, G. Orefice, M. Palmieri, N. Nocerino, G. Malgieri, L.D. D’Andrea, R. Capparelli, R.

Fattorusso, A. Romanelli BBA general Subjects (2013) 1813, 3767.


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Antiviral activity of a peptido-dendrimerLucia Lombardi1, Annarita Falanga2,3, Eleonora Mignogna1, Novella Incoronato1, Rossella Tarallo4,

Emiliana Perillo2, Marcus Weck4, Massimiliano Galdiero1 and Stefania Galdiero2,3

1 Division of Microbiology, Department of Experimental Medicine, II University of Naples, Via De Crecchio 7, 80138 Naples, Italy

2 Department of Pharmacy, University of Naples Federico II, Via Mezzocannone 16, 80134 Naples, Italy3 DFM Scarl, University of Naples Federico II, Via Mezzocannone 16, 80134, Napoli, Italy 4 Department of Chemistry, New York University, New York, NY 10003, United States

Herpes simplex viruses (HSVs) are responsible for a wide variety of clinical manifestations and represent a significant worldwide disease and economic burden. There are two serotypes of HSV, HSV-1 and HSV-2, which can infect either oral or genital sites, respectively. HSV infections are often subclinical, but their incidence and severity have increased over the past decades due to the increasing number of immunocompromised patients. The HSV entry pathway is thought to be determined by both virus and host cell factors. In particular, HSV-1 enters cells through fusion of the viral envelope with a cellular membrane in a cascade of molecular interactions involving multiple viral glycoproteins and cellular receptors. The envelope glycoproteins gH/gL, gB, and gD are all essential for the entry process[1]. gB, the most conserved within the herpes virus family, is involved in virus attachment, penetration, and cell-to-cell spread and has proved to function as a membrane fusogen. The authors of the present abstract previously reported that a modified peptide corresponding to central helical domain gB is very active as fusion inhibitor[2]. Here a poly(amide)-based dendrimers (PAMAM) terminated with this peptide are shown to perform higher antiviral activities than the peptide alone. While the mechanisms of antiviral activity is not fully understood, it is thought that the interactions used by viruses to infect cells can be inhibited due to the dendrimer physically blocking or interfering with the fusion mechanism between the virus and the cell. The peptide-dendrimer is very active in all kinds of inhibition experiments and in particular in the virus pretreatment experiment, indicating that it is able to interact with the virus in its prefusogenic structure.

References1. S.A. Connolly, J.O. Jackson, T.S. Jardetzky, R. Longnecker Nat. Rev. Microbiol. (2011), 9, 369−381. 2. M. Cantisani, A. Falanga, N. Incoronato, L. Russo, A. De Simone, G. Morelli, R. Berisio, M. Galdiero, S. Galdiero,

J. Med. Chem. 2013, 56, 8366−8376


83

P10

Dissection of the structural features and fungicidal activity of an antibody-derived peptide

T.A. Pertinhez1, T. Ciociola2, L. Giovati2, M. Sperindè2, W. Magliani2, L. Polonelli2, S. Conti2 and A. Spisni3

1 C.I.M. Laboratory - Technopole Parma, University of Parma,2 Dept. Biomedical, Biotechnological and Translational Sciences (S.Bi.Bi.T.), University of Parma,3 Dept. Surgical Sciences, University of Parma, Parma, Italy

The peptide T11F (TCRVDHRGLTF), derived from the constant region of human IgM, proved to display significant in vitro fungicidal activity, inclusive against multidrug resistant strains [1]. The functional contribution of each residue has been highlighted using alanine scanning. The substitution of positively charged residues was associated to a decrease in the candidacidal activity, a feature typical of cationic AMPs. A dramatic reduction in activity was also caused by the replacement of Cys2, responsible for the formation of a disulphide bridge [1]. In this work, we investigated the conformational properties of T11F, by means of circular dichroism (CD) and nuclear magnetic resonance (NMR), and the structure-function relationships of its alanine-substituted derivatives (asd). CD spectrum of T11F in aqueous solution is characterized by a negative band around 200 nm and by a weaker positive band at ≈ 218 nm, characteristic of the polyproline II (PPII) helix. Given that PPII helix is generally more stable at low temperatures, we tested the peptide at various temperatures and we found that the fraction of PPII helix increases as the temperature decreases, confirming the presence of that secondary structure motif. Noteworthy the process is reversible. The difference spectrum between 5 °C and 90 °C is reminiscent of a b-structure, suggesting, as pointed out by Shi et al. [2] for polypeptides in PPII conformation, that heating favours a partial transition of the peptide conformation to a b-structure organization. The measured NMR coupling constants (3JNH-a) are in agreement with a structural model for T11F where PPII and b-conformation already coexist at 5 °C.Interestingly, CD analysis of T11F asd showed that only Phe11 is essential to preserve the PPII conformation. In fact, the F11A mutant exists in a random coil conformation, as opposed to all other asd. Based on the knowledge that the supercoil organization of collagen is stabilized also by inter-chain interactions of the few Phe residues, our data indicate that these peptides form extended coiled-coil dimeric structures stabilized, in N-terminus by the Cys disulphide bond, and in C-terminus, by interaction of the Phe side chains.As for the peptides, biological activity the data indicate that the crucial feature is the existence of a dimeric structure. In fact, C2A while it preserves a PPII helix conformation, it exhibits a significantly reduced fungicidal activity. The S-S bond at position 2 appear to provide the required robustness to make the peptides fungicidal.Recognizing that more and more data indicate that the PPII conformation is involved in many biological functions, our data point to an additional functionality for this relevant conformation.

References1. Polonelli L, et al PLoS ONE (2012) 7, e34105.2. Shi Z, et al. Proc. Natl. Acad. Sci. USA (2007) 99, 9190


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P11

Dimerization of HBHA adhesion from Mycobacterium tuberculosis, insights into bacterial agglutination

C. Esposito,1,3, M. Cantisani2,3, E. Pedone3 S. Galdiero3 and R. Berisio3

1 DFM scarl,via Mezzocannone 16,80134 – Napoli2 Istituto Italiano di Tecnologia, Largo Barsanti e Matteucci 53, 80125 Napoli – Napoli3 CNR, Istituto di Biostrutture e Bioimmagini, via Mezzocannone 16,80134 – Napoli

HBHA is a cell-surface protein implicated in the dissemination of Mycobacterium tuberculosis (Mtb)from the site of primary infection. Similar to other adhesins, HBHA is able to promote bacterial agglutination by its N-terminal coiled-coil region[1]. It was shown that HBHA coiled coil domain is responsible for protein oligomerization. More precisely, HBHA has an elongated shape characterized by a dimeric coiled coil structure that is the key to its structural integrity[2,3]. As coiled coil proteins are notably capable of dynamic switching of monomer subunits, it’s likely that HBHA forms reversible bridge-like structures connecting bacteria through the N-terminal coiled coil domain[3]. However, HBHA mediated aggregation of bacteria leading to clumping of bacilli, generating a robust spatial structure with a high local cell density. These multicellular aggregates give resistance for a toxic agents such as antibiotics[4]. The existing correlation between Mtb clumping and HBHA oligomerization has prompted us to investigate the interaction mode between HBHA subunits to form dimers. We identified critical regions for HBHA dimerization and used site directed mutagenesis to produce mutants with altered dimerization capacities. Together with mapping critical regions for protein dimerization, we discovered peptide molecules able to disaggregate HBHA dimers, with the production of a well-structured monomeric protein–peptide hybrids. Our findings provide the first molecular entities able to interfere with HBHA dimerization and, likely, with Mtb agglutination, providing a strong contribution to the formulation of anti-agglutination molecular entities of therapeutic interest[5]. Results of these studies will be described in the poster.

References1. G. Delogu, and M.J.Brennan. (1999) J Bacteriol 181, 7464–7469.2. C. Esposito, M.V. Pethoukov, D.I. Svergun, A. Ruggiero, C. Pedone, E. Pedone and R. Berisio. J Bacteriol (2008)

190,4749–4753.3. C. Esposito, P.Carullo, E. Pedone, G. Graziano, P. Del Vecchio, and R. Berisio.(2010) FEBS Lett.584, 1091–1096.4. N.Mittal, E.O. Budrene,M.P. Brenner and A.Van Oudenaarden. (2003) PNAS.100, 13259–13263.5. C. Esposito, M. Cantisani, G. D’Auria, L. Falcigno, E. Pedone,S.Galdiero and R. Berisio. (2012) FEBS

Lett.586,659–667.


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P12

Structure and Function of RNase AS, a Polyadenylate-Specific Exoribonuclease Affecting Mycobacterial Virulence In Vivo

M. Romano1,2, F. Squeglia2, A. Ruggiero2, P. De Simone2, B. Appelmelk3 and R. Berisio2

1 Second University of Naples, 81100 – Caserta2 Institute of Biostructure and Bioimaging - CNR, 80134 – Naples3 VU University Medical Center, 1081- Netherlands

The cell-envelope of Mycobacterium tuberculosis plays a key role in bacterial virulence and antibiotic resistance [1-4]. Little is known about the molecular mechanisms of regulation of cell-envelope formation. We elucidate functional and structural properties of RNase AS, which modulates M. tuberculosis cell-envelope properties and strongly impacts bacterial virulence in vivo [5]. The structure of RNase AS reveals a resemblance to RNase T from Escherichia coli, an RNase of the DEDD family involved in RNA maturation [6-8]. We show that RNase AS acts as a 3’-5’-exoribonuclease that specifically hydrolyzes adenylate-containing RNA sequences. Also, crystal structures of complexes with AMP and UMP reveal the structural basis for the observed enzyme specificity. Notably, RNase AS shows a mechanism of substrate recruitment, based on the recognition of the hydrogen bond donor NH2 group of adenine. Our work, presented in the poster, opens a field for the design of drugs able to reduce bacterial virulence in vivo.

References1. M. Daffe´, Tuber. Lung Dis. (1999) 79, 153–169.2. Stokes, R.W. Infect. Immun. (2004) 72, 5676– 5686.3. M.C. Gagliardi, Cell. Microbiol. (2007) 9, 2081–2092.4. R. Berisio, Curr. Protein Pept. Sci. (2012) 13, 697–698.5. M. Romano, Structure (2014) 22, 1–12.6. Y. Y. Hsiao, Nat. Chem. Biol. (2011) 7, 236–243.7. Y. Y. Hsiao, Nucleic Acids Res. (2012) 40, 8144–81548. Y. Zuo, Structure (2007) 15, 417–428.


86

P13

The identification and characterization of a novel CAMP from Stf76, a Sulfolobus islandicus plasmid-virus pSSVx transcription

factor

L. Pirone1, P. Contursi2, A. Zanfardino2, S. Fusco2, M. Varcamonte2, E. Notomista2, A. Falanga3, S. Galdiero3, 4, E. Pedone3, 5

1 Istituto di Cristallografia-CNR, Bari, Italy2 Dipartimento di Biologia, Università degli Studi di Napoli “Federico II”, Napoli, Italy3 Centro Interuniversitario di Ricerca sui Peptidi Bioattivi, Università degli Studi di Napoli “Federico II”, Napoli, Italy 4 Dipartimento di Farmacia, Università degli Studi di Napoli “Federico II”, Napoli, Italy5 Istituto di Biostrutture e Bioimmagini-CNR, Naples, Italy

The increasing resistance of bacteria to classical types of antibiotics has become a serious problem in the health community dealing with infectious diseases. As more strains of pathogenic bacteria become resistant to antibiotics, the need to develop new antimicrobial agents becomes more critical. Antimicrobial peptides have been viewed as possible alternatives to classical antibiotics1, however, their use has yet to become widespread because more research on discovering their exact mechanism of action is needed. The cationic antimicrobial peptides (CAMPs) are an interesting class of antibacterial molecules that could overcome the problem of multi-drug resistance because they are an essential component of the innate immune response of multicellular eukaryotes. Furthermore they show very advantageous properties: they are relatively small molecules with a broad spectrum of activity (Gram +, Gram -, yeasts and fungi) on both dividing and resting cells; they are able to damage bacterial membranes, thus reducing the onset of resistant strains. Moreover they have an effective action on mature biofilms (another important resistant factor) and possess additional immunomodulatory properties that include chemotaxis, induction of cytokine release, regulation of angiogenesis and anti-inflammatory activity. Thus, CAMPs could be considered as a bridge between innate and adaptive immunity. A quantitative prediction of antibacterial activity and the localization of a CAMP within the sequence of a well characterized DNA-binding protein Stf76 from the Sulfolobus islandicus plasmid-virus pSSVx2 has been performed by means of bioinformatic tools. Once identified the peptide, here named PepC, it has been characterized from a structural and a functional point of view. Antimicrobial assays on a wide range of microorganisms have been performed. Far-UV circular dichroism spectra of PepC alone and in the presence of different detergents have been registered highlighting a propensity to form helical structure. Moreover, PepC showed a significantly greater fusion activity in DOPE/DOPG LUVs compared with the fusion activity in DOPC/DOPG LUVs. In addition leakage assays showed a capability of PepC-induced pore formation. All the results will be widely discussed.

References1. Rezansoff AJ, et al J Pept Res. 2005 May;65(5):491-501..2. Contursi P., Pirone L.; Farina B. et al. Nucleic Acids Res. 2014 Mar 25.3. Galdiero S, et al. Int J Mol Sci. 2013 Sep12;14(9):18758-89.


87

P14

Structure-Activity Relations of Myxinidin, an Antibacterial Peptide Derived from the Epidermal Mucus of Hagfish

M. Cantisani1,2,3, A. Falanga1,2, M. Leone2, E. Mignogna4, E. Finamore4, M. Vitiello,4 G. Morelli1,2,S. Galdiero1,2 and M. Galdiero4

1 Department of Pharmacy, CIRPEB and DFM, University of Naples “Federico II,” Naples, Italy2 Istituto di Biostrutture e Bioimmagini, CNR, Naples, Italy3 Center for Advanced Materials for Health Care IIT@CRIB, Italian Institute of Technology, Naples, Italy4 Department of Experimental Medicine, II University of Naples, Naples, Italy

The structure-activity relations of myxinidin[1], a peptide derived from epidermal mucus of hagfish, Myxine glutinosa L., were investigated. Analysis of key residues allowed us to design new peptides with increased efficiency. SAR studies indicate the key role of several parameters that can influence the potency and spectrum of activity: size, sequence, percent helical content, charge, overall hydrophobicity, amphipathicity, and widths of the hydrophobic and hydrophilic faces of the helix. These parameters are intimately related and are the key to designing novel peptides with increased potency and directed antimicrobial activity. Antimicrobial activity of native and modified peptides demonstrated the key role of uncharged residues in the sequence; the loss of these residues reduces almost entirely myxinidin antimicrobial activity, while insertion of arginine at charged and uncharged position increases antimicrobial activity compared with that of native myxinidin. Particularly, we designed a peptide capable of achieving a high inhibitory effect on bacterial growth. Experiments were conducted using both Gram-negative and Gram-positive bacteria. Nuclear magnetic resonance (NMR) studies showed that myxinidin is able to form an amphipathic a-helical structure at the N-terminus and a random coil region at the C terminus. Our data support the hypothesis that the ability of our peptide analogues to structure into a well-defined, amphipathic a-helix is strongly correlated to antimicrobial activity. On the other hand, formation of a-helical structure before membrane interaction is not beneficial. Also, the net positive charge, the type of cationic residues, and the percentage of hydrophobic residues were key factors in determining their antibacterial and hemolytic activities[2].

References1. Subramanian S, Ross NW, MacKinnon SL. Mar Biotechnol (NY). 2009 Nov-Dec;11(6):748-57. 2. Cantisani M, Leone M, Mignogna E, Kampanaraki K, Falanga A, Morelli G, Galdiero M, Galdiero S. Antimicrob

Agents Chemother. 2013 Nov;57(11):5665-73.


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P15

Synthesis and Biological Activity of Temporin L Analogues F. Merlino1, G. I. Lopez Cortés2, A. M. Yousif1, B. Casciaro2, A. Carotenuto1, P. Campiglia3, I. Monterrey-Gomez1,

A. Di Grazia2, E. Novellino1, P. Grieco1, M. L. Mangoni2

1 Department of Pharmacy, University of Naples Federico II, 80131 – Naples2 Department of Biochemistry Sciences, University of Rome La Sapienza, 00185 – Rome3 Department of Pharmacy, University of Salerno, 84084, Fisciano

Temporins are antimicrobial peptides (AMP’s) isolated from the skin of Red European frog “Rana temporalis”. They are active particularly against Gram-positive bacteria, Candida species, fungi. They have the ability to bind and permeate both artificial and biological membranes. Temporins are short, linear 10-14 residues long peptides, with a net charge positive and an amidate C-terminal.We have recently synthesized an interesting Temporin L analogue, named TL34 (FVPWFSKFLGRIL-NH2), endowed with a higher antimicrobial activity and a lower hemolytic activity than the native peptide TL. New analogues of TL34 have been synthesized and tested, and preliminary results are here reported.

References1. Carotenuto, A.; Malfi, S.; Saviello, M.R.; Campiglia, P.; Gomez-Monterrey, M.I.; et.al. J. Med. Chem., 51, 2354

(2008). 2. Saviello, M.R.; Malfi, S.; Campiglia, P.; Cavalli, A.; Grieco, P. Novellino, E.; Carotenuto, A. Biochemistry, 49, 1477

(2010).


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P16

EGF analog peptide functionalized micelles for target-selective sorafenib delivery

P. Righieri1, F. D’Agosto1, A. Cusimano2, M. Cervello2, G. Morelli1 and D. Tesauro1

1 Department of Pharmacy & CIRPeB University of Naples “Federico II”, IBB CNR 80134 - Napoli2 Institute of Biomedicine and Molecular Immunology “Alberto Monroy” CNR 90146 - Palermo

Target delivery to a desirable site of action is one of the most crucial issue in cancer therapy. Nanoparticles are regarded to be ideal vehicles for antitumor drug because their hydrophobic inner core is an appropriate reservoir for hydrophobic anticancer drugs, moreover their hydrophilic outer shell facilitates long blood circulation, and the improvement of enhanced permeation and retention [EPR] effect in tumor tissue. Delivery improvement can be obtained by labelling nanoparticles with bioactive moiety like antibodies, organic molecules and peptides able to recognize tumor cells overexpressing receptors[1]. Epidermal growth factor receptor (EGFR) is a promising target site for cancer therapy since it is high-expressed in several kinds of tumors such as lung cancer, ovarian cancer and hepatocellular carcinoma[3].The aim of this study is to construct and evaluate GE11 peptide modified micelles for targeted delivery of sorafenib to EGFR-positive cells. Sorafenib is a high hydrophobic chemotherapeutic agent that inhibits tumor cell proliferation and vascularization[3]. The amino acid sequence YHWYGYTPQNVI (designated as GE11 peptide) was selected in literature as ligand with specific binding capabilities to EGFR by phage display peptide library[4] and it was conjugated to hydrophobic moiety thought ethoxylic linkers. The amphiphilic surfactant pluronic F127 was added to the peptide derivative in order to obtain mixed peptide-labelled micelles. Sorafenib payload was fine tuning with emulsion method achiving high loading ratio. The physicochemical properties of these targeted micelles, including unloaded and loaded drug, such as particle size and polydispersity index, were examined by dynamic light scattering (DLS) measurements. Micelle cell uptake was investigated on human hepatocellular carcinoma (HCC) PLC/PRF/5 cells overexpressing EGF receptors and related to HCC EGFR-negative HepG2 cells. The future aims will be to evaluate the antitumor activity of sorafenib-incorporated nanoparticles on the same cell lines.

References1. A Accardo, L Aloj, et al. Int. J Nanomedicine (2014); 9, 15372. N Normanno, MR Maiello, et al. J Cell Physiol (2003);194, 133. SM Wilhelm, L Adnane et al. Mol Cancer Ther (2008); 7, 31294. Z. Li, R Zhao et al. The Faseb J. (2005), 1978


90

P17

Multi-Target Peptides for chronic pain control: opioid agonists / w-conotoxin analogues

A. Mollica1, A. Stefanucci1, R. Costante1

1 Department of Pharmacy, University “G’ d’ Annunzio” of Chieti-Pescara, Via Dei Vestini 31, 66100 – Chieti

Pharmacological management of severe and chronic pain is still a challenging task. The currently available analgesic drugs are not always efficacious, and pain control remains a large unmet therapeutic need. Although opioids are considered the standards of analgesic cares for acute nociceptive pain, they have limited efficacy for neuropathic pain at tolerable doses. Chronic opioid dosing can be associated with serious unwanted side-effects, finally resulting in a significantly decreasing of the patients’ quality of life. [1]

1: Tyr-D-Ala-Gly-Phe-Ser-Arg-Leu-Met-Lys-Tyr-NH2

Opioid portion Conotoxin portion

2: Tyr-D-Ala-Gly-Phe-Arg-Leu-Tyr-NH2

Multi-target approach

Figure 1. Design of multi-target analgesic peptides 1 and 2

Recently, the synthetic peptide Ziconotide has been approved by the FDA and the European Medicines Agency for treatment of patients with severe chronic pain refractory to standard protocols. Ziconotide can also be used in combination with other drugs by i.t. administration, and clinical trial results suggested an additive or synergistic analgesic effect with opioids, with reduced risk of dependency and tolerance development. Recently, two studies demonstrated the efficacy morphine/Ziconotide combination in reducing pain, inadequately controlled by only one of these two drugs in patients with chronic pain. Recent literature on bivalent opioid/CCK peptides [2], prompts us to design a series of analogues based on the adjacent pharmacophores concept (Figure 1), in which the opioid agonist pharmacophore is linked at the N-terminus and w-conotoxin pharmacophore (products 1 and 2). In vitro and in vivo bioactivity has been tested [3].

References1. Mollica, A.; Pinnen, F.; Stefanucci, A.; Costante, R. Curr. Bioact. Compd. 2014, in press, 10.2174/15734072096661

31227162147.2. Mollica, A.; Pinnen, F.; Costante, R.; Locatelli, M.; Stefanucci, A.; Pieretti, S.; Davis, P.; Lai, J.; Rankin, D.;

Porreca, F.; Hruby, V. J. J. Med. Chem. 2013, 56, 3419.3. Mollica, A.; Pinnen, F.; Feliciani, F.; Stefanucci, A.; Lucente, G.; Davis, P.; Porreca, F.; Ma, S. W.; Lai, J.; Hruby, V.

J. Amino acids 2011, 40, 1503.


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P18

Target-selective theranostic micelles for Bombesin receptors incorporating Au(III)-dithiocarbamate complex

R. Iannitti,1 A. Accardo,1,2 P. Ringhieri,2 R. Palumbo,1 C. Nardon,3 D. Fregona3 and G. Morelli1,2

1 Institute of Biostructures and Bioimaging (IBB-CNR), Via Mezzocannone 16, I-80134 Napoli , Italy2 Department of Pharmacy and CIRPeB, University of Naples “Federico II”, Via Mezzocannone 16, I-80134 Napoli,

Italy3 Department of Chemical Sciences, University of Padua, Via F. Marzolo, 1-35131 Padova, Italy

Cisplatin and its analogues are currently used in the treatment of a large number of solid tumours. Anyway high toxicity, rapid inactivation and frequent occurrence of platinum resistance restrict their clinical use. Gold(III) dithiocarbamato complexes have proved to be very promising anticancer drugs for their biological behaviour notwithstanding their low water solubility. However, their toxicological profile is so far encouraging in animal models, likely resulting from a good selectivity toward the cancerous cells [1,2]. Sterically stabilized micelles (SSM) and sterically stabilized mixed micelles (SSMM) based on DSPE-PEG2000 have been extensively investigated as drug nanovectors encapsulating hydrophobic drugs such as Camptothecin, Diazepam and Paclitaxel [3, 4]. In order to increase their solubility and thus their bioavailability, here we studied the incorporation of an active gold(III) dithiocarbamate complex AuL12 in mixed micelles based on DSPE-PEG2000 phospholipid. The effect of PC or DOPC commercial phospholipids (5%, 10% and 20% mol/mol) on the AuL12 loading and on the physicochemical properties of micelles was also investigated. Target-selective theranostic micelles were also prepared by adding a small amount of the amphiphilic peptide derivative MonY-BN-AA1 in micelle composition [5]. BN-AA1 peptide sequence is an analogue of [7–14]Bombesin peptide fragment able to selectively target GRP receptors, overexpressed by several cancer cells, such as prostate cancer and ovarian cancer cells. Preliminary in vitro cytotoxicity studies on PC-3 cells overexpressing GRP receptors are also reported. References1. L. Ronconi, D. Fregona, Dalton Transctions. 2009, 48, 10670-10680.2. C. Nardon, et al. Inter. J. Cancer Res. and Treat. 2014, 34, 487-492.3. A. Krishnadas, et al. Pharmaceutical Research,2003, 20, 297-3024. O.M. Koo, et al. Nanomedicine: Nanotechnology, Biology, and Medicine, 2005, 1, 77- 845. A. Accardo, et al. J. Drug Targeting, 2013, 21(3), 240-249


92

P19

RGD Peptide–Conjugated Silica Coated PbS Nanocrystals with Tunable Emission in the Near Infrared Region

for Molecular Targeted Imaging

N. Depalo1, M. Corricelli2, I. de Paola3, E. Fanizza2, R. Comparelli1, M. Striccoli1, A. Agostiano1,2, N. Denora4, V. Laquintana4, R. M. Iacobazzi4, M. Saviano5, A. Del Gatto3, L. Zaccaro3, M. L. Curri1

1 CNR-IPCF UOS Bari, Via Orabona 4, 70125 - Bari, Italy 2 Università degli Studi di Bari Aldo Moro, Dipartimento di Chimica, Via Orabona 4, 70125 - Bari, Italy3 Istituto di Biostrutture e Bioimmagini-CNR, Via Mezzocannone, 16, Napoli 80134, Italy 4 Università degli Studi di Bari Aldo Moro, Dipartimento di Farmacia, Via Orabona 4, 70125 - Bari, Italy 5 Istituto di Cristallografia-CNR,Via Amendola 122/O, 70126 Bari

Near-infrared (NIR) fluorescence imaging is most attractive and rapidly progressing area for early detection, accurate diagnosis, and targeted therapy of various diseases, especially cancer.[1] NIR emitting semiconductor nanocrystals (NCs) are emerging as revolutionary labelling materials for in vivo and deep-tissue imaging of biological targets, due to their high photostability, versatile surface modification and unique tunability in the optical properties.

Figure 1. Vis-NIR absorption (black line, a) and PL emission spectra (red line, a) of organic capped PbS NCs with two different sizes. TEM micrograph of PbS NCs before (b) and after growth of silica shell (c). Molecular structure of cyclic RGD peptide.

Here, the synthesis of uniform silica coated PbS NCs with emission properties conveniently tunable from the first to the second ‘biological window’ has been attained.[2] Active targeting has been achieved by coupling the silica coated PbS NCs with a designed cyclic RGD peptide, providing NIR fluorescent nanoprobes able to interact with integrin αvß3 expressed on the tumor vasculature. [3] The NP/peptide bioconjugates, characterized by a high colloidal stability in physiological media and preservation of the relevant optical properties in the NIR region of electromagnetic spectrum, are promising candidates for targeted NIR molecular labelling and in vivo NIR tissue imaging applications.

References1. M. Wang, C. Mi, Y. Zhang, J. Liu, F. Li, C. Mao, S. Xu J. Phys. Chem. C (2009), 113, 19021.2. M. Corricelli, D. Altamura, et al. Cryst Eng Comm. (2011), 13, 3988.3. G. Scarì, F. Porta, U. Fascio, S. Avvakumova, V. Dal Santo, et al. Bioconjugate Chem. (2012), 23, 340.


93

P20

Nanocrystalline Semiconducting-Magnetic Heterostructures decorated with cyclic RGD peptide for Integrin Targeting

N. Depalo1, G. Valente2, I. de Paola3, R. Comparelli1, E. Fanizza2, M. Striccoli1, A. Agostiano1,2, N. Denora4, V. Laquintana4, R. M. Iacobazzi4, M. Saviano5, A. Del Gatto3, L. Zaccaro3, M. L. Curri1

1 CNR-IPCF UOS Bari, Via Orabona 4, 70125 - Bari, Italy2 Università degli Studi di Bari Aldo Moro, Dipartimento di Chimica, Via Orabona 4, 70125 - Bari, Italy3 Istituto di Biostrutture e Bioimmagini-CNR, Via Mezzocannone, 16, Napoli 80134, Italy 4 Università degli Studi di Bari Aldo Moro, Dipartimento di Farmacia , Via Orabona 4, 70125 - Bari, Italy 5 Istituto di Cristallografia-CNR,Via Amendola 122/O, 70126 Bari

Theranostic nanosystems offer new and improved opportunities to overcome limitations associated with conventional cancer diagnosis and therapy. In particular, multifunctional nanoparticles based on inorganic heterostructures, able to integrate several features within a single construct, can be successfully conjugated with targeting ligands to achieve multi-targeting nanoplatforms potentially useful for selective drug delivery to the tumor cells. [1,2]

Figure 1. TEM micrograph of BNCs before (A) and after PEG-modified phospholipid functionalization (B). Molecular structure of cyclic RGD peptide (C). Scheme for BNC micelles bioconjugated with peptide for targeting of αvß3 integrin (D).

Here, asymmetric binary nanocrystals (BNCs), composed of a semiconductor TiO2 nanorod joined to a magnetic γ-Fe2O3 spherical domain, have been embedded in polyethylene glycol (PEG) modified phospholipid micelles. A designed peptide containing the RGD motif for targeting of αvß3, expressed on several types of cancer cells, has been successfully conjugated with the BNC incorporated in lipid micelles. The BNC bioconjugation process has been monitored by using optical and structural techniques. In vitro investigation has also been performed to assess the cytotoxicity of the peptide/BNC conjugates. These systems have a large potential for cancer treatment, since the RGD motif can be used to drive the nanostructures toward tumor area, where magnetically induced hyperthermia can be combined with TiO2-based photodynamic therapy.

References1. G. Scarì, F. Porta, U. Fascio, S. Avvakumova, V. Dal Santo, M. De Simone, M. Saviano, M. Leone, A. Del Gatto, C.

Pedone, L. Zaccaro Bioconjugate Chem. (2012), 23, 340.2. N. Depalo, P. Carrieri, R. Comparelli, M. Striccoli, A. Agostiano, L. Bertinetti, et al. Langmuir (2011), 27, 6962


94

P21

Identifying IgE-Binding Sites of Cor a 8 by SPOT-Synthesis on PU-Modified Cellulose Membranes

B. Ay1, N. Offermann1, S. Gauck1, V. Cardona2, S. Dölle3, M. Worm3 and M. Fooke1

1 DR. FOOKE LABORATORIEN GMBH, 41468 Neuss, Germany2 Allergy Section, Department of Internal Medicine, Hospital Vall d’Hebron, Barcelona, Spain3 Allergy-Center-Charité, Department of Dermatology and Allergy, Charité-Universitätsmedizin, 10117 Berlin,

Germany

Allergy to hazelnut (Corylus avellana) occurs worldwide. Currently 11 allergens with 21 homologues are known (allergome.org). Among hazelnut allergy patients, the frequency and the type of allergic reaction varies geographically depending on the presence of PR-10 like proteins (birch, alder or hazel trees) or LTP-containing food[1,2]. Hazelnut allergy caused by Cor a 8 sensitization is often associated with allergic reactions to other LTP-containing food such as peach and cherry[3]. LTP proteins are extremely resistant to proteolysis and heat denaturation, which enables the survival in the digestive tract environment, leading to strong IgE sensitisation capability and more severe symptoms[4]. A characterisation (IgE epitope mapping) at amino acid sequence level is helpful for a better understanding of IgE reactivity, especially to distinguish cross-reactivities to other LTPs in food.SPOT-synthesis is a useful and inexpensive technique for exploring protein-peptide interactions[5]. One disadvantage is the single use of the spotted peptide membranes. The standard membrane type (ß-Ala or Gly) is not stable during regeneration procedures (de-esterification of peptides). The innovative PU-(polyurethane) membrane eliminates this hindrance due to their inert polyurethane bonds. This new kind of membrane is stable during regeneration and may be used several times. Identification of IgE-binding sites by means of SPOT-synthesis is a common method for allergen characterisation. In recent years several allergens were investigated and many IgE-epitopes were found[6]. We performed hazelnut LTP (Cor a 8) epitope mapping by using the PU-modified cellulose membranes. 12-mer peptides with a shift of 3 amino acids from hazelnut LTP were synthesised on the newly developed PU-membrane and incubated with sera from Spanish and German patients. Our investigations resulted in the detection of two IgE-binding regions in the hazelnut LTP. We found two peptide sequences that are recognised by hazelnut LTP-sensitised individuals (in total 18) with a frequency of 80% and 60%, respectively. A better understanding of the reactive parts of allergenic proteins supports the development of innovative diagnostics and therapeutics.

References1. A.E. Flinterman et al., J Allergy Clin Immunol. (2006), 118(5): 1186. 2. C. Hartz et al., Int Arch Allergy Immunol. (2010); 153(4): 335. 3. F. Schocker et al., J Allergy Clin Immunol. (2004), 113(1): 141.4. F. Ferreira1, T. Hawranek, P. Gruber,N. Wopfner, A. Mari, Allergy (2004), 59: 243.5. R. Frank, Journal of Immunological Methods (2002), 267: 13.6. R.C. Aalberse, R. Crameri, Allergy, (2011); 66: 1261.


95

P22

Investigating the substrate specificity and use of M-TGase to graft peptide synthons onto proteins

A. Caporale1, F. Selis2, A. Sandomenico1, 3, G. Tonon2 and M. Ruvo1, 3

1 CIRPeB, Via Mezzocannone, 16, 80134 – Napoli2 BIOKER c/o CNR IGB, Via Pietro Castellino, 80131- Napoli3 IBB-CNR, via Mezzocannone, 16, 80134 – Napoli

Transglutaminases catalyze transglutamination or deamidation reactions on glutamine residues embedded in surface exposed consensus sequences [1]. The enzyme is largely exploited for modifying proteins in pharmaceutical, food and other biotechnological applications. Following a combinatorial approach, we have identified here a new optimized substrate (LQSP) that is recognized and processed by the enzyme with a strikingly higher efficiency compared to the well-known TQGA sequence [2]. LQSP has a Vmax of 0.158 x 10-2 mM/min compared to the Vmax of 0.185 x 10-5 mM/min exhibited by TQGA. Surprisingly, Kms, which reflect the affinity of enzymes for their substrates, are inverted with TQGA displaying a much lower Km (0.0027 mM) compared to LQSP (0.0731 mM). The new substrate has been introduced in model peptides, which have been used to modify with high selectivity prototypical bioactive peptides and proteins with fluoresceine or recognition motifs, allowing them to acquire new properties.

References1. N. M. Rachel, J. N. Pelletier, Biomolecules (2013), 3, 870 - 8882. C. Maullu, D. Raimondo, F. Caboi, A. Giorgetti, M. Sergi, M. Valetini, G. Tonon, A. Tramontano, FEBS (2009),

6741-6750.


96

P23

RGD: SPPS and on-resin functionalization suitable for Nuclear Medicine application

B. Monaco1, 2, A. Yousif1, C. Bolzati3, P. Grieco1, M. Ruvo4 and A. Caporale2

1 Univ. di Napoli “Federico II” , Dept Pharmacy, 80131 – Napoli2 Univ. di Napoli “Federico II”, CIRPeB, 80134 – Napoli3 CNR Padova, Dept Pharmacy, 35131 – Padova4 IBB-CNR, CIRPeB, 80134 – Napoli

Angiogenesis, the formation of new blood vessels, is the first essential step for pathologic processes in the growth of solid tumors, therefore Angiogenesis Imaging is gaining an ever increasing importance for the early diagnosis of the disease [1, 2]. Due to their relevant affinity and specificity for various αVß3 integrin receptors, several cyclic Arg-Gly-Asp (RGD) peptides have been used to inhibit pathologic angiogenesis and for the detection of the corresponding receptors [3]. Efficient syntheses of labelled RGD-containing cyclic peptides via SPPS is thus the target to obtain the molecular tools for these purposes, trying to reduce laborious synthetic steps and to improve the purity of final compounds. Taking advantage of the various new protocols already reported for the assembly of several of these derivatives [4, 5, 6], we have elaborated and tested some approaches whereby on-resin cyclization - with or without microwave assistance - have been introduced. Probes for Nuclear Medicine application, i.e. SPECT-Imaging, have also been successfully introduced exploiting side chain functions, including the εNH2 of lysines.

References1. F. G. Gaertner, H. Kessler, H.-J. Wester, M. Schwaiger, A.J. Beer, Eur J Nucl Med Mol Imaging (2012), 39 (Suppl 1),

S126- S138. 2. M. Trajkovic-Arsic, P. Mohajerani, A. Sarantopoulos, E. Kalideris, K. Steiger, I. Esposito, X. Ma, G. Themelis,

N. Burton, C. W. Michalski, J. Kleeff, S. Stangl, A. J. Beer, K. Pohle, H.-J. Wester, R. M. Schmid, R. Braren, V. Ntziachristos, J. T. Siveke, Journal of Nuclear Medicine (2014), 55, 8A

3. K.-E. Gottschalk, H. Kessler, Angew Chem Int ed (2002) 41, 3767-37744. A. Del Gatto, M. De simone, I. de Paola, M. Saviano, L. Zaccaro, Int J Pept Res Ther (2011), 17, 39-455. K. Yamada, I. Nagashima, M. Hashisu, I. Matsuo, H. Shimizu, Tetrahedron Lett. (2012), 53, 1066-10706. R. Hassert, P.-G. Hoffmeister, M. Pagel, M. Hacker, M. Schulz-Siegmund, A. Beck-Sickinger, Chemistry &

Biodiversity (2012), 9, 2648-2658


97

P24

Solid phase synthesis and nucleic acids binding studies of a thymine-functionalized oligolysine

D. Musumeci1, G. N. Roviello2, L. Rozza1, C. Pedone3

1 Dipartimento di Scienze Chimiche, Università di Napoli Federico II, 80126 – Napoli, Italy2 Istituto di Biostrutture e Bioimmagini – CNR, 80134 – Napoli, Italy3 Centro Regionale di Competenza in diagnostica e Farmaceutica Molecolari, 80134 – Napoli, Italy

In the last decades peptide-based DNA analogs emerged as a promising class of DNA mimics able, in some cases, to interact with natural nucleic acids by forming complexes characterized by high thermal stability and specificity and a significant cell-membrane permeability.[1-3] Diamino acids represent a class of natural building blocks frequently used for the realization of peptide-like analogs of nucleic acids, offering the possibility to introduce the nucleobases on one of the two amino groups, and employ the other for the oligomerization.[3,4] Taking into account all these considerations, we here reported the synthesis of an oligonucleotide analog, characterized by a peptide backbone comprising both nucleobase-functionalized amino acids and underivatized L-lysine moieties in an alternate sequence. The underivatized L-lysine residues conferred positive charges to the molecule, especially valuable for their potential in improving the water-solubility of the nucleopeptide, as well as its ability to recognize the negatively-charged natural nucleic acids.

Figure 1. Hexathymine nucleopeptide and its building blocks

The ability of the nucleopeptide to interact with DNA and RNA was investigated by circular dichroism, UV and surface plasmon resonance. Human serum stability of the thymine-bearing nucleopeptide was also studied.

References1. G.N. Roviello, E. Benedetti, C. Pedone, E.M. Bucci, Amino Acids (2010), 39, 45.2. A. Calabretta, T. Tedeschi, G. Di Cola, R. Corradini, S. Sforza, R. Marchelli, Mol. BioSyst. (2009), 5, 1323.3. G.N Roviello, D. Musumeci, C. Pedone, E.M. Bucci, Amino Acids (2010), 38, 103.4. G.N Roviello, D. Musumeci, M. Castiglione, E.M. Bucci, C. Pedone, E. Benedetti, J. Pept. Sci. (2009), 15, 155.


98

P25

Liposomes doubly functionalized with the CCK8 and gH625 peptides for intracellular drug delivery

A. Accardo, P. Ringhieri, A. Falanga, S. Galdiero and G. Morelli

Department of Pharmacy and CIRPeB, University of Naples “Federico II”, Institute of Biostructures and Bioimaging (IBB-CNR), Via Mezzocannone 16, I-80134 Napoli , Italy One of the key challenges in medicine is the creation of therapeutic agents able to reach the intended target organ at full concentration, where they act selectively on diseased cells and tissues only, without creating undesired side effects [1]. Unfortunately, current therapies fail to attain this ideal behavior. Accumulation of the drug to the target organs can be achieved by using two classes of peptide molecules: homing peptides (HPs) and cell-penetrating peptides (CPPs). Here we report the synthesis and the characterization of a prototype of antitumor liposomal drug derivatized with two different peptide sequences: an HP able to bind, with high affinity and selectivity, membrane receptors over-expressed by cancerous cells; and a membrane-perturbing domain able to promote membrane bilayer translocation and to transport a cargo into the cytoplasm. CCK8 was chosen as HP for its capability to recognize both CCK-1 and CCK-2 cholecystokinin receptors, both of them overexpressed in several human tumours [2]. gH625 peptide, previously identified as a membrane-perturbing domain in the gH protein of Herpes simplex virus type I, was chosen for its capability to traverse the membrane bilayer and to increase the cytosolic translocation of the drug loaded nanocarriers [3]. Liposomes doubly decorated were obtained by using simultaneously a pre-liposomal and a post-liposomal derivatization method. In details, (C18)2-L5-SPDP-CCK8 monomer containing two alkyl chains at the N-terminus of the CCK8 sequence, a cleavable disulphide bond (SPDP) and a PEG spacer (L5), was introduced directly during the aggregation process [4]. On the other hand, gH625 modified at the C-terminus with a propagyl glycine residue (gH625-Pra) was bound to liposomes containing azido function on the external surface using copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition (click-chemistry) reaction [5]. Doxorubicin was loaded in liposomes by using the well-known ammonium gradient method. Before and after functionalization, targeted liposomal doxorubicin were structurally characterized by dynamic light scattering (DLS).

References1. Kim, T. H. et al. Expert Review of Molecular Diagnostics, 2013, 13(3), 257. 2. Silvente-Poirot, S, et al. Eur. J. Biochem, 1993, 215, 513.3. A. Falanga, et al.Nanomedicine, 2011, 7(6), 925.4. A. Morisco,et al. J. Pept. Sci., 2009, 15, 242.5. R. Tarallo, et al. Chem.–Eur. J.,2011, 17, 12659.


99

P26

Dendrimer’s functionalized with the membrane-interacting peptide gH625: mechanism of interaction with liposomes

A. Falanga1, R. Tarallo2, M. Galdiero3, M. Weck2, E. Perillo1, Lucia Lombardi1, G. Morelli1 and S. Galdiero1

1 Department of Pharmacy University of Naples Federico II & CIRPEB & DFM Scarl, 80134-Naples 2 Molecular Design Institute and Department of Chemistry New York University, 10003-New York3 Department of Experimental Medicine Second University of Naples 80134-Naples

We have demonstrated that amide-based dendrimers functionalized with the membrane-interacting peptide gH625 derived from the herpes simplex virus type 1 (HSV-1) envelope glycoprotein H enter cells mainly through a non-active translocation mechanism.[1] Herein, we investigate the interaction between the peptide-functionalized dendrimer and liposomes composed of PC/Chol using fluorescence spectroscopy, isothermal titration calorimetry and surface plasmon resonance to get insights into the mechanism of internalization. The affinity for the membrane bilayer resulted very high and the interaction between the peptide-dendrimer and liposomes took place without evidence of pore formation. These results suggest that the presented peptidodendrimeric scaffold may be a promising material for efficient drug delivery.

Figure 1.Interaction of peptide-dendrimer with liposome

References1. D. Guarnieri, A. Falanga, O. Muscetti, R. Tarallo, S. Fusco, M. Galdiero, S. Galdiero, P. A. Netti, Small (2013), 9,

853.


100

P27

In vitro investigation on cancer cell uptake and toxicity of liposomes functionalized with the membranotropic peptide gH625

E. Perillo*1, S. Porto*2, A. Falanga1, M.Galdiero3, P. Grieco1, G. De Rosa1, S. Galdiero1 and M. Caraglia2

1 Department of Pharmacy, University of Naples “Federico II,” 80134 Naples, Italy2 Department of Biochemistry, Biophisics and General Pathology, Second University of Naples, 80134 Naples, Italy 3 Department of Experimental Medicine, Second University of Naples, 80134 Naples, Italy

Liposomes are used as biocompatible carriers of different anti-cancer agents from classical chemotherapy agents to peptides and nucleic acids. Both versatility in particle size and physical characteristics of lipids make them attractive for building vehicles for a wide range of applications. In particular, associating a drug with liposomes markedly changes its pharmacokinetic and pharmacodynamic properties and lowers systemic toxicity; moreover, the drug is prevented from early degradation and/or inactivation.[1] To enhance the antitumor efficacy of liposomal drugs, we have prepared liposomes externally decorated with the nineteen residues of gH625 peptide, previously identified as a membrane-perturbing domain in the gH glycoprotein of Herpes simplex virus type I.[2] Several copies of the hydrophobic peptide gH625 were bound to the external surface of PEGylated liposomes (Soy Phospholipids/Cholesterol/DSPE-PEG) in a controlled way, using an easy and versatile synthetic strategy based on click chemistry.Physicochemical characterization of the liposome system revealed a size of 140 nm with uniform distribution and high doxorubicin encapsulation efficiency. The gH625 decorated liposomes loaded with doxorubicin (Doxo), the anticancer chemotherapy drug (Lipo-gH625Doxo) were evaluated for their uptake as well as cytotoxicity against lung adenocarcinoma parental and Doxo-resistant cells, A549 and A549-Doxo, respectively. The maximal cellular uptake was recorded at 6h in A549-Doxo cells. Interestingly, Doxo uptake was 50% higher if compared to that one of LipoDoxo and it was about four-fold higher than free Doxo as evaluated with a spectrophotometric assay. Moreover, the cytotoxic effects induced by Lipo-gH625Doxo were about two-fold higher than those induced by LipoDoxo, but two-fold lesser that one induced by free drug. In conclusion, we have demonstrated that the functionalization of liposomes with gH625 increase the Doxo uptake in Doxo-resistant cell line and this effect is paralleled by an increased cytotoxicity.

References1. M. B. Bally, R. Nayar, D. Masin, M. J. Hope, P. R. Cullis, L. D. Mayer, Biochim. Biophys. Acta Biomembr. (1990),

1023, 1332. A. Falanga, M. Vitiello, M. Cantisani, R. Tarallo, D. Guarnieri, E. Mignogna, P. Netti, C. Pedone, M. Galdiero, S.

Galdiero, Nanomedicine (2011), 7, 925


101

P28

Peptide-based siRNA for the treatment of cancer cellsNunzia Migliaccio1, Annalisa Lamberti1, Camillo Palmieri2, Giuseppe Fiume2, Nicola M. Martucci1,

Immacolata Ruggiero1, Ileana Quinto2, Giuseppe Scala2, Paolo Arcari1,3

1. Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Via S. Pansini 5, I-80131 Naples, Italy

2. Department of Experimental and Clinical Medicine, University of Magna Graecia, Viale Europa, I-88100 Germaneto, Catanzaro, Italy

3. CEINGE Advanced Biotechnologies, Via G. Salvatore 486, I-80145, Naples, Italy

Tumorigenic B-cell lymphomas, likewise other cancer diseases, show an incomplete response to clinical treatments such as conventional chemotherapeutic treatments, radiation therapy and corticosteroids. As result, a minimal residual disease (MRD) occurs, where few residual neoplastic cells undetected in vivo replenish the cancer cell reservoir. This scenario requires the development of new strategies for the selective targeting toward the tumorigenic cells that survive to the anticancer treatments. In this work, we have settled an anti cancer strategy based on the selected delivery of electrostatic-based peptide-siRNA complex, taking advantage of the therapeutic properties of an idiotype specific peptide (A2036) that specifically binds murine B-lymphoma cells [1]. Two engineered arginine rich peptides containing the A2036 targeting sequence were designed to bind fluorescent-labelled siRNA. One peptide contained 9 Arg at the C-terminal of A2036 whereas the other one included 5 Arg at the N- and C-terminus, respectively. Similar peptides containing a random sequence were used as controls. Both A2036-siRNA complexes were endowed with the selective delivering of fluorescent-labelled siRNA (siGLO) toward the A20 murine B-cell lymphoma, as evaluated by cytofluorimetry (Figure 1) and confocal microscopy, whereas fluorescent-labelled siRNA (siGLO) alone was not internalized in the selected cells.

Figure 1. Fluorescence analysis of A20 cells after treatment with peptide-siRNA. Peptide-siRNA complexes were incubated with A20 cells for the indicated times and after washes cells were analyzed at cytofluorimeter. Samples: 0, untreated control cells; 1, fluorescent siRNAsiGLO alone; 2, A2036-9R-siRNAsiGLO; 3, 5R-A2036-5R-siRNAsiGLO; 4, RND-9R-siRNAsiGLO; 5, 5R-RND-5R-siRNAsiGLO; 6, A2036-siRNAsiGLO.

In addition, with respect to control peptides, the use of the modified A2036 peptides complexed with siRNA anti-GAPD once internalized showed a reduction in the enzyme expression levels.This strategy is expected to provide a safe and non-invasive approach for the delivery of therapeutic molecules.

Reference1. C. Palmieri, C. Falcone, E. Iaccino, F.M. Tuccillo, M. Gaspari, F. Trimboli, A. De Laurentiis, L. Luberto, M.

Pontoriero, A. Pisano, E. Vecchio, O. Fierro, M.R. Panico, M. Larobina, S. Gargiulo, N. Costa, F. Dal Piaz, M. Schiavone, C. Arra, A. Giudice, G. Palma, A. Barbieri, I. Quinto, G. Scala Blood (2010), 2, 226.


102

P29

Development of new fluorescent probes for RNA imaging in cellL.Piras1, E. Novellino2, M. Saviano1 and A. Romanelli3*

1 National Research Council-CNR , Institute of Crystallography-IC, 70126 – Bari2 University of Naples “Federico II”, Department of Pharmacy, 80131 – Naples3 University of Naples “Federico II”, Department of Pharmacy, 80134 – Naples

Neuroblastoma (NB) is one of the most challenging malignancies of childhood, being associated with the highest death rate in paediatric oncology. Typically, patients with high risk disease undergo an initial remission in response to treatment, followed by disease recurrence that has become refractory to further treatment.[1] In NB cells MYCN and miR-17-5p polycistronic cluster members, are both expressed at high levels and may therefore be considered as markers for NB.In this work we report the design, synthesis and characterization of a sequence of a PNA (Peptide Nucleic Acids) based probe for miRNA. The PNA was designed complementary to miR-17-5p, conjugated to a “tetracysteine tag” that has a high affinity for membrane-permeant , fluorogenic biarsenicals FlAsH-EDT2.

[2,3] Unique feature of the biarsenicals is that their fluorescence is quenched until bound by “tetracysteine tag” which allows the PNA to be imaged without exhaustive washing to remove unbound dye. Also the FlAsH-EDT2 is easily uptaken by cells.In principle we should have a very selective tool for diagnostic of cancer or other diseases.

Figure 1. Figure caption example

References1. L.M. Wagner, M.K. Danks J. Cell Biochem. (2009), 107, 46.2. S. R. Adams, R. Y. Tsien Nat Protoc. (2008), 3, 1527.3. B. Krishnan, L. M. Gierasch Chem Biol. (2008), 15, 1104.


103

P30

Novel peptide based CXCR4 antagonists for cancer imagingL. Aloj1, A. Trotta2, M. Aurilio1, E. Squame1, S. Santagata2, G. Morelli3, S. Scala2

1 SC Medicina Nucleare, Istituto Nazionale Tumori, Fondazione G. Pascale - IRCCS, 80131-NAPOLI, ITALY, 2 Immunologia Oncologica, Istituto Nazionale Tumori, Fondazione G. Pascale - IRCCS, 80131-NAPOLI, ITALY3 CIRPeB, Universita “Federico II”, 80131-NAPOLI, ITALY

Background: The CXCR4/CXCL12 axis appears to play an important role in cancer metastases. A number of CXCR4 inhibitors are being evaluated for anticancer therapy as well as for the development of novel radiopharmaceuticals. Recent work from our institution has identified small peptide molecules containing a three amino acid motif (Ar-Ar-X or X- Ar-Ar) that inhibit CXCR4 dependent cell migration and the formation of lung metastases in animal models. Peptide R (Arg-Ala-[Cys-Arg-Phe-Phe-Cys]) and peptide S (Arg-Ala-[Cys-Arg-His-Trp-Cys]) have shown the best CXCR4 inhibition properties Aim: to evaluate binding properties of DTPA coupled derivatives of peptides R and S for use as radiopharmaceuticals for targeting CXCR4 receptors in vivo. Methods: Peptides containing different spacers to distance the chelators from the active molecule were synthesized. Four derivatives of peptide R (DTPA-R, DTPA-Dioxa-R, DTPA- Dioxa3-R and DTPA-PEG-R) and one derivative of peptide S (DTPA-Ahoh-S) were coupled on the N-terminus. R-bAla- DTPA and S-bAla-DTPA were coupled on the C terminus. Binding inhibition by the new peptides of the PE-labeled anti- CXCR4 12G5 antibody was assessed by flow cytometry. Displacement experiments were performed with cold peptides against the high affinity CXCR4 ligands 125I SDF1alpha or 111In-DTPA-T140 on CEM lymphoblastic leukemia cells cells. All peptides were labeled with 111In in citrate buffer for saturation binding experiments. Results: All R and S peptide derivatives showed IC50 between in the 10-100 µM range in flow cytometry experiments, whereas the native peptides under the same conditions showed better inhibition (IC50 in the 1-10 µM range). Displacement experiments performed against SDF1alpha/CXCL12 or labeled T140 peptide showed no significant displacement of radioactivity of all new derivatives tested at concentrations up to 10 µM. Similarly, saturation binding experiments performed with 111In labeled peptides showed no saturable binding up to 100 µM with the exception of R-bAla-DTPA where saturable binding was obtained with a dissociation constant in the order of 10 µM. Discussion and Conclusions: All derivatives tested show affinities too low to be assessed with the described radio assays. The derivative of the R peptide containing the chelator at the C terminus did show saturable binding although with very low affinity. While the currently described derivatives may be of little value for imaging CXCR4 in vivo the R-bAla-DTPA derivative may be a starting point for development of higher affinity CXCR4 ligands based on these peptide sequences.

References1. L. Portella, R. Vitale et al. PLOS One. (2013), 8, e74548.2. A. Muller, B. Horney et al. Nature. (2001), 410, 50.


104

P31

Harnessing the potential of idiotypic peptides to design smart drug delivery system

E. Iaccino1, S. Mimmi1, E. Vecchio1, A. de Laurentiis1, M. Pontoriero1, F. Fasanella Masci1,C. Falcone1, A. Pisano1, S. Ceglia1, G. Fiume1, A. Scialdone1, C. Palmieri1, I. Quinto1 and G. Scala1.

1. Department of Experimental and Clinical Medicine, University “Magna Graecia” of Catanzaro - Italy

Background: The adoption of macromolecular drugs in routine clinical practice is limited by lack of efficient, safe, and specific delivery strategies. Due to their ability in overcoming plasma membrane barrier and transport molecules into cytoplasm of live cells, peptides represent an attractive option for drug delivery, gene therapy and cancer treatment. In the recent years we developed a novel experimental approach to identify peptide ligands for the idiotypic determinant (Id) of the B cell Receptor (BCR), named “Id-peptides”. Using the same validated approach we identified a pool of specific binders for the IM9 multiple myeloma cell line that if opportunely engineered are able to vehiculate GFP as a cargo into target tumour cells. Results: Tumour-specific Id-peptides were selected by screening phage-displayed random peptide libraries using the cognate IM9 IgG paraprotein as bait. In vitro functional analysis of the identified peptides highlights a consistent activity of a specific binding of pIM9-31 to target IM9 cells followed by the internalization into the cognate neoplastic cells. A GFP fusion protein displayed pIM9-31 peptide was designed via E. Coli, produced and assayed both in vitro and in vivo: results confirm the capability of identified Id-peptide to specifically vehiculate GFP and to induce selective protein internalization by target tumour cells. Conclusion: The application of peptides as carriers for the design and development of targeted drug-delivery systems for cancer therapeutics represents a newly emerging field with a high commercial potential. Here, we have reported the targeting specificity and the cargo properties of an idiotype specific peptide toward a human myeloma cell line. As a whole, using a validated phage-display approach, the presented work endorses Id-peptides as a fruitful field of interest in the direction of new smart drug delivery systems design for the enforcement of the current anticancer therapeutics arsenal.

References1. C. Palmieri et al. Blood (Jul, 2010)2. F. De Angelis et al. Nanoscale (Oct, 2010).3. A. W. Tong, Y. A. et al. Current opinion in molecular therapeutics (Apr, 2005).4. W. M. Pardridge et al. Advanced drug delivery reviews. (Mar, 2007).5. M. C. Morris et al. Nature biotechnology (Dec, 2001).6. Bramsen JB, Kjems J. Methods Mol Biol (2013)


105

P32

Aldehyde modification and alum synergize to enhance anti-Tnfα vaccination and mitigates arthritis in rats

A.Ostuni1, A. Bavoso1, J. De Vendel1, A. Bracalello1, T. Shcheglova2 , S. Makker2, and A. Tramontano2

1 University of Basilicata, Department of Sciences, 85100 Potenza, Italy2 University of California, Davis-Medical School, Davis, CA USA 95616

Cytokine antagonism by autoantibodies induced through vaccination show promise as a novel immunotherapy for chronic inflammatory diseases[1-2]. A polypeptide comprised of residues 141-235 of rat TNFa fused to the C-terminus of glutathione-S-transferase was developed and used as an auto-vaccine against the self-protein TNFa. Reaction of the hybrid protein with glycolaldehyde to introduce aldehyde residues was expected to enhance immunogenicity[3]. Whereas the modified protein was essentially non-immunogenic in rats in the absence of adjuvant, elevated anti-TNFa antibody and cytokine neutralizing activities were produced in response to the modified fusion protein adsorbed on alum. These activities were also significant enhanced relative to the unmodified protein on alum. Antibodies to glutathione-S-transferase were augmented by a similar factor. Efficacy in therapeutic vaccination was evaluated in two rat models of rheumatoid arthritis. Adjuvant arthritis and collagen-induced arthritis (CIA) progressed similarly in groups immunized with the unmodified fusion protein or a control protein, whereas the disease induced in rats vaccinated with glycolaldehyde-modified fusion protein was significantly attenuated. Anti-collagen IgG antibodies titers in CIA did not significantly deviate in autovaccinated and control groups, suggesting that immunizations did not skew subsequent responses for immune-mediated disease induction. These results provide support for aldehyde adduction of protein antigens as an alternative or auxiliary to conventional adjuvants in development of safe and effective molecular vaccines. References1. I. Dalum, D.M. Butler, M.R. Jensen, P. Hindersson, L. Steinaa, A.M. Waterston, S.N. Grell, M. Feldmann, H.I.

Elsner, S. Mouritsen Nat. Biotechnol. (1999) , 17,666. 2. H. Le Buanec, L. Delavallee, N. Bessis, S. Paturance, B. Bizzini, R. Gallo, D. Zagury, M.C. Boisser Proc. Natl. Acad.

Sci. USA (2006), 103, 19442.3. M.E. Allison, D.T. Fearon Eur. J. Immunol. (2000), 30, 2881.


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Gliadin peptide P31-43: structure and biological effectsR. Aitoro1, M. Nanyakkara1, M. V. Barone1, G. D’Auria2, L. Falcigno2, M. Sanseverino3, A.L. Tornesello3,

L. Calvanese4 and G. Morelli2

1 University Federico II, Department of Traslational Medical Science (section of Pediatrics) and ELFID (European Laboratory for the Investigation of Food Induced Diseases), 80131 – Naples

2 University Federico II, Department of Pharmacy, 80134 – Naples3 INBIOS srl Via P. Castellino, 111 80131 - Napoli4 CIRPEB, University Federico II, 80134 - Naples

In the intestine and particularly in the enterocytes, nutrients are modulators of various cellular functions and may be involved in tissue immune response and inflammation. Dietary proteins are often not completely digested by the intestinal proteases and residual peptides can have biological effects. An example of an intestinal inflammatory and remodelling response of the intestine to poorly digested proteins is the small intestinal celiac lesion induced by gluten – an alimentary protein present in wheat and other cereals. Celiac disease is a genetic disease characterized by inflammation, due to the adaptive and innate immune responses (with IL-15 as a major mediator of the innate immune response), and structural changes resulting in remodelling of the small intestinal mucosa with an inversion of the differentiation and proliferation compartment leading to villi atrophy and crypts hyperplasia. Undigested A-gliadin peptides P31-43 is central to Celiac Disease (CD) pathogenesis, entering enterocytes in vesicular compartments by endocytosis and inducing an innate immune response in CD intestinal mucosa[1, 2]. P31-43 shares a sequence homology with HRS (Hepatocytes growth factor Regulated tyrosine kinase Substrate) a key protein that regulates endocytic maturation and can activate several signaling molecules including ERK (extracellular responsive kinase). In this study we focus on structural and biological properties of P31-43 peptide and a series of its mutants obtained by alanine scanning. Our analysis has highlighted the crucial role of three central aminoacids for P31-43 effect on ERK phosphorylation. Interestingly these mutants are able to reduce ERK phosphorylation also in not treated cells indicating that they might act as P31-43 antagonists. HPLC profiles of P31-43 and several mutants signal the occurrence of conformational equilibria in a slow exchange regime (min). NMR structure analyses in water confirm that the peptides, due to cis-trans isomerism of the proline residues, are involved in multiple conformational equilibria with interconversion kinetics from slow to fast. Globally the experimental data so far collected, highlight the relevance of few central residues in determining both the conformation distributions and function abilities of the peptides.

References1. M.V. Barone et al GUT. (2007), 56,4.2. M. Nanayakkara et al AJCN (2213), 8, 4.


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Small peptide inhibitors of Protein-Protein interactions essential in JAK-STAT pathway

Carmen Aiello1,2, Pasqualina Liana Scognamiglio1,3, Concetta Di Natale1,3, Domenico Riccardi1, Sabatino Pacia1, and Daniela Marasco1

1 Department of Pharmacy, CIRPEB, DFM, University of Naples “Federico II”, 80134, Naples, Italy2 Institute of Biostructures and Bioimaging, National Research Council, 80134, Naples, Italy3 IIT Italian Institute of Technology, 80125, Naples, Italy

Protein-protein interactions (PPIs) play an essential role in biological systems: indeed, often proteins interact each other to express their biological activity, creating a network of PPIs that results essential for virtually all cellular processes. Alterations of these interactions are often at the basis of several pathologies, thus the identification of molecules, primarily peptides and peptidomimetics, able to modulate, inhibit or promote certain interactions represents a valuable therapeutic approach [1,2]. Suppressor Of Cytokine Signalling (SOCS) proteins are negative feedback regulators of several pathways involved in immune response [3]. SOCS1 and SOCS3 have many similarities as well as some intriguing differences: both can block signalling by direct inhibition of JAK2 enzymatic activity yet apparently require different anchoring points within the receptor complex. Recently [4] new structural and functional studies have been carried out on SOCS-3/JAK2 complex: they showed that SOCS-3 simultaneously binds JAK2 and the cytokine receptor to which it is attached, revealing how specificity is generated in SOCS action and explaining why SOCS-3 inhibits only a subset of cytokines. They showed that SOCS-3 interacts with both JAK2 and the gp130 receptor simultaneously by utilizing two adjacent binding surfaces and that ATP binding by JAK is unaffected. Here, by means of protein dissection and overlapping sequences approaches and we have identified small peptide and peptidomimetics able to bind to JAK2 and inhibit JAK2/SOCS3 through surface plasmon resonance (SPR) technique.

References1. Marasco D., et al., Past and future perspectives of synthetic peptide libraries. (2008) Curr Protein Pept Sci. 9(5):447-

67.2. Scognamiglio P.L., et al., From peptides to small molecules: an intriguing but intricated way to new drugs. (2013)

Curr Med Chem. 20(31):3803-17.3. Kershaw NJ, et al, Regulation of Janus kinases by SOCS proteins. (2013) Biochem Soc Trans.41(4):1042-7 4. Kershaw NJ, et al., SOCS3 binds specific receptor-JAK complexes to control cytokine signaling by direct kinase

inhibition. (2013) Nat Struct Mol Biol. 20(4):469-76.


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Cripto recognition by the Loop-Helix Motif [44-67] of Nodal: an AlaScan Analysis

B. Carfora1, A. Caporale2, A. Focà1,4, G. Focà1,4, A. Sandomenico2,3, L. Calvanese2, G. D’Auria 1-3, L. Falcigno1-3 , M. Ruvo2,3

1 Dept. of Pharmacy, University of Naples Federico II, via Mezzocannone, 16, 80134 Napoli, Italy2 CIRPeB, University of Naples Federico II, via Mezzocannone, 16, 80134 Napoli, Italy3 CNR-IBB, via Mezzocannone, 16, 80134 Napoli, Italy4 BIOKER c/o CNR IGB, Via Pietro Castellino, 80131- Napoli

Nodal is an extracellular growth factor with oncogenic properties. It belongs to the TGF-ß superfamily of proteins, which share a common topology characterized by the “cysteine-knot cytokines” fold, comprising a conserved “cysteine-knot” motif, four pairs of antiparallel ß-strands and an α-helix (α3). Nodal has powerful transforming properties exerted mostly through the activation of the ALK4/Cripto/Nodal/ActRIIB receptor complex. We have previously reported the molecular model of the ternary complex containing ALK4/Cripto/Nodal, generated using a combination of homology modelling, docking simulations and biochemical binding data of Nodal-derived peptides with both Cripto and Alk4[1]. This study also identified several Nodal residues potentially involved in the interaction with the co-receptor Cripto and to be used as target hot-spots for the development of antagonists. Most of these residues reside within the Nodal 43-69 region, corresponding to the α3-wrist helix along with the pre-helix loop3, which is directly involved in binding to the Cripto CFC domain. Here, we have designed (Fig. 1) and synthesized a set of Ala-scan peptides to explore the effect of alanine mutations on the α3 helix loop structure and on the Cripto recognition properties. These peptides have been structurally characterized by dichroism circular and comparative affinity binding measurements for Cripto have been carried out by SPR assays.

Figure 1. Set of modified Nodal peptides prepared and tested in this study

References1. L. Calvanese, et al., Biopolymers (2010), 93, 1011-1021.


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Comparative binding to VEGF165 of PEGylated Bevacizumab fragments

G. Focà1-3, F. Selis3, S. Scaramuzza3, A. Focà1-3, R. Schrepfer3, A. Sandomenico2,4, G. Tonon3 and M. Ruvo2,4

1 Dept. of Pharmacy, University of Naples Federico II, via Mezzocannone, 16, 80134 Napoli, Italy2 CIRPeB, University of Naples Federico II, via Mezzocannone, 16, 80134 Napoli, Italy3 BIOKER c/o CNR IGB, Via Pietro Castellino, 80131- Napoli4 CNR-IBB, via Mezzocannone, 16, 80134 Napoli, Italy

PEGylation is the most clinically validated method to improve the efficacy and the half-life of biotherapeutics, of which monoclonal antibodies represent an important class[1]. To date, at least 25 antibodies and a smaller number of antibody fragments (principally Fabs) have been approved for human therapy[2] and an ever increasing interest is addressed to new smaller antibody fragments having different formats and increased affinities[3]. For developing these molecules, pharmacokinetic properties have to be improved to ensure maximal efficacy and safety profiles. Fab antibody fragments or otherwise engineered antibody fragments (scFv, diabodies ect) reduce the occurrence of Fc derived side effects, but show rapid clearance from blood circulation, thereby, as such, they are optimal candidates for PEGylation. On this basis, we have focused our attention on the marketed monoclonal antibody anti-VEGF Bevacizumab, a very potent angiogenesis inhibitor also used in the treatment of age-related macular degeneration (AMD) [4]. Given the reported short half-life of both Bevacizumab and Ranibizumab (Bevacizumab-related Fab fragment) within the eye following intravitreal injection[4], we have explored the possibility to prepare and test a set of PEGylated Bevacizumab fragments, potentially employable for intravitreal or systemic administration. Bevacizumab Fabs and F(ab)2 were prepared by proteolytic pepsin digestion, then a set of PEGylated fragments were obtained by site-specific chemical conjugation. Such products have been biochemically characterized and the effect of PEG of different sizes on affinity for the VEGF165 antigen has been evaluated by surface plasmon resonance analysis.

References1. Pasut G1, Veronese FM., Drugs Today (Barc). 2009 Sep;45(9):687-95. doi: 1396674/dot.2009.45.9.1416421.2. http://www.immunologylink.com/FDA-APP-Abs.html 3. Aaron L Nelson, MAbs. 2010 Jan-Feb; 2(1): 77–83.4. Meyer CH, Holz FG. Eye (Lond). 2011 Jun;25(6):661-72. doi: 10.1038/eye.2011.66. Epub 2011 Apr 1.


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Study of the interaction of a salivary proline-rich peptide with SH3 domains from the SRC kinases family

A. Arcovito1, M. Castagnola1, 2, V. Trapè2, M.T. Sanna3, I. Messana3, T. Cabras3,V. Marzano1 and A. Vitali2

1 Catholic University of Sacred Heart , Institute of Biochemistry and Clinical Biochemistry, 00168 – Rome, Italy2 CNR, Institute for the Chemistry of Molecular Recognition, 00168 – Rome, Italy3 University of Cagliari, Dep. of Life Sciences and Environment, 09042 Monserrato (CA), Italy

Protein kinases playing a key role in cell signal transduction pathways represent ideal targets for new natural and designed drugs to be applied in many diseases. Among kinases, the Src protein-tyrosine kinases family has been found to be implied in several types of human diseases from cancer and even in the pathogenic mechanisms of AIDS [1]. A drug target structure of such kinases is represented by their SH3 domains that are specific for recognizing proline-rich sequences with the Pro-x-x-Pro characteristic consensus sequence. A human 1932 Da salivary proline-rich peptide (p1932) previously identified as anti-retroviral agent [2] has been structurally characterized by means of circular dichroism and ATR-FTIR spectroscopic techniques. The peptide assumed a polyproline-II structure thus resulting an ideal partner for SH3 domains. To verify this hypothesis, 8 different SH3 domains have been tested by means of Surface Plasmon Resonance and the affinity constants have been measured. Three of them (c-SRC, Hck and Fyn) gave positive result showing Kd ranging from nanomolar to micromolar values being Fyn SH3 domain the best performer. It is noteworthy that all the interacting domains belong to the same SH3-SRC family, indicating a possible selectivity of p1932. These results may explain the retroviral activity of the proline-rich peptide being Fyn, c-SRC and Hck implied in the mechanism of HIV-1 infection [3].

References1. R. Roskoski Jr., Biochem. Biophys. Res. Comm. (2004), 324, 1155.2. [Pat. n° PCT/IB2012/050415].3. J. Kakkar, K.K. Chaudhary, C.V. Prasad. Bioinformation. (2013), 28, 777-781.


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PEGylated Trastuzumab FabsS. Scaramuzza1, F. Selis1, A. Focà1,4, G. Focà1,4, A. L. Politano1, A. Sandomenico2,3, G. Orsini1, R. Schrepfer1,

M. Ruvo2,3 and G. Tonon1

1 BIO-KER c/o CNR IGB, via Pietro Castellino, 111, 80131 - Napoli 2 IBB-CNR, via Mezzocannone, 16, 80134 – Napoli3 CIRPeB, via Mezzocannone, 16, 80134 – Napoli4 Dept. of Pharmacy, University of Naples Federico II, via Mezzocannone, 16, 80134 – Napoli

The antigen-binding antibody fragments (Fabs) of monoclonal antibodies (mAbs) are powerful tools for many diagnostic and therapeutic applications. The main drawback for systemic use is their rapid clearance from blood circulation, due to the lack of the fragment crystallisable (Fc). PEGylation technology is currently widely used to extend their short half-life in vivo and to increase their efficacy in clinical treatments where long serum persistence is required. Trastuzumab (trade name Herceptin) is a mAb approved for the treatment of early-stage breast cancer that are Her2-positive and their Fab fragments are intensively investigated to broaden its applicability as both therapeutic and diagnostic [1,2].We have successfully obtained Trastuzumab Fab and Fab’ fragments by proteolytically digesting the whole antibody with papain and pepsin enzymes [3] and prepared a set of PEG-conjugated derivatives to preliminarily explore their pharmacological profile. 20kDa mPEG-propionaldehyde was conjugated to the purified Fab fragment at the N-terminus to give the mono-PEG20kDa-Fab. Site-specific conjugation of two 10kDa or 20kDa mPEG-maleimide molecules to the C-terminal hinge free cysteines of the Fab’ fragment, gave the di-PEGylated derivatives. All products were characterised by SDS-PAGE and HPLC analysis to evaluate their identity, purity and concentration whereas binding studies to Her2 were performed by surface plasmon resonance (SPR) using Biacore, immobilizing the receptor on suitable sensor chip [4]. Remarkably, Fab and Fab’ binding affinities were similar to that observed with the whole antibody whereas PEGylated fragments showed ten to one hundred eighty-fold less affinity, depending on polymer attachment site and PEGs molecular weight. Data thus suggest that the PEGylation of Trastuzumab and likely other therapeutic mAb fragments needs an accurate evaluation of conjugation sites and of PEG size and shape to retain the target recognition properties.

References1. D. A. Scollard, C. Chan, C. M. B. Holloway, R. M. Reilly, Nucl. Med. Biol. (2011), 38, 129-136.2. J. M. Scheer, W. Sandoval, J. M. Elliott, L. Shao, E. Luis, S. Lewin-Koh, G. Schaefer, R. Vandlen, PLos One

(2012), 7(12), e51817. 3. S. M. Andrew, J. A. Titus, Curr. Protoc. Immunol (1997), 2.8.1-2.8.104. H. Khalili, A. Godwin, J. Choi, R. Lever, S. Brocchini, Bioconjugate Chem. (2012), 23, 2262-2277.


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Characterization of Herceptin antibody fragments in in vitro assays

L. Sanguigno1, M. Cantile1, R. Sanna1, F. Selis1, E. Truppo1, A. Sandomenico2,3, M. Ruvo2,3, G. Tonon1, R. Schrepfer1, G. Orsini1

1 BIOKER c/o CNR IGB, Via Pietro Castellino, 80131- Napoli2 CNR, Institute of Biostructure and Bioimaging, via Mezzocannone 16, 80134 – Naples, Italy3 CIRPeB, University of Naples “Federico II”, via Mezzocannone 16, 80134 – Naples, Italy

PEGylation of therapeutic proteins and antibody fragments is used to increase their half-life and to suppress immune response in vivo. We have prepared PEG-conjugated antibody fragments deriving from the drug Herceptin[1] and performed a preliminary in vitro characterization with the aim of investigating their biological properties prior to in vivo testing. The Herceptin Fab’ fragment obtained by digestion with pepsin (BK0501) and its analogue BK0504, obtained by chemical pegylation with methoxy-PEG(20kDa)-maleimide on a C-terminal cysteine freed by pepsin digestion, have been fully characterized analytically and for their ability to recognize the ErbB2 antigen (See Poster n°38, Scaramuzza et al.). The two products have been tested in in vitro assays of binding to ErbB2 by ELISA, and in vitality and internalization assays on ErbB2-positive SKBR3 carcinoma cells. Also a localization study on the same cell line has been comparatively performed with the two proteins.We found that BK0501 bound to ErbB2 with a 2-fold reduced affinity compared to the whole Herceptin, whereas BK0504 was 4 times less active. Viability MTT assays of the two products on SKBR3 cells showed that the two new products BK0501 and BK0504 had a 142 and 44 times reduced efficacy, respectively. Internalization assay on SKBR3 cells showed instead that both BK0501 and BK0504 were internalized at 3h and 6h, respectively, likely through a mechanism of receptor-mediated endocytosis and that both molecules, after 3h incubation, colocalized with lysosomal markers. In conclusion, the two new Herceptin-derived fragments are not suitable as surrogate of the whole antibody for direct cell killing.

References1. Garnock-Jones KP1, Keating GM, Scott LJ. Drugs. 2010;70(2):215-39.


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Spectroscopic investigation of auranofin binding to zinc finger nucleocapsidic domains

A.Bavoso1, A. Ostuni1 , D. Tesauro2 and M. A. Castiglione Morelli1

1 University of Basilicata, Department of Sciences, 85100 Potenza, Italy2 CIRPeB, Department of Pharmacy and IBB CNR, University of Naples “Federico II” Napoli, Italy

The gold-based compound auranofin [2,3,4,6-tetra-O-acetyl-1-thio-b-D-glucopyranose-S-triethylphosphine gold (I)] is an orally administrable drug that has been first successfully used in the treatment of rheumatoid arthritis [1] and later used in other immuno-mediated diseases. [2,3]

Auranofin demonstrated also promising in the treatment of several other diseases: leukaemia, carcinomas, and parasitic, bacterial and viral infections.[4]

Furthermore, it has been shown to decrease the viral load in HIV-infected cells in patients infected with human immunodeficiency virus HIV-1[5] and proposed to be useful in the treatment of HIV in combination with antiretroviral agents. [6]

We report here a study on two peptides corresponding to different isolates of the C-terminal zinc finger Cys-X2-Cys-X4-His-X4-Cys domain of the HIV-2 nucleocapsid protein NCp8. Their interaction with auranofin has been studied with different spectroscopic techniques.

References1. A.E. Finkelstein, D.T. Walz, V. Batista, M. Mizraji, Roisman, A, Misher Ann Rheum Dis (1976), 35(3), 2512. P.N. Fonteh, F.K. Keter, D. Meyer Biometals (2010), 23, 185.3. S.J. Berners-Price, A. Filipovska. Metallomics (2011), 3, 863.4. J.M. Madeira, D.L.Gibson, W.F. Kean, A. Klegeris Inflammopharm. (2012), 20, 297.5. D.L. Shapiro, J.R. Masci J. Rheumatol (1996), 23,18186. M.G. Lewis, S. DaFonseca, N. Chomont, A.T. Palamara, M. Tardugno, A. Mai, M. Collins, W.L. Wagner, J.Yalley-

Ogunro, J. Greenhouse, B. Chirullo, S. Norelli, E. Garaci, A. Savarino, AIDS (2011), 25, 1347.


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NMR structural investigation of the membrane proteins involved in the dimerization of the mitochondrial ATP-synthase

J. Tolchard1, A. Barras2, D. Brèthes2, MF. Giraud2 and B. Odaert1

1. CBMN-UMR 5248, bât 14bis, allées Saint Hilaire, 33600 Pessac, France2. IBGC-UMR 5095, 1 rue Camille Saint Saëns, 33077 Bordeaux, France

ATP, the universal fuel molecule of any cell, is mainly produced by the F1Fo-ATP synthase. The yeast enzyme, like mammalian ones, is not only involved in ATP synthesis but also forms dimers essential for the organization of the mitochondrial membrane into cristae vesicles. Three subunits are involved in ATP synthase oligomerization: g, e and the N-terminal extremity of the subunit 4 (S4t). Deletion or partial mutation of these subunits lead to anomalous mitochondrial morphologies. No information has been obtained on the three-dimensional structure of these small hydrophobic proteins that create the membrane interface of ATP synthase oligomerization. A production of these proteins, in the presence or not of isotopically labelled amino acids, has been performed with the precipitate-based method using cell-free expression system. Solubilization of Nter4 has been achieved with zwitterionic detergent (DPC, LMPC) or with anionic detergents (SDS, LMPG). Circular dichroïsm spectra have indicated that the solubilized proteins contain mainly alpha helicoïdal structures. However, well dispersed 1H15N-HSQC NMR spectra of 15N-alanine labeled S4t were only obtained with SDS or LMPG. Assignment of resonances of 15N13C S4t has allowed us to solve its three-dimensional structure with the Rosetta program by using chemical shifts as restraints. S4t adopts in LMPG micelle a three helical fold, composed of a short helix at its N-terminus and a long helical hairpin. In a real membrane environment, this hairpin may be embedded in the membrane as two transmembrane segments, while the short helix may be located in the matrix.


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Investigation of the bioactive conformation of 26RFa, an orexigenic peptide

L. Guilhaudis1, A. Marotte1, I. Buchet1, B. Lefranc2, C. Neveu2, N. Chartrel2, H. Vaudry2, A. Ganesan3, J. Leprince2 and I. Ségalas-Milazzo1

1 COBRA, UMR CNRS 6014, IRIB, Université de Rouen, 76821 – Mont-Saint Aignan2 DC2N, INSERM U982, IRIB, Université de Rouen, 76821 – Mont-Saint Aignan3 School of Pharmacy, University of East Anglia, NR4 7TJ– Norwich

26RFa, a RFamide neuropeptide, is the ligand of the G-protein-coupled-receptor GPR103 and the system 26RFa/GPR103 is involved in feeding behaviour.[1] SAR studies on 26RFa truncated fragments indicated that, the deletion of the first nine residues does not markedly alter the biological activity (EC50: 26RFa = 10,4nM; 26RFa10-26 = 37,5nM) while the suppression of additional residues (EC50: 26RFa13-26 = 95,3nM; 26RFa16-26 =237nM; 26RFa20-26 =739 nM) provokes more important effects.[2]

In order to explain these differences, we determined the solution structures of 26RFa and several N-terminal truncated fragments (26RFa7-26, 26RFa11-26, 26RFa13-26 and 26RFa20-26) in a membrane mimetic medium. 26RFa possesses an alpha-helix (P4-K19), followed by a mixture of turns (S23-F26). Deletion of N-terminal residues led to destabilization (26RFa11-26) then loss of the helical conformation (26RFa13-26), the C-terminal structuration being conserved. In contrast, in 26RFa20-26, the structure of the region (S23-F26) is altered, the nature of the turns being modified.These results show that (i) a correct C-terminal structuration is necessary for GPR103 activation; (ii) part of the N-terminal helix is required to maintain the C-terminal conformations observed in 26RFa.To gain further insight into the importance of the helix on the biological activity, we used the Hydrogen Bond Surrogate method to synthesize a modified 26RFa11-26 in which this structural motif is stabilized by replacing a hydrogen bond by a hydrazone covalent link.[3] NMR studies showed that the modified peptide possesses a stabilized helix which is longer than in the original peptide, while the C-terminal region is unstructured. Nevertheless, 26RFa11-26 hydrazone linked analogue still exhibits a moderate biological activity (EC50: [JLNGhyd]26RFa11-26 = 233nM). This suggests that the binding and activation of GPR103 are not only due to a correct C-terminal structuration but also to the presence of an N-terminal helical motif.

References1. J.C. do Rego J. Leprince, N. Chartrel, H. Vaudry, J. Costentin, Peptides, 27, 2715 (2006); S. Takayasu, T. Sakurai,

S. Iwasaki, H. Teranishi, A. Yamanaka, S.C. Williams, H. Iguchi, Y.I. Kawasawa, Y. Ikeda, I. Sakakibara, K. Ohno, R.X. Ioka, S. Murakami, N. Dohmae, J. Xie, T. Suda, T. Motoike, T. Ohuchi, M. Yanagisawa, J. Sakai, Proc. Natl. Acad. Sci. U S A. (2006), 103, 7438.

2. O. Le Marec, C. Neveu, B. Lefranc, C. Dubessy, J.A. Boutin, J.C. Do-Rego JC, J. Costentin, M.C. Tonon, M. Tena-Sempere, H. Vaudry, J. Leprince, J. Med. Chem. (2011), 54, 4806.

3. E. Cabezas and and A. C. Satterthwait J. Am. Chem. Soc. (1999), 121, 3862.


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BMP-2 fragments: synthesis, structural characterization, binding properties and biological activity

L. Falcigno,1,2 G. D’Auria,1,2,3 L. Calvanese,3 D. Marasco,1,2 P. Brun,4 I. Castagliuolo4, R. Danesin5, A. Zamuner5 and M. Dettin5

1 Department of Pharmacy, University of Naples Federico II, 80134 Naples, Italy.2 Institute of Biostructure and Bioimaging (IBB) - CNR, 80134 Naples, Italy.3 CIRPEB - University of Naples Federico II, 80134 Naples, Italy.4 Department of Molecular Medicine, University of Padua, 35131 Padua, Italy.5 Department of Industrial Engineering, University of Padua, 35131 Padua, Italy.

Bone morphogenetic proteins (BMPs) play a key role in bone and cartilage formation and for this reason are employed in the field of tissue engineering to induce bone regeneration in damaged tissues.[1,2] To overcome drawbacks due to the use of entire proteins, synthetic peptides, derived from their parent BMPs and able to mimic their osteogenic properties, are widely exploited in the field of tissue engineering and have come out as promising molecules for biomaterial design.[3]

Here we report three peptides designed on the basis of experimental BMP-2:ActRIIB receptor complexes, reported in the literature.[4,5] These peptides were characterized by NMR and the structural features of the peptide:receptor binding interface were highlighted by docking simulations. Peptide-receptor binding affinities were evaluated by ELISA and Surface Plasmon Resonance (SPR) experiments. Moreover, cellular assays were performed to assess their osteoinductive properties. Among the peptides here proposed, a chimera peptide shows good binding affinity for the cognate ActRIIB receptor and osteoinductive properties.

Figure 1. Molecular model of chimera peptide:ActRIIB receptor complex.

References1. A. H. Reddi Cytokine Growth Factor Rev (2005), 16, 249.2. P. C Bessa, M. Casal, R. L. Reis J Tissue Eng Regen Med (2008), 2, 1.3. H. Senta, O. Drevelle, H. Park, N. Faucheux The Canadian Journal of Chemical Engineering (2011), 89, 227.4. G. P. Allendorph, W. W. Vale, S. Choe Proc Natl Acad Sci U S A (2006), 103, 7643.5. D. Weber, A. Kotzsch, J. Nickel, et al. BMC Struct Biol (2007), 7, 6.


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Lipidated peptides via post-synthetic thioalkylation promoted by molecular sieves

E. Calce1, M. Leone1, L. Monfregola2 and S. De Luca1

1 Institute of Biostructures and Bioimaging, National Research Council, 80134 Naples, Italy2 Department of Chemistry and Biochemistry, University of Colorado, Boulder, 80309 Colorado, United States

A chemoselective, convenient and mild synthetic strategy to modify peptides on a cysteine sulfhydryl group is described. It simply requires activated molecular sieves to selectively promote S-alkylation in presence of peptide nucleophilic functionalities. The procedure is easy to perform, fast and provides high yields even in case of poor electrophilic groups. In particular, it was exploited to provide peptides with useful functional groups (lipidic moieties), naturally occurring on proteins as post-translational modifications. The procedure was further implemented to synthesize tailor-made lipidated peptides, interesting tools to investigate biological processes involving their Ras parent proteins. Moreover, the one-pot preparation of multi-alkylated peptides confirms the versatility and flexibility of the employed methodology.

Figure 1. S-lipidation of peptide sequences

References1. L. Monfregola, M. Leone, E. Calce, S. De Luca Org. Lett. (2012) 14, 1664.2. E. Calce, M. Leone, L. Monfregola, S. De Luca Org. Lett. (2013), 15, 5354.3. M. Lumbierres, J.M. Palomo, G. Kragol, S. Roehrs, O. Müller, H. Waldmann Chem. Eur. J. (2005) 11,7405.4. K. Kuhn, D.J. Owen, B. Bader, A. Wittinghofer, J. Kuhlmann, H. Waldmann J. Am. Chem. Soc. (2001) 123, 1023.


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Cytotoxicity and interaction with cellular membranes of Trichogin GA IV analogs

M. De Zotti1, A. Dalzini2, M. Bortolus2, R. Fato3, C. Bergamini3, C. Peggion1, B. Biondi1 and A.L. Maniero2

1 Institute of Biomolecular Chemistry, CNR, Padova Unit, Department of Chemistry, University of Padova, 35131 - Padova2 Dipartimento di Scienze Chimiche, Università degli Studi di Padova, 35131 - Padova 3 Dipartimento di Farmacia e Biotecnologie FaBiT, Università di Bologna, 40126 - Bologna

Antimicrobial peptides (AMPs) have been recently studied for their potential use as anticancer agents. Their mechanism of action seems to be tied to membrane binding and disruption. Among AMPs, peptaibols,[1] rich in Cα-tetrasubstituted residues, are particularly attractive for their resistance to enzyme hydrolysis and their remarkable bioactivity.[2] The peptaibol trichogin GA IV (TG)[3] and its synthetic analogs showed promising anticancer activity. Here, we present the first results of our investigation aimed to explore the mechanism of action and the selectivity of TG analogs towards healthy or cancer cell lines. The analogs, synthesized by SPPS, were designed to examine the effects of charge, amphipathicity, helicity and conformation on their citotoxicity and selectivity towards different cell lines. The toxicity of all the analogs on a variety of healthy and cancer cell lines was tested using the MTT assay. The insertion of the fluorescein isothiocyanate probe at the N- or C-terminus enabled to visualize the peptide distribution in the cells using fluorescence microscopy. The insertion of the paramagnetic amino acid TOAC in the peptide sequence allowed the investigation of the peptide/membrane interaction via EPR spectroscopy.[4] EPR experiments have been performed on the TG analogs interacting with small unilamellar vesicles of different composition and with reconstituted cell membranes obtained from the cell lines tested in the MTT assay. For different membrane compositions we obtained different binding equilibria and insertion depth of the labeled peptides into the phospholipid bilayer. Those information could be related to different mechanisms of membrane disruption.

References1. C. Toniolo et al., Cell. Mol. Life Sci. (2001), 58, 1179.2. M. De Zotti et al., Amino Acids (2012), 43, 1761.3. M. De Zotti et al., Org. Biomol. Chem. (2012), 10, 1285.4. R.A. Jose et al., J. Pept. Sci (2011), 17, 377; V.N. Syryamina et al., J. Phys. Chem. (2010), 114, 1227; D. Marsh et al.,

Biophys. J. (2007), 92, 473.


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Functional binding surface of a β-hairpin VEGF receptor targeting peptide determined by NMR in living cells

D. Diana1, A. Russomanno1, R. Di Stasi1, L. D. D’Andrea1 and R. Fattorusso2

1 Istituto di Biostrutture e Bioimmagini , C.N.R., 80134 – Napoli (Italy).2 Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, Seconda Università di Napoli, 81100 – Caserta (Italy).

Nowadays, in cell NMR represents a very useful technique in system biology, allowing structural and functional study of biological macromolecules in their physiological environment.[1-3] To target membrane-bound receptors, several homonuclear NMR experiments, such as saturation transfer difference (STD) and transferred NOESY (trNOESY) have been extensively used on living cells, detecting binding events and providing information on the bound conformation of the ligands.[4,5] However, these techniques produce not sufficiently informative spectra when cells remain in suspension for few hours or membrane receptors lose soon their fold or activity. Finally, the epitope mapping analysis is hampered if the peptide presents numerous overlapping NMR signals. Herein, we structurally characterize in cellular environment the functional interaction of HPLW peptide[6] with VEGFRs, by using fast 15N-edited NMR experiments. To this aim, we produced 15N uniformly labelled recombinant HPLW peptide, which has been added to Porcine Aortic Endothelial Cells, overexpressing VEGFR2 (PAEC-VEGFR2). This allowed the acquisition of isotope-edited NMR experiments, including 15N relaxation measurements, to finely determine the in cell HPLW epitope recognized by the VEGFR2.

Figure 1. Schematic representation of peptide/receptor binding studies by means of NMR in living cell

References1. Y. Ito, P. Selenko Curr. Opin. Struct. Biol. (2010), 20, 640; 2. Z. Serber, A.T. Keatinge-Clay, A.E. Kelly, S.M. Miller, V. Dötsch J. Am. Chem. Soc. (2001), 123, 2446;3. D. Diana, G. Smaldone, P. De Antonellis, L. Pirone, M.e Carotenuto, A. Alonzi, S. Di Gaetano, M. Zollo, E. Pedone,

R. Fattorusso Chem. Eur. J. (2013), 19, 12217;4. B. Claasen, M. Axmann, R. Meinecke, B. Meyer J. Am. Chem. Soc. (2005), 127, 916;5. D. Potenza, F. Vasile, L. Belvisi, M. Civera, E. M. V. Araldi, ChemBioChem, 2011, 12, 695-699;6. D. Diana, G. A. Basile, L. De Rosa, R. Di Stasi, S. Auriemma, C. Arra, C. Pedone, M.C. Turco, R. Fattorusso, L.D.

D’Andrea J Biol Chem (2011), 286, 41680.


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Synthesis of modified peptides containing P1 arginine mimetics

I. Małuch, T. Łepek, P. Najda, M. Lewandowska, E. Sikorska and A. Prahl

University of Gdańsk, Faculty of Chemistry, Department of Organic Chemistry, 80-308 Gdańsk, Poland

Serine proteases play a crucial role in many physiological functions, such as protein activation, angiogenesis and immune response. Moreover, these enzymes are also involved in the development of a wide range of diseases, from bacterial and viral infections, to cancer or cardiovascular and neurodegenerative disorders. This suggests proteases to be potential drug targets for those diseases. The general strategy for therapeutically targeting serine proteases is to determine a specific inhibitor that interacts with the active site, potently blocking the activity of this enzyme. It has been proved, that peptidomimetics exhibit the pharmacokinetic properties needed to be proper as a drug[1].L-Arginine (Arg) residue is widely used for specific recognition of biomolecules, e.g. interactions between protein and DNA, RNA or even different protein[2,3]. There is known a variety of arginine mimetics, that incorporated in position P1 of the peptide chain form potent inhibitors of serine proteases. Moreover, these modified inhibitors are more stable in plasma because of their lower susceptibility to enzymatic degradation. Therefore, the replacement of Arg in position P1 of the inhibitor’s peptide chain with its decarboxylated mimetic - 4-amidinobenzylamine (Amba), is a promising concept for the development of effective strategy of blocking the activity of chosen serine proteases[4-6]. Those compounds could be used as potential therapeutic agents in the future.Here we present different ways of synthesis of peptides modified in the C-terminal part with Amba. According to obtained results we were able to select the most effective and convenient procedure of synthesis of analogues containing mentioned arginine mimetic in position P1.

References1. M. Drag, G.S. Salvesen Nat. Rev. Drug Discov. (2010), 9, 690.2. S. Balakrishnan, M.J. Scheuermann, N.J. Zondlo ChemBioChem (2012), 13, 259. 3. T.A. Dzimbova, P.B. Milanov, T.I. Pajpanowa J. Amino Acids (2013), doi: 10.1155/2013/407616.4. H. Gagnon, S. Beauchemin, A. Kwiatkowska, F. Couture, F. D’Anjou, C. Levesque, F. Dufour, A.R. Desbiens, R.

Vaillancourt, S. Bernard, R. Desjardins, F. Malouin, Y.L. Dory, R. Day J. Med. Chem. (2014), 51, 29. 5. M.Z. Hammamy, C. Haase, M. Hammami, R. Hilgenfeld, T. Steinmetzer ChemMedChem (2013), 8, 231.6. D. Meyer, F. Sielaff, M. Hammami, E. Böttcher-Friebertshäuser, W. Garten, T. Steinmetzer Biochem. J. (2013), 452,

331.


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De novo design of Nodal mimetic peptides L. Calvanese1, A. Sandomenico1,2, B. Carfora2,3, G. Focà 2,3, A. Focà 2,3, A. Caporale1, G. D’Auria,1,2,3

L. Falcigno,1,2,3 and M. Ruvo 1,2

1 CIRPEB - University of Naples Federico II, 80134 Naples, Italy.2 Institute of Biostructure and Bioimaging (IBB) - CNR, 80134 Naples, Italy.3 Department of Pharmacy, University of Naples Federico II, 80134 Naples, Italy.

Nodal, a member of the TGF-ß superfamily, is a potent embryonic morphogen also implicated in tumor progression.[1-3] Nodal signals through a receptor complex comprising a type I (ALK4) and a type II (ActRIIB) transmembrane serine/threonine kinase receptor. At variance with other proteins of the same family, Nodal can bind to the ALK4/ActRIIB receptor complex only in the presence of Cripto, the founding member of the epidermal growth factor-Cripto, FRL-1, and Cryptic (EGF-CFC) family.[4-6] Several studies have established that the Cripto EGF-like domain[5] binds to Nodal while the CFC binds to ALK4 [6] thus forming a large ALK4/Cripto/Nodal/ActRIIB receptor complex. Using the molecular model of the ternary complex containing ALK4/Cripto/Nodal, obtained by homology modeling and docking simulations,[7] we identified several Nodal residues potentially involved in the interaction with Cripto. On the basis of such information, synthetic peptides reproducing the Nodal binding regions have been designed, synthesized and structurally characterized in solution. Peptide-Cripto binding affinities have been comparatively evaluated by Surface Plasmon Resonance (SPR) experiments. The data so far collected by both conformational analysis and binding measurement provide useful information that confirm the reliability of the theoretical models and pave the way to the design of suitable antagonists.

Figure 1. Molecular model of ALK4/Cripto/Nodal complex.

References1. J. Brennan, D.P. Norris, E.J. Robertson Genes Dev.(2002), 16, 2339.2. P.M. Eimon, R.M. Harland Development (2002), 129, 3089.3. S. Nonaka, H. Shiratori,Y. Saijoh, H. Hamada Nature (2002), 418, 96.4. S. Parisi, D. D’Andrea, C.T. Lago, E. D. Persico, M. G. Minchiotti J Cell Biol (2003), 163, 303-14 5. S. G. Schiffer, S.Foley, A. Kaffashan, X. Hronowski, A. E. Zichittella, C. Y. Yeo, K. Miatkowski, H. B. Adkins, B.

Damon, M. Whitman, D. Salomon, M. Sanicola, K. P. Williams, J Biol Chem (2001), 276, 37769-78.6. C. Yeo, M. Whitman Mol Cell 2001, 7, 949-57.7. L. Calvanese, D. Marasco, N. Doti, A. Saporito, G. D’Auria, L. Paolillo, M. Ruvo, L. Falcigno Biopolymers (2010),

93, 1011-1021.


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Thermodynamics of interaction between a small peptide derived from glycoprotein gp36 of Feline Immunodeficiency Virus and model

membrane systemsR. Oliva1, L. Petraccone1, M. I. Stellato1, G. D’Errico1,2, L.Paduano1,2, A. D’Ursi3 and P. Del Vecchio1

1 University of Naples “Federico II” , Department of Chemical Sciences, 80126 – Naples - Italy2 CSGI (Consorzio per lo Sviluppo dei Sistemi a Grande Interfase) – Florence - Italy 3 University of Salerno, Department of Pharmaceutical Sciences, 84084 – Fisciano (SA) -Italy

The interactions between peptides and lipid bilayers are fundamental in a variety of key biological processes and among these the fusion process by viral proteins is one of the most important. A great number of viruses (e.g. human immunodeficiency virus) have a lipid bilayer rich in glycoproteins, which through a series of fission and fusion events, allows the infection of host cell to occur[1]. It is known that the glycoprotein gp 36 of the feline immunodeficiency virus (FIV) has a key role in the fusion process. Particularly, it was found that a small region of the membrane proximal external region (MPER) is of fundamental importance[2]. In this study, we report a physicochemical characterization of the interaction process between an octapeptide of that region, named C8, and some biological membrane models (liposomes) by using calorimetric and circular dichroism measurements. CD studies have shown that the peptide conformation changes upon binding to the liposomes. Interestingly the peptide folds from a disordered structure (in the absence of liposomes) to a more ordered structure with a low but significant helix-content. ITC measurements have shown that the interaction between C8 and liposomes strongly depends on the lipid composition. Particularly, it interacts with liposomes composed by POPC, POPC/POPG and POPC/SM whereas no interaction was observed with the Chol-containing membranes. DSC studies with liposomes of DPPC, DPPC/Chol and POPC/SM indicated that the peptide interacts preferentially with zwitterionic polar headgroups affecting dramatically the thermotropic properties of the liposomes. In conclusion this study can provide interesting insights into the role of this short fragment in the infection process.

POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPG: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol; DPPC: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; SM: sphingomyelin;Chol: cholesterol.

References1. A. Merlino, G. Vitiello, G. Grimaldi, F. Sica, E. Busi, R. Basosi, A.M. D’Ursi, G. Fragneto, L. Paduano, G. D’Errico

J. Phys. Chem. B 2012, 116, 401-4122. G. Vitiello, G. Fragneto, A.A. Petruk, A. Falanga, S. Galdiero, A.M. D’Ursi, A. Merlino, G. D’Errico Soft Matter,

2013, 9, 6442-6456


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Capillary electrophoresis as a tool for analysis of the Conus geographus venom profile: proof-of-concept study

A. Walewska1, T. Łepek1, G. Bulaj2, A. Prahl1

1 University of Gdańsk , Faculty of Chemistry, Department of Organic Chemistry, 80-308 Gdańsk, Poland 2 University of Utah, Department of Medicinal Chemistry, L.S. Skaggs Pharmacy Institute, Salt Lake City, USA

The cone snails (genus Conus) are venomous marine molluscs that use peptide toxins for rapid prey immobilization, defense and competitor deterrence. The venoms of cone snails encloses bioactive peptides and small proteins, called conotoxins or conopeptides, that selectively and potently modulate a broad range of biological targets including ligand- and voltage-gated ion channels, G protein coupled receptors and transporters[1]. Conus sp. contain up to a few hundred peptides, with <1% of these conopeptides presently pharmacologically characterized. Their structural diversity and relatively small size, enhanced by the presence of a large number of post-translational modifications, contribute to the value of conopeptides as both research tools and leads to new therapeutics[2].Over the last years capillary electrophoresis (CE) has become an efficient separation technique to perform analysis of diverse compounds, including peptides in biological samples, blood, plasma, urine, cerebral-spinal fluid and tissues[3].In this preliminary study, we selected venom of Conus geographus to analyze its profile using capillary zone electrophoresis (CZE). Different pH of separation buffers and synthesized markers permitted us to optimize conditions for the analysis of the crude venom. The initial results of this project, allow continuing studies of cone snail venoms for tracking and profiling conotoxin-based drug candidates using capillary electrophoresis.

References1. H. Terlau, B.M. Olivera, Physiol Rev. (2004), 84, 41-68.2. B.M. Olivera, R.W. Teichert, Mol Interv. (2011), 7, 251-260.3. A.C. Moser, D.S. Hage, Electrophoresis (2008), 29, 3279–3295.


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Dynamic interplay of the N- and the C-terminal domains in KCTD5D. Barone1,2, N. Balasco, 1,2, L. Esposito1 and L. Vitagliano1

1 Institute of Biostructures and Bioimaging, C.N.R., 80134 Napoli, Italy.2 Second University of Napoli, 81100 Caserta, Italy.

Recent investigations have highlighted a key role of the proteins of the KCTD family in several fundamental biological processes. Despite the growing importance of KCTDs, our current understanding of their biophysical and structural properties is very limited. Biochemical characterizations of these proteins have shown that most of them act as substrate adaptors in E3 ligases during protein ubiquitination. In the last few years, we have undertaken a biophysical characterization of these proteins by combining computational and experimental approaches [1-4]. In this framework, we have recently characterized the interactions between the BTB domain of KCTD5 and Cullin3 [5]. Here we report molecular dynamics studies on the KCTD5 C-terminal domain and on the full-length protein. The analysis of the C-terminal domain indicates that it is intrinsically able to form stable pentameric assemblies. The inspection of the dynamics of the full-length protein unveils a twisting relative motion of the two domains which holds interesting functional implications. Moreover, molecular modeling of KCTD5 with its numerous partners leads to a crowded pentameric assembly in which the dynamical behaviour of all components is essential for function.

Figure 1. Model of KCTD5 pentamer (red) in complex with its biological partners : Cullin3 (blu), Cand1 (yellow), Rbx1 (Orange), E2 (green), and Ubiquitin (magenta).

References1. G. Canettieri et al. Nature Cell Biology (2010) 12:132-42.2. S. Correale et al. Biochimie (2011) 93:715-243. L. Pirone et al. Biomed Res Int. (2013) 2013:1626744. S. Correale et al. J Mol Recognit (2013) 26:488-955. N. Balasco et al. BBA Proteins and Proteomics (2014). In press.


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Loop insertions in helices: a novel structural motifN. Balasco 1,2, R. Riccio1 , D. Barone1,2, A. De Simone3, A. Ruggiero1 and L. Vitagliano1

1 Institute of Biostructures and Bioimaging, C.N.R., 80134 Napoli, Italy.2 Second University of Napoli, 81100 Caserta, Italy.3 Imperial College, London, UK.

Hydrogen bonding interactions play a crucial role in the stabilization of both global and local structures of proteins. Each secondary structure element is characterized by a specific hydrogen bonding pattern that connects either consecutive (helices) or distant residues (sheets) within the sequence. The well known H-bond pattern of the α-helix is characterized by the repetitive bonding of the CO group of the residue i with the NH group of the residues i+4. In order to identify novel motifs in protein structures, we here evaluated the occurrence of H-bondings between two pairs of residues whose distance in the sequence is larger than that observed in α- or pi-helices. This analysis reveals the occurrences of some previously un-described motifs. In particular, the survey highlights the occurrence of fifty-eight large insertions within helices in an ensemble of non-redundant PDB structures. These insertions were located either at the middle or at the termini of the helices. Interestingly, the inserted residues could adopt different type of structures. In particular, they could (a) present an irregular loop structure, (b) form ß-hairpins or (c) be completely disordered in the crystal structure. The analysis of the conservation of these typically exposed motifs in classes of homologous proteins indicates that they are generally well preserved although their length and/or amino-acid composition are characterized by a significant variability. On the basis of these findings innovative protein scaffolds may be designed.

Figure 1. Examples of different structural motifs inserted in protein helices


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Research of potent inhibitors of furin modified in position P5M. Lewandowska1, A. Kwiatkowska2, C.Levesque2, R. Desjardins2, R. Day2, A. Prahl1

1 University of Gdańsk, Faculty of Chemistry, Laboratory of Biopolymers, Gdansk, Poland2 Universite de Sherbrooke, Institut de Pharmacologie de Sherbrooke, Sherbrooke, Canada

Many cellular proteins including receptors, hormones, zymogens, enzymes are synthesized as inactive precursors. These proforms are transform into biologically active compound by furin and furin-like proteinases (proprotein convertases, PCs). Moreover, the PC family is implicated with activation of protoxins secreted by various bacteria and viruses (including Pseudomonas aeruginosa, anthrax toxins, pathogenic Ebola strains, the avian influenza virus hemagglutinin, HIV gp160).[1,2,3] A more information about the cleavage preferences of furin would assist in a better understanding of the functional significance of furin proteolysis in normal development relative to disease. No natural protein inhibitors of furin are known so the only way to found potent and selective inhibitors is modified derivatives of original inhibitory peptide.The Ac-RARRRKKRT-NH2 inhibitor is based on modified sequences derived from the hemagglutinin H5N1 avian influenza virus.[4] This model peptide compound is furin inhibitor against HA H5N1, antrax and Pseudomonas aeuriginosa. The positions P5-P8 in this modelled inhibitor is responsible for the selectivity and the interaction of substrates and inhibitors of PCs. The position P5 is substituted with one of the nineteen encoded amino acid residues. Modification at this position dramatically changing the activity of the enzyme.The series of peptide analogues were synthesized on a continuous flow peptide synthesizer with standard Fmoc/tBu strategy. We believed that our work will provide useful information about the specificity of proprotein convertases.

References1. G. Thomas Nat Rev Mol Cell Biol (2002), 3, 753-7662. A.Basak, M. Zhong, J. S. Munzer, M. Chretien, N. G. Seidah Biochem J (2001), 353, 537-5453. R.J. Collier, J.A. Young Annu Rev Cell Dev Biol (2003), 19, 45-704. A.G. Remacle, K. Gawlik, V.S. Golubkov, G.W. Cadwell, R.C. Liddington, P. Cieplak, S.Z. Millis, R. Desjardins, S.

Routhier, X.W. Yuan, W. A.Neugebauer, R. Day, A.Y. Strongin Int J Biochem Cell Biol (2010), 42, 987-995

AcknowledgementThis work was supported by the National Science Center in Poland with the grant no. UMO-2012/07/N/ST5/01998.


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Mimicking SERPINH1 chaperone function in collagen type I biosynthetic pathway via the TASP approach

S. Pascarella1,3, F. Melani1, L. Giovannelli2, P. Rovero1,3

1 Dept. NEUROFARBA, Section of Pharmaceutics and Nutraceutics, I-50019 Sesto Fiorentino, Italy2 Dept. NEUROFARBA, Section of Pharmacology and Toxicology, I-50139 Florence, Italy3 Laboratory of Peptide and Protein Chemistry and Biology, University of Florence, I-50019 Sesto Fiorentino, Italy

Collagen superfamily comprises twenty-eight collagen types involved in a broad range of functions, whose molecular hallmarks are the multiple repetition of the Gly-X-Y motif and the unique triple helical structure; among collagens, type I can be considered as the archetypal fibrillar collagen, characterized by ubiquitous occurrence and provided with a predominant structural role. The 47 kDa serine protease inhibitor Hsp47 plays a key role in collagen superhelix folding process, recognizing and transferring procollagen molecules from the ER to the Golgi apparatus.[1,2] The ability of Hsp47 to bind selectively its triple helical client resides on a long deep cleft whose base is formed by a ß-sheet,[3,4] termed B according to Huber and Carrell labelling system.[5] We are proposing a Template Assembled Synthetic Protein (TASP) as an innovative pharmacological tool to explore Hsp47 actual impact on collagen turnover, with the purpose to investigate its role in collagen-related diseases, such as Ehlers-Danlos syndrome and Osteogenesis Imperfecta. According to the TASP approach,[6,7] an appropriate synthetic scaffold should act as a built-in device for suitably selected peptide blocks from the functional part of Hsp47, mimicking the native folding of the protein chain. To this aim, with the support of molecular modelling techniques, we designed a small library of TASPs, each possibly mimicking the ß-sheet B conformation and function, and we selected the most promising molecule either in terms of RMSD values, or in terms of docking energy estimation, employing a synthetic homotrimeric collagen model.[4] The next steps will be the systematic synthesis of the selected TASP, followed by in vitro biological activity evaluation, taking into account both the collagen binding ability and the modifications in collagen type I production induced in cultured Normal Human Dermal Fibroblasts. The latter will be measured through Western Blot analysis of cell lysates, and solid-phase ELISA on culture media.

References1. T. Koide, K. Nagata, Top. Curr. Chem. (2005), 247, 85.2. A. Nakai, M. Satoh, K. Hirayoshi, K. Nagata, J. Biol. Chem. (1992), 117, 903. 3. J.W. Davids, T.S. El-Thaher, A. Nakai, K. Nagata, A.D. Miller, Bioorg. Chem. (1995), 23, 227.4. C. Widmer, J.M. Gebauer, E. Brunstein, S. Rosenbaum, F. Zaucke, C. Drögemüller, T. Leeb, U. Baumann, Proc. Nat.

Acad. Sci. USA (2012), 109, 13243.5. R. Huber, R.W. Carrell, Biochemistry (1989), 28, 8951.6. M. Mutter, S. Vuilleumier, Angew. Chem. Int. Ed. Engl. (1989), 28, 535.7. M.Mutter, P. Dumy, P. Garrouste, C. Lehman, M. Mathieu, C. Peggion, S. Peluso, A. Razaname, G. Tuchscherer,

Angew. Chem. Int. Ed. Engl. (1996), 35, 13.


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Peptides mimicking a discontinuos VEGF binding epitope

L. De Rosa1, F. Finetti2, L. Morbidelli2, M. Ziche2 and L.D. D’Andrea1

1 Istituto di Biostrutture e Bioimmagini, CNR, 80134 – Napoli2 Dipartimento di Science della vita, Università di Siena, 53100 – Siena

All biological processes are finely regulated by a network of protein-protein interactions whose characterization at the molecular level can promote the design of novel drug therapeutics working as protein binding modulators. The development of such molecules remains a challenging task because of the complex nature of protein binding sites, which often consist of multiple discontinuous epitopes. Peptide-based mimics of protein binding sites designed to reproduce multiple discontinuous epitopes are promising candidates for this purpose. Here we describe the design, synthesis, structural and biological characterization of a series of peptides mimicking two binding epitopes of Vascular Endothelial Growth Factor (VEGF) with its receptor (VEGFR1).VEGF is the main regulator of angiogenesis, a fundamental process for healing, reproduction and embryonic development. It involves the growth of new blood vessels from pre-existing vessels and it is intimately associated with endothelial cells migration and proliferation. Angiogenesis is strictly tuned by several pro- and anti-angiogenic factors. An imbalance between such factors contributes to the onset, development and progression of several common and lethal human diseases, including cancer, cardiovascular disorders, retinal degeneration and chronic inflammation.[1, 2] Thus, the design of new, safe and effective angiogenic modulators is gaining a big interest for therapeutic and diagnostic applications.[3, 4, 5] On the basis of the crystal structure of VEGF in complex with VEGFR[6], we designed six peptides mimicking two discontinuous binding epitopes of VEGF (helix 17-25 and ß-hairpin 79-92). The two linear epitopes were synthesized by solid-phase peptide synthesis and conjugated by chemical ligation trough amino acid spacers of variable length. Their biological activity has been investigated and they will be structurally characterized by NMR.

References1. Y. Cao, Sci. Signal. (2009), 2(59).2. L.D. D’Andrea et al. Curr. Pharm. Des. (2009), 15, 2414-2429.3. L.D. D’Andrea et al. PNAS (2005), 102(40), 14215-20.4. D. Diana et al. J. Biol. Chem. (2001), 286(48), 41680-91.5. L. De Rosa et al. Eur. J. Med. Chem. (2014) 73, 210-6.6. C. Wiesmann et al. Cell. (1997) 91(5), 695-704.


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Conformational studies of nucleophosmin C-terminal leukemia-associated regions: new insights from protein dissection approach

Concetta Di Natale1,2, Pasqualina Liana Scognamiglio1,2, Valentina Punzo1, Anne Lise Ferrara1, Marilisa Leone3,

Luigi Vitagliano3 and Daniela Marasco1

1 Department of Pharmacy, CIRPEB, DFM, University of Naples “Federico II”, 80134, Naples, Italy2 Center for Advanced Biomaterials for Healthcare@CRIB, Istituto Italiano di Tecnologia (IIT), Largo Barsanti e Matteucci 53, 80125 Naples, Italy3 Institute of Biostructures and Bioimaging, National Research Council, 80134, Naples, Italy

Nucleophosmin (NPM1, B23) is a multifunctional protein that is involved in a variety of fundamen-tal biological processes. NPM1/B23 deregulation is implicated in the pathogenesis of several human malignancies. This protein exerts its functions through the interaction with a multiplicity of biological partners [1]. Notably, NPM1/B23 has been identified as the most frequently mutated gene in acute my-eloid leukemia (AML) patients, accounting for approximately 30% of cases [2]. Structural characteriza-tions of NPM1 have shown that the protein is endowed with a modular structure.The C-terminal domain (CTD) forms a globular structure consisting of a three helix bundle. The de-stabilization of this structural unit abolishes the nucleolar localization of the protein and has been the subject of a number of intriguing investigations aimed at unveiling its folding mechanism [3]. Recently we have highlighted portions mainly involved in G-quadruplex DNA recognition mechanism [4]. Here we have further investigated the structural determinants of CTD folding process focusing on the region encompassing the second helix of the bundle.To this purpose we have designed and synthesized several peptides corresponding to the entire wt se-quences and other truncated forms and analyzed their structural properties by means of circular dichro-ism (CD), nuclear magnetic resonance (NMR) and fluorescence spectroscopies.

References1. Scaloni F. et al. Deciphering the folding transition state structure and denatured state properties of nucleophosmin

C-terminal domain. (2010) Proc Natl Acad Sci USA. 107:5447-52. 2. Falini B. et al. Altered nucleophosmin transportin acute myeloid leukaemia with mutated NPM1: molecular basis

and clinical implications. (2009) Leukemia. 10:1731-43.3. MarascoD. Et al. Role of mutual interactions in the chemical and thermal stability of nucleophosmin NPM1

domains. (2013) Biochem Biophys Res Commun. 430:523-528.4. Scognamiglio P.L. et al. G-quadruplex DNA recognition by nucleophosmin: New insights from protein dissection.

(2014) Biochim Biophys Acta. 6:2050-2059.


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Local backbone geometry and conformational preferences of amino acids

N. Balasco1,2, L. Esposito1, A. De Simone3 and L. Vitagliano1

1 Institute of Biostructures and Bioimaging , CNR, via Mezzocannone 16, I – 80134 Napoli, Italy2 Second University of Naples, Via Vivaldi 43 – 81100 Caserta, Italy3 Division of Molecular Biosciences, Imperial College South Kensington Campus London SW7 2AZ, UK

The classic experiments of Anfinsen have established that the 3D structure of proteins is dictated by their amino acid sequence [1]. The definition of the structural basis of the conformational preferences of the genetically encoded amino acids is crucial to decipher the folding code and would have a huge impact on our understanding of protein structure. Indeed, although a large number of computational and experimental investigations have highlighted that the different amino acid residues are endowed with distinct conformational propensities, none of the current hypotheses is able to satisfactorily explain these preferences [2-4]. In order to gain insights into this intricate issue, we here determined and compared the amino acid propensity scales for different (φ,ψ) regions of the Ramachandran plot and for different secondary structure elements (E, G, H, PPII). Propensities of each residue were calculated by using the Chou-Fasman approach on a dataset of 4731 non-redundant protein chains (sequence identity ≤ 25%, resolution better than 2.2 Å, R-factor lower than 0.20). Similarities between propensity scales of different (φ,ψ) regions were evaluated by linear regression analyses in terms of the correlation coefficient r. One of the most striking and unexpected findings emerged from this study is that distant (φ, ψ) regions of the Ramachandran plot occasionally exhibit significantly similar propensity scales. On the other hand, contiguous regions of the Ramachandran plot present anti-correlated propensities. These results are corroborated by the analysis of the propensity scales for different secondary structure elements such as helices and ß-sheets. These comparisons unveiled some previously undetected (anti)correlations. In order to provide an interpretative background to these results, we evaluated the role that the local variability of protein backbone geometry plays in this context. Our analysis indicates that (dis)similarities of propensity scales between different regions of the Ramachandran plot are coupled with (dis)similarities in the local geometry. In recent years, many investigations have highlighted the influence of the conformation on the protein local geometry [5] . The present findings reverse this concept by showing that the local geometric requirements have a significant impact on the preference of individual amino acids for specific conformational states.

References1. CB. Anfinsen, HA. Scheraga Adv Protein Chem. (1975), 29:205-300.2. TP. Creamer, GD. Rose Proc. Natl.Acad. Sci. USA. (1992), 89(13):5937-41. 3. AG. Street, SL. Mayo Proc. Natl.Acad. Sci. USA. (1999), 96(16):9074-6.4. F. Avbelj, RL. Baldwin Proc. Natl.Acad. Sci. USA. (2003), 100(10):5742-7.5. L. Esposito, N. Balasco, A. De Simone, R. Berisio, L. Vitagliano Biomed Res Int. (2013) 2013:326914.


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Structured-based optimization of AIF(370-394), an inhibitor of the AIF/CypA lethal complex

F. Mascanzoni1,2, B. Farina1, A. Caporale1, G. Di Sorbo1,3, R. Fattorusso3, M. Ruvo1and N. Doti1

1 IBB-CNR, CIRPEB University of Naples “Federico II”, 80134, Naples, Italy2 Faculty of Pharmacy, University of Naples “Federico II”, 80131, Naples, Italy3 Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, 81100, Caserta, Italy

Apoptosis inducing factor (AIF) is a bifunctional mitochondrial flavoprotein critical for energy metabolism and induction of caspase-independent apoptosis in neurons.[1] AIF is inserted into the inner mitochondrial membrane where it exerts oxidoreductase activity.[1] Upon apoptotic stimuli, the membrane linker of AIF becomes proteolyzed and the released Δ1–101 domain moves to nuclei, where it triggers chromatin condensation, large-scale DNA fragmentation and cell death by its direct interaction with the protein cyclophilin A (CypA).[2] Once released from mitochondria, AIF binds CypA in the cytosol and then they together translocate into nuclei, generating a lethal DNA-degrading complex.[2]

In a previous study, we showed that the aminoacid region of AIF spanning residues 370-394 mediates the protein complex between AIF and CypA.[3] The synthetic AIF(370-394) peptide inhibits the complex formation in vitro by binding CypA with a KD of 12 µM. Moreover, the peptide provides neuroprotective effects to a similar extent as CypA-siRNA in a model of AIF-mediated neuronal cell death.[3] Data obtained clearly demonstrated that the selective inhibition of AIF-CypA complex formation is an effective strategy of neuroprotection.

In the present study, using the peptide AIF(370-394) as starting template, a structure-based approach was applied for the design and optimization of new selective inhibitors of the AIF-CypA complex. In particular, by means of 1H Saturation Transfer Difference NMR experiments, the AIF(370-394) residues crucial association to CypA were identified. These data provided the rationale for restricting the template to a shorter peptide, which showed a dose-dependent binding to CypA with a KD value in the µM range as detected by biochemical assays. The peptide here identified represents an improved template for the design of higher affinity compounds more suitable for cell-based and in vivo assays.

References1. I.F. Sevrioukova J Mol Biol. (2009), 390, 924–38.2. JE. Slemmer Am J Pathol (2008), 173, 1795–1805.3. N. Doti, et al. Cell death and Diseas. (2014) e993. doi: 10.1038/cddis.2013.518.


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Structural insights on the recognition of AIF by CypA revealed by NMR spectroscopy

B. Farina1, G. Di Sorbo1,2, F. Mascanzoni1,3, R. Fattorusso2, M. Ruvo1, N. Doti1

1 IBB-CNR, CIRPEB University of Naples “Federico II”, 80134, Naples, Italy2 Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, 81100, Caserta, Italy3 Faculty of Pharmacy, University of Naples “Federico II”, 80131, Naples, Italy

Apoptosis-inducing factor (AIF) is a highly conserved, phylogenetically old mitochondrial flavoprotein implicated in embryonic development, cardiac cell survival, carcinogenesis and neurodegenerative disorders.[1] In healthy mitochondria, the mature form of AIF consists of residues 102–612, named AIF(Δ1–101), which exerts vital functions.[1] Upon apoptotic stimuli, AIF is released from mitochondria in its lethal form, AIF(Δ1–121), and moves to the nucleus where triggers chromatin condensation and large scale DNA fragmentation.[2] This process is mediated by the interaction of AIF with other proteins including cyclophilin A (CypA). Previous data demonstrated that the inhibition of AIF/CypA complex formation in neuronal cells is an effective strategy to block the lethal action of AIF without interfering with its vital functions.[2,3] Therefore, the development of antagonists could be a promising approach for the pharmacotherapy of neurodegenerative diseases. In order to define the molecular basis of the recognition of AIF by CypA, we have characterized, by means of NMR spectroscopy, the interaction of CypA with a smaller region of AIF, encompassing residues 370-394, named AIF(370-394).[3] This peptide has strong affinity for CypA and provides neuroprotective effects in a model of AIF-mediated neuronal cell death by inhibiting the formation of the AIF-CypA lethal complex.[3] In particular, Chemical Shift Perturbation analyses were performed by acquiring 2D [15N, 1H] HSQC spectra of 15N-labeled CypA in presence of the AIF peptide allowing us to define the AIF(370-394) binding site on CypA. Detailed insights on the structural determinants underlying the interaction of AIF with CypA provide the basis for building an AIF-CypA complex model and for the development of new compounds able to modulate this relevant molecular interaction.

References1. B. M. Polster, Neurochem Int. (2013), 62, 695–702.2. N. Plesnila, et al, J Cereb Blood Flow Metab. (2004), 24, 458–466.3. N. Doti, et al, Cell death and Diseas. (2014), e993. doi: 10.1038/cddis.2013.518.


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Cis-trans prolyl isomerase activity: new internally quenched fluorogenic substrates for HTS assay

G. Di Sorbo1,2, A. Caporale1, B. Farina1, F. Mascanzoni1,3, M. Ruvo1 and N. Doti1


Peptidyl prolyl cis-trans isomerases (PPIases) catalyses the slow, rate-limiting cis→trans isomerisation of peptidyl-prolyl bonds.[1] PPIases have been associated with many physiological and pathophysiological processes, including protein folding, cancer, neurodegeneration, inflammation and infectious diseases.[1] Great efforts are spent for the development of robust and sensitive assays, useful for selecting specific PPIase modulators for therapeutic approaches. The catalytic activity of PPIases is normally monitored spectrophotometrically by using the chymotrypsin-coupled assay. This assay is based on the conformational specificity of chymotrypsin, which cleaves the 4-p-nitroanilide (pNA) moiety from succinyl-Ala-Xaa-Pro-Phe-p-NA only when the Xaa-Pro is in the trans conformation. In the presence of PPIases, the Xaa-Pro bond is more rapidly converted to the trans conformation, which is readily cleaved by chymotrypsin leading to the formation of the colored product p-nitroaniline.[1] This assay has several limitations. For example, since only 10% of the total substrate in solution is in the cis conformation, the signal-to-noise (S/N) ratio is low, and potentially causes false negatives with poorly active compounds. This background is generally minimised by dissolving the substrate in organic solvent such as TFE.[1]

Here we set up a chymotrypsin-coupled HTS assay using a new internally quenched fluorogenic substrate. The new substrate, mostly in cis conformation in aqueous solution, as detected by NMR, incorporates the fluorophore EDANS at one end and an EDANS-quenching moiety (Dabcyl) on the other. Following the cis-trans isomerization catalyzed by a PPIase and the subsequent cleavage by chymotrypsin, the peptide-EDANS product is released interrupting the FRET effect and yielding bright fluorescence, which can be detected using excitation wavelengths at 335-345 nm and emission wavelengths at 485-510 nm.[2] In order to assess its usefulness in High Throughput Screenings, we have optimized the method on 384-well plates and automated the screening process on a HTS platform. The automated method has been optimized and validated by testing known PPIase inhibitors and will be used for the screening of chemical libraries to find out new inhibitors of PPIases of therapeutic interest.

References1. J.L. Kofron, et al. Biochemistry (1991), 30, 6127-6134.2. N. Doti et al. Mol Biotechnol. (2013), 54, 283-29.


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Defining the minimal interacting region in Cul3-BTB complexesSmaldone G.1, Pirone L.2, Balasco N.1,3, Vitagliano L.1, Di Gaetano S.1, Pedone E.1

1 Institute of Biostructures and Bioimaging, C.N.R., 80134 Napoli, Italy.2 Institute of Crystallography, C.N.R., 70126 Bari, Italy 3 Second University of Napoli, 81100 Caserta, Italy

Cullin 3 (Cul3) is a major player in protein ubiquitylation and regulation. Cul3 operates by binding protein substrate adaptors through their BTB domain. Among the potential adaptors recognized by Cul3 a great attention has been recently devoted to the class of proteins denoted as KCTDs. Although the structural and the biochemical characterization of these proteins is still very limited, several important studies have shown the implication of these proteins in the insurgence of severe human diseases [1, 2, 3, 4]. Although some members of the family are involved in processes not related to protein degradation[5], it has been suggested that many other KCTDs should be able to interact with Cul3 [6]. In order to gain new insights into KCTD functionality we performed computational and biophysical studies on the interaction between Cul3 and BTB domains of different KCTD proteins. Our findings clearly indicate that different members of the family present distinct affinity for Cul3. Some of the members present high affinity for the cullin (KD in the range of 40-80 nM). This high affinity has been related to the large surface of interaction of Cul3 with KCTDs. On the other hand, other members of the family do not interact with the cullin. Interestingly, the ability of KCTDs to form tight complexes with Cul3 is not related to a specific oligomeric state. Moreover, KCTD proteins that bind Cul3 do not display an increased sequence similarity compared to those that do not interact with the cullin. This indicates that KCTDs have lost the ability to bind Cul3 in different times along their evolution. A comparative analysis of different KCTD sequences have highlighted a crucial role played by the valuable and flexible loop α2ß3. Indeed, the characterization of KCTD chimeras demonstrates that swapping of the α2ß3 loop between KCTDs that are Cul3 binders and KCTDs that do not bind Cul3 is sufficient to abolish their ability to bind to cullin.

References1. Canettieri G. et al. Nat. Cell Biol. 2010 2. Golzio C. et al. Nature 2013.3. Tong X. Et al. Nature comm. 20134. Pfeiffenberger C. et al. Plos Genetics 20125. J. Schwenk et al. Nature 20106. Skoblov M. et al. Bioessays 2013


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Dissecting domain swapping in the Arginine Binding Protein isolated from Thermotoga maritima

A. Ruggiero1, R. Berisio1 , S. D’Auria2, and L. Vitagliano1

1 Institute of Biostructures and Bioimaging, C.N.R., 80134 Napoli, Italy.2 Institute of Protein Biochemistry, C.N.R., Napoli, Italy.

The Arginine Binding Protein isolated from T. maritima (ArgBP) is an intriguing protein endowed with striking biophysical and structural properties [1,2] . It presents an extraordinary thermal/chemical stability that extends to the protein dimer, whose dissociation requires harsh denaturing conditions. Moreover, the three-dimensional of ArgBP represents a unique example in which domain swapping is coupled with large variations of tertiary and quaternary structures (Figure) [2]. Therefore, ArgBP constitutes a remarkable model for studying basic concepts related to domain swapping, a widespread event among globular proteins. With this aim, we designed and characterized different mutants of the protein. In particular, we dissected and individually characterized two portions of the protein (a) its main body devoid of the swapping fragment body (ΔC-234ArgBP) and (b) the C-terminal peptide corresponding to the swapping element. Although the removal of the C-terminal helix produces a significant destabilization of the protein, we could perform both thermodinamical and structural studies on the truncated ΔC-234ArgBP. The structural characterization of the C-terminal peptide shows that it presents a very limited structure in solution. These analyses, coupled with binding experiments performed on ΔC-234ArgBP and the C-terminal peptide, provide interesting clues on the recognition mechanism of the swapping element with the main body of the protein.

Figure 1. Large tertiary and quaternary structural rearrangements of ArgBP upon ligand binding

References1. Scirè et al. Mol. Biosyst. , (2010), 6, 687.2. A. Ruggiero et al. Plos One (2014), in the press.


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Inhibition of APEH with a clickA. Focà1, V. Celentano2, A. Sandomenico2, D.L. D’Andrea2 and M. Ruvo2,3

1 University of Naples “Federico II”, Department of Pharmacy, 80134 – Naples, Italy2 CNR, Institute of Biostructure and Bioimaging, via Mezzocannone 16, 80134 – Naples, Italy3 CIRPeB, University of Naples “Federico II”, via Mezzocannone 16, 80134 – Naples, Italy

Acyl peptide hydrolase (APEH), one of four members of the prolyl oligopeptidase family of serine proteases (POP, clan SC, family S9)[1], is an enzyme that catalyzes the removal of acyl groups from N-terminally acylated peptides and proteins[2] to generate an acylamino acid and a peptide with a free NH2 terminus that is shortened by one amino acid. APEH plays a role in the protein degradation machinery and as secondary antioxidant defence system for proteins damaged by oxidative stress[3], however the precise biological activity of APEH is unknown[6]. Recent studies suggest that APEH is involved in several diseases, including cancer, inflammation, cardiovascular, haematological, neurological and urological disease[5], therefore this enzyme holds the promise to become an innovative, multipurpose, therapeutic target. To identify novel compounds able to inhibit the APEH enzyme activity, we screened click-generated peptide libraries (I° generation library), containing 1,4-disubstituted 1,2,3-triazole-derivatives[4] with a common Trpzip2 like-peptide structure[5]. Using a simple enzyme inhibition assay[6], we previously selected a clicked peptide named NHB3-3, which is able to reduce in a dose-dependent manner the enzyme activity with an IC50 of 10.5 ± 1.7 µM [7] through a non-competitive inhibitory mechanism. To better investigate the structure-activity relationship, trying at the same time to identify new more potent inhibitors, we planned a size-reduction strategy and designed a number of new analogues with shortened flanking peptide sequences. We found that all shorter NHB3-3 clicked peptides (II° generation of NHB peptides) are not so efficient as the precursor compound to inhibit enzyme activity. These findings confirm that the 1,2,3-triazole ring is minimally required to inhibit APEH enzyme and suggest that chemical or conformational modifications of the Trpzip2 –like peptide portion might contribute to the inhibitory activity of NHB3-3[5].

References1. Rea D, Fulop V (2006). Cell Biochem Biophys 44: 349–365.2. Palmieri, G.; Bergamo, P.; Luini, A.; Ruvo, M.; Gogliettino, M.; Langella, E.; Saviano, M.; Hegde, R.; Sandomenico,

A.; Rossi, M. PLoS One 2011, 6(10), e25888. 3. Olmos, C.; Sandoval, R.; Rozas, C.; Navarro, S.; Wyneken, U.; Zeise, M.; Morales, B.; Pancetti, F.. Toxicol. Appl.

Pharmacol. 2009, 238, 37-46. 4. Adibekian A, Martin BR, Wang C, Hsu KL, Bachovchin DA, Niessen S, Hoover H, Cravatt BF. s. Nat. Chem. Biol.

2012 Mar;8(3):318.5. Celentano V., et al Chem. Commun. (2012), 48, 762-764.6. Sandomenico A, et al., J. Med. Chem. 2012, Mar 8;55(5):2102-217. Sandomenico A. , 13th Naples Workshop, 7-10 june 2012 Poster P32


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Site-specific mono-pegylation of glucagon-like peptide 1 and its mutants using prokaryotic microbial transglutaminase

Carla Marra1*, Fabio Selis1, Silvia Scaramuzza 1, Pierluigi Onali2, Rodolfo Schrepfer1, and Giancarlo Tonon1

1 Bioker srl-Multimedica Group, c/o CNR-IGB, Via P. Castellino 111, 80131 Naples, Italy 2 Section of Biochemical Pharmacology, Department of Neuroscience, University of Cagliari, 09042 Monserrato (Cagliari), Italy

Several classes of protein drugs, such as enzymes, cytokines and antibodies, are significantly improved by PEGylation, a successfull technological approach to increase the drug plasma half life, to reduce the immunogenicity and to enhance the stability towards metabolic enzymes.Enzymatic strategy is fully described such as a site specific pegylation methodology, developed to modify a specific aminoacid of polypeptide chain and to obtain a site-specific conjugated protein[1]. Moreover, in the case only one aminoacid residue of the polypeptide chain is affected by the enzymatic pegylation, a site-specific mono-conjugated protein is obtained.This approach was used in our studies to reduce the rapid proteolytic degradation by ubiquitous dipeptidyldipeptidase IV (DPP IV) towards the Human glucagon-like peptide-1 (GLP-1), a physiological gastrointestinal peptide with glucose-dependent insulinotropic effects, considered an interesting antidiabetic agent[2]. We demonstrated that GLP-1-(7-36)-amide and its mutants were covalently conjugated with amine-reactive poly (ethylene glycol) (PEG) reagent via microbial transglutaminase (MTGase), a peculiar enzyme of prokaryotic origin which catalyze an acyl transfer reaction between a g-carboxyamido group of protein-bound glutamine residue (acyl donor) and an e-amino groups of a lysine residue (acyl acceptor). The compounds tested, resulting from site-specific mono-PEGylation mediated by bacterial transglutaminase, exhibit successfully an increased in vitro resistance to DDP IV and the ability to activate the GLP-1 receptor expressed in the rat ß-cell line RIN-m5F[3]. Therefore the results of our laboratory indicate that the enzymatic method using prokaryotic microbial transglutaminase is a strong strategy to improve pharmacological properties of therapeutic molecules for their potential clinical application.

References1. Francesco M. Veronese and Gianfranco Pasut. Pegylation, successful approach to drug delivery, Review Drug

Discovery Today (2005), Volume 10, Number 21. 2. Filip K. Knop, Tina Vilsbøll and Jens J. Holst. Incretin-Based Therapy of Type 2 Diabetes Mellitus. Current Protein

and Peptide Science, 10, 46-55, 2009.3. Selis F., Schrepfer R., Sanna R., Scaramuzza S., Tonon G., Onali P., Orsini G., Genovese S. Enzymatic mono-

pegylation of glucagon-like peptide 1 towards lost lasting treatment of type 2 diabetes. Results in Pharma Sciences, 2, 58-65, 2012.


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Conformational and binding studies of peptides spanning the EphA2 interacting region of the first Sam domain of Odin

F. A. Mercurio1, C. Di Natale5, L. Pirone4, P. L. Scognamiglio3,5, D. Marasco1,2,3, E. M. Pedone1,3, M. Saviano3,4 and M. Leone1,3

1 Institute of Biostructures and Bioimaging, National Research Council, 80134 - Naples, Italy2 Department of Pharmacy, University of Naples “Federico II”, 80134 - Naples, Italy3 Centro Interuniversitario di Ricerca sui Peptidi Bioattivi (CIRPEB), 80134 - Naples, Italy4 Institute of Crystallography, National Research Council, 70126 - Bari, Italy5 IIT Italian Institute of Technology, 80125 - Naples, Italy

Sam domains are small modules which mediate protein-protein interactions in a wide number of physiological and pathological processes.[1] Odin is a protein belonging to the ANKS (Ankyrin repeat and Sterile alpha motif domain protein) family with two Sam domains in tandem in its primary sequence. The first Sam domain of Odin (Odin-Sam1) is able to interact with the Sam domain of the tyrosine kinase receptor EphA2 (EphA2-Sam). Odin Sam domains are important to regulate EphA2 endocytosis, an event that has an impact in receptor related diseases such as cancer.[2] We have recently characterized the heterotypic Sam-Sam association Odin-Sam1/EphA2-Sam, showing that this complex forms via the “Mid-Loop/End-Helix” topology, in which Odin represents the Mid-Loop interface.[3] Here we have designed and synthesized two peptides, Sam2 and Sam3, encompassing the EphA2 interacting portion of Odin-Sam1, and analyzed their structural properties by means of circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Sam2 and Sam3 conformational properties have been studied in aqueous buffer and in presence of a structuring co-solvent. Moreover, peptide abilities to interact with EphA2-Sam have been investigated by means of surface plasmon resonance (SPR) techniques.

References1. C. A. Kim, J. U. Bowie. Trends Biochem. Sci. (2003)28:625-28.2. J. Kim, H. Lee, Y. Kim, S. Yoo, E. Park, S. Park. Mol. Cell. Biol. (2010)30:1582-92.3. F. A. Mercurio, D. Marasco, L. Pirone, E. M. Pedone, M. Pellecchia, M. Leone. Biochemistry (2012)51:2136-45.


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Inhibition of Prep1-p160 complex by peptides targeting the Prep1 binding site on p160

V. Lorenzo1,2, N. Doti1, F. Mascanzoni1,3, L. Vitagliano1, M. Ruvo1


The Pbx-regulating protein-1 (PREP1)-p160 Myb-binding protein (p160) complex plays a key role in glucose homeostasis.[1] Prep1-hypomorphic (Prep1i/i) mice exhibit enhanced sensitivity to insulin action and are protected from developing streptozotocin-induced diabetes through a p160-dependent mechanism. When p160 binds Prep1, the former gets stabilized, and represses the expression of Glucose Transporter 4 (GLUT4) reducing insulin sensitivity.[1] Available data therefore strikingly indicate the PREP1-p160 complex as an important target for diseases associated with diabetes and selective inhibitors of this complex should be seen as novel useful therapeutic approaches. To enhance at both structural and functional levels our understanding of the PREP1/p160 complex, we expressed in E. coli and purified to homogeneity the helix-rich domains 20-160 of p160 (p16020-160) and 45-155 of PREP1 (PREP145-155) involved in mutual binding. Direct binding ELISA-like assays between PREP145-155 and p16020-160 indicated that the two domains tightly associate, showing a KD value of 15x10-8 M. Moreover, to identify inhibitors of the biological target we designed and synthesized two peptides targeting the PREP1-binding site on p-160. The effect of these peptides on the complex formation has been evaluated by competitional and direct ELISA-like binding assays and results show that both peptides are able to disrupt the complex by interacting with p160. Further studies are underway to gain insights on the effects these peptides may have on cellular models of p160-mediated insulin sensitivity and on the structural features of the interacting protein. Peptides will also be used to develop new compounds modulating the p160 activity.

References1. F. Oriente, et al. Mol Cell Biol. (2008), 28(18), 5634–5645.


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APEH-mediated downregulation of proteasome by a potential anticancer peptide

R. Palumbo1, M. Balestrieri2, R. Iannitti1, M. Gogliettino2, M. Ruvo1, A. Sandomenico1, M. Rossi2, V. Cecere Palazzo2, E Cocca and G. Palmieri2

1. CNR-IBB , 80136-Naples2. CNR-IBBR, 80131-Naples

SsCEI represents the first archaeal phosphatidylethanolamine-binding protein (PEBP)-serine proteinase inhibitor, reported to date, that is able to efficiently inhibit Acylpeptide hydrolase (APEH) from mammalian sources with IC50 values in the nanomolar range. Homology modeling and point mutation experiments allowed us to identify the “re-active site loop”(RSL) on the inhibitor, responsible for the interaction with the protease target. This site includes an unusual amino acid sequence which cannot be classified in any of the canonical motifs of serine protease inhibitors so far characterized[1]. APEH is one of the four members of the POP family, which removes acylated amino acid residues from the N terminus of blocked peptides and has been recently recognized as having a role in protein-degradation machinery in coordination with the proteasome, and in the modulation of cancer progression[2,3]. In light of this, our study shows that proteasome functions can be upstream regulated by APEH, and that inhibition of APEH activity ap-pears to be an important event in controlling the proteasome dysfunction associated with pathological conditions. On this background, we have investigated the molecular mechanisms that underlie the interrelationship between APEH and the proteasome, and their eventual regulation by a synthetic peptide, SsCEI4, reproducing the RSL of SsCEI pro-tein. SsCEI4 shows a selective and specific inhibitory activity against mammalian APEHs with IC50 and Ki values of 84±16 mM and 4.0±0.8 mM, respectively. Therefore, to further study the biological role of APEH, the basal activity levels of APEH and proteasome were preliminarily measured in several cancer cell lines and a strong positive corre-lation was revealed, suggesting that the two enzymes could cooperate in the protein turnover processes. Moreover, on the basis of gene expression analysis and protein/activity levels, the different cancer cells could be divided into two groups displaying low (Group I) or high (Group II) levels of APEH and proteasome. These findings suggested that cells belonging to Group II could be highly dependent on these enzyme functions and hence more sensitive to their specific down-regulation. The susceptibility of these cell lines to the growth inhibitory effects of SsCEI4 was inve-stigated and results indicated that the peptide molecule efficiently reduced APEH activity/protein levels in a dose-dependent manner in two cancer cell lines (Caco-2 and U2OS cells) without necrosis, inducing a marked decrease of cell viability. Surprisingly, in both cell lines its effects were paralleled by a dose-dependent reduction of proteasome activity with a concomitant accumulation of known cytoplasmic proteasome substrates.

Hence, it appears reasonable to hypothesize that an enzyme machinery, such as APEH/proteasome system, could be involved in the marked anti-proliferative activity exerted by SsCEI4 through its specific capacity to down-regulate APEH. Finally, our study supports a previously unrecognized role of APEH as a negative effector of proteasome activity by an unknown mechanism and opens new perspectives for the development of promising strategies aimed at modulation of cancer progression, involving a new class of molecules.

References1. G. Palmieri, G. Catara, M. Saviano, E. Langella, M. Gogliettino and M. Rossi, J. Prot Res (2009), 8, 3272. G. Palmieri, P. Bergamo, A. Luini, M. Ruvo, M. Gogliettino, E. Langella, M. Saviano, R. N. Hegde, A. Sandomeni-

co, M. Rossi, Plos One (2011), 6, e258883. P. Bergamo, E. Cocca, R. Palumbo, M. Gogliettino, M. Rossi, G. Palmieri, Plos One (2013), 8, e80900


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Characterization of the cholesterol-lowering effects of soy and lupin peptides at HepG2 cell line

Carmen Lammi, Chiara Zanoni, Anna Arnoldi

Department of Pharmaceutical Sciences, University of Milan, via Mangiagalli 25, 20133 Milano

The investigations on the mechanism of action of bioactive components of functional foods and nutraceutics are not very frequent and in general not very detailed. We have recently established a cell culture lab aimed to fill this gap, starting our work from grain legumes. In fact, the seeds of grain legumes, besides being major foodstuffs in most countries, provide also some health benefits, in particular in the area of hypercholesterolemia and hypertension prevention[1, 2]. The main objective of the present work was to provide evidences that peptides generated by the digestion of soy and lupin proteins may be responsible of the hypocholesterolemic activity observed in vivo and to investigate the mechanism of action. In particular, a deepened study was carried out in order to characterize the hypocholesterolemic effects of pure peptides from soy proteins as well as peptide mixtures obtained by pepsin and trypsin hydrolysis of lupin proteins. The hepatic cell line HepG2 was treated with these peptides and molecular and functional investigations were performed on the LDL receptor / SREBP2 pathway. For the first time, this report provides the experimental evidence that, interfering with the HMGCoAR activity, legume peptides are able to up-regulate the LDL receptor and SREBP2 proteins via the activation of specific phosphorylation pathways and increasing the LDL-uptake at HepG2 cell line.

References1. Bouchenak M, Lamri-Senhadji M. Nutritional quality of legumes, and their role in cardiometabolic risk prevention:

a review. J Med Food. 2013 Mar;16(3):185-98.2. Arnoldi A, Zanoni C, Lammi C, Boschin G. THE ROLE OF GRAIN LEGUMES IN THE PREVENTION OF

HYPERCHOLESTEROLEMIA AND HYPERTENSION. Critical Reviews in Plant Sciences.


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Conformational study of bioactive peptides finalized to design of tailored delivery systems

E. Fenude

ICB, Department Chemical Science and Material Technology, CNR, 07100 – Sassari, Italy

Biologically active sequences deriving from food proteins are of particular interest in food science and nutrition because they have been shown in play physiological roles. Hidden or inactive in the amino acid sequence of proteins, they can be released or activated in vivo during gastrointestinal digestion, or upstream during food processing via specific, enzyme mediated proteolysis. This peculiar characteristics permit us to explore the properties of synthetic analogues that assume ‘hollow’ secondary structures and can, potentially, function as transport systems.[1] Structure-activity relationship of linear peptides in solution is ruled by conformational effects because of the peptide’s main chain flexibility. D,L-alternating peptides are able to adopt specific, symmetric, secondary structures containing an internal cavity[2] and they can be used as ‘models’ for conformational studies of biologically active peptides. We have used this approach for a study of the solution conformational properties of biologically active sequences deriving from food proteins. In this paper we report about synthesis and NMR investigation of the conventionally protected: natural peptides, containing only L-residues and analogues ‘models’ containing D-residue(s) in well defined position(s) of the main chain. The results obtained by conformational analysis of this compounds are compared and discussed.

References1. E. Fenude, S.Dedola, M. Fais VII Congress “Complex Systems: structure, properties, reactivity and dynamics”

(2005), Alghero, Italy2. E. Navarro, E. Fenude, B. Celda, Biopolymers (2004), 73, 229.


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Probing the helical stability in a proangiogenic peptideV. Celentano1, D. Diana1, D. Milardi2, R. Fattorusso3 and L.D. D’Andrea1

1 Istituto di Biostrutture e Bioimmagini, CNR, 80134 – Napoli2 Istituto di Biostrutture e Bioimmagini, CNR, 95125 – Catania3 Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche -Seconda Università di Napoli, 81100-Caserta

Protein folding, the process by which a protein assumes its three-dimensional shape, is one of the basic unsolved problems of biophysical and biochemical research, in fact represents one of the most important studied phenomena of recent times.In the last years, we reported the structural and biological characterization of a designed, a-helical, 15 mer peptide, named QK, which corresponds to the helix region 17-25 of the VEGF.[1] The peptide QK interacts with VEGF receptors and has a proangiogenic biological profile with potential therapeutic applications in reparative medicine. It shows an unusual high thermal stability, which structural determinants were investigated.[2,3] In particular, we identified the hydrophobic interaction between Leu7 and Leu10 as crucial for stabilizing the helical fold of QK.In order to correlate the relationships between hydrophobic interaction and helical stability in peptide QK, we synthesized eight QK analogue peptides where Leu10 was replaced by amino acids with different side chain hydrophobicity and steric hindrance, such as phenylalanine (F), isoleucine (I), valine (V), phenylglycine (Phg), norleucine (Nle), norvaline (Nva), tert-leucine (Tle) and amino butyric acid (Abu).The structure and the effect of the temperature on the QK analogues was examined using circular dichroism, NMR spectroscopy and differential scanning calorimetry.

References1. L. D. D’Andrea , G. Iaccarino, R. Fattorusso, D. Sorriento, C. Carannante, D. Capasso, B. Trimarco, C. Pedone

Proc. Natl. Acad. Sci U S A (2005), 102, 14215-20.2. D. Diana, B. Ziaco, G. Colombo, G. Scarabelli, A. Romanelli, C. Pedone, R. Fattorusso, L. D. D’Andrea Chemistry

(2008) 14, 4164-4166.3. D. Diana, B. Ziaco, G. Scarabelli, C. Pedone, G. Colombo, L. D. D’Andrea, R. Fattorusso, Chemistry (2010) 16,

5400- 5407.


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VEGF/VEGF receptor interaction: a structural analysis on living cellsR. Di Stasi1, D. Diana1, R. Fattorusso2 & L. D. D’Andrea1

1 Istituto di Biostrutture e Bioimmagini-CNR, 80134-Napoli2 Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche -Seconda Università di Napoli, 81100-Caserta

VEGF-A is a highly specific mitogen for vascular endothelial cells and plays a fundamental role in regulating both physiologic and pathological angiogenesis [1]. Its biological activity is mediated through the binding to two membrane receptors belonging to the tyrosine kinase family (VEGFR1 and VEGFR2) and to a co-receptor, neuropilin1, that enhances VEGF-A chemotactic and mitogenic activity [2]. Both VEGFR1 and VEGFR2 bind VEGF with high affinity, although only VEGFR2 modulates the mitogenic/angiogenic response [3]. In 1997, the crystal structures of free VEGF receptor-binding domain (VEGF8–109)

[4] and in complex with VEGFR1 domain 2 [5] were obtained. In the same year, the structural analysis in solution of VEGF fragment 11-109 was reported [6]. Up to know, no structural information on the interaction between VEGF-A and VEGFR2 has been reported. In order to fill this gap we intend to characterize the structure of VEGF-A in complex with VEGFR2 in the cellular environment. To this aim, the double labeled hVEGF11-109 protein was expressed in minimal medium, in presence of 15NH4Cl and 13C-glucose as unique sources of nitrogen and carbon. Fast 15N/13C-edited NMR experiments were carried out by adding the isotopically enriched 15N/13C- hVEGF11_109 to a suspension of Porcine Aortic Endothelial cells overexpressing VEGFR2 receptor. As negative control, 2D[1H-15N-13C] HSQC spectra of 15N/13C- hVEGF11_109 in the presence of WS1 fibroblasts lacking VEGF receptors were also performed. The resulting data showed the power of a direct spectroscopic approach in the identification of VEGF region immobilized upon binding to the receptors localized on cellular membrane, representing the first step towards the analysis of the receptor-bound VEGF structure. References1. Dvorak H. F. et al., Am. J. Pathol. (1995) 146, 1029-1039.2. Soker S. et al., Cell (1998) 92, 735-745.3. Waltenberger J. et al., J. Biol. Chem. (1994) 269, 26988-26995. 4. Muller Y. A. et al., P. N. A. S. USA (1997) 94,7192-7197 . 5. Wiesmann C. et al., Cell (1997) 91, 695-704.6. Starovasnik M. A. et al. Protein Science (1997) 6, 2250-2260.


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An intein-based strategy for the preparation of isotopically labelled peptides

A. Russomanno,1 L. De Rosa,1 D. Diana,2 A. Romanelli,3 R. Fattorusso,2 L. D. D’Andrea 1

1. Istituto di Biostrutture e Bioimmagini, CNR, Napoli, Italy2. Dip. di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, Seconda Università di Napoli, Caserta, Italy3. Dip. di Farmacia, Università di Napoli “Federico II”, Napoli, Italy

NMR spectroscopy is a powerful method to perform structural studies on peptides. To completely fulfill the potential of NMR, peptides labeled with stable isotopes (15N,13C,2H) are essential [1]. Peptides are usually prepared on solid-phase but chemical synthesis becomes prohibitively expensive when applied to the incorporation of isotopes. An alternative cost-effective strategy is the recombinant expression of peptides in E. coli as fusion constructs with carrier proteins[2]. We used the self-cleaving MxeGyrA mini-intein as fusion partner for the preparation by recombinant means of two isotope labeled peptides, HPLW and QK[3,4]. The two peptides target Vascular Endothelial Growth Factor Receptor (VEGFR) and have been described to modulate VEGF-dependent angiogenesis. Our strategy allows to prepare homogeneously isotope labeled peptides in free or acetylated/amidated form. The availability of isotope labeled HPLW and QK opens the way to further NMR studies aimed to characterize the folding dynamics of the two peptides and their structures in complex with VEGFR. Furthermore, the presented protocol can be applied to the preparation of any isotope labeled peptide.

References1. Koenig B. et al, J Biomol. NMR (2003) 26, 193-2022. Li Y., Protein Expr. Purif. (2011) 80, 260-673. Diana D. et al, J Biol. Chem. (2011) 286(48), 41680-914. D’Andrea et al., PNAS (2005), 102(40), 14215-20


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RGDechi-hCit peptide: new insights into biological behavior on melanoma metastatic cells

Ivan de Paola1, Annamaria Liguoro3, Daniela Comegna2, Annarita Del Gatto1, Sonia Di Gaetano1, Domenica Capasso2, Daniela Guarnieri4, Paolo A. Netti4, Michele Saviano5,

Laura Zaccaro1

1. Istituto di Biostrutture e Bioimmagini-CNR, Napoli, Italy; 2. Università degli Studi di Napoli “Federico II”, Napoli, Italy; 3. Diagnostica e Farmaceutica Molecolari Scarl, Napoli, Italy; 4. Istituto Italiano di Tecnologia, Center for Advanced Biomaterials for Health Care IIT@CRIB – Napoli, Italy;5. Istituto di Cristallografia-CNR, Bari, Italy

Integrins are important tumour markers as they are widely expressed on the surface of tumour cells [1]. Previously, we reported some features of RGDechi-hCit peptide, a selective avb3 antagonist [2-4]. Here we aimed at evaluating the in vitro biological behaviour of this peptide on WM-266 cell, human malignant melanoma cells expressing high levels of avb3 integrin. Firstly we evaluated the ability of the peptide to inhibit the adhesion to different matrices and the growth of WM266. Later we performed apoptosis activation studies using Cilengitide[5] as positive control. Finally cellular uptake studies were carried out by cytometry analysis and confocal microscopy using the peptide opportunely labelled. The results of all experiments gave deep insight into the bio-logical behavior and the potential of the peptide in the therapy and diagnosis of metastatic melanoma.

References1. Hynes R.O.,2002, Cell,110: 673-687.2. Del Gatto A, Zaccaro L, Grieco P, Novellino E, Zannetti A, et al. 2006, J. Med. Chem. 49, 3416-3420.3. Zannetti A, Del Vecchio S, Iommelli F, Del Gatto A, De Luca S, et al. ,2009, Clinical Cancer Research, 15: 5224-

5233.4. Santulli G, Basilicata MF, De Simone M, Del Giudice C, Anastasio A, et al. 2011, J Transl Med. 9:7.5. Oliveira-Ferrer L, Hauschild J, Fiedler W, Bokemeyer C, Nippgen J, et al. 2008 , Journal of experimental & clinical

cancer research : CR 27: 86


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Design and synthesis of new Integrase inhibitors

A. Spensiero1, M. Sala1, E. Vernieri1, M. C. Scala1, E. Novellino2, P. Grieco2, P. Campiglia1 and I. M. Gomez-Monterrey2

1 Department of Pharmacy, University of Salerno, 84084-Fisciano (SA), Italy2 Department of Pharmacy, University of Napoli “Federico II”, 80131-Napoli, Italy

Human immunodeficiency virus type 1 Integrase (IN) is one of three virally encoded enzymes essential for viral replication without counterpart in the host cell, and therefore, a rational specific choice as a drug target for the treatment of HIV-1 infection. IN performs both the viral processing step as well as the strand transfer step to insert viral DNA into the host DNA[1]. The structure of IN consists of three domains: the N-terminal domain has a HHCC motif which chelates zinc; the core domain has the catalytic DDE motif which is required for its enzymatic activity; and the C-terminal domain has an SH3-like fold which binds DNA not specifically. In the present study, we have focused on the N-terminal domain, which plays also a key role for the formation of specific IN–DNA contacts[2]. Therefore, in this communication we report the preliminary results obtained with a small library of peptides derived from N-terminal domain.

References1. Y. Pommier et al. Nat. Rev. Drug Discov. (2005), 4, 236-248. 2. K. Carayon et al. Nucleic Acids Research (2010), 38, 3692-3708.


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Solution conformational features of intrinsically disordered phosphopeptides: a new class of potential targets in drug discovery

M. Vincenzi1, F. Mercurio2, A. Accardo1,2, D. Tesauro1,2, J.Guillon3, L. Ronga3, M. Leone2 and F. Rossi1,2

1 Department of Pharmacy and CIRPeB, University of Naples “Federico II”, Via Mezzocannone 16, I-80134 Naples, Italy

2 Institute of Biostructures and Bioimaging (IBB-CNR), Via Mezzocannone 16, I-80134 Naples , Italy3 Univ. Bordeaux, UFR des Sciences Pharmaceutiques and INSERM U869, ARNA Laboratory, F-33076 Bordeaux,

France

In the last few years intrinsically disordered proteins (IDPs) have received great attention from the scientific community as they participate in several important biological processes and diseases[1,2]. The intrinsic disorder and flexibility of IDPs grant them a number of advantages with respect to ordered proteins, such as conformational plasticity to bind several targets, a large interaction surface, involvement in high specificity/low affinity interactions, enhanced binding kinetics. It is assumed that post-translational modifications such as phosphorylation can stimulate structural rearrangement in IDPs and facilitate their binding to partners[3]. For a better comprehension at structural level of the multifaceted mechanisms that govern molecular recognition processes involving IDPs, we designed the amino acid sequence AQIREASSPSLQVDNQSDQT containing a low content of “order-promoting” amino acids.This sequence was modified by inserting at either the N- or C-terminal end different phosphorylated residues (serine or threonine) to mimic the effects of phosphorylation. In addition, the conformational behavior of the unphosphorylated peptide obtained by adding at the C terminus an Allyl-glycine, was also investigated. All peptides were synthesized by solid phase methods, and characterized with physico-chemical methodologies. The lack of one single ordered conformation in these peptides was established experimentally by CD and NMR analyses that were conducted under different solution conditions (i.e.: aqueous buffer, water/trifluoroethanol (TFE) mixtures, dimethylsulfoxide (DMSO)).

References1. V. Uversky, Int J Biochem Cell Biol (2011), 43, 1090.2. M. M. Babu, Mol Biosyst (2012), 8, 21.3. V. Uversky, A. K. Dunker, Biochim Biophys Acta (2010), 1804, 1231.


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Use of a novel Transglutaminase Glutamine-containing consensus sequence for the identification of conjugation site of proteins by mass

spectroscopy

F. Selis1, A. Sandomenico2,3, A. Caporale2, G. Tonon1, R. Schrepfer1, M. Ruvo2,3

1 Bioker -Multimedica group c/o CNR IGB, Via Pietro Castellino, 80131 Napoli 2 CIRPeB, University of Naples Federico II, via Mezzocannone, 16, 80134 Napoli3 CNR-IBB, via Mezzocannone, 16, 80134 Napoli

Microbial transglutaminase (MTGase) is extensively used in the field of food and pharmaceutical preparation for cross-linking or conjugating exogenous compounds to proteins. Unlike other TGases, the activity of MTGase is Ca2+-independent, rendering its applicability much easier from a regulatory point of view and broader. MTGase catalyzes cross-linking reactions in peptides and proteins by forming an epsilon-(gamma-glutamyl)lysine bond, for that reason it is used to modify protein and peptide drugs at glutamine or lysine residues[1]. Here, a new optimized TGase substrate consisting of a Gln-containing consensus sequence (LQSP), identified by a combinatorial approach, is proposed to identify new Lys-containing conjugation sites on a set of therapeutic peptide and protein drugs, including Interferon Alfa-2b, human granulocyte colony-stimulating factor and human growth hormone. We have developed a fluoresceine-linked short peptide substrate (LQSP-Fluo) which is conjugated to the proteins of interest via MTAGse and the site of attachment is identified by LC-coupled electrospray ionization mass spectroscopy[2]. Interestingly, data show that in the tested proteins the use of MTGase in the enzymatic conjugation leads to the specific modification of only one lysine, producing mono-derivatized products. Results here reported suggest that the MTGase-mediated conjugation can be extremely selective and thus can be used for the site-specific introduction of different types of synthons, including orthogonal reactive groups (click- or chemical ligation-prone groups), PEGs, binding motifs and reporter moieties.

References1. Fontana, A., Spolaore, B., Mero, A., Veronese, F. M. Adv.Drug Deliv. Rev. (2008), 60, 13–28. 2. Mero A, Spolaore B, Veronese FM, Fontana A. Bioconjug Chem. (2009), 20, 384-9.


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4-(1-Piperidinyl)aspartate formation during the preparation of lactam constrained cyclic peptides

M. C. Scala1, E. Vernieri1, A. Spensiero1, E. Novellino2, A. Carotenuto2, I. M. Gomez-Monterrey2, P. Campiglia1 and M. Sala1

1 Department of Pharmacy, University of Salerno, 84084-Fisciano (SA), Italy2 Department of Pharmacy, University of Napoli “Federico II”, 80131- Napoli, Italy

The synthesis of large peptides by SPPS can be achieved by chemical ligation; however, side reactions can hamper the overall synthesis leading to decreased yields and/or quality. Several side reactions have been detected for aspartic acid containing peptides [1-2].In this communication we present the synthesis of a lactam peptide, using allyl ester as a protection for the aspartic acid side chain, that gave a mixture of desired compound and 4-(1-Piperidinyl) aspartate derivative. HPLC and NMR analysis were employed to characterize the two major components and were useful technique to determine this phenomenon.

References1. S.C. Vigil-Cruz, J.V. Aldrich. Lett. Peptide Sci. (1999), 6, 71-75.2. J.L. Lauer, C.G. Fields, G.B. Fields. Lett. Peptide Sci. (1994), 1,197-205.


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Biophysical characterization of the Helicobacter pylori A1 toxin

Gilmar Salgado, Dursun Nizam Korkut, Sandrine Chabas, Arnion Hélène, Alves Isabel, De Reuse Hilde, and Fabien Darfeuille

Toxin-Antitoxin (TA) systems are present in almost all bacterial chromosomes; these regulatory systems are composed by two genes coding for a toxin and an antitoxin molecules. Among the TA systems, the type I system is characterized by a peptidic toxin which translation is inhibited by a non-coding sRNA. Following the transcriptome of Helicobacter pylori, it was possible to establish that this human pathogen presents a major regulation at transcriptomic level, leading to the discovery of a new TA type I system; the AapA1/IsoA1. The AapA1 gene codes for hydrophobic peptide, which encompasses a predicted α-helix motif between TRP10 and LEU28. It also possesses a global positive charge of +7: MATK(H)GKNSWKTLYLKISFLGCKVVVLLKR. In this study we present the structure of A1 peptide obtained from NMR spectroscopy. Biophysical studies allow us to determine its membrane localization on H. pylori. We found out that PepA1 induces high rates of H. pylori cell mortality and erythrocyte cellular lysis. Additionally we tested the affinity of PepA1 for different membrane models using Plasmon Waveguide Resonance spectroscopy, evidencing a strong affinity in the order of 25 nM.


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Gp41 MPER in membrane mimetics characterized by mixed lipid composition

A.M.D’Ursi1, M. Scrima1, M. Grimaldi1 and G. Della Sala2

1 University of Salerno , Department of Pharmacy , 84084 – Fisciano (SA)2 University of Florence Department of Neuroscience, Psychology, Drug Research and Child Health

NEUROFARBA, Florence ITALY.

Human immunodeficiency virus (HIV), effects cell entry via a mechanism involving surface glycoprotein named gp41. This protein includes a tryptophan-rich Membrane Proximal External Region(MPER) domain, whose action has been indicated to be crucial in the membrane fusion process[1]. In order to comprehend the molecular mechanism favoring the membrane tropism of MPER, a plethora of biophysical studies have been carried out. NMR structure of GP41 MPER has been determined in membrane mimicking environment composed of dodecyl phosphocholine (DPC) micelle solution[2] . Several structural investigations based on different biophysical techniques have shown that the lipid composition has an important role in the definition of the structural and functional properties of MPER at membrane level. To investigate the effect of the lipid composition on the interaction with bio-membrane, here we present the NMR conformational analysis of MPER in DPC micelles doped with little amount of negatively charged sodium dodecyl sulfate (SDS) tensioactive. In an integrated approach, we report a confocal microscopy imaging analysis providing evidence of MPER ability of modifying the surface and the shape of liposomes characterized by mixed phospholipid composition.

References1. A. S Dimitrov, S. S. Rawat, S. Jiang, and R. Blumentha. Biochemistry (2003) 42,14150–141582. Z.Y. J. Sun, K. Joon Oh, M. Kim, J. Yu, V. Brusic, L. Song, Z. Qiao. Immunity (2008) 28, 52–63.


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Design, synthesis and efficacy of lactam-constrained GRK2 peptide inhibitors

E. Vernieri1, M. Sala1, M. C. Scala1, A. Spensiero1, E. Cipolletta2, A. Limatola3, A. Carotenuto3, P. Grieco3,

E. Novellino3, I. M. Gomez-Monterrey3, G. Iaccarino4 and P. Campiglia1

1 Department of Pharmacy ,University of Salerno, 84084-Fisciano (SA), Italy2 Department of Clinical Medicine Cardiovascular and Immunological Sciences, University of Napoli “Federico II”

80131- Napoli, Italy3 Department of Pharmacy, University of Napoli “Federico II”, 80131- Napoli, Italy4 Department of Medicine and Surgery, University of Salerno,84084-Fisciano (SA), Italy

G protein-coupled receptor kinase 2 (GRK2) is a relevant signaling node of the cellular transduction network, playing major roles in the physiology of various organs/tissues including the heart and blood vessels. Emerging evidence suggests that GRK2 is up regulated in pathological situations such as heart failure, hypertrophy and hypertension, and its inhibition offers a potential therapeutic solution to these diseases. As reported in literature[1,2], two short peptides KRX107 (GLLRrHS) and KRX124 (GLLRrH-SI) derived from HJ loop of GRK2/3 inhibit GRK2 activity and emerge as a valuable starting point for the development of a novel class of GRK2 inhibitors. Based on these findings we identified, the cyclic peptide [KLLRrHD]I-NH2, that inhibits potently and selectively the GRK2 activity[3].Thus, in this communication we report the preliminary results obtained with a small library of cyclic peptides, containing a lactam bridge, derived from KRX107 and KRX124.

References1. Y. Anis et al. Diabetologia (2004), 47, 1232-1244.2. I.M Gomez Monterrey et al. Biopolymers. (2014), 101(1), 121-128.3. A. Carotenuto et al. Eur. J. Med. Chem. (2013), 69, 384-392.


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Nuclear transport factors Importin Kapβ2 and Exportin CRM1 interact with the tumor enzyme hCA IX

M. Buonanno1,2, A. Di Fiore1, A. Sandomenico1, A. Meccariello1,3, V. Alterio1, G. De Simone1 and S.M. Monti1

1 Istituto di Biostrutture e Bioimmagini-CNR, Via Mezzocannone 16, 80134 Naples, Italy 2 Seconda Università di Napoli (SUN), 81100 Caserta3 Università di Napoli Federico II, 80100, Napoli

The superfamily of kariopherin-β is composed by several transport receptors which are involved in the regulation of the traffic between nucleus and cytosol. They are characterized by HEAT repeats arranged in superhelical, or ring-like structures, all sharing similar molecular weights (90-150 kDa) and isoelectric points (pI= 4.0-5.0) [1]. Among these, two members such as hCRM1 (a nuclear export factor, also called XPO1) and hKapβ2 (a nuclear import factor, also called TNPO1) have been described as interactors of the human Carbonic Anhydrase IX (hCA IX), a tumor enzyme associated to the cell membrane[2]. This enzyme consists of four different regions: a N-terminal proteoglycan (PG)-like region, a carbonic anhydrase catalytic domain (CA), a transmembrane segment (TM) and an intracellular tail (IC), which contains three phosphorilation sites (T443, S448 and Y449) [3,4]. The last two regions have been recognized as the portion interacting with the above mentioned nuclear transport factors. hCRM1 and hKapβ2 have been expressed in E.coli, purified and characterized by means of Circular Dichroism and Dinamic Light Scattering. At the same time, peptides of different length, mimicking the TM and IC sequences of hCAIX, either phosphorilated or not at residues T443 and Y449, have been designed, synthesized, purified and used for SPR assays.

References1. D. Xu, A.Farmer and Y.M. Chook Curr. Opin. Struct. Biol. (2010), 20, 782.2. P. Buanne, G. Renzone, F. Monteleone, M. Vitale, S.M. Monti, et al. J. Proteome Res. (2013), 12, 282.3. C.T. Supuran, A. Di Fiore, V. Alterio, S.M. Monti and G. De Simone Curr. Pharm. Des. (2010), 16, 3246.4. V. Alterio, M. Hilvo, A. Di Fiore, et al. Proc. Natl. Acad. Sci. USA, (2009) 106, 16233.


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Carbonic anhydrases as new targets against the bacterial pathogen Brucella suis

A. Meccariello1,2, V. Alterio1, M. Buonanno1,3, K. D’Ambrosio1, D. Vullo4, C.T. Supuran4, S.M. Monti1 and G. De Simone1

1 Istituto di Biostrutture e Bioimmagini-CNR, Via Mezzocannone 16, 80134 Naples, Italy2 Università di Napoli Federico II, 80100, Naples, Italy3 Seconda Università di Napoli (SUN), 81100, Caserta, Italy4 Università degli studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della

Lastruccia 3, 50019 Sesto Fiorentino, Italy

Brucella sp. is a facultative intracellular coccobacillus responsible for brucellosis in a variety of mammals including ruminants and humans (“Malta Fever”)[1]. Brucellosis represents the major bacterial zoonosis worldwide and in humans can become chronic, eventually leading to death. So far, no vaccine has been developed for this pathogen which is treated by antibiotics. However, since it can develop antibiotic resistance, there is a renewed interest in the discovery of antibacterials able to act on novel molecular targets, circumventing the drug resistance problems[2]. The bacterial pathogen Brucella suis encodes two isoforms of Carbonic Anhydrases (CAs), named BsCA I and BsCA II which are considered as potential targets for the development of anti-Brucella agents. In order to perform structural studies to be used for drug design, BsCA I and BsCA II have been cloned, expressed in E. coli and purified. The two isoforms are highly active catalyzing the rapid interconversion of carbon dioxide and water to bicarbonate and proton, BsCA II being more active than BsCA I. Both enzymes are inhibited by the well-studied inhibitor acetazolamide, a sulfonamide drug[3]. Several inhibitors have been tested in order to identify lead compounds to be used as anti-infective agents in place of classical antibiotics.

References1. M. Lopez, S. Köhler and J.Y. Winum J. Inorg. Biochem. (2012), 111, 138.2. P. Joseph, F. Turtaut, S. Ouahrani-Bettache, et al. J. Med. Chem. (2010) 53, 2277.3. J.Y. Winum, S. Köhler and C.T. Supuran Curr Pharm Des (2010), 16, 3310.


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Carbonic anhydrase inhibitors: X-ray crystallographic studies for the binding of molecules containing a sulfamide moiety

A. Di Fiore1, V. Alterio1, K. D’Ambrosio1, M. Buonanno1,2, A. Meccariello1,3, S. M. Monti1, C. T. Supuran4 and G. De Simone1

1 Istituto di Biostrutture e Bioimmagini-CNR, Via Mezzocannone 16, 80134 Naples, Italy 2 Seconda Università di Napoli (SUN), 81100 Caserta3 Università di Napoli Federico II, 80100, Napoli4 Università degli Studi di Firenze, Via della Lastruccia 3, 50019 - Sesto Fiorentino (Firenze)

Carbonic anhydrases (CAs) are ubiquitous metalloenzymes, which catalyze the reversible hydration of carbon dioxide to bicarbonate ion and proton. These proteins are present in prokaryotes and eukaryotes, and are encoded by five evolutionarily unrelated gene families.[1] Human CAs are widely distributed in many tissues and organs. Since at these sites CAs play a crucial role in various physiological processes, they have recently become interesting targets for pharmaceutical research. Indeed, several CA inhibitors (CAIs) incorporating a sulfonamide/sulfamate/sulfamide moieties are currently clinically used for the treatment or prevention of a multitude of diseases such as glaucoma, solid tumors, and epilepsy.[2] However, most of the CAI based drugs present various non-desired side-effects, mainly because of their lack of selectivity for the different CA isoforms. Thus, the identification of selective CAIs is one of the main purpose for the development of new pharmacological agents. Here we report a new series of compounds containing a sulfamide moiety as zinc-binding group (ZBG) (Figure 1).

Figure 1. Schematic representation of chemical structures of CA inhibitors 1-2

These compounds have been synthesized and tested for determining their inhibitory action against human CA I and II. The X-ray structures of isoform hCA II in complex with 1-2 have also been solved providing further insights into sulfamide binding mechanism and confirming that such ZBG, if conveniently derivatized, can be usefully exploited for obtaining new effective and selective CAIs.

References1. C.T. Supuran. Nat. Rev. Drug Discov. (2008) 7, 168.2. V. Alterio, A. Di Fiore, K. D’Ambrosio, C.T. Supuran and G. De Simone. Chem. Rev. (2012), 112, 4421.


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Synthesis and biological activity of novel peptides active on urokinase system

A.M.Yousif 1, M.V. Carriero 2, F. Merlino 1, V. Ingangib 1, E. Novellino 1, P. Grieco1

1 Dept. of Pharmacy, University of Naples Federico II, Italy; IRCCS National Institute of Cancer “Fondazione G. Pascale”, Naples, Italy

The urokinase-type plasminogen activator receptor (uPAR) plays a central role in sustaining the malignant phenotype, promoting tumor metastasis and modulating the development of both innate and adaptive immunity. Recently, a number of studies have analyzed the involvement of the uPA/uPAR system in inflammatory bowel disease (IBD) pathogenesis. Thereby, studies devoted to characterize the role of uPAR in IBD pathogenesis could allow to generate new anti-inflammatoryagents devoted to block the signaling pathways triggered by uPAR. The uPAR is formed by three domains connected by short linker region. The Ser88-Arg-Ser-Arg-Tyr92 is the minimum chemotactic sequence of uPAR required to induce the same intracellular signaling as its ligand urokinase (uPA). With aim to perform a SAR study on this sequence we have synthesized a peptide library using phosphorylated amino acids, non-coded amino acids, D-amino acid scan and Nmethylated amino acid scan. Here we will report preliminarily SAR studies performed on theminimum chemotactic sequence of uPAR.

Key words: uPAR, peptide synthesis, SAR studiesApplication type: Poster presentationTopic: Synthetic Chemistry of Peptides and Amino Acids

Reference:1. K. Bifulco, I. Longo-Cattani, L. Gargiulo, O. Maglio, M. Cataldi, M. De Rosa, M.P. Stoppelli, V. Pavone and M.V.

Carriero,FEBS Letters 582, 1141–1146(2008).2. L. Gargiulo, I. Longo-Cattani, K. Bifulco, P. Franco, R. Raiola, P. Campiglia, P. Grieco, G. Peluso, M.P. Stoppelli

and M.V. Carriero, J. Biol. Chem 280, 25225-25232(2005).


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A novel and facile synthesis of tetra branched derivatives of nociceptin/orphanin FQ

Remo Guerrini1,2, Erika Marzola1,2, Claudio Trapella1, Michela Pela’1, Stefano Molinari3, Maria Camilla Cerlesi3, Davide Malfacini3, Anna Rizzi3, Severo Salvadori1,2, and Girolamo Calo’3

1 Department of Chemical and Pharmaceutical Sciences, 44121 Ferrara, Italy2 Laboratorio per le tecnologie delle terapie avanzate (LTTA), 44121 Ferrara, Italy3 Department of Medical Science, Section of Pharmacology and National Institute of Neuroscience, 44121 Ferrara Italy

Branched peptides have been found to be useful in several research fields however their synthesis and purification is complicated. Here we present a novel and facile synthesis of tetra branched derivatives of nociceptin/orphanin FQ (N/OFQ). Three N/OFQ tetra branched derivatives were prepared using novel cores (PWT1, PWT2 and PWT3) containing a maleimido moiety. [Cys18]N/OFQ-NH2 was linked to the cores via thiol-Michael reaction characterized by high yield and purity of the desired final product. In the electrically stimulated mouse vas deferens PWT-N/OFQ derivatives mimicked the inhibitory action of the natural sequence showing similar maximal effects and 3 fold higher potencies. The NOP selective antagonist SB-612111 antagonized the effects of N/OFQ and PWT derivatives with similar pKB values (8.02 – 8.48). In vivo after supraspinal administration PWT2-N/OFQ (Figure 1) stimulated food intake in mice mimicking the action of N/OFQ. Compared to the natural peptide PWT2-N/OFQ was 40 fold more potent and elicited larger effects. These findings suggest that the

N

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Figure 1. Chemical structure of PWT2-N/OFQ

PWT chemical strategy can be successfully applied to biologically active peptides to generate, with unprecedented high purity and yield, tetra branched derivatives displaying an in vitro pharmacological profile similar to that of the natural sequence associated, in vivo, to increased potency and duration of action.


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The BIORICE European project: BIO technology for the recovery of valuable peptides from industrial RICE by-products and production of added value ingredients for nutraceuticals, functional foods and

cosmetics

Lucilla Scarnato, Maura Ferri,, Annalisa Tassoni

Department of Biological, Geological and Environmental Sciences, University of Bologna, Via Irnerio 42, 40126 Bologna, Italy

The nutraceutical, functional food and cosmetic EU and worldwide markets are rapidly evolving sectors always in need to develop new classes of products. One of the most important market trends is the use of new bio-based ingredients obtained by environment-friendly extraction processes and testing methods. Of particular importance is the re-cycling and valorization of agro-food industry by-products as feedstocks for the isolation of bioactive molecules. The BIORICE project aims to fill the gap of knowledge of the involved Small and Medium Enterprises (SMEs) on rice protein by-product pre-treatment, peptide isolation and relative bioactivity and safety testing. In particular, BIORICE research activities will produce added value bioactive ingredients (semi-purified digestates and small molecular weight peptides) starting from protein by-products contained in the processing water of the rice starch production stream. The protein by-products will be pre-treated via biotechnological approaches (enzymatic proteolysis, microbial treatments) and different small molecular weight (5-15 kDa) peptides will be isolated by means of innovative eco-sustainable and not degrading techniques. A new method for in vitro evaluation of the skin sensitization potency will be developed so that the products will be tested without using laboratory animals. The BIORICE project brings together 6 partners distributed in 3 EU Member States and 1 Associated Country. In BIORICE 3 RTD Performers will make their multidisciplinary and complementary expertise in the areas of plant biotechnology, downstream processing and human tissue engineering available to 3 SMEs operating in the nutraceutical, food and cosmetic markets. The aim of the present project is to obtain and to characterise peptides from rice protein by-products and evaluate their bioactive properties. The project results are also expected to have a significant impact on the competitiveness of SME Participants that will be able to expand their business by adding to their product range new bioactive ingredients and protocols enabling new product formulations applicable in food, cosmetic and nutraceuticals sectors.The BIORICE project started on 1st November 2013 and will last 24 months. The project is co-funded by the Seventh Framework Programme (FP7/2007- 2013) Capacities, Research for the Benefit of SMEs of the European Union and is coordinated by Dr. Annalisa Tassoni, Department of Biological, Geological and Environmental Sciences, University of Bologna, Italy


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Molecular Dynamics studies of PMMA polymer film- c02 peptide interfacial phenomena

M. Scrima, A. De Nicola, G. Milano

Department of Chemistry and Biology, University of Salerno, 84084 Fisciano (SA) Italy

To understand and regulate biological phenomena such as protein adsorption and cell adhesion with a molecular level on the surface of synthetic polymers is extremely crucial for developing novel polymer materials utilized in biomedical fields [1].Elucidations about peptides interacting with polymer films to study interfacial phenomena are generally quite difficult. We present a computational study using Molecular Dynamics approach, at atomistic level, of c02 peptide[2] interacting with a polymer film of PMMA (Poly methylmethacrylate) in water solution.To correct reproduction of main structural and dynamic properties of both, PMMA film and c02 peptide in water, have been preliminary tested and compared with experimental data[2-3].

Our preliminary results could be open the way to all atom description of these systems.

References1. D.L. Elbert, J.A. Hubbell. Rev. Mater. Sci. (1996), 26, 365–394.2. T. Serizawa, T. Sawada, H. Matsuno, T. Matsubara, T. Sato JACS (2005), 127, 13780-137813. N.L. Jarvis, R.B. Fox, and W.A. Zisman. American Chemical Society, Washington, DC, 1964, p. 317-340


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Design, production and characterization of new protein sweeteners

F. Donnarumma1, M. F. Rega1, R. Di Monaco2, S. Cavella2 and D. Picone1

1 University of Naples Federico II, Department of Chemical Sciences, I-80126 – Naples, Italy2 University of Naples Federico II, Food Science and Agricultural Department, I-80055 Portici (NA), Italy

There is an increasing attention, in the more developed countries, to the identification of new sweeteners directed to people affected by diabetes and obesity, which ideally should combine a low caloric content and the absence of side-effects. However, none of the current low-calories sweeteners available in the market matches sucrose in sweet taste quality or temporal characteristics. In fact sensory performances of sweeteners change over time and, when added to a foodstuff, they can interact with other stimuli (taste, odour, texture) and elicit undesirable effects. Sweet proteins, isolated from tropical fruits [1], represent a promising alternative to other natural or artificial sweeteners, because they combine an extremely low caloric intake with a natural origin, which at least in principle may reduce the risks of health side-effects. Among the proteins so far identified, Monellin, Brazzein and Thaumatin show a very strong sweet taste, which is thousand times the one of sucrose. However, their diffusion for commercial purposes is limited by the difficulty to extract significant protein amount from the fruits of origin and from the low stability they have in the conditions used for food processing. Structural modification have been introduced in the native molecules in order to increase their stability and to widen the potential applications of these substances as food ingredients and/or drug excipients. For instance Monellin is a natively dimeric protein which readily unfolds at temperatures above 50 °C and loses the intrinsic sweetness, but its single chain derivative have a much higher thermal stability and keep a native 3D-structure, and a very high sweetness, even at temperatures of about 80 °C. However, prolonged treatment at high temperatures induces protein aggregation and even precipitation [2]. In this framework the use of known natural sweet proteins as templates for the rational design of new sweeteners, with an higher stability to the conditions required for food processing, and efficient production procedures, represents an interesting goal. We have already shown that, based on the detailed knowledge of the 3D structure of a single chain monellin, it is possible to design mutants with increased sweetness [3]. Here, we report a detailed characterization of the aggregation properties and of the sensory performance of a super-sweet single chain derivative of Monellin, which shows promising features as component of model beverages.

References1. Picone, D. and Temussi, P.A. Plant Sci. (2012), 195, 135.2. Esposito, V. et al., , Biochemistry (2010), 49, 2805.3. Esposito, V. et al., , J. Mol. Biol. (2006), 360, 448.Acknowledgments: Financial support from “Fondazione con il Sud” is greatly acknowledged.


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SAR Study on P5U and Urantide by replacing Tyr9 with uncoded and constrained amino acids

F. Merlino,1 AM. Yousif,1 P. Campiglia,2 I.M. Gomez-Monterrey,1 L. Piras1, P. Santicioli,3 S. Meini,3 C.A. Maggi,3 E. Novellino,1 A. Carotenuto 1 and P. Grieco1

1 Dept. of Pharmacy, University of Naples Federico II, 80131 – Naples, Italy2 Dept. of Pharmacy, University of Salerno, 80131 – Naples, Italy3 Dept. of Pharmacology, Menarini Ricerche SpA, 50131 – Florence, Italy

Urotensin II (hU-II), a potent vasoconstrictor, is found in diverse species, including human. Several biological studies indicate that hU-II is the most potent mammalian peptide vasoconstrictor reported to date, and it appears to be involved in the regulation of cardiovascular homeostasis and pathology. Previously, we have reported the first superagonist (P5U) and the full antagonist (Urantide) at UT receptor (Figure 1) [1,2]. Recently, we have highlighted that the residues of Trp7 and Lys8 are important for agonist activity. In fact, when they are replaced with DTrp7 and Orn8 the activity change from Agonist to Antagonist.With the aim to shed light on the most important substructural features responsible for agonist/antagonist activity of these important peptides we decided to explore the position 9 replacing the Tyr residue with several uncoded and constrained amino acids (Figure 1).

H-Asp-c[Pen-Phe-Trp-Lys-Tyr-Cys]-Val-OH P5U H-Asp-c[Pen-Phe-DTrp-Orn-Tyr-Cys]-Val-OH Urantide

H-Asp-c[Pen-Phe-Xaa-Yaa-Zaa-Cys]-Val-OH

Xaa= Trp, DTrp; Yaa= Lys, Orn; Zaa= unconventional amino acids

Figure 1. New Urotensin-II ligands

The peptides were tested for their ability to induce efficacious contractions in the rat isolated thoracic aorta. Here we report the preliminarily biological and conformational data on these new ligands.

References1. P. Grieco, A. Carotenuto, P. Campiglia, E. Zampelli, R. Patacchini, C.A. Maggi, E. Novellino, P. Rovero, Journal of

Medicinal Chemistry, (2002), 45, 4391. 2. R. Patacchini, P. Santicioli, S. Giuliani, P. Grieco, E. Novellino, P. Rovero, C.A. Maggi, British Journal of

Pharmacology. (2003), 140, 1155.


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Conformational Analysis of Urotensin-II Related Peptide in Membrane Mimetic Environment

A. Carotenuto1, D. Brancaccio1, F. Merlino1, A.M. Yousif1, P. Campiglia2, I.M. Gomez-Monterrey1, E. Novellino1, P. Grieco1

1 Dept. of Pharmacy, University of Naples Federico II, 80131 – Naples, Italy2 Dept. of Pharmacy, University of Salerno, 80131 – Naples, Italy

The urotensin-II receptor (UT) has long been studied mainly for its involvement in the cardiovascular homeostasis in either health or disease state [1]. The biological activities associated with UT activation are modulated by two endogenous ligands, i.e. urotensin-II (UII) and urotensin -I-related peptide (URP). These peptides share the C-terminal sequence and differ for the exocyclic N-terminus (Figure 1). Extensive expression of the two ligands revealed the multiple pathophysiological effects mediated by the urotensin-II system such as cardiovascular disorders (heart failure, cardiac remodelling, hypertension), smooth muscle cell proliferation, renal disease, diabetes, and tumour growth.

H-Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH h-UII H-Ala-c[Cys-Phe-Trp-Orn-Tyr-Cys]-Val-OH URP

Figure 1. Sequences of human Urotensin-II (h-UII) and URP

As recently demonstrated, UII and URP could exert different actions on transcriptional activity, cell proliferation, and myocardial contractile activities supporting the idea that UII and URP interact with UT in a distinct manner (biased agonism) [2]. To shed light on the origin of the divergent activities of the two endogenous ligands, we performed a conformational studies on URP by solution NMR in SDS micelle solution and compared the obtained NMR structure of URP with that of h-UII previously determined [3]. Finally, we undertook docking studies between URP, h-UII and an UT receptor model.

References1. H. Vaudry, J.C. Do Rego, J.C. Le Mevel, D. Chatenet, H. Tostivint, A. Fournier, M.C. Tonon, G. Pelletier, J.M.

Conlon, J. Leprince Ann. N. Y. Acad. Sci. (2010), 200, 53. 2. Chatenet, T.T. Nguyen, M. Létourneau, A. Fournier Front. Endocrinol. (2013), 3, 1.3. A. Carotenuto, P. Grieco, P. Campiglia, E. Novellino, P. Rovero J. Med. Chem. (2004), 47, 1652.


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Investigating the oxidative refolding mechanism of Cripto CFC domainG. Corvino1, V. Severino2,3, E. Iaccarino3, A. Sandomenico2,3, A. Chambery3,4 and M. Ruvo2,3

1 Bio-Ker s.r.l. 80128-Napoli2 Istituto di Biostrutture e Bioimmagini-IBB, CNR 80134-Napoli3 Centro Interuniversitario di Ricerca sui Peptidi Bioattivi-CIRPEB 80134-Napoli 4 Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, Seconda Università degli Studi di

Napoli 81100- Caserta

Disulfide bonds play an essential role in the folding and assembly of secretory proteins. The mechanism underlying the acquisition of the native state by unfolded proteins is one of the most challenging issues in structural biology. Understanding the sequence of protein folding events may provide useful information in studying protein misfolding associated with pathological diseases or in assisting protein structure prediction and design. CFC domains (Cripto Frl-1 Cryptic domain) are C-terminal disulfide rich modules only found in proteins of the EGF-CFC family, whose founding member Cripto, is an established oncogene. The CFC domain appears to play a crucial role in the Cripto tumorigenic activity by directly engaging the ALK4 receptor and mediating its downstream signaling [1]. The domain contains six cysteinyl residues engaged in three disulfide bonds arranged in a C1-C4, C2-C6, C3-C5 pattern [2].Here, we characterized the kinetic of the oxidative folding of the CFC domain of human Cripto by MALDI-TOF mass spectrometry. CFC folding proceeds through the sequential formation of three disulfide species to reach the native form. Oxidative refolding of CFC domain comprises two main independent folding events involving the formation of two intermediates. A first species containing the Cys115-Cys133 (intermediate I) disulfide bridge is detected at the beginning of the folding process, whose formation is likely driven by the occurrence of a partially structured region at the CFC N-terminus. Folding then progresses through the formation of a second disulfide pair between Cys131-Cys140 (intermediate II) mainly detected within the time window between 30 and 90 min. Data also show the coexistence, starting at 30 min, of intermediate I and II with the fully refolded domain containing the third Cys128-Cys149 disulfide pair. At 180 min, only the natively-paired disulfide assembly is detected, suggesting that the stable intermediates I and II funnel the more flexible C-terminal region of the CFC domain toward the acquisition of the native state. In conclusion, data are supportive of a highly concerted refolding mechanism driven by the formation of the Cys115-Cys133 bridge, but involving the interplay between all 6 cysteine residues.

References1. S.F. Foley et al. Eur J Biochem (2003), 270, 3610. 2. L. Calvanese et al. J Pept Sci (2008), 15, 175.

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SATELLITE MEETING MicroRNA:

Potential for Cancer Detection and Diagnosis


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Targeting biological functions of disease-associated microRNAs: novel frontiers in miRNA-Therapeutics

Roberto Gambari

University of Ferrara, Department of Life Sciences and Biotechnology, Italy

The issue of targeting microRNAs (miRNA Therapeutics) is one of the most relevant fields of applied biomedicine. In view of the role of miRNAs in epigenetic regulation of gene expression, miRNas have been proposed as possible candidates for drug targeting with the objective of interfere with their biological functions, altering the expression of mRNAs specifically regulated by the targeted miRNAs. Within the name “miRNA therapeutics”, we can consider two major field of intervention, i.e. the “miRNA replacement therapy” and the “miRNA targeting therapy”. In the first field, the biological activity of miRNAs which are down-regulated during onset and progression of a given disease (for instance tumor-suppressor miRNAs in the case of cancer) is replaced with ectopically added, suitably delivered pre-miRNA or using recombinant viral molecules carrying the therapeutic miRNA gene enabling high-level production of the miRNA within transfected cells. In the second field of “miRNA therapeutics”, disease associated up-regulated miRNAs are targeted by a variety of potentially therapeutic molecules (antagomiRs). In miRNA replacement therapy down-regulation of the expression of miRNA targeted mRNAs is expected. Vice-versa, in miRNA targeting the mRNA regulated by the targeted miRNAs are expected to be up-regulated. Using the development of anti-cancer therapies as a representative field of investigation, studies concerning the “miRNA replacement therapy” were performed mimicking the activity of several tumor-suppressor microRNAs in different tumor types, including miR-27a (acute leukemias), miR-569 (squamous cell carcinoma), miR-302 (breast cancer), miR-33a (CML and colon carcinoma), miR-145 (colon carcinoma), miR-34a (lung cancer), miR-let7 (lung cancer), miR-29b (non-small cell lung adenocarcinoma and acute myeloid leukemia).The effects of molecules against microRNA (antagomiRs) have been the object of several studies aimed at the inhibition of the activity of several onco-microRNAs or metasta-miRNAs in different tumor types, including miR-27a (glioblastoma), miR-155 (cutaneous squamous cell carcinoma), miR-335 (malignant astrocytoma), miR-92 (neuroblastoma), miR-381 (glioma), miR-10b (breast cancer) and miR-221/222 (prostate cancer).In the field of “miRNA targeting” peptide nucleic acids (PNAs) have been recently demonstrated to be of great interest. PNAs are DNA analogues in which the sugar-phosphate backbone is replaced by N-(2-aminoethyl)glycine units and efficiently hybridize with complementary DNA and RNA, forming double helices with Watson-Crick base pairing. PNAs have been demonstrated to inhibit the biological activity of several miRNAs involved in human pathologies, including miR-221/222, miR-122, miR-155. We have found that PNAs directed against miR-221 inhibit miR-221 function, causing increase of expression of the miR-221 target p27-Kip1 and TIMP3 genes and induction of apoptosis [1].

References1. E. Brognara, E. Fabbri et al. J. of Neurooncol. 2014

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Modulation of Chemotherapeutic Drug Resistance by miRNA

Isabella Bray, Harry Harvey, Olga Piskareva, Ray Stallings

MiRNAs are a short, non-coding RNA capable of post transcriptional regulation of gene expression. They are involved in every aspect of cellular function, from the control of differentiation, to metabo-lism, cell growth and cell death. More recently it has been established that miRNA can alter the cellular response to chemotherapeutic agents through modulation of drug targets, drug transporters, DNA repair systems and the distortion of the apoptotic and survival pathways. This discovery is of major interest as the acquisition of drug resistance poses a significant impediment to the successful treatment of many forms of cancer. This is particularly true for neuroblastoma, a clinically heterogeneous cancer account-ing for ~15% of all paediatric cancer deaths. In order to elucidate the mechanisms involved with drug resistance in neuroblastoma, an SK-N-AS subline (SK-N-AsCis24) that is significantly resistant to cis-platin and cross resistant to several other chemotherapeutics was developed through a pulse-selection process. High resolution aCGH analysis of SK-N-AsCis24 revealed a focal gain on chromosome 5 containing the coding sequence for the neural apoptosis inhibitory protein (NAIP). Significant over-expression of NAIP mRNA and protein was documented, while experimental modulation of NAIP levels in both SK-N-AsCis24 and in parental SK-N-AS cells confirmed that NAIP was responsible for the drug resistant phenotype by apoptosis inhibition. Furthermore, a decrease in the NAIP targeting microRNA, miR-520f, was also demonstrated to be partially responsible for increased NAIP levels in SK-N-AsCis24. Interestingly, miR-520f levels were determined to be significantly lower in post-che-motherapy treatment tumours relative to matched pre-chemotherapy samples, consistent with a role for this miRNA in the acquisition of drug resistance in vivo, potentially through decreased NAIP targeting. This novel insight into the mechanism of drug-resistance in neuroblastoma has implications for NAIP as a future therapeutic target in cancer. Moreover, the current pursuit of miRNA as therapeutic tools in cancer makes miR-520f an ideal candidate for the development of a NAIP inhibitor.

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Fingerprinting of ultra conserved long noncoding RNAs in bladder can-cer analysis reveals a network between non-coding RNA and microRNA

Amelia Cimmino1

1 Institute of Genetics and Biophysics “A. Buzzati Traverso”, National Research Council (CNR), Naples, Italy

Transcribed ultraconserved regions (T-UCRs) are pieces of human genome located both intra- and in-ter-genic that are conserved between orthologous regions of the human, rat and mouse genomes. They are frequently located at fragile sites and genomic regions involved in cancers [1]. By using genome-wide profiling, we identified 289 ucRNAs de-regulated in patients with bladder cancer (BC) compared to normal control (24 tumors, 4 control). The greatest change was noted for ultraconserved element 8 (uc.8+), which was increased in expression by 5.45 ± 0.9-fold (P = 0.001), and for ultraconserved element 388 (uc.388+), which was decreased in expression by 0.23-fold. Expression level of the most deregulated T-UCRs was validated in 60 patients and 16 normal donors. We showed that uc.8+ func-tions as a natural decoy RNA for miR-596 in BC. As result, matrix metallopeptidase 9 (MMP9), a direct target of these microRNAs is up-regulated, promoting cancer growth and migration. We also observed that either the up regulated, as well as the most down-regulated T-UCRs in BC, showed puta-tive binding sites for the mir-596 with a mfe < -30 kcal/mol for each duplex formed. Our studies have found and experimentally validated an extensive and dynamic regulatory network based on the RNA signaling that explains how the perturbation of a single T-UCR can affect on whole T-UCRs network of interactions.

References1. Calin GA, Liu CG, Ferracin M, Hyslop T, Spizzo R, Sevignani C, Fabbri M, Cimmino A, Lee EJ, Wojcik SE, Shimizu

M, Tili E, Rossi S, Taccioli C, Pichiorri F, Liu X, Zupo S, Herlea V, Gramantieri L, Lanza G, Alder H, Rassenti L, Volinia S, Schmittgen TD, Kipps TJ, Negrini M, Croce CM. 2007. Ultraconserved regions encoding ncRNAs are altered in human leukemias and carcinomas. Cancer Cell 12:215-229.

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Molecular Dynamics simulations of two PNA-based systems using new parameters implemented in the GROMACS package

Ida Autiero1, Michele Saviano2 and Emma Langella1

1. National Research Council, Institute of Biostructures and Bioimaging, 80138-Naples 2. National Research Council, Institute of Crystallography, 70126-Bari

Peptide Nucleic Acids (PNAs), nucleic acid analog, represent a promising challenging of the biomedical research. Currently, PNA finds useful applications in a wide range of important fields concerning the diagnostics and therapeutics. Among others, this system is used for gene induction, for inhibition of the translation and also as probe to identify specified gene sequences or to recognize even a single gene mutation. Although great efforts have been spent to explore its biochemical properties, either experimentally or theoretically, many structural properties of the PNA remain yet to be fully defined. In these regards, we have properly derived new force field parameters to describe PNA molecules. These parameters were implemented in the widely used GROMACS simulation package, and have been used to perform all-atoms Molecular Dynamics (MD) simulations on a PNA-PNA duplex and a PNA-DNA hetero-duplex systems. The trajectories obtained from the performed MD simulations reveal interesting conformational and binding features, showing an important agreement with the experimental observations, available for both systems studied. The new parameters and the approach used have shown a good validity, these data will be useful for the rational design of new PNA-based molecules and for the improvement of this powerful system.[1]

References1. Autiero I1, Saviano M, Langella E. Phys Chem Chem Phys. (2014), 7, 16, 1868-74

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Synthesis and characterization of g-thiazole orange mono and bifunctionalized PNA to light up PNA targets

R. Ummarino1, C. Avitabile2, L. D. D’Andrea1, M. Saviano1 and A. Romanelli3

1 Istituto di Biostrutture e Bioimmagini, via Mezzocannone 16, Napoli, Italy2 Diagnostica e Farmaceutica Molecolari Scarl, via Mezzocannone 16, Napoli, Italy3 Università di Napoli “Federico II”, Dipartimento di Farmacia, via Mezzocannone 16, Napoli, Italy

Fluorescent probes which emit only upon recognition of their targets have been recently investigated: thiazole orange (TO) has been employed for the modification of nucleotides, the functionalization of DNA and PNA oligomers terminal positions and to build Forced Intercalation (FIT) probes[1]. Such modified oligonucleotides, after hybridization of their targets, allowed the detection of SNPs and mRNA in living cells. Recently exciton controlled hybridization-sensitive fluorescent (ECHO) oligonucleotides containing a pair of TO units linked to the base thymidine were reported to intercalate into DNA duplexes and produce an increase in the fluorescence signal upon hybridization higher than that observed for FIT and TO ending oligonucleotides[2]. The selective recognition of mRNA or DNA fully matched complementary sequences over mismatched ones still remains an issue. In this work we describe the design of PNA oligomers bearing one or two TO units on the side chain of a g-modified PNA. PNA monomers bearing the lysine side chain were obtained following standard procedures and incorporated by solid phase synthesis into PNA oligomers. The PNA sequence was designed to be complementary to miR-210, an evolutionarily conserved and ubiquitously expressed miRNA in hypoxic cell and tissue[3]. Fluorescence and Circular Dichroism experiments were carried out using complementary and mismatched DNA oligomers.

References

1. F. Hövelmann, I. Gaspar, A. Ephrussi, O. Seitz, J. Am. Chem. Soc., (2013), 135, 19025.2. Y. Kimura, T. Hanami, Y. Tanaka, M. J. L. de Hoon, T. Soma, M. Harbers, A. Lezhava, Y. Hayashizaki, K. Usui,

Biochemistry, (2012), 51, 6056.3. S. Y. Chan, , J. Loscalzo, Cell Cycle, (2010), 9 (6), 1072.

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LIST OF PARTICIPANTS


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AAccardo Antonella ItalyAiello Carmela ItalyAitoro Rosita ItalyAliberti Anna ItalyAloj Luigi ItalyAlterio Vincenzo ItalyAlves Isabel FranceAndreu David SpainArcovito Alessandro ItalyArnoldi Anna ItalyAutiero Ida ItalyAvitabile Concetta ItalyAy Bernhard Germany

BBalasco Nicole ItalyBánóczi Zoltán HungaryBarone Daniela ItalyBarone Maria Vittoria ItalyBavoso Alfonso ItalyBechinger Burkhard FranceBehrendt Raymond SwitzerlandBerisio Rita ItalyBianchi Elisabetta ItalyBianco Alberto FranceBotti Paolo SwitzerlandBracci Luisa ItalyBray Isabella IrelandBuonanno Martina Italy

CCai Minying UsaCalce Enrica ItalyCaliendo Giuseppe ItalyCalvanese Luisa ItalyCantisani Marco ItalyCapasso Domenica ItalyCaporale Andrea ItalyCaraglia Michele Italy


173

Carfora Bianca ItalyCarotenuto Marianeve ItalyCarotenuto Alfonso ItalyCarvajal-Rondanelli Patricio ChileCeci Adriana ItalyCelentano Veronica ItalyChambery Angela ItalyCilurzo Felisa ItalyCimmino Amelia ItalyCiociola Tecla ItalyColavita Irene ItalyComegna Daniela ItalyCorvino Giusy Italy

DD’Agosto Filomena ItalyD’Ambrosio Katia ItalyD’Andrea Luca ItalyD’Auria Gabriella ItalyDe Angelis Teresa ItalyDe Falco Sandro ItalyDe Luca Luca ItalyDe Luca Stefania Italyde Paola Ivan ItalyDe Rosa Lucia ItalyDe Rosa Giuseppe ItalyDe Rosa Raffaella Italy De Simone Paola ItalyDe Simone Giuseppina ItalyDe Spiegeleer Bart BelgiumDe Zotti Marta ItalyDel Gatto Annarita ItalyDel Zoppo Luisa Italy Depalo Nicoletta ItalyDi Fiore Anna ItalyDi Gaetano Sonia ItalyDi Maro Salvatore ItalyDi Natale Concetta ItalyDi Sorbo Gianluigi ItalyDi Stasi Rossella ItalyDiaferia Carlo ItalyDiana Donatella ItalyDinarvand Rassoul IranDonnarumma Federica ItalyDoti Nunzianna Italy


174

EEsposito Luciana ItalyEsposito Carla Italy

FFalanga Annarita ItalyFalcigno Lucia ItalyFarina Biancamaria ItalyFarrotti Andrea ItalyFattorusso Roberto ItalyFenude Emma ItalyFerrara Anne Lise ItalyFiorino Ferdinando ItalyFocà Giuseppina ItalyFocà Annalia ItalyFormaggio Fernando ItalyFrecentese Francesco Italy

GGaldiero Stefania ItalyGaldiero Massimiliano ItalyGambari Roberto ItalyGiovati Laura ItalyGobbo Marina ItalyGogliettino Marta ItalyGrieco Paolo ItalyGuerrini Remo ItalyGuilhaudis Laure France

HHan Guoxia CinaHancock Robert CanadaHruby Victor UsaHudecz Ferenc Hungary


175

IIaccarino Emanuela ItalyIaccino Enrico ItalyIannitti Roberta ItalyIncoronato Novella ItalyIngenito Raffaele ItalyIsernia Carla Italy

JJensen Knud Denmark

LLammi Carmen ItalyLangella Emma ItalyLeone Marilisa ItalyŁepek Teresa PolandLewandowska Monika PolandLombardi Lucia ItalyLorenzo Virginia ItalyLubell William CanadaLucietto Pierluigi Italy Luongo Veronica ItalyLuzzietti Nicholas Germany

MMałuch Izabela PolandMangoni Maria Luisa ItalyMarasco Daniela ItalyMarra Carla ItalyMascanzoni Fabiola ItalyMazzaferro Rocco ItalyMeccariello Angela ItalyMelo Manuel NetherlandsMercurio Flavia Anna ItalyMerlino Francesco ItalyMigliaccio Nunzia ItalyMignogna Eleonora Italy


176

Mollica Adriano ItalyMonaco Barbara ItalyMonti Simona Maria ItalyMorelli Giancarlo ItalyMoroder Luis GermanyMusumeci Domenica Italy

NNovellino Ettore Italy

OOdaert Benoit FranceOliva Rosario ItalyOnyuksel Hayat UsaOstuni Angela Italy

PPacia Sabatino ItalyPalmieri Gianna ItalyPalumbo Rosanna ItalyPapini Anna Maria ItalyPascarella Simona ItalyPatel Arvind UKPedone Emilia ItalyPedone Carlo ItalyPedone Paolo ItalyPellegrino Sara ItalyPerillo Emiliana ItalyPertinhez Thelma ItalyPessi Antonello ItalyPicone Delia ItalyPini Alessandro ItalyPiras Linda ItalyPirone Luciano ItalyPolonelli Luciano ItalyProvesiero Massimo ItalyPunzo Valentina Italy


177

RRentero Rebollo Inmaculada SwitzerlandRentier Cedric FranceRicciardi Domenico Rocco ItalyRiccio Raffaele ItalyRinghieri Paola Italy Romanelli Alessandra ItalyRomano Maria ItalyRovero Paolo ItalyRoviello Giovanni ItalyRuggiero Alessia ItalyRussomanno Anna ItalyRuvo Menotti Italy

SSala Marina ItalySalgado Gilmar FranceSandomenico Annamaria ItalySanguigno Luca ItalySanseverino Marina Italy Santagada Vincenzo ItalySantos Nuno PortugalSarmientos Luca Italy Sarmientos Paolo ItalySaviano Michele ItalyScala Maria Carmina ItalyScaramuzza Silvia ItalyScarnato Lucilla ItalyScognamiglio Pasqualina Liana ItalyScrima Mario ItalySelis Fabio ItalySeverino Renato ItalySeverino Beatrice ItalySmaldone Giovanni ItalySpensiero Antonia ItalySperindè Martina ItalySqueglia Flavia ItalyStella Lorenzo ItalyStrandberg Erik GermanySwiecicki Jean-Marie FranceSzabo Ildiko Hungary


178

TTesauro Diego ItalyToniolo Claudio ItalyTornesello Anna Lucia Italy

UUmmarino Raffaella Italyvan der Meijden Benjamin Switzerland

VVancolen Annick GermanyVenanzi Mariano ItalyVernieri Ermelinda ItalyVincenzi Marian ItalyVitagliano Luigi ItalyVitali Alberto Italy

WWimley William UsaWynendaele Evelien Belgium

YYousif Ali Munaim Italy

ZZaccaro Laura ItalyZehender Fabian GermanyZollo Massimo Italy


179

LIST OF AUTHORS


180

AAccardo A. 89, 96, 146 Accordi B. 61Agostiano A. 90, 91Aiello C. 105Aiese N. 61Aisenbrey C. 40Aitoro R. 104Albericio F. 41Aleman C. 51Almeida F. 32Aloj L. 101Alterio V. 152, 153, 154Alves I. 149Alves I. D. 64Ambati J. 66Andreasen P. 35Andreu D. 27Apicella I. 66Appelmelk B. 83Arcari P. 99Arcovito A. 108Arnion H. 149Arnoldi A. 139Aróstica M. 41Assunção-Miranda I. 32Attanasio C. 61Atyabi F. 26Aureli S. 77Aurilio M. 101Autiero I. 167Avitabile C. 79, 169Ay B. 92

BBalasco N. 122, 128, 132Balestrieri M. 138Bánóczi Z. 50, 67Barone D. 122, 123Barone M. V. 104Barras A. 112


181

Basso G. 61Battezzati P. M. 52Bavoso A. 103, 111Bechinger B. 40, 44Bergamini C. 116Berisio R. 72, 82, 83, 133Bettati S. 60Bianco A. 25Bianco R. 66Biondi B. 43, 116Blanco E. 27Bobone S. 53Bocchinfuso G. 44, 51, 53Bolzati C. 94Bortolus M. 116Bősze Sz. 50, 59Botti P. 37Bouhss A. 55Bracalello A. 103Bracci L. 69Bracke M. 70Braguer D. 64Branca D. 78Brancaccio D. 161Bray I. 165Bressollier J-M. 55Brèthes D. 112Brun P. 114Brunetti J. 56, 69Buchet I. 113Bulaj G. 121Buonanno M. 152, 154Bürck J. 54Burlina F. 63Burvenich C. 70

C Cabras T. 108Cai M. 62Calce E. 115Calo’ G. 156Calvanese L. 104, 106, 114, 119Campiglia P. 86, 145, 148, 151, 160, 161Cantile M. 110


182

Cantisani M. 72, 82, 85Capasso D. 68, 144Caporale A. 93, 94, 106, 119, 129, 131, 147Caraglia M. 58, 98Cardona V. 92Carfora B. 106, 119Carneiro F. A. 32Carotenuto A. 86, 148, 151, 160, 161Carotenuto M. 61Carré M. 64Carriero M.V. 155Caruso M. 51Carvajal-Rondanelli P. 41Carvalho F. A. 32Casciaro B. 86Castagliuolo I. 114Castagnola M. 108Castanho M. 32Castiglione Morelli M. A. 111Causon R. 37Cavella S. 159Cecere Palazzo V. 138Ceglia S. 102Celentano V. 134, 141Cerlesi M.C. 156Cervello M. 87Chabas S. 149Chambery A. 162Chartrel N. 113Chassaing G. 63Chemtob S. 28Chen S. 38Chiarolla M.C. 61Chwetzoff S. 63Cicatiello V. 66Cilurzo F. 49Cimmino A. 166Ciociola T. 42, 73, 75, 81Cipolletta E. 151Cocca E. 138Coman D. 74Comegna D. 68, 144Comparelli R. 90, 91Conti S. 42, 73, 75, 81Contini A. 60Contursi P. 84


183

Corricelli M. 90Corvino G. 162Cosco D. 49Costante R. 88Craik D. 62Csik G. 50Curri M. L. 90, 91Cusimano A. 87

DD’Agosto F. 87D’Ambrosio K. 152, 154D’Andrea D. L. 134D’Andrea L. D. 79, 117, 126, 141, 142, 143, 168D’Auria G. 104, 106, 114, 119D’Auria S. 133D’Errico G. 120D’Hondt M. 70D’Ursi A. 120 D’Ursi A. M. 150Da Poian A. T. 32Dalzini A. 116Damiani V. 61Danesin R. 114Darfeuille F. 149Day R. 124De Falco S. 66de la Torre B. G. 27de Laurentiis A. 102De Luca S. 115De Nicola A. 158de Paola I. 65, 68, 90, 91, 144De Reuse H. 149De Rosa G. 98De Rosa L. 126, 143De Simone A. 72, 123, 128De Simone G. 152, 153, 154De Simone P. 83De Spiegeleer B. 45, 70De Vendel J. 103De Wever O. 70De Zotti M. 43, 53, 76, 116Del Gatto A. 68, 90, 91, 144Del Vecchio P. 120


184

Della Sala G. 150Denora N. 90, 91Depalo N. 90, 91Depau L. 69Desbat B. 55Desjardins R. 124Dettin M. 114Di Fiore A. 152, 154Di Gaetano S. 65, 68, 132, 144Di Grazia A. 86Di Marcotullio L. 65Di Monaco R. 159Di Natale C. 105, 127, 136Di Sorbo G. 129, 130, 131Di Stasi R. 117, 142Diana D. 117, 141, 142, 143Dinarvand R. 26Doan N. D. 28Dókus L. E. 67Dölle S. 92Donnarumma F. 159Dosselli R. 47Doti N. 129, 130, 131, 137Dufourc EJ. 55

EEsposito C. 82Esposito L. 122, 128

F Falanga A. 72, 80, 84, 85, 96, 97, 98Falciani C. 56, 69Falcigno L. 104, 106, 114, 119Falcone C. 102Fanizza E. 90, 91Farina B. 68, 129, 130, 131Farrotti A. 44, 53Fasanella Masci F. 102Fato R. 116Fattorusso R. 68, 79, 117, 129, 130, 141, 142, 143Faustino A. F. 32


185

Federico A. 58Fenude E. 140Ferrara A. L. 127Ferri M. 157Finamore E. 85Finetti F. 126Fiume G. 99, 102Focà A. 106, 107, 109, 119, 134Focà G. 106, 107, 109, 119Fooke M. 92Formaggio F. 43, 51, 53, 74, 76Franco R. 58Fregona D. 89Fresta M. 49Fusco S. 84

GGaldiero M. 72, 80, 85, 97, 98Galdiero S. 72, 80, 81, 84, 85, 96, 97, 98Gallay J. 55Gambari R. 164Ganesan A. 113Ganges Ll. 27Garcia-Ramos Y. 28Gatto E. 51Gauck S. 92Gelmi M. L. 60Giovannelli L. 125Giovati L. 42, 73, 75, 81Giraud MF. 112Glattard E. 40Gobbo M. 47Gogliettino M. 138Gomez-Monterrey I. M. 145, 148, 151, 160, 161Grau-Campistany A. 54Grieco P. 58, 86, 94, 98, 145, 151, 155, 160, 161Grimaldi M. 150Guarnieri D. 144Guerrini R. 156Guilhaudis L. 113Guillon J. 146Guzman F. 41


186

H Hancock R. E.W. 24Harvey H. 165Heinis C. 38Hendrix A. 70Hilma G. 74Hosseini M. 35Hruby V. J. 36, 62Huang M. 35Hudecz F. 50, 67

IIaccarino E. 162Iaccarino G. 151Iaccino E. 102Iacobazzi R. M. 90, 91Iannitti R. 89, 138Ibba E. 56Iftemi S. 76Imbimbo C. 61Incoronato N. 72, 80Ingangi V. 155Isernia C. 79

J Jensen K. J. 35

KKessler H. 62Kwiatkowska A. 124

LLamberti A. 99Lammi C. 139Langella E. 167


187

Laquintana V. 90, 91Lavielle S. 63, 64Lefranc B. 113Leone M. 85, 115, 127, 136, 146Łepek T. 118, 121Leprince J. 113Levesque C. 124Lewandowska M. 118, 124Liguoro A. 144Limatola A. 151Liquori A. 68Lombardi L. 80, 97Lopez Cortés G. I. 86Lorenzo V. 137Lozzi L. 56, 69Lubell W. D. 28Luca V. 77Luce A. 58Luchian T. 76Luker T. 37

MMaggi C.A. 160Magliani W. 42, 73, 75, 81Makker S. 103Malfacini D. 156Malgieri G. 65, 79Małuch I. 118Mangoni M. L. 77, 86Maniero A.L. 116Mansuy C. 63Marasco D. 105, 114, 127, 136Marotte A. 113Marquette A. 40Marra C. 135Marrink S. J. 46Marshall SH. 41Martins Ivo C. 32Martora F. 72Martucci N. M. 99Marzano V. 108Marzola E. 156Mascanzoni F. 129, 130, 131, 137Mazzà D. 65


188

Mazzacuva M. 69Meccariello A. 152, 153, 154Meini S. 160Melani F. 125Melo M.N. 46Mercurio F. 146Mercurio F. A. 136Merlino F. 86, 155, 160, 161Mero A. 49Messana I. 108Migliaccio N. 99Mignogna E. 80, 85Milano G. 158Milardi D. 141Mimmi S. 102Mohana-Borges R. 32Molinari S. 156Mollica A. 88Monaco B. 94Monfregola L. 115Monterrey-Gomez I. 86Monti S.M. 152, 153, 154Morbidelli L. 126Morelli G. 85, 87, 89, 96, 97, 101, 104Moret F. 47Mozzarelli A. 60Murray F. 37Musumeci D. 95

N Najda P. 118Nanyakkara M. 104Nardon C. 89Netti P. A. 144Neveu C. 113Nizam Korkut D. 149Nonell S. 47Notomista E. 84Novellino E. 58, 86, 100, 145, 148, 151, 155, 160, 161Nuti F. 52


189

O Oancea S. 74Oda R. 55Odaert B. 55, 112Offermann N. 92Ojeda C. 41Oliva R. 120Onali P. 135Ong H. 28Onyuksel H. 31Orbán E. 50Orlandin A. 74Orsini G. 109, 110Ostuni A. 103, 111

P Pacia S. 105Pacini G. 52Padfield A. 37Paduano L. 120Palleschi A. 44, 51, 53Palmieri C. 99, 102Palmieri G. 138Palmieri M. 65, 79Palumbo R. 89, 138Paolino D. 49Papini A.M. 34, 52Papini E. 43Paredi G. 60Park Y. 53, 77Parker A. 37Pascarella S. 125Pasut G. 49Patel A. H. 33Pedone C. 95Pedone E. 65, 82, 84, 132Pedone E. M. 136Peggion C. 43, 74, 116Pela’ M. 156Pellegrino S. 60Peremans K. 70


190

Perillo E. 80, 97, 98Pertinhez T.A. 73, 75, 81Pessi A. 57Petraccone L. 120Piano R. 60Picone D. 159Pini A. 56, 69Piras L. 100, 160Pirone L. 65, 84, 132, 136Pisano A. 102Piskareva O. 165Pohankova P. 28Politano A. L. 109Pollaro L. 38Polonelli L. 42, 73, 75, 81Pontoriero M. 102Porto S. 98Prahl A. 118, 121, 124Punzo V. 127

QQuercini L. 56Quibell M. 37Quinto I. 99, 102

R Rabanal F. 54Reddi E. 47Rega M. F. 159Reichert J. 54Rentero Rebollo I. 38Rentier C. 52Reza Mahmoudi A. 26Riazi Esfahani M. 26Riccardi D. 105Riccio R. 123Righieri P. 87Ringhieri P. 89, 96Rizzi A. 156Rojas R. 41Romanelli A. 79, 100, 143, 168


191

Romano M. 83Ronda L. 60Ronga L. 146Roodbeen R. 35Rosato N. 44Roscia G. 56, 69Rossi F. 146Rossi M. 138Rovero P. 52, 125Roversi D. 53, 77, 78Roviello G. N. 95Rozza L. 95Ruggiero A. 83, 123, 133Ruggiero I. 99Russomanno A. 117, 143Ruvo M. 66, 93, 94, 106, 107, 109, 110, 119, 129, 130, 131, 134, 137, 138, 147, 162

S Sala M. 145, 148, 151Salgado G. 149Salnikov E. S. 40Salvadori S. 156Sandomenico A. 66, 93, 106, 107, 109, 110, 119, 134, 138, 147, 152, 162Sanguigno L. 110Sanna M.T. 108Sanna R. 110Sanseverino M. 104Santagata S. 101Santicioli P. 160Santinoli C. 42, 75Santos Nuno C. 32Saviano M. 68, 90, 91, 100, 136, 144, 167, 168Scala G. 99, 102Scala M. C. 145, 148, 151Scala S. 101Scali S. 69Scaramuzza S. 107, 109, 135Scarnato L. 157Schlosser G. 50Schmitter J-M. 55Schrepfer R. 107, 109, 110, 135, 147Schulz-Utermoehl T. 37Scialdone A. 102


192

Scognamiglio P. L. 105, 127, 136Scrima M. 150, 158Ségalas-Milazzo I. 113Selis F. 93, 107, 109, 110, 135, 147Selmi C. 52Severino V. 162Shcheglova T. 103Sikorska E. 118Smaldone G. 132Sobrino F. 27Spensiero A. 145, 148, 151Sperindè M. 42, 73, 75, 81Spisni A. 73, 75, 81Squame E. 101Squeglia F. 83Stallings R. 165Stalmans S. 45Stefanucci A. 88Stella L. 44, 53, 76, 77, 78Stellato M. I. 120Strandberg E. 54Striccoli M. 90, 91Su J. 46Supuran C. T. 153, 154Swiecicki J.-M. 63Szabó I. 59Szabó R. 50

T Tailhades J. 63Tantos Á. 67Tarallo R. 80, 97Tarallo V. 66Tassoni A. 157Tavano R. 43Tchertchian S. 37Tesauro D. 87, 111, 146Theurillat D. 37Thiebaut F. 63Tolchard J. 112Toniolo C. 51, 53, 76Tonon G. 93, 107, 109, 110, 135, 147Tornesello A.L. 104Toupé J. 55


193

Tramontano A. 103Traore M. 28Trapè V. 108Trapella C. 156Trotta A. 101Trugnan G. 63Truppo E. 110Tudisco L. 66Turcotte S. 28

UUlrich A. S. 54Ummarino R. 79, 168Urdaci MC. 55

V Vaezi Z. 78Valente G. 91Van De Wiele C. 70Varcamonte M. 84Varshochian R. 26Vaudry H. 113Vecchio E. 102Venanzi M. 51Verbeke F. 70Vernieri E. 145, 148, 151Vincenzi M. 146Vitagliano L. 65, 122, 123, 127, 128, 132, 133, 127Vitali A. 108Vitiello M. 85Vitiello M. T. 72Vullo D. 153

W Wadhwani P. 54Walewska A. 121Weck M. 80, 97Wimley WC. 30Worm M. 92Wynendaele E. 45, 70


194

Y Yousif A. 94Yousif A. M. 86, 155, 160, 161

Z Zaccaro L. 65, 68, 90, 91, 144Zamuner A. 114Zanfardino A. 84Zanoni C. 139Zanuy D. 51Zappavigna S. 58Zehender F. 48Zhang J. 28Ziche M. 126Zollo M. 61

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Centro Congressi d’Ateneo “Federico II” – Via Partenope ... · Centro Congressi d’Ateneo “Federico II” – Via Partenope, Napoli June 12-14, 2014 14th Naples Workshop