Comité d`organisation : Avec la participation locale de

Transcription

19ème Congrès du Groupe Français des Peptides et des Protéines
Portbail, Normandie, France 17-22 mai 2015
Comité d’organisation :
Jérôme LEPRINCE, président
Didier BOTURYN
Inserm U982
Institut de Recherche et d’Innovation Biomédicale
Université de Rouen
Place Emile Blondel
76821 Mont-Saint-Aignan
DCM – Département de Chimie Moléculaire
UMR CNRS 5250, ICMG FR2607
Université Joseph Fourier
570 Rue de la chimie, BP 53
38041 Grenoble
Samuel COUVE-BONNAIRE, vice-président
Florine CAVELIER
COBRA UMR CNRS 6014
IRCOF – INSA de Rouen
Rue Tesnières
76821 Mont-Saint-Aignan
Institut des Biomolécules Max Mousseron
CNRS UMR 5247
Place Eugène Bataillon
34095 Montpellier
Marie-Christine AVERLANT-PETIT,
secrétaire
Céline DOUAT
CBMN – CNRS UMR 5248
Institut Européen de Chimie et de Biologie
2 Rue Robert Escarpit
33607 Pessac
Laboratoire de Chimie-Physique
Macromoléculaire
Université de Lorraine
1 Rue Grandville, BP 20451
54001 Nancy
Nicolas FLOQUET
Institut des Biomolécules Max Mousseron
CNRS UMR 5247
15 Avenue Charles Flahault, BP 14491
34093 Montpellier
Arthur COLAU, trésorier
51 Boulevard Blanqui
Hall C
75013 Paris
Emeric MICLET
CNRS UMR 7203
ENS - Département de chimie
24 Rue Lhomond
75005 Paris
David AITKEN
Laboratoire de Synthèse Organique et
Méthodologie
ICMMO - Bât 420
Université Paris Sud
15 Rue Georges Clemenceau
91405 Orsay
Françoise OCHSENBEIN
iBiTec-S
Commissariat à l’Energie Atomique
91191 Gif sur Yvette
Yannick ARLOT
CNRS UPR 41
Université de Rennes
Faculté de Médecine
2 Avenue Pr. Léon Bernard, CS 34317
35043 Rennes
Avec la participation locale de :
Joël HENRY
UMR BOREA
MNHN, UPMC, UCBN, CNRS-7208, IRD-207
Institut de Biologie Fondamentale et Appliquée
Esplanade de la Paix
14032 Caen
Vincent AUCAGNE
CNRS UPR 4301
Centre de Biophysique Moléculaire
Rue Charles Sadron
45071 Orléans
Marc BLONDEL
Inserm U1078
Faculté de Médecine et des Sciences de la
Santé
22 Avenue Camille Desmoulins
29200 Brest
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Table des matières
Informations pratiques........................................................................................................ 3
Introduction......................................................................................................................... 4
Sponsors .............................................................................................................................. 5
Planning ............................................................................................................................... 12
Programme détaillé ............................................................................................................. 13
Conférenciers Invités (C1-C14) ............................................................................................ 19
Communications Orales (O1-O43) ...................................................................................... 35
Présentations d’Industriels (I1-I10) ..................................................................................... 81
Communications par Affiche (P1-P48) ................................................................................ 93
Liste des Participants........................................................................................................... 143
INFORMATIONS PRATIQUES
Lieu du congrès:
VVF Portbail
3, avenue Pasteur
50580 Portbail - France
Site web : http://www.vvf-villages.fr/villages-vacances/vacances-port-bail-vvf-villages.html
Code WIFI (partie commune) : 15vvf512 – login : internet
Code d’accès à la piscine : 1418A – ouverture de 10h à 19h30. Attention seuls les maillots de
bain sont autorisés (les shorts et bermudas de bain sont strictement interdits).
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INTRODUCTION
Chers collègues,
Au nom du "Groupe Français des Peptides et des Protéines" (GFPP), nous vous souhaitons la
bienvenue à Portbail pour ce 19ème Congrès du GFPP qui se déroule pour la première fois en
Normandie.
Le lieu du 19ème congrès du GFPP est le Village Vacances Familiales « La Porte des Isles »
situé à Portbail, une charmante ville située sur la côte ouest du Cotentin, face aux îles anglonormandes de Jersey et Guernesey.
Le Comité d'organisation a préparé, comme toujours, un programme attractif qui couvrira
les aspects scientifiques fondamentaux et appliqués ayant trait aux peptides et aux
protéines. Le programme comprendra une conférence inaugurale, une conférence grandpublic, 12 conférences plénières, 43 communications orales sélectionnées et plusieurs
dizaines de posters ainsi que de nombreux stands d’exposition de nos partenaires
industriels.
Une fois de plus, notre association a distribué des bourses, 21 cette année, pour les jeunes
chercheurs (étudiants et stagiaires postdoctoraux) leur permettant de participer
gratuitement au congrès et de nous présenter leurs travaux de recherche.
Nous vous remercions chaleureusement pour votre participation à ce 19ème congrès du GFPP
où, nous l’espérons, vous aurez l'occasion de découvrir les avancées les plus récentes dans
les domaines des peptides et des protéines, de rencontrer des amis, des collègues et
d'établir de nouveaux contacts dans des disciplines connexes aux vôtres.
Merci à tous les conférenciers qui ont accepté notre invitation à ce congrès.
Un grand merci aussi à nos sponsors industriels, habituels et nouveaux venus, qui nous
permettent d’organiser ce congrès dans les meilleures conditions.
Enfin nous remercions nos institutions pour leur support financier. La Normandie est une
région qui soutient fortement la recherche et nous en avons eu encore la preuve durant
l’organisation de ce congrès avec des aides conséquentes de diverses institutions et instituts
de recherche normands.
Nous vous souhaitons à toutes et à tous un excellent congrès du GFPP et un bon séjour en
Normandie à Portbail.
Pour le comité d’organisation, Samuel COUVE-BONNAIRE, Joël HENRY et Jérôme LEPRINCE
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SPONSORS INSTITUTIONNELS
Nous tenons à remercier l’ensemble de nos partenaires institutionnels qui, en grand
nombre, ont répondu présent pour ce congrès GFPP normand.
Merci à l’INSERM, le CNRS, l’INSA de Rouen, l’université de Rouen, l’université de Caen,
l’Institut de Recherche et d’Innovation Médicale (IRIB), le labex SYNORG, la Société Chimique
de France (SCF) ainsi qu’ à nos deux écoles normandes doctorales de chimie (EDNC) et de
biologie (EDNBise) pour leurs supports financiers. Un grand merci également à la région
Basse-Normandie et au département de la Manche pour les aides financières allouées pour
l’organisation du congrès. Enfin, merci à la mairie de Portbail pour son aide logistique.
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SPONSORS INDUSTRIELS
Nous tenons également à remercier l’ensemble de nos sponsors industriels pour leur soutien
à notre communauté scientifique. Leur aide financière conséquente est primordiale à
l’organisation de ce congrès bisannuel. Un grand merci à nos partenaires habituels pour leur
fidélité et bienvenue à nos nouveaux sponsors.
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PLANNING
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PROGRAMME DETAILLE
Dimanche 17 Mai 2015
Navette à 14h57 en gare de Valognes.
A partir de 15h : Accueil des participants
19h40 : Apéritif de bienvenue
20h30 : Dîner
21h30 : Ouverture (Jérôme Leprince) puis conférence inaugurale
C1
Alain PROCHIANTZ : Signalisation par transduction protéique
Lundi 18 mai 2015
Session 1 – Modérateurs : David AITKEN et Vincent AUCAGNE
C2
8h40
O1
9h20
O2
9h40
O3
10h00
O4
10H20
Claire LOISON
Modeling of Antimicrobial Lipopeptides in interaction with lipidic membranes
Céline LANDON
Highlight of four consecutive residues in plant defensins involved in their ability
to tolerate zinc
Valérie CORNET
Les SepECPs : Une nouvelle classe de protéines capsulaires impliquées dans la
protection de l’œuf chez Sepia officinalis
Thibaut ROSAY
The C-type natriuretic peptide (CNP) prevents Pseudomonas aeruginosa biofilm
formation using AmiC: identification of a new bacterial target
Dominique BURNOUF
Bacterial replicative sliding clamps as new antibacterial targets
10h40
C3
11h00
O5
11h40
O6
12h00
I1
12h20
Pause café
Christian HACKENBERGER
Chemoselective reactions for the synthesis of functional proteins
Laurent RAIBAUT
Design and synthesis of luminescent lanthanide zinc fingers for biological
applications
Victor TERRIER
A straightforward strategy for automated Fmoc-based synthesis of peptide
crypto-thioesters. Application to the chemical synthesis of disulfide rich peptides
Nicolas RAYNAL (société CEM)
Liberty BlueTM et HE-SPPS: Rapidité et efficacité
13
I2
12h30
Aymeric AUDFRAY (société MALVERN)
La microcalorimétrie renforce le portfolio de Malvern pour caractériser les
protéines
12h40
Déjeuner puis temps libre
Session 2 – Modérateurs : Nicolas FLOQUET et Emeric MICLET
C4
15h00
O7
15h40
O8
16h00
O9
16h20
I3
16h40
I4
16h50
Sébastien GRANIER
Structural studies of opioid receptor activation
Sami MARROUN
Structural and enzymatic probing of S-glycosyltransterases with a joint molecular
modeling and NMR approach
Julia KAFFY
19
F NMR monitoring of the eukaryotic 20S proteasome Chymotrypsin-like activity
by FABS and FAXS
Anaïs TERRIEN
Structural analysis of a collagen triple-helix model by liquid state NMR
Caroline DELMOTTE (société GILSON)
La purification par chromatographie liquide préparative : un savoir-faire Gilson
historique renforcé depuis l’acquisition de la société Armen
Philippe SOMMER (société PROTEOGENIX)
Présentation de PROTEOGENIX
17h00
Pause café
O10
17h20
O11
17h40
O12
18h00
James TOLCHARD
Initial steps in describing the F1Fo ATP synthase dimer interface and modelling
the small hydrophobic subunits of the Fo region with solution state NMR
Nicolo-Michele TONALI
Synthetic mimics of β sheets: design, synthesis and evaluation of their ability to
modulate the aggregation of the β-amyloid 1-42 peptide
Romain TROUILLARD
Production of uniformly 15N/13C labeled glycosylated proteins by hairy roots in
view to NMR structural studies
18h20
SESSION POSTERS PAIRS
20H00
C5
21h00
Dîner
Raphaël HAUMONT (Conférence GRAND PUBLIC)
Innovations en cuisine moléculaire
Mardi 19 Mai 2015
Session 3 – Modérateurs : Gilles SUBRA et Marc BLONDEL
C6
8h40
Annemieke MADDER
Modified peptides for non-covalent and covalent recognition of biological
targets
14
O13
9h20
O14
9h40
O15
10h00
O16
10H20
Cécile ECHALIER
From hybrid peptide trialkoxysilane units to biocompatible hydrogels
Elisabeth GARANGER
Auto-assemblage de polypeptides recombinants à base de motifs élastine
Charlotte MARTIN
Rational design of injectable hexapeptide hydrogelators for controlled-release
drug delivery
Eugénie ROMERO
Efficient synthesis of cyclo 1:1-[α/α-Nα-Bn-hydrazino]mers for the elaboration of
nanotubular self-organization in solution and solid state
10h40
C7
11h00
O17
11h40
O18
12h00
I5
12h20
Pause café
Robin FAHRAEUS
Alternative mRNA translation events for antigenic peptides and for full length
proteins
Ignacio LOPEZ
p53 control of gene expression via mRNA translation during endoplasmic
reticulum stress
Maria-José LISTA
Identification of cellular factors involved in the mechanism of the immune
evasion of the Epstein-Barr virus mediated by the GAr domain of the virallyencoded EBNA1 protein
Antoine BABIN (société BIOPHARMATEC)
Acheter un lyophilisateur n’est pas lyophiliser !
I6
12h30
Aurore FIESCHI (société POLYPEPTIDE)
PolyPeptide Group - The Ultimate Peptide Partner
12h40
Session 4 – Modérateurs : Samuel COUVE-BONNAIRE et Grégory CHAUME
C8
15h20
O19
16h00
I7
16h20
I8
16h30
Philippe JUBAULT
Synthesis of highly functionalized fluorocyclopropanes. Applications to the
synthesis of constrained amino-acid analogs
François HALLE
Palladium-catalyzed synthesis of bridged Phe-Gly dipeptide to access novel
ligands of Translocator Protein 18kDa (TSPO)
Luc ARNAUD (société AGILENT)
Solutions pour les analyses de protéines, peptides en spectrométrie de masse
Alain OMASSON (société PROTEIGENE/PROTEOMICS SOLUTION)
La synthèse de peptides, Systèmes et services
16h40
O20
17h00
Pause café
Olga OVDIICHUK
New
Variety
of
Potential
Amidoxime
Prodrugs
[cyano(hetero)aryl]carbonyl Substituted α-Amino Acids
15
From
(2S)-
O21
17h20
O22
17h40
I9
18h00
David GUERIN
New synthetic pathway to fluoropseudopeptides cisoid conformers via
fluorinated olefin Ring Closing Metathesis (RCM)
Xiaofei ZHANG
Acides aminés α, α-disubstitués de type « super-aspartique » : de nouvelles
«briques élémentaires » pour la synthèse peptidique
Thierry CASCALES (société CS Bio)
La solution pour vos synthétiseurs automatisés et façonnage de peptides
I10
18h10
Jean-Philippe WENCKER (société JASCO France)
Présentation de JASCO France
18h20
SESSION POSTERS IMPAIRS
20H00
Dîner
Mercredi 20 mai 2015
Session 5 – Modérateurs : Didier BOTURYN et Jérôme LEPRINCE
C9
8h40
O23
9h20
O24
9h40
O25
10h00
Norbert SEWALD
Cytotoxic peptide-drug conjugates based on cryptophycins
Jérémie CICCIONE
Direct synthesis of multifunctional NPs for cancer cells imaging
Clément SANCHEZ
Vectorization of pepstatin: a cathepsin D inhibitor as potential antiproliferative
agent
Mélissa DEGARDIN
Development of peptidic bioconjugates featuring RGD ligands for specific cell
capture on functionalized gold surface
10h20
C10 10h40
O26
11h20
Pause café
Olivier BERTEAU
Radical SAM enzymes: Novel catalysts for peptide modification and
engineering
Alhosna BENJDIA
Novel cyclization reactions in bacteriocin biosynthesis
11h40
ASSEMBLEE GENERALE
12h40
Jeudi 21 mai 2015
Session 6 – Modérateurs : Florine CAVELIER et Céline DOUAT
C11 8h40
Sophie FAURE
Peptoïdes : peptidomimétiques à architecture modulable
16
O27
9h20
O28
9h40
O29
10h00
Stéphanie ANTUNES
Innovative oligourea foldamers with antimicrobial effects: application to Bacillus
anthracis infection
Claire GRISON
New β/γ-peptide manifolds designed as the 9/8-helix and the 13-helix
Clément BONNEL
Design, synthesis and structural studies of a new 1:1 α/γ heterogeneous peptide
foldamer: identification of a ribbon-like structure mimicking α-helix
10h20
Pause café
O30
10h40
O31
11h00
O32
11h20
O33
11h40
Khoubaib BEN HAJ SALAH
How does the 1,2,3-triazole amide bond mimetic influences the secondary
structure of peptides? Case study on peptaibols
Grégory CHAUME
Peptaibols actifs sur les membranes : analyse structure-fonction par RMN de
l'état solide à l'aide d'aminoacides fluorés synthétisés sur-mesure
Roberto FANELLI
TES-Dpg, a new Cα-tetrasubstituted silylated amino acis : synthesis and impact
on peptide conformation
Mimoun AYOUB
Technologies for cost effective peptide manufacture, environmental and
economical considerations
12h00
Déjeuner
Session 7 – Modérateurs : Yannick ARLOT et Marie-Christine AVERLANT-PETIT
C12 14h00
O34
14h40
O35
15h00
O36
15h20
Patrick LEGEMBRE
Non-apoptotic role of CD95 in lupus and its disruption using a small peptide
Rhita LAMTAHRI
The octadecaneuropeptide ODN exacerbates focal cerebral ischemia while
exerting protective action in vitro
Joël HENRY
Caractérisation du neuropeptidome de la seiche Sepia officinalis : identification
des neuropeptides impliqués dans la ponte
Maxime ROSSATO
Quantification de peptides modifiés par spectrométrie de masse MALDI-ToF –
Mesures d’interactions récepteur-ligand
15h40
Pause café
C13 16h00
Anthony ROMIEU
O37
Nouveaux outils chimiques pour le marquage fluorescent et la bioconjugaison Applications aux agents d'imagerie moléculaire
Thomas BORDENAVE
Novel contrast agents for in vivo molecular imaging of matrix metalloproteinase12 (MMP-12)
16h40
17
O38
17h00
O39
17h20
17h40
Fabien THOREAU
Conception et synthèse de vecteurs peptidiques pour le ciblage de la périphérie
tumorale : aide à la chirurgie
Ibai VALVERDE
Optimization of bombesin based radiotracers for tumor targeting
Temps libre – Mini-marché d’exposants locaux à partir de 18h30: dégustation et
vente de produits régionaux
Banquet – Soirée de GALA
20H00
Vendredi 22 mai 2015
Session 8 – Modérateurs : Gilles GUICHARD et Françoise OCHSENBEIN
C14 9h20
O40
10h00
O41
10h20
Tamas MARTINEK
Protein mimicry with peptidic foldamers, structure and function
Frédéric BIHEL
Metal-catalyzed synthesis of a new chemical family of unnatural basic amino
acids leading to orally-active NPFF receptor antagonists preventing opioidinduced hyperalgesia
Emmanuelle REMOND
Synthèse diastéréosélective de dérivés thiazoles α-aminoacides et applications
pour la synthèse de nouveaux analogues de la neurotensine
10h40
Pause café
O42
11h00
O43
11h20
Mathieu MAINGOT
Ligands du récepteur de la ghréline. Du peptide au peptido-mimétique : vers une
sélectivité fonctionnelle
Thibault TINTILLIER
Synthèse d'inhibiteurs sélectifs et réversibles de iNOS : chimie sur support,
biologie et modélisation
11h40
Clôture
12h00
Déjeuner
Départ de la navette à 13 h vers la gare de Valognes pour le train de 13h55 en
direction de Paris-St-Lazare.
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CONFERENCIERS
INVITES
C1 – C14
19
20
C1
Signalisation par transduction protéique
Alain Prochiantz
Collège de France, Chair des Processus Morphogénétiques, 11 place Marcelin Berthelot,
75005 Paris, France.
La découverte des peptides transducteurs est née de l’observation que certains
facteurs de transcription, ou certains de leurs domaines, sont internalisés par les cellules vivantes et
adressés directement au cytoplasme et au noyau de ces cellules. C’est le cas du facteur TAT du virus
HIV et d’un grand nombre d’homéoprotéines, probablement la très grande majorité d’entre elles. En
fait, les homéoprotéines ne sont pas seulement internalisées par les cellules, mais aussi sécrétées,
révélant de ce fait l’existence d’un mécanisme inattendu de signalisation cellulaire. La conservation
des séquences permettant internalisation et sécrétion et la présence d’homéoprotéines chez tous les
métaphytes et métazoaires, et même chez les unicellulaires eucaryotes, suggère qu’il s’agit là d’un
mécanisme très ancien, probablement actif chez les premiers organismes multicellulaires.
Au cours de cette présentation, des exemples seront donnés qui illustreront pour quelques
homéoprotéines l’importance de cette voie de signalisation au cours du développement et chez
l’adulte. Leur possible utilisation comme protéines thérapeutiques dans des modèles murins de la
maladie de Parkinson sera discutée.
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C2 Modeling of Antimicrobial Lipopeptides in interaction with
lipidic membranes
Claire Loison,1 Mehmet Nail Nasir,2 Françoise Besson,3 Emmanuel Benichou1
and Pierre-François Brevet1
1
2
Institut Lumière Matière, CNRS, Claude Bernard Lyon1 University, Lyon, France; Liège
3
University, Liège, Belgium; Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, CNRS, Claude
Bernard Lyon1 University, INSA Lyon, CPE Lyon, Lyon, France.
The resistance of bacteria and fungi to available antibiotics is a major concern worldwide, leading to
enormous effort to develop innovative drugs with new modes of actions. Two families of natural
peptides are in this respect promising : host-defense cationic antimicrobial peptides, and
lipopeptides. Iturinic lipopeptides, in particular Mycosubtilin, shows interesting biocide activity
against fungi. Its interactions with the lipidic membranes of the pathogens seem to be at the origin of
the biological activities. Cholesterol or ergosterol have been shown to play a role in Mycosubtilin
activities, but the precise molecular mechanisms of the interactions need to be further elucidated. In
our theoretical approach, using molecular dynamics simulations, we investigated the interaction
between iturinic lipopeptides and various interfaces. We have performed molecular dynamics
simulation of the peptide in water, at the air/water interface, and in interaction with phospholipids
or sterols monolayers. Conformations of the lipopeptide were generated using replica-exchange
molecular dynamics (REMD) [1,2]. Using metadynamics simulations, the free energy of adsorption
and insertion of the lipopeptide in lipid membranes was estimated. Specific interactions of the
lipopeptide with sterols are observed, they may be decisive in the selectivity of its antimicrobial
activity. These results are discussed along with experimental data describing physico-chemical
properties of lipopeptides at various interfaces [2,3,4].
1. Loison C. et al. (2014) Phys. Chem. Chem. Phys. 16 ,2136
2. Nasir M. N. et al. (2014) Phys. Chem. Chem. Phys. 15, 19919
3. Nasir M. N. and Besson F. (2011) Langmuir 27, 10785
4. Nasir M. N. et al. (2013) Phys. Chem. Chem. Phys. 15, 19919
22
C3 Chemoselective reactions for the synthesis of functional
proteins
Christian P. R. Hackenberger
Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Roessle-Str. 10, 13125 Berlin,
Germany; Humboldt Universität zu Berlin, Department Chemie, Brook-Taylor-Str.2, 12489
Berlin, Germany.
In our laboratory we constantly aim to develop new organic transformations for the chemoselective
modification and ligation of proteins (see Scheme)1. In this, our main focus is to apply these novel
transformations as well as other established reactions in combination with biochemical methods to
study functional consequences of natural protein modifications as well as to generate novel peptideand protein-conjugates or pharmaceutical and medicinal applications.
Recently, we have identified Staudinger reactions with phosphites, phosphonites and silylated
phosphinic acids as such chemoselective transformations for the functionalization of azides under
mild conditions in high yields2. In this presentation selected examples for the synthesis of P(III)building blocks and their applications to deliver functional peptide and protein-conjugates are given.
These applications include a PEGylation strategy for the intracellular stabilization of peptides3 and
the study of site-specifically phosphorylated Lys-peptides4.
1. Hackenberger C. P. R. & Schwarzer D. (2008) Angew. Chem. Int. Ed. 47, 10030-10074
2. a) Serwa R. et al. (2009) Angew. Chem. Int. Ed. 48, 8234-8239; b) Vallée M. R. J. et al. (2013) Angew. Chem. Int. Ed. 52,
9504-9508; c) Wilkening I. et al. (2011) Chem. Commun. 47, 349-351
3. Nischan N. et al. (2013) Angew. Chem. Int. Ed. 52, 11920-11924
4. Bertran-Vicente J. et al. (2014) J. Am. Chem. Soc. 136, 13622-13628
23
C4
Structural studies of opioid receptor activation
Sébastien Granier
Institut de Génomique Fonctionnelle, Inserm U1191, CNRS UMR 5203, Université de
Montpellier, 141 rue de la cardonille, 34094 Montpellier, France.
Opioid receptors (OR), members of the G protein-coupled receptor (GPCR)
superfamily, constitute the major and the most effective target for the treatment of
pain. Both beneficial and adverse effects of illicit opioid drugs (opium, heroin) as well as approved
therapeutics (morphine and codeine) are mediated by the activation of the mu-opioid receptor
(µOR).
We recently described the structure of an inactive conformation of the µOR. It provided important
information regarding the binding site of small morphinan antagonists, revealed a largely exposed
binding pocket, and demonstrated key molecular determinants for antagonist binding preferences
for OR. However, much remains to be learned about the mechanisms by which different agonists can
induce distinct levels of Gi protein activation and/or arrestin recruitment upon activation of µOR.
In this study, we propose to analyse the activation mechanism of the µOR using liquid-state NMR
spectroscopy and X-ray crystallography. Our goal is to provide insights into opioid receptor activation
upon binding of ligands presenting distinct efficacy and/or biased signaling properties. A better
knowledge of the structural basis for opioid drug efficacy may lead to new therapeutic approaches
with limited side effects.
24
C5
Innovations en cuisine moléculaire
Raphaël Haumont
1
ICMMO-CFIC, Chaire « cuisine du futur », Faculté des sciences d’Orsay, Université Paris Sud,
91405 Orsay, France
« En un mot, la cuisine, sans cesser d'être un art, deviendra scientifique et devra
soumettre ses formules, empiriques trop souvent encore, à une méthode et à une précision qui ne
laisseront rien au hasard ». Escoffier 1907 (Guide culinaire)
100 ans après, la « cuisine moléculaire » entre dans le dictionnaire de la langue française.
La cuisine moléculaire n’est ni une mode, ni une tendance. Cette cuisine innovante n’est en aucun
cas en rupture avec la tradition. Voyons simplement cela comme une cuisine de ce début de siècle,
utilisant les techniques et les connaissances d’aujourd’hui.
Les avancées scientifiques (Pasteur, Appert,…) ont toujours contribués à faire avancer la cuisine, et il
est finalement naturel que cuisiniers et scientifiques s’associent pour aller plus loin et faire
différemment. La cuisine moléculaire est moins empirique et demande plus de rigueur, en vue de
réaliser des préparations mieux maîtrisées, sur mesure, pour le plus grand respect des produits, -de
leurs cuissons et de leurs transformations-, à l’origine de jeux de textures et de températures,
d’innovations, et dans le but ultime de procurer des sensations et émotions nouvelles aux clients. En
effet, celui qui cherche à comprendre les phénomènes qui se produisent pendant qu’il prépare et
associe des produits, pourra prétendre maîtriser, reproduire à l’exactitude, anticiper, et de ce fait
aller plus loin : prédire, donc pouvoir créer des choses nouvelles et innovantes: De la cuisine un peu
moins empirique, certes, mais plus créative et toujours plus gourmande !
La conférence présentera, démonstrations à l’appui, comment la physico-chimie peut aider à
l’innovation en cuisine, mais aussi à répondre aux enjeux de l’alimentation de demain (gaspillage,
emballage, déchets etc…). Mousses de fruits effervescences, canette comestible, cryoconcentration,
marmelade instantanée, mousse de chocolat…à vos éprouvettes, et à vos fourchettes !
25
C6 Modified peptides for
recognition of biological targets
non-covalent
and
covalent
Abhishek Iyer, Yara Ruiz, Lieselot Carrette, Kurt Hoogewijs, Willem Vannecke
and Annemieke Madder
Organic and Biomimetic Chemistry Research Group, Department of Organic Chemistry, Ghent
University, Krijgslaan 281 S4, B-9000 Gent, Belgium.
The study of DNA-protein interactions, important for many cellular processes, has so far not allowed
unraveling the exact mechanism behind their unique selectivity and affinity. We have been studying
these interactions through the use of DNA binding protein mimics.1 Next to an earlier described noncovalent GCN4 mimicking peptide dimer as standard;2 a new type of steroid-based dipodal DNA
binders has been investigated.3 Though synthetically challenging in view of the close distance
between two peptide chains immobilized on the steroid template, improved properties in terms of
binding, biostability and bio-availability have been observed. In a subsequent attempt to further
reduce the size of the synthetic DNA binders, we have been looking into existing stapling methods to
enhance the DNA binding potential of a single chain peptide containing the binding region of the
GCN4 protein. Two selectively positioned, non-binding amino acids on the GCN4 peptide were
replaced by Cysteine residues and covalently connected using bisalkylators resulting in stapled
peptides. Unlike the synthetic bZip peptides where α helicity is induced via a dimeric structure, we
here stabilize a single α helix via peptide stapling. CD analysis of the peptides has shown
improvement in helicity. Subsequently, the DNA binding of the peptides to the CRE binding site was
studied by Gel electrophoresis.4
In a further study, synthetic miniaturized peptides were modified with furan moieties, which trigger
crosslinking upon oxidation into a reactive ketobutenal5 and allow the conversion of transient
supramolecular interactions into stable covalently bound complexes. Proximity being a prerequisite
for this novel selective DNA-peptide crosslinking methodology, it allows for distance probing to
interacting functionalities and offers further opportunities for therapeutic developments.6 The furanoxidation based crosslink methodology was then transferred to and applied on the peptide-protein
interface and some examples thereof will be discussed.
1. Carrette L. L. G. et. al. (2010) Drug. Discov. Today: Tech 7, 115
2. Ueno M. Et al. (1993) J. Amer. Chem. Soc. 115, 125752
3. Verzele D. & Madder A. (2013) Eur. J. Org. Chem. 4, 673
4. Iyer A. & Madder A. (2015) Org. Biomol. Chem. Early edition
5. Op de Beeck M. & Madder A. (2011) J. Amer. Chem. Soc. 133, 796
6. Carrette L. L. G. et al. (2013) Bioconj. Chem. 24, 2008
26
C7 Alternative mRNA translation events for antigenic
peptides and for full length proteins
Sébastien Apcher2, Chrysoula Daskalogianni1, Guy Millot1 and Robin
Fahraeus1
1
Inserm UMRS 1162, 27 rue Juliette Dodu, 75010 Paris, France; 2Gustave Roussy,
Université Paris Sud, Département d’immunologie, INSERM U1015, 114 Rue
Édouard Vaillant, Villejuif, F-94805, France.
The presentation of antigenic peptides (AP) on MHC class I molecules allows the immune system to
detect and eliminate cells that are infected by pathogens or are transformed. Until more recently
little attention was given to the source AP and it was widely assumed that these were derived from
the degradation of full length proteins. But based on observations showing a poor correlation
between antigenic peptides and protein turnover rate challenged this concept1. More recently we
made the observation that mRNAs targeted for the nonsense-mediated degradation pathway which
prevents faulty mRNAs from translating full length proteins still produced the same amount of AP
substrates2. We could also show that mRNAs stop producing AP substrates long before they stop to
encode the full length proteins. Most surprisingly was the observation that APs are equally well
presented when inserted into introns3. Our observations suggest that AP and full length proteins are
produced during two spatiotemporally different events and several different data indicate that the
former might take place in the nuclear compartment.
1. Yewdell J. W., et al. (1996) J. Immunol. 157, 1823-1826
2. Apcher S. et al. (2011) Proc. Natl. Acad. Sci. USA 108, 11572-11577
3. Apcher S. et al. (2013) Proc. Natl. Acad. Sci. USA 110, 17951-17956
27
C8 Synthesis of highly functionalized fluorocyclopropanes.
Applications to the synthesis of constrained amino-acid analogs
Philippe Jubault, Pavel Ivashkin, Gérald Lemonnier, Gaëlle Milanole, Samuel
Couve-Bonnaire and Xavier Pannecoucke
Normandie Univ., COBRA, UMR 6014 et FR 3038; Univ. Rouen; INSA Rouen; CNRS, 1 rue
Tesnière, 76821 Mont Saint-Aignan Cedex, France.
Synthesis of biologically active peptides is a central goal in medicinal chemistry. However, major
drawbacks in this area are their low metabolic stability and poor ligand-receptor interactions eroding
the bioavailability of peptide-based drugs. To overcome these disadvantages, considerable interest
has been devoted to synthesize peptidomimetics by modifying the natural sequence of amino acids
of bioactive peptides.
In our ongoing project, we decided to focus on the design of peptidomimetics including a fluorinated
cyclopropane moiety. Incorporation of a three-membered ring influences the secondary structure,
leading to conformationally constrained peptide chains. Thus, the ligand affinity for the enzyme
binding site would be improved, as well as the orientation of amino acid side chains. Moreover,
many studies show that the presence of a fluorine atom induces modifications of various parameters
(lipophilicity, acidity/basicity, electronic distribution or bond lengths).
We report here an efficient, general, asymmetric synthesis of highly functionalized fluorinated
cyclopropanes and especially the synthesis of fluorinated cyclopropyl analogs of four natural amino
acids (methionine, leucine, arginine and lysine), and the incorporation of these scaffolds in peptides.
We also report the diastereoselective synthesis of glutamic acid analogs and the evaluation of their
agonist activity towards metabotropic glutamate receptor sub-type 4 (mGluR4). Finally the synthesis
of fluorinated analog of TMC 435 (which incorporates within its structure a cyclopropyl amino-acid
unit), a NS3/4A serine protease inhibitor for HCV treatment will be described.
Conférencier sponsorisé par le Labex SYNORG
28
C9
Cytotoxic peptide-drug conjugates based on cryptophycins
Norbert Sewald,1 Tobias Bogner,1 Stefan Eißler,1 Markus Nahrwold,1 Benedikt
Sammet,1 Arvydas Stoncius,1 Soledad Royo Gracia,1 Christine Weiß1 and Ralf
Palmisano2
1
Bielefeld University, Department of Chemistry, Organic and Bioorganic Chemistry, Bielefeld,
2
Germany; Department of Biology, Friedrich-Alexander Universität Erlangen-Nürnberg,
Erlangen, Germany.
Anticancer chemotherapeutics like paclitaxel interfere with microtubule dynamics and prevent
microtubules from forming correct mitotic spindles, which causes cell-cycle arrest and apoptosis.
Cryptophycins are a class of 16-membered highly cytotoxic macrocyclic depsipeptides isolated from
cyanobacteria.1 The biological activity is based on their ability to interact with tubulin. Strong
antiproliferative activities with 100- to 1000-fold increased potency compared to paclitaxel and
vinblastine have been observed.2 Cryptophycins are highly promising drug candidates, since their
biological activity is not negatively affected by P-glycoprotein, a drug efflux system commonly found
in multdrug resistant cancer cell lines and solid tumors. Cryptophycin-52 had been investigated in
phase II clinical trials, but failed because of its high neurotoxicity.3
We have developed efficient strategies for the synthesis of cryptophycins and their analogues [2] for
structure-activitiy relationship data, taking specific emphasis on the synthetically most challenging
unit A.4 In addition, new interesting functionalities have been introduced in different positions for
SAR studies and application in bioconjugation for targeted delivery.4,5
The quasi-isosterism of 1,4-disubstituted 1H-1,2,3-triazoles and trans-amide bonds is still under
debate. Therefore, we additionally synthesized an analogue of cryptophycin-52 where the transamide bond between units B and C is replaced by a 1,4-disubstituted 1H-1,2,3-triazole. The cytotoxic
activity is largely retained for this “clicktophycin”, generated by a [3+2] “click” cycloaddition reaction.
Consequently, this proves the bio-equivalence of 1,4-disubstituted 1H-1,2,3-triazoles and transamide bonds even in complex compounds.6
An azide-functionalized cryptophycin was connected by copper(I)-catalyzed azide-alkyne
cycloaddition (CuAAC) to a fluorescently labeled cyclic RGD-peptide for internalization studies. The
cyclic RGD-peptide was designed to act as the homing device, because it binds to integrin αVβ3, which
is highly expressed e.g. on some tumor cells. Confocal fluorescence microscopy proves the
internalization and final lysosomal localization of the cryptophycin conjugate.7
1. Schwartz R. E. et al. (1990) J. Ind. Microbiol. 5, 113-124
2. a) Eissler S. et al. (2006) Synthesis 3747-3789; b) Weiss C. et al. (2013) Nat. Prod. Rep. 30, 924-940
3. D’Agostino G. et al. (2006) Int. J. Gynecol. Cancer 16, 71-76
4. a) Eissler S. et al. (2007) Org. Lett. 9, 817-819; b) Eissler S. et al. (2007) Synlett 273-277; c) Sammet B. et al. (2009) Synlett
417-420
5. Eissler S. et al. (2009) Chem. Eur. J. 15, 11273-11287; Sammet B. et al. (2010) J. Org. Chem. 75, 6953-6960
6. Nahrwold M. et al. (2010) Org. Lett. 12, 1064-1067
7. Nahrwold M. et al. (2013) J. Med. Chem. 56, 1853-1864
29
C10 Radical SAM enzymes: Novel catalysts for peptide
modification and engineering
Olivier Berteau
INRA and AgroParisTech, ChemSyBio, Institut Micalis (UMR 1319), 78350 Jouy-en-Josas, France.
Radical SAM enzymes are an emerging class of enzymes only recently recognized as a
super-family (1). These metallo-enzymes which catalyze chemically challenging reactions use a
radical-based mechanism to have access to chemistry and transformations not otherwise reachable.
Recently, in connection with the progresses in genomic and metagenomic, radical SAM enzymes
have been shown to be widespread in natural product biosynthetic pathways including many
antibiotics, bacteriocins and anticancer agents. They have been shown to catalyze a broad range of
reactions, some of which having no counterparts in synthetic chemistry.
In the last years, radical SAM enzymes have been shown to be instrumental for the biosynthesis of an
emerging class of natural products called RiPPs "Ribosomally synthesized and post-translationally
modified peptides" (2). RiPPs are characterized notably by unconventional post-translational
modifications many of which including cyclization, epimerization or methyl transfer reactions are
catalyzed by radical SAM enzymes.
Recent progresses have been made on these challenging enzymes leading to a deeper understanding
of these novel reactions which, in most cases, remain elusive. Notably, we have demonstrated novel
enzymatic strategies for peptide methylation (3) and identified novel post-translational modifications
leading to the formation of complex peptide structures. These fascinating enzymes represent thus an
emerging class of catalysts with an outstanding potential for peptide engineering.
1. Sofia H. J. et al. (2001) Nucleic Acids Res. 29, 1097
2. Arnison P. G. et al. (2013) Nat. Prod. Rep. 30, 108
3. Pierre S et al. (2012) Nat. Chem. Biol. 8, 957
30
C11 Peptoïdes : peptidomimétiques à architecture modulable
Sophie Faure
ICCF, UMR 6296, 24 avenue des Landais, BP 80026, 63171 Aubière.
Un enjeu majeur dans le domaine des foldamères est de combiner au sein
d’oligomères synthétiques de longueur modeste, la fonctionnalisation nécessaire
à l’activité et la présence de structure secondaire stable en milieu biologique. Les peptoïdes, mimes
de peptides résultant du déplacement des chaînes latérales des carbones Cα vers les atomes d’azote
des amides adjacents, de par leurs propriétés intrinsèques semblent être de bons candidats pour
relever ce challenge.1 Les peptoïdes possèdent des avantages indéniables, notamment en termes de
facilité de synthèse, de diversité des chaînes latérales accessible et de stabilité protéolytique. Par
contre, d’un point de vue conformationnel, cet oligoamide étant constitué d’amides N,Ndisubstitués, les isomères cis et trans peuvent exister en solution.2 Ce degré de liberté
supplémentaire comparé aux peptides, est souvent considéré comme un désavantage car cette
flexibilité rend les peptoïdes plus difficiles à structurer. Pourtant les recherches actuelles menées
dans ce domaine montrent que cette isomérie peut être mise à profit pour concevoir de nouvelles
architectures contrôlées.3
1. a) Zuckermann R. N. (2011) Biopolymers 96, 545; b) Fowler S. H. & Blackwell H. (2009) Org. Biomol. Chem. 7, 1508
2. Szekely T. et al. (2013) C. R. Chimie 16, 318
3. a) Gorske C. et al. (2009) J. Am. Chem. Soc. 131, 16555; b) Caumes C. et al. (2012) J. Am. Chem. Soc. 134, 9553; c) Crapster,
J. A. et al. (2013) Angew. Chem. Int. Ed. 52, 5079; d) Roy O. et al. (2013) Org. Lett. 15, 2246
31
C12 Non-apoptotic role of CD95 in lupus and its disruption
using a small peptide
Doriane Sanséau,1,2,3 Aubin Penna,1,2,3 Marine Malleter,1,2,3 Patrick
Blanco,4,5,6 Alain Dupuy,7 Florence Poizeau,7 Alain Fautrel,8 Roselyne Viel,8
Julien Séneschal,4,5 Cécile Contin,4,5,6 Thomas Ducret,4,9 Anne-Marie
Vacher,4,10 Nicolas Levoin,1,2,3 Robin Flynn,11 Pierre Vacher,4,10 and Patrick
Legembre1,2,3
1
2
Université de Rennes-1, Inserm U1085, 2 avenue du Prof Léon Bernard, 35043 Rennes cedex, France; Centre
3
Eugène Marquis, rue bataille Flandres Dunkerque, 35042 Rennes, France; Equipe Labellisée Ligue Contre Le
Cancer “Death Receptors and Tumor Escape”, 2 avenue du Professeur Léon Bernard, 35043, Rennes, France;
4
5
Université de Bordeaux, 146 rue Léo Saignat, 33076 Bordeaux, France; CHU Bordeaux, Bordeaux, France;
6
7
8
UMR CNRS 5164, 146 rue Léo Saignat, 33076 Bordeaux, France; CHU Rennes, Rennes, France; Université de
Rennes-1, Plateforme H2P2, Biosit, Biogenouest, 2 avenue du Prof Léon Bernard, 35043 Rennes Cedex, France;
9
Inserm U1045, Centre de recherche cardiothoracique de Bordeaux, 146 rue Léo Saignat, 33076 Bordeaux,
10
11
France; Inserm U916, VINCO, 33000 Bordeaux, France; School of Veterinary Medicine and Science, University
of Nottingham, Nottingham, United Kindom.
The death receptor CD95 (also known as Fas) plays a pivotal role in immune surveillance and immune
tolerance. Interaction of CD95 with its ligand, CD95L, leads to the formation of a molecular complex
named death inducing signaling complex (DISC), which orchestrates the implementation of a
caspase-driven apoptotic signaling pathway. CD95L is a transmembrane protein that can be cleaved
by metalloprotease. Unlike membrane-bound CD95L, metalloprotease-cleaved CD95L (cl-CD95L) fails
to trigger DISC formation and rather promotes cell migration through the induction of a PI3K/calcium
(Ca2+) cue (Tauzin, PLoS Biol, 2011 & Malleter, Cancer Res, 2013).
We demonstrated that the concentration of cl-CD95L is correlated with the severity of the pathology
in systemic lupus erythematosus (SLE) patients. This soluble CD95L is able to enhance extravasation
of activated T cells, a cellular phenomenon contributing to the accumulation of lymphocytes in
inflamed tissues through the formation of an unconventional CD95-containing receptosome termed
the motility-inducing signaling complex (MISC). Formation of this complex is instrumental in evoking
a Ca2+ response. By selectively interfering with this CD95-mediated Ca2+ signal using a cellpenetrating peptide, we prevented in vitro and in vivo endothelial transmigration of T lymphocytes.
In conclusion, our study provides novel insights into the cellular and molecular mechanisms by which
cl-CD95L contributes to SLE pathogenesis. Moreover, neutralizing the CD95/CD95L signaling pathway
may turn out to be a future therapeutic approach in the treatment of SLE.
32
C13 Nouveaux outils chimiques pour le marquage fluorescent et la
bioconjugaison - Applications aux agents d'imagerie moléculaire
Anthony Romieu
Université de Bourgogne - UFR Sciences et Techniques - Faculté des Sciences Mirande ; ICMUB UMR CNRS 6302 - Equipe "P2DA", 9, avenue Alain Savary BP 47870, 21078 Dijon Cedex, France ;
Institut Universitaire de France, 103, boulevard Saint-Michel, 75005 Paris, France.
Le développement de nouveaux outils chimiques innovants destinés à des applications en imagerie
moléculaire in cellulo ou in vivo, selon différentes modalités incluant notamment la résonance
magnétique, les ultrasons, les radionucléides et l'optique, est aujourd'hui un domaine de recherche
en pleine expansion. En effet, l'essor de ces modalités d'imagerie dans des applications biomédicales
de pointe (diagnostic et théranostic) est indissociable des améliorations à apporter aux sondes
moléculaires qui sont aujourd'hui les réactifs de choix pour leur mise en œuvre.
C'est dans ce cadre que s'inscrit cette présentation dont l'accent sera plus particulièrement mis sur
les agents d'imagerie optique. Elle s'articulera autour deux thèmes centraux. Le premier concernera
la (bio)chimie des colorants fluorescents et les stratégies récemment développées afin de les rendre
biocompatibles, facilement conjugables à des vecteurs biologiques et "intelligents" du point de vue
photophysique. Le second traitera des avancées récentes en chimie des plateformes moléculaires
multivalentes. En effet, ces outils s'imposent actuellement comme des réactifs de choix dans des
applications qui requièrent un accès simple et rapide à des bioconjugués toujours plus sophistiqués
(comme c'est le cas notamment avec les agents d'imagerie). Quelques réalisations récentes émanant
des acteurs du domaine et de notre laboratoire seront présentées.
33
C14 Protein mimicry with peptidic foldamers, structure and
function
Tamás A. Martinek
Institute of Pharmaceutical Chemistry, University of Szeged, Somogyi u. 4., H-6720, Szeged,
Hungary.
Solvent exposed flat regions are responsible for many of the known protein–protein
and protein-membrane interaction interfaces.1 It is challenging, however, to construct artificial
protein mimetic sequences, which fold (foldamers) and are able to cover these relatively large flat
surfaces with programmable anchor points.2 Peptidic sequences with unnatural building blocks (e.g.,
β-amino acids) are known to form compact secondary structures, which can be controlled through
the stereochemical pattern along the peptidic backbone.3 It will be shown how this principle affords
the de novo desing of foldameric secondary structures.
In the second part of the presentation, examples will be given for the application of these protein
mimetic sequences. We have successfully synthesized multivalent helical foldamer-dendrimer
conjugates that recognize and inhibit the neurotoxic properties of the oligomeric β-amyloid (Aβ).4
These foldamer conjugates can functionally mimic the molecular recognition properties of the antiAβ monoclonal antibodies in an ELISA setup.
Water soluble β-sandwich mimetic foldamers can have sufficiently large and flat surface, but their
stabilization in water is a great current challenge. Here we present the design, main structural
features and biological effects of the foldameric analogs of anginex, a 33-mer antiangiogenic peptide
with a tendency to form β-sandwich. The effects of the β-amino acid substitutions in this β-sheet
structure will be discussed.5
1. Li B. et al. (2008) Proteins 71, 670
2. Wilson A. J. (2009) Chem. Soc. Rev. 38, 3289
3. Martinek T. A. & Fulop F. (2012) Chem. Soc. Rev. 41, 687
4. Fulop L. et al. (2012) Plos One 7, e39485
5. Hegedus Z. et al. (2013) J. Am. Chem. Soc. 135, 16578
34
COMMUNICATIONS
ORALES
O1 - O43
35
36
O1 Highlight of four consecutive residues in plant defensins involved in
their ability to tolerate zinc
Françoise Paquet,1 Fanny Meindre,1 Dominique Lelièvre,1 Pierre Berthomieu,2 Laurence
Marquès,2 Agnès F. Delmas1 and Céline Landon1
1
Centre de Biophysique Moléculaire, CNRS UPR4301, Rue Charles Sadron 45071 Orléans Cedex 2, France ;
Laboratoire de Biochimie & Physiologie Moléculaire des Plantes, INRA/SupAgro, 2 Place P. Viala 34060
Montpellier Cedex 2, France;
2
Plant defensins (PDF) are cysteine-rich peptides identified in most of the plant tissues. Their
sequences (about 50 amino acids) harbour eight highly conserved cysteines involved in four
conserved disulphide bonds (C1–C8, C2–C5, C3–C6 and C4–C7). Despite a weak sequence identity,
the 13 3D structures of plant defensins available in the Protein Data Bank share a common globular
cysteine stabilized α-helix β-sheet motif (CSαβ motif) also found in scorpion toxins and insect
defensins.
PDFs are major actors in the innate immunity in plants, largely documented for their antifungal
activity [1]. However, besides these antifungal properties, new unrelated activities have been
reported more recently. For example, some PDFs, such as Arabidopsis halleri PDF1.1b, have been
shown to confer zinc tolerance in yeast and in plant cells when overexpressed [2].
In the present work [3], we first report an efficient protocol for the production AhPDF1.1b by solidphase peptide synthesis. SPPS had never been used for the production of plant defensins before
(rather long peptide, conserved C-terminal cysteine, presence of 4 disulfide bridges). Our controlled
oxidative folding leads to the highly pure native form of AhPDF1.1b.
Then we determined the 3D NMR structure of AhPDF1.1b to gain initial insights into the structural
requirement for zinc tolerance. Based on our detailed structural analysis, and in combination with
previous biological data for antifungal and zinc tolerance activities, we established the essential role
of four consecutive residues [Val39-Phe40-cisPro41-Ala42] strictly conserved for plant defensins able to
tolerate zinc.
1. Lacerda A. F. et al. (2014) Front. Microbiol. 5, 116
2. Mirouze M. et al. (2006) Plant J. 47, 329-342
3. Meindre F. et al. (2014) Biochemistry 53,7745-7754
37
O2 Les SepECPs : Une nouvelle classe de protéines capsulaires impliquées
dans la protection de l’œuf chez Sepia officinalis
Valérie Cornet,1,2 Joël Henry,1,2,3 Didier Goux,4 Elise Duval,1 Erwan Corre,5 Gildas Le
Corguillé,5 Benoît. Bernay1,3 et Céline Zatylny-Gaudin1,2
1
2
Université de Caen Basse-Normandie, F-14032 Caen, France, UMR 7208 BOREA (Unicaen - CNRS - MNHN 3
UPMC – IRD) F-14032 Caen, France, Plateforme Post-génomique PROTEOGEN, Université de Caen Basse4
Normandie, F-14032 Caen, France, CMAbio, Université de Caen Basse-Normandie, F-14032 Caen Cedex,
5
France, Plateforme ABiMs, Station biologique de Roscoff (UPMC-CNRS), F-29688 Roscoff, France.
Chez Sepia officinalis, la protection de l’œuf est assurée par une enveloppe capsulaire complexe
secrétée par les glandes génitales femelles et la poche du noir. Les principaux constituants de cette
capsule sont d’origine protéique et ont fait l’objet de cette étude. Les transcriptomes de novo
réalisés en Illumina à partir des glandes génitales femelles ont permis d’obtenir les transcrits
nécessaires à l’identification des protéines de cette capsule. Une approche protéomique classique en
SDS-PAGE couplée à des analyses en spectrométrie de masse a permis d’identifier une nouvelle
famille de protéines capsulaires : les SepECPs (Sepia officinalis Egg Case Proteins). La glycosylation de
ces protéines a été démontrée à partir d’une coloration à l’acide périodique de Schiff sur SDS-PAGE.
Ces glycoprotéines ont pour origine les glandes nidamentaires principales, où leurs transcrits sont
très largement exprimés. Les SepECPs possèdent de fortes homologies de séquences (54 %
d’identité) particulièrement au niveau du peptide signal et au niveau de trois régions riches en
cystéines. L’analyse de l’assemblage des séquences obtenues en Illumina a démontré une homologie
de séquence de 100% au niveau de la partie 5’ correspondant au peptide signal, suggérant
l’existence d’un épissage alternatif à partir d’un gène unique. Les SepECPs présentent un nombre
élevé de Cystéines avec des motifs conservés impliqués dans des liaisons intramoléculaires mais
également intermoléculaires. La dimérisation de ces SepECPs observée en SDS-PAGE a été confirmée
par des observations en microscopie électronique révélant la formation d’un réseau protéique. Ce
réseau est semblable à celui observé au niveau des coupes histologiques de capsule, où ces protéines
de structure associées à des polysaccharides, de la mélanine et des bactéries forment un maillage
étroit. Ce maillage assure la protection physique de l’embryon par sa résistance et son élasticité.
Outre leur rôle dans la protection physique, les SepECPs possèdent également une activité
antimicrobienne de type bactériostatique sur des bactéries à GRAM-. Les complexes SepECP/Vibrio
aestuarianus observés en MEB démontrent la capacité de ces protéines à agglomérer les bactéries
inhibant ainsi leur croissance. Ces protéines originales identifiées à partir de l’enveloppe externe de
l’œuf assurent la pérennité de l’espèce en prodiguant une protection physique et chimique aux
embryons laissés dans l’environnement sans protection maternelle.
38
O3 The C-type natriuretic peptide (CNP) prevents Pseudomonas aeruginosa
biofilm formation using AmiC: identification of a new bacterial target
Thibaut Rosay,1 Alexis Bazire,2 Suraya Diaz,3 Brice Hoffmann,4 Jérôme Leprince,5 Julien
Vieillard,6 Alain Dufour,2 Nicholas J. Harmer,3 Marc GJ Feuilloley1 and Olivier Lesouhaitier1
1
Laboratory of Microbiology Signals and Microenvironment EA 4312, Normandie Univ., Univ. Rouen; IRIB,
2
3
Evreux, France; LBCM EA 3884, IUEM, Univ. Bretagne-Sud, Lorient, France; School of Biosciences, University of
4
5
Exeter, Exeter, UK; IMPMC, UMR7590, CNRS, Université Pierre et Marie Curie - Paris 6, Paris, France; Inserm
6
U982, Normandie Univ., IRIB, Univ. Rouen, Mt St Aignan, France; UMR CNRS 6014 COBRA, Normandie Univ.,
Univ. Rouen; Evreux, France.
There is now ample evidence that bacterial virulence is modulated by detection of eukaryotic
messengers including both neurotransmitters and peptide hormones1. We have previously shown
that the C-type Natriuretic Peptide (CNP), a member of the mammalian natriuretic peptide family
produced by lung endothelial cells, enhances both Pseudomonas cytotoxicity activity2 and
Pseudomonas global virulence3. Since virulence factor production and biofilm formation are usually
inversely regulated, we investigated the effect of CNP on biofilm formation.
In the present study, we studied the effect of CNP on bacterial biofilm formation and looked for a
putative P. aeruginosa protein that could explain the effect of CNP on bacteria. We used several
peptide agonists to characterize pharmacologically the bacterial CNP sensor.
We used in silico and in vitro approaches to identify a bacterial sensor for CNP. Additionally,
pharmacological tools (i.e. synthetic peptides) were used as an innovative strategy to characterize
the binding site in P. aeruginosa.
We observed that CNP inhibits P. aeruginosa biofilm formation. This effect is totally prevented by
Isatin, an antagonist of natriuretic peptide receptors in eukaryotic cells. The action of CNP on P.
aeruginosa virulence is mimicked by the eukaryotic receptor NPR-C agonist cANF4-23. An in silico
comparative three-dimensional screen of human natriuretic peptide receptors and Pseudomonas
proteins revealed that the bacterial protein AmiC shows significant homology with the human C-type
natriuretic peptide receptor (hNPR-C). Docking of CNP and Isatin into AmiC suggests that both ligands
bind the same sites as in hNPR-C. AmiC was purified recombinantly and the protein interactions
assessed using MicroScale Thermophoresis. The results showed that both CNP and hNPR-C agonist
bind to AmiC with a KD of 2 µM and less than 100 nM respectively, whereas hNPR-A agonist has poor
affinity for AmiC. Finally, we observed that an amiC- mutant strain failed to appropriately respond to
CNP. This highlights the critical role of AmiC in regulating P. aeruginosa virulence and biofilm
formation in humans.
Taken together, our work provides the first demonstration that the bacterial protein AmiC may be an
ortholog of the eukaryotic receptor hNPR-C, acting as a C-type natriuretic peptide sensor in P.
aeruginosa. AmiC appears to modulate a switch between chronic and acute infection phenotypes
depending on exposition to host factors. The observation that CNP strongly decreases bacterial
biofilm formation should have major consequences for cystic fibrosis treatment.
1. Lesouhaitier O. et al (2009) Sensors 9, 6967-6990
2. Varon W. et al. (2007) FEBS J. 274, 5852-5864
3. Blier A. S. et al. (2011) Microbiology 157, 1929-1944
39
O4
Bacterial replicative sliding clamps as new antibacterial targets
Philippe Wolff,1 Vincent Oliéric,2 Olivier Chaloin,3 Gilles Guichard,4 Jérôme Wagner,5
Annick Dejaegere6 and Dominique Y. Burnouf1
1
Université de Strasbourg, UPR9002, Architecture et Réactivité de l’ARN, Institut de Biologie Moléculaire et
2
Cellulaire du CNRS, 15 rue René Descartes, 67084 Strasbourg, France; Swiss Light Source (SLS), Paul-Scherrer3
Institute (PSI), Villigen, Switzerland; CNRS, Institut de Biologie Moléculaire et Cellulaire, Immunologie et Chimie
4
Thérapeutiques, 15 rue René Descartes, 67084, Strasbourg, France; Université de Bordeaux, CNRS, IPB, UMR
5
5248, CBMN, Institut Européen de Chimie et de Biologie, 2 rue Robert Escarpit, 33607 Pessac, France; CNRS
6
UMR 7242, ESBS, Université de Strasbourg, BP 10413, 67412 Strasbourg, France; IGBMC, Département de
Biologie Structurale et Génomique, 1 rue Laurent Fries, BP10142, 67404 Illkirch, France.
Bacterial sliding clamps are molecular hubs that interact with many proteins involved in DNA
metabolism through their binding, via a conserved peptidic sequence, into a universally conserved
pocket1. This interacting pocket is acknowledged as a potential molecular target for the development
of new antibiotics. Using a structure-based approach, we designed short peptides with chemical
modifications at specific residues resulting in an improved affinity (10-8 M) for the Escherichia coli
binding pocket2 as measured by SPR and ITC. We further showed that these peptides differentially
interact with other bacterial clamps, although binding pockets are phylogenetically conserved and
structurally similar3. This led to differentiate between two categories of clamps: group I clamps
interacts efficiently with our designed peptides and gathers the E. coli and related orthologs clamps,
while group II poorly interact with the same peptides and gathers Bacillus subtilis and other Gram+
clamps. Thermodynamic and modeling analyses reveal that conservative substitutions of pocket
residues subtly modulate peptide interaction. These studies suggest that the peptide binding process
occurs via different mechanisms depending on which type of clamp it binds. This opens the possibility
for the development of strain specific antibiotics.
1. Burnouf D. et al. (2004) J. Mol. Biol. 335, 1187-1197
2. Wolff P. et al. (2011) J. Med. Chem. 54, 4627-4637
3. Wolff P. et al. (2014) J. Med. Chem. 57, 7565-7576
40
O5 Design and synthesis of luminescent lanthanide zinc fingers for
biological applications
Laurent Raibaut,1 William Vasseur,1 Manon Isaac,1 Céline Cépeda,1 Svetlana Eliseeva,2
Stéphane Petoud,2 Jean-Marc Latour,1 and Olivier Sénèque1
1
2
PMB/LCBM/iRTSV, Univ. Grenoble Alpes/CNRS/CEA, 38054 Grenoble; Centre de Biophysique Moléculaire, UPR
4301 CNRS, 45000 Orléans
Metal ions are essential for life. In particular Zn2+ is widely required in cellular functions, and its
dysregulation is implicated in neurodegenerative diseases or cancer1. A major comprehension of the
physiological role of zinc required new tools to image zinc and its flux in living organisms.
Interestingly, a class of proteins named zinc fingers binds specifically Zn2+ in which the metal plays a
structural role contributing to the stability of the domain. Advantageously, we can exploited this
property to design new smart zinc probes operating on the basis of Ln3+ ions emitting in the nearinfrared and of zinc finger peptides for the selective binding of Zn2+ to ensure specific and sensitive
response of the probe. Remarkably, the probe benefits from the Zn2+-induced folding properties of
zinc fingers to modulate the energy transfer between the sensitizing antenna and the emitting
lanthanide2 (scheme).
In this communication, we will describe the conception of theses probes and a proof of principle by
using a visible-emitting lanthanide ion Tb3+. In particular, we will present the powerfulness of native
ligation methods3 for building different variety of probes.
1. Fukada T. et al. (2011) Biol. Inorg. Chem. 16, 1123-1134
2. Heffern M C. et al. (2014) Chem. Rev. 114, 4496−4539
3. Raibaut L., et al. (2012) Chem. Soc. Rev, 41, 7001-7015
41
O6 A straightforward strategy for automated Fmoc-based synthesis of
peptide crypto-thioesters. Application to the chemical synthesis of disulfide
rich peptides
Victor Terrier, Hélène Adihou, Mathieu Arnould, Agnès Delmas and Vincent Aucagne
Centre de Biophysique Moléculaire - UPR CNRS 4301 - Rue Charles Sadron 45071 ORLEANS Cedex 2, France.
The importance of proteins in biology and medicine has inspired chemists to consider methods that
bring about their synthesis for over a century. The native chemical ligation reaction (NCL)1 plays a
pivotal role in contemporary approaches, since it provides access to small proteins by coupling two
unprotected peptide segments, containing a Cα-thioester and an N-terminal cysteine, respectively.
However, the synthesis of peptide thioesters by Fmoc-based SPPS remains a real bottleneck to
expand the limits of chemical protein synthesis. Recently, a number of very promising methods2-4
that exploit an intramolecular N→S acyl shift for the in situ formation of thioesters from β-mercaptoamides during the NCL reaction (“crypto-thioesters”) have emerged. Nevertheless, these approaches
are still impaired by the complex synthesis of the linker, the difficulty of the N-acylation of the
secondary amine to introduce the first amino acid of the peptide segment, or slow kinetics of the
NCL reaction. Moreover, peptides containing these β-mercapto-amides can be unstable in water due
to premature N→S acyl shift, making the crypto-thioester difficult to handle and to purify. We have
developed an N-(2-hydroxybenzyl)cysteine C-terminal linker for the synthesis of peptide cryptothioesters, which can be readily synthesized on solid phase from inexpensive commercially available
compounds. This linker was designed to facilitate the N-acylation step thanks to an intramolecular
assistance based on an O→N acyl shift mechanism (Scheme A), as well as to promote fast kinetics of
the NCL through an intramolecular catalysis by the phenol functionality. Importantly, the synthesis of
the target crypto-thioester, including the assembly of the linker and its N-acylation, can be fully
automated on a peptide synthesizer as we have demonstrated for the 20 proteogenic amino acids.
Moreover, the use of a tert-butylsulfanyl (StBu) group for the protection of the thiol of the cysteine
makes the product stable and simplify its purification, so that no post-synthetic steps are required.
Thus, the method is easily usable by non-specialists. We have successfully synthesized a cryptothioester of 56 amino acid residues bearing our linker, which makes it one of the longest thioester
ever synthesized by Fmoc-SPPS. The method has been successfully applied to the synthesis of several
naturally occurring disulfide-rich peptides (scheme B) which exhibit interesting pharmacologic
profiles, such as the green mamba venom toxin MT7, a highly selective and potent antagonist of the
M1 subtype of muscarinic receptor that acts by binding to an allosteric site.
S
FmocHN
OH
OH HN
+
S
O
O
(A)
PG
PG
PG
R
S
FmocHN
FmocHN
O-acylation
O
O
HN
R
N
N-acylation
R
HO
NO2
S-N shift
O
NO2
NO2
PG : Protecting group
HS
R'
O
(B)
PEPTIDE 1
R' = -StBu
in situ removal
of the -StBu
protecting group
R' = H
S
N
N-S shift
PEPTIDE 1
R
PEPTIDE 2
H2N
O
R
S
PEPTIDE 1
HN
NO2
O
S-S shift
HO
HO
NO2
1. Dawson P. et al. (1994) Science 266, 776-779
2. Kawakami T. & Aimoto S. (2009) Tetrahedron 65, 3871-3877
3. Tsuda S. et al. (2009) Org. Lett. 11, 823-826
4. Ollivier N. et al. (2010) Org. Lett. 12, 5238-5241
42
S
PEPTIDE 2
NH2
PEPTIDE 1
HS
N
H
PEPTIDE 2
O7 Structural and enzymatic probing of S-glycosyltransterases with a joint
molecular modeling and NMR approach
Sami Marroun,1 Gaël Coadou,1 Pierre Lafite,2 Richard Daniellou2 and Hassan Oulyadi1
1
COBRA (Laboratoire de chimie organique et analytique), Mont Saint-Aignan, France; 2ICOA (Institut
de Chimie Organique et Analytique), Orléans, France.
Thio-glycosyltransferases (S-GTs) catalyze the transfer of a sugar donor’s glycosidic moiety upon an
acceptor’s sulfur atom. These enzymes are the rarest and the least studied of the
glycosyltransferases but hold great potential in the production of novel glycomimetic therapeutics. In
fact, thioglycosides are less sensitive to chemical hydrolysis in vivo while maintaining similar
conformations to the corresponding O-glycosides1. Among these S-GTs, two Arabidopsis thaliana
enzymes hold particular promise: UGT74B12 and UGT74C13. Though their role in the production of a
particular class of thioglycosides called glucosinolates from thiohydroxamates has been well
established, much remains elusive about their structure, mechanism, donor and acceptor as well as
heteroatom selectivity. Our ongoing work aims to investigate these two proteins using molecular
modeling techniques (homology modeling, molecular docking and density functional theory
calculations) as well as ligand based NMR methods (STD -saturation transfer difference- and
transferred NOE) in order to obtain a clearer view of their function and and understand their
selectivities at the atomic level. The exploration of these enzymes would allow us to produce novel
thio-glycosyltransferase activities to be used in sustainable chemistry and drug discovery through the
means of directed evolution.
Figure 1-Top: Homology model of UGT74B1; Bottom: Enzymatic reaction catalyzed by S-GTs UGT74B1
and UGT74C1
1. Guillotin L. et al. (2014) in Carbohydrate Chemistry
2. Guo L. & Poulton J. E. (1994) Partial purification and characterization of Arabidopsis thaliana UDPG thiohydroximate
glucosyltransferase.
3. Gachon C. M. et al. (2005) Plant Mol. Biol. 58, 229-245
43
O8 19F NMR monitoring of the eukaryotic 20S proteasome Chymotrypsinlike activity by FABS and FAXS
Julia Kaffy,1 Massaba Keita,1 Claire Troufflard,2 Martial Piotto,3 Estelle Morvan,2 Benoît
Crousse1 and Sandrine Ongeri1
1
2
Molécules Fluorées et Chimie Médicinale, Plateforme de RMN, BioCIS UMR-CNRS 8076, LabEx LERMIT,
Université Paris-Sud, Faculté de Pharmacie, 5 rue Jean-Baptiste Clément, 92296, Châtenay-Malabry Cedex,
3
France; Bruker Biospin, 34 rue de l’industrie, 67166 Wissembourg, France.
Regulator of a vast array of vital cellular processes including cell-cycle progression, apoptosis and
antigen presentation, the proteasome represents a major therapeutic target.1 Therefore, selective
inhibitors of the proteasome are promising candidates to develop new treatments for diseases such
as cancer. Bortezomib, the first proteasome inhibitor approved by the FDA to treat multiple
myeloma, and many described proteasome inhibitors interact covalently with the active site of the
enzyme through an electrophilic reactive function. However, non-covalent inhibitors have been less
widely investigated.2 We recently reported that α- and β-hydrazino acid-based pseudopeptides
inhibited non covalently the chymotrypsin-like (ChT-L) activity of eukaryotic 20S proteasome.3
A powerful role of fluorine atom as a tag for 19F NMR, lies in the high sensitivity of 19F NMR
spectroscopy, the lack of background signal for 19F and its ability to detect weak intermolecular
interactions. Therefore, the 19F NMR spectroscopy has been used to study enzymatic reactions and
protein-ligand interactions with respectively the FABS (Fluorine Atoms for Biochemical Screening)
and the FAXS (Fluorine chemical shift Anisotropy and eXchange for Screening) methods.4 We will
present here both experiments using the 20S proteasome as the biological target.
First, we developed an original fluorinated substrate of the ChT-L proteasome active site and used
the FABS experiment for the detection of the starting and enzymatically modified substrates.5 The
method allowed us to measure reliable IC50 values of synthesized compounds. We lifted a major lock
existing with the actual fluorescent substrates that prevent totally simultaneous monitoring of the
three proteolytic activities. Indeed, we demonstrated that the ChT-L activity can be modulated in
presence of the T-L or the PA substrates.
R2
R1
N
H
O
H
N
O
R3
R4
N
H
H
N
O
O
ChT-L active site of H2N
N
H
CF3
proteasome
CF3
OH
Secondly, we will present preliminary results on the development of the FAXS method in order to
screen a library of potential inhibitors of proteasome. We have already identified a "spy" fluorinated
molecule that showed a different 19F NMR signal when it is in interaction with proteasome.
Molecules having a greater affinity should displace the fluorinated spy which will recover the 19F NMR
signal of its free form.
From Dalvit 4
1. Ciechanover A. (1998) EMBO J. 17, 7151-7160
2. Kaffy J. et al. (2013) Curr. Pharm. Des. 19, 4115-4130
3. a) Formicola L. et al. (2009) Bioorg. Med. Chem. Lett., 19, 83-86; b) Bordessa A. et al. (2013) Eur. J. Med. Chem. 70, 505524
4. Dalvit C. (2007) Prog. Nucl. Magn. Res. Spect. 51, 243-271
5. Keita M. et al. (2014) Org. Biomol. Chem. 12, 4576-4581
44
O9
Structural analysis of a collagen triple-helix model by liquid state NMR
Anaïs Terrien,1 Lauriane Devaux,1 Grégory Chaume2 and Emeric Miclet1
1
Laboratoire des biomolécules, Université Pierre et Marie Curie/Ecole Normale Supérieure, UMR 7203, 4 place
2
Jussieu, 75252 Paris cedex 05, France; Laboratoire SOSCO, Université́ de Cergy-Pontoise, EA 4505, 5 mail Gay
Lussac, 95000 Cergy-Pontoise.
The collagen is the most abundant protein in the animal kingdom. It is also an important biomaterial
owing to its excellent thermal stability and its great mechanical strength. This last decade, both the
industry and the academia have been involved in the development of new collagen-related
biomaterials. A common strategy consists in using Collagen Model Peptides (CMP) as collagen
surrogates. One characteristic of the collagen sequence is the repeat of the Xaa-Yaa-Gly triplet where
Xaa and Yaa are often Proline and Hydroxyproline residues, respectively (occurrence of 28% and
38%). CMPs are generally 15-45 residue-long peptides that mimic the (Pro-Hyp-Gly)n collagen
primary sequence1. It has been shown that such sequences can adopt a polyproline II conformation
and undergo a supramolecular assembly in triple helix.
We have synthesized seven collagen model peptide (Pro-Hyp-Gly)7 , each of them incorporating one
C-enriched Glycine residue. These peptides were synthetized on solid support and purified by HPLC
with good yields (50-80%). The formation of the triple helix has then been studied using 1D 1H and
2D 1H-13C NMR experiments and displayed a strong dependence on both the temperature and the
CMP concentration. Triple helix and minor conformations populations have been quantified using
each Gly residue as precise local probes. Comparative studies have been performed by circular
dichroism, which showed a good agreement with our NMR results.
13
More specifically, a CH2-TROSY experiment (Transverse Relaxation-Optimised SpectroscopY) was
developed to accurately determine the 14 3JHN-Hα constants characterizing the backbone
conformation of the peptide.2 Measurements have been performed both when the CMP is a part of
the triple helix and when free in solution. Structural features of the two states will be discussed in
details.
Figure 1: Structuration of collagen model peptide
1. Shoulders M. D. & Raines R. T. (2009) Annu. Rev. Biochem. 78, 929-958
2. Aliev A.-E. & Courtier-Murias D. (2010) J. Phys. Chem. B 114, 12358-12375
45
Initial steps in describing the F1Fo ATP synthase dimer interface and
modelling the small hydrophobic subunits of the Fo region with solution
state NMR.
O10
James Tolchard,1 Daniel Brethes,2 Marie-France Giraud2 and Benoit Odaert1
1
2
CBMN, UMR 5248, 14 bis, allée Saint-Hilaire, 33607, Talence, France; IBGC, UMR 5095, 1 rue Camille Saint
Saens, 33077, Bordeaux, France.
The multi-subunit complex of F1Fo ATP synthase is the principle source of cellular ATP throughout
the biological kingdom. Although variability exists in the precise F1Fo composition between
species, the most studied protein is from Saccharomyces cerevisiae where the complex is
comprised of 17 core subunits. Two of the Yeast F1Fo subunits (e and g) and the N-terminal
membrane domain of subunit 4, which all locate to the mitochondrial membrane Fo region, have
recently been shown [1] to be absolutely required for the dimerisation of ATP synthase which
facilitates the formation of the mitochondrial cristae [2]. Currently, no high-resolution structural
information for the interface of dimeric ATP synthase exists and as a result, the interactions which
stabilise the ATP dimer are unknown; with prior investigations typically hampered by poor yields
of the membrane domain subunits.
To this end, we have optimised a home-made cell-free expression system for the production and
isotopic enrichment of these subunits - with final yields >1 mg which allows for investigation with
nuclear magnetic resonance (NMR) spectroscopy. Initial studies, using unlabelled and 15N-Ala
samples, focussed on validating the incorporation of samples into LMPG micelles and optimising
the quality of NMR spectra for an 83 residue (8.7 kDa) construct comprising the N-terminal region
of subunit 4 from the Yeast F1Fo ATP synthase (S4T). From synthesising single (15N) and double
(13C, 15N) labelled samples of S4T, two and three-dimensional NMR spectra (15N and 13C HSQC,
HNCACB, CBCACONH, HNCA, HNCOCA and , HCACO) were acquired at 800 MHz enabling us to
assign 95% of the S4T backbone nuclei. In silico predictions from primary sequence alone
suggested that S4T would have a predominantly alpha helical conformation and this agrees with
both chemical shift analysis and a preliminary 3D model from CS-ROSETTA [3] which describes the
structure of S4T as a bundle of 3 trans-membrane helices. Furthermore, recent
Hydrogen:Deuterium exchange experiments on the S4T construct have given insight into the
residues which are shielded from amide exchange.
Future work will focus on optimising the acquisition of TOCSY and NOESY-based NMR
experiments for classical distance-based structure determinations of S4T before beginning NMR
investigations into subunits e and g. Studies into the interactions which stabilise the dimeric state
of ATP synthase will then be undertaken through a number of biophysical techniques to
investigate the different affinities and sites of interaction amongst the three subunits.
1. Davies K. M. et al. (2012) PNAS 109, 13602-13607
2. Paumard P. et al. (2002) EMBO J. 21, 221-230
3. Shen Y. et al. (2008) PNAS, 105, 4685-4690
46
O11 Synthetic mimics of β sheets: design, synthesis and evaluation of their
ability to modulate the aggregation of the β-amyloid 1-42 peptide
Nicolò Tonali,1 Julia Kaffy,1 Jean-Louis Soulier,1 Dimitri Brinet,1,2 Myriam Taverna,2 Benoit
Crousse,1 Sandrine Ongeri,1 Sara Pellegrino,3 Emanuela Erba,3 Alessandro Contini3 and
Maria-Luisa Gelmi3
1
Molécules Fluorées et Chimie Médicinale, BioCIS UMR-CNRS 8076, Université Paris Sud, Châtenay-Malabry,
2
France; Protéines et Nanotechnologies en Sciences Séparatives, Institut Galien de Paris Sud, UMR-CNRS 8612,
3
Université Paris Sud, Châtenay-Malabry, France; DISFARM Sezione di Chimica Generale e Organica "A.
Marchesini", Università Degli studi di Milano, Milano Italy.
Alzheimer’s disease is a neurodegenerative disorder linked to oligomerization and fibrillization of
amyloid β peptides. Aβ fibrils present a cross-β structure in which the individual β-strands are
oriented perpendicular to the fibril axis. In Aβ1-42, the more aggregative and neurotoxic amyloid
peptide, the N-terminus is thought to be unstructured, and there are two largely hydrophobic βstrand segments within fibrils, formed by residues 18-26 (β1) and 31-42 (β2), that have a parallel and
in-register orientation. An attractive strategy for treating AD could be the inhibition of the
oligomerization of soluble Aβ monomers, by destabilizing the β-sheets formed within the monomer
and in the peptide-peptide interactions. The use of peptidomimetics able to bind the nucleation site
(K16L17V18F19F20) of Aβ1-42 peptide has been considered. Recently, macrocyclic β-sheet mimics have
been described as inhibitors of Aβ1-42 aggregation by forming a 3-strands β-sheet structure in
presence of the amyloid protein.1 Relying to this hypothesis, we designed and synthesized novel
acyclic mimics of β-sheets based on a piperidine-pyrrolidine scaffold.2 Preliminary results on a short
peptide containing the above scaffold showed its ability to induce a β turn conformation. We have
linked to the scaffold pentapeptides designed to bind specifically Aβ1-42 peptide. Conformational
studies of two molecules were performed by circular dichroism (CD) and NMR in order to confirm
their capacity to form a β-sheet structure. According to the pentapeptide sequences, we observed
differences in flexibility and stability of the β-sheet conformation. The capacity of this novel series of
peptidomimetics to interfere with the aggregation process of Aβ1-42 was evaluated by Thioflavin-T
fluorescence assay and TEM. Very efficient inhibition of the aggregation was obtained even at a low
ratio of Aβ1-42: peptidomimetic (1:1). Moreover, since experimental evidences support the hypothesis
that low molecular weight oligomers are primarily responsible for the neurodegeneration, the
laboratory recently proposed an improved method based on capillary electrophoresis (CE) to monitor
overtime easily, reproducibly and efficiently the very early steps of oligomerization process.3 In order
to study the effect of our molecules on the oligomers formation, the compound that displayed the
most promising activity on Aβ1-42 aggregation according to the previous assays, was studied by CE and
demonstrated its ability to stabilize the monomeric Aβ1-42 peptide and thus to prevent its
aggregation. Thanks to these promising results, we report here the synthesis and the evaluation of
these β-sheets mimics which can be considered as novel tools for the study of the prevention and/or
modulation of the aggregation process of Aβ1-42 peptide. Pharmacomodulations of the molecules are
in course with the aim to decrease the peptidic character of the molecule and to increase their
hydrosolubility in order to obtain compounds more resistant to proteolysis and bioavailable.
General scheme of the β-sheets mimics
47
O12 Production of uniformly 15N/13C labeled glycosylated proteins by hairy
roots in view to NMR structural studies
Romain Trouillard,1 Marie Hubert-Roux,2 Vincent Tognetti,2 Laure Guilhaudis,1 Carole
Plasson,3 Laurence Menu-Bouaouiche,3 Laurent Coquet,4 François Guérineau,5 Julie
Hardouin,4 Jean-Pierre Ele Ekouna,5 Pascal Cosette,4 Patrice Lerouge,3 Michèle BoitelConti,5 Carlos Afonso2 and Isabelle Ségalas-Milazzo1
1
Normandie Université, Université de Rouen, UMR 6014 CNRS COBRA, IRIB, 76821 Mont-saint-Aignan Cedex,
2
France; Normandie Université, Université de Rouen, UMR 6014 CNRS COBRA, FR3038 INC3M, 76821 Mont3
saint-Aignan Cedex, France; Normandie Université, Université de Rouen, EA 4358 Glyco-MEV, IRIB, 76821
4
Mont-Saint-Aignan Cedex, France; Normandie Université, Université de Rouen, UMR 6270 CNRS PBS, IRIB,
5
Plateforme Protéomique PISSARO, FR3038 INC3M, 76821 Mont-Saint-Aignan Cedex, France; Université de
Picardie Jules Verne, BioPI, 80039 Amiens, France.
Almost half of eukaryote proteins are glycosylated1. Glycosylation plays a role in the protein folding,
stability and activity. However, glycosylation impact on the protein structures remains little studied.
Actually, heterogeneous glycan chains of glycoproteins often hinder the crystal growth, preventing
the use of crystallography, the most widespread method for structural biology.2 Nuclear magnetic
resonance (NMR) spectroscopy is an appealing alternative technique, but it requires proteins
exhibiting 15N or 15N/13C isotopic labeling for overcoming resonance overlap and assignment
difficulties. Hairy roots may constitute a good candidate for the production of uniformly labeled
glycoproteins in view to NMR structural studies. Indeed, these transformed roots can be cultivated in
vitro in a confined and controlled medium allowing easy supply of labeled molecules.3 They can
perform glycosylation of their proteins and can produce exogenous proteins in high amount (about
120 mg L-1). Furthermore, they are able to secrete the protein of interest thanks to the fusion of a
signal peptide, making protein purification easier to achieve.
In this context, we have initiated a program aiming at exploring the possibility to produce, into hairy
roots, labeled glycoproteins. In a first step, we focused on the 15N isotopic incorporation in the
enhanced green fluorescent protein (eGFP), a non glycosylated model protein already produced in
high amount by the system.3 Heterogeneous isotopic distributions constituted of different labeled
protein populations were obtained. Thus, we developed a new strategy for the isotopic
measurement, from mass spectrometry analyses, able to calculate the proportion of these different
labeled populations. Such isotopic measurement allowed us to demonstrate that hairy roots were
able to produce 15N labeled proteins with a relatively high labeling ratio.
Following these first encouraging results, the protocol was extended to 13C labeling and then to the
15
N/13C eGFP production. In parallel, we have recently started the production of an artificial
glycosylated eGFP in order to investigate the abilities of hairy roots to produce a glycosylated 15N/13C
labeled protein. These studies would allow validating hairy roots as good candidates for uniformly
labeled glycosylated protein production in view to NMR structural studies.
1. Zafar S. et al. (2011) Bioinformation 6, 352-355
2. Davis S. J. & Crispin M. (2010) In Functional and Structural Proteomics of Glycoproteins; Owens, R.; Nettleship, J., Eds.;
Springer Netherlands: Dordrecht, pp. 127–158
3. Huet Y. et al. (2013) Biotechnol. Lett. 36, 181-190
48
O13 From hybrid peptide trialkoxysilane units to biocompatible hydrogels
Cécile Echalier,1,2 Said Jebors,1 Jérémie Cicionne,1,2 Xavier Garric,1 Hélène Van Den Berghe,1
Estelle Jumas Bilak,3 Jean Martinez,1 Ahmad Mehdi2 and Gilles Subra1
1
2
Institut des Biomolécules Max Mousseron, UMR 5247, Faculté de pharmacie, Montpellier, France; Institut des
3
Matériaux Charles Gerhardt, UMR 5253, Faculté des Sciences, Montpellier, France; Ecologie des systèmes
marins côtiers, UMR 5119, Faculté de pharmacie, Montpellier, France.
The design of biocompatible materials is important in tissue engineering, reconstructive surgery and
drug release.1,2 One of the main challenge is to introduce covalently attached bioactive molecules, in
particular peptide ligands, in these biomaterials. In this context, we developed a sol-gel process for
preparing hybrid hydrogels by a bottom-up approach.3
We first functionalized polyethylene glycol-based units by trialkoxysilane groups. These building
blocks were used to form hydrogels in biocompatible conditions. The influence of temperature,
building block concentration and additives on gelification and gel properties was studied. Finally,
different biological properties were given to hydrogels by incorporation of active hybrid peptides.
1.Kim B.-S. et al. (2011) Progress in Polymer Science 36, 238-268
2. Vashist A. et al. (2014) J. Mater. Chem. B 2, 147-166
3. Jebors S. et al. (2013) J. Mater. Chem. B 1, 6510-6515
49
O14 Auto-assemblage de polypeptides recombinants à base de motifs
élastine
Elisabeth Garanger,1,2 Sarah MacEwan,3 Olivier Sandre,1 Annie Brûlet,4 Ashutosh Chilkoti3
et Sébastien Lecommandoux1
1
2
Laboratoire de Chimie des Polymères Organiques (LCPO), Pessac, France; Institut Européen de Chimie et
3
Biologie (IECB), Pessac, France; Department of Biomedical Engineering, Duke University, Durham, NC, USA;
4
Laboratoire Léon Brillouin, CEA Saclay, Gif-sur-Yvette, France.
Notre groupe s’intéresse depuis quelques années aux polypeptides recombinants à base de motifs
élastine (ELPs, Elastin-like polypeptides).1 Dérivés de la séquence élastomère de la tropoélastine, les
ELPs sont constitués de répétitions de séquences pentapeptidiques (Val-Pro-Gly-Xaa-Gly)n, le résidu
hôte (Xaa) pouvant être tout acide aminé naturel ou non-naturel autre que Pro. Ceux-ci présentent
une température critique basse de solubilité (LCST), pouvant être modulée par les paramètres
macromoléculaires, et qui constitue un double avantage (i) pour leur extraction et purification des
lysats bactériens et (ii) pour le contrôle de leurs propriétés d’auto-assemblage.
Afin d’étudier finement les mécanismes d’auto-assemblage de ces polypeptides, nous avons produit
chez E. Coli une série de copolypeptides diblocs possédant un bloc ELP thermosensible (VPGVG)n de
longueur variable (n= 40, 60, 80, 120, 200), suivi d’un bloc « hydrophile permanent » (LCST>60°C)
(VPGXG)60 (X=A,G (1:1)). Au moyen de techniques complémentaires de diffusion de la lumière et des
neutrons aux petits angles, nous avons pu mettre en évidence un mécanisme en deux étapes dans
lequel les chaînes polypeptidiques s’auto-assemblent au voisinage de la LCST pour former des
nanoparticules très régulières, dont le cœur se déshydrate régulièrement par chauffage au dessus de
la LCST.2 Dans cette présentation, nous expliciterons les méthodes de production et de purification
de ces polymères parfaitement monodisperses et l’étude physico-chimique de leur auto-assemblage.
1. Garanger E. & Lecommandoux S. (2012) Angew. Chem. Int. Ed. 51, 3060-3062
2. Garanger E. et al. En préparation
50
O15 Rational design of injectable hexapeptide hydrogelators for controlledrelease drug delivery
Charlotte Martin,1 Mathieu Bibian,1 Jeroen Mangelschots,1 James Gardiner,2 Lynne
Waddington,2 M.M. Diaz Acevedo,3 Bruno Van Mele,3 Annemieke Madder,4 Richard
Hoogenboom5 and Steven Ballet1
1
Research Group of Organic Chemistry, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels,
Belgium; 2CSIRO Materials Science & Engineering, Bayview Ave, Clayton, VIC 3169, Australia; 3Vrije
Universiteit Brussel, Physical Chemistry and Polymer Science, Pleinlaan 2, B-1050 Brussels, Belgium;
4
Organic and Biomimetic Chemistry Research Group, Ghent University, Krijgslaan 281, 9000 Ghent,
Belgium; 5Supramolecular Chemistry Group, Ghent University, Krijgslaan 281, 9000 Ghent, Belgium.
The interest in hydrogels has rapidly grown due to their potential in the development of drug
delivery systems, cell growth media and many other material-related applications.1,2 The design of
hydrogels using peptides is of particular interest since hydrogelation is driven by physical (noncovalent) interactions, eliminating the need for chemical cross linking. Moreover, the shear thinning
behavior of physical hydrogels renders them very attractive for drug delivery applications by
subcutaneous injection. Additionally, peptide hydrogels can possess high drug loading capacities in
comparison to conventional drug delivery polymer hydrogels. In this work, a new family of simple,
short, linear, amphiphilic peptides was designed inspired by the previously reported sequence 1.3
Our recently discovered lead sequence H-Phe-Glu-Phe-Gln-Phe-Lys-OH (2 in Figure. 1)4 was utilized as
basis for a family of peptides to systematically study the influence of peptide sequence on
hydrogelation conditions.
hydrophobic side
(internal side of the fibril)
amide-amide
interactions
for β-sheets
O
H
N
H 2N
N
H
O
O
H
N
O
O
N
H
O
H
N
N
H
O
OH
O
H
N
O
O
H
N
H2N
O
OH
O
H
N
N
H
O
O
NH 2
1
OH
N
H
O
OH
O
H
N
OH
O
NH 2
NH 2
NH2
hydrophilic side
with complementary
side chain charges
O
2
amide group: both H-acceptor
and donor, more hydrophobic
than NH 3+ or COO - at
physiological pH
Figure 1: Design of novel peptide hydrogelators inspired by H-FEFKFEFK-OH
Several of these peptides proved to induce hydrogel formation in water-phosphate buffered saline
(PBS) mixtures, which are considered as physiologically compatible conditions that allow PBS
triggered gelation of peptide solutions in water. The hydrogels were fully characterized at
macroscopic and microscopic level by rheology, circular dichroism, Fourier transform infrared
spectroscopy, cryogenic transmission electron microscopy (TEM) and negative staining TEM analysis.
The release properties of several hydrogels were examined for various cargoes in order to determine
and compare the release profiles. The release of fluorescein from a peptide 2 hydrogel is shown in
Figure 2 as illustrative example.
Figure.2: Static release experiment of fluorescein (0.1% w/v) from a H-FEFQFK-OH
(2) hydrogel (1% w/v in water/PBS 1:1 v/v)
1. Bowerman C. J. & Nilsson B. L. (2012) Biopolymers 98, 169
2. Tomasini C. & Castellucci N. (2013) Chem. Soc. Rev. 42, 156
3. Mohammed A. et al. (2007) Macromol. Symp. 251, 88-95
4. Bibian M. et al. (2015) J. Mater. Chem. B DOI : 10.1039/C4TB01294A
51
O16 Efficient synthesis of cyclo 1:1-[α/α-Nα-Bn-hydrazino]mers for the
elaboration of nanotubular self-organization in solution and solid state
Eugénie Romero, Ralph-Olivier Moussodia, Samir Acherar and Brigitte Jamart-Grégoire
Laboratoire de Chimie Physique Macromoléculaire, FRE CNRS-UL 3564, ENSIC, 1 rue Grandville 54001 Nancy
cedex, France
Recently, a new family of foldamers has been identified. Their backbone, enriched in nitrogen atoms,
is locally structured by short-distance hydrogen bonds. These compounds have conformational
properties that enable efficient cyclisation. As short-distance intramolecular contacts within the
cyclic molecules may be kept. Cyclofoldamers can design perfectly defined conformation thanks to
secondary structuration by hydrogen bonding. We expected that under favourable conditions, these
compounds could give rise to nanotube formation, of chosen pore diameter, due to piling up
(stacking) of cycles through π-π-stacking type interactions and/or intramolecular H-bonding. Cyclo
1:1-[α/α-Nα-Bn-hydrazino]mers series can be obtained very efficiently from the corresponding linear
oligomers. X-ray analyses confirm their nanotubular organization in solid state. Moreover, we
demonstrated that these cyclopseudopeptides lead to organogel in the presence of toluene. FTIR and
NMR analyses confirm that nanotubular organization is maintained in gel state. SEM analysis shows
thatthe corresponding aerogel is composed of linear fibres.
Boc
H
N
O
*
R
R1 O
N *
N
OMe
H
n
R2
H
N
O
*
R
R1 O
N *
N
H
R2
n
Figure1: Mixed 1:1[α/α-Nα-hydrazino]linear and cyclic oligomer synthesis
Figure2: a) X-ray analysis of self-assembling from the 1:1[α/α-Nα-hydrazino]cyclic tetramer;
b) SEM analysis of the aerogel
1. Hoffmann R. & Kim H. O. (1990) Tetrahedron Lett. 31, 2953-2956
2. Acherar S. & Jamart-Grégoire B. (2009) Tetrahedron Lett. 46, 6277-6279
3. Moussodia R.-O. et al. (2012) Tetrahedron 68, 4682-4692
52
O17 p53 control of gene expression via mRNA translation during
endoplasmic reticulum stress
Ignacio López, Anne-Sophie Tournillon and Robin Fåhraeus
1
Équipe Labellisée Ligue Contre le Cancer, Université Paris 7, INSERM UMR 1162 “Génomique fonctionnelle des
tumeurs solides”, Hôpital St Louis, 27 Rue Juliette Dodu, 75010 Paris, France.
Several conditions that induce accumulation of unfolded or misfolded proteins, such as poor tissue
perfusion, nutrient deprivation, or underglycosylation of proteins among others, can trigger the
Endoplasmic Reticulum (ER) stress. These conditions may appear during physiological fluctuations of
protein production/folding or pathological scenarios, and some have been associated with viral
infection, aging and notably those cancers where tumour cells are suffering from poor perfusion and
high rate of protein synthesis. Cells respond to ER stress through the unfolded protein response
(UPR), a three-branched pathway that inhibits global cap-dependent protein synthesis by PERK
activity, and promotes induction of ER chaperons and favours the elimination of misfolded proteins
by activation of ATF6 and IRE-1. Our group showed that during ER stress, a selective induction of the
p53 isoform p53/47 by PERK leads to increased 14-4-4σ production causing G2 arrest1. Later on, our
group showed that in order to arrest cells at G2 phase, p21CDKN1A must be suppressed to avoid COP-1mediated degradation of 14-3-3σ. In addition, suppression of p21CDKN1A was shown to be ER stressinduced p53/47-dependent acting at both transcriptional and translational levels2. Considering that
p21CDKN1A-dependent G1 arrest is caused by p53 under DNA damage, the work from our lab
demonstrates how p53 isoforms control cell cycle progression in response to different insults, and
how they target different levels of gene expression.
Aimed at contributing to unravel the expression pattern of p53 target genes in the UPR we focused
on its main cellular regulator; MDM2. These two proteins are closely interlinked and understanding
the underlying molecular mechanisms could contribute to identify targets for therapeutic
interventions.
We could show that MDM2 is down-regulated in p53-positive HCT116 and A549 cells following ER
stress. A similar UPR-dependent suppression of MDM2 levels was observed in the p53–null H1299
and Saos-2 cell lines following expression of ectopic p53, but not in the non-p53 expressing cells.
Interestingly, the mdm2 mRNA expression pattern was induced by p53, irrespectively of the UPR
status of the cells. Increasing the levels of p53 in H1299 cells resulted in a dose-dependent downregulation in MDM2 expression, whereas the mdm2 mRNA levels augmented in accordance with
increasing levels of p53 as determined using RT-qPCR.
Suppression of p21CDKN1A mRNA translation by p53 was shown to be mediated via its CDS and not on
the more commonly described mechanisms of mRNA translation initiation control acting via the
UTRs2. This is also the case for MDM2 and co-transfection of an HA-tagged MDM2 from an mRNA
that lacks mdm2’s UTRs resulted in a similar p53 dose-dependent suppression of HA-MDM2, as
determined by immunoblotting using an anti-HA antibody. Repression of translation by p53 has been
shown to depend on a direct interaction with the mRNA, and we tested whether this is also the case
for mdm2 and we could show using RNA co-immunoprecipitation of p53 produced in bacteria and in
vitro-synthesized mdm2 mRNA that p53 binds mdm2’s mRNA but to the CDS and not to the UTRs,
suggesting that p53 indeed plays a role in controlling MDM2 expression at a post-transcriptional
level.
That two of the major up-regulated target genes of p53 during DNA damage are actually downregulated at the posttranslational step under ER stress suggests that mRNA translation control is
favoured by p53 under these conditions.
1. Bourougaa K. et al. (2010) Mol. Cell. 38, 78-88
2. Mlynarczyk C. et al. (2014) Nat. Commun. 5, 5067
53
O18 Identification of cellular factors involved in the mechanism of the
immune evasion of the Epstein-Barr virus mediated by the GAr domain of the
virally-encoded EBNA1 protein
Maria-José Lista,1 Marie-Astrid Contesse,1 Chrysoula Daskalogianni,2 Robin Fåhraeus,2
Cécile Voisset1 and Marc Blondel1
1
Institut National de la Santé et de la Recherche Médicale UMR1078; Université de Bretagne Occidentale,
Faculté de Médecine et des Sciences de la Santé; Etablissement Français du Sang (EFS) Bretagne; CHRU Brest,
2
Hôpital Morvan, Laboratoire de Génétique Moléculaire, Brest, F-29200, France; Cibles Thérapeutiques, Institut
National de la Santé et de la Recherche Médicale UMRS940, Institut de Génétique Moléculaire, Université Paris
7, Hôpital St. Louis, F-75010 Paris, France.
The Epstein-Barr Virus (EBV) is the first oncogenic virus described in humans. It is a ubiquitous virus
which infects over 90% of the human population.
EBV causes a latent infection of B lymphocytes which remains asymptomatic in most of the patients.
However, in some conditions like immunosuppression, EBV can cause uncontrolled cell proliferation
responsible for some types of cancers like Burkitt lymphoma, Hodgkin lymphoma and
nasopharyngeal carcinoma.
During latency, EBNA1 is the only protein expressed in all EBV-infected cells, as it is essential for EBV
genome replication and maintenance. EBNA1 is highly antigenic and T-cells raised against EBNA1
exist, nevertheless EBV-infected cells are not eliminated by the host immune system. This is due to
the central GAr (Gly-Ala repeat) domain of EBNA1 which is able to inhibit the translation of its own
mRNA in cis, in that way it prevents the recognition of EBV-infected cells by the immune system of
the host.
The mechanisms involved in the GAr domain regulation of translation are still undeciphered.
Recently, we set up a yeast-based assay that recapitulates the effect of the GAr domain on the
regulation of translation.
The main objective of my PhD project was to identify cellular factors involved in the GAr-domain
regulation of translation. To carry out this task, we performed a genetic screening based on the
yeast-based assay that recapitulates the GAr domain regulation of translation. This genetic screening
allows the identification of host cell components that affect the regulation of translation by the GAr
domain. Through this methodology, we isolated several candidate genes conserved from yeast to
human that regulate the activity of the GAr-domain in a positive (exacerbation of translation
inhibition) or negative (suppression of translation inhibition) manner. We are now analyzing the
candidate genes to decipher their roles in the GAr-based mechanisms of the immune evasion.
54
O19 Palladium-catalyzed synthesis of bridged Phe-Gly dipeptide to access
novel ligands of Translocator Protein 18kDa (TSPO)
François Hallé,1 Imane lejri,2 Irene Marginedas,3 Christelle Doebelin,1 Séverine Schneider,1
Christian Klein,4 Michel Maitre,4 Martine Schmitt,1 Guy Mensah,4 Mariano Ostuni,3 Anne
Eckert2 and Frédéric Bihel1
1
2
CNRS, University of Strasbourg, UMR7200, Faculté de pharmacie, 74, route du Rhin, 67401 Illkirch; University
of Basel, Dept of Biomedicine, Psychiatric University Clinics, Wilhelm Klein-Strasse 27, CH-4012 Basel;
3
4
UMRS665 - Institut National de la transfusion sanguine, 6, rue Alexandre Cabanel, 75015 Paris; INSERM,
University of Strasbourg, U1119, Faculté de medicine, 11 rue Humann, 67000 Strasbourg.
We identified compound NCS1008 (1) as a good ligand of the Translocator Protein 18kDa (TSPO). As
many ligands of this protein, the druggability of NCS1008 is pretty poor as this compound is planar
and contains a high number of aromatic rings leading to low water solubility.
With the goal to improve druggability, we designed a bridged Phe-Gly dipeptide (2) which presents
the same pharmacophoric pattern as NCS 1008. This non-natural rigidified dipeptide constitutes a
new unplanar scaffold for TSPO. The bridged dipeptide is obtained through an intramolecular
Buchwald-Hartwig cross-coupling reaction starting from a 2-bromophenylalanine-glycine derivative
(3), and we developed conditions that induce chemoselectivity leading to either indoline (4) or 3,4dihydroquinolinone (5) derivatives. Performed under mild conditions, no racemization was observed
during cyclization. A preliminary mechanistic study was performed to identify the parameters leading
to the cyclization chemoselectivity.
Following an unconventional strategy, we started from a planar heterocyclic compound to design a
novel unplanar bridged dipeptide, affording a new class of TSPO ligands.
55
O20 New variety of potential amidoxime prodrugs from (2S)[cyano(hetero)aryl]carbonyl substituted α-amino acids
Olga V. Ovdiichuk,1,2 Olga V. Hordiyenko,1 Marie-Christine Averlant-Petit2 and Axelle
Arrault2
1
Department of Chemistry, Kyiv National Taras Shevchenko University, 64/13, Volodymyrska str., 01601, Kyiv,
2
Ukraine; Laboratoire de Chimie Physique Macromoléculaire, Université de Lorraine, 1 rue Grandville, BP 20451,
54001 Nancy, France.
Chemical modification of drugs into prodrugs potentially can improve physicochemical properties
such as water solubility and lipophilicity, transport of drug to the site of action, and presystemic
degradation and hence can improve the oral bioavailability.1 Recently it has been shown that the
mARC-containing enzyme system is responsible for the activation of amidoxime prodrugs so that
compounds of this class are of great interest.2
The used approach to the synthesis of the target prodrugs includes transformation of nitrile
functionality of 2-cyanoheteroaromatic acid (1) into corresponding amidoxime 4. The latter was
obtained by consequent coupling of 1 with L-α-amino acid methyl esters followed by hydroxylamine
treatment of (2S)-[cyano(hetero)aryl]carbonyl substituted amino acids 2 or their cyclic intermediates
3. When proline ester was taken, unclosed product 2’ was isolated as alone, and both Pro derivatives
2’ and 4’ demonstrated conformational isomerism in the NMR spectra with prevalence of trans-form.
Described the above strategies were applied as well for the synthesis of pseudo-tripeptides (2”, 4”).
In addition, we included some AA-amidoximeesters 5 and 1,2,4-oxadiazole derivatives 6 as masked
prodrugs in our investigations(Scheme).
O
X
O
X
Cl H3N
OH
N
N
1
R
N
COOMe
EDCI, HOBt, Et3N
X
R
X
N
2
+
O
N
O
.
NH2OH HCl, Et3N
MeOH
X
R
NH CO2Me
N
OH
NH2
4
N
R
N
CO2Me
N
NH
3
X = C, N
Y = CN (2'; 2''), C(NH2)(NOH) (4'; 4'')
R = Me (Ala), i-Pr (Val), i-Bu (Leu), Bn (Phe), CH2STr (CysTr)
HN
N
NH2
Y
2', 4'
trans
H
N
N
X
O
Bn
CO2Me
O
Y
trans
2'', 4''
R
CO2Me
O
N
O
N
R
X
CO2Me
N
NH CO2Me
HN
X
O
NHPG
O
R'
N
N
CO2Me
O
N
O
R''
5
6
Scheme
1. a) Bundgaard H. (1985) in Design of Prodrugs (Ed.: H. Bundgaard), Elsevier, Amsterdam, The Netherlands, pp 1-92 ; b)
Bundgaard H. (1991) in A Textbook of Drug Design and Development (Eds.: P. Krogsgaard-Larsen, H. Bundgaard), Harwood
Academic Publ., Switzerland, pp 113-191
2. Plitzko B. et al. (2013) J. Biol. Chem. 288, 20228-20237
56
O21 New synthetic pathway to fluoropseudopeptides cisoid conformers via
fluorinated olefin Ring Closing Metathesis (RCM)
David Guérin,1 Isabelle Dez,1 Annie-Claude Gaumont,1 Samuel Couve-Bonnaire2 and Xavier
Pannecoucke2
1
Laboratoire de Chimie Moléculaire et Thio-organique, CNRS UMR 6507 & FR 3038, ENSICAEN and University of
2
Caen-Basse Normandie, 14050 Caen; France Laboratoire COBRA, CNRS UMR 6014 & FR 3038, INSA and
University of Rouen, IRCOF, 1 rue Tesnière, 76821 Mont-Saint-Aignan, France.
The fluoroolefin moiety can be used as an efficient peptide bond mimic. Indeed, several studies have
demonstrated the isosteric and isoelectronic nature of this function regarding the amide bond. It is
furthermore characterized by a greater enzymatic degradation resistance and a more pronounced
lipophilicity. However, despite the interest in the peptidomimetics synthesis, there are only few
methods relating to the preparation of these fluorinated pseudopeptides.1
As part of our project dedicated to the development of new pseudopeptide synthetic methodologies,
we have recently described several asymmetric strategies towards these fine fluorine chemicals.2
Nevertheless, it is noteworthy that very few articles report the access to the fluoroolefin cisoid
conformers, which could be applied to bioactive conformation evaluations for biomolecules.
A new synthetic pathway to fluoroolefin cisoid conformers, based on the Ring-Closing-Metathesis
(RCM) reaction, is currently underway in our group. Examples that describe the fluoroalkene RCM
reaction are very scarce in the literature3 as it remains a major synthetic challenge, mainly in regards
to the inherent low reactivity of the fluoroalkene involved in the metathesis reaction.
A recent methodological study, that allowed us to reach excellent results for the synthesis of
fluorinated lactams through fluoroalkene RCM reaction will be first displayed.4 Then, the
development of the so obtained fluorinated compounds, for access to a wide range of
fluoropseudopeptides will be presented.
O
O
N
R1
GP
RCM
N
F
Ar
Fluorinated bisolefin:
RCM Substrate
GP
HOOC
R1
R1
Gly Ψ[(E)CH=CF] Aa
NHGP
F
F
Aa = Amino acid
RCM = Ring Closing Metathesis
Scheme 1: Strategy for access to cicoid fluoropseudopeptides via RCM reaction
1. Couve-Bonnaire S. et al. (2007) Org. Biomol. Chem. 5, 1151-1157
2. a) Couve-Bonnaire S. et al. (2007) Angew. Chem. Int. Ed. 46, 1290-1292 ; b) Pierry C. et al. (2011) Org. Biomol. Chem. 9,
2378-2386 ; c) Pierry C. et al. (2013) ChemBioChem 14, 1620-1633 ; d) Dutheuil G. et al. (2013) New J. Chem. 37, 1320-1325
3. a) Brown R. C. D. et al. (2003) Org. Lett. 19, 3403-3406; b) Haufe G. et al. (2004) Tetrahedron Lett. 45, 57-60; c) Rutjes F.
P. J. T. et al. (2004) Tetrahedron Lett. 45, 959-963; (2006) Eur. J. Org. Chem. 1166-1176; (2007) Eur. J. Org. Chem. 26672675; (2008) Synlett 3, 351-354
4. Guérin D. et al. (2014) ACS Catal. 4, 2374-2378
57
O22 Acides aminés α, α-disubstitués de type « super-aspartique » : de
nouvelles « briques élémentaires » pour la synthèse peptidique
Xiaofei Zhang,1 Gilles Dujardin,2 Sandrine Py,3 Mathieu Y. Laurent4 et Arnaud Martel5
1
Département de Chimie Moléculaire (SERCO) UMR-5250, CNRS, Université Joseph Fourier, BP 53, 38041
2
Grenoble Cedex 09, France; LUNAM Université du Maine, IMMM UMR 6283 CNRS, Equipe Méthodologie et
Synthèse Organique, 72085 Le Mans Cedex 09, France.
Les acides aminés α,α- disubstitués (AAD, acides aminés quaternaires) sont des blocs de construction
de grande valeur pour la synthèse de peptidomimétiques. Dans cette communication, nous
présenterons la synthèse asymétrique de nouveaux dérivés d'acides aminés quaternaires équipés de
deux chaînes latérales fonctionnelles. Celle-ci est basée sur une cycloaddition 1,3-dipolaire entre
divers éthers vinyliques et des cétonitrones1 originales équipées d’un auxiliaire chiral sur l’atome
d'azote.
Cette étude a permis de sélectionner un auxiliaire dérivé du mannose précédemment introduit par
Vasella2 qui permet d’obtenir un cycloadduit avec un double diastéréocontrôle élevé (exo et facial).3
Cette stratégie donne accès à des acides aminés de type « super-aspartique » équipés de deux
chaines fonctionnelles différenciées.
Man*
N
O
OR2
Man*
N O
HN
OR2
R1O2C
R1O2C
CO2R1
CO2R1
Man* = auxiliaire chiral mannosyl
O
OEt
t-BuO2C
CO2t-Bu
1
Acide aminé
"super-aspartique"
R2 = Et, ou t-Bu
R1 = Me, ou t-Bu
Afin de valider l’introduction de 1 en synthèse peptidique fonctions esters ont été différenciées : (i)
l’hydrolyse sélective du diester 2 permet d’accéder en peu d’étapes à un dipeptide couplé en
position β (ii) l’ouverture réductive de 2 donne un ester d’éthyle 4, précurseur direct de l’anhydride
cyclique 5 qui conduit après ouverture régiosélective au même dipeptide couplé en β.
HN
F3COC
O
OEt
t-BuO2C
N
F3COC
O
OEt
t-BuO2C
CO2t-Bu
1
N
O
OEt
t-BuO2C
HOOC
CO2t-Bu
COOH
3
2
NHCOCF3
CO2Et
t-BuO2C
NHCOCF3
CO2Et
CO-AA-OR
β- dipeptides
NHCOCF3
CO2Et
O
O
CO2t-Bu
O
4
5
En conclusion, cette communication présentera l’accès asymétrique inédit à des dérivés énantiopurs
d’AAD bi-fonctionnels de type « super-aspartique » ainsi que leur introduction dans une séquence
peptidique. Des développements synthétiques très récents pour l’accès à des AAD contraints par un
cycle lactamique pourront également être présentés.
1. a) Pfeiffer J. Y. & Beauchemin A. M. (2009) J. Org. Chem. 74, 8381; b) Hoods T. S. et al. (2012) Tetrahedron Lett. 53, 4679;
c) Nguyen T. B. et al. (2008) Org. Lett. 10, 4493; d) Nguyen T. B. et al. (2010) J. Org. Chem. 75, 611
2. Vasella A. (1977) Helv. Chim. Acta 60, 1273
3. Zhang X. F. et al. (2014) Org. Lett. 16, 19362
58
O23 Direct synthesis of multifunctional NPs for cancer cells imaging
Jérémie Ciccione,1,2 Said jebors,1,2 Ahmad Mehdi,2 Tao Jia,3 Jean-Luc Coll,3 Gilles Subra1 and
Jean Martinez1
1
2
Institut des Biomolécules Max Mousseron, Montpellier, France; Institut Charles Gerhardt, Montpellier, France;
Institut Albert Bonniot, La Tronche, France.
3
To improve diagnosis and cancer treatment, conjugation of targeting elements to the drug and/or
the detection probe is one of the most investigated approaches. In this field, functional particles of
nanometric scale can be seen as a promising diagnostic therapeutic or theragnostic tool1. In the
family of inorganic particles, silica nanoparticles (NpSi) are very attractive due to their ease of
synthesis by of sol-gel process, size control, biocompatibility2, and their ability to eventually
encapsulate different types of cargo molecules if they are porous.
O
O
Si
O
O
= c[RGD]-Si(OEt)3
= NRP-Si(OEt) 3
FITC-Si(OEt)3
In this context, we developed tunable multifunctional NpSi, presenting at their surface multiple
ligands, containing imaging probes and eventually a system of drug release. We first describe a
straightforward strategy relying on the synthesis of trialkoxysilyl hybrid building blocks3,4 (i.e
peptides, fluorophores). On the contrary of existing methods that require orthogonal protecting
strategies, click or ligation methods and surface modification, our method yields in one step
multifunctional NpSi with various ratios of ligands targeting receptors overexpressed in cancer cells
( ,  in the scheme). Starting fluorescent NPSi were obtained by co-condensation of TEOS with
hybrid fluoresceine, thus avoiding leaking of the fluorophore from the NP.
1. Zongxi L. et al. (2012) Chem. Soc. Rev. 41, 2590-2605
2. Tewodros A. & Zhimin T. (2012) Chem. Res. Toxicol. 25, 2265-2284
59
O24 Vectorization of pepstatin: a cathepsin D inhibitor as potential
antiproliferative agent
Clément Sanchez, Virginie Bellet, Lubomir L. Vezenkov, Vincent Martin, Marie Maynadier,
Jean Martinez, Muriel Amblard, Vincent Lisowski, Marcel Garcia and Jean-François
Hernandez
Institut des Biomolécules Max-Mousseron, UMR 5247 CNRS, Université de Montpellier, Faculté de Pharmacie,
15 av. Charles Flahault, 34093 Montpellier cedex 05, France.
To decrease the significant side toxicity of standard chemotherapies, using a specific feature of
tumors, a peptidase for example, could be a relevant approach. It has been shown that cathepsin D
(CathD), a lysosomal aspartyl protease overexpressed in many solid tumors [1], plays an important
role in cell proliferation and in the metastatic process.[2],[3] Pepstatin is a potent inhibitor of CathD
but its effectiveness is hampered by poor cellular internalization. Therefore, we developed a
conjugate in which pepstatin was attached to a new family of cell penetrating vector based on
oligomers of constrained dipeptide mimetics (AMPA) at its C-terminus, and a hydrophilic moiety to
increase hydro-solubility at its N-terminus. This conjugate was shown to enter cells, to reach the
endolysosomal compartment, and to strongly inhibit the growth of several tumor cell lines.[4] We
now study the structure-activity relationships of this conjugate to understand its mechanism of
action and to improve its properties. Thus, various points have been explored: (i) length and nature
of the vector, (ii) modification of the CathD inhibitor, (iii) modification of the hydrophilic moiety, (iv)
position of each element in the conjugate. In particular, a comparison with classical cell penetrating
peptides (penetratin and polyarginine) showed that these conjugates were not cytotoxic, indicating
that these vectors did not allow pepstatin to reach the right compartment. All compounds were
tested for their ability to inhibit isolated CathD, intracellular CathD, and tumor cell growth.
1. Rochefort H. et al. (2001) J. Steroid Biochem. Mol. Biol., 76, 119
2. Kute T.E. et al. (1992) Cancer Res., 52, 5198
3. Brouillet J.P. et al. (1993) Eur. J. Cancer, 29A, 1248
4. Maynadier M. et al. (2013) J. Control Release, 171, 251
60
O25 Development of peptidic bioconjugates featuring RGD ligands for
specific cell capture on functionalized gold surface
Mélissa Degardin, Céline Cépeda, Dhruv Thakar, Jérôme Dejeu, Liliane Guerente and Didier
Boturyn
Département de Chimie moléculaire (DCM), UMR CNRS-UJF 5250, ICMG FR-2607, Université Joseph Fourier,
Grenoble, France.
Further progress in tissue engineering requires an accurate knowledge of the cell response to their
interactions with their surrounding material. To properly seed cells on synthetic substrates through
immobilization of cell adhesion ligands on a surface, the study of the interactions between the
ligands and the substrate as well as their influence on cell adhesion is of high interest.
Supramolecular chemistry, allowing tunable molecular recognition, is particularly attractive for the
attachment of biomolecules to solid surfaces, which is a key issue in nanotechnology.
Our group has developed a regioselectively addressable cyclodecapeptide scaffold featuring in a
spatially controlled manner two independent functional domains using chemoselective ligations. The
structural feature allows a guest domain for the anchoring onto self-assembled monolayers βcyclodextrin surface (β-CD SAM) and a domain devoted to cell adhesion (RGD cell-adhesive ligands).1
Here we describe the convergent synthesis of macromolecules featuring both clustered guest motifs
and clustered RGD cell-adhesive ligands, achieved using chemoselective ligations (oxime, Huisgen
cycloaddition).2 The strength of the ligand interaction with the surface is modulated by varying the
chemical nature of the guest motifs (ferrocenyl or adamantyl) and the valency. The ability of the
obtained bioconjugates for selective cell capture is demonstrated by characterization of the
supramolecular bioassemblies on β-CD SAM substrates by quartz crystal microbalance with
dissipation monitoring (QCM-D) and complementary optical and fluorescence microscopy.
Asp G ly
Gly
Asp
Phe
Phe
Arg
Asp
Gly
Arg
Phe Asp
Arg
Lys
Lys
Gly
Phe
Lys
Eukaryotic cell
Arg
Lys
N
N
N
O
O
Lys
Lys
O
G ly
Lys Lys
Lys
Pro
N
O
Pro
Lys
Gly
N
N
O
Pro
Gly
Lys
O
G ly
Lys
Lys
Lys
Lys
N
Pro
N
Lys
N
N
N
N
N
N
N N
N N
Fe
Fe
Fe
Fe
Gold surface
1. Thakar D. et al. (2014) ChemBioChem 15, 377-381
2. Galibert M. et al. (2009) Angew. Chem. Int. Ed. 48, 2576-2579
61
O26 Novel cyclization reactions in bacteriocin biosynthesis
Alhosna Benjdia,1,2 Alain Guillot,1,2 Benjamin Lefranc,3,4 Hubert Vaudry,3,4 Jérôme
Leprince3,4 and Olivier Berteau1,2
1
2
INRA, ChemSyBio, Institut Micalis (UMR 1319), 78350 Jouy-en-Josas, France; AgroParisTech, ChemSyBio,
3
Institut Micalis (UMR), F-78350 Jouy-en-Josas, France; INSERM U982, 76821 Mont-Saint-Aignan France;
4
Institute for Research and Innovation in Biomedicine (IRIB), Regional Platform for Cell Imaging, PRIMACEN,
University of Rouen, 76821 Mont-Saint-Aignan, France.
Genomic and metagenomic investigations have recently led to delineate a novel class of natural
products called: ribosomally-synthesized and post-translationally-modified peptides (RiPPs).1 RiPPs
are ubiquitous among living organisms and include pharmaceutically relevant compounds such as
antibiotics, anti-cancer agents or toxins. Nevertheless, their biosynthetic pathways are poorly
understood and many RiPPs such as Thiostrepton A,2 Polytheonamides3 or Bottromycin,4 were
believed, until very recently, to be non-ribosomal peptides or polyketides rather than being of
ribosomal origin.
Among distinct features, some RiPPs possess unusual thioether bonds which formation has been
recently shown to be catalyzed by novel radical SAM enzymes and likely to result from the radical
activation of a peptidyl Cα−atom.5 We have undertaken the detailed investigation of AlbA, a key
enzyme in the biosynthesis of Subtilosin A, a higly cyclized antibiotic produced by Bacillus subtilis6,7
and containing three unusual thioether bonds. Combining high-resolution mass spectrometry, kinetic
and labeling experiments, we establish, in opposite to current paradigm, that the activity of AlbA is
peptide-leader independent and we propose a novel mechanism for the formation of thioether bond
in biosynthetic peptides.
2. Kelly W. L. et al. (2009) J. Am. Chem. Soc. 131, 4327
3. Freeman M. F. et al. (2012) Science 338, 387
4. Huo L et al. (2012) Chem. Biol. 19, 1278
5. Flühe L. K. et al. (2012) Nat. Chem. Biol. 8, 350
6. Babasaki K et al. (1985) J. Biochem. 98, 585
7. Zheng G et al. (1999) J. Bacteriol. 181, 7346
62
O27 Innovative oligourea foldamers with antimicrobial effects: application
to Bacillus anthracis infection
Stéphanie Antunes,1 Céline Douat,1 Jean-Philippe Corre,2 Pierre Goossens2 and Gilles
Guichard1
1
University of Bordeaux – CNRS UMR5248, IECB European Institute of Chemistry and Biology, CBMN, 2 rue
2
Robert Escarpit, Pessac, France; Laboratories of Pathogenesis of Bacterial Toxi-Infections, Pasteur Institute,
Paris, France.
The design of unnatural oligomers with predictable folding patterns (i.e. foldamers) has attracted
considerable attention over the last fifteen years. Potential applications in biology include the
development of anti-microbial agents, cell-penetrating agents, and inhibitors of protein–protein
interactions. One of the main interests for exploring biological applications of foldamers stems from
their high resistance to proteolytic degradation1.
Our group is developing peptidomimetic oligomer incorporating ethylene diamine units linked by
urea bond ((NH-CH(R)-CH2-NH-CO)n, Figure 1a) that have a strong helix propensity in solution and to
the solid state. More particularly, oligoureas of eight residues designed to mimic the amphipathic
character of antimicrobial peptides and maintaining a helical conformation in lipidic environment
(eg. OL1, Figure 1b) display a significant activity in vitro against Gram negative and positive bacteria,
with certain selectivity towards the eucaryote cells2. Recent data demonstrate that OL1 also exhibits
promising activity on capsulated and non capsulated pathogenic bacterium Bacillus anthracis.
Figure 1: a) N, N’-linked oligourea; b) Sequence of the oligourea OL1 designed to adopt an helical
amphipathic structure
To gain additional insight into the mechanism of action of antibacterial oligoureas and optimize first
generation molecules, the bactericidal efficiency in vitro of OL1 derivatives on Bacillus anthracis by
introducing variations in the primary sequence (nature of the side chains, charge distribution,
hydrophobic content…) has been evaluated and physicochemical studies on lipids membrane models
with the best candidates are being investigated. Herein we report the data gathered during this first
structure-activity relationship study.
1. Guichard G. & Huc I. (2011), Chem. Commun. 47, 5933-5941
2. Claudon P. et al. (2010), Angew. Chem. Int. Ed. Engl. 49, 333-336
63
O28 New β/γ-peptide manifolds designed as the 9/8-helix and the 13-helix
Claire M. Grison,1 Sylvie Robin1,2 and David J. Aitken1
1
Institut de Chimie Moléculaire et des Matériaux d’Orsay – UMR 8182 CNRS - Université Paris Sud, Orsay,
2
France; Université Paris-Descartes, Paris, France.
Oligopeptides containing β- or γ-amino acids are well known to adopt regular secondary structures
whilst being resistant to proteolytic degradation, making them of considerable interest as
peptidomimetic foldamers. Mixed β/γ-peptides are rather less studied but two types of helical
structures have been demonstrated for such derivatives, a mixed 11/13-helix1 and a 13-helix2 which
correlates to the well-known α-helix of native proteins. Mimetics of the α-helix displaying functional
side chains are attractive manifolds for the inhibition of protein-protein interactions. But the
backbones of the β- and γ-amino acids used in those examples were densely substituted restricting
the introduction of functional side chains and further biological applications.
We describe here our investigations of the folding behaviour of new mixed β/γ-peptides, designed to
show folding propensity with only a minimum of sterically-imposed conformational constraints. The
governing principle is to target selected residues for bearing minimal-bulk folding constraints, and
allow this to induce cooperative participation of the remaining residues, which are otherwise highly
flexible. Using this approach, we have been able to target the previously unknown (and unpredicted)
9/8-helix, which is stabilized by i,i+2 hydrogen-bonding contacts.
By introducing side chains in appropriate positions of the β/γ-peptide backbone, we are able to
modify the folding behaviour targeting the first functional 13-helix, which is stabilized by i,i+4
hydrogen-bonding contacts.
We will present the synthetic approaches for obtaining the appropriate β/γ-peptides, as well as the
theoretical and experimental studies (notably 1D, 2D NMR and CD). Collectively, the data suggest
that with specific design features included in the constituent amino acid building blocks, a 9/8-helix
or a 13-helix folding can be imparted to the β/γ-peptide manifolds, opening the way to a new α-helix
mimetic paradigm.
O
a)
O
H
N
1 γ β α
O
b)
O
O
H
N
1 γ β α
2
N
H
β
O
α
O
3
N
H
γ
β α
O
H
N
4 β α
O
2
N β α
H
3
N γ β α
H
O
H
N
5 γ β α
O
O
H
α
N
4 β
6 β α
N
H
O
7
N
H
ROE correlation:
H-bonds: 9/8-helix:
13-helix:
NH: bonded
free
H
N
5 γ β α
O
O
6 β α
N
H
O
7
N
H
Figure 1: a) The basis of the 9/8-helix secondary structure; b) The basis of the 13-helix secondary
structure
1. Sharma G. V. et al., (2006) J. Am. Chem. Soc., 128, 14657
2. Guo L. et al., (2010) J. Am. Chem. Soc., 132, 7868
3. Baldauf C. et al., (2006) J. Org. Chem., 71, 1200
64
O29 Design, synthesis and structural studies of a new 1:1 α/γ heterogeneous
peptide foldamer: identification of a ribbon-like structure mimicking α-helix
Clément Bonnel,1 Baptiste Legrand,1 Jean Martinez,1 Nicolas Masurier1 and Ludovic T.
Maillard1
1
Institut des Biomolécules Max Mousseron, CNRS UM1-UM2 UMR5247, Faculté de Pharmacie, 15 Av. Charles
Flahault, 34093, Montpellier, France.
The realm of foldamers is in continual expansion thanks to the design of new synthetic sequences
containing non-natural amino acids. Foldamers are defined as "any oligomer that folds into a
conformationally ordered state in solution, the structures of which are stabilized by a collection of
noncovalent interactions between nonadjacent monomer units".1 These architectures constitute a
promising approach to develop protein-protein interactions inhibitors. While heterogeneous
oligomers containing α- and β-amino acids have been widely investigated for many years,2 those
incorporating γ-amino acids drew far less attention due to the difficulty to access the corresponding
stereochemically pure γ-building blocks. In this context, we recently described a new class of
constrained heterocyclic γ-amino acids named ATCs (1) built around a thiazole ring. According to its
restricted conformational freedom, ATC oligomers adopt predictable helical secondary structures
stabilized by a C9-intramolecular hydrogen bonding network.3 Incorporation of this block in a αγαα
tetrapeptide induced turn-like structures depending on the ATC absolute stereochemistry.4 In this
work, we evaluate the folding propensity of ATCs in α/γ hybrid oligomers. We synthesized two
heterogeneous oligomers alternating (S)-α-amino acids with (S)- or (R)-ATCs to generate homochiral
and heterochiral platforms and access different structures, thus diversifying the spatial distribution
of the side chains. This communication will present the NMR structural studies of the two oligomers
and the discovery of a unique ribbon molecular shape for the heterochiral sequence. This unusual
folding pattern offers a new template to mimic α-helix secondary structure to modulate proteins
recognition in pathological contexts.
1. a) Appella D. H. et al. (1996) J. Am. Chem. Soc. 118, 13071-13072; b) Hill D. J. et al. (2001) Chem. Rev. 101, 3893-4011
2. Horne W. S. et al. (2008) Acc. Chem. Res. 41, 1399-1408
3. Mathieu L. et al. (2013) Angew. Chem. Int. Ed. 52, 6006-6010
4. Legrand B. et al. (2014) Chem. Eur. J. 20, 6713–6720
65
O30 How does the 1,2,3-triazole amide bond mimetic influences the
secondary structure of peptides? Case study on peptaibols
Khoubaïb Ben Haj Salah,1 Baptiste Legrand,2 Nicolas Ruiz,3 Jean Martinez2 and Nicolas
Inguimbert1
1
Université de Perpignan Via Domitia, CRIOBE USR CNRS 3278, centre de phytopharmacie, batiment T, 58
2
avenue P. Alduy, 66860 Perpignan, France; IBMM, Faculté de Pharmacie, 15 avenue Charles Flahault, BP
3
14491, 34093 Montpellier Cedex 5, France; Département Bioproduits, Service des Sciences Végétales et
Fongiques, UFR de Sciences pharmaceutiques et biologiques, MMS / EA 2160, IUML FR3473 CNRS Université de
Nantes, 9 rue Bias, BP 53508, 44035 Nantes Cedex 1, France.
α-Helices and strand account for respectively 30% and 20% of the secondary structures found in
proteins. Due to the possibility of designing de novo small peptides that folded into helices this
secondary structure has attracted considerable interest for application in biology, biomedicine, drug
discovery and also for the design of nanomaterials.1-2-3 For example helical structured peptides could
found applications as protein/protein interaction disruptors. Future studies concerning the
development of structures able to self-associate or to adopt a stable helical structure led us to
consider peptaibols as a template. Peptaibols are a class of non-ribosomally synthesized peptides
from a diverse set of fungi that include Trichoderma, Gliocladium and Stibella.4 Usually linear, these
peptides contain 4 to 20 residues and are characterized by a high content of α,α-dialkyl α-amino
acids such as α-aminoisobutyric acid (Aib) and D or L-isovaline (Iva). They possess an acetylated Nterminus along with a β-amino alcohol generally phenylalaninol (Fol) or leucinol (Lol) at their Cterminus. Alamethicin F50/5 and Bergofungin D are two representatives of these antimicrobial
peptides with a helical structure.5 These two peptaibols were chosen to insert a presupposed
isosteric 1,2,3-triazole motif in place of the amide bond on the Cter side of Aib residues and to assess
its impact on their secondary structure.6 For this purpose an optimized solid phase synthetic strategy
for preparing peptaibols and analogues of varying lengths has been developed that allows their
syntheses on L-Phenylalaninol 2-chlorotrityl resin using DIC/Oxyma as coupling reagents under
microwave irradiation.7 Introduction of the 1,2,3-triazole ring in the peptaibols sequence was
accomplished thanks to the prior synthesis of dipeptide like units embedding the heterocycle. The
synthesis and results of NMR and CD studies on eight Alamethicin and five Bergofungin analogues
containing triazoles will be discussed along with the preliminary biological effects of this insertion on
their biological activities.
Figure 1: Chemical structure of Alamethicin F-50 and different possible amendments
1. Seebach D. et al. (2006) Pept. Sci. 84, 23-37
2. Hosseinkhani H. et al. (2013) Chem. Rev. 113, 4837-4861
3. Zhao X. et al. (2010) Chem. Soc. Rev. 39, 3480-3498
4. Brückner H. & Toniolo C. (2013) Chem. Biodivers. 10, 731-733
5. Stoppacher N. et al. (2013) Chem. Biodivers. 10, 734-743
6. Fox R. O., Jr, & Richards F. M. (1982) Nature 300, 325-330
7. Ben Haj Salah K. & Inguimbert N. (2014) Org. Lett. 16, 1783-1785
66
O31 Peptaibols actifs sur les membranes : analyse structure-fonction par
RMN de l'état solide à l'aide d'aminoacides fluorés synthétisés sur-mesure
Véronique Doan,1 Andréa Bordessa,1 Grégory Chaume,1 Fabio Rizzolo,1,2 Elisa Peroni,1
Anna Maria Papini,1,2 Erik Strandberg,3 Sezgin Kara,3 Sergiy Afonin,3 Anne Ulrich3 et Thierry
Brigaud1
1
Laboratoire de Chimie Biologique (LCB), EA4505, Université de Cergy-Pontoise, 5 mail Gay-Lussac, Neuville sur
2
Oise, 95031 Cergy-Pontoise cedex, France; French-Italian Laboratory of Peptide & Protein Chemistry & Biology 3
PeptLab - www.peptlab.eu;. Karlsruhe Institute of Technology (KIT), Institut für Organische Chemie, Fritz-HaberWeg 6, 76131 Karlsruhe, Germany.
L’objectif de ce projet est de comprendre le mécanisme moléculaire de formation de canaux dans les
biomembranes par les « peptaibols » qui sont supposés perméabiliser les membranes bactériennes
par assemblage en paquets transmembranaires.
Ces peptides antimicrobiens comportent une forte proportion d’acide aminoisobutyrique (Aib) qui
les rend difficile à synthétiser. De ce fait, leur mécanisme d’action est assez mal connu. C’est le cas en
particulier de séquences peptidiques très courtes qui ne sont pas susceptibles de traverser la
membrane mais qui sont aussi actives que les séquences peptidiques typiques plus longues. Afin
d’élucider le mécanisme d’action de ces peptides, nous avons envisagé d’effectuer une analyse
structurale comparative par RMN 19F de l’état solide de deux peptaibols actifs sur les membranes de
tailles différentes : l’harzianine HK-VI (HZ, 11 résidus) qui est un peptide court et l’alaméthicine F30/3
(ALM, 20 résidus) qui est un peptide plus long. Pour ces mesures RMN très sensibles, il faut concevoir
des aminoacides trifluorométhylés énantiopurs très spécifiques et les incorporer dans les peptides.1
Notre choix s’est tout d’abord porté sur les (R)- et (S)-Tfm-alanines qui sont des isostères de l’Aib.2
H3C CH3
F3C CH3
H3C CF3
H 2N
H2N
H2N
CO2H
Aib
CO2H
(R)-α-TfmAla-OH
CO2H
(S)-α-TfmAla-OH
Ac-Aib-Asn-Ile-Ile-Aib-Pro-Leu-Leu-Aib-Pro-Leu-ol
1
2 3 4 5
6
7 8 9 10 11
Harzianine HK-VI
Ac-Aib-Pro-Aib-Ala-Aib-Ala-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu-Gln-Phe-ol
1
2 3 4 5 6
7 8 9 10 11 12 13 14 15 16 17 18 19 20
Alaméthicine F30/3
Les synthèses par SPPS de l’Harzianine et de l’Alaméthicine ainsi que leurs analogues fluorés
incorporant une (R)- ou (S)-α-Tfm-alanine en lieu et place d’un résidu Aib seront détaillées.
L’incorporation de ces aminoacides trifluorométhylés a constitué un défi en soi compte tenu de leur
faible réactivité et de leur encombrement stérique. Les résultats préliminaires concernant les études
de dichroïsme circulaire, d’activité antimicrobienne ainsi que l’analyse structurale par RMN 19F de
l’état solide seront également présentés.
Ces études sont susceptibles de fournir des informations pertinentes sur l’étape critique de
formation des canaux; c’est à dire la liaison avec la membrane, l’insertion dans la membrane,
l’oligomérisation et la réorientation.
1. a) Kubyshkin V. S. et al. (2011) In Gouverneur V. & Müller K. (Eds) Fluorine in Pharmaceutical and Medicinal Chemistry:
From Biophysical Aspects to Clinical Applications; Imperial College Press; b) Maisch D. et al. (2009) J. Am. Chem. Soc. 131,
15596-15997
2. a) Chaume G. et al. (2009) Eur. J. Org. Chem. 5717-5724; b) Huguenot F. & Brigaud T. (2006) J. Org. Chem. 71, 7075-7078
67
O32 TES-Dpg, a new Cα-tetrasubstituted silylated amino acis : synthesis and
impact on peptide conformation
Roberto Fanelli,1 Aurélien Lebrun,1 Dorothée Berthomieu,2 Jean Martinez1 and Florine
Cavelier1
1
IBMM, UMR-CNRS 5247, Université de Montpellier, Place Eugène Bataillon, 34095 Montpellier cedex 05,
2
France; ICG, UMR-CNRS 5253, Université de Montpellier, Place Eugène Bataillon, 34095 Montpellier cedex 05,
France.
Cα-tetrasubstituted amino acids (CTAAs) are α-amino acids in which the hydrogen atom at the αposition of the α−amino acid is replaced with an alkyl substituent. The introduction of an α-alkyl
substituent changes the properties of amino acids, with the conformational freedom of the side
chain and the secondary structure of peptides being especially restricted.1
The synthesis of silicon-containing amino acids represents a very promising target.2 They can be
obtained by hydrosilylation reaction, this methodology also enabling the synthesis of α,α’disubstituted amino acids with one silylated side chain.3 Silicon-containing amino acids can be useful
tools in the synthesis of modified bioactive peptides in order to increase their lipophilic character and
their resistance toward enzymatic degradation.4
Combining the properties of CTAAs and Si-containing amino acids, we have prepared Triethylsilyl
dipropylglycine (TES-Dpg), the first example of bis-sylilated CTAAs. Synthesis and conformational
studies will be presented.
1. Tanaka M. (2007) Chem. Pharm. Bull. 55, 349-358
2. Marchand D. et al. (2008) Eur. J. Org. Chem. 3107-3112
3. Cavelier F. et al. (2008) Chem. Biodiv. 1279-1287
4. a) Cavelier F. et al. (2002) J. Am. Chem. Soc. 124, 2917-2923; b) Cavelier F. et al. (2004) J. Pept. Res. 63, 290-296 ;.c) Pujals
S. et al. (2006) J. Am. Chem. Soc. 128, 8479-8483 ;.d) Dalkas G. A. et al. (2010) J. Peptide Sci. 16, 61-97
68
O33 Technologies for cost effective peptide manufacture, environmental
and economical considerations
Mimoun Ayoub
Director, Global Peptides, Carbohydrates, Lipids and Sterile Injectable Drug Products. CordenPharma
International.
Key and proprietary technologies in peptide synthesis enabling cost-effective production by
increasing yield and purity, reducing the number of chemical steps and avoiding racemisation. The
technologies offered by CordenPharma are highly flexible, and are not limited to one typical product
line, but can be used for a wide range of peptide APIs spanning all therapeutic areas. A review of the
most innovative proprietary technologies and applications will be presented.
Case studies of peptide synthesis at large scale will be presented. A special focus on challenges
associated with scale-up of peptide products from few grams to several 100’s of kg with the impact
of starting materials, synthesis and purification on cost of manufacturing.
69
O34 The octadecaneuropeptide ODN exacerbates focal cerebral ischemia
while exerting protective action in vitro
Rhita Lamtahri, Julie Maucotel, Benjamin Lefranc, David Vaudry, Jérôme Leprince and
Julien Chuquet
INSERM U982, Laboratoire de Différenciation et Communication Neuronale et Neuroendocrine, Université de
Rouen, Institut de Recherche et d’Innovation Biologique, C.U.B.I.R., Place Emile Blondel, 76821 Mont-SaintAignan, France.
Octadecaneuropeptide (ODN) is one of the biologically active fragments of diazepam-binding
inhibitor (DBI33-50). This endozepine is synthesized by astrocytes and is involved in several
neurobiological processes such as neurogenesis and central glucose sensing. We have previously
shown that ODN is a potent protector of neurons or astrocytes coping with a lethal oxidative or
apoptotic stress. To test the hypothesis that ODN is also protective in experimental stroke, we used a
model of brain ischemia consisting in the introduction of a nylon filament into one carotid artery and
push it forward the middle cerebral artery to occlude it for 1 h. This resulted in a 60% reduction of
blood flow and the development of a brain infarction within 24 h. ODN (1µg/3µl) or its vehicle
solution was injected into the brain lateral ventricle 20 min before the onset of ischemia.
Surprisingly, administration of ODN resulted in a significant increase in the infarction volume
accompanied by an increase in the brain edema volume. The monitoring of various physiological
parameters shows that this deleterious effect of ODN can neither be explained by a hyperthermic, a
cerebrovascular or a cardiovascular effect of the peptide. In order to test if the ODN aggravation
accounts for an amplification of excitotoxic processes, the glutamatergic agonist NMDA (20 nmol)
and ODN (1 µg/0.5 µl) or its vehicle solution was injected into the right striatum. The coadministration of ODN and NMDA also resulted, 48 h later, in an increase in the excitotoxic lesion
volume. To further explore the role of endogenous ODN in stroke we induced brain ischemia in
knockout (KO) mice for DBI (DBI-/-). Consistent with our previous observations, brains from KO mice
showed smaller infarct size compared with wild type mice. Thus, our results suggest that the
neuroprotective potential of ODN seen in vitro may be overtaken in vivo by an exacerbation of
excitotoxic processes likely caused by its inverse agonist effect on the GABAA receptor.
70
O35 Caractérisation du neuropeptidome de la seiche Sepia officinalis :
identification des neuropeptides impliqués dans la ponte.
Joël Henry,1,2,5 Valérie Cornet,1,2 Alexandre Leduc,1,2 Bruno Zanuttini,3 Erwan Corre,4 Gildas
Le Corguillé,4 Benoît Bernay,5 Alexandra Kraut6 et Céline Zatylny-Gaudin1,2
1
2
Université de Caen Basse-Normandie, Caen, France; Université de Caen Basse-Normandie, UMR BOREA
3
MNHN, UPMC, UCBN, CNRS-7208, IRD-207, F-14032 Caen, France; GREYC, Université de Caen Basse4
Normandie, F-14032 Caen, France; UPMC, CNRS, FR2424, ABiMS, Station Biologique, 29680, Roscoff, France;
5
Plateforme postgénomique PROTEOGEN, Université de Caen Basse-Normandie, SF ICORE 4206, F-14032 Caen,
6
France; Plateforme de protéomique platform, EDyP / BGE / iRTSV / CEA / INSERM U1038 / UJF, F-38054
Grenoble, France.
Les céphalopodes possèdent une grande variété de comportements associés à la prédation, à la
communication, au camouflage et à la reproduction. Grâce à un système nerveux central complexe
divisé en plusieurs lobes fonctionnels, ils sont capables d’exprimer une grande diversité de
neuropeptides intervenant dans le comportement et dans la régulation des mécanismes
physiologiques associés aux principales étapes du cycle de vie. Dans ce contexte, pour identifier et
caractériser le neuropeptidome impliqué dans le contrôle des différentes étapes de la ponte – ponte
ovulaire, transport des ovocytes dans l’oviducte, sécrétion des capsules internes et externes,
fécondation - nous avons réalisé une approche transcriptomique globale du SNC en RNAseq de novo
combinée à une approche peptidomique destinée à identifier et à localiser les neuropeptides
matures chez la femelle en ponte. L’identification des neuropeptides a été réalisée à partir d’une
analyse in silico des données transcriptomiques complétée par l’analyse exhaustive des données de
spectrométrie de masse. Les neurohormones ont été identifiées par l’analyse du peptidome de l’aire
neurohemale de la veine cave et de l’hémolymphe. Quant aux neurotransmetteurs, ils ont été
recherchés au niveau des terminaisons nerveuses des glandes annexes et de l’ovaire des femelles en
ponte.
Parmi les 53 précurseurs de neuropeptides identifiés correspondant à 37 familles, 12 sont très
fortement surexprimés chez la femelle en ponte versus mâle mature et les neuropeptides de 8
familles sont retrouvés dans les terminaisons nerveuses de l’appareil génital femelle. Par ailleurs,
certains transcrits de neuropeptides sont présents en quantité importante dans les glandes annexes
de l’appareil génital femelle sans que nous puissions encore déterminer s’il s’agit d’une réelle
expression tissulaire ou d’ARN messagers mobilisés dans les terminaisons nerveuses. Cette analyse
réalisée tant au niveau du transcriptome que du peptidome permet finalement de cibler 8 familles
candidates potentiellement impliquées dans la régulation des différentes étapes de la ponte:
APGWamide, Crustacean Cardioactive Peptides (CCAPs), FLGamide, FaRPs 1, Insulin, Myomodulin 1,
PXXIamide et Small Cardioactive peptides (SCPs). Les neuropeptides appartenant à ces familles
devront donc faire l’objet d’une étude fonctionnelle approfondie.
Par ailleurs, plusieurs nouvelles familles de neuropeptides ont été identifiées pour la première fois
grâce à l’analyse in silico du transcriptome de S. officinalis combinée à l’analyse par spectrométrie de
masse du SNC et de l’appareil génital femelle. Ces familles ont été retrouvées par Blast chez d’autres
mollusques gastéropodes (Lottia gigantea) et bivalves (Crassostrea gigas), chez les annélides
(Capitella tellata), chez les arachnides (Stegodyphus mimosarum), chez les insectes (Acromyrmes
echinatior) et chez les crustacés (Daphnia pulex) alors que les transcrits étaient annotés
« uncharacterized protein » ou « hypothetical protein ». Leur conservation structurale dans les 3
principaux phyla de protostomiens, mollusques, annélides et arthropodes permet de spéculer sur
leur implication dans des mécanismes de régulation majeurs qu’il reste encore à déterminer.
L’approche transcripto-peptidomique développée dans cette étude a donc permis (1) d’identifier et
de caractériser le neuropeptidome de l’espèce S. officinalis ce qui constitue donc le premier
neuropeptidome caractérisé chez un céphalopode, (2) de définir une liste restreinte de
neuropeptides candidats pour la régulation de la ponte et (3) d’identifier plusieurs familles de
neuropeptides totalement nouvelles.
71
O36 Quantification de peptides modifiés par spectrométrie de masse
MALDI-ToF – Mesures d’interactions récepteur-ligand
Maxime Rossato, Sonia Cantel, Didier Gagne, Gilles Subra, Christine Enjalbal et Jean
Martinez
Institut des Biomolécules Max Mousseron (IBMM), UMR5247, Université Montpellier 1 et 2, France.
Mesurer l'affinité d'un ligand pour sa cible est une question d'importance capitale en pharmacologie
et plus précisément dans le développement de médicaments. En particulier, comprendre la façon
dont un médicament interagit avec sa cible (récepteur, enzyme, ADN...) fournit des informations
précieuses dans la quête de nouveaux composés pharmaceutiques. Concernant l’interaction ligandrécepteur, La liaison d'un candidat-médicament se caractérise par des tests de liaison compétitive
dans lequel un ligand de référence marqué (dont affinité vers le récepteur est connue) est mis en
concurrence avec la molécule d'intérêt. Étant donné que l'affinité du ligand de référence pour le
récepteur cible est généralement élevée, la mesure de ces interactions non-covalentes nécessite une
excellente sensibilité (<10-8 M).
La radioactivité est encore aujourd’hui la méthode universelle pour ces études pharmacologiques.
Cependant, cette dernière implique beaucoup de contraintes liées à la manipulation des
radioéléments et leur stockage, et limite donc fortement son application. Dans ce contexte, nous
visons à développer une technologie s'appuyant sur la détection ultrasensible et la quantification par
spectrométrie de masse.
Des agents de dérivation chimiques de peptides ont été développés au laboratoire, jouant le rôle
d’exhausteur d’ionisation en spectrométrie de masse moléculaire dans l’optique d’augmenter la
sensibilité de détection de biomolécules, problématique particulièrement délicate lors de l’étude de
milieux complexes.1-2 Une méthodologie analytique basée sur la spectrométrie de masse MALDI-ToF
a été mise en place avec l’utilisation conjointe du marquage spécifique des peptides par HCCA (acide
α-cyano-4-hydroxycinnamique) qui constitue l’une des matrices les plus couramment employées en
MALDI-Tof pour la détection de ces biomolécules et de la molécule HCCE (ester méthylique de
l’HCCA) en tant que matrices de dépôt MALDI. Cette combinaison «marqueur HCCA / matrice HCCE»
a démontré sur des peptides modèles une efficacité accrue de la réponse en spectrométrie de masse
MALDI-Tof de ces molécules portant la dérivation par rapport aux peptides non marqués présents en
mélange.
L’optimisation méthodologique a permis d’améliorer significativement la sensibilité de l’analyse
MALDI en atteignant des seuils de détection de l’ordre de 10-12 M. Après validation de la
méthodologie de quantification, des tests de liaison ont été effectués sur le modèle CCKR/AVP.
1. Lascoux D. et al. (2007) Angew. Chem. Int. Ed. 46, 5594-5597
2. Paramelle D. et al. (2010) Angew. Chem. Int. Ed. 49, 8240-8243
72
O37 Novel contrast agents for in vivo molecular imaging of matrix
metalloproteinase-12 (MMP-12)
Thomas Bordenave,1 Yupeng Ye,2 Mahmoud Razavian,2 Laurent Devel,1 Mehran Sadeghi2
and Vincent Dive1
1
CEA (Commissariat à l'Energie Atomique), iBiTec-S, Service d'Ingénierie Moléculaire de Protéines (SIMOPRO),
2
CE Saclay 91191 Gif/Yvette, Cedex, France; Cardiovascular Molecular Imaging Laboratory, Veterans Affairs
Medical Center, 950 Campbell Avenue, 06516 West Haven, CT, USA.
Matrix Metallo proteinases (MMPs) are zinc-dependent endo peptidases that form a family of 24
members in mammals. These proteases are overexpressed during tissue remodeling and their
uncontrolled activity is often associated to inflammatory disorders.1 Within this family of enzyme,
MMP-12 or macrophage metallo elastase is mainly secreted by macrophages in inflammatory
conditions. Under its active form, this protease plays a central role in various pathologies including
lung inflammation and different cardiovascular diseases such as atherosclerosis and aneurysm. More
particularly, MMP-12 is directly involved in the development and rupture of atheroma plaques and
was shown to be a prognostic indicator of the pathology advancement.2 In this context, MMP-12 can
be considered as a biomarker of monocyte-macrophage activation and consequently targeting
activated MMP-12 in vivo would allow imaging vessel wall inflammation and remodelling in various
inflammatory pathologies.
RXP470.1 a phosphinic pseudo peptide as a selective and potent inhibitor of MMP-123 demonstrated
its ability to target active form of MMP-12 in vivo. Thus, in a mouse model of atherosclerosis, this
inhibitor proved its efficiency to block atheroma plaques development and rupture4. More recently,
this compound was shown to be able to block MMP-12 activity in a mouse model of rheumatoid
arthritis5 or during viral infection6. In this respect, we consider that RXP470.1 may serve as a valuable
starting point for the development of specific tracers to image and monitor MMP 12 activity in vivo.
Based on the 3D-structure of RXP470.1 in interaction with MMP-12 catalytic domain we recently
developed contrast agents harbouring a selective ligand toward MMP-12, a polyethylene glycol
spacer and various reporter groups according to the imaging modalities desired.
O
H
N
O
O
O
Imaging
reporter
spacer
SPECT imaging
O
O
O
O
n
FMT
N
H
99m Tc
O
NIR
O
n
N
H
SPECT
FMT imaging
In this series, the synthesis of several original tracers will be described. Spacer length, nature of
reporter groups and global net charge of the tracers may impact the tracers ability to selectively
interact with MMP-12 in complex media. Influence of such parameter will be then commented.
Tracer kinetics and bio distribution will be also presented. Finally, through competition experiments
and MMP-12 gene invalidation, their ability to specifically interact with their prime target will be
addressed in several mouse models of inflammation.
1. Vandenbroucke R. E. et al. (2014) Nat. Rev. Drug. Discov. 13, 905-927
2. Sholtes V. P. et al. (2012) J. Am. Heart. Assoc. 6, 1-12
3. Matusiak N. et al. (2013) Curr Pharm Des. 19, 4647-4672
4. Devel L. et al. (2006) J. Biol. Chem. 281, 11152-11160
5. Johnson J. L. et al. (2011) Arterioscler. Thromb. Vasc. Biol. 31, 528-535
6. Lim N. H. et al. (2014) Arthritis and Rheumatology. 66, 589-598
7. Marchant D. J. et al. (2014) Nature Med. 20, 493-502
73
O38 Conception et synthèse de vecteurs peptidiques pour le ciblage de la
périphérie tumorale : aide à la chirurgie
Fabien Thoreau,1,2 Tao Jia,2 Jungyoon Choi,2 Jean-Luc Coll2 et Didier Boturyn1
1
Département de Chimie Moléculaire, UMR CNRS-UJF 5250, équipe I2BM, Université Joseph Fourier – Grenoble
2
1, Grenoble, France; Institut Albert Bonniot, CRI INSERM/UJF U823, Grenoble, France.
Avec la chimiothérapie et la radiothérapie, la chirurgie fait partie des trois techniques majeures pour
lutter contre le cancer. Elle est la plus utilisée dans le cas de tumeurs solides accessibles. Retirer une
tumeur par opération chirurgicale implique de pouvoir distinguer le tissu tumoral du tissu sain. Le
chirurgien dispose alors de différentes techniques d’imagerie telles que l’IRM ou encore le PET scan.
Ces techniques sont néanmoins préopératoires. Or, lors d’une intervention chirurgicale, une imagerie
« en temps réel » serait préférable. Il est alors concevable d’utiliser un composé fluorescent
permettant d’imager la tumeur.
Dans ce contexte, l’objectif de ma thèse est de vectoriser un fluorophore proche infra-rouge jusqu’à
la périphérie tumorale afin de mieux guider le geste chirurgical. Nous proposons de cibler
spécifiquement la périphérie plutôt que la tumeur entière pour obtenir un meilleur contraste. Nous
avons identifié deux récepteurs dont le ciblage permettra de véhiculer le fluorophore vers la
périphérie tumorale : l’intégrine αvβ3 et la Neuropiline-1 (NRP1). Ces deux récepteurs sont
surexprimés dans beaucoup de cancers et sont impliqués dans les phénomènes d’angiogénèse, ce
qui explique leur présence plus importante au niveau de la périphérie tumorale. Des ligands de ces
récepteurs ont déjà été décrits : la séquence peptidique cyclique -RGD- qui a une forte sélectivité
pour l’intégrine αvβ3, et le ligand peptidique ATWLPPR qui lui est spécifique de la neuropiline-1.
Nous avons ainsi synthétisé des systèmes macromoléculaires sophistiqués présentant les ligands de
ciblage -RGD- et ATWLPPR conjugués ensemble à un fluorophore du type Cyanine 5.5®. Pour cela,
nous avons utilisé des châssis moléculaires cyclodécapeptidiques [1] comportant diverses fonctions
chimiosélectives orthogonales permettant des ligations via des cycloadditions de Huisgen ou des
formations de liens oxime.[2] Ceci a permis de concevoir des vecteurs fluorescents avec un double
ciblage de la périphérie tumorale (figure 1). Plusieurs structures ont été réalisées pour tester l’impact
du double ciblage. Des tests in vitro (cytométrie de flux, Western Blot, microscopie) et in vivo
(biodistribution) permettront de valider le/les composés candidats pour l’aide à la chirurgie.
RGD
ATWLPPR
ligand
Cy 5.5
Peptidic scaffold
Figure 1 : Schéma d’un vecteur fluorescent avec double
ciblage
1. Boturyn D. et al. (2008) J. Pept. Sci. 14, 224-240
74
O39 Optimization of bombesin based radiotracers for tumor targeting
Ibai E. Valverde, A. Mascarin, S. Vomstein and T. L. Mindt
Division of Radiopharmaceutical Chemistry, Clinic of Radiology and Nuclear Medicine, University of Basel
Hospital, Petersgraben 4, 4301 Basel, Switzerland.
The gastrin-releasing peptide receptor (GRPr) is a bombesin receptor overexpressed in a variety of
clinically relevant tumors including prostate and breast cancer. Thus, bombesin-based
radiopharmaceuticals hold great promise for imaging and peptide receptor radionuclide therapy of
GRPr-expressing tumors. In recent studies, we have shown that the use of 1,4-disubstituted 1,2,3triazoles as protease resistant amide bond surrogates leads to an enhanced stability of a radiolabeled
bombesin derivative and to an improved tumor uptake in vivo.1 Despite the excellent
pharmacokinetic profile of our peptidomimetic lead compound, we wanted to further improve its
tumor uptake in vivo. For further optimization, we set out to conduct a structure activity relationship
(SAR)-guided study on the bombesin binding domain BBN(6-14) by variation of the amino acid
sequence. We herein wish to report the synthesis and in vitro and in vivo evaluation of a series of
novel radiolabeled bombesin analogs with promising biological characteristics for radiotracer
development.
Peptide and triazolopeptide sequences were synthesized by a convenient and efficient solid phase
synthesis approach as previously reported. Conjugation of the chelator DOTA via a N-terminal
tetraethylene glycol spacer provided the means for radiolabeling of the peptides with Lu-177. Cell
internalization kinetics and GRPr binding affinity (KD) of the radioconjugates were determined using
GRPr-expressing PC-3 cells. Metabolic stability of the radioconjugates was evaluated by incubation in
blood serum. Biodistribution experiments were performed in PC3-xenografted Foxn1 nude mice.
We have successfully synthesized a series of novel bombesin conjugates and labeled them with Lu177 in excellent radiochemical yields and purity (>95%). The influence of different amino acid and
amide bond substitutions within the binding sequence of BBN(6-14) on critical parameters such as
cell internalization kinetics, metabolic stability, receptor affinity and occupancy, as well as tumor
uptake in xenografted nude mice will be presented and discussed.
1. Valverde I. E. et al. (2013) Angew. Chem. Int. Ed. 52, 8957-8960
75
O40 Metal-catalyzed synthesis of a new chemical family of unnatural basic
amino acids leading to orally-active NPFF receptor antagonists preventing
opioid-induced hyperalgesia
Séverine Schneider,1 Hussein Ftouni,1 Khadija Elhabazi,2 Jean-Paul Humbert,2 Martine
Schmitt,1 Jean-Jacques Bourguignon1, Frédéric Simonin2 and Frédéric Bihel1
1
2
UMR7200, CNRS, Université de Strasbourg, Illkirch, France; UMR7242, CNRS, Université de Strasbourg, Illkirch,
France.
The development of drugs that can more effectively and safely treat both acute and chronic pain
(resulting from post-operative surgery, cancer, neuropathies, etc) remains a major unmet challenge
in pharmaceutical industry. Opiate analgesics, such as morphine or fentanyl, continue to be the
cornerstone medication for treating moderate to severe pain, but their use upon chronic
administration suffers from important side-effects such as opioid-induced tolerance, addiction and
hyperalgesia. Several anti-opioid systems have been reported as valuable targets for blocking opioidinduced hyperalgesia (OIH). Among them, the NPFF-receptors belonging to the GPCR family were
recently identified as one of the keystone of the opioid-induced hyperalgesia. In a first approach, we
developed the first NPFF-receptor antagonist (RF9), and its co-administration with opioid analgesics
(fentanyl) led to block the delayed and long lasting paradoxical opioid induced-hyperalgesia and
prevent the development of associated tolerance.1,2 However the dipeptidic nature of RF9 limited its
application to subcutaneous or intravenous administration. Based on an extensive SAR study, we
were able to develop a peptidomimetic of RF9 containing only one unnatural amino-acid.3,4 The
development of this novel basic amino acid was obtained by using a specific metal-catalyzed
reduction of amide applied for the first time to amino acids. Generally, metal-catalysis reduction of
amides required harsh conditions incompatible with peptide stability. Using mild conditions, we were
able to optimize the reaction to afford in very high yields novel families of chiral α, β or γ amino acids
exhibiting a large diversity of basic side chains. Obtained at a gram-scale with a Fmoc protection at
the N-terminus, these unnatural amino acids are directly suitable for peptide synthesis in liquid or
solid phase in order to develop new unnatural peptides.5,6 When applied to the development of new
ligand of NPFF receptors, we were able to design and synthesize RF313 as the first orally-active NPFF
receptor antagonist able to reverse opioid-induced hyperalgesia in several pain models (postoperative pain, neuropathic pain, inflammatory pain). Active at low dose (1 mg/kg p.o. in rat), RF313
is also able to extend the duration of the analgesic effect induced by the opiates.7
NH2
HN
R1
N
R2
NH
α-amino acid
O
Fmoc
N
H
COOH
NH
H
N
R1
O
NH2
O
Analgesia
RF9
N
R2
Fentanyl
± RF313
β-amino acid
COOH
Fmoc
NH
Hyperalgesia
N
R1
N
R2
γ-amino acid
O
H
N
N
H
Fmoc
NH
O
COOH
RF313
1. Simonin F. et al. (2006) P. Natl. Acad. Sci. USA 103, 466-471
2. Elhabazi K. et al. (2012) Brit. J. Pharmacol. 165, 424-435
3. Gealageas R. et al. (2012) Bioorg. Med. Chem. Lett. 22, 7471-7474
4. Bihel F et al, (2012). Patent WO2013178810
5. Bihel F. et al. (2014) Patent EP14306411
6. Schneider S. et al. (2015) submitted manuscript
7. Elhabazi K. et al. (2015) submitted manuscript
76
O41 Synthèse diastéréosélective de dérivés thiazoles α-aminoacides et
applications pour la synthèse de nouveaux analogues de la neurotensine
Emmanuelle Rémond,1 Denisa Hapău,2 Adeline René,1 Jean Martinez,1 Philippe Sarret,3
Valentin Zaharia2 et Florine Cavelier1
1
2
IBMM, UMR-CNRS-5247, Universités Montpellier I and II, Place Eugène Bataillon, 34095, Montpellier, France;
Iuliu Haţieganu University of Medicine and Pharmacy, Department of Organic Chemistry, Victor Babes 41, Ro400012, Cluj-Napoca, Romania.
Le traitement de la douleur représente un marché considérable et le développement de
médicaments impliquant des effets secondaires limités est indispensable. Dans ce domaine, la
neurotensine [8-13] possède des propriétés hypothermiques, antipsychotiques et exerce des
réponses analgésiques puissantes induites par l’activation des récepteurs NTS1 et NTS2. Cependant,
sa faible biodisponibilité est un obstacle à son activité biologique et ses effets thérapeutiques. De
nombreux travaux portent sur les relations structure-activité de la neurotensine en introduisant des
acides aminés non naturels ou en apportant des modifications structurales.1 En particulier, des
études de relation structure-activité de la NT [8-13] ont montré que le résidu en position 11 doit
posséder une chaine latérale aromatique, la substitution de la tyrosine par une chaine aliphatique
(leucine ou alanine) supprimant son activité biologique centrale ou périphérique.2 Parmi les acides
aminés modifiés, les dérivés thiazoles présentent un intérêt de tout premier plan pour la synthèse et
l’étude de peptides bioactifs.3
Ainsi, nous décrivons la synthèse diastéréosélective de nouveaux dérivés thiazoles α-aminoacides 3 à
partir de la Base de Schiff dérivée de l’hydroxypinanone 1. Ces acides aminés ont été obtenus avec
de bons rendements et énantiosélectivités (e.e. ˃ 99%), puis introduits en position 11 de la NT[8-13]
(Schéma 1). Les études de radioliaison et de relations structure-activité ont permis d’identifier des
analogues sélectifs pour le récepteur NTS2 afin de réaliser des études in-vivo dans différents modèles
d’évaluation de la douleur.
Y
I
Ar
N
X
CO2PG1
OH
n
G2PHN
2
3
1
CO2PG1
Y
Ar
X
Ar
X, Y = N,S
Y
H2N
N
9
10
O
n
O
4
8
N
H
NH
2
4
X
NH2
O
n
N
H
H
N 12
11
O
O
N
H
OH
13
O
Schéma 1 : Synthèse asymétrique des dérivés thiazoles α-aminoacides 3 et analogues de la NT [8-13].
1. a) Cavelier F. et al. (2002) J. Am. Chem. Soc. 124, 2917-2923;.b) Bredeloux P. et al. (2008) J. Med. Chem. 6, 1610-1616; c)
Kleczkowska P. & Lipkowski A. W. (2013) Eur. J. Pharmacol. 716, 54-60
2. a) Quirion R. et al. (1980) Brit. J. Pharmacol. 69, 689-700; b) Einsiedel J. et al. (2011) J. Med. Chem. 54, 2915-2923
3. a) Davidson B. S. (1993) Chem. Rev. 93, 1771-1791; b) Cragg D. J. et al. (2012) Natural Products, Second Edition, 451-496
77
O42 Ligands du récepteur de la ghréline. Du peptide au peptido-mimétique :
vers une sélectivité fonctionnelle
Mathieu Maingot, Céline N’Kadmi, Jacky Marie, Jean-Louis Banères et Jean-Alain Fehrentz
Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, CNRS – Université Montpellier 1, BP 14491,
Faculté de Pharmacie, 15 avenue Charles Flahault, 34093, Montpellier Cedex 5, France.
La ghréline, hormone peptidique de 28 acides aminés, d’abord identifiée comme un sécretagogue de
l'hormone de croissance, joue également un rôle central dans la prise alimentaire ainsi que dans
certains processus liés à l’addiction. Ces effets sont médiés par son récepteur : le GHS-R1a (Growth
Hormon Secretagogue Receptogue). En tant que membre de la famille des RCPG, ce récepteur couple
à la fois aux protéines Gq, Go, Gi et à l’arrestine.
Compte tenu de leurs multiples rôles physiologiques, la ghréline et son récepteur sont des cibles
pharmacologiques de premier plan dans le domaine biomédical avec de potentielles applications
cliniques: traitement de la cachexie et de l'obésité, du diabète, de l’addiction à certaines
substances... Dans ce contexte, nous avons développé à travers différentes approches des séries de
ligands du GHS-R1a.
La première, une approche pseudo-peptidique développée à partir d’un tripeptide (EP 51389), nous a
permis d’identifier un puissant agoniste actif par voie orale chez l’homme. Ce composé (JMV 1843) a
passé les phases cliniques et devrait bientôt être commercialisé pour le diagnostic de déficience en
GH.
O
H2N
N
H
H
N
NH
O
NH
NH
O
NH2
O
H 2N
N
H
H
N
O
H
N
CHO
O
NH
Peptide
EP 51389
O
NH
Pseudo-peptide
JMV 1843
H2 N
N
H
N
N N
Peptido-mimétique
JMV 2959
Nous avons également développé une approche peptido-mimétique basée sur le motif triazole 1,2,4trisubstitués. Des tests pharmacologiques effectués sur ces composés hétérocycliques nous ont
permis d’identifier un ligand particulièrement prometteur : le JMV 2959. Une de ses caractéristiques
est de posséder une sélectivité à la fois fonctionnelle (in vitro) et à la fois physiologique (in vivo).
Effectivement, in vitro, il présente un caractère agoniste partiel sur Gq et un caractère antagoniste
sur Gi, Go et l’arrestine. In vivo, il agit sélectivement sur les différents processus biologiques
contrôlés par la ghréline : il inhibe la prise alimentaire et la dépendance aux drogues et à l'alcool
mais reste sans effet sur la sécrétion GH induite par l’hexaréline. Cet aspect de sélectivité
fonctionnelle observé sur certains de nos ligands apparait donc comme un résultat extrêmement
encourageant, puisqu’il fournit des informations primordiales pour la conception de nouvelles
molécules thérapeutiques possédant des effets secondaires limités.
78
O43 Synthèse d'inhibiteurs sélectifs et réversibles de iNOS : chimie sur
support, biologie et modélisation
Thibault Tintillier,1 Nicolas Floquet,1 Anne-Dominique Lajoix,2 Jérémy Leroy,2 Jean-Luc
Boucher,3 Jean Martinez1 et Jean-François Hernandez1
1
Institut des Biomolécules Max Mousseron, UMR 5247, CNRS, Université de Montpellier, Faculté de Pharmacie,
2
15 avenue Charles Flahault, 34093, Montpellier, France; Centre de Pharmacologie & Innovation dans le
3
Diabète, EA7288, Université de Montpellier, Faculté de Pharmacie, Montpellier, France; Laboratoire de Chimie
et Biochimie Pharmacologiques et Toxicologiques, UMR 8601, CNRS, Université Paris Descartes, 45 rue des
Saints Pères, 75006, Paris, France.
Les oxydes nitriques synthases (NOS) sont des enzymes qui catalysent la conversion de la L-arginine
en L-citrulline avec formation d'oxyde nitrique (NO). Chez l'homme, les NOS existent sous trois
isoformes différentes : la NOS neuronale (nNOS) et la NOS endothéliale (eNOS) sont calciumdépendantes et sont impliquées respectivement dans la transmission neuronale et dans la régulation
de fonctions vasculaires ; la NOS inductible (iNOS), produite par les macrophages, est impliquée dans
la réponse immunitaire innée. Cependant, une surproduction de NO par la iNOS est impliquée dans
de nombreuses maladies à caractère inflammatoire telles que les maladies neurodégénératives,
l'arthrite, le diabète, … L'inhibition de cette enzyme apparait donc comme une approche
intéressante, mais elle doit être sélective pour préserver les fonctions des deux autres NOS.
Cependant, il n’existe actuellement aucun inhibiteur de iNOS utile cliniquement, principalement
parce que la très haute conservation des résidus du site actif pour les trois isoformes rend la
découverte d'inhibiteurs sélectifs très difficile.
Notre but est de synthétiser de tels inhibiteurs en alliant synthèse, tests biologiques et modélisation
moléculaire. Ce poster présente les résultats obtenus dans les trois domaines.
Les composés développés sont constitués de : i) un analogue de substrat interagissant au niveau du
site actif, auquel est ajoutée sur sa fonction carboxylique ii) une extension pouvant interagir au
niveau du canal d’accès du substrat où existent des différences entre les 3 isoformes. Ces deux
parties sont jointes par un lien qui peut être de type amide ou hétérocyclique. Une stratégie sur
support solide a été développée au laboratoire afin de synthétiser ces molécules et a permis de
synthétiser environ 200 molécules à ce jour. Deux inhibiteurs intéressants ont été obtenus pour la
iNOS et la nNOS et font actuellement l’objet de modifications pour tenter d’augmenter l’affinité et la
sélectivité.
L’activité des molécules est ensuite mesurée sur les trois enzymes recombinantes puis l’IC50 est
calculée pour les ligands les plus intéressants. De plus, des tests ont été mis en place sur des cellules
RAW 264.7 (macrophages) et INS-1 (cellules β-pancréatiques) afin d’appréhender la capacité de nos
molécules à traverser la membrane.
Enfin, nous développons un protocole de docking en nous basant sur les structures
cristallographiques de NOS seules ou en complexe avec divers inhibiteurs, présentes dans la Protein
Data Bank (PDB). Nous avons d’abord comparé des logiciels de docking (VINA, PLANTS et DOCK) puis
optimisé celui qui fournissait les meilleurs résultats (PLANTS). Nous avons ensuite appliqué ce modèle
aux inhibiteurs co-cristallisés qui se rapprochaient le plus de nos ligands. Un pourcentage de bonne
prédiction suffisant (65 %) a été obtenu. Nous discuterons aussi de la manière d’améliorer ce modèle,
notamment en s’intéressant à la question des molécules d’eau. A terme, nous voulons créer un
modèle capable de prédire la pose et l’affinité du ligand, modèle qui nous aiderait dans la conception
de nouvelles molécules.
79
80
PRESENTATIONS
D’INDUSTRIELS
I1 – I10
81
82
Liberty BlueTM et HE-SPPS: Rapidité et efficacité
Nicolas Raynal1 et Jon Collins2
1
2
CEM µWaves, 4 Rue René Razel 91400 Saclay, France; CEM Corp, Matthews NC, USA.
La synthèse peptidique en phase solide (SPPS) développée par Merryfield1 a connu une première
révolution en 2006 avec le développement par CEM du premier synthétiseur de peptide assisté par
micro-ondes, le Liberty. La communauté scientifique a vite compris les avantages d’une telle
innovation. Fort de cette expérience et dans un souci d’innovation et d’excellence, CEM a conçu un
nouveau synthétiseur de peptide sous champs micro-onde le Liberty Blue™ et optimisé la chimie. La
Synthèse Peptidique en Phase Solide à Haute Efficacité (HE-SPPS)2 ainsi développée par CEM permet
de minimiser les temps de couplage et de déprotection sous micro-ondes pour arriver à un cycle
total de 4 minutes (déprotection, lavages, couplage) avec des rendements accrus en travaillant à
90°C. En parallèle, CEM participe au développement d’une nouvelle résine Speritide Resin™ qui offre
des bénéfices pour réduire les agrégations intra et intermoléculaires. Cette résine qui possède une
structure à base de poly-lysine apporte des nombreux avantages pour un grand nombre de peptides.
La convergence des ces innovations permet entre autres, des résultats très intéressants pour
l’incorporation des Arginines en s’affranchissant de la formation intramoléculaire de δ-lactame.
CEM propose aussi un passeur automatique de résine 12 ou 24 positions pour le Liberty Blue afin de
réaliser des synthèses de manière séquentielle. Deux autres appareils viennent compléter la gamme,
un dispositif manuel, le Discover Bio SPS et le Liberty XL avec un réacteur de 3 litres pour une montée
en échelle. CEM demeure présent dans la chimie sous micro-onde avec le Discover SP et le Mars 6
synthèse.
1. Merrifield R. B. (1963) J. Am. Chem. Soc. 85, 2149-2154
2. Collins J. M. et al. (2014) Org. Lett. 16, 940
83
La microcalorimétrie renforce le portfolio de Malvern pour caractériser les
protéines
Aymeric Audfray et Michel Terray
Malvern Instruments, 30 rue Jean Rostand, 91893 Orsay cedex France.
En 2014 Malvern Instruments a racheté la société MicroCal à GE Healthcare et est à présent
responsable de vendre, installer et dépanner les systèmes de micro ITC (Titration Calorimétrique
Isotherme) et micro DSC (Calorimétrie Différentielle à Balayage). Malvern a aussi innové dans ce
domaine en lançant en Janvier 2015 un nouvel ITC nommé PEAQ, acronyme de Performance et de
Qualité.
Ce nouveau calorimètre de titration isotherme présente une belle alternative à la SPR lorsque l’on
préfère travailler en solution. Il est équipé d’une nouvelle station de lavage, de seringues incassables
et connectables sans risquer de les endommager et d’un hardware permettant d’améliorer le
rapport signal sur bruit et donc le niveau de détection. Le logiciel est assisté par des tutoriels sous
forme de vidéos pour les utilisateurs occasionnels, tandis que l'analyse de données permet aux
experts d’interpréter des séries de résultats en toute confiance avec des diagnostics en ligne et des
représentations intuitives de l’entropie, de l’enthalpie et de l’énergie d’interaction assurant ainsi une
plus grande pertinence des paramètres thermodynamiques et d'affinité. Le PEAQ permet un criblage
des Leads et une validation plus rapide des Hits. Cet appareil manuel peut être automatisé
ultérieurement et transformé en PEAQ-ITC Automatisé. Son nouveau concept d’automatisation très
robuste autorise l’utilisation dans les laboratoires, mais également dans des unités de production
industrielles de médicaments à base de petites molécules où l'identification des cibles
thérapeutiques est effectuée à grande échelle.
Malvern est également très impliquée dans de nouvelles technologies à destination des Biosciences
avec 5 nouveaux appareils qui ont déjà été présentés sous forme de poster ou de communications
orales, lors des éditions précédentes du GFPP :
Viscosizer mesure avec seulement quelques microlitres, la viscosité et la taille de peptides en
solution jusqu’à 2 angström dans des milieux complexes par dispersion de Taylor.
Morphologi G3 ID combine l’analyse d’images et la spectroscopie Raman pour classifier les plus gros
agrégats de protéines et identifier les corps étrangers.
Archimedes utilise la mesure de masse par résonnance (RMM) mise au point par le MIT, pour
caractériser les agrégats protéiques dans les vaccins.
Helix, combine quant à lui diffusion de la lumière et spectroscopie Raman pour identifier les liaisons
chimiques impliquées dans les interactions entre protéines ou lors de leur dénaturation lors de
rampe de température.
Et enfin depuis le rachat de la société Nanosight en 2013, la technologie NTA (Nanoparticle Tracking
Analysis) permet de compter et de caractériser la taille des nanoparticules dans des milieux
complexes, en utilisant si nécessaire différents filtres adaptés à des marqueurs fluorescents et
permet de jongler entre 4 longueurs d’onde d’excitation différentes.
Votre contact :
Michel Terray
Tel. 06 30 23 45 50
[email protected]
84
La purification par chromatographie liquide préparative : un savoir-faire
Gilson historique renforcé depuis l’acquisition de la société Armen
Caroline Delmotte,1 Luke Roenneburg,2 Grégoire Audo3 et Céline Le Quémener3
1
2
3
Gilson International France SAS, Villiers-le-Bel, France; Gilson Inc., Middleton (WI), Etats Unis; Armen Instrument SAS,
Saint Avé, France.
Le Dr Warren Gilson crée la société portant son nom en 1952. En 1960, il développe le premier
collecteur de fractions grand volume à détection de niveau. Depuis lors, la gamme s’étoffe en
systèmes de pompage et de détection pour permettre l’automatisation de la purification par
chromatographie liquide dans les laboratoires. La philosophie et le savoir-faire de Gilson se sont
toujours focalisés sur la modularité des systèmes proposés afin de pouvoir répondre au mieux aux
besoins des utilisateurs. Pour renforcer son expertise dans le domaine de la purification, Gilson Inc.
fait l’acquisition de la société française Armen SAS en 2013. Les deux sociétés ont certains points
communs : elles développent elles-mêmes leurs produits dans leur bureau d’étude et les fabriquent
dans leurs propres usines. Aujourd’hui, elles mettent leurs atouts en commun. Le catalogue des
produits dédiés à la purification permet d’apporter des solutions allant du milligramme aux quantités
pilote, des solutions compactes individualisées aux plateformes multi-service, couvre les marchés
tels que la chimie thérapeutique, les produits naturels, les peptides et protéines, etc… Gilson a aussi
acquis le savoir-faire Armen dans le domaine de la CPC (Centrifugal Partition Chromatography), cette
technique de séparation peut être une bonne alternative à la chromatographie « classique » lorsque
celle-ci ne donne pas de bons résultats en pureté et/ou rendement, ou tout simplement pour réduire
les coûts de production ou être plus écologique.
Cette présentation montrera quelques exemples concrets de solutions personnalisées permettant
d'améliorer les rendements de purification par HPLC et autres techniques...
85
Présentation de PROTEOGENIX
Philippe Sommer
ProteoGenix, Schiltigheim, France.
ProteoGenix is a leading life sciences company providing solutions and services in molecular biology
and immunology. Our customers have been witnessing our proven expertise in the fields of
antibodies, proteins, peptides and genes since 2003. Our commitment is to give them access to
comprehensive and integrative solutions to develop the tools they need to efficiently conduct their
research work.
Our broad skills in all the complementary service disciplines we are focusing on help us to better
design production strategies including all the requirements and specifications of the different
disciplines involved.
We also provide the Life sciences community with a wide range of commercial products available on
our website such as antibodies, proteins, ELISA kits, density gradient medium, molecular biology
products, 2100 retriever (IHC unmasking tool), etc.
Besides, we research and develop innovative solutions to improve our production processes.
Votre contact :
Philippe Sommer
Tel. 06 66 09 23 75
[email protected]
86
Acheter un lyophilisateur n’est pas lyophiliser !
Antoine Babin
Directeur de Biopharma Technologies France, Lyon, France.
Nombres de projets de lyophilisations auxquels nous sommes confrontés démontrent une approche
purement quantitative au moment de concrétiser l’achat d’un lyophilisateur. Certes, les appels
d’offres spécifient souvent le volume à traiter, le type de produit (protéine, plantes, vaccin…), le ou
les solvants, la masse sèche finale. Mais, de ces informations nous ne sommes généralement en
mesure que de calibrer une taille de lyophilisateur généralement exprimée en surface d’étagère.
L’idée ensuite qui consisterait à penser, qu’une fois installé, l’utilisation est réduite à la simple
programmation d’une recette standard est par contre source de nombreuses frustrations car de fait,
acheter un lyophilisateur n’est pas lyophiliser !
N’oublions jamais que le but est de lyophiliser et non d’acheter un lyophilisateur ; à ce titre, le
support applicatif en amont et aval de l’achat du lyophilisateur est infiniment plus important que
l’achat du lyophilisateur en soit.
Quid des températures critiques du produit, est-il amorphe ou cristallin, quelle est la température
d’effondrement. Ma capacité de glace/24hr sera-t-elle suffisante par rapport au profil de
dégagement de vapeur de mon produit ? Dois-je utiliser des thermocouples ou des PT100 ? Porte
acrylique/verre/inox ?... Les questions à se poser à minima afin de faire le bon choix sont avant tout
qualitatives ! Et pas seulement sur des critères de constructions ou de matériaux. Même les
applications qui peuvent sembler les plus simples cachent de potentielles mauvaises surprises :
combien d’utilisateurs ont acheté un lyophilisateur en -50C pour sécher des légumes sur la base d’un
contenu aqueux pour s’apercevoir rapidement qu’ils ne réussissent pas à lyophiliser des fruits avec la
même machine ! Le fructose abaissant radicalement le point de transition vitreuse ! Combien
d’utilisateurs ont acheté un lyophilisateur à température de congélation unique de -40C pour ensuite
s’apercevoir qu’un « annealing » est absolument nécessaire !
Forte de ce constat et soucieuse de proposer ses lyophilisateurs via une plateforme experte dans le
domaine applicatif; la société SP scientific, fabricant des lyophilisateurs Virtis, FTS et Hull a confié la
distribution de ses lyophilisateurs en France à la société Biopharma via une nouvelle entité
Biopharma Technologies France (BTF) depuis le 1er août 2014.
Ma présentation tentera d’apporter quelques éléments à considérer pour les personnes ayant soient
des problèmes sur leur processus en cours soient un projet d’achat de lyophilisateur.
87
PolyPeptide Group - The Ultimate Peptide Partner
Aurore Fieschi
Polypeptide Laboratories France, 7 rue de Boulogne, 67100 Strasbourg.
The PolyPeptide Group is a privately-held group of manufacturing companies which focus on
proprietary and generic GMP-grade peptides for the pharmaceutical and biotechnological market.
With more than 60 years of experience, the Group is committed to the highest quality of peptide
manufacturing, irrespective of whether this is for approved drug substances, GMP peptides in clinical
trials, or small-scale non-GMP custom syntheses.
Since its inception the PolyPeptide Group has grown by selective acquisition of existing expertise,
culminating in its position today as the undisputed number two peptide manufacturing organization
in the world. The Group has manufacturing facilities in Denmark (Hillerød), Sweden (Malmo), France
(Strasbourg), India (Ambernath) and two sites in the USA (San Diego CA & Torrance CA). As a
multinational company with about 470 employees worldwide, its diversity brings breadth and depth
of knowledge and experience to the Group.
The Group's long-established core strength in GMP manufacturing and broad range of services
supports peptide & peptide-like projects, including conjugation to non-peptide moieties, from the
bench through to commercialization. With continually increasing capacity for GMP manufacturing,
the PolyPeptide Group is stronger and better equipped to serve the needs of its customers at all
stages of pharmaceutical peptide development. With its multinational organization, strict focus on
peptides and solid financial base, the Group offers an almost unique security of supply to its
customers.
88
Solutions pour les analyses de protéines, peptides en spectrométrie de masse
Luc Arnaud
Agilent Technologies, 91978 Les Ulis, France.
Agilent Technologies est un leader mondial pour les analyses en Sciences de la Vie et Analyses
Chimiques. Les technologies en spectrométrie de masse couplée haute résolution Tof ou QTof à la
chromatographie Liquide Ultra Haute Performance (UHPLC) permettront d’identifier ou de
confirmer l’identification de peptides naturels ou peptides synthétisés mais aussi de pouvoir les
quantifier avec un grand niveau de sensibilité.
Outre la spectrométrie de masse, Agilent Technologies propose des systèmes de Chromatographie
Liquide Infinity 1200 Bio-inert qui permettent avec détection intégrée UV, Indice de réfraction (RI),
Viscosimètre et Détecteur à diffusion de lumière (ELSD) la caractérisation de macromolécules.
89
La synthèse de peptides, Systèmes et services
Alain Omasson1 et Benoit Sarazin2
1
2
Protéigène, St Marcel, France; Proteomic Solutions, St Marcel, France.
Proteigene distribue les synthétiseurs de peptides Intavis Respep SL, MultipepRSI et Multipep CF.
Faciles à utiliser et polyvalents, ces synthétiseurs de peptides sont particulièrement destinés aux
biologistes.
Différentes échelles de synthèse : de 1µmol à 2mmol (selon le système et le module choisi) :
Sur Résine :
- En colonne de 0,4 ml à 40 ml.
- En plaque de 1 à 4 plaques 96 puits.
Sur Membrane :
- Jusqu’à 2400 peptides sur 4 membranes selon la méthode Spot1 de Ronald Frank.
Ils automatisent les différentes étapes de la synthèse : Déprotection, coupling, capping ainsi que tous
les lavages.
Différentes options selon modèles : Agitation, Inertage, Chauffage, Monitoring UV en temps réel…
Notre laboratoire Proteomic Solutions propose en partenariat avec la société Intavis de réaliser la
synthèse de vos peptides pour vos études biologiques.
Ces peptides sont utilisables pour l'obtention d'anticorps ou pour les études de peptides actifs :
épitopes mapping, étude des protéases, recherche de site de phosphorylation de kinases ...
Nous pouvons :
- synthétiser de nombreux peptides en faible quantité, (set de 384 peptides : échelle de
synthèse 2 µmol, pureté d'environ 70 %),
- proposer des peptides arrays
(CelluspotsTM: peptides sur lame de microscope : jusqu'à 2 x 384 peptides par lame),
- générer des banques de peptides sur membrane (technologie Spot).
La technologie Celluspot est une alternative aux banques de peptides sur membranes : les peptides
sont fixés sur une lame de microscope.
Son avantage principal réside dans la possibilité d'avoir de nombreuses copies identiques de la même
synthèse ce qui permet de réaliser rapidement et en parallèle plusieurs études.
La forte densité de peptides sur la lame rend la possibilité de détecter des interactions de faible
affinité.
Aucun équipement sophistiqué n'est nécessaire pour visualiser les résultats : la détection peut se
faire par autoradiographie, par réaction enzymatique colorimétrique ou avec des sondes
fluorescentes.
1. Frank R. (1992) Tetrahedron 48, 9217–9232
www.proteigene.com ; www.proteomicsolutions.fr
90
La solution pour vos synthétiseurs automatisés et façonnage de peptides
Thierry Cascales
Directeur CHEMGO SARL, Représentant exclusif de CS Bio en Europe (en collaboration avec CHEMGO Organica
AG).
CS Bio, créé en 1993 en Californie, s’est d’abord positionné sur la production et la commercialisation
de synthétiseurs automatisés capables de reproduire des synthèses de peptides longues chaines du
mg au kg en phase solide, à travers une gamme complète d’outils : du CS-136 au CS-936. Grâce à une
conception robuste et accessible, la maintenance est aisée et rend l’exploitation très fiable. Les
protocoles d’automatisation ont eux aussi été conceptualisés afin de faciliter l’usage tout en
garantissant un fonctionnement c-GMP/US-FDA.
Afin d’assoir son leadership sur le marché des synthétiseurs et démontrer l’efficacité de ses produits,
CS Bio fut l’un des premiers à compléter son offre par la fabrication à façon de peptides du mg au kg.
C’est le meilleur moyen de garantir l’excellence de nos outils préalablement à votre investissement.
De plus ce service permet d’augmenter vos capacités de production lorsque vos équipes sont
saturées, tout en partageant notre expertise.
Quel que soit votre besoin dans le domaine des peptides, CS-Bio aura la solution.
CS Bio is a rapidly growing biotechnology company located in Menlo Park, CA. As the world of
biotechnology continues to utilize peptides in the fields of therapeutics, vaccines, drug discovery, and
bioengineering, CS Bio provides high quality clinical-grade compounds to these dynamic industries.
We provide a seamless supply of peptide drugs and APIs to support the growth of these programs.
With our new, state of the art, large scale cGMP manufacturing facility we can produce multi-kg
quantities for our clients. No matter your requirements, CS Bio will provide high quality service to
ensure your peptide program's best chance for success.
91
Présentation de JASCO France
Jean Philippe WENCKER
JASCO France, 17 Rue Gutenberg 44340 Bouguenais, France. Tél :02 40 43 00 43.
Spectroscopie des peptides et protéines.
JASCO est un fabricant de matériel de laboratoire en HPLC, UHPLC , SFC/SFE, FT-IR, UV/Visible/NIR,
Fluorescence, Polarimétrie, Spectropolarimètrie (CD), Dichroisme vibrationnel ( VCD), Raman et
Champ proche avec plus de 50 ans d’expérience et d’innovations au service de la recherche.
Nous sommes particulièrement présents dans le domaine de l’analyse de peptides et protéines par
dichroïsme circulaire, IRTF, UV/Vis et fluorescence.
Présentation :
Analyse de structures secondaires de peptides et protéines par spectroscopie CD et IR, VCD IR .
Nouveaux modèles et dernières avancées instrumentales ATR CD, HTCD.
Analyse micro volumes UV, Fluo , CD et nouvelles techniques d’échantillonnage
Votre contact :
Jean Philippe WENCKER
Tel. 06 08 93 14 65
[email protected]
92
COMMUNICATIONS
PAR AFFICHE
P1 – P48
93
94
Demonstration of the occurrence of an α-helix in the extracellular loop
2 of GPR103
Karima Alim,1 Laure Guilhaudis,2 Isabelle Ségalas-Milazzo,2 David Vaudry,1 Julien Chuquet,1
Nicolas Chartrel,1 Ronan Bureau,3 Hubert Vaudry,1 Jana Sopkova-de Oliveira Santos3 and
Jérôme Leprince1
1
INSERM U982, Laboratoire de Différenciation Neuronale et Neuroendocrine, Institut de Recherche et
d’Innovation Biomédicale de Haute Normandie (IRIB), Université de Rouen, 76821 Mont-Saint-Aignan, France;
2
3
UMR CNRS 6014 COBRA, IRIB, Université de Rouen, 76821 Mont-Saint-Aignan, France; CERMN, Université de
Caen Basse-Normandie, boulevard Becquerel, 14032 Caen, France.
The neuropeptide 26RFa, the latest member of the Arg-Phe-NH2 peptide family discovered in
vertebrates, and its cognate receptor GPR103 are involved in the control of food intake and bone
mineralization.1 26RFa also stimulates the gonadotrope axis, regulates glucose homeostasis,
increases locomotor activity, and induces analgesic effects.1 Elucidating the structures of G proteincoupled receptors (GPCRs) and characterizing the mechanisms controlling ligand/receptor binding
are required for rational drug design. Among the GPCRs whose structures were solved at the
beginning of this study, GPR103 exhibits the highest sequence homology with the β2-adrenergic
receptor. Starting from the X-ray structure of this template, we have recently built a 3-D molecular
homology model of human GPR103.2 As expected, the GPR103 model presents a short α-helix (Ile188Glu197) in the second extracellular loop, ECL2 (Gln184-Gln211) in very much the same as in the β2adrenergic receptor. Here, we have experimentally investigated the occurrence of this putative αhelix in GPR103 ECL2.
Prediction of secondary structure and determination of helical propensity per residue for various
length GPR103 fragments encompassing ECL2 (Leu166-Phe223; Trp170-Ile213; Leu171-Lys212; Met180-Ile199
and Met180-Lys212) were performed using the Agadir program.3 A helical tendency was detected for all
the segments from residue Pro179 to Glu197 depending on the length of the fragment. Taking into
account the correlation between the position of the α-helix in the GPR103 3-D model and the Agadir
prediction, we have selected the Met180-Ile199 segment (MWHVQQLEIKYDFLYEKEHI) for far-UV
circular dichroism (CD) spectroscopy in phosphate buffer (10 mM, pH 6), containing trifluoroethanol
(TFE) (0%, 20%, 30%, and 50%) or dodecylphosphocholine (DPC, 20 mM). The CD spectrum obtained
in phosphate buffer was characterized by the presence of a negative band around 195 nm and a
small positive signal upper to 218 nm, indicating a mainly unordered peptide. In contrast, CD spectra
recorded in the presence of 20%, 30% and 50% TFE exhibited patterns characteristic of a mixture of
an α-helical and random conformations with a positive band at 190 nm, a negative band around 205
nm and second negative band close to 218 nm. Although the segment was poorly soluble in DPC
micelles, CD spectra recorded in this medium exhibited several features suggesting the presence of a
structure similar to the one observed in the presence of TFE. Our data from CD spectroscopy confirm
the occurrence of an α-helix in GPR103 ECL2 as previously suggested in the 3-D homology model of
the receptor and predicted by the Agadir algorithm.
Since the short extracellular ECL2 α-helix in the GPR103 model displays an amphiphilic feature like
that of 26RFa,2 we propose the setting of a helix-helix interaction between the ECL2 and the
endogenous ligand which could be investigated by surface plasmon resonance. This surface
interaction may be involved in the selectivity process of 26RFa for GPR103.
1. Leprince J. et al. (2013) Handbook of Biologically Active Peptides. 917-923
2. Neveu C. et al. (2014) Br. J. Pharmacology. 171, 4425-4439
3. Lacroix E. et al. (1998) J. Mol. Biol. 284, 173-191
95
Rational design of triazolo-lipopeptide analogues of kisspeptin inducing
a long-lasting increase of gonadotrophins
Vincent Aucagne,1 Mathieu Galibert,1 Jean-Baptiste Madinier,1 Philippe Marceau,1 Vincent
Robert,2 Hugues Dardente,2 Didier Lomet,2 Agnès F. Delmas,1 Alain Caraty2 and
Massimiliano Beltramo2
1
UMR Physiologie de la Reproduction et des Comportements (INRA, UMR85 ; CNRS, UMR7247, Université
2
François Rabelais Tours, IFCE), Nouzilly, France; Centre de Biophysique Moléculaire (CNRS UPR 4301), Orléans,
France.
The reduced activity of the hypothalamus-pituitary system leading to insufficient secretion of
gonadotropins (luteinizing hormone (LH) and follicule-stimulating hormone (FSH)) is the main cause
of severe dysfunctions of the reproductive system. It has recently been shown that deficiency in the
neuropeptide kisspeptin (KP)-kisspeptin receptor (KISS1R, also called GPR54) system leads to
reduced LH and FSH plasma concentration and infertility.1,2 Corroborating this finding, injection of
the endogenous 10 amino acid-long form of KP (KP10) produces a rapid, albeit transient, increase of
LH and FSH in several mammals including human.3-5 This makes kisspeptin system an appealing target
for treating human reproductive system dysfunction as well as to cope with problems in
reproduction management of livestock. We previously showed that continuous administration of
KP10 can induce ovulation in anoestrus ewe.4 This result was a breakthrough unveiling essential
mechanisms controlling reproduction. However, the short lasting effect of KP blunts its usefulness to
treat a deficient reproductive system. Despite the major importance of the KP system as a
pharmacological target, surprisingly few synthetic KISS1R agonists have been disclosed so far. Takeda
pharmaceutical generated KISS1R agonists by designing KP10 pseudopeptide analogs chiefly to treat
hormone-dependent cancers.6,7 The design of these analogs was aimed at preventing proteasemediated degradation,8,9 that together with fast renal excretion are the main factors accounting for
KP timely restricted action. However, to our knowledge no KP analogs have been evaluated in vivo
for their capacity to restore reproductive system activity either in human or in seasonal breeding
livestock. We report here the design of new, potent and selective KISS1R agonists based on a
combination of rational chemical modifications of KP10.10,11 Improved resistance to degradation and
reduced renal clearance were obtained by introducing a 1,4-disubstitued 1,2,3-triazole as a
proteolysis-resistant amide mimic and a serum albumin-binding motif, respectively. These triazololipopeptides are highly potent full agonist of KISS1R and are selective over the closely related NPFF1
receptor. When injected in ewes with a quiescent reproductive system, they induced a much
prolonged increase of LH release compared to KP10 and also an augmentation of FSH plasma
concentration. Hence, these KISS1R agonists are new valuable pharmacological tools to explore the
potential of KP system in reproduction control. Furthermore they represent the first step to develop
drugs treating reproductive system disorder due to a reduced activity of the hypothalamus-pituitarygonad axis such as delayed puberty, hypothalamic amenorrhea, and hypogonadotropic
hypogonadism.
1. de Roux N. et al. (2003) Proc. Natl. Acad. Sci. USA 100, 10972-10976
2. Seminara S. B. et al. (2003) New Engl. J. Med. 349, 1614-1627.
3. Navarro V. M. et al. (2005) Endocrinology 146, 156–163
4. Caraty A. et al. (2005) Endocrinology 148, 5258–5267
5. Chan Y. M. et al. (2011) Clin. Endocr. Metab. 96, E908–E915
6. Matsui H. et al. (2012) Endocrinology 153, 5297–5308
7. Scott G. et al. (2013) J. Clin. Pharmacol. 75, 381–391
8. Asami T. et al. (2012) Bioorg. Med. Chem. Lett. 22, 6391–6396
9. Curtis A. E. et al. (2010) Am. J. Physiol. Endocrinol. Metab. 298, E296-E303
10. Beltramo M. et al. (2014) patent WO2014118318
11. Beltramo M. et al. (2015) submitted manuscript
96
The neuroprotective effect of the ODN and the PACAP in experimental
Parkinson disease model
Seyma Bahdoudi,1,2 David Vaudry,2,3 Hadhemi Kaddour,1 Yosra Hamdi,1 Salma Douiri,1
Jérôme Leprince,2,3 Marie-Christine Tonon,2 Mohamed Amri1 and Olfa Masmoudi-Kouki1
1
Laboratory of Functional Neurophysiology and Pathology, 00/UR/08-01, Department of Biological Sciences,
2
Faculty of Science of Tunis, University Tunis El Manar, 2092 Tunis, Tunisia; Inserm U982, Laboratory of
Neuronal and Neuroendocrine Communication and Differentiation, University of Rouen, 76821 Mont-Saint3
Aignan, France; Regional Platform for Cell Imaging of Haute-Normandie (PRIMACEN), IRIB, University of Rouen,
76821 Mont-Saint-Aignan, France.
The pituitary adenylate cyclase-activating polypeptide (PACAP) exerts potent neuroprotective effects
in models of neurodegenerative diseases, traumatic brain injury and stroke. Besides its direct
neuroprotective action, PACAP may also act indirectly on astrocytes to stimulate the release of
neurotrophic factors that prevent neuronal cell death. We have previously shown that PACAP
stimulates the biosynthesis and the release of endozepines, a family of biologically active peptides
that are exclusively produced by astroglial cells. Since the endozepine octadecaneuropeptide (ODN)
is a neurotrophic factor regulating proliferation and/or survival of neurons, we have investigated the
ability of PACAP alone or in association with ODN to counteract the neurotoxic effects of 6hydroxydopamine (6-OHDA) on cerebellar granule neurons (CGN). Incubation of cultured CGN with
6-hydroxidopamine (6-OHDA) provoked a biphasic mirror effect on neuronal survival measured by
FDA and LDH release after 72 h of treatment. Co-incubation of CGN with 6-OHDA and graded
concentrations of PACAP (10-11M - 10-6 M) for 72 prevented in a dose-dependent manner the
deleterious effect of 6-OHDA on CGN. At very low concentrations (10-14 M), ODN prevented the
deleterious effect of 6-OHDA on CGN survival and suppressed the stimulatory effect of the toxin.
Kinetic studies revealed that PACAP inhibited 6-OHDA-evoked cell apoptosis within 24 h with a
maximal effect occurring 72 h after the onset of the treatment. Thereafter, the neuroprotective
action of PACAP gradually declined and vanished after 96 h. Addition of the gliopeptide ODN in the
culture medium prolonged the neuroprotective effect of PACAP up to at least 120 h, suggesting that
PACAP and ODN act through complementary mechanisms.
We next investigated the signaling cascade involved in the neuroprotective action of ODN and PACAP
on CGN. The PKC inhibitor chelerythrine (10-7 M) only abrogated the neuroprotective action of ODN
against 6-OHDA-evoked cell death. In contrast, the selective PKA inhibitor H89 did not modify the
effect of ODN on 6-OHDA-induced cell death but totally blocked the effect of PACAP, confirming that
PACAP and ODN use additive signaling pathways in the regulation of neuronal survival.
Cells treated with 6-OHDA exhibited a high level of reactive oxygen species (ROS) and a stimulation
of caspase-3 activity. These effects of 6-OHDA were blocked by PACAP and co-administration of
PACAP and ODN resulted in a synergistic up-regulation of the inhibitory effect of PACAP on 6-OHDAevoked caspase-3 stimulation and antioxidant enzymes expression.
Supported by research Unit UR11ES09, INSERM U982, PeReNE, a France-Tunisia exchange programs InsermDGRST and Erasmus Mendus Battuta Program.
97
Implication des peptides exprimés par les cellules de Purkinje au cours
de l'histogenèse du cervelet chez la souris
Magali Basille-Dugay,1 Hélène Blanchon,1,2 Isabelle Tournier,3 David Vaudry1,2 et Delphine
Burel1
1
2
Laboratoire DC2N, INSERM U982, IRIB, Normandie Université, Mont-Saint-Aignan, France; PRIMACEN, Mont3
Saint-Aignan, France; INSERM U1079, IRIB, Normandie Université, Cancéropôle Nord-Ouest, Rouen, France.
L’ensemble des études portant sur l’histogenèse du cortex cérébelleux démontre une
communication bidirectionnelle étroite entre les cellules de Purkinje et les cellules en grain lors de la
mise en place des couches cérébelleuses. Cependant, la plupart des acteurs de cette interaction sont
encore méconnus. Nous avons donc entrepris d’étudier le transcriptome des cellules de Purkinje
chez la souris entre la naissance et l’âge adulte afin de déterminer les peptides/protéines exprimés et
sécrétés transitoirement par ces cellules qui pourraient être impliqués dans le contrôle de la
prolifération, migration et/ou différenciation des cellules en grain. Dans un premier temps, nous
avons optimisé le prélèvement tissulaire des cellules de Purkinje par microdissection laser en
évaluant le nombre de cellules nécessaire à l’amplification des ARNm. Puis, nous avons réalisé un
panel de gènes afin de déterminer les peptides/protéines régulés dans la couche des cellules de
Purkinje et de rechercher les récepteurs de ces facteurs dans les précurseurs des cellules en grain de
la couche granulaire externe. L’étude de l’expression des différents gènes en fonction du stade de
développement est actuellement en cours par PCR quantitative et permettra de s’assurer que le
système ligand/récepteur est présent simultanément dans le cortex cérébelleux à un instant donné.
A titre d'exemple, nos résultats préliminaires ont déjà démontré que les ARNm du pituitary
adenylate cyclase activating polypeptide, de la cérébelline, de la somatostatine et de leurs
récepteurs sont fortement exprimés aux stades P10 et P12 respectivement dans la couche de cellules
de Purkinje et dans la couche granulaire externe, alors qu’ils sont peu ou non présents dans le
cervelet aux stades P21 et adulte.
Par ailleurs, afin d’avoir une vue d’ensemble des facteurs intervenant dans la communication entre
les cellules en grain et les cellules de Purkinje, nous amorçons actuellement une étude de
séquençage à très haut débit pour définir le transcriptome global des cellules de Purkinje pendant les
différentes étapes de l’histogenèse du cortex cérébelleux. Les peptides exprimés transitoirement
pendant les deux premières semaines postnatales seront ensuite sélectionnés pour rechercher leurs
possibles effets in vitro, ex vivo et in vivo sur la prolifération, la migration et la différenciation des
cellules en grain. Un protocole de chimiotactisme entre les cellules de Purkinje et les cellules en grain
est également en cours de développement ce qui nous permettra, par la suite, d'obtenir un modèle
d'étude in vitro de communication cellulaire et de mieux appréhender les mécanismes cellulaires mis
en jeu au cours de l’histogenèse du cortex cérébelleux.
Travail soutenu par le projet Interreg PeReNE et la région Haute-Normandie.
98
Structural studies of peptidomimetic ligands of TRAIL complexed to
pro-apoptotic death receptors
Antoine Baudin,1 Gavin Collie,2 Cameron Mackereth,2 Gilles Guichard2 and Benoît Odaert1
1
2
Institut de Chimie et Biologie des Membranes et Nanoobjets, Pessac, France; Institut Européen de Chimie et
Biologie, Pessac, France.
Apoptosis, the programmed death of cells, plays a protective role against tumor formation. This
phenomenon is either regulated by the intrinsic pathway or by the extrinsic pathway. The latter is
stimulated through the activation of cell-surface death receptors (DR) by Tumor necrosis factorRelated Apoptosis Inducing Ligand, or TRAIL. The ligand binding leads to the trimerization of the
receptors, which is a necessary step for the apoptosis answer. This TRAIL ligand has the interesting
particularity to bind only the DR4 and DR5 receptors of tumor cells. This unique property is at the
center of several therapeutic assays for cancer cell targeting.
The aim of this study is to use a structural approach in order to find new peptidomimetic ligands of
TRAIL, or TRAILmim/DR5, that can enhance activation of the extrinsic pathway of apoptosis. Several of
these different peptides have been produced through solid-phase synthetic chemistry in monomeric,
dimeric or trimeric states, and have been shown to selectively bind to DR5 [1]. Some of these
peptides have been studied with NMR spectroscopy and show a β-hairpin motif [2]. We plan to use
two-dimensional and three-dimensional NMR spectroscopy to study the molecular details of the
interaction between these new ligands and Death Receptor 5. We have already begun the
production of 15N-labelled of the protein in minimal medium, and we plan to assign the backbone
using a 13C-15N double-labelling strategy. The other part of this project is to crystallize the complex
formed by the receptor and either the monomeric, dimeric or trimeric peptides, then to use X-ray
diffraction on the crystals obtained, in order to get atomic scale information. These data should help
us to have a better understanding of extrinsic apoptosis, and thus designate a model candidate to
trigger the TRAIL apoptosis pathway for cancer therapy.
1. Pavet V. et al. (2010) Cancer Res. 70, 1101-1110
2. Pulka-Ziach K. et al. (2013) ChemBioChem DOI: 10.1002/cbic.200
99
Novel cyclization reactions in bacteriocin biosynthesis
Alhosna Benjdia,1,2 Alain Guillot,1,2 Benjamin Lefranc,3,4 Hubert Vaudry,3,4 Jérôme
Leprince3,4 and Olivier Berteau1,2
1
2
INRA, ChemSyBio, Institut Micalis (UMR 1319), 78350 Jouy-en-Josas, France; AgroParisTech, ChemSyBio,
3
Institut Micalis (UMR), F-78350 Jouy-en-Josas, France; INSERM U982, 76821 Mont-Saint-Aignan France;
4
Institute for Research and Innovation in Biomedicine (IRIB), Regional Platform for Cell Imaging, PRIMACEN,
University of Rouen, 76821 Mont-Saint-Aignan, France.
Genomic and metagenomic investigations have recently led to delineate a novel class of natural
products called: ribosomally-synthesized and post-translationally-modified peptides (RiPPs).1 RiPPs
are ubiquitous among living organisms and include pharmaceutically relevant compounds such as
antibiotics, anti-cancer agents or toxins. Nevertheless, their biosynthetic pathways are poorly
understood and many RiPPs such as Thiostrepton A,2 Polytheonamides3 or Bottromycin,4 were
believed, until very recently, to be non-ribosomal peptides or polyketides rather than being of
ribosomal origin.
Among distinct features, some RiPPs possess unusual thioether bonds which formation has been
recently shown to be catalyzed by novel radical SAM enzymes and likely to result from the radical
activation of a peptidyl Cα−atom.5 We have undertaken the detailed investigation of AlbA, a key
enzyme in the biosynthesis of Subtilosin A, a higly cyclized antibiotic produced by Bacillus subtilis6,7
and containing three unusual thioether bonds. Combining high-resolution mass spectrometry, kinetic
and labeling experiments, we establish, in opposite to current paradigm, that the activity of AlbA is
peptide-leader independent and we propose a novel mechanism for the formation of thioether bond
in biosynthetic peptides.
2. Kelly W. L. et al. (2009) J. Am. Chem. Soc. 131, 4327
3. Freeman M. F. et al. (2012) Science 338, 387
4. Huo L. et al. (2012) Chem. Biol. 19, 1278
5. Flühe L. K. et al. (2012) Nat. Chem. Biol. 8, 350
6. Babasaki K. et al. (1985) J. Biochem. 98, 585
7. Zheng G. et al. (1999) J. Bacteriol. 181, 7346
100
Study of the foldameric potential of ATCs γ-amino acids included into
heterogeneous oligoamide sequences
Clément Bonnel, Baptiste Legrand, Jean Martinez, Nicolas Masurier and Ludovic T.
Maillard
Institut des Biomolécules Max Mousseron, CNRS UM1-UM2 UMR5247, Faculté de Pharmacie, 15 Av. Charles
Flahault, 34093, Montpellier, France.
The inhibition of protein-protein interactions represents an attractive strategy to develop biomedical
applications. In this context, the rational design of peptidic or non-peptidic oligomers mimicking
protein secondary structures (α-helix, β-sheet and turns), named foldamers, constitutes a promising
approach. Foldamers are defined as "any oligomer that folds into a conformationally ordered state in
solution, the structures of wich are stabilized by a collection of noncovalent interactions between
nonadjacent monomer units".1 The key advantages of such non-natural architectures are their
enhanced structural and proteolytic stability.2 Thus, we recently described a new class of constrained
heterocyclic γ-amino acids named ATCs (1) built around a thiazole ring. According to its restricted
conformational freedom, ATC oligomers adopt predictable helical secondary structures stabilized by
C9-intramolecular hydrogen bonding network.3
In continuation of this work, we synthesized heterogeneous backbone oligomers alternating both αor β-amino acids with ATCs building blocks. The variations in the patterns of the subunit
combinations, but also in the ATC stereochemistry to generate heterochiral platforms, may allow us
to access subsequent molecular shapes diversifying the spatial distribution of the side chains. The
NMR structural studies of homo- and heterochiral sequences are reported.
1. Appella D. H. et al. (1996) J. Am. Chem. Soc. 118, 13071-13072; Hill D. J. et al. (2001) Chem. Rev. 101, 3893-4011
2. Koyack M. & Cheng R. (2007) In Biological Applications of Foldamers. In Foldamers: Structure, Properties and
Applications, Hecht S. & Huc I., Wiley-VCH, Weinheim, pp 229-265
3. Mathieu L. et al. (2013) Angew. Chem. Int. Ed. Engl. 52, 6006-6010
101
Novel contrast agents for in vivo molecular imaging of matrix
metalloproteinase-12 (MMP-12)
Thomas Bordenave,1 Yupeng Ye,2 Mahmoud Razavian,2 Laurent Devel,1 Mehran Sadeghi2
and Vincent Dive1
1
CEA (Commissariat à l'Energie Atomique), iBiTec-S, Service d'Ingénierie Moléculaire de Protéines (SIMOPRO),
2
CE Saclay 91191 Gif/Yvette, Cedex, France; Cardiovascular Molecular Imaging Laboratory, Veterans Affairs
Medical Center, 950 Campbell Avenue, 06516 West Haven, CT, USA.
Matrix Metallo proteinases (MMPs) are zinc-dependent endo peptidases that form a family of 24
members in mammals. These proteases are overexpressed during tissue remodeling and their
uncontrolled activity is often associated to inflammatory disorders.1 Within this family of enzyme,
MMP-12 or macrophage metallo elastase is mainly secreted by macrophages in inflammatory
conditions. Under its active form, this protease plays a central role in various pathologies including
lung inflammation and different cardiovascular diseases such as atherosclerosis and aneurysm. More
particularly, MMP-12 is directly involved in the development and rupture of atheroma plaques and
was shown to be a prognostic indicator of the pathology advancement.2 In this context, MMP-12 can
be considered as a biomarker of monocyte-macrophage activation and consequently targeting
activated MMP-12 in vivo would allow imaging vessel wall inflammation and remodelling in various
inflammatory pathologies.
RXP470.1 a phosphinic pseudo peptide as a selective and potent inhibitor of MMP-123 demonstrated
its ability to target active form of MMP-12 in vivo. Thus, in a mouse model of atherosclerosis, this
inhibitor proved its efficiency to block atheroma plaques development and rupture4. More recently,
this compound was shown to be able to block MMP-12 activity in a mouse model of rheumatoid
arthritis5 or during viral infection6. In this respect, we consider that RXP470.1 may serve as a valuable
starting point for the development of specific tracers to image and monitor MMP 12 activity in vivo.
Based on the 3D-structure of RXP470.1 in interaction with MMP-12 catalytic domain we recently
developed contrast agents harbouring a selective ligand toward MMP-12, a polyethylene glycol
spacer and various reporter groups according to the imaging modalities desired.
In this series, the synthesis of several original tracers will be described. Spacer length, nature of
reporter groups and global net charge of the tracers may impact the tracers ability to selectively
interact with MMP-12 in complex media. Influence of such parameter will be then commented.
Tracer kinetics and bio distribution will be also presented. Finally, through competition experiments
and MMP-12 gene invalidation, their ability to specifically interact with their prime target will be
addressed in several mouse models of inflammation.
1. Vandenbroucke R. E. et al. (2014) Nat. Rev. Drug. Discov. 13, 905-927
2. Sholtes V. P. et al. (2012) J. Am. Heart. Assoc. 6, 1-12
3. Matusiak N. et al. (2013) Curr Pharm Des. 19, 4647-4672
6. Lim N. H. et al. (2014) Arthritis and Rheumatology. 66, 589-598
7. Marchant D. J. et al. (2014) Nature Med. 20, 493-502
102
Synthesis of a library of protected dipeptides featuring
enantiomerically pure cyclobutane β-amino acids: a key role for
photochemistry
France Boyaud, Florian Hernvann, Elisa Parades, Sandra Muñoz Piña, Thomas Boddaert,
Valérie Declerck and David J. Aitken
CP3A Organic Synthesis Group, ICMMO, Université Paris-Sud, Orsay, France.
Interest in cyclic β-amino acids has increased in the past few years, and these molecules have
become valuable, restricted-conformation building blocks in synthetic and medicinal chemistry, with
applications ranging from peptide chemistry through heterocyclic chemistry to combinatorial
chemistry.1 Our group has developed a particular interest in cyclobutane β-amino acids and the
parallel development of photochemical methodologies in order to access these non-canonical amino
acids.2
In the context of a collaborarative programme we recently required the establishment of a viable,
gram-scale synthesis of a series of dipeptides of general structure Fmoc-Arg(Pbf)-Xaa-OH, where Xaa
is a stereochemically defined cyclobutane β-amino acid bearing an appropriately protected
substitutent on the ring. These peptides are required to be amenable for use in SPPS.
In this communication we will present the photochemical synthetic strategies developed and applied
for the preparation of a library of appropriate cyclobutane β-amino acid derivatives in
sterechemically pure form3, and hence the preparation of the corresponding dipeptide series. Among
the substituents considered are hydroxy- and hydroxymethyl- groups at the C3 and C4 positions of
the cyclobutane, which are conceived as mimetics for the alcohol-bearing canonical α-amino acids
Thr and Ser.4
h
h
Access to cyclobutane β-amino acids by photochemical strategies and preparation of target
dipeptides
1. a) Fülöp F. et al. (2006) Chem. Soc. Rev.36, 323-334; b) Kiss & Fülöp F. (2014) Chem. Rev. 114, 1116-1169
2. Declerck V. et al. (2011) Amino Acids 41, 587-595
3. Hassoun A. et al. (2015) New J. Chem DOI: 10.1039/c4nj01929f
4. Hernvann F. et al (2014) J. Org. Biomol. Chem. 12, 8212-8222
103
Glycopeptides modulate Aβ1-42 oligomerization; structure-activity
and in vitro evaluations
Dimitri Brinet,1,2 Julia Kaffy,1 Jean-Louis Soulier,1 Katia Fabiana Fera,1 Chiara Bernardi,1
Olivier Lequin,3 Anaïs Hoffmann,3 Isabelle Correia,3 Lucie Khemtémourian,3 Benoit
Crousse,1 Myriam Taverna2 and Sandrine Ongeri1
1
Molécules Fluorées et Chimie Médicinale, BioCIS UMR-CNRS 8076, LabEx LERMIT, Université Paris-Sud, Faculté
2
de Pharmacie, 5 rue Jean-Baptiste Clément, 92296, Châtenay-Malabry Cedex, France, Pays; Protéines et
nanotechnologies en sciences séparatives (LPNSS), Institut Galien, UMR 8612 CNRS, Université Paris-Sud,
3
Faculté de Pharmacie, 5 rue Jean-Baptiste Clément, 92296, Châtenay-Malabry Cedex, France; UPMC
Laboratoire des Biomolécules, UMR 7203 CNRS-UPMC-ENS, 4 place Jussieu, 75252 Paris Cedex 05, France.
The aggregation of amyloid-β (Aβ) peptides is a major player in Alzheimer’s disease (AD). The result
of this aggregation is the formation of morphologically distinct species, in particular oligomers of
different size, protofibrils and fibrils. It appears that soluble and small oligomeric forms of Aβ1-42 are
primarily responsible for the neurotoxicity associated with AD.1 We described previously a novel
series of glycopeptides able to inhibit the aggregation of Aβ1-40.2 We report here the
pharmacomodulations and the relationship studies on this series of glycopeptides3 as well as the
replacement of a peptide sequence by a peptidomimetic unit.4 We also report here the capacity of
the glycopeptides and glycopeptidomimetics to modulate the aggregation of the more neurotoxic
Aβ1-42, by Thioflavin-T fluorescence and transmission electronic microscopy. With the aim to resolve
the small oligomeric states and to allow the monitoring of this dynamic process in solution, we
developed an in vitro model based on capillary electrophoresis (CE) to evaluate the synthesized
compounds.5 Indeed, only very few techniques to monitor the neurotoxic oligomers are described in
the literature. The separation improvement by CE allowed to monitor also intermediate species
which were discriminated from the small oligomeric or the large and soluble oligomeric ones.5 Taylor
diffusion and SDS-PAGE were employed to estimate the average hydrodynamic radius and the weight
of the separated species. CE was used to establish that the glycopeptides and glycopeptidomimetic
have a dramatic effect on the early state species of the oligomerization process.3,4 The overall results
suggest also that CE is a innovative physicochemical technique that allows a reliable evaluation of the
effect of potential drugs on the most neurotoxic species of Aβ1-42, and that should contribute to
accelerate the development of novel small Aβ kinetics modulator with potential application in AD.
1. Dahlgren K. N. et al. (2002) J. Biol. Chem. 277, 32046-32053; b) D. B. Teplow (2009) Proc. Natl. Acad. Sci. U.S.A. 106,
14745-14750
2. Dorgeret B. et al. (2011) Eur. J. Med. Chem. 46, 5959-5969
3. Kaffy J. et al. (2014) Eur. J. Med. Chem. 86, 752-758
4. Results to be published.
5. Brinet D. et al. (2014) Electrophoresis 35, 3302–3309
104
Etudes des propriétés de translocation des homéoprotéines et des
peptides pénétrants
Sébastien Cardon, Ludovic Carlier, Laura Molina, Alain Joliot, Fabienne Burlina, Olivier
Lequin et Sandrine Sagan
Laboratoire des biomolécules, UMR7203 UPMC-CNRS-ENS, Paris, France.
Les homéoproteines sont des facteurs de transcription à homéodomaine qui ont pour particularité
d'être secrétés puis internalisés par les cellules eucaryotes1, agissant ainsi comme des messagers
cellulaires de type paracrine2. Le mécanisme d'internalisation de ces homéoprotéines n'est pas
encore connu et constitue l'un des objectifs de ce travail.
Au niveau moléculaire, la troisième hélice de l’homéodomaine de ces protéines, composée de 16
acides aminés représente la séquence minimale d'internalisation et a permis l'identification, il y a
une vingtaine d'années, du premier peptide vecteur (Cell-penetrating peptide, CPP), la pénétratine.
Cette hélice est la partie la plus conservée des homéodomaines et présente, en particulier des
acides aminés comme le tryptophane ou l'arginine, cruciaux pour l'internalisation4.
La première étape d'internalisation correspond au recrutement des homéoprotéines par des
composants de la membrane, tels les glycosaminoglycanes5 (GAG) qui pourraient être à l'origine de
l'initiation du processus d'internalisation avant que la protéine puisse interagir avec la membrane
lipidique. Actuellement il existe en effet deux mécanismes plausibles d’internalisation qui sont
l’endocytose et la translocation directe. Le processus de translocation est une perturbation de la
membrane lipidique dans lequel le potentiel de la membrane pourrait servir de force motrice pour
l'entrée de ces molécules6 dans le compartiment intracellulaire. En revanche, la voie de l'endocytose
implique que la protéine se retrouve dans un premier temps piégée dans des vésicules à l'intérieur
des cellules. Quel que soit le mécanisme d'entrée, une fois internalisées les homéoproteines sont
entièrement actives afin d'effectuer leur activité de transcription ce qui tend à démontrer que la
translocation n'est pas associée à des modifications irréversibles et que la protéine atteint le noyau
des cellules.
Les premières étapes de ce travail ont consisté à produire et purifier l'homéoprotéine entière
Engrailed 2 ainsi que deux fragments de la protéine, l'homéodomaine, et l'homéodomaine étendu en
N-terminal pour englober un motif de localisation nucléaire (NLS). Après purification, les structures
secondaires adoptées par ces protéines, libres en solution, ou en interaction avec des modèles de
membranes ou des carbohydrates ont été déterminées par dichroïsme circulaire. En parallèle la
capacité d'internalisation de ces trois séquences a été étudiée et quantifiée par spectrométrie de
masse sur des cellules présentant ou non des GAG à leur surface.
1. Prochiantz A & Joliot A. (2003) Nat. Rev. Mol. Cell. Biol. 4, 814-819
2. Spatazza J et al. (2013) Pharmacol. Rev. 65, 90-104
3. Derossi D et al. (1994) J. Biol. Chem. 269, 10444-10450
4. Tassetto M et al. (2005) EMBO Rep. 6, 885-890
5. Jiao C. Y. et al. (2009) J. Biol. Chem. 284, 33957-33965
6. Rothbard J. B. et al. (2005) Adv. Drug. Deliv. Rev. 57, 495-504
105
SEA SPPS : Protein total synthesis by sequential solid phase
chemoselective peptide ligation
Marine Cargoët, Rémi Desmet, Annick Blanpain and Oleg Melnyk
UMR CNRS 8161, Université de Lille, Institut Pasteur de Lille, 59021, Lille, France.
Numerous methodologies have been developed for the chemical synthesis of peptides or small
proteins. The solid phase peptide synthesis (SPPS) introduced by Merrifield in the 1960s has
simplified considerably the access to synthetic peptides.1 Among other advantages, the use of a solid
support facilitates the intermediate purifications, which are realized by simple washing procedures,
and the automation of the process. Automation increases the rapidity and the reproducibility of
peptide synthesis and provides an efficient method to access to short peptides (< 50 amino acids).
The aim to access to larger polypeptides has stimulated the development of chemoselective amide
bond forming reactions which allow the assembly of unprotected peptide segments in water. Among
these, the Native Chemical Ligation (NCL) introduced by Kent in the 1990s2 is certainly the most
popular chemoselective amide bond forming reaction. Several one-pot three peptide segments
sequential ligation methods relying on NCL reaction or related methodologies have been developed
during the last decade.3,4 These processes simplify the access to proteins composed of less than 150
amino acids, considering the actual limits of the SPPS technique. The routine assembly of more than
three peptide segments might extend considerably the actual limits of protein chemical synthesis.
Unfortunately, solution methods are faced with time-consuming intermediate isolation steps,
accompanied by significant material losses.
The implementation of NCL chemistry on a water compatible solid support is a potential solution to
this challenging problem. We have recently introduced a novel solid phase peptide segment
chemoselective elongation cycle5 which relies on the chemical properties of the bis(2sulfanylethyl)amido (SEA)6-12 group. The optimization of the different chemical steps involved in the
SEA SPPS process will be discussed in this poster.
2. Dawson P.E. et al. (1994) Science 266, 776-779
3. Raibaut L. et al. (2012) Chem. Soc. Rev. 41, 7001-7015
4. Ollivier N. et al. (2012) Angew. Chem. Int. Ed. 51, 209
5. Raibaut L. et al. (2013) Chem. Sci. 4, 4061-4066
7. Dheur J. et al. (2011) J. Org. Chem. 76, 3194-202
8. Raibaut L. et al. (2013) Bioorg. Med. Chem. 21, 3486-3494
9. Boll E. et al. (2014) Chem. Sci. 5, 2017-2022
10. Ollivier N. et al. (2014) J. Pept. Sci. 20, 92-97
11. Boll E. et al (2014) Nature Protocols 10.1038/nprot.2015.013
12. Boll E. et al. (2015) Org. Lett. 17, 130-133
106
Luminescent peptide probes for biological imaging of metal cations
Céline Cépeda,1 Manon Isaac,1 Jean-Marc Latour,1 Didier Boturyn2 and Olivier Sénèque1
1
2
iRTSV/LCBM, CEA Grenoble, 17 rue des Martyrs, 38 054 Grenoble Cedex, France; DCM/I2BM, Université Joseph
Fourier - Grenoble 1, UFR de Chimie, BP 53, 38 041 Grenoble cedex 9, France.
Metal ions are essential for life. Complex machineries at both the cellular and the whole organism
levels regulate finely their concentrations. A disruption of their homeostasis can lead to disorders
and serious diseases [1-4]. In order to better understand the biological processes involving these
metals, it is important to be able to detect, track and quantify in cellulo and in vivo.
We aim at developing luminescent probes for Zn2+ and Cu+ that can be used in cells or in small
animals. These probes are based on a peptide backbone inspired by metalloprotein metal binding
sites. Their peptidic nature makes them soluble and biocompatible. A lanthanide complex is
conjugated to the peptide to benefit from their fantastic luminescence properties (large Stokes shift,
fine lines and independent of the environment, long luminescence lifetime to overcome the intrinsic
fluorescence of biological environment).[5, 6, 7]
Up to now we have developed Cu+ and Zn2+ sensors operating in vitro. To improve further their
efficiency, we want to build ratiometric systems, for quantification purpose, and makes probes
penetrate cells. The former point can be achieved by the conjugation of a second emitting lanthanide
complex to the existing probes and the second by conjugation of CPP sequences. We will present our
strategy and first results on this project.
Figure : Cu+ bidding site of the metalloprotein CusF
1. Maret W. (2009) Biometals 22, 149-157
2. Fukada T. et al. (2011) J. Biol. Inorg. Chem. 16, 1123-1134
3. Franklin R. B. & Costello L. C. (2009) J. Cell. Biochem. 106, 750-757
4. Costello L. C. & Franklin R. B. (2012) Expert Rev. Anticancer Ther. 12, 121-128
5. Eliseeva S. V. & Bünzli J. C. G. (2010) Chem. Soc. Rev. 39, 189-227
6. Bünzli J. C. G. (2010) Chem. Rev. 110, 2729-2755
7. Thibon A. & Pierre V. C. (2009) Anal. Bioanal. Chem. 394, 107-120
107
Metalloproteins sustaining Direct Electron Transfer in Ionic Liquids
media: DET-structure relationship
Soizic Chevance,1 Corinne Lagrost,2 Gérard Simonneaux1 and Joëlle Rault-Berthelot2
1
2
ICMV, ISCR UMR CNRS 6226, Rennes, France ; MaCSE, ISCR UMR CNRS 6226, Rennes, France.
Electron transfer (ET) reactions involving metalloproteins play a fundamental role in the regulation of
biological processes as photosynthesis and cellular respiratory. The fine understanding of the
mechanisms of ET and their reproduction, in particular when an electrode replaces natural redox
partners, represent important stakes, both fundamental and applicative. An interesting particular
case that we develop in the laboratory is where the electrode becomes the only donor or an
acceptor of electrons of proteins (Direct Electron Transfer), without using any redox mediator. To
develop easily biodevices (biosensors, biofuel cells), an effective DET between the protein and the
electrode is required. Generally, the activity of proteins is studied in aqueous environment, which is
their "natural" environment. However, in terms of applications, water is not necessarily the most
relevant medium, the temperature domain of functioning of biodevices being limited to it as well as
the stability of proteins in time. In this context, ionic liquids (IL) constitute a very attractive
alternative. They were successfully used in the field of the biotechnologies, allowing increasing the
protein stability, even the enzymatic activity of certain proteins. In this context, our work aims at a
better fundamental understanding of the structure / redox activity relationship of metalloproteins in
media containing IL.
We investigated the DET of some model proteins at a bare glassy carbon electrode in an aqueous
ionic liquid mixture.1 We used the water-miscible 1-butyl-3-methyl imidazolium tetrafluoroborate
(BMI.BF4) as IL. Detailed analyses of the electrochemical response were carried out with cyclic
voltammetry and thin-layer cyclic voltammetry. We particularly focused on the transport properties,
the nature and the kinetics of the electron transfer, in order to evidence the influence of the ionic
liquid. We demonstrated that the electron transfer of Myoglobin or Hemoglobin is fast in the
aqueous ionic liquid medium without the need to build a sophisticated structured electrode. In
contrast, Cytochrome c doesn’t give any electrochemical signal under identical experimental
conditions. Several hypotheses could be made for explaining this striking difference: the orientation
of the protein towards the electrode (importance of the charges repartition at the protein surface),
accessibility of the electrochemical active center, protein conformational changes induced by IL, …
Aiming at testing this last hypothesis, we studied by different spectroscopic techniques (CD and
NMR) the conformational stability of Myoglobin and Cytochrome c in presence of increasing
concentration of 1-R-3-methyl imidazolium tetrafluoroborate (where R represents a ethyl, butyl or
hexyl group). Results obtained will be presented and discussed.
1. G. Loget et al. (2011) ChemPhysChem 12, 411-418
108
Sélénaproline: un "nouvel" acide aminé sélénié en synthèse
peptidique?
Emmanuelle Cordeau, Gilles Subra, Sonia Cantel, Jean Martinez et Christine Enjalbal
Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, Université Montpellier, France.
La sélénaproline (acide sélénazolidine-4-carboxylique; SCA) est un analogue de la proline comportant
un atome de sélénium en β de l'amine. La synthèse de cet acide aminé modifié est décrite pour la
première fois par De Marco en 19761 et repose sur la condensation de la sélénocystéine avec une
molécule de formaldéhyde.
La sélénaproline, en tant qu’aminoacide, a fait l'objet de quelques études. On peut citer par exemple
son activité antibactérienne vs Escherichia coli2 ou son role en tant que reservoir de sélénium,
micronutriment et antioxidant important3. Elle n'a, en revanche, jamais été incorporée dans un
peptide. Dans le cadre d’un projet de recherche visant à introduire différents hétéro éléments dans
des séquences peptidiques pour faciliter leur quantification en milieu complexe (ANR MTaQ), nous
nous sommes intéressés à l'incorporation de la sélénaproline dans des peptides modèles. La Fmoc- et
la Boc-sélénaproline ont été obtenues. Les conditions de couplage de ces analogues et leur
déprotection ont été étudiées. Une attention particulière a été portée à la stabilité de ce résidu lors
des diverses étapes des protocoles opératoires. A la lumière de ces résultats, les possibilités
d’incorporation de sélénaproline dans une séquence peptidique seront débattues que ce soit en
solution ou sur support solide, en stratégie Fmoc/tBu ou Boc/Bzl.
1. De Marco C. et al. (1977) Ital. J. Biochem. 26, 51
2. Deutch C. E. et al. (2014) J. Applied Microbiol. 117, 1487
3. Short M. D. et al. (2003) J. Med. Chem. 46, 3308
109
Asymmetric synthesis of fluoropseudopeptides towards constrained
prolines and analogue of neuropeptide 26RFa
Samuel Couve-Bonnaire,1 Camille Pierry,1 Emilie Villiers,1 Guillaume Dutheuil,1 Laure
Guilhaudis,1 Cindy Neveu,2 Amélie Marotte,1 Benjamin Lefranc,3 Dominique Cahard,1
Isabelle Ségalas-Milazzo,1 Jérôme Leprince2,3 and Xavier Pannecoucke 1
1
Normandie Univ., COBRA, UMR 6014 et FR 3038; Univ. Rouen; INSA Rouen; CNRS, 1 rue Tesnière, 76821 Mont
2
Saint-Aignan Cedex, France ; INSERM U982, IRIB, University of Rouen, 76821 Mont-Saint-Aignan, France;
3
PRIMACEN, IRIB, University of Rouen, 76821 Mont-Saint-Aignan, France.
Despite the growing interest in the synthesis of peptidomimetics (increased bioavailability, enzymatic
resistance), there are very few methods of preparation of the fluorinated pseudopeptides whereas
the fluoroolefin moiety can be used as an effective peptide bond mimic.1 Indeed, many studies
showed than the fluoroolefin moiety is both isosteric and isoelectronic to the amide function with,
moreover, better resistance over enzymatic degradation compared to the peptidic bond.
From many years, we have developed several asymmetric methodologies allowing us to synthesize
various fluoropseudodipeptides as depicted below.2
We have synthesized four fluorinated pseudodipeptides and used them in peptide synthesis to
design new fluorinated analogues of the 26RFa(20-26) heptapeptide. These fluorinated pseudopeptides
have been submitted to biological evaluation, enzymatic degradation and conformational analysis.
The results suggested that the fluoroolefin moiety can be employed as an effective mimic of the
peptide bond with great enhancement of the peptide stability.3
H
N
H2N
O
O
N
H
H
N
O
O
N
H
OH
H
N
O
26RFa(20-26) : Gly-Gly-Phe-Ser-Phe-Arg-Phe-NH 2
O
N
H
N
H
NH2
O
NH
NH 2
1. Couve-Bonnaire S. et al. (2007), Org. Biomol. Chem. 5, 1151
2. Dutheuil G. et al. (2007) Angew. Chem. Int. Ed. 46, 1290; b) Pierry C. et al. (2011) Org. Biomol. Chem. 9, 2378; c) Dutheuil
G. et al. (2013) New J. Chem. 37, 1320
3. Pierry C. et al. (2013) ChemBioChem 14, 1620
110
Spatiotemporal and reversible photocontrol of PDZ domain-mediated
interactions involved in the trafficking of synaptic glutamate receptors
Sara Crespillo,1 Isabel Gauthereau,1 Charlotte Rimbault,1 Dolors Grillo-Bosh,1 Christel
Poujol,2 Daniel Choquet1,2 and Matthieu Sainlos1
1
2
Interdisciplinary Institute of Neuroscience (IINS) UMR 5297, Bordeaux, France; Bordeaux Imaging Center (BIC),
Bordeaux, France.
The dynamic regulation of AMPA glutamate receptors (AMPARs) trafficking into and out of the
synapse is a major mechanism underlying synaptic plasticity, a fundamental basis of learning and
memory1,2. Recent studies have shown that the PDZ domain mediated multivalent interactions
between Stargazin, an intrinsic AMPAR auxiliary subunit, and PSD-95, one of the major multi domain
scaffold proteins of the postsynaptic density (MAGUK), constituted a dominant factor contributing to
AMPAR synaptic retention3,4. However, the detailed molecular mechanisms governing these
interactions and their role on the dynamics of AMPARs are still not fully understood, stressing the
need for new methods to modulate and monitor these specific protein-protein interactions (PPIs).
Our principal aim is to develop innovative approaches based on the use of light-sensitive groups to
reversibly control these interactions, in their complex cellular context. Photoactivatable competing
ligands have been designed within this project by following three different strategies: 1) taking
advantage of the oligomerization properties of coiled coils-based ligands incorporating a
photoisomerizable molecule (azobenzene group)5, 2) using naturally occurring photoreceptor
modules such as LOV domains or cryptochrome proteins6-8 and 3) using of a light-inducible
fluorescent protein (DRONPA)9. These non–invasive tools will allow us to increase the spatiotemporal
resolution of the perturbation to study the effect of glutamate receptors in synaptic regulation.
In an early stage, we have designed and produced control sequences to validate the general
approach and confirm their functionality. These control ligands incorporate the dimeric coiled-coil
motif (CCdi) without the light-sensitive unit, the natural photoreceptor constructs including
mutations maintaining a dimeric form or DRONPA in a monomeric form, and a PDZ domain-binding
motif (C-terminal sequence of Stargazin). Here we show the design and production of these ligands
as well as their biophysical characterization. We have studied their interaction with PSD-95 by SPR.
Moreover, we have characterized these control ligands with the Stargazin-PSD-95 interaction in
transfected eukaryotic cells (COS-7) as a model for a complex cellular system by FRET. Based on these
studies we have selected the best suitable construct to produce the photoactivatable ligands with
the final goal of studying the local effect of the perturbation of AMPAR-MAGUK complexes in
hippocampal neurons.
1. Shepherd J. D. & Huganir RL (2007) Annu. Rev. Cell. Dev. Biol. 23, 613-643
2. Derkach V. A. et al. (2007) Nat. Rev. Neurosci. 8, 101-113
3. Opazo P. et al. (2012) Curr. Opin. Neurobiol. 22, 453-460
4. Bats C. et al. (2007) Neuron 53, 719-734
5. Woolley G. A. et al. (2006) Biochemistry 45, 6075-6084
6. Grusch M. et al. (2014) EMBO J. 33, 1713-1726
7. Gautier A. et al. (2014) Nat. Chem. Biol. 10, 533-541
8. Baker A. S. & Deiters A. (2014) ACS Chem. Biol. 9, 1398-1407
9. Zhou X. X. et al. (2012) Science 338, 810-814
111
Expanding the solid phase chemical ligation approach to oxime and
triazole ligations
Agnès F. Delmas, Mathieu Galibert, Ibai E. Valverde, Isidore E. Decostaire, Dominique
Lelièvre, Philippe Marceau, Véronique Piller, Friedrich Piller and Vincent Aucagne
Centre de Biophysique Moléculaire, UPR 4301 CNRS, Rue Charles Sadron, 45071 Orléans cedex 2, France.
In the last two decades, the solid phase approach has been combined with successive chemical
ligation reactions to minimize the laborious chromatographic purification and handling steps
encountered during multiple chemoselective couplings. The potential of solid phase chemical ligation
(SPCL) was initially shown for native chemical ligation.[1] To extend the repertoire of chemical
ligation reactions operable on solid phase, we focused on the use of multiple successive triazole and
oxime ligations, and the protein MUC1 as a biologically relevant example.
As starting material, SPCL requires an unprotected segment grafted onto a water-compatible solid
support through a dedicated linker. By grafting this segment at its N-terminus through the use of the
base-labile N3-Esoc linker, the subsequent N-to-C elongation through triazole ligation benefits from a
self-purification effect.[2] A second generation azido linker cleavable under neutral conditions
enabled the synthesis of a glycosylated 160 mer analogue of the MUC1 glycoprotein.[3] The
exploitation of the solid support used for the SPCL to facilitate post-ligation transformations was
illustrated here by the enzymatic introduction of 24 GalNAc residues through enzymatic glycosylation
(Galibert et al., soumis).
Alternatively, using oxime ligation, we explored a C-to-N SPCL elongation to simplify the overall
synthetic scheme, which can be designed to exclusively include solid phase operations and a final
acid release.[3] The high purity of the resulting triazole- and oxime-containing polypeptides
highlights the efficiency of our approaches.
1. Canne L. E. et al. (1999) J. Am. Chem. Soc. 121, 8720; b) Brik A. et al. (2000) J. Org. Chem. 65, 3829 c) Cotton G. J. & Muir
T. W. (2000) Chem. Biol. 7, 253
2. Aucagne V. et al. (2012) Angew. Chem. Int. Ed. Engl. 51, 11320
3. Decostaire I. E. et al. (2014) Org. Biomol. Chem. 12, 5536–5543
112
Multifunctional nanoparticles for targeting atherosclerotic vulnerable
plaques
Laurent Devel,1 Boris Garnier,2 Marion Helle,1 Mylène Bernes,1 Robert Thai,1 Fabrice Beau,1
Elias Fattal,2 Sylviane Lesieur2 and Vincent Dive1
1
2
CEA, iBiTec-S, CE Saclay, 91191 Gif sur Yvette, Cedex, France; Institut Galien Paris-Sud, UMR CNRS 8612,
Université Paris-Sud 11, 5 rue J.B. Clément, 92296 Châtenay-Malabry Cedex, France.
Atherosclerosis is a chronic systemic inflammatory disease in which the arterial wall thickens as a
result of the accumulation of fatty material (atherosclerotic plaques) within the vessel. One of the
most severe complications arises from the plaque rupture in coronary arteries, which accounts for
the majority of fatal myocardial infarction, and in carotid arteries that causes ischemic stroke. In such
a context, the identification and treatment of rupture-prone plaques in patients before clinical
events occur would result in substantial health benefits.
Matrix Metallo Protease 12 (MMP-12), a zinc endopeptidase also termed macrophage metallo
elastase is involved both in atherosclerotic plaque growth by driving the macrophages recruitment
and in plaque rupture by weakening the fibrous cap [1,2]. In this context, our laboratory has
developed the first potent and highly selective inhibitors for MMP-12 as a phosphinic pseudo
peptide, the RXP470.1 [3]. In a mice model of atherosclerosis with established plaques, this inhibitor
was recently shown to fully block further plaques growth and prevent their rupture [4]. Remarkably,
these results reproduce those observed in MMP-12 knockout mice suggesting that this compound is
indeed able to target MMP-12 within the plaque. Based on these compelling results, we
hypothesized that MMP-12 as a biomarker as well as a therapeutic target could be exploited for
active targeting of the vulnerable plaque with nanoparticles decorated with RXP470.1 ligand with
imaging and therapeutic applications in perspective.
Here, we report the design of multifunctional liposomes combining imaging (Cy5.5), specific sitetargeting (RXP470.1 ligand) and drug delivery properties. Their controlled fabrication and their full
characterization will be presented. Their in vitro evaluation towards a set of MMPs as well as their
ability to selectively interact with MMP-12 will be discussed. Finally, influence of RXP470.1 surface
density on their in vivo properties (pharmacokinetics and bio distribution) will be presented.
1. Johnson J. L. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 15575-15580
2. Liang J. et al. (2006) Circulation 113, 1993-2001
113
Phéromones sexuelles de seiche (Sepia officinalis) : diversité des
produits d’expression
Maxime Endress,1,2 Jérémy Enault,1,2 Valérie Cornet,1,2 Céline Zatylny-Gaudin,1,2 Benoît
Bernay3 et Joël Henry1,2,3
1
2
Université de Caen Basse-Normandie, Caen, France; Université de Caen Basse-Normandie, UMR BOREA
3
MNHN, UPMC, UCBN, CNRS-7208, IRD-207, F-14032 Caen, France; Post Genomic platform PROTEOGEN,
Université de Caen Basse-Normandie, SF ICORE 4206, Caen, France.
Le mollusque céphalopode Sepia officinalis peut être considéré comme un modèle très pertinent
pour étudier les stratégies de reproduction associées aux migrations saisonnières. C’est pourquoi,
par la combinaison de multiples approches, transcriptomique, peptidomique et fonctionnelle, nous
cherchons à identifier les phéromones sexuelles peptidiques et polypeptidiques impliquées dans le
comportement des géniteurs en période de reproduction : agrégation dans les zones côtières de
ponte, accouplement et ponte.
Une première approche transcriptomique ciblée sur les transcrits majeurs des glandes annexes de
l’appareil génital femelle a permis d’identifier 3 transcrits très redondants que nous avons appelés
SPα, SPα’ et SPβ (SP pour Sex Pheromone) et dont une partie des produits de clivage a été identifiée
structuralement et fonctionnellement (Enault et al, 2012).
Une seconde approche transcriptomique réalisée en RNAseq de novo a permis d’identifier un 4ème
transcrit que nous avons nommé SPγ en raison de son homologie structurale avec SPα, SPα’ et SPβ.
Ces phéromones sont co-exprimées et co-sécrétées avec les protéines capsulaires par la glande de
l’oviducte et sont associées à la couche la plus interne de la future enveloppe de l’œuf. Les transcrits
SP ont une expression maximale chez les femelles à maturité sexuelle, strictement localisée dans la
glande de l’oviducte.
L’analyse peptidomique de la glande l’oviducte a permis de retrouver une partie des produits de
clivage prédits par la structure du précurseur et dont les masses moléculaires sont comprises entre 1
et 9 kDa. Par ailleurs, l’analyse protéomique de la glande de l’oviducte et de la capsule des œufs
révèle la présence de polypeptides de masse moléculaire élevée issus des précurseurs SP. Ces
résultats analytiques démontrent la cohabitation de deux modes de clivage au niveau des
précurseurs SP générant d’une part des peptides de relative faible masse moléculaire issus des
clivages de type prohormone convertase et d’autre part des polypeptides/protéines de masse
moléculaire voisine de 22kDa. L’analyse de l’eau de balnéation des œufs pondus depuis moins d’une
semaine révèle la présence des produits de masse moléculaire élevée dans le milieu démontrant
ainsi la capacité des œufs à libérer des phéromones polypeptidiques dans le milieu environnant.
La perfusion d’organes tels que le pénis, l’oviducte, les branchies avec de l’eau de balnéation des
œufs induit une modification importante de l’activité contractile. Ces tests devront être répétés avec
les protéines recombinantes pures. Les tests fonctionnels permettent de comprendre le
comportement des géniteurs en milieu naturel vis-à-vis des casiers à seiches utilisés au printemps
par les pêcheurs professionnels. En effet, plus la densité de pontes est importante sur l’armature du
casier, plus le casier est « pêchant », sans doute en raison de la quantité de phéromones libérées
dans le milieu environnant.
La production de ces phéromones en système recombinant permettra de réaliser des tests
comportementaux en aquarium de type « open space » et « labyrinthe Y » afin de vérifier les
fonctions supposées d’attraction et de stimulation de la ponte à l’origine des agrégations massives
de géniteurs sur les aires de ponte côtières normandes au printemps.
114
Molecular dynamics with excited normal modes reveals the role of the
activation loop of cyclin-dependent kinases in their open/closed
conformational equilibrium
Nicolas Floquet,1 Mauricio G.S. Costa,2 Paulo R. Batista,2 Pedro Renault,3 Paulo M. Bisch,3
Florent Raussin,1 Jean Martinez,1 May C. Morris1 and David Perahia4
1
Institut des Biomolécules Max Mousseron (IBMM), CNRS UMR 5247, Université de Montpellier, Ecole Normale
Supérieure de Chimie de Montpellier, Faculté de Pharmacie, 15 Av. Charles Flahault, BP 14491, 34093
2
Montpellier, Cedex 05, France; Programa de Computação Científica, Fundação Oswaldo Cruz, 21949900, Rio de
3
Janeiro, Brazil; Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21949-901,
4
Rio de Janeiro, Brazil; Laboratoire de Biologie et de Pharmacologie Appliquée, Ecole Normale Supérieure de
Cachan, Centre National de la Recherche Scientifique, 61, F-94235 Cachan, France.
Cyclin-dependent kinases are central for timely regulation of cell cycle progression and constitute
attractive pharmacological targets. Characterization of the structural and dynamic features of these
kinases is essential to gain in-depth insight into their structure-activity relationships. Structural
studies of CDK2/Cyclin A have yielded a wealth of information concerning the conformations of the
monomeric and heterodimeric forms of this kinase. There is however much less structural
information available for other CDK/Cyclin complexes, including CDK4/Cyclin D complexes. In this
study, we performed Normal Modes Analyses on CDK2/Cyclin A and CDK4/Cyclin D1 X-ray structures,
which display a different position of the Cyclin partner relative to the CDK, CDK2/Cyclin A being in a
“closed” conformation in contrast to the “open” conformation of CDK4/Cyclin D1. Interestingly, we
observed that the lowest frequency NM computed for each of these complexes described the
transition between the « open » and the « closed » conformations. Exploration of this motion with
an explicit representation of the solvent using the Molecular Dynamics with Excited Normal Modes
(MDeNM) method confirmed that the closed conformation was the most stable for the CDK2/Cyclin
A complex, in agreement with all available structures in the PDB. On the contrary, we clearly show
that an open ↔ closed equilibrium may exist in CDK4/Cyclin D1, with closed conformations
resembling that captured for CDK2/Cyclin A. Using the same approach, the putative roles of both the
phosphoryl group on Thr160 and of the conformation of the T-loop on this conformational
equilibrium were investigated. These results provide, for the first time, a dynamic view of Cyclindependent kinases and reveal the existence of intermediate conformations which have not yet been
characterized for CDK members other than CDK2, and which will be useful for the design of
inhibitors, targeting critical conformational transitions.
115
Stabilization of an α-helix by short adjacent accessory foldamers
Juliette Fremaux,1,2 Laura Mauran,1,2 Karolina Pulka-Ziach,1 Brice Kauffmann,3 Benoit
Odaert4 and Gilles Guichard1
1
Université de Bordeaux, CNRS, Institut Polytechnique de Bordeaux, UMR 5248 CBMN, Institut Européen de
2
Chimie et Biologie, 2 rue Robert Escarpit, 33607 Pessac, France; UREKA Sarl, 2 rue Robert Escarpit, 33607
3
Pessac, France; Université de Bordeaux, CNRS, UMS 3033, INSERM US001, Institut Européen de Chimie et de
4
Biologie, 2 rue Robert Escarpit, 33607 Pessac, France; Université de Bordeaux, CNRS, Institut Polytechnique de
Bordeaux, UMR 5248 CBMN, All. Geoffroy Saint-Hilaire, 33607 Pessac, France.
α-Helices often play the key role of recognition elements between proteins. In recent years, much
attention has been paid to the potential of foldamers as α-helix mimetics to target protein-protein
interactions (PPI) [1]. However, one difficulty to the use of foldamers for mimicking protein surfaces
resides in reproducing the spatial arrangement of the side chains found in the cognate α-helix. One
solution could be to fuse foldamer and peptide backbones in one molecule to preserve a portion of
the original α-helical segment and fully optimize contacts with the protein recognition surface. In
addition, the possibility for the foldamer backbone to exert a dual effect by also nucleating an αhelical structure (short synthetic peptides are barely helical) in the contiguous α-peptide segment is
appealing. Despite the potential interest, there are relatively few examples of such αpeptide/foldamer chimeras in the literature [2].
In this work, the ability of helical oligourea foldamers to nucleate α-helices when fused to short αpeptide segments has been explored. A variety of chimeric oligomers obtained by joining aliphatic
oligoureas either to the C- or N-terminus of peptides of different chain-lengths have been designed
and their conformation investigated. NMR spectroscopy and X-ray diffraction studies indicate that
short oligourea/peptide chimeras can adopt well-defined helical structures with a continuous
intramolecular H-bond network spanning the entire sequence and connecting two geometrically
distinct helices, (i.e. an α- (or 310) helix in the peptide segment and a canonical 2.5-helix in the
oligourea segment [3, 4]). Our results also point to the remarkable capacity of short tri-urea segments
to nucleate the formation of an α-helical conformation in the fused peptide segment.
These results suggest a general approach to stabilize and mimic peptide and protein helices and pave
the way for exploration and future development of oligourea/peptide chimeras as modulators of
protein-protein interactions
1. Estieu-Gonnet K. & Guichard G., (2011) Expert Opin Drug Discov. 6, 937-963
2. Sadowsky J. D. et al. (2005) J. Am. Chem. Soc. 127, 11966-11968
3. Fischer L. et al. (2010) Org. Biomol. Chem. 8, 3101-3117
4. Fischer L. et al. (2010) Angew. Chem. Int. Ed. 49, 1067-1070
116
Design of new RGD-containing NPs for imaging and drug targeted
delivery
Adrien Grassin1, Damien Duret2, Arnaud Favier2, Jean-Luc Coll3, Muriel Jourdan1, Sabrina
Valetti4, Didier Desmaële4, Simona Mura4, Pascal Dumy5, Patrick Couvreur4 and Didier
Boturyn1
1
2
Département de Chimie Moléculaire, UMR CNRS 5250, Université de Grenoble, Grenoble ; Laboratoire
d’Ingénierie des Matériaux Polymères, UMR CNRS 5223, INSA de Lyon | Laboratoire Joliot-Curie, USR CNRS
3
4
3010, Lyon ; Inserm U823, Institut Albert Bonniot, Université de Grenoble, Grenoble ; CNRS UMR 8612, Institut
5
Galien Paris-Sud, Châtenay-Malabry ; Institut des Biomolécules Max Mousseron, Montpellier.
Over the last decade, our laboratory has designed non-viral peptidic vectors that are able to
specifically target tumor microenvironment through the recognition of an overexpressed
transmembrane receptor, the αvβ3 integrin.1 These vectors are based on a cyclic decapeptide
scaffold that presents in a spatially controlled manner two independent functional domains: i) a
domain encompassing clustered RGD ligands for the integrin recognition and cell targeting, ii) a
separate domain that may contain a label (fluorophore, radiotracer, biotin), and/or a cytotoxic
element (drugs, radioelements), and/or an anchoring system for imaging, and/or therapy
applications. The benefit of these compounds encompassing a near-infrared fluorophore was
demonstrated for the optical guided surgery of tumors in cats.2 Moreover, a palmitoylated
tetrameric RGD containing scaffold was recently exploited to design liposomes that were used for
selective gene transfection in cancer cells.3
During our continuing work in developing new imaging and drug delivery systems, we decided to
design new biocompatible nanoparticles (NPs) that contain RGD compounds. Herein, we will describe
the preparation of new NPs and the preliminary biological experiments. We first studied the
influence of the linker length between the peptidic scaffold and the RGD ligands for cell recognition.
For this purpose, biological and molecular dynamic experiments were carried out in parallel. The
results indicate that slight changes in length affect the cell binding. We then chose to design two
different NPs by using either (1) multifunctional polymers encompassing several copies of RGD
clusters and NIR fluorophores with a controlled structure and architecture designed for imaging
applications, or (2) squalenoylated tetrameric RGD containing scaffold synthesized and exploited for
the preparation of NPs for therapeutic application.
1. Boturyn D. et al. (2004) J. Am. Chem. Soc. 126, 5730-5739
2. Wenk C. H. F. et al. (2013) Cancer Lett. 334, 188-195
3. Misra S. K. et al. (2014) J. Mat. Chem. B 2, 5758-5767
117
Comparative structural analyses of urotensinergic peptides by CD
spectroscopy, NMR and molecular modelling
Laure Guilhaudis,1 Riham Najjar,1 Nicolas Perzo,2 Hélène Castel2 and Isabelle SégalasMilazzo1
1
UMR 6014 CNRS COBRA, Institut de Recherche et d’Innovation Biomédicale de Haute Normandie (IRIB),
2
Université de Rouen, 76821 Mont-Saint-Aignan, France; INSERM U982, Laboratoire de Différenciation
Neuronale et Neuroendocrine (DC2N), Institut de Recherche et d’Innovation Biomédicale de Haute Normandie
(IRIB), Université de Rouen, 76821 Mont-Saint-Aignan, France.
UII (11 amino acids, aa) and its paralog URP (8 aa) are considered as the most potent vasoactive
molecules known so far. Both peptides are endogenous ligands of a 7-transmembrane GPCR, the UT
receptor. Little is known regarding the potential differential physiological roles of UII and URP. It was
shown that UII and URP might exert common but also divergent physiological actions in rat
astrocytes.1,2 Both peptides were able to reduce myocardial injury through creatine kinase but only
UII was able to reduce atrial natriuretic peptide (ANP) production3 and distinct pathophysiological
roles for UII and URP in hypertension have been suggested.4 More recently, UII unlike URP, has been
shown to stimulate invasion of macrophages and vascular smooth muscle cells in mice-transplanted
matrigel sponges and to promote in vitro angiogenesis (submitted).
These functional differential effects of UII and URP toward UT activation could be due to differences
in their 3D structures, but further experimental data are needed to confirm this point. NMR studies
of several UT ligands were carried out in different solvents and showed several discrepancies related
to secondary structure. Flohr et al. reported that, in aqueous solution, at ambient temperature
(T=300K), the cyclic region of human UII (hUII) is well defined but do not contain a canonical turn5,
while Lescot et al. showed that, at lower temperature (T=280K), hUII is structured in two successive
inverse γ-turns centered on W7-K8-Y9 and K8-Y9-C10 residues, respectively.6 We showed that URP
contains an inverse γ-turn centered on W4-K5-Y6 residues in water.7 In a SDS micellar medium, a
truncated form of hUII, hUII4-11, corresponding to the minimal biological active sequence of 8 aa,
contains a type II’ β-turn between F6 and Y9 residues.8
Such differences suggest that the structure of UII and URP may depend on the solvent and raise the
question of the bioactive structure in close proximity with membranes and GPCRs. In order to
confirm this hypothesis, we performed systematic structural analyses of hUII, hUII4-11 and URP using
CD spectroscopy in aqueous solution (phosphate buffer) and in the presence of micelles (SDS and
DPC). The obtained CD spectra indicated that the structure of all peptides was medium-dependent
and suggested differences in their secondary structures. We, thus, decided to compare their 3D
solution structures in a common medium and performed our study in the presence of DPC micelles, a
medium often used to mimic the eukaryotic cell membrane in NMR studies. First results indicate that
the encountered secondary structure elements in this medium are different from the previous
studies, confirming the environmental importance in the conformational state of the peptides and
suggesting an adaptability of urotensinergic peptides that could explain their differential
physiological roles.
1. Jarry M. et al. (2010) Biochem. J. 428, 113-124
2. Desrues L. et al. (2012) PLoS One 7, e36319
3. Prosser H. C. et al. (2008) Peptides 29, 770-777
4. Hirose T. et al. (2009) Peptides 30, 1124-1129
5. Flohr S. et al. (2002) J. Med. Chem. 45, 1799-1805
6. Lescot E. et al. (2007) J. Chem. Inf. Model 47, 602-612
7. Chatenet D. et al. (2004) Peptides 25, 1819-1830
8. Carotenuto A. et al. (2004) J. Med. Chem. 47, 1652-1661
118
Different experimental approaches to analyse the anti-proliferative
effect of chemical compounds on solid tumors
Florence Jouan1, Cathy Le Goff1, Michelle Baudy-Floc’h2, Franck Camerel3 and Yannick
Arlot1
1
Institut Génétique et Développement de Rennes (IGDR), UMR CNRS 6290, University of Rennes 1, Rennes,
2
France; Ciblage et auto-Assemblages Fonctionnels (ICMV), UMR CNRS 6226, University of Rennes 1, Rennes,
3
France ; Institut des Sciences Chimiques de Rennes (ISCR), UMR CNRS 6226, University of Rennes 1, Rennes,
France.
Tumor growth is associated with cell proliferation and vascularization. Tumor angiogenesis is
recognised as an essential hallmark for tumor growth, invasion and metastasis. In the case of kidney
cancer, the clear cell renal cell carcinoma (ccRCC) is highly vascularised and represents the most
important subtype with about 80% of renal carcinoma. The advent of molecular targeted therapy in
the last few years has changed treatment of patients with metastatic ccRCC with benefits in terms of
overall survival. The main therapy consist in treating the patients with anti-angiogenic drugs but
some patients become resistant. New strategies need to be investigated to find more efficient
treatment and to predict drug response in cancer patients.
In this study, we developed a set of experiments to study the effect of chemical compounds alone or
in association with others signal transduction inhibitors in order to characterize the anti-angiogenic
and anti-proliferative effects.
119
From unfolded peptides to cell-penetrating α-amino γ-lactam ribbons
Baptiste Legrand,1 Vincent Martin,1 Lubomir Vezenkov,1 Jean-Louis Bantignies,2 Gilles
Subra,1 Monique Calmès,1 Virginie Bellet,1 Marcel Garcia,1 Jean Martinez1 and Muriel
Amblard1
1
Institut des Biomolécules Max Mousseron (IBMM), UMR CNRS 5247, Université de Montpellier, 15 avenue
2
Charles Flahault, BP 14 491, 34093 Montpellier Cedex 5, France; Equipe Nanostructures Laboratoire Charles
Coulomb, UMR CNRS 5221, Université de Montpellier, place Eugène Bataillon, 34095 Montpellier Cedex 5,
France.
Peptides afford a large functional diversity and attractive routes of syntheses but they do not allow
the control of the molecular shape to access well-organized scaffolds. In this work, we developed a
strategy to convert peptide sequences into γ-lactam-containing oligomers and showed the
propensity of these molecules to adopt ribbon like secondary structure. The periodic distribution of
the functional groups on either side of the plane of the ribbon is simply encoded by the peptide
sequences. We evaluated the ability of these scaffolds to cross the cell membrane and successfully
identified series of efficient cell penetrating compounds. These oligomers represent promising new
vectors for the translocation of drugs.
120
A native chemical ligation strategy to overcome side reactions during
Fmoc-based synthesis of C-terminal cysteine-containing peptides. Application
to the plant β-defensin AhPDF1.1
Dominique Lelièvre, Victor Terrier, Agnès F. Delmas and Vincent Aucagne
Centre de Biophysique Moléculaire, UPR 4301 CNRS, Rue Charles Sadron, 45071 Orléans cedex 2, France.
Fmoc-based solid phase peptide synthesis (SPPS) of peptides bearing a C-terminal cysteine is a longlasting problem due to several specific side reactions raised by the enhanced acidity of the Cys ester
Cα proton. Besides racemization during the esterification step for anchoring the C-terminal cysteine
on the solid support, the repeated piperidine treatments used for successive Fmoc deprotection also
leads to Cys epimerization during the course of SPPS. A second frequent side-reaction is 3-(1piperidinyl) alanine formation arising from piperidine-mediated β-elimination followed by Michaeltype addition to the resulting dehydroalanine residue (scheme 1). These side-products become
particularly problematic when synthesizing long peptides. Several methodologies have been
developed to overcome these limitations, such as the use of a 2-chloro-trityl linker, side-chain
anchoring strategies, specifically-developed S-protecting groups, or use of an orthoester-based
building block.
PG
N
HS
S
Fmoc SPPS
Fmoc
N
H
O
linker
resin
peptide
N
H
O
OH
+
peptide
O
N
H
OH
O
Scheme 1: side reactions commonly observed during the synthesis of peptides bearing a C-terminal cysteine
We were recently confronted with these problems during the synthesis of the C-terminal Cyscontaining disulfide-rich miniprotein AhPDF1.1, a 51 amino acid plant defensin that is implied in zinc
tolerance of Arabidopsis thaliana. Even when using a chloro-trityl linker, about 30% epimerization
and 10% piperidylalanine formation were observed in the final product, that considerably
complicated the purification of the target peptide and leaded to moderate yields. To overcome these
problems and improve the overall yield, we designed and optimized a strategy based on the postsynthetic introduction of the C-terminal cysteine through native chemical ligation (NCL). Due to the
C-terminal Pro-Cys sequence of the defensin, this peptide constitutes a highly challenging example to
apply this methodology, as NCL at proline is known to lead to the slowest kinetics amongst the 20
proteogenic amino acids. We recently introduced a straightforward methodology for the Fmoc
synthesis of peptide crypto-thioesters, that can be fully-automatized using non-expensive reagents
and leads to C-terminal N-2-nitro-4-hydroxy benzyl (Hnb) cysteine peptide β-mercapto amides. They
can directly be used in NCL reactions with fast kinetics. Here we will discuss the preparation of a [150]AhPDF1.1-(Hnb)Cys(StBu)-Gly-NH2 crypto-thioester and the optimization of its NCL with cysteine,
that allowed to improve the yield for the AhPDF1.1 chemical synthesis. Since many naturally
occurring peptides and proteins contains a C-terminal cysteine, this strategy should be of a wide
interest for the peptide chemist community.
SPPS
H2 N- resin
NCL
[1-50]AhPDF1.1 -(Hnb)Cys(StBu)-Gly-NH2
[1-50]AhPDF1.1 -Cys-OH
H-Cys-OH
[1-51)]AhPDF1.1
AHPDF1.1: H-ZRLCEKPSGT WSGVCGNNGACRNQCIRLEK ARHGSCNYVF PAHKCICYFP C-OH, Z = pyroGlu
Scheme 2: epimerization-free synthesis of AhPDF1 by a NCL-based strategy
121
The C-type natriuretic peptide (CNP) acts as an active peptide on
Pseudomonas aeruginosa bacteria: Identification of the binding site, the
mechanism of entrance and the bacterial effectors
Olivier Lesouhaitier,1 Thibaut Rosay,1 Thomas Clamens,1 Suraya Diaz,2 Jérôme Leprince,3
Julie Hardouin,4 Pascal Cosette,4 Nicholas J. Harmer2 and Marc G.J. Feuilloley1
1
Laboratory of Microbiology Signals and Microenvironment EA 4312, Normandie Univ., Univ. Rouen, IRIB,
2
3
Evreux, France; School of Biosciences, University of Exeter, Exeter, UK; Inserm U982, Normandie Univ., IRIB,
4
Univ. Rouen, Mont-Saint-Aignan, France; Laboratoire Polymères, Biopolymères, Surfaces, UMR 6270 CNRS,
Proteomic Platform PISSARO, Univ. Rouen, Mont-Saint-Aignan, France.
There is now ample evidence that bacterial virulence is modulated by eukaryotic messengers
including both neurotransmitters and peptide hormones1. We have previously shown that the C-type
Natriuretic Peptide (CNP), a member of the mammalian natriuretic peptide family produced by lung
endothelial cells, enhances Pseudomonas global virulence2 and strongly decreases biofilm formation.
Since it is considered that virulence factor production and biofilm formation are inversely regulated,
we investigated the mechanism of action of CNP on P. aeruginosa.
In the present study, we used several peptide agonists to characterize pharmacologically the
bacterial CNP sensor. In parallel, using numerous bacterial mutant strains we identify how the
peptide is entering into bacterial cytosol and which transcriptional regulator is involved.
We observed that CNP inhibits P. aeruginosa biofilm formation. This effect is totally prevented by
Isatin, an antagonist of natriuretic peptide receptors in eukaryotic cells. The action of CNP on P.
aeruginosa virulence is mimicked by the eukaryotic receptor NPR-C agonist cANF4-23. Additionally, an
in silico comparative three-dimensional screen of human natriuretic peptide receptors and
Pseudomonas proteins revealed that the bacterial protein AmiC shows significant homology with the
human C-type natriuretic peptide receptor (hNPR-C). AmiC was purified recombinantly and the
protein interactions assessed using MicroScale Thermophoresis. The results showed that both CNP
and an hNPR-C agonist bind to AmiC with a KD of 2 µM and less than 100 nM respectively, whereas
an hNPR-A agonist has poor affinity for AmiC. We observed that an amiC- mutant strain failed to
appropriately respond to CNP. Since AmiC is located into the bacterial cytosol, we identified, in silico,
the protein PA4858, an amino-acid transporter located in the periplasmic membrane as an AmiC
partner. Using a PA4858 mutant strain, we validated the involvement of this protein in CNP effect on
P. aeruginosa. AmiC belongs to the ami operon in which the amiR gene encodes for a transcriptional
regulator. We built a mutant strain that overexpresses the AmiR protein in order to identify the
wider effect of AmiR on P. aeruginosa and to identify the AmiR targets using a global proteomic
quantification.
Taken together, our work provides the first demonstration that the bacterial protein AmiC may be an
ortholog of the eukaryotic receptor hNPR-C, acting as a C-type natriuretic peptide sensor in P.
aeruginosa. We demonstrated that after entry into the bacterium, CNP binds AmiC triggering the
release of the active AmiR protein which finally modulates a bacterial switch between chronic and
acute infection phenotypes. The understanding of this key role of AmiR in CNP bacterial effects
should have major consequences for cystic fibrosis treatment.
1. Lesouhaitier O. et al. (2009) Sensors 9, 6967-6990
2. Blier A. S. et al. (2011) Microbiology 157, 1929-1944
122
Development and optimization of amphipathic peptides for siRNA
delivery
Mattias Lindberg, Karidia Konate, Sébastien Deshayes, Gilles Divita, Gundrun AldrianHerrada, Carole Jourdan and Prisca Boisguerin
Centre de Recherches de Biochimie Macromoléculaire, CRBM-CNRS, UMR-5237, Chemical Biology and
Nanotechnology for therapeutic tools, Montpellier, France.
Since twenty years, Cell-Penetrating Peptides (CPPs) have been widely developed to enhance the
cellular delivery of therapeutics. Among the huge number of existing CPPs, amphipathic peptides
showed a real efficiency both in cellulo and in vivo. To date, our group has developed three kinds of
carriers, the MPG, Pep-1 and CADY peptides which are able to transfer nucleic acids, proteins and
siRNA, respectively.
CADY is a 20-residue secondary amphipathic peptide as it adopts an α-helical conformation in the
presence of lipids or siRNA, thereby distributing the residues all along the helix to form distinct
aromatic, hydrophobic and hydrophilic domains. This particularity makes these peptide suitable for a
non-covalent strategy for the delivery of cargo, especially oligonucleotides, into cells through the
formation of Peptide-Based-Nanoparticles (PBN)1. Thus, by combining both electrostatic and
hydrophobic interactions, CADY is able to spontaneously self-associate with siRNA with a strong
affinity, forming stable nanoparticles (mean diameter ≈ 150nm, zeta potential ≈ +45mV) which are
able to deliver siRNAs into a wide variety of cell lines by a mechanism independent of any
endocytotic pathway.2-4
Here, we will present our actual developments of CPP-based nanoparticles for the delivery of siRNA
in cellulo.
1. Crombez L. et al. (2008) Curr. Pharm. Des. 14, 3656-3665
2. Crombez L. et al. (2009) Mol. Ther. 17, 95-103
3. Deshayes S. et al. (2012) Small 8, 2184-2188
4. Rydström A. et al. (2011) PLoS One 6(10)
123
Adrénomedulline et vaccination thérapeutique
Kamel Mabrouk,1 Caroline Berenguer-Daize,2 Nadège Dussault,2 Didier Gigmes,1 Denis
Bertin,1 Pierre-Marie Martin2 et L’Houcine Ouafik2
1
AMU ICR, CNRS, UMR 6517, Equipe CROPS « Peptides A Visée Thérapeutique », Site de Saint-Jérôme, Avenue
2
Escadrille Normandie Niemen, 13397 Marseille, Cedex 20; AMU, INSERM UMR 911 CRO2, Angiogénèse,
invasivité et micro-environnement tumoral, Faculté de Médecine Secteur Nord, Boulevard Pierre Dramard,
13916 Marseille, Cedex 20.
Parmi les nouvelles stratégies de traitement des tumeurs solides à l’aide des anti-angiogéniques, des
travaux de recherches intenses concernent actuellement l’immunothérapie active spécifique. Dans le
groupe des antigènes vaccinaux le VEGF a gagné un intérêt particulier grâce aux succès
thérapeutiques cliniques de l’anticorps anti-VEGF bevacizumab, les inhibiteurs de kinases Sorafénib
et le sunitinib. Ainsi par exemple, la formulation vaccinale du CIGB-247 (VEGF 121, isoforme de 121
acides aminés) inhibe la croissance de mélanome agressif (B16-F10) chez la souris et réduit
l’incidence des métastases de poumons chez différents modèles tumoraux.1 De manière
intéressante, les résultats de l’essai clinique de phase 1 « CENTAURO » réalisé avec CIGB-247 sur 30
patients ont montré le bénéfice chez 12 d’entre eux. Le CIGB-47 semble être immunogène et non
toxique.2
Notre projet vise à développer des anticorps anti-peptides dérivés de l’adrénomédulline (AM) et de
ses récepteurs (AMR) à des fins thérapeutiques. L’AM est un peptide vasoactif de 52 acides aminés
qui exerce de multiples actions physiologiques. Le récepteur AMR est formé d’un complexe entre
une protéine à 7 domaines transmembranaires le CLR (Calcitonin Receptor Like Receptor) et une
protéine ayant un seul domaine transmembranaire appartenant à la famille des RAMPs (RAMP2,
RAMP3) (Receptor Activity Modifying Protein). Ces récepteurs sont présents non seulement dans les
cellules de la plupart des tissus, tels que le cœur, le rein, le cerveau, le poumon et la glande
surrénale, mais aussi dans les cellules tumorale et dans les cellules qui constituent le stroma tumoral.
Nous avons montré que i) l’administration intrapéritonéale in vivo chez des souris xénogreffées
(glioblastome, prostate, colon, poumon), des anticorps dirigés contre AM ou des peptides dérivés de
AMR, induit une disparition quasi totale de la tumeur après 25 semaines de traitement ; ii) les
anticorps obtenus contre des peptides chimériques dérivés de AM et AMR inhibent la croissance
tumorale in vitro et in vivo ; iii) l’utilisation de ces anticorps pourrait être d’un intérêt considérable
dans le diagnostic et le traitement des cancers dont celui de la prostate homono-indépendant, iv) ces
anticorps exercent leur effet anti-angiogénique et anti-vasculaire à travers l’inhibition rapide du
complexe VE-cadherin/b catenin, ce dernier qui est nécessaire à l’adhésion et à la survie des cellules
endothéliales lors de l’assemblage des nouveaux vaisseaux.3-9
Ainsi l’utilisation de l’AM et AMR pour inhiber la croissance tumorale constitue une approche
pertinente en raison de son mécanisme d’action qui porte à la fois sur les cellules endothéliales mais
aussi, à la différence du VEGF, sur la cellule tumorale et sur l'ensemble des cellules du stroma,
particulièrement les péricytes impliquées dans l’établissement d’une angiogenèse tumorale stable et
fonctionnelle. Tous ces résultats ont fait l’objet de dépôt de 3 demandes de brevets. Forts des
résultats obtenus avec les anticorps dirigés contre AM et ses récepteurs nous projetons de
développer une stratégie de vaccination thérapeutique en utilisant comme antigènes les peptides
synthétiques dérivés de l’AM et AMR.
1. Bequet-Romero et al. (2012) Vaccine 30, 1790-1796
2. Gavilondo et al. (2014) Vaccine 32, 2241-2250
3. Ouafik et al. (2002) Am. J. Pathol. 160, 1279-1285
4. Fernandez-Sauze et al. (2004) Int. J. Cancer 108, 797-804
5. Berenguer et al. (2008) Oncogene 27, 506-518, (2013) Clin. Cancer Res. 15, 6138-6150
6. Kaafarani et al. (2009) FASEB J. 23, 3424-3435
7. Metellus et al. (2011) Eur. J. Cancer 47, 1727-1735
8. Nouguerede et al. (2013) Cancer Med. 2, 196-207
9. Khalfaoui et al. (2015) Oncotarget (sous presse)
124
Alkoxyamine peptide ligation (APL): a straightforward method for nonnative peptide conjugation using intermolecular radical 1,2-addition
Kamel Mabrouk, Thomas Trimaille, Laurent Autissier, Rakotonirina Mama Daniel, Yohann
Guillaneuf, Denis Bertin and Didier Gigmes
AMU ICR, CNRS, UMR 6517, Equipe CROPS « Peptides A Visée Thérapeutique », Site de Saint-Jérôme, Avenue
Escadrille Normandie Niemen, 13397 Marseille, Cedex 20.
Many chemoselective ligation methods have emerged over the two past decades for coupling of
peptide fragments or for peptide modification with biomolecules of interest. These methods include
native chemical ligation, imine or oxime ligation, Staudinger ligation, triazole formation through
alkyne-azide addition, decarboxylative condensations of N-alkylhydroxylamines and -ketoacids or,
more recently, bis(2-sulfanylethyl)amino native peptide ligation. However, to our knowledge, free
radical chemistry based reactions have not been exploited yet as a peptide conjugation/ligation
strategy. In this study we describe a novel conjugation method, namely Alkoxyamine Peptide Ligation
(APL), based on intermolecular radical 1,2-addition (IRA) of SG1 nitroxide based alkoxyamines onto
double bonds 1. This strategy implies simple peptide pre-derivatization to obtain (i) a SG1 nitroxide
functionalized resin peptide at its N–terminus (SG1-peptide alkoxyamine), (ii) a vinyl functionalized
peptide (either at its C-terminus or N-terminus), and does not require any coupling agents. This is the
first example of chemoselective peptide conjugation based on a radical reaction.
1. Trimaille T. et al. (2014) Chem. Commun. 50, 2744-2747
125
Développement d’une stratégie multi-échelle combinant des données
expérimentales et in silico pour la reconstruction de complexes impliquant la
dystrophine
Anne-Elisabeth Molza,1 Khushdeep Mangat,2 Olivier Delalande,1 Nicolas Férey,3 Marc
Baaden,4 Chantal Prévost,4 Sébastien Fiorucci,5 Mirjam Czjzek,6 Nick Menhart,2 Denis
Chrétien,7 Elisabeth Le Rumeur 1 et Jean-François Hubert 1
1
Equipe SIM- CNRS UMR 6290, Institut de Génétique et de Développement de Rennes (IGDR), Rennes, France;
3
Illinois Institute of Technology, Chicago IL, United States; Laboratoire d'Informatique pour la Mécanique et les
Sciences de l'Ingenieur (LIMSI)- CNRS UPR 3251- Institut Universitaire et Technologique d'Orsay, Université
4
PARIS XI, Paris, France; Laboratoire de Biochimie Théorique - CNRS UPR 9080 - Institut de Biologie Physico5
Chimique (IBPC), Université PARIS 7, Paris, France; Institut de Chimie de Nice, CNRS UMR 7272, Université de
6
7
Nice-Sophia Antipolis, Nice, France ; CNRS UMR 8227, Station Biologique de Roscoff, Roscoff, France; Equipe
TIPs- UMR CNRS 6290, Institut de Génétique et de Développement de Rennes (IGDR), Rennes, France TeamInstitut de Génétique et Développement de Rennes (IGDR), Université de Rennes 1, Rennes, France.
2
La dystrophine est une très grande protéine sub-sarcolemmique de 427kDa codée le gène DMD.
Cette protéine joue un rôle essentiel dans le maintien de l’intégrité de la cellule musculaire lors des
cycles de contraction/relaxation.
La dystrophine est composée de quatre principaux domaines structuraux dont le domaine central
composé de 24 répétitions et quatre charnières. Chaque répétition est organisée en faisceau de trois
α-hélices homologues à la spectrine et appelé « coiled-coil ».
Des mutations du gène DMD sont à l’origine des myopathies de Duchenne et de Becker, lesquelles
s’accompagnent de ruptures fréquentes de la membrane des cellules musculaires.
Malgré le rôle important que joue la dystrophine, il existe peu de données sur sa structure et ses
interactions avec ses principaux partenaires cellulaires.
Afin de pallier à ce manque, nous avons développés une approche multi-échelle combinant des
données expérimentales et des données in silico1.
Cette méthodologie nous a permis d’une part d’obtenir des modèles tout-atome de fragments de la
dystrophine native et mutée, en se basant sur des données de diffusion des rayons X aux petits
angles (SAXS) et des méthodes de simulations interactives.
D’autre part, nous avons utilisés des méthodes d’amarrage moléculaires afin d’obtenir des modèles
de complexes impliquant l’actine filamenteuse (actine-F) ou le domaine PDZ de la nNOS (nNOS-PDZ)
avec les répétitions R11-15 ou R16-17 de la dystrophine native ou de mutants BMD de la protéine.
Cette stratégie a permis de reconstruire un modèle tout-atome du complexe macromoléculaire
impliquant les répétitions R11-17 de la dystrophine, l’actine-F, nNOS-PDZ et les phospholipides
membranaires.
1. Molza A.-E. et al. (2014) Faraday Discuss. 169, 45–62
126
New antimicrobial marine cyclolipopeptides
Irène Nicolas,1 Eric Balnois,2 Yannick Fleury2 and Michèle Baudy-Floc'h1
1
2
UMR 6226 CNRS, Université de Rennes 1, Rennes, France; LUBEM, EA 3882, Quimper, France.
Multidrug-resistant Gram-negative bacteria (Acinetobacter baumannii, Pseudomonas aeruginosa,
and Klebsiella pneumoniae) are a serious threat to human health and need the development of new
drugs to combat microbial infection. The antimicrobial peptides (AMPs) have been proposed as an
1
alternative.
Novel antibacterial cyclic lipopeptides have recently been characterized from.marine micro2,3
organisms. They contain unnatural amino acids such as didehydro aminobutyric acid (Dhb), α, γdiamino butyric acid (Dab) and β-hydroxy Dab (β-OH Dab). The latter are commercially unavailable
and need to be synthesized to allow solid phase peptide synthesis.
We will present the synthesis of β-hydroxy Dab (β-OH Dab) conveniently substituted to realize the
preparation of new cyclolipopeptides as well as new analogs of didehydro aminobutyric acid, α, γdiamino butyric acid (Dab) and β-hydroxy Dab. All these new synthetic monomers could be
subsequently introduced in peptide synthesis by SPPS using a Fmoc/t-Bu strategy.
A set of synthetic analogs has been assayed for antimicrobial activity to clarify structure activity
relationships. The crucial role of Dhb and β-OH Dab in antibacterial activity was highlighted.
n
O
NH
HO
H2N
O
HN
O
NH
O
HO
NH
HN
NH2
O
HO
HN
NH
H2N
N
H
O
O
HN
H2N
NH2
1. Plaza A. & Bewley C. A. (2006) J. Org. Chem. 71, 6898-6907
2. Desriac F. et al. (2010) Mar. Drugs. 8, 1153-1177
3. Desriac F. et al. (2013) BFF 13P0392/EG, 24 mai 2013
127
O
Selenopeptide transamidation and methathesis
Nathalie Ollivier, Annick Blanpain, Emmanuelle Boll, Laurent Raibaut, Hervé Drobecq and
Oleg Melnyk
Selenocysteine (Sec/U), i.e. the selenium analog of cysteine (Cys/C), is a naturally occurring amino
acid found in selenoproteins. Most of the 25 human selenoproteins are redox enzymes which feature
a Sec residue in their catalytic site. These proteins are collectively essential for living organisms as
they participate in a large array of biological processes such as the control of the cellular redox
balance. The loss of activity observed upon mutation of catalytic Sec residue into Cys in some
selenoenzymes originates, at least in part, from the large difference in pKa and in reducing potential
between Sec and Cys. Indeed, although selenium and sulfur are neighboring members of the
chalcogen family, the pKa of the selenol group in Sec (pKa ~5.5) is about 3 units less than the pKa of
the thiol group (pKa 8.3) in Cys. Also, the redox properties of disulfides and diselenides differ
significantly, the redox potential of diselenides being significantly lower than those of disulfides.
Accordingly, the replacement of Cys by Sec is increasingly used in protein engineering approaches as
a tool for modulating the stability, the physico-chemical properties or the folding pathways of
polypeptides.
Recent studies have shown that the properties of selenopeptides have not been fully explored.
Indeed, we report here a novel property for this important class of biomolecules by showing that
selenopeptides featuring an internal or C-terminal Sec residue can participate in a transamidation
reaction with cysteinyl peptides or in a metathesis reaction.1,2 Both processes rely on an aminecarboxamide exchange reaction which proceeds chemoselectively in water at mildly acidic pH. We
show also that SEA native peptide ligation3,4 is a useful method for accessing selenopeptides.
2. Melnyk O. & Agouridas V. (2014) Curr. Opin. Chem. Biol. 22, 137-145
3. Raibaut L. et al. (2013) Org. Lett. 15, 5516-5519
128
A new linker for solid phase protein synthesis
Nathalie Ollivier, Raphaël Loval, Annick Blanpain, Rémi Desmet and Oleg Melnyk
Protein chemical synthesis is made possible by the combination of several essential chemical tools.
Solid phase peptide synthesis (SPPS)1 enables the synthesis of peptide segments by the iterative
coupling of protected amino acids to a solid support. Other major tools are chemoselective peptide
bond forming reactions which enable the coupling of unprotected peptide segments in water.
Among these reactions, Native Chemical Ligation (NCL)2 which is based on the coupling of a Cterminal peptide thioester with an N-terminal cysteinyl peptide is undoubtedly the most popular
reaction for protein total synthesis.
The ligation of two peptide segments gives access to polypeptides composed of up to 100 amino acid
residues, considering the current limits of SPPS. The synthesis of larger polypeptides usually requires
the chemical ligation of three or more peptide segments either using iterative or convergent
assembly schemes. We have recently developed an efficient one-pot three peptide segments
assembly method3-8 relying on a sequential NCL/bis(2-sulfanylethyl)amido (SEA)9,10 native peptide
ligation process. The concepts behind this one-pot process were recently extended to the solid phase
using the Esoc linker11 developed by Aucagne and coworkers12. In this work, up to five peptide
segments were ligated sequentially to produce a 15 kDa polypeptide. Performing the concatenation
of a large number of peptide segments on a water compatible solid support might extend
considerably the limits of protein total synthesis.
One substantial bottleneck to the development of these approaches is the design of linkers for the
immobilization of the growing peptide chain through either from the N- or C-terminus.
Here, we describe the design of a new traceless linker technology for the chemoselective attachment
of the N-terminal segment on a water compatible resin. It allows the elongation of the peptide chain
in the N-to-C direction using the SEA SPPS method.
2. Dawson P. E. et al (1994) Science 266, 776-779
3. Ollivier N. et al (2012) Angew. Chem. Int. Ed. 51, 209-213
4. Raibaut L. et al (2012) Chem. Soc. Rev. 41, 7001-7015
5. Raibaut L. et al (2013) Bioorg. Med. Chem.21, 3486-3494
6. Boll E. et al (2014) Chem. Sci. 5, 2017-2022
7. Boll E. et al (2015) Nature Protocols, in press
8. Boll E. et al (2015) Org. Lett., in press
9. Ollivier N. et al (2010) Org. Lett. 12, 5238-5241
10. Ollivier N. et al (2014) J. Pept. Sci. 20, 92-97
11. Aucagne V. (2012) Angew. Chem. Int. Ed. 51, 11320-11324
12. Raibaut L. et al (2013) Chem. Sci. 4, 4061-4066
129
Nitrones dérivées du L-gulose et synthèse asymétrique d’amino-acides
énantiopurs
Sandrine Py,1 Moustapha Fortunato,1 Xiaofei Zhang,1,2 Gilles Dujardin,2 Mathieu Y.
Laurent2 et Arnaud Martel2
1
Département de Chimie Moléculaire (SERCO) UMR-5250 CNRS, Université Joseph Fourier, BP 53, 38041
2
Grenoble Cedex 09, France; LUNAM, Université du Maine, IMMM, UMR 6283 CNRS, Equipe Méthodologie et
Synthèse Organique, 72085 Le Mans Cedex 09, France.
Nous avons récemment montré que l’introduction d’un auxiliaire chiral dérivé du D-mannose
(auxiliaire de Vasella)1 sur des cétonitrones « aspartiques »2 de type 1 permet de contrôler la
stéréosélectivité de leur cycloaddition avec des éthers d’énols, pour l’obtention d’isoxazolidines
énantiopures 2. Cette stratégie a été exploitée avec succès pour la synthèse d’amino acides α,αdisubstitués tels que 3.3
D’autres équipes ont utilisé des dérivés du gulose comme auxiliaires chiraux pour des cycloadditions
diastéréosélectives impliquant des nitrones. Les avantages de ces derniers seraient une meilleure
disponibilité en séries L et D, ainsi que la formation d’adduits plus cristallins.4 Cependant, les nitrones
formées par réaction de l’auxiliaire 4 avec des aldéhydes ne sont en général ni isolées, ni
caractérisées, elles ont été directement transformées in situ par réaction avec des dipolarophiles. La
préparation et la caractérisation dans nos laboratoires de nitrones comportant un auxilaire gulosyle
nous a réservé quelques surprises. En effet, la réaction de la gulosyl-hydroxylamine 4 avec le DMAD
(ou le DTAD) a conduit à deux nitrones isomères, chacune d’elle formant des cycloadduits différents
par réaction avec les éthers de vinyle.
Une fois les isomères de nitrones et de cycloadduits obtenus caractérisés, leur transformation en
amino acides et oxaprolines α,α-disubstitués énantiopurs sera présentée, ainsi que leur couplage
avec d’autres acides aminés. L’incorporation de tels motifs dans des systèmes peptidiques constitue
un objectif majeur de notre projet.
1. a) Vasella A. (1977) Helv. Chim. Acta 60, 1273; b) Huber R. & Vasella A. (1990) Tetrahedron 46, 33
2. a) Nguyen T. B. et al. (2008) Org. Lett. 10, 4493; b) Nguyen T. B. et al. (2010) J. Org. Chem. 75, 611
3. Zhang X. F. et al. (2014) Org. Lett. 16, 1936
4. a) Kasahara K. et al. (1989) J. Org. Chem. 54, 2225; b) Yu S. et al. (2010) Chem. Sci. 1, 637; c) Gerfaud T. et al. (2012) Org.
Proc. Res. Dev. 16, 687; d) Pattabiraman V. R. et al. (2012) Angew. Chem. Int. Ed. 51, 5114
130
BactPepDB: a database of predicted peptides from an exhaustive survey
of complete prokaryote genomes
Julien Rey,1,2,3 Patrick Deschavanne1,2 and Pierre Tuffery1,2,3
1
2
INSERM, U973, MTi, F-75205 Paris, France; Univ Paris Diderot, Sorbonne Paris Cité, F-75205 Paris, France;
RPBS, F-75205 Paris, France.
3
With the recent progress in complete genome sequencing, mining the increasing amount of genomic
information available should in theory provide the means to discover new classes of peptides.
However, annotation pipelines often do not consider small reading frames likely to be expressed.
BactPepDB, available online at http://bactpepdb.rpbs.univ-paris-diderot.fr, is a database that aims at
providing an exhaustive re-annotation of all complete prokaryotic genomes – chromosomal and
plasmid DNA - available in RefSeq for coding sequences ranging between 10 and 80 amino acids. The
identified peptides are classified as (i) previously identified in RefSeq, (ii) entity-overlapping
(intragenic) or intergenic, (iii) potential pseudogenes - intergenic sequences corresponding to a
portion of a previously annotated larger gene. Additional information is related to homologs within
order, predicted signal sequence, transmembrane segments, disulfide bonds, secondary structure,
and the existence of a related 3D structure in the Protein Databank. As a result, BactPepDB provides
insights about candidate peptides, and provides information about their conservation, together with
some of their expected biological/structural features. The BactPepDB interface allows to search for
candidate peptides in the database, or to search for peptides similar to a query, according to the
multiple properties predicted or related to genomic localization.
131
Modulation of PDZ domain-mediated interactions by a directed
molecular evolution approach
Charlotte Rimbault,1 Isabel Gauthereau,1 Camille Genuer,1 Dolors Grillo-Bosch,1 Coraline
Thibaut,1 Daniel Choquet,1,2 Christelle Breillat1 and Matthieu Sainlos1
1
Université de Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, Bordeaux, France;
Bordeaux Imaging Center, Bordeaux, France.
2
Identification of the cellular and molecular mechanisms involved in the regulation of glutamate
receptor trafficking is crucial to our understanding of synaptic maturation and plasticity. In this
context, the PDZ domain-mediated interactions of the AMPA- and NMDA-type glutamate receptors
with the synaptic scaffold proteins from the PSD-95 family have been identified over the last decade
as critical for their synaptic stabilization and their function. However, the mechanisms that
dynamically govern their respective synaptic retention remain poorly understood.
In order to investigate these PDZ domain-mediated interactions, our aim is to develop small synthetic
antibodies that will allow us to specifically study these protein complexes either by detection or
modulation.
Our strategy relies on a selection approach and is based on the Fibronectin Type III domain (FN3) - a
robust scaffold for antibody-like binding proteins - in order to target the PDZ domain partners of the
receptor complexes or their own binding motifs (e.g., stargazin for the AMPARs and GluN2A for the
NMDARs). To this end, the FN3 domain is used either alone, or fused to a PDZ domain PSD-95 (the
second or the third) to generate an “affinity clamp”1.
Using a recently developed strategy, we generated large combinatorial phage-display libraries in
which the two main surface loops of FN3 were diversified2. After multiple rounds of selection against
peptide or protein targets (derived from the synaptic proteins of interest), we could isolate strong
binding clones and extensively characterize them by various biophysical techniques in particular with
respect to their affinity and specificity. The clones presenting the most promising properties were
next evaluated for their capacity to bind their target in live cells. Candidates that maintain their
activity in such a complex environment are currently engineered to be used as specific binders of PDZ
domain-containing proteins or modulators of intracellular PDZ domain-mediated interactions.
1. Huang et al. (2008) PNAS 105, 6578-6583
2. Firnberg et al. (2012) PLOS One 7, e5031
132
Caractérisation structurale et fonctionnelle de deux hydrolysats
protéiques destinés à l’alimentation d’espèces aquacoles
Marie Robert,1,2 Alexandre Leduc,1,2,5 Céline Zatylny-Gaudin,1,2 Benoit Bernay,3 Erwan
Corre,4 Gildas Le Corguillé,4 Vincent Fournier5 et Joël Henry1,2,3
2
1
Université de Caen Basse-Normandie, UMR BOREA, IBFA, F-14032 Caen, France; UMR BOREA Biologie des
3
ORganismes et Ecosystèmes Aquatiques" MNHN, UPMC, UCBN, CNRS-7208, IRD-207; Plateforme post4
génomique PROTEOGEN, Université de Caen Basse-Normandie, SF ICORE 4206, Caen, France; ABiMS, FR2424
5
CNRS-UPMC, Station Biologique, 29680 Roscoff, France; Aquativ (DIANA SPF, Membre du groupe Symrise) ZA
du Gohelis - 56250 Elven, France.
Le développement de l’aquaculture se traduit par un accroissement de la demande en aliments
aquacoles et par une forte tension au niveau de la disponibilité et des prix des matières premières
d’origine marine. Les farines de poissons, bases de la formule alimentaire pour poissons et crevettes
d’aquaculture, sont devenus une ressource rare. La société DIANA SPF a développé une nouvelle
génération de matières premières, les hydrolysats protéiques de coproduits d’origine marine, pour
permettre le remplacement des farines de poissons dans les formulations aquacoles. En effet, du fait
de l’importance des composés azotés de faible masse moléculaire pour les espèces d’intérêt
aquacoles dans le comportement alimentaire, la nutrition et la santé, les hydrolysats constituent un
substitut à la fois performant et économique.
Il apparaît par ailleurs que les propriétés nutritionnelles et fonctionnelles d’un hydrolysat de
coproduits dépendent essentiellement de l’abondance, de la diversité et de la masse moléculaire des
peptides qui le composent. C’est pourquoi, dans le cadre d’une collaboration associant l’UMR BOREA
et la société DIANA SPF, une étude structurale portant sur deux hydrolysats a été réalisée pour la
première fois. Ces deux hydrolysats ont été obtenus à partir des coproduits de la crevette Litopeneus
vannamei, et du poisson téléostéen Oreochromis nilocus aussi connu sous le nom de tilapia.
La caractérisation par une approche peptidomique s’est révélée particulièrement performante grâce
au séquençage préalable du transcriptome en RNAseq de novo des organes et tissus soumis à
l’hydrolyse. La mise en œuvre d’un protocole de séparation associant l’électrophorèse préparative
OFF-GELL (Agilent) et la nanoLC en phase inversée a permis de fractionner des échantillons
extrêmement complexes. Afin de couvrir au mieux la gamme de masses 200-5000Da, les échantillons
ont été analysés successivement en ESI-IT-MS/MS (Orbitrap) et en MALDI-TOF/TOF. 1010 et 1374
peptides hydrolytiques ont ainsi été identifiés respectivement chez L. vannamei et O. niloticus
révélant deux profils de masses moléculaires très distincts (Robert et al, 2014, 2015).
Des tests antibactériens réalisés sur des souches de pathogènes rencontrés dans les élevages
aquacoles ont permis de mettre en évidence le potentiel antibactérien des hydrolysats, lequel
pourrait expliquer au moins partiellement, le gain en termes de survie observé lors de tests
zootechniques réalisés sur le bar d’élevage Dicentrarchus labrax. Par ailleurs, il a également été
démontré par des essais nutritionnels in vivo sur cette même espèce, que l’incorporation dans les
aliments de 5% d’hydrolysats permettait le remplacement de 15% de farine de poissons. Le suivi de
biomarqueurs spécifiques de l’absorption intestinale et de l’immunité par RNAseq, devrait permettre
d’établir un lien entre le niveau d’hydrolyse, la croissance et le statut sanitaire des bars d’élevage.
Dans ce contexte, l’application de process d’hydrolyse différentielle et contrôlée à partir de multiples
origines de matières premières marines permettra de générer des profils peptidiques différents aux
propriétés fonctionnelles variées et pour lesquels il subsistera un besoin de caractérisation des
produits et de compréhension des mécanismes d’action, leviers d’optimisation de leurs
performances.
133
Quantification de peptides modifiés par spectrométrie de masse
MALDI-ToF – Mesures d’interactions récepteur-ligand
Maxime Rossato, Sonia Cantel, Didier Gagne, Gilles Subra, Christine Enjalbal et Jean
Martinez
Institut des Biomolécules Max Mousseron (IBMM), UMR5247, Université Montpellier 1 et 2, France.
Mesurer l'affinité d'un ligand pour sa cible est une question d'importance capitale en pharmacologie
et plus précisément dans le développement de médicaments. En particulier, comprendre la façon
dont un médicament interagit avec sa cible (récepteur, enzyme, ADN...) fournit des informations
précieuses dans la quête de nouveaux composés pharmaceutiques. Concernant l’interaction ligandrécepteur, La liaison d'un candidat-médicament se caractérise par des tests de liaison compétitive
dans lequel un ligand de référence marqué (dont affinité vers le récepteur est connue) est mis en
concurrence avec la molécule d'intérêt. Étant donné que l'affinité du ligand de référence pour le
récepteur cible est généralement élevée, la mesure de ces interactions non-covalentes nécessite une
excellente sensibilité (<10-8 M).
La radioactivité est encore aujourd’hui la méthode universelle pour ces études pharmacologiques.
Cependant, cette dernière implique beaucoup de contraintes liées à la manipulation des
radioéléments et leur stockage, et limite donc fortement son application. Dans ce contexte, nous
visons à développer une technologie s'appuyant sur la détection ultrasensible et la quantification par
spectrométrie de masse.
Des agents de dérivation chimiques de peptides ont été développés au laboratoire, jouant le rôle
d’exhausteur d’ionisation en spectrométrie de masse moléculaire dans l’optique d’augmenter la
sensibilité de détection de biomolécules, problématique particulièrement délicate lors de l’étude de
milieux complexes.1-2 Une méthodologie analytique basée sur la spectrométrie de masse MALDI-ToF
a été mise en place avec l’utilisation conjointe du marquage spécifique des peptides par HCCA (acide
α-cyano-4-hydroxycinnamique) qui constitue l’une des matrices les plus couramment employées en
MALDI-Tof pour la détection de ces biomolécules et de la molécule HCCE (ester méthylique de
l’HCCA) en tant que matrices de dépôt MALDI. Cette combinaison «marqueur HCCA / matrice HCCE»
a démontré sur des peptides modèles une efficacité accrue de la réponse en spectrométrie de masse
MALDI-Tof de ces molécules portant la dérivation par rapport aux peptides non marqués présents en
mélange.
L’optimisation méthodologique a permis d’améliorer significativement la sensibilité de l’analyse
MALDI en atteignant des seuils de détection de l’ordre de 10-12 M. Après validation de la
méthodologie de quantification, des tests de liaison ont été effectués sur le modèle CCKR/AVP.
1. Lascoux D. et al. (2007) Angew. Chem. Int. Ed. 46, 5594-5597
2. Paramelle D. et al. (2010) Angew. Chem. Int. Ed 49, 8240-8243
134
In silico prediction of peptide structure and peptide-protein
interactions: Where are we ?
Adrien Saladin, Julien Rey, Pierre Thévenet, Gautier Moroy, Alexis Lamiable and Pierre
Tufféry
INSERM UMR-S 973, University Paris-Diderot, Sorbonne-Paris-Cité, Paris, France.
Peptide-protein interactions are important to many processes of life, particularly for signal
transmission or regulatory mechanisms. When no information is known about the interaction
between a peptide and a protein of known structure, in silico approaches can provide means to (i)
propose candidate sites of interaction on the surface of the protein (ii) propose binding modes for
the peptide-protein interaction.
In the recent years, important progress has been achieved for each of these topics. Several
approaches such as PEP-FOLD (1-2) have been developed to predict peptide conformation from
sequence. These approaches start to reach maturity, assist peptide characterization and design (3,4)
and and now can undergo large scale prediction. The blind identification of candidate binding sites
remains more challenging. Based on a modified version of PEP-FOLD that samples sub-optimal
conformations, we have recently introduced PEP-SiteFinder (5). Given the structure of a protein and
the sequence of a peptide, it identifies protein residues likely to be at peptide-protein interface. PEPSiteFinder starts with the 3D de novo generation of peptide sub optimal conformations. These
conformations then undergo a fast blind rigid docking on the complete protein surface. We have
found that PEP-SiteFinder best poses overlap to some extent the experimental patch of interaction
for close to 90% of the complexes. Finally, once known the binding site, the more accurate
characterization of the binding mode of the peptide can be assimilated to folding a peptide in the
vicinity of the protein.
Importantly however, we are not yet at the end of the game. Difficult matters are still to be solved.
Among these, considering non standard amino-acids to better assist the design of therapeutic
peptides, modeling disulfide bond rich peptides (among which venom peptides) for which no
satisfactory approach presently exists, or the efficient scoring of peptide binding affinities on a large
scale.
1. Maupetit J. et al. (2010) J Comput Chem. 31, 726-738
2. Thévenet P. et al. (2012 ) Nucleic Acids Res. 40, W288-293
3. Sterpone F. et al. (2014) Chem. Soc. Rev. 43, 4871-4893
4. Thévenet P. et al. (2015 ) Methods Mol. Biol. 1268:1-13
5. Saladin A. et al. (2014) Nucleic Acids Res. 42, W221-226
135
Characterization of cyclolipopeptides from a marine bacteria
Pseudoalteromonas
Matthieu Simon,1 Florie Desriac,2 Sandrine Pottier,1 Liza Mouret,1 Michèle Baudy-Floc'h,1
Yannick Fleury2 and Arnaud Bondon1
2
1
UMR CNRS 6226 - Université de Rennes 1, Rennes, France; Université de Brest, LUBEM EA3882, Quimper,
France.
Bacterial resistance to antibiotics constitutes a major public health issue. Despite intensive research
only a few new structural classes of new antibiotic compounds have been described. Among those,
cyclolipopeptides such as daptomycin represent a new class of AMP.1
Several compounds displaying antibacterial activities have been isolated from oyster hemolymph.2
These compounds have been shown to be produced by bacteria belonging to Pseudoalteromonas
genera. NMR characterization permits to identify a common cyclopeptide with various fatty acid side
chains. The peptidic part comprises "unnatural" amino acids such as dehydro aminobutyric acid
(DHB), α,γ-diaminobutyric acid (Dab) and β-hydroxy diaminobutyric acid. Further analyses are in
progress attempting to determine the stereochemistry of the various asymetric carbons.
1. Desriac F. et al. (2013) Mar. Drugs 11, 3632-3660
2. Desriac F. et al. (2013) BFF 13P0392/EG, 24 mai 2013
136
From a Staphylococcus aureus toxin to cyclic pseudopeptide antibiotics
Olivia Solecki,1 Amor Mosbah,2 Brice Felden1 and Michèle Baudy Floc'h2
2
1
U835 Inserm, Pharmaceutical Biochemistry, Rennes, France; UMR 6226 CNRS, Université de Rennes 1, Rennes,
France.
Staphylococcus aureus produces peptide toxins that it uses to respond to environmental cues. We
previously characterized PepA1,1 a peptide toxin from S. aureus, that induces lytic cell death of both
bacterial and host cells. That led as to suggest that PepA1 has an antibacterial activity. Here, we
demonstrate that exogenously provided PepA1 has activity against both Gram positive and Gram
negative bacteria. We also see that PepA1 is significantly hemolytic thus limiting its use as an
antibacterial agent. To overcome these limitations, we convert PepA1 into non-hemolytic derivatives.
Our most promising derivative is a cyclic heptapseudopeptide with inconsequential toxicity to human
cells, enhanced stability in human sera, and sharp antibacterial activity. Mechanistically, linear and
helical PepA1 derivatives form pores at the bacterial and erythrocyte surfaces while the cyclic
peptide induces bacterial envelope reorganization, with insignificant action on the erythrocytes. Our
work demonstrates that bacterial toxins might be an attractive starting point for antibacterial drug
development.
N H2
OH
HO
O
O
H
N
NH
N
HN
O
O
NH
HN
NH
HN
O
NH
H2N
O
N
H
O
HN
NH
HN
NH2
1. Sayed N. et al. (2012) J. Biol. Chem. 287, 43454-43463
137
Determining the stability and structure of therapeutic proteins
Michel Terray1 and Stacy Kenyon2
2
1
Malvern Instruments, 30 rue Jean Rostand, 91893 Orsay, France; Malvern Instruments, 7221 Lee Deforest Dr,
Columbia, MD, USA.
Aggregation and native-state unfolding of therapeutic proteins is of critical importance to
manufacturers and patients alike. Product quality and safety concerns require both identification and
understanding of aggregation, and careful monitoring for long-term stability. The use of hybrid
techniques lends itself to more thorough characterization of therapeutics. This paper will introduce a
case study to illustrate the advantages of using combined dynamic light scattering and Raman
spectroscopy for protein characterization.
The ability to identify aggregates and understand the underlying mechanisms of protein aggregation
can help to mitigate their formation. An approach that can measure high-order structure of a
protein, while concurrently monitoring for aggregation, can provide insight into both the molecular
and physical state of the protein in solution. Dynamic light scattering (DLS), which is inherently
sensitive to large particles, is capable of detecting particulates in protein formulations, including
protein aggregates, while Raman spectroscopy is ideal for elucidating the secondary and tertiary
structure of proteins at high concentration (up to 150 mg/mL and higher). Combining DLS and Raman
spectroscopy into a single instrument allows for elucidation of protein structure and size changes
from a single sample, providing complimentary tests on the same sample, at the same time, under
identical conditions. This combination of characterization techniques could provide additional
mechanistic insight into the kinetics or thermodynamics of aggregation and denaturation. DLS and
Raman was used to monitor the thermally induced unfolding and refolding of a 30 mg/mL lysozyme
sample at pH 4 (to ensure reversibility of the unfolding). The unfolding process was initiated by
thermally stressing the sample in the temperature-controlled sample compartment. The sample was
heated from 20-80°C, and then the temperature was reduced back to 20°C. Both DLS and Raman
data were collected at each of the three temperature points. As the temperature is increased from
20°C to 80°C, the Z-average size of the lysozyme increases from ~4 nm with a PDI of 0.057 to ~6 nm
with a PDI of 0.058. The modest size increase and the unchanged PDI indicate an unfolding event,
rather than an aggregation event, upon heating. The corresponding Raman spectra indicate changes
in secondary structure (Amide I and Amide III), tertiary structure (tryptophan and tyrosine), as well as
the disulfide region upon heating. When the temperature is decreased back to 20°C, the size
decreases to ~4 nm, while the PDI increases slightly to 0.080. This small increase in the size and PDI
indicates the possibility of limited aggregation or oligomer formation during the cooling process;
however, the mass percentage of these large particles is estimated to be less than 0.2%. The Raman
spectrum corresponding the second measurement at 20°C indicates any structural changes that
occurred during heating are reversible, as the spectra are identical before and after the temperature
jump to 80°C. The combined DLS and Raman data reveals that upon heating, the lysozyme undergoes
an unfolding event, as indicated by the increase in size and changes in the secondary, tertiary, and
disulfide regions of the Raman spectra. Additionally, the results indicate that the unfolding process is
reversible.
In this study, a model protein was used to illustrate the benefits of combining Raman spectroscopy
with dynamic light scattering to elucidate the thermally-induced unfolding behavior. As Raman
spectroscopy is ideal for high concentrations, samples can be evaluated at formulation conditions
without the need to dilute as with more traditional techniques that are used to study protein
structure. DLS, with exquisite sensitivity to small amounts of large particles, is ideal for detecting
aggregate formation. Combining these techniques into a single platform permits for simultaneous
monitoring of size and structure changes, allowing for the elucidation of protein unfolding and
aggregation pathways, ultimately providing information regarding the stability of the therapeutic
product.
1. Lewis N. & Kenyon S. (2014) Molecules, 19, 20888-20905
138
Conception et synthèse de vecteurs peptidiques pour le ciblage de la
périphérie tumorale : aide à la chirurgie
Fabien Thoreau,1,2 Tao Jia,2 Jungyoon Choi,2 Jean-Luc Coll2 and Didier Boturyn1
1
Département de Chimie Moléculaire, UMR CNRS-UJF 5250, équipe I2BM, Université Joseph Fourier – Grenoble
2
1, Grenoble, France ; Institut Albert Bonniot, CRI INSERM/UJF U823, Grenoble, France.
Avec la chimiothérapie et la radiothérapie, la chirurgie fait partie des trois techniques majeures pour
lutter contre le cancer. Elle est la plus utilisée dans le cas de tumeurs solides accessibles. Retirer une
tumeur par opération chirurgicale implique de pouvoir distinguer le tissu tumoral du tissu sain. Le
chirurgien dispose alors de différentes techniques d’imagerie telles que l’IRM ou encore le PET scan.
Ces techniques sont néanmoins préopératoires. Or, lors d’une intervention chirurgicale, une imagerie
« en temps réel » serait préférable. Il est alors concevable d’utiliser un composé fluorescent
permettant d’imager la tumeur.
Dans ce contexte, l’objectif de ma thèse est de vectoriser un fluorophore proche infra-rouge jusqu’à
la périphérie tumorale afin de mieux guider le geste chirurgical. Nous proposons de cibler
spécifiquement la périphérie plutôt que la tumeur entière pour obtenir un meilleur contraste. Nous
avons identifié deux récepteurs dont le ciblage permettra de véhiculer le fluorophore vers la
périphérie tumorale : l’intégrine αvβ3 et la Neuropiline-1 (NRP1). Ces deux récepteurs sont
surexprimés dans beaucoup de cancers et sont impliqués dans les phénomènes d’angiogénèse, ce
qui explique leur présence plus importante au niveau de la périphérie tumorale. Des ligands de ces
récepteurs ont déjà été décrits : la séquence peptidique cyclique -RGD- qui a une forte sélectivité
pour l’intégrine αvβ3, et le ligand peptidique ATWLPPR qui lui est spécifique de la neuropiline-1.
Nous avons ainsi synthétisé des systèmes macromoléculaires sophistiqués présentant les ligands de
ciblage -RGD- et ATWLPPR conjugués ensemble à un fluorophore du type Cyanine 5.5®. Pour cela,
nous avons utilisé des châssis moléculaires cyclodécapeptidiques [1] comportant diverses fonctions
chimiosélectives orthogonales permettant des ligations via des cycloadditions de Huisgen ou des
formations de liens oxime.[2] Ceci a permis de concevoir des vecteurs fluorescents avec un double
ciblage de la périphérie tumorale (figure 1). Plusieurs structures ont été réalisées pour tester l’impact
du double ciblage. Des tests in vitro (cytométrie de flux, Western Blot, microscopie) et in vivo
(biodistribution) permettront de valider le/les composés candidats pour l’aide à la chirurgie.
RGD
ATWLPPR
ligand
Cy 5.5
Peptidic scaffold
Figure 1 : schéma d’un vecteur fluorescent avec double
ciblage
1. Boturyn D. et al. (2008) J. Pept. Sci. 14, 224-240
139
Quantification of the octadecaneuropeptide ODN in complex biological
matrices
Marie-Laure Walet-Balieu,1,2 Philippe Chan,1 Rhita Lamtahri,2 Julien Chuquet,2 Jérôme
Leprince2 and David Vaudry1,2
1
PISSARO Platform, Institut de Recherche et d’Innovation Biologique (IRIB), University of Rouen, 76821 Mont2
Saint-Aignan, France; INSERM U982, Laboratoire de Différenciation et Communication Neuronale et
Neuroendocrine, IRIB, Université de Rouen, Place Emile Blondel, 76821 Mont-Saint-Aignan, France.
Octadecaneuropeptide (ODN, QATVGDVNTDRPGLLDLK) is an endogenous peptide with potent in
vitro neuroprotective properties1. Its quantification at very low levels in biological tissues was
needed to study its effects in vivo. Nevertheless, identification and quantification of peptides in
complex matrices such as cerebrospinal fluid (CSF) is still challenging and requires development. For
ODN quantification, a heavy form of the peptide ([U-13C,15N-Leu15]ODN) was added to samples which
were then purified by extraction on C18 ziptips before elution with H2O/CH3CN 40/60 v/v. Then,
samples were analysed on an Agilent 6490 Triple Quadrupole using the MRM (Multiple Reaction
Monitoring)-based detection system. The Optimizer software was used to determine MRM
parameters of the compounds, especially transitions (couple precursor ion – product ion) and
combined collision energies. The MRM acquisition allowed to achieve quantification ranges of 1
fmol/µL to 500 fmol/µL in the presence of BSA (Bovin Serum Albumin) and of 5 fmol/µL to 500
fmol/µL in rodent CSF. Despite the complexity of the matrix, the limit of quantification was very low
through the Agilent 6490 LC-QQQ system due to its ion funnel technology. The quantification of
other peptides such as PACAP and small organic compounds in different complex matrices (CSF,
plasma, biological tissues) is now being implemented.
Supported by the Interreg PeReNE project, INSERM and the Région Haute-Normandie.
1. Kaddour H. et al. (2013) J. Neurochem. 125, 620-633.
140
Arginine-rich cell penetrating peptides: the role of number and position
of tryptophan residues
Astrid Walrant,1 Natpasit Chaianantakul ,1,2 Manjula Pallerla,1 Fabienne Burlina1 and
Sandrine Sagan1
1
Sorbonne Universités - UPMC Univ Paris 06, École Normale Supérieure-PSL Research University, Département
2
de Chimie, CNRS UMR 7203 LBM, Paris, France; Department of Medical Technology, Faculty of Allied Health
Science, Naresuan University, Phitsanulok, Thaïlande.
Cell-penetrating peptides (CPPs) have attracted much attention in the recent years for their unique
ability to enter cells in a receptor-independent manner and their potential to convey biologically
active molecules into cells. They are short sequences containing a great number of positively charged
residues. Their uptake mechanism is still heavily debated, but it is now accepted that different
pathways (endocytosis or direct translocation through the membrane) can be used in parallel for
peptide internalisation. The use of these different entry routes suggests that molecular partners at
the plasma membrane, such as membrane phospholipids or cell surface glycosaminoglycans (GAGs),
bind CPPs with different affinity or selectivity. Thus the interaction between the CPP and the cell
membrane is a preliminary key point to the entry of the peptide into the cell, and the analysis of the
peptide/membrane interactions should lead to a better understanding of the molecular mechanisms
underlying CPP internalisation.
R6W3-1, also called RW9, (RRWWRRWRR-NH2) is a potent and well characterised peptide which was
derived from a structure/function study of the CPP Penetratin. Previous studies showed that the
tryptophan residues in Penetratin are essential for its efficient internalisation.1 Similarly, substitution
of one or more tryptophan residues in R6W3-1 by leucines2 or phenylalanines3 strongly compromises
its cell uptake efficacy.
In order to further study the role of tryptophan residues, three nonapeptides containing arginine and
tryptophan residues at different positions were designed: R6W3-2 (RRWRRRRWW-NH2), R7W2-1
(RRRWRRWRR-NH2) and R7W2-2 (RWRWRRRRR-NH2). R6W3-1 and 2 have three tryptophans
whereas R7W2-1 and 2 have only two. Additionally R6W3-1 and R7W2-1 both have a potential
amphipathic helical secondary structure whilst the tryptophan residues on R6W3-2 and R7W2-2 are
distributed evenly around the putative helix.
Internalisation of these peptides in CHO cell lines possessing or not cell surface GAGs is evaluated
and preliminary results suggest that sequences with more tryptophans are internalised more
efficiently regardless of the position of these residues. The interactions of these four model peptides
with model lipid membranes and heparin are characterised using a set of biophysical techniques to
get more insight on the different molecular interactions underlying the differences in internalisation
levels between the four peptides.
1. Derossi D. et al. (1994) J. Biol. Chem. 269, 10444-10450
2. Walrant A. et al. (2011) BBA Biomembranes. 1808, 382-393
3. Jobin M. L. et al. (2015) BBA Biomembranes. 1848, 593-602
141
Study and development of robust and widely applicable chemical
strategies for toxin production
Claude Zoukimian,1 Michel De Waard,2 Rémy Béroud2 and Didier Boturyn1
1
Département de Chimie Moléculaire, UMR CNRS 5250, Université de Grenoble, Grenoble;
Biotechnology, 570 rue de la chimie, Saint-Martin-d’Hères.
2
Smartox
Venoms result from thousands years of evolution and provides venomous species with different
cocktails of hundreds of peptides and proteins, named toxins. Taking the number of venomous
species (more than 100,000) into account, venoms constitute a bank of natural molecules with a
wide range of pharmacological properties. Indeed, each toxin shows a particular affinity regarding its
target, generally an ion channel, and so a high efficiency to inhibit it. Considering this, toxins are
knowing an increasing interest in the medical field since several decades.
Smartox Biotechnology is a company specialized in studying venoms, identifying toxins of interest
and providing corresponding synthetic molecules. A part of its activity relies on its ability to
synthesize properly and quickly peptides of different sizes showing a wide range of physicochemical
properties. The increasing need for longer and more complex toxins leads Smartox Biotechnology to
settle a R&D project in order to develop a robust and widely applicable method to synthesize toxins
from 50 to more than a hundred of amino acids.
Naturally, this project turns towards the study of several methods of fragment ligations via an amide
bond. One of the possible strategies is the Native Chemical Ligation (NCL), introduced by Kent and
co-workers in 1994.1 NCL permits to chemoselectively link a thioester fragment with the terminal
cysteine residue of a second fragment, thanks to a trans-thioesterification followed by an S-to-N acyl
shift. The main advantage is the chemoselectivity of this method, permitting the use of unprotected
fragments, however requiring a well-placed cysteine in the final peptide. Another strategy is to use
protected fragments to complete the ligation reaction, for instance by using an activated carboxyl
group and an amine terminated fragment, in solution2 or on a solid support.3
In this context, herein we present the comparison of different chemical ligation (yields, costs,
robustness, and setting up facility) to access to toxins.
1. Dawson P. E. et al. (1994) Science 266, 776-779
2. Schneider S. E. et al. (2005) J. Peptide Sci. 11, 744-753
3. Vasileiou Z. et al. (2010) Biopolymers 94, 339-349
142

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